diff --git a/medical/md/PMC10028668.md b/medical/md/PMC10028668.md new file mode 100644 index 0000000000000000000000000000000000000000..990ab54c25174c66fd8bc053880c52ffdb0c28be --- /dev/null +++ b/medical/md/PMC10028668.md @@ -0,0 +1,400 @@ +# Artifact Removal From Semg Signals Recorded During Fully Unsupervised Daily Activities + +Digital Health + +![0_image_0.png](0_image_0.png) Volume 9: 1–16 © The Author(s) 2023 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/20552076231164239 journals.sagepub.com/home/dhj Álvaro Costa-García1 , Shotaro Okajima1, Ningjia Yang1 and Shingo Shimoda1 + +## Abstract + +Objective: In this study, we propose a method for removing artifacts from superficial electromyography (sEMG) data, which have been widely proposed for health monitoring because they encompass the basic neuromuscular processes underlying human motion. + +Methods: Our method is based on a spectral source decomposition from single-channel data using a non-negative matrix factorization. The algorithm is validated with two data sets: the first contained muscle activity coupled to artificially generated noises and the second comprised signals recorded under fully unsupervised conditions. Algorithm performance was further assessed by comparison with other state-of-the-art approaches for noise removal using a single channel. + +Results: The comparison of methods shows that the proposed algorithm achieves the highest performance on the noiseremoval process in terms of signal-to-noise ratio reconstruction, root means square error, and correlation coefficient with the original muscle activity. Moreover, the spectral distribution of the extracted sources shows high correlation with the noise sources traditionally associated to sEMG recordings. + +Conclusion: This research shows the ability of spectral source separation to detect and remove noise sources coupled to sEMG signals recorded during unsupervised daily activities which opens the door to the implementation of sEMG recording during daily activities for motor and health monitoring. Keywords Electromyography, daily logging, muscle activity, health monitoring, artifact removal Submission date: 10 October 2022; Acceptance date: 1 March 2023 + +## Introduction + +The adoption of new technologies by large population groups has paved the way for the emergence of what is known as the "digital health era", in which people are encouraged to take up new digital media technologies to engage in health self-monitoring.1,2 This trend has been further accentuated since 2020, when the COVID-19 pandemic triggered a situation in which people were requested to socially distance and reduce visits to hospitals and other health centers.3,4 Governments and health organizations have promoted the development and implementation of self-monitoring networks to reduce health care system burden and improve societal health.5–7 Feasible self-monitoring is usually based on cheap and easy-to-use technologies that actually target societal needs. Some examples are self-measurement of blood glucose by people with diabetes8 and heart beat monitoring for athletes and people with heart disease.9,10 It is likely that new self-monitoring technologies will be adopted by the general populace in the coming years. 1Intelligent Behavior Control Unit, RIKEN Institute, Nagoya, Japan Corresponding author: Álvaro Costa-García, Intelligent Behavior Control Unit, RIKEN Institute, Nagoya, Japan. Email: alvaro.costagarcia@riken.jp Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/ +open-access-at-sage). + +![1_image_0.png](1_image_0.png) + +![1_image_1.png](1_image_1.png) + +The monitoring of muscle activity during daily life has been proposed for the early diagnosis and treatment of the age-related loss of muscle mass known as sarcopenia,11 head, neck, and back pain,12 and anxiety.13 Moreover, superficial electromyography (sEMG) has been extensively studied and correlated to the basic neuromuscular processes underlying human motion.14 For these reasons, daily logging of sEMG signals will likely play an important role in the future of health monitoring. + +Although the recording of muscle activity during daily life presents several challenges,15 sEMG can now be acquired using wearable and increasingly inexpensive devices.16–19 In general, sEMG signals present a high signal-to-noise ratio (SNR) compared with other physiological signals such as electroencephalography and magnetic resonance images. + +In the laboratory, it is relatively simple to create experimental conditions in which artifacts affecting sEMG are + +![2_image_0.png](2_image_0.png) + +suppressed or controlled. However, it becomes more challenging in the analysis of large amounts of data recorded during unsupervised daily tasks. The wide range of environmental and behavioral changes during daily activities generates huge variability in signal quality because the noises affecting muscle recordings during these conditions present heterogeneous distributions and are difficult to predict. Therefore, the identification and removal of noises during unsupervised recordings are one of the main challenges facing the implementation of daily health logging systems based on this technology. + +Noise sources for sEMG signals are mainly divided into three groups: white Gaussian noise (WGN), which has an equally distributed spectrum; power line interference (PLI), which mainly affects 50/60 Hz (depending on the country); +and low-frequency artifacts (LFAs), which include electrocardiography (for electrodes around the chest) and a range of disturbances produced by motion (e.g. electrode vibration, hanging wires, and wireless data loss).20,21 Raw sEMG signals have a bandwidth in the approximate range of 10–200 Hz.22,23 Regular bandpass filtering is normally accepted to reduce LFAs and WGN while a + +![3_image_0.png](3_image_0.png) + +common technique to remove PLI is the application of a 50/60-Hz Notch filter.24,25 However, this approach is not time discriminant; that is, it requires a priori knowledge of the noises affecting the signals and also assumes that the noises have a homogeneous distribution and temporal modulation. These assumptions can be true under controlled conditions but should not be made during unsupervised recordings. + +Previous studies with high-density sEMG (HD-EMG) +arrays have proposed noise removal methods based on source separation approaches such as canonical correlation analysis21,26 and independent component analysis (ICA).27 Their aim was to separate sEMG from noise components by using redundant information recorded from several electrodes placed on the same muscle. These approaches are strongly dependent on the amount of spatial information available. + +For daily logging of sEMG data, the use of HD arrays is not always a feasible option. Apart from the increased cost associated with HD-EMG arrays, not all muscles are large enough to allow multiple simultaneous sEMG recordings. Aware of this issue, Mijovic et al. proposed a generalized noise source separation method from single-channel biological signal recordings that combines empirical-mode decomposition (EMD) (to decompose a signal into spectrally separated sources) and ICA (to extract statistically independent sources).28 This technique was successfully validated with experimental data recorded under laboratory conditions and compared with other similar approaches. However, EMD decomposition assumes that the spectral components extracted are present throughout the signal under analysis. In this case, the limitations of EMD analysis during fully unsupervised conditions are unclear. + +However, it is clear that the development and validation of a method for sEMG artifact removal during fully unsupervised activities is currently lacking a state-of-the-art application of this technology. For this reason, the current work proposes a novel algorithm for the analysis of sEMG data with the two following main goals: (a) the effective detection of noise sources from data recorded during fully unsupervised conditions, and (b) the development of a noise removal technique that allows the discrimination of valid sEMG data from large and highly contaminated signals. + +Our approach targets the separation of muscle activity from noise sources by using a non-negative matrix factorization (NMF) over the signal spectrum. To perform such a decomposition, the NMF algorithm extracts common spectral patterns from a number of signals. Redundancy is achieved by the temporal segmentation of the signal into overlapping epochs. The data used for source extraction are the normalized spectral magnitude of those epochs. + +This article describes the proposed methodology and compares its performance with that of regular bandpass filtering and of the method proposed by Mijovic et al.28 Two different data sets were used to validate methodology performance. A +set of sEMG signals combined with artificially created noises was used to compare the signal reconstruction performance + +![4_image_0.png](4_image_0.png) + +among methods. In addition, to show the effects of each method on real data, a second set of signals recorded during fully unsupervised conditions was used. + +## Materials And Methods Conceptual Source Decomposition + +In general, most of the power of raw sEMG signals is contained in a frequency range between 10 and 200 Hz,22,23 with an approximate energy distribution as presented in Figure 1(A). Although the noise sources might overlap the spectral distribution of sEMG data, they present independent modulation. This means that, with enough information, the spectrum of sEMG signals contaminated with different noise sources (Figure 1(B)) can be decomposed into signal and noise components. Figure 1(C) shows an approximate graphical representation of the spectral distribution associated with the three main noise sources affecting sEMG signals +(LFAs, PLI, and WGN).21 The present work focuses on the detection and removal of these sources by using redundant information extracted from temporally shifted signal epochs. + +## General Workflow + +Figure 2 shows the general workflow of the methodology presented in this work. First, single-/multi-channel signals + +![5_image_0.png](5_image_0.png) + +are segmented (Figure 2—segmentation) and the spectrum of each epoch is computed and normalized (Figure 2—spectral computation). Standardized spectrums undergo a NMF +where the noise sources are decoupled from the sEMG spectrum (Figure 2—source separation). Then, noise source contributions are removed from each epoch, leaving only the contribution related to sEMG activation (Figure 2—filter design/noise removal). Clean epochs are shifted back to the time domain and the final signal is reconstructed by the temporal arrangement of all of the clean epochs. The technical details of each stage are provided in Appendix 1. + +## Noise Removal + +After source decomposition, the spectrum of each epoch +(Epe(f)) can be reconstructed (REpe(f)) as the linear combination of one sEMG source and three noise sources, as in: +REpe(f) = sEMGSourcee(f) + NoiseSourcese(f); (1) +where + +$$s E M G S o u r c e_{e}(f)=s E M G(f)\cdot W_{e}^{s E M G};$$ +e ; (2) +$$N o i s e S o u r c e s_{e}(f)=W G N(f)\cdot W_{e}^{W G N}+L F A(f)$$ +$$\cdot W_{\epsilon}^{L F A}+P L I(f)\cdot W_{\epsilon}^{s E M G},$$ +e ; (3) +where sEMG(f), WGN(f), LFA(f), and PLI(f) are the sources associated with sEMG and each noise, respectively, and WsEMG +e , WWGN +e , WLFA +e , and WPLI +e are the weights of each source associated with the epoch e. From this source-based reconstruction, it is possible to define an epoch-specific filter that removes the noise contributions from each frequency of the original epoch (Epe(f)). The filter is described by the following equation: + +$$S F_{e}(f)=1-{\frac{N o i s e S a u r c e s_{e}(f)}{R E p_{e}(f)}}$$ +$$(4)$$ + +Finally, the clean spectrum of each epoch e is computed as the element-by-element multiplication of the original epoch spectrum Epe(f) by the epoch-specific filter SFpe(f), as in: + +$$C l e a n E p_{e}(f)=E p_{e}(f)\cdot S F_{e}(f)$$ +CleanEpe(f) = Epe(f) · SFe(f) (5) +Method comparison +To test the proposed methodology, the extraction and removal of noise sources was compared among three different methods. The first comprised a regular bandpass and notch filtering (hereinafter, RFilt), the second was based on the approach introduced in this work (hereinafter, FFT-NMF), +and the third used the EMD-ICA approach previously introduced in the literature.28 The technical details of the parameter tuning for each method can be found in Appendix 2. + +The NMF and ICA source separation techniques require a priori selection of the number of sources from which data will be decomposed. Four sources were selected in both methods (FFT-NMF and EMD-ICA) (one source representing the sEMG data and three sources representing WGN, PLI, and LFA noises). Both source separation algorithms return + +![6_image_0.png](6_image_0.png) + +randomly arranged components. Therefore, to avoid biases generated by a visual component arrangement, the sources extracted were rearranged according to their similarity to the model spectrums shown in Figure 3. These signals are a simplifi cation of the spectral magnitudes associated with each expected spectral source. Similarity was tested by computing + +![7_image_0.png](7_image_0.png) + +the coefficient correlation matrix between the sources extracted from each session and the modeled spectral distributions. The highest coefficient values were used to rearrange the data. + +Two data sets were used to validate and compare methodology performance. The first data set included single-channel sessions in which sEMG data recorded under laboratory conditions were combined with different distributions of artificially generated noise sources (including LFAs, PLI, and WGN). The second data set comprised sessions of sEMG data recorded during unsupervised daily activities. + +## Data Set 1: Artificial Noises Artificial Noise Data Generation + +Figure 4 describes the process followed to include artificial noises into sEMG data recorded under laboratory conditions. A 10-min session of sEMG recordings was combined with three types of noises with spectral distributions corresponding to those associated with LFA, PLI, and WGN +noises. + +Noise source modulation and their temporal features were randomized to achieve the unstable behavior expected from unsupervised recordings. Figure 5 describes the details for noise generation and sEMG data acquisition. Figure 5(A) shows a 10-min sEMG signal recorded from the sternocleidomastoid muscle of a healthy subject under laboratory conditions. The subject was instructed to sit in a chair and wait for 10 min. During the waiting period, the subject was allowed to perform any desired postural changes and head motions. The fast-Fourier transform +(FFT) of the data presented in the right graph of Figure 5(A) shows the spectral distribution of the power of the data. + +Figure 5(B) shows the elements required for noise generation. Each noise source was described by two vectors. + +The first was a 10-min normalized noise source (NNS) in which the spectral distribution corresponded with one of the three main noises affecting sEMG data. The second was a 10-min vector defining the noise appearance periods and modulating its amplitude (NAV) during the 10-min session. The element-by-element multiplication of both vectors defined the features and behavior of a single noise source during a single session. Figure 5(C) shows the process followed to generate the NNS. First, a random 10-min vector was generated with values between 1 and +−1 (top-left graph—Figure 5(C)). The spectrum of this vector was computed through a FFT and divided into its magnitude and phase (note that its magnitude behaves as WGN with an equally distributed spectrum). The noise source spectral distributions modeled in Figure 3 were used to fit the computed magnitude into the spectral distribution expected for LFAs, PLI, or WGN. The modified magnitude and its original phase were used to reconstruct the signal in the temporal domain by an inverse FFT (IFFT). Finally, the temporal signal was normalized between 1 and −1. + +For NAV generation (Figure 5(D)), several parameters were considered. Each noise source was set to be active for 80% of the total session duration (8 out of 10 min). + +Moreover, to decide the length and amplitude of each activation period, two vectors were used. These vectors, + +![8_image_0.png](8_image_0.png) + +amplitude vector (AV), and length vector (LV), were defined as: + +$$(6)$$ + +REpe(f) = [MaxAmp, MaxAmp − incA, ... , MinAmp]; +(6) + +$AV=[MaxAmp,\ MaxAmp-incA,\ \ldots,\ MinAmp];$ (6) +$$M i n A m p=\frac{3}{4}\cdot s E M G_{p95};$$ $$M a x A m p=1.5\cdot s E M G_{p95};$$ +incA = MaxAmp − MinAmp 99 ; (9) +LV = [MaxLen, MaxLen − incL, ... , MinLen]; (6) +$$\begin{array}{l}\mbox{MinAmp}=\frac{3}{4}\cdot\mbox{sEMG}_{p95};\\ \mbox{MaxAmp}=1.5\cdot\mbox{sEMG}_{p95};\\ \mbox{incL}=\frac{\mbox{MaxLen}-\mbox{MinLen}}{99};\end{array}\tag{9}$$ +(7) $\frac{1}{2}$ . +$${\mathrm{(8)}}$$ +$$(9)$$ +where sEMGp95 is the 95th percentile of the rectified amplitude from the sEMG signal and EMGLen is the length of + +![9_image_0.png](9_image_0.png) + +the session (10 min). Both vectors contain 100 values arranged in a descending order for AV and an ascending order for LV. Figure 5(D) summarizes the algorithm work-flow to generate the NAV. First, a random amplitude and length are chosen from AV and LV, respectively. In addition, a random starting sample is chosen from the number of samples within the total length of the data. If these randomly chosen values allow the creation of an activation period that fits within the session time and that do not overlap with any other activation period, it gets included in the NAV. The algorithm keeps repeating this loop, adding new activation periods, until the total time of noise activation reaches 80% of the signal length. In addition, a stability index (SI) was defined as a number between 1 and 100. The SI was used to tune the range of available values in AV and LV as: + +$$A V_{\small{\small{available}}}=A V[S I{\small{:}}e n d];$$ $$L V_{\small{\small{available}}}=L V[S I{\small{:}}e n d];$$ + +Therefore, for an SI=100, there will be a single value available for both vectors (AVavailable = AV[100] = MinAmp and LVavailable = LV[100] = MaxLen) and for SI=1, all amplitude and length values defined in equations (6) and +(10) will be available. Modification of this parameter allows tuning of the noise variability both in amplitude and active time. + +## Data Set Generation + +The process described in Figure 4 was repeated 100 times for each SI = [10, 20, 30, 40, 50, 60, 70, 80, 90, 100], +giving a total amount of 1000 10-min sessions in which different distributions of LFA, PLI, and WGN sources were added to the sEMG signal. + +## Method Performance Validation + +$$(10)$$ +$$(11)$$ + +The results obtained from this data set were used to compare the performances of each method regarding the separation of sEMG signals from noise sources. For that purpose, the original SNR data were compared to the SNR predicted from each method after source decomposition. Efficiency in signal and noise reconstruction was quantified by two parameters. The first parameter was the root mean square error (RMSE), computed as: + +$$R M S E={\sqrt{a v g((S_{0}-S_{r})^{2})}};$$ +$$(12)^{\frac{1}{2}}$$ +; (12) + +![10_image_0.png](10_image_0.png) + +where avg((S0 − Sr) +2) is the averaged value of the square difference between the original signal So and the reconstructed signal Sr. This parameter quantifies the energy difference between two signals. The second parameter was the correlation coefficient between the original and reconstructed recordings. This coefficient quantifies, with a value between +−1 and 1, the similarity in the shapes of the two vectors. + +## Data Set 2: Unsupervised Semg Recordings Experimental Set-Up + +Unsupervised sEMG data were acquired using a medium density band recording 8 channels from 16 bipolar electrodes (Figure 6(B)) with a sampling frequency of 2000 Hz. A single electrode located in the area of the sternocleidomastoid muscle was selected for analysis. The system included a belt pouch with a battery providing autonomy of around 10 h. Every 10 min, a data file was stored in a SD card (Figure 6(C)). + +## Participants + +Five patients with neck pain were requested to wear the sEMG band shown in Figure 6(A). They were instructed on how to set up the recording device and all of them provided signed informed consent according to the Declaration of Helsinki. No further instructions were given, and the participants were asked to continue with their normal daily activities. + +## Data Set + +Each patient recorded around 5 h of muscle activity. Thirty 10-min recordings were extracted from each patient, giving a total of 150 10-min sessions (30 sessions × 5 patients). + +## Data Evaluation + +This data set was used to visualize the spectral distribution of sEMG and the noise sources extracted by the RFilt, FFT-NMF, and EMD-ICA methods during real unsupervised sEMG recordings. Moreover, the separability between spectral component pairs was quantified for each method as their Bhattacharyya distance (Bdist), first introduced by Bhattacharyya et al.29 This parameter is closely related to the Bhattacharyya coefficient, which measures the amount of overlap between two statistical samples or populations. + +Bdist can provide any value in the range 0 < Bdist < ∞, +with larger values associated with higher class separability. + +## Details Of The Study + +Patients' sEMG data was fully recorded by them during their daily lives in Saitama and Tokyo Prefectures in Japan during periods that varied between 4 and 17 days. + +The conceptualization of the experiment, generation of artificial noises, and recordings of clean sEMG signals were performed at the same time in the facilities of Riken in Nagoya. Data analysis, results extraction, and interpretation were done during the following months also in Riken's laboratory in Nagoya. + +## Results Semg Reconstruction Performance + +The signal/noise decomposition performance for each method was assessed by using the data set including real sEMG data coupled to artificially created noises (data set 1) in which the original sources and SNR were known. Figure 7(A) compares the SNR of the original data with that predicted from source decomposition using the RFilt, FFT-NMF, and EMD-ICA methods. Each boxplot represents the SNRs of 100 10-min sessions associated with different values of the SI, as shown on the x-axis (original SNR, +blue; FFT-NMF-predicted SNR, green; EMD-ICA-predicted SNR, red; RFilt-predicted SNR, orange). As designed, the real SNR (blue) decreases with an increasing variability of noise appearance. The predicted SNRs presented significant differences for the RFilt, FFT-NMF, and EMD-ICA methods, both compared with original SNR and among each other. However, the SNR predicted by the FFT-NMF algorithm exhibited higher accuracy and less variability that predicted by the RFilt and EMD-ICA methods, which showed a significantly higher standard deviation for all values of the SI. Moreover, the RFilt method presented significantly lower performance in SNR prediction and was also more affected by noise stability than the methods based on source decomposition. + +Figure 7(B) and (C) uses the same boxplot format to show the RMSE and correlation coefficients between the original sEMG data and the sEMG data reconstructed by each method (FFT-NMF, green; EMD-ICA, red; and RFilt, orange). For each SI value, the FFT-NMF method showed less RMSE (error recovering the energy of the original data) and a higher correlation coefficient (shape similarity to original data) than the RFilt and EMD-ICA +methods. The increase in the noise variability (a decrease in the SI) produced, in the three methods, a decrease in the energy and shape reconstruction performance. This phenomenon was stronger in the sEMG data extracted using the RFilt method. Moreover, the FFT-NMF algorithm showed a more stable reconstruction (lower standard deviation) +for lower stability values (0.7≥SI≥0.1). + +## Real Data Analysis + +Extracted sources. Data set 2 was used to compare the signal decomposition results from the FFT-NMF and EMD-ICA algorithms applied to data recorded from the sternocleidomastoid muscle during unsupervised tasks. In Figure 8(A) and (B), the four spectral components extracted using each method are presented according to their median +(red), 25th and 75th percentiles (blue), and maximum and minimum values (back) computed from the set of 150 sessions (30 10-min session×5 subjects). Spectral components were arranged according to their correlation coefficient with the sources modeled in Figure 3 that represent a simplification of the spectral distributions expected for the sEMG +and LFA, WGN, and PLI sources. Figure 8(C) shows the Bhattacharyya distances computed between each pair of components extracted using each method. The spectral distributions of sources extracted using the FFT-NMF method showed higher similarity with the conceptual decomposition of the data into four sources representing the sEMG data and WGN, PLI, and LFA sources. Moreover, Bhattacharyya distances exhibited higher class separability for component pairs extracted using the FFT-NMF method. Figure 8(D) shows an example of the FFT-NMF algorithm reconstructing the original sEMG signals recorded under a controlled environment from versus their version where with the addition of artificial noises were added. + +Clean data and sEMG estimation. Figure 9 shows the results of the use of the FFT-NMF algorithm to remove the noise contributions from two 10-min recordings from the sternocleidomastoid muscle during fully unsupervised tasks. The graphs on the left show the raw data (top graph), the clean sEMG signal (blue) +original signal reconstruction (OSR) (orange) (middle graph), and the epoch-wise predicted SNR (bottom graph). The right graphs show a zoom-in of selected periods that allows a clear visualization of the effects of the noise removal technique. Both for raw signals (two upper graphs) and clean data (two lower graphs), a time/amplitude and spectrogram representation are shown. Three signal periods labeled as 1∗, 2∗, and 3∗ are highlighted from both examples to discuss algorithm effects. 1∗ shows an example in which the main contribution of the algorithm is the removal of WGN (affecting mainly the higher frequency bands), allowing a clear visualization of the muscle activity after cleaning. 2∗ shows an example in which the strong coupling of PLI is removed by the algorithm, leaving only the part in which a small sEMG activation happened. 3∗ shows an example of the ability of the FFT-NMF algorithm to removing the appearance of a LFA that produced low-frequency variations in the raw data. The OSR ratio and SNR provide extra information about the relative power between the predicted sEMG signals and noises. Both ratios can be used for a time-wise estimation of the physiological meaning of cleaned signals. + +Finally, Figure 10 shows an example of the RFilt, FFT-NMF, and EMD-ICA methods applied to a highly contaminated session where most of the recorded data was noise. + +The blue data represent the raw recorded data while the + +## Discussion + +orange data represent the signal after noise removal for each method. The effects on the spectral domain can be seen in the respective FFTs presented in the right graphs. + +should carefully check the origins of the LFA noises coupled to their signals. Given the multi-origin nature of these noises, the fine-tuning of their reference spectral distribution could be vital for the proper detection and removal of these artifacts. + +## Conclusion + +This research shows the ability of spectral source separation to detect and remove noise sources coupled to sEMG signals recorded during unsupervised daily activities. The algorithm presented showed a better performance than traditional and other state-of-the-art methodologies for noise removal, which opens the door to the implementation of sEMG recording during daily activities for motor and health monitoring. + +As mentioned in the Introduction, social trends toward subject engagement with self-health monitoring and the development of increasingly cheap and wearable sEMG devices put this technology in the early mass adoption stage. Under this scenario, the first big challenge of people in charge of sEMG big data analysis will be artifact removal and discrimination of valid muscle activity. In this regard, the current methodology was developed as a tool for the treatment of large sets of sEMG data recorded during fully unsupervised daily activities. Our method provides a robust basis for the preliminary treatment and classification of data recorded under highly variable conditions. + +Nevertheless, there are still several challenges that remain to be addressed to further improve this implementation. One of the outstanding challenges is in the development and inclusion of hardware-specific modules in the amplification stage of sEMG sensors that apply the proposed algorithm during recording. Moreover, future researches must address the characterization of the spectral distribution of other potential noises affecting sEMG signals. + +Finally, in future works, this methodology will be used to treat sEMG signals recorded during the daily lives of people in age ranges where muscle conditions such as back and neck pain, and sarcopenia present their first symptoms, potentially enabling for their early detection and treatment. The effects of artifact reduction on the later stage of signal processing and analysis of sEMG signals must also be discussed according to the results of future works. Acknowledgments: The authors would like to thank the Toyota Motor Corporation for their promotion of this work and the Saitama Neuropsychiatric Institute for assisting with recruitment of participants for this research. + +Authors' Note: Álvaro Costa-García, National Institute of Advance Industrial Science and Technology, Chiba, Kashiwa, Japan; Shotaro To the best of our knowledge, the current work presents the first method for the effective detection and removal of noise sources from sEMG recordings during fully unsupervised daily activities. The method can be fully automated after establishment of the number of sources (four in the case of this article) and their spectral distributions (Figure 3). Depending on the data set under analysis, the number of sources and the model of their spectral distribution can be tuned to increase method performance. + +The method was compared to traditional signal filtering and to a state-of-the-art approach (EMD-ICA) previously introduced in the literature.28 The FFT-NMF method showed higher performance both when reconstructing the original sEMG data and when predicting the SNR +(Figure 7). The segmentation of the original data into overlapping epochs generates sufficient signal redundancy for the extraction of common spectral sources, which allows its application to both single- and multi-channel recordings. Moreover, epoching decouples time and frequency domains, which improves the detection of sources whose contributions are not stable in time. The results also show that traditional noise removal (RFilt) is more affected by higher noise variabilities than FFT-NMF and EMD-ICA methods (Figure 7). The sources extracted with the FFT-NMF algorithm present a spectral distribution that correlates with the main noises affecting sEMG data +(Figure 8(A)).20 Such a correlation was not found in the sources extracted using the EMD-ICA method, at least when applied to fully unsupervised recordings +(Figure 8(B)). The epoch-specific filters generated by the FFT-NMF method allow fine-tuning of the spectral bands and power reduction in accordance with the noises affecting the data in each time period (Figure 9). + +In addition, OSR and SNR indices allow the detection of temporal periods of interest that can be used to estimate the physiological meaning of the cleaned signals. Finally, the FFT-NMF algorithm allows a signal reconstruction for illustrating which spectral features present a similar distribution to the one expected from sEMG signals (Figure 10). + +The effectiveness of the current algorithm is strongly related to the number of noises coupled to the sEMG signals because the redundancy in their spectral distributions over time allows the extraction of the spectral pattern associated with each noise. The algorithm might present limitations if applied to large data sets that present a very low amount of coupled noises because there will not be enough redundant noise information to extract common spectral patterns between epochs. Moreover, researchers interested in applying the current methodology Okajima, Nagoya University, Aichi, Nagoya, Japan; Shingo Shimoda, Nagoya University, Aichi, Nagoya, Japan. + +Contributorship: ACG and SS researched the literature and conceived the study. ACG, NY, and SO were involved in methodology development, data recording, and analysis. SO and SS contributed to the recruitment of participants. ACG wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript. + +Declaration of conflicting interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. + +Ethical approval: The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Riken (Reference Wako3 28-13). Informed consent was obtained from all participants involved in the study. + +Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the Toyota Motor Corporation and a JSPS KAKENHI grant (number 18K18431). + +Guarantor: ACG accepts responsibility for the overall integrity of the manuscript (including ethics, data handling, reporting of results, and study conduct). + +ORCID iDs: Álvaro Costa-García https://orcid.org/0000-00030097-2793 References 1. Lupton D. The digitally engaged patient: self-monitoring and self-care in the digital health era. Soc Theory Health 2013; 11: +256–270. + +2. Lancaster K, Abuzour A, Khaira M, et al. The use and effects of electronic health tools for patient self-monitoring and reporting of outcomes following medication use: systematic review. J Med Internet Res 2018; 20: e294. + +3. Chakraborty I and Maity P. COVID-19 outbreak: migration, effects on society, global environment and prevention. Sci Total Environ 2020; 728: 138882. + +4. Bayoumy AB, Veugen CC, van Rijssen LB, et al. + +Self-monitoring of the tympanic membrane: an opportunity for telemedicine during times of COVID-19 and beyond. J Otol 2021; 16: 120–122. + +5. Wahlqvist ML. Self-monitoring networks for personal and societal health: dietary patterns, activities, blood pressure and Covid-19. Asia Pac J Clin Nutr 2020; 29: 446–449. + +6. Lim HM, Teo CH, Ng CJ, et al. An automated patient selfmonitoring system to reduce health care system burden during the COVID-19 pandemic in Malaysia: development and implementation study. JMIR Med Inform 2021; 9: +e23427. + +7. Whiting P and Elwenspoek M. Accuracy of self-monitoring heart rate, respiratory rate and oxygen saturation in patients with symptoms suggestive of covid infection. + +8. Benjamin EM. Self-monitoring of blood glucose: the basics. + +Clin Diabetes 2002; 20: 45–47. + +9. Dooley EE, Golaszewski NM and Bartholomew JB. + +Estimating accuracy at exercise intensities: a comparative study of self-monitoring heart rate and physical activity wearable devices. JMIR Mhealth Uhealth 2017; 5: e7043. + +10. Forerunner G and Fuse M. Comparison of wearables for selfmonitoring of heart rate in coronary rehabilitation patients. + +Georgian Medical 2021; 315: 78–85. + +11. Caroppo A, Rescio G, Leone A, et al. A surface electromyographybased platform for the evaluation of sarcopenia. + +12. Hart JD and Cichanski KA. A comparison of frontal EMG biofeedback and neck EMG biofeedback in the treatment of musclecontraction headache. Biofeedback Self Regul 1981; 6: 63–74. + +13. Viens DA. The effects of self-monitoring, emg biofeedback, and relaxation tapes in the treatment of tension headaches. + +14. Merletti R and Farina D (eds). Surface electromyography: +physiology, engineering, and applications. Hoboken, New Jersey, USA: John Wiley & Sons, 2016. + +15. Milosevic B, Benatti S and Farella E. Design challenges for wearable EMG applications. InDesign, Automation & Test in Europe Conference & Exhibition (DATE), 2017 2017 Mar 27 (pp. 1432-1437). IEEE. + +16. Cerone GL, Botter A and Gazzoni M. A modular, smart, and wearable system for high density sEMG detection. IEEE Trans Biomed Eng 2019; 66: 3371–3380. + +17. Pino EJ, Arias Y and Aqueveque P. Wearable EMG shirt for upper limb training. In2018 40th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) 2018 Jul 18 (pp. 4406-4409). IEEE. + +18. Yamaguchi T, Mikami S, Maeda M, et al. Portable and wearable electromyographic devices for the assessment of sleep bruxism and awake bruxism: a literature review. CRANIO® +2020; 41(1): 69–77. + +19. Michelsen JS, Lund MC, Alkjær T, et al. Wearable electromyography recordings during daily life activities in children with cerebral palsy. Dev Med Child Neurol 2020; 62: 714–722. + +20. Clancy EA, Morin EL and Merletti R. Sampling, noisereduction and amplitude estimation issues in surface electromyography. J Electromyogr Kinesiol 2002; 12: 1–6. + +21. Al Harrach M, Boudaoud S, Hassan M, et al. Denoising of HD-sEMG signals using canonical correlation analysis. Med Biol Eng Comput 2017; 55: 375–388. + +22. Wakeling JM and Rozitis AI. Spectral properties of myoelectric signals from different motor units in the leg extensor muscles. J Exp Biol 2004; 207: 2519–2528. + +23. Von Tscharner V and Goepfert B. Estimation of the interplay between groups of fast and slow muscle fibers of the tibialis anterior and gastrocnemius muscle while running. J +Electromyogr Kinesiol 2006; 16: 188–197. + +24. De Luca CJ, Gilmore LD, Kuznetsov M, et al. Filtering the surface EMG signal: movement artifact and baseline noise contamination. J Biomech 2010; 43: 1573–1579. + +25. Wang J, Tang L and Bronlund JE. Surface EMG signal amplification and filtering. Int J Comput Appl 2013; 82: 15–22. + +26. Anand D, Bhateja V, Srivastava A, et al. An approach for the preprocessing of EMG signals using canonical correlation analysis. InSmart Computing and Informatics: Proceedings of the First International Conference on SCI 2016, Volume 2 2018 (pp. 201-208). Springer Singapore. + +27. Azzerboni B, Carpentieri M, La Foresta F, et al. Neural-ICA +and wavelet transform for artifacts removal in surface EMG. In2004 IEEE International Joint Conference on Neural Networks (IEEE Cat. No. 04CH37541) 2004 Jul 25 (Vol. 4, pp. 3223-3228). IEEE. + +28. Mijović B, De Vos M, Gligorijević I, et al. Source separation from single-channel recordings by combining empirical-mode decomposition and independent component analysis. IEEE Trans Biomed Eng 2010; 57: 2188–2196. + +29. Bhattacharyya A. On a measure of divergence between two multinomial populations. Sankhyā: The Indian J Stat 1946; 7(4): 401–406. + +30. Zhang Y and Fang Y. A NMF algorithm for blind separation of uncorrelated signals. In2007 International Conference on Wavelet Analysis and Pattern Recognition 2007 Nov 2 (Vol. 3, pp. 999-1003). IEEE. + +## Appendix 1 Fft-Nmf Method Stages + +Segmentation. Signals are divided into L sample epochs with O sample overlap. The length of the window (L) can be modified depending on the bandwidth of interest and the sampling frequency and following the Nyquist criteria +(Fmax < Fsamp/2). The selection of the overlap size (O) +will depend on the desired temporal accuracy. Wider overlaps will result in a larger number of epochs, which leads to an increase in the amount of redundant information for the source separation stage by also increasing the computational demands. + +Spectrum computation. The spectrum of each epoch is computed through the FFT and divided into its magnitude +(MAG) and phase (PHA). Phases are kept for the temporal reconstruction of the data (see the "Signal reconstruction" section) and the magnitudes are normalized as: + +$$N M a g_{e}={\frac{M a g_{e}}{\sum_{i=1}^{n}M a g_{e}^{i}}}$$ +$$(17)$$ + +where Mage is a vector containing the spectral magnitude of the epoch e, NMage is a vector containing i normalized frequencies, and n is the total number of frequencies under analysis (i = 1, 2, 3, ... , n Hz). This process normalizes the NMage area to 1. The goal is to remove the amplitude variations and emphasize the shape of the spectrum. While the amplitude of noises might experience large variations during unsupervised recordings, their spectral shape will present higher stability. Therefore, it is expected that the extraction of components by the source separation algorithm should perform better when applied to normalized spectral shapes. The amplitude information associated with each epoch e (ae = n i=1 Magie) will be used for filter design during the noise removal stage (see the +"Noise removal" section). + +Source separation. To decompose the data into x = 1 + N +sources (one sEMG component and the N noise components), +the set of normalized spectral epochs was described as: + +$$E=W\cdot S\tag{18}$$ $E\in R^{\rm c\!xf},\ \ W\in R^{\rm c\!cx},\ \ S\in R^{\rm x\!cf}$ (19) +where E is an e × f matrix of normalized spectrums (with e the number of epochs and f the frequencies under analysis), W +is an e × x matrix containing the weights used to reduce the e epochs to an x-dimensional space, and S is an x × f matrix containing the shape of the x spectral sources. Matrices W and S can be calculated from E through the NMF algorithm by fixing the x-dimensionality. The NMF algorithm extracts the S component matrix by minimizing the correlation between them.30 Signal reconstruction. Signal reconstruction is divided into two stages. In the first stage, the clean spectral magnitude +(CMage) and the original phase (PHAe) of each epoch e are used to reconstruct their temporal information through an IFFT. This inverse transformation applied to discrete data results in the appearance of amplitude spikes at the beginning and end of each epoch that were not present in the original signal (Figure 11(A)). In the second stage, the time series of the clean epochs are recombined to reconstruct the sEMG signal. To remove the spikes appearing in the temporal reconstruction of each epoch, two recombination strategies are proposed (Figure 11(B) and (C)). + +The first strategy (Figure 11(B)) requires that, during the segmentation process, the overlap length is selected as half of the window length (O =L/2). This allows two reconstructions of the original signal, either using the even epochs or using the odd epochs. The spikes of each reconstructed signal will be shifted O =L/2 samples. The final sEMG +data are reconstructed by substituting the spike samples of the signal reconstructed from even epochs with the same temporal samples of the signal reconstructed from odd epochs (which do not include the spikes). The second reconstruction strategy requires larger overlaps between segments +(O > L/2) so that the sEMG signal can be reconstructed an X +number of times, as presented in Figure 11(C). Once all of the signals are aligned in time, the final sEMG reconstruction is obtained by computing the sample-by-sample median value across all of the extracted vectors. Although this second strategy uses more information for an accurate signal reconstruction, the increased overlap produces an increased number of epochs, which leads to higher computational demands during the source separation stage. + +Estimation of the sEMG contribution. To perform a time-wise identification of the SNR, the following index was computed: + +## Snre = Cmage Nmage(20) + +where SNRe is the ratio of the epoch e between the nonnormalized spectral magnitude CMage after noise removal and the summation of the non-normalized spectral magnitudes of the noise sources (see NoiseSourcese(f) in equation +(3)). Moreover, the ratio between the signal before and after denoising was computed as: + +$$O S R_{e}={\frac{C M a g_{e}}{M a g_{e}}}$$ +Mage(21) +where OSRe is the ratio of the epoch e between the nonnormalized spectral magnitudes CMage (after noise removal) and Mage (before noise removal). This index represents the percentage of the contribution of the predicted muscle activity to the original signal. + +## Appendix 2 Method Parameters + +Rfilt method. Each 10-min session was bandpass-filtered between 10 and 200 Hz to isolate the frequency range commonly associated with sEMG activity.22,23 In addition, a Notch filter was applied at 50, 100, and 150 Hz to remove the main contribution and harmonics of PLI. + +FFT-NMF method. The parameters for the application of the FFT-NMF method are the following: + +$$(21)$$ + +Segmentation stage. Window size (L) of 2000 samples +(1 s) and overlap (O) of 1000 samples (0.5 s). + +Spectral computation stage. Evaluation of the spectral range between 1 and 1000 Hz to meet Nyquist criteria. + +Normalized spectral magnitudes were submitted to the source separation stage. + +Source separation stage. Signals were decomposed into four different sources (x=4). One source represented muscle activity while the other three sources represented the main types of noise affecting sEMG signals (LFAs, PLI, and WGN). + +Reconstruction stage. To keep the computational demands low during the source separation stage, the first reconstruction strategy (see Appendix 1) was applied. + +sEMG identification. SNR and OSR (see Appendix 2) +were computed by comparing the original data and the clean data to provide a quantifiable threshold for the discrimination of valid sEMG signals. + +EMD-ICA method. Temporal data from each 10-min session was decomposed into physically significant components based on EMD. ICA was applied over these modes to obtain four statistically significant sources (one representing the sEMG data and three representing WGN, PLI, and LFA noises). The spectral magnitude of the four sources extracted using the ICA were computed through a FFT with a resolution of one sample per frequency (from 1 to 1000 Hz) to compare the extracted sources in the same domain as the ones extracted using the FFT-NMF method. \ No newline at end of file diff --git a/medical/md/PMC10099623.md b/medical/md/PMC10099623.md new file mode 100644 index 0000000000000000000000000000000000000000..9fd45350dd4f08fb65006e3e2f4d6fa977e968e8 --- /dev/null +++ b/medical/md/PMC10099623.md @@ -0,0 +1,286 @@ +DOI: 10.1111/ene.15629 + +# Sex Differences In Imaging And Clinical Characteristics Of Patients From The Wake-Up Trial + +Anke Wouters1,2,3 | Lauranne Scheldeman2,3,4 | Hannelore **Liessens4** | Patrick Dupont5,6 | Florent Boutitie7 | Bastian Cheng8 | Martin **Ebinger9,10** | Matthias Endres9,11,12,13,14 | Jochen B. Fiebach9 | Christian Gerloff8 | Keith W. **Muir15** | Norbert Nighoghossian16 | Salvador Pedraza17 | Claus Z. **Simonsen18** | Vincent Thijs19,20 | Götz Thomalla8 | Robin **Lemmens2,3,4** +1Neurology, Amsterdam University Medical Centers location AMC, Amsterdam, the Netherlands 2Department of Neurosciences, Experimental Neurology, KU Leuven - University of Leuven, Leuven, Belgium 3Center for Brain & Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium 4Department of Neurology, University Hospitals Leuven, Leuven, Belgium 5Department of Neurosciences, Laboratory for Cognitive Neurology, KU Leuven - University of Leuven, Leuven, Belgium 6Leuven Brain Institute, Leuven, Belgium 7Hospices Civils de Lyon, Service de Biostatistique, Université Lyon, Lyon, France 8Klinik und Poliklinik für Neurologie, Kopf- und Neurozentrum, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany 9Center for Stroke Research Berlin (CSB), Charité - Universitätsmedizin Berlin, Berlin, Germany 10Klinik für Neurologie, Medical Park Berlin Humboldtmühle, Berlin, Germany 11Klinik und Hochschulambulanz für Neurologie, Charité– Universitätsmedizin Berlin, Berlin, Germany 12German Center for Cardiovascular Research (DZHK), partner site Berlin, Berlin, Germany 13German Center for Neurodegenerative Diseases (DZNE), partner site Berlin, Berlin, Germany 14ExcellenceCluster NeuroCure, Berlin, Germany 15Institute of Neuroscience & Psychology, University of Glasgow, Glasgow, UK 16Department of Stroke Medicine, Université Claude Bernard Lyon 1; Hospices Civils de Lyon, Lyon, France 17Department of Radiology, Institut de Diagnostic per la Image (IDI), Hospital Dr Josep Trueta, Institut d'Investigació Biomedica de Girona (IDIBGI), Parc Hospitalari Marti i Julia de Salt - Edifici M2, Girona, Spain 18Department of Neurology, Aarhus University Hospital, Aarhus, Denmark 19Stroke Theme, Florey Institute of Neuroscience and Mental Health, University of Melbourne, Heidelberg, Victoria, Australia 20Department of Neurology, Austin Health, Heidelberg, Victoria, Australia Correspondence Anke Wouters, Herestraat 49, 3000 Leuven, Belgium. Email: anke.wouters@kuleuven.be + +## Abstract + +Background and purpose: Sex-based differences in acute ischemic stroke are a wellknown phenomenon. We aimed to explore these differences between women and men in the Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke (WAKE-UP) trial. Methods: We compared baseline demographic and imaging characteristics (visual fluidattenuated inversion recovery [FLAIR] positivity, relative FLAIR signal intensity, collateral status) between women and men in all screened patients. In randomized patients (i.e., those with Anke Wouters and Lauranne Scheldeman contributed equally. + +This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2022 The Authors. *European Journal of Neurology* published by John Wiley & Sons Ltd on behalf of European Academy of Neurology. + +diffusion-weighted imaging (DWI)–FLAIR mismatch), we evaluated a modifying role of sex on the treatment effect of alteplase in multivariable logistic regression, with treatment adjusted for National Institute of Health Stroke Scale (NIHSS) score and age. Dependent variables were modified Rankin Scale (mRS) score of 0–1 at 90 days and distribution of mRS scores at 90 days. Results: Of 1362 screened patients, 529 (38.8%) were women. Women were older than men, had higher baseline NIHSS scores and smoked less frequently. FLAIR positivity of the DWI lesion was equally present in women (174/529, 33.1%) and men (273/833, 33.3%; +p = 1.00) and other imaging variables also did not differ between the sexes. In a total of 503 randomized patients, of whom 178 were women (35.4%), sex did not modify the treatment effect of alteplase on mRS score 0–1 or on the total distribution of mRS scores. + +Conclusion: As in many other stroke trials, more men than women were included in the WAKE-UP trial, but the presence of a visual DWI–FLAIR mismatch and the relative FLAIR +signal intensity did not differ between the sexes. The treatment effect of alteplase was not modified by sex. + +KEYWORDS +ischemic stroke, sex, MRI, wake-up + +## Introduction + +Sex-based differences in the acute stroke setting have been described. Women more often present with non-typical and more severe stroke symptoms and with stroke mimics [1–4]. Furthermore, they may have longer delays from stroke onset to emergency department arrival as well as longer door-to-imaging times, and receive treatment with alteplase less often, despite having no difference in overall treatment eligibility [5–15]. Visual fluid-attenuated inversion recovery (FLAIR) positivity and the relative FLAIR (rFLAIR) signal intensity within the area of a diffusion-weighted imaging (DWI) lesion are related to the time from symptom onset [16, 17]. Therefore differences between men and women with regard to these imaging variables in unknown stroke onset patients could be assumed but are unknown. Furthermore, men and women have similar outcomes after treatment with intravenous recombinant tissue plasminogen activator, but a stronger treatment effect may exist in women and they might have worse outcomes if acute stoke is untreated [18–21]. However, there is substantial between-study variability and more recent studies suggest that the gap between men and women has narrowed over recent years [6, 10]. In this post hoc analysis of the Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke (WAKE-UP) trial [22], we explored sex-based differences in baseline imaging (FLAIR lesion, DWI lesion, rFLAIR signal intensity and quality of collateral circulation) and clinical characteristics in screened patients, and outcome in randomized women versus men. + +## Patients And Methods Study Design And Patient Population + +In this post hoc analysis, we studied sex-based differences in demographic, clinical, and imaging baseline characteristics and clinical outcome in patients screened and randomized in the WAKE-UP trial. The WAKE-UP trial was a multicenter, randomized, doubleblind, placebo-controlled trial, designed to investigate if patients with an unknown time of stroke onset benefit from thrombolysis in the presence of a DWI–FLAIR mismatch [22]. Patients or their legal representatives provided written informed consent according to national and local regulations. The trial was approved for each study site by the competent authorities and the corresponding ethics committee. Patients were screened for the presence of a DWI– FLAIR mismatch with magnetic resonance imaging (MRI; screened patients) and randomized to placebo or alteplase if this mismatch was present (randomized patients). Only randomized patients received follow-up with imaging at 24–36 hours and documentation of clinical outcome. + +## Analysis Of Imaging Characteristics + +As in the original WAKE-UP trial, acute MRI images of the screened patients were visually rated for the presence of a DWI-positive lesion on the B1000 images and for a FLAIR hyperintense signal (i.e., FLAIR-positive) within the region of the acute stroke. The DWI– FLAIR mismatch was defined as a DWI-positive lesion with no signal alterations on the FLAIR [22]. Furthermore, we investigated the maturation of the FLAIR lesion in men versus women in screened patients in the WAKE-UP trial who had FLAIR of sufficient quality at baseline available. The rFLAIR signal intensity was calculated as described previously [23]. To summarize, the rFLAIR signal intensity was calculated as the ratio of the FLAIR signal intensity in one voxel and median FLAIR signal intensity of all voxels within a sphere with radius of 15 mm around a homologue voxel in the contralateral hemisphere. We defined the ischemic core lesion and its volume using RAPID software, which calculates the ischemic core lesion based on the apparent diffusion coefficient threshold <620 × 10−6mm2/s. + +Only patients with a core lesion volume >10 ml were included for further analysis. + +To investigate sex-based differences in the quality of the collateral circulation, we studied the hypoperfusion intensity ratio (HIR) +in patients screened in the WAKE-UP trial who also underwent perfusion-weighted imaging [24, 25]. We used RAPID software (iSchemaView) to calculate the perfusion lesion, defined as tissue with a time to the maximum of the residue function (Tmax) of >6 s. + +For patients with a minimal perfusion lesion of 10 ml, we calculated the HIR as the ratio of the lesion volume with Tmax >10 s and the lesion volume with a Tmax >6 s. We dichotomized the HIR at a threshold of 0.5 to define collateral status: <0.5 as an indicator of good and +≥0.5 of poor collaterals [25]. + +## Statistical Analysis + +The baseline characteristics were compared between men and women in the screened and randomized population using twosample t-tests for parametric data, Wilcoxon rank-sum tests for nonparametric data, and chi-squared tests for categorical data. + +In randomized patients (i.e., those with the DWI–FLAIR mismatch), we evaluated the influence of sex on treatment effect of alteplase in multivariable logistic regression with treatment, sex and their interaction term as independent variables (adjusted for NIHSS +score >10 and age >60 years, as in the original trial) [22] to predict good outcome, defined as a score on the modified Rankin Scale (mRS) of 0–1 at 90 days, and mortality at 90 days. The categorical shift in distribution of the mRS scores was studied with proportional odds logistic regression analysis, adjusted for the same variables. p values < 0.05 were taken to indicate statistical significance. All the analyses were conducted in R. + +## Results Baseline Demographic, Clinical And Imaging Characteristics In Screened Patients + +In the population of 1,362 screened patients, more men (833; 61.2%) were screened than women (p< 0.01). Women were older, had higher baseline NIHSS score, higher baseline mRS score and smoked less frequently (Table 1). Furthermore, they presented more often with aphasia compared to men (9.3% vs. 5.3%; p = 0.01). + +A trend towards a lower proportion of women being randomized compared to men was not statistically significant (178/529 women, 33.6% vs. 325/833 men, 39.0%; p = 0.05). The presence of a hyperintense FLAIR signal within the DWI lesion did not differ between women (174/529, 33.1%) and men (273/833, 33.3%; p = 1.00). Also, the proportion of screened patients without a DWI-positive lesion was similar between the sexes (27.6% of women vs. 24.6% of men; p = 0.52; Table 1). + +TABLE 1 Baseline characteristics of the screened patients. + +Women + +(n = **529)** + +Men + +(n = 833) p **value** + +Age, years 70 (61–76) 67 (57–74) <0.01 + +Arterial hypertension 288 (54.9%) 421 (50.9%) 0.30 Atrial fibrillation 47 (9.0%) 60 (7.3%) 0.53 Hypercholesterolemia 157 (29.9%) 272 (32.9%) 0.52 Type 2 diabetes 75 (14.3%) 149 (18.3%) 0.20 + +Smoking 89 (18.4%) 223 (29.6%) <0.01 + +Pre-stroke mRS score 0.04 + +0 422 (80.1%) 698 (84.9%) 1 99 (18.8%) 112 (13.6%) 2 2 (0.4%) 7 (0.9%) 3 3 (0.6%) 1 (0.1%) + +4 1 (0.2%) 4 (0.5%) + +Median NIHSS score 7 (4–12) 5 (3–9) <0.01 + +Reason for unknown + +time of symptom onset + +Night-sleep 445 (85.1%) 734 (88.9%) Day-sleep 20 (3.8%) 24 (2.9%) Aphasia 49 (9.3%) 44 (5.3%) Other 5 (1.0%) 16 (1.9%) + +Randomization rate 178 (33.6%) 325 (39.0%) 0.05 FLAIR positivity 174 (33.1%) 273 (33.3%) 1.00 DWI negativity 97 (27.6%) 125 (24.6%) 0.52 + +Note: Data are median (IQR) or n (%). + +Abbreviations: DWI, diffusion-weighted imaging; FLAIR, fluidattenuated inversion recovery; IQR, interquartile range; mRS, Modified Rankin Scale; NIHSS, National Institute Health Stroke Scale. + +Analysis of the rFLAIR signal intensity (as a proxy of time from symptom onset to imaging) was successful in 307 patients with min- + +| 0.03 | +|--------| + +imal core lesion of 10 ml. The mean rFLAIR signal intensity did not differ in women (ratio 1.08, interquartile range [IQR] 1.05–1.13) versus men (ratio 1.07, IQR 1.03–1.12; p = 0.31). + +Of 1,362 screened patients, 186 had successful RAPID analysis of perfusion-weighted imaging and a minimal Tmax >6 s lesion of 10 ml. An HIR ≥0.5 suggestive of poorer collateral status did not differ significantly between men and women (40/110 women, 36.3%, vs. + +22/76 men, 28.9%; p = 0.37). + +## Patient Characteristics, Outcome And Treatment Effect In Randomized Patients + +Of 503 randomized patients, 178 were female (35.4%). Baseline and outcome characteristics for men and women are presented in Table 2. Similarly to the screened population, women were older, had higher baseline NIHSS scores and smoked less frequently. Of all TABLE 2 Baseline and outcome characteristics of the randomized patients. + +Women + +(n = 178) + +Men + +(n = 325) p **value** + +Age, years 70 (63–76) 67 (58–73) 0.02 Arterial hypertension 101 (56.7%) 165 (50.8%) 0.20 Atrial fibrillation 29 (16.3%) 30 (9.2%) 0.06 Hypercholesterolemia 67 (37.6%) 111 (34.2%) 0.38 Type 2 diabetes 24 (13.5%) 58 (17.8%) 0.13 Smoking 42 (24.1%) 91 (29.2%) 0.01 Median NIHSS score 7 (4–11) 5 (3–9) 0.01 Reason for unknown + +time of symptom onset + +| randomized patients. Reason for unknown time of symptom onset Core volume at baseline, mla Hemorrhage type PH1c Hemorrhage type PH2c | +|-------------------------------------------------------------------------------------------------------------------------------------------| + +Night-sleep 156 (87.6%) 293 (90.2%) Day-sleep 10 (5.6%) 13 (4.0%) Aphasia 9 (5.1%) 12 (3.7%) Other 3 (1.7%) 7 (2.1%) + +Treatment with IV rtPA 89 (50.0%) 165 (50.8%) 0.94 Core volume at + +baseline, mla + +2.5 (0.96–11) 2.1 (0.73–7.5) 0.14 + +mRS scoreb 2 (1–3) 2 (1–3) 0.69 mRS score 0–1b 76 (44.2%) 157 (49.4%) 0.32 + +Mortalityb 5 (2.9%) 8 (2.5%) 1.00 + +Overall 6 (3.4%) 7 (2.2%) 0.36 Group with IV rtPA 3 (3.4%) 4 (2.4%) 0.83 Overall 2 (1.1%) 8 (2.5%) 0.29 Group with IV rtPA 1 (1.1%) 8 (4.8%) 0.28 + +Note: Data are median (IQR) or n (%). Abbreviations: IQR, interquartile range; IV rtPA, intravenous recombinant tissue plasminogen activator; mRS, modified Rankin Scale; NIHSS, National Institute Health Stroke Scale. + +aFourteen patients had missing data. + +bThirteen patients were lost to follow-up. Outcome parameters were assessed at Day 90. + +cSeven patients had missing data. + +| 0.67 | +|--------| + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +randomized patients, 249 received placebo (35.7% women, 64.3% men) and 254 received recombinant tissue plasminogen activator (35.0% women, 65.0% men). No day 90 outcome was available for 13 patients lost to follow-up. There was no difference between women and men in the rate of good outcome (44.2% vs. 49.4%; p = 0.32), in mortality (2.9% vs. 2.5%; p = 1.00) or in the distribution of mRS +scores at 90 days (adjusted odds ratio [aOR] 1.14, 95% confidence interval [CI] 0.82–1.60; Figure 1). Although the effect of thrombolysis on mRS score 0–1 was significant in men (aOR 1.79 95% CI +1.10–2.91; p = 0.02) but not in women (aOR 1.36, 95% CI 0.74–2.59; p = 0.35), sex did not modify the treatment effect of alteplase on mRS score 0–1 (p = 0.57 for interaction) or on the total distribution of the mRS scores (p = 0.75 for interaction; Figure 2). + +## Discussion + +In this post hoc analysis of the WAKE-UP trial, we found that the proportion of women screened for inclusion in the trial was lower compared to men. Moreover, women were older, had increased stroke severity at admission and smoked less often than men. Presence of DWI–FLAIR mismatch was similar in women and men and there were similar proportions of patients that had no DWI lesion on baseline imaging. Also, we found no difference in other imaging variables, such as rFLAIR signal intensity and quality of the collateral circulation. Outcomes were similar between men and women, and sex did not modify the treatment effect of alteplase on clinical outcome. + +The literature consistently reports that women are older at stroke onset [2, 4, 26]. The most obvious explanation is that women live longer than men, in concert with a higher incidence of stroke with aging. The higher severity of stroke on admission has been attributed to women more often presenting with proximal occlusions, anterior circulation stroke and cardio-embolic strokes, which are more frequently severe compared to other stroke mechanisms [13, 27, 28]. Another explanation might be older age and women more often having worse premorbid functional status [8, 28]. In our population of screened patients, women were indeed older and presented with a higher pre-stroke mRS score. Furthermore, women presented more often with aphasia, possibly reflected in the higher NIHSS score. This sex disparity regarding language disturbance at presentation has been described previously [29, 30]. A smaller stroke lesion volume threshold to cause aphasia for women as compared to men has been postulated as a potential cause [31]. The lower incidence of smoking in female WAKE-UP patients is also consistent with previous literature [32]. Despite the non-significant results in our study, there was also a trend towards a higher frequency of hypertension and atrial fibrillation in women, as described in the literature [5, 8]. + +Collateral circulation as assessed by the HIR was not different between women and men. A recent study showed better collaterals in women, associated with better functional outcome. Women had smaller perfusion lesions, less ischemic growth, and smaller final infarct volumes [33]. Similar results of better collateral circulation, smaller baseline core volumes, and slower ischemic core growth among women were reported in DEFUSE 3 [34]. In WAKE-UP, we included a more heterogeneous population. Many patients had small DWI and perfusion lesions and only approximately one-third of patients presented with large vessel occlusions, hampering a robust analysis of collateral status by sex [22]. + +Fewer women than men were screened with MRI for the presence of a DWI–FLAIR mismatch. One explanation for this could be that women are older when presenting with stroke, exceeding the upper age limit of 80 years as defined by the inclusion criteria in the original WAKE-UP trial. Furthermore, poorer premorbid functionality similarly could have led to exclusion of more women than men because WAKE-UP excluded patients with an mRS score >1 [28]. + +Also the number of women presenting with a wake-up stroke could have been lower compared to men due to different stroke mechanisms. However, because data are lacking on the total number of women presenting to the sites during the trial, this remains speculative. Since sites did not document reasons for not screening patients with unknown stroke onset, the possibility of more atypical symptoms and more risk averseness in women compared to men, although reported by others, could not be assessed [26, 35]. A higher frequency of stroke mimics could be a possible explanation for fewer women being randomized in an MRI-based stroke study. However, in our analysis, lack of DWI lesion as a reason for exclusion was equal between the sexes, which suggests similar presentation of stroke mimics (although some of these patients could have had transient ischemic attacks without a DWI lesion). An overview of sex representation in different fields of stroke research found a similar under-representation of women [36]. The average percentage of female participants is approximately 40% but the reasons for this under-representation in randomized controlled stroke trials has not been extensively investigated [32]. More equal sex representation in randomized controlled trials is desirable but in order to achieve this, better understanding of the factors that limit participation of women is needed. Our data indicate no difference in eligibility based on imaging features. + +The presence of a DWI–FLAIR mismatch was equal in women and men. As reports exist on longer times from stroke onset to emergency department arrival in women, we explored the visual FLAIR positivity and rFLAIR signal intensity within the DWI lesion because this is known to increase with time after symptom onset [9, 17]. However, we did not identify more FLAIR-positive lesions, neither did we identify an increase in the rFLAIR signal intensity in women, arguing against a delay between stroke onset to time of imaging in women with wake-up stroke. + +The treatment effect of alteplase on clinical outcome in patients with unknown onset of stroke and a DWI–FLAIR mismatch was not modified by sex. The rate of excellent functional outcome and mortality was comparable between men and women. There is heterogeneity in the literature about the differences in outcome or effect of alteplase between men and women. Some authors report similar outcomes at hospital discharge and 90-day survival [4, 19], whereas others report worse functional outcome [27] and higher mortality rate in women [5], or even a more favourable in-hospital recovery in women compared to men and higher likeliness of favourable functional outcome at discharge [13]. Different pathophysiological and hormonal-based theories are described to explain the sex differences [19, 20, 27]. Interestingly, sex differences were no longer present in many studies after correction for age, stroke severity and premorbid function [28]. In the WAKE-UP trial, an upper age limit of 80 years and minimal premorbid functionality state were predefined inclusion criteria, possibly reducing the confounding effect of these variables. Furthermore, the analysis of the interaction between sex and treatment effect was adjusted for age and NIHSS score. This revealed the lack of a modifying effect of sex on clinical outcomes in this population. + +Our study has some limitations. First of all, we had no insights into the rationale of each local investigator to decide not to screen patients with unknown stroke onset time with MRI, nor on the total number of female stroke patients presenting to the different study sites. Furthermore, we only had outcome data from the population of randomized patients because screened patients had no follow-up after documentation of the absence of a DWI–FLAIR mismatch. Therefore, we cannot exclude sex-based variation in outcome for wake-up stroke patients without the DWI–FLAIR mismatch at admission. In the population of randomized patients, subgroups may have been too small to study the treatment effect of alteplase in men and women separately. + +In conclusion, in the WAKE-UP trial, fewer women than men were included in MRI screening. Women were older, had higher prestroke mRS scores, higher NIHSS scores at admission, and smoked less frequently. Imaging characteristics did not differ between the sexes. In this post hoc analysis, there was no effect of sex on the clinical outcome after treatment with alteplase. + +## Author Contributions + +lecturer from Acandis, Alexion, Amarin, Bayer, Boehringer Ingelheim, BristolMyersSquibb, Daichii Sankyo, Portola and Stryker. K.W.M. reports fees as a consultant from Boehringer Ingelheim, Abbvie and Biogen, and institutional support from Boehringer Ingelheim for the ATTEST-2 trial, all outside the submitted work. V.T. reports personal fees and non-financial support from Boehringer Ingelheim, Pfizer/ BMS, Bayer, Sygnis, Amgen and Allergan outside the submitted work. All remaining authors report nothing to disclose. + +## Data Availability Statement + +The data that support the findings of this study are available from the corresponding author upon reasonable request. + +## Orcid + +Anke Wouters https://orcid.org/0000-0001-5229-2699 Lauranne Scheldeman https://orcid.org/0000-0002-5263-3550 Keith W. Muir https://orcid.org/0000-0001-9535-022X +Norbert Nighoghossian https://orcid.org/0000-0003-0594-4409 Anke Wouters: Conceptualization; Investigation; Writing—original draft; Methodology; Visualization. Lauranne Scheldeman: Investigation; Writing—original draft; Methodology; Conceptualization; Visualization. Hannelore Liessens: Writing—original draft; Conceptualization. Patrick Dupont: Writing—review and editing; Methodology. Florent Boutitie: Writing—review and editing; Methodology. Bastian Cheng: Writingreview and editing; Data curation. Martin Ebinger: Writing—review and editing. Matthias Endres: Writing—review and editing. Jochen B Fiebach: Writing—review and editing. Christian Gerloff: Writingreview and editing. Keith W Muir: Writing—review and editing. Norbert Nighoghossian: Writing—review and editing. Salvador Pedraza: Writing—review and editing. Claus Z Simonsen: Writing—review and editing. Vincent Thijs: Writing—review and editing. Götz Thomalla: Writing—review and editing. Robin Lemmens: Writing—review and editing; Supervision; Methodology; Conceptualization; Investigation. + +## Funding Information + +The WAKE-UP trial was supported by a grant (278276) from the European Union Seventh Framework Program. M.E. received funding from DFG under Germany´s Excellence Strategy—EXC- 2049—390688087, BMBF, DZNE, DZHK, EU, Corona Foundation and Fondation Leducq. R.L. is a senior clinical investigator for Research foundation Flanders. + +## Conflicts Of Interest + +L.S. reports grants from Research Foundation Flanders during the conduct of the study (L.S. is supported by Research Foundation Flanders, PhD fellowship fundamental research 1193620N) and other (congress participation) from Daiichi Sankyo outside the submitted work. R.L. has no personal disclosures, but reports consultancy fees paid to KU Leuven from Ischemaview and Boehringer Ingelheim. J.B.F. reports outside the submitted work personal fees from Abbvie, AC Immune, Artemida, Bioclinica/Clario, Biogen, BMS, Brainomix, Cerevast, Daiichi-Sankyo, Eisai, F.Hoffmann-La Roche AG, Eli Lilly, Guerbet, Ionis Pharmaceuticals, IQVIA, Janssen, Julius clinical, jung diagnostics, Lysogene, Merck, Nicolab, Premier Research and Tau Rx. C.Z.S. reports research grants from Novo Nordisk Foundation and Health Research Foundation of Central Denmark Region. M.End reports grants from Bayer and fees paid to the Charité from AstraZeneca, Bayer, Boehringer Ingelheim, BMS, Daiichi Sankyo, Amgen, GSK, Sanofi, Covidien, Novartis and Pfizer, all outside the submitted work. G.T. reports fees as a consultant or + +## References + +1. Stuart-Shor EM, Wellenius GA, Dellolacono DM, Mittleman MA. + +Gender differences in presenting and prodromal stroke symptoms. Stroke. 2009;40:1121-1126. + +2. Gall SL, Donnan G, Dewey HM, et al. Sex differences in presentation, severity, and management of stroke in a population-based study. *Neurology*. 2010;74:975-981. + +3. Artto V, Putaala J, Strbian D, et al. Stroke mimics and intravenous thrombolysis. *Ann Emerg Med*. 2012;59:27-32. + +4. Fraticelli L, Freyssenge J, Claustre C, et al. Sex-related differences in management and outcome of acute ischemic stroke in eligible patients to thrombolysis. *Cerebrovasc Dis*. 2019;47:196-203. + +5. Park SJ, Do SS, Ro YS, Song KJ, Oh J. Gender differences in emergency stroke care and hospital outcome in acute ischemic stroke: a multicenter observational study. *Am J Emerg Med*. 2013;31:178-184. + +6. Reeves M, Bhatt A, Jajou P, Brown M, Lisabeth L. Sex differences in the use of intravenous rt-PA thrombolysis treatment for acute ischemic stroke: a meta-analysis. *Stroke*. 2009;40:1743-1749. + +7. Burton KR, Kapral MK, Li S, et al. Predictors of diagnostic neuroimaging delays among adults presenting with symptoms suggestive of acute stroke in Ontario: a prospective cohort study. *Can Med Assoc* Open Access J. 2016;4:E331-E337. + +8. Asdaghi N, Romano JG, Wang K, et al. Sex disparities in ischemic stroke care: FL-PR CReSD study (Florida-Puerto Rico Collaboration to Reduce Stroke Disparities). *Stroke*. 2016;47:2618-2626. + +9. Mainz J, Andersen G, Valentin JB, Gude MF, Johnsen SP. + +Disentangling sex differences in use of reperfusion therapy in patients with acute ischemic stroke. *Stroke*. 2020;51:2332-2338. + +10. Strong B, Lisabeth LD, Reeves M. Sex differences in IV thrombolysis treatment for acute ischemic stroke: a systematic review and meta-analysis. *Neurology*. 2020;95:e11-e22. + +11. Fredwall M, Sternberg S, Blackhurst D, Lee A, Leacock R, +Nathaniel TI. Gender differences in exclusion criteria for recombinant tissue-type plasminogen activator. *J stroke Cerebrovasc Dis*. + +2016;25:2569-2574. + +12. Nagaraja N, Olasoji EB, Patel UK. Sex and racial disparity in utilization and outcomes of t-PA and thrombectomy in acute ischemic stroke. *J stroke Cerebrovasc Dis*. 2020;29:104954. + +13. Bonkhoff AK, Karch A, Weber R, Wellmann J, Berger K. Female stroke: sex differences in acute treatment and early outcomes of acute ischemic stroke. *Stroke*. 2021;52:406-415. + +27. Reeves MJ, Bushnell CD, Howard G, et al. Sex differences in stroke: +epidemiology, clinical presentation, medical care, and outcomes. Lancet Neurol. 2008;7:915-926. + +28. Manwani B, McCullough LD. On the basis of sex. *Stroke*. + +2019;50:2285-2287. + +29. Hier DB, Yoon WB, Mohr JP, Price TR, Wolf PA. Gender and aphasia in the stroke data bank. *Brain and Language*. 1994;47:155-167. + +30. Di Carlo A, Lamassa M, Baldereschi M, et al. Sex differences in the clinical presentation, resource use, and 3-month outcome of acute stroke in Europe: Data from a multicenter multinational hospitalbased registry. *Stroke*. 2003;34:1114-1119. + +31. Bonkhoff AK, Schirmer MD, Bretzner M, et al. Outcome after acute ischemic stroke is linked to sex-specific lesion patterns. *Nat* Commun. 2021;12:1-14. + +32. Carcel C, Woodward M, Wang X, Bushnell C, Sandset EC. Sex matters in stroke: a review of recent evidence on the differences be-tween women and men. *Front Neuroendocrinol*. 2020;59:100870. + +33. Demeestere J, Christensen S, Mlynash M, et al. Effect of sex on clinical outcome and imaging after endovascular treatment of largevessel ischemic stroke. *J Stroke Cerebrovasc Dis*. 2021;30:105468. + +34. Dula AN, Mlynash M, Zuck ND, Albers GW, Warach SJ, DEFUSE 3 Investigators. Neuroimaging in ischemic stroke is different between men and women in the DEFUSE 3 cohort. *Stroke*. 2020;51:481-488. + +35. Kapral MK, Devon J, Winter A-L, Wang J, Peters A, Bondy SJ. Gender differences in stroke care decision-making. *Med Care*. 2006;44:70-80. + +36. Tsivgoulis G, Katsanos AH, Caso V. Under-representation of women in stroke randomized controlled trials: Inadvertent selection bias leading to suboptimal conclusions. *Ther Adv Neurol Disord*. + +2017;10:241-244. + +14. De Silva DA, Ebinger M, Davis SM. Gender issues in acute stroke thrombolysis. *J Clin Neurosci*. 2009;16:501-504. + +15. Tsivgoulis G, Katsanos AH, Malhotra K, et al. Thrombolysis for acute ischemic stroke in the unwitnessed or extended therapeutic time window. *Neurology*. 2020;94:e1241-e1248. + +16. Thomalla G, Cheng B, Ebinger M, et al. DWI-FLAIR mismatch for the identification of patients with acute ischaemic stroke within 4·5 h of symptom onset (PRE-FLAIR): a multicentre observational study. *Lancet Neurol*. 2011;10:978-986. + +17. Cheng B, Brinkmann M, Forkert ND, et al. Quantitative measurements of relative fluid-attenuated inversion recovery (FLAIR) signal intensities in acute stroke for the prediction of time from symptom onset. *J Cereb Blood Flow Metab*. 2013;33:76-84. + +18. Lindley RI, Wardlaw JM, Whiteley WN, et al. Alteplase for acute ischemic stroke: outcomes by clinically important subgroups in the Third International Stroke Trial. *Stroke*. 2015;46:746-756. + +19. Hametner C, Macisaac RL, Kellert L, Abdul-Rahim AH, Ringleb PA, +Lees KR. Sex and stroke in thrombolyzed patients and controls. Stroke. 2017;48:367-374. + +20. Kent DM, Price LL, Ringleb P, Hill MD, Selker HP. Sex-based differences in response to recombinant tissue plasminogen activator in acute ischemic stroke: a pooled analysis of randomized clinical trials. *Stroke*. 2005;36:62-65. + +21. Shobha N, Sylaja PN, Kapral MK, Fang J, Hill MD. Differences in stroke outcome based on sex. *Neurology*. 2010;74:767-771. + +22. Thomalla G, Simonsen CZ, Boutitie F, et al. MRI-guided thrombolysis for stroke with unknown time of onset. *N Engl J Med*. + +2018;379:611-622. + +23. Scheldeman L, Wouters A, Dupont P, et al. Reversible edema in the penumbra correlates with severity of hypoperfusion. *Stroke*. 2021;52:2338-2346. + +24. Olivot JM, Mlynash M, Inoue M, et al. Hypoperfusion intensity ratio predicts infarct progression and functional outcome in the DEFUSE 2 Cohort. *Stroke*. 2014;45:1018-1023. + +25. Guenego A, Mlynash M, Christensen S, et al. Hypoperfusion ratio predicts infarct growth during transfer for thrombectomy. *Ann* Neurol. 2018;84:616-620. + +26. Berglund A, Schenck-Gustafsson K, von Euler M. Sex differences in the presentation of stroke. *Maturitas*. 2017;99:47-50. + +How to cite this article: Wouters A, Scheldeman L, +Liessens H, et al. Sex differences in imaging and clinical characteristics of patients from the WAKE-UP trial. *Eur* J *Neurol*. 2023;30:641-647. doi:10.1111/ene.15629 \ No newline at end of file diff --git a/medical/md/PMC10398594.md b/medical/md/PMC10398594.md new file mode 100644 index 0000000000000000000000000000000000000000..8de80e86c9f513edb3f0c7bd651ae879bcab49cc --- /dev/null +++ b/medical/md/PMC10398594.md @@ -0,0 +1,444 @@ + + +![0_image_0.png](0_image_0.png) + +# Optimizing Point-Of-Care Testing Strategies For Diagnosis And Treatment Of Hepatitis C Virus Infection In Australia: A Modelbased Cost-Effectiveness Analysis + +Sophy T. F. Shih,a,∗ Qinglu Cheng,a Joanne Carson,a Heather Valerio,a Yumi Sheehan,a Richard T. Gray,a Evan B. Cunningham,a Jisoo A. Kwon,a Andrew R. Lloyd,a Gregory J. Dore,a Virginia Wiseman,a,b and Jason Grebelya aThe Kirby Institute, University of New South Wales, Level 6, Wallace Wurth Building, Sydney 2052, Australia bDepartment of Global Health and Development, London School of Hygiene and Tropical Medicine, London, UK +Summary Background Timely diagnosis and treatment of hepatitis C virus (HCV) is critical to achieve elimination goals. This study evaluated the cost-effectiveness of point-of-care testing strategies for HCV compared to laboratory-based testing in standard-of-care. Methods Cost-effectiveness analyses were undertaken from the perspective of Australian Governments as funders by modelling point-of-care testing strategies compared to standard-of-care in needle and syringe programs, drug treatment clinics, and prisons. Point-of-care testing strategies included immediate point-of-care HCV RNA testing and combined point-of-care HCV antibody and reflex RNA testing for HCV antibody positive people (with and without consideration of previous treatment). Sensitivity analyses were performed to investigate the cost per treatment initiation with different testing strategies at different HCV antibody prevalence levels. + +Findings The average costs per HCV treatment initiation by point-of-care testing, from A$890 to A$1406, were up to 35% lower compared to standard-of-care ranging from A$1248 to A$1632 depending on settings. The average costs per treatment initiation by point-of-care testing for three settings ranged from A$1080 to A$1406 for RNA, A$960– +A$1310 for combined antibody/RNA without treatment history consideration, and A$890–A$1189 for combined antibody/RNA with treatment history consideration. When HCV antibody prevalence was <74%, combined pointof-care HCV antibody and point-of-care RNA testing were the most cost-effective strategies. Modest increases in treatment uptake by 8%–31% were required for immediate point-of-care HCV RNA testing to achieve equivalent cost per treatment initiation compared to standard-of-care. + +Interpretation Point-of-care testing is more cost-effective than standard of care for populations at risk of HCV. Testing strategies combining point-of-care HCV antibody and RNA testing are likely to be cost-effective in most settings. + +Funding National Health and Medical Research Council. Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: HCV; Health economics; Modelling; Antibody testing; RNA testing Introduction The ability to meet the World Health Organization's hepatitis C virus (HCV) elimination targets is threatened by low diagnosis (23%)1 and treatment (5% of people diagnosed) globally.2 Increasing HCV testing uptake is hampered by current diagnostic pathways requiring multiple visits, contributing to loss to follow-up.3 In Australia, modelling suggests that HCV RNA testing must increase by 50% annually to achieve elimination by 2030.4 Traditional testing pathways involve an HCV antibody test to confirm exposure and an HCV RNA test to detect active infection. Point-of-care HCV RNA testing for detection of current HCV infection in 1 h has changed HCV clinical management,5,6 enabling diagnosis and treatment in a single-visit,7–11 increasing testing acceptability,12,13 reducing loss to follow-up7–11 and increasing treatment uptake.7–11 Point-of-care HCV +RNA tests have good technical accuracy5,6,14 and clinical utility, with high treatment uptake following testing in needle and syringe programs (NSPs) (81%),7 supervised consumption sites (89%),9 mobile outreach models (74%),8 and prisons (control: 22%; intervention 93%).11 +*Corresponding author. University of New South Wales, Level 6, Wallace Wurth Building, Sydney 2052, Australia. + +E-mail address: sshih@kirby.unsw.edu.au (S.T.F. Shih). + +The Lancet Regional Health - Western Pacific 2023;36: 100750 Published Online 4 April 2023 https://doi.org/10. + +1016/j.lanwpc.2023. 100750 + +## Research In Context + +Evidence before this study We searched several databases, including MEDLINE, Scopus, Web of Science, Cochrane Central Register of Controlled Trials, Embase, Cost-Effectiveness Analysis Registry, International HTA database, Econlit, and PsycINFO, up to 7 April 2022 by the search terms "Hepatitis C" OR "HCV" OR "hepacivirus", and "screen* OR "test" OR "tested" OR "testing" OR "treat*" OR "diagnos*", and "costs and cost analysis" OR "cost*" OR "healthcare resource*" OR "economic*" OR "QALY* OR "ICER* OR "life year*" OR "LY" OR "DALY". Collectively, four protocols of randomized controlled trials, feasibility and cohort studies involving point-of-care HCV testing were identified. Before the empirical evidence and trial-based cost-effectiveness were available, many modelling cost-effectiveness analyses suggested that HCV point-of-care testing could identify more cases, achieve more cures, lead to greater life expectancy with QALY gains. Most modelling studies indicated point-of-care testing was more expensive in the short-term but has the potential to lower the long-term lifetime costs by reducing HCV incidence and prevalence. Two papers reported results of HCV point-of-care testing combined with HIV testing or offered concomitantly to COVID-19 vaccination showing the prospect of point-of-care testing to identify undiagnosed infections at a reasonable cost and a feasible way to implement in practice. Added value of this study This study evaluated the cost-effectiveness of point-of-care testing strategies compared to standard of care in key Australian service settings for HCV elimination, including prisons, needle and syringe programs, and drug treatment clinics. Our analyses determined the cost-effective point-ofcare testing strategies for different HCV antibody prevalence However, point-of-care HCV RNA testing is more expensive than point-of-care antibody testing and has a longer time to result (60 min for an RNA result vs. 1–20 min for a negative antibody result and 1–15 min for a positive antibody result). Combining point-of-care HCV antibody and RNA testing could optimise testing outcomes, enhance cost-effectiveness and acceptability, without impact on effectiveness. + +Many modelling cost-effectiveness studies suggested that HCV point-of-care testing could identify more cases, achieve more cures, lead to greater life expectancy with QALY gains.15–24 However, studies exploring the cost-effectiveness of HCV point-of-care testing strategies are limited to particular populations (mostly low- and middle-income countries),18,19,24 have lacked detailed costing information,17–20 and have not included realworld epidemiological and clinical data to inform analyses.17–20 Understanding the HCV prevalence and settings where different point-of-care testing strategies should be used is critical to inform implementation, levels and the treatment uptake required for point-of-care testing to be more cost-effective than standard of care. This study identifies which point-of-care testing strategies are more cost-effective to implement across service settings and HCV prevalence and provides economic evidence to guide point-of-care testing to optimize treatment outcomes. This information is critical for the implementation and scale-up of point-of-care testing for HCV infection globally and will therefore inform clinical guidelines, clinical practice, and health policy, including national and international guidelines. Implications of all the available evidence Our findings suggested that all point-of-care HCV testing strategies had a lower average cost per treatment initiation compared to standard of care across all settings evaluated and HCV antibody prevalence. Based on HCV prevalence, different testing strategies should be used to optimize the testing and treatment outcomes. When HCV antibody prevalence was +<74%, combined point-of-care HCV antibody and point-ofcare RNA testing (with and without consideration of treatment history) is shown to be the most cost-effective strategies. Modest increases in treatment uptake by 8%–31% +were required for immediate point-of-care HCV RNA testing to achieve equivalent cost per treatment initiation compared to standard of care. Combining point-of-care HCV antibody and RNA testing could optimise testing outcomes, enhance cost-effectiveness by linking more people with current HCV to treatment, and improve patient acceptability by reducing time to receipt of results. This study is important for informing the future implementation and evaluation of point-of-care testing strategies to achieve HCV elimination targets. + +funding, and integration into policy/practice. There is a lack of economic evidence to guide implementation of different testing strategies to optimize the outcomes. + +This study evaluated the cost-effectiveness of pointof-care testing strategies including immediate point-ofcare HCV RNA testing and combined point-of-care HCV antibody and RNA testing for HCV antibody positive people (with and without consideration of previous treatment) compared to standard of care in key service settings for HCV elimination. This study determined the most cost-effective point-of-care testing strategies for different HCV antibody prevalence levels and the treatment uptake required for point-of-care testing to be more cost-effective than standard of care. + +## Methods Economic Evaluation + +This economic evaluation followed the Consolidated Health Economic Evaluation Reporting Standards 2022 +(CHEERS 2022) Statement.25 The PICO (Population, Intervention, Comparator, Outcomes) statement and evaluation parameters are presented in Supplementary Table S1. + +## Study Perspective + +Cost-effectiveness analysis was undertaken from the perspective of the Australian Governments as the funders responsible for the delivery and funding of HCV +testing and treatment, recognising settings where healthcare provision is segregated (e.g., testing in prison is paid by the State government only and testing in the community is paid by the State and Federal governments). + +Study population and setting The target population was people at risk of HCV infection. Study settings included prisons, NSPs, and drug treatment clinics. + +Diagnostic strategies A. Point-of-care HCV RNA testing. + +B. Combined point-of-care HCV antibody and reflex point-of-care RNA testing (for antibody positive people). + +C. Combined point-of-care HCV antibody and reflex point-of-care RNA testing (for antibody positive people) in those self-reporting no previous HCV +treatment and immediate point-of-care RNA testing in people self-reporting previous HCV treatment. + +D. Standard of care (on-site sample collection by venepuncture and laboratory-based HCV antibody followed by RNA testing if antibody positive). +We assumed that people with detectable HCV RNA +following point-of-care testing were offered same-visit treatment and people receiving standard of care required 2–3 visits for testing and treatment.26 + +## Outcome Measured + +Outcomes included: 1) current HCV infection (detectable RNA); and 2) direct-acting antiviral (DAA) treatment initiation in people with current HCV infection. + +The time horizon of analysis was one year, thus discounting was not applied. + +## Analytic Methods + +Decision analytic models were constructed to assess the four HCV diagnostic strategies using the TreeAge software (Fig. 1).27 Further modelling details are provided in the Supplementary Materials. Costs and outcomes for point-of-care HCV testing strategies were compared to standard of care, considering HCV prevalence, treatment history, and test accuracy. Costs and outcomes among the three point-of-care testing strategies were also compared to investigate the most efficient way to conduct point-of-care HCV testing in each setting. + +Cost-effectiveness analyses estimated the average costs of diagnosis and treatment initiation by the four testing strategies in prisons, NSPs, and drug treatment clinics. + +Model parameters for outcomes Model parameters and estimate sources are presented in Table 1. HCV antibody and RNA prevalence, and selfreported treatment history were estimated based on Australian studies evaluating HCV testing, diagnosis and treatment in prisons, NSPs, and drug treatment clinics.28,29 Health outcomes were assessed under each testing strategy, including true positives identified and people with current infection linked to treatment. Assay diagnostic performance was sourced from peerreviewed literature and manufacturer specifications. + +## Costs + +An ingredient approach of costing was used to identify all resources required to perform testing and clinical assessment prior to treatment initiation. Costs of pointof-care RNA testing were estimated at three levels: 1) +variable costs only (device, testing cartridge, consumables, and staff time); 2) direct costs including variable costs and fixed costs of IT/connectivity, quality assurance, and training; 3) total program costs including direct variable and fixed costs, and indirect program coordination/communication costs (Table 2 and Supplementary Table S2). Direct costs (variable and fixed costs for the operation of HCV detection, diagnosis, assessment and reporting as per clinical and public health requirements) were used in the base case analysis and sensitivity analysis were performed considering costs ranging from variable costs only (testing) to total program costs. + +Variable costs (tests and labour costs involved in testing/clinical assessment) were measured from clinical studies.7,11,28 Labour costs included nursing staff time (at different seniority levels according to tasks performed),30 infectious disease specialists, and correctional officers (in prisons). Capital cost for point-of-care testing device was proportionated to unit cost per test (annuitization discounted 3% per annum with 10 years effective life). Testing kit, testing cartridge, and consumables for point-of-care testing and IT/connectivity, quality assurance, training, and program coordination were estimated based on the financial information from the National Australian Hepatitis C Point-of-Care Testing Program (Supplementary Table S2). + +Costs associated with tests and clinical assessments prior to treatment initiation were considered, thus DAA pharmaceutical costs were excluded (Table 2). Clinical assessments costs included labour costs and costs of serology tests for hepatitis B virus and human immunodeficiency virus, liver function tests, full blood count, urea, electrolytes and creatine, pregnancy test (women only) and FibroScan. The Australian Medical Benefit Schedule was used for testing costs and services + +![3_image_0.png](3_image_0.png) + +| Parameter | Setting | Range for sensitivity Distribution in probabilistic | Source | | | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|-------------------------------------------------------|-------------------|-------------|------------|----------------------| +| analyses | sensitivity analyses | | | | | | +| Prison (95% CI) | Needle Syringe | Drug treatment clinic | | | | | +| Program (95% CI) | (95% CI) | | | | | | +| Proportion of people tested | 1 | 1 | 1 | 0.50–1.00 | Triangular | Assumptiona | +| HCV Antibody prevalence | 0.37 (0.33, 0.40) | 0.61 (0.54, 0.68) | 0.69 (0.66, 0.72) | 0.10–0.90 | Beta | 28,29 | +| HCV RNA (+) in HCV Antibody (+) | 0.32 (0.26, 0.38) | 0.30 (0.22, 0.38) | 0.20 (0.17, 0.23) | 0.20–0.40 | Beta | 28,29 | +| HCV RNA prevalence | 0.12 (0.09, 0.14) | 0.18 (0.13, 0.24) | 0.14 (0.12, 0.16) | 0.10–0.20 | Beta | 28,29 | +| HCV Antibody (+) in HCV RNA (−) | 0.28 (0.25, 0.32) | 0.53 (0.45, 0.60) | 0.64 (0.61, 0.68) | 0.20–0.70 | Beta | 28,29 | +| Point-of-care RNA invalid test due to | 0.03 | 0.03 | 0.03 | 0.01–0.1 | Triangular | Assumption | +| operation error Sensitivity of test - Point-of-care HCV antibody | 0.993 | 0.993 | 0.993 | 0.961–0.999 | Beta | Bioline HCV, Abbottb | +| - Point-of-care HCV RNA | 1.00 | 1.00 | 1.00 | 0.84–1.00 | Beta | 5,6 | +| - Laboratory HCV antibody | 1.00 | 1.00 | 1.00 | | | | +| - Laboratory HCV RNA | 1.00 | 1.00 | 1.00 | | | | +| Specificity of test - Point-of-care HCV antibody | 0.981 | 0.981 | 0.981 | 0.945–0.994 | Beta | Bioline HCV, Abbottb | +| - Point-of-care HCV RNA | 1.00 | 1.00 | 1.00 | 0.81–1.00 | Beta | 5,6 | +| - Laboratory HCV antibody | 1.00 | 1.00 | 1.00 | Assumptionc | | | +| - Laboratory HCV RNA | 1.00 | 1.00 | 1.00 | Assumptionc | | | +| Proportion lost to follow-up - Point-of-care RNA re-test | 0.25 | 0.25 | 0.25 | 0.20–0.30 | Triangular | Assumption | +| - Standard of care RNA test | 0.31 | 0.31 | 0.31 | 0.30–0.32 | Beta | 3,11 | +| Treatment uptake - Point-of-care | 0.93 (0.78, 0.99) | 0.81 (0.62, 0.94) | 0.81 (0.62, 0.94) | 0.70–0.99 | Beta | 7,11 | +| - Standard of care | 0.60 (0.57, 0.63) | 0.59 (0.56, 0.61) | 0.59 (0.56, 0.61) | 0.20–0.65 | Beta | 3,11 | +| Self-report history of HCV treatment | 0.14 (0.11, 0.17) | 0.18 (0.07, 0.33) | 0.26 (0.19, 0.34) | 0.10–0.30 | Beta | 28,29 | +| history in RNA (+) | 0.09 (0.08, 0.10) | 0.39 (0.31, 0.46) | 0.43 (0.39, 0.46) | 0.05–0.50 | Beta | 28,29 | +| Self-report history of HCV treatment history in RNA (−) Treatment-naïve in HCV Antibody (+) | 0.74 (0.72, 0.77) | 0.43 (0.35, 0.52) | 0.42 (0.38, 0.46) | 0.30–0.80 | Beta | 28,29 | +| Treatment-naïve in HCV RNA (+) | 0.86 (0.83, 0.88) | 0.83 (0.67, 0.93) | 0.74 (0.66, 0.81) | 0.60–0.90 | Beta | 28,29 | +| Self-report history of HCV treatment | 0.10 (0.09, 0.11) | 0.35 (0.28, 0.41) | 0.40 (0.37, 0.43) | 0.10–0.40 | Beta | 28,29 | +| HCV Antibody (+) and RNA (−) in TxNaïve | 0.17 (0.15, 0.18) | 0.23 (0.15, 0.32) | 0.38 (0.33, 0.42) | 0.10–0.40 | Beta | 28,29 | +| a Proportion of people tested: Assume to be 100% in the base case and tested in the ranges 0.5–1.0 in sensitivity analysis. b Sensitivity and specificity of POC HCV-Antibody test: by Bioline HCV, Abbott, product specification insert. c Sensitivity and specificity of lab HCV Antibody and RNA tests: assume 100% as gold standard tests. Table 1: Decision analytical model epidemiological parameters. | | | | | | | + +rendered by private healthcare providers, and government employment pay rates were used for public healthcare providers.31,32 Costs were valued in 2021 Australian Dollar (A$). + +## Cost-Effectiveness Analysis Incremental Analysis + +Costs and outcomes were compared to standard of care laboratory-based HCV diagnosis for each point-of-care testing strategy. Pairwise comparisons (testing strategy A vs. B, A vs. C, B vs. C) were carried out for the three point-of-care testing strategies in three settings. + +Incremental costs were compared to incremental outcomes to generate incremental cost-effectiveness ratios +(ICERs). + +## Sensitivity Analysis + +Deterministic univariate sensitivity analysis was performed to investigate the cost per treatment initiation for each testing strategy by HCV antibody prevalence and treatment uptake. The most efficient and costeffective testing strategies by HCV antibody prevalence were identified for each setting. Threshold analyses were undertaken to identify the minimum treatment uptake for point-of-care testing strategies to achieve equivalent average cost per treatment initiation compared to standard of care. One-way sensitivity analysis was performed with all model parameters with value ranges presented in Table 1 (effectiveness variables) and Supplementary Table S2 (cost variables). + +Probabilistic sensitivity analysis with 1000 iterations was + +![5_image_0.png](5_image_0.png) + +| Test | Costs | Source | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------|------------------------|------------------------------------------------------------| +| Base case estimate (low, high) | | | | +| Point-of-care RNA test | Testing variable cost | Direct cost (including | Total cost | +| variable and fixed costs) | | | | +| $96 ($77, $165) | $129 ($109, $199) | $153 ($133, $222) | Cepheid 2019, 2020a Cepheid 2019b National Programc TEMPOd | +| Point-of-care combined HCV Ab and RNA tests | Anti-HCV only | Anti-HCV Reflex RNA | RNA onlye | +| $34 ($30, $86) | $163 ($139, $285) | $129 ($109, $199) | 7 | +| Standard of care laboratory HCV tests | Anti-HCV only | Anti-HCV & RNA | RNA onlye | +| $38 ($34, $90) | $153 ($117, $191) | $115 ($103, $174) | 31 | +| Treatment initiation assessment | $180 ($162, $198) | 28,31 | | +| a GeneXpert IV 4sites Laptop model with lifespan 10 years for 375 screened annually per site, including annual service and maintenance fees. Discount 20%∼50% applied for sensitivity analysis. b Costs include cartridge, reagent, delivery fee, and consumables. c National Point-of-care Testing Program budget 2021. d Ranges are based on TEMPO pilot data - 2 patients per hour by CNS, additional 15 min for reflex RNA after positive HCV antibody. e Immediate RNA tests with treatment history. Table 2: Cost estimates for point-of-care HCV RNA, point-of-care anti-HCV and standard of care laboratory HCV tests. (Costs in 2021 A$). | | | | + +conducted to illustrate the impact of parameter variability on costs and outcomes. + +## Role Of The Funding Source + +The study was supported by the Point of Care Research Consortium for Infectious Disease in the Asia Pacific +(RAPID) which is funded through a National Health and Medical Research Council Centre for Research Excellence Grant in Australia. The funders did not have any role in study design, data collection, data analysis, interpretation, writing of the report. + +## Results Average Cost-Effectiveness For Diagnosis Of Hcv Infection + +With standard of care HCV testing, the average costs per HCV-infected person detected were $748 in prisons, $630 in NSPs, and $857 in drug treatment clinics (base case) (Table 3). The expected average cost of HCV diagnosis by point-of-care testing varied by setting and testing strategy, A$689–A$1140 per person detected in prisons, A$575–A$729 in NSPs, and A$817–A$957 in drug treatment clinics, depending on testing strategies +(Table 3). Across all settings, diagnosis by immediate point-of-care HCV RNA testing was the most expensive testing strategy (A$729–A$1140), while combining point-of-care HCV antibody and reflex point-of-care RNA testing with consideration of treatment history achieved the lowest average cost per HCV-infected person detected (A$575–A$817). + +## Average Cost-Effectiveness For Hcv Treatment Initiation + +With standard of care HCV testing, the average cost per treatment initiation was $1426 in prisons, $1248 in NSPs, and $1632 in drug treatment clinics (base case) +(Table 3). Across all settings, the average cost per treatment initiation with point-of-care testing was up to 35% less than the average cost in standard of care. The expected average cost per treatment initiation by pointof-care testing ranged from A$921–A$1406 in prisons, A$890–A$1080 in NSPs, to A$1189–A$1362 in drug treatment clinics, depending on testing strategies +(Table 3). The average cost of immediate point-of-care HCV RNA testing per treatment initiation in the base case analysis was A$1406 in prison, A$1080 in NSP, and A$1362 in drug treatment clinics. The average cost per treatment initiation with combined point-of-care HCV +antibody and reflex RNA testing was lower than immediate point-of-care RNA testing, with the lowest average cost per treatment initiation when considering previous treatment history (prisons A$921 vs. A$960, NSP A$890 vs. A$970, and drug treatment clinics A$1189 vs. A$1310). + +## Incremental Cost-Effectiveness Ratios + +Compared to standard of care, point-of-care HCV testing strategies detected more infections and increased treatment uptake with higher cost per person tested, resulting in ICERs from A$408 to A$2409 per detectable RNA and A$424–A$1386 per treatment initiation. Among the three point-of-care testing strategies, combined antibody and reflex RNA testing with consideration of treatment history was always the most efficient (lowest average cost per treatment initiation). + +Immediate RNA testing was more expensive but detected more people with HCV and associated with higher treatment uptake, resulting in ICERs from A$33,764 to A$89,757 for each additional person initiating HCV +treatment, compared to the most efficient combined point-of-care testing strategy considering treatment history (strategy A vs. C in Table 4). When compared to the combined point-of-care antibody and reflex RNA +tests irrespective of treatment history, immediate RNA +testing produced ICERs from A$8698 in drug treatment + +| Number to test for one person diagnosed or treated | | | | | | | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------|-----|--------|--------|-------------|-------|------| +| Detection of active HCV infection Prison setting POC HCV RNA for all | $132 | $66 | 0.1159 | 0.0274 | $2409 $1140 | 8.6 | | +| POC HCV-Antibody for all with reflex HCV-RNA | $83 | $17 | 0.1151 | 0.0266 | $650 | $725 | 8.7 | +| POC HCV Antibody for treatment-naive with Reflex HCV-RNA | $79 | $13 | 0.1153 | 0.0268 | $493 $689 | 8.7 | | +| Standard of Care | $66 | - | 0.0885 | - | - | $748 | 11.3 | +| Needle Syringe Program Setting POC HCV RNA for all | $132 | $47 | 0.1813 | 0.0453 | $1027 | $729 | 5.5 | +| POC HCV-Antibody for all with reflex HCV-RNA | $115 | $29 | 0.1800 | 0.0441 | $669 | $640 | 5.6 | +| POC HCV Antibody for treatment-naive with reflex HCV-RNA | $104 | $18 | 0.1802 | 0.0443 | $408 | $575 | 5.5 | +| Standard of Care | $86 | - | 0.1359 | - | - | $630 | 7.4 | +| Drug Treatment Setting POC HCV RNA for all | $132 | $40 | 0.1381 | 0.0308 | $1308 | $957 | 7.2 | +| POC HCV-Antibody for all with Reflex HCV-RNA | $126 | $34 | 0.1371 | 0.0299 | $1127 | $916 | 7.3 | +| POC HCV Antibody for Tx-naive with Reflex HCV-RNA | $112 | $20 | 0.1740 | 0.0301 | $676 | $817 | 7.3 | +| Standard of Care | $92 | - | 0.1072 | - | - | $857 | 9.3 | +| HCV detected and linked to treatment Prison setting POC HCV RNA for all | $152 | $76 | 0.1078 | 0.0547 | $1386 $1406 | 9.3 | | +| POC HCV-Antibody for all with Reflex HCV-RNA | $103 | $27 | 0.1071 | 0.0539 | $500 $960 | 9.3 | | +| POC HCV Antibody for Tx-naive with Reflex HCV-RNA | $99 | $23 | 0.1072 | 0.0541 | $424 | $921 | 9.3 | +| Standard of Care | $76 | - | 0.0531 | - | - | $1426 | 18.8 | +| Needle Syringe Program Setting POC HCV RNA for all | $159 | $59 | 0.1468 | 0.0666 | $879 $1080 | 6.8 | | +| POC HCV-Antibody for all with Reflex HCV-RNA | $141 | $41 | 0.1458 | 0.0656 | $630 | $970 | 6.9 | +| POC HCV Antibody for Tx-naive with Reflex HCV-RNA | $130 | $30 | 0.1460 | 0.0658 | $455 $890 | 6.9 | | +| Standard of Care | $100 | - | 0.0802 | - | - | $1248 | 12.5 | +| Drug Treatment Setting POC HCV RNA for all | $152 | $49 | 0.1118 | 0.0486 | $1010 $1362 | 8.9 | | +| POC HCV-Antibody for all with Reflex HCV-RNA | $146 | $42 | 0.1111 | 0.0478 | $884 $1310 | 9.0 | | +| POC HCV Antibody for Tx-naive with Reflex HCV-RNA | $132 | $29 | 0.1113 | 0.0480 | $605 $1189 | 9.0 | | +| Standard of Care | $103 | - | 0.0633 | - | $1632 | 15.8 | | +| HCV: hepatitis C, ICER: incremental cost-effective ratio, POC: point-of-care, Tx: treatment. - The comparator to generate incremental cost/effectiveness. Table 3: Average cost per person tested, HCV detected, and people treated by three point-of-care testing strategies and incremental cost-effectiveness ratio compared to standard of care by laboratory testing. Strategy Average cost per person tested Incremental Effectiveness cost (HCV diagnosed or treated) Incremental ICER Average cost per effectiveness person diagnosed or treated | | | | | | | | + +clinics to A$64,706 in prisons (strategy A vs. B in Table 4). Pairwise comparisons of the two combined point-of-care antibody and reflex RNA testing showed that, in all settings, the testing strategy without consideration of treatment history was dominated (more expensive and less people treated) by the combined tests with consideration of treatment history. Costeffectiveness frontier presented in a cost-effectiveness plane in Fig. 2 illustrates the points of base case corresponding to four testing strategies with the gradient of the line representing the ICER of the testing strategy comparison between two alternatives. The testing strategy of point-of-care antibody testing for everyone and reflex RNA test for antibody positives sit on the left side of frontier lines, meaning this strategy was dominated by the expansion of intervention undominated lines and would not be a choice for consideration from costeffectiveness point of view. + +## Sensitivity Analysis + +To assess the impact of HCV antibody prevalence on cost-effectiveness of different testing strategies, one-way sensitivity analysis was performed (Fig. 3). Combined point-of-care HCV antibody testing with reflex point-ofcare RNA testing with consideration of previous treatment was the most cost-effective testing strategy across all settings up to an HCV antibody prevalence of 79% in prisons and 86% in NSPs and drug treatment clinics. + +When HCV antibody prevalence was <74%, combined point-of-care HCV antibody and point-of-care RNA +testing, both with and without consideration of treatment history, were more cost-effective than immediate + +| Pairwise | Strategy | Average | | | | | +|------------------------------------------------------------------|---------------------------------------------------|----------------------------------------------------------------------|------|--------|---------|------------| +| comparison | cost per person tested | Incremental Effectiveness cost (HCV detected or treated) Incremental | ICER | | | | +| effectiveness | | | | | | | +| Prison setting Detection of active HCV infection | | | | | | | +| A | POC HCV RNA for all | $132 | $49 | 0.1159 | 0.0008 | $60,000 | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $83 | - | 0.1151 | - | - | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $83 | 4 | 0.1151 | −0.0002 | dominateda | +| C | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $79 | - | 0.1153 | - | - | +| A | POC HCV RNA for all | $132 | $53 | 0.1159 | 0.0006 | $83,310 | +| C | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $79 | - | 0.1153 | - | - | +| HCV detected & linked to treatment | | | | | | | +| A | POC HCV RNA for all | $152 | $49 | 0.1078 | 0.0008 | $64,700 | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $103 | - | 0.1071 | - | - | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $103 | $4 | 0.1071 | −0.0002 | dominated | +| C | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $99 | - | 0.1072 | - | - | +| A | POC HCV RNA for all | $152 | $53 | 0.1078 | 0.0006 | $89,800 | +| C | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $99 | - | 0.1072 | - | - | +| Needle Syringe Program Setting Detection of active HCV infection | | | | | | | +| A | POC HCV RNA for all | $132 | $17 | 0.1813 | 0.0013 | $13,400 | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $115 | - | 0.1800 | - | - | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $115 | $11 | 0.1800 | −0.0002 | dominated | +| C | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $104 | - | 0.1802 | - | - | +| A | POC HCV RNA for all | $132 | $28 | 0.1813 | 0.0010 | $27,200 | +| C | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $104 | - | 0.1802 | - | - | +| HCV detected & linked to treatment | | | | | | | +| A | POC HCV RNA for all | $159 | $17 | 0.1468 | 0.0010 | $16,800 | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $141 | - | 0.1458 | - | - | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $141 | $11 | 0.1458 | −0.0002 | dominated | +| C | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $130 | - | 0.1460 | - | - | +| A | POC HCV RNA for all | $159 | $29 | 0.1468 | 0.0008 | $33,800 | +| C | POC HCV-Antibody for Tx-naive with Reflex HCV-RNA | $130 | - | 0.1460 | - | - | +| Drug Treatment Setting Detection of active HCV infection | | | | | | | +| A | POC HCV RNA for all | $132 | $7 | 0.1381 | 0.0010 | $6900 | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $126 | - | 0.1371 | - | - | +| B | POC HCV-Antibody for all with Reflex HCV-RNA | $126 | $13 | 0.1371 | −0.0003 | dominated | +| C | POC HCV Antibody for Tx-naive with Reflex HCV-RNA | $112 | - | 0.1374 | - | - | +| A | POC HCV RNA for all | $132 | $20 | 0.1381 | 0.0007 | $27,900 | +| C | POC HCV Antibody for Tx-naive with Reflex HCV-RNA | $112 | - | 0.1374 | - | - | +| (Table 4 continues on next page) | | | | | | | + +![8_image_0.png](8_image_0.png) + +![8_image_1.png](8_image_1.png) + +A POC HCV RNA for all $152 $7 0.1118 0.0008 $8700 B POC HCV-Antibody for all with Reflex HCV-RNA $146 - 0.1111 - – +B POC HCV-Antibody for all with Reflex HCV-RNA $146 $13 0.1111 −0.0002 dominated C POC HCV Antibody for Tx-naive with Reflex HCV-RNA $132 - 0.1113 - – +A POC HCV RNA for all $152 $20 0.1118 0.0006 $34,600 C POC HCV Antibody for Tx-naive with Reflex HCV-RNA $132 - 0.1113 - – +ICER: incremental cost-effective ratio. - The comparator to generate incremental cost/effectiveness. aDominated: meaning more costly but less effective. + +Table 4: Pairwise comparison between point-of-care testing strategies for HCV detection and treatment initiation for the base cases in three settings. +point-of-care RNA testing and lab-based standard of care testing. At an HCV antibody prevalence <35% in prison, +<45% in NSP, and <49% in drug treatment clinics, immediate point-of-care RNA testing was the least costeffective strategy; while at an antibody prevalence >79% +in prisons and >86% in NSPs and drug treatment clinics, immediate point-of-care RNA testing became the most cost-effective testing strategy because the cost of the HCV antibody test is not wasted on people who are highly likely to be HCV antibody positive. + +The key factor determining the cost-effectiveness per person treated was the difference in treatment uptake between point-of-care and standard of care testing. + +Threshold analyses demonstrated that for immediate point-of-care RNA testing, treatment uptake levels at 67%–91% would achieve an equivalent average cost per treatment initiation as those in standard of care (59%– +60%, depending on setting) (Table 5 and Fig. 4). For combined point-of-care HCV antibody and reflex RNA +testing, treatment uptake of 54%–57% with consideration of prior treatment and 58%–64% without consideration of prior treatment, were required to achieve an equivalent average cost per treatment initiation as in standard of care. + +One-way sensitivity analyses suggested that point-ofcare RNA test costs, number of annual tests per site, and number of samples collected in 1 h played a role in determining the ICERs of point-of-care testing compared to standard of care (Supplementary Fig. S1). Standard of care treatment uptake also impacted ICERs by determining the magnitude of improvements in treatment uptake with point-of-care testing. + +Accounting for model input variability by simultaneously changing all parameters with plausible ranges +(including effectiveness and costs variables) in the probabilistic sensitivity analysis, the three point-of-care testing strategies showed an increased treatment initiation compared to standard of care at a higher cost, albeit with great uncertainly (scatterplots of cost and effectiveness in Supplementary Fig. S2). Additionally, cost-effectiveness planes of incremental cost and incremental effectiveness of pairwise comparisons between the three point-of-care testing strategies are presented in Supplementary Fig. S3, which further validates the robustness of our analyses and strengthen the base case results presented in Table 4 and Fig. 2. That is, strategy B was dominated by strategy C, i.e., POC antibody testing for everyone without treatment history consideration and reflex with RNA testing (strategy B) less effective and more costly than the same testing with treatment history consideration (strategy C). + +## Discussion + +This study demonstrated that all point-of-care HCV +testing strategies had a lower average cost per treatment initiation compared to standard of care across settings and HCV antibody prevalence. When HCV antibody prevalence was below 74%, combining point-of-care HCV antibody and reflex point-of-care RNA testing +(with or without consideration of previous treatment experience) is most cost-effective. Only modest improvements in treatment uptake are required for pointof-care RNA testing to be cost-effective compared to standard of care. Our study findings are important to inform optimal strategies for the scale-up of point-ofcare testing in settings critical to support HCV +elimination. + +Point-of-care RNA testing was more cost-effective than standard of care, consistent with studies in the US, Canada, and Africa.15–21 Limitations of previous studies included limited cost inclusions,15,17 underestimated point-of-care RNA testing costs,20 and simplified testing pathways assuming no loss to followup.19 The strengths of this study are the availability of detailed costing data, the inclusion of costs for critical aspects of point-of-care testing (e.g., operator training, + +![9_image_0.png](9_image_0.png) + +quality assurance, and IT/connectivity), and real-world data for key parameters related to HCV prevalence and treatment. The superior cost-effectiveness of point-ofcare testing was primarily driven by a higher treatment uptake compared to standard of care (despite the higher costs related to point-of-care testing). Most costs related to standard of care were attributed to wasted testing costs due to loss of follow-up in the care cascade compared to point-of-care testing. + +When the HCV antibody prevalence was below 74%, combined point-of-care HCV antibody with reflex point-of-care HCV RNA testing was most cost-effective, with consideration of previous treatment history further improving cost-effectiveness. This testing strategy was more cost-effective because people who were HCV antibody negative received a less expensive antibody test rather than an RNA test. The improved cost-effectiveness of this strategy is primarily driven by the decreased costs of point-of-care antibody testing (A$34 including test kit, consumables, labour) +compared to immediate point-of-care RNA testing +(A$96 including GeneXpert machine, test cartridge, consumables, labour). Point-of-care HCV antibody testing also has a shorter time to result compared to point-of-care HCV RNA testing (60 min). Combined point-of-care HCV antibody and RNA testing strategies are likely to improve patient and staff acceptability by reducing time to result for those who are HCV antibody negative (1–20 min vs. 60 min), while not considerably increasing the total time to result for those who are positive (61–65 min in total). It is unclear whether a combined point-of-care and RNA +Fig. 3: Average cost per HCV treatment initiation in relation to HCV antibody prevalence in three settings (a) Prison (b) Needle Syringe + +![10_image_0.png](10_image_0.png) + +Program and (c) Drug treatment clinics. + +testing strategy has any impact on treatment uptake or response to therapy. Further research is needed to understand the effectiveness, acceptability, implementation challenges, and long-term cost-effectiveness to inform implementation, funding, and integration into policy and practice. + +This study suggested that only modest increases in treatment uptake from 59% to 60% in standard of care + +| Setting | Treatment uptake in the base case | Threshold of point-of-care treatment uptake for equal average cost per person treated compared to standard of care | | | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------|----------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------|--------------------------------------------------|-------------------| +| SOC (95% CI) | POC (95% CI) | Combined POC HCV antibody and reflex RNA with treatment history consideration, base case (range) | Combined POC HCV antibody and reflex RNA without treatment history consideration, base case (range) | Immediate POC HCV RNA for all, base case (range) | | +| Prison | 0.60 (0.57, 0.63) | 0.93 (0.78, 0.99) | 0.55 (0.52, 0.59) | 0.58 (0.55, 0.59) | 0.91 (0.88, 0.95) | +| Needle Syringe Program | 0.59 (0.56, 0.61) | 0.81 (0.62, 0.94) | 0.54 (0.52, 0.56) | 0.60 (0.57, 0.62) | 0.68 (0.66, 0.70) | +| Drug treatment clinic | 0.59 (0.56, 0.61) | 0.81 (0.62, 0.94) | 0.57 (0.53, 0.59) | 0.64 (0.60, 0.66) | 0.67 (0.62, 0.68) | +| POC: Point-of-Care, SOC: Standard of Care. Difference in treatment linkage between POC and SOC: base case 0.93–0.60, worst case 0.78–0.63, best case 0.99–0.57 in prison; base case 0.81–0.59, worst case 0.62–0.61, best case 0.94–0.56 in NSP and drug treatment clinics. Table 5: Threshold of point-of-care treatment uptake for equal average cost per person treated compared to standard of care in three settings. | | | | | | + +![11_image_0.png](11_image_0.png) + +to 67%–91% with immediate point-of-care HCV RNA +testing is needed for the higher-cost point-of-care HCV RNA testing to become a preferred strategy compared to standard of care. Clinical studies have demonstrated that high treatment uptake (81%–93%) following pointof-care HCV RNA testing can be achieved in various settings.7–11 In our analyses, treatment uptake in the standard of care was based on the proportion of diagnosed people in 2016–2017 in New South Wales, providing an accurate estimate of HCV treatment uptake.3 The benefit of point-of-care testing with same-visit treatment is the ability to detect current infection and initiate treatment in a timely manner.7,11 This study demonstrates that if real-world treatment uptake following point-of-care RNA testing is lower than pilot studies to date, point-of-care testing is still likely to be more cost-effective than standard of care. Lower treatment uptake is required by the point-of-care combined testing with treatment history consideration to achieve equivalent average costs per treatment initiation in standard of care, suggesting the relative advantage of point-of-care HCV antibody testing in cost terms. + +This study has several limitations. Our models are based on empirical data from clinical trials and studies in Australian service settings. Thus, diagnosis outcomes for the base cases in each setting are mainly built on the epidemiological profiles of the population in these Australian settings.28,29 The results are driven by the attributes of these studies, particularly the HCV antibody and RNA prevalence and treatment history among the study population. The findings may not be comparable to other populations with different epidemiological characteristics from these Australian studies. Interpretation and application of our study results for other populations and settings should be undertaken cautiously as HCV prevalence and testing costs would vary widely (e.g., homeless people compared to patients of mental health services or attendees of general practice). + +Treatment costs for people diagnosed with HCV +were excluded as the end of testing pathway for this cost-effectiveness analysis was initiation, rather than completion, of DAA treatment. The DAA reimbursement agreement between the Australian government and the pharmaceutical manufacturers stipulates the prices by a fixed budget regardless of number of patients treated.33,34 Thus, the additional benefits of increasing treatment did not incur additional costs; therefore, the DAA pharmaceutical cost was regarded as a sunk cost and was excluded from this analysis. Lastly, lack of modelling the long-term outcomes for people living with HCV is a key limitation of the current analysis. The one-year time horizon means that potential long-term costs and benefits of treatment with respect to prevention of HCV transmission and reductions in morbidity/mortality are not captured. + +Our work in the near future will include the measurement of quality-adjusted life-years (QALYs) and mathematical modelling to estimate expected transmission and long-term disease burden benefits to guide implementation and enable comparison with other public health interventions and programs. In the interim, this study lays the foundations for future modelling by providing useful information on costs and benefits that can be expected in the short term and by identifying uncertain estimates requiring more detailed investigation for future extrapolation beyond the trial follow-up period. This study also provides a model that could be used to assess scale-up of point-ofcare testing strategies in other countries that could be parametrised to the local epidemiology, including the Western Pacific. This is important given the recent inclusion of point-of-care testing in the new recommendations for simplified service delivery and diagnostic testing by the World Health Organization.35 + +## Conclusion + +Point-of-care HCV testing enables diagnosis and treatment in a single-visit, increases testing acceptability, and reduces loss to follow-up, addressing the drop-off in the HCV care cascade. Combining point-of-care HCV +antibody and RNA testing could optimise testing outcomes, enhancing cost-effectiveness and patient acceptability. This study is important for informing the future implementation of point-of-care testing strategies to achieve HCV elimination targets. + +Contributors S.T.F.S., V.W., and J.G. conceived the study. S.T.F.S., R.T.G., A.R.L., V.W., and J.G. obtained funding. S.T.F.S., Q.C., and R.T.G. contributed to model development. S.T.F.S. and J.G. designed the analyses, interpreted the results, and drafted the manuscript. J.C., H.V., Y.S., E.B.C., +J.A.K., A.R.L., G.J.D., and J.G. provided data and validated model inputs. V.W. and J.G. supervised the project. All authors reviewed, edited, and approved the manuscript. Data sharing statement Models parameters are presented in Table 1 and Supplementary Table S2. Data sources for model parameters will be made available on request. + +Declaration of interests R.T.G. has received funding for his research from WHO and has provided non-funded project advice to Gilead and ViiV. Y.S. is a coinvestigator on investigator-initiated research grants from AbbVie and Gilead Sciences. A.R.L. has received investigator-initiated research grants from AbbVie, Gilead Sciences, and Sequiris. G.J.D. is a consultant/advisor and has received research grants from Abbvie, Abbot Diagnostics, Gilead Sciences, Bristol Myers Squibb, Cepheid, GlaxoSmithKline, Merck, Janssen and Roche. J.G. is a consultant/ +advisor and has received research grants from AbbVie, Biolytical, Camurus, Cepheid, Gilead Sciences, Hologic, Indivor, and Merck/MSD +and has received honoraria from AbbVie, Cepheid, Gilead Sciences, and Merck. No input into this work was provided by any of the above listed organisations or institutions. + +All other authors have no conflicts of interest to declare. + +Acknowledgement We would like to acknowledge the contributors to the studies which provided data that informed the analyses presented in this paper. + +Funding: This work was supported by the Point of Care Research Consortium for Infectious Disease in the Asia Pacific (RAPID) +which is funded through a National Health and Medical Research Council Centre for Research Excellence Grant [grant number 1153647]. The funding body has no role in the writing of the manuscript or the decision to submit the manuscript for publication. The Kirby Institute is funded by the Australian Government Department of Health and Ageing. The views expressed in this publication do not necessarily represent the position of the Australian Government. G.J.D. is supported through a National Health and Medical Research Council Investigator Grant [2008276]. J.G. is supported through a National Health and Medical Research Council Investigator Grant [1176131]. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi. + +org/10.1016/j.lanwpc.2023.100750. References + +![12_image_0.png](12_image_0.png) + +1 WHO. Global health sector strategy on viral hepatitis 2016-2021. + +Geneva: WHO; 2017. + +2 Polaris Observatory HCV Collaborators. Global change in hepatitis C virus prevalence and cascade of care between 2015 and 2020: a modelling study. Lancet Gastroenterol Hepatol. 2022;7(5):396–415. + +3 Yousafzai MT, Alavi M, Valerio H, Hajarizadeh B, Grebely J, +Dore GJ. Timely hepatitis C RNA testing and treatment in the era of direct-acting antiviral therapy among people with hepatitis C in New South Wales, Australia. Viruses. 2022;14(7):1496. + +4 Scott N, Sacks-Davis R, Wade AJ, et al. Australia needs to increase testing to achieve hepatitis C elimination. Med J Aust. 2020;212(8):365–370. + +5 Grebely J, Lamoury FMJ, Hajarizadeh B, et al. Evaluation of the Xpert HCV Viral Load point-of-care assay from venepuncturecollected and finger-stick capillary whole-blood samples: a cohort study. Lancet Gastroenterol Hepatol. 2017;2(7):514–520. + +6 Lamoury FMJ, Bajis S, Hajarizadeh B, et al. Evaluation of the Xpert HCV Viral Load finger-stick point-of-care assay. J Infect Dis. + +2018;217(12):1889–1896. + +7 Grebely J, Gilliver R, McNaughton T, et al. Single-visit hepatitis C +point-of-care testing, linkage to nursing care, and peer-supported treatment among people with recent injecting drug use at a peer-led needle and syringe program: The TEMPO Pilot Study. Int J Drug Pol. 2023;114:103982. + +8 MacIsaac M, Whitton B, Anderson J, et al. Rapid point of care HCV +testing allows high throughout HCV screening and rapid treatment uptake among PWID attending a medically supervised injecting room. International Conference on Hepatitis Care in Substance Users. 2021. + +9 O'Loan J, Young M, Mooney M, O'Flynn M. Same day delivery! + +HCV point of care testing in South East Queensland mar-ginalised communities simplifies diagnosis and ensures rapid access to treatment. International Conference on Hepatitis Care in Substance Users. 2021. + +10 Ralton L, McCartney E, Ferguson C, et al. Prompt - point of care testing for hepatitis C in the priority settings of mental health, prisons and drug & alcohol facilities. International Conference on Hepatitis Care in Substance Users. 2021. + +11 Sheehan Y, Cunningham E, Cochrane A, et al. A 'one-stop-shop' point-of-care hepatitis C RNA testing intervention to enhance treatment uptake in a reception prison: the PIVOT study. J Hepatol. + +(Submitted 27 September 2022, Manuscript number JHEPAT-D22-01849). + +12 Bajis S, Maher L, Treloar C, et al. Acceptability and preferences of point-of-care finger-stick whole-blood and venepuncture hepatitis C +virus testing among people who inject drugs in Australia. Int J Drug Policy. 2018;61:23–30. + +13 Lafferty L, Cochrane A, Sheehan Y, Treloar C, Grebely J, Lloyd AR. + +"That was quick, simple, and easy": patient perceptions of acceptability of point-of-care hepatitis C RNA testing at a reception prison. + +Int J Drug Policy. 2022;99:103456. + +14 Catlett B, Hajarizadeh B, Cunningham E, et al. Diagnostic accuracy of assays using point-of-care testing or dried blood spot samples for the determination of HCV RNA: a systematic review. J Infect Dis. 2022;226:1005–1021. + +15 Assoumou SA, Nolen S, Hagan L, et al. Hepatitis C management at federally qualified health centers during the opioid epidemic: a cost-effectiveness study. Am J Med. 2020;133(11):e641–e658. + +16 Assoumou SA, Tasillo A, Leff JA, et al. Cost-effectiveness of onetime hepatitis C screening strategies among adolescents and young adults in primary care settings. Clin Infect Dis. 2018; 66(3):376–384. + +17 Duchesne L, Dussault C, Godin A, Maheu-Giroux M, Kronfli N. + +Implementing opt-out hepatitis C virus (HCV) screening in Canadian provincial prisons: a model-based cost-effectiveness analysis. + +Int J Drug Policy. 2021;96:103345. + +18 Duchesne L, Hejblum G, Njouom R, et al. Model-based costeffectiveness estimates of testing strategies for diagnosing hepatitis C virus infection in Central and Western Africa. PLoS One. + +2020;15(8):e0238035. + +19 Duchesne L, Hejblum G, Toure Kane NC, et al. Model-based costeffectiveness estimates of testing strategies for diagnosing hepatitis C virus infection in people who use injecting drugs in Senegal. Int J Drug Policy. 2020;75:102613. + +20 Koo V, Tian F, Wong WWL. Cost-effectiveness analysis of hepatitis C virus (HCV) point-of-care assay for HCV screening. Liver Int. + +2022;42(4):787–795. + +21 Malaisamy M, Nagarajan K, Kirti T, et al. Economic evaluation of implementing a rapid point-of-care screening test for the identification of hepatitis C virus under National Viral Hepatitis Control Programme in Tamil Nadu, South India. J Glob Infect Dis. 2021;13(3):126–132. + +22 Mohamed Z, Scott N, Nayagam S, et al. Cost effectiveness of simplified HCV screening-and-treatment interventions for people who inject drugs in Dar-es-Salaam, Tanzania. Int J Drug Policy. 2022;99:103458. + +23 Scott N, Doyle JS, Wilson DP, et al. Reaching hepatitis C virus elimination targets requires health system interventions to enhance the care cascade. Int J Drug Policy. 2017;47:107–116. + +24 Chhatwal J, Chen Q, Wang X, et al. Assessment of the feasibility and cost of hepatitis C elimination in Pakistan. JAMA Netw Open. 2019;2(5):e193613. + +25 Husereau D, Drummond M, Augustovski F, et al. Consolidated Health Economic Evaluation Reporting Standards 2022 (CHEERS 2022) statement: updated reporting guidance for health economic evaluations. Value Health. 2022;25(1):3–9. + +26 Palmer AY, Wade AJ, Draper B, et al. A cost-effectiveness analysis of primary versus hospital-based specialist care for direct acting antiviral hepatitis C treatment. Int J Drug Policy. 2020;76:102633. + +27 TreeAge Pro Healthcare. In: © 2021 TreeAge Software LLC. 2022. 28 Hajarizadeh B, Grebely J, Byrne M, et al. Evaluation of hepatitis C +treatment-as-prevention within Australian prisons (SToP-C): a prospective cohort study. Lancet Gastroenterol Hepatol. 2021;6(7):533–546. + +29 Valerio H, Alavi M, Silk D, et al. Progress towards elimination of hepatitis C infection among people who inject drugs in Australia: +the ETHOS Engage Study. Clin Infect Dis. 2020;73(1):e69–e78. + +30 Palmer A, Papaluca T, Stoove M, et al. A costing analysis of a statewide, nurse-led hepatitis C treatment model in prison. Int J Drug Policy. 2021;94:103203. + +31 Medicare benefit schedule book [internet] [cited Oct 2020]. Available from: http://www.mbsonline.gov.au/internet/mbsonline/ +publishing.nsf/Content/Home; 2018. + +32 Public health system nurses' & midwives' (state) award 2021 [internet]. + +2021. [cited Nov 15, 2021]. + +33 Drug Utilisation Sub-Committee PBAC. Direct acting antiviral medicines for the treatment of chronic hepatitis C. In: Department of Health. Canberra, Australia: Australian Commonwealth Governement; 2018. + +34 Moon S, Erickson E. Universal medicine access through lump-sum remuneration - Australia's approach to hepatitis C. N Engl J Med. + +2019;380(7):607–610. + +35 World Health Organization. Updated recommendations on HCV +simplified service delivery and HCV diagnostics: policy brief. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO. Geneva, Switzerland. \ No newline at end of file diff --git a/medical/md/PMC10523130.md b/medical/md/PMC10523130.md new file mode 100644 index 0000000000000000000000000000000000000000..98c91ac413897771dc3ffed1d373e7d5939c4c05 --- /dev/null +++ b/medical/md/PMC10523130.md @@ -0,0 +1,360 @@ + +## Review Article + +Prostate cancer + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +pISSN: 2287-4208 / eISSN: 2287-4690 World J Mens Health 2023 Oct 41(4): 759-768 https://doi.org/10.5534/wjmh.220202 + +# Emerging Relationship Between The Gut Microbiome And Prostate Cancer + +Makoto Matsushita1 , Kazutoshi Fujita1,2 , Koji Hatano1 , Marco A. De Velasco2,3 , Akira Tsujimura4 , Hirotsugu Uemura2 , Norio Nonomura1 1 Department of Urology, Osaka University Graduate School of Medicine, Suita, Departments of 2Urology and 3Genome Biology, Kindai University Faculty of Medicine, Osakasayama, 4Department of Urology, Juntendo University Urayasu Hospital, Urayasu, Japan The human gut microbiota changes under the influence of environmental and genetic factors, affecting human health. Extensive studies have revealed that the gut microbiome is closely associated with many non-intestinal diseases. Among these, the influence of the gut microbiome on cancer biology and the efficacy of cancer therapy has attracted much attention. Prostate cancer cells are affected by direct contact with the microbiota of local tissues and urine, and a relationship between prostate cancer cells and the gut microbiota has been suggested. In the human gut microbiota, bacterial composition differs depending on prostate cancer characteristics, such as histological grade and castration resistance. Moreover, the involvement of several intestinal bacteria in testosterone metabolism has been demonstrated, suggesting that they may affect prostate cancer progression and treatment through this mechanism. Basic research indicates that the gut microbiome also plays an important role in the underlying biology of prostate cancer through multiple mechanisms owing to the activity of microbial-derived metabolites and components. In this review, we describe the evidence surrounding the emerging relationship between the gut microbiome and prostate cancer, termed the "gut-prostate axis." Keywords: Bacteria; Gut microbiome; Host microbial interactions; Prostate cancer; Testosterone This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. + +## Introduction + +Various bacterial species are present on every surface of the human body and form unique flora, depending on their location. In particular, there are approximately 1013–1014 total bacteria in the colon, making the gut microbiota the largest flora in the body [1]. It is now known that the bacteria that make up the gut microbiota interact with us through various factors. Lifestyle factors such as diet and exercise are the primary influencers of the composition of the gut microbiota [2]. In turn, intestinal bacterial components and their metabolites can influence human biological systems such as the immune system [3]. + +Some bacteria-induced diseases have long been known and studied; however, very few commensal bacteria have been targeted for the treatment and prevention of these diseases [4]. Recent advances in bacterial analysis have revealed that numerous diseases are affected by local or distant microbiomes. The study of the relationship between humans and their microbiota first focused on intestinal diseases, such as inflammatory bowel disease (IBD), because the local environment is rich in bacteria [5-8]. In addition, intervention with the gut microbiota by fecal microbiota transplantation and probiotics is becoming a therapeutic approach for these intestinal diseases [9]. More recently, the systemic effects of intestinal bacteria have been analyzed, and these symbiotic bacteria have been found to influence the state of diseases, including diabetes, dementia, hepatic steatosis, and non-intestinal cancers in distant organs [10-12]. Because the interactions between the intestinal tract and other distant organs are connected via their microbiota and believed to play an important role in human health, these major relationships have been termed, for example, the "gut-liver axis, gut-brain axis, and gut-kidney axis" [11,13,14]. + +Diseases involving the prostate have also been reported in several bacterial studies. Prostate cancer is strongly associated with lifestyle, factors, including diet, and and genetic factors, including race; in particular, animal fat intake and African-American race are respective risk factors for prostate cancer [15,16]. These factors modulate the risk of prostate cancer by altering the gut microbiome. In this review, we investigate the relationship between the gut microbiota and prostate cancer, using findings from previous studies. + +## Clinical Research On Gut Microbiota And Prostate Cancer + +The gut microbiota can affect the incidence and progression of various types of cancers, but fewer reports consider its relationship with prostate cancer than other cancers. Recent studies in the USA have reported that the composition of the gut microbiota differs depending on the status of prostate cancer. In 2018, Liss et al [17] analyzed the gut microbiota of 133 American men who underwent prostate biopsy and reported a relationship between human prostate cancer and the gut microbiome for the first time. Their cohort primarily included African-Americans and Caucasians. The composition of intestinal bacteria was significantly different between men with positive and those with negative biopsy results (p<0.01), and patients with prostate cancer had a higher ratio of *Bacteroides* and Streptococcus species in their gut microbiota (p<0.04). + +The researchers also performed a gene-based functional analysis of gut microbiota, revealing that the folate and arginine metabolism pathways were enriched in patients with prostate cancer. Because of the increased dependency on folate in prostate cancer cells, intensive research has been conducted on the relationship between them, and it has been shown that blood folate levels are correlated with prostate cancer risk and proliferation rate [18,19]. Furthermore, in a mouse model of prostate cancer, oral folate intake affected both the blood and prostate folate levels, and dietary folate deficiency improved cancer pathology [20]. Risk scoring based on 10 microbiome-derived metabolic pathways can detect prostate cancer with higher accuracy than the standard prostate-specific antigen (PSA) test. These results suggest that specific intestinal bacteria and their metabolic functions could play a role in the risk of prostate cancer, similar to that observed in several other diseases. + +In another report, *Bacteroides massiliensis* was more common in the gut microbiota of Caucasian males with prostate cancer than in those with benign prostatic hyperplasia (BPH), whereas *Faecalibacterium prausnitzii* and *Eubacterium rectalis* were less common [21]. F. + +prausnitzii is a common anti-inflammatory bacterium, and its decrease is associated with a higher risk of Crohn's disease recurrence [22]. In addition, culture supernatants of this bacterium exhibit anti-inflammatory effects by reducing NF-κB activity in intestinal epithelial cells, both *in vitro* and *in vivo* [22]. These results suggest that some metabolites secreted by *F. prausnitzii* may contribute to the prevention of prostate cancer and IBD; therefore, this species is potentially beneficial as a probiotic. The composition of the gut microbiota of non-Hispanic Caucasians did not differ significantly between patients with and without prostate cancer [23]. + +In Caucasian-centric communities, these results suggest that there may be no major specificity in the gut microbiota of patients with prostate cancer, but some Bacteroides species may predominate. + +A few reports on the gut microbiome profile of patients with prostate cancer living outside the USA +show different results. Our group has previously analyzed the gut microbiota composition of 152 Japanese patients who underwent prostate biopsy [24]. In this study, patients with high-grade prostate cancer (grade group >2) showed a significantly higher abundance of Alistipes and Lachnospira. *Alistipes* was enriched in the gut microbiota of patients with colorectal cancer, and *Alistepes* administration induced colitis in rodents by activating IL-6 signaling and promoting tumorigenesis [25,26]. The same mechanism may be responsible for the progression of prostate cancer. On the other hand, *Lachnospira* belongs to the family Lachnospiraceae, which is considered to contain beneficial bacteria that have anti-inflammatory effects and locally inhibit colon cancer [27,28]. However, as discussed later, short-chain fatty acids (SCFAs) produced by this family promote prostate cancer growth [29]. Moreover, a new index calculated from the abundance of 18 intestinal bacteria characteristic of patients with highgrade prostate cancer could discriminate the cancer status with greater accuracy than the PSA test (area under the curve [AUC]=0.81 vs. 0.67). Despite the different bacteria identified in studies in the USA, our functional analysis of the gut microbiome of patients with high-grade prostate cancer showed high starch and sucrose metabolism pathways, which are also common pathways seen in Americans. These results support previous reports that the profile of gut microbiota varies by region and race [30,31]. It also suggests that although different intestinal bacteria are involved in prostate cancer risk by region, their functions may be common across regions. + +Since the microbiota, which consists of foreign elements, interacts with the immune system, it is often associated with inflammation. Urinary and prostatic tissue-residing microbiota can directly cause prostatitis and prostatic hyperplasia through inflammatory processes [32,33]. Gut microbiota are involved locally in the pathogenesis of these inflammatory diseases, but may also play a role in more distant organs via immune and inflammatory processes [34]. In a previous study, we found that men with enlarged prostates had significantly higher Firmicutes/Bacteroidetes ratios in the gut microbiota than those without enlarged prostates [35]. + +An imbalance between Firmicutes and Bacteroidetes in the human gut microbiota is considered an indicator of dysbiosis, and has also been observed in patients with obesity, IBD, nonalcoholic steatohepatitis, and myocarditis, suggesting a link to systemic inflammation [3640]. Thus, our results indicate that inflammation caused by intestinal bacteria may also affect distant prostate cancers. Furthermore, gut microbiota may be associated not only with hypertrophy but also with inflammatory carcinogenesis in the prostate. In the future, there is a need to investigate the independent impact of these bacterial factors on prostate cancer risk, as both gut microbiota and prostate cancer development are affected by various lifestyle factors, such as diet and exercise. + +## The Regulation Of Testosterone By Intestinal Bacteria + +Some bacteria reportedly affect testosterone levels in humans through deglucuronidation of testosterone in the gut, wherein the biologically active form is subsequently reabsorbed into the host [41]. Conversely, testosterone can alter the composition of gut microbiota +[42,43]. We have previously shown that the fraction of the Firmicutes phylum in the gut microbiota of elderly Japanese men (median age, 71.0 y) was significantly correlated with serum testosterone levels (r=0.3323, p=0.01) [44]. A similar trend was observed in younger cohorts. Shin et al. also found that in 31 men with a median age of 37.45 years, serum testosterone levels were positively correlated with several bacteria belonging to the phylum Firmicutes, including Megamonas +(r=0.4161, p=0.02) and *Ruminococcus* (r=0.4589, p=0.01) +[45]. Similar to our study, in a group of adolescents +(median age, 10.99 y), testosterone levels were positively correlated with *Ruminococcus* (r=0.371, p=0.040) and Dorea (r=0.471, p=0.007), both of which belong to Firmicutes [46]. + +Despite age-related changes in the gut microbiota composition, bacteria belonging to the phylum Firmicutes may be responsible for testosterone regulation in hosts. Fecal transplantation from adult male mice into immature female mice elevated their testosterone levels, indicating that the gut microbiome of males produces testosterone [47]. Oral administration of *Lactobacillus*, which belongs to Firmicutes, also increased testosterone levels in mice [48,49]. These results suggest that probiotics may improve testosterone-related health conditions and diseases in men. + +The gut microbiota may also influence the development of breast cancer because of its ability to deconjugate conjugated estrogen excreted in the intestinal tract [50]. In addition, intestinal bacteria can metabolize estrogen-like compounds such as enterodiol [51]. + +Similarly, bacterial-derived testosterone analogs may be relevant in prostate cancer, and further studies are needed in this category. + +The gut microbiota has recently been described as an androgen-producing "organ." Accordingly, androgen deprivation therapy administered as a systemic treatment for prostate cancer can alter the gut microbiota. + +Sfanos et al [52] analyzed the gut microbiota of 30 American men with various stages of prostate cancer and healthy controls. The results showed that patients with prostate cancer had a significantly different microbial composition from healthy controls (p=0.02). + +In addition, treatment with anti-androgen therapy reduced the diversity of the gut microbiota in these patients and increased the proportion of *Akkermansia muciniphila* (0.002 vs. 0.055, p=0.002), which is also known to increase responses to immunotherapy [53]. + +In an *in vivo* study using a mouse model, Akkermansia muciniphila decreased in the gut microbiota after castration, and testosterone propionate supplementation increased this bacterium [54]. These conflicting results may be due to the underlying differences in the composition of the gut microbiota in humans and rodents. + +Further studies are needed to clarify the interaction between testosterone and *Akkermansia muciniphila*. + +Thus, during treatment selection, we should consider the possibility that androgen deprivation therapy influences the efficacy of other types of cancer treatments through gut microbiome-mediated mechanisms. + +## The Role Of The Gut Microbiome In Prostate Cancer Pathogenesis + +Although human microbiome studies suggest that the gut microbiota plays a role in prostate cancer progression, few basic studies have elucidated the mechanisms by which this can occur. In other diseases, including non-intestinal diseases, basic research has revealed that intestinal immunity or bacterial-derived metabolites influenced by the gut microbiota can have augmenting or suppressive effects. Thus, the two-sided mechanisms by which the intestinal microbiota affects human health are complex, but have become clearer in recent studies. + +Previous findings have shown that animal fat intake promotes cancer growth in a prostate cancer mouse model, specifically in prostate-specific *Pten* knockout mice, through various mechanisms [55-57]. In our previous study, oral administration of antibiotics significantly reduced prostate cancer growth in mice fed a high-fat diet (HFD) (p=0.015) [29]. This study provides the first firm evidence that the gut microbiome is involved in prostate cancer progression. We found that this inhibitory effect involved a reduction in SCFAs produced by intestinal bacteria. SCFAs, major metabolites of the gut microbiota, stimulate systemic as well as local prostatic insulin-like growth factor 1 (IGF-1) +production and activate the IGF-1 signaling pathway in prostate cancer cells, which in turn promotes cancer growth [29]. The results of our study suggest that influencing the gut microbiota by diet and medications may play a role in the pathogenesis of prostate cancer. + +In contrast, butyrate, an SCFA, exerts anti-inflammatory effects through the activation of Gpr109a, inhibits carcinogenesis in the gut, and is a beneficial metabolite in humans [58]. Interestingly, this metabolite has opposing functions in the prostate and gut, indicating that the profile of healthy and beneficial gut microbiota may differ for each disease. For example, cohort studies have shown that some dairy products increase the risk of prostate cancer, whereas the opposite is observed in colorectal cancer [59-61]. The cause has not been identified, although the high butyrate content in milk suggests that this compound may be responsible for this discrepancy [62]. Since obese persons are at an increased risk of high-grade prostate cancer and have been found to have high levels of SCFAs in their stool, SCFAs are likely to be involved in prostate cancer risk +[63,64]. + +Other mechanisms also suggest the involvement of gut microbiota in prostate cancer. We found that HFD-induced dysbiosis of the gut microbiota in mice elevated intestinal permeability and promoted lipopolysaccharide (LPS) leakage from this tract [65]. Serum LPS levels were significantly higher in prostate cancer model mice fed HFD than in those fed the control diet +(p=0.005). Prostate local histamine signaling activated by LPS can promote inflammatory prostate cancer growth by stimulating the interleukin-6 (IL6)-signal transducer and activator of transcription 3 (STAT3) +axis and increasing myeloid-derived suppressor cells +(MDSCs); however, the LPS/toll-like receptor 4 (TLR4) signaling block inhibited HFD-induced prostate cancer growth [65]. In another report, it was found that gut dysbiosis and increased intratumoral LPS promoted prostate cancer progression and docetaxel resistance via the IL6-STAT3 axis *in vivo* and *in vitro* [66]. Intestinal bacterial components have also been reported to cause a variety of diseases in distant organs via inflammatory and immune mechanisms. The influx of gut microbiota-derived endotoxins in the portal circulation promotes TLR4 expression and is associated with hepatic inflammation and steatosis [67,68]. Obesityinduced hepatic translocation of lipoteichoic acid, one of the components of the cell wall of gram-positive bacteria, accelerates the senescence of hepatic stellate cells, promoting HCC progression via prostaglandin E2-mediated suppression of antitumor immunity [69]. + +Endotoxins and amyloids from gram-negative bacteria can penetrate the blood-brain barrier and induce amyloid β aggregation and neuroinflammation in the central nervous system, suggesting that bacterial molecules and metabolites may be involved in the onset and progression of Alzheimer's disease [70]. Bacterial components other than LPS may also play key roles in the inflammatory and immune-related development and progression of prostate cancer. + +Modulation of the immune system and other factors by intestinal bacteria is associated with the efficacy of various prostate cancer treatments. In an in vitro gut microbiota-simulated model, Daisley et al [71] +demonstrated that abiraterone acetate, a hormonebased chemotherapeutic used to treat prostate cancer, increased *Akkamansia muciniphila* and suggested that the species enhanced the supply of bacteria-derived vitamin K2, which in turn increased the efficacy of this medication. *Akkamansia* in the gut microbiota can also improve the efficacy of immunotherapeutic checkpoint inhibitors in several cancer types [53]. In prostate cancer, extracellular vesicles from this species activate antitumor immunity by interacting with CD8-positive T +cells and macrophages [72]. Gut microbiota can also affect immunity in the prostate, a distant organ, through bacterial components that circulate in the body. + +Other mechanisms of prostate cancer regulation by androgens in the gut microbiome have also been demonstrated. Pernigoni et al [73] found that the gut microbiota of patients and murine models with castration-resistant prostate cancer (CRPC) was enriched with Ruminococcus and *Bacteroides* genera. These intestinal bacteria transform androgens into their active forms and are involved in prostate cancer progression by supplying the active androgens. Indeed, elimination of the microbiota by antibiotics delayed the acquisition of castration resistance in prostate cancer, but transplantation with stools of patients with CRPC in mice accelerated their prostate cancer progression. Conversely, transplantation of stools of patients with castrationsensitive prostate cancer or the particular species *Prevotella stercorea* inhibited cancer progression. + +The reliable presence of multiple mechanisms, acting via metabolites, immunity, and androgens, suggests that a complex signaling network mediated by the microbiome is formed between the gut and prostate (Fig. + +![4_image_0.png](4_image_0.png) + +Fig. 1. Overview of the "Gut-Prostate Axis". Specific taxa and dysbiosis in the gut microbiome regulate crucial features of prostate cancer through multiple mechanisms. + +1). Future studies can help provide a more complete profile of this "gut-prostate axis." Understanding this axis may lead to improved prevention and treatment of prostate cancer using specialized probiotics or prebiotics. In addition, fecal microbiota transplantation, as a more direct treatment method, has been in clinical trials for the treatment of some intestinal diseases [74], +and its application in prostate cancer is expected soon. + +## Prostate Cancer And The Local Microbiota + +In addition to the gut microbiome, the relationship between prostate cancer risk and the local microbiota has been studied. In the microbiota of human prostate tissue, *Cutibacterium acnes*, previously classified as Propionibacterium acnes, which commonly colonizes the skin, is more abundant in patients with prostate cancer. + +Chen and Wei [75] investigated the genomic sequence of *C. acnes* in three sets of RNA-seq data from prostatic tissues of Australian, Caucasian, and Chinese patients and detected *C. acnes* in both cancer and adjacent tissue samples, but not in non-cancerous samples. Cavarretta +[76] also showed by bacterial genome sequencing of 16 prostatectomy specimens that *Propionibacterium* was more common in cancerous and peri-cancerous areas than in non-cancerous areas (60% vs. 49%). + +In a study of 100 samples from patients with prostate cancer and 50 samples from men without cancer, C. acnes was found to be significantly more prevalent in patients with prostate cancer (60% vs. 26%, p=0.001) +[77]. This is important, given that *C. acnes* can colonize prostate tissue and cause chronic inflammation in a rat model [78]. In addition, *Escherichia* has been detected in prostate cancer tissues in several studies, and is a genus of interest [79,80]. This genus mainly colonizes the gut, and the colibactin produced by some strains promotes carcinogenesis through DNA damage caused by double-strand breaks [81]. *Escherichia*-colonized prostate tissue may also have a similar oncogenic effect. + +The association of prostate cancer with the microbiota present in urine and prostatic fluid that comes in close contact with the prostate gland has also been investigated, and several small-scale studies using metagenomic analysis have shown a common abundance of *Streptococcus* in patients with prostate cancer +[23,82,83]. Although the mechanism underlying this **Table 1.** List of bacteria-related factors with potential impacts on prostate cancer**Bacterial factor** + +![5_image_0.png](5_image_0.png) + +remains unclear, these results suggest that the microbiome present in urine and prostatic fluid may be more directly involved in prostate cancer than in the gut. + +However, urine and prostate tissue contain far fewer numbers of bacteria than stool, and sequencing-based methods for detecting microbes in samples with low microbial concentrations are susceptible to contamination. Further verification, including contamination-free sample collection methods and appropriate sequencing controls, is required to obtain reliable results. + +The gut microbiome may be indirectly involved in the carcinogenesis and progression of prostate cancer, but the local microbiome also appears to be directly involved. The composition of the microbiota in the prostate is different from that seen in stool, and it is likely that different bacterial associations and activities exist locally compared to indirect actions from the gut microbiota. + +## Conclusions + +In addition to the importance of the local microbiome, the relationship between the gut microbiota and prostate cancer has been extensively investigated in both basic and clinical studies, and these suggest that the gut-prostate axis houses various bacterial associations through different mechanisms (Table 1). Furthermore, since the gut microbiome affects androgen production, it may be associated with the pathogenesis of several predominant male health conditions, such as the late onset of hypogonadism, erectile dysfunction, and BPH. However, the overall complexity of the axes remains unclear. Both genetic predisposition and environmental factors play a role in the pathogenesis of prostate cancer [84,85]. The gut microbiota is known to be susceptible to the same host factors [86,87]. Further studies should examine the gut-prostate axis as well as relevant genetic and environmental factors to determine the true impact of the gut microbiome on prostate cancer development and progression. These studies are expected to provide further evidence for the importance of the gut microbiome and support its modification for the prevention, diagnosis, and treatment of prostate cancer. + +## Conflict Of Interest + +The authors have nothing to disclose. + +## Funding + +This work was supported by Japan Society for the Promotion of Science KAKENHI (Grant No. JP21K09421). + +## Author Contribution + +Conceptualization: MM, KF, KH, MADV, AT, HU, NN. Data curation: MM. Formal analysis: MM. Funding acquisition: MM. + +Investigation: MM. Methodology: MM. Project administration: +MM, KF, KH, MADV, AT, HU, NN. Resources: MM. Software: MM. Supervision: AT, HU, NN. Validation: MM, KF. Visualization: MM. Writing - original draft: MM. Writing - review & +editing: KF, KH, MADV, AT, HU, NN. + +## References + +1. Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, et al. Metagenomic analysis of the human distal gut microbiome. Science 2006;312:1355-9. + +2. Strasser B, Wolters M, Weyh C, Krüger K, Ticinesi A. The effects of lifestyle and diet on gut microbiota composition, inflammation and muscle performance in our aging society. Nutrients 2021;13:2045. + +3. Rooks MG, Garrett WS. Gut microbiota, metabolites and host immunity. Nat Rev Immunol 2016;16:341-52. + +4. de Brito BB, da Silva FAF, Soares AS, Pereira VA, Santos MLC, +Sampaio MM, et al. Pathogenesis and clinical management of *Helicobacter pylori* gastric infection. World J Gastroenterol 2019;25:5578-89. + +5. Tilg H, Adolph TE, Gerner RR, Moschen AR. The intestinal microbiota in colorectal cancer. Cancer Cell 2018;33:954-64. + +6. Nishida A, Inoue R, Inatomi O, Bamba S, Naito Y, Andoh A. + +Gut microbiota in the pathogenesis of inflammatory bowel disease. Clin J Gastroenterol 2018;11:1-10. + +7. Ohkusa T, Sato N, Ogihara T, Morita K, Ogawa M, Okayasu I. Fusobacterium varium localized in the colonic mucosa of patients with ulcerative colitis stimulates species-specific antibody. J Gastroenterol Hepatol 2002;17:849-53. + +8. Lupp C, Robertson ML, Wickham ME, Sekirov I, Champion OL, Gaynor EC, et al. Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae. Cell Host Microbe 2007;2:204. + +9. Imdad A, Nicholson MR, Tanner-Smith EE, Zackular JP, Gomez-Duarte OG, Beaulieu DB, et al. Fecal transplantation for treatment of inflammatory bowel disease. Cochrane Database Syst Rev 2018;11:CD012774. + +10. Teng NMY, Price CA, McKee AM, Hall LJ, Robinson SD. + +Exploring the impact of gut microbiota and diet on breast cancer risk and progression. Int J Cancer 2021;149:494-504. + +11. Cryan JF, O'Riordan KJ, Cowan CSM, Sandhu KV, Bastiaanssen TFS, Boehme M, et al. The microbiota-gut-brain axis. Physiol Rev 2019;99:1877-2013. + +12. Gurung M, Li Z, You H, Rodrigues R, Jump DB, Morgun A, +et al. Role of gut microbiota in type 2 diabetes pathophysiology. EBioMedicine 2020;51:102590. + +13. Albillos A, de Gottardi A, Rescigno M. The gut-liver axis in liver disease: pathophysiological basis for therapy. J Hepatol 2020;72:558-77. + +14. Stavropoulou E, Kantartzi K, Tsigalou C, Konstantinidis T, +Romanidou G, Voidarou C, et al. Focus on the gut-kidney axis in health and disease. Front Med (Lausanne) 2021;7:620102. + +15. Matsushita M, Fujita K, Nonomura N. Influence of diet and nutrition on prostate cancer. Int J Mol Sci 2020;21:1447. + +16. Gann PH. Risk factors for prostate cancer. Rev Urol 2002;4(Suppl 5):S3-10. + +17. Liss MA, White JR, Goros M, Gelfond J, Leach R, Johnson- +Pais T, et al. Metabolic biosynthesis pathways identified from fecal microbiome associated with prostate cancer. Eur Urol 2018;74:575-82. + +18. Price AJ, Travis RC, Appleby PN, Albanes D, Barricarte Gurrea A, Bjørge T, et al. Circulating folate and vitamin B12 and risk of prostate cancer: a collaborative analysis of individual participant data from six cohorts including 6875 cases and 8104 controls. Eur Urol 2016;70:941-51. + +19. Tomaszewski JJ, Cummings JL, Parwani AV, Dhir R, Mason JB, Nelson JB, et al. Increased cancer cell proliferation in prostate cancer patients with high levels of serum folate. Prostate 2011;71:1287-93. + +20. Bistulfi G, Foster BA, Karasik E, Gillard B, Miecznikowski J, +Dhiman VK, et al. Dietary folate deficiency blocks prostate cancer progression in the TRAMP model. Cancer Prev Res (Phila) 2011;4:1825-34. + +21. Golombos DM, Ayangbesan A, O'Malley P, Lewicki P, Barlow L, Barbieri CE, et al. The role of gut microbiome in the pathogenesis of prostate cancer: a prospective, pilot study. Urology 2018;111:122-8. + +22. Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermúdez-Humarán LG, Gratadoux JJ, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A 2008;105:16731-6. + +23. Alanee S, El-Zawahry A, Dynda D, Dabaja A, McVary K, Karr M, et al. A prospective study to examine the association of the urinary and fecal microbiota with prostate cancer diagnosis after transrectal biopsy of the prostate using 16sRNA gene analysis. Prostate 2019;79:81-7. + +![7_image_0.png](7_image_0.png) + +24. Matsushita M, Fujita K, Motooka D, Hatano K, Fukae S, +Kawamura N, et al. The gut microbiota associated with high- Gleason prostate cancer. Cancer Sci 2021;112:3125-35. + +25. Dai Z, Coker OO, Nakatsu G, Wu WKK, Zhao L, Chen Z, et al. Multi-cohort analysis of colorectal cancer metagenome identified altered bacteria across populations and universal bacterial markers. Microbiome 2018;6:70. + +26. Moschen AR, Gerner RR, Wang J, Klepsch V, Adolph TE, +Reider SJ, et al. Lipocalin 2 protects from inflammation and tumorigenesis associated with gut microbiota alterations. Cell Host Microbe 2016;19:455-69. + +27. Meehan CJ, Beiko RG. A phylogenomic view of ecological specialization in the Lachnospiraceae, a family of digestive tract-associated bacteria. Genome Biol Evol 2014;6:703-13. + +28. Yachida S, Mizutani S, Shiroma H, Shiba S, Nakajima T, Sakamoto T, et al. Metagenomic and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer. Nat Med 2019;25:968-76. + +29. Matsushita M, Fujita K, Hayashi T, Kayama H, Motooka D, +Hase H, et al. Gut microbiota-derived short-chain fatty acids promote prostate cancer growth via IGF1 signaling. Cancer Res 2021;81:4014-26. + +30. Nishijima S, Suda W, Oshima K, Kim SW, Hirose Y, Morita H, +et al. The gut microbiome of healthy Japanese and its microbial and functional uniqueness. DNA Res 2016;23:125-33. + +31. Chen L, Zhang YH, Huang T, Cai YD. Gene expression profiling gut microbiota in different races of humans. Sci Rep 2016;6:23075. + +32. Lee HY, Wang JW, Juan YS, Li CC, Liu CJ, Cho SY, et al. The impact of urine microbiota in patients with lower urinary tract symptoms. Ann Clin Microbiol Antimicrob 2021;20:23. + +33. Miyake M, Tatsumi Y, Ohnishi K, Fujii T, Nakai Y, Tanaka N, +et al. Prostate diseases and microbiome in the prostate, gut, and urine. Prostate Int 2022;10:96-107. + +34. Al Bander Z, Nitert MD, Mousa A, Naderpoor N. The gut microbiota and inflammation: an overview. Int J Environ Res Public Health 2020;17:7618. + +35. Takezawa K, Fujita K, Matsushita M, Motooka D, Hatano K, +Banno E, et al. The Firmicutes/Bacteroidetes ratio of the human gut microbiota is associated with prostate enlargement. Prostate 2021;81:1287-93. + +36. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature 2006;444:1022-3. + +37. Stojanov S, Berlec A, Štrukelj B. The influence of probiotics on the Firmicutes/Bacteroidetes ratio in the treatment of obesity and inflammatory bowel disease. Microorganisms + +## 2020;8:1715. + +38. Vallianou N, Christodoulatos GS, Karampela I, Tsilingiris D, +Magkos F, Stratigou T, et al. Understanding the role of the gut microbiome and microbial metabolites in non-alcoholic fatty liver disease: current evidence and perspectives. Biomolecules 2021;12:56. + +39. Sawicka-Śmiarowska E, Moniuszko-Malinowska A, Kamiński KA. Which microbes like my diet and what does it mean for my heart? Nutrients 2021;13:4146. + +40. Malesza IJ, Malesza M, Walkowiak J, Mussin N, Walkowiak D, Aringazina R, et al. High-fat, Western-style diet, systemic inflammation, and gut microbiota: a narrative review. Cells 2021;10:3164. + +41. Cross TL, Kasahara K, Rey FE. Sexual dimorphism of cardiometabolic dysfunction: gut microbiome in the play? Mol Metab 2018;15:70-81. + +42. Barroso A, Santos-Marcos JA, Perdices-Lopez C, Vega-Rojas A, Sanchez-Garrido MA, Krylova Y, et al. Neonatal exposure to androgens dynamically alters gut microbiota architecture. J Endocrinol 2020;247:69-85. + +43. Moreno-Indias I, Sánchez-Alcoholado L, Sánchez-Garrido MÁ, Martín-Núñez GM, Pérez-Jiménez F, Tena-Sempere M, et al. Neonatal androgen exposure causes persistent gut microbiota dysbiosis related to metabolic disease in adult female rats. Endocrinology 2016;157:4888-98. + +44. Matsushita M, Fujita K, Motooka D, Hatano K, Hata J, Nishimoto M, et al. Firmicutes in gut microbiota correlate with blood testosterone levels in elderly men. World J Mens Health 2022;40:517-25. + +45. Shin JH, Park YH, Sim M, Kim SA, Joung H, Shin DM. + +Serum level of sex steroid hormone is associated with diversity and profiles of human gut microbiome. Res Microbiol 2019;170:192-201. + +46. Yuan X, Chen R, Zhang Y, Lin X, Yang X. Gut microbiota: effect of pubertal status. BMC Microbiol 2020;20:334. + +47. Markle JG, Frank DN, Mortin-Toth S, Robertson CE, Feazel LM, Rolle-Kampczyk U, et al. Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Science 2013;339:1084-8. + +48. Lee J, Yang W, Hostetler A, Schultz N, Suckow MA, Stewart KL, et al. Characterization of the anti-inflammatory Lactobacillus reuteri BM36301 and its probiotic benefits on aged mice. BMC Microbiol 2016;16:69. + +49. Dardmeh F, Alipour H, Gazerani P, van der Horst G, Brandsborg E, Nielsen HI. Lactobacillus rhamnosus PB01 (DSM 14870) supplementation affects markers of sperm kinematic parameters in a diet-induced obesity mice model. PLoS One 2017;12:e0185964. + +50. Yang J, Tan Q, Fu Q, Zhou Y, Hu Y, Tang S, et al. Gastrointestinal microbiome and breast cancer: correlations, mechanisms and potential clinical implications. Breast Cancer 2017;24:220-8. + +51. Parida S, Sharma D. The microbiome-estrogen connection and breast cancer risk. Cells 2019;8:1642. + +52. Sfanos KS, Markowski MC, Peiffer LB, Ernst SE, White JR, +Pienta KJ, et al. Compositional differences in gastrointestinal microbiota in prostate cancer patients treated with androgen axis-targeted therapies. Prostate Cancer Prostatic Dis 2018;21:539-48. + +53. Cani PD, Depommier C, Derrien M, Everard A, de Vos WM. + +Akkermansia muciniphila: paradigm for next-generation beneficial microorganisms. Nat Rev Gastroenterol Hepatol 2022;19:625-37. Erratum in: Nat Rev Gastroenterol Hepatol 2022;19:682 54. Song CH, Kim N, Nam RH, Choi SI, Jang JY, Lee HN. Changes in gut microbiome upon orchiectomy and testosterone administration in AOM/DSS-induced colon cancer mouse model. Cancer Res Treat 2022. doi: 10.4143/crt.2022.080 [Epub]. + +55. Hayashi T, Fujita K, Matsushita M, Hayashi Y, Uemura M, +Nonomura N. Metformin inhibits prostate cancer growth induced by a high-fat diet in Pten-deficient model mice. Int J Urol 2019;26:307-9. + +56. Hayashi T, Fujita K, Nojima S, Hayashi Y, Nakano K, Ishizuya Y, et al. High-fat diet-induced inflammation accelerates prostate cancer growth via IL6 signaling. Clin Cancer Res 2018;24:4309-18. + +57. Narita S, Nara T, Sato H, Koizumi A, Huang M, Inoue T, et al. + +Research evidence on high-fat diet-induced prostate cancer development and progression. J Clin Med 2019;8:597. + +58. Singh N, Gurav A, Sivaprakasam S, Brady E, Padia R, Shi H, +et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity 2014;40:128-39. + +59. Orlich MJ, Mashchak AD, Jaceldo-Siegl K, Utt JT, Knutsen SF, +Sveen LE, et al. Dairy foods, calcium intakes, and risk of incident prostate cancer in adventist health study-2. Am J Clin Nutr 2022;116:314-24. + +60. Aune D, Navarro Rosenblatt DA, Chan DS, Vieira AR, Vieira R, Greenwood DC, et al. Dairy products, calcium, and prostate cancer risk: a systematic review and meta-analysis of cohort studies. Am J Clin Nutr 2015;101:87-117. + +61. Collatuzzo G, Seyyedsalehi MS, Rezaeianzadeh A, Marzban M, Rashidian H, Hadji M, et al. Consumption of yoghurt and other dairy products and risk of colorectal cancer in Iran: the IROPICAN study. Nutrients 2022;14:2506. + +62. Harrison S, Lennon R, Holly J, Higgins JPT, Gardner M, +Perks C, et al. Does milk intake promote prostate cancer initiation or progression via effects on insulin-like growth factors (IGFs)? A systematic review and meta-analysis. Cancer Causes Control 2017;28:497-528. + +63. Fujita K, Hayashi T, Matsushita M, Uemura M, Nonomura N. Obesity, inflammation, and prostate cancer. J Clin Med 2019;8:201. + +64. Schwiertz A, Taras D, Schäfer K, Beijer S, Bos NA, Donus C, +et al. Microbiota and SCFA in lean and overweight healthy subjects. Obesity (Silver Spring) 2010;18:190-5. + +65. Matsushita M, Fujita K, Hatano K, Hayashi T, Kayama H, Motooka D, et al. High-fat diet promotes prostate cancer growth through histamine signaling. Int J Cancer 2022;151:623-36. + +66. Zhong W, Wu K, Long Z, Zhou X, Zhong C, Wang S, et al. + +Gut dysbiosis promotes prostate cancer progression and docetaxel resistance via activating NF-κB-IL6-STAT3 axis. Microbiome 2022;10:94. + +67. Henao-Mejia J, Elinav E, Jin C, Hao L, Mehal WZ, Strowig T, +et al. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature 2012;482:179-85. + +68. Rivera CA, Adegboyega P, van Rooijen N, Tagalicud A, Allman M, Wallace M. Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis. J Hepatol 2007;47:571-9. + +69. Loo TM, Kamachi F, Watanabe Y, Yoshimoto S, Kanda H, +Arai Y, et al. Gut microbiota promotes obesity-associated liver cancer through PGE2-mediated suppression of antitumor immunity. Cancer Discov 2017;7:522-38. + +70. Kim HS, Kim S, Shin SJ, Park YH, Nam Y, Kim CW, et al. + +Gram-negative bacteria and their lipopolysaccharides in Alzheimer's disease: pathologic roles and therapeutic implications. Transl Neurodegener 2021;10:49. + +71. Daisley BA, Chanyi RM, Abdur-Rashid K, Al KF, Gibbons S, +Chmiel JA, et al. Abiraterone acetate preferentially enriches for the gut commensal Akkermansia muciniphila in castrateresistant prostate cancer patients. Nat Commun 2020;11:4822. Erratum in: Nat Commun 2020;11:6394. + +72. Luo ZW, Xia K, Liu YW, Liu JH, Rao SS, Hu XK, et al. Extracellular vesicles from *Akkermansia muciniphila* elicit antitumor immunity against prostate cancer via modulation of CD8+ T cells and macrophages. Int J Nanomedicine 2021;16:2949-63. + +73. Pernigoni N, Zagato E, Calcinotto A, Troiani M, Mestre RP, +Calì B, et al. Commensal bacteria promote endocrine resistance in prostate cancer through androgen biosynthesis. Science 2021;374:216-24. + +74. Wortelboer K, Nieuwdorp M, Herrema H. Fecal microbiota transplantation beyond Clostridioides difficile infections. EBioMedicine 2019;44:716-29. + +75. Chen Y, Wei J. Identification of pathogen signatures in prostate cancer using RNA-seq. PLoS One 2015;10:e0128955. + +76. Cavarretta I, Ferrarese R, Cazzaniga W, Saita D, Lucianò R, +Ceresola ER, et al. The microbiome of the prostate tumor microenvironment. Eur Urol 2017;72:625-31. + +77. Davidsson S, Mölling P, Rider JR, Unemo M, Karlsson MG, +Carlsson J, et al. Frequency and typing of Propionibacterium acnes in prostate tissue obtained from men with and without prostate cancer. Infect Agent Cancer 2016;11:26. + +78. Olsson J, Drott JB, Laurantzon L, Laurantzon O, Bergh A, +Elgh F. Chronic prostatic infection and inflammation by Propionibacterium acnes in a rat prostate infection model. PLoS One 2012;7:e51434. Erratum in: PLoS One 2013;8:10.1371/ annotation/2160e616-aa79-4097-96ab-e143d2a4d136. + +79. Feng Y, Jaratlerdsiri W, Patrick SM, Lyons RJ, Haynes AM, +Collins CC, et al. Metagenomic analysis reveals a rich bacterial content in high-risk prostate tumors from African men. Prostate 2019;79:1731-8. + +80. Feng Y, Ramnarine VR, Bell R, Volik S, Davicioni E, Hayes VM, et al. Metagenomic and metatranscriptomic analysis of human prostate microbiota from patients with prostate cancer. BMC Genomics 2019;20:146. + +81. Nougayrède JP, Homburg S, Taieb F, Boury M, Brzuszkiewicz E, Gottschalk G, et al. Escherichia coli induces DNA doublestrand breaks in eukaryotic cells. Science 2006;313:848-51. + +82. Ma X, Chi C, Fan L, Dong B, Shao X, Xie S, et al. The microbiome of prostate fluid is associated with prostate cancer. Front Microbiol 2019;10:1664. + +83. Shrestha E, White JR, Yu SH, Kulac I, Ertunc O, De Marzo AM, et al. Profiling the urinary microbiome in men with positive versus negative biopsies for prostate cancer. J Urol 2018;199:161-71. + +84. Demichelis F, Stanford JL. Genetic predisposition to prostate cancer: update and future perspectives. Urol Oncol 2015;33:75-84. + +85. Bostwick DG, Burke HB, Djakiew D, Euling S, Ho SM, +Landolph J, et al. Human prostate cancer risk factors. Cancer 2004;101(10 Suppl):2371-490. + +86. Wang J, Tang M, Jiang H, Wu B, Cai W, Hu C, et al. The role of adrenergic activation on murine luteal cell viability and progesterone production. Theriogenology 2016;86:1182-8. + +87. Claesson MJ, Jeffery IB, Conde S, Power SE, O'Connor EM, +Cusack S, et al. Gut microbiota composition correlates with diet and health in the elderly. Nature 2012;488:178-84. \ No newline at end of file diff --git a/medical/md/PMC10790021.md b/medical/md/PMC10790021.md new file mode 100644 index 0000000000000000000000000000000000000000..f20d2368bd140b44ae145974913a7f06c8991b8c --- /dev/null +++ b/medical/md/PMC10790021.md @@ -0,0 +1,549 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +Contents lists available at ScienceDirect + +![0_image_3.png](0_image_3.png) + +# Critical Care And Resuscitation + +![0_Image_4.Png](0_Image_4.Png) + +journal homepage: www.elsevier.com/locate/ccrj Original Article + +![0_image_2.png](0_image_2.png) + +The impact of body mass index on long-term survival after ICU admission due to COVID-19: A retrospective multicentre study Ashwin Subramaniam, MBBS MMed FRACP FCICM a, b, c, d, *, Ryan Ruiyang Ling, MBBS e, Emma J. Ridley, PhD d, f, David V. Pilcher, MBBS MRCP(UK) FRACP FCICM d, g, h a Department of Intensive Care, Peninsula Health, Frankston, Victoria, Australia; b Department of Intensive Care, Dandenong Hospital, Monash Health, Dandenong, Victoria, Australia; c Peninsula Clinical School, Monash University, Frankston, Victoria, Australia; d Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), School of Public Health and Preventive Medicine, Monash University, Melbourne, Victoria, Australia; e Yong Loo Lin School of Medicine, National University of Singapore, Singapore; f Department of Nutrition and Dietetics, Alfred Hospital, Melbourne, Victoria, Australia; g Department of Intensive Care, Alfred Hospital, Melbourne, Victoria, Australia; h Centre for Outcome and Resource Evaluation, Australian and New Zealand Intensive Care Society, Melbourne, Victoria, Australia article information + +## Abstract + +| Article history: Received 16 October 2023 Accepted 18 October 2023 | +|----------------------------------------------------------------------| + +| Keywords: Body mass index Obesity Obesity paradox COVID-19 ANZICS adult patient database Long-term survival | +|---------------------------------------------------------------------------------------------------------------| + +Objective: The impact of obesity on long-term survival after intensive care unit (ICU) admission with severe coronavirus disease 2019 (COVID-19) is unclear. We aimed to quantify the impact of obesity on time to death up to two years in patients admitted to Australian and New Zealand ICUs. + +Design: Retrospective multicentre study. Setting: 92 ICUs between 1st January 2020 through to 31st December 2020 in New Zealand and 31st March 2022 in Australia with COVID-19, reported in the Australian and New Zealand Intensive Care Society adult patient database. + +Participants: All patients with documented height and weight to estimate the body mass index (BMI) +were included. Obesity was classified patients according to the World Health Organization recommendations. + +Interventions and main outcome measures: The primary outcome was survival time up to two years after ICU admission. The effect of obesity on time to death was assessed using a Cox proportional hazards model. Confounders were acute illness severity, sex, frailty, hospital type and jurisdiction for all patients. + +Results: We examined 2,931 patients; the median BMI was 30.2 (IQR 25.6e36.0) kg/m2. Patients with a BMI 30 kg/m2 were younger (median [IQR] age 57.7 [46.2e69.0] vs. 63.0 [50.0e73.6]; p < 0.001) than those with a BMI <30 kg/m2. Most patients (76.6%; 2,244/2,931) were discharged alive after ICU admission. The mortality at two years was highest for BMI categories <18.5 kg/m2 (35.4%) and 18.5e24.9 kg/m2 (31.1%), while lowest for BMI 40 kg/m2 (14.5%). After adjusting for confounders and with BMI +18.5e24.9 kg/m2 category as a reference, only the BMI 40 kg/m2 category patients had improved survival up to 2 years (hazard ratio ¼ 0.51; 95%CI: 0.34e0.76). + +Conclusions: The obesity paradox appears to exist beyond hospital discharge in critically ill patients with COVID-19 admitted in Australian and New Zealand ICUs. A BMI 40 kg/m2 was associated with a higher survival time of up to two years. + +© 2023 The Authors. Published by Elsevier B.V. on behalf of College of Intensive Care Medicine of Australia and New Zealand. This is an open access article under the CC BY-NC-ND license (http:// +creativecommons.org/licenses/by-nc-nd/4.0/). + +Abbreviations: ANZ, Australia and New Zealand; ANZICS, Australia and New Zealand Intensive Care Society; ANZROD, Australia and New Zealand risk of death; APACHE, +Acute Physiology and Chronic Health Evaluation; APD, Adult Patient Database; BMI, body mass index; COPD, chronic obstructive pulmonary disease; COVID-19, coronavirus disease 2019; CFS, Clinical Frailty Scale; ECMO, extracorporeal membrane oxygenation; ED, emergency department; HR, hazard ratio; ICU, intensive care unit; IQR, interquartile range; LOS, length of stay; MET, medical emergency team; MI, myocardial infarction; NIV, non-invasive ventilation; n, number; OR, odds ratio; RRT, renal replacement therapy; SD, standard deviation. + +* Corresponding author at: Intensive care specialist, Department of Intensive Care, Frankston Hospital, 2 Hastings Road, Frankston, Victoria, 3199, Australia. Tel.: þ61 3 9784 7422. + +E-mail address: ashwin.subramaniam@monash.edu (A. Subramaniam). + +@catchdrash (A. Subramaniam) +https://doi.org/10.1016/j.ccrj.2023.10.004 1441-2772/© 2023 The Authors. Published by Elsevier B.V. on behalf of College of Intensive Care Medicine of Australia and New Zealand. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +## Key Points + +Question: What is the association between BMI and survival time up to 2 years after ICU admission due to COVID-19? + +Findings: In this bi-national retrospective study, we found that only patients with a BMI 40 kg/m2 with severe COVID19 were independently associated with better survival time up to two years following an ICU admission in Australia and New Zealand. The obesity paradox appears to exist beyond hospital discharge in critically ill patients with COVID-19. + +Meaning: Our findings suggest that future studies should explore the reasons why there is a survival advantage for morbidly obese patients with COVID-19. Tweet: A retrospective multicentre study from ANZ found that only patients with a BMI 40 kg/m2 with severe COVID19 were independently associated with better survival time up to two years following an ICU admission. The obesity paradox appears to exist beyond hospital discharge in critically ill patients with COVID-19. + +## 1. Introduction + +The prevalence of obesity is increasing globally, with approximately 20 % of patients admitted to the intensive care unit (ICU) +obese.1e3 While obesity is typically associated with morbidity and mortality and poses additional care challenges in managing the critically ill, emerging literature has paradoxically reported higher short-term survival for patients with critical illness who are obese, also known as the obesity paradox.2,4e7 A large epidemiological study reporting on patients in ICUs from Australia and New Zealand observed that 35 % of the patients were obese and confirmed that some level of obesity was associated with overall lower in-hospital mortality.8 A recent study observed the obesity paradox in survivors of critical illness up to 4 years after their ICU +admission.9 There are however no accepted physiological mechanistic models to explain why morbid obesity could be protective. + +Although the causal mechanisms are unknown, some pathophysiologic mechanisms such as higher energy reserves, anti-inflammatory immune profile, the role of adipose tissue, prevention of muscle wasting and an association between increased BMI and lower risk of hypoglycaemia may all provide explanations for this obesity paradox.4,8e10 Apart from chronic disease, sarcopenia, malnutrition and smoking status, other non-physiological methodological explanations have been postulated.8,11,12 Furthermore, many biases such as selection bias, confounder bias, collider stratification bias, reporting bias, treatment bias and publication bias may be added to explain this phenomenon.1,8 A large retrospective study demonstrated that the obesity paradox is more than just a simple association between body mass index (BMI) and mortality and emphasized the importance of acute illness severity.13 Several studies have found an association between coronavirus disease 2019 (COVID-19) severity and obesity, 14e21 such that obesity was labelled a risk factor for severe COVID-19, ICU +admission and mechanical ventilation.18 As excess body fat mass results in various hormonal, metabolic, and inflammatory changes,22 it was hypothesized that adipose tissue may play a vital role in the mechanism of progression of COVID-19.23,24 In patients with severe COVID-19, there is a linear association between body mass index (BMI) and hospitalization, ICU admission and the need for mechanical ventilation and death among patients with COVID19.14,17e20,25e39 Furthermore, the Health Outcome Predictive Evaluation for COVID-19 Registry revealed no evidence of obesity paradox in patients with COVID-19.40 However, a recent study found that obese patients with COVID-19 admitted to hospital had lower survival, but amongst those who have been admitted to ICU, +there is a higher survival associated with obesity.25 These discrepancies in findings may be due to differences in study population characteristics, and most meta-analyses showed significant heterogeneity.39 To our knowledge, the impact of obesity on long-term survival after intensive care unit (ICU) admission with severe COVID-19 has never been investigated. Therefore, our primary aim was to investigate the association between BMI and long-term survival after ICU +admission due to COVID-19. We hypothesized that after adjusting for confounders, increasing levels of obesity would be associated with higher survival for up to two years. + +## 2. Methods: 2.1. Study Design And Participants + +We performed a retrospective multicentre study of all critically ill adult (age 16 years) patients admitted to Australian and New Zealand ICUs for their index ICU admission with a diagnosis of COVID-19. The patients were identified as suspected or confirmed COVID-19 based on the diagnostic code allocated as the cause of ICU +admission (Supplementary Table 1). We only included the first ICU +admission per hospitalization. Patients transferred to another ICU +were excluded. We also excluded patients if there were no documented height and weight to estimate BMI, if the outcome was missing or if admitted for organ donation purposes. + +## 2.2. Data Sources And Measurement + +Data were extracted from the Australian and New Zealand Intensive Care Society (ANZICS) adult patient database, a binational clinical quality registry dataset, collected by the ANZICS +Centre for Outcomes and Resources Evaluation, that contains information on all admissions to 98 % of adult ICUs in Australia and 67 % of ICUs in New Zealand. ICU admission records between 1st January 2020 through 31st December 2020 in New Zealand and 31st March 2022 in Australia, were linked to the date of death recorded in the national death registers in each country using an encoded linkage key. This provided a maximum follow-up of 27 months. Data collectors receive regular training and quality assurance reviews and data is collected using a standardized data dictionary. In addition, regular automated data checks further ensure the validity of recorded data.41 Apart from each patient's demographic details, the registry also captures their diagnostic, biochemical, physiological, and chronic health parameters from the first 24 h of ICU admission as required to calculate illness severity 184 A. Subramaniam et al. / Critical Care and Resuscitation 25 (2023) 182e192 scores. The definitions are described in the ANZICS adult patient database data dictionary.42 + +## 2.3. Variables + +We extracted data on patient demographics (age, sex, comorbidities, ethnicity, ICU admission source, smoking status), frailty status using the clinical frailty scale (CFS), BMI (based on patient's weight and height which could have been estimated at ICU admission), ICU organ supports (receipt of invasivemechanical ventilation, non-invasive ventilation, vasopressors, extracorporeal membrane oxygenation, and/or renal replacement therapy), ICU and hospital mortality, ICU and hospital length of stay and discharge destinations (home, chronic care facility or rehabilitation). + +## 2.4. Bmi Definition + +BMI was classified according to the World Health Organization recommendations:43 BMI <18.5 kg/m2 'underweight'; BMI between 18.5 24.9 kg/m2 'healthy weight'; BMI 25.0 and 29.9 kg/m2 +'overweight' and BMI30 kg/m2 'obese'. Obesity was further classified into three severity categories: class-I (BMI 30.0e34.9 kg/m2), +class-II (BMI 35.0e39.9 kg/m2) and class-III (BMI 40 kg/m2).44 BMI +was calculated based on the available weight and height in the ANZICS adult patient database (body weight (kg) divided by height squared (m2)). + +## 2.5. Confounding Variables + +Based on a previous study, the confounding variables included in the model pertain to the ICU (location, type, and size), patient (frailty, age, gender, and comorbidities), ICU admission (care type, source, treatment limitations and pre-ICU length of stay) and patient acute illness severity.45 Illness severity was determined using the ANZROD which is a highly predictive mortality prediction model used for benchmarking ICU performance in Australia and New Zealand.46,47 ANZROD includes components of the APACHE IV +scoring system, such as age, chronic illnesses, acute physiological disturbance and diagnosis, and the presence of treatment limitation on admission to ICU and provides an accurate estimate of the severity of illness in the first 24 h of ICU admission. Recent studies demonstrated that ANZROD was predictive of longer-term outcomes.48,49 Frailty was included as a confounder as 20 % of patients with COVID-19 admitted to ICU were frail.49e51 + +## 2.6. Outcomes + +The primary outcome was a survival time of up to two years and is reported as observed mortality at one and two years after ICU +admission. Secondary outcomes included ICU and hospital mortality, ICU and hospital length of stays, and discharge destinations. + +## 2.7. Subgroup Analysis + +Predefined subgroup analyses based on those who survived hospitalisation, and those receiving mechanical ventilation were performed. + +## 2.8. Statistical Analysis + +For categorical variables, we reported counts with percentage (n +(%)) and made comparisons between BMI categories using Chisquared tests. For continuous data, we report normally distributed data using means (standard deviation) and non-parametric data using median (interquartile range [IQR]). We made comparisons using the student's t-test for normally distributed data and the ManneWhitney U test otherwise. All analyses were performed with the BMI 18.5e24.9 kg/m2 category as the reference. + +We treated BMI both as a continuous and categorical variable in separate regression models. As estimates can cluster around centres, we used a robust-variance sandwich-type estimator to derive the standard errors and account for this clustering.52 Overall survival estimates are displayed using KaplaneMeier curve plots. The effect of BMI on time to death was assessed using a Cox proportional hazards model, adjusting for ANZROD, male sex, CFS, hospital type (tertiary, metropolitan, rural/regional, and private) and jurisdiction (Australian states and New Zealand), clustered by site, and site treated as a random effect. The results were reported using hazard ratios (HR, 95%CI). We assigned CFS and ANZROD as nonlinear predictors in the regression analysis where the estimation algorithm arranges the predictors based on significance to determine the best fitting fractional-polynomial function for each predictor while assuming all other predictors are linear using the mfp package on R. We conducted an additional post hoc analysis looking at an interaction effect of age*obesity treated both as continuous and categorical variables while adjusting for illness severity, hospital type, jurisdiction, and frailty. We performed data analysis using R4.2.2 (The R Foundation).53 We used a two-sided p-value of +<0.05 to indicate statistical significance. + +## 2.9. Ethics Approval + +All experimental protocols were approved by The Alfred Hospital Ethics Committee (local reference 413/19). + +## 3. Results + +During the study period, the ANZICS adult patient database listed 5834 patients with COVID-19 hospitalizations which were linked with the National Death Index. After excluding: 64 patients from New Zealand admitted in 2021; 413 patients who were transferred from another ICU; and 2423 patients who had insufficient information to estimate BMI, the final study population comprised 2931 patients from 87 Australian and 5 New Zealand ICUs (Supplementary Fig. 1). Compared to patients with estimated BMI, those without BMI were of similar age, frailty status and illness severity, less commonly were mechanically ventilated and had lower 2-year mortality after ICU admission (Supplementary Table 2). + +Of the total 2931 patients, 50.9 % (n ¼ 1493) had a BMI of 30 kg/m2 (median [IQR] BMI 30.2 [25.6e36.0]) kg/m2). Patients with obesity were younger (median [IQR] age 57.7 [46.2e69.0] vs. + +63.0 [50.0e73.6]; p < 0.001), a higher proportion had diabetes mellitus and a lower proportion of solid organ or haematological malignancies or immunosuppressive conditions or treatment limitations at ICU admission. Patients with obesity were more likely to receive invasive and non-invasive ventilatory and vasopressor supports than those without obesity. No difference in the receipt of other organ supports was observed. The baseline characteristics of patients with COVID-19 based on BMI categories are summarized in Table 1, while Supplementary Table 3 summarizes the comparison based on obesity status. + +## 3.1. Primary Outcome + +The mortality at two years progressively decreased with increasing BMI, highest for BMI <18.5 kg/m2 (35.4 %) and lowest for BMI 40 kg/m2 (14.5 %; p < 0.001) (Table 2, Fig. 1, Supplementary Table 4). The median [IQR] survival time for patients with obesity was 6.4 [4.5e16.8] months when compared to 7.1 [4.0e20.6] months for patients with a BMI <30 kg/m2 (p < 0.001). After adjusting for + +| Table 1 Baseline characteristics, illness severity and ICU management of patients with COVID-19 based on BMI categories. BMI <18.5 kg/m2 18.5e24.9 kg/m2 25.0e29.9 kg/m2 | 30.0e34.9 kg/m2 | 35.0e39.9 kg/m2 | 40.0 kg/m2 | p-value | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------|-------------------|-------------------|-------------------|-------------------|-------------------|---------| +| (Underweight) | (Healthy) | (Overweight) | (Class 1 obesity) | (Class 2 obesity) | (Class 3 obesity) | | | +| Number | 48 (1.6 %) | 595 (20.3 %) | 793 (27.1 %) | 659 (22.5 %) | 375 (12.8 %) | 459 (15.7 %) | e | +| Male sex | 20 (41.7 %) | 370 (62.2 %) | 534 (67.3 %) | 390 (59.2 %) | 206 (54.9 %) | 212 (46.2 %) | < 0.001 | +| Indigenous status | 4 (8.9 %) | 34 (5.9 %) | 27 (3.6 %) | 18 (2.8 %) | 9 (2.5 %) | 20 (4.7 %) | 0.030 | +| CFS score | 5 (4, 6) | 3 (2, 4) | 3 (2, 4) | 3 (2, 4) | 3 (2, 4) | 3 (3, 4) | < 0.001 | +| Age (years) | 63.5 (50.1, 76.4) | 63.0 (49.0, 73.6) | 62.7 (50.7, 73.5) | 60.0 (49.7, 70.1) | 58.0 (45.9, 70.0) | 52.7 (41.7, 64.4) | < 0.001 | +| Hospital classification - Public Tertiary | 16 (33.3 %) | 312 (52.4 %) | 430 (54.2 %) | 347 (52.7 %) | 205 (54.7 %) | 216 (47.1 %) | < 0.001 | +| - Public Metropolitan | 17 (35.4 %) | 167 (28.1 %) | 250 (31.5 %) | 231 (35.1 %) | 118 (31.5 %) | 177 (38.6 %) | | +| - Public rural/regional | 13 (27.1 %) | 100 (16.8 %) | 100 (12.6 %) | 77 (11.7 %) | 48 (12.8 %) | 63 (13.7 %) | | +| - Private | 2 (4.2 %) | 16 (2.7 %) | 13 (1.6 %) | 4 (0.6 %) | 4 (1.1 %) | 5 (1.1 %) | | +| ICU admission source - Emergency department | 29 (60.4 %) | 372 (62.5 %) | 420 (53.0 %) | 341 (51.7 %) | 186 (49.6 %) | 250 (54.5 %) | < 0.001 | +| - Ward | 12 (25.0 %) | 187 (31.4 %) | 329 (41.5 %) | 282 (42.8 %) | 164 (43.7 %) | 189 (41.2 %) | | +| - Other hospital | 4 (8.3 %) | 29 (4.9 %) | 38 (4.8 %) | 33 (5.0 %) | 22 (5.6 %) | 16 (3.5 %) | | +| - Operating theatre/ Recovery | 0 (0 %) | 1 (0.2 %) | 1 (0.1 %) | 0 (0 %) | 1 (0.3 %) | 1 (0.2 %) | | +| - Direct admit | 3 (6.3 %) | 6 (1.0 %) | 5 (0.6 %) | 3 (0.5 %) | 2 (0.5 %) | 4 (0.9 %) | | +| Documented co-morbidities - Chronic respiratory condition | 24 (50.0 %) | 95 (16.0 %) | 88 (11.1 %) | 82 (12.4 %) | 46 (12.3 %) | 80 (17.4 %) | < 0.001 | +| - Chronic cardiovascular condition | 5 (10.4 %) | 55 (9.2 %) | 57 (7.2 %) | 57 (8.6 %) | 29 (7.7 %) | 40 (8.7 %) | 0.83 | +| - Chronic renal failure | 1 (2.1 %) | 19 (3.2 %) | 24 (3.0 %) | 18 (2.7 %) | 13 (3.5 %) | 14 (3.1 %) | 0.017 | +| - Chronic liver disease | 4 (8.3 %) | 4 (0.7 %) | 2 (0.3 %) | 8 (1.2 %) | 5 (1.3 %) | 6 (1.3 %) | < 0.001 | +| - Diabetes mellitus | 8 (16.7 %) | 126 (21.2 %) | 199 (25.1 %) | 228 (34.6 %) | 120 (32.0 %) | 148 (32.2 %) | < 0.001 | +| - Cancer | 2 (4.2 %) | 33 (5.5 %) | 30 (3.8 %) | 14 (2.1 %) | 8 (2.1 %) | 7 (1.5 %) | 0.003 | +| - Immunosuppression | 5 (10.0 %) | 40 (7.1 %) | 51 (6.1 %) | 35 (5.3 %) | 14 (3.6 %) | 9 (1.8 %) | < 0.001 | +| - Lymphoma | 0 (0 %) | 7 (1.2 %) | 5 (0.6 %) | 2 (0.3 %) | 3 (0.8 %) | 1 (0.2 %) | 0.44 | +| - Leukaemia | 0 (0 %) | 11 (1.8 %) | 15 (1.9 %) | 8 (1.2 %) | 2 (0.5 %) | 2 (0.4 %) | 0.19 | +| Miscellaneous - ICU admission post-MET call | 8 (16.7 %) | 129 (21.9 %) | 238 (30.1 %) | 198 (30.3 %) | 127 (34.0 %) | 133 (29.2 %) | < 0.001 | +| - Treatment limitations | 16 (33.3 %) | 103 (17.3 %) | 102 (12.9 %) | 70 (10.6 %) | 42 (11.2 %) | 44 (9.6 %) | < 0.001 | +| - Cardiac arrest 24 h prior | 2 (4.2 %) | 14 (2.4 %) | 11 (1.4 %) | 6 (0.9 %) | 4 (1.1 %) | 2 (0.4 %) | 0.004 | +| - Pre-ICU (hours) | 9.0 (3.7, 19.4) | 7.3 (4.0, 24.0) | 9.8 (4.5, 40.4) | 10.6 (4.5, 39.3) | 10.0 (4.1, 46.2) | 8.6 (4.4, 42.4) | 0.033 | +| Organ failure scores - APACHE-II | 17.7 [6.1] | 16.5 [7.7] | 15.9 [6.7] | 15.8 [6.4] | 15.2 [6.6] | 15.0 [6.3] | < 0.001 | +| - APACHE-III | 56.4 [21.0] | 53.7 [15.7] | 53.2 [21.8] | 51.9 [20.1] | 50.0 [20.0] | 48.3 [19.0] | < 0.001 | +| - ANZROD (%) | 8.6 (4.4, 18.4) | 6.3 (2.5, 14.3) | 5.8 (2.5, 13.2) | 5.0 (2.6, 11.9) | 4.8 (2.3, 9.7) | 3.9 (2.0, 8.7) | < 0.001 | +| Organ supports - Mechanical ventilation | 6 (12.5 %) | 158 (27.0 %) | 277 (35.4 %) | 239 (36.5 %) | 144 (38.6 %) | 190 (42.4 %) | < 0.001 | +| - MV duration (hours) | 326 (85, 548) | 105 (26, 226) | 128 (27, 308) | 152 (49, 315) | 167 (42, 290) | 142 (42, 256) | 0.09 | +| - Non-invasive ventilation | 15 (31.3 %) | 162 (28.0 %) | 284 (36.4 %) | 289 (44.4 %) | 152 (41.0 %) | 235 (52.6 %) | < 0.001 | +| - Vasopressor and inotropes | 13 (27.1 %) | 209 (35.7 %) | 307 (39.1 %) | 270 (41.3 %) | 158 (42.2 %) | 187 (41.6 %) | 0.047 | +| - Renal replacement therapy | 2 (4.3 %) | 23 (4.0 %) | 52 (6.7 %) | 38 (5.9 %) | 20 (5.5 %) | 17 (3.9 %) | 0.018 | +| - ECMO | 0 (0 %) | 5 (0.9 %) | 10 (1.3 %) | 12 (1.9 %) | 3 (0.8 %) | 8 (1.8 %) | 0.61 | +| - Tracheostomy | 2 (4.3 %) | 14 (2.4 %) | 35 (4.6 %) | 26 (4.0 %) | 19 (5.2 %) | 15 (3.4 %) | 0.40 | +| Data are n (%), mean [SD] or median (IQR). CFS e clinical frailty scale, SD e standard deviation, IQR e interquartile range, BMI e body mass index, MET e medical emergency team, APACHE e Acute Physiology and Chronic Health Evaluation, ICU e intensive care unit, | A. Subramaniam et al. / Critical Care and Resuscitation 25 (2023) 182 e192 | | | | | | | + +186 + +![4_image_0.png](4_image_0.png) + +This retrospective study examined the impact of obesity +(defined via BMI) on long-term survival time up to two years after ICU admission among patients with severe COVID-19 in Australia and New Zealand. We found that more than half the patients had obesity, which was higher than pre-pandemic. Patients with Table 2Raw outcomes of patients with COVID-19 based on BMI categories.obesity were younger and had lower illness severity scores than +confounders, the Cox proportional hazards regression demonstrated higher survival time up to two years only for patients with BMI 40 kg/m2 (HR ¼ 0.51; 95%CI: 0.34e0.76) (Table 3). When BMI +was modelled as a continuous variable, a non-linear relationship with time to death was noted. Patients with BMIs between BMI 32 and 60 kg/m2 had the best survival (Fig. 2). + +## 3.2. Subgroup Analyses + +Patients who survived hospitalization: 2570 patients were discharged alive from the hospital; 52.2 % (n ¼ 1342) were obese. + +Kaplan Meier survival curves estimated that patients with BMI +40 kg/m2 had the highest survival compared to other BMI categories (p < 0.001; Supplementary Fig. 2, Supplementary Table 5). + +Similar findings of higher survival time up to two years only for patients with BMI 40 kg/m2 were observed in the adjusted Cox proportional hazards regression (Supplementary Table 6). The log hazards 2-year survival demonstrated a non-linear survival advantage for patients with BMI >25 kg/m2 (Supplementary Fig. 3). + +Patients receiving mechanical ventilation: 1015 patients received mechanical ventilation, of which 56.6 % (n ¼ 574) were obese. The duration of mechanical ventilation was also similar across BMI +categories (Supplementary Table 7). Kaplan Meier survival curves estimated that patients with BMI category <18.5 kg/m2 had the lowest 2-year survival, while BMI 40 kg/m2 had the highest +(Supplementary Fig. 2). The adjusted Cox proportional hazards regression demonstrated a higher survival time of up to 2 years for all patients with BMI 25 kg/m2 (Supplementary Table 8). The log hazards' 2-year survival demonstrated a non-linear survival advantage beyond BMI 25 kg/m2 (Supplementary Fig. 3). + +## 3.3. Secondary Outcomes + +The hospital mortality was highest for BMI 18.5e24.9 kg/m2 +(15 %, n ¼ 89), and lowest for patients with BMI 40 kg/m2 (8.0 %, +n ¼ 37). Compared to patients with BMI <18.5 and 18.5e24.9 kg/ +m2, patients with BMI 25.0e29.9 kg/m2 and patients with all three obesity classes had longer ICU and hospital length of stays +(p < 0.001 and p ¼ 0.015, respectively). There was no difference in the discharge destinations between BMI categories among patients discharged to their usual residence, to rehabilitation or a nursing home, respectively. The raw secondary outcomes are summarized in Table 2. + +## 3.4. Post Hoc Analysis + +When the mortality was standardised by age and obesity in the population, there was no interaction effect between age and obesity (pinteraction ¼ 0.32), when both were treated as continuous variables, adjusted for confounders (Supplementary Table 9). + +However, when BMI was categorised, age significantly interacted with only BMI<18.5 kg/m2 (pinteraction ¼ 0.004) and BMI +35.0e35.9 kg/m2 (pinteraction ¼ 0.026) categories, when adjusted for the same confounders (Supplementary Table 10). + +## 4. Discussion 4.1. Executive Summary + +![5_image_0.png](5_image_0.png) + +those who were within the normal or underweight range. Secondly, in the adjusted analysis, only patients with BMI 40 kg/m2 were associated with higher survival times up to two years. Third, most of the patients who survived hospitalization were alive at 2 years, the highest for patients with BMI 40 kg/m2. Fourth, all three obesity classes had higher two-year survival among those needing mechanical ventilation. Finally, our findings suggest that the obesity paradox exists beyond hospital discharge and up to 2 years in critically ill patients with COVID-19 admitted to Australian and New Zealand ICUs. + +| ANZROD, male sex, frailty (CFS), hospital type and jurisdiction for all patients with COVID-19. BMI was treated as a categorical variable. Predictor HR (95%CI) p-value BMI categories <18.5 kg/m2 0.69 (0.41e1.19) 0.18 18.5e24.9 kg/m2 Reference 25.0e29.9 kg/m2 0.90 (0.67e1.20) 0.46 30.0e34.9 kg/m2 0.84 (0.66e1.05) 0.13 35.0e39.9 kg/m2 0.95 (0.65e1.36) 0.78 40.0 kg/m2 0.51 (0.34e0.76) 0.001 Sex Male sex 1.17 (0.99e1.39) 0.06 Patient factors log (ANZROD/10) 2.02 (1.79e2.27) < 0.001 log (CFS/10) 6.30 (5.19e7.26) < 0.001 Hospital classification Metropolitan Reference Private 1.12 (0.67e1.88) 0.65 Rural/Regional 0.86 (0.68e1.09) 0.21 Tertiary 0.90 (0.73e1.12) 0.39 Jurisdiction New South Wales Reference Northern Territory 0.88 (0.07e2.09) 0.27 New Zealand 1.55 (0.58e4.15) 0.39 Queensland 0.60 (0.37e0.98) 0.041 South Australia 0.26 (0.19e0.34) < 0.001 Tasmania 0.00 (0.00e0.00) < 0.001 Victoria 0.81 (0.67e0.99) 0.035 Western Australia 0.98 (0.30e3.20) 0.98 CFS e Clinical Frailty Scale, BMI e body mass index, ANZROD e Australia New Zealand risk of death. | +|---| + +## 4.2. Relationship To Previous Studies + +The in-hospital mortality rates for patients with COVID-19 in Australia and New Zealand were lower than in other countries.50,54 Recent studies from Australia have found that in-hospital mortality rates in patients with severe COVID-19 reached nearly 15 %51,54 and that this was highest in the third wave of COVID-19 (26th June to 1st November 2021).54 While one study reported that 6-month survival was less than 50 % following ICU admission,55 others have found that more than 50 % of patients receiving mechanical ventilation for COVID-19 survived to 180 days.56 However, no studies reported long-term outcomes while taking BMI into account. + +Previous evidence suggests that being overweight and moderately obese was protective with lower mortality when compared with underweight BMI, normal BMI, or more severe obesity.25 Our + +![6_image_0.png](6_image_0.png) + +study observed that patients with obesity were younger, had fewer comorbidities, and had lower CFS and acute illness severity scores when compared to the underweight group who were relatively older patients, had more comorbidities, frailer with higher acute illness severity scores. A recent study found that ICU survivors demonstrated greater annual increases in lean and fat mass.57 Contrarily, although the point estimates for the other obese groups were also in the same direction, raising the possibility that this an overall trend to greater survival with progressively increasing BMI, our study found that survival time up to 2 years was independently associated only for patients with BMI 40 kg/m2. + +Our findings, therefore, suggest that an obesity paradox exists among survivors of COVID-19 beyond hospital discharge up to two years. Furthermore, a study that looked at the mortality trends between three COVID-19 surges among 1868 patients in the USA found that low BMI was associated with higher hospital mortality in patients admitted to ICU.58 In contrast, our study did not identify any not independent association with long-term survival. + +Previous evidence suggests that COVID-19 patients with a BMI of 30 kg/m2 were independently associated with needing mechanical ventilation.19,59 In contrast to the findings of a pre-pandemic large multicentre study which found that patients with class-II +and class-III obesity had longer durations of mechanical ventilation and ICU care, we found similar durations of mechanical ventilation across all BMI categories. + +## 4.3. Study Implications + +We found an over-representation of patients with severe COVID-19 who were obese in Australian and New Zealand ICUs when compared to the large Australian epidemiological study pre- +COVID-19.8 Our findings suggest the existence of obesity paradox existed beyond hospitalization for up to two years in critically ill patients. These findings will help determine the mortality risk associated with obesity in patients with severe COVID-19 to individualize patient intervention, such as healthy eating, minimizing ultra-processed foods and lifestyle modification.60 Furthermore, the COVID-19 vaccine's effectiveness in inducing protective humoral immunity is possibly reduced among obese individuals, therefore will require timely booster doses to improve their neutralizing immunity.61 Furthermore, priority should be given to exploring the reasons why there is a survival advantage for morbidly obese patients with COVID-19. + +## 4.4. Strengths + +Our study has several notable strengths. Firstly, our study spans numerous ICUs that enrolled patients across Australia and New Zealand. Secondly, the relatively larger sample of high-quality data increased the precision of our estimates. Thirdly, we incorporated several pre-specified secondary analyses to assess the association of obesity with survival of up to two years. + +## 4.5. Limitations + +There are a few limitations to this study. First, the retrospective study design meant that data collection was reliant on existing datasets and medical records. As a result, patients who did not have a recorded height or weight were excluded, reducing the sample size of our study. However, despite some group differences compared to those without BMI, the illness severity was similar, suggesting that our study is representative. In addition, there is a 189 possibility of data coding inaccuracy, and without site-based auditing of diagnostic codes, we cannot be certain about the degree of misclassification if any, and what its effects are on our findings. While we believe that our cohort is broadly representative of the larger population, this cannot be confirmed. Moreover, as a retrospective registry-based study, it is only possible to highlight associations and no causal inferences can be drawn. Furthermore, pre-ICU factors used to estimate illness severity may be colliders or confounders. This is an inherent limitation of the registry which does not capture pre-ICU factors that may influence ICU admission. + +Second, we did not have any information regarding the number of patients that were referred for and denied ICU admission. Third, the Australian and New Zealand healthcare system has been very fortunate with the magnitude of COVID-19 infections being largely under control, therefore the results may not be generalizable in resource-constrained healthcare systems. Fourth, the BMI was estimated only once at ICU admission. Although the median time spent outside the ICU was 7e10 h, fluid management acutely and the loss of lean mass (over days to weeks in the hospital) could have affected the weight and BMI. Fifth, although there is evidence that patients with obesity were more prone to have pathological pulmonary limitation and pulmonary gas exchange impairment to exercise compared with nonobese COVID-19 patients,62 we did not have the functional outcome following hospitalisation. Sixth, after discharge, the database did not record any ongoing healthcare needs following discharge. As a result, it is challenging to determine the precise impact of BMI after discharge on long-term survival. Seventh, COVID-19, its treatments, and in many places the composition of patients admitted to the ICU with COVID-19 rapidly evolved during the pandemic. Although this could have accounted for era effects in their analyses, a recent study from Australia did not show any difference based on the year of admission.50 Eighth, although many biases are related to the obesity paradox,1,8 collider stratification bias could be highly likely to underlie our study's findings and the decreased risk of mortality associated with obesity when in fact there is no biological basis for this relationship.63 Finally, the results from this dataset cannot be translated to a non-critically ill population. The existing public health message that the healthy-weight BMI is 18.5e24.9 kg/m2 has evidential support, including an association with a range of chronic diseases and early death, and the over-representation of patients with obesity and above in this dataset may speak to the increased burden of disease associated with obesity.8 + +## 5. Conclusion + +In a large bi-national retrospective study, we confirmed the existence of the obesity paradox existed beyond hospital discharge in critically ill patients with COVID-19 admitted in Australian and New Zealand ICUs. Patients with BMI 40 kg/m2 were associated with a higher survival time of up to two years. Future studies should explore reasons why there is a survival advantage for morbidly obese patients with COVID-19. + +## Disclaimer + +Ethics approval and consent to participate: + +- All experimental protocols were approved by The Alfred Hospital Ethics Committee (Project No: 413/19, Project Title BMI and frailty in Critical Illness, approved 11/01/2022) approved this study with a waiver of informed consent. This was a sub-study of the larger study titled "BMI and frailty in Critical Illness". +- ANZICS Centre for Outcome and Resource Evaluation Management Committee granted access to the ANZICS-APD following standing protocols on 11/16/2022. + +- All methods were carried out following the relevant guidelines and regulations of the Declaration of Helsinki. + +- Consent for publication e Not applicable Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available as these are linked from two registries (ANZICS, and the National Death Index), but are available from the corresponding author upon reasonable request. + +## Funding + +No financial support, including any institutional departmental funds, was used for the study. + +## Statement Of Authors' Contributions To The Manuscript + +1. Designed Research: + Study conception: AS + Study design: AS, RRL, ER, DP + Development of overall research plan: AS, RRL, ER, DP + Study oversight: AS + +## 2. Conducted Research: N/A. 3. Provided Essential Reagents/Materials: Dp Is The Custodian Of The Anzics Dataset. 4. Analysis Of Data: + + Data analysis, performed statistical analysis: AS, RRL Long-term survival statistical analysis and interpretation: DP + Tables and figures: RRL, AS + +## 5. Wrote Paper: + + Original drafting of the manuscript: AS Critical revision of the manuscript for intellectually important content: AS, RRL, ER, DP + +## 6. Primary Responsibility For Final Content: As. + +7. Other: AS and DP were responsible for the decision to submit the manuscript. + +8. All authors provided critical conceptual input, interpreted the data analysis, and read, and approved the final draft. + +## Conflict Of Interest + +The authors declare the following financial interests/personal relationships that may be considered as potential competing interests: n/a If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. + +## Acknowledgement + +The authors and the ANZICS CORE management committee would like to thank clinicians, data collectors and researchers at the following contributing sites: + +## Appendix A. Supplementary Data + +Supplementary data to this article can be found online at https://doi.org/10.1016/j.ccrj.2023.10.004. + +| Australian Capital Territory Calvary Hospital (Canberra) ICU | Canberra Hospital ICU | | +|-------------------------------------------------------------------------|-----------------------------------------------|-----------------------------------------------| +| New South Wales Bankstown-Lidcombe Hospital ICU | Hornsby Ku-ring-gai Hospital ICU | Shoalhaven Hospital ICU | +| Bathurst Base Hospital ICU | John Hunter Hospital ICU | St George Hospital (Sydney) ICU | +| Blacktown Hospital ICU | Lismore Base Hospital ICU | St Vincent's Hospital (Sydney) ICU | +| Calvary Mater Newcastle ICU | Maitland Hospital ICU | Goulburn Base Hospital ICU | +| Campbelltown Hospital ICU | Nepean Hospital ICU | Sydney Adventist Hospital ICU | +| Coffs Harbour Health Campus ICU | Northern Beaches Hospital ICU | Tamworth Base Hospital ICU | +| Concord Hospital (Sydney) ICU | Orange Base Hospital ICU | The Chris O'Brien Lifehouse ICU | +| Dubbo Base Hospital ICU | Port Macquarie Base Hospital ICU | Tweed Heads District Hospital ICU | +| Fairfield Hospital ICU | Prince of Wales Hospital ICU | Westmead Hospital ICU | +| Gosford Hospital ICU | Royal North Shore Hospital ICU | Wyong Hospital ICU | +| Sutherland Hospital & Community Health Services ICU | Royal Prince Alfred Hospital ICU | Wollongong Hospital ICU | +| Northern Territory Alice Springs Hospital ICU | Royal Darwin Hospital ICU | | +| Queensland Caboolture Hospital ICU | Mater Adults Hospital (Brisbane) ICU | Redcliffe Hospital ICU | +| Cairns Hospital ICU | Mater Health Services North Queensland ICU | Royal Brisbane and Women's Hospital ICU | +| Gold Coast University Hospital ICU | The Prince Charles Hospital ICU | St Vincent's Private Hospital Northside ICU | +| Hervey Bay Hospital ICU | Mater Private Hospital (Brisbane) ICU | St Vincent's Private Hospital (Toowoomba) ICU | +| Logan Hospital ICU | Noosa Hospital ICU | Sunshine Coast University Hospital ICU | +| Mackay Base Hospital ICU | Princess Alexandra Hospital ICU | Toowoomba Hospital ICU | +| South Australia Calvary Adelaide Hospital ICU | Flinders Medical Centre ICU | Royal Adelaide Hospital ICU | +| Tasmania Launceston General Hospital ICU | Royal Hobart Hospital ICU | | +| Victoria Alfred Hospital ICU | Epworth Freemasons Hospital ICU | Mulgrave Private Hospital ICU | +| Austin Hospital ICU | Epworth Geelong ICU | Royal Melbourne Hospital ICU | +| Box Hill Hospital ICU | Footscray Hospital ICU | St John of God Hospital (Bendigo) ICU | +| Cabrini Hospital ICU | Frankston Hospital ICU | St Vincent's Hospital (Melbourne) ICU | +| Casey Hospital ICU | Goulburn Valley Health ICU | Sunshine Hospital ICU | +| Central Gippsland Health Service ICU | Holmesglen Private Hospital ICU | The Northern Hospital ICU | +| Dandenong Hospital ICU | Knox Private Hospital ICU | University Hospital Geelong ICU | +| Bendigo Health Care Group Hospital ICU | Monash Medical CentreeClayton Campus ICU | | +| Western Australia Fiona Stanley Hospital ICU | Joondalup Health Campus ICU | Royal Perth Hospital ICU | +| Sir Charles Gairdner Hospital ICU New Zealand Christchurch Hospital ICU | Nelson Hospital ICU | Waikato Hospital ICU | +| Middlemore Hospital ICU | Whangarei Area Hospital ICU, Northland Health | | + +## References + +[1] Decruyenaere A, Steen J, Colpaert K, Benoit DD, Decruyenaere J, +Vansteelandt S. The obesity paradox in critically ill patients: a causal learning approach to a casual finding. Crit Care 2020;24(1):485. https://doi.org/ +10.1186/s13054-020-03199-5. + +[2] Schetz M, De Jong A, Deane AM, Druml W, Hemelaar P, Pelosi P, et al. Obesity in the critically ill: a narrative review. Intensive Care Med 2019;45(6):757e69. + +https://doi.org/10.1007/s00134-019-05594-1. + +[3] Sakr Y, Alhussami I, Nanchal R, Wunderink RG, Pellis T, Wittebole X, et al. + +Being overweight is associated with greater survival in ICU patients: results from the intensive care over nations audit. Crit Care Med 2015;43(12): +2623e32. https://doi.org/10.1097/ccm.0000000000001310 (In eng). + +[4] Karampela I, Chrysanthopoulou E, Christodoulatos GS, Dalamaga M. Is there an obesity paradox in critical illness? Epidemiologic and metabolic considerations. Curr Obes Rep 2020;9(3):231e44. https://doi.org/10.1007/s13679020-00394-x. + +[5] Pepper DJ, Sun J, Welsh J, Cui X, Suffredini AF, Eichacker PQ. Increased body mass index and adjusted mortality in ICU patients with sepsis or septic shock: a systematic review and meta-analysis. Crit Care 2016;20(1):181. https:// doi.org/10.1186/s13054-016-1360-z. + +[6] Hutagalung R, Marques J, Kobylka K, Zeidan M, Kabisch B, Brunkhorst F, +et al. The obesity paradox in surgical intensive care unit patients. + +Intensive Care Med 2011;37(11):1793. https://doi.org/10.1007/s00134011-2321-2. + +[7] Acharya P, Upadhyay L, Qavi A, Naaraayan A, Jesmajian S, Acharya S, et al. The paradox prevails: outcomes are better in critically ill obese patients regardless of the comorbidity burden. J Crit Care 2019;53:25e31. https://doi.org/ +10.1016/j.jcrc.2019.05.004 (In eng). + +[8] Secombe P, Woodman R, Chan S, Pilcher D, van Haren F. Epidemiology and outcomes of obese critically ill patients in Australia and New Zealand. Crit Care Resusc 2020;22(1):35e44. https://www.ncbi.nlm.nih.gov/pubmed/ +32102641. + +[9] Zhou D, Wang C, Lin Q, Li T. The obesity paradox for survivors of critically ill patients. Crit Care 2022;26(1):198. https://doi.org/10.1186/s13054-02204074-1. + +[10] Plecko D, Bennett N, Martensson J, Bellomo R. The obesity paradox and hypoglycemia in critically ill patients. Crit Care 2021;25(1):378. https://doi.org/ +10.1186/s13054-021-03795-z. + +[11] Tobias DK, Hu FB. Does being overweight really reduce mortality? Obesity 2013;21(9):1746e9. https://doi.org/10.1002/oby.20602. + +[12] Schooling CM, Au Yeung SL. "Selection bias by death" and other ways collider bias may cause the obesity paradox. Epidemiology 2017;28(2):e16e7. https:// doi.org/10.1097/EDE.0000000000000591. + +[13] Jagan N, Morrow LE, Walters RW, Plambeck RW, Wallen TJ, Patel TM, et al. + +Sepsis and the obesity paradox: size matters in more than one way. Crit Care Med 2020;48(9):e776e82. https://doi.org/10.1097/ccm.0000000000004459 +(In eng). + +[14] Williamson EJ, Walker AJ, Bhaskaran K, Bacon S, Bates C, Morton CE, et al. + +Factors associated with COVID-19-related death using OpenSAFELY. Nature 2020;584(7821):430e6. https://doi.org/10.1038/s41586-020-2521-4 (In eng). + +[15] Caussy C, Pattou F, Wallet F, Simon C, Chalopin S, Telliam C, et al. Prevalence of obesity among adult inpatients with COVID-19 in France. Lancet Diabetes 191 Endocrinol 2020;8(7):562e4. https://doi.org/10.1016/s2213-8587(20) +30160-1 (In eng). + +[16] Petrilli CM, Jones SA, Yang J, Rajagopalan H, O'Donnell L, Chernyak Y, et al. + +Factors associated with hospital admission and critical illness among 5279 people with coronavirus disease 2019 in New York City: prospective cohort study. BMJ 2020;369:m1966. https://doi.org/10.1136/bmj.m1966 (In eng). + +[17] Goyal P, Ringel JB, Rajan M, Choi JJ, Pinheiro LC, Li HA, et al. Obesity and COVID-19 in New York city: a retrospective cohort study. Ann Intern Med 2020;173(10):855e8. https://doi.org/10.7326/m20-2730 (In eng). + +[18] Simonnet A, Chetboun M, Poissy J, Raverdy V, Noulette J, Duhamel A, et al. + +High prevalence of obesity in severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) requiring invasive mechanical ventilation. Obesity 2020;28(7):1195e9. https://doi.org/10.1002/oby.22831 (In eng). + +[19] Kalligeros M, Shehadeh F, Mylona EK, Benitez G, Beckwith CG, Chan PA, et al. + +Association of obesity with disease severity among patients with coronavirus disease 2019. Obesity 2020;28(7):1200e4. https://doi.org/10.1002/oby.22859 +(In eng). + +[20] Palaiodimos L, Kokkinidis DG, Li W, Karamanis D, Ognibene J, Arora S, et al. + +Severe obesity, increasing age and male sex are independently associated with worse in-hospital outcomes, and higher in-hospital mortality, in a cohort of patients with COVID-19 in the Bronx, New York. Metabolism 2020;108: 154262. https://doi.org/10.1016/j.metabol.2020.154262 (In eng). + +[21] Rebelos E, Moriconi D, Virdis A, Taddei S, Foschi D, Nannipieri M. Letter to the Editor: importance of metabolic health in the era of COVID-19. Metabolism 2020;108:154247. https://doi.org/10.1016/j.metabol.2020.154247 (In eng). + +[22] Huttunen R, Syrjanen J. Obesity and the risk and outcome of infection. Int J +Obes 2013;37(3):333e40. https://doi.org/10.1038/ijo.2012.62. + +[23] Luzi L, Radaelli MG. Influenza and obesity: its odd relationship and the lessons for COVID-19 pandemic. Acta Diabetol 2020;57(6):759e64. https://doi.org/ +10.1007/s00592-020-01522-8. + +[24] Malavazos AE, Corsi Romanelli MM, Bandera F, Iacobellis G. Targeting the adipose tissue in COVID-19. Obesity 2020;28(7):1178e9. https://doi.org/ +10.1002/oby.22844. + +[25] Chetboun M, Raverdy V, Labreuche J, Simonnet A, Wallet F, Caussy C, et al. BMI +and pneumonia outcomes in critically ill COVID-19 patients: an international multicenter study. Obesity 2021;29(9):1477e86. https://doi.org/10.1002/ +oby.23223. + +[26] Bello-Chavolla OY, Bahena-Lopez JP, Antonio-Villa NE, Vargas-V azquez A, +Gonzalez-Díaz A, M arquez-Salinas A, et al. Predicting mortality due to SARSCoV-2: a mechanistic score relating obesity and diabetes to COVID-19 outcomes in Mexico. J Clin Endocrinol Metab 2020;105(8). https://doi.org/ +10.1210/clinem/dgaa346 (In eng). + +[27] Busetto L, Bettini S, Fabris R, Serra R, Dal Pra C, Maffei P, et al. Obesity and COVID-19: an Italian snapshot. Obesity 2020;28(9):1600e5. https://doi.org/ +10.1002/oby.22918 (In eng). + +[28] Cai Q, Chen F, Wang T, Luo F, Liu X, Wu Q, et al. Obesity and COVID-19 severity in a designated hospital in shenzhen, China. Diabetes Care 2020;43(7): +1392e8. https://doi.org/10.2337/dc20-0576 (In eng). + +[29] Gao M, Piernas C, Astbury NM, Hippisley-Cox J, O'Rahilly S, Aveyard P, et al. + +Associations between body-mass index and COVID-19 severity in 6.9 million people in England: a prospective, community-based, cohort study. Lancet Diabetes Endocrinol 2021;9(6):350e9. https://doi.org/10.1016/S22138587(21)00089-9. + +[30] Sanchis-Gomar F, Lavie CJ, Mehra MR, Henry BM, Lippi G. Obesity and outcomes in COVID-19: when an epidemic and pandemic collide. Mayo Clin Proc 2020;95(7):1445e53. https://doi.org/10.1016/j.mayocp.2020.05.006 (In eng). + +[31] Anderson MR, Geleris J, Anderson DR, Zucker J, Nobel YR, Freedberg D, et al. + +Body mass index and risk for intubation or death in SARS-CoV-2 infection : a retrospective cohort study. Ann Intern Med 2020;173(10):782e90. https:// +doi.org/10.7326/m20-3214 (In eng). + +[32] Tartof SY, Qian L, Hong V, Wei R, Nadjafi RF, Fischer H, et al. Obesity and mortality among patients diagnosed with COVID-19: results from an integrated health care organization. Ann Intern Med 2020;173(10):773e81. + +https://doi.org/10.7326/m20-3742 (In eng). + +[33] Sharma A, Garg A, Rout A, Lavie CJ. Association of obesity with more critical illness in COVID-19. Mayo Clin Proc 2020;95(9):2040e2. https://doi.org/ +10.1016/j.mayocp.2020.06.046 (In eng). + +[34] Lavie CJ, Coursin DB, Long MT. The obesity paradox in infections and implications for COVID-19. Mayo Clin Proc 2021;96(3):518e20. https://doi.org/ +10.1016/j.mayocp.2021.01.014. + +[35] Docherty AB, Harrison EM, Green CA, Hardwick HE, Pius P, Norman L, et al. + +Features of 20 133 UK patients in hospital with covid-19 using the ISARIC +WHO Clinical Characterisation Protocol: prospective observational cohort study. BMJ 2020;369:m1985. https://doi.org/10.1136/bmj.m1985 (In eng). + +[36] Foldi M, Farkas N, Kiss S, Z € adori N, V ancsa S, Szak o L, et al. Obesity is a risk factor for developing critical condition in COVID-19 patients: a systematic review and meta-analysis. Obes Rev 2020;21(10):e13095. https://doi.org/ +10.1111/obr.13095. + +[37] Huang Y, Lu Y, Huang YM, Wang M, Ling W, Sui Y, et al. Obesity in patients with COVID-19: a systematic review and meta-analysis. Metabolism 2020;113:154378. https://doi.org/10.1016/j.metabol.2020.154378 (In eng). + +[38] Liu M, Deng C, Yuan P, Ma J, Yu P, Chen J, et al. Is there an exposure-effect relationship between body mass index and invasive mechanical ventilation, severity, and death in patients with COVID-19? Evidence from an updated meta-analysis. Obes Rev 2020;21(11):e13149. https://doi.org/10.1111/ obr.13149. + +[39] Soeroto AY, Soetedjo NN, Purwiga A, Santoso P, Kulsum ID, Suryadinata H, +et al. Effect of increased BMI and obesity on the outcome of COVID-19 adult patients: a systematic review and meta-analysis. Diabetes Metabol Syndr 2020;14(6):1897e904. https://doi.org/10.1016/j.dsx.2020.09.029 (In eng). + +[40] Abumayyaleh M, Núnez Gil IJ, El-Battrawy I, Estrada V, Becerra-Mu ~ noz VM, El- ~ +Aparisi A, et al. Does there exist an obesity paradox in COVID-19? Insights of the international HOPE-COVID-19-registry. Obes Res Clin Pract 2021;15(3): +275e80. https://doi.org/10.1016/j.orcp.2021.02.008 (In eng). + +[41] ANZICS Centre for Outcomes and Resource Evaluation. Centre for resource and outcomes evaluation 2020 report. 2021. https://www.anzics.com.au/wpcontent/uploads/2020/11/2019-CORE-Report.pdf. + +[42] ANZCIS Centre for Outcomes and Resource Evaluation. Adult Patient Database Data Dictionary. (https://www.anzics.com.au/wp-content/uploads/2021/03/ ANZICS-APD-Dictionary.pdf). + +[43] Obesity: preventing and managing the global epidemic. Report of a WHO +consultation. World Health Organ Tech Rep Ser 2000;894(i-xii):1e253 [In eng)]. + +[44] Poirier P, Giles TD, Bray GA, Hong Y, Stern JS, Pi-Sunyer FX, et al. Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight loss. Arterioscler Thromb Vasc Biol 2006;26(5):968e76. https://doi.org/10.1161/ +01.ATV.0000216787.85457.f3 +(In eng). + +[45] Darvall JN, Bellomo R, Bailey M, Young PJ, Rockwood K, Pilcher D. Impact of frailty on persistent critical illness: a population-based cohort study. + +Intensive Care Med 2022;48(3):343e51. https://doi.org/10.1007/s00134022-06617-0. + +[46] Pilcher D, Paul E, Bailey M, Huckson S. The Australian and New Zealand Risk of Death (ANZROD) model: getting mortality prediction right for intensive care units. Crit Care Resusc 2014;16(1):3e4 [In eng)]. + +[47] Paul E, Bailey M, Kasza J, Pilcher D. The ANZROD model: better benchmarking of ICU outcomes and detection of outliers. Crit Care Resusc 2016;18(1):25e36 +[In eng)]. + +[48] Subramaniam A, Ueno R, Tiruvoipati R, Srikanth V, Bailey M, Pilcher D. + +Comparison of the predictive ability of clinical frailty scale and hospital frailty risk score to determine long-term survival in critically ill patients: a multicentre retrospective cohort study. Crit Care 2022;26(1):121. https://doi.org/ +10.1186/s13054-022-03987-1. + +[49] Subramaniam A, Ueno R, Tiruvoipati R, Darvall J, Srikanth V, Bailey M, et al. + +Comparing the clinical frailty scale and an international classification of diseases-10 modified frailty index in predicting long-term survival in critically ill patients. Crit Care Explor 2022;4(10):e0777. https://doi.org/10.1097/ +CCE.0000000000000777. + +[50] Subramaniam A, Shekar K, Anstey C, Tiruvoipati R, Pilcher D. Impact of frailty on clinical outcomes in patients with and without COVID-19 pneumonitis admitted to intensive care units in Australia and New Zealand: a retrospective registry data analysis. Crit Care 2022;26(1):301. https://doi.org/10.1186/ +s13054-022-04177-9. + +[51] Subramaniam A, Anstey C, Curtis JR, Ashwin S, Ponnapa Reddy M, +Aliberti MJR, et al. Characteristics and outcomes of patients with frailty admitted to ICU with coronavirus disease 2019: an individual patient data meta-analysis. Crit Care Explor 2022;4(1):e0616. https://doi.org/10.1097/ +CCE.0000000000000616. + +[52] Wei LJ, Lin DY, Weissfeld L. Regression analysis of multivariate incomplete failure time data by modeling marginal distributions. J Am Stat Assoc 1989;84(408):1065e73. https://doi.org/10.2307/2290084. + +[53] Team RC. R: A language and environment for statistical computing. . R +Foundation for Statistical Computing. (https://www.R-project.org/). + +[54] Begum H, Neto AS, Alliegro P, Broadley T, Trapani T, Campbell LT, et al. People in intensive care with COVID-19: demographic and clinical features during the first, second, and third pandemic waves in Australia. Med J Aust 2022;217(7):352e60. https://doi.org/10.5694/mja2.51590 (In eng). + +[55] Taniguchi LU, Avelino-Silva TJ, Dias MB, Jacob-Filho W, Aliberti MJR. Association of frailty, organ support, and long-term survival in critically ill patients with COVID-19. Crit Care Explor 2022;4(6):e0712. https://doi.org/10.1097/ +CCE.0000000000000712. + +[56] Penuelas O, del Campo-Albendea L, de Aledo ALG, A ~ n~on JM, Rodríguez- +Solís C, Mancebo J, et al. Long-term survival of mechanically ventilated patients with severe COVID-19: an observational cohort study. Ann Intensive Care 2021;11(1):143. https://doi.org/10.1186/s13613-02100929-y. + +[57] Thackeray M, Kotowicz MA, Pasco JA, Mohebbi M, Orford N. Changes in body composition in the year following critical illness: a case-control study. J Crit Care 2022;71:154043. https://doi.org/10.1016/j.jcrc.2022.154043. + +[58] Auld SC, Harrington KRV, Adelman MW, Robichaux CJ, Overton EC, CaridiScheible M, et al. Trends in ICU mortality from coronavirus disease 2019: a tale of three surges. Crit Care Med 2022;50(2):245e55. https://doi.org/ +10.1097/ccm.0000000000005185 (In eng). + +[59] Martino JL, Stapleton RD, Wang M, Day AG, Cahill NE, Dixon AE, et al. Extreme obesity and outcomes in critically ill patients. Chest 2011;140(5):1198e206. + +https://doi.org/10.1378/chest.10-3023 (In eng). + +[60] Popkin BM, Du S, Green WD, Beck MA, Algaith T, Herbst CH, et al. Individuals with obesity and COVID-19: a global perspective on the epidemiology and 192 + +biological relationships. Obes Rev 2020;21(11):e13128. https://doi.org/ 10.1111/obr.13128. + +[61] Faizo AA, Qashqari FS, El-Kafrawy SA, Barasheed O, Almashjary MN, +Alfelali M, et al. A potential association between obesity and reduced effectiveness of COVID-19 vaccine-induced neutralizing humoral immunity. J Med Virol 2023;95(1):e28130. https://doi.org/10.1002/jmv.28130 +(In eng). + +[62] Lacavalerie MR, Pierre-Francois S, Agossou M, Inamo J, Cabie A, Barnay JL, et al. + +Obese patients with long COVID-19 display abnormal hyperventilatory response and impaired gas exchange at peak exercise. Future Cardiol 2022;18(7):577e84. https://doi.org/10.2217/fca-2022-0017 (In eng). + +[63] Banack HR, Kaufman JS. Does selection bias explain the obesity paradox among individuals with cardiovascular disease? Ann Epidemiol 2015;25(5): +342e9. https://doi.org/10.1016/j.annepidem.2015.02.008. \ No newline at end of file diff --git a/medical/md/PMC10882963.md b/medical/md/PMC10882963.md new file mode 100644 index 0000000000000000000000000000000000000000..5cce508841d6daf9e31bbeb4b3b5e56b77f9b8d6 --- /dev/null +++ b/medical/md/PMC10882963.md @@ -0,0 +1,402 @@ +pubs.acs.org/est Article + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +# A U.S. Lead Exposure Hotspots **Analysis** + +Valerie G. Zartarian,* Jianping Xue, Antonios G. Poulakos, Rogelio Tornero-Velez, Lindsay W. Stanek, Emily Snyder, Veronica Helms Garrison, Kathryn Egan, and Joseph G. Courtney Cite **This:** Environ. Sci. *Technol.* 2024, 58, 3311−3321 Read **Online** +ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: To identify U.S. lead exposure risk hotspots, we expanded upon geospatial statistical methods from a published Michigan case study. The evaluation of identified hotspots using five lead indices, based on housing age and sociodemographic data, showed moderate-to-substantial agreement with state-identified higher-risk locations from nine public health department reports +(45−78%) and with hotspots of children's blood lead data from Michigan and Ohio (e.g., Cohen's kappa scores of 0.49−0.63). Applying geospatial cluster analysis and 80th−100th percentile methods to the lead indices, the number of U.S. census tracts ranged from ∼8% (intersection of indices) to ∼41% (combination of indices). Analyses of the number of children <6 years old living in those census tracts revealed the states (e.g., Illinois, Michigan, New Jersey, New York, Ohio, Pennsylvania, Massachusetts, California, Texas) and counties with highest potential lead exposure risk. Results support use of available lead indices as surrogates to identify locations in the absence of consistent, complete blood lead level (BLL) data across the United States. Ground-truthing with local knowledge, additional BLL data, and environmental data is needed to improve identification and analysis of lead exposure and BLL hotspots for interventions. While the science evolves, these screening results can inform "deeper dive" analyses for targeting lead actions. KEYWORDS: children's health, environmental health, metals, indices, mapping, *biomonitoring* +- **INTRODUCTION** +While average blood lead levels in the U.S. have declined, millions of children and adults are still exposed to various sources of lead.1−3 Although there are known sources of lead exposure (e.g., paint in older homes, drinking water from lead pipes, soil, and consumer products),4 it is difficult to identify communities that may have disproportionate exposures because of limitations in children's blood lead surveillance data and gaps in environmental and other exposure data.5 There is no known level of lead exposure to be without risk,6−8 and many communities are disproportionately impacted.9,10 Identifying and addressing remaining lead exposure risk hotspots are priorities in the United States. The Federal Lead Action Plan10 and the U.S. Environmental Protection Agency +(EPA) Lead Strategy (e.g., Goal 2, "Identify Communities with High Lead Exposures and Improve Their Health Outcomes")9 highlight the need for lead mapping as part of whole-ofgovernment efforts to address high exposure risk locations and disparities. Data mapping can inform screening and prioritization efforts to guide interventions and "deeper dive" analyses +(such as enhancing children's blood lead level (BLL) surveillance data analyses and lead source apportionment analyses). These analyses can assist in efforts around primary prevention; lead-based paint mitigation; lead remediation, enforcement, education, and outreach.11 Federal agencies are collaborating to identify geographic locations and populations at risk for lead exposure so that they can be addressed proactively. Examples include targeting HUD remediation grants, EPA environmental cleanup actions, and CDC primary prevention and enhanced blood lead testing programs for children.5 The EPA, U.S. Department of Housing and Urban Development (HUD), Centers for Disease Control and Prevention (CDC), and other organizations are applying geospatial statistical methods with available data (blood lead surveillance data, lead indices, and environmental data) to identify locations that are disproportionately at risk. However, there are remaining gaps and challenges such as cross-agency data collection and integration.5 The interagency lead mapping state-of-the-science paper5 presented a data integration roadmap to identify places for public health action: + +| Received: | September 22, 2023 | +|-----------------------------|----------------------| +| Revised: | December 7, 2023 | +| Accepted: | December 8, 2023 | +| Published: February 9, 2024 | | + +![0_image_2.png](0_image_2.png) + +"For national scale, one could use lead indices and *evaluate* identified geographic locations by available BLL data and local knowledge...While the science is evolving to *compare* available lead indices and models against each other and evaluate or ground-truth them against states'-measured surveillance BLL data...an analyst or decision-maker may want to use them collectively to identify high-risk locations to cast a wider net or to use their intersecting locations for a more focused *list...".* +This paper provides a next step in the research to identify U.S. lead exposure hotspots using existing data. + +- MATERIALS AND **METHODS** +The general approach taken for this analysis consists of the following four steps: +1. Statistically evaluated hotspots identified with lead indices against children's BLL surveillance data: +(a) from Michigan (MI) and Ohio (OH), using the BLL +data and statistical methods described in Xue et al.12 and Stanek et al.,13 respectively, and an expanded set of national lead indices; +(b) from matching hotspots identified using lead indices with community hotspots identified in 9 state health department public reports (listed in Zartarian et al.5) +and quantifying the percent; a match is defined here as a community with at least one census tract identified by the lead indices in our analyses; 2. Compared existing national indices against each other and against available BLL surveillance data using sensitivity, specificity, and Cohen's kappa score to determine which indices are the statistically strongest predictors of hotspots for the national-scale analysis; 3. Produced census tract-level maps for the United States that visualize the intersection and collective combination of hotspots based on the two methods discussed in Xue et al.,12 top 20 (i.e., 80th−100th) percentiles and GetisOrd Gi*14 geospatial cluster hotspots analysis methods; 4. Conducted national-scale analyses to identify states and counties with the highest potential lead exposure risk, based on the considered indices and the number of children younger than six years old in the identified 2010 census tracts (n = 73,086 census tracts containing at least one child <6 years old in the 50 states). + +In this paper, hotspots refer to geographic locations with a higher prevalence of children's lead exposures, based on the percentage of higher BLLs among children and/or identification by lead exposure indices using two statistical methods: +top 20 (80th−100th) percentile and Getis-Ord Gi* geospatial cluster hotspot analysis (ESRI ArcGIS Desktop v.10.8.2 and Pro 3.1 Hot Spot Analysis tool15). The top 20 (80th−100th) +percentile method uses a direct percentile calculation conducted with traditional statistical software (i.e., Microsoft Excel). The Getis-Ord Gi* geospatial cluster hotspot analysis method uses the ArcGIS Hot Spot tool15 which is a spatial interpolation function that calculates the Getis-Ord Gi* statistic for the exposure value of each census tract within the context of neighboring ones. To be identified as a geographic location with a higher prevalence of children's lead exposures, a given census tract will have higher BLL values and/or high lead exposure index values that are also adjacent to other census tracts with higher BLL values and/or high lead +exposure index values. More detail on the application of this method can be found in Xue et al.12 In our analysis, "higher BLLs" (referred to previously as elevated BLLs or EBLLs) are defined as greater than or equal to CDC's previous blood lead reference value of 5 μg/dL.16 We elected to use CDC's 2012 blood lead reference value +(BLRV)16 of 5 μg/dL for this analysis to remain consistent with the 2010 census data, MI and OH BLLs acquired through EPA's data use agreements, and the public state health department reports used in this analysis (all of which contain data predating 2020). The BLRV was updated to 3.5 μg/dL in 2021.17 The five lead exposure indices and models (referred to collectively as lead indices) used in this national-scale analysis are based on publicly available data on older housing (prior to 1980) and sociodemographic variables. We included the following indices: (1) the EPA EJSCREEN Lead Paint EJ +Index ("EJSCREEN Index");18 (2) Schultz et al., 2017-based BLL model ("Schultz Model") developed by the EPA Office of Research and Development (ORD);19 (3) U.S. Department of Housing and Urban Development Deteriorated Paint Index +(HUD DPI);20 (4) the Vox U.S. Lead Risk Exposure score +("Vox");21 and Random Forest (RF) Regression EBLL +Prediction Model ("RF Model"). The first four were summarized by Zartarian et al.5 Two versions of the new Random Forest (RF) Regression EBLL Prediction Model developed by EPA ORD22,23 were also included: RF Model version 1 (v1) and RF Model version 2 (v2). "RF Model v1" refers to version 1 of the regression model built using Ohio 2007−2011 BLL data and the 2013 OH DH report model.24 It includes the following five variables: percent of homes built prior to 1940, percent of homes built prior to 1950, percent of families whose income-to-poverty ratio was greater than 2, percent of population with either high school or higher education, and percent of non-Hispanic African Americans in each census tract. Demographic data originate from the American Community Survey 2013−2017 5-year summary file.25 "RF Model v2" refers to a modified version that includes the following variables: percent of homes built prior to 1940, percent of homes built prior to 1950, and percent of families whose income-to-poverty ratio was greater than 2. Note that RF Model v2, which is a reduced form of v1, is included in a subset of tables and figures in the Results and Discussion, as it was developed after the initial analyses were conducted. Please see the Supporting Information for more details. + +We used BLL data from Michigan (∼1.9 million BLL data points; 2006−2016 among children <6 years old) and Ohio +(∼2.3 million BLL data points; 2005−2018 among children <6 years old) acquired through data use agreements between the state health departments and the EPA/ORD. More detail on these data can be found in Xue et al. (MI data)12 and Stanek et al. (OH data).13 For steps 1 and 2, indices were compared together and with children's BLL data from MI and OH and state-identified locations in public health department reports. The identified lead exposure indices hotspots were evaluated by applying Cohen's kappa26 agreement statistic (<0.4 = low; >0.4−0.6 = +moderate; >0.6−0.8 = substantial; >0.8−0.99 = near perfect agreement) and sensitivity and specificity analyses. Note that some reports refer to these locations as "high risk communities" while others refer to them differently, e.g., "selected community". In the context of this application, sensitivity is the percentage of true positives (i.e., the rate at + +![2_image_0.png](2_image_0.png) + +![2_image_1.png](2_image_1.png) + +which a model or index correctly predicts a hotspot for BLLs) and specificity is the percentage of true negatives (i.e., the rate at which a model or index correctly predicts a nonhotspot for BLLs). + +To compare hotspots identified by the lead indices with +"high risk locations" identified by state health departments based on their BLL surveillance data, we assessed the summary of available data and reports listed in Zartarian et al. figure B +(left panel) and table D.5 Note that 9 of 32 public reports identified hotspots at the community/city/town level; the rest reported them at other geographic scales (i.e., state, county, parish, health jurisdiction, zip code). The 9 states used for evaluation were: Connecticut (city/town), Maine (city/town), Massachusetts (city/town, census tracts), Michigan (city/ town), New Hampshire (city/town, census tracts), New Jersey (municipality), Ohio (city/town, county), Pennsylvania (municipality), and Rhode Island (city). As Table D in Zartarian et al.5 shows, states used different criteria, geographic scales, time periods, and blood lead reference values for identifying higher risk locations. + +After comparing and evaluating the indices using the statistical methods (i.e., top 20th percentile and Getis-Ord Gi* geospatial clustering) described in Xue et al.,12 the intersection and collective combinations were mapped with ESRI ArcGIS Desktop version 10.8.2. This data intersection and combination was executed in ArcGIS by performing spatial data joining using census tract geographic identification numbers (GEOIDs). + +We summarized the results into tables by U.S. states and + +![2_image_2.png](2_image_2.png) + +counties, ranking them by total number of children <6 years old (per 2010 Census27) in identified census tracts based on the lead exposure indices results. Corresponding county and state ranking tables are displayed by index or model identification (1 = identified as a lead exposure risk hotspot by index or model; 0 = not identified by index or model as a lead exposure risk hotspot; Total = total number of indices and models that identified the respective location). For Tables S1−S8 identifying states and counties with the highest potential lead exposure risk (as defined by total number of children <6 years old identified by the indices based on housing age and sociodemographics), we featured the Schultz Model, Vox, and RF Model v1 based on the evaluation results. Our intent of identifying states and counties is to inform further tailored analyses for lead mitigation efforts. + +- RESULTS AND DISCUSSION +National-Scale Lead Exposure Hotspot Maps **Based** +on 5 Indices and **Models.** Figure 1 shows national analyses of high lead exposure locations with five available indices/ +models, using the Getis-Ord Gi* geospatial cluster method and the top 20 percentile (80th−100th) method. The top panels show the intersection of the five indices, and the bottom shows the combination. Figures S-1 and S-2 show the individual hotspot maps for the five indices using the two statistical methods. The maps look different due to the nuances in underlying science and data as discussed in Zartarian et al.,5 i.e., national-scale lead indices constructed with different + +![3_image_0.png](3_image_0.png) + +Figure 2. National scale convergence analyses of the highest potential lead (Pb) exposure risk locations identified using 5 available indices and models with the Getis-Ord Gi* geospatial cluster and top 20 percentile (80th−100th) methods. + +| state | EJSCREEN Index | Schultz Model | HUD Index | Random Forest Model v1 | Vox | totala | +|--------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|-----------------|-------------|--------------------------|-------|----------| +| Illinois | 1 | 1 | 1 | 1 | 1 | 5 | +| Michigan | 1 | 1 | 1 | 1 | 1 | 5 | +| New Jersey | 1 | 1 | 1 | 1 | 1 | 5 | +| New York | 1 | 1 | 1 | 1 | 1 | 5 | +| Ohio | 1 | 1 | 1 | 1 | 1 | 5 | +| Pennsylvania | 1 | 1 | 1 | 1 | 1 | 5 | +| Massachusetts | 0 | 1 | 1 | 1 | 1 | 4 | +| California | 1 | 1 | 0 | 1 | 1 | 4 | +| Indiana | 0 | 0 | 1 | 1 | 1 | 3 | +| Texas | 1 | 1 | 1 | 0 | 1 | 4 | +| Connecticut | 0 | 0 | 1 | 0 | 0 | 1 | +| Florida | 1 | 1 | 0 | 0 | 0 | 2 | +| Maryland | 1 | 0 | 0 | 0 | 0 | 1 | +| Wisconsin | 0 | 0 | 0 | 1 | 0 | 1 | +| a Total = total number of indices and/or models that identified the respective location. 1 = identified by index or model. 0 = not identified by index | | | | | | | + +methods, variables, and geographic scales. For example, different years of housing age (% pre-1940 vs pre-1960 vs pre-1980 homes) or different sociodemographic variables (e.g., % people of color vs % non-Hispanic African American) can produce different looking maps with differences in hotspots identified, depending on the location and population. + +Figure 2 shows the intersection and combination of the two statistical methods on the same maps, along with the number of U.S. census tracts identified. Considering the intersection of the five indices, the number of census tracts is 562 (0.8%) +based on Getis-Ord Gi* identified only, 1869 (2.6%) based on top 20 percentiles identified only, and 3430 (4.7%) identified by both analyses (for a total of ∼8%). Considering the combination of the five indices, there is much more coverage over the entire United States, with the number of census tracts as 2107 (2.9%) based on Getis-Ord Gi* identified only, 9234 +(12.6%) based on top 20 percentiles identified only, and 18 867 (25.8%) identified by both analyses (for a total of +∼41%). + +States and Counties with the Most Children **Living** in Hotspot Census Tracts Based on Lead Exposure **Indices.** Utilizing five indices and the two geospatial statistical methods +(Getis-Ord Gi* and top 20 percentiles), states and counties with the most children <6 years old residing in hotspot census tracts include: +- IL, MI, NJ, NY, OH, PA (identified by 5 indices); MA, +CA, TX (identified by 4 indices) (Getis-Ord Gi* geospatial cluster method) as shown in Table 1 along with other results (i.e., states identified by 1, 2, or 3 indices); +- IL, MI, NJ, NY, OH, PA, TX (identified by 5 indices); +CA (identified by 4 indices) (top 20, 80th−100th, percentile method) as shown in Table S-1 along with other results (i.e., states identified by 1, 2, or 3 indices); + +| state | county | EJSCREEN Index | Schultz Model | HUD Index | Random Forest Model v1 | Vox | totala | +|--------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------|------------------|-----------------|-------------|--------------------------|-------|----------| +| Illinois | Cook County | 1 | 1 | 1 | 1 | 1 | 5 | +| Michigan | Wayne County | 1 | 1 | 1 | 1 | 1 | 5 | +| New York | Bronx County | 1 | 1 | 1 | 1 | 1 | 5 | +| New York | Kings County | 1 | 1 | 1 | 1 | 1 | 5 | +| New York | New York County | 1 | 1 | 1 | 1 | 1 | 5 | +| New York | Queens County | 1 | 1 | 1 | 1 | 1 | 5 | +| Pennsylvania | Philadelphia County | 1 | 1 | 1 | 1 | 1 | 5 | +| New Jersey | Essex County | 0 | 1 | 1 | 1 | 1 | 4 | +| California | Los Angeles County | 1 | 1 | 0 | 1 | 1 | 4 | +| Maryland | Baltimore City | 0 | 1 | 0 | 1 | 1 | 3 | +| Massachusetts | Suffolk County | 0 | 0 | 1 | 1 | 1 | 3 | +| Ohio | Cuyahoga County | 0 | 1 | 0 | 1 | 1 | 3 | +| Pennsylvania | Allegheny County | 0 | 0 | 1 | 1 | 1 | 3 | +| Texas | Harris County | 1 | 1 | 1 | 0 | 1 | 4 | +| Wisconsin | Milwaukee County | 0 | 1 | 1 | 1 | 1 | 4 | +| Arizona | Maricopa County | 1 | 1 | 0 | 0 | 1 | 3 | +| California | Alameda County | 1 | 0 | 0 | 1 | 1 | 3 | +| Florida | Miami-Dade County | 1 | 1 | 0 | 0 | 1 | 3 | +| Indiana | Marion County | 0 | 1 | 1 | 0 | 0 | 2 | +| New Jersey | Hudson County | 0 | 0 | 1 | 1 | 0 | 2 | +| New York | Erie County | 0 | 0 | 1 | 1 | 0 | 2 | +| Connecticut | New Haven County | 0 | 0 | 1 | 0 | 0 | 1 | +| Massachusetts | Middlesex County | 0 | 0 | 1 | 0 | 1 | 2 | +| New York | Westchester County | 0 | 0 | 1 | 0 | 0 | 1 | +| Ohio | Hamilton County | 0 | 1 | 0 | 1 | 0 | 2 | +| Rhode Island | Providence County | 0 | 0 | 0 | 1 | 0 | 1 | +| Tennessee | Shelby County | 0 | 1 | 0 | 0 | 0 | 1 | +| Texas | Dallas County | 1 | 1 | 0 | 0 | 0 | 2 | +| California | Fresno County | 1 | 0 | 0 | 0 | 0 | 1 | +| California | Kern County | 1 | 0 | 0 | 0 | 0 | 1 | +| California | Orange County | 1 | 0 | 0 | 0 | 0 | 1 | +| California | Riverside County | 1 | 0 | 0 | 0 | 0 | 1 | +| California | San Bernardino County | 1 | 0 | 0 | 0 | 0 | 1 | +| California | San Diego County | 1 | 0 | 0 | 0 | 0 | 1 | +| California | San Francisco County | 0 | 0 | 0 | 0 | 1 | 1 | +| District of Columbia | District of Columbia | 0 | 1 | 0 | 0 | 0 | 1 | +| Missouri | St. Louis city | 0 | 0 | 0 | 1 | 0 | 1 | +| New Jersey | Middlesex County | 0 | 0 | 1 | 0 | 0 | 1 | +| New York | Suffolk County | 0 | 0 | 1 | 0 | 0 | 1 | +| Texas | Bexar County | 1 | 0 | 0 | 0 | 0 | 1 | +| a Total = total number of indices and/or models that identified the respective location. 1 = identified by index or model. 0 = not identified by index | | | | | | | | + +- Cook County, IL, Wayne County, MI, Bronx, Kings, New York, Queens counties, NY, Philadelphia County, PA (identified by 5 indices); Essex County, NJ, Los Angeles County, CA, Harris County, TX, Milwaukee County, WI (identified by 4 indices); others shown in Table 2 (Getis-Ord Gi* geospatial cluster method); +- Cook County, IL, Wayne County, MI, Bronx, Kings, Queens counties, NY, Philadelphia County, PA, Los Angeles County, CA, Harris County, TX, Milwaukee County, WI (identified by 5 indices); Essex County, NJ, New York County, NY, Dallas County, TX (identified by 4 indices); others shown in Table S-2 (top 20, 80th− +100th, percentile method). + +Details of states and counties identified based on the number of children in lead indices hotspot census tracts are +shown in Tables S-3, S-4, and S-5 and S-6, S-7, and S-8, respectively. Of the three featured indices (RF Model v1, Schultz Model, and Vox), the Vox score yielded the highest number of children living in identified lead exposure risk hotspots in New York state (627 045; Table S-4) and in Los Angeles County (279 113) (Table S-7). + +Evaluation of Lead Indices and Hotspots **Identified.** +Statistical evaluation of hotspots identified using five lead indices showed good agreement with OH and MI BLL data hotspots. Comparing the indices vs BLLs, Cohen's kappa scores demonstrate moderate to substantial statistical agreement (0.49−0.63) between hotspots (Table 3). Generally, the RF Model v1 and v2 had the strongest agreement results (0.63 and 0.54/0.52 for MI and OH, respectively). Tables S-9 and S10 further illustrate strong statistical agreement with sensitivity Table 3. Statistical Agreement Analyses (Cohen's **Kappa)** of the Highest Potential Lead (Pb) Exposure Risk **Hotspots** Identified: Michigan and Ohio Blood Lead Levels **(BLLs)** vs Pb Exposure Indices and **Models**a + +| Michigan BLLs | Ohio BLLs | | | | | | +|---------------------------------------------------------------------------------------------------------------------------|-------------|-------------|------|------|-------------|------| +| GetisOrd | GetisOrd | | | | | | +| top 20 | top 20 | | | | | | +| N | Gi* | percentiles | N | Gi* | percentiles | | +| kappa | kappa | | | | | | +| EJSCREEN | 2401 | 0.56 | 0.53 | 2850 | 0.59 | 0.50 | +| Index HUD DPI | 2401 | 0.50 | 0.43 | 2850 | 0.49 | 0.42 | +| Random Forest | 2401 | 0.63 | 0.52 | 2850 | 0.54 | 0.51 | +| Model version 1 Schultz Model | 2401 | 0.49 | 0.49 | 2850 | 0.46 | 0.51 | +| Vox | 2401 | 0.50 | 0.55 | 2850 | 0.44 | 0.51 | +| Random Forest | 2401 | 0.63 | 0.52 | 2850 | 0.52 | 0.51 | +| Model version 2 | | | | | | | +| a Cohen's kappa agreement statistic: <0.4 low; >0.4−0.6 moderate; >0.6−0.8 substantial; >0.8−0.99 near perfect agreement. | | | | | | | + +and specificity analyses: sensitivity of 91−96% for three of the indices (RF Model v1, Schultz Model, and Vox − Getis-Ord Gi*); specificity of 78−94% for all indices (top 20 percentiles and Getis-Ord Gi*). Compared against each other, the lead indices demonstrate low to near-perfect statistical agreement +(e.g., kappa scores of 0.37−0.87 as shown in Table S-11). + +The hotspots identified with the five considered lead indices match well with community hotspots identified in the available state health department reports (Tables 4 and 5). For the U.S., the results were 45% (EJSCREEN Index), 62% (Schultz Model), 78% (HUD DPI), 58% (RF Model v1), 72% (Vox), and 61% (RF Model v2). For OH and MI, the results were 74% (EJSCREEN Index), 95% (Schultz Model), 89% (HUD DPI), 89% (RF Model v1), 100% (Vox), and 95% (RF Model v2). Since the convergence analyses of RF Model v1, Schultz Model, and Vox show better statistical agreement with each other (Cohen's kappa of ∼0.7) than with EJSCREEN Index and HUD DPI (kappa ranged from 0.47 to 0.59 with the GetisOrd Gi* method) for the national scale analysis (Table S-11), +we focused on these three indices for identifying states and counties with the highest potential lead exposure risk (as mentioned in Materials and Methods). + +- DISCUSSION +Strengths. This paper presents an innovative, state-of-thescience geospatial data analysis and screening approach to help identify and address U.S. lead exposure hotspot locations. + +Current limitations and gaps in environmental, sociodemographic, and blood lead data make it challenging to develop a single U.S. blood lead map or blood lead prediction model accounting for all sources of lead exposure. Thus, our methodology is to utilize a convergence and collective set of currently available information, considering uncertainties and different data sets and approaches. The results are displayed visually with U.S. hotspot map figures showing the intersection and combination of five available lead indices and corresponding tables with locations they identified. The accompanying statistical analyses in the tables presented show the strength of the indices used and verification of locations they identified. + +Our results support the use of available lead indices in the absence of consistent, complete BLL surveillance data across Environmental Science & Technology **pubs.acs.org/est** Article the United States. This analysis utilizes peer-reviewed, published lead exposure hotspot analysis methods in EPA's Michigan lead analysis paper12 and builds off an interagency +(EPA/HUD/CDC) lead mapping state-of-the-science paper.5 Statistical evaluation of the available lead indices was conducted in multiple ways: with MI and OH children's BLL surveillance data and locations identified from 9 state health department reports. + +We used census tract level analyses to identify, on a national scale, the states and counties with the highest potential lead exposure risk based on the number of children living in lead exposure hotspots. These results can inform further tailored analyses for lead mitigation efforts. For example, these screening analysis results could include identifying the most disproportionately impacted communities that might be eligible for federal or state lead mitigation programs; determining where resources should be focused to reduce lead-based paint exposures, replace lead service lines, and address lead-contaminated soils and other sources; assessing additional data needs and priority locations for collecting more environmental and biomonitoring data to identify lead exposure hotspots and their drivers. The analysis presented in this paper to advance the science for identifying high lead exposure locations supports U.S. efforts including the Federal Lead Action Plan,10 EPA Lead Strategy,9 Biden-Harris Lead Pipe and Paint Action Plan,28 and Biden-Harris "Get the Lead Out" Partnership.29 This science can guide other lead hotspot identification and verification efforts and inform lead reduction actions through collaborations with state and local health departments.30 Not only are these national-scale maps and results informative to help target U.S. efforts, but the approaches of this paper could also inform lead discussions and efforts in other countries. For example, a report to G7 ministers31 on the outcomes of the November 2022 workshop entitled, "Lead as a Major Threat for Human Health and the Environment: An Integrated Approach Strengthening Cooperation toward Solutions," discussed options for future work to reduce sources of lead and minimize lead exposure in low- and middle-income countries (LMICs) that included: "helping LMICs conduct initial diagnostic assessments about the prevalence of lead poisoning and identification and ranking of sources of lead exposure." In the U.S. and other countries, while data collection and hotspot mapping science evolves to help pinpoint the highest risk locations, short-term national and community actions can be informed by existing best practices and toolkits (e.g., EPA's Local Lead Action Plan Guide;32 United Nations Environment Programme, Toolkit for establishing laws to eliminate lead paint33). + +Limitations. There are a number of limitations of this analysis and opportunities to enhance data and methods to identify lead exposure risk hotspots. The lead indices used in this analysis include 2010 census data, geographic identifiers, and boundaries. The 2020 census data, geographic identifiers, and boundaries cannot be fully incorporated at this time due to incomplete census data or index incompatibility. As mentioned in the Materials and Methods, this analysis uses 5 μg/dL rather than the current CDC blood lead reference value of 3.5 μg/dL +to be consistent with the underlying data in the lead indices and the data used to evaluate them (public reports and BLL data from MI and OH). Although this may be considered a limitation of the analysis, Xue et al.12 found that, when comparing MI census tract hotspots using 5 μg/dL vs + +Table 4. State-Identified Locations in 9 Public Health Department Reports (Based on Blood Lead Levels ≥5 μg/dL) **Across** + +the United States That Were Also Identified by the Getis-Ord Gi* Analysis of Lead Exposure Indices and **Models**a + +| the United States That Were Also Identified by the Getis-Ord Gi* Analysis of Lead Exposure Indices and Modelsa state health department community hotspotb EJSCREEN Schultz HUD Random Forest Model community index-model identification count Index Model Index v1 Vox Akron, OH 1 1 1 1 1 5 Atlantic City, NJ 1 1 1 1 1 5 Bridgeport, CT 1 1 1 1 1 5 Brockton, MA 1 1 1 1 1 5 Canton, OH 1 1 1 1 1 5 Central Falls, RI 1 1 1 1 1 5 Cincinnati, OH 1 1 1 1 1 5 Cleveland, OH 1 1 1 1 1 5 Columbus, OH 1 1 1 1 1 5 Dayton, OH 1 1 1 1 1 5 Detroit, MI 1 1 1 1 1 5 East Cleveland, OH 1 1 1 1 1 5 East Orange, NJ 1 1 1 1 1 5 Flint, MI 1 1 1 1 1 5 Hamtramck, MI 1 1 1 1 1 5 Harrisburg, PA 1 1 1 1 1 5 Hartford, CT 1 1 1 1 1 5 Highland Park, MI 1 1 1 1 1 5 Irvington, NJ 1 1 1 1 1 5 Lancaster, PA 1 1 1 1 1 5 Meriden, CT 1 1 1 1 1 5 Muskegon, MI 1 1 1 1 1 5 New Haven, CT 1 1 1 1 1 5 Paterson, NJ 1 1 1 1 1 5 Plainfield, NJ 1 1 1 1 1 5 Reading, PA 1 1 1 1 1 5 Toledo, OH 1 1 1 1 1 5 Trenton, NJ 1 1 1 1 1 5 Waterbury, CT 1 1 1 1 1 5 York, PA 1 1 1 1 1 5 Youngstown, OH 1 1 1 1 1 5 Jackson, MI 0 1 1 1 1 4 Lewiston, ME 0 1 1 1 1 4 New Bedford, MA 0 1 1 1 1 4 Portland, ME 0 1 1 1 1 4 Providence, RI 0 1 1 1 1 4 Scranton, PA 0 1 1 1 1 4 Springfield, OH 0 1 1 1 1 4 Woonsocket, RI 0 1 1 1 1 4 Grand Rapids, MI 0 1 1 1 1 4 Bangor, ME 0 1 1 0 1 3 Adams, MA 0 0 1 0 1 2 Auburn, ME 0 0 1 0 1 2 Gloucester, MA 0 0 1 0 1 2 Newark, NJ 0 1 1 0 0 2 Newport, RI 0 0 1 0 1 2 North Adams, MA 0 0 1 0 1 2 Total 31 43 54 40 50 Percentc 45 62 78 58 72 | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +a1 = identified by index or model; 0 = not identified by index or model. bState health department reports and their respective community hotspots are sourced from Table D in Zartarian, V.; Poulakos, A.; Garrison, V. H.; Spalt, N.; Tornero-Velez, R.; Xue, J.; Egan, K.; Courtney, J. Lead Data Mapping to Prioritize US Locations for Whole-of-Government Exposure Prevention Efforts: State of the Science, Federal Collaborations, and Remaining Challenges. Am. J. Public *Health*. 2022, 112 (S7), S658−S669. DOI: 10.2105/AJPH.2022.307051 (ref 5). cPercent refers to the percentage of community hotspots identified by each respective index or model. Percentages were calculated by taking the values in the "Total" row and dividing them by 69 (i.e., the total number of state health department community hotspots in the aforementioned 9 public health department reports). 57 community hotspots were identified by at least one index/model. 47 of the 69 are shown in the table as these are the communities identified by two or more indices/models (this was also done due to table formatting and spacing limitations). + +Table 5. State-Identified Locations in Michigan and Ohio Public Health Department Reports (Based on Blood Lead **Levels** ≥5 μg/dL) That Were Also Identified by the Getis-Ord Gi* Analysis of Lead Exposure Indices and **Models**a + +| Pb exposure indices and models | | | | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------|----------|---------|-----|---------------------|--------------------------------------------|----| +| state health dept. community hotspotb | EJSCREEN | Schultz | HUD | Random Forest Model | community index-model identification count | | +| Index | Model | Index | v1 | Vox | | | +| Akron, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Canton, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Cincinnati, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Cleveland, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Columbus, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Dayton, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Detroit, MI | 1 | 1 | 1 | 1 | 1 | 5 | +| East Cleveland, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Flint, MI | 1 | 1 | 1 | 1 | 1 | 5 | +| Hamtramck, MI | 1 | 1 | 1 | 1 | 1 | 5 | +| Highland Park, MI | 1 | 1 | 1 | 1 | 1 | 5 | +| Muskegon, MI | 1 | 1 | 1 | 1 | 1 | 5 | +| Toledo, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Youngstown, OH | 1 | 1 | 1 | 1 | 1 | 5 | +| Jackson, MI | 0 | 1 | 1 | 1 | 1 | 4 | +| Springfield, OH | 0 | 1 | 1 | 1 | 1 | 4 | +| Grand Rapids, MI | 0 | 1 | 1 | 1 | 1 | 4 | +| Lansing, MI | 0 | 1 | 0 | 0 | 1 | 2 | +| Adrian, MI | 0 | 0 | 0 | 0 | 1 | 1 | +| Total | 14 | 18 | 17 | 17 | 19 | | +| Percentc | 74 | 95 | 89 | 89 | 100 | | +| a 1 = identified by index or model; 0 = not identified by index or model. b State health department reports and their respective community hotspots | | | | | | | + +3.5 μg/dL, a Cohen's kappa value of ∼0.94 was calculated, demonstrating near perfect statistical agreement. Furthermore, the use of 3.5 μg/dL as a blood lead reference value has yet to be adopted by all state health departments, which presently limits and complicates its use in a national scale analysis. + +This research focuses on children because of available blood lead data, but reducing lead exposure is also important for adults.1 We note that some researchers have questioned the interpretability of agreement statistics such as Cohen's kappa.34,35 Only nine state health department reports identified community-level locations; this, along with most of them not reporting at the census tract level, limited the extent to which we could evaluate the indices across the country. + +Ideally, more blood lead data would be available for groundtruthing of index-identified hotspots. + +The indices utilized are moderate to substantial predictors of higher BLLs and do not include environmental data and sources that could likely improve the identification of lead exposure hotspots (e.g., prevalence of lead service lines; drinking water and soil lead concentration data in residences, schools, and daycare centers; other data discussed in Zartarian et al.5). We emphasize that, because the indices and models used in this paper are based on publicly available housing age and sociodemographic data, the lead exposure risk hotspots identified in this national-scale analysis are most relevant to old housing-related sources of lead (e.g., lead-based paint, residential soil and dust, and lead in drinking water in old homes with lead pipes). Identification of high-risk locations and their underlying sources may change as environmental data, and ideally also food and consumer products data, are incorporated into the indices and finer scales are considered. Understanding which lead sources and pathways are contributing the most to BLLs within lead exposure hotspots identified is important for prioritizing exposure reduction actions and resources. However, source apportionment can vary by location and scenarios,36,37 and quantification is challenging without an integrated database of multimedia environmental lead and BLL data.5 Table 1 results, based on Pb indices constructed with old housing and sociodemographic data from the census, may potentially be skewed toward states with higher population densities because of significantly more old housing stock (i.e., in northeastern and north-central U.S. in comparison to the south and other parts of the U.S). Rural counties may appear less than urban areas in this analysis because of smaller populations and potentially lower quantity of census data from those counties used in the indices. Prior research with Michigan data showed that the HUD DPI, EJSCREEN Index, and Schultz Model predominantly identify urban hot spots, particularly with the geospatial cluster analysis (Xue et al.12). This U.S. analysis paper and the ones it builds upon focus on potential lead exposure risk from old housing-related sources, based on population level (census tract, county, and state results) not at the individual or household levels. + +As a screening approach to identify locations with an increased potential of high lead exposure or higher BLLs, this national-scale analysis cannot identify sources at particular addresses or risk at an individual level. Our methodology focuses on the census tract scale for reasons discussed in Xue et al..12 To target resources for local-scale source and risk mitigation, further investigation of hotspots identified should be conducted to identify individuals who may have been exposed.30 More verification or "ground-truthing" of lead hotspots is needed using more blood lead surveillance data, as it becomes available, and information from local partners with on-the-ground knowledge of their communities.30 We also note that counties with high population densities were identified by most or all of the indices used, which may raise the question of whether population density alone may be sufficient in determining high lead exposure risk. As described in the U.S. EPA Integrated Science Assessment for Lead,4 old housing�a key environmental variable included in all lead exposure indices considered in this paper�is a widely known and acknowledged indicator of potential lead exposures. This reinforces its use over simple population-based metrics. + +Furthermore, lead exposure risk has been observed to disproportionately impact specific communities.9,10 From a social and environmental justice perspective, population density data alone cannot account for this. + +Research **Needs.** More research is needed to understand the differences in high potential lead exposure risk locations identified and the choice of the most appropriate index or indices for different geographic locations. In addition, while some indices statistically agree more with higher BLL hotspots for MI and OH (as shown in Table 3), the requisite BLL data to be able to preferentially select one over another are not available across the entire U.S. Thus, our national approach is to intersect or combine indices (as shown in Figure 2) to narrow or broaden the set of locations for lead-related efforts, depending on the intended purpose and available resources. + +Future research can also include enhancing and expanding the current lead indices to address the various limitations as more data become available. Research is underway to enhance the Random Forest Model by incorporating environmental data in EPA/OECA's POST (EPA Office of Enforcement and Compliance Assurance Pb Occurrence and Source Tool described in Zartarian et al.5). For example, analyses have been conducted to determine the relative impact of lead service line (LSL) prevalence vs old housing in two cities where LSL data are available22 and to geospatially analyze linkages between commercial sources of lead and childhood BLLs.38 CDC is currently developing a web-based Lead Exposure Risk Index (LERI) that will incorporate multiple demographic, geographic, and environmental risk factors at the census tract level. This tool is intended for use by health agencies and providers to identify high-risk areas where children should be tested for lead and to target lead-reduction interventions.39 In the next two years, HUD plans to geocode the historic database of HUD grants' production of lead-safe housing units and update the Deteriorated Paint Index in 2024 after the 2023 American Housing Survey, Healthy Homes supplement is fielded. + +Due to the current data limitations discussed in Zartarian et al.,5 development of lead indices as surrogates in the absence of complete and accurate population-level BLL surveillance data requires a whole-of-government approach. EPA, HUD, and CDC continue to collaborate and evolve the research and address data gaps. Various groups are working to collect more representative BLL surveillance data and detailed environmental lead sources and measurement data that could help evaluate and improve future indices.30 Collaborative interagency case studies applying the EPA Lead Strategy Goal 2 +"blueprint"9,30 to identify and prioritize potential places for action are further developing and applying these methods to determine drivers of lead exposure hotspots. While science in this area evolves, these U.S. screening results identifying states, counties, and census tracts with potential high lead exposure risk can inform further efforts to target lead actions (such as those described in Breysse et al.11 and Zartarian et al.5) for public health protection. + +The overarching public health questions this research is addressing to help prioritize actions are as follows: Where are exposure risk hotspots and what sources are driving them? The lead-focused methodologies in this paper and previous research it expands upon, using available blood lead surveillance data and surrogate exposure indices, could potentially be applied to other chemicals (and countries) depending on available data. + +- ASSOCIATED **CONTENT** + +## Data Availability **Statement** + +Data from this paper are available in EPA's Environmental Dataset Gateway at https://doi.org/10.23719/1529907. + +*sı Supporting **Information** +The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.3c07881. + +Additional maps and analyses of high lead exposure risk locations based on available lead indices and blood lead data (PDF) Detailed background information and method details on Random Forest Model v1 and v2; table showing variables used; figure showing model validation and performance (PDF) +- AUTHOR INFORMATION + +## Corresponding **Author** + +Valerie G. **Zartarian** − U.S. Environmental Protection Agency, Office of Research and Development, Research Triangle Park, North Carolina 27711, United *States;* orcid.org/00090003-7788-7033; Phone: 617-620-3148; Email: Zartarian.valerie@epa.gov + +## Authors + +Jianping Xue − U.S. Environmental Protection Agency, Office of Research and Development, Research Triangle Park, North Carolina 27711, United *States* Antonios G. **Poulakos** − U.S. Environmental *Protection* Agency, Office of Research and Development, Research Triangle Park, North Carolina 27711, United States; LinTech Global, Inc., Boston, Massachusetts 02109, United States; orcid.org/0000-0001-5755-3595 Rogelio **Tornero-Velez** − U.S. Environmental *Protection* Agency, Office of Research and Development, Research Triangle Park, North Carolina 27711, United *States;* +orcid.org/0000-0003-2102-0548 Lindsay W. **Stanek** − U.S. Environmental Protection *Agency,* +Office of Research and Development, Research Triangle Park, North Carolina 27711, United *States;* orcid.org/00000002-9921-7458 Emily **Snyder** − U.S. Environmental Protection Agency, *Office* of Research and Development, Research Triangle Park, *North* Carolina 27711, United *States* + +Veronica Helms **Garrison** − U.S. Department of Housing and Urban Development, Office of Policy Development and Research, Washington, D.C. 20410, United *States* Kathryn **Egan** − Centers for Disease Control and Prevention, National Center for Environmental Health, Agency for Toxic Substances and Disease Registry, Office of Science, *Atlanta,* +Georgia 30329, United *States* Joseph G. **Courtney** − Centers for Disease Control and Prevention, National Center for Environmental *Health,* Division of Environmental Health Science and *Practice,* Atlanta, Georgia 30329, United *States* Complete contact information is available at: https://pubs.acs.org/10.1021/acs.est.3c07881 + +## Notes + +The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency, U.S. Department of Housing and Urban Development, Centers for Disease Control and Prevention, or state partners. Any mention of trade names, products, or services does not imply an endorsement by the U.S. Government or EPA, HUD, or CDC. EPA, HUD, and CDC do not endorse any commercial products, services, or enterprises. In addition, the contractor's role did not include establishing agency policy. Michigan Department of Health and Human Services (MDHHS) provided blood lead data used in this paper, pursuant to Data Use Agreement 201909-157. EPA assumes full responsibility for the analysis and interpretation of the data. This paper also includes analyses with blood lead data provided by the Ohio Department of Health (ODH), through the Ohio Public Health Information Warehouse under an approved Data Use Agreement. The use of these data should not be considered an endorsement of this study or its conclusions by ODH. Research included in this analysis was approved under Institutional Review Boards (IRBs) through University of North Carolina at Chapel Hill (UNC; 16-2302), MDHHS (201703-12-EA), and ODH (2019-41). The authors declare no competing financial interest. J.X. is now retired from the EPA. + +- **ACKNOWLEDGMENTS** +We gratefully acknowledge the technical reviewers; EPA/ORD lead mapping team, managers, and support staff; EPA ORD and Region 5 coauthors of the MI paper; EPA, HUD, and CDC collaborators; coauthors of state-of-the-science lead mapping paper; partners in Michigan MDHHS, Michigan EGLE, and Ohio DOH. This article has been reviewed by EPA, HUD, and CDC and approved for publication. The coauthors appreciate technical reviewers and managers from our agencies who supported this article and related research. We gratefully acknowledge the EPA's Office of Research and Development; the CDC Lead Exposure Risk Index Working Group, Agency for Toxic Substances and Disease Registry, Office of Community Health and Hazard Assessment and the National Center for Environmental Health, Division of Environmental Health Science and Practice; the HUD Office of Policy Development and Research. The mapping and analysis work has been funded in part by the U.S. EPA under the East Coast Help Desk Operations (ECHO)−Multi Region Information Technology (IT) Services Support Contract and the U.S. EPA Region 1 IT Services Support Task Order; specifically, LinTech Global, Inc.'s ECHO Contract No. GS35F-0343W/68HE0319F0020 with the U.S. Environmental Protection Agency, Regions 1, 2, & 3. + +- **REFERENCES** +(1) Lamas, G. A.; Bhatnagar, A.; Jones, M. R.; Mann, K. K.; Nasir, K.; Tellez-Plaza, M.; Ujueta, F.; Navas-Acien, A. the American Heart Association Council on Epidemiology and Prevention; Council on Cardiovascular and Stroke Nursing; Council on Lifestyle and Cardiometabolic Health; Council on Peripheral Vascular Disease; Council on the Kidney in Cardiovascular Disease. Contaminant Metals as Cardiovascular Risk Factors: A Scientific Statement from the American Heart Association. J. Am. Heart *Assoc.* **2023**, 12, No. e029852. + +(2) Egan, K. B.; Cornwell, C. R.; Courtney, J. G.; Ettinger, A. S. + +Blood lead levels in US children ages 1−11 years, 1976−2016. + +Environ. Health *Perspect.* **2021**, 129 (3), 37003. + +(3) Hauptman, M.; Niles, J. K.; Gudin, J.; Kaufman, H. W. + +Individual- and community-level factors associated with detectable and elevated blood lead levels in US children: results from a national clinical laboratory. JAMA *Pediatr* **2021**, 175 (12), 1252−1260. + +(4) Integrated Science Assessment (ISA) for Lead (Final Report, *July* 2013); EPA-600-R-10-075F; U.S. Environmental Protection Agency: Washington, DC, 2013. + +(5) Zartarian, V.; Poulakos, A.; Garrison, V. H.; Spalt, N.; TorneroVelez, R.; Xue, J.; Egan, K.; Courtney, J. Lead Data Mapping to Prioritize US Locations for Whole-of-Government Exposure Prevention Efforts: State of the Science, Federal Collaborations, and Remaining Challenges. Am. J. Public *Health.* **2022**, 112 (S7), S658− +S669. + +(6) Centers for Disease Control and Prevention. Childhood Lead Poisoning Prevention Program: Lead Poisoning *Prevention*; https:// +www.cdc.gov/nceh/lead/prevention/default.htm (updated September 2, 2022; accessed 2023-09-01). + +(7) Agency for Toxic Substances and Disease Registry. Toxicological profile for *lead*; U.S. Department of Health and Human Services, Public Health Service: Atlanta, GA, 2020;https://wwwn.cdc.gov/ TSP/ToxProfiles/ToxProfiles.aspx?id=96&tid=2web2 (accessed 2023-09-01). + +(8) U.S. Environmental Protection Agency. EPA Lead (Pb) *Research*; +https://www.epa.gov/land-research/epa-lead-pb-research (updated November 7, 2022; accessed 2023-09-01). + +(9) U.S. Environmental Protection Agency. EPA strategy to *reduce* lead exposures and disparities in US *communities*; November 16, 2021; https://www.epa.gov/system/files/documents/2021-11/updatedpublic-comment-draft-lead-strategy-11-16-2021.pdf (accessed 202309-01). + +(10) President's Task Force on Environmental Health Risks and Safety Risks to Children. Federal Action Plan to Reduce Childhood *Lead* Exposures and Associated Health *Impacts*; December 2018; https:// www.epa.gov/sites/default/files/2018-12/documents/fedactionplan_ lead_final.pdf (accessed 2023-09-01). + +(11) Breysse, P. N.; Cascio, W. E.; Geller, A. M.; Choiniere, C. J.; +Ammon, M. Targeting Coordinated Federal Efforts to Address Persistent Hazardous Exposures to Lead. Am. J. Public *Health.* **2022**, +112 (S7), S640−S646. + +(12) Xue, J.; Zartarian, V.; Tornero-Velez, R.; Stanek, L. W.; +Poulakos, A.; Walts, A.; Triantafillou, K.; Suero, M.; Grokhowsky, N. A generalizable evaluated approach, applying advanced geospatial statistical methods, to identify high lead (Pb) exposure locations at census tract scale: Michigan case study. Environ. Health *Perspect.* **2022**, 130 (7), 77004. + +(13) Stanek, L. W.; Xue, J.; Zartarian, V. M.; Poulakos, A.; TorneroVelez, R.; Snyder, E.; Walts, A.; Triantafillou, K. Identification of *High* Lead Exposure Locations in Ohio at the Census Tract Scale *using* a Generalizable Geospatial Hotspot *Approach*. **2024**, in journal review. + +(14) Ord, J. K.; Getis, A. Local spatial autocorrelation statistics: +distributional issues and an application. Geogr. *Anal.* **1995**, 27 (4), +286−306. + +(15) ESRI ArcGIS Pro. How Hot Spot Analysis (Getis-Ord Gi*) +works; 2023; https://pro.arcgis.com/en/pro-app/latest/toolreference/spatial-statistics/h-how-hot-spot-analysis-getis-ord-gispatial-stati.htm (accessed 2023-09-01). + +(16) Centers for Disease Control and Prevention. Childhood Lead Poisoning Prevention Program: Blood Lead Reference *Value*; https:// +www.cdc.gov/nceh/lead/data/blood-lead-reference-value.htm (updated December 2, 2022; accessed 2023-09-01). + +(17) Ruckart, P. Z.; Jones, R. L.; Courtney, J. G.; LeBlanc, T. T.; +Jackson, W.; Karwowski, M. P.; Cheng, P. Y.; Allwood, P.; Svendsen, E. R.; Breysse, P. N. Update of the blood lead reference value − United States, 2021. Morb. Mortal. Wkly. *Rep.* **2021**, 70 (43), 1509− +1512. + +(18) U.S. Environmental Protection Agency; Office of Environmental Justice EJScreen: Environmental Justice Screening and *Mapping* Tool; https://www.epa.gov/ejscreen (accessed 2023-09-01). + +(19) Schultz, B. D.; Morara, M.; Buxton, B. E.; Weintraub, M. + +Predicting blood-lead levels among US children at the census tract level. Environ. *Justice.* **2017**, 10 (5), 129−136. + +(20) Garrison, V. E.; Ashley, P. J. Identifying jurisdictions at risk of containing housing units with deteriorated paint: results and targeting implications for the U.S. Department of Housing and Urban Development. J. Public Health Manag. *Pract.* **2021**, 27 (6), 546−557. + +(21) Vox. The risk of lead poisoning isn't just in Flint. So we mapped the risk in every neighborhood in *America*; 2016; https://www.vox.com/ a/lead-exposure-risk-map (accessed 2023-09-01). + +(22) Tornero-Velez, R.; Zartarian, V.; Poulakos, A.; Shao, N.; +Stanek, L.; Buckley, T.; Goldberg, M. Tapping into Lead Service Line Information: Two City Case Study. In National Environmental *Health* Association (NEHA) Conference Lead Mapping Session. Lead Data Mapping: Methods and Tools for Lead Prioritization, Prevention, and Mitigation, New Orleans, LA, August 1, 2023. + +(23) Zartarian, V.; Xue, J.; Poulakos, A.; Tornero-Velez, R.; Stanek, L.; Geller, A. Identifying High Lead (Pb) Exposure Locations in the US With an Environmental Justice Focus. Oral presentation at the Society of Toxicology (SOT) conference, Session on Translating *Lead* Toxicology; Engaging Stakeholders and *Communities*, San Diego, CA, March 26, 2022. + +(24) Ohio Department of Health. Ohio Healthy Homes and Lead Poisoning Prevention Program prepared by the Ohio State University Statistical Consulting Service. Final Report on Targeted Testing Plan for Childhood Lead *Poisoning*, April 30, 2013. + +(25) U.S. Census Bureau. Detailed Tables, 2013−2017 American Community Survey 5-year *estimates*; https://data.census.gov/all?g= 010XX00US$1400000=2017=ACS+5- Year+Estimates+Detailed+Tables (accessed 2023-09-01). + +(26) Cohen, J. A coefficient of agreement for nominal scales. *Educ.* +Psychol. *Meas.* **1960**, 20 (1), 37−46. + +(27) U.S. Census Bureau. 2010 Decennial *Census*; https://data. + +census.gov/advanced?y=2010 (accessed 2023-09-01). + +(28) The White House. Biden-Harris Lead Pipe and Paint Action Plan; December 16, 2021; https://www.whitehouse.gov/briefingroom/statements-releases/2021/12/16/fact-sheet-the-biden-harrislead-pipe-and-paint-action-plan/ (accessed 2023-09-01). + +(29) The White House. Biden-Harris "Get the Lead Out" *Partnership*; +January 26, 2023; https://www.whitehouse.gov/build/briefing-room/ 2023/01/26/biden-harris-get-the-lead-out-partnership/ (accessed 2023-09-01). + +(30) Zartarian, V.; Oliveri, A.; Gross-Davis, C.; Triantafillou, K.; +Walts, A.; Spalt, N.; Poulakos, A.; Tornero-Velez, R.; Lytle, D.; +Christian, M.; Stanek, L.; Snyder, E. Work-in-Progress Whole-ofGovernment Lead (Pb) "Blueprint" Examples for Michigan and West Virginia. In National Environmental Health Association (NEHA) Conference Lead Mapping Session. Lead Data Mapping: Methods and Tools for Lead Prioritization, Prevention, and *Mitigation*, New Orleans LA, August 1, 2023. + +Environmental Science & Technology **pubs.acs.org/est** Article +(31) German Federal Ministry for the Environment, Nature Conservation, Nuclear Safety and Consumer Protection. G7 Workshop. Lead as a Major Threat for Human Health and the *Environment:* +An Integrated Approach Strengthening Cooperation toward *Solutions*; +Report to G7 ministers on Key Workshop Outcomes; November 9− +10, 2023; https://www.bmuv.de/fileadmin/Daten_BMU/ Download_PDF/Europa___International/outcome_g7_workshop_ lead_2022_bf.pdf (accessed 2023-09-01). + +(32) U.S. Environmental Protection Agency. Build Your Local *Lead* Action Plan: A Guide for Local Leaders, Local Lead Action Plan *Guide*; +https://www.epa.gov/lead/llap-guide (updated July 25, 2023; accessed 2023-09-01). + +(33) United Nations Environment Programme. Toolkit for establishing laws to eliminate lead *paint*; https://www.unep.org/toolkitestablishing-laws-eliminate-lead-paint (accessed 2023-09-01). + +(34) Pontius, R. G., Jr.; Millones, M. Death to Kappa: birth of quantity disagreement and allocation disagreement for accuracy assessment. Int. J. Remote *Sens.* **2011**, 32 (15), 4407−29. + +(35) McHugh, M. L. Interrater reliability: the kappa statistic. + +Biochem. Med. *(Zagreb)* **2012**, 22 (3), 276−282. + +(36) Stanek, L. W.; Xue, J.; Lay, C. R.; Helm, E. C.; Schock, M.; +Lytle, D. A.; Speth, T. F.; Zartarian, V. G. Modeled impacts of drinking water Pb reduction scenarios on children's exposures and blood lead levels. Environ. Sci. *Technol.* **2020**, 54 (15), 9474−9482. + +(37) Zartarian, V.; Xue, J.; Tornero-Velez, R.; Brown, J. Children's lead exposure: a multimedia modeling analysis to guide public health decision-making. Environ. Health *Perspect.* **2017**, 125 (9), 097009. + +(38) Kulikowski, N.; Tornero-Velez, R.; Lamichhane, A.; Kim, H.; +Gohari, Z.; Hall, A.; Spalt, N.; DeFlorio-Barker, S. Geospatial Analysis to Assess Linkage between Commercial Sources of Lead (Pb) and Childhood Blood Lead Levels. Get the Lead Out, Beyond Gas and Paint: Databases, Crowdsourcing, and Other Approaches to Understand Modern Day Lead Exposures. In International Society of Exposure Science (ISES) Conference. Symposium: Track *Vulnerabilities* in Environmental Exposure and *Health*, August 30, 2023. + +(39) Egan, K.; Courtney, J. CDC-ATSDR Lead Exposure Risk Index +(LERI). In Lead Exposure and Prevention Advisory *Committee* (LEPAC) *meeting*; December 3, 2021; https://www.cdc.gov/nceh/ lead/advisory/lepac-meeting-12-3-21.html (accessed 2023-09-01). \ No newline at end of file diff --git a/medical/md/PMC10954656.md b/medical/md/PMC10954656.md new file mode 100644 index 0000000000000000000000000000000000000000..05c5a6a6d4f4bb2a5abd9393f8b399e3f0214e93 --- /dev/null +++ b/medical/md/PMC10954656.md @@ -0,0 +1,128 @@ +# A Tale Of Three Chambers: Cor Triatriatum Sinistrum + +Maliha Butt, MD, Husban Halim, MD, Ataul Qureshi, MD, Ryan Koene, MD, Su Aung, MD, +Tara Karamlou, MD, and Ashish Saini, MD, Canton, Cleveland, and Akron, Ohio INTRODUCTION +Cor triatriatum sinistrum (CTS) is one of the rarest congenital abnormalities of the heart (0.1%-0.4%) that is defined by the presence of an abnormal fibromuscular septum dividing the left atrium (LA) into two chambers.1 CASE PRESENTATION +A 40-year-old patient with a history of sinus bradycardia who had undergone dual chamber pacemaker implantation at 37 years of age, with morbid obesity, type 2 diabetes mellitus, and hyperlipidemia, presented with palpitations, exertional dyspnea, and chest pain. The patient also reported having ''three chambers in the heart'' found at the age of 18 years during evaluation of a syncopal episode. Interestingly, the patient did not follow up with any cardiologist, although they did undergo two transthoracic echocardiograms +(TTE) during hospital admissions for noncardiac reasons that showed possible cor triatriatum and a left ventricular ejection fraction (EF) of 50% in 2014 (Figure 1). On presentation to our hospital, the patient was diagnosed with heart failure with reduced EF of 30% and started on guideline-directed medical therapy, which resulted in improved EF (Figure 2). The patient was morbidly obese and referred for bariatric surgery, resulting in significant reduction in weight. They also had recurrent episodes of symptomatic paroxysmal atrial fibrillation and were referred to an electrophysiologist. The patient underwent direct-current cardioversion two times as well as radiofrequency pulmonary vein isolation ablation. Transesophageal echocardiography +(TEE) was done to further evaluate cardiac anatomy and revealed CTS with a small opening of 0.8 cm2 (Figures 3-5, Videos 1 and 2). + +The interatrial septum was normal. There was no anomalous pulmonary venous return. There was concern for mitral stenosis–like physiology caused by the CTS membrane, so the patient was referred to an adult congenital heart disease clinic. Right heart catheterization showed pulmonary artery pressure of 50/30 mm Hg, pulmonary capillary wedge pressure of 30.0 mm Hg, and pulmonary venous resistance of 0.8 Wood unit. The cardiac index was 3.5 L/min/m2. + +Coronary angiography showed no significant coronary artery disease. + +The patient underwent surgical correction of CTS. Intraoperative findings included a small patent foramen ovale and a large LA membrane with a tiny aperture near the septum. Surgical LA membrane resection, patent foramen ovale closure, and biatrial maze and LA +appendage ligation was performed. Antiarrhythmics were continued, and anticoagulation was stopped after 3 months. The patient continued to do well at 6-month follow-up, with no further atrial fibrillation or symptoms of congestive heart failure. + +DISCUSSION CTS is a congenital heart disease in which the LA is divided into two compartments by a fold of tissue, a membrane, or a fibromuscular band. The exact pathophysiology is unknown, but several theories have been proposed, including malseptation (abnormal growth of septum primum that bisects the atrium), entrapment (embryonic sinus venosus traps the common pulmonary vein and prevents its incorporation into the LA), and the most widely accepted theory, malincorporation, in which the pulmonary vein fails to incorporate into the LA, +forming a septumlike appendage that divides the LA into two chambers. Classically, the true atrial chamber is in contact with the atrioventricular valve and contains the atrial appendage as well as the true atrial septum that bears the fossa ovalis, while the accessory portion of the atrium receives venous blood. The membrane that separates the affected atrium into two parts has been documented to be of various sizes and shapes and may be with or without any openings. + +This determines the severity of symptoms and complications of the disease. The Loeffler classification divides CTS into three groups: +group 1, absence of connection between the superior and inferior (true atrium) chambers; group 2, one or a few small openings in the intra-atrial membrane; and group 3, wide opening between superior and inferior chamber. + +Although the first two groups cause severe symptoms in childhood and result in high mortality, group 3 usually presents in adulthood, as it may remain asymptomatic for years. The spectrum of symptoms largely depends on the severity of obstruction caused by the membrane. Patients are likely to be asymptomatic when the aperture or fenestration is larger than 1 cm.2 In a systematic review of literature, Rudiene_ et al. + +3 found only 170 reported cases of CTS in adults, with a mean age at diagnosis of 43 years. Forty-one percent of the patients had restrictive physiology and resulting complications of CTS, +including atrial fibrillation (32.8%), congestive heart failure (44.3%), +and pulmonary hypertension (27.1%). Pulmonary edema during labor has also been reported in young adult women.4 It is unclear why patients with Loeffler group 3 CTS who are asymptomatic for years later develop complications. It is thought that there might be electrical and mechanical cardiac remodeling of the tissue predisposing to atrial fibrillation or mitral stenosis–like physiology. Another explanation is the degenerative changes and calcification that can occur over years to create a restrictive physiology of the From the Department of Cardiology, Aultman Hospital, Canton, Ohio (M.B., H.H., A.Q., R.K., S.A.); the Department of Cardiothoracic Surgery, Cleveland Clinic Main Campus, Cleveland, Ohio (T.K.); and the Adult Congenital Heart Service, Akron Children's Hospital Heart Center, Akron, Ohio (A.S.). + +Keywords: Cor triatriatum sinistrum, Atrial fibrillation, Systolic heart failure, Congenital heart disease, Surgical membrane resection Correspondence: Maliha Butt, MD, Department of Cardiology, Aultman Hospital, 2600 6th Street SW, Canton, OH, 44710. (E-mail: *maliha.butt@aultman.com*). + +Published by Elsevier Inc. on behalf of the American Society of Echocardiography. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 2468-6441 https://doi.org/10.1016/j.case.2023.12.019 221 VIDEO HIGHLIGHTS +Video 1: Two-dimensional TEE, midesophageal view, without +(left) and with (right) color flow Doppler, demonstrates flow acceleration across the opening in the cor triatriatum membrane. + +Video 2: Three-dimensional TEE, midesophageal view, demonstrates the CTS membrane. + +View thevideo contentonlineatwww.cvcasejournal.com. + +membrane. However, this theory was disputed by a retrospective study of patients with CTS who were followed for 6 years and showed no significant increase in membrane gradient in patients who were managed conservatively.5 Regrettably, there are no available studies following patients with CTS for a longer duration to confirm the findings of Fuchs et al. + +5 Presenting symptoms of complicated CTS may include syncope, neurologic deficits, chest pain, and shortness of breath. Subtle cardiac auscultatory findings suggestive of CTS may be present on physical examination. Cardiac murmur is typically diastolic with a loud P2 when pulmonary hypertension is present. The absence of an opening snap or a loud S1 marks the difference between CTS and mitral stenosis. + +Signs of right heart failure, pulmonary hypertension, and pulmonary congestion may also be present. + +Diagnostic evaluation begins with TTE. On the four-chamber view, the membrane of CTS is seen as a thin linear structure bisecting the atria distally to the mitral valve and the LA appendage. In the long-axis view, the superior part of the membrane is usually parallel to the aortic wall, while its inferior part connects to the LA posterior wall. One or more foramina may be seen within the membrane, with an increased velocity on color flow Doppler. TTE is also useful in identifying other associated congenital cardiac anomalies, such as atrial septal defects, ventricular septal defects, and tricuspid or mitral stenosis. Spectral Doppler interrogation is used to measure the mean and maximal transmembrane pressure gradients and maximal velocity as well as the pulmonary artery pressure. It has been reported that severe obstruction is indicated by a maximum Doppler velocity >2 m/sec. Although three-dimensional TTE may be useful to delineate the size and anatomy of the orifice, TEE is often required for this purpose. TEE also helps determine the relation of the membrane to other cardiac structures, especially to differentiate CTS from supravalvular ring, the latter being located below the LA appendage. Three-dimensional matrix-array TEE can also provide detailed visualization of the orifice or fenestrations of CTS. A retrospective analysis showed a 50% higher detection rate of CTS on preoperative computed tomographic imaging compared with echocardiography. In 80% of patients, complex cardiac anomalies were either missed or misdiagnosed on echocardiography.6 Thus, echocardiography alone may not be sufficient to describe the anatomic presentation of the defect and an integrative diagnosis may be helpful. Cardiovascular magnetic resonance (CMR) can be helpful in the assessment of hemodynamics, valve disease, and chamber size and function. Sakamoto et al. + +7 described two cases of CTS in adults in which the use of CMR provided additional anatomic information that was not evident on TTE, particularly regarding the location of atrial communication and anatomy of the pulmonary veins. + +Figure 1 Two-dimensional TTE, apical four-chamber view, systolic phase performed 8 years before the current presentation, + +![1_image_0.png](1_image_0.png) + +demonstrates the cor triatriatum membrane (CTM) dividing the left atrium (LA) into true and accessory chambers. LV, Left ventricle; MV, mitral valve; RA, right atrium; RV, right ventricle. + +According to the updated 2018 American Heart Association and American College of Cardiology guidelines for the management of adult congenital heart diseases, adults presenting with CTS should be evaluated for other congenital abnormalities, particularly atrial septal defect, ventricular septal defect, and anomalous pulmonary venous connection (Class 1). In adults with previous repair of CTS and recurrent symptoms, they recommend evaluating for pulmonary vein stenosis (Class 2a).8 The management of CTS requires a multidisciplinary approach. + +Early involvement of the congenital heart disease team, electrophysiologists, and the cardiac surgery team will help accurately diagnose and understand the extent of the condition, tailor treatment plan on the basis of age, overall health, and severity of disease, as well as provide ongoing follow-up and monitoring for complications. + +Cardiopulmonary exercise testing can be useful for baseline functional assessment and serial testing in adult congenital heart diseases +(2018 American Heart Association/American College of Cardiology Class 2a recommendation), but there is scarce evidence of its use in patients with CTS.9 Exercise stress echocardiography can also be useful to determine the functional severity of the membrane.10 An incidental echocardiographic finding of LA extra membrane with no pressure gradient in an asymptomatic patient requires monitoring only. When exertional dyspnea and signs of pulmonary congestion occur, diuretics and preload reduction are the mainstays of medical therapy. + +Atrial arrhythmias should be adequately treated with ventricular rate control and appropriate anticoagulation, as they may quickly lead to cardiac decompensation. As with other congenital heart diseases, it is not uncommon for patients with CTS to have other complex interatrial arrhythmias aside from atrial fibrillation.11 When performing catheter ablation for atrial fibrillation, additional electrophysiologic testing (e.g., rapid atrial pacing) should be considered to assess for atrial flutters and atrial tachycardias. Successful management of atrial fibrillation in patients with CTS with catheter ablation is reported, but long-term outcomes have not been reported.12 However, catheter ablation can be challenging in these patients with an altered LA anatomy. First, when performing transseptal access to the LA, there needs to be specific focus on entering the ''accessory atrium'' receiving blood from the pulmonary veins. As CTS can + +![2_image_0.png](2_image_0.png) + +![2_image_1.png](2_image_1.png) + +involve the interatrial septum, there may be less interatrial septum available for transeptal access. Second, once access into the ''accessory atrium'' is achieved, catheter manipulation may be constrained because there is a smaller than usual space in which to perform catheter ablation of the pulmonary veins.13 In our patient, the CTS membrane was noted to originate from the posterolateral ridge but was mostly out of the way. + +According to the 2018 American Heart Association and American College of Cardiology guidelines, surgical repair is indicated for adults with CTS for symptoms attributable to the obstruction or a substantial gradient across the membrane $8 mm Hg (mean, 17 mm Hg; range, 8-40 mm Hg; Class 1).8 Complete resection of the membrane and closure of the atrial septum with a pericardial patch is a common approach. Associated congenital defects need to be corrected at the same time. Surgical outcomes are usually favorable at well-experienced centers, with nearly all patients becoming asymptomatic at follow-up and an overall reported survival 10 years of 83%.14 Percutaneous strategies have also been explored as a treatment modality. There are no guidelines for selecting patients for percutaneous treatment. However, Li et al. + +15 suggested that balloon + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +angioplasty can be considered as a bridge to surgery or as definitive therapy in patients with acute heart failure who are unfit for surgery at the time or symptomatic pregnant patients who have compatible anatomic variants of CTS. They concluded that anatomic characteristics, determined using advanced imaging such as CMR, should be considered to predict the technical success of catheter-based interventional strategies and that percutaneous strategies can be recommended only for isolated forms of CTS in which all pulmonary veins ultimately drain into the LA. Given that the long-term efficacy of percutaneous strategies remains to be confirmed, surgery is still the mainstay of treatment. CONCLUSION +CTS is an important differential diagnosis in young patients with systolic heart failure, even if they have multiple comorbidities. Early multidisciplinary involvement is important for timely diagnosis and intervention. + +ETHICS STATEMENT The authors declare that the work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. + +CONSENT STATEMENT +The authors declare that since this was a non-interventional, retrospective, observational study utilizing de-identified data, informed consent was not required from the patient under an IRB exemption status. + +FUNDING STATEMENT The authors declare that this report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. + +DISCLOSURE STATEMENT +The authors report no conflict of interest. ACKNOWLEDGMENT We thank sonographers Sylvia V. McAninch and Nicole Miller for their assistance in the acquisition of images. + +## Supplementary Data + +Supplementary data related to this article can be found at https://doi. org/10.1016/j.case.2023.12.019. + +REFERENCES +1. Al Sabri AR, Joshi N, Al Kindi H, Al Senaidi KS. Cor Triatriatum Sinistrum: a rare disease with a common presentation. Sultan Qaboos Univ Med J 2021;21:e324-6. + +2. Nassar PN, Hamdan RH. Cor triatriatum sinistrum: classification and imaging modalities. Eur J Cardiovasc Med 2011;1:84-7. + +3. Rudiene V, Hjortshøj CMS, Glaveckait _ e S, Zakarkait _ e D, Petrulionien _ e_ Z, - +Gumbiene L, et al. Cor triatriatum sinistrum diagnosed in the adulthood: a _ systematic review. Heart 2019;105:1197-202. + +4. Davlouros PA, Koutsogiannis N, Karatza A, Alexopoulos D. An unusual case of cor triatriatum sinister presenting as pulmonary edema during labor. Int J Cardiol 2011;150:e92-3. + +5. Fuchs M, Connolly H, Said S, Egbe A. Outcomes in patients with cor triatriatum sinister. Congenit Heart Dis 2018;13:628-32. 6. Goel A, Viswamitra S, Reddy BN, Gaduputi J. Computed tomography features of cor triatriatum: an institutional review. Br J Radiol 2021; 94:20201252. + +7. Sakamoto I, Matsunaga N, Hayashi K, Ogawa Y, Fukui J. Cine-magnetic resonance imaging of cor triatriatum. Chest 1994;106:1586-9. + +8. Stout KK, Daniels CJ, Aboulhosn JA, Bozkurt B, Broberg CS, Colman JM, +et al. 2018 AHA/ACC guideline for the management of adults with congenital heart disease: a report of the American College of Cardiology/American Heart Association task force on clinical practice guidelines +[published correction appears in J Am Coll Cardiol. 2019 may 14;73(18):2361-2362]. J Am Coll Cardiol 2019;73:e81-192. + +9. Tuan SH, Chen GB, Chen CH, Liou I, Sun SF, Wu SY, et al. Serial cardiopulmonary exercise testing in an asymptomatic young female receiving percutaneous balloon dilatation for cor triatriatum sinistrum at an early age. J Formos Med Assoc 2021;120:1143-7. + +10. Cotrim CA, Cafe H, Jo~ao I, Cotrim N, Guardado J, Cordeiro P, et al. Exercise stress echocardiography: where are we now? World J Cardiol 2022;14: +64-82. + +11. Okada A, Kato K, Shoda M, Tabata H, Yoshie K, Kuwahara K. Successful catheter ablation of atrial tachycardia in cor triatriatum sinister: a figureof-8 reentry in the left atrial membrane. HeartRhythm Case Rep 2020; 7:109-11. + +12. Minciuna IA, Cismaru G, Puiu M, Roșu R, Amet D, Anghelina D, et al. + +Atrial fibrillation ablation in a patient with cor triatriatum sinister and left common pulmonary vein: impact of left atrium anatomy on ablation approach. Life (Basel) 2022;12:992. + +13. Borne RT, Gonzalez J, Khanna A, Sauer WH, Thai Nguyen D. Getting to the right left atrium: catheter ablation of atrial fibrillation and mitral annular flutter in cor triatriatum. HeartRhythm Case Rep 2016;2:502-5. + +14. Saxena P, Burkhart HM, Schaff HV, Daly R, Joyce LD, Dearani JA. Surgical repair of cor triatriatum sinister: the Mayo Clinic 50-year experience. Ann Thorac Surg 2014;97:1659. + +15. Li WW, Koolbergen DR, Bouma BJ, Hazekamp MG, de Mol BA, de Winter RJ. Cathether-based interventional strategies for cor triatriatum in the adult - feasibility study through a hybrid approach. BMC Cardiovasc Disord 2015;15:68. \ No newline at end of file diff --git a/medical/md/PMC2196312.md b/medical/md/PMC2196312.md new file mode 100644 index 0000000000000000000000000000000000000000..675893b51a56e0a7a0ead67eaeabc79a7c35c901 --- /dev/null +++ b/medical/md/PMC2196312.md @@ -0,0 +1,367 @@ +# A Novel Inhibitory Receptor (Ilt3) Expressed On + +## Monocytes, Macrophages, And Dendritic Cells Involved In Antigen Processing + +By Marina Cella,* Christian Döhring,* Jacqueline Samaridis,* Mark Dessing,* Manfred Brockhaus,‡ Antonio Lanzavecchia,* and Marco Colonna* From the *Basel Institute for Immunology, CH-4005 Basel, Switzerland; and ‡*Hoffmann-La Roche* AG, CH-4002 Basel, Switzerland + +## Summary + +Immunoglobulin-like transcript (ILT) 3 is a novel cell surface molecule of the immunoglobulin superfamily, which is selectively expressed by myeloid antigen presenting cells (APCs) such as monocytes, macrophages, and dendritic cells. The cytoplasmic region of ILT3 contains putative immunoreceptor tyrosine-based inhibitory motifs that suggest an inhibitory function of ILT3. Indeed, co-ligation of ILT3 to stimulatory receptors expressed by APCs results in a dramatic blunting of the increased [Ca21]i and tyrosine phosphorylation triggered by these receptors. Signal extinction involves SH2-containing protein tyrosine phosphatase 1, which is recruited by ILT3 upon cross-linking. ILT3 can also function in antigen capture and presentation. + +It is efficiently internalized upon cross-linking, and delivers its ligand to an intracellular compartment where it is processed and presented to T cells. Thus, ILT3 is a novel inhibitory receptor that can negatively regulate activation of APCs and can be used by APCs for antigen uptake. + +Engagement of B cell antigen receptor, TCR, and several Fc receptor (FcR) complexes by cognate ligands results in tyrosine phosphorylation of cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs)1 (1–6). + +Phosphorylated ITAMs recruit and activate protein tyrosine kinases, which trigger a cascade of intracellular phosphorylation events leading to cellular activation. Immune cell responses are negatively regulated when stimulatory receptors are co-ligated to inhibitory receptors. These include the low affinity FcR for IgG, FcgRIIB, and CD22 in B +cells (7–10), the Ly-49 molecules, the killer cell inhibitory receptors (KIRs), the NKG2A/CD94 heterodimer in NK cells (11–13), and glycoprotein (gp)49B1 in mast cells and NK cells (14–16). All of these receptors are characterized by the presence of cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs; 17). Upon receptor engagement, cytoplasmic ITIMs are tyrosine phosphorylated, and subsequently bind the SH2 domain(s) of SHP-1 protein tyrosine phosphatase (18–23) and/or SHIP inositol phosphatase (24), which mediate downregulation of cell activation. + +Recently, we have described two new members of the immunoglobulin superfamily (Ig-SF), called immunoglobulin-like transcript 1 (ILT1) and ILT2 (25). These are homologous to bovine Fcg2 receptor (Fcg2R; 26), murine cell surface antigen gp49 (27, 14), human KIRs (28–30), human Fca receptor (FcaR; 31) and map to human chromosome 19, as do the FcaR and the KIRs. ILT1 and ILT2 are characterized by similar extracellular regions consisting of four Ig-SF domains. Their transmembrane and cytoplasmic domains, however, differ substantially. ILT1 has a charged residue of arginine within the transmembrane region, followed by a short cytoplasmic tail with no ITAMs or ITIMs. Similar characteristics are found in human FcaRI and bovine Fcg2R, which trigger cell responses via associated signaltransducing, ITAM-containing subunits. Conversely, ILT2 has a long cytoplasmic tail, which contains putative ITIMs similar to those known to bind SHP-1 in KIRs. ILT1 and ILT2 also display different tissue distributions. Although ILT1 is predominantly expressed by myeloid cells, ILT2 is expressed by B cell lines. Thus, ILT1 and ILT2 may mediate activation of myeloid cell responses and inhibition of B cell activation, respectively. + +Here we characterize a third member of the ILT family, called ILT3, which, similarly to ILT2, displays a long cytoplasmic tail containing putative ITIMs. By using an antiILT3 mAb, we have found that ILT3 is selectively expressed on myeloid APCs. Functional studies show that ILT3 behaves as an inhibitory receptor when cross-linked to a stimulatory receptor. A cytoplasmic component of the ILT3-mediated negative signaling pathway is the SH2containing phosphatase SHP-1, which becomes associated with ILT3 upon cross-linking. Furthermore, ILT3 is internalized and ILT3 ligands are efficiently presented to specific T cells, suggesting that ILT3 may be involved in antigen uptake and presentation. + +## Materials And Methods + +Cells. THP1 is a human myeloid cell line. C1R and 721.221 are MHC class I–deficient, EBV-transformed human B cell lines. Jurkat is a human T cell line. All of these cells were grown in RPMI, 10% FCS. Monocytes were prepared from PBMCs by adhesion to plastic. In brief, PBMCs were isolated from whole blood by Ficoll-Hypaque density gradient, washed with RPMI, +resuspended at 5 3 106/ml in RPMI, 10% FCS and cultured in six-well plates for 1 h at 378C. Nonadherent cells were removed by washing twice with RPMI. Adherent cells were cultured overnight in RPMI, 10% FCS supplemented with 50 ng/ml GM-CSF and 1,000 U/ml IL-4. Dendritic cells (DCs) were obtained from monocytes as previously described (32). Macrophages were obtained by culturing monocytes for 10 d in six-well plates at a concentration of 106 cells/ml in RPMI, 20% human serum supplemented with 1 ng/ml macrophage CSF (M-CSF) (33, 34). + +cDNA Synthesis, Oligonucleotide Primers, PCR, and Cloning. + +The 39 end of ILT3 was cloned by rapid amplification of cDNA 39 end (35). In brief, RNA was prepared from 721.221 and C1R +cells as described (36) and single-strand cDNA was synthesized by reverse transcription of 1 mg of total RNA using Moloney murine leukemia virus reverse transcriptase and a (dT)17 adaptor in a reaction volume of 20 ml. cDNA was amplified by two nested PCRs. The 59 oligonucleotides were CCCCCAGCGACCCCCTGGA (nucleotide 926–944 of ILT2) and ACGGTGGCCTCAGGAGAGA (nucleotide 1003–1021 of ILT2) (25). In both PCRs, the 39 primer corresponded to the adaptor primer. + +PCRs (50 ml volume) contained 1 ml of total cDNA sample, PCR primers (0.5 mM each), 29-deoxynucleoside 59-triphosphates (125 mM each), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM +MgCl2, 0.01% gelatin, and 0.25 units of Taq polymerase. PCR +was carried out for 35 cycles, each consisting of 1-min steps at 94, 55, and 728C. Amplified products were gel purified in 1.2% agarose gel, cloned into pCRII (Invitrogen, San Diego, CA), and sequenced. The 59 end of ILT3 was cloned by two consecutive cycles of 59 rapid amplification of cDNA ends (35). + +Production of ILT3 Human IgG1 Fusion Protein. To produce ILT3 as a soluble fusion protein, we constructed a chimeric gene consisting of ILT3 extracellular domains and human IgG1 constant regions. The cDNA fragment encoding the ILT3 extracellular region was amplified by PCR from cloned plasmid DNA. The 59 primer (GATC*GAATTC*ATGATCCCCACCTTCACGGCT) contained an EcoRI restriction site (*italic*) and the ILT3 start codon. The 39 primer (CTGATC*AAGCTT*ATACTTACCCTCCCAGTGCCTTCTCAGACC) provided a HindIII restriction site (*italic*), a splice donor sequence, and seven ILT3 codons preceding the transmembrane domain. The z800-bp PCR product was cut with EcoRI and HindIII, and ligated into an expression vector containing the exons for hinge, CH2 and CH3 region of human IgG1, the guanosine phosphotransferase gene conferring resistance to mycophenolic acid, and the k promoter for the expression in mouse myeloma (37). Transfections, selection of secreting transfectants, and purification of fusion protein from culture supernatants were performed as described (38). + +Production of anti-ILT3 mAbs. 10-wk-old, female BALB/c mice +(Iffa-Credo, L'Arbresle, France) received an initial injection of 100 mg of ILT3 human IgG1 fusion protein (ILT3-HuIgG1), +mixed 1:1 (vol/vol) with Alu-Gel-S (Serva Biochemicals, Paramus, NJ), behind the neck. 4 wk later, they were given a booster immunization with the same immunogen, followed after 2 wk by a final injection of 100 mg of purified ILT3-HuIgG1. 3 d later, mice were killed and draining lymph node cells were isolated and fused with the myeloma fusion partner, Ag8.653, using polyethylene glycol 4000. Hybridoma supernatants were screened in two steps. First, an ELISA was performed using ILT3-HuIgG1 in the coating step and human-adsorbed alkaline phosphatase–labeled goat anti–mouse IgG as secondary antibody. Supernatants from clones that were positive in ELISA were then tested by FACS +analysis for staining C1R cells by flow cytometry. + +Transfections. ILT1, ILT2, and ILT3 cDNAs were subcloned into pCDNA3 (Invitrogen) and transfected into COS7 cells by lipofectin (GIBCO BRL, Gaithersburg, MD). Cell surface expression of ILTs was assessed 48 h after transfection by FACS analysis. A stable transfectant of ILT3 in Jurkat T cells was obtained as previously described (28). + +Antibodies and FACS *Staining.* Before staining, all the cells were preincubated with PBS, 20% human serum for 30 min on ice, to block Fc receptors. In two- and three-color staining experiments, cells were stained with the following primary mAbs: OKT3 +(anti-CD3, IgG2a; American Type Culture Collection, Rockville, MD), CD16 (IgG2a; Milan Analytica AG, La Roche, Switzerland), 3C10 (anti-CD14, IgG2b; American Type Culture Collection), 1F5.4 (anti-CD20, IgG2a; American Type Culture Collection), M-T102 (anti-CD1a, IgG2b; PharMingen, San Diego, CA), L243 (anti-HLA-DR, IgG2a; American Type Culture Collection), and HB15a (anti-CD83, IgG2b; provided by Dr. T. Tedder, Duke University, Durham, NC). As secondary antibodies, we used human-adsorbed FITC- or PE-conjugated goat anti–mouse IgG1, IgG2a, and IgG2b (Southern Biotechnology Assoc., Inc., Birmingham, AL). In three-color stainings, biotin-labeled goat anti–mouse IgG2a was followed by streptavidin-allophycocyanin (SBA). Stained cells were analyzed by flow cytometry on a FACStar Plus (Becton Dickinson, Mountain View, CA) using the LYSYS II software. + +Immunoprecipitations. Cells were surface labeled with 1 mCi of Na125I using the sulfosuccinimidyl-3-(4-hydroxyphenyl)propionate method (39). For metabolic labeling with [32P]orthophosphate, cells were incubated in phosphate-free DMEM (Sigma Chemical Co., St. Louis, MO) containing 10% Tris-buffered saline–dialyzed FCS for 1 h. [32P]orthophosphate (Amersham Corp., Arlington Heights, IL) was then added at 0.5 mCi/ml and incubation was continued for 5 h. After surface or metabolic labeling, cells were lysed in 1% Triton X-100, 100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. After overnight preclearing with protein G–sepharose, lysates were incubated with culture supernatants of either ZM3.8 (anti-ILT3, IgG1) or control IgG1 mAbs +(5.133, anti-NKAT4 [40] and 1B7.11, anti-2,4,6 TNP, American Type Culture Collection) at 148C for 4 h, and immune complexes were precipitated by addition of protein G–Sepharose for 1 h at 148C. Precipitates were washed 3 times with lysis buffer, followed by a final wash with 10 mM Tris-HCl, pH 7.4, and resuspended in nonreducing or reducing sample buffer. SDS-PAGE +analysis was performed according to a standard procedure. After the run, gels were dried and exposed to autoradiography film +(Amersham Corp.) for 2–5 d. + +Measurement of Cytosolic Calcium in Macrophages and Monocytes. Monocytes and macrophages were loaded with Indo-1 AM (Sigma Chemical Co.) as described (41). In stimulation experiments, anti-CD11b mAb (44aabc; American Type Culture Collection) and anti-CD16 mAb (B73.1; American Type Culture Collection) were added to monocytes and macrophages, respectively, followed by a F(ab9)2 goat anti–mouse IgG (Milan Analytica) +as cross-linker. Cells were then analyzed on a flow cytofluorimeter (FACS Vantage or Coulter Elite Enhanced Sort Performance +[Coulter Corp., Hialeah, FL]) to detect Ca21 fluxes. Stimulation of monocytes with the anti-HLA-DR mAb 3.8B1 (Cella, M., and A. Lanzavecchia unpublished data) was performed without a cross-linker. In inhibition experiments, cells were preincubated for 10 min at room temperature either with ZM3.8 mAb, or with the anti–MHC class I W6/32 mAb. After washing with PBS, either anti-CD11b, anti-HLA-DR, or anti-CD16 mAbs were added followed by the cross-linking antibody, and cells were analyzed by flow cytometry to detect Ca21 fluxes. Only live (based on forward scatter criteria) and Indo-1–loaded cells (based on 405 nM +versus 525 nM emission spectra) were included in the analysis. + +Immunoblotting. For antiphosphotyrosine blots, monocytes (2 3 106) were incubated for 2 min at 378C with medium, ZM3.8 mAb, 3.8B1 mAb, or with both mAbs in the absence or in the presence of a secondary cross-linker. Cells were washed in icecold PBS and lysed as previously described (20). Cell lysates were boiled, separated on SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase (HRP)-coupled 4G10 +(500 ng/ml; Upstate Biotechnology Inc., Lake Placid, NY). Immunoblotted proteins were visualized by chemiluminescence using the enhanced chemiluminescence detection reagents (Amersham Corp.). For anti-SHP blots, 107 monocytes were plated on six-well tissue culture plates previously coated with purified ZM3.8 or 5.133 mAbs at 50 mg/ml, centrifuged for 2 min, shifted to 378C for 2 min, collected in cold PBS, 0.5 mM EDTA, +and washed twice. Identical number of cells stimulated with ZM3.8 or 5.133 mAbs were lysed and immunoprecipitated with ZM3.8 and protein G as described above. Precipitated samples were boiled for 5 min, separated by SDS-PAGE, and transferred to nitrocellulose (Hybond-C extra; Amersham Corp.). Membranes were blocked with PBS, 3% nonfat, dry milk and probed anti-SHP-1 rabbit antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) followed by HRP-labeled goat anti–rabbit Ig (SBA). Immunoblotted proteins were visualized by chemiluminescence. Membranes were stripped of antibody by incubating for 30 min at 508C in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM TrisHCL, pH 6.7, according to the protocol supplied by Amersham Corp. Stripped blots were reprobed after blocking with antiSHP-2 rabbit antiserum (Santa Cruz Biotechnology). + +Internalization Assay. Monocytes were cultured for 24–48 h in culture medium containing GM-CSF and IL-4, as described above. One aliquot of monocytes was fixed for 1 min at room temperature in RPMI, 0.05% glutaraldehyde. Another aliquot was used without fixation. Both samples were stained with ZM3.8 mAb for 40 min on ice, washed twice with ice-cold PBS, +1% FCS, and subsequently stained with biotin-labeled F(ab9)2 goat anti–mouse IgG for 1 h on ice. After washing with ice-cold PBS, 1% FCS, cells were incubated at 378C in prewarmed RPMI, 10% FCS for various times to allow internalization. Cells were then cooled on ice, washed once with PBS, 0.1% NaN3, stained with PE-conjugated streptavidin for 30 min on ice, washed, and analyzed by FACS. As a measure of internalization in nonfixed cells, we used the percental decrease of the median fluorescence intensity (MFI) as compared to control samples kept at 48C. The percentage decrease of MFI observed in fixed cells was taken as a measure of the off-rate of the antibody at 378C. + +Antigen Presentation Assay. 4 3 104 irradiated monocytes (3,000 rad) were co-cultured with 8 3 104 cells of the B13 T cell clone +(42) in 96-well flat-bottom microplates in the presence of serial dilutions of IgG1 mAbs. mAbs used in the assay were the following: ZM3.8 (anti-ILT3, IgG1) or 5.133 (anti-NKAT4 KIR, +IgG1), 1B7.11 (anti-2,4,6 TNP, IgG1; American Type Culture Collection), and 32.2 (anti-FcgRI, IgG1; American Type Culture Collection). After 48 h, the cultures were pulsed with [3H]thymidine (1 mCi/well, specific activity 5 Ci/mmol), and the radioactivity incorporated was measured after additional 16 h. The data were plotted against the concentration of mAbs determined by ELISA using a purified mouse IgG1 (anti-CD68, Dako Corp., +Carpinteria, CA) as a standard. + +Figure 1. Amino acid sequence of human ILT3, aligned with ILT1 and ILT2. Since ILT1 and ILT2 consist of four Ig-SF +domains, only the two domains with higher homology to ILT3 were included in the alignment. + +The alignment was generated by the Clustal method. Gaps (*dashes*) +were introduced to maximize homologies. Amino acids identical to the consensus are indicated by dots. Conserved cysteines involved in disulfide bonds in the extracellular domains are identified by asterisks. Cytoplasmic tyrosine-based motifs potentially involved in signal transduction and/or endocytosis are boxed. + +ILT3 has no N-linked glycosylation sites (N-X-S/T). Amino acid residues are numbered on the right side, beginning with the first residue of each of the predicted domains. ss, signal sequence; *Ig1–4*, Ig-SF extracellular domains; cp, connecting peptide; tm, transmembrane domain; cy, cytoplasmic domain. + +ILT3 cDNA sequence is available from EMBL/GenBank/DDBJ under accession number U82979. + +## Results + +![3_Image_0.Png](3_Image_0.Png) + +ILT3 Is a Member of the ILT Multigene Family Located on Human Chromosome 19. During the process of cloning the 39 end of ILT2 cDNA from EBV-B cell lines (25), we obtained an additional cDNA sequence that differed from ILT2 by several amino acid residues. Amplification of the 59 end of this fragment yielded a new cDNA, called ILT3, which contains a single open reading frame encoding a transmembrane protein of 447 amino acids with a predicted molecular mass of z47 kD (Fig. 1). The amino acid sequence begins with a hydrophobic signal peptide of 23 amino acids followed by an extracellular region composed of two C2 type Ig-SF domains (43). Each domain shows two characteristic cysteines that are 49 and 50 residues apart, flanked by conserved residues (Val-x-Leu/Ile-x-Cys and His/Tyr-x-Gly-x-Tyr-x-Cys-Tyr/Phe, respectively, where x is any amino acid). The putative transmembrane domain of ILT3 consists of 21 amino acids, followed by a long cytoplasmic region of 167 amino acids, which is characterized by the presence of one Tyr-x-x-Val motif followed by two Tyr-x-x-Leu motifs spaced by 26 amino acid residues. These Tyr-x-x-Leu pairs and their spacing are reminiscent of the Tyr-x-x-Leu motifs identified in KIRs as binding sites for protein tyrosine phosphatase SHP-1 +(19–23). + +Comparison of the predicted amino acid sequence of ILT3 with Ig-SF protein sequences, revealed that ILT3 is closely related to ILTs (64% similarity with ILT2), bovine Fcg2R (45%), murine cell surface antigen gp49 isoforms +(39–43%), and, to a lesser extent, to KIRs (23–32%) and human FcaR (28%). Genomic DNA analysis of human– +hamster hybrid cell lines, each with a different partial complement of human chromosomes, showed hybridizing bands only in samples containing human chromosome 19 (data not shown). Thus, ILT3 belongs to the ILT family which maps to human chromosome 19, as do the FcaR +and the KIR genes. Expression of ILT3 by RNA blot analysis revealed a major transcript of z1.6 kb (data not shown). Interestingly, this transcript was weakly expressed in the B cell lines C1R or 721.221, from which ILT3 was initially cloned, whereas it was predominantly expressed in monocytes purified from PBMCs and in the myeloid cell line THP1. No transcripts were detected in the T cell line Jurkat. + +ILT3 Is Expressed on Monocytes, Macrophages, and Dendritic Cells. To characterize the ILT3-encoded cell surface mol- + +![4_image_0.png](4_image_0.png) + +ecule, we produced anti-ILT3 mAbs using a soluble ILT3- +HuIgG1 fusion protein as an immunogen. The ZM3.8 mAb specifically recognized ILT3 on transiently transfected COS cells (data not shown). In PBMCs, ZM3.8 mAb stained only CD141 monocytes, whereas no staining was observed in CD31 T cells, CD161 NK cells, or CD201 B cells (Fig. 2 A). Interestingly, in monocyte-enriched populations derived from peripheral blood, ZM3.8 also stained CD831 cells (Fig. 2 A), as well as CD14-/HLA-DRhigh cells (Fig. 2 B). Both of these cell populations correspond to circulating primary DCs (44–46). In addition, ILT3 was expressed by both DCs and macrophages derived from monocytes cultured with GM-CSF plus IL-4 or M-CSF, respectively +(Fig. 2, C and D; 32–34). + +We also tested ILT3 expression in several cell lines. + +Among four myelo-monocytic cell lines (U937, Monomac6, THP1, and HL-60), only THP1 reacted with ZM3.8. All tested EBV-B cell lines (n 5 10) were negative, with the exception of C1R. No cell surface expression was detected either on purified fresh B cells or on B cells activated by CD40L +in the presence of IL-4, IL-2, and IL-10 (data not shown). Finally, ZM3.8 did not stain neutrophils (data not shown). Thus, ILT3 is selectively expressed on myeloid APCs. + +To define the biochemical characteristics of ILT3, we carried out immunoprecipitations from ILT3-transfected Jurkat T cells and monocytes (Fig. 3). A prominent band of z55 kD in ILT3-transfected T cells was detected under nonreducing and reducing conditions, while a z58–60 kD +band was detected in monocytes (Fig. 3 A) and in THP1 +(data not shown). N- and O-deglycosylation of immunoprecipitates did not produce any change in the size of the bands. After labeling cells with [32P]orthophosphate, ILT3 was immunoprecipitated as a constitutively phosphorylated molecule (Fig. 3 B). The discrepancy in apparent molecular mass of ILT3 between ILT3-transfected T cells and monocytes may be due to a different degree of phosphorylation in distinct cell types. + +Intracellular Ca21 *Mobilization Responses Triggered by* CD11b, MHC Class II, and CD16 in APCs Are Negatively Regulated by Co-ligation with ILT3. The presence of putative ITIMs suggested an inhibitory role of ILT3 on APC +activation. To test this hypothesis, we triggered monocytes and macrophages with mAbs that have been shown to induce Ca21 mobilization, such as anti-CD11b (47, 48), antiHLA-DR (49–51), and anti-FcgRIII (CD16; 52) mAbs, and tested whether recruitment of ILT3 to the triggering receptors inhibits Ca21 mobilization. As shown in Fig. 4 A, +ligation of surface CD11b with a specific mAb followed by a secondary cross-linking antibody elicited a rapid and transient rise in intracellular calcium concentration ([Ca21]i). + +On the contrary, preincubation of monocytes with ZM3.8 mAb, followed by addition of anti-CD11b and co-crosslinking of CD11b and ILT3 by a secondary antibody, resulted in complete inhibition of the [Ca21]i increase (Fig. 4 D). In control experiments, the [Ca21]i increase induced by CD11b was not affected by co-ligation of CD11b with MHC class I. Also, cross-linking of ILT3 alone did not evoke Ca21 mobilization, indicating that ILT3 is not a stimulatory receptor (data not shown). + +It has been reported that some anti–MHC class II mAbs can trigger functional responses in B lymphocytes (49–51). We have recently developed an anti-HLA-DR mAb, called 3.8B1, which triggers a rapid rise in [Ca21]i, even in the absence of a secondary cross-linking antibody (Fig. 4 B). Co-ligation of ILT3 and HLA-DR resulted in significant inhibition of [Ca21]i increase (Fig. 4 E). Interestingly, HLA-DR–mediated stimulation of monocytes was not affected by preincubation of monocytes with anti-ILT3 anti- + +![4_image_1.png](4_image_1.png) + +Figure 5. (A) Protein tyrosine + +![5_image_0.png](5_image_0.png) + +phosphorylation stimulated by treatment of monocytes with the anti-HLA-DR mAb 3.8B1 is inhibited by co-ligation of HLADR with ILT3. Monocytes were incubated with medium alone +(lane 1), with ZM3.8 mAb (antiILT3; lane 2), with 3.8B1 mAb +(anti-HLA-DR; lane 3) or with both mAbs in the absence (lane 4) or in the presence (lane 5) of a secondary cross-linking antibody. Cell lysates were separated on SDS-PAGE, transferred to nitrocellulose, and probed with HRP-coupled antiphosphotyrosine mAb 4G10. + +(B) Association of SHP-1 with ILT3 is increased upon ILT3 cross-linking. Monocytes were stimulated with ZM3.8 (lane 2) +or with a control IgG (5.133 mAb) (lane 1) coated on plastic plates. Cells were kept at 378C for 2 min, harvested, and lysed. ILT3 was immunoprecipitated with ZM3.8. Proteins were separated on SDSPAGE, transferred to nitrocellulose, and probed with anti-SHP-1, followed by HRP-conjugated goat anti–rabbit Ig. The migration of SHP-1 is marked by the arrow. +body in the absence of the secondary cross-linking antibody, indicating that recruitment of ILT3 to the stimulatory receptor is necessary to block the [Ca21]i increase (Fig. 4 G). + +The low affinity receptor for IgG, FcgRIII, is expressed on NK cells and macrophages and has been shown to trigger Ca21 mobilization in NK cells (52, 53). Similarly, we showed that cross-linking of FcgRIII in macrophages evokes a strong increase of [Ca21]i (Fig. 4 C). Despite the low expression of ILT3 on macrophages (see Fig. 2 D), co-ligation of ILT3 and FcgRIII inhibited the [Ca21]i increase, especially at early time points (Fig. 4 F). Together, these results demonstrate that co-ligation of ILT3 with a triggering receptor results in signal extinction. + +SHP-1 Is Recruited to ILT3 upon Cross-linking. To test whether inhibition of Ca21 mobilization responses was paralleled by inhibition of tyrosine phosphorylation, we performed antiphosphotyrosine immunoblotting on whole cell lysates of monocytes stimulated via HLA-DR in the absence or in the presence of co-ligation with ILT3. As shown in Fig. 5 A, monocytes triggered via HLA-DR displayed a substantial increase of tyrosine phosphorylation (lane 3), as compared to monocytes incubated with medium alone (lane 1) +or with anti-ILT3 mAb (lane 2). Co-ligation of ILT3 with HLA-DR resulted in a dramatic reduction of the intensity and number of tyrosine phosphorylated species (lane 5). In the absence of secondary cross-linking antibody, ILT3 did not block HLA-DR–triggered tyrosine phosphorylation increase (lane 4). + +Since in NK cells, KIRs downregulate calcium mobilization and tyrosine phosphorylation by recruiting SHP-1 and SHP-2 (19–23), we tested whether these phosphatases could be also involved in the negative signaling pathway Figure 6. ZM3.8 mAb is internalized upon cross-linking. + +![5_image_1.png](5_image_1.png) + +ZM3.8 bound to monocytes on ice did not disappear from the cell surface when the cells were shifted at 378C, in the absence of a cross-linking antibody (m). After cross-linking with a F(ab9)2 biotin-labeled secondary antibody, ZM3.8 was rapidly internalized, becoming undetectable by FACS analysis in 30–60 min +(h). In fixed cells, only a minimal decrease of the median fluorescence intensity was detected over time (j). Cell surface– +bound ZM3.8 was revealed by PE-conjugated goat anti–mouse IgG1 (m) or by PE-conjugated streptavidin (h, j). The MFI +was determined by FACS analysis. The percentage decrease of MFI as compared to a control sample kept at 48C was used as a measure of internalization. + +mediated by ILT3. ILT3 was cross-linked on monocytes by ZM3.8 mAb attached to the plastic surface of tissue culture plates and subsequently immunoprecipitated from cell lysates. In control experiments, ILT3 was immunoprecipitated from unstimulated cells. Immunoblotting of immunoprecipitates with anti-SHP-1 antibodies demonstrated association of SHP-1 with ILT3 that significantly increases upon ILT3 cross-linking (Fig. 5 B). On the contrary, no association with SHP-2 was detected (data not shown). + +These results implicate SHP-1 as a cytosolic component of the signal extinction mediated by ILT3. + +Cross-linking of ILT3 on Monocytes Results in Receptor Internalization and Delivery into an Antigen-processing Compartment. Since ILT3 is selectively expressed on APCs and displays putative tyrosine-x-x-valine/leucine internalization motifs in the cytoplasmic tail (54–56), we analyzed the ability of ILT3 to endocytose and deliver its ligand to an antigen-processing and loading compartment. As shown in Fig. 6, ZM3.8 mAb bound at 08C to monocytes was not internalized when the cells were shifted to 378C. We therefore examined whether internalization occurs in the presence of a secondary cross-linker. ZM3.8 mAb was bound on ice to monocytes and cross-linked by a biotinylated F(ab9)2 secondary antibody. Cells were subsequently warmed to 378C to allow internalization. After various time points, cells were returned to ice and the amount of ZM3.8 mAb remaining on the cell surface was determined by flow cytometry using PE-conjugated streptavidin. Freshly isolated monocytes efficiently internalized bound ZM3.8 mAb, as demonstrated by a 60–80% reduction after 30 min at 378C and by a complete disappearance after 60–90 min +(Fig. 6). In control experiments, no significant decrease of surface-bound ZM3.8 was observed using fixed monocytes, ruling out a detachment of the primary or secondary antibodies from the cell surface at 378C (Fig. 6). In addi1748 An Inhibitory Receptor Expressed in Myeloid Antigen-presenting Cells + +![6_image_0.png](6_image_0.png) + +tion, when 125I-labeled streptavidin was added as a third step reagent before incubation at 378C, no significant decrease of cell-bound radioactivity was observed over time, thus excluding shedding of ILT3 from the cell surface (data not shown). + +To further investigate a possible role of ILT3 in antigen capture, we evaluated the ability of monocytes to present ZM3.8 mAb to a CD41 class II–restricted T cell clone specific for a mouse IgG1 peptide epitope (42). The presentation of the ZM3.8 mAb was compared to that of IgG1 mAbs that bind to other receptors (anti-FcgRI mAb) or do not bind to surface molecules and are taken up in the fluid phase [anti-TNP and anti-NKAT4 KIR mAbs]. As shown in Fig. 7, monocytes presented ZM3.8 mAb to T cells 50– +100-fold more efficiently than the anti-FcgRI mAb, and 400–500-fold more efficiently than anti-TNP and antiNKAT4 KIR mAbs. Together, these experiments indicate that ILT3 can efficiently deliver its ligand to an intracellular compartment where class II loading can occur. + +## Discussion + +Previous work has extensively shown that inhibitory receptors regulate cell activation in B, NK, mast cells, and subsets of T cells when co-ligated with ITAM-containing stimulatory receptor complexes. Our data extend this concept to professional APCs, showing that ILT3 negatively regulates APC functional responses triggered via stimulatory receptors, such as CD11b, CD16, and even MHC +class II. In addition to its inhibitory function, ILT3 is involved in antigen uptake. It is noteworthy that ILT3 is expressed on DCs. These are a unique type of leukocytes whose primary function is to efficiently capture antigens, process them, and present them to naive T cells (57). Although the uptake and processing of antigen is a major function of DCs, only a few mechanisms and receptors specialized for antigen capture have been identified on DCs +(58–60). Our data indicate that ILT3 is a unique receptor expressed in DCs, able to target the ligand into processing and peptide-loading compartments. Thus, ILT3 displays a dual function of inhibitory receptor and antigen-capturing molecule. A similar dual function has been previously shown for the low affinity receptor for IgG, FcgRIIB (7, 8). + +The ligand of ILT3 is still unknown. ILT3 is closely related to bovine Fcg2R and, to a lesser extent, to human FcaR, suggesting that ILT3 may be an Fc receptor. However, we did not detect any significant binding of human monomeric and heat-aggregated IgM, IgG, IgA, and IgE to ILT3-transfected Jurkat T cells (data not shown). ILT3 also shows homology to KIRs, which suggests that it may be a receptor for MHC class I molecules. We have investigated this possibility by analyzing binding of soluble ILT3- +HuIgG1 fusion protein to MHC class I transfectants in the class I–deficient mutant 721.221. No significant binding could be detected by FACS analysis under the same experimental conditions in which KIR-HuIgG fusion proteins bind to MHC class I molecules (data not shown). We are investigating the possibility that ILT3 is a receptor for cell surface molecules related to MHC class I molecules, such as nonclassical class I molecules (CD1 [61], MR1 [62], +MIC [63]), for MHC class II molecules, or for serum factors such as complement components. ILT3 is a member of the ILT subfamily of Ig-SF molecules, which is located on chromosome 19, most likely in close linkage with KIRs. Another member of this family, ILT2, is characterized by similar cytoplasmic tyrosine-based motifs such as ILT3, and is expressed in B cell lines, suggesting that this may be a putative inhibitory resceptor in B cells. We have also cloned additional cDNAs which encode novel molecules homologous to ILT2 and ILT3 +(ILT4 and ILT5, GenBank accession numbers AF000574 and AF000575). Thus, the number of Ig-SF inhibitory receptors with different tissue distribution and specificity is likely to increase. + +Received for publication 30 January 1997. + +## References + +1. Cambier, J.C., C.M. Pleiman, and M.R. Clark. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. *Annu. Rev. Immunol.* 12:457–486. + +2. Weiss, A., and D.R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. *Cell.* 76:263–274. + +3. Letourneur, F., and R.D. Klausner. 1991. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor z family proteins. *Proc. Natl. Acad. Sci. USA.* 88:8905–8909. + +4. Letourneur, O., I.C. Kennedy, A.T. Brini, J.R. Ortaldo, J.J. + +O'Shea, and J.P. Kinet. 1991. Characterization of the family of dimers associated with Fc receptors (Fce RI and Fcg RIII). + +J. Immunol. 147:2652–2656. + +5. Bonnerot, C., S. Amigorena, D. Choquet, R. Pavlovich, V. + +Choukroun, and W.H. Fridman. 1992. Role of associated g-chain in tyrosine kinase activation via murine FcgRIII. + +EMBO (Eur. Mol. Biol. Organ.) J. 11:2747–2757. + +6. Daeron, M., O. Malbec, C. Bonnerot, S. Latour, D.M. Segal, and W.H. Fridman. 1994. Tyrosine-containing activation motifdependent phagocytosis in mast cells. *J. Immunol.* 152:783–792. + +7. Amigorena, S., C. Bonnerot, J.R. Drake, D. Choquet, W. + +Hunziker, J.G. Guillet, P. Webster, C. Sautes, I. Mellman, and W.H. Fridman. 1992. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. *Science (Wash. DC).* 256:1808–1812. + +8. Muta, T., T. Kurosaki, Z. Misulovin, M. Sanchez, M.C. + +Nussenzweig, and J.V. Ravetch. 1994. A 13-amino-acid motif in the cytoplasmic domain of FcgRIIB modulates B-cell receptor signalling. *Nature (Lond.).* 368:70–73. + +9. Daeron, M., S. Latour, O. Malbec, E. Espinosa, P. Pina, S. + +Pasmans, and W.H. Fridman. 1995. The same tyrosine-based inhibition motif, in the intracytoplasmic domain of FcgRIIB, +regulates negatively BCR-, TCR-, and FcR-dependent cell activation. *Immunity.* 3:635–646. + +10. Doody, G.M., L.B. Justement, C.C. Delibrias, R.J. Matthews, J. Lin, M.L. Thomas, and D.T. Fearon. 1995. A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP. *Science (Wash. DC).* 269:242–244. + +11. Yokoyama, W.M., and W.E. Seaman. 1993. The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex. *Annu. Rev. Immunol.* +11:613–635. + +12. Lanier, L.L., and J.H. Phillips. 1996. Inhibitory MHC class I +receptor on NK cells and T cells. *Immunol. Today.* 17:86–91. + +13. Lazetic, S., C. Chang, J.P. Houchins, L.L. Lanier, and J.H. + +Phillips. 1996. Human natural killer cell receptors involved in MHC class I recognition are disulfide linked heterodimers of CD94 and NKG2 subunits. *J. Immunol.* 157:4741–4745. + +14. Katz, H.R., E. Vivier, M.C. Castells, M.J. McCormick, J.M. + +Chambers, and F.K. Austen. 1996. Mouse mast cell gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs and suppresses mast cell activation when coligated with the high-affinity Fc receptor for IgE. Proc. Natl. Acad. Sci. USA. 93:10809–10814. + +15. Wang, L.L., I.K. Mehta, P.A. LeBlanc, and W.M. Yokoyama. + +1997. Mouse natural killer cells express gp49B1, a structural homologue of human killer inhibitory receptors. *J. Immunol.* +158:13–17. + +16. Rojo, S., D.N. Burshtyn, E.O. Long, and N. Wagtmann. + +1997. Type I transmembrane receptor with inhibitory function in mouse mast cells and NK cells. *J. Immunol.* 158:9–12. + +17. Thomas, M.L. 1995. Of ITAMs and ITIMs: turning on and off the B cell antigen receptor. *J. Exp. Med.* 181:1953–1956. + +18. D'Ambrosio, D., K.L. Hippen, S.A. Minskoff, I. Mellman, G. Pani, K.A. Siminovitch, and J.C. Cambier. 1995. Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by FcgRIIB1. *Science (Wash. DC).* +268:293–297. + +19. Burshtyn, D.N., A.M. Scharenberg, N. Wagtmann, S. Rajagopalan, K. Berrada, T. Yi, J.P. Kinet, and E.O. Long. 1996. + +Recruitment of tyrosine phosphatase HCP by the killer cell inhibitor receptor. *Immunity.* 4:77–85. + +20. Campbell, K.S., M. Dessing, M. Lopez Botet, M. Cella, and M. Colonna. 1996. Tyrosine phosphorylation of a human killer inhibitory receptor recruits protein tyrosine phosphatase 1C. *J. Exp. Med.* 184:93–100. + +21. Olcese, L., P. Lang, F. Vely, A. Cambiaggi, D. Marguet, M. + +Blery, K.L. Hippen, R. Biassoni, A. Moretta, L. Moretta, J.C. Cambier, and E. Vivier. 1996. Human and mouse killercell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases. *J. Immunol.* 156:4531–4534. + +22. Fry, A.M., L.L. Lanier, and A. Weiss. 1996. Phosphotyrosines in the killer cell inhibitory receptor motif of NKB1 are required for negative signaling and for association with protein tyrosine phosphatase 1C. *J. Exp. Med.* 184:295–300. + +23. Binstadt, B.A., K.M. Brumbaugh, C.J. Dick, A.M. Scharenberg, B.L. Williams, M. Colonna, L.L. Lanier, J.-P. Kinet, R.T. Abraham, and P.J. Leibson. 1996. Sequential involvement of Lck and SHP-1 with MHC-recognizing receptors on NK cells inhibits FcR-initiated tyrosine kinase activation. Immunity. 5:629–638. + +24. Ono, M., S. Bolland, P. Tempst, and J.V. Ravetch. 1996. + +Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor FcgRIIB. *Nature* +(Lond.). 383:263–266. + +25. Samaridis, J., and M. Colonna. 1997. Cloning of novel immunoglobulin superfamily receptors expressed on human myeloid and lymphoid cells: structural evidence for new stimulatory and inhibitory pathways. *Eur. J. Immunol.* 27:660–665. + +26. Zhang, G., J.R. Young, C.A. Tregaskes, P. Sopp, and C.J. + +Howard. 1995. Identification of a novel class of mammalian Fcg receptor. *J. Immunol.* 155:1534–1541. + +27. Arm, J.P., M.F. Gurish, D.S. Reynolds, H.C. Scott, C.S. + +Gartner, K.F. Austen, and H.R. Katz. 1991. Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily. *J. Biol. Chem.* 266:15966–15973. + +28. Colonna, M., and J. Samaridis. 1995. Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. *Science* (Wash. DC). 268:405–408. + +1750 An Inhibitory Receptor Expressed in Myeloid Antigen-presenting Cells 29. Wagtmann, N., R. Biassoni, C. Cantoni, S. Verdiani, M.S. + +Malnati, M. Vitale, C. Bottino, L. Moretta, A. Moretta, and E.O. Long. 1995. Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular domains. *Immunity.* 2:439–449. + +30. D'Andrea, A., C. Chang, K. Franz-Bacon, T. McClanahan, J.H. Phillips, and L.L. Lanier. 1995. Molecular cloning of NKB1. A natural killer cell receptor for HLA-B allotypes. J. Immunol. 155:2306–2310. + +31. Maliszewski, C.R., C.J. March, M.A. Schoenborn, S. Gimpel, and L. Shen. 1990. Expression cloning of a human Fc receptor for IgA. *J. Exp. Med.* 172:1665–1672. + +32. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. *J. Exp. Med.* 179:1109–1118. + +33. Bender, A., M. Sapp, G. Schuler, R.M. Steinman, and N. + +Bhardwaj. 1996. Improved methods for the generation of dendritic cells from non proliferating progenitors in human blood. *J. Immunol. Methods.* 196:121–135. + +34. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, D. Niederwieser, and G. Schuler. 1996. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. *J. Immunol. Methods.* 196:137–151. + +35. Frohman, M.A., M.K. Dush, and G.R. Martin. 1988. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. + +Proc. Natl. Acad. Sci. USA. 85:8998–9002. + +36. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 8.11. + +37. Traunecker, A., F. Oliveri, and K. Karjalainen. 1991. Myeloma based expression system for production of large mammalian proteins. *Trends Biotechnol.* 9:109–113. + +38. Dohring, C., and M. Colonna. 1996. Human natural killer cell inhibitory receptors bind to HLA class I molecules. *Eur.* J. Immunol. 26:365–369. + +39. Reid, P.A., and C. Watts. 1990. Cycling of cell-surface MHC glycoproteins through primaquine-sensitive intracellular compartments. *Nature (Lond.).* 346:655–657. + +40. Dohring, C., D. Scheidegger, J. Samaridis, M. Cella, and M. + +Colonna. 1996. A human killer inhibitory receptor specific for HLA-A. *J. Immunol.* 156:3098–3101. + +41. Valitutti, S., M. Dessing, and A. Lanzavecchia. 1993. Role of cAMP in regulating cytotoxic T lymphocyte adhesion and motility. *Eur. J. Immunol.* 23:790–795. + +42. Lanzavecchia, A., S. Abrignani, D. Scheidegger, R. Obrist, B. Dorken, and G. Moldenhauer. 1988. Antibodies as antigens. The use of mouse monoclonal antibodies to focus human T cells against selected targets. *J. Exp. Med.* 167:345–352. + +43. Williams, A.F., and N.A. Barclay. 1988. The immunoglobulin superfamily-domains for cell surface recognition. Ann. + +Rev. Immunol. 6:381–405. + +44. Zhou, L.J., and T.F. Tedder. 1995. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. *J. Immunol.* 154:3821–3835. + +45. Zhou, L.J., and T.F. Tedder. 1996. CD141 blood monocytes can differentiate into functionally mature CD831 dendritic cells. *Proc. Natl. Acad. Sci. USA.* 93:2588–2592. + +46. O'Doherty, U., R.M. Steinman, M. Peng, P.U. Cameron, S. + +Gezelter, I. Kopeloff, W.J. Swiggard, M. Pope, and N. + +Bhardwaj. 1993. Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium. *J. Exp. Med.* 178:1067–1076. + +47. Altieri, D.C., S.J. Stamnes, and C.G. Gahmberg. 1992. Regulated Ca21 signalling through leukocyte CD11b/CD18 integrin. *Biochem. J.* 288:465–473. + +48. Crockett-Torabi, E., B. Sulenbarger, C.W. Smith, and J.C. Fantone. 1995. Activation of human neutrophils through L-selectin and Mac-1 molecules. *J. Immunol.* 154:2291–2302. + +49. Cambier, J.C., D.C. Morrison, M.M. Chien, and K.R. Lehmann. 1991. Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia crosslinking. J. Immunol. 146:2075–2082. + +50. Cambier, J.C., and K.R. Lehmann. 1989. Ia-mediated signal transduction leads to proliferation of primed B lymphocytes. J. Exp. Med. 170:877–886. + +51. Charron, D., S. Brick-Ghannam, R. Ramirez, and N. + +Mooney. 1991. HLA class-II-mediated B-lymphocyte activation: signal transduction and physiologic consequences. Res. + +Immunol. 142:467–474. + +52. Cassatella, M.A., I. Anegon, M.C. Cuturi, P. Griskey, G. + +Trinchieri, and B. Perussia. 1989. FcgR(CD16) interaction with ligand induces Ca21 mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca21 in FcgR +(CD16)-induced transcription and expression of lymphokine genes. *J. Exp. Med.* 169:549–567. + +53. Ravetch, J.V., and J.P. Kinet. 1991. Fc receptors. *Annu. Rev.* +Immunol. 9:457–492. + +54. Trowbridge, I.S., J.F. Collawn, and C.R. Hopkins. 1993. + +Signal-dependent membrane protein trafficking in the endocytic pathway. *Annu. Rev. Cell. Biol.* 9:129–161. + +55. Letourneur, F., and R.D. Klausner. 1992. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. *Cell.* 69:1143–1157. + +56. Sandoval, I.V., and O. Bakke. 1994. Targeting of membrane proteins to endosomes and lysosomes. *Trends Cell Biol.* 4: 292–297. + +57. Steinman, R.M. 1991. The dendritic cell system and its role in immunogenicity. *Annu. Rev. Immunol.* 9:271–296. + +58. Jiang, W., W.J. Swiggard, C. Heufler, M. Peng, A. Mirza, R.M. Steinman, and M.C. Nussenzweig. 1995. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. *Nature (Lond.).* 375:151–155. + +59. Sallusto, F., M. Cella, C. Danieli, and A. Lanzavecchia. 1995. + +Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. *J. Exp. Med.* 182:389–400. + +60. Bieber, T., H. de la Salle, A. Wollenberg, J. Hakimi, R. + +Chizzonite, J. Ring, D. Hanau, and C. de la Salle. 1992. Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (FceRI). *J. Exp. Med.* 175:1285–1290. + +61. Porcelli, S.A. 1995. The CD1 family: a third lineage of antigen-presenting molecules. *Adv. Immunol.* 59:1–98. + +62. Hashimoto, K., M. Hirai, and Y. Kurosawa. 1995. A gene outside the human MHC related to classical HLA class I genes. *Science (Wash. DC).* 269:693–695. + +63. Bahram, S., M. Bresnahan, D.E. Geraghty, and T. Spies. + +1994. A second lineage of mammalian major histocompatibility complex class I genes. *Proc. Natl. Acad. Sci. USA.* 91: +6259–6263. \ No newline at end of file diff --git a/medical/md/PMC2529247.md b/medical/md/PMC2529247.md new file mode 100644 index 0000000000000000000000000000000000000000..3b29d036c9e8a5f75cd523507089c8806d7634cb --- /dev/null +++ b/medical/md/PMC2529247.md @@ -0,0 +1,506 @@ +Journal of Experimental Botany, Vol. 59, No. 12, pp. 3485–3499, 2008 doi:10.1093/jxb/ern201 Advance Access publication 06 August, 2008 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) + +# Research Paper Arabidopsis Sterol Carrier Protein-2 Is Required For Normal Development Of Seeds And Seedlings + +Bing Song Zheng1,2, Elin Ro¨nnberg1, Lenita Viitanen3, Tiina A. Salminen3, Krister Lundgren4, Thomas Moritz4 and Johan Edqvist5,* 1 Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, PO Box 7080, 750 07 Uppsala, Sweden 2 School of Forestry and Biotechnology, Zhejiang Forestry University, 311300, Lin An, China 3 Department of Biochemistry and Pharmacy, A˚bo Akademi University, Artillerigatan 6 A III, FIN-20520 Turku, Finland 4 Umea˚ Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umea˚, Sweden 5 IFM Biology, Linko¨ping University, 581 83 Linko¨ping, Sweden Received 25 April 2008; Revised 27 June 2008; Accepted 4 July 2008 + +## Abstract + +The Arabidopsis thaliana sterol carrier protein-2 (AtSCP2) is a small, basic and peroxisomal protein that in vitro enhances the transfer of lipids between membranes. AtSCP2 and all other plant SCP-2 that have been identified are single-domain polypeptides, whereas in many other eukaryotes SCP-2 domains are expressed in the terminus of multidomain polypeptides. The AtSCP2 transcript is expressed in all analysed tissues and developmental stages, with the highest levels in floral tissues and in maturing seeds. + +The expression of AtSCP2 is highly correlated with the multifunctional protein-2 (MFP2) involved in b-oxidation. + +A. thaliana Atscp2-1 plants deficient in AtSCP2 show altered seed morphology, a delayed germination, and are dependent on an exogenous carbon source to avoid a delayed seedling establishment. Metabolomic investigations revealed 110 variables (putative metabolites) that differed in relative concentration between Atscp2-1 and normal A. thaliana wild-type seedlings. + +Microarray analysis revealed that many genes whose expression is altered in mutants with a deficiency in the glyoxylate pathway, also have a changed expression level in Atscp2-1. + +Key words: Arabidopsis, b-oxidation, germination, glyoxylate + +![0_image_0.png](0_image_0.png) cycle, lipid, lipid transport, metabolomics, microarray, peroxisomes, SCP-2. + +## Introduction + +Sterol carrier protein-2 (SCP-2) is an intracellular, small, basic protein domain that in vitro stimulates the transfer of lipids between membranes (Ritter et al., 1971; Bloj et al., 1978). In mammals, SCP-2 is implicated to have role in peroxisomal b-oxidation. The exact function of SCP-2 in b-oxidation is unclear, but it might facilitate the presentation and solubilization of the substrates or stabilizing the enzymes involved in catalysing the reaction cycles +(Seedorf et al., 2000). Such suggestions are mainly based on studies of the mammalian peroxisomal proteins sterol carrier protein-X (SCP-X) and D-bifunctional protein +(DBP), which both contain C-terminal SCP-2 domains. + +The human gene SCPX (also known as SCP2) encodes SCP-X, which consists of a 3-ketoacyl-CoA thiolase domain connected to a C-terminal SCP-2 domain (Ohba et al., 1994). Due to the existence of dual promoters, as well as proteolytic cleavage sites the SCPX encoded SCP-2 domain is also expressed as a single-domain protein +(Ohba et al., 1995). SCP-X and the single-domain SCP-2 are both predominantly located to peroxisomes. Gene + +## 3486 Zheng Et Al. + +targeting in mice revealed that complete deficiency of SCPX resulted in an impaired catabolism of 2-methyl branched-chain fatty acyl CoAs as shown by a 10-fold accumulation of phytanic acid in SCPX(–/–) mice (Seedorf et al., 1998). Further, it has been demonstrated with FRET microscopy that the single-domain SCP-2 interacts in peroxisomes with enzymes involved in b-oxidation such as acyl-CoA oxidase, bifunctional protein, and 3-ketoacylCoA thiolase (Wouters et al., 1998). It may be noteworthy that plants are not encoding any gene orthologous to the 3-ketoacyl-CoA thiolase in SCP-X (Edqvist and Blomqvist, 2006). + +A SCP-2 domain is also present in the C-terminus of the mammalian DBP (also referred to as MFE-2), which in the human genome is encoded from HSD17B4. DBP has domains for D-3 (equivalent to 3R)-hydroxyacyl-CoA +dehydrogenase, 2-enoyl-CoA hydratase, and SCP-2 +(Adamski et al., 1995; Leenders et al., 1998) and catalyses the second and third steps of the b-oxidation pathway. Reactions catalysed by the mammalian DBP +proceed through D-3-hydroxyl-CoA esters. DBP is suggested to be required for the peroxisomal b-oxidation of the enoyl-CoA esters of very long chain fatty acids, pristanic acid, and of dihydroxycholestanoic acid (DHCA) +(Wanders, 2004). The role of the SCP-2 domain in DBP is unclear, but it contains a peroxisomal targeting signal (PTS1) that locates DBP to the peroxisomes. The structural and functional conservation may indicate that the SCP-2 domain in DBP also has additional functions, such as to interact with enzymatic domains of DBP to form an extended hydrophobic cavity for the hydrophobic tails of some b-oxidation substrates (Haapalainen et al., 2001). + +A. thaliana do not encode DBP, and there are no plant genes identified orthologous to the D-3-hydroxyacyl-CoA +dehydrogenase domain of mammalian DBP (Edqvist and Blomqvist, 2006). Rather, the A. thaliana multifunctional proteins AIM1 and MFP2 each share domain structure and approximately 50% amino acid sequence similarity to the human peroxisomal L-bifunctional protein (LBP) (also referred to as MFE-1) (Kiema et al., 2002). LBP and DBP +lack significant sequence similarity and LBP, as well as AIM1 and MFP2, are not carrying any C-terminal SCP-2 domain. Furthermore, reactions catalysed by LBP, AIM1, and MFP2 proceeds through L-3-hydroxyl-CoA esters. + +Thus, plants do not encode DBP or SCP-X, but it has recently been shown that plants also encode and express SCP-2 (Eklund and Edqvist, 2003; Edqvist et al., 2004; Edqvist and Blomqvist, 2006). The gene AtSCP2 (At5g42890) on chromosome 5 encodes the sole SCP-2 domain in the A. thaliana genome. AtSCP2 is a 13.6 kDa protein with a pI of 9.2, which localizes to peroxisomes through its C-terminal PST1 targeting signal. It has lipid transfer activity in vitro (Edqvist et al., 2004). The known crystal structures of SCP-2 from rabbit (Choinowski et al., 2000), of the SCP-2 domain of the human DBP +(Haapalainen et al., 2001) and of yellow fever mosquito SCP-2 (Dyer et al., 2003) have an a/b-fold consisting of a five stranded b-sheet and four or five a-helices. A +C-terminal segment, together with part of the b-sheet and four a-helices form a hydrophobic tunnel, which is very suitable for binding of lipids or other hydrophobic ligands. + +According to generated models, AtSCP2 has a similar a/b-fold forming a hydrophobic tunnel (Edqvist et al., +2004). AtSCP2 and also all other plant SCP-2 that have been identified are single-domain polypeptides (Edqvist and Blomqvist, 2006; Viitanen et al., 2006), whereas, as indicated above, SCP-2 domains in animals and many other eukaryotes are often present in the terminal of polypeptides which carry multiple protein domains. + +The only SCP-2 domain encoded in A. thaliana is the single-domain protein AtSCP-2. As described above and in Edqvist and Blomqvist (2006), the situation is more complex in animals, with larger SCP-2 gene families and often quite complicated arrays of protein domain fusions. + +We reason that this turns A. thaliana into a very suitable model organism for studying the function of the still enigmatic SCP-2 domain. Here, an initial investigation on the biological function of AtSCP2 is presented. It is shown that the activity of the peroxisomal protein AtSCP2 is important for the metabolism in A. thaliana seeds and seedlings. + +## Materials And Methods + +Plant materials and growth conditions A. thaliana ecotype Columbia (Col-0) was used as the wild-type plant. Seeds of the T-DNA insertion lines Sail_1231_F11 were purchased from the European Arabidopsis Stock Centre (NASC) +(Loughborough, UK). The Sail_1231_F11 line is referred to as Atscp2-1. Seeds were surface-sterilized (washed in 70% ethanol for 1 min and in 15% chlorine and 0.5% SDS for 10 min followed by at least four washes in sterile distilled water) and sown on halfstrength Murashige and Skoog medium (1/2 MS). Before cultivation, seed dormancy was broken by 72 h of cold treatment (4 C). + +The synthetic auxin 2,4-dichlorophenoxybutyric acid (2,4-DB) +(0.1 lM, 4 lM) and indole-3-butyric acid (IBA) (3 lM, 30 lM) +were added to the autoclaved medium where indicated. Ten-day-old plants grown under sterile conditions were transplanted on soil mixed with vermiculite (2:1 v/v). The plants were cultivated under controlled conditions in environmental chambers at 20–22 C under long day (16/8 h light/dark) conditions. The Atscp2-1 mutant was back-crossed to wild-type A. thaliana Col-0. + +For expression of AtSCP2 in Atscp2-1 under the control of its own promoter, a DNA fragment carrying the AtSCP2 gene including the promoter was obtained through amplification of A. thaliana genomic DNA with primers ATSCP2promattB1F (5\#- +GGGGACAAGTTTGTACAAAAAAGCAGGCTCACACCTCCTATTTATCGGACAT-3\#) and AtSCP2attB2R (5\#-GGGGACCACTTTGTACAAGAAAGCTGGGTTCACAACTTTGAAGGTTTACGGAAGAT-3\#). The PCR fragment was recombined into the destination vector pMDC99 (Curtis and Grossniklaus, 2003) +resulting in the plasmid pJE602. For expression of AtSCP2 cDNA +under control of the cauliflower mosaic virus (CaMV) 35S promoter, a fragment carrying a cDNA copy of AtSCP2 was amplified from A. thaliana cDNA with ATSCP2attB1F (5\#- +GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCGAATACCCAACTCAAATC-3\#) and ATSCP2attB2R. The PCR fragment was recombined into destination vector pMDC32 (Curtis and Grossniklaus, 2003) yielding plasmid pJE601. Recombination events were done with the Gateway technology from Invitrogen +(Carlsbad, CA, USA). pJE601 and pJE602 were transformed into Agrobacterium tumefaciens C58. The floral dip method (Clough and Bent, 1998) was used to transform A. thaliana Atscp2-1 with A. tumefaciens C58 carrying pJE601 or pJE602. Transformations and selection of transformants were done at the Uppsala Transgenic Arabidopsis Facility. The transformants obtained were denoted Atscp2-1(35S::AtSCP2) for transformation with pJE601 and Atscp21(AtSCP2::AtSCP2) for transformation with pJE602. + +## Phenotypic Assays + +The hypocotyl and root lengths were measured on 3-d-old and 7-dold seedlings grown on ½ MS medium with 0% or 1% sucrose. At least 20 seedlings were measured from each growth condition and time point. All experiments were performed in triplicate. Rosette diameter was measured at the widest point of the plant without disturbing any leaves under light. The developing seeds were counted after self-pollination. The percentage of seed germination was scored every 12 h after transferring stratified seeds on the MS media to a growth chamber. Germination was defined as an obvious protrusion of the radicle through the seed coat. Several seed lots were tested with similar results. + +## Histochemical And Quantitative Gus Activity Assays + +A DNA fragment carrying the AtSCP2 promoter was amplified from the A. thaliana Col-0 genome by the use of primers SCPPrU2 +(5\#-CACACCTCCTATTTATCGGACAT-3\#) and SCPPrN2 (5\#- +GATTTTTGTTAGAGACTGGCACG-3\#). The PCR primers were designed such that a fragment was amplified stretching from the untranslated region of the nearest gene upstream of AtSCP2 to the 5' untranslated region of AtSCP2. The obtained 1.4 kb AtSCP2 promoter fragment was inserted into vector PCR2.1-TOPO (Invitrogen) to yield the plasmid pER2. The AtSCP2 promoter fragment was released from pER2 by restriction enzymes XbaI+BamH1, and subsequently fused to the b-glucuronidase (GUS) reporter gene by ligation to the XbaI+BamH1 sites of vector pBI101 resulting in plasmid pER1. The plasmid pER1 was transformed to A. tumefaciens C58. + +Histochemical GUS-assays were performed as described by Jefferson et al. (1987). Plant tissues were incubated in a substrate solution containing 50 mM Na-phosphate buffer (pH 7.0), 1 mM +5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid cyclohexyl ammonium salt (X-GlcA CHA) (Duchefa Biochemie, Haarlem, The Netherlands), 0.5 mM K4Fe(CN)6, 0.5 mM K3Fe(CN)6, and 0.01% +(w/v) Triton X-100 at 37 C overnight. Stained samples were incubated in 95% ethanol at room temperature to extract the chlorophyll. + +## Quantitative Real-Time Reverse Transcriptase-Pcr, Reverse Transcriptase-Pcr And Genomic Pcr + +RNA was extracted from A. thaliana using the Qiagen RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Five lg RNA was used for cDNA synthesis using oligo dT-primer and Superscript II +Rnase-Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Amplification of the cDNA was performed in the presence of gene-specific primers and the SYBR Green PCR +master mix (Applied Biosystems, Foster City, CA, USA) in MicroAmp Optical 96-well reaction plates with optical covers using an ABI Prism 7000 Sequence Detector (Applied Biosystems). + +Reaction conditions were 50 C for 2 min, 94 C for 10 min, followed by 40 cycles of 94 C for 15 s and 60 C for 1 min. All cDNA-samples were included in triplicate in all assays. Primers were designed using Primer express software (Applied Biosystems). + +Relative quantification of gene expression data was carried out with the 2–DDCT or comparative CT method (Livak and Schmittgen, 2001). The threshold cycle (CT) indicates the cycle number at which the amount of amplified transcript reaches a fixed threshold. + +Expression levels were normalized with the CT values obtained for the A. thaliana tubulin b-2/b-3 chain (At5g62690). The following gene-specific primers were used: AtSCP2: SCPF1 (5\#-GCCGGGAAAGAGGTCACA-3\#) and SCPR1 (5\#-TGTAAGTAACCTCTTCAAACCCTAATTTCT-3); A. thaliana tubulin b-2/b-3 chain +(At5g62690): Tub1 (5\#-ACACCAGACATAGTAGCAGAAATCAAG-3\#) and Tub2 (5\#-GAGCCTTACAACGCTACTCTGTCTGTC-3\#); At3g21720: 3g21720F (5\#-TTGCACAGATCGCAGACATCA-3\#) and 3g21720R (5\#-GAATTGGGTGCATTCATTGAGA-3\#); At1g06570: 1g06570F (5\#-CTCCGCCAAATCCGATCTT-3\#) and 1g06570R (5\#-GGAGGTCACCGGAGGTGAGT-3\#); +At3g47340: 3g47340F (5\#-AACGTCTTGCCGTCATCGAT-3\#) and 3g47340R (5\#-CAATGGTCTTGTCCTCGTTGAA-3\#); At1g08630: +1g08630F (5\#-CGATGCGAGAAGCAATGTGTA-3\#) and 1g08630R +(5\#-CGTCTAGCCGTTGGGTCATATC-3\#); At1g21400: 1g21400F +(5\#-AGACACAGGCTGATCATTTGGTT-3\#) and 1g21400R (5\#- +TTTGCCTCCTGGGAAATCC-3\#); At3g45140: 3g45140F (5\#-AGCCCCAATGGAAACAAGTCT-3\#) and 3g45140R (5\#-AGCAAGATTCCATAGCCAGCA-3\#); At5g55730: 5g55730F (5\#-GGCACCAGAGGATGGTGATG-3\#) and 5g55730R (5\#-TCTTTCCTTTCGCTTTCCCTTT-3\#); At5g04960: 5g04960F (5\#-AAACGCGAGCCGATCAAG-3\#) and 5g04960R (5\#-AGAGCTCCGTGAT-GGTGACTTT3\#); At3g14210: 3g14210F (5\#-CAGGAGGAAATGGCTCATCTTCTA-3\#) and 3g14210R (5\#-AGCTCACGGACC-GTCATTG-3\#); +At1g52050: 1g52050F (5\#-TTCCTAAGCTACAGAAGTTTGTTCATGCATG-3\#) and 1g52050R (5\#-CATAGTTGACAACATCGGAATCG-3\#). + +Reverse transcriptase-PCR (RT-PCR) was done as described previously (Edqvist et al., 2004). The oligonucleotides ATSCPRT1 and ATSCPRT2 were used for expression analysis of AtSCP2 cDNA, while UBL1 and UBL2 were used for amplification of cDNA for the ubiquitin-conjugating enzyme E2-21 kD (At5g41340) (Edqvist et al., 2004). + +For isolation of A. thaliana genomic DNA, 500 ll of an extraction buffer consisting of 200 mM TRIS, 250 mM NaCl, and 25 mM EDTA was added to plant tissues. After addition of six glass beads (3 mm in diameter), the tissues were disrupted by incubation in a FAST-prep instrument (MP Biomedicals, Irvine, CA, USA) for 30 s. 25 ll of 10% SDS was added, and the samples were incubated at 65 C for 10 min and then centrifuged for 5 min. + +450 ll of the supernatant was removed and precipitated with an equal volume of cold isopropanol. After incubation for 2 min at room temperature, samples were centrifuged for 5 min. The supernatant was discarded, and the DNA pellet obtained was dried, dissolved in water, and used for PCR. The following primers were used for analysing genomic DNA from Atscp2-1: SAIL_1231_F11_RP +(5\#-AACATTGCTCCAAAGGTTGGT-3\#), SAIL_1231_F11_LP +(5\#-GGACCAAATCCAAGTCACACA-3\#) and SAIL_LB1 (5\#- +GCCTTTTCAGAAATGGATAAATAGCCTTGC-3\#). + +## Expression Profiling Through Microarray Analysis + +Two-day-old seedlings of the Atscp2-1 and wild-type plants grown under light conditions on 1/2 MS without sucrose were harvested and frozen quickly in liquid nitrogen. Two-day-old seedlings were selected for the expression analysis due to the high AtSCP-2 expression levels and the manifested phenotype of young Atscp2-1 + +## 3488 Zheng Et Al. + +seedlings when grown on 1/2 MS without exogenous carbon source. RNA samples were extracted using RNeasy Plant Mini Kit +(Qiagen). Five lg of total RNA was converted to double-stranded cDNA using the SuperScript polymerase II (Invitrogen) with a T7-dT primer incorporating a T7 RNA polymerase promoter +(Ambion, Austin, TX, USA). The double-stranded cDNA was amplified to cRNA through in vitro transcription using Megascript T7 kit (Ambion). To obtain aminoallyl-labelled cDNA the amplified cRNA was used as template for cDNA synthesis performed with SuperScript III (Invitrogen) in the presence of dATP, dCTP, dGTP, +and aminoallyl-dUTP (aa-dUTP). After completion of cDNA +synthesis unincorporated aa-dUTP was removed by using Qiagen QIAquick PCR purification kit. The purified aminoallyl-labelled cDNA (aa-cDNA) was lyophilized and subsequently dissolved in 4.5 ll of 0.1 M Na2CO3-buffer, pH 9.0. To label the aa-cDNA, +4.5 ll of the NHS-ester Cy3 or Cy5 dye, prepared in DMSO, was added and the mixture was incubated at room temperature for 1 h in darkness. The two resulting dye-labelled cDNA pools were mixed and then co-hybridized to the same A. thaliana CATMA microarray slide (Allemeersch et al., 2005) for 2 d at 42 C in a water bath. + +After that, the arrays were washed and then scanned using a GenePix 4000B array scanner (Molecular Devices, Sunnyvale, CA, USA). The raw data were stored and analysed using BASE +(https://base.lcb.uu.se) and the Linnaeus Centre for Bioinformatics +(LCB) Data Warehouse (https://dw.lcb.uu.se) at LCB and the WCN +Expression Array Facility at Uppsala University and The Swedish University of Agricultural Sciences, Uppsala, Sweden. Four replicates were made. The empirical Bayes methods B-statistics (Filtering Bstat1 >7.21) (Lo¨nnstedt and Speed, 2002; Smyth et al., 2003; Lo¨nnstedt and Britton, 2005) was applied to rank the genes in order of evidence for differential expression, from the strongest to the weakest evidence. + +## Metabolomics + +Metabolites were extracted from 3-d-old seedlings of A. thaliana wild-type and Atscp2-1. Seedlings were grown on 1/2 MS without sucrose. The young seedlings were selected for metabolome profiling due to the high AtSCP-2 expression levels in wild-type and the manifested phenotype in young Atscp2-1 seedlings when grown on 1/2 MS without sucrose. From each genotype, 10 samples of 20 mg fresh weight were used for the analysis. Extraction of metabolites, GC/MS analysis, and data processing were done as described previously (Gullberg et al., 2004; Jonsson et al., 2005). All multivariate statistical investigations (PCA, PLS-DA) were performed using Simca software 10.5.0.0 (Umetrics, Umea˚, Sweden). The following statistics for the PLS-DA models are discussed throughout this paper: R2X is the cumulative modelled variation in X, R2Y is the cumulative modelled variation in Y and Q2Y is the cumulative predicted variation in Y, according to cross-validation. The range of these parameters is 0–1, where 1 indicates a perfect fit. + +## Docking Of Auxin Precursors Into The Homology Model Of Arabidopsis Thaliana Scp-2 + +The docking program GOLD 3.2 (Jones et al., 1995, 1997) was used to dock the auxin precursors IBA and 2,4-DB into the previously created homology model of AtSCP-2 (Edqvist et al., 2004), which is based on the crystal structure of the SCP-2-like domain of human DBP (Haapalainen et al., 2001). The IBA and 2,4-DB structures were generated using the program SYBYL 8.0 +(Tripos International, St Louis, MO, USA). Ten independent genetic algorithm runs with the default docking parameters were made in GOLD for the ligands. The binding site was restricted within a 15 A˚ radius of the side-chain hydrogen (HZ) of Phe112 in the AtSCP2 model. The docking was stopped if the three best scoring solutions were within 1.5 A˚ rmsd of each other. The cavity in the AtSCP2 model was identified using the program SURFNET +(Laskowski, 1995). The docking results were visualized and examined in the BODIL modelling environment (Lehtonen et al., +2004). + +## Results Expression Of Atscp2 During Development + +To gain insight on the expression pattern of AtSCP2, we analyzed large amount of data accessible in public databases (i.e. www.weigelworld.org, www.arabidopsis.org, and www.genevestigator.ethz.ch) from microarray analysis of gene expression during A. thaliana development. Figure 1 shows the expression of AtSCP2 in 63 samples from different tissues or stages of development. + +The data are from the AtGenExpress expression atlas +(www.weigelworld.org) (Schmid et al., 2005) and shows that the AtSCP2 mRNA is present in all tissues and at all stages of the life of the plant. The highest levels of the AtSCP2 transcript was found in seeds, such as in green cotyledons of maturating seeds (Fig. 1, sample 63), and in floral tissues, such as the stamens of stage 12 flowers (Fig. 1, sample 51) and petals (Fig. 1, sample 50) and stamens of stage 15 flowers (Fig. 1, sample 52). The accumulation of the AtSCP2 transcript in roots, stems, buds, siliques, inflorescences, and leaves of mature A. thaliana plants was analysed with quantitative real-time RT-PCR +(data not shown). The results obtained confirmed that the AtSCP2 mRNA is abundant in most A. thaliana tissues. + +The A. thaliana AtSCP2 promoter was fused to the b-glucuronidase (GUS) reporter gene and the temporal and spatial patterns of expression were assessed during plant growth and development (Fig. 2). Endosperm (Fig. 2B), +embryo (Fig. 2C), and 2-d-old seedlings (Fig. 2D) showed very high GUS activity. Staining was also detected in vascular tissues and hydathodes of cotyledons (Fig. 2E), +in trichomes of the rosette leaves (Fig. 2F), in the receptacles (Fig. 2G, H), in vascular tissues of sepals and petals (Fig. 2G, I, J), in the style and stigma of the carpels (Fig. 2G, K), and in anthers, filaments, and pollen +(Fig. 2G, L). The funiculi of the siliques were also shown to contain GUS activity (Fig. 2M). + +The performance of the AtSCP2 promoter-GUS fusion was also assayed in response to light, darkness, and sucrose in 1–7-d-old seedlings (Fig. 3). The 1-d-old seedlings showed high levels of GUS-activity, indicating high levels of activity of the AtSCP2 promoter early during germination. There was a decrease in expression from the AtSCP2 promoter with time, as 7-d-old seedlings showed lower GUS activity compared with 1-d-old and 3-d-old seedlings. Dark-grown seedlings showed a more intense staining, indicating a higher activity of the AtSCP2 promoter. Interestingly, the GUS activity increased with + +![4_image_0.png](4_image_0.png) + +increasing amounts of sucrose when grown under constant illumination as well as in darkness. The microarray data compiled in Fig. 1 also revealed a slight increase in the accumulation of the AtSCP2 transcript in seedlings in response to sucrose (compare samples 4 and 5, and samples 27 and 28). + +## Correlated Expression Patterns Of Atscp2 To Other Genes + +To establish a functional context of the expression pattern of AtSCP2, it was analysed whether its expression is correlated to that of other genes. To calculate Pearson correlation coefficients (R) from microarray analysis of gene expression, the gene correlator function of Genevestigator (www.genevestigator.ethz.ch) (Zimmermann et al., +2004, 2005) and the ExpressionAngler function of the Botany Array Resource in the University of Toronto +(bbc.botany.utoronto.ca) (Toufighi et al., 2005) were used. Results from such investigations using ExpressionAngler on data from the AtGenExpress experiments are shown in Fig. 4. At the ExpressionAngler, the data are divided into four groups: tissue, hormone, stress, and pathogen for samples collected from various tissues and developmental stages, after hormone treatments, after abiotic stresses or biotic stresses, respectively. The 100 genes from each group giving the highest R to AtSCP2 were listed. The R cut-off values required for genes to appear on the TOP100-lists were R¼0.923 (hormone), R¼0.800 (stress), R¼0.656 (pathogen), and R¼0.686 (tissue). R >0.7 is generally considered a rule-of-thumb threshold for true correlation and used in various analysis (Lee et al., 2004; Ren et al., 2005). Figure 4 shows the R of the 26 genes that were identified in the lists from at least two of the four groups. Firstly, it can be noted that no gene was found in all the lists, and that only one gene (At4g07390 encoding a PQ-loop repeat family protein) appeared in three of the lists. Of the 25 remaining genes there are three genes (MFP2, PED1, and PMDH1) which encode proteins involved in the b-oxidation cycle (Hayashi et al., +1998; Rylott et al., 2006; Pracharoenwattana et al., 2007). The correlation of expression between MFP2 and AtSCP2 is particularly strong, with R¼0.82 for tissue samples and R¼0.99 for the hormone samples. + +## Characterization Of An Atscp2 T-Dna Insertion Mutant + +To elucidate the biological role of AtSCP2 further, the TDNA insertion line Sail_1231_F11 was obtained from NASC (Fig. 5A). The Sail_1231_F11 line is referred to as Atscp2-1. In Atscp2-1 the T-DNA insertion is in the second intron of AtSCP2. A homozygous insertion line was obtained as confirmed by PCR analysis (Fig. 5B). Transcript analysis using RT-PCR showed that no AtSCP2 transcript was detected in Atscp2-1 seedlings (Fig. 5C). + +Adult plants of Atscp2-1 were normal in appearance (data not shown). Since the expression analysis revealed the highest levels of the AtSCP2 transcript in floral tissues and during seed maturation, seed development in Atscp2-1 was investigated. Thirty-three siliques were removed from wild-type and Atscp2-1 plants, and the developing seeds were examined under a microscope and counted. For + +![5_image_0.png](5_image_0.png) + +wild-type plants, the average silique contained 56 green seeds, whereas the average Atscp2-1 silique contained 49 green seeds. According to the performed t test, the seed per silique numbers of Atscp2-1 were significantly different from wild-type at a P-value of 0.000155. When the morphology of the seeds was examined after harvest, it was revealed that seeds from the Atscp2-1 plants had an imperfect appearance (Fig. 6). Seeds from Atscp2-1 plants complemented with wild-type AtSCP-2 from either pJE602 containing a genomic fragment carrying AtSCP2 including the AtSCP2 promoter [Atscp2-1(AtSCP2::AtSCP2)] or pJE601 carrying a cDNA copy of AtSCP2 connected to the CaMV 35S promoter [Atscp2-1(35S::AtSCP2)] showed wild-type morphology (data not shown). + +The germination of Atscp2-1 seeds was tested by scoring the radicle emergence every 12 h after transferring stratified seeds on 1/2 MS media to a growth chamber. The germination kinetics was significantly slower for Atscp2-1 seeds compared to wild-type seeds (Fig. 7). 75% of the Atscp2-1 seeds were germinated after 72 h + +![6_image_0.png](6_image_0.png) + +incubation showing that the mutant seeds also had a lower germination frequency than the wild-type. Neither the lowered frequency nor the slower kinetics of germination could be rescued by the addition of 1% sucrose to the media. Germination kinetics resembling A. thaliana wildtype were obtained when Atscp2-1 was complemented with wild-type AtSCP-2 (Fig. 7). + +In order to investigate whether the lack of SCP-2 had an effect on seedling establishment, seeds from Atscp2-1 and wild-type were placed on agar media with or without sucrose and germinated under light or in darkness (Fig. 8). + +After 3 d on media without sucrose, the Atscp2-1 seedlings were clearly distinguishable from the wild-type. + +The insertion line showed smaller cotyledon rosettes and shorter hypocotyls than wild-type and the root elongation was also retarded (Fig. 8A–F). Seven days after sowing the root length was still retarded compared with the wildtype when grown without a carbohydrate supplement +(Fig. 8E, F). Also in the dark, root elongation was inhibited on media lacking sucrose as the length of the primary root in Atscp2-1 was reduced on average to 67% and 48% of the wild-type length when assayed after 3 d and 7 d, respectively. The length of the mutant hypocotyls were slightly reduced in darkness on media lacking sucrose, as they showed a 20% and 14% reduction in length after 3 d and 7 d, respectively. Growth and development of Atscp2-1 on 1% sucrose closely matched the wild-type control, in light (Fig. 8A–F) as well as in darkness (data not shown). + +## Effect Of 2,4-Db And Iba On Germination Of Atscp2 Mutants + +2,4-DB is b-oxidized to 2,4-dichlorophenoxyacetic acid, which inhibits root elongation. Several A. thaliana mutants with deficiencies in b-oxidation have been identified in screens for plants that elongated roots on normally inhibitory concentrations of 2,4-DB (Hayashi et al., 1998). + +To investigate whether the AtSCP2 protein may have a role in b-oxidation of auxin precursors and analogues, seeds of Atscp2-1 were germinated on media containing 0.1 lM and 4 lM 2,4-DB and root elongation was followed. The growth inhibition of root elongation of Atscp2-1 was indistinguishable from the control wild-type plants on any tested concentration of 2,4-DB indicating that the AtSCP2 is not involved in catabolism of 2,4-DB (data not shown). Several b-oxidation mutants also show resistance to the growth inhibition caused by IBA (Zolman et al., 2001; Adham et al., 2005). However, Atscp2-1 also showed wild-type sensitivity to IBA (data not shown). + +The binding mode of IBA and 2,4-DB into the homology model of AtSCP2 was studied by the automated docking program GOLD, which gave four docking results for IBA and three results for 2,4-DB. The ligands were positioned quite similarly in all the dockings and only the results with the highest fitness are presented in Fig. 9. Both IBA and 2,4-DB were positioned with their carboxyl groups in one of the cavity openings (near Gln92, Gln110, Ile102, and Leu106). The indole of IBA + +![7_image_0.png](7_image_0.png) + +and the dichlorophenyl group of 2,4-DB were positioned towards the hydrophobic cavity (near Ile37, Phe74, Phe76, Phe81, Met100, and Phe112). The SURFNET plot shows that the AtSCP2 model has a very large tunnel-like cavity. Based on the docking results, the IBA and 2,4-DB molecules take up only a part of the cavity volume and, thus, do not extensively interact with the large hydrophobic cavity, indicating that they are not optimal AtSCP2 ligands (Fig. 9). + +## Expression Profiling Of Atscp2-1 + +To reveal the molecular mechanisms underlying the delayed root elongation of Atscp2-1 seedlings, expression profiling was performed using the CATMA microarray slides (Allemeersch et al., 2005). RNA was isolated from 2-d-old seedlings of wild-type and Atscp2-1 germinated in the light on media lacking sucrose. The RNA was used as the template for the synthesis of cDNA, which subsequently was labelled with Cy5 and Cy3 and hybridized to the gene-specific tags of 150–500 bp on the microarray slide. The empirical Bayes methods B-statistics (Lo¨nnstedt and Speed, 2002; Smyth et al., 2003; Lo¨nnstedt and Britton, 2005) was applied to identify and rank the 100 reporters that, among about 25 000 reporters on the slide, showed the most significant changes in expression pattern in Atscp2-1. These 100 reporters corresponded to 94 different genes from the A. thaliana genome of which 54 were up-regulated and 40 were down-regulated (including AtSCP2) in Atscp2-1 (see Supplementary Table S1 at JXB +online). The expression pattern of 10 genes identified as up- or down-regulated in the microarray analysis was tested with quantitative real-time RT-PCR using gene specific primers. The results confirmed that these genes had a modified expression in Atscp2-1 (see Supplementary Table S2 at JXB online). + +Those genes with a significantly altered expression represent a large range of functional categories, such as carbohydrate and amino acid metabolism, transport, and stress response. Interestingly, several genes that previously were shown to have an altered expression in seedlings of A. thaliana mutants icl-2 and mls-2 with mutations in genes encoding the key enzymes in the glyoxylate cycle, isocitrate lyase (ICL), and malate synthase (MLS) (Cornah et al., 2004), also showed an altered expression in Atscp2-1 + +![8_image_0.png](8_image_0.png) + +![8_image_1.png](8_image_1.png) + +(Table 1). In fact, of the 20 genes listed showing the most significant overexpression in icl-2 mutants, six were among the 54 genes that were up-regulated on our Top 100 list. + +Two out of 12 genes that showed a 2-fold repression in mls-2, and 2 out of 10 genes showing a 2-fold overexpression in mls-2 were also identified in our Top 100 list. + +Three genes, ASN1 (At3g47340), At2g05540, and THA1 +(At1g08630), are overexpressed in Atscp2-1, icl-2, and mls2. ASN1 is a glutamine-dependent asparagine synthase +(Lam et al., 1994), At2g055490 is a Gly-rich protein, and THA1 is a Thr aldolase, which catalyse the formation of Gly from Thr (Joshi et al., 2006). + +## Metabolome Analysis Of Atscp2-1 + +We were interested in identifying metabolites that showed either significantly increased or decreased levels in Atscp2-1. Metabolome analyses were performed on 3-dold seedlings of Atscp2-1 and A. thaliana wild-type. The samples were extracted, derivatized, and analysed by GCMS according to Gullberg et al. (2004). The GC-MS data were analysed using hierarchical multivariate curve resolution (H-MCR; (Jonsson et al., 2005)). The data were centred and scaled to unit variance prior to partial least squares discriminant analysis (PLS-DA) classification of the genotypes. The obtained PLS-DA model (three components; R2X¼0.48; R2Y¼0.99; Q2Y¼0.93) shows a clear separation of the genotypes for the first two components (data not shown). The identification of differences was performed by interpretation of the loadings (as described in Trygg and Wold, 2002) from the PLS-DA +model together with the 99% confidence intervals calculated using jack-knifing. In seedlings, 421 variables were detected, 110 of those showed a significant difference between wild-type and Atscp2-1 according to PLS-DA +analysis (one component; R2X¼0.20; R2Y¼0.91; Q2Y¼0.81) and interpretation of first loading vector as described above. The significant metabolites were identified by comparison of retention index and mass spectra with retention index and mass spectra libraries (Schauer et al., 2005). The 20 metabolites that showed the most significant difference in accumulation between Atscp2-1 and wild-type were ranked. Twelve different metabolites + +![9_image_0.png](9_image_0.png) + +![9_image_1.png](9_image_1.png) + +were identified on the ranking list (Table 2). There were higher levels of Gln, pyroglutamic acid (a putative derivatization artefact from glutamate), Asp, b-Ala, fumaric acid, glyceraldehyde, and ribose in wild-type seedlings compared to Atscp2-1 (Table 2). The Atscp2-1 seedlings contained enhanced levels of Ser, Gly, Asn, 3cyanoalanine, and 5-methylthiopentanitrile (tentative identification). Comparison with the previously published metabolome analyses of icl-2 and mls-2 seedlings (Cornah et al., 2004) revealed several similarities as these mutants were also shown to have lowered amounts of Gln, and that mls-2 and Atscp2-1 seedlings both contained increased levels of Ser and Gly. The increased levels of Asn in Atscp2-1 coincide with increased expression of the asparagine synthase ASN1, as shown by the microarray analysis of Atscp2-1 seedlings. Elevated levels of Gly correlated with increased expression of the Thr aldolase THA1 in Atscp2-1. + +## Discussion + +It has been shown that AtSCP2 is ubiquitously expressed in maturing seeds, young seedlings, and floral tissues, although the transcript is present throughout all developmental stages of A. thaliana. The expression of AtSCP2 is correlated to the expression of enzymes of the b-oxidation machinery, such as MFP2, PED1, and PMDH1. The AtSCP2-deficient Atscp2-1 show altered seed morphology, compromised germination, and require exogenous carbohydrates to avoid delayed seedling establishment. Transcriptome and metabolome analysis of AtSCP2-1 revealed similarities to the glyoxylate cycle mutants icl-2 and mls-2, such as increased expression of the genes ASN1 and THA1, decreased levels of Gln, and elevated levels of Gly and Ser. + +According to current knowledge on b-oxidation and the glyoxylate cycle in A. thaliana (reviewed in Baker et al., +2006), fatty acids are delivered into the peroxisomes by the peroxisomal ABC-transporter CTS (also referred to as PED3 or PXA1) (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al., 2002). The delivered fatty acids are activated to acyl-CoA esters through the activity of peroxisomal acyl-CoA synthetases such as the long-chain acyl-CoA synthetases LACS6 and LACS7 (Fulda et al., 2004). The acyl-CoA esters then go through the repeated cleavage of acetate units from the thiol end through the activities of the b-oxidation enzymes: acyl-CoA oxidases ACX1-6, the multifunctional proteins MFP2 or AIM1, and the 3-ketoacyl-CoA thiolases (PED1, KAT1 and + +![10_image_0.png](10_image_0.png) + +PKT2) (Hayashi et al, 1998, 1999; Richmond and Bleecker, 1999; Germain et al., 2001; Adham et al., 2005; Rylott et al., 2006). To allow for conversion of lipids into sugar via gluconeogenesis, the acetate obtained + +## Functional Analysis Of Atscp2 3495 + +may be fed into the glyoxylate cycle for the synthesis of succinate, malate, and oxaloacetate. The reactions in the glyoxylate cycle are catalysed by citrate synthase (CSY), aconitase, ICL, MLS, and malate dehydrogenase. + +Seedling establishment of the Atscp2-1 mutant is not dependent on an exogenous supply of sucrose, but sucrose is required to avoid a delay. Sucrose-dependent seedling establishment has been shown for many A. thaliana mutants deficient in proteins with roles in b-oxidation or the glyoxylate cycle, such as the acx1acx2 mutant deficient in the ACX1 long chain acyl-CoA oxidase and the ACX2 very long chain acyl CoA oxidase (Adham et al., 2005), the aim1 mutant (Richmond and Bleecker, 1999), the mfp2 mutant (Rylott et al., 2006), the pex6 mutant deficient in a putative peroxisomal ATPase (Zolman and Bartel, 2004), the pxa1/ped3/cts mutant (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al., 2002), the lacs6lacs7 mutant +(Fulda et al., 2004), the ped1/kat2 mutant (Hayashi et al., 1998; Germain et al., 2001), the csy2csy3 mutant (Pracharoenwattana et al., 2005), the mls-2 mutant (Cornah et al., 2004), and the sdp2 mutant deficient in peroxisomal monohydroascorbate reductase (Eastmond, 2007). The sucrose dependency of these mutants is considered to reflect a lack of gluconeogenesis as a consequence of reduced fatty acid b-oxidation or a blocked glyoxylate cycle. + +Many of the above sucrose-dependent mutants with defects in b-oxidation are able to grow in toxic levels of 2,4-DB or IBA. The phenotype of the Atscp2-1 mutant resembles that of the mfp2, lacs6lacs7, acx1acx2, and sdp2 mutants, which are sucrose-dependent but still sensitive to 2,4-DB and IBA (Fulda et al., 2004; PinfieldWells et al., 2005; Rylott et al., 2006; Eastmond, 2007). + +The phenotype of the lacs6lacs7 mutant may be explained by the fact that probably another CoA-synthetase is capable of activating 2,4-DB and IBA to the + +| to icl-2 and mls-2. NA, not applicable (see Cornah et al., 2004). Gene Descriptiona | Atscp2-1b | icl-2bc | mls-2bc | | +|---------------------------------------------------------------------------------------|--------------------------------|-----------|-----------|----| +| THA1 | Threonine aldolase | + | + | + | +| (At1g08630) At1g21400 | 2-Oxoisovalerate dehydrogenase | + | + | NA | +| At2g05540 | Gly-rich protein | + | + | + | +| ASN1 | Gln-dependent Asn-synthetase | + | + | + | +| (At3g47340) At5g20230 | Blue-copper binding protein | + | + | NA | +| At5g50600 | Hydroxysteroid dehydrogenase 1 | + | + | NA | +| At3g14210 | Myrosinase-associated protein | - | NA | - | +| At1g52060 | Jasmonate-inducible protein | - | NA | - | + +| d-old seedlings of wild-type and Atscp2-1 Compound Wild-type Relativea mean area 699% confidence interval | Atscp2-1 Relativea mean area 699% confidence interval | | +|-------------------------------------------------------------------------------------------------------------|---------------------------------------------------------|---------------| +| Asp | 14 12263572 | 880862170 | +| b-Ala | 7986374 | 396665 | +| Fumaric acid | 15826412 | 8876163 | +| Gln | 174 860637561 | 89 282610191 | +| Glyceraldehyde | 8156266 | 323671 | +| Pyroglutamic acid | 312 447655162 | 182 199618855 | +| Ribose | 8846265 | 458654 | +| Asn | 647761874 | 11 38061560 | +| 3-Cyanoalanine | 7526100 | 1171697 | +| Gly | 22 28963940 | 50 760611217 | +| 5-Methylthiopentanenitrileb | 060 | 8766136 | +| Ser | 27 87969060 | 58 18965825 | +| a Corrected for internal standards and weight. b Tentative identification. | | | + +corresponding CoA-thioester before b-oxidation. Moreover, IBA and 2,4-DB sensitivity shows that the deficiency of the two long chain acyl-CoA synthetases does not cause a general block of the b-oxidation cycle in the lacs6lacs7 mutant. The 2,4-DB sensitivity of mfp2 mutants is probably due to that the other A. thaliana multifunctional protein AIM1 is active on 2,4-DB in a mfp2 background (Rylott et al., 2006). In the case of the acx1acx2 mutants, the 2,4-DB sensitivity could be due to ACX1 and ACX2 showing specificity for medium to very long chain acyl-CoA species, whereas 2,4-DB is metabolized by ACXs with short-chain specificity as suggested by Pinfield-Wells et al. (2005). The 2,4-DB sensitivity shown for sdp2 was hypothesized to be due to many peroxisomal matrix and membrane proteins remaining functional in the mutant (Eastmond, 2007). The IBA and 2,4-DB sensitivity of the Atscp2-1 mutant show that AtSCP2 is not required for b-oxidation of those compounds, and that the b-oxidation cycle is not blocked in the mutant. Furthermore, the docking analysis of IBA and 2,4-DB shows that they are not the most favourable ligands for AtSCP2, indicating that AtSCP2 would not be involved in the binding or transfer of the auxin precursors. + +The Atscp2-1 mutant shows a compromised germination, with slower germination kinetics as well as a lower germination frequency. Unlike the post-germinative growth phenotype, the germination phenotype of Atscp2-1 is not rescued by the addition of exogenous sugar. The slow germination phenotype of Atscp2-1 resembles the phenotype of the lac6lac7 double mutant which reached 50% +germination in 4.5 d compared to 1.5 d for wild-type (Footitt et al., 2006). Sucrose-independent germination phenotypes have also been shown for acx1acx2, ped1/ kat2, and cts mutants (Pinfield-Wells et al., 2005). Seeds from these mutants showed germination frequencies below 30%, indicating a more severe block in germination compared to Atscp2-1. Seeds from ped1/kat2 and cts mutants contain significant amounts of sucrose (Footitt et al., 2002; Pritchard et al., 2002) suggesting that the germination phenotypes are not due to a limited supply of soluble sugars. Rather, as suggested recently (Pinfield-Wells et al., 2005) the b-oxidation pathway may, during germination, be utilized for the synthesis of a specific signal molecule promoting germination, or to degrade a molecule that inhibits germination. This capacity may then be compromised in Atscp2-1, lac6lac7, acx1acx2, ped1/kat2, and cts mutants leading to an inhibition of germination. + +During development and maturation of A. thaliana seeds, the total fatty acid levels in the seeds increase, until the later stages of seed maturation when the levels drop to close to 30% relative to the peak levels (Baud et al, 2002). Metabolic studies of developing embryos of Brassica napus showed that at least 10% of the fatty acids stored as triacylglycerol was lost during the desiccation phase of seed development. Interestingly, metabolic labelling of the embryos revealed that b-oxidation was not associated with net gluconeogenic activity (Chia et al., 2005). The function of fatty acid breakdown during seed development remains unclear, nevertheless, the activity during seed maturation may explain the altered morphology of the Atscp2-1 seeds if AtSCP2 is involved in peroxisomal lipid utilization. We were somewhat surprised to find that sucrose stimulated the expression of AtSCP2. Rather, it had been expected that sucrose would repress the expression, as increased levels of sucrose could be signalling a limited need for lipid utilization. Further investigations of the function of AtSCP2 may possibly reveal the significance of this observation. + +The Atscp2-1 seedlings had elevated levels of Asn, Ser, and Gly, and decreased levels of Asp, Glu, and Gln. + +Changed levels of Gln, Ser, and Gly were previously reported for icl-2 and mls-2 seedlings (Cornah et al., 2004). Asn accumulates in response to sugar starvation, probably due to an increase in expression of Asn synthase +(Lam et al., 1994; Azevedo et al., 2006), which catalyses the formation of Asn through the transfer of an amide group from Gln to Asp. Our microarray experiments revealed overaccumulation of the ASN1 transcript in Atscp2-1 seedlings (see Supplementary Table S1 at JXB +online) which suggests that the modified levels of Asn, Asp, Gln, and Glu in the Atscp2-1 mutant are consequences of increased accumulation of Asn synthase. Interestingly, ASN1 had elevated expression levels also in icl-2 and mls-2 seedlings (Cornah et al., 2004). Why are deficiencies in AtSCP2, ICL, and MLS leading to increased expression of ASN1? Possibly, when an exogenous carbon source is lacking, these mutant seedlings are in a condition suggestive of sugar starvation. Sugar starvation triggers ASN1 expression and leads to increased levels of Asn. Atscp2-1 also had increased levels of Gly and Ser. This was also seen for the mls-2 seedlings. In the case of mls-2, Cornah et al. (2004) hypothesized that when the glyoxylate cycle is blocked at MLS, glyoxylate feeds into the photorespiratory pathway. The increased activity of the photorespiratory pathway then leads to elevated levels of Gly and Ser. The elevated levels of Gly and Ser in Atscp2-1 possibly indicate that AtSCP2 also functions in the glyoxylate cycle. This hypothesis may also be supported by the decreased levels of fumarate in Atscp2-1. An underperforming glyoxylate cycle in Atscp2-1 may lead to lowered levels of fumarate, as fumarate is produced in the citric acid cycle from succinate, which is a product of the glyoxylate cycle. The elevated Gly levels may also, at least partially, be due to the demonstrated overexpression of THA1 in Atscp2-1. THA1, which is mainly expressed in seeds and young seedlings, encodes a Thr aldolase functioning in Thr catabolism where it is catalysing the conversion of Thr to Gly (Joshi et al., +2006). A tha1 mutant has a 50% decrease in Gly content (Joshi et al., 2006), which indicates that it is not unlikely that the overexpression of THA1 in Atscp2-1 would result in elevated Gly levels. It is interesting that the expression of THA1 was also induced in mls-2 and icl-2 seedlings +(Cornah et al, 2004).Thus, it can not be excluded that the elevated Gly levels in mls-2 seedlings is, to some extent, related to increased THA1 activity. + +We have presented data here showing that the activity of the peroxisomal protein AtSCP2 is important for metabolism in A. thaliana. A function in b-oxidation is supported from the co-expression with MFP2, PED1, and PMDH1. The common pattern seen in animals, fungi, and protists with fusions of SCP-2 domains to catalytic domains involved in b-oxidation could also support a direct involvement in b-oxidation for AtSCP2. However, the 2,4-DB-sensitivity shown for Atscp2-1 may suggest that AtSCP2 is not required for b-oxidation, at least not for all compounds. A function related to the glyoxylate cycle is supported by the similarities between Atscp2-1 and the glyoxylate cycle mutants icl-2 and mls-2, as revealed by transcriptome and metabolome analyses. Another possibility that cannot be excluded is that the function of AtSCP2 is not directly connected to a specific metabolic pathway. Rather, AtSCP2 could be involved in the transport, solubilization, and presentation of hydrophobic reaction intermediates from several metabolic pathways. + +Further detailed investigations, including lipid profiling of Atscp2-1 seedlings, as well as AtSCP2-protein and AtSCP2-ligand interactions may provide us with additional clues to the biological function of AtSCP2. + +## Supplementary Data + +Supplementary data are available at JXB online. + +Table S1. Ranking of genes with significantly altered expression in Atscp2-1 relative to wild-type. + +Table S2. Comparison of results from microarray and quantitative real-time reverse transcriptase PCR (qRTPCR) analysis of gene expression in wild-type and Atscp2-1 seedlings. + +## Acknowledgements + +We thank Maria Andersson, Gunilla Swa¨rd, and Gun Ro¨nnqvist for technical assistance, Kristina Blomqvist for comments on the manuscript, and Jens Sundstro¨m for tubulin primers and advice on real-time PCR. We thank Anette Hagberg for help with designing and analysing microarray experiments. This work was supported by The Swedish Research Council (JE), Carl Trygger Foundation (JE), +China Scholarship Council (BSZ), and the Natural Science Foundation of Zhejiang Province (Y305314) (BSZ). + +## References + +Adamski J, Normand T, Leenders F, Monte D, Begue A, +Stehelin D, Jungblut PW, de Launoit Y. 1995. Molecular cloning of a novel widely expressed human 80 kDa 17 b-hydroxysteroid dehydrogenase IV. Biochemical Journal 311, 437–443. + +Adham AR, Zolman BK, Millius A, Bartel B. 2005. Mutations in Arabidopsis acyl-CoA oxidase genes reveal distinct and overlapping roles in beta-oxidation. The Plant Journal 41, 859–874. + +Allemeersch J, Durinck S, Vanderhaeghen R, et al. 2005. + +Benchmarking the CATMA microarray. A novel tool for Arabidopsis transcriptome analysis. Plant Physiology 137, 588–601. + +Azevedo RA, Lancien M, Lea PJ. 2006. The aspartic acid metabolic pathway, an exciting and essential pathway in plants. + +Amino Acids 30, 143–162. + +Baker A, Graham IA, Holdsworth M, Smith SM, +Theodoulou FL. 2006. Chewing the fat: b-oxidation in signalling and development. Trends in Plant Science 11, 124–132. + +Baud S, Boutin JP, Miquel M, Lepiniec L, Rochat C. 2002. An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiology and Biochemistry 40, 151–160. + +Bloj B, Hughes ME, Wilson DB, Zilversmit DB. 1978. Isolation and amino acid analysis of a nonspecific phospholipid transfer protein from rat liver. FEBS Letters 96, 87–89. + +Chia TY, Pike MJ, Rawsthorne S. 2005. Storage oil breakdown during embryo development of Brassica napus (L.). Journal of Experimental Botany 56, 1285–1296. + +Choinowski T, Hauser H, Piontek K. 2000. Structure of sterol carrier protein 2 at 1.8 A˚ resolution reveals a hydrophobic tunnel suitable for lipid binding. Biochemistry 39, 1897–1902. + +Clough SJ, Bent AF. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735–743. + +Cornah JE, Germain V, Ward JL, Beale MH, Smith SM. 2004. + +Lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis mutants lacking the glyoxylate cycle enzyme malate synthase. Journal of Biological Chemistry 279, 42916–42923. + +Curtis MD, Grossniklaus U. 2003. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiology 133, 462–469. + +Dyer DH, Lovell S, Thoden JB, Holden HM, Rayment I, +Lan Q. 2003. The structural determination of an insect sterol carrier protein-2 with a ligand-bound C16 fatty acid at 1.35A +resolution. Journal of Biological Chemistry 278, 39085–39091. + +Eastmond PJ. 2007. MONODEHYROASCORBATE +REDUCTASE4 is required for seed storage oil hydrolysis and postgerminative growth in Arabidopsis. The Plant Cell 19, 1376–1387. + +Edqvist J, Blomqvist K. 2006. Fusion and fission, the evolution of sterol carrier protein-2. Journal of Molecular Evolution 62, 292–306. + +Edqvist J, Ro¨nnberg E, Rosenquist S, Blomqvist K, Viitanen L, +Salminen TA, Nylund M, Tuuf J, Mattjus P. 2004. Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2. Journal of Biological Chemistry 279, 53544–53553. + +Eklund DM, Edqvist J. 2003. Localization of non-specific lipid transfer proteins correlate with programmed cell death responses during endosperm degradation in Euphorbia lagascae seedlings. + +Plant Physiology 132, 1249–1259. + +Footitt S, Slocombe SP, Larner V, Kurup S, Wu Y, Larson T, +Graham I, Baker A, Holdsworth M. 2002. Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALDP. EMBO Journal 21, 2912–2922. + +Footitt S, Marquez J, Schmuths H, Baker A, Theodoulou FL, +Holdsworth M. 2006. Analysis of the role of COMATOSE and peroxisomal b-oxidation in the determination of germination potential in Arabidopsis. Journal of Experimental Botany 57, 2805–2814. + +Fulda M, Schnurr J, Abbadi A, Heinz E, Browse J. 2004. + +Peroxisomal acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana. The Plant Cell 16, 394–405. + +Germain V, Rylott EL, Larson TR, Sherson SM, Bechtold N, +Carde JP, Bryce JH, Graham IA, Smith SM. 2001. Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid b-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. The Plant Journal 28, 1–12. + +Gullberg J, Jonsson P, Nordstro¨m A, Sjo¨stro¨m M, Moritz T. + +2004. A strategy for identifying differences in large series of metabolomic samples analysed by GC/MS. Analytical Chemistry 76, 1738–1745. + +Haapalainen AM, van Aalten DM, Merilainen G, Jalonen JE, +Pirila P, Wierenga RK, Hiltunen JK, Glumoff T. 2001. Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 A˚ resolution. + +Journal of Molecular Biology 313, 1127–1138. + +Hayashi H, De Bellis L, Ciurli A, Kondo M, Hayashi M, +Nishimura M. 1999. A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes. Journal of Biological Chemistry 274, 12715–12721. + +Hayashi M, Nito K, Takei-Hoshi R, Yagi M, Kondo M, +Suenaga A, Yamaya T, Nishimura M. 2002. Ped3p is a peroxisomal ATP-binding cassette transporter that might supply substrates for fatty acid b-oxidation. Plant and Cell Physiology 43, 1–11. + +Hayashi M, Toriyama K, Kondo M, Nishimura M. 1998. 2,4- +Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid b-oxidation. The Plant Cell 10, 183–195. + +Jefferson RA, Kavanagh TA, Bevan MW. 1987. GUS fusions: bglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6, 3901–3907. + +Jones G, Willett P, Glen RC. 1995. Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation. Journal of Molecular Biology 245, 43–53. + +Jones G, Willett P, Glen RC, Leach AR, Taylor R. 1997. + +Development and validation of a genetic algorithm for flexible docking. Journal of Molecular Biology 267, 727–748. + +Joshi V, Laubengayer KM, Schauer N, Fernie AR, Jander G. + +2006. Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool. + +The Plant Cell 18, 3564–3575. + +Jonsson P, Johansson A, Gullberg J, Trygg J, Jiye A, Grung B, +Marklund S, Sjo¨stro¨m M, Antti H, Moritz T. 2005. High through-put data analysis for detecting and identifying differences between samples in GC/MS-based metabolomic analyses. Analytical Chemistry 77, 5635–5642. + +Kiema TR, Taskinen JP, Pirila PL, Koivuranta KT, +Wierenga RK, Hiltunen JK. 2002. Organization of the multifunctional enzyme type 1: interaction between N- and Cterminal domains is required for the hydratase-1/isomerase activity. Biochemical Journal 367, 433–441. + +Lam HM, Peng SS, Coruzzi GM. 1994. Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana. Plant Physiology 106, 1347–1357. + +Laskowski RA. 1995. SURFNET: a program for visualizing molecular surfaces, cavities, and intermolecular interactions. + +Journal of Molecular Graphics 13, 323–330. + +Lee HK, Hsu AK, Sajdak J, Qin J, Pavlidis P. 2004. Coexpression analysis of human genes across many micro array data sets. Genome Research 14, 1085–1094. + +Leenders F, Dolez V, Begue A, Moller G, Gloeckner JC, de Launoit Y, Adamski J. 1998. Structure of the gene for the human 17b-hydroxysteroid dehydrogenase type IV. Mammalian Genome 9, 1036–1041. + +Lehtonen JV, Still DJ, Rantanen VV, et al. 2004. BODIL: +a molecular modelling environment for structure-function analysis and drug design. Journal of Computer-Aided Molecular Design 18, 401–419. + +Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCT +method. Methods 25, 402–408. + +Lo¨nnstedt I, Britton T. 2005. Hierarchical Bayes models for cDNA microarray gene expression. Biostatistics 6, 279–291. + +Lo¨nnstedt I, Speed TP. 2002. Replicated microarray data. + +Statistica Sinica 12, 31–46. + +Ohba T, Holt JA, Billheimer JT, Strauss 3rd JF. 1995. Human sterol carrier protein x/sterol carrier protein 2 gene has two promoters. Biochemistry 34, 10660–10668. + +Ohba T, Rennert H, Pfeifer SM, He Z, Yamamoto R, Holt JA, +Billheimer JT, Strauss 3rd JF. 1994. The structure of the human sterol carrier protein X/sterol carrier protein 2 gene +(SCP2). Genomics 24, 370–374. + +Pinfield-Wells H, Rylott EL, Gilday AD, Graham S, Job K, +Larson TR, Graham IA. 2005. Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism. The Plant Journal 43, 861–872. + +Pracharoenwattana I, Cornah JE, Smith SM. 2005. Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. The Plant Cell 17, 2037–2048. + +Pracharoenwattana I, Cornah JE, Smith SM. 2007. Arabidopsis peroxisomal malate dehydrogenase functions in b-oxidation but not in the glyoxylate cycle. The Plant Journal 50, 381–390. + +Pritchard SL, Charlton WL, Baker A, Graham IA. 2002. + +Germination and storage reserve mobilization are regulated independently in Arabidopsis. The Plant Journal 31, 639–647. + +Ren XY, Fiers MW, Stiekema WJ, Nap JP. 2005. Local coexpression domains of two to four genes in the genome of Arabidopsis. Plant Physiology 138, 923–934. + +Richmond TA, Bleecker AB. 1999. A defect in b-oxidation causes abnormal inflorescence development in Arabidopsis. The Plant Cell 11, 1911–1924. + +Ritter MC, Dempsey ME. 1971. Specificity and role in cholesterol biosynthesis of a squalene and sterol carrier protein. Journal of Biological Chemistry 246, 1536–1539. + +Rylott EL, Eastmond PJ, Gilday AD, Slocombe SP, Larson TR, +Baker A, Graham IA. 2006. The Arabidopsis thaliana multifunctional protein gene (MFP2) of peroxisomal b-oxidation is essential for seedling establishment. The Plant Journal 45, 930–941. + +Schauer N, Steinhauser D, Strelkov S, et al. 2005. GC-MS +libraries for the rapid identification of metabolites in complex biological samples. FEBS Letters 579, 1332–1337. + +Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, +Vingron M, Scholkopf B, Weigel D, Lohmann JU. 2005. A gene expression map of Arabidopsis thaliana development. + +Nature Genetics 37, 501–506. + +Seedorf U, Ellinghaus P, Nofer JR. 2000. Sterol carrier protein-2. + +Biochimica et Biophysica Acta 1486, 45–54. + +Seedorf U, Raabe M, Ellinghaus P, et al. 1998. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-X +gene function. Genes and Development 12, 1189–1201. + +Smyth GK, Yang YH, Speed T. 2003. Statistical issues in cDNA +microarray data analysis. Methods in Molecular Biology 224, 111–136. + +Toufighi K, Brady SM, Austin R, Ly E, Provart NJ. 2005. The Botany Array Resource: e-Northerns, Expression Angling, and promoter analyses. The Plant Journal 43, 153–163. + +Trygg J, Wold S. 2002. Orthogonal projections to latent structures +(O-PLS). Journal of Chemometrics 16, 119–128. + +Viitanen L, Nylund M, Eklund DM, Alm C, Eriksson AK, +Tuuf J, Salminen TA, Mattjus P, Edqvist J. 2006. Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity. FEBS +Journal 273, 5641–5655. + +Wanders RJ. 2004. Peroxisomes, lipid metabolism, and peroxisomal disorders. Molecular Genetics and Metabolism 83, 16–27. + +Wouters FS, Bastiaens PIH, Wirtz KWA, Jovin TM. 1998. + +FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes. EMBO Journal 17, 7179– +7189. + +Zimmermann P, Hennig L, Gruissem W. 2005. Gene-expression analysis and network discovery using Genevestigator. Trends in Plant Science 10, 407–409. + +Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W. + +2004. GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiology 136, 2621–2632. + +Zolman BK, Bartel B. 2004. An Arabidopsis indole-3-butyric acid-response mutant defective in PEROXIN6, an apparent ATPase implicated in peroxisomal function. Proceedings of the National Academy of Sciences, USA 101, 1786–1791. + +Zolman BK, Silva ID, Bartel B. 2001. The Arabidopsis pxa1 mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta-oxidation. Plant Physiology 127, 1266–1278. \ No newline at end of file diff --git a/medical/md/PMC2687925.md b/medical/md/PMC2687925.md new file mode 100644 index 0000000000000000000000000000000000000000..5e63e7e51492228d36bc4533014eab0254e41bff --- /dev/null +++ b/medical/md/PMC2687925.md @@ -0,0 +1,28 @@ + +## Book Review + +The first essay in the collection is an autobiographical piece by Bliss himself. In it, he describes the course that his career prospects and world-view have taken since his childhood. The son of a physician, Bliss reports that "from the time I could talk I would automatically say that I wanted to grow up to be a doctor." However, he changed his mind at the age of fourteen: + +## Book Review By Christine + +Zadorozny, McGill University, by Haeman, Li and McKeller, eds University of Toronto Press, March 2008,464 pages ISBN-10: 0802090974 ISBN-13: 978-0802090973 Price: $75.00 +… there was a Sunday afternoon when Dad's and my Scrabble game was interrupted by the appearance at the office door of a policeman with a drunk in tow, the drunk having been in a fight and suffering a badly slashed face. Dad had to sew him up, suturing both inside and outside the cheek, and invited me to watch what would be a demonstration of his surgical skill […] with blood and alcohol fumes everywhere, reflecting on my own complete disinterest in and lack of manual skills, I decided that this was not what I wanted to do in life. And that was the end of my ambition to be a doctor. + +Michael Bliss taught and wrote upon a few basic historical themes - medicine, business, and politics - in analyses that were alternatively framed as large-scale events or as individual life stories. In his own academic career, he moved from one special subject to another, showing a certain defiance of narrow academic boundaries. + +Thus reads the introduction to Figuring the Social, a collection of essays published by the University of Toronto Press in honour of Canadian historian Michael Bliss. This anthology, edited by E.A. Heaman, Alison Li and Shelley McKellar, consists of dissertations written by students and colleagues of Bliss, who has produced notable works such as The Discovery of Insulin, Plague: A Story of Smallpox in Montreal and a biography of Sir William Osler, entitled William Osler: A Life in Medicine. In addition to his work in the area of medical history in Canada, Bliss has also written extensively about the business and political history of this country. It is, perhaps, due to the wide scope of Bliss's work that the essays presented in this anthology resist categorization under any particular theme. Rather, they vary quite widely in both style and subject matter, from one author's reflection on his professional relationship with Bliss to another's examination of the relationship between Charles Gordon (also known as novelist "Ralph Conner") and Gordon's son, King. While Bliss' dexterity with a wide variety of subjects is to be lauded, however, the attempt of the editors to mirror this diversity results in a compilation of essays that can seem out of place next to one another - a little more thematic cohesion would have given the collection more strength. That being said, there are some thought-provoking essays in this book that are well worth a read by anyone who is interested in Bliss' work, in his philosophies, or in the history of medicine in Canada. + +Bliss parallels this moment of revelation with a similar decision made by famous physician, teacher and essayist Sir William Osler. Bliss reports that Osler, the son of a Minister, also forsook his father's trade for another - with the belief that "man's redemption of man" could mean more on earth than the type of redemption that a Minister offered to his parishioners. Bliss situates himself firmly within this tradition, saying that "in looking at the history of medicine we are studying, revealing, and, when it is appropriate, celebrating human achievement aimed at the redemption and salvation of humankind. This is the great satisfaction I find in doing medical history." An excellent essay by Allison Li on the transformation of endocrinology brought on by insulin further highlights Bliss' fascination with the revivifying possibilities afforded by medicine, quoting him as saying that the effects of insulin were the "closest approach to the resurrection of the body that our secular society can achieve." +Figuring the Social contains essays on the subject of Michael Bliss' career path and influence as well as dissertations on subjects in politics, business, religion, family, health policy and the practice of medicine in Canada. This latter section contains historical pieces on subjects such as the response of the Canadian 94 + +![0_image_0.png](0_image_0.png) + +government to the thalidomide tragedy, the origins of the Connaught Medical Research Laboratories in Toronto and their influence on Canadian Public Health Policy, and an essay entitled "Comfort, Security, Dignity: Home Care for Canada's Aging Veterans, 1977-2004," which provides an overview on Canada's policies on veteran health care during this time period. Other articles on the subject of health include a history of lobotomy in Ontario, a discussion of medical biography and autobiography as a genre, and an account of the pursuit to develop a mechanical heart undertaken by William Kolff, inventor of the artificial kidney. + +This anthology might have been stronger if it had focused on one particular aspect of Bliss' work. For instance, Bliss' discussion of medical history in his essay is well paired with the historical accounts found in the later chapters on health in Canada. If, as Bliss describes, medicine is an attempt at "man's redemption of man" on earth, which is more successful: the practice of medicine itself or its figuration and chronicling in history? Medicine can prolong the life of individuals - history prolongs our memory of them and can figure them in new ways, beyond what is possible in a lifetime. + +According to Bliss, There is the immensely frustrating fact that health care offers only temporary salvation. It buys time, but the time always runs out - even for those virtually resurrected children who outlived every one of the discoverers of insulin. They're all dead now. We can assault morbidity, we can postpone mortality, but we can't change the absolute mortality rate, which is permanently stuck at 100 per cent. Which means, of course, that the deal Osler offers us, of salvation through physical health and health care - he called it a "ministry of health" - is hugely unsatisfactory compared to what Osler's father peddled, which was life everlasting. + +The strength of this anthology lies in its exploration of medicine's pursuit to preserve life. This is a goal that is both absolutely worth striving for and, as is illustrated by the dramatic outcomes of the discovery of insulin, is one that medicine can definitely achieve. And yet, as Bliss points out, it is eventually bound to fail as human life cannot be prolonged indefinitely. This is where the historian steps in to record and present these achievements to the generation to come, endowing them with a "life everlasting" of their own. + +Christine Zadorozny (M.D.C.M., 2011) received her B.A. (Hons) in English Literature from the University of Ottawa and her M.A. at Queen's University. She is currently a medical student at McGill University. \ No newline at end of file diff --git a/medical/md/PMC2933849.md b/medical/md/PMC2933849.md new file mode 100644 index 0000000000000000000000000000000000000000..ee1bc7651ab8a6fc59488a3ec4c82d294cb95e4f --- /dev/null +++ b/medical/md/PMC2933849.md @@ -0,0 +1,354 @@ +# Systems Microscopy Approaches To Understand Cancer Cell Migration And Metastasis + +Sylvia E. Le De´ve´dec - Kuan Yan - Hans de Bont • +Veerander Ghotra - Hoa Truong - Erik H. Danen - Fons Verbeek - Bob van de Water Received: 23 December 2009 / Revised: 21 April 2010 / Accepted: 14 May 2010 / Published online: 18 June 2010 +- The Author(s) 2010. This article is published with open access at Springerlink.com Abstract Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration. + +S. E. Le De´ve´dec - H. de Bont - V. Ghotra - H. Truong - +E. H. Danen - B. van de Water Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands K. Yan - F. Verbeek Imaging and BioInformatics, Leiden Institute of Advanced Computer Science, Leiden University, Leiden, The Netherlands B. van de Water (&) +Leiden/Amsterdam Center for Drug Research, Gorleaus Laboratories, Leiden University, Einsteinweg 55, P.O. Box 9502, 2300 RA Leiden, The Netherlands e-mail: water_b@lacdr.leidenuniv.nl Keywords Cancer - Cell migration - Matrix adhesions - +Dynamic imaging - Multiparametric analysis + +## Introduction + +Cell migration, both single and collective, is a highly integrated multistep process that is essential in embryonic morphogenesis, tissue homeostasis and immune surveillance. While collective migration requires the movement of cohesive groups of cells [1], the single migrating cell is highly polarised with complex regulatory pathways that are spatiotemporally controlled [2]. Migration contributes to several important pathological processes, including cancer progression and metastasis formation. Metastasis, dissemination of malignant tumours to a distant organ, is the major cause of cancer mortality. Tumour cell motility is the hallmark of invasion and is an essential step in metastasis. + +Cell migration can be seen as a cyclic process. The initial response of a cell to a chemotactic signal is to polarise and extend protrusions in the direction of movement. These protrusions are usually driven by actin polymerisation, and are stabilised by adhering to the extracellular matrix (ECM). These adhesions serve as traction sites for migration as the cell moves forward on top of them, and they are disassembled at the cell rear, allowing it to detach. This process depends on the cell type and environment. Matrix adhesion turnover is clearly visible in slow-moving cells such as fibroblasts or epithelial cells which show large protrusions, and is less visible in fast-moving cells such as neutrophils or cancer cells, which display small protrusions with a rapid turnover [2]. Interestingly, the movement of cell sheets shows some features of single-cell migration; however, the polarisation extends over the entire sheet [1]. In addition to typical matrix adhesions, typically referred to as focal adhesions, cells can form another variety of adhesive structures, namely podosomes and invadopodia, also termed podosome-type adhesions (PTAs) [3]. Those unique actin-rich adhesion structures are typically associated with sites of proteolytic degradation of the extracellular matrix components. This is thought to contribute to cellular invasiveness in physiological and pathological situations. Cell types that form podosomes include monocytic, endothelial and smooth muscle cells, whereas invadopodia are mostly observed in carcinoma cells [4–6]. In this review, we will focus on the study of matrix adhesions in relation to tumour cell migration and invasion; however, most of what we will discuss here is also relevant for the study of PTAs [7, 8]. + +Although matrix adhesion dynamics is very important for understanding cell migration behaviour, little is known about the molecular mechanisms that regulate adhesion dynamics and signalling during tumour cell migration and invasion. Advances in microscopic imaging techniques including spatiotemporally resolved imaging, fluorescent reporter reagents and multiparametric image analysis will contribute to a better insight into these processes. In this review, we will focus only on single cell migration and discuss the current and emerging imaging technologies that can be implemented to study adhesion dynamics and signalling in migrating cells. Tables 1 and 2 summarise all the existing techniques to study, respectively, protein dynamics and modes of tumour migration and can be used as guidelines. In parallel, we discuss the different multiparametric image analysis tools that can be applied after image acquisition to generate more detailed and reliable cellular and molecular measurements. Table 3 gives a non-exhaustive list of challenges and potential solutions for improving the 'throughput' of this systems microscopy approach. Finally, studying the fundamental mechanisms underlying cell migration and invasion using physiologically relevant experimental models together with dynamic imaging and multiparametric image analysis will help in discovering new interesting targets and therapeutics for inhibition of tumour invasion and consequently cancer metastasis formation. + +## Adhesion Proteins + +Matrix adhesion complexes are cytoplasmic structures that have been originally identified by electron microscopy [9] +or by interference reflection microscopy [10]. They are the closest site of contact between the cell and the underlying substratum. Integrins are the principal cell surface adhesion receptors mediating cell–matrix adhesions [11]. Integrins are heterodimeric receptors that directly bind extracellular matrix molecules and couple them to the actin cytoskeleton (for reviews, see [12, 13]). Integrin cytoplasmic domains form multi-molecular complexes with proteins involved in cell adhesion signalling and with adaptors that provide a connection to the cytoskeleton [11]. Approximately 150 proteins, which form the so-called 'integrin adhesome', +have been retrieved to date to be part of the matrix adhesions including kinases, phosphatases and structural proteins (for review, see [14]). Even a larger number of over 600 proteins are defined to be involved in the spatiotemporal regulation of matrix adhesions (van Roosmalen et al., unpublished data). Upon attachment, integrins will cluster and promote local recruitment of structural proteins like vinculin, paxillin, talin, a-actinin and tensin, and signalling molecules including tyrosine kinases such as focal adhesion kinase (FAK), serine/threonine kinases and various adapter proteins. The molecular complexity of cell– +matrix adhesions enables them to fulfill their dual role as modulators of both mechanical cell anchorage and transmembrane signalling [12]. Integrins are not the unique transmembrane receptors that have been described to control and regulate cell adhesion co-signalling; syndecans, discoidin domain receptors (DDR), CD44 and dystroglycan can also bind directly to the ECM, while growth factor receptors such as the epidermal growth factor receptor +(EGFR) can crosstalk with the integrins to regulate the recruitment of cytoplasmic proteins to adhesion sites (for reviews, see [15, 16]). Syndecans as well as DDRs can bind to ECM molecules but most of all they synergise with integrins [17]. DDRs are a novel classs of receptor tyrosine kinases that bind to several collagens and stimulate matrix metalloproteinases (MMP) production. CD44, ICAM-1 and ICAM-2 and L-selectin are other adhesion receptors that are able to interact with ECM ligands as well as with ezrin, radixin, moesin (ERM) family proteins. Dystroglycan is a ubiquitously expressed heterodimeric adhesion receptor that can localise at matrix adhesions [18]. Dystroglycan in concert with integrins is involved in the spatial and temporal remodelling of adhesion numbers, types and distribution. It has emerged as a multifunctional adhesion platform with many interacting partners (for review, see +[19]). One emerging area is the role of dystroglycan in cancer. Dystroglycan function appears to be disrupted in numerous epithelial-derived cancers [20]. A lot is known on the role of integrins in cell migration; in contrast, the mechanistic contribution of distinct adhesion systems such as syndecans, DDRs, CD44 and dystroglycan still remains incompletely understood. Advanced methods are necessary to systematically study the mechanism of regulation of these diverse adhesion systems. + +## Adhesion Types In 2D And 3D + +Matrix adhesions are diverse in molecular composition, size and shape. Their distribution over the whole cell body is also heterogeneous and depends on cell type and environment. Typically adherent cultured cells on two-dimensional substratum in vitro show a large number of matrix adhesions ranging in size from less than 1 to several lm2. + +Detailed analysis based on morphology, molecular composition and method of formation of mainly fibroblasts and epithelial cells allowed those adhesions to be classified in three different classes (Fig. 1a). The most common forms of integrin-mediated cell–matrix adhesions in cultured cells are focal adhesions (FA), fibrillar adhesions (FB) and focal complexes (FC) [12, 13]. Focal adhesions are oval structures, usually a few lm2 in area, and are associated with the termini of actin stress fibres. Fibrillar adhesions, which are derived from FAs, are elongated contact sites, associated with fibronectin fibrils. Focal complexes are small, dot-like adhesions that are mainly found at the cell edge and apparently nucleate FA formation (reviewed by [12]). + +Based on intensity-based segmentation, matrix adhesions Fig. 1 Matrix adhesions + +![2_image_0.png](2_image_0.png) diversity and composition. + +a Schematic view of the three classes of matrix adhesions found in adherent cells in vitro. b Image analysis of matrix adhesions. Confocal picture of epithelial cell stained for Hoechst (blue), P-Tyr (green) +and F-actin (red) (a); scale bar 10 lm. Confocal picture of focal adhesions only (b). Matrix adhesions segmentation (c) and clustering according to size (d). Distribution of the matrix adhesions according to their size +(e) and clustering according to matrix adhesions intensity and length (f). c Matrix adhesions differ in size and shape according to their environment: in 2D rigid versus soft and in 3D; scale bar 10 lm can be analysed using fluorescence microscopy images obtained after immunofluorescence staining of cells for focal adhesion marker such as vinculin or paxillin. Indeed, the morphological and intensity parameters of adhesions can be quantified by analysing images following waterline image segmentation ([21] and Yan et al., unpublished data) +(Fig. 1b). This segmentation defines adhesions as +''objects'' (Fig. 1b, part c), based on their fluorescence intensity and size range (Fig. 1b, part d). The image analysis provides various features such as size (Fig. 1b, part e), +elongation, mean fluorescence-signal intensity and cell localisation with respect to the nucleus which can be used for further clustering (Fig. 1b, part f). Furthermore, matrix adhesions are heterogeneous in their molecular composition which can be studied using fluorescence ratio imaging. For example, FC do not contain zyxin whereas FB do not contain b3 integrin or phosphorylated FAK [22] but is highly enriched with tensin [13]. In general, quantitative multicolour fluorescence imaging can be used to unravel the molecular complexity of the various types of adhesions [23]. Matrix adhesion complexity also depends on the environmental stimuli. FAs are mechanosensors that sense the matrix physical properties whether it is rigid or soft and whether it is two- or three-dimensional [24]. Distinctive 3D +matrix adhesions were described for the first time when cells were cultured in cell-derived 3D matrices: they appear like fibrillar adhesions also enriched in tensin but also rich in tyrosine phosphorylated proteins [25]. Epithelial cells stably expressing an alternative 'reporter' for protein tyrosine phophorylation GFP-dSH2 [26] show large peripheral and ventral focal adhesions on a rigid substrate, whereas on a soft substrate, the adhesions become smaller, dot-like and only localised in the periphery of the cell (Fig. 1c). In addition, the GFP-dSH2 construct localised in a fibrilla pattern alongside the elongated cell protrusions, in a 3D collagen matrix (Fig. 1c, part c). In conclusion, matrix adhesion complexity and diversity can be studied with static immunofluorescent images, but those sites are obviously very dynamic during cell migration in 2D and 3D +environments. + +## Adhesion Turnover During Cell Migration + +Cell migration is an integrated process that requires the coordinated regulation of various structural and signalling molecules, including distinct kinases and phosphatases +[27, 28]. Cell migration requires the establishment of cell polarity to create a leading edge and a trailing edge. The leading edge undergoes membrane protrusive activities driven by actin polymerisation that establish new matrix contacts, whereas at the trailing edge, cell adhesions are disassembled to promote retraction of the cell rear and forward cell movement. The rate of cell migration can be limited by the rate of rear retraction, and thus the dynamic formation and disassembly of cell–matrix adhesions are critical to cell migration [2, 28]. + +## Formation Of Adhesions + +The mechanism by which adhesions assemble in migrating cells is still under investigation. Some cells, particularly rapidly migrating ones such as leukocytes, have few visible integrin clusters, and thus very small submicroscopic adhesions are probably important for their migration. In other cells, small adhesions known as focal complexes can be observed at the leading edge. Formation of these adhesions depends on Rac- and Cdc42-GTPases, and these adhesions stabilise the lamellipodium by mediating attachment to the ECM, thereby contributing to efficient migration. However, cells with large integrin clusters +(''focal adhesions'') are tightly adherent and are typically either non-migratory or move very slowly. The assembly of focal adhesions involves Rho-GTPase as well as myosininduced contractility. During their formation, some protein components enter adhesions with similar kinetics, which suggests that they exist in preformed cytoplasmic complexes. However, other components enter adhesions with very distinct kinetics, which is consistent with a model in which a regulatory event initiates the serial addition of different proteins [27]. Adhesion disassembly at the front and the rear Adhesion disassembly is observed both at the leading edge, where it accompanies the formation of new protrusions, and at the cell rear, where it promotes tail retraction. At the front of migrating cells, adhesions at the base of a protrusion disassemble as new adhesions form at the leading edge (for review, see [27]). However, some adhesions persist and mature into larger, more stable structures. Little is known about adhesion disassembly versus maturation; however, targeting of microtubules has been implicated as one factor that promotes adhesion disassembly (for review, see [29]). Both protein kinases and phosphatases also appear to be central to the regulation of adhesion turnover and stability [30]. For example, cells lacking the tyrosine kinases FAK or Src have more and larger adhesions and migrate poorly [31]. Adhesion turnover in migrating cells is also regulated by a complex of Rac-associated proteins +[32, 33]. Also, the calpain family of intracellular proteases is implicated in regulating cell migration [28]. Calpain activity is tightly controlled in vivo as this intracellular protease irreversibly cleaves numerous signalling and structural proteins with widespread impacts on cell functioning and viability. The ubiquitous calpains can cleave a large number of adhesion complex components including talin, paxillin, vinculin, ezrin, alpha-actinin, FAK, E-cadherin, and the cytosolic tails of some of the beta-integrins +[28, 34–36]. Calpain activity which relies on the removal of a protein or elimination of an enzymatic activity is a key regulator of matrix adhesion dynamics. For instance, the study of Franco and co-workers demonstrates that calpainmediated proteolysis of talin is a rate-limiting step in adhesion disassembly [37]. In conlusion, functional studies that will sytematically determine the role of individual proteins involved in cell–matrix adhesions using dynamic imaging of the protein localisation, kinetics and interactions will shed a more complete light on the mechanisms of cell migration. RNAi (live) cell imaging-based screening approaches are key to such an analysis. + +## Cell Migration/Matrix Adhesion And Cancer + +Metastasis is the most frequent cause of death for patients with cancer. Tumour cell motility is the hallmark of invasion and is an initial step in metastasis. In order to metastasise, cancer cells must first detach from the primary tumour, migrate, invade through tissues, and attach to a second site. Invasive carcinoma cells acquire a migratory phenotype associated with increased expression of several genes involved in cell motility such as matrix adhesionassociated genes. Many metastatic human carcinomas are characterised by the overexpression or constitutive activation of ErbB tyrosine kinase receptor (EGFR) family members involving activating mutations of the receptor kinases or an autocrine loop with EGF family ligands. High expression of EGFR and ErbB2, as well as another tyrosine kinase receptor c-Met, are associated with poor prognosis of breast cancer patients. In addition to stimulating cell differentiation and proliferation, EGF promotes tumour cell motility, invasion and metastasis [38–40]. Recent data provide evidence for a requirement for certain focal adhesion protein expression (e.g. integrin, FAK/Pyk2, paxillin, ILK, Ezrin) in metastatic dissemination. Integrins are known to regulate a diverse array of cellular functions crucial to the initiation, progression and metastasis of solid tumours. The importance of integrins in several cell types that affect tumour progression has made them an appealing target for cancer therapy [41]. Increased activity or overexpression of Src is a frequent occurence in many types of human cancer. Indeed, a prominent role of Src is played in invasion, epithelial-mesenchymal transition (EMT) and development of metastasis. Inhibitors of Src are promising drugs for cancer therapy (for reviews, see [42, 43]). Focal adhesion kinase (FAK), another well-studied non receptor tyrosine kinase, plays a key role in cancer progression. FAK expression and activity are enhanced in metastatic tumours of diverse origin [44–46]. FAK is considered as a promising gene candidate, and inhibitors of FAK are currently in clinical trial (for reviews, see [47, 48]). Not only activity of kinases but also activity of proteases such as calpain is enhanced upon transformation induced by the v-Src, v-Jun, v-Myc, k-Ras, and v-Fos oncoproteins. + +Elevated calpain activity commonly promotes focal adhesion remodelling, disruption of actin cytoskeleton, morphological transformation, and cell migration, although proteolysis of target substrates (such as focal adhesion kinase, talin, and spectrin) is differently specified by individual oncoproteins [34, 35]. Therefore, studying the motility mechanisms used by cancer cells would clarify some of the key events influencing metastasis in cancer. In addition, identification of the molecular pathways that play a role in cancer cell motility will provide new diagnostic approaches and targets for the treatment of metastatic cancer. + +## Studying Migration And Adhesion Dynamics In Living Cells + +The above indicates a general requirement for advanced technologies to systematically study both cell adhesion dynamics and tumour cell migration. Recent advances in fluorescence probes and microscopy technologies have provided powerful approaches that present advantages over the traditional biochemical approaches. In particular, the newly developed recombinant fluorescent proteins (FPs) +and genetically encoded biosensors are useful tools for imaging protein distribution, dynamics and interactions in live cells with high temporal and spatial resolutions enabling the elucidation of molecular mechanisms behind adhesion turnover responsible for the cell migration [49–54]. + +## Phase Contrast/Normaski Imaging + +The starting point for many investigations of cell movment is to characterise the behaviours of cells, by recording shape changes, direction and persistence of movement and the dynamics of subcellular membrane structures such as lamellipodia, filopodia, protrusions and membrane ruffling. + +This can be done with wide-field microscopy in either phase-contrast or Normarski Interference Contrast modes which is fast, not photo-toxic to cells and can be performed over a long period of time. Many software packages are now available that automatically track moving cells visualised in this way: e.g. CellTrack, ImageJ (plug-in MTrack2), Imaris, Image Pro and in-house-developed cell track systems [55, 56]. The tracking rates of those different softwares are very good, although application of fluorescence microscopy instead of phase contrast can improve the tracking efficiency [56]. Furthermore, due to high signal to noise ratio in phase-contrast or Normaski imaging, it remains difficult to analyse other cell features. + +Fluorescent imaging is an answer to that problem. + +## Fluorescent Live Cell Imaging Cell Migration: Wide-Field + +Wide-field fluorescent microscopes equipped with the appropriate excitation and emission filters and sensitive CCD camera are fast and sensitive enough to detect GFP stably expressing migrating cells. The dynamic imaging of 2D cell migration includes various assays such as directed cell migration (chemotaxis and woundhealing) and random cell migration [57]. The dynamic imaging of migrating cells which implies tracking of fast objects needs high temporal resolution. Indeed, to track fast-moving objects, the interval between two sequential frames should be short enough to ensure an overlap of the moving object between frame t and frame t ? 1. Using cell-lines stably expressing GFP together with advanced image analysis tools allow the simultaneous detection of multiple cellular characteristics that define phenotypic response such as EGF response in rat mammary carcinoma cell-line (Fig. 2b). Application of advanced bioinformatics and statistical methods to multiparametric image data generates a non-biased phenotypic fingerprint that describes the effect of genetic or pharmacological manipulation (Fig. 2c). The extension of multiparametric image analysis to live cell studies can reveal further mechanistic insight (Yan K et al., unpublished data). Epi-fluorescent microscopy can also be used to visualise adhesion dynamics. The quantification of adhesion dynamics was first described by Webb and colleagues [58] using epi-fluorescence microscopy. Adhesion assembly and disassembly rate constants can be calculated by measuring the incorporation or loss of fluorescence +(e.g. GFP-paxillin). Assembly will provide an increase in fluorescence intensity whereas disassembly will lead to a loss in intensity. Intensity values at various time points are plotted on a semi-logarithmic scale representing fluorescent intensity ratios over time. These ratios are calculated using the formula ln(I/I0) for assembly and ln(I0/I) for disassembly (where I0 is the initial fluorescent intensity and I is the fluorescent intensity at the indicated time). Rate constants can then be calculated from the slope of the resulting line of best fit. The study conducted by Webb et al. [58] revealed for the first time that fibroblasts with a knockout of FAK, Src and p130Cas decreased the disassembly rate constant of 1.2 9 10-1 min-1 in wildtype cells to between 6 9 10-3 and 9 9 10-3 min-1 in knockout cells. Confocal laser scanning microscopy (CLSM) +While epi-fluorescence microscopy provides the high degree of time resolution which is required to visualise fast processes, confocal laser scanning microscopy (CLSM) offers several advantages over conventional + +![5_image_0.png](5_image_0.png) + +![5_image_1.png](5_image_1.png) + +optical microscopy, including controlled depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. It provides high spatial resolution, but still many physiological processes and events take place faster than they can be captured by most CLSMs, which have image acquisition rates typically in the order of one frame per second. CLSMs using acousto-optical devices and a slit for scanning are faster than the galvanometer-driven pointscanning systems, and are more practical for physiological studies. These quicker designs combine good spatial resolution with good temporal resolution, which may be 30 frames per second at full screen resolution, or near video rate. The slower point-scanning microscope systems can achieve the best temporal resolution only by scanning a much reduced area on the specimen. If full spatial resolution is required, the frames must be collected less frequently, losing some temporal resolution. The confocal systems using disk-scanning or oscillating mirror-scanning methods are also capable of imaging rapid physiological or other transient events. Nowadays, new developments in confocal microscopy provide both high-speed and highresolution imaging to capture intracellular biological processes. A resonant confocal scan head dramatically improves time, spatial and spectral resolutions and allows high-speed imaging up to 230 frames per second (512 9 64 pixels) which make it an ideal technique for bleaching and protein kinetic experiments (photo-activation and photo-bleaching, see below). A major drawback of confocal microscopy is photo-bleaching and photo-damage from the illuminating laser beam that can be cumulative over multiple scans. So the exposure to the beam should be kept to the minimum necessary to acquire the image. Another recent development is controlled light exposure microscopy (CLEM) which should be ideal for live imaging as it helps to reduce photo-bleaching and phototoxicity; the two main limitations in live-cell confocal microscopy [59]. In CLEM illumination excitation light is reduced using two strategies. The first is based on the principle that if there is no signal, then no illumination is required (for example, when imaging the background). The second detects whether there is sufficient signal to acquire an image. If so, illumination is stopped. Total internal reflection fluorescence (TIRF) microscopy One key advance in the field of dynamic imaging of matrix adhesions is the recent commercial development of total internal reflection fluorescence (TIRF) microscopy. TIRF was first described almost 30 years ago (for review, see [60]): cell-substrate contacts of human skin fibroblasts, labelled with fluorescent lipids, were investigated by TIRF +microscopy, which exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices. In practice, the most commonly utilised interface is the contact area between a specimen and a glass coverslip. In a typical experimental setup, fluorophores located in the vicinity of the glass–liquid or plastic–liquid surface can be excited by the evanescent field, provided they have potential electronic transitions at energies within or very near the wavelength bandwidth of the illuminating beam. Because of the exponential fall-off of evanescent field intensity, the excitation of fluorophores is restricted to a region that is typically less than 100 nm in thickness. By comparison, this optical section thickness is approximately one-tenth that produced by confocal fluorescence microscopy techniques. Because excitation of fluorophores in the bulk of the specimen is avoided, confining the secondary fluorescence emission to a very thin region, a much higher signal-to-noise ratio is achieved compared to conventional wide-field epifluorescence illumination. This enhanced signal level makes it possible to detect single-molecule fluorescence by the TIRF +microscopy method. In Fig. 3a, we show the improvement of TIRF imaging above wide-field and confocal microscopy by eliminating out of focus background fluorescence. + +Another example of the application of high speed and resolution TIRF imaging of focal adhesion turnover is shown in Fig. 3b. The matrix adhesion turnover can be consequently quantified with multiparametric image analysis +(Fig. 3c) (Yan K et al., unpublished data). + +## Studying Protein Dynamics And Interactions In Adhesion + +While imaging matrix adhesion proteins using fluorescent microscopy provides information about spatial localisation, it does not allow further measurements of protein modification that regulate the adhesion signalling. The goal of imaging matrix adhesions in migrating cells is not only to visualise their localisation but also to determine their function, the interaction of the different adhesome components, and the mechanisms that regulate adhesion formation and disassembly. To be able to quantify and model protein dynamics in adhesions, many quantitative measurements are needed: interaction between adhesion components, kon and koff of individual components and complexes, diffusion values, protein concentration in both the cytosol and the focal adhesions. The challenge is to develop methods for measuring these parameters during adhesion complex formation and disassembly in migrating cells and preferentially in a high-throughput manner +(Table 1). + +![7_image_0.png](7_image_0.png) + +Many fluorescent proteins (FPs) have been designed by mutagenesis to change their fluorescence intensity (photoactivation) or colour (photo-conversion) upon illumination by lights with specific wavelength, intensity and duration (for review, see [52]). Those FPs can be grouped into three classes: (1) irreversible changes in colour upon illumination which includes photo-activatible (PA)-GFP [61] that can be photo-activated with UV illumination and emits green fluorescence; (2) irreversible change in colour from green to red upon UV illumination which includes Kaede +[62] and Dendra [63]; and (3) reversible change in intensity/colour upon UV illumination such as Dronpa [64] +which is a very valuable marker for photo-conversion studies and fast cellular processes. Until now, mainly PA-GFP has been successfully used to investigate actin dynamics [65], protein adhesion movement [66] and trafficking of integrin receptors [67]. Those photo-switchable fluorophores are valuable probes than can be used in live cells to monitor with high resolution diffusion, trafficking and stability of protein targets. + +Fluorescence recovery after photo-bleaching is a commonly used technique to measure protein kinetics. In a + +| Table 1 Imaging techniques to study protein dynamics and interactions in adhesion Technology Biology What is next | References | | | +|---------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------|----------------------| +| Photoactivation | Measure high resolution diffusion, trafficking and stability of protein Actin dynamics, protein movement, integrin receptor trafficking | For in vivo imaging, to track in the long term photoconverted cells and study protein dynamics | [65–67] | +| FRAP or FLIP | Measure t1/2, kon, koff, diffusion coefficient but individual measurements Integrin, FAK, paxillin, zyxin, vinculin, and actin | Comined FRAP and/or FLIP with | [70–81] | +| TIRF and/or FRET | | | | +| FRAP–FLIP | Measure t1/2, kon, koff, diffusion coefficient and protein mobility parameters. Include all bleached and unbleached matrix adhesions FAK, paxillin, vinculin, zyxin and actin | Comined FRAP-FLIP with TIRF | Le De´ve´dec et al., | +| and/or FRET | unpublished data | | | +| FCS, ICS, RICS | Determine rates of diffusion, degree of aggregation, number of fluorescent entities and flow velocities (mainly used in solution) a5-integrin, a-actinin, FAK, paxillin and actin | In living cells to study protein distribution, dynamics and interactions at high time and spatial resolution | [87–91] | +| FSM | Movement of structure, assembly dynamics, and subunit turnover Actin, integrin, FAK, talin and a-actinin | [82–86] | | +| FRET | Protein–protein interaction and protein activity Src, FAK, Rho GTPases, and matrix adhesion proteins | Combined with FRAP and/or TIRF | [93, 97–102, 106] | +| and in vivo | | | | + +![8_image_0.png](8_image_0.png) + +typical FRAP experiment, a small defined region within a larger volume (for instance, cell nucleus or a focal adhesion) is briefly illuminated at high laser intensity (Fig. 4a) +[68, 69]. Immediately after the bleach-pulse, the majority of the GFP-tagged proteins within the region have irreversibly lost their fluorescent properties: they are photobleached. Proteins from outside will then diffuse into the bleached region resulting in the increase of the fluorescence signal in the region until it reaches a defined signal intensity depending on the (transiently) immobile protein fraction. In summary, FRAP experiments provide information on protein mobility parameters including diffusion coefficient, immobile fraction and residence time. + +Several variants of FRAP have been developed including spot-FRAP, strip-FRAP, FLIP (Fluorescence loss in photobleaching), combined FLIP-FRAP. Spot-FRAP is based on photo-bleaching of a small spot within a focal adhesion +(Fig. 4B), whereas in a strip-FRAP, a narrow strip spanning the cytoplasm in between focal adhesions is bleached. + +FRAP is widely and successfully used in adhesion biology. + +Several FRAP studies have addressed the dynamic properties of numerous adhesion properties [70–81]. + +In a FLIP experiment, the loss of fluorescence in a region or structure far from the bleached region is monitored. FRAP and FLIP can also be combined (FLIP– +FRAP): two regions are monitored simultaneously after bleaching only one of them. We successfully developed a FLIP–FRAP bleaching procedure so that in one experiment we can analyse all the focal adhesions distributed over the whole cell body (Le De´ve´dec et al., unpublished data). + +Together with Monte Carlo simulation, we observed that FAK and paxillin had an equal diffusion rate but a differential residence time that is related to adhesion size and strength. With this technique, we are able to extract mobility parameters of adhesion proteins as well as a mapping of the protein kinetics according to focal adhesion size, type and localisation in the cell (Fig. 4b). + +## Fluorescence Speckle Microscopy (Fsm) + +Another fluorescent technique that detects protein dynamics, turnover and interaction is a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera [82]. In FSM, the fraction of fluorescently labelled molecules in the cell, relative to the level of endogenous unlabelled molecules, has to be very low (typically 0.5% or less). Labellled and unlabelled molecular subunits stochastically co-assemble into structures, giving a random and sparse distribution of fluorescent subunits with a +'speckled' appearance in high-resolution fluorescence images. The low level of fluorescent subunits reduces background fluorescence. Translation of the fluorescent speckle distribution indicates movement of structures whereas changes in speckle intensity and pattern reveal assembly dynamics and subunit turnover. Keys to successful FSM are the ability to image diffraction-limited regions (*0.25 mm) containing few (2–10) fluorophores, and the capacity to inhibit photo-bleaching which is only possible with sensitive imaging system that includes a low noise/high quantum efficiency camera. Extensive characterisation of actin dynamics using FSM has revealed two spatially, kinetically and kinematically distinct actin networks, with the local expansion of the lamella network being a source of persistent cell protrusion [83, 84]. There is also evidence that the actin network is dynamically coupled to adhesions [85]. A recent study using FSM measured the coupling of focal adhesion proteins to actin filament. Their FSM analysis of the dynamic interactions between matrix adhesion components and F-actin in living cells revealed that there is a hierarchy of motion from fast to slow, from actin-binding proteins to adhesion proteins within matrix adhesions and to integrins [86]. Those FSM-mediated molecular measurements provided considerable knowledge on the mechanism behind matrix adhesion dynamics. Fluorescence correlation spectroscopy (FCS) and variants Fluorescence correlation spectroscopy (FCS) analyses concentration fluctuations as a function of time to determine kinetic parameters, molecular associations and concentrations. This technique requires laser excitation of small focal volume and measure fluctuations in fluorescence intensity over many time intervals. Single or cross-correlation analysis (in the case of two different fluorophores) is applied in local areas across a time course to determine rates of diffusion, degree of aggregation, number of fluorescent entities and flow velocities. When two different fluorescent proteins are used, the crosscorrelation function provides estimates of their fractional association and rates of co-transport. FCS is used most widely to study molecules in solution; only a few groups have applied FCS to analyse adhesion protein movements in intact living cells. A variant of FCS, image correlation spectroscopy (ICS), was implemented by Wiseman and co-workers [87] and allowed them to investigate the distribution, dynamics and interactions of a5-integrin, and a-actinin in the context of the formation and disassembly of adhesions during cell migration. Integrins are clustered throughout the cell and in nascent adhesions get 1.4 times more concentrated and 4.5 times more clustered and less mobile than in surrounding regions. Although FCS has a high temporal resolution (microseconds) but low spatial resolution, ICS has a low temporal resolution (seconds) but a high spatial resolution. A new analysis method, termed raster imaging correlation spectroscopy (RICS), can be applied on any confocal microscope [88] and bridges the timescales of FCS and ICS, as well as providing spatially resolved dynamic information such as the diffusion and binding of paxillin-GFP stably expressed in CHOK1 cells +[89] and of FAK-GFP in MEFs [90]. Another variant of FCS, a general velocity-mapping technique termed spatio-temporal image correlation spectroscopy (STICS) has been described and provided new insights into the protein mobilities within the focal adhesions: while integrins were mostly immobile, paxillin and FAK immobile fractions were equal (74%), and actin was more rapidly diffusing (24%) [91]. Finally, a recent study that combined different fluorescence fluctuation approaches demonstrated that paxillin-GFP shows a heterogeneous dynamic within the cell [92]. In the cytoplasm, paxillin is uniformly distributed and diffuses freely as a monomer. Near adhesions, paxillin binds to protein partners and so its dynamics is reduced. These dynamic were different from assembling to disassembling adhesion regions, even within a single adhesion [92]. The implementation of complementary fluctuation methods will provide new data on the dynamics of protein adhesions during cell migration. + +## Fluorescence Resonance Energy Transfer (Fret) + +A powerful imaging method to study protein–protein interactions in living cells is fluorescence resonance energy transfer (FRET) [93, 94]. FRET is the non-radiative transfer of energy from a donor fluorophore in an excited state to a nearby acceptor fluorophore to allow energy transfer if within only 10 nm. Because this distance is in the range of protein sizes, FRET can also be used to study conformational changes of proteins tagged with a FRET +donor and FRET acceptor. The most frequently used FRET +methods are sensitised emission, ratio imaging, and acceptor photo-bleaching FRET [95] but the latter is not appropriate for studying rapid changes of protein interactions over time. The sensitised emission approach detects the emission of the acceptor fluorophore (often cyan fluorescent protein, CFP) while the donor fluorophore (often yellow fluorescent protein, YFP) is excited. Although still widely used, sensitised emission requires careful data processing [96] and due to signal-to-noise ratio can be poorly sensitive. Cross-talk and bleed through from one fluorophore to another makes the analysis highly dependent on control measurements of cells in which only one of the two fluorophores is present. An alternative approach to determine FRET is acceptor/donor ratio imaging (e.g. YFP/ +CFP) where both donor and acceptor emission are detected simultaneously when excited at the excitation wavelength of the donor. However, this method can be only applied when donor and acceptor are equally expressed in a cell system which is always the case when using FRET biosensors. In the study of adhesions, a few FRET biosensors have been designed to monitor in live cells the activity of a number of kinases, e.g. Src and FAK [97, 98] and GTPases, e.g. Rho, Rac and Cdc42 [99–102]. A fourth method to detect FRET is based on the reduced lifetime of excited donor molecules when they are in the proximity of acceptors [103, 104]. This technique is considerably more sensitive and accurate than intensity-based methods, but is slower and requires specific detector and often a pulsed light source such as a two-photon laser. Consequently, this has limited the application of FLIM in live cell studies. However, improvements in microscope design detector technology have reduced the time for data acquisition. Indeed, the group of P. French has developed a time domain optically sectioned FLIM microscope developed for high-speed live cell imaging. This single photon excited system utilises a spinning Nipkow disc microscope and can acquire fluorescence lifetime images of live cells at up to 10 frames per second, permitting high-speed FLIM of cell dynamics and protein interactions with potential for high throughput cell imaging and screening applications [105]. + +When correctly applied, FRET is a useful tool for investigating the molecular mechanisms that regulate integrinmediated signalling in migrating cells [93, 97, 102, 106]. + +## Optical Imaging Towards Understanding Tumour Cell Migration And Intravasation + +Of course, studying and understanding adhesion dynamics in cells migrating onto a rigid 2D substrate still does not reflect the in vivo situation. The critical comparison of 2D and 3D in vitro migration methods brought to light that there are differences in the observations made with these two systems. + +A combination of methods and environments is needed to investigate cell migration with regard to metastasis development. The biological relevance of focal adhesions was initially questioned, since equivalent structures to these prominent 2D adhesion structures were not easily observed in most tissues. However, focal adhesions have been found at point of high fluid shear stress in blood vessels [107] and in proximal tubular cell–ECM contact sites in kidney tissue +[108]. Imaging migration and adhesions in 3D culture systems in vitro and in vivo are still in development. Despite the recent advances in dynamic imaging, a number of technical challenges remain to be overcome to allow functional and biochemical study of the tumour invasion mechanism in 3D ECM substrates and in vivo (Table 2). + +Imaging adhesions and tumour invasion in 3D culture systems Although studying the role of matrix adhesion in migrating cells in a 2D environment has provided a wealth of data, it is not surprising that there is an ongoing effort on setting up 3D invasion models adapted for dynamic live cell imaging that allow a more robust understanding of tumour cell invasion (for recent review, see [109]). A number of + +| Table 2 Modelling distinct modes of tumour migration Environment Models Microscopy | Obtained information | References | | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------|------------------------------------------------------------------------------------------------|-----------------------------| +| In vitro/2D | Matrix coating | Wide-field, confocal | Insights into the organisation of molecular machineries underlying cell adhesion and migration | [24, 53, 120, 129, 159–163] | +| Patterned | | | | | +| In vitro/3D | Matrigel | Wide-field, confocal, confocal reflection microscopy, SHG | [25, 109, 110, 112–114, 164] | | +| On/in collagen gel | 116–118, 121, 132, 159, | | | | +| Distinguish aspects of cell movement/ invasion (collective/individual; mesenchymal/amoeboid). Visualise interactions cell. ECM (in particular, collagen fibers I). | | | | | +| In vivo | Zebrafish | Confocal, confocal reflection, Multiphoton (SHG and FLIM) | [1, 49, 120, 123, 125–135, | | +| Mouse | 165–171] | | | | +| Rat | Aspects of cell movement in the primary environment. Visualise interaction between tumour cells and tumour environment (ECM, host cells and blood vessels). Visualise intravasation event when blood vessels are counterstained. | | | | + +experimental methods incorporating different types of ECM substrates (Type I collagen, fibronectin, Matrigel, matrix polymers and tumour associated 3D matrices) have been developed to study 3D tumour invasion in vitro [110–115]. For instance, a recent patent application (US 20100056390) +for a 3D cell culture system adapted for high-throughput drug screening has been accepted. Despite these recent advances in 3D substrates, it remains a technical challenge to visualise first the entire cell body of moving cells and second the matrix adhesions self. Several handicaps include background due to the matrix itself, signal to noise and the depth of the sample which limit the choice of objectives. + +Phase contrast imaging is a convenient imaging approach if signal to noise is not an issue for further data processing +(Fig. 5a). It provides cellular and temporal resolution which is already enough to characterise the type of 3D invasion which can be depicted as either single (mesnchymal or amoeboid) or collective migration [1, 109, 110, 116]. + +Confocal microscopy is the most suitable imaging technique to collect fixed endpoints or time-lapse sequences of threedimensional data of migrating cells and matrix adhesion activities [117–120]. Advanced bioinformatics is needed to further process the data and apply multiparametric image analysis to describe cell behaviour with several parameters [117, 121, 122]. In Table 3, we recapitulate the challenges and potential solutions to make the 3D culture system a versatile model for understanding tumour invasion mechanisms. Next to the dynamic imaging of cell migration, imaging of matrix adhesion turnover in a 3D environment is technically extremely challenging. In Fig. 5a, we show how confocal imaging allow high resolution imaging of the GFPdSH2 reporter overexpressed in epithelial cells that have the ability to form tubulogenesis. Thanks to this reporter combined with fast confocal microscopy, we will be able to monitor tyrosine phosphorylation at adhesion sites when cells are migrating collectively through the collagen gels +(Le De´ve´dec et al., unpublished data). Furthermore, by using our established 3D invasion assay for 4T1 mammary tumour cells together with GFP-tagged adhesion protein overexpression we are able to image adhesion dynamics in a 3D environment during tumour cell migration (Fig. 5b). + +Imaging migration and adhesions in vivo Studying adhesion dynamics in migrating cells in an in vitro 3D matrix is already technically challenging. Obviously, it is even more challenging to do so within intact organisms. Suitable in vivo models for both cancer progression and high resolution imaging are necessary. + +## Zebrafish Model For Metastasis Analysis + +Recently, the zebrafish and its transparent embryos became a new model system to investigate tumour development, cancer cell invasion and metastasis formation [123–125]. + +In the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Moreover, a number of unique features make this animal model very attractive: zebrafish are inexpensive to maintain and breed in large numbers, develop rapidly ex vivo, and can be maintained in small volumes of water. Several independent studies have now shown that human melanoma cells and other cancer cell lines are able to induce neovascularisation when xenografted in the zebrafish [125–127].The role of the small GTPase RhoC in tumour formation, angiogenesis and cell invasion was investigated in real-time in 1-monthold immunosuppressed zebrafish xenografted with the human breast cancer cell line MDA-435 [125]. This study achieved high-resolution imaging of the dynamic cell– +vascular interface in transparent juvenile zebrafish. All these innovative studies established the use of the zebrafish xenotransplantation model for the analysis of cancer cell lines as we also did in our laboratory (Fig. 6). We screened + +![12_image_0.png](12_image_0.png) + +a number of cancer cell-lines and established that very aggressive phenotype after injection in the yolk (Fig. 6a, part a), were able to outgrow and disseminate throughout the animal body (Fig. 6a, part b) (Ghotra et al., +unpublished data). We also demonstrated that certain celltypes could trigger the angiogenesis process (Fig. 6a, parts c and d) (Ghotra et al., unpublished data). In a very recent study, it was shown that zebrafish embryos can even be used to directly transplant human tumour tissue and primary human tumour cells [128]. Zebrafish embryos thus provide a simple, fast and cost-effective method to test the metastatic behaviour of human cell-lines and primary tumours in an in vivo vertebrate animal model that also permits high throughput drug screening (see later section). Mouse metastasis model for imaging of tumour cell migration The availability of multi-photon intravital microscopy has allowed researchers to visualise the dynamic behaviour of cancer cells in vivo [119, 120, 129, 130]. Multiphoton microscopy uses longer wavelengths (up to 1,200 nm) that are able to penetrate deeper into tissues and allow us to visualise more than 100 lm deep into the primary tumour. + +Multi-photon excitation also causes less photo-damage, permits good optical sectioning and 3D resolution [131] and non-invasive visualisation of the extra-cellular matrix thanks to the second harmonic generation phenomenon +[132, 133]. In the past years, intravital imaging has been mainly used to follow up individual or group of cells fluorescently labelled within the primary tumour and study the interaction of moving cells with their microenvironment such as collagen matrix (Fig. 6b) [130, 134]. Recently, a new technical development, the mammary imaging window (MIW) has been shown to be advantageous for studying cell movement and adhesion with high resolution. The use of photo-convertible fluorophores such as Kaede or Dendra2 allows a precise monitoring of cellular movement in vivo no longer just over hours but over days [135], [136]. Despite all these technological advances, it was still not possible to visualise adhesions in migrating cells in vivo. Just recently, for the first time, a study on E-cadherin dynamics in living animals has been reported [137]. Photo-bleaching and photo-activation was used to compare the mobility of cell adhesion and plasma membrane probes in vitro and in tumours grown in mice and consequently demonstrate critical differences in molecular dynamics in vitro and in vivo. + +## Future Directions And Concluding Remarks Frap/Fret, Frap/Tirf And Fret/Tirf + +An interesting alternative to the FRAP method is the combination of both FRAP and FRET that has already been applied in the nucleus [95, 138]. In the acceptor photobleaching FRET methodology, the bleaching of the acceptor results in an increase of the donor intensity. By applying FRAP on cells expressing adhesion proteins tagged with both + +![13_image_0.png](13_image_0.png) + +FRET partners, e.g. CFP and YFP, and simultaneously recording in the bleached region the acceptor recovery and the redistribution of the increased donor signal, it is possible to compare the mobility of the interacting proteins (donor redistribution) relative to the mobility of the total pool of proteins (the YFP recovery as in a conventional FRAP experiment). Of course, it would be even more attractive to apply our previously described FLIP/FRAP methodology together with FRET to understand the complexity of both protein dynamics and interactions. Another very attractive combination of imaging technique is TIRF together with FRAP and/or FRET, a technique that is currently experiencing rapid growth in application [139–141]. This combination of imaging technologies will ensure an improved insight into adhesion protein dynamics and complexation in migrating cells since TIRF microscopy enormously enhances spatial resolution of fast-moving matrix adhesions. + +## High Through Put Techniques (2D And 3D) For Target Identification + +To explore the mechanisms underlying the regulation of cell migration by matrix adhesion dynamics, we described various qualitative and quantitative approaches that study adhesion dynamics and cell migration in a 2D and 3D +environment. Such dynamic studies are of particular relevance to understand cancer cell motility. To provide a systematic analysis of genes that regulate cell migration or to study effect of potential drugs on tumour cell migration, high throughput image-based screening (HTS) is the most recent advance in imaging technology which has been made possible thanks to the availability of high-precision robotic liquid handling machinery, automated fluorescence microscopy and high-performance computing (for reviews, see [142] [143–146]). The capability of screening and segmenting diverse cellular populations combined with the possibility to detect protein–protein interactions or protein dynamics can offer a significant advantage in the fields of cellular proteomics and interactomics [147]. A highthroughput screening campaign includes the following: +assay design (cell line, probes, kinetic, or end point), image acquisition (confocal vs wide-field, hardware specifications), image analysis (canned or custom algorithms), and data interpretation and image management (storage space, database, and visualisation). With the proper tools, HTS +can be an invaluable tool to exploit the complexity of adhesion signalling pathways at a single cell level in a high-throughput manner (Fig. 7). The most recent image analysis packages provide capabilities that cover many microscopic applications suitable for HTS experiments, including the documentation of phenotypic readouts, the analysis of subcellular localisation and morphological changes, and the quantification of individual molecular modifications. Automated, cluster-based data analysis tools play an important role in analysing highly complex multiplexed HTS datasets into specific classes of phenotypic responses and assigning statistical significance to the changes in cellular processes that comprise the overall response [148]. However, first, not all microscopic applications are suitable for HTS experiments (e.g. FRAP, FCS, +intravital imaging using mouse models), and second, kinetic HTS is still a technical and intellectual challenge +(see Table 3 for detailed overview of the existing challenges and potential solutions). + +High-throughput fluorescence microscopy using fixed assays The Geiger laboratory published two different screens which provide helpful methodologies and data on cell adhesion and migration: the first used high resolution microscopy to profile the effect of a library of natural extract on cell adhesion [149], while the second used a Fig. 7 Steps for high content + +![14_image_0.png](14_image_0.png) + +systems microscopy approach to understand cancer metastasis. Overview of imaging techniques that allow the phenotypic profiling of proteomics and cellomics (fixed multicolour and time-lapse) and the understanding at systems level (FRET, FRAP and FCS). To enhance our understanding of cancer metastasis, the highthroughput fluorescence microscopy should be applied in 2D, 3D and finally in vivo. a Sample preparation including cell transfection, exposure or immunostaining is nowadays conducted in multi-well dishes using robotics. b Automated image acquisition of fixed or living cells is done using automated microscopes (see Table 1, Available High Content Screening (HCS) instrumentation in [157]). Images can be acquired using different fluorescence microscopy techniques +(e.g. fixed multicolour, timelapse, FRET, FRAP, FCS. + +c Image data storage requires specialised software and hardware for data handling. d Automated image analysis which needs to be adapted or developed for each assay and is currently a challenge in HTS +field. e Another big challenge in the field is the data mining and modelling which requires different disciplines such as statistics and bioinformatics (see Table 2 and available HCS informatics tools in [157]) (adapted from [142]) +modified phagokinetics tracks with MCF7 and identified novel pro-migratory, cancer-associated genes [150]. Very recently, a third screen using high-throughput, high-resolution, microscopy-based assay together with human kinases, phosphatases and adhesome libraries was also performed and it provided a model for the molecular hierarchy of FA formation [151]. Another very elegant study used the traditional wound-healing assay with MCF10A breast epithelial and screened siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion [152]. + +Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes were not previously associated with motility or adhesion [152]. Although, the results of these screens are very promising and provide new data on cell migration, the analysis was performed with fixed samples. + +High-throughput time-lapse fluorescence microscopy Time-resolved live imaging would provide the highest content for assays on complex dynamic processes such as cell migration and invasion. Indeed, despite the recent described advances, a number of technical and analytical challenges remain to be solved to allow functional + +| Table 3 Challenges and potential solutions to increase throughput of imaging techniques Challenge Type Potential solutions Sample preparation 2D Development of predictive screening assays (? use of primary or embryonic stem cells (ESCs) instead of easy to culture tumour cell-lines but genetically aberrant) 3D Development and validation of relevant 3D scaffolds (? characterise ECM of patient tumour material) Improve 3D cell culture techniques for automated liquid handling robotics (? collaboration between academia and pharmaceutical companies) In vivo Automated microinjection of tumour cells in ZF (? automatic microinjector based on pattern recognition) Automated filling of the microwells plates with ZF preferably all similarly orientated (? make use of adapted mould) Automated image acquisition 2D Autofocus combined with z-scans for 3D imaging (? image-based or reflectionbased autofocusing) Pre-optimisation of the acquisition settings (? autoexposure algorithm to adjust integration time of detectors) Automated object (cells of matrix adhesion) localisation (? autoexposure algorithm to adjust integration time of detectors) Intelligent microscope [158, 172] 3D/in vivo Higher throughput kinetic imaging microscopes suitable for automated 3D invasion studies (? see commercially available kinetic imaging systems such as Incucyte or Cell-IQ [117]) Higher throughput kinetic imaging microscopes suitable for FRET, FRAP or FCS (? in the future, intelligent microscope that recognises the object to be visualised) Data handling 2D Storing terabytes of data (? storage area network (SAN) which has multiterabyte to tens of terabytes capacity; commonly, data on the SAN are backed up on tape as 3D/in vivo well) Data management (? development of databases retrieved [157, Table 2]) Image analysis 2D Image segmentation (? depending on imaging quality, choose between regionbased, edge-based or region-growing method) 3D/in vivo Multiparametric image analysis (? phenotypic profiling which involves computer vision methods) Object tracking (? high time resolution for imaging; adapted tracking algorithms for 3D imaging [173] and HTS data) Data mining and modeling 2D Screening reproducibility and estimators (? quality standards, e.g. coefficient of variations (CVs) and zscores should not exceed 5% and should be higher than 0.5, respectively) 3D/in vivo Significant behaviour changes detection Automated classification (? supervised machine learning) Development of computational models | +|---| + +genomics screening together with dynamic imaging in a 2D and 3D environment (see Table 3). The major technical challenge is the need for (ultra-) fast image acquisition as well as to obtain ''high content'' information about the migratory behaviour of many cells, including dynamic features such as migration velocity. Only fast microscopes with high sensitive CCD camera and adapted bioinformatics can fulfill those requirements. In our laboratory, we are setting up a screen based on dynamic imaging of fastmoving cells to obtain cell behaviour measurements. Postimage acquisition, we also fix the plate and stain for focal adhesion markers so that we can correlate changes in cell motility with altered focal adhesion morphometry. Very recently, for the first time, a large study provided timeresolved profiles of RNAi-induced loss-of-function phenotypes resulting from siRNAs targeting the entire genome +[153]. Here, a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, 2-day time-lapse microscopy and computational image processing was established. This was applied for a genome-wide phenotypic profiling of mitototic events for each of the approximately 21,000 human protein-coding genes. Cellular phenotypes were scored quantitatively by computational image processing, which allowed the identification of hundreds of genes involved in diverse biological functions including cell division, migration and survival. Such a systems microscopy approach will also be applicable in profiling tumor cell migration and invasion. + +The next step is the development of high-throughput methods that make use of functionally tagged transgenes in live cells to analyse protein–protein interactions and posttranslational modifications. Their subcellular localisation and dynamics are needed to go from phenotypic profiling of the human genome to a systems-level understanding of the functional principles of the encoded protein machinery of human cells[143]. Indeed, in the future, it might be possible to analyse living cells using high-throughput approaches to determine intermolecular interactions using FRET, or protein dynamics using FRAP or FCS. These techniques are extremely powerfull in single-cell experiments since they provide quantitative data on the dynamics of biochemical reactions in living cells. However, except for FRET, none of these techniques have been successfully applied to systematic studies using HTS. As already mentioned, French reported an automated optically sectioning fluorescence lifetime imaging (FLIM) multiwell plate reader for high content analysis (HCA) in drug discovery and accelerated research in cell biology [105, 154]. Finally, the ultimate goal would be to perform those highthroughput screenings using time-lapse imaging in a relevant 3D culture environment and, even more challenging, in the zebrafish. + +Perspectives and challenges to drug discovery and clinical treatment of cancer metastas formation The above systems microscopy approach integrates different imaging techniques and relevant models for all stages of target-based drug discovery. This involves the study of cells, including target discovery, drug screening in cell-based assays, early safety evaluation, mode-of-action studies and in vivo studies to monitor cell fate. A prerequisite for reaching conclusions with low failure rates in drug discovery is the improvement of throughput that provides reliable data. The level of complexity comprising both changes in cellular morphology and macromolecular subcellular localisation and expression level makes HTS +ideal among current approaches used for drug screening +[155, 156]. Automated microscopy platforms that run in medium- or high-throughput mode to screen thousands of cDNAs, siRNAs, aptamers, proteins and antibodies using endpoints 2D migration and 3D invasion would benefit target discovery and validation [122, 148]. For the lead identification and optimisation, endpoint and time-lapse microscopy combined with multiparametric kinetic analysis incorporating both cell invasion assays and assessment of target activity with FAST/FLIM would be the next step [117]. However, as previously mentioned, kinetic HTS is still a technical and intellectual challenge which requires adapted fluorescent labelling techniques, automated +'intelligent' microscopes and well-developed software platforms [157, 158] (Table 3). In the later stages of the drug discovery trajectory, intravital imaging of tumour invasion in various relevant in vivo models can be used to evaluate the response of the drug in development and assess its activity when combined with FRET/FLIM. In the future, the combination of other microscopy techniques such as FRAP or FCS will help to obtain additional mechanistic information so that the drug development process can be more efficient. Nonetheless, a number of significant challenges remain related to the use of more biology- and disease-relevant predictive cell systems and the development of biosensors and bioreporters to measure and manipulate cellular events. Finally, the identification of relevant biomarkers specific to distinct invasion mechanisms will facilitate the development of clinical and preclinical studies and help administrate appropriate antiinvasive therapies to cancer patients [117]. + +## Concluding Remarks + +Here, we presented a systems microscopy approach which integrates different imaging techniques developed over the past years to study adhesion signalling in relation to tumor cell migration and cancer metastasis [145]. In Table 1, we summarised the different techniques to study protein dynamics and interactions in matrix adhesions. In Table 2, an overview of the techniques used to model the various modes of tumour migration was given. In Table 3, we recapitulated the challenges and potential solutions to increase the throughput of imaging and multiparametric analysis. Understanding the movement of single cells and cells in tissue requires the analysis of complex processes under normal and perturbed conditions. Ideally, one would like to quantitatively determine movement and shape changes of cells and correlate these with the spatiotemporal dynamics of the cytoskeletal elements and the extra- and intracellular signalling pathways controlling these behaviours in isolated cells as well as in cells in tissue. Imaging together with multiparametric analysis and data modelling is the most appropriate method to reach that ultimate goal. Optimisation of current techniques and systems that combine diverse techniques will improve both our spatial and temporal resolutions of the role of matrix adhesions in migrating cells. Also, more and more bioinformatics tools will be generated for rapid and detailed image analysis and data processing. The additional knowledge obtained will hopefully provide insights into the molecular mechanisms behind tumour cell migration and help in developing new anticancer therapies. + +Acknowledgments The authors would like to thank Leo Price for technical support for the 3D invasion assays. This work was financially supported by grants from the Dutch Cancer Society (UL 20073860), the EU FP7 Health Program Metafight (Grant agreement no.201862) and ZF Cancer (Grant agreement no.201862), and the Netherlands Organization for Scientific Research (902-21-229 and 911-02-022). + +Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. + +## References + +1. Friedl P, Gilmour D (2009) Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10:445–457 2. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, +Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: +integrating signals from front to back. Science 302:1704–1709 3. Linder S (2007) The matrix corroded: podosomes and invadopodia in extracellular matrix degradation. Trends Cell Biol 17:107–117 4. Destaing O, Saltel F, Geminard JC, Jurdic P, Bard F (2003) +Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein. Mol Biol Cell 14:407–416 5. Yamaguchi H, Lorenz M, Kempiak S, Sarmiento C, Coniglio S, +Symons M, Segall J, Eddy R, Miki H, Takenawa T, Condeelis J +(2005) Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J Cell Biol 168:441–452 6. Yamaguchi H, Pixley F, Condeelis J (2006) Invadopodia and podosomes in tumor invasion. Eur J Cell Biol 85:213–218 7. Baldassarre M, Ayala I, Beznoussenko G, Giacchetti G, +Machesky LM, Luini A, Buccione R (2006) Actin dynamics at sites of extracellular matrix degradation. Eur J Cell Biol 85:1217–1231 8. Chan KT, Cortesio CL, Huttenlocher A (2009) FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion. J Cell Biol 185:357–370 9. Heath JP, Dunn GA (1978) Cell to substratum contacts of chick fibroblasts and their relation to the microfilament system. + +A correlated interference-reflexion and high-voltage electronmicroscope study. J Cell Sci 29:197–212 10. Izzard CS, Lochner LR (1976) Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique. J Cell Sci 21:129–159 11. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110:673–687 12. Geiger B, Bershadsky A, Pankov R, Yamada KM (2001) +Transmembrane crosstalk between the extracellular matrix– cytoskeleton crosstalk. Nat Rev Mol Cell Biol 2:793–805 13. Zamir E, Geiger B (2001) Molecular complexity and dynamics of cell-matrix adhesions. J Cell Sci 114:3583–3590 14. Zaidel-Bar R, Itzkovitz S, Ma'ayan A, Iyengar R, Geiger B +(2007) Functional atlas of the integrin adhesome. Nat Cell Biol 9:858–867 15. Huveneers S, Danen EH (2009) Adhesion signaling—crosstalk between integrins, Src and Rho. J Cell Sci 122:1059–1069 16. Schmidt S, Friedl P (2010) Interstitial cell migration: integrindependent and alternative adhesion mechanisms. Cell Tissue Res 339:83–92 17. Shintani Y, Fukumoto Y, Chaika N, Svoboda R, Wheelock MJ, +Johnson KR (2008) Collagen I-mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1. J Cell Biol 180:1277–1289 18. Thompson O, Moore CJ, Hussain SA, Kleino I, Peckham M, +Hohenester E, Ayscough KR, Saksela K, Winder SJ (2010) Modulation of cell spreading and cell-substrate adhesion dynamics by dystroglycan. J Cell Sci 123:118–127 19. Moore CJ, Winder SJ (2010) Dystroglycan versatility in cell adhesion: a tale of multiple motifs. Cell Commun Signal 8:3 20. Cross SS, Lippitt J, Mitchell A, Hollingsbury F, Balasubramanian SP, Reed MW, Eaton C, Catto JW, Hamdy F, Winder SJ +(2008) Expression of beta-dystroglycan is reduced or absent in many human carcinomas. Histopathology 53:561–566 21. Zamir E, Katz BZ, Aota S, Yamada KM, Geiger B, Kam Z +(1999) Molecular diversity of cell-matrix adhesions. J Cell Sci 112:1655–1669 22. Zaidel-Bar R, Cohen M, Addadi L, Geiger B (2004) Hierarchical assembly of cell-matrix adhesion complexes. Biochem Soc Trans 32:416–420 23. Zamir E, Geiger B, Kam Z (2008) Quantitative multicolor compositional imaging resolves molecular domains in cellmatrix adhesions. PLoS ONE. doi:10.1371/journal.pone. + +0001901 24. Geiger B, Spatz JP, Bershadsky AD (2009) Environmental sensing through focal adhesions. Nat Rev Mol Cell Biol 10:21–33 25. Cukierman E, Pankov R, Stevens DR, Yamada KM (2001) +Taking cell-matrix adhesions to the third dimension. Science 294:1708–1712 26. Kirchner J, Kam Z, Tzur G, Bershadsky AD, Geiger B (2003) +Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption. J Cell Sci 116:975– 986 +27. Webb DJ, Parsons JT, Horwitz AF (2002) Adhesion assembly, disassembly and turnover in migrating cells–over and over and over again. Nat Cell Biol 4:E97–E100 28. Carragher NO, Frame MC (2004) Focal adhesion and actin dynamics: a place where kinases and proteases meet to promote invasion. Trends Cell Biol 14:241–249 29. Small JV, Kaverina I (2003) Microtubules meet substrate adhesions to arrange cell polarity. Curr Opin Cell Biol 15:40–47 30. Larsen M, Tremblay ML, Yamada KM (2003) Phosphatases in cell-matrix adhesion and migration. Nat Rev Mol Cell Biol 4:700–711 31. Schlaepfer DD, Mitra SK, Ilic D (2004) Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochim Biophys Acta 1692:77–102 32. Tomar A, Schlaepfer DD (2009) Focal adhesion kinase: +switching between GAPs and GEFs in the regulation of cell motility. Curr Opin Cell Biol 21:676–683 33. Turner CE, West KA, Brown MC (2001) Paxillin-ARF GAP +signaling and the cytoskeleton. Curr Opin Cell Biol 13:593–599 34. Carragher NO (2006) Calpain inhibition: a therapeutic strategy targeting multiple disease states. Curr Pharm Des 12:615–638 35. Carragher NO, Fonseca BD, Frame MC (2004) Calpain activity is generally elevated during transformation but has oncogenespecific biological functions. Neoplasia 6:53–73 36. Carragher NO, Frame MC (2002) Calpain: a role in cell transformation and migration. Int J Biochem Cell Biol 34:1539–1543 37. Franco SJ, Rodgers MA, Perrin BJ, Han J, Bennin DA, Critchley DR, Huttenlocher A (2004) Calpain-mediated proteolysis of talin regulates adhesion dynamics. Nat Cell Biol 6:977–983 38. Xue C, Wyckoff J, Liang F, Sidani M, Violini S, Tsai KL, +Zhang ZY, Sahai E, Condeelis J, Segall JE (2006) Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. Cancer Res 66:192–197 39. Le Devedec SE, van Roosmalen W, Maria N, Grimbergen M, +Pont C, Lalai R, van de WB (2009) An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2(-/-) gammac (-/-) mouse. Clin Exp Metastasis 26:673–684 40. Takeuchi K, Ito F (2010) EGF receptor in relation to tumor development: molecular basis of responsiveness of cancer cells to EGFR-targeting tyrosine kinase inhibitors. FEBS J 277:316– 326 41. Desgrosellier JS, Cheresh DA (2010) Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 10:9–22 42. Kim LC, Song L, Haura EB (2009) Src kinases as therapeutic targets for cancer. Nat Rev Clin Oncol 6:587–595 43. Guarino M (2010) Src signaling in cancer invasion. J Cell Physiol 223:14–26 44. Golubovskaya VM, Kweh FA, Cance WG (2009) Focal adhesion kinase and cancer. Histol Histopathol 24:503–510 45. Owens LV, Xu L, Craven RJ, Dent GA, Weiner TM, Kornberg L, Liu ET, Cance WG (1995) Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 55:2752–2755 46. Zhao J, Guan JL (2009) Signal transduction by focal adhesion kinase in cancer. Cancer Metastasis Rev 28:35–49 47. van Nimwegen MJ, van de WB (2007) Focal adhesion kinase: +a potential target in cancer therapy. Biochem Pharmacol 73:597–609 48. Yafeng M, van de Water B, Le De´ve´dec SE (2009) Focal Adhesion Kinase inhibition as a potential strategy for anticancer therapies. Drugs Future 34:477–484 49. Chudakov DM, Lukyanov S, Lukyanov KA (2005) Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol 23:605–613 50. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909 51. Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120:4247–4260 52. Wang Y, Shyy JY, Chien S (2008) Fluorescence proteins, livecell imaging, and mechanobiology: seeing is believing. Annu Rev Biomed Eng 10:1–38 53. Dormann D, Weijer CJ (2006) Imaging of cell migration. + +EMBO J 25:3480–3493 54. Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224 55. Sun YN, Lin CH, Kuo CC, Ho CL, Lin CJ (2009) Live cell tracking based on cellular state recognition from microscopic images. J Microsc 235:94–105 56. Hand AJ, Sun T, Barber DC, Hose DR, MacNeil S (2009) +Automated tracking of migrating cells in phase-contrast video microscopy sequences using image registration. J Microsc 234:62–79 57. Entschladen F, Drell TL, Lang K, Masur K, Palm D, Bastian P, +Niggemann B, Zaenker KS (2005) Analysis methods of human cell migration. Exp Cell Res 307:418–426 58. Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, +Parsons JT, Horwitz AF (2004) FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat Cell Biol 6:154–161 59. Hoebe RA, Van Oven CH, Gadella TW Jr, Dhonukshe PB, Van Noorden CJ, Manders EM (2007) Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging. Nat Biotechnol 25:249–253 60. Axelrod D, Thompson NL, Burghardt TP (1983) Total internal inflection fluorescent microscopy. J Microsc 129:19–28 61. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877 62. Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A +(2002) An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci USA 99:12651–12656 63. Gurskaya NG, Verkhusha VV, Shcheglov AS, Staroverov DB, +Chepurnykh TV, Fradkov AF, Lukyanov S, Lukyanov KA +(2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24:461–465 64. Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373 65. Osborn EA, Rabodzey A, Dewey CF Jr, Hartwig JH (2006) +Endothelial actin cytoskeleton remodeling during mechanostimulation with fluid shear stress. Am J Physiol Cell Physiol 290:C444–C452 66. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, +Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF +(2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645 67. Caswell PT, Spence HJ, Parsons M, White DP, Clark K, Cheng KW, Mills GB, Humphries MJ, Messent AJ, Anderson KI, +McCaffrey MW, Ozanne BW, Norman JC (2007) Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments. Dev Cell 13:496–510 68. Houtsmuller AB, Vermeulen W (2001) Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching. Histochem Cell Biol 115:13–21 69. van Royen ME, Farla P, Mattern KA, Geverts B, Trapman J, +Houtsmuller AB (2009) Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells. + +Methods Mol Biol 464:363–385 70. Ballestrem C, Hinz B, Imhof BA, Wehrle-Haller B (2001) +Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior. + +J Cell Biol 155:1319–1332 71. Hamadi A, Bouali M, Dontenwill M, Stoeckel H, Takeda K, +Ronde P (2005) Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397. J Cell Sci 118:4415–4425 72. Cluzel C, Saltel F, Lussi J, Paulhe F, Imhof BA, Wehrle-Haller B (2005) The mechanisms and dynamics of (alpha)v(beta)3 integrin clustering in living cells. J Cell Biol 171:383–392 73. Geuijen CA, Sonnenberg A (2002) Dynamics of the alpha6beta4 integrin in keratinocytes. Mol Biol Cell 13:3845–3858 74. Lele TP, Pendse J, Kumar S, Salanga M, Karavitis J, Ingber DE +(2006) Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells. J Cell Physiol 207:187–194 75. Edlund M, Lotano MA, Otey CA (2001) Dynamics of alphaactinin in focal adhesions and stress fibers visualized with alphaactinin-green fluorescent protein. Cell Motil Cytoskeleton 48:190–200 76. von Wichert G, Haimovich B, Feng GS, Sheetz MP (2003) Forcedependent integrin-cytoskeleton linkage formation requires downregulation of focal complex dynamics by Shp2. EMBO J +22:5023–5035 77. Lele TP, Thodeti CK, Pendse J, Ingber DE (2008) Investigating complexity of protein-protein interactions in focal adhesions. Biochem Biophys Res Commun 369:929–934 78. Wolfenson H, Lubelski A, Regev T, Klafter J, Henis, YI, Geiger B (2009) A role for the juxtamembrane cytoplasm in the molecular dynamics of focal adhesions. PLoS ONE. doi: +10.1371/journal.pone.0004304 79. Fraley TS, Pereira CB, Tran TC, Singleton C, Greenwood JA +(2005) Phosphoinositide binding regulates alpha-actinin dynamics: mechanism for modulating cytoskeletal remodeling. + +J Biol Chem 280:15479–15482 80. Chandrasekar I, Stradal TE, Holt MR, Entschladen F, Jockusch BM, Ziegler WH (2005) Vinculin acts as a sensor in lipid regulation of adhesion-site turnover. J Cell Sci 118:1461–1472 81. Goetz JG, Joshi B, Lajoie P, Strugnell SS, Scudamore T, Kojic LD, Nabi IR (2008) Concerted regulation of focal adhesion dynamics by galectin-3 and tyrosine-phosphorylated caveolin-1. + +J Cell Biol 180:1261–1275 82. Waterman-Storer CM, Desai A, Bulinski JC, Salmon ED (1998) +Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Curr Biol 8:1227–1230 83. Ponti A, Machacek M, Gupton SL, Waterman-Storer CM, +Danuser G (2004) Two distinct actin networks drive the protrusion of migrating cells. Science 305:1782–1786 84. Ponti A, Matov A, Adams M, Gupton S, Waterman-Storer CM, +Danuser G (2005) Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative Fluorescent Speckle Microscopy. Biophys J 89:3456–3469 85. Gupton SL, Waterman-Storer CM (2006) Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration. Cell 125:1361–1374 86. Hu K, Ji L, Applegate KT, Danuser G, Waterman-Storer CM +(2007) Differential transmission of actin motion within focal adhesions. Science 315:111–115 87. Wiseman PW, Brown CM, Webb DJ, Hebert B, Johnson NL, +Squier JA, Ellisman MH, Horwitz AF (2004) Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy. J Cell Sci 117:5521–5534 88. Brown CM, Dalal RB, Hebert B, Digman MA, Horwitz AR, +Gratton E (2008) Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J Microsc 229:78–91 89. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E (2005) Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys J 89:1317–1327 90. Digman MA, Gratton E (2009) Analysis of diffusion and binding in cells using the RICS approach. Microsc Res Tech 72:323–332 91. Brown CM, Hebert B, Kolin DL, Zareno J, Whitmore L, +Horwitz AR, Wiseman PW (2006) Probing the integrin-actin linkage using high-resolution protein velocity mapping. J Cell Sci 119:5204–5214 92. Digman MA, Brown CM, Horwitz AR, Mantulin WW, Gratton E (2008) Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy. Biophys J +94:2819–2831 93. Ballestrem C, Erez N, Kirchner J, Kam Z, Bershadsky A, Geiger B (2006) Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer. J Cell Sci 119:866–875 94. Parsons M, Vojnovic B, Ameer-Beg S (2004) Imaging proteinprotein interactions in cell motility using fluorescence resonance energy transfer (FRET). Biochem Soc Trans 32:431–433 95. van Royen ME, Dinant C, Farla P, Trapman J, Houtsmuller AB +(2009) FRAP and FRET methods to study nuclear receptors in living cells. Methods Mol Biol 505:69–96 96. van Rheenen J, Langeslag M, Jalink K (2004) Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission. Biophys J 86:2517–2529 97. Cai X, Lietha D, Ceccarelli DF, Karginov AV, Rajfur Z, +Jacobson K, Hahn KM, Eck MJ, Schaller MD (2008) Spatial and temporal regulation of focal adhesion kinase activity in living cells. Mol Cell Biol 28:201–214 98. Wang Y, Chien S (2007) Analysis of integrin signaling by fluorescence resonance energy transfer. Methods Enzymol 426:177–201 99. Itoh RE, Kurokawa K, Ohba Y, Yoshizaki H, Mochizuki N, Matsuda M (2002) Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells. Mol Cell Biol 22:6582–6591 100. Nakamura T, Kurokawa K, Kiyokawa E, Matsuda M (2006) +Analysis of the spatiotemporal activation of rho GTPases using Raichu probes. Methods Enzymol 406:315–332 101. Pertz O, Hahn KM (2004) Designing biosensors for Rho family proteins–deciphering the dynamics of Rho family GTPase activation in living cells. J Cell Sci 117:1313–1318 102. Pertz O, Hodgson L, Klemke RL, Hahn KM (2006) Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440:1069–1072 103. Parsons M, Messent AJ, Humphries JD, Deakin NO, Humphries MJ (2008) Quantification of integrin receptor agonism by fluorescence lifetime imaging. J Cell Sci 121:265–271 104. Bastiaens PI, Squire A (1999) Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol 9:48–52 105. Grant DM, McGinty J, McGhee EJ, Bunney TD, Owen DM, +Talbot CB, Zhang W, Kumar S, Munro I, Lanigan PM, Kennedy GT, Dunsby C, Magee AI, Courtney P, Katan M, Neil MA, French PM (2007) High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events. Opt Express 15:15656–15673 106. Fan Y, Gong Y, Ghosh PK, Graham LM, Fox PL (2009) Spatial coordination of actin polymerization and ILK-Akt2 activity during endothelial cell migration. Dev Cell 16:661–674 107. Romer LH, Birukov KG, Garcia JG (2006) Focal adhesions: +paradigm for a signaling nexus. Circ Res 98:606–616 108. Alderliesten M, de Graauw M, Oldenampsen J, Qin Y, Pont C, +van Buren L, van de WB (2007) Extracellular signal-regulated kinase activation during renal ischemia/reperfusion mediates focal adhesion dissolution and renal injury. Am J Pathol 171:452–462 109. Wolf K, Alexander S, Schacht V, Coussens LM, von Andrian UH, van Rheenen J, Deryugina E, Friedl P (2009) Collagenbased cell migration models in vitro and in vivo. Semin Cell Dev Biol 20:931–941 110. Sahai E, Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5:711–719 111. Yates C, Shepard CR, Papworth G, Dash A, Beer SD, +Tannenbaum S, Griffith L, Wells A (2007) Novel three-dimensional organotypic liver bioreactor to directly visualize early events in metastatic progression. Adv Cancer Res 97:225–246 112. Kenny HA, Krausz T, Yamada SD, Lengyel E (2007) Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells to the omentum. Int J Cancer 121: +1463–1472 113. Abhyankar VV, Toepke MW, Cortesio CL, Lokuta MA, +Huttenlocher A, Beebe DJ (2008) A platform for assessing chemotactic migration within a spatiotemporally defined 3D +microenvironment. Lab Chip 8:1507–1515 114. Benton G, George J, Kleinman HK, Arnaoutova IP (2009) +Advancing science and technology via 3D culture on basement membrane matrix. J Cell Physiol 221:18–25 115. Zaman MH, Trapani LM, Siemeski A, Mackellar D, Gong H, +Kamm RD, Wells A, Lauffenburger DA, Matsudaira P (2006) Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proc Natl Acad Sci USA 103:10889–10894 116. Hooper S, Marshall JF, Sahai E (2006) Tumor cell migration in three dimensions. Methods Enzymol 406:625–643 117. Carragher NO (2009) Profiling distinct mechanisms of tumour invasion for drug discovery: imaging adhesion, signalling and matrix turnover. Clin Exp Metastasis 26:381–397 118. Gobel W, Kampa BM, Helmchen F (2007) Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat Methods 4:73–79 119. Wolf K, Friedl P (2005) Functional imaging of pericellular proteolysis in cancer cell invasion. Biochimie 87:315–320 120. Friedl P (2004) Dynamic imaging of cellular interactions with extracellular matrix. Histochem Cell Biol 122:183–190 121. Nystrom ML, Thomas GJ, Stone M, Mackenzie IC, Hart IR, +Marshall JF (2005) Development of a quantitative method to analyse tumour cell invasion in organotypic culture. J Pathol 205:468–475 122. Adanja I, Debeir O, Megalizzi V, Kiss R, Warzee N, Decaestecker C (2010) Automated tracking of unmarked cells migrating in three-dimensional matrices applied to anti-cancer drug screening. Exp Cell Res 316:181–193 123. Goessling W, North TE, Zon LI (2007) New waves of discovery: modeling cancer in zebrafish. J Clin Oncol 25:2473–2479 124. Langenau DM, Traver D, Ferrando AA, Kutok JL, Aster JC, +Kanki JP, Lin S, Prochownik E, Trede NS, Zon LI, Look AT (2003) Myc-induced T cell leukemia in transgenic zebrafish. Science 299:887–890 125. Stoletov K, Montel V, Lester RD, Gonias SL, Klemke R (2007) +High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. Proc Natl Acad Sci USA 104:17406–17411 126. Haldi M, Ton C, Seng WL, McGrath P (2006) Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish. Angiogenesis 9:139–151 127. Nicoli S, Presta M (2007) The zebrafish/tumor xenograft angiogenesis assay. Nat Protoc 2:2918–2923 128. Marques IJ, Weiss FU, Vlecken DH, Nitsche C, Bakkers J, +Lagendijk AK, Partecke LI, Heidecke CD, Lerch MM, Bagowski CP (2009) Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model. BMC Cancer 9:128 129. Sahai E (2007) Illuminating the metastatic process. Nat Rev Cancer 7:737–749 130. Condeelis J, Segall JE (2003) Intravital imaging of cell movement in tumours. Nat Rev Cancer 3:921–930 131. Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: +multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377 132. Campagnola PJ, Loew LM (2003) Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol 21:1356–1360 133. Kedrin D, Wyckoff J, Sahai E, Condeelis J, Segall JE (2007) +Imaging tumor cell movement in vivo. Curr Protoc Cell Biol 19.7.1–19.7.17 134. Kedrin D, van Rheenen J, Hernandez L, Condeelis J, Segall JE (2007) Cell motility and cytoskeletal regulation in invasion and metastasis. J Mammary Gland Biol Neoplasia 12:143–152 135. Kedrin D, Gligorijevic B, Wyckoff J, Verkhusha VV, Condeelis J, Segall JE, van Rheenen J (2008) Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods 5:1019–1021 136. Gligorijevic B, Kedrin D, Segall JE, Condeelis J, van Rheenen J +(2009) Dendra2 photoswitching through the Mammary Imaging Window. J Vis Exp. doi:10.3791/1278 137. Serrels A, Timpson P, Canel M, Schwarz JP, Carragher NO, +Frame MC, Brunton VG, Anderson KI (2009) Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development. Cancer Res 69:2714–2719 138. van Royen ME, Cunha SM, Brink MC, Mattern KA, Nigg AL, +Dubbink HJ, Verschure PJ, Trapman J, Houtsmuller AB (2007) Compartmentalization of androgen receptor protein-protein interactions in living cells. J Cell Biol 177:63–72 139. Bal M, Zaika O, Martin P, Shapiro MS (2008) Calmodulin binding to M-type K? channels assayed by TIRF/FRET in living cells. J Physiol 586:2307–2320 140. Wang L, Bittner MA, Axelrod D, Holz RW (2008) The structural and functional implications of linked SNARE motifs in SNAP25. Mol Biol Cell 19:3944–3955 141. Raveh A, Riven I, Reuveny E (2008) The use of FRET +microscopy to elucidate steady state channel conformational rearrangements and G protein interaction with the GIRK channels. Methods Mol Biol 491:199–212 142. Pepperkok R, Ellenberg J (2006) High-throughput fluorescence microscopy for systems biology. Nat Rev Mol Cell Biol 7:690–696 143. Neumann B, Held M, Liebel U, Erfle H, Rogers P, Pepperkok R, +Ellenberg J (2006) High-throughput RNAi screening by timelapse imaging of live human cells. Nat Methods 3:385–390 144. Conrad C, Gerlich DW (2010) Automated microscopy for highcontent RNAi screening. J Cell Biol 188:453–461 145. Megason SG, Fraser SE (2007) Imaging in systems biology. Cell 130:784–795 146. Glory E, Murphy RF (2007) Automated subcellular location determination and high-throughput microscopy. Dev Cell 12:7–16 147. Esposito A, Dohm CP, Bahr M, Wouters FS (2007) Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening. Mol Cell Proteomics. 6:1446–1454 148. Denner P, Schmalowsky J, Prechtl S (2008) High-content analysis in preclinical drug discovery. Comb Chem High Throughput Screen 11:216–230 149. Paran Y, Ilan M, Kashman Y, Goldstein S, Liron Y, Geiger B, +Kam Z (2007) High-throughput screening of cellular features using high-resolution light-microscopy; application for profiling drug effects on cell adhesion. J Struct Biol 158:233–243 150. Naffar-Abu-Amara S, Shay T, Galun M, Cohen N, Isakoff SJ, +Kam Z, Geiger B (2008) Identification of novel pro-migratory, cancer-associated genes using quantitative, microscopy-based screening. PLoS ONE. doi:10.1371/journal.pone.0001457 151. Winograd-Katz SE, Itzkovitz S, Kam Z, Geiger B (2009) +Multiparametric analysis of focal adhesion formation by RNAimediated gene knockdown. J Cell Biol 186:423–436 152. Simpson KJ, Selfors LM, Bui J, Reynolds A, Leake D, Khvorova A, Brugge JS (2008) Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat Cell Biol 10:1027–1038 153. Neumann B, Walter T, Heriche JK, Bulkescher J, Erfle H, +Conrad C, Rogers P, Poser I, Held M, Liebel U, Cetin C, +Sieckmann F, Pau G, Kabbe R, Wunsche A, Satagopam V, +Schmitz MH, Chapuis C, Gerlich DW, Schneider R, Eils R, +Huber W, Peters JM, Hyman AA, Durbin R, Pepperkok R, +Ellenberg J (2010) Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature 464:721–727 154. Talbot CB, McGinty J, Grant DM, McGhee EJ, Owen DM, +Zhang W, Bunney TD, Munro I, Isherwood B, Eagle R, Hargreaves A, Dunsby C, Neil MA, French PM (2008) High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis. J Biophotonics 1:514–521 155. Loo LH, Wu LF, Altschuler SJ (2007) Image-based multivariate profiling of drug responses from single cells. Nat Methods 4:445–453 156. Lang P, Yeow K, Nichols A, Scheer A (2006) Cellular imaging in drug discovery. Nat Rev Drug Discov 5:343–356 157. Zanella F, Lorens JB, Link W (2010) High content screening: +seeing is believing. Trends Biotechnol 28:237–245 158. King RD, Rowland J, Oliver SG, Young M, Aubrey W, Byrne E, +Liakata M, Markham M, Pir P, Soldatova LN, Sparkes A, +Whelan KE, Clare A (2009) The automation of science. Science 324:85–89 159. Bershadsky AD, Ballestrem C, Carramusa L, Zilberman Y, +Gilquin B, Khochbin S, Alexandrova AY, Verkhovsky AB, Shemesh T, Kozlov MM (2005) Assembly and mechanosensory function of focal adhesions: experiments and models. Eur J Cell Biol 85:165–173 160. Lauffenburger DA, Horwitz AF (1996) Cell migration: a physically integrated molecular process. Cell 84:359–369 161. Roy P, Rajfur Z, Pomorski P, Jacobson K (2002) Microscopebased techniques to study cell adhesion and migration. Nat Cell Biol 4:E91–E96 162. Webb DJ, Brown CM, Horwitz AF (2003) Illuminating adhesion complexes in migrating cells: moving toward a bright future. Curr Opin Cell Biol 15:614–620 163. Wouters FS, Verveer PJ, Bastiaens PI (2001) Imaging biochemistry inside cells. Trends Cell Biol 11:203–211 164. Sahai E (2005) Mechanisms of cancer cell invasion. Curr Opin Genet Dev 15:87–96 165. Peter M, Ameer-Beg SM (2004) Imaging molecular interactions by multiphoton FLIM. Biol Cell 96:231–236 166. Condeelis JS, Wyckoff J, Segall JE (2000) Imaging of cancer invasion and metastasis using green fluorescent protein. Eur J Cancer 36:1671–1680 167. Friedl P, Wolf K (2003) Tumour-cell invasion and migration: +diversity and escape mechanisms. Nat Rev Cancer 3:362–374 168. Hoffman RM, Yang M (2006) Subcellular imaging in the live mouse. Nat Protoc 1:775–782 169. Pinner S, Sahai E (2008) Imaging amoeboid cancer cell motility in vivo. J Microsc 231:441–445 170. Hoffman RM (2009) Imaging cancer dynamics in vivo at the tumor and cellular level with fluorescent proteins. Clin Exp Metastasis 26:345–355 171. Provenzano PP, Eliceiri KW, Keely PJ (2009) Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment. Clin Exp Metastasis 26:357–370 172. King RD, Rowland J, Oliver SG, Young M, Aubrey W, Byrne E, +Liakata M, Markham M, Pir P, Soldatova LN, Sparkes A, +Whelan KE, Clare A (2009) Make way for robot scientists. + +Science 325:945 173. Rabut G, Ellenberg J (2004) Automatic real-time three-dimensional cell tracking by fluorescence microscopy. J Microsc 216:131–137 \ No newline at end of file diff --git a/medical/md/PMC2948382.md b/medical/md/PMC2948382.md new file mode 100644 index 0000000000000000000000000000000000000000..27e8b0c1439215d1d4f1c0e63cec87d64e34bce1 --- /dev/null +++ b/medical/md/PMC2948382.md @@ -0,0 +1,131 @@ +# Diagnosing Malabsorption In The Intensive Care Unit + +![0_Image_0.Png](0_Image_0.Png) Martijn B Keur, Albertus Beishuizen And Adriaan A Van Bodegraven* + +Address: Departments of Intensive Care and Gastroenterology, VU University Medical Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands +* Corresponding author: Adriaan A van Bodegraven (v.bodegraven@vumc.nl) +2010, 2:7 (doi:10.3410/M2-7) +for non-commercial purposes provided the original work is properly cited. You may not use this work for commercial purposes. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/3.0/legalcode), which permits unrestricted use, distribution, and reproduction in any medium, F1000 Medicine Reports The electronic version of this article is the complete one and can be found at: http://f1000.com/reports/medicine/content/2/7 + +## Abstract + +Malabsorption as a result of decreased intestinal function is a frequently occurring problem in intensive care units. Small bowel dysfunction may lead to malnutrition and may predispose patients to infectious complications (sepsis) and may be linked to increased hospitalization duration, morbidity and mortality. There are several small bowel function tests, such as faecal fat excretion and sugar absorption tests, but data specifically applicable to the intensive care setting are limited. Moreover, there are multiple confounders such as altered intestinal permeability, changed transit time and renal dysfunction. A more specific test, fasting plasma citrulline concentration, has poor test characteristics for detection of decreased intestinal function in patients with dysfunctional enterocytes. Therefore, a citrulline generation test was recently developed in order to measure the conversion of glutamine (administered either enterally or intravenously) into citrulline. This conversion takes place exclusively in a properly functioning enterocyte; therefore, citrulline generation upon glutamine stimulation may be a promising biomarker for assessment of the small bowel function. + +## Introduction And Context + +Malabsorption in intensive care unit (ICU) patients is arbitrarily defined as an intestinal absorption capacity of 85% or less. It is believed to be a frequently occurring problem in ICUs, particularly in patients suffering from sepsis or multiple organ dysfunction [1,2]. Malabsorption is the most important clinical sign of intestinal dysfunction or failure, reflecting various altered aspects of intestinal physiology due to a diseased state. Some of these altered aspects are coordinated intestinal transport +(motility) and coordinated exocrine digestive functions of various organs like the pancreas, liver and jejunum (enterocytes). Other factors involve the intestinal wall function, like mucosal immunological response and intestinal barrier function, as well as visceral blood flow. + +Critically ill patients may be prone to a reduced enterocyte function due mainly to a reduced splanchnic blood flow. This dysfunction may subsequently contribute to (the alleged) bacterial translocation and ultimately sepsis and multi-organ dysfunction syndrome +(MODS) [3,4]. Simple, clear-cut and validated tests to assess these various aspects of coordinated physiological functions of digestion are not available at the bedside. + +Nevertheless, adequate absorption of food is paramount to the improvement of the patient's condition and to the outcome of the critically ill patient [5]. This concise review aims to emphasize the importance of diagnosing malabsorption and to give new insights into how to assess intestinal function. + +Current tests In critically ill patients, it may be cumbersome to assess the various aspects of digestion as there are no validated tests to quantify the small bowel function. To meet this challenge, we tend to approach the problem as a failure of the intestinal tract comparable with other organ failures as a part of MODS (such as renal failure). + +However, a simple blood test or marker such as creatinine or BUN (blood urea nitrogen) levels is lacking in the evaluation of intestinal function. Thus, secondary variables, such as measurement of intestinal barrier function and intestinal absorption, are usually applied. Relatively common small bowel function tests are the sugar absorption test (SAT), quantification of fat excretion in stools (FES) and bomb calorimetry (BC). + +Sugar absorption test Gastrointestinal permeability can be measured by the SAT. During this test, a solution of multiple saccharides is orally administered [6-8]. The applied saccharides have different molecule sizes and are specifically chosen as these cannot be metabolized in the human body. The tight junctions in the tip of the villi allow translocation of small saccharides, whereas translocation of larger saccharides is restricted to the crypts [6]. In a normally functioning small bowel, the permeability for small saccharides is higher than for larger saccharides. However, under pathological conditions, the permeability for small saccharides decreases whereas the permeability for larger saccharides increases [7]. The amount of renallyexcreted saccharides (particularly the large saccharide/ +small saccharide ratio) is an indicator of small bowel absorptive function [7,8]. There are some flaws in this test. By means of SAT, intestinal (paracellular) permeability rather than (metabolic) enterocyte function is assessed [7]. In addition, Oudemans-van Straaten et al. + +[9] reported that the SAT is unreliable when used on ICU patients with multiple organ failure because there are various non-permeability-related confounders, such as impaired renal function and diminished intestinal motility. Also, oliguria/anuria limits its use in daily ICU practice. + +Quantification of fat excretion in stools This test aims to quantify the FES. The patient is given a diet containing a known high amount of fat to saturate the intestinal mechanisms and to challenge the intestinal maximal absorptive capacity. During this diet, the patient's faeces are sampled for 24 hours or, preferably, 72 hours. + +The amount of faecal fat can be measured by the Van de Kamer method, which allows the calculation of fat absorption capacity [10]. The underlying concept of FES +is that malabsorption of fat correlates with malabsorption in general. However, Wierdsma and van Bodegraven [11] +found that not all malabsorption states are detected by measuring fat in faeces. Faecal carbohydrate loss significantly contributed to the malnutrition state, and this contribution is overlooked by looking merely at FES [11]. + +Bomb calorimetry Faecal energy is measured by combustion of a faecal sample and subsequent calculation of 72 hours of faecal energy loss, which is considered the (alleged) gold standard for intestinal digestive function [12,13]. Combining the energy (caloric) intake and the faecal energy loss into a formula allows the calculation of intestinal energy absorption (percentage). Again, data are scarce, specifically in the critically ill, in whom BC is hampered by problems with reliable collection of faeces during a full 3-day period [1]. + +## Recent Advances + +Citrulline is an amino acid that is produced almost entirely from glutamine in the enterocytes. Because this conversion can take place only in a functioning enterocyte, citrulline has been proposed as an attractive and promising biomarker to assess the function of the small intestine [14,15]. Previous studies reported the value of fasting plasma citrulline concentration as a biomarker for enterocyte (intestinal) mass in a wide array of intestinal diseases such as coeliac disease, Crohn's disease, short bowel syndrome and radiation enteritis, and in recipients of intestinal transplants [16-21]. + +Recently, however, fasting plasma citrulline concentration was found to be a poor predictor of the clinical relevance of decreased intestinal function in patients with short bowel syndrome and, in other words, failed to predict long-term dependence on total parenteral nutrition [22,23]. + +Citrulline generation test The citrulline generation test (CGT) is a recently developed test to dynamically measure the conversion of glutamine into citrulline over time, following administration of glutamine as a dipeptide with alanine +(Dipeptiven). Through the administration of a large quantity of glutamine-alanine, the capacity of this enterocytic metabolic pathway is challenged, and this challenge is manifested by a decreased conversion in patients with a decreased enterocyte mass. The CGT is validated in various intestinal diseases [14]. In practice, following a slow intravenous injection of 100 mL of glutamine-alanine, multiple blood samples are drawn at different times to determine the increment (in other words, generation) in serum level of citrulline by using reverse-phase high-performance liquid chromatography +[24]. The CGT is easily applied in patients with suspected small bowel dysfunction. The citrulline generation [as defined by either the citrulline increase or the AUC (area under the curve)] correlates closely with enterocyte mass and enterocyte function and reflects the amount of functioning enterocytes to generate citrulline. In stable ICU patients, preliminary results from our own group indicate a reduced enterocyte mass that was associated with disease severity. + +## Implications For Clinical Practice + +The small intestine has many functions, two of which are of major importance in the setting of critical illness: nutrient and energy absorption and function of the intestinal barrier against antigens. The absorption of nutrients, water and drugs is an essential part of improving or regaining health [5]. Malnutrition and insufficient bioavailability of drugs can increase hospitalization stay, morbidity, mortality and costs [5]. The intestinal barrier function is a very important intestinal feature that keeps bacteria from translocating into the blood and thus prevents sepsis from originating from the gut [3,4]. It is obvious that dysfunction of the small intestine may have serious consequences and that identifying those patients who are at risk for developing malabsorption or those who are already malnourished is therefore pivotal. + +The most common applied intestinal function tests - faecal, urine and blood tests alike - all have several major flaws (in particular in very ill patients), which prevent these tests from being used and render it very difficult to accurately quantify intestinal function in the ICU setting. + +The CGT, a more functional test, measures the enterocyte function and is likely to play an important role in the assessment of intestinal function in the future. The CGT +is relatively easy to perform but warrants further validation in several ICU patient cohorts by comparing it with one or more of the other small bowel function tests and by taking nutritional status into account. + +## Abbreviations + +BC, bomb calorimetry; BUN, blood urea nitrogen; CGT, citrulline generation test; FES, fat excretion in stools; ICU, intensive care unit; MODS, multi-organ dysfunction syndrome; SAT, sugar absorption test. + +## Competing Interests + +The authors declare that they have no competing interests. + +## References + +1. Strack van Schijndel RJ, Wierdsma NJ, van Heijningen EM, Weijs PJ, de Groot SD, Girbes AR: Fecal energy losses in enterally fed intensive care patients: an explorative study using bomb calorimetry. Am J Gastroenterol 2007, 102:1-6. + +2. Carlson GL: Surgical management of intestinal failure. Proc Nutr Soc 2003, 62:711-8. + +3. Doig CJ, Sutherland LR, Sandham JD, Fick GH, Verhoef M, +Meddings JB: Increased intestinal permeability is associated with the development of multiple organ dysfunction syndrome in critically ill ICU patients. Am J Respir Crit Care Med 1998, 158:444-51. + +4. Deitch EA: Multiple organ failure: pathophysiology and potential future therapy. Ann Surg 1992, 216:117-34. + +5. Martindale RG, McClave SA, Vanek VW, McCarthy M, Roberts P, +Taylor B, Ochoa JB, Napolitano L, Cresci G; American College of Critical Care Medicine; A.S.P.E.N. Board of Directors: Guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: Society of Critical Care Medicine and American Society for Parenteral and Enteral Nutrition: Executive Summary. Crit Care Med 2009, 37:1757-61. + +6. Bjarnason I, MacPherson A, Hollander D: Intestinal permeability: +an overview. Gastroenterology 1995, 108:1566-81. + +7. van Elburg RM, Uil JJ, Kokke FT, Mulder AM, van de Broek WG, +Mulder CJ, Heymans HS: Repeatability of the sugar-absorption test, using lactulose and mannitol, for measuring intestinal permeability for sugars. J Pediatr Gastroenterol Nutr 1995, 20:184-8. + +8. Hessels J, Eidhof HH, Steggink J, Roeloffzen WW, Wu K, Tan G, van de Stadt J, van Bergeijk L: Assessment of hypolactasia and sitespecific intestinal permeability by differential sugar absorption of raffinose, lactose, sucrose and mannitol. Clin Chem Lab Med 2003, 41:1056-63. + +9. Oudemans-van Straaten HM, van der Voort PJ, Hoek FJ, Bosman RJ, +van der Spoel JI, Zandstra DF: Pitfalls in gastrointestinal permeability measurement in ICU patients with multiple organ failure using differential sugar absorption. Intensive Care Med 2002, 28:130-8. + +10. Van de Kamer JH, Ten Bokkel Huinink H, Weyers HA: Rapid method for determination of fat in feces. J Biol Chem 1949, 177:347-55. + +11. Wierdsma NJ, van Bodegraven AA: Energy losses from a short bowel: not only fat. Ned Tijdschr Geneeskd 2005, 149:381-4. + +12. Miller DS, Payne PR: A ballistic bomb calorimeter. Br J Nutr 1959, 13:501-8. + +13. Lovelady HG, Stork EJ: An improved method for preparation of feces for bomb calorimetry. Clin Chem 1970, 16:253-4. + +14. Peters JHC, Wierdsma NJ, Teerlink T, van Leeuwen PAM, Mulder CJJ, +van Bodegraven AA: The citrulline generation test: proposal for a new enterocyte function test. Aliment Pharmacol Ther 2008, 27:1300-10. + +15. Crenn P, Messing B, Cynober L: Citrulline as a biomarker of intestinal failure due to enterocyte mass reduction. Clin Nutr 2008, 27:328-39. + +16. Crenn P, Vahedi K, Lavergne-Slove A, Cynober L, Matuchansky C, +Messing B: Plasma citrulline: a marker of enterocyte mass in villous atrophy-associated small bowel disease. Gastroenterology 2003, 124:1210-9. + +17. Miceli E, Poggi N, Missanelli A, Bianchi P, Moratti R, Corazza GR: Is serum citrulline measurement clinically useful in coeliac disease? Intern Emerg Med 2008, 3:233-6. + +18. Rhoads JM, Plunkett E, Galanko J, Lichtman S, Taylor L, Maynor A, +Weiner T, Freeman K, Guarisco JL, Wu GY: Serum citrulline levels correlate with enteral tolerance and bowel length in infants with short bowel syndrome. J Pediatr 2005, 146:542-7. + +19. Lutgens LC, Blijlevens NM, Deutz NE, Donnelly JP, Lambin P, de Pauw BE: Monitoring myeloablative therapy-induced small bowel toxicity by serum citrulline concentration: a comparison with sugar permeability tests. Cancer 2005, 103:191-9. + +20. Papadia C, Sherwood RA, Kalantzis C, Wallis K, Volta U, Fiorini E, +Forbes A: Plasma citrulline concentration: a reliable marker of small bowel absorptive capacity independent of intestinal inflammation. Am J Gastroenterol 2007, 102:1474-82. + +21. Pappas PA, Saudubray JM, Tzakis AG, Rabier D, Carreno MR, GomezMarin O, Huijing F, Gelman B, Levi DM, Nery JR, Kato T, Mittal N, +Nishida S, Thompson JF, Ruiz P: Serum citrulline as a marker of acute cellular rejection for intestinal transplantation. Transplant Proc 2002, 34:915-7. + +22. Peters JHC, Wierdsma NJ, Teerlink T, van Leeuwen PAM, Mulder CJJ, +van Bodegraven AA: Poor diagnostic accuracy of a single fasting plasma citrulline concentration to assess intestinal energy absorption capacity. Am J Gastroenterol 2007, 102:2814-9. + +23. Luo M, Fernández-Estívariz C, Manatunga AK, Bazargan N, Gu LH, +Jones DP, Klapproth JM, Sitaraman SV, Leader LM, Galloway JR, +Ziegler TR: Are plasma citrulline and glutamine biomarkers of intestinal absorptive function in patients with short bowel syndrome? J Parenter Enteral Nutr 2007, 31:1-7. + +24. Teerlink T, van Leeuwen PA, Houdijk A: Plasma amino acids determined by liquid chromatography within 17 minutes. Clin Chem 1994, 40/2:245-9. + +Page 3 of 3 diff --git a/medical/md/PMC3165116.md b/medical/md/PMC3165116.md new file mode 100644 index 0000000000000000000000000000000000000000..ea7965e088a845a5032c3c07b189617a6d7d2bff --- /dev/null +++ b/medical/md/PMC3165116.md @@ -0,0 +1,26 @@ +# Comment On Cho Et Al.: Usefulness Of Fdg Pet/Ct In Determining Benign From Malignant Endobronchial Obstruction + +Astrid A. M. van der Veldt & Mark Lubberink & Adriaan A. Lammertsma & Egbert F. Smit Received: 26 March 2011 /Accepted: 23 April 2011 / Published online: 18 June 2011 +\# The Author(s) 2011. This article is published with open access at Springerlink.com Dear Sir, With interest we read the study by Cho et al [1], evaluating the usefulness of FDG PET/CT for differentiating malignant endobronchial lesions with distal atelectasis from benign bronchial stenosis. In this retrospective study, they adequately demonstrated the additional value of FDG PET/ +CT in this challenging clinical problem. Besides differentiation of endobronchial lesions, accurate discrimination between malignant tumours and atelectatic lung tissue can facilitate the definition of radiotherapy target volumes. As a result, the size of radiotherapy fields can be decreased [2], +potentially reducing radiotherapy-induced side-effects. + +A reply to this letter can be found at doi:10.1007/s00330-011-2170-y. + +A. A. M. van der Veldt (*) : M. Lubberink : A. A. Lammertsma Department of Nuclear Medicine & PET Research, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands e-mail: aam.vanderveldt@vumc.nl E. F. Smit Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands For these purposes, the PET technique may be of additional value to discriminate the obstructive lesion from the atelectatic lung tissue. Although Cho et al [1] +mentioned different patterns of FDG uptake in atelectatic lung tissue, we wonder whether the FDG uptake in the obstructive lesions could be adequately distinguished from the FDG uptake in the atelectatic lung tissue. As FDG +uptake is dependent on perfusion for its delivery to tissue, variation in tissue vascularity may contribute to variable FDG uptake in obstructive atelectasis. In a previous PET/ +CT study, we quantified tumour perfusion in lung cancer patients using radiolabelled water ([15O]H2O) [3]. The measured perfusion values in atelectatic lung tissue differed significantly as compared with perfusion values in malignant tumours. Figure 1 shows an example of a patient with relatively high perfusion in atelectatic lung tissue as compared with the malignant tumour. These observations suggest that perfusion measurements may have additional value to discriminate atelectatic lung tissue from malignant lesions. Therefore, we believe that additional perfusion measurements of atelectatic lung tissue may improve diagnosis and radiotherapy planning of malignant endobronchial lesions. + +![1_image_0.png](1_image_0.png) + +Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. + +## References + +1. Cho A, Hur J, Kang WJ, Cho HJ, Lee JH, Yun M, Lee JD (2011) +Usefulness of FDG PET/CT in determining benign from malignant endobronchial obstruction. Eur Radiol 21:1077–1087. doi:10.1007/ +s00330-010-2006-1 2. Nestle U, Walter K, Schmidt S, Licht N, Nieder C, Motaref B, +Hellwig D, Niewald M, Ukena D, Kirsch CM, Sybrecht GW, +Schnabel K (1999) 18F-deoxyglucose positron emission tomography (FDG-PET) for the planning of radiotherapy in lung cancer: +high impact in patients with atelectasis. Int J Radiat Oncol Biol Phys 44:593–597 3. Van der Veldt AA, Hendrikse NH, Harms HJ, Comans EF, Postmus PE, Smit EF, Lammertsma AA, Lubberink M (2010) Quantitative parametric perfusion images using 15O-labeled water and a clinical PET/CT scanner: test-retest variability in lung cancer. J Nucl Med 51:1684–1690 \ No newline at end of file diff --git a/medical/md/PMC3168093.md b/medical/md/PMC3168093.md new file mode 100644 index 0000000000000000000000000000000000000000..291c68d3366131abe060f5622c366fbfd8f83645 --- /dev/null +++ b/medical/md/PMC3168093.md @@ -0,0 +1,45 @@ +# Case Report + +www.ijpm.info + +# An Atypical Presentation Of Obsessive Compulsive Disorder With Difficulty In Hearing + +Indrapal Singh, Anshul K. Rana, Malay K. Singh, Rakesh K. Tripathi + +## Abstract + +Obsessive compulsive disorder (OCD) is a common psychiatric disorder which is easily recognized. However, sometimes patients of OCD present in such an atypical or bizarre way that their problem comes to notice as being a psychiatric disorder after multiple consultations in different specialties. We are reporting a case of a man who had first sought opinion in the Department of Ear, Nose and Throat (ENT) for hearing impairment. He was then referred to a neurologist and a general physician for evaluation of neurological cause of his symptom. As no pathology related to ENT or neurology could be detected, he was referred to the Department of Psychiatry. The patient's chief complaints were difficulty in hearing and inability to understand at once. He could be diagnosed as a case of OCD after meticulous evaluation and studying his response to treatment. There was significant improvement in all the presenting symptoms over a period of 6 weeks on 60 mg of fluoxetine. + +Key words: *Atypical presentation, hearing impairment, obsessive compulsive disorder* + +## Introduction + +Obsessive compulsive disorder (OCD) is a common psychiatric disorder (2% - 3% of the general population) which is diagnosed easily most of the time by the psychiatrist or even the physician.[1] It is sometimes very difficult to properly diagnose a person for OCD based only on an interview.[2] The presentation of OCD may be so atypical or unusual that the patients may be referred to different specialties before the actual diagnosis can be made. Neuropsychological studies have consistently found cognitive impairment in the domains of memory and attention in patients of OCD. Anxiety, lack of confidence, indecisiveness, associated clinical symptoms of OCD, along with impaired memory and attention, may further complicate or interfere in the delivery of information.[2,3,4] + +## Case Report + +A 36-year-old man used to request multiple repetitions of spoken words in order to understand what was said. Relatives and family members advised him to consult an ENT surgeon for hearing impairment. No organic cause of hearing impairment was found. He was then referred to a neurologist and a general physician for evaluation of neurological cause of his symptoms. Even after multiple consultations with the ENT, neurology and medicine specialists, no organic pathology to explain his symptoms could be detected. The patient lost considerable time and money because of delay in diagnosis caused by the atypical presentation to specialties other than Psychiatry. At last, the patient was referred to the Department of Psychiatry for further evaluation. The patient's chief complaints were difficulty in hearing, grasping or comprehending the components of conversations, interactions or clinical interviews at once. He had this problem for the last 6 months. During the interview, almost every question or the instruction had to be repeated slowly 2 to 3 times or more to make him understand. He was anxious and expressed regret repeatedly for the inconvenience caused to the doctor because of his disability. Initially the patient was put on mirtazapine 15 mg/day and clonazepam 0.75 mg/day, keeping in mind the possibility of anxiety disorder NOS (not otherwise specified). There was no significant improvement even after 4 weeks of Department of Geriatric Mental Health, CSM Medical University, Consultant Psychiatrist, RML Hospital, Lucknow, India Address for correspondence: Dr. Anshul K. Rana, A-144, Indira Nagar, Lucknow - 226016, UP, India. E-mail: drakrana@yahoo.com DOI: 10.4103/0253-7176.63581 therapy. The case was reviewed from the diagnosis point of view. Mental state examination of the patient revealed impaired attention and concentration with depressive and anxiety symptoms. Detailed evaluation of the patient revealed that his mind remained full of unnecessary thoughts. However, the patient could not express further details to enable definite diagnosis. The possibility of OCD, predominantly obsessive type, was kept on the basis of answers of leading questions, and he was put on fluoxetine 60 mg/day (titrated from 40 to 60 mg) and clonazepam 0.5 mg/day. There was drastic improvement in all the presenting symptoms, including his ability to grasp and comprehend conversation, along with improvement in anxiety symptoms and clarity in thinking over a period of 6 weeks. The patient was then able to report his pre-treatment problem and the level of improvement comprehensively. He accepted that he was unable to understand or express his own problem before treatment. According to him, there were thoughts keeping his mind busy all the time, to the extent that he was unable to think or communicate his problem effectively. According to him, initially he used to make frequent unsuccessful attempts to get rid of these excessive and unnecessary thoughts. According to the wife of the patient, after treatment the patient was able to hear clearly. The diagnosis could be confirmed as OCD on the basis of patient's spontaneous elaboration of symptoms and the level of improvement. + +## Discussion + +In this case, the presentation of OCD is atypical, as the chief complaint was problem in hearing. He was able to hear on normal volume of sound, but the sentences needed to be repeated multiple times. He was not hard of hearing. Moreover, no ENT-related or neurological cause of hearing impairment was found in the patient. + +Though hearing difficulty was not his chief subjective problem, he consulted the ENT doctor only after repeated and forced advice by his wife and relatives. The hearing problem as perceived by the patient's friends and relatives was basically a misinterpretation of the patient's problem secondary to his psychiatric illness. Also the patient was unable to express his actual problem at the time of presentation. The patient used to ask for repetition of the spoken sentences multiple times in order to grasp the components of interaction because of his significantly impaired attention. However, there could be the other possibility that this behavior could be a compulsive act of the patient to confirm his doubts. The patient could not confirm the latter. The patient could not express his problem elaborately, which was one of the reasons for not being able to make an early diagnosis. + +There were multiple factors that had complicated and hindered the narration of history by the patient. Lack of clarity of thoughts and associated clinical symptoms of OCD (especially predominantly obsessive type), indecisiveness, anxiety, lack of confidence, along with impaired memory and attention, were the possible factors in this case, as also reported in different studies.[2,3,4] The patient could express details of his illness comprehensively once his above-mentioned signs and symptoms improved significantly. His wife and relatives also reported improvement in his hearing, which was actually the improvement in his attention and OCD symptoms. Such an atypical presentation may lead to misdiagnosis, delay in diagnosis, loss of time and money as the patient may present to other specialties instead of Psychiatry. High suspicion for diagnosis of OCD and response to treatment may help in managing such cases. + +## Conclusion + +Obsessive compulsive disorder is usually easily recognized, but sometimes its presentation is so atypical or bizarre that the problem comes to notice as being a psychiatric disorder after multiple consultations in different specialties. But if proper evaluation is done, such cases can easily be recognized and treated effectively. + +## References + +1. Crino R, Slade T, Andrews G. The changing prevalence and severity of OCD criteria from DSM-III to DSM-IV. Am J Psychiatry 2005;162:876-82. + +2. Penzel F. Obsessive compulsive disorders. New York: Oxford University; 2000. p. 308. + +3. Muller J, Roberts JE. Memory and attention in obsessivecompulsive disorder: A review. J Anxiety Disord 2005; 19:1-28. + +4. Deckersbach T, Otto MW, Savage CR, Baer L, Jenike MA. The relationship between semantic organization and memory in obsessive-compulsive disorder. Psychother Psychosom 2000;69:101-7. + +Source of Support: Nil, **Conflict of Interest:** None. \ No newline at end of file diff --git a/medical/md/PMC4120424.md b/medical/md/PMC4120424.md new file mode 100644 index 0000000000000000000000000000000000000000..4448c2801571ea4a7982fa8b027966b8e3eda16e --- /dev/null +++ b/medical/md/PMC4120424.md @@ -0,0 +1,370 @@ +# Is There A Causal Link Between Knee + +loading and knee osteoarthritis progression? A systematic review and meta-analysis of cohort studies and randomised trials Marius Henriksen,1 Mark W Creaby,2 Hans Lund,3 Carsten Juhl,3,4 Robin Christensen1,3 To cite: Henriksen M, +Creaby MW, Lund H, et al. Is there a causal link between knee loading and knee osteoarthritis progression? A +systematic review and metaanalysis of cohort studies and randomised trials. BMJ Open 2014;4:e005368. + +doi:10.1136/bmjopen-2014005368 +▸ Prepublication history and additional material is available. To view please visit the journal (http://dx.doi.org/ 10.1136/bmjopen-2014005368). Received 31 March 2014 Revised 16 June 2014 Accepted 23 June 2014 For numbered affiliations see + +![0_image_0.png](0_image_0.png) end of article. Correspondence to Dr Robin Christensen; robin.christensen@frh. regionh.dk + +## Abstract + +Objective: We performed a systematic review, metaanalysis and assessed the evidence supporting a causal link between knee joint loading during walking and structural knee osteoarthritis (OA) progression. + +Design: Systematic review, meta-analysis and application of Bradford Hill's considerations on causation. + +Data sources: We searched MEDLINE, Scopus, AMED, CINAHL and SportsDiscus for prospective cohort studies and randomised controlled trials (RCTs) from 1950 through October 2013. + +Study eligibility criteria: We selected cohort studies and RCTs in which estimates of knee joint loading during walking were used to predict structural knee OA progression assessed by X-ray or MRI. + +Data analyses: Meta-analysis was performed to estimate the combined OR for structural disease progression with higher baseline loading. The likelihood of a causal link between knee joint loading and OA progression was assessed from cohort studies using the Bradford Hill guidelines to derive a 0–4 causation score based on four criteria and examined for confirmation in RCTs. + +Results: Of the 1078 potentially eligible articles, 5 prospective cohort studies were included. The studies included a total of 452 patients relating joint loading to disease progression over 12–72 months. There were very serious limitations associated with the methodological quality of the included studies. The combined OR for disease progression was 1.90 (95% +CI 0.85 to 4.25; I2=77%) for each one-unit increment in baseline knee loading. The combined causation score was 0, indicating no causal association between knee loading and knee OA progression. No RCTs were found to confirm or refute the findings from the cohort studies. + +Conclusions: There is very limited and low-quality evidence to support for a causal link between knee joint loading during walking and structural progression of knee OA. + +Trial registration number: CRD42012003253 + +## Strengths And Limitations Of This Study + +- We performed the first systematic evaluation of the evidence for a causal link between mechanical loading of the knee during walking and structural progression of knee osteoarthritis. + +- This systematic review, meta-analysis, and causation analysis find very limited evidence of a causal link between knee joint loading during walking and structural progression of knee osteoarthritis. + +- Few studies were included in the meta-analysis since only a small number of studies have been conducted. Further research is needed. + +## Background + +Osteoarthritis (OA) is the most common form of arthritis affecting a large section of the population, and is a major cause of illness and disability.1 2 The knee joint is most frequently affected,3 and due to the knee's crucial role in independent ambulation, knee OA leads to considerable disability affecting an individual's participation in society and independent living. Because no cure is available and many people are affected, healthcare costs associated with knee OA are enormous—even higher than for the more high-profile diseases such as diabetes, cancer and cardiovascular diseases.4 With its prevalence on the rise, knee OA poses a substantial socioeconomic and public health burden. + +Walking is the most common form of human locomotion, and most people walk every day, resulting in millions of steps per year. Biomechanical loading on the knee joint during walking is estimated to exceed 2–3 times body weight5; thus, knee joint loading is unavoidable component of an independent lifestyle. One widely accepted theory is that higher knee joint loading during walking is causally linked with accelerated structural knee OA progression.6–8 In accordance with this theory, unloading is advocated in an attempt to slow or halt disease progression.9 Unspecific measures of high joint loading, such as excess body mass and body mass index (BMI), have been associated with OA development,10 11 and obesity is believed to act mainly through high mechanical loads,12 although systemic effects have also been indicated.13 However, higher body mass does not necessarily lead to higher joint loading during walking,14 and more specific measures of joint loading are necessary to apply. + +Objective estimates of joint loading during walking can be obtained by three-dimensional gait analysis. + +Typically, the knee adduction moment (KAM) or biomechanical modelling of compression forces is used. + +The KAM has been of particular interest because it reflects the medial to lateral joint load distribution,15 with good face validity with respect to the relative prevalence of medial, as opposed to lateral compartment, tibiofemoral knee OA.16 Despite its common usage, a recent systematic review did not find consistent evidence to the fact that the KAM differs between those with and without knee OA.17 The relationship between knee joint loading during walking and knee OA has been a focus of knee OA-related biomechanical research for 10–20 years. + +Studies reveal that symptomatic treatment of knee OA results in increases in knee joint loads during walking,5 18–20 which are unwanted according to the hypothesised structural consequences of increased joint loading. This may generate confusion among researchers, clinicians and patients, who are interested in this information to aid them in prevention, palliation and treatment strategies. Establishing causal relationship among modifiable factors, such as knee joint loading, and disease-specific measures, such as structural disease progression, is imperative in the generation of effective strategies. + +Seminal epidemiological work by Hill proposed a set of considerations (the Bradford Hill criteria) to systematically evaluate the existence of a causal link between an exposure and a health outcome.21 These criteria have been previously employed to demonstrate the causal link between smoking and lung cancer,22 and between dietary factors and coronary heart disease,23 and have the capacity to evaluate the link between knee joint loading and structural knee OA progression. + +Therefore, the objectives of this study were (1) to systematically evaluate the evidence supporting a causal link between exposure to knee joint loading during walking and structural progression of knee OA based on Hill's considerations on causality21 and (2) to determine which knee joint load variables had been studied sufficiently in randomised controlled trials (RCTs) and found to support the findings of prospective cohort studies. + +## Methods + +The methods of the study search strategy, inclusion criteria and data analysis were prespecified in a protocol +(see online supplementary file 1) and pre-registered +(PROSPERO 2012:CRD42012003253). + +We searched MEDLINE, Scopus, AMED, CINAHL and SportsDiscus for prospective cohort studies and RCTs from 1950 through October 2013. Search strategies are available as an online supplementary file 2. The reference lists of the retrieved articles were also searched for additional cohort studies and RCTs. Two reviewers (MH and MWC) independently assessed study eligibility. Excluded studies and reasons for exclusion were recorded. Disagreement was resolved by discussion and consensus. We included original full-length articles pertaining to the association between knee joint loading during walking, measured by three-dimensional gait analysis, and structural disease progression assessed quantitatively or semiquantitatively by X-ray or MRI. We considered only those studies that included participants diagnosed with knee OA that were followed for at least 1 year (necessarily because of the sensitivity of imaging and slow disease progression24). Cohort studies had to include estimates of knee joint loading during walking at baseline and imaging-based assessment of structural disease progression. Clinical trials had to be randomised and compare image-based structural disease progression among different knee joint loading interventions (eg, increase vs decrease), placebo or control. + +## Data Extraction And Analysis + +The following data were extracted from the studies: (1) +Study design; (2) Country of origin; (3) Number of subjects; (4) Characteristics of the subjects; (5) Type of knee joint loading estimate; (6) Magnitudes of the estimate; (7) Description of the interventions (if any) and +(8) Structural disease progression outcome (ie, semiquantitative or quantitative measures on X-ray and/or MRI). + +For prospective cohort studies, we extracted estimates of the association between baseline knee joint loading and structural disease progression from baseline to follow-up. A progression group is typically compared to a non-progression group at baseline and the results are typically reported as ORs for disease progression for a one-unit higher baseline knee joint loading. If no OR was reported, we either calculated it from the reported data (if possible), or contacted the study authors and requested for the OR. For the RCTs we aimed to compare relative risks of structural disease progression between knee joint loading modification and control groups. + +The individual study results (ie, log OR values and their corresponding SEs) were combined in a random effects meta-analysis model using the generic inverse variance outcome type in Review Manager.25 Heterogeneity between trials was assessed using the standard Q-test statistic (testing the hypothesis of homogeneity), and we present the I2 value, which can be interpreted as the percentage of total variation across the studies due to heterogeneity.26 Methodological quality and risk of bias assessment The methodological quality of the cohort studies was assessed using a published check list.27 Two of the authors (MH and MWC) assessed this individually and judged each criterion to be 'Adequately described', +'Unclear', or 'Inadequately described'–corresponding to +'low risk of bias', 'unclear risk of bias', and 'high risk of bias', respectively. Disagreement was resolved by discussion. If an included study was authored by one or more of the current authors, a third reviewer (HL) was asked to perform a quality assessment. One quality assessment item was omitted ("Was a dose–response relationship between exposure and outcome demonstrated?") +because this item relates to the findings of the study and not the methodological quality, and assessment of dose– +response forms part of the Bradford Hill criteria for causation (see below). The overall extent of risk of bias and methodological quality within each study was assessed using the GRADE approach to evaluate study limitations.28 Evaluation of evidence for causality Based on the Bradford Hill considerations on causality,21 a causation score was developed and used to systematically evaluate the evidence of a causal link between knee joint loading during walking and structural progression of knee OA. A similar score has previously been derived from the Bradford Hill considerations to assess the causal relationship between dietary factors and coronary heart disease.23 The following criteria were used in the review of the cohort studies and given a score of 1 (criterion satisfied) or 0 (criterion not satisfied): +1. Strength of association: Associations quantified as a pooled OR ≥5.0, with lower 95% CI above 2.0; the expected direction was defined as 'strong association.' 'Moderate association' is defined as any statistically significant pooled OR (p<0.05). A statistically non-significant pooled OR was defined as 'no association.' +2. Consistency across studies: An association requires replication in other studies. Consistency is defined as +≥75% of associations being strong or moderate. + +3. Temporality: Refers to temporal relationship of association between exposure and outcome; exposure has to precede outcome. It is difficult to ensure temporal correctness because study participants are assumed to walk daily and are, therefore, exposed to knee joint loading throughout observation periods. We retained this criterion because temporality is necessary to infer causation; absence of temporal relationship between exposure and outcome precludes a causal link. Our analysis accepted studies with a temporally correct design defined as baseline knee joint loading related to disease progression over time from that baseline. The temporality criterion was satisfied when temporal correctness was accepted in ≥75% of the included studies. + +4. Biological gradient: When rate of progression increases +(or decreases) incrementally as dose of exposure increases; provides strong evidence of causal relationship. This criterion is satisfied when ≥75% of tests for a trend pertaining to structural progression outcomes are statistically significant in the expected direction for knee joint loading. + +The aforementioned four criteria scores were applied to derive one causation score for each knee joint loading variable. The scores were computed as the unweighted sum of the scores from each of the above criteria, for a possible range of 0–4. A score of 4 is considered strong evidence of a cause-and-effect relationship. A score of 3 is deemed to indicate moderate evidence. A score of 2 or less is considered weak evidence of causation. + +A fifth criterion, experimental evidence, was used to examine whether the evidence from the prospective cohort studies was consistent with that from RCTs. + +Experimental evidence enhances the probability of causation, and may be used to upgrade or downgrade the calculated causation score. + +The following four criteria were omitted: +Coherence: Causation is more likely if what is observed is supported by and in agreement with the natural history of the disease. This criterion is usually applied when the outcome is assessed by surrogate outcomes. + +This criterion is omitted because it is satisfied by default since imaging modalities are surrogate outcomes. + +Plausibility relates to the assessment of whether the association is plausible or not. This criterion is omitted because of the highly subjective nature of this criterion. + +Specificity relates to the specific response to the exposure. This criterion is omitted because OA is a multistructure disease; an association between knee joint loading and disease progression in multiple structures does not preclude a possible causal relationship. + +Analogy relates to the possibility that existing similar association can support causation (eg, Does the same association exist for hip or hand OA? If so, causation may be supported). This criterion is omitted because this review focuses specifically on knee OA. + +## Results Study Selection And Characteristics + +The search yielded 1078 potentially eligible studies +(figure 1; references available in online supplementary file 3). Of those, we included five prospective cohort studies published between 2002 and 2013 and involving a total of 452 patients with knee OA29–33 and 0 RCTs. + +The diagnostic criteria for knee OA used to determine participant inclusion in individual studies varied. The American College of Rheumatology (ACR) criteria34 were used in one study,32 whereas a combination of + +![3_image_0.png](3_image_0.png) + +clinical and radiographic signs of knee OA was used as criteria in three studies,29 31 33 and one study did not report the diagnostic criteria used.30 Study characteristics are summarised in table 1. + +Joint loading and structural progression measures Four cohort studies29–32 focused on the same knee joint loading estimate: the peak KAM. In these studies, peak KAM was assessed at baseline and related to structural medial disease progression assessed after a median of 15 months from that baseline (12–72 months). In one study,29 the baseline KAM impulse (area under the curve) was also related to medial disease progression. In the fifth study,33 overall knee compression loading was used as load exposure. + +Two studies30 31 defined structural disease progression as any worsening in semiquantitative radiographic medial joint space grade.36 One study32 defined structural disease progression as any loss of medial femoral cartilage volume (quantitative MRI) above a measurement error previously established. In another study,33 structural disease progression was assessed from semiquantitative grading of tibiofemoral cartilage loss and bone marrow lesions. In one study,29 structural progression was assessed semi-quantitatively as medial tibiofemoral cartilage loss and medial tibiofemoral bone marrow lesions, and quantitatively as medial tibial cartilage volume loss. For the meta-analysis, we extracted the association (OR) between peak KAM and semiquantitative progression in medial tibiofemoral cartilage loss. No ORs were available for the association between KAM and cartilage volume loss because both were continuous variables. However, the linear regression analysis was extracted for the assessment of the biological gradient criteria. + +Association between joint loading and progression ORs were not reported in three studies,31–33 but in32 individual patient data were reported and an OR was calculated. We contacted the authors of ref. 31 asking them to provide an OR if possible. The authors returned a conference abstract including an OR published 4 years prior to the article.35 The abstract recorded 64 patients in contrast to the 57 patients included in ref. 31 The authors could not account for the difference in number of the subjects between the full paper and conference abstract. One study33 grouped participants based on changes in joint loading following weight loss (increased vs reduced loads) and found no between group differences in structural disease progression. Owing to the use of an intervention (weight loss), it was not appropriate to extract OR for baseline loading. + +The ORs for structural disease progression ranged from 0.40 to 6.46 for each unit higher baseline peak KAM magnitudes. From the random effects model, the combined OR was estimated to 1.90 (95% CI 0.85 to 4.25). The confidence in this estimate was downgraded due to heterogeneity (I2=77%). Figure 2 summarises the individual and pooled estimates. + +In ref 29, the OR for progression of bone marrow lesions was 1.31 (95% CI 0.86 to 1.98) with every unit increment in baseline peak KAM; ORs for progression of cartilage defects and bone marrow lesions were 0.42 (95% CI 0.12 to 1.48) and 1.8 (95% CI 0.63 to 5.17), respectively, with every unit increment in baseline KAM impulse. Methodological quality of the studies One or more criteria were limited in the assessments of risk of selection, detection and attrition biases. Individual risk assessments are presented in online + +| Table 1 | Summary of the characteristics of the included studies | | | | | | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------|---------------------------|----------------------------------------|-----------|-----------|----|----| +| Author (year) | Knee loading | Structural | | | | | | +| country | variable | progression measure | Number of patients (total/progressors/ | Females | | | | +| non-progressors) | (%) | Reference | | | | | | +| Miyazaki et al (2002) Japan | Follow-up time (months) | | | | | | | +| Peak KAM (unit: | ≥1 grade | | | | | | | +| %BW×HT) | according to Altman atlas | sq | 72 | 74/42/32 | 78 | 30 | | +| X-ray | 18 | 56 (64*)/41/15 | 59 | 31 | | | | +| sq | 35* | | | | | | | +| X-ray | | | | | | | | +| Chang et al | Peak KAM (unit: | ≥1 grade | | | | | | +| (2007) USA | %BW×HT) | according to Altman atlas | | | | | | +| Bennell et al (2011) Australia | Peak KAM (unit: | ≥1 grade | | | | | | +| %BW×HT) | medial tibiofemoral cartilage defects | sq | 12 | 138/45/93 | 56 | 29 | | +| MRI | | | | | | | | +| KAM impulse | Cartilage | | | | | | | +| (unit: %BW×HT) | volume loss (mm3 ) | q | 144/NA/NA | | | | | +| MRI | 12 | 13/6/7 | 23 | 32 | | | | +| q MRI | | | | | | | | +| Woollard et al (2011) USA | Peak KAM (unit: | Cartilage | | | | | | +| Nm/kg)† | volume loss (mm3 ) | | | | | | | +| Henriksen et al (2013) Denmark | Peak overall knee compression force (unit: N) | Cartilage loss | sq | 12 | 157/NA/NA | 89 | 33 | +| MRI | Median: 12 | Totals: 452/134/147 | Mean: 67 | | | | | +| *On request, the authors forwarded a conference abstract with additional data based on 64 subjects. The number of progressors/ non-progressors was not available from that abstract. †We converted the data into %BW×HT after requesting for body weight and height data from the authors. KAM, knee adduction moment; BW, body weight; HT, height; q, quantitative; sq, semiquantitative grading; NA, non-applicable. | | | | | | | | + +supplementary appendix A. Across the studies, the comparability of the groups at baseline was generally inadequately or unclearly described (selection bias). Also, descriptions of how reliably the joint load exposures were ascertained were not included in four of the five studies (detection bias), and group differences in loss of subjects were not accounted for (attrition bias). The discrepancy in subject numbers in reports from one cohort,31 35 induced a high risk of selection and attrition bias. Table 2 presents summaries of the quality assessment. Agreement between quality raters was good +(κ=0.73 (95% CI 0.48 to 0.97)). + +## Causality Assessment + +The non-significant combined OR indicates no association between knee joint load during walking and structural disease progression. Thus, a score of 0 in the Strength of Association criterion was assigned. The associations were not consistently replicated (50%), +resulting in a score of 0 in the Consistency criterion. This finding was further supported by the heterogeneity between the studies (I2=77%; figure 2), and the discrepancies between baseline peak KAM magnitudes among progressors and non-progressors across the cohorts +(figure 3). Temporal correctness was accepted in one cohort.30 After requesting for additional information from the authors of one cohort study,31 we discovered that the article reported only part of the study. The additional information retrieved35 showed that the study consisted of three measurements taken every 18 months. Joint space and Kellgren-Lawrence grades (X-rays) were recorded at all three time points, whereas the KAM was obtained at time points 2 and 3 (18 and 36 months). KAM at time point 2 (18 months) was associated with structural disease progression from 18 to 36 months and structural disease progression from time points 1 to 2 + + et al. 5 + +| Figure 2 | Forest plot of the | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------| +| individual ORs of structural disease progression with every increment in baseline peak knee adduction moment. Weights are from a random effects analysis. Individual and pooled estimates are shown with 95% CIs. | | + +| Table 2 | Summary of the quality assessment (risk of bias) | | | | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------|---------------------------------------------|---------|----------|-----------|----| +| Bias | Miyazaki | Chang | Bennell | Woollard | Henriksen | | +| et al30 | et al31 35 | et al29 | et al32 | et al33 | | | +| type | Quality criteria | | | | | | +| Selection | Were the descriptions of the groups and the distribution of prognostic factors sufficient? | A | I | I | A | A | +| Were the groups assembled at a similar point in their disease progression? | A | I | I | A | A | | +| Were the groups comparable on all important confounding factors? | I | I | I | U | A | | +| Detection | Was the joint load estimate | U | U | U | U | I | +| reliably ascertained? Was adequate adjustment made for the effects of these confounding variables? | A | I | A | I | I | | +| Was outcome assessment | A | A | I | I | A | | +| blind to exposure status? Was follow-up long enough | A | A | A | A | A | | +| for the outcomes to occur? What proportion of the | U | A | U | A | A | | +| cohort was followed-up?* | | | | | | | +| Attrition | Were dropout rates and reasons for drop-out similar across groups? | I | I | I | A | A | +| Overall risk of bias† | Very serious limitations; high risk of bias | Very serious limitations; high risk of bias | | | | | +| Methodological quality | Low | Low | Low | Low | Low | | +| *Adequate=follow-up proportion >80%; unclear 50–80%; inadequate=<50%. †Risk of bias within studies is assessed using GRADE's approach to study limitations: No serious limitation defined as all criteria being adequately described (high methodological quality); serious limitations defined as one criterion being inadequately described or >1 criterion being unclearly described (moderate methodological quality); very serious limitation defined as >1 criterion being inadequately described (low methodological quality). A, adequately described; I, inadequately described; U, unclear. Very serious limitations; high risk of bias Very serious limitations; high risk of bias Very serious limitations; high risk of bias | | | | | | | + +(baseline to 18 months) was associated with increased KAM from 18 to 36 months.35 Thus, temporal correctness cannot be assigned to the observed associations in this study, and the overall Temporality criterion was not satisfied (a 0 score). Biological Gradient was investigated in one cohort29 that showed no positive association between baseline peak KAM and loss of medial tibial cartilage volume over 12 months, resulting in a score of 0. Consequently, the causation score based on the included cohort studies was 0 for the association between peak KAM during walking and structural disease progression in patients with knee OA (table 3). + +For the KAM impulse and overall knee compression loading, only one study was available for each exposure,29 33 and therefore no causation scores were calculated for these loading exposures. + +As the systematic literature search did not identify any RCTs, the findings from the cohort studies can neither be confirmed nor refuted by experimental evidence. + +Hence, the fifth criterion could not be used to upgrade or downgrade the causation scores. + +## Discussion + +This review is the first to systematically assess—using systematic review, meta-analysis, and the Bradford Hill considerations on causality—whether a causal link exists between knee joint loading during walking and structural disease progression of knee OA. The systematic literature search identified five prospective cohort studies focusing on three estimates of knee joint loading: the peak KAM, KAM impulse and overall tibiofemoral compression force. Using a predefined algorithm, a causation score of 0 was reached, showing that there is no evidence of a causal link between knee joint loading during walking and structural progression of knee OA. + +![6_image_0.png](6_image_0.png) + +Furthermore, no experimental data from RCTs exist to support or oppose the findings from the cohort studies. + +One of the prospective cohort studies33 compared increased versus decreased loads, without demonstrating differences in structural disease progression opposing a causal link. + +Indirectly supporting the current findings, a longitudinal randomised trial comparing laterally wedged insoles (previously demonstrated to reduce loading37 38) +with placebo insoles, showed no group differences in structural disease progression.39 While that study seemingly provided experimental evidence, no joint load exposure data were included to document the assertion that joint loadings were in fact reduced in the 'wedged insole' group. Thus, the study did not fulfil the criteria for inclusion in this review. A recent meta-analysis on symptomatic effects of laterally wedged insoles showed no beneficial effects over neutral insoles, indicating limited effects of laterally wedged insoles.40 However, the null outcome is supported by the results in ref. 33, included in this review, showing neither detrimental effects of increased loading nor beneficial effects of decreased loading over 12 months in a group of knee OA patients. + +The quality assessment of the included studies showed high risks of selection, detection and attrition biases (table 3). An important precondition for valid results in cohort studies is that groups should be comparable at baseline. Unless the groups are balanced for relevant baseline characteristics, differences in structural disease progression cannot confidently be attributed to the effects of interest ( joint loads during walking). The included studies revealed significant inadequacies when assessing the comparability of the groups on relevant baseline characteristics, leading to prognostic imbalance. + +Four of the five cohort studies did not report how reliably the joint loading estimates were ascertained (detection bias), which could influence the individual results. + +Moreover, the loss of subjects at follow-up in two of the five cohort studies was beyond 20%, with the dropout rates and reasons for dropout across groups not adequately accounted for (attrition bias). Finally, the discrepant numbers of subjects presented in different reports from the same cohort31 35 introduced a serious risk of bias and low confidence in the results. The overall low methodological quality of the included studies gives little confidence in the reported estimates of causation. + +Despite the shortage of studies reporting a causal link between joint loading and structural disease progression, the conjectured relationship underlies the rationale for many clinical efforts to reduce knee loading during walking in attempts to retard disease progression. + +Weight loss, insoles, modified footwear, strengthening exercises, knee braces and gait modifications have been + +| Table 3 | Causation criteria and scores for the identified knee joint loading exposures | Causation score (number of criteria met) | | | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------|--------------------------------------------|------------|------------|-----| +| Peak KAM | 1.90 (0.85 to 4.25) | 2/4 (50%) | 1/4 (25%) | 0/4 (0%) | 0 | +| KAM impulse | 0.42 (0.12 to 1.48)¶ | −** | 0/1 (0%) | 1/1 (100%) | −†† | +| Peak overall compression | No group difference | −** | 1/1 (100%) | 0/1 (0%) | −†† | +| *Strong association is defined as a pooled OR ≥5 with lower 95% CI excluding 2.0. Moderate association is any statistically significant association. Strength* Summary Consistency† Temporality‡ or OR (95% CI) N (%) N (%) Biological gradient§ N (%) Knee joint load exposure | | | | | | + +Causation score (number of criteria met) +Peak KAM 1.90 (0.85 to 4.25) 2/4 (50%) 1/4 (25%) 0/4 (0%) 0 KAM impulse 0.42 (0.12 to 1.48)¶ −** 0/1 (0%) 1/1 (100%) −†† Peak overall compression No group difference −** 1/1 (100%) 0/1 (0%) −†† +*Strong association is defined as a pooled OR ≥5 with lower 95% CI excluding 2.0. Moderate association is any statistically significant association. + +†Consistency is defined as ≥75% of associations showing strong or moderate associations. + +‡In cohort studies it is difficult if not impossible to ensure temporal correctness because participants in the studies are expected to walk daily and are, therefore, exposed to knee joint loading throughout the observation period. In the current analysis, the temporality criterion is satisfied in studies that relate baseline knee joint loading exposures to disease progression over time from that baseline and demonstrate a statistically significant association with structural disease progression. §Biological gradient is defined as demonstrated when the rate of progression increases (or decreases) incrementally as dose of exposure increases. + +¶Estimate based on one cohort study29 assessing baseline KAM impulse to progression of cartilage defects assessed by semiquantitative grading of MRI. **Consistency not possible to assess with only one cohort. + +††Causality score not calculated because only one study available. + +KAM, knee adduction moment. + +suggested as means to lower knee joint loads and thus slow structural disease progression through mechanical pathways,41–46 with effects yet to be proven. The rationale for such efforts is diluted by the current results, not only by the low causality score, but also by the inconsistency across studies in the loading magnitudes between individuals who progress and those that do not. Figure 3 illustrates that patients with a certain baseline peak KAM magnitude in one study progress, whereas those with similar baseline peak KAM magnitudes in other studies do not. These differences in knee joint loading magnitudes between studies may be due to differences in gait analysis protocols across the involved gait labs, for example, in the selection of reference frame convention for the calculation of the KAM47 and footwear condition.44 However, including diversity of measures of the same variable can strengthen the confidence in the results of a systematic review. + +The results from the included studies exhibited a high degree of heterogeneity (I2 from the meta-analysis) +(figure 2). The two cohorts with significant OR30 31 35 both used radiography as the structural outcome assessment, whereas the studies reporting non-significant ORs used MRI.29 32 Furthermore, the two studies reporting significant associations had longer follow-up periods (72 and 18 months), whereas the studies without significant associations assessed disease progression after 12 months. This concurrence of follow-up time and imaging modality precludes conclusions about whether the differences in results are based on follow-up time or imaging modality. Radiography is the current clinical gold standard for assessing structural disease progression, but it has low temporal sensitivity, making long follow-up times necessary. MRI—with its shorter temporal sensitivity—is possibly the more promising tool for assessing single structures and should be used in studies with longer follow-up times. The total number of patients was 452 in five cohorts; that is a very small number of patients when considering the large population and when compared to similar reviews in other chronic diseases. For instance, a recent review of the causal relationship between dietary factors and coronary heart disease included several millions of individuals in 361 cohorts and a multitude of dietary exposures.23 Our study has strengths because we undertook several measures to minimise bias, restricting our review to studies with the strongest causal inference (cohort studies and RCTs), conducted independent assessments of study eligibility, and using predefined criteria to evaluate causation and methodological quality. We may be criticised for using arbitrary OR cut-offs to define strong and moderate association, but identifying the true cut-off for defining clinically meaningful OR values is impossible in a field that has few longitudinal studies to consult. An important limitation is the causality score that applied. The score is a modification of a previous similar score,23 thus it has limited empirical evidence to support its validity. Also, the causality score omitted several of the original causation considerations as proposed by Hill,21 yet the applied method was prespeci-fied. We may also be criticised for analysing any image-based structural deterioration, rather than separating the analyses in X-ray and MRI-based assessments. + +However, the analysis plans were also prespecified, without prior knowledge of the number of studies and specifics of the outcomes. + +Current guidelines on interpretation of data from observational studies and data combined from repeated observational studies (with a reasonable internal validity) +would correspond to confidence in the estimate's being +'Low-quality evidence', but could be upgraded if there is great confidence in the estimates and the effects are large.48 However, the current analysis revealed a large inconsistency among study results, poor precision of the combined analysis, and low methodological quality. + +Thus, we downgraded further to 'very low quality evidence'. The inconsistency of the available studies may limit the appropriateness of meta-analyses, yet support the overall conclusion of this systematic literature review. + +In conclusion, our systematic review, meta-analyses, and application of Bradford Hill's considerations on causation shows that there is no evidence of a causal link between estimates of knee joint loading during walking and structural progression of knee OA. Future large welldesigned prospective cohorts with subsequent confirmation from randomised trials are strongly recommended. + +The current study findings question the rationale behind clinical efforts to reduce knee joint loading during walking as such efforts are currently supported by very low-quality evidence showing no causal link between knee joint loading during walking and progression of knee OA. + +## Author Affiliations + +1The Parker Institute, Copenhagen University Hospitals Bispebjerg and Frederiksberg, Copenhagen, Denmark 2School of Exercise Science, Australian Catholic University, Brisbane, Queensland, Australia 3Faculty of Health Sciences, Institute of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark 4Department of Rheumatology and Gerontology, Copenhagen University Hospital Gentofte, Copenhagen, Denmark Contributors MH, MWC, HL, CJ and RC were responsible for study concept and design. RC and MH supervised the study. HL and CJ performed the literature search. MH and MWC acquired the data, which were analysed and interpreted by MH, MWC, CJ, HL and RC. MH drafted the manuscript, which was critically revised for important intellectual content by MWC, HL, CJ and RC. RC did the statistical analysis. MH is the guarantor. Funding The Parker Institute holds a grant from The Oak Foundation. Competing interests MH was supported by a grant from The Danish Council for Independent Research|Medical Sciences (Grant number: 10-093704). Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement Extra data can be accessed via the Dryad data repository at http://datadryad.org/ with the doi:10.5061/dryad.b40hm. Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/ + +## References + +1. Nguyen US, Zhang Y, Zhu Y, et al. Increasing prevalence of knee pain and symptomatic knee osteoarthritis: survey and cohort data. + +Ann Intern Med 2011;155:725–32. + +2. Felson DT, Naimark A, Anderson J, et al. The prevalence of knee osteoarthritis in the elderly. The Framingham Osteoarthritis Study. + +Arthritis Rheum 1987;30:914–18. + +3. Lawrence RC, Felson DT, Helmick CG, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum 2008;58:26–35. + +4. Aspden RM. Osteoarthritis: a problem of growth not decay? + +Rheumatology (Oxford) 2008;47:1452–60. + +5. Henriksen M, Simonsen EB, Alkjaer T, et al. Increased joint loads during walking—a consequence of pain relief in knee osteoarthritis. Knee 2006;13:445–50. + +6. Andriacchi TP, Mündermann A. The role of ambulatory mechanics in the initiation and progression of knee osteoarthritis. Curr Opin Rheumatol 2006;18:514–18. + +7. Felson DT. Osteoarthritis as a disease of mechanics. Osteoarthritis Cartilage 2013;21:10–15. + +8. Andriacchi TP. Valgus alignment and lateral compartment knee osteoarthritis: a biomechanical paradox or new insight into knee osteoarthritis? Arthritis Rheum 2013;65:310–13. + +9. Lafeber FP, Intema F, Van Roermund PM, et al. Unloading joints to treat osteoarthritis, including joint distraction. Curr Opin Rheumatol 2006;18:519–25. + +10. Lohmander LS, Gerhardsson dV, Rollof J, et al. Incidence of severe knee and hip osteoarthritis in relation to different measures of body mass: a population-based prospective cohort study. Ann Rheum Dis 2009;68:490–6. + +11. Coggon D, Reading I, Croft P, et al. Knee osteoarthritis and obesity. + +Int J Obes Relat Metab Disord 2001;25:622–7. + +12. Pottie P, Presle N, Terlain B, et al. Obesity and osteoarthritis: more complex than predicted! Ann Rheum Dis 2006;65:1403–5. + +13. Richette P, Poitou C, Garnero P, et al. Benefits of massive weight loss on symptoms, systemic inflammation and cartilage turnover in obese patients with knee osteoarthritis. Ann Rheum Dis 2011;70:139–44. + +14. DeVita P, Hortobagyi T. Obesity is not associated with increased knee joint torque and power during level walking. J Biomech 2003;36:1355–62. + +15. Zhao D, Banks SA, Mitchell KH, et al. Correlation between the knee adduction torque and medial contact force for a variety of gait patterns. J Orthop Res 2007;25:789–97. + +16. Ledingham J, Regan M, Jones A, et al. Radiographic patterns and associations of osteoarthritis of the knee in patients referred to hospital. Ann Rheum Dis 1993;52:520–6. + +17. Mills K, Hunt MA, Ferber R. Biomechanical deviations during level walking associated with knee osteoarthritis: a systematic review and meta-analysis. Arthritis Care Res (Hoboken) 2013;65:1643–65. + +18. Shrader MW, Draganich LF, Pottenger LA, et al. Effects of knee pain relief in osteoarthritis on gait and stair-stepping. Clin Orthop 2004;421:188–93. + +19. Schnitzer TJ, Popovich JM, Andersson GB, et al. Effect of piroxicam on gait in patients with osteoarthritis of the knee. Arthritis Rheum 1993;36:1207–13. + +20. Hurwitz DE, Sharma L, Andriacchi TP. Effect of knee pain on joint loading in patients with osteoarthritis. Curr Opin Rheumatol 1999;11:422–6. + +21. Hill AB. The environment and disease: association or causation? + +Proc R Soc Med 1965;58:295–300. + +22. DOLL R, Hill AB. Smoking and carcinoma of the lung; preliminary report. BMJ 1950;2:739–48. + +23. Mente A, de KL, Shannon HS, et al. A systematic review of the evidence supporting a causal link between dietary factors and coronary heart disease. Arch Intern Med 2009;169:659–69. + +24. Bellamy N, Kirwan J, Boers M, et al. Recommendations for a core set of outcome measures for future phase III clinical trials in knee, hip, and hand osteoarthritis. Consensus development at OMERACT +III. J Rheumatol 1997;24:799–802. + +25. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7:177–88. + +26. Higgins JP, Thompson SG, Deeks JJ, et al. Measuring inconsistency in meta-analyses. BMJ 2003;327:557–60. + +27. Khan K, ter Riet G, Popay J, et al. Undertaking systematic reviews of research on effectiveness. STAGE II—conducting the review. PHASE 5—study quality assessment. Undertaking systematic reviews of research on effectiveness: CRD's guidance for carrying out or commissioning reviews. 2nd edn. York: Centre for Reviews and Dissemination, 2001. + +28. Guyatt GH, Oxman AD, Vist G, et al. GRADE guidelines: 4. Rating the quality of evidence—study limitations (risk of bias). J Clin Epidemiol 2011;64:407–15. + +29. Bennell KL, Bowles KA, Wang Y, et al. Higher dynamic medial knee load predicts greater cartilage loss over 12 months in medial knee osteoarthritis. Ann Rheum Dis 2011;70:1770–4. + +30. Miyazaki T, Wada M, Kawahara H, et al. Dynamic load at baseline can predict radiographic disease progression in medial compartment knee osteoarthritis. Ann Rheum Dis 2002;61:617–22. + +31. Chang A, Hurwitz D, Dunlop D, et al. The relationship between toe-out angle during gait and progression of medial tibiofemoral osteoarthritis. Ann Rheum Dis 2007;66:1271–5. + +32. Woollard JD, Gil AB, Sparto P, et al. Change in knee cartilage volume in individuals completing a therapeutic exercise program for knee osteoarthritis. J Orthop Sports Phys Ther 2011;41:708–22. + +33. Henriksen M, Hunter DJ, Dam EB, et al. Is increased joint loading detrimental to obese patients with knee osteoarthritis? A secondary data analysis from a randomized trial. Osteoarthritis Cartilage 2013;21:1865–75. + +34. Altman R, Asch E, Bloch D, et al. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 1986;29:1039–49. + +35. Sharma L, Dunlop D, Andriacchi T, et al. The adduction moment and knee osteoarthritis (OA), a longitudinal study. Arthritis Rheum 2003;48(9 Suppl):S452. + +36. Altman RD, Hochberg M, Murphy WA Jr, et al. Atlas of individual radiographic features in osteoarthritis. Osteoarthritis Cartilage 1995;3 +(Suppl A):3–70. + +37. Hinman RS, Bowles KA, Bennell KL. Laterally wedged insoles in knee osteoarthritis: do biomechanical effects decline after one month of wear? BMC Musculoskelet Disord 2009;10:146. + +38. Hinman RS, Bowles KA, Metcalf BB, et al. Lateral wedge insoles for medial knee osteoarthritis: effects on lower limb frontal plane biomechanics. Clin Biomech (Bristol, Avon) 2012;27:27–33. + +39. Bennell KL, Bowles KA, Payne C, et al. Lateral wedge insoles for medial knee osteoarthritis: 12 month randomised controlled trial. BMJ 2011;342:d2912. + +40. Parkes MJ, Maricar N, Lunt M, et al. Lateral wedge insoles as a conservative treatment for pain in patients with medial knee osteoarthritis: a meta-analysis. JAMA 2013;310:722–30. + +41. Gross KD, Hillstrom HJ. Noninvasive devices targeting the mechanics of osteoarthritis. Rheum Dis Clin North Am 2008;34:755–76. + +42. Bennell KL, Kean CO, Wrigley TV, et al. Effects of a modified shoe on knee load in people with and those without knee osteoarthritis. + +Arthritis Rheum 2013;65:701–9. + +43. Bennell KL, Egerton T, Wrigley TV, et al. Comparison of neuromuscular and quadriceps strengthening exercise in the treatment of varus malaligned knees with medial knee osteoarthritis: a randomised controlled trial protocol. BMC Musculoskelet Disord 2011;12:276. + +44. Kemp G, Crossley KM, Wrigley TV, et al. Reducing joint loading in medial knee osteoarthritis: shoes and canes. Arthritis Rheum 2008;59:609–14. + +45. Hinman RS, Bennell KL. Advances in insoles and shoes for knee osteoarthritis. Curr Opin Rheumatol 2009;21:164–70. + +46. Simic M, Hinman RS, Wrigley TV, et al. Gait modification strategies for altering medial knee joint load: a systematic review. Arthritis Care Res (Hoboken) 2011;63:405–26. + +47. Schache AG, Baker R, Vaughan CL. Differences in lower limb transverse plane joint moments during gait when expressed in two alternative reference frames. J Biomech 2007;40:9–19. + +48. Guyatt G, Oxman AD, Akl EA, et al. GRADE guidelines: 1. + +Introduction-GRADE evidence profiles and summary of findings tables. J Clin Epidemiol 2011;64:383–94. \ No newline at end of file diff --git a/medical/md/PMC4368931.md b/medical/md/PMC4368931.md new file mode 100644 index 0000000000000000000000000000000000000000..2ef3f8c788350cfa8a397e6561698e2b0d118fff --- /dev/null +++ b/medical/md/PMC4368931.md @@ -0,0 +1,253 @@ +# 25 Mg Versus 50 Mg Dose Of Rectal Diclofenac For Prevention Of Post-Ercp Pancreatitis In Japanese Patients: A Retrospective Study + +Takeo Yoshihara,1 Masayoshi Horimoto,1 Tetsuhisa Kitamura,2 Naoto Osugi,1 Tatsuro Ikezoe,1 Kaori Kotani,1 Toru Sanada,1 Churi Higashi,1 Daisuke Yamaguchi,1 Makiyo Ota,1 Tatsunori Mizuno,1 Yasukazu Gotoh,1 Yorihide Okuda,1 Kunio Suzuki1 To cite: Yoshihara T, Horimoto M, Kitamura T, +et al. 25 mg versus 50 mg dose of rectal diclofenac for prevention of post-ERCP pancreatitis in Japanese patients: a retrospective study. BMJ Open 2015;5: +e006950. doi:10.1136/ bmjopen-2014-006950 +▸ Prepublication history for this paper is available online. To view these files please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2014-006950). Received 18 October 2014 Revised 26 January 2015 Accepted 27 January 2015 1Department of + +![0_image_0.png](0_image_0.png) + +Gastroenterology, Osaka Saiseikai Senri Hospital, Suita, Osaka, Japan 2Division of Environmental Medicine and Population Sciences, Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Correspondence to Dr Masayoshi Horimoto; horimotomasa@hotmail.com + +## Abstract + +Objectives: The aim of the present study was to assess the appropriate administration dose of nonsteroidal anti-inflammation drugs to prevent pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP). Importantly, the 100 mg dose of diclofenac recommended in Western countries has not been permitted in Japan. + +Design: A retrospective study. Settings: A single centre in Japan. Participants: This study enrolled patients who underwent ERCP at the Department of Gastroenterology, Osaka Saiseikai Senri Hospital, from April 2011 through June 2013, and who received either a 25 or a 50 mg dose of rectal diclofenac after ERCP. + +Primary outcome measure: The occurrence of postERCP pancreatitis (PEP). A multivariate regression model was used to assess the effect of the 50 mg dose (the 50 mg group) of rectal diclofenac and to compare it to the occurrence of PEP referring to the 25 mg group. + +Results: A total of 155 eligible patients received either 25 mg (84 patients) or 50 mg (71 patients) doses of rectal diclofenac after ERCP to prevent PEP. The proportion of PEP was significantly lower in the 50 mg group than in the 25 mg group (15.5% (11/71) vs 33.3% (28/84), p=0.018). In a multivariate analysis, the occurrence of PEP was significantly lower in the 50 mg group than in the 25 mg group even after adjusting potential confounding factors (adjusted OR=0.27, 95% CI 0.11 to 0.70). + +Conclusions: From this observation, the occurrence of PEP was significantly lower among ERCP patients with the 50 mg dose of rectal diclofenac than among those with the 25 mg dose. + +## Introduction + +Endoscopic retrograde cholangiopancreatography (ERCP) is widely conducted as a therapeutic and diagnostic procedure for hepatobiliary-pancreatic diseases.1–6 However, + +## Strengths And Limitations Of This Study + +- This study showed that the occurrence of postendoscopic retrograde cholangiopancreatography +(ERCP) pancreatitis was significantly lower among ERCP patients with the 50 mg dose of rectal diclofenac than among those with the 25 mg dose. + +- This study was a retrospective study, so there was the potential of selection bias. + +- This study did not assess the effect of non-steroidal anti-inflammation drugs other than diclofenac for prevention of post-ERCP pancreatitis. + +the prevalence of ERCP adverse events has become high, and post-ERCP pancreatitis +(PEP) has occurred in 1–30% of patients who have undergone ERCP. PEP can become severe and lead to death among these patients.1–6 Therefore, many studies on risk factors, preventive manoeuvres and drug administrations for PEP have been conducted worldwide.1–11 Recently, numerous reports have been published on the effectiveness of rectal administration of non-steroidal anti-inflammation drugs (NSAIDs) to prevent PEP.11–15 Several randomised controlled trials revealed that the 100 mg dose of rectal diclofenac/indomethacin after ERCP significantly reduced PEP occurrence compared with nonadministration,11–15 and the rectal administration of NSAIDs after ERCP has been widely accepted worldwide, including in Japan.16 A +meta-analysis demonstrated that NSAID +administration for PEP prevention was effective compared with non-administration, and its effectiveness is currently being established.11 However, little is known about the appropriate NSAID dose to prevent PEP.17 In addition, the 100 mg dose of diclofenac recommended in Western countries has not been legally permitted in Japan.16–18 The aim of this study was to compare a 25 mg dose of diclofenac to the 50 mg dose for the prevention of PEP +among ERCP patients. Our hypothesis is that the occurrence of PEP would be lower in the 50 mg group than in the 25 mg group. + +## Methods Study Design + +This observation was a single-centre, retrospective study. We enrolled patients who underwent ERCP at the Department of Gastroenterological Medicine, Osaka Saiseikai Senri Hospital, from April 2011 through June 2013, who then received either a 25 or 50 mg dose of rectal diclofenac. + +## Settings And Data Collection + +Osaka Saiseikai Senri Hospital, located in the northern area of Suita City, Osaka, Japan, has 343 beds, seven attending doctors and four residents in its Department of Gastroenterology (50 beds). Approximately 250 patients with gastroenterological diseases annually undergo ERCP in this hospital. The side-viewing endoscopes used for ERCP were either TJF-260V or JF-260V +(Olympus Corporation), and the appliance for cannulation was Tandem XL TAPERED TIP and Jagwire (0.035 inch; Boston Scientific Corporation). + +Among patients undergoing ERCP, either a 25 or 50 mg dose of rectal diclofenac was administered within 10 min immediately after ERCP for those more likely to develop PEP, based on the judgment of a main ERCP +operator, and the dose (25 or 50 mg) of rectal diclofenac was decided comprehensively based on patient age, body mass index (BMI) and serum creatinine. In our hospital, during the study period, both sulbactam sodium/cefoperazone sodium 3.0 g/day and gabexate mesilate 0.3 g/day were administered to all patients for at least 2 days after ERCP, and blood tests such as serum pancreatic-type amylase and creatinine were also conducted the day after ERCP. In addition, when a doctor in charge diagnosed or suspected PEP in a patient who underwent ERCP, contrast-enhanced CT scanning and blood tests were conducted.17 Information on characteristics and outcomes of ERCP +patients was retrospectively extracted from medical records, and the following data were included: gender, age, height, body weight, BMI, serum creatinine concentration; history of hepatobiliary, pancreatic, or gastrointestinal cancers; history of pancreatitis, first conducted ERCP, type of operator (resident or not), duration of ERCP examination, difficult cannulation and ERCP individual procedures (wire insertion into pancreatic duct, cytology of pancreas duct, pancreatography, cytology of bile duct), endoscopic sphincterotomy, endoscopic papillary balloon dilation, choledocholithotomy and stenting to pancreatic duct. Duration of ERCP examination was defined as the time interval from the side-viewing endoscope insertion to its removal,19 and the difficulty of cannulation was determined by a main operator or an attending doctor. The procedure for pancreatic duct was defined as any one of wire insertions into the pancreatic duct, its cytology or pancreatography. + +## Study End Point + +The primary outcome measure was the occurrence of PEP, defined as the development of both new or worsened abdominal pain and hyperamylasaemia based on the Cotton criteria.1 6 The secondary outcome measure was the occurrence of post-ERCP hyperamylasaemia, defined as a rise in the serum pancreatic-type amylase concentration to more than three times the upper limit of the normal laboratory value (>123 IU/mL) within 24 h after ERCP.1 6 In addition, death, severe acute pancreatitis, acute renal failure and gastrointestinal bleeding were defined as severe adverse events after rectal diclofenac administration.1 6 Severe acute pancreatitis was defined based on the Japanese guideline.17 Acute renal failure was defined as an increase of the serum creatinine ≥3 times over that before ERCP.20 + +## Statistical Analyses + +Patient characteristics and outcomes between the two groups with 25 and 50 mg doses of rectal diclofenac were compared after ERCP. Continuous data were presented as the median (interquartile), differences were analysed by Mann-Whitney test, categorical data were expressed in percentages (n/N) and differences were analysed by χ2 test or Fischer's exact test. + +The multivariate logistic regression was used to assess the effect of 50 mg of rectal diclofenac (the 50 mg group) and to compare it to the occurrence of PEP and hyperamylasaemia referring to the 25 mg group. + +The ORs and their CIs were calculated. Potential confounding factors based on biological plausibility and previous studies were included in the multivariate analysis.5 6 21 These variables were gender (male, female), +age (<65, ≥65 years old), BMI (<22, ≥22 kg/m2), type of operator (resident or not), difficult cannulation +(yes, no), and procedure in pancreatic duct (yes, no). All statistical analyses were performed using SPSS V.22.0J (IBM Corp, Armonk, New York). All tests were two tailed, and p values of <0.05 were considered statistically significant. + +## Results + +During the study period, a total of 166 patients received either a 25 or 50 mg dose of rectal diclofenac after ERCP to prevent PEP and hyperamylasaemia. Excluding three patients who had pancreatitis and eight who were given NSAIDs before ERCP, 155 patients (84 in the 25 mg group and 71 in the 50 mg group) were eligible for our analysis. + +| Table 1 | Characteristics of patients administered a 25 versus 50 mg dose of rectal diclofenac after ERCP Rectal diclofenac Total 25 mg 50 mg | p Value | | | | | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------|------------------|------------------|---------|------|---------|-------| +| Men, % (n/N) | 54.8 | (85/155) | 46.4 | (39/84) | 64.8 | (46/71) | 0.022 | +| Age (years), median (IQR) | 71.0 (63.0–76.0) | 73.5 (68.0–80.0) | 67.0 (57.0–74.0) | <0.001 | | | | +| Elderly aged ≥65, % (n/N) | 71.0 | (110/155) | 82.1 | (69/84) | 57.7 | (41/71) | 0.001 | +| BMI (kg/m2 ), median (IQR) | 22.5 (19.5–25.3) | 23.2 (21.2–25.6) | 21.9 (19.2–24.4) | 0.019 | | | | +| BMI <22 | 59.6 | (90/151) | 50.0 | (40/80) | 70.4 | (50/71) | 0.011 | +| Cancer, % (n/N) | 32.9 | (50/155) | 34.1 | (28/82) | 31.4 | (22/70) | 0.722 | +| History of pancreatitis, % (n/N) | 16.2 | (25/154) | 13.3 | (11/83) | 19.7 | (14/71) | 0.278 | +| First conducted ERCP, % (n/N) | 34.8 | (54/155) | 33.3 | (28/84) | 36.6 | (26/71) | 0.669 | +| Resident as a main operator, % (n/N) | 63.9 | (99/155) | 71.4 | (60/84) | 54.9 | (39/71) | 0.033 | +| Serum creatinine before ERCP (mg/dL) | 0.80 (0.70–0.90) | 0.80 (0.70–0.90) | 0.80 (0.70–1.00) | 0.642 | | | | +| Duration of examination, median (IQR) | 36.5 (26.0–51.3) | 37.0 (28.0–50.0) | 35.0 (23.0–53.0) | 0.568 | | | | +| Duration of examination >30 min, % (n/N) | 63.6 | (98/154) | 66.3 | (55/83) | 60.6 | (43/71) | 0.463 | +| Difficult cannulation, % (n/N) | 28.4 | (44/155) | 27.4 | (23/84) | 29.6 | (21/71) | 0.763 | +| Procedures for pancreatic duct, % (n/N) | 68.4 | (106/155) | 69.0 | (58/84) | 67.6 | (48/71) | 0.847 | +| Pancreatic duct wire, % (n/N) | 65.2 | (101/155) | 65.5 | (55/84) | 64.8 | (46/71) | 0.929 | +| Cytology of pancreas duct, % (n/N) | 19.4 | (30/155) | 17.9 | (15/84) | 21.1 | (15/71) | 0.608 | +| Pancreatography, % (n/N) | 31.6 | (49/155) | 34.5 | (29/84) | 28.2 | (20/71) | 0.397 | +| Cytology of bile duct, % (n/N) | 18.7 | (29/155) | 20.2 | (17/84) | 16.9 | (12/71) | 0.596 | +| EST, % (n/N) | 26.5 | (41/155) | 23.8 | (20/84) | 29.6 | (21/71) | 0.417 | +| EPBD, % (n/N) | 5.8 | (9/155) | 6.0 | (5/84) | 5.6 | (4/71) | 0.605 | +| Choledocholithotomy, % (n/N) | 38.1 | (59/155) | 41.7 | (35/84) | 33.8 | (24/71) | 0.315 | +| Stenting to pancreatic duct, % (n/N) | 2.6 | (4/155) | 2.4 | (2/84) | 2.8 | (2/71) | 0.624 | +| BMI, body mass index; EPBD, endoscopic papillary balloon dilation; ERCP, endoscopic retrograde cholangiopancreatography; EST, endoscopic sphincterotomy. | | | | | | | | + +Table 1 shows the baseline characteristics and ERCPrelated procedures of eligible patients with the 25 vs 50 mg of rectal diclofenac after ERCP. The proportion of males was significantly higher in the 50 mg group than in the 25 mg group (64.8% vs 46.4%, p=0.022), +whereas median age was significantly lower in the 50 mg group than in the 25 mg group (67.0% vs 73.5%, +p<0.001). The 50 mg group had significantly lower BMI +than the 25 mg group (21.9 vs 23.2, p=0.019). The proportion of ERCP conducted by a resident as main operator was significantly lower in the 50 mg group than in the 25 mg group (54.9% vs 71.4%, p=0.022). Other factors did not differ between the groups. + +Among eligible patients receiving rectal diclofenac after ERCP, 25.2% (39/155) had PEP and 43.2% +(67/155) had post-ERCP hyperamylasaemia. The proportion of PEP was significantly lower in the 50 mg group than in the 25 mg group (15.5% (11/71) vs 33.3% (28/84), p=0.018, figure 1), and the proportion of post-ERCP hyperamylasaemia was also significantly lower in the 50 mg group than in the 25 mg group +(31.0% (22/71) vs 53.6% (45/84), p=0.005, figure 2). + +The concentration of serum pancreatic-type amylase after ERCP was significantly lower in the 50 mg group than in the 25 mg group (54.0 (30.8–189.3) vs 148.0 (40.0–502.0) IU/mL, p=0.006). As for severe events after ERCP, there were no deaths, acute renal failures or gastrointestinal bleeding in either group, but one patient in the 25 mg group had severe acute pancreatitis. + +In a multivariate analysis, the occurrence of PEP was + +![2_image_0.png](2_image_0.png) + +significantly lower in the 50 mg group than in the 25 mg group even after adjusting for potential confounding factors such as sex, age, BMI, the type of operator, difficult cannulation and procedures for pancreatic duct +(adjusted OR=0.27, 95% CI 0.11 to 0.70, table 2). The occurrence of post-ERCP hyperamylasaemia was also significantly lower in the 50 mg group than in the 25 mg group (adjusted OR=0.35, 95% CI 0.15 to 0.84, table 3). + +![2_image_1.png](2_image_1.png) + +![3_image_0.png](3_image_0.png) + +## Discussion + +From a single-centre observational study, we demonstrated that the occurrence of PEP and post-ERCP hyperamylasaemia was significantly lower among ERCP +patients on the 50 mg dose of rectal diclofenac than among those on the 25 mg dose, which suggested that the effectiveness of diclofenac for PEP was dose dependent. Importantly, the dose of diclofenac administration for patients was limited to a single maximum of 50 mg in Japan,16 17 but the preceding studies could not sufficiently compare the effects of 25 and 50 mg doses of rectal diclofenac for the prevention of PEP among ERCP patients. Therefore, our study provides helpful new clues for actual prevention of PEP at gastroenterological clinical settings in Japan. + +The present study showed that the prevention of PEP +among ERCP patients by diclofenac was dosedependently effective. Previous randomised trials and meta-analyses indicated that 100 mg (not 50 mg) rectal NSAID administration was more effective for PEP prevention than non-administration.11–13 In a randomised + +| Table 2 | Association between 25 and 50 mg dose of | | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------|----------------|-------| +| rectal diclofenac and post-ERCP pancreatitis OR (95% CI) | p Value | | | +| Post-ERCP pancreatitis Univariate | 0.37 | (0.17 to 0.80) | 0.018 | +| Multivariate Model 1 | 0.31 | (0.13 to 0.75) | 0.009 | +| Model 2 | 0.27 | (0.11 to 0.70) | 0.007 | +| ORs were calculated for the 50 mg group referring to the 25 mg group. Model 1: Adjusted for sex, age and BMI. Model 2: Adjusted for sex, age, BMI, type of operator, difficult cannulation and procedures for pancreatic duct. BMI, body mass index; ERCP, endoscopic retrograde cholangiopancreatography. | | | | + +control trial in Japan, the authors showed that low doses +(either 25 or 50 mg) of rectal diclofenac were more effective in preventing PEP compared with the placebo group. + +16 From a subanalysis of the trial, the occurrence of PEP among patients administered 50 mg of rectal diclofenac tended to be lower than among those given 25 mg, although it was statistically insignificant (0% (0/29) vs 9% +(2/22), p=0.101). This result was almost consistent with ours. No randomised trials directly compared a 25 mg of rectal diclofenac to a 50 mg dose for the prevention of PEP, but conducting randomised trials to determine the appropriate dose of NSAID administration for the prevention of PEP is important in Japan. + +Why does the administration of NSAIDs for ERCP +patients prevent PEP? Although much of its preventive mechanism is unclear, it is suggested that the activation of trypsin/trypsinogen in acinar cells of the pancreas causes acute pancreatic inflammation by factors such as mechanical stimulus of the pancreatic duct, intestinal fluid flow into the pancreatic duct, increase in pancreatic duct internal pressure or pancreatic tissue pressure by pancreatography and pancreatic fluid congestion by Vater papilla oedema.11 22 23 In particular, phospholipase A2 synthesised in the pancreas regulates inflammation mediators such as prostaglandins, leukotrienes and thromboxanes by arachidonic acid cascade24 and NSAIDs, the cyclo-oxigenase inhibitors, prevent PEP by inhibiting its cascade.12 22–25 The efficacy of NSAIDs including diclofenac is reportedly dose dependent.26–28 This study, showing the dose-dependent effect of diclofenac for PEP prevention, suggested that sufficient doses of NSAIDs are needed to inhibit arachidonic acid cascades for PEP prevention. + +Gastrointestinal bleeding and acute renal failure were among the severe adverse events after NSAID administration.15 Preceding studies underscored the very low occurrence of severe adverse events such as gastrointestinal bleeding and acute renal failure after NSAID +administration for PEP prevention.13 15 16 Even in the present study, there was no gastrointestinal bleeding or acute renal failure regardless of the dose of diclofenac, + +| Table 3 | Association between 25 and 50 mg dose of | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------| +| rectal diclofenac and post-ERCP hyperamylasaemia OR (95% CI) p Value Post-ERCP hyperamylasaemia Univariate 0.39 (0.20 to 0.75) 0.005 Multivariate Model 1 0.44 (0.21 to 0.92) 0.028 Model 2 0.35 (0.15 to 0.84) 0.018 ORs were calculated for the 50 mg group referring to the 25 mg group. Model 1: Adjusted for sex, age and BMI. | | + +and diclofenac administration would thus be acceptable for PEP prevention. However, gastroenterologists in Japan tend to hesitate to use the 50 mg dose of rectal diclofenac for PEP prevention16 17 29 because of concerns such as gastrointestinal adverse events, acute renal failure, and decrease in blood pressure after NSAID +administration.27–29 However, administering the 50 mg dose of rectal diclofenac for patients without problems such as renal dysfunction before ERCP should be recommended. + +NSAID administration for PEP prevention is given a grade A recommendation by the European Society of Gastrointestinal Endoscopy guideline, grade C1 by the Japanese guideline, and a moderate rating by the American College of Gastroenterology (ACG) guideline, respectively.17 30 31 Recently, the number of patients with acute pancreatitis has been excessively increasing,17 32 and its medical cost has also been climbing. Considering the limited medical resources in Japan, the 50 mg dose of rectal diclofenac for PEP prevention will surely become an established method to be administered without hesitation, because NSAIDs including diclofenac are inexpensive and have few adverse effects, and their administration method is also simple and easy. However, large-scale prospective multicentre observational or randomised trials are warranted to confirm our results. + +There are several limitations to this study. First, it was conducted retrospectively, and there was the potential of selection bias. For example, the proportion of PEP occurrence in this study was higher than that in previous studies,13 16 because either a 25 or 50 mg dose of diclofenac was selectively administered to ERCP patients who were more likely to develop PEP based on the judgment of a main ERCP operator. Second, our study did not assess the effect of NSAIDs other than diclofenac for PEP prevention. Third, unmeasured confounding factors may have influenced the association between the occurrence of PEP and rectal diclofenac administration among ERCP patients. + +## Conclusion + +From a single-centre observation in Osaka, this study demonstrated that the occurrence of PEP and post-ERCP hyperamylasaemia was significantly lower among ERCP patients with the 50 mg dose of rectal diclofenac than among those with the 25 mg dose. Further large-scale prospective multicentre observational or randomised controlled trials are warranted to confirm our results. + +Acknowledgements The authors are deeply indebted to the staff and physicians of Osaka Saiseikai Senri Hospital. Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Competing interests None. Ethics approval The study was approved by the institutional review board of Osaka Saiseikai Senri Hospital. + +Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement No additional data are available. Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http:// creativecommons.org/licenses/by-nc/4.0/ + +## References + +1. Cotton PB, Eisen GM, Aabakken L, et al. A lexicon for endoscopic adverse events: report of an ASGE workshop. Gastrointest Endosc 2010;71:446–54. + +2. Freeman ML, Nelson DB, Sherman S, et al. Complications of endoscopic biliary sphincterotomy. N Engl J Med 1996;335: +909–18. + +3. Rabenstein T, Hahn EG. Post-ERCP pancreatitis: new momentum. + +Endoscopy 2002;34:325–9. + +4. Vandervoort J, Soetikno RM, Tham TC, et al. Risk factors for complications after performance of ERCP. Gastrointest Endosc 2002;56:652–6. + +5. Freeman ML, DiSario JA, Nelson DB, et al. Risk factors for post-ERCP pancreatitis: a prospective, multicenter study. + +Gastrointest Endosc 2001;54:425–34. + +6. Kubiliun NM, Adams MA, Akshintala VS, et al. Evaluation of Pharmacologic Prevention of Pancreatitis Following Endoscopic Retrograde Cholangiopancreatography: a Systematic Review. Clin Gastroenterol Hepatol 2015. Published Online 8 Jan 2015. doi:10.1016/j.cgh.2014.11.038 7. Fazel A, Quadri A, Catalano MF, et al. Does a pancreatic duct stent prevent post-ERCP pancreatitis? A prospective randomized study. + +Gastrointest Endosc 2003;57:291–4. + +8. Andriulli A, Leandro G, Federici T, et al. Prophylactic administration of somatostatin or gabexate does not prevent pancreatitis after ERCP: an updated meta-analysis. Gastrointest Endosc 2007;65:624–32. + +9. Tarnasky PR, Palesch YY, Cunningham JT, et al. Pancreatic stenting prevents pancreatitis after biliary sphincterotomy in patients with sphincter of Oddi dysfunction. Gastroenterology 1998;115:1518–24. + +10. Singh P, Das A, Isenberg G, et al. Does prophylactic pancreatic stent placement reduce the risk of post-ERCP acute pancreatitis? A meta-analysis of controlled trials. Gastrointest Endosc 2004;60:544–50. + +11. Akbar A, Abu Dayyeh BK, Baron TH, et al. Rectal nonsteroidal anti-inflammatory drugs are superior to pancreatic duct stents in preventing pancreatitis after endoscopic retrograde cholangiopancreatography: a network meta-analysis. + +Clin Gastroenterol Hepatol 2013;11:778–83. + +12. Murray B, Carter R, Imrie C, et al. Diclofenac reduces the incidence of acute pancreatitis after endoscopic retrograde cholangiopancreatography. Gastroenterology 2003;124:1786–91. + +13. Elmunzer BJ, Waljee AK, Elta GH, et al. A meta-analysis of rectal NSAIDs in the prevention of post-ERCP pancreatitis. Gut 2008;57:1262–7. + +14. Zheng MH, Xia HH, Chen YP. Rectal administration of NSAIDs in the prevention of post-ERCP pancreatitis: a complementary meta-analysis. Gut 2008;57:1632–3. + +15. Elmunzer BJ, Scheiman JM, Lehman GA, et al. A randomized trial of rectal indomethacin to prevent post-ERCP pancreatitis. N Engl J +Med 2012;366:1414–22. + +16. Otsuka T, Kawazoe S, Nakashita S, et al. Low-dose cholangiopancreatography pancreatitis: a randomized controlled trial. + +J Gastroenterol 2012;47:912–17. + +17. Arata S, Takada T, Hirata K, et al. Post-ERCP pancreatitis. + +J Hepatobiliary Pancreat Sci 2010;17:70–8. + +18. Summary of product characteristics of Voltaren® at Novartis company homepage. http://product.novartis.co.jp/vol/pi/VS1402.pdf (accessed 13 Jun 2014) (In Japanese). + +19. Mehta PP, Sanaka MR, Parsi MA, et al. Association of procedure length on outcomes and adverse events of endoscopic retrograde cholangiopancreatography. Gastroenterol Rep 2014;2: +140–4. + +20. Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for acute kidney injury. Kidney Int Suppl 2012;2:19–36. + +21. Day LW, Lin L, Somsouk M. Adverse events in older patients undergoing ERCP: a systematic review and meta-analysis. + +Endoscopy Int Open 2014;02:E28–36. + +22. Pezzilli R, Romboli E, Campana D, et al. Mechanisms involved in the onset of post-ERCP pancreatitis. JOP 2002;3:162–8. + +23. Tsujino T, Kawabe T, Omata M. Antiproteases in preventing post-ERCP acute pancreatitis. JOP 2007;8:509–17. + +24. Mäkelä A, Kuusi T, Schröder T. Inhibition of serum phospholipase-A2 in acute pancreatitis by pharmacological agents in vitro. Scand J Clin Lab Invest 1997;57:401–7. + +25. Gross V, Leser HG, Heinisch A, et al. Inflammatory mediators and cytokines—new aspects of the pathophysiology and assessment of severity of acute pancreatitis? Hepatogastroenterology 1993;40:522–30. + +26. Giagoudakis G, Markantonis SL. Relationships between the concentrations of prostaglandins and the nonsteroidal antiinflammatory drugs indomethacin, diclofenac, and ibuprofen. + +Pharmacotherapy 2005;25:18–25. + +27. Richy F, Bruyere O, Ethgen O, et al. Time dependent risk of gastrointestinal complications induced by non-steroidal anti-inflammatory drug use: a consensus statement using a meta-analytic approach. Ann Rheum Dis 2004;63: +759–66. + +28. Rostom A, Goldkind L, Laine L. Nonsteroidal anti-inflammatory drugs and hepatic toxicity: a systematic review of randomized controlled trials in arthritis patients. Clin Gastroenterol Hepatol 2005;3:489–98. + +29. Warning and precautions of Voltaren® at Novartis company homepage. http://www.voltaren.jp/m_kinki/kinki_a11.html (accessed 13 Jun 2014) (In Japanese). + +30. Dumonceau JM, Andriulli A, Deviere J, et al. European Society of Gastrointestinal Endoscopy (ESGE) Guideline: prophylaxis of post-ERCP pancreatitis. Endoscopy 2010;42:503–15. + +31. Tenner S, Baillie J, DeWitt J, et al. American College of Gastroenterology guideline: management of acute pancreatitis. Am J +Gastroenterol 2013;108:1400–15. + +32. American Gastroenterological Association (AGA) Institute on +"Management of Acute Pancreatits" Clinical Practice and Economics Committee; AGA Institute Governing Board. AGA Institute Medical Position Statement on Acute Pancreatitis. Gastroenterology 2007;132:2019–21. \ No newline at end of file diff --git a/medical/md/PMC4652769.md b/medical/md/PMC4652769.md new file mode 100644 index 0000000000000000000000000000000000000000..62dfdff04e30684a94c6524ce64ee78cea1a308c --- /dev/null +++ b/medical/md/PMC4652769.md @@ -0,0 +1,537 @@ +# Nucleotide Binding Interactions Modulate Dntp Selectivity And Facilitate 8-Oxo-Dgtp Incorporation By Dna Polymerase Lambda + +Matthew J. Burak, Kip E. Guja and Miguel Garcia-Diaz* Received March 17, 2015; Revised July 13, 2015; Accepted July 15, 2015 + +## Abstract + +8-Oxo-7,8,-dihydro-2--deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP +at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol -) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol -**. We have solved high resolution** +crystal structures showing how Pol - accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn**-conformation, and is** +stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 **facilitate incorporation of 8-oxo-dGMP opposite dA and** +dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol - and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases. + +## Introduction + +DNA is vulnerable to damage by reactive oxygen species +(ROS). Of the four nucleobases, guanine is the most susceptible to oxidative damage owing to its low redox potential (1). Moreover, guanine oxidation products have a high mutagenic potential (2–6). 8-oxo-7,8-dihydro-2-- +deoxyguanosine (8-oxo-dG) is a major oxidative product present in cells and can arise in either DNA or the free nucleotide pool (7). Accordingly, oxidation of dGTP results in the formation of 8-oxo-7,8,-dihydro-2--deoxyguanosine triphosphate (8-oxo-dGTP). Experimental measurements indicate that 8-oxo-dGTP may account for up to 10% of the total dGTP pool (8). In order to prevent the accumulation of 8-oxo-dGTP, cells encode a pyrophosphohydrolase, MutT/MTH1, which degrades the oxidized nucleotide (9– 11). Despite expression of MutT/MTH1, nucleotide pools can accumulate enough 8-oxo-dGTP to promote mutagenesis and drive cancer progression (12,13). 8-oxo-dGTP is capable of adopting either an anti or syn conformation and thus can base-pair with either cytosine (dC) or adenine +(dA), respectively (14–17). As a consequence of this, 8-oxodGTP is frequently misincorporated opposite dA *in vitro* by several DNA polymerases (18–25). Thus, *in vivo*, incorporation of 8-oxo-dGMP can lead to an A to C transversion mutation if left unrepaired (9). Conversely, 8-oxo-dGTP incorporation opposite dC can also eventually result in G to T tranversion mutations after subsequent replication (26). + +All cells have specialized repair systems to deal with DNA modifications, and the base excision repair (BER) +pathway is the main mechanism for purging the genome of oxidative lesions such as 8-oxo-dG (27). A specialized form of the BER pathway, initiated by the glycosylase MutY/MTYH, specifically recognizes and repairs 8-oxodG:dA mispairs by removing dA and replacing it with dC, +enabling subsequent repair of the 8-oxo-dG lesion (28– +31). However, no repair mechanism exists to cope with misincorporated 8-oxo-dGMP. Significantly, 8-oxo-dGMP +misincorporation would lead to mutagenic repair by the MutY/MTYH-dependent BER pathway, directing removal of dA and not 8-oxo-dGMP. Thus, 8-oxo-dGMP misincorporation would unavoidably lead to mutagenesis, resulting an A to C transversion (26). + +Therefore, preventing misincorporation of 8-oxo-dGMP +outright is crucial to avoid mutagenesis. In addition to the hydrolytic activity of MutT/MUTH1, discrimination against 8-oxo-dGTP by DNA polymerases is an important factor in the prevention of mutagenesis. Therefore, understanding the mechanistic basis of this discrimination is of significant interest. With the exception of the recent characterization of the interaction between DNA polymerase beta +(Pol -) and 8-oxo-dGTP (32,33), there is a lack of structural information that might help explain how DNA polymerases avoid 8-oxo-dGTP-induced mutagenesis. + +*To whom correspondence should be addressed. Tel: +1 631 444 3054; Fax: +1 631 4449749; Email: miguel.garcia-diaz@stonybrook.edu The family X DNA polymerase lambda (Pol ) plays an important role in mediating cellular tolerance of guanine oxidation (34–36). A key element of the MutY/MTYH +BER pathway is the ability to readily incorporate dCMP opposite a templating 8-oxodG. In the context of this repair reaction, Pol is uniquely capable of mediating faithful incorporation of dCMP to complete the repair (23). Pol also takes advantage of its unique catalytic properties to mediate additional roles in other DNA repair pathways related to oxidative damage, such as non-homologous end joining (NHEJ) (37–41). + +Considering that Pol operates under conditions where oxidative damage is prevalent, it is expected to frequently encounter 8-oxo-dGTP. Moreover, due to its function in NHEJ, Pol may be responsible for mediating 8-oxodGTP-driven mutagenesis. Curiously, despite its relatively error-free behavior when faced with a templating 8-oxodG, +Pol readily misinserts 8-oxo-dGMP opposite dA (23). The contrast between its behavior when incorporating 8-oxodGTP and opposite 8-oxo-dG indicates that the active site of Pol is asymmetrical, but the structural factors influencing 8-oxo-dGMP misincorporation by Pol are not well understood. + +Here, we report high resolution pre- and post-catalytic crystal structures of Pol in complex with 8-oxo-dGTP +and 8-oxo-dGMP, respectively. The structures demonstrate that the oxidized nucleotide can be accommodated in the Pol active site, where it is stabilized in the pro-mutagenic syn-conformation. Kinetic analysis demonstrates that two residues that partially conform the dNTP binding pocket influence 8-oxo-dGMP incorporation. Ala510 makes a Van der Waals interaction with the base of the incoming nucleotide, whereas Asn513 hydrogen-bonds with the C8carbonyl. Our results indicate that Ala510 and Asn513 differentially impact 8-oxo-dGMP incorporation as well as the balance of purine/pyrimidine incorporation, confirming the importance of these residues in mediating nucleotide selectivity in family X DNA polymerases. + +## Materials And Methods Protein Purification + +The sequence corresponding to residues 242–575 of human Pol was previously cloned into the bacterial expression vector pET-22b (42). Site-directed mutagenesis was performed on the Pol expression vector to generate the A510D, N513A and A510D/N513A mutants. WT, A510D, +N513A and A510D/N513A Pol were subsequently expressed in the *E. coli* strain BL21-CodonPlus(DE3)-RIL +and purified as described (42). + +## Oligonucleotides For Crystallography + +Oligonucleotides used for the pre-catalytic ternary and post-catalytic nick complexes are shown in Supplementary Figure S1A and B respectively. Oligos were synthesized by solid-state synthesis methods using an automated DNA +synthesizer. Oligos were subsequently purified by HPLC, ethanol precipitated and quantified by UV absorbance +(A260). P/T were annealed as previously described in 25 mM Tris–HCl pH 7.5 and 40 mM MgCl2. + +## Crystallization + +Pre-catalytic ternary complexes were formed using an upstream primer (UP5), downstream primer containing a 5-- +phosphate group (DP4), and template (T8-A) (Supplementary Figure S1A). UP5, DP4 and T8-A (1:1:1) were heated to 80◦C for 10 min before slowly cooling to room temperature in 25 mM Tris–HCl pH 7.5 and 50 mM MgCl2. The resulting primer/templates (P/T) were mixed with WT Pol +. However, due to the low melting temperature of DP-4 +(16◦C), a second annealing step in the presence of protein was used to ensure the proper formation of a 1-nt gapped substrate. Accordingly, the mixture was heated to 15◦C for 5 min before slowly cooling to 4◦C to ensure proper annealing of DP4. ddCTP and 8-oxo-dGTP/dTTP were sequentially added to the mixture in order to generate a pre-catalytic ternary complex. The resulting mixture (100 l) contained 15 mM Tris–HCl pH 7.5, 75 mM NaCl, 10 mM MgCl2, 1 mM DTT, P/T (0.5 mM), WT Pol (0.4 mM), 1 mM ddCTP and 3 mM 8-oxo-dGTP/dTTP. Crystals containing 8oxo-dGTP were formed using the hanging drop method by mixing 2 l of the protein/DNA solution containing DNA with 1 l of the reservoir solution containing 0.1 M sodium cacodylate trihydrate pH 7.4 and 1.9 M sodium acetate trihydrate. The crystal was transferred to a solution containing 0.1 M sodium cacodylate trihydrate pH 7.4 and 1.9 M +sodium acetate trihydrate and 25% (w/v) glycerol and cryocooled in liquid nitrogen prior to data collection. dTTPcontaining crystals were formed as previously stated in solution containing 0.1 M sodium acetate trihydrate pH 4.6 and 2.0 M sodium formate. The crystal was transferred to a solution containing 0.1 M sodium acetate trihydrate pH 4.6 and 2.0 M sodium fomate and 25% w/v glycerol, harvested with a Mitegen MicroMount and cryo-cooled in liquid nitrogen prior to data collection. + +Post-catalytic nick complexes were formed using an upstream primer (UP6), DP4 and template T8-A (Supplementary Figure S1B). Pre-hybridized UP6, DP4 and T8-A were mixed with WT Pol as previously described. 8-Oxo-dGTP +(4 mM) was subsequently added and the resulting mixture +(100 l) was equilibrated for 60 min on ice in order to generate a post-catalytic nick complex. Crystals containing 8oxo-dGMP were grown using the hanging drop method by mixing 1 l of the protein/DNA solution containing DNA +with 1 l of the reservoir solution containing 0.1 M sodium citrate pH 5.6 and 1 M ammonium phosphate. The crystal was transferred to a solution containing 0.1 M sodium citrate pH 5.6, 1.2 M ammonium phosphate and 30% (w/v) +glycerol, harvested with a Mitegen MicroMount and cryocooled in liquid nitrogen prior to data collection. + +## X-Ray Data Collection And Structure Determination + +Diffraction data were collected on beamline X25 of the National Synchrotron Light Source at Brookhaven National Laboratory for the dTTP pre-catalytic complex and the 8oxo-dGTP post-catalytic complex, and on beamline 5.0.2 of the Advanced Light Source at Lawrence Berkeley National Laboratory for the 8-oxo-dGTP post-catalytic complex. All datasets were collected at 100 K using a wavelength of 1.0 A. Diffraction data were processed using XDS ( ˚ 43) +and Aimless (44) as implemented in the autoPROC pipeline +(45). Phases were obtained by molecular replacement using Phaser (46); search models for the pre- and post-catalytic complexes were created from 1XSN (47) and 1XSP (47), +respectively. Model building was carried out in Coot (48), +followed by refinement in Phenix (49), Refmac (50) and BUSTER (51). Diffraction data for the dTTP pre-catalytic complex were strongly anisotropic, with resolution limits +(defined by CC1/2 and I/σI (44,52) of 2.4 A in ˚ a* and c* reciprocal space directions, but only 2.9 A along the ˚ b* direction. For this reason, an ellipsoidal truncation was carried out using the Anisotropy Diffraction Server (53). Briefly, data were truncated that fell outside an ellipse centered at the reciprocal lattice origin and having vertices at 1/2.4, 1/2.9, and 1/2.4 A along ˚ a*, b*, and c* axes, respectively. + +Isotropy was approximated by applying a negative scale factor along b* (−16.8 A˚ 2) with no correction along a* or c*. + +These anisotropically scaled data then were used for refinement. In all three structures a small region of the protein surface, far removed from the active site (Loop 3) was disordered. The geometric quality of the refined models was assessed with MolProbity (54) and the structure validation tools in the Phenix suite. Data collection and refinement statistics are shown in Supplementary Table S1. + +In order to facilitate more precise structural comparisons, a statistical analysis of atomic coordinate accuracy was carried out using SFCHECK (55). Following the method of Cruickshank (56), the standard deviation of the atomic coordinates can be derived from the properties of the electrondensity map: + +## Σ(X) = Σ(Slope)/Curvature + +where σ(slope) and curvature are the standard deviation of the slope and curvature of the electron density map at the atomic center. The curvature is approximated by: + +$$\mathrm{curvature}=2\pi^{2}\Bigl(\sum h^{2}F_{o b s}\Bigr)\Bigl/a^{2}\,V_{\mathrm{unit,cell}}$$ + +where a is the unit-cell length and h is the Miller index +(57,58). The maximal expected coordinate error is calculated by expressing σ(slope) as a function of the difference between Fobs and Fcalc + +$$\sigma(\mathrm{slope})=2\pi\left\{\sum\left[h^{2}\left(F_{\mathrm{obs}}-F_{\mathrm{calc}}\right)^{2}\right]\right\}^{1/2}\int a\,V_{\mathrm{unit\_cell}}$$ + +For any missing reflections, the program uses the average value of σ(F) for the corresponding resolution shell instead of (Fobs - Fcalc). The expected minimal coordinate error is estimated using the experimental σ(F) instead of the difference between the observed and calculated structure factors. + +In this case σ(slope) is computed as + +$$\sigma(\mathrm{slope})=2\pi\left\{\sum\left[h^{2}\sigma(F)^{2}\right]\right\}^{1/2}\int\!a\,V_{\mathrm{unit\_cell}}$$ + +If there is no value for an observed structure factor, the program uses = 0.04Fobs as the default value, which is roughly the error magnitude usually encountered. The both minimal and maximal estimated errors were calculated to account for the fact that the local quality of the electron density will amost always vary across different regions of a given crystal structure. These values are listed in Supplementary Table S2. + +## Oligonucleotides For Steady-State Primer Extension Assays + +Oligonucleotides used for steady-state primer extension assays were obtained from Invitrogen and are shown in Supplementary Figure S1C and 1D. Oligos were purified by HPLC and polyacrylamide gel-electrophoresis (PAGE), +quantified by UV absorbance (A260), and heated to 80◦C for 10 min before slowly cooling to room temperature overnight in 20 mM Tris–HCl pH 7.5 and 150 mM MgCl2. + +## Steady-State Primer Extension Assays + +Steady-state experiments were performed with an upstream primer 5- labeled with a Cy3 fluorophore (UP20), downstream primer containing a 5--phosphate group (DP14), +and a template (T35-A/T35-C) (Supplementary Figure S1C +and 1D). The core sequence of P/T used was the same as the crystallography experiments. Pre-hybridized UP20, DP14 and T35-A/T35-C (1:1.2:1.2) were mixed with either WT +A510D, N513A or A510D/N513A Pol . The resulting mixture (18 l) contained 50 mM Tris–HCl pH 8.5, 10 mM +MgCl2, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, P/T (200 nM) and either WT (0.3–5.0 nM), A510D (0.3–5.0 nM), +N513A (0.3–5.0 nM) or A510D/N513A (0.5–10.0 nM) Pol +. The protein/DNA mixture was directly added to varying amounts of either dTTP (0–500 M), dGTP (0–500 M) or 8-oxo-dGTP (0–1000 M) to start the polymerization reaction. + +Reaction mixtures (20 l) were quenched by the addition of 95% (v/v) formamide, 10 mM EDTA, 0.001% xylene cyanol, 0.001% bromophenol blue (10 l). Extended primers were separated by denaturing (8 M urea) 18% (v/v) +PAGE. The fluorescence intensity of the bands was quantified using a Typhoon FLA 9000 imager and ImageQuant software. + +## Kinetic Analysis Of Primer Extension Assays + +The observed rate of nucleotide incorporation (extended primer) was plotted as a function of nucleotide concentration. Steady-state kinetic parameters, Vmax and KM, were determined by fitting the data to the Michaelis–Menten equation: V = Vmax[S]/(KM + [S]). *kcat* was determined with the equation: kcat = Vmax/[E]. + +## Results And Discussion Pre-Catalytic Structure Of Pol - **With An Incoming 8-Oxodgtp** + +In order to understand how 8-oxo-dGTP is misincorporated into DNA, we decided to solve a structure of Pol in complex with an incoming 8-oxo-dGTP in a precatalytic state. Thus, we crystallized Pol with a 1-nt gapped, dideoxy-terminated DNA substrate and an incoming 8-oxo-dGTP (Figure S1A). We obtained crystals that diffracted to a resolution of 1.95 A. We were able to solve ˚ +the structure using molecular replacement, and subsequent refinement converged to an Rwork and Rfree of 0.1999 and + +![3_image_0.png](3_image_0.png) + +0.2273 respectively (Supplementary Table S1). The electron density was of sufficient quality to build protein residues 252–575 and all DNA bases, including the incoming 8-oxodGTP. Inspection of the structure immediately reveals that, as expected, 8-oxo-dGTP is bound in the active site (Figure 1A). A Mg2+ atom is present in the dNTP-binding metal (B) +site. However, a water molecule is occupying the site of the catalytic metal (A), consistent with the absence of the 3-OH +group from the nucleotide at the primer terminus (47) (Figure 1A). + +The density for the 8-oxo-dGTP residue is of high quality, and the C8 carbonyl is clearly observed (Figure 1B). The structure indicates that the incoming 8-oxo-dGTP adopts a syn-conformation and forms a Hoogsteen base-pair with the templating adenine base. Hydrogen-bonds are established between N6 of the adenine and O6 of the guanine +(3.0 A) and N1 of the adenine and N7 of the guanine (2.9 ˚ A). An intramolecular hydrogen-bond between the N2 and ˚ +a non-bridging oxygen on the -phosphate of 8-oxo-dGTP +may stabilize the syn-conformation. These interactions and the geometry of 8-oxo-dGTP in the active site of Pol are consistent with the conformation observed in existing structures of Pol - in complex with 8-oxo-dGTP (32,33). + +Interestingly, the geometry of the 8-oxo-dGTP:dA mispair differs from a canonical Watson-Crick base-pair. The R angle for 8-oxo-dGTP is 44.3◦ which is consistent with the syn-conformation of the base in other reported structures (Pol - - 44.2◦) (32,33). However, this differs from that of a dGMPCPP:dC base-pair (54.2◦) (59). The angle for dA is 55.7◦ and is consistent with an ddTTP:dA base-pair +(1XSN) (47). This is in agreement with the idea that 8-oxodG adopts an alternate conformation in order to mispair with an adenosine (15,16). Also, the C1-–C1- width of the mispair is 10.7 ± 0.015–0.091 A (where 0.015 and 0.091 ˚ A˚ +correspond to the minimum and maximum estimated errors respectively, Supplementary Table S2) which is larger than for a dTTP:dA base-pair (10.5 ± 0.021–0.094 A, Supple- ˚ +mentary Table S2). Interestingly, this larger base pair width appears to be correlated with the presence of a mispair in the active site in Pol (dGMPCPP:dT, 10.8 A) ( ˚ 59). However, despite these distortions, Pol can accommodate the 8oxo-dGTP:dA mispair in the active site without any major conformational rearrangements, as evidenced by the small RMSD between our structure and a reference pre-catalytic complex containing a ddTTP:dA base-pair in the active site +(1XSN, RMSD of 0.320 A over 261 C ˚ atoms) (47). + +## Post-Catalytic Structure Of Pol - **With Incorporated 8-Oxodgmp** + +The absence of large-scale subdomain conformational motions during catalysis in Pol implies that completion of the nucleotidyl transfer reaction and advancement from precatalytic to the post-catalytic complex involves no significant conformational changes, other than stereochemical inversion of the -phosphate (47). We therefore took advantage of this fact in order to determine whether our structure indeed represents a catalytically relevant conformation of 8oxo-dGTP. We decided to crystallize a post-catalytic complex of Pol with a nicked DNA substrate containing an 8-oxo-dG moiety at the primer terminus (Figure 2A). The crystals of the post-catalytic complex diffracted to a resolution of 2.15 A. The structure was solved by molecular re- ˚ +placement, and refinement converged to an Rwork and Rfree of 0.2144 and 0.2450, respectively (Supplementary Table S1). The electron density was of sufficient quality to build protein residues 251–575 and all DNA bases, including the incorporated 8-oxo-dGMP. + +The post-catalytic structure contains both products of the phosphoryl transfer reaction (47). In the structure, 8oxo-dGTP has been turned over, and the incorporated 8oxo-dGMP maintains the syn-conformation. However, the pyrophosphate released by the reaction has diffused away from the active site and has been replaced by a citrate ion. + +Citrate is present in high concentration in the crystallization buffer and mimics the conformation of pyrophosphate in other post-catalytic structures (47). Additionally, the citrate is coordinating a Na+ ion that is present in the metal B site (Figure 2A). The metal A is absent in the structure, + +![4_image_0.png](4_image_0.png) + +consistent with the notion that it dissociates after nucleotide incorporation (47). Superimposition of the pre- and postcatalytic structures reveals that both complexes adopt similar conformations (RMSD of 0.238 A over 236 C ˚ atoms). + +Furthermore, the geometry of all active site atoms is conserved (Figure 2B). This confirms that the conformations seen in our structures are catalytically relevant. + +## 8-Oxo-Dgtp Is Stabilized By Numerous Interactions + +Close inspection of the dNTP binding pocket reveals five residues that interact with 8-oxo-dGTP (Supplementary Figure S2). Three residues form hydrogen-bonding interactions with non-bridging phosphate oxygen atoms in the incoming nucleotide. Arg386 forms a bidentate hydrogenbond (3.0 and 3.2 A) with the ˚ -phosphate while Ser417 and Arg420 each form a hydrogen-bond with the -phosphate +(2.6 A) and the ˚ --phosphate (3.0 A), respectively. Together, ˚ +these three residues form the phosphate-binding pocket for the incoming nucleotide. However, these interactions are unlikely to confer specificity, since they are established irrespective of the identity of the incoming nucleotide. + +Ala510 provides a Van der Waals stacking interaction with the oxidized nucleobase (Figure 3A). This interaction is analogous to what is observed in a complex with undamaged substrates, and is thought to be responsible for the high dNTP binding affinity of Pol (60). It is however conceivable that the strength of the Ala510 interaction depends on the identity of the incoming dNTP. Consistent with this idea, Ala510 was shown to help discriminate against incorrect nucleotide binding (61). Thus, Pol may rely on Ala510 to probe for proper nascent base-pair geometry of 8-oxodGTP. + +In addition to Ala510, another residue recognizes a unique structural characteristic of 8-oxo-dGTP. Asn513 forms a hydrogen-bonding interaction (3.1 A) with the C8-carbonyl ˚ +of 8-oxo-dGTP (Figure 3B). In the Pol active site, Asn513 establishes a sequence independent hydrogen-bond with the O2/N3 hydrogen-bond acceptors in the minor groove of the incoming nucleotide (42). These types of minor groove interactions are thought to be crucial to the ability of DNA polymerases to discriminate against non-Watson– Crick base pairs and thus promote fidelity of synthesis (62,63). Similar to what is observed with 8-oxo-dG in the active site of different polymerases (64–67), 8-oxo-dGTP +may hijack this fidelity checkpoint mechanism in order to stabilize syn-dependent misincorporation. The O8 of 8-oxodGTP (syn) in the DNA minor groove is in a position similar to that of O2 of dTTP for a canonical Watson-Crick base-pair. This suggests that Asn513 stabilizes the lesion in the syn-conformation and thus mediates misincorporation opposite dA. Asn513 is conserved in another family X DNA +polymerase, human Pol -, where it plays a critical role in facilitating incorporation of 8-oxo-dGMP opposite template dA (19,32,33). Our structural data suggest that this residue could play a similar role in Pol . + +## Steady-State Kinetic Analysis Of The Relevance Of Ala510 **For 8-** Oxo-Dgtp Incorporation + +As mentioned above, our structural results reveal that 8oxo-dGTP is stabilized by two specific interactions. One of these, Ala510, provides a Van der Waals stacking interaction with the nucleobase of 8-oxo-dGTP. Interestingly, Ala510 in Pol is replaced by an aspartic acid in Pol - (Asp276), which is believed to restrict dNTP binding (68,69). Thus, to further characterize the role of Ala510 on 8-oxo-dGTP misincorporation in Pol , we constructed an A510D active-site substitution and performed a steady-state kinetic analysis. + +Previous data had determined the catalytic efficiency of incorporation of 8-oxo-dGMP opposite dA and dC (23). + +However, these studies lacked a comparison to dTMP incorporation. This is relevant, since 8-oxo-dGMP competes with dTTP for binding opposite a templating dA, and thus the relative incorporation efficiency of the two nucleotides ultimately determines the mutagenic potential of 8-oxodGTP *in vivo*. In order to avoid possible sequence-related effects, the core sequence of the P/T used was identical to that of the oligos used for crystallography (Supplementary Figure S1C and 1D). Using these substrates we determined the kinetic parameters [kcat and Km(app)] and catalytic efficiencies [kcat/Km(app)] for both WT and A510D Pol (Table 1). + +A comparison of the catalytic efficiencies for WT Pol revealed a 150-fold difference between dGMP and 8oxodGMP incorporation opposite dC. Conversely, incorporation of 8-oxo-dGMP was only 13-fold less efficient than dTMP opposite dA. The relatively small difference in catalytic efficiencies for 8-oxo-dGMP and dTMP incorporation opposite dA highlights the mutagenic potential of this lesion. Furthermore, comparison of the dA/dC selectivity for 8-oxo-dGTP also revealed that the pro-mutagenic synconformation is preferred in WT Pol (5:1). + +Interestingly, the A510D substitution had a differential effect on all nucleotides tested. With respect to WT protein, dTTP and 8-oxodGTP incorporation opposite dA was reduced by 4.8-fold (Figure 3C) and 5.1-fold (Figure 3D), respectively. This suggests that the A510D substitution may affect 8-oxo-dGTP(syn) in a similar manner to dTTP. However, the same does not hold true for dGTP/8-oxo-dGTP +opposite dC. Notably, the A510D substitution had the largest effect on the incorporation of 8-oxo-dGTP opposite dC (7.6-fold, Figure 3E) as opposed to dGTP (2.8-fold, Figure 3F). This suggests that Ala510 may preferentially stabilize the *anti*-conformation of 8-oxo-dGTP, promoting its incorporation opposite dC. Accordingly, Ala510 may enhance the ability of Pol to incorporation 8-oxo-dGMP opposite dC, which could ultimately increase the frequency of G to T tranversion mutations (26). + +## Steady State Kinetic Analysis Of The Relevance Of Asn513 **On 8-** Oxo-Dgtp Incorporation + +In addition to Ala510, Asn513 also makes a specific interaction with the oxidized nucleobase. To characterize the role of Asn513 on 8-oxo-dGTP misincorporation in Pol , we generated a N513A active-site substitution. Steady-state kinetic parameters [kcat and Km(app)] and catalytic efficiencies +[kcat/Km(app)] for dTTP/8-oxo-dGTP incorporation opposite dA and dGTP/8-oxo-dGTP opposite dC were determined using N513A Pol (Table 1). Given the importance of Asn513 during normal polymerization, the N513A substitution expectedly affected the catalytic efficiency for all nucleotides tested. Interestingly, similar to what was observed with Ala510, the effects were differential for all nucleotides tested. With respect to the WT protein, the catalytic efficiency for dTTP decreased by 5.8-fold (Figure 3C) +in N513A Pol . However, the N513A substitution resulted in a much more drastic effect in the context of 8-oxo-dGMP +incorporation opposite dA (25-fold, Figure 3D). Also, the catalytic efficiencies of dGMP and 8-oxo-dGMP incorporation opposite dC were reduced by 1.3 (Figure 3E) and 6.4fold (Figure 3F) respectively. Taken together, these results suggest that Asn513 plays an important role in facilitating 8oxo-dGMP incorporation opposite dA, implying that this residue selectively stabilizes the *syn-*conformation of 8-oxodGTP. + +Our results suggest that dNTP binding by Pol is influenced by multiple amino acid residues. Moreover, both Ala510 and Asn513 appear to play critical roles for anti/syndiscrimination. To examine if these residues behave individually or cooperatively, we created a A510D/N513A double substitution (Table 1, Figure 3C–F). As expected, the effects of both substitutions synergistically impacted all nucleotides tested. Interestingly, the drop in catalytic efficiency was nearly identical for 8-oxo-dGMP incorporation opposite dA and dC. This is consistent with the opposite effects of Ala510 and Asn513, facilitating incorporation of the *anti-*and syn-conformations, respectively. + +![6_image_0.png](6_image_0.png) + +| Table 1. Steady-state kinetic parameters of incorporation of 8-oxo-dGTP opposite dA and dC by DNA polymerase lambda Protein Template dNTP KM (M) kcat (min−1) | kcat/KM (min−1 M−1) | | | | +|-------------|------------|-------------|---------------|---------------| +| WT | dTTP | 4.2 ± 0.7 | 131 ± 7 | 32 ± 4 | +| 8-oxo-dGTP | 20 ± 3 | 45.2 ± 3.9 | 2.4 ± 0.3 | | +| A510D | dTTP | 35 ± 4 | 234 ± 10 | 6.7 ± 0.6 | +| A | 8-oxo-dGTP | 120 ± 8 | 56.8 ± 2.8 | 0.47 ± 0.01 | +| N513A | dTTP | 21 ± 4 | 113 ± 1 | 5.5 ± 1.0 | +| 8-oxo-dGTP | 41 ± 1.6 | 3.99 ± 0.06 | 0.097 ± 0.003 | | +| A510D N513A | dTTP | 120 ± 16 | 71.8 ± 5.6 | 0.62 ± 0.07 | +| 8-oxo-dGTP | 71 ± 2 | 3.00 ± 0.41 | 0.043 ± 0.005 | | +| WT | dGTP | 1.7 ± 0.1 | 140 ± 22 | 80 ± 10 | +| 8-oxo-dGTP | 8.4 ± 0.8 | 4.39 ± 0.04 | 0.53 ± 0.05 | | +| A510D | dGTP | 2.8 ± 0.5 | 80.0 ± 6.8 | 29 ± 5 | +| C | 8-oxo-dGTP | 24 ± 6 | 1.53 ± 0.05 | 0.070 ± 0.014 | +| N513A | dGTP | 2.0 ± 0.2 | 121 ± 2 | 60 ± 4 | +| 8-oxo-dGTP | 16 ± 0.47 | 1.31 ± 0.05 | 0.083 ± 0.005 | | +| A510D N513A | dGTP | 6.3 ± 0.8 | 67.2 ± 1.3 | 11 ± 2 | +| 8-oxo-dGTP | 120 ± 24 | 1.06 ± 0.04 | 0.009 ± 0.002 | | + +## Structural Features In Pol - **Discourage 8-Oxo-Dgtp Misincorporation** + +Although the 8-oxo-dGTP:dA mispair is tolerated in the active site of Pol , kinetic analysis indicates that the insertion efficiency is reduced relative to that of a Watson–Crick basepair (Table 1). In order to determine the structural features discouraging incorporation of 8-oxo-dGTP we crystallized Pol in complex with undamaged DNA in the exact same sequence context (Supplementary Table S1). We rationalized that this would provide a more accurate basis for comparison to the 8-oxo-dGTP containing structure, since existing structural models either lacked a 3--OH on the incoming nucleotide (1XSN) (47), or used a non-hydrolyzable dNTP +analog to trap the ternary complex (2PFO) (70). The crystals diffracted with strong anisotropy, to a resolution limit of 2.4 A in ˚ a* and c* reciprocal space directions, but only 2.9 A along the ˚ b* direction. For this reason, an anisotropic correction was carried out using the Anisotropy Diffraction Server [PMID: 24203335]. The structure was solved with molecular replacement and the model was refined to convergence (Rwork/Rfree: 0.1874/0.2358). The electron density was of sufficient quality to build protein residues 252–575 and all DNA bases, including the incoming dTTP. + +Comparing our precatalytic structure for 8-oxo-dGMP +incorporation with this structure indicates that the overall fold of the polymerase is conserved (RMSD of 0.418 over 289 C atoms). However, closer inspection of the active site reveals some important differences. First, the 8-oxo-dGTP +does not appear to stack as well as dTTP with the base at the primer terminus (Figure 4A). In an attempt to maximize this stacking interaction and accommodate 8-oxo-dGTP in the active site, the templating adenine shifts 0.3 A away ˚ +from the incoming nucleotide (Figure 4B). The direct consequence of this distortion places the templating base 0.4 A away from the minor groove interacting residue, Arg ˚ 517 +(Figure 4B). + +Arg517 plays an important role interrogating the nascent base-pair conformation. This residue forms a hydrogenbond with the templating base and adopts a base-pair checking conformation in response to dNTP binding (47). + +Figure 4. Structural features that discourage incorporation. (A) Comparison of dTTP and 8-oxo-dGTP stacking interactions in the pre-catalytic structures. The stacking interaction between 8-oxo-dGTP(syn) (magenta) and ddC (cyan) at the primer terminus of a complex is shown in the left panel. Comparison with dTTP (magenta) and the ddC (cyan) in the right panel demonstrates that 8-ox-dGTP stacks more poorly (yellow). (B) Overlay of the nascent base-pair in the pre-catalytic 8-oxo-dGTP and dTTP +complexes. 8-oxo-dGTP (magenta) appears to displace the templating dA +away from its canonical position. The direct consequence of this is that dA +shifts further away from Arg517. The hydrogen-bonding distances for the pre-catalytic 8-oxo-dGTP and dTTP complexes are shown in black and red respectively. + +This mechanism appears to be critical for fidelity of synthesis in family X polymerases. Consistently, substitution of Arg517 in Pol sharply decreases the fidelity of the polymerase as well as the catalytic efficiency of dNTP incorporation (61). Similarly, even subtle alterations of this residue in Pol - result in substantial reduction in the catalytic efficiency (71). Thus, repositioning of the templating adenosine away from Arg517 as observed in our structure would likely cause a loss of insertion efficiency. This provides a structural explanation for the decreased incorporation efficiency observed in our kinetic analysis (Table 1). + +## Ala510 And Asn513 Influence The Purine/**Pyrimidine Balance In** Pol - + +While Asn513 enhances 8-oxo-dGTP incorporation in Pol +, this residue also had a differential effect on dTTP and dGTP incorporaton. Steady-state kinetic analysis demonstrated that the catalytic efficiency for dTTP incorporation by N513A Pol was 5.8-fold (Figure 3C) lower than WT, +whereas the decrease observed in the case of dGTP incorporation was just 1.3-fold (Figure 3E). Interestingly, the equivalent residue in the family X DNA polymerase from the heat-stable organism *T. thermophilus* (TthPolX) appears to also influence the balance of purine/pyrimidine incorporation (72). Asn513 appears to play a similar role in balancing the purine/pyrimidine bias in Pol . + +Strikingly, Ala510 also appears to have a greater effect on pyrimidines than purines. A510D substitution results in a 4.8-fold (Figure 3C) decrease in catalytic efficiency for dTTP incorporation but only a 2.8-fold (Figure 3E) +decrease for dGTP. In agreement with these results, the A510D/N513A double mutant displays a clear synergistic effect. The double substitution affects dTTP incorporation to a far greater extent (52-fold, Figure 3C) than dGTP +(7.3-fold, Figure 3E), demonstrating that both residues contribute to balancing purine/pyrimidine incorporation in Pol +. This indicates that residues in addition to Asn513 are important to achieve the correct purine/pyrimidine balance. + +## Conclusion + +The free nucleotide pool is susceptible to oxidative damage +(73). To cope with this oxidative burden, cells encode an enzyme (MutT/MTH1) to sanitize the nucleotide pools by degrading oxidized nucleotides such as 8-oxo-dGTP (10,11). + +Despite the presence of this protein, residual 8-oxo-dGTP +is still capable of promoting mutagenesis (8,12,13). As a result, DNA polymerases such as Pol may encounter and incorporate this oxidized nucleotide, ultimately leading to mutagenesis. + +The structural and kinetic studies presented here reveal how 8-oxo-dGTP evades fidelity checkpoint mechanisms in Pol . Active site residues Ala510 and Asn513 establish critical interactions that help stabilize the incoming nucleotide during polymerization. Asn513 forms a minor groove interaction with the incoming nucleotide. While this interaction is used to ensure that a base-pair with the proper geometry is formed in the active site of the polymerase (42,47), +this mechanism is co-opted by 8-oxo-dGTP and appears to drive its mutagenic incorporation opposite dA. Asn513 is capable of forming a hydrogen-bond with the C8-carbonyl of 8-oxo-dGTP, thereby stabilizing the oxidized nucleotide in a syn-conformation. Consistently, substitution of asparagine to an alanine eliminates the stabilizing hydrogen-bond and results in a 25-fold reduction in the catalytic efficiency for 8oxo-dGTP misincorporation opposite dA. Conversely, the N513A substitution has only a minor effect on dTTP incorporation opposite dA (5.8-fold). Thus, Asn513 appears to selectively facilitate 8-oxo-dGMP misincorporation. + +Asn513 is also conserved in another family X DNA polymerase, Pol - (Asn279). Accordingly, Asn279 also appears to play a role mediating 8-oxo-dGTP-induced mutagenesis. + +Strikingly, in comparison to Pol - (1000-fold), the effects of Asn513 on 8-oxo-dGMP misincorporation in Pol are far less pronounced (25-fold) (19). This implies that minor groove interactions are less critical to stabilize 8-oxo-dGTP +in a syn-conformation in the Pol active site. The lesser relative importance of Asn513 with respect to Asn279 is perhaps owed to the differential interactions established by Pol with the incoming nucleotide. Pol has a higher binding affinity for nucleotides than Pol - (60), which is thought to be largely the result of a Van der Waals contact established between Ala510 and the incoming nucleobase. Conversely, the equivalent residue in Pol -, Asp276, is believed to restrict dNTP binding (68,69). + +Our results demonstrate that Ala510 substitution differentially affects incorporation of the nucleotides tested. This implies that Ala510 establishes distinct interactions with each nucleotide and therefore is unequally responsible for their stability in the active site. While an A510D substitution had an nearly equivalent effect on both dTTP (4.8fold) and 8-oxo-dGTP(syn) (5.1-fold), one consequence of this behavior is that substitution of Ala510 selectively impacts 8-oxo-dGTP misincorporation opposite dC. Substitution of Ala510 to aspartic acid results in a greater effect on 8-oxo-dGMP incorporation (7.6-fold) opposite dC than dGMP (2.8-fold). This may be due to an unfavorable electrostatic interaction between the side chain and the C8carbonyl of the nucleotide. As a consequence, Ala510 selectively enhances the incorporation of 8-oxo-dGMP opposite dC. These results are consistent with the increased propensity of 8-oxo-dGTP incorporation opposite dA relative to dC by Pol (5:1) in comparison to Pol - (24:1) (19,23). Furthermore, this implies that Pol may be more susceptible to G to T transversion mutations induced by 8-oxo-dGMP incorporation opposite dC than Pol -. + +Curiously, the effects of Ala510 and Asn513 on normal nucleotide incorporation appear to have a more pronounced effect on dTMP than dGMP incorporation. The A510D +substitution results in a greater effect for dTTP (4.8-fold) +incorporation than dGTP (2.8-fold). Likewise, a N513A +substitution has a more prominent effect on dTTP (5.8fold) incorporation than dGTP (1.3-fold). Interestingly, the family X DNA polymerase from the heat-stable organism T. thermophilus, TthPolX, contains a serine in the equivalent position of Asn513. The lack of the asparagine in TthPolX restricts the error-prone insertion of 8-oxo-dGTP opposite dA by 10-fold (72). This is thought to be an adaptation to the higher levels of 8-oxo-dGTP in the dNTP +pool expected at high temperatures. However, the absence of an asparagine residue at this position results in an imbalance in the efficiency of incorporation of purine and pyrimidine nucleotides (72), suggesting that acquiring an asparagine at this position is important for fidelity of incorporation when dealing with undamaged substrates. Accordingly, Asn513 appears to play a role in improving the purine/pyrimidine balance in Pol . Furthermore, our results demonstrate that Ala510 is also important for reducing the purine/pyrimindine bias. Owing to their larger surface area, purines have more stable base-stacking interactions than pyrimidines. Thus, direct interactions with the nucleobase appear to be relatively more important to stabilize the smaller pyrimidines. In that context, the energetics of the Ala510 interaction are likely to depend on the identity of the incoming nucleotide. At the same time, the hydrogen bond established by Asn513 appears to be critical to properly orient the incoming base. Thus, altering the Asn513 interaction in turn affects the Van der Waals contacts established by Ala510. Accordingly, the effect of the double substitution on 8-oxo-dGMP/dTMP incorporation opposite dA is markedly different than the product of both individual single substitutions. + +In all DNA polymerases, interactions with the incoming dNTP are essential for catalysis and selectivity. In the Pol active site, Ala510 and Asn513 cooperate to stabilize the incoming nucleotide. Our results highlight that dNTP +binding involves multiple interactions and the relative importance of each interaction depends on the specific base being incorporated. Both Ala510 and Asn513 influence 8oxo-dGMP incorporation as well as the balance between purine/pyrimidine incorporation. Thus, the increased catalytic efficiency of incorporation of undamaged nucleotides and purine/pyrimidine balance conferred by these residues comes thus at the price of an increased mutagenic risk in the presence of a significant pool of 8-oxo-dGTP. The described structures and kinetic results provide a framework for understanding 8-oxo-dGTP incorporation by DNA polymerases, and specifically in the context of the Pol active site. These new structural insights into the catalytic cycle for 8-oxo-dGTP incorporation by Pol will contribute to our understanding of the mutagenic potential of oxidized nucleotide pools, and the mechanisms by which DNA polymerases cope with and discriminate against these mutagenic events. + +## Accession Codes + +Atomic coordinates and structure factors for the precatalytic ternary complex and the post have been deposited in the Protein Data Bank with accession codes 4XA5, 4X5V +and 4XUS. + +## Supplementary Data + +Supplementary Data are available at NAR Online. + +## Acknowledgements + +The authors wish to thank all members of the Garcia-Diaz laboratory for insightful discussions and support, as well as Drs. Mark Lukin and Elena Yakubovskaya for oligonucleotide synthesis. + +## Funding + +National Institutes of Health (NIH) [T32 GM092714 to M.J.B.]; NIH [ES022930 to K.E.G.]; NIH [GM100021 to M.G.D.]; Use of beamline X25 at the National Synchrotron Light Source; Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-98CH10886]; +Use of beamline 5.0.2 at the Advanced Light Source was supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy +[DE-AC02–05CH11231]. Funding for open access charge: +NIH [GM100021]; Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). + +Conflict of interest statement. None declared. + +## References + +1. Neeley,W.L. and Essigmann,J.M. (2006) Mechanisms of formation, genotoxicity, and mutation of guanine oxidation products. Chem. + +Res. Toxicol., 19, 491–505. + +2. Kamiya,H., Miura,K., Ishikawa,H., Inoue,H., Nishimura,S. and Ohtsuka,E. (1992) c-Ha-ras containing 8-hydroxyguanine at codon 12 induces point mutations at the modified and adjacent positions. + +Cancer Res., 52, 3483–3485. + +3. Moriya,M. (1993) Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C–>T.A transversions in simian kidney cells. *Proc. Natl. Acad. Sci.* U.S.A., 90, 1122–1126. + +4. Kamiya,H., Murata-Kamiya,N., Koizume,S., Inoue,H., Nishimura,S. + +and Ohtsuka,E. (1995) 8-Hydroxyguanine +(7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene: effects of sequence contexts on mutation spectra. *Carcinogenesis*, 16, 883–889. + +5. Le Page,F., Margot,A., Grollman,A.P., Sarasin,A. and Gentil,A. + +(1995) Mutagenicity of a unique 8-oxoguanine in a human Ha-ras sequence in mammalian cells. *Carcinogenesis*, 16, 2779–2784. + +6. Kamiya,H., Murata-Kamiya,N., Fujimuro,M., Kido,K., Inoue,H., +Nishimura,S., Masutani,C., Hanaoka,F. and Ohtsuka,E. (1995) +Comparison of incorporation and extension of nucleotides in vitro opposite 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene. *Jpn. J. Cancer Res.: Gann*, 86, 270–276. + +7. De Bont,R. and van Larebeke,N. (2004) Endogenous DNA damage in humans: a review of quantitative data. *Mutagenesis*, 19, 169–185. + +8. Pursell,Z.F., McDonald,J.T., Mathews,C.K. and Kunkel,T.A. (2008) +Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma replication fidelity. *Nucleic Acids Res.*, 36, 2174–2181. + +9. Maki,H. and Sekiguchi,M. (1992) MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. *Nature*, 355, 273–275. + +10. Gad,H., Koolmeister,T., Jemth,A.S., Eshtad,S., Jacques,S.A., +Strom,C.E., Svensson,L.M., Schultz,N., Lundback,T., +Einarsdottir,B.O. *et al.* (2014) MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. *Nature*, 508, 215–221. + +11. Huber,K.V., Salah,E., Radic,B., Gridling,M., Elkins,J.M., +Stukalov,A., Jemth,A.S., Gokturk,C., Sanjiv,K., Stromberg,K. *et al.* (2014) Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. *Nature*, 508, 222–227. + +12. Colussi,C., Parlanti,E., Degan,P., Aquilina,G., Barnes,D., +Macpherson,P., Karran,P., Crescenzi,M., Dogliotti,E. and Bignami,M. (2002) The mammalian mismatch repair pathway removes DNA 8-oxodGMP incorporated from the oxidized dNTP +pool. *Curr. Biol.: CB*, 12, 912–918. + +13. Satou,K., Kawai,K., Kasai,H., Harashima,H. and Kamiya,H. (2007) +Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells. Free Radic. Biol. Med., 42, 1552–1560. + +14. Oda,Y., Uesugi,S., Ikehara,M., Nishimura,S., Kawase,Y., +Ishikawa,H., Inoue,H. and Ohtsuka,E. (1991) Nmr-Studies of a DNA Containing 8-Hydroxydeoxyguanosine. *Nucleic Acids Res.*, 19, 1407–1412. + +15. Kouchakdjian,M., Bodepudi,V., Shibutani,S., Eisenberg,M., +Johnson,F., Grollman,A.P. and Patel,D.J. (1991) NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-Oxo-7H-dG(syn).dA(anti) +alignment at lesion site. *Biochemistry*, 30, 1403–1412. + +16. McAuley-Hecht,K.E., Leonard,G.A., Gibson,N.J., Thomson,J.B., +Watson,W.P., Hunter,W.N. and Brown,T. (1994) Crystal structure of a DNA duplex containing 8-hydroxydeoxyguanine-adenine base pairs. + +Biochemistry, 33, 10266–10270. + +17. Lipscomb,L.A., Peek,M.E., Morningstar,M.L., Verghis,S.M., +Miller,E.M., Rich,A., Essigmann,J.M. and Williams,L.D. (1995) +X-ray structure of a DNA decamer containing 7,8-dihydro-8-oxoguanine. *Proc. Natl. Acad. Sci. U.S.A.*, 92, 719–723. + +18. Einolf,H.J., Schnetz-Boutaud,N. and Guengerich,F.P. (1998) +Steady-state and pre-steady-state kinetic analysis of 8-oxo-7,8-dihydroguanosine triphosphate incorporation and extension by replicative and repair DNA polymerases. *Biochemistry*, +37, 13300–13312. + +19. Miller,H., Prasad,R., Wilson,S.H., Johnson,F. and Grollman,A.P. + +(2000) 8-oxodGTP incorporation by DNA polymerase beta is modified by active-site residue Asn279. *Biochemistry*, 39, 1029–1033. + +20. Shimizu,M., Gruz,P., Kamiya,H., Kim,S.R., Pisani,F.M., +Masutani,C., Kanke,Y., Harashima,H., Hanaoka,F. and Nohmi,T. + +(2003) Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases. *EMBO Rep.*, 4, 269–273. + +21. Hanes,J.W., Thal,D.M. and Johnson,K.A. (2006) Incorporation and replication of 8-oxo-deoxyguanosine by the human mitochondrial DNA polymerase. *J. Biol. Chem.*, 281, 36241–36248. + +22. Yamada,M., Nunoshiba,T., Shimizu,M., Gruz,P., Kamiya,H., +Harashima,H. and Nohmi,T. (2006) Involvement of Y-family DNA polymerases in mutagenesis caused by oxidized nucleotides in Escherichia coli. *J. Bacteriol.*, 188, 4992–4995. + +23. Brown,J.A., Duym,W.W., Fowler,J.D. and Suo,Z. (2007) +Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2--deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta. J. Mol. + +Biol., 367, 1258–1269. + +24. Shimizu,M., Gruz,P., Kamiya,H., Masutani,C., Xu,Y., Usui,Y., +Sugiyama,H., Harashima,H., Hanaoka,F. and Nohmi,T. (2007) Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta. *Biochemistry*, 46, 5515–5522. + +25. Katafuchi,A., Sassa,A., Niimi,N., Gruz,P., Fujimoto,H., +Masutani,C., Hanaoka,F., Ohta,T. and Nohmi,T. (2010) Critical amino acids in human DNA polymerases eta and kappa involved in erroneous incorporation of oxidized nucleotides. *Nucleic Acids Res.*, 38, 859–867. + +26. Zaccolo,M., Williams,D.M., Brown,D.M. and Gherardi,E. (1996) An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. *J. Mol. Biol.*, 255, 589–603. + +27. Svilar,D., Goellner,E.M., Almeida,K.H. and Sobol,R.W. (2011) Base excision repair and lesion-dependent subpathways for repair of oxidative DNA damage. *Antioxid. Redox Signal.*, 14, 2491–2507. + +28. Aburatani,H., Hippo,Y., Ishida,T., Takashima,R., Matsuba,C., +Kodama,T., Takao,M., Yasui,A., Yamamoto,K. and Asano,M. (1997) Cloning and characterization of mammalian 8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue. *Cancer Res.*, 57, 2151–2156. + +29. Williams,S.D. and David,S.S. (1998) Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA. *Nucleic Acids Res.*, 26, 5123–5133. + +30. Zharkov,D.O. and Grollman,A.P. (1998) MutY DNA glycosylase: +base release and intermediate complex formation. *Biochemistry*, 37, 12384–12394. + +31. Williams,S.D. and David,S.S. (1999) Formation of a Schiff base intermediate is not required for the adenine glycosylase activity of Escherichia coli MutY. *Biochemistry*, 38, 15417–15424. + +32. Batra,V.K., Beard,W.A., Hou,E.W., Pedersen,L.C., Prasad,R. and Wilson,S.H. (2010) Mutagenic conformation of 8-oxo-7,8-dihydro-2--dGTP in the confines of a DNA polymerase active site. *Nat. Struct. Mol. Biol.*, 17, 889–890. + +33. Freudenthal,B.D., Beard,W.A., Perera,L., Shock,D.D., Kim,T., +Schlick,T. and Wilson,S.H. (2014) Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. *Nature*, 517, 635–639. + +34. Maga,G., Villani,G., Crespan,E., Wimmer,U., Ferrari,E., Bertocci,B. + +and Hubscher,U. (2007) 8-oxo-guanine bypass by human DNA +polymerases in the presence of auxiliary proteins. *Nature*, 447, 606–608. + +35. Markkanen,E., Castrec,B., Villani,G. and Hubscher,U. (2012) A +switch between DNA polymerases delta and lambda promotes error-free bypass of 8-oxo-G lesions. *Proc. Natl. Acad. Sci. U.S.A.*, +109, 20401–20406. + +36. Markkanen,E., Dorn,J. and Hubscher,U. (2013) MUTYH DNA +glycosylase: the rationale for removing undamaged bases from the DNA. *Front. Genet.*, 4, 18. + +37. Fan,W. and Wu,X. (2004) DNA polymerase lambda can elongate on DNA substrates mimicking non-homologous end joining and interact with XRCC4-ligase IV complex. *Biochem. Biophys. Res. Commun.*, +323, 1328–1333. + +38. Lee,J.W., Blanco,L., Zhou,T., Garcia-Diaz,M., Bebenek,K., +Kunkel,T.A., Wang,Z. and Povirk,L.F. (2004) Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts. J. Biol. + +Chem., 279, 805–811. + +39. Nick McElhinny,S.A., Havener,J.M., Garcia-Diaz,M., Juarez,R., +Bebenek,K., Kee,B.L., Blanco,L., Kunkel,T.A. and Ramsden,D.A. + +(2005) A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. *Mol.* Cell, 19, 357–366. + +40. Capp,J.P., Boudsocq,F., Bertrand,P., Laroche-Clary,A., Pourquier,P., +Lopez,B.S., Cazaux,C., Hoffmann,J.S. and Canitrot,Y. (2006) The DNA polymerase lambda is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells. *Nucleic* Acids Res., 34, 2998–3007. + +41. Akopiants,K., Zhou,R.Z., Mohapatra,S., Valerie,K., +Lees-Miller,S.P., Lee,K.J., Chen,D.J., Revy,P., de Villartay,J.P. and Povirk,L.F. (2009) Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts. *Nucleic* Acids Res., 37, 4055–4062. + +42. Garcia-Diaz,M., Bebenek,K., Krahn,J.M., Blanco,L., Kunkel,T.A. + +and Pedersen,L.C. (2004) A structural solution for the DNA polymerase lambda-dependent repair of DNA gaps with minimal homology. *Mol. Cell*, 13, 561–572. + +43. Kabsch,W. (2010) Xds. *Acta Crystallogr. Sect. D, Biol. Crystallogr.*, +66, 125–132. + +44. Evans,P.R. and Murshudov,G.N. (2013) How good are my data and what is the resolution? *Acta Crystallogr. Sect. D, Biol. Crystallogr.*, 69, 1204–1214. + +45. Vonrhein,C., Flensburg,C., Keller,P., Sharff,A., Smart,O., +Paciorek,W., Womack,T. and Bricogne,G. (2011) Data processing and analysis with the autoPROC toolbox. *Acta Crystallogr. Sect. D, Biol.* +Crystallogr., 67, 293–302. + +46. McCoy,A.J., Grosse-Kunstleve,R.W., Adams,P.D., Winn,M.D., +Storoni,L.C. and Read,R.J. (2007) Phaser crystallographic software. + +J. Appl. Crystallogr., 40, 658–674. + +47. Garcia-Diaz,M., Bebenek,K., Krahn,J.M., Kunkel,T.A. and Pedersen,L.C. (2005) A closed conformation for the Pol lambda catalytic cycle. *Nat. Structu. Mol. Biol.*, 12, 97–98. + +48. Emsley,P., Lohkamp,B., Scott,W.G. and Cowtan,K. (2010) Features and development of Coot. *Acta Crystallogr. Sect. D, Biol.* +Crystallogr., 66, 486–501. + +49. Zwart,P.H., Afonine,P.V., Grosse-Kunstleve,R.W., Hung,L.W., +Ioerger,T.R., McCoy,A.J., McKee,E., Moriarty,N.W., Read,R.J., +Sacchettini,J.C. *et al.* (2008) Automated structure solution with the PHENIX suite. *Methods Mol. Biol.*, 426, 419–435. + +50. Murshudov,G.N., Skubak,P., Lebedev,A.A., Pannu,N.S., +Steiner,R.A., Nicholls,R.A., Winn,M.D., Long,F. and Vagin,A.A. (2011) REFMAC5 for the refinement of macromolecular crystal structures. *Acta Crystallogr. Sect. D, Biol. Crystallogr.*, 67, 355–367. + +51. Smart,O.S., Womack,T.O., Flensburg,C., Keller,P., Paciorek,W., +Sharff,A., Vonrhein,C. and Bricogne,G. (2012) Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. *Acta Crystallogr. Sect. D, Biol. Crystallogra.*, 68, 368–380. + +52. Karplus,P.A. and Diederichs,K. (2012) Linking crystallographic model and data quality. *Science*, 336, 1030–1033. + +53. Sawaya,M.R. (2014) Methods to refine macromolecular structures in cases of severe diffraction anisotropy. *Methods Mol. Biolo.*, **1091**, +205–214. + +54. Chen,V.B., Arendall,W.B. 3rd, Headd,J.J., Keedy,D.A., +Immormino,R.M., Kapral,G.J., Murray,L.W., Richardson,J.S. and Richardson,D.C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D, Biol. Crystallogr., 66, 12–21. + +55. Vaguine,A.A., Richelle,J. and Wodak,S.J. (1999) SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. + +Acta Crystallogra. Sect. D, Biol. Crystallogr., 55, 191–205. + +56. Cruickshank,D. (1949) The accuracy of electron-density maps in X-ray analysis with special reference to dibenzyl. *Acta Crystallogr.*, 2, 65–82. + +57. Agarwal,R. (1978) A new least-squares refinement technique based on the fast Fourier transform algorithm. *Acta Crystallogr. Sect. A*, +34, 791–809. + +58. Murshudov,G.N., Vagin,A.A. and Dodson,E.J. (1997) Refinement of Macromolecular Structures by the Maximum-Likelihood Method. + +Acta Crystallogr. Sect. D, 53, 240–255. + +59. Bebenek,K., Pedersen,L.C. and Kunkel,T.A. (2011) Replication infidelity via a mismatch with Watson-Crick geometry. *Proc. Natl.* Acad. Sci. U.S.A., 108, 1862–1867. + +60. Garcia-Diaz,M., Bebenek,K., Sabariegos,R., Dominguez,O., +Rodriguez,J., Kirchhoff,T., Garcia-Palomero,E., Picher,A.J., +Juarez,R., Ruiz,J.F. *et al.* (2002) DNA polymerase lambda, a novel DNA repair enzyme in human cells. *J. Biol. Chem.*, 277, 13184–13191. + +61. Brown,J.A., Pack,L.R., Sherrer,S.M., Kshetry,A.K., Newmister,S.A., +Fowler,J.D., Taylor,J.S. and Suo,Z. (2010) Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase lambda. *J. Mol. Biol.*, 403, 505–515. + +62. Eom,S.H., Wang,J. and Steitz,T.A. (1996) Structure of Taq polymerase with DNA at the polymerase active site. *Nature*, 382, 278–281. + +63. Kiefer,J.R., Mao,C., Braman,J.C. and Beese,L.S. (1998) Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. *Nature*, 391, 304–307. + +64. Krahn,J.M., Beard,W.A., Miller,H., Grollman,A.P. and Wilson,S.H. + +(2003) Structure of DNA polymerase beta with the mutagenic DNA lesion 8-oxodeoxyguanine reveals structural insights into its coding potential. *Structure*, 11, 121–127. + +65. Hsu,G.W., Ober,M., Carell,T. and Beese,L.S. (2004) Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA +polymerase. *Nature*, 431, 217–221. + +66. Brieba,L.G., Eichman,B.F., Kokoska,R.J., Doublie,S., Kunkel,T.A. + +and Ellenberger,T. (2004) Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase. + +EMBO J., 23, 3452–3461. + +67. Vasquez-Del Carpio,R., Silverstein,T.D., Lone,S., Swan,M.K., +Choudhury,J.R., Johnson,R.E., Prakash,S., Prakash,L. and Aggarwal,A.K. (2009) Structure of human DNA polymerase kappa inserting dATP opposite an 8-OxoG DNA lesion. *PLoS One*, 4, e5766. + +68. Beard,W.A. and Wilson,S.H. (2000) Structural design of a eukaryotic DNA repair polymerase: DNA polymerase beta. *Mutat. Res.*, 460, 231–244. + +69. Vande Berg,B.J., Beard,W.A. and Wilson,S.H. (2001) DNA structure and aspartate 276 influence nucleotide binding to human DNA +polymerase beta. Implication for the identity of the rate-limiting conformational change. *J. Biol. Chem.*, 276, 3408–3416. + +70. Garcia-Diaz,M., Bebenek,K., Krahn,J.M., Pedersen,L.C. and Kunkel,T.A. (2007) Role of the catalytic metal during polymerization by DNA polymerase lambda. *DNA Repair*, 6, 1333–1340. + +71. Werneburg,B.G., Ahn,J., Zhong,X., Hondal,R.J., Kraynov,V.S. and Tsai,M.D. (1996) DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity. + +Biochemistry, 35, 7041–7050. + +72. Garrido,P., Mejia,E., Garcia-Diaz,M., Blanco,L. and Picher,A.J. + +(2014) The active site of TthPolX is adapted to prevent 8-oxo-dGTP misincorporation. *Nucleic Acids Res.*, 42, 534–543. + +73. Fraga,C.G., Shigenaga,M.K., Park,J.W., Degan,P. and Ames,B.N. + +(1990) Oxidative damage to DNA during aging: 8-hydroxy-2--deoxyguanosine in rat organ DNA and urine. Proc. + +Natl. Acad. Sci. U.S.A., 87, 4533–4537. \ No newline at end of file diff --git a/medical/md/PMC5083326.md b/medical/md/PMC5083326.md new file mode 100644 index 0000000000000000000000000000000000000000..74500977d3893ac1c45ba8f0cfd40c91b3452ae5 --- /dev/null +++ b/medical/md/PMC5083326.md @@ -0,0 +1,52 @@ +# The Earl Of Feversham + +Chairman of the Council vffjlfj, National Association for Mental Health, 1946-1"' + +# A Personal Tribute + +Lord Feversham has % +after a short illness at the ea j age of 56. Tributes have & +paid to his career in P? V.j3l agriculture, the Arts and s?\y reform. I want to Pay,hair' tribute to him as the first Co . + +man of The National Ass? + +tion for Mental HealthHe was no figure-head c" ^ +man; he saw clearly the sC j of the Association's work { +as he judged appropriate 8 t an impetus to develops , +Often he backed his judgO1 ^ +with gifts from his own P.?c 0f He was behind the opening the Northern Office in 1952'^ +creation of the Mental He National Appeal in 1954, appointment of a public formation officer in 1956 a0<* ^ +a local associations organise^ +1962. In 1961 he began a s* , +of discussions on positive me ^ +health and as a result oi ^ +personal invitation there is J +a Seminar in Bristol ai end of September on Counselling Services in Schools. + +As he gained confidence, he began to assume a national role as for the Mental Health Services. In 1958 he initiated the first debate Jn to House of Lords on the Royal Commission's Report on the Law relating Mental Illness and Mental Deficiency. He attended all the sittings w^^nr of Mental Health Act of 1959 was before the Lords, speaking on a nurn amendments. In February 1960 it was at his instigation that the YounghusDtj,e Report was first debated. Last year, in July he initiated a debate on working of the Mental Health Act. .. + +? 1 1 (1 These solemn milestones in his career as our Chairman give very little { +of the kind of man he was. He had fastidious tastes and everything a y$ +him was of the best; he was never satisfied with second best. In some ^ ^ +he was delighted to play his part as a peer and he did it beautifully, was always approachable; he always minded about those who worked foT i a His relationship with all his staff was one of warm friendship. He gift of fun which acted as a leaven to any meeting. Nobody could be + +![0_image_0.png](0_image_0.png) + +he are the competing of the competing of the competing of the wide vistes as a sense of the came to the vistas of his Yorkshire moors, plus a feeling that he had just come from a from of his Yorkshire mors, plus a feeling room. But he had probably come from a local associations meeting or from conversations with his friends the Probation Officers. Nothing was too large or too small for his attention. + +His interest in mental health—not mental ill-health—inspired all his work for social reform. He drove himself to make speeches because he felt this as something he alone could do. It did not come easily to him. He was e on trable chairman, courteous, patient, humorous, kind : always with an cye on larger issues. + +All the world loves a Lord, but very few Lords are as many-sided in their interests or as worthy of love as Lord Feversham. I have written this tribute to my chief and my friend. + +## Mary Applebey Editorial The Space Between + +It is a common error to assume that the "teenage problem" is that of the present generation and age only. Yet Keats described adolesence as "the space in between in rhich the soul is in a ferment, the if. acter undecided and the way of life uncertain". And, in Henry IV, +d shakespeare gives a vivid clinical description of adolescent rebellion as parental authority and expectation in his picture of Prince denry of Monmouth. The present dy problem differs in detail and degree rather than in basic principle. + +Critical adults There are many reasons, too, why the adult world is basically so critical of the up-and-coming generation; and why we are often reluctant to ne the problem as one for rather than solely of, the adolescent. Not the least of these reasons is that this up-and-coming generation reminding us of our own steadily advancing years; and of the fact that we will be challenged by, and in rivalry with, this new generation when they shortly reach adulthood themselves. + +## Try To Understand + +The adult need not, and indeed often cannot and should not, agree with the teenager's views on life; but at least he must try to understand the meaning of these views, and the problems which often lie behind them for the adolescent. + +The aim of the contributors to this issue has been to highlight and explain some of the specific and special problems of the present-day teenage generation. \ No newline at end of file diff --git a/medical/md/PMC5136409.md b/medical/md/PMC5136409.md new file mode 100644 index 0000000000000000000000000000000000000000..d5b1fcc07046763d062616ffe72df41993e56a1d --- /dev/null +++ b/medical/md/PMC5136409.md @@ -0,0 +1,273 @@ +# Original Article The Effect Of Lucite Glass Reinforcement On The Properties Of Conventional Glass-Ionomer Filling Materials + +Haleh Kazemi Yazdi 1, Richard Van Noort 2, Mona **Mansouri** 3 1 Dept. of Restorative Dentistry, Dental Branch, Islamic Azad University, Tehran, Iran. + +2 Academic Unit of Restorative Dentistry, University of Sheffield, Sheffield, UK. + +3 Residence of Dept. Operative and Cosmetic Restorative, Dental Branch, Islamic Azad University, Tehran Branch, Tehran, Iran. + +![0_image_0.png](0_image_0.png) + +## Abstract + +Statement of the **Problem:** The usage of glass ionomer cements (GICs) restorative materials are very limited due to lack of flexural strength and toughness. Purpose: The aim of this study was to investigate the effect of using a leucite glass on a range of mechanical and optical properties of commercially available conventional glass ionomer cement. + +Materials and **Method:** Ball milled 45μm leucite glass particles were incorporated into commercial conventional GIC, Ketac-Molar Easymix (KMEm). The characteristics of the powder particles were observed under scanning electron microscopy. The samples were made for each experimental group; KMEm and lucite- modified KetacMolar easy Mix (LMKMEm) according to manufacturer's instruction then were collected in damp tissue and stored in incubator for 1 hour. The samples were divided into two groups, one stored in distilled water for 24 hours and the others for 1 week.10 samples were made for testing biaxial flexural strength after 1 day and 1 week, with a crosshead speed of 1mm/min, calculated in MPa. The hardness (Vickers hardness tester) of each experimental group was also tested. To evaluate optical properties, 3 samples were made for each experimental group and evaluated with a spectrophotometer. The setting time of modified GIC was measured with Gillmore machine. + +Result: The setting time in LMKMEm was 8 minutes. The mean biaxial flexural strength was LMKMEm/ 1day: 24.13±4.14 MPa, LMKMEm/ 1 week: 24.22±4.87 MPa KMEm/1day:28.87±6.31 MPa and KMEm/1 week: 26.65±5.82 MPa which were not statistically different from each other. The mean Vickers hardness was LMKMEm: 403±66 Mpa and KMEm: 358±22 MPa; though not statistically different from each other. The mean total transmittance (Tt) was LMKMEm: 15.9±0.7, KMEm: 22.3±1.2, the mean diffuse transmittance (Td) was LMKMEm: 12.2±0.5, KMEm: 18.0±0.5 which were statistically different from each other. Conclusion: Leucite glass can be incorporated with a conventional GIC without interfering with setting time. Yet, it did not improve the mechanical and optical properties of the GIC. + +Corresponding **Author:** Mansouri M., Dept. Operative and Cosmetic Restorative, Dental Branch, Islamic Azad University, Tehran Branch, Tehran, Iran. Tel: +98-2532942742 Email: dr_monamansouri@yahoo.com Cite this article as: Yazdi H., Van Noort R., Mansouri M. The Effect of Lucite Glass Reinforcement on the Properties of Conventional Glass-Ionomer Filling Materials. J *Dent* Introduction One major advantage of glass ionomer cements (GICs) +as a restorative material is bonding to dental tissue and due to this, the prepared cavity for this material can be very conservative. [1] In addition to this, glassionomer has many other advantages such as fluoride release, coefficient of thermal expansion close to tooth structure, durable bond to tooth tissue and biocompatibility. [2-3] In spite of all these advantages, the clinical performance of the material are limited due to other drawbacks: low flexural and fracture toughness, early water sensitivity, low fatigue strength, high surface roughness and poor wear resistance. [2-4] +Many investigators have attempted to modify the formulation of GICs to overcome these problems. The modifications have been done on the powder and liquid composition and configuration. [2-6] Modification in powder was different in many aspects, from refinement of glass particle size to incorporation of different particles such as metal, ceramics and fibres. [1-2, 714] Along with modifications in the glass powder of GICs, the matrix has been changed. Some of these modifications are: use of copolymer (carboxylic aciditaconic acid), incorporation of tartaric acid in the liquid part, use of the copolymer with high molecular weight, concentration of poly acid, incorporation of freeze- dried acid copolymer in the cement powder and the use of N-vinylpyrrolidone containing polyacids +(NVP). [15-16] Culberston et al. [7] showed that acrylic acid-itaconic acid- NVP (AA/IA/NVP) polymers with diverse molar ratio can produce GICs with increased mechanical properties. Hydroxy apatite (HA) can react with GICs matrix by polyacid hydroxyl group, so incorporation of HA into GICs may improve physical properties and biocompatibility of set cement. Besides, it contributes to the bond strength of GICs due to a similar composition to tooth structure. [10] Lucas et al. [10] illustrated that incorporation of HA in GICs powder increased the fracture toughness significantly so it made more durable bond to dental tissues. Moreover, according to Moshavernia et al. [5] addition of nano HA enhanced the mechanical properties of commercial GIC Fuji II (Fuji II LC, GC Corp.; Tokyo, Japan) and it seems that the bonding property was also enhanced. + +Micro-sized Yttria stabilized zirconia (YSZ) +powder was added to Miracle Mix (GC America Inc.; Alsip, IL, USA) powder to improve the mechanical properties. The study showed that the uniform distribution of the glass and YSZ particle in the matrix, which ensured a high packing density of GICs, gave relatively high mechanical properties to YSZ GIC. [17] However, these two last modifications have been done as a patent and there are no commercial brands with these modifications. + +High viscosity conventional GICs like KetacMolar-easy mix (3M ESPE I.D. No.70201119107, Germany), and Fuji IX (GC America Inc.; Alsip, IL, +USA) have been introduced recently, designed as an alternative to amalgam for posterior preventive restorations. These show significant improvements in compressive strength and fracture toughness. [15, 18] They can be used as a core build up material under a crown, permanent filling for primary teeth and class I restorations in non-occluded regions. [15, 19] Nevertheless, they are not suitable for high stress areas and still their main problems are low fracture toughness and flexural strength compared to other restorative materials. [18] +The aim of this study was to evaluate physical and mechanical properties of a conventional glass ionomer material after substitution a leucite glass for the acid soluble glass in the powder of this material. + +## Materials And Method + +The material used in this study was a conventional glassionomer material, Ketac-Molar Easymix(KMEm) (3M ESPE I.D. No.70201119107, Germany), shade +(A3). The principle composition of the powder is aluminum-calcium-lanthanum-fluorosilicate glass and the liquid is an aqueous solution of polycarbonic acid (a copolymer from acrylic and maleic acid) and tartaric acid. + +In order to modify the GIC powder, a glass powder with a composition of a leucite glass ceramic (PS4) which was made by a PhD student in Sheffield University Dental School Adult Dental care, was added to KMEm glass ionomer powder. The modified glass powder was ball milled for 10 min and 400 rpm in ball milling machine (Retsch GmbH; PM 100, Germany) to reduce the particles size. To achieve a uniform size of glass filler the leucite glass ceramic was sieved manually with a 45μ sieve (Retsch GmbH; Germany), thus most of the glass had a particle size less than 45 μm. + +The leucite glass ceramic was added to the glassionomer powder in 20 vol%. This addition has been done manually by dry mixing on a paper plate using a metal spatula until an even distribution was achieved. + +The original and modified glass powder was observed under scanning electron microscope (SEM) +(X1-20 SEM, Holland). + +A silicone rubber mould was used for fabrication of the samples. The samples were made in a disc with a thickness of 2.5mm and diameter of 12mm.Mixing of the modified powder and the cement liquid was carried out using a metal spatula according to manufacturer's instructions. The mixing time did not exceed 1 min and working time was 3-3.5 min. The cement paste was carried into the mould by metal spatula and pressed using a glass slab. After the material was set, the specimens were collected in a damp tissue and were stored in an incubator (LEEC Compact Incubator LEEC Limited; Nottingham, UK) at a temperature of 37ºC for 1 hour. This was followed by storing the specimens in distilled water for 24 hours. For the biaxial flexural strength test, the specimens were ground and polished down to 600 grit using SiC (Buehler-Met; Metallographic Grinding paper, UK) paper. The final dimensions of specimens were measured with digital micrometer with a precision of 0.01 mm on four central axes and thickness of all samples was 2±0.2mm. + +For the hardness test, the specimens were ground and polished down to 1μm using SiC papers and diamond paste. + +Three samples were made for each experimental group for spectrophotometer test in the silicon rubber mold. The samples were made with a thickness of 2mm and diameter of 16mm. Mixing the modified powder and the cement liquid was carried out by using a metal spatula according to manufacturer's instructions. The mixing time did not exceed 1 min and working time was 3-3.5 min. The cement paste was carried into the mould by metal spatula and was pressed using a glass slab. After the materials were set, the specimens were collected in damp tissue and were stored in an incubator at a temperature of 37ĉ for 1 hour. After that the specimens were stored in distilled water for 24 hours. The specimens were ground and polished down to 600 grit using SiC papers. The thickness of specimens was measured with digital micrometer with a precision of 0.01 mm on three central axes and it was 1±0.1 mm. The specimens were stored in distilled water until the spectrophotometer test was carried out. + +The setting time of modified glass ionomer was measured with Gillmore needle apparatus (Impact Test Equipment Ltd; Scotland, UK) and digital timer. The Gilmore needle works on the principle of two different sized needles with different weight being placed onto the surface of setting cement. Initially a large needle (10 mm) with a weight of (28g) is applied and the working time is determined when the needle no longer indents the surface. A second needle with a smaller diameter (1mm) and larger weight (400g) is then applied to the surface and time when this needle no longer indents the surface is designated as the setting time. Both tests were carried out at ambient temperature 21ºC. + +To evaluate flexural strength, ten discs (12mm diameter and 2±0.2 thickness) were made for each experimental group. The biaxial flexural strength was measured after 1day and 1 week. + +The thickness of each specimen was measured in the centre and three peripheral points using a digital micrometer before testing. Flexural property was quantified by biaxial flexural strength test (LLOYD Instrument, LRX 103648, Farham, UK) at a crosshead speed of 1 mm/min (Figure 1). The flexural strength was obtained by measuring the load at fracture in Newton and then calculated in MPa. + +![2_image_0.png](2_image_0.png) + +Ten discs were made for each experimental group (N=10). The discs were mounted on the Vickers hardness machine (Vickers Hardness VX Series Tester) and indented with Vickers diamond pyramid at 1 Kg load and the resulting diagonal length was measured. The Vickers hardness in MPa is then obtained using the following equation: HV=1.854 P/d2 Where p is the applied load in Newtons and d is the mean diagonal length in mm. [21] + +![3_image_0.png](3_image_0.png) + +Figure 2: Schematic picture of the mechanism of light transmittance detection through the integrating sphere. + +Three sample of each group (N=3) were analyzed by Perkin Elmer Lambda 2 spectrophotometer + +![3_image_2.png](3_image_2.png) with an integrating sphere sensor (Figure 2). The spectral range of the colorimeter was between 380 and 700 nm at 1 nm interval. The data were analyzed by UV +WinLab (version 2) software. One-way ANOVA followed by Tukey's pairwise comparison was used for analyzing the data. + +![3_image_3.png](3_image_3.png) + +## Result + +Characterizations of **powders** +The SEM of glass powder in KMEm (3M ESPE, I.D. + +No.70201119107, Germany) (control) and LMKMEm + +![3_image_1.png](3_image_1.png) (experimental) groups are illustrated in Figure 3 and 4. + +The particle size in LMKME was larger as a result of adding 45μm Leucite glass ceramic. However the particle size of the powder was significantly decreased after ball milling and it can be clearly seen in Figure 5. + +Setting **time analysis** +The setting time in KMEm GICs (3M, ESPE, I.D. No.70201119107, Germany) is 7 min. Adding 20 vol% leucite glass ceramic to the powder of KMEm GICs increased the setting time. After ball milling, the setting time was decreased to 8 min which was very close to original cements. + +Biaxial **Flexural Strength** +The mean biaxial flexural strength of the control (KMEm; 3M, ESPE, I.D. No.70201119107, Germany) and experimental (LMKMEm) groups is summarized in Table 1. + +Table 1: Summary of result of biaxial flexural strength of two glass ionomer cement and storage time (MPa). + +KMEm after 1 day storage recorded a mean biaxial flexural strength of 28.87 MPa, which was the highest. After 1 week storage, the biaxial flexural strength decreased in this group and a mean of 26.65 MPa was recorded for this group. However the differ- + +| Materials and | (MPa) | Min-Max | SD | | +|--------------------------------------------------------------------------------------------------------------|---------|-----------|------|----| +| Mean | (MPa) | N | | | +| storage time | | | | | +| KMEm,1 day | 28.87 | 19.2-37.1 | 6.31 | 10 | +| LMKMEm,1 day | 24.13 | 17.4-31.5 | 4.14 | 10 | +| KMEm,1 week | 26.65 | 17.6-36.1 | 5.82 | 10 | +| LMKMEm,1 week | 24.22 | 18.6-32.7 | 4.87 | 10 | +| KMEm= Ketac-Molar Easymix (Control group), LMKMEm= Leucite Modified Ketac-Molar Easymix(Experimental group) | | | | | + +ence was not significant. (p> 0.05) For LMKMEm, the biaxial flexural strength has not changed after 1 week storage and a mean of 24.13 MPa and 24.22 MPa was recorded for 1day and 1 week storage respectively. + +One-way ANOVA followed by Tukey's pairwise comparison showed no significant statistical difference between the control and experimental groups(p> 0.05). + +Vickers **Hardness** +The calculated Vickers hardness values are summarized in Table 2. + +| Materials | Mean (MPa) | Min-Max | SD | | +|------------------------------|---------------|-----------|------|----| +| (MPa) | N | | | | +| KMEm (Control group) | 358 | 325-399 | 22 | 10 | +| LMKMEm (Experimental group) | 403 | 275-533 | 66 | 10 | + +Table 2: Summary of the calculated Vickers hardness in two glass ionomer cements groups in MPa. + +LMKMEm GIC recorded a mean Vickers hardness of 402 MPa which is higher than KMEm GIC + +![4_image_0.png](4_image_0.png) + +with 358 MPa. One-way ANOVA followed by Tukey's pairwise comparison showed no significant statistical difference between the control and experimental groups (p> 0.05). + +![4_image_1.png](4_image_1.png) + +## Optical Properties + +Three optical properties of two glass ionomers were analyzed in this study. The result is graphically presented in Figures 6, 7. + +The means of 22.3 for total transmittance (Tt) +and 18 for diffuse transmittance (Td) and 4.3 for direct transmittance (Tdir) were recorded for KMEm GIC (control group) and the means of Tt, Td and Tdir were recorded 15.9, 12.2 and 3.7 respectively for LMKMEm GIC (experimental group). A decrease in the mean of all optical values was observed in the experimental group according to these data. + +One-way ANOVA followed by Tukey's pairwise comparison showed significant statistical difference of Tt, Td between the control and experimental groups (p< 0.05). Regarding Tdir, one-way ANOVA followed by Tukey's pairwise showed no statistical difference between the control and experimental groups(p> 0.05). + +## Discussion + +In this experimental study, 20% volume fraction of a conventional GIC (like KMEm) was replaced with non +-reactive leucite glass to try and produce a conventional GIC with improved mechanical properties. Leucite glass was chosen to add to the powder because it is a strong glass with an affinity to poly acrylic acid. [22] +Simultaneous with preparing and working with glass ionomer formulation, it is really critical to consider working and setting time. [7] Substitution of reactive powder with non-reactive produced a modified conventional glass ionomer (LMKMEm) with significantly increased setting time (14 minute). The proper setting time for restorative GIC is 6-8 minutes. [23] So to overcome this problem, the powder was ball milled to produce finer grain size to reduce the setting time. + +Finer grain size can speed up the setting reaction because of greater surface. [16] +Modification of conventional glass ionomer with leucite glass was a novel approach. Hence the proper milling time and rotating speed for ball milling of the powder had to be done by trial and error. The powder was ball milled with increasing time till the mixing cement had set in proper setting time and was ended up by 10 minute rotating time and 400 rotating speed. The SEM micrographs clearly showed finer grain size of the LMKMEm GICs powder after ball milling (Figure 3, 4). Regarding manipulation properties, LMKMEm- GICs has a setting time of 8 minute which is a + +![5_image_0.png](5_image_0.png) + +proper setting time for restorative GIC. Additionally, the LMKMEm GICs powder is mixed to the liquid as the same amount as the KMEm-GICs powder and the viscosity of the paste is the same as well. (Figure 8) +In this study, flexural strength test was used to evaluate the mechanical strength of the new cement. Mechanical strength of many brittle dental materials such as cements is evaluated by tensile rather than compressive strength. Crack propagation is the main reason for the failure of these materials which is favored by tensile rather than compressive loading. + +Prosser et al. [24] have recommended that the flexural strength is the most appropriate measurement method for strength evaluation of GICs. They elucidated that, as the fracture would occur in atomic level in GICs matrix by tensile or shear failure; compressive strength basically had no meaning. [24] +Although tensile strength is the best method to estimate the mechanical properties of GICs, direct measurement of tensile strength for brittle material has technical problems. Tensile strength measurement by loading in compression (diametrical tensile strength) is only valid if a material does not have significant plastic deformation. So, diametrical tensile strength test usually gives higher values to polyacrylic acid base cements which have some degrees of plasticity. According to these reasons, it has been suggested that the flexural strength tests are the most practical and reliable way for evaluation of tensile strength of brittle materials. However, measurement of compressive and diametrical tensile strength has been reported for GICs, recently. [25] +The results in Table 1 show that the mechanical properties of KMEm have not improved with this modification. A mean biaxial flexural strength of 24.13±4.14 was recorded for LMKMEm after 1 day storage in distilled water. In comparison it was 28.87± 6.31 MPa for KMEm. However this difference was not statistically significant. + +It is suggested that different microstructure and variation in composition may have affected the mechanical properties of the GICs. [25] The smaller particle size, smaller and lesser voids and proper bonding of glass fillers and matrix in KMEm GIC appear to have resulted in higher flexural strength. The somewhat larger particle size, lack of proper bond between the filler and matrix and larger and more voids in LMKMEm GIC have caused opposite trends value for flexural strength. Mitsuhashi et al. [3] suggested that the direct tensile strength of resin modified GIC increased significantly by reduction of filler particle size to 5μm. They claimed that the particle size in KMEm - GIC powder is mainly less than 9.6μm, which means that 90% of the particle having a diameter of less than 9.6μm. Additionally 50% of the glass particles are 2.8μm or smaller. [3] Moreover, it has been reported that proper bonding between filler particles and glass matrix is contributing to mechanical properties of set cement by modifying the character of local stress and crack propagation in the matrix. [2] As a result of improper bond between the leucite glass particles and matrix of GIC, the large leucite glass particles might act as a void and contribute to crack propagation in LMKMEm GIC. It can be another reason for decreasing the flexural strength in LMKMEm GIC. Treatment of leucite glass particles with a coupling agent might improve the bonding of this glass particle to the matrix and consequently can improve the mechanical properties of GIC. However further research is necessary to establish this. + +The biaxial flexural strength of KMEm had decreased after 1 week storage in distilled water. Although this reduction was not significant, it was an interesting finding. In this GIC, 5% dried polycarbonate (a copolymer from acrylic and maleic acid) is incorporated into the powder. This leads to greater overall acid concentration in the cement which increases cross-linking and improves mechanical values without a considerable increasing in the initial viscosity. It was reported that GICs based on copolymer of acrylic and maleic acid demonstrate deterioration in flexural strength after water storage. [26] It seems in the glass ionomers with polyacrylic matrix compressive and flexural strength increases after storage in the water, whereas that with copolymers acid does not. + +[27] Generally, the mechanical strength of GIC increases with time. This has been attributed to the maturation of the cement matrix due to further cross-linking because of the formation of new aluminum and calcium polyacrylates and the slow build up of a silica matrix from acid solution of glass particles. [26] +The fracture mode of flexural strength samples was also different in KMEm and LMKMEm. All the samples in LMKMEm (experimental groups) regardless to the storage time have been fractured into two pieces. While in KMEm (control groups) a few samples have been fracture into three pieces. It can be attributed to higher flexural strength of control groups comparing to experimental groups. + +In this study, traditional Vickers hardness was used to test the hardness of KMEm-GIC and LMKMEm-GIC. As it was mentioned in the literature review, this was not the best method for testing hardness of GICs because this material is a heterogeneous, biphasic and weak. The applying load was 1 kg which might be very heavy for these materials. But it was the only available method for testing the hardness of the new material. + +According to the result in Table 2, the Vickers hardness of LMKMEm was 403±66 MPa which was higher than KMEm (358 ± 22). This greater value could be attributed to use of extremely hard leucite glass fillers; however, this difference was not statistically significant. Guggenberger et al. [28] reported that the hardness of KMEm reached the level of hybrid composite with the value of 420 MPa. In their study they did not mention the test method they used. Their result was represented in a diagram without showing the standard deviation. According to product pamphlet from ESPE, the surface hardness of KMEm GIC was recorded 420±82 MPa with DIN 53456 standard methods. + +The result of the above mentioned study is different from the outcome of the present study. The difference in the hardness value can be explained by using different testing methods and numerous porosities in our samples. + +The reading of indention was really difficult in this study due to indistinct border of pyramid in the micrograph of Vickers hardness tester (Figures 9 and 10). This phenomenon was explained by Yap AU. et al. [29]They suggested that the pile- up of the material around the indenter due to plasticity of the material is the main cause of this problem. Therefore, to record exact mechanical properties from instrumented indention, a good understanding of the relationship between indentational load and the true area is necessary. [29] +The optical properties of new GIC along with the mechanical properties were evaluated. Table 3 shows the mean Tt of 22.3±1.2 and Td of 18.0±0.5 for KME +m-GIC compared to 15.9±0.7 and 12.2±0.5 respectively for LMKMEm GIC. A reduction in Tt value and Td value was observed in the LMKMEm GIC which was statistically significant p< 0.05. According to this result LMKMEM GIC is more opaque comparing to KMEm GIC. One of the reasons for opacity of GIC is the mismatch of refractive index of the fillers and matrix. [30] Hence, this reduction can be attributed to more porosity and imperfection in LMKMEM GIC which can increase the refractive index of the matrix. + +Table 3: Summary of calculated three optical properties in two glass-ionomer cements. + +| Optical properties | Tt | Td | Tdir | +|------------------------------|---------------------------|------|--------| +| Materials | | | | +| KMEm (Control group) | 22.3±1.2 18.0±0.5 4.3±1.3 | | | +| LMKMEm (Experimental group) | 15.9±0.7 12.2±0.5 3.7±0.2 | | | + +Filler composition and content also can change the optical properties of direct restorative materials. [31] By very careful evaluation of Figures 6 and 7, a shifting down of Tt and Td can be observed in LMKMEm GIC. The curvature of Tt and Td is the same in both materials but it seems that the curve shifted down in LMKMEm GIC group. It showed that color does not change in the new material because light absorption did not change in all reflected wavelengths. The leucite glass has low refractive index so this opacity could be attributed to different refractive indices of leucite glass and glass ionomer matrix. + +Using leucite glass composition with refractive index closer to the matrix might preclude this problem and more lucent GICs could be produced. However, KMEm is a posterior restorative material and improvement in mechanical properties might have priority compared to optical properties. + +The samples with numerous porosities were the prominent problem in this study. The depth and size of the voids is related to microstructure of the GIC. Some factors such as size and shape of fillers, wet ability of fillers and mixing procedure can affect the number of porosity. [25] Mechanical mixing and using angled nozzle for accurate placement can produce samples with less porosity. + +In this study, new GIC with proper manipulation properties was produced successfully. The mechanical properties of the new material have not improved as a result of problems which were mentioned in the discussion. However, this study could be a good basic study for future investigations. + +## Conclusion + +Within the limitations of the present study, the addition of leucite glass into the KMEm GIC produced a modified conventional GIC with reasonable setting time and manipulation properties. The addition of leucite glass to the KMEm GIC did not improve the biaxial flexural strength and hardness of the material. + +The addition of leucite glass to the KMEm GIC cement produced a new material with a higher opacity comparing to the original material. + +## Acknowledgments + +This article is based on thesis submitted to the Academic Unit of Restorative dentistry, University of Sheffield, Sheffield, UK in fulfillment of the requirements for master degree. Special gratitude is forwarded to Prof. Richard Van Noort, for his expert advice and academic devotion. + +## Conflict Of Interest + +The authors of this manuscript certify that they have no conflict of interest. + +## References + +[1] Nagaraja Upadhya P, Kishore G. Glass ionomer cement: The different generations. Trends Biomater Artif Organs. 2005; 18: 158–165. + +[2] Hammouda IM. Reinforcement of conventional glassionomer restorative material with short glassfibers. J Mech Behav Biomed Mater. 2009; 2: 73-81. + +[3] Mitsuhashi A, Hanaoka K, Teranaka T. Fracture toughness of resin-modified glass ionomer restorative materials: effect of powder/liquid ratio and powder particle size reduction on fracture toughness. Dent Mater. 2003; 19: 747-757. + +[4] Ana ID, Matsuya S, Ohta M, Ishikawa K. Effects of added bioactive glass on the setting and mechanical properties of resin-modified glass ionomer cement. Biomaterials. 2003; 24: 3061-3067. + +[5] Moshaverinia A, Ansari S, Moshaverinia M, Roohpour N, Darr JA, Rehman I. Effects of incorporation of hydroxyapatite and fluoroapatite nanobioceramics intoconventional glass ionomer cements (GIC). Acta Biomater. 2008; 4: 432-440. + +[6] Smith DC. Development of glass-ionomer cement systems. Biomaterials. 1998; 19: 467-478. + +[7] Culberston BM. Glass-ionomer dental restoratives. Prog + +Polym Sci. 2001; 26: 577–604. + +[8] McLean JW. Glass-ionomer cements. Br Dent J. 1988; 164: 293-300. +[9] Lohbauer U, Walker J, Nikolaenko S, Werner J, Clare A, Petschelt A, Greil P. Reactive fibre reinforced glass ionomer cements. Biomaterials. 2003; 24: 2901-2907. + +[10] Lucas ME, Arita K, Nishino M. Tough-ness, bonding and fluoride-release properties of hydroxyapatite-added glassionomer cement. Biomaterials. 2003; 24: 37873794. +[11] Kim DA, Abo-Mosallam HA, Lee HY, Kim GR, Kim HW, Lee HH. Development of a novel aluminum-free glass ionomer cement based onmagnesium/strontiumsilicate glasses. Mater Sci Eng C Mater Biol Appl. 2014; 42: 665-671. + +[12] Shinonaga Y, Arita K, Nishimura T, Chiu SY, Chiu HH, Abe Y, et al. Effects of porous-hydroxyapatite incorporated into glass-ionomer sealants. Dent Mater J. 2015; 34: 196-202. + +[13] Lohbauer U. Dental Glass Ionomer Cements as Permanent Filling Materials?- Properties, Limitations and Future Trends. Materials. 2010; 3: 76-96. + +[14] Moshaverinia A, Roohpour N, Chee WL, Schricker SR. + +A review of powder modifications in conventional +glass- ionomer dental cements. J Mater Chem. 2011; 21: 1319-1328. + +[15] Major C. Advance in Glass-Ionomer Cements. 1th ed. + +Berlin, Chicago:Quintessence Publishing Co;1999.p.97. + +[16] Alan D, Wilson J. Glass-Ionomer Cement. 1th ed., Chicago: Quintessence books; 1988. p.247. +[17] Gu YW, Yap AUJ, Cheang P, Koh YL, Khor KA. Development of zirconia-glass ionomer cement composites. J Non-Crystalline Solids. 2005; 351: 508-514. + +[18] Hickel R, Dasch W, Janda R, Tyas M, Anusavice K. + +New direct restorative materials. FDI Commission Project. Int Dent J. 1998; 48: 3-16. + +[19] Peez R, Frank S. The physical-mechanical performance of the new Ketac Molar Easymix compared tocommercially available glass ionomer restoratives. J Dent. 2006; 34: 582-587. + +[20] Moshaverinia A, Ansari S, Movasaghi Z, Billington RW, Darr JA, Rehman IU. Modification of conventional glass-ionomer cements with N-vinylpyrrolidone containing polyacids, nano-hydroxy and fluoroapatite to improve mechanical properties. Dent Mater. 2008; 24: 1381-1390. + +[21] Pittayachawan P, McDonald A, Petrie A, Knowles JC. + +The biaxial flexural strength and fatigue property of Lava Y-TZP dental ceramic. Dent Mater. 2007; 23: 1018-1029. + +[22] Jivraj SA, Kim TH, Donovan TE. Selection of luting agents, part 1. J Calif Dent Assoc. 2006; 34: 149-160. + +[23] Robert G. Craig JM. Powers. Restorative Dental Mate rials. 11th ed. St. Louis, Missouri: Mosby; 2002. p.92. + +[24] Prosser HJ, Powis DR, Wilson AD. Glass-ionomer + +cements of improved flexural strength. J Dent Res. 1986; 65: 146-148. +[25] Xie D, Brantley WA, Culbertson BM, Wang G. Mechanical properties and microstructures of glassionomer cements. Dent Mater. 2000; 16: 129-138. + +[26] Khouw-Liu VH, Anstice HM, Pearson GJ. An in vitro investigation of a poly(vinyl phosphonic acid) based cement with four conventional glass-ionomer cements. Part 1: Flexural strength and fluoride release. J Dent. 1999; 27: 351-357. + +[27] Williams JA, Billington RW. Changes in compressive strength of glass ionomer restorative materials withrespect to time periods of 24 h to 4 months. J Oral Rehabil. 1991; 18: 163-168. + +[28] Guggenberger R, May R, Stefan KP. New trends in glass-ionomer chemistry. Biomaterials. 1998; 19: 479483. + +[29] Yap AU, Wang X, Wu X, Chung SM. Comparative hardness and modulus of tooth-colored restoratives: a depth-sensing micro in dentation study. Biomaterials. 2004; 25: 2179-285. + +[30] Noort RV. Introduction to Dental Materials. 3th ed. + +Sheffield UK: Mosby Elsevier; 2007. p. 90. + +[31] Engqvist H, Lööf J, Uppström S, Phaneuf MW, Jonsson JC, Hermansson L, et al. Transmittance of a bioceramic dental restorative material based on calciumaluminate. J + +Biomed Mater Res B Appl Biomater. 2004; 69: 94-98. \ No newline at end of file diff --git a/medical/md/PMC5183298.md b/medical/md/PMC5183298.md new file mode 100644 index 0000000000000000000000000000000000000000..8ece0a0f9fc4a186f4a0e8b6d2ba94e0ccc59617 --- /dev/null +++ b/medical/md/PMC5183298.md @@ -0,0 +1,107 @@ +does not regard the tetrachloride as being a perfectly good anesthetic ; but he thinks that, in several instances, it may bo advantageously mixed with chloroform, as it has some properties which are antagonistic to those of the latter substance, and may, therefore, modify its action. The use of tetrachloride of carbon may be made to precede that of chloroform ; and, although he invites further experiments upon the point, Dr. Sansom thinks that a mixture of one part of the tetrachloride of carbon and six of chloroform will constitute a safe, as well as agreeable, anesthetic.— British Medical Journal, Sept. 7, 1867. + + On the Resin of the Veratrum viride and its Therapeutical action. By Dr. Srom , of Wurzburg.—The resin of the Veratrum viride employed by Dr. Stohr is an extract from the root of the plant, which in the dry state ought to yield a bright brown, faintly-smelling powder, with a slight taste. The cases in which Dr. Stohr employed this resin included several of pneumonia, one of acute rheumatism, one of measles, and one of emphysema, in the last of which there were very distressing palpitations of the heart. In this last case the patient took four pills, each containing one-sixth of a grain of the resin with one-eighth of a grain of morphia, at intervals of half-an-hour. + +the third dose the temperature of the extremities was reduced, the frequency of the pulse was lowered, the breathing became quieter, and the palpitations ceased in two hours, and after the fourth pill nausea supervened. The general effects produced by the resin of the Veratrum viride were similar to those just mentioned—namely, reduction of the temperature, of the pulse, and of the frequency of respiration. Dr. Stohr considers that the use of the resin is indicated only in those diseases in which the pulse and the temperature are unusually high, because in such cases a very rapid remedial action is desirable, and the unpleasant effects are outweighed by a quickly successful result. When the medicine is employed, its rapid and energetic action must be recollected, and the patient should be carefully watched. Schmidt's Jahrbiicher der Gesammten Medicin, December 10, 1866. + +REPORT ON PATHOLOGY AND PRINCIPLES AND +PRACTICE OF MEDICINE. + +BY FRANCIS C. WEEB, M.D., F.L.S., +Member of the Royal College of Physicians, Physician to the Great Northern Hospital. + +On the Pathogeny of Fatty Transformations, with Experimental Researches on the Action of Phosphorus on the Living Tissues. By Dr. L. Ranvier.—The author combats the idea accepted by Virchow, that in the case of fatty transformation following inflammation, the former may be considered a part of the inflammatory process. + +Numberless facts show that inflammation and fatty degeneration, Dstead of being connected, are in reality opposed. In inflammation 0 the subcutaneous cellular tissue fatty cells lose the fat which they c9ntain; their nuclei and the little mass of enclosed protoplasm 8ne place by division to an abundant production of cells, which fill old adipose vesicle. In acute osteitis we And the fatty medulla transformed in like manner into proliferous tissue. In acute or +]oriC ^u^ammations of joints, the cartilage cells, which, physioogically, contain fat, are deprived of it as long as cellular prolifera-continues. ^1IS ^appearance of fat in elements which physiog'cally contain it is met not only iu inflammatory processes, u a also in all active new formations. Thus, when new growths te their point of departure from cellulo-adipose tissue, or from niedulla of bone, they determine the disappearance of fat in t^e ce^3 which they invade. But a more important fact still is at when, under the influence of a pathological cause, fatty traus- +?rmation has invaded certain cells, it is observed that these +_ e is may free themselves from the fat which they contain under fi !nfluence inflammation, provided it supervene before the e lular elements are completely destroyed by the degeneration, evertheless, at the end of the inflammatory process, and in the last age of every new formation, a fatty transformation is observed of per abundant elements. This transformation must not be attricd to the original irritative action, but rather to an alteration of u rxtion, for it never supervenes when the cells are in full prolifera-It occurs only when the former movement is arrested, and +"en nutritive changes go on with difficulty in those elements of +!ch the number is excessive in proportion to the vascular supP J' The first day of a catarrhal inflammation the exudation is lansparent, and the numberless cellules which it contains afford t Jundant evidence of active multiplication, but do not enclose a ngfe fatty granule. Later, when the exudation becomes yellow a|1(| ?paque, the contents of the cells are nearly all granules and g ooules of fat. The author sums up the results of his experiments on animals and observations in the following conclusions: The pro-oplasm of cells appears to be the seat of changes in, and of the e aboration of the materials brought by the blood; it is also in the protoplasm that fat is at first deposited. The presence of fat in a ce which does not apparently contain it in the normal state proceeds from a diminution of the nutritive movement of that cell. If e movement is rendered active by irritation, fat disappenrs. Cer-ain cells have considerable activity in the elaboration of fat, but it +?es not follow that they form it from other materials with which ney are brought in contact, for blood contains fat in a metamor-phosed state, that is to say, in a state of combination or of saponifi-cation. Fat-cells, the cells of the liver, &c., seem then to bring back such metamorphosed fat to the neutral or insoluble state. In dead +?tuses retained some weeks in the uterine cavity, metamorphosed at becomes apparent to microscopic observation in the liver, the Sidneys, the heart, and cartilage cells. Phosphorus determines fatty ransformations, because it enfeebles the nutrition of histological e ements.? Gazette Medicate de Paris, July 6 and 13, 1867. + +On a Form of Fever ivhich Prevailed throughout certain Districts of Western Malwa in 1865. By H. C. Brodrick, M.D., 1st Regiment Central India Horse.?The author was employed by the Indian Government to report on a very fatal form of fever known in the districts of Western Malwa by the name of Oujrati Beemari, and amongst the Mussulmans as Yerkan or Feelia, both terms indicating jaundice. The disease is described as an ardent fever, without remissions, attended with congestion of the liver, pain in the side, and jaundice, and with congestion of the lungs or pneumonia, indi-cated by difficulty of breathing, cough, and bloody expectoration. + +The skin and conjunctivae were yellow, and the perspiration stained the clothes of a yellow colour. The disease prevailed pari passu with cholera, and the symptoms of both seem to have been con-founded by the native population. The author believes it to have been a form of jungle fever, without remissions. Relapses, however, were observed. The disease was generally fatal on the third day. In one village visited, which originally contained fifty houses, twenty-five had been desolated by the fever. There was no evidence that it was contagious. The author has treated several cases in the Cavalry Hospital at Augger of a fever which he believes to be identical with the Gujrati Beemari. They were very unpromising cases, but yielded to quinine in doses of from ten to twenty grains, with animal broths and alcohol. The author suggests that this disease may be identical with Palee plague.?Madras Quarterly Journal, October, 1866. + +On Leucocythoemia and the Pathogeny of Hemorrhages and Thromboses which supervene in that affection. By MM. Auguste Olltvier and Louis Ranyier.?The authors observe that Yirchow, and the principal writers of Germany who have discussed the pathological changes in leucocythcemia, have considered the presence of white globules between the hepatic cells or the renal tubuli as the result of hyperplasie of the interstitial connective tissue of the organs. This interpretation seems at first sight to account for the enormous pro-duction of white globules, although it does not explain the manner in which they penetrate the circulatory system. Since the researches of Reeling hausen there has been a disposition to consider the plas-matic network of the connective tissue as the origin of the lymphatics, and to explain the presence of globules in their final ramifications, by supposing that they are penetrated by the cells of that tissue. It follows that every multiplication of these cells would throw a great number of globules into the lymph, and thence into the blood. On the other hand, we know that lymphatic tumours may develope in a large number of organs without determining leucocythoemia. The observations of the authors have led them to give a different inter-pretation to the facts. In a case of leucocythoemia they believe they have observed, both in the liver and in the kidney, a connection of continuity between the interior of the bloodvessels and the islets formed in the stroma of these organs by an accumulation of white globules. Hence the presence of white globules in these islets results from capillary haemorrhage, and moreover, in the kidney they cceeded in discovering some red globules mixed, in small propor-ar?n''Wl^ wkite. As to organized lymphatic new formations, they +^ +e inclined to consider them as having two origins?the one a yperpJasie 0f the connective tissue, the other the formation of a ymphatic tissue developed at the expense of extravasated white ca? !!i They ^??k 011 ^e white globules as embryonic cells, pable of forming a new tissue. In the majority of cases the esS?u.ce red globules in the meshes of the connective tissue is v P j by the rapid destruction of these globules when extra-ceh i Case examined by the authors exhibited multiple o-ra h?morrhages, and also coagulations of blood in the crural and er arteries. The latter are frequent in leucocythoemia. According to tl? } l61"' Wou^ be due to a chemical alteration of the plasma of 6 b*??d, although it is difficult to conceive how the same modifica-on could produce at once haemorrhage and thrombosis. The authors er the following interpretation : The red globules of the blood re i ;re easily in the capillaries because they are smooth and the11 White globules are larger in man, rugous, and possess property of adhering. If the white globules become very merous capiHary circulation will be difficult; and if, as in the of tf ^ ?kservedj the number of white globules is larger than that h< +le re and their size attains ^ of a millimetre, as frequently +^ouw\S ^ ^eucocJ^oemia> they think that the capillary circulation jj u be extremely slackened, and even suppressed in some organs. + +a _,nce there will be a detention of globules in the capillary network, St t>a s.u?c^ent tension to determine the rupture of the vessels. + +, ??ati?n of blood will equally account for coagulation in the erial or venous trunks. The most powerful of the causes pro-thrombosis is arrest or slackening of the circulation.?Gaz. + +e lca^G Paris, June 15, 1867. + +^le influence of Disease upon the Weight of the Body. By Thos. + +a^T10^' Sterne, Paris.?The author's observations were undertaken +?o a Charite, under the direction of Prof. Monneret. In regard acute diseases, two principal periods are noticed?those of loss as reP,arati?n- Whilst the period of loss lasts the weight diminishes, an +^ +] +8' raPidly> and from the proportion of its diurnal variations ' ea may be formed of the relative gravity of the case. The dail +?r ^aS S6en a decrease in typhoid fever of more than 500 grammes x'1 ^ reparation the increase of weight is at times sudden and ti exPected, and is ushered in before other symptoms would warrant littl au.uoVnc?meiit of convalescence. This fact is so striking that but wl 6 ati?n need be felt in declaring convalescent the patient u os? weight begins to ascend, even though, from the indications s ^a y accepted, this announcement may seem premature. The ries of weights taken during convalescence furnish the means of +^ +no wing whether this state of reparation will be short or tedious, as hpS if k rapid augmentation accompanies a speedy return to a h, while the contrary obtains when the intermediary condition aut^601? health and disease is destined to drag on slowly. The or has made these observations on weight coincide with thermometric investigation, and has found that an elevation of temperature is attended by a diminution of weight; or in other terms, the higher the fever the lower is the figure given by the scales. The author's paper contains a number of observations of the variations of weight in different acute and chronic diseases.?Southern Journal of Medical Sciences, August, 1867. + +Case of Cholesteatoma. By Dr. F. Bate man.?T. H?, zet. 2 6, a tall, well-made farm labourer; admitted into Norwich and Norfolk Hospital on July 7,1866. History.?Been married four years ; had one child twenty-one months old; never had syphilis; had always been a steady man, and had enjoyed good health until nine months after marriage. First morbid symptom, a shaking first of right hand, then of left; this was followed in a few months by frontal headache. About a year ago weakness of legs set in and inability to guide himself, but intelligence and memory remained unimpaired; about this time slight embarrassment of speech was observed, this was followed by dimness of vision, which resulted in complete blind-ness, the right eye being first affected. Symptoms observed on admis-sion.?Staggering gait; no actual paralysis of limbs, but perhaps a little less muscular power on right than on left side; sensation un-impaired ; difficult micturition ; no want of power over rectum. He complains of severe frontal headache, is deaf on the right side ; there is double amaurosis, the pupils act, though sluggishly; no apparent disease of the eye itself. Intelligence and memory unimpaired; he converses coherently, with well-selected words, but he has an un-natural jerking, spasmodic manner of speaking, as if the same want of power of co-ordination in the act of volition which is apparent in his gait extends to the expression of his ideas. After a few days the frontal headache increased in intensity, he became drowsy, and died suddenly on the eleventh day after admission. Necropsy, thirteen hours after death.?Body well nourished ; lungs sligthly adherent at both apices ; heart healthy; kidneys, liver, and spleen intensely con-gested. Brain congested, but extremely firm. Arachnoid contained five and a half ounces of cerebro-spinal fluid ; ventricles dilated with fluid. On removing the brain, " a glistening tumour was found at the base of the cranium, adherent to and apparently springing from the posterior surface of the petrous portion of the right temporal, and from the basilar process of the occipital bones." " Tumour was flattened from above downwards; two and a quarter inches in its longest, one and a quarter in its shortest diameter; its surface uneven, of a white silvery appearance, resembling mother-o'-pearl." +It was invested with a membrane of the thickness of ordinary paper, which enclosed, as in a cyst, a substance of the thickness and consistence of wax, the central portion being of a dirty straw-colour, not unlike tallow, but becoming paler, of an almost pure white, and firmer as it approached the circumference of the tumour. It had no farther adhesion, and there was no trace of vessels in its structure. + +It lay against the under-surface of the right lobe of the cerebellum, where an excavation existed for its reception; it pressed on this lobe, also on the right side of the medulla oblongata, the right crus cerebelli, and the right side of the poos varolii in its whole extent. + +A portion examined microscopically exhibited a number of cells, crystals of cholesterine, and fat-globules. A portion was examined by Dr. Beale and pronounced cholesteatoma. The author refers the unsteadiness of gait to impairment of the functions of the cerebellun by pressure; the amaurosis to pressure by the anterior extremity ol the growth upon the posterior and inferior surface ol the right tha-lamus opticus, and, consequently, upon the origin of the optic nerve +(the double amaurosis being explicable by the decussation of the fibres of the optic nerve at the commissure); and the deafness to pressure on the portio mollis, as its winds round the corpus resti-forme, and also at its entrance into the meatus auditorius internus. + +The author believes this tumour to be the largest of this description and in this situation recorded. The ataxia ot articulation he thinks Was probably due to pressure on the origin of the glossopharyngeal and hypoglossal nerves.?JBeetle's Archives, No. xvi, vol. iv. + +Anatomico-pathological Considerations on Abscess of the Brain. + +?By M. E. GiNTitAC.?Suppuration and ramollissement are the modes of alteration by which encephalitis terminates, or under which it most frequently presents itself, since out ot 410 cases there were only 29 in which these conditions were not established; suppuration numbers rather more examples than inflammatory ramollissement. Kamol-lissement is notably more frequent in the cortical substance, in the anterior lobes of the brain, in the corpora striata and optic thalami, and in the pons varolii. Suppuration has been more frequently observed in the middle and posterior lobes of the brain, and in the cerebellum. It is also the most frequent termination of cerebral or cerebellar inflammation accompanying alteration ot the bones of the skull, and principally of the petrous portion of the temporal^ Pus which forms in the brain may be exuded on its surface, or disseminated io its tissue, or collected at one or more points ol its extent. 1 he first mode is rare; the second is sometimes observed with ramollissement, to which it gives a yellow-greenish tinge, great fluidity, and a peculiar odour. The third (abscess) is the most common. Lebert divides abscesses of the brain into multiple and solitary. But in circumscribed localities abscesses are not always solitary?sometimes two, but rarely a larger number are met with in the middle or posterior lobe, or in the cerebellum. Xiebert has noted that the left hemisphere was more often affected than the right, twenty-thiee to eighteen. M. Gintrac has collected ninety-three cases of abscess ot the right half of the encephalon, and eighty-five of the left. It is principally in the medullary substance of the different lobes ot the cerebrum that purulent collections are found, whilst the cortical substance is almost exempt. But the cerebellum, which is abun antly provided with grey substance, is pre-eminently subject to abscess, and the pons very little so, although principally formed ot white matter. Cerebral abscesses,however,arise principally in the me-dullary substance, which, although less vascular than the g, ey> is not, therefore,less apt to inflame and suppurate. 1 he volume of the abscess is sometimes considerable, varying from the size of a nut to that ot a 80?xl. + +pigeon's egg, a hen's egg, and an orange. The author has seen the hemi-sphere converted almost entirely into a sac full of pus. The form of the abscess is usually rounded or ovoid. When two or more abscesses are contiguous, they generally remain separated by partitions of medullary substance. But communications may be established between them. + +In some instances several foci communicate and run together, pro-ducing a cavity of irregular form, and of anfractuous or multilocular appearance. The pus contained in cerebral abscesses is usually greenish-yellow or olive-green, thick, creamy, or liquid and ropy. + +It may contain a pultaceous reddish matter; it may be inodorous, but in a large number of instances it is very fetid, although it has had no communication with the air. It is generally alkaline; under the microscope it presents ordinary characters, but granules are more abundant than the proper pus cells. The matter is sometimes in immediate contact with the cerebral substance, the surface of which may be softened or hardened, injected, red, or greyish, or yellowish, or granular; or sometimes it is enclosed in a cyst. + +This cyst is formed of a vascular membrane of variable thickness, white, reddish, or greyish in colour. The more recent the cyst is, the thinner and softer it is. From the thirtieth to the sixtieth day and later it becomes thick and dense, and appears formed of two or three membranes. Of these, the external has a cellular appearance, the middle may be dense, resistant, fibrous, or cellulo-fibrous and vascular ; the internal is soft, and like a mucous membrane. This is sometimes covered by a sort of thick concretion of pus. The cyst is sometimes easily detached from the cerebral substance, at other times it is closely united. The cysts differ from those which are formed round hasmorrhagic clots. The latter are agents of secretion, absorption, and resolution. Those which enclose abscesses do not fulfil like functions. Their membrane is rather pyogenic than absorbent. Abscesses are rarely found diminishing and on the road.to cure; but although such a termination is exceedingly rare, it is not impossible. Cerebral abscesses have a tendency to open towards the points where there is the least resistance. Pus generally makes its way towards the ventricles, and these cavities are generally found more or less filled with it. Very rarely the matter appears at the surface of the brain. Cerebral abscesses evacuated by the nostrils have been reported, but the minute details fail, or pua is said to have been evacuated by the ear. But it is worthy of note that in certain cases where the cavities of the internal ear enclosed pus, the cerebral abscess itself did not communicate with the interior of the petrous bone, of which the surface was entire, and the dura mater intact. Nevertheless, it is more common to trace the con-nection between the contemporaneous affections of the ear, the membranes, and the brain.?Journal de MSdecine de Bordeaux. + +Gazette MSdicale de Paris, July 27. + +On Convulsive Tremor. By Dr. William A. Hammond.?Under this term Dr. Hammond describes a paroxysmal affection, of which the following case may be taken as an example :?J. S?, a gentle-man, set. 35. Ordinarily had nothing to complain of on the score of health. Appetit? was good, and all functions performed with regularity; but two or three times during the day he would be seized with severe and unrestrainable muscular tremor, involving the head and all the muscles of the trunk and arms. At the same time there would be slight headache and vertigo, and an intense feeling of anxiety. There was no I03S of consciousness, not even for an instant, nor inability to walk or to direct any muscle, and no confusion of thought. After the paroxysm had lasted fifteen or twenty minutes it gradually passed off, leaving him in a profuse perspiration. During his first interview with Dr. Hammond he was seized with a paroxysm which is thus described :?" He was seized with as much suddenness as though he were struck with an epileptic fit. His head shook violently, the muscles of his face were convulsed, his arms and hands trembled, and his gluteal muscles contracted so powerfully as to cause him to move convulsively up and down in the chair. His lower extremities were altogether free from spasm or convulsion. Upon putting my hand on his wrist, 1 found that every tendon was in action, and in the arm, hand, neck, and face, the vibration of the muscular fibres could be distinctly seen and felt. The thermometer applied to the axillae marked 101? + +Fahr., and the sesthesiometer showed an increased sensibility of the skin of the face, neck, hands, and ail the upper parts of the body examined. The respiration was quickened and the pulse was increased from 80 to 95 per minute. During the paroxysm he con-versed rationally, but with some difficulty, owing to the action in the muscles of the neck, mouth, and chest." In this case the attacks seem to have been brought on by venereal excesses, the first paroxysm having supervened during coitus. He rapidly improved under treat-ment, which consisted of a seton in the nape of the neck (to counteract supposed cerebellar irritation), thirty grains of bromide of potas-sium three times a day, and the application of the constant galvanic current. Two other cases of somewhat similar character are related, in which a similar treatment proved also of use.?New York Medical Journal, June, 1867. + +On the Influence of Narrowing of the Pulmonary Orifice on the Formation of Pulmonary Tubercles. By M. Lebeet. Presented to the Academy of Sciences.?The author, in the course of his re-searches on mechanical influences in the etiology of tuberculous affections, has been struck by the frequency of the occurrence of pulmonary tubercle in cases of congenital narrowing, either of the cone or the orifice of the pulmonary artery. This coincidence has been observed, in isolated cases, by Favre, Travers, Gregory, Louis, and Creveld, and during the last twenty years it has been noticed in one third of the observed cases, often under circumstances in which no other etiological element of tuberculisation could be sup-posed to exist. The author has been able to collect twenty-four facts of this kind, a large number if the relative rarity of this affection be taken into account. The frequent development of tubercles in connection with this condition is the more striking, as nothing is more rare than to meet with pulmonary tubercles in alterations of the orifices of the left heart. Three forms of congenital narrow-ing of the pulmonary artery are to be distinguished?primitive narrowness of the vessel, which is furnished only with two valves, narrowing of the pulmonary arterial cone, and narrowing of the orifice. The last two are the results of endocarditis or myocarditis, and as ordinarily the interventricular septum is deficient, this inflammation must take place before the end of the third month of intra-uterine life. The foramen ovale remains often also open, the arterial duct more rarely. Hence, the circulation through the lungs is irregular and incomplete. Dilatation of the bronchial, cesophagean and coronary arteries of the heart, also of the sub-clavian, furnishes only an incomplete collateral circulation, whence arises imperfect nutrition of the lungs, so that they are observed to be small and incompletely developed. This irregular, unequal, incomplete circulation in places, gives rise to pulmonary tubercle, whilst the most intense and extended pulmonary hyperemia from alteration of the bicuspid and tricuspid orifices does not conduce to its development. The author knows neither age nor disease which presents this proportion, i. e. of one third affected with pulmonary tubercle, and hence he can only refer it to pulmonary stenosis. + +The character of the tubercular attack is protracted, progressive, and fatal. Haemoptysis is frequent, and it is ordinarily the left lung that is first attacked, and not the right, which the author thinks is the lung which ordinarily suffers first. The anatomical characters are the same as in other forms of tuberculisation.? + +Gazette Medicale de Paris, July 27, 1867. + +Rupture of the Tricuspid Valves of the Heart from Fright. By Alfred Hitchcock, M.D.?M. H?, set. 2 years and 9 months, of a sanguine and very nervous temperament, was frightened in the middle of the night of July 16th, 1865, by an alarm suddenly sounded by the steam fire-whistle in the immediate vicinity of the house where she was sleeping. She awoke in terror, screaming and panting for breath, with suffocating paroxysms. She died seventy-eight hours after the fright. The symptoms present were vomiting, dyspnoea, lividity of skin, absence or great feebleness and irregu-larity of the radial pulse. The heart sounds were too feeble to be heard, or too tumultuous and irregular to be identified. There was loud moist bronchial and tracheal rattle. + +Autopsy.?"The right auriculo-ventricular valves were lacerated and broken in their substance ; also several of the carnece columnce and chordae tendinece; and the endocardiac serous membrane in the vicinity was ecchymosed and elevated by the infiltration of blood underneath and around the points of ruptured tissue. The right auricle and ventricle, by the destruction of the tricuspid valves, had become one continuous pouch, wThich contained a small quantity of coagula."?Boston Med. and Surg. Journ. New York Med. Journal, May, 1867. + +The Condition of the Blood after Death from SnaJce Bite. By Professor G-. B. Hautord, M.D.?In a case of poisoning by the bite of the cobra-di-capella, and in animals bitten by poisonous snakes, Dr. Halford has found that the blood contains a number of nucleated cells. In a dog killed by the injection of cobra poison under the skin, these cells were of a perfectly circular form, with a diameter on the +: +average of 1707 of an inch. The nucleus was nearly round, and 7707 of an inch broad. On the second day, on applying magenta, a minute spot became visible at some part of the circumference of the cell. + +This was visible in all. Of these cells there were many millions ; +they were present in the blood of every part of the body, but in much greater quantity in the dark congested parts of the lungs. In the poison of the snake itself, Dr. Halford found, besides epithelium and much molecular matter, nucleated cells of the same size as those seen in the blood of the dog, but no spot or macula was seen in the circumference. Dr. Halford believes that when a person is mortally bitten by the cobra-di-capella, molecules of living germinal matter are thrown into the blood, and speedily grow into cells, and as rapidly multiply, so that in a few hours millions upon millions are produced at the expense, probably, of the oxygen absorbed into the blood during respiration ; hence the gradual decrease and ultimate extinction of combustion, and chemical change in every other part of the body, followed by coldness, sleepiness, insensibility, slow breathing, and death. Dr. Halford suggests that cholera may be due to the presence of foreign cells in the blood, and refers to an observation by Dr. James Cowan, published in the +' Edinburgh Monthly Review,' of some peculiar bodies in the blood of a cholera patient, and also to an observation by Virchow that the white corpuscles are increased in cholera, in support of the hypothesis.— Pamphlet, Melbourne. - Int On the Formation of the so-called Cells the By J. GA. G B. Horton, M.D. Londen, Essentials of the Principles and Practice of Medicine. By H. Hartshorne, M.D. + +An Practical Treatise on Shock after Surgical Operations and Injuries, with +special reference to Shock caused by +Hardwicke, London. 1867. pp. 88. + +Royal Society,' Nos. 89 and 94.) 1867. + +Lectures on Clinical Medicine. By A. + +Trousseau. Of Translated fand o Edited by P. V. Bazire, M.D. Hardwicke, London. + +Iluncan, M.D. (From ' Transactions of The Treatment of Aneurysm by Electrolysis. 18 By Drs. J. Duncan and T. R. \ No newline at end of file diff --git a/medical/md/PMC5280726.md b/medical/md/PMC5280726.md new file mode 100644 index 0000000000000000000000000000000000000000..7fb3459cd02323db8f3183eed046b1726969efb7 --- /dev/null +++ b/medical/md/PMC5280726.md @@ -0,0 +1,36 @@ +lP" Applied Anatomy of the Nervous System: Being a Study of this Portion of the Human Body from a Standpoint of its general Interest and Practical Utility in Diagnosis, designed for as a lext-book and a Work of Reference. By Ambrose L. + +-^anney, A.M., M.D., Professor of the Anatomy and Physiology o the Nervous System in the New York Post-Graduate Medical c iool and Hospital, etc., etc. Second Edition. London: H. + +1^. Lewis: 1888. + +an^T ^ diagnosis disease of the nervous system, more than of an t group of morbid conditions, that a precise knowledge of the rp,0lny and physiology of the structures involved is essential, lab lau recently devised methods of research, and to the an (|UIS ?^a ^ar?e body of able investigators, our knowledge of the cor- ^ie nervous system is being daily extended and pi ? f ' as a necessary consequence, our knowledge of its Cai!?Sy is likewise widening and becoming more precise. There Sei> V10 ^ou^t that it is largely due to this advance of our purely infl knowledge?although we must also fully recognise the tion-^T better clinical and pathological methods of investigar is ^le diagnosis of many diseased conditions of the brain aPt)r VV- S? Wonderfully accurate as to enable us to operate with +^?Xln?a*;e certainty of success. + +l?gical +? ^ie most recent results of anatomical and physiotliese researc^) and at the same time showing the bearing of beCOrng^0n explanation of various pathological conditions, thus is Well08.a de.s^eratum with every practitioner; and such a want second edition of Dr Ranney's admirable chana-e -^naiomy and Physiology of the Nervous System. The must al m t^le second edition are so radical and extensive that it +^hi/11^ regai'ded as a new work. + +*endenr - \n mos^ t'ie recent works upon the nervous system the finical been somewhat to magnify the pathological and P'^iolo^?8 ^ie subject a* the expense of the anatomical and +*'le Srea?1^ ^ I^nney's treatise the reverse holds good; and ll0i'mal' 61 J-a.rt ^ie W01'k is devoted to the elucidation of the divero-e,,00110!1^0118' structural and functional, while the possible After tC6S m ^ese are merely sketclied in outline. + +brain are ^nera introduction, the anatomy and physiology of the nerves ar ilsc|lssed w^b great fulness, in Part II. the cranial fUncti0nse f? Wlt^' while in Parts III. and IV. the structure and +^ full and? 116 C01"d and spinal nerves are carefully described, corisid a j?Urate index accompanies the book?a fact which must The field a Stron? rec?mmendation to such a work of reference. + +and the ai tl C?^eied *3 necessarily very wide, and ever extending; in brinp-ino-T-18-t0 be COngr?' . we do not possess, and it must necessarily be faulty. On the other +? hand, the artificial arrangement of Willan, notwithstanding its imperfections and inconveniences, can be at once understood by, and is tolerably easy for, the practical student; and this classifica-tion, certainly rendered more perfect by the modifications of Biett, is the one which best answers the purposes both of teacher and student, and will enable the latter to acquire a knowledge of forms and varieties more readily than any other. + +Mr Wilson denominates his classification a natural one. It is founded on a mixture of anatomy, physiology, and pathology. For instance, the four great divisions refer to the anatomy of the skin. + +They are, 1st, Diseases of the Derma; 2d, of the Sudoriparous Glands ; 3d, of the Sebiparous Glands; 4th, of the Hairs and Hair Follicles. We do not object to this primary anatomical division; on the contrary, it may be considered good, if it were possible to separate precisely the diseases belonging to each. Most of the subdivisions, however, are very unphysiological and unpathological. + +YVc arc at once struck with the following extraordinary arrangement of inflammatory diseases of the derma:? + +" inflammationCongestive? + +Specific?Rubeola?Scarlatina?V ariola?V aricella?V accinia. + +Non- Specific?Erysipelas?U rticaria?Roseola?Erythema. + +Effusive? + +A sthcnic?Pem phigus?Rupia. + +Sthcnic?Herpes?Eczema?Sudamina. + +Suppurative?Impetigo?Ecthyma. + +Depositive?Strophulus?Lichen?Prurigo. + +Squamous?Lepra?Psoriasis?Pityriasis. + +From Parasitic Animalcules?Scabies."?Pref., p. xxii. + +mations, arc novel. Let us endeavour to understand their mean-ing?and, first, what are we to comprehend by a congestive inflammation ? All pathologists, whilst they allow that congestion precedes inflammation, have been most careful to separate the two lesions. Does Mr Wilson affirm that a congestion, that is, an un-usual distension of the vessels with blood, is an inflammation ? We cannot think so. On the other hand, does he mean to assert, that there can be any acute inflammation without congestion? We hope not. Surely, then, the term congestive inflammation is some-what contradictory. Under this head he has arranged Variola, Varicella, and Vaccinia. What, then, we should like to know, is suppurative inflammation ? On turning to page 220, we are told it is characterized by the formation of pus occupying the surface of the derma, and producing an elevation of it to a limited extent. + +Why, then, are variola and vaccinia not placed in this group ? Be- ..." +cause, says Mr Wilson, they are torn from their natural affinities, +" and, for this reason, I have thought it proper to transfer the genus ; ^ +to the group of eruptive fevers."?P. 221. Now, we do not object * .. * +to Mr Wilson's forming a group of eruptive fevers, but we strongly . y,i. + +protest against his placing pustular diseases under such an unmean- +'? + +. * '?' +ing head as Congestive Inflammation. + +Is there any method of drawing a pathological distinction between an effusive and a suppurative inflammation? We think not. Examine the contents of a herpetic vesicle, and of an ecthymatous pustule, and both will be found to contain pus. True, the vesicular and the pustula? have been long separated, and we understand by the former a disease in which the vesicles present a stage in which the liquor sanguinis has not yet fully passed into pus. It must be evident, however, that such a distinction can have no real patholo-gical value, for, as regards their inflammatory phenomena, that is, the formation of small collections of pus, they are the same. Why the pus of Herpes should be longer in forming than that of Ecthyma, or why the latter should leave a cicatrix, whilst the former does not, we are ignorant. Again, the suppurative action of Herpes is identical with that of Variola or Vaccinia; but why a pus globule of these last should have the power of regenerating a similar disease, whilst one of the latter has not, we know nothing. But it is exactly on account of our ignorance of such points, which consti-tute the true nature of these affections, that it is impossible to found a classification on a pathological basis. + +The term depositive inflammation, we are told, p. 236, has been selected only in the absence of a more suitable word. Assuredly that chosen is not a good one, inasmuch as every inflammation is depositive. It is employed by Mr Wilson, however, " to express that condition of the inflamed membrane in which plastic lymph is exuded by the capillary rete into the tissue of the derma, so as to give rise to the production of small hard elevations of the skin, or pimples." We beg leave to deny that, in strophulus, lichen, or prurigo, a deposit is exuded in any way analogous to plastic lymph, as Mr Wilson may easily satisfy himself by a microscopic examination. + +What a squamous inflammation is the author has not thought proper to inform us; for, on turning to p. 261, we find no attempt at definition. The scales are composed of epidermic cells, which, not being vascular, cannot become inflamed. A squamous inflam-mation, therefore, cannot mean inflammation of the scales. Are we to understand by it an inflammation of the skin, in which the scales peel off in increased quantity ? If so, Scarlatina and Erysipelas ought to be classified under this head. The nature and meaning of this newly discovered inflammation is a perfect mystery to us, and we therefore strongly object to it. We are told " that hypertrophy of the derma in the squamous diseases is very evident." Why, then, not place them with callosities and corns, which are also hypertrophies of the derma ? + +Another subdivision of inflammation is that from parasitic animalcules. But is the inflammation produced in this way different from that originating in any other ? Or is it consistent with the principles of classification to divide inflammation in some cases according to its results, as effusive or suppurative, and in others according to its causes ? Again, if animal parasites are to consti-tute a ground of distinction, why may not vegetable parasites claim a similar notice? Do these, in the course of their development, never cause inflammation of the derma ? Most assuredly they do, as we have proved innumerable times. + +With regard to vegetable parasites, however, we have to charge Mr Wilson with a total unacquaintance with the subject. Whilst he has personally examined into diseases caused by animal para-sites, he seems to have entirely neglected those of vegetable origin. It is true he quotes passages from the writings of Gruby and Hughes Bennett, but his own opinion seems to be, that the structures described have no analogy with those of the vegetable wrorld. Speaking of the favus crust, in the preface, p. xx., he says, +" Its vegetable nature is hypotheticaland, when treating of the pathology of favus, he remarks,? + +" However closely the fungous growth here described may resemble a plant, its vegetable nature is very far from being established. The simplest forms of animals are composed, like the mycodermis, of cells, variously connected to-gether ; and subsequent research may prove the growth under consideration to be of a similar nature. To my mind there is nothing improbable in the sup-position of the origin of the growth from morbidly developed epidermal cells of the hair-follicle, or from those of the sebaceous substance. In a preceding section of this work, I have shown that the latter are susceptible of considerable alteration, and that in this state they assume an appearance widely different from that of their normal condition. Mr Busk also entertains doubts with regard to the vegetable nature of the mycodermis, and deduces an opinion favourable to his opinion, from the chemical analysis of the crusts of favus given by Tlienard, who found them composed of ? + +``` +Albumen +Gelatine +Phosphate of Lime +Water and loss + +``` + +70 +.17 +. 5 +. 8 100 +" Dr Carpenter, in his ' Principles of Physiology' (p. 453), speaking on the same subject, remarks,?' It has been assumed that the organization is vegetable, because it (mycodermis) consists of a mass of cells capable of extending themselves by the ordinary process of multiplication. But it must be remembered that the vesicular organization is common to animals as well as to plants, being the only form that manifests itself at an early period of development in either kingdom, and remaining throughout life in those parts which have not undergone a metamorphosis for special purposes. Hence, to speak of porrigo favosa, or any similar disease, as produced by the growth of a vegetable within the animal body, appears to the author a very arbitrary assumption; the simple fact being, in regard to this and many other structures of a low type, that they present the simplest or most general kind of organization.'"?Pp. 430, 431. + +Mr Wilson has taught anatomy, and is the author of an ana-tomical manual of no small repute. How any one possessing a knowledge of the elementary structures of the animal tissues could have published such a passage surpasses our comprehension. It immediately follows an accurate description of the vegetations by Gruby, in which we are told that they consist of roots and branches dividing dichotornously, composed of jointed tubes with transverse septa, containing granules, and giving off at their extremities seeds or sporules. + +We have placed this passage in italics purposely. Where, we ask Mr Wilson, is there an animal tissue which presents a similar struc-ture, and where is the vegetable conferva that does not ? Knowing, as Mr Wilson seems to do, that such accurate observers and laborious inquirers as Remak, Schonlein, Gruby, Fuchs, Henle, Vogel, and many others, have pronounced in favour of the vegetable nature of these growths, we can only regard his citing as an authority, in favour of his own opinion, a writer like Dr Carpenter, to be either a piece of innocent badinage, or a specimen of tine irony. The fact is, that there is no animal tissue either healthy or diseased, which in any way resembles the structure of the favus crust. + +In the present edition, Mr Wilson has separated ichthyosis from warts, corns, and other hypertrophies of the derma. He assigns, as a reason for this, his conviction that the disease is a concretion of altered sebaceous substance? + +" In an instance of this affection which fell under my observation about ten years since, I had the opportunity of examining the skin after the death of the patient from visceral disease. In this case, the scales were remarkable for their thickness ; after being well washed, they were greyish in colour upon the surface, but white beneath, and evidently consisted of concreted sebaceous substance. On removing a portion of the epiderma by maceration, the ducts of the sebiparous glands and hair-follicles were found distended with inspissated white secretion, and had a very beautiful and brilliant appearance, projecting like cones of pearl from the under surface of the membrane. The derma presented a number of small deep pits, corresponding with these dilated ducts. + +The mouths of the distended excretory ducts opened upon the surface of the epiderma, some immediately beneath, and in the middle of the scales, and others by their borders. In the former situation, they could be seen as small white points through the scale, and still more evidently when the epiderma was separated by maceration. + +From the careful examination of this case, of which a preparation is now before me, and of others which I have subsequently observed, I have been led to the conclusion, that the scales, in this disorder, increase in thickness, in two ways, firstly, by additions to the free surface, by means of the secretion poured out in the linear furrows of the skin, and, consequently, between the scales ; and, secondly, by additions successively made to the attached surface by the effusion of inspissated secretion beneath them. In the preparation before me, the growth of the scales by both of these processes is distinctly evident." +Supposing Mr Wilson's view to be correct, which, as regards all the forms of ichthyosis, we are disposed to doubt, it must be ac-knowledged that the disorder is in every way allied to the squama?. + +Thus, while under the head of congestive inflammation, he mixes up the exanthemata and pustulae on account of their natural affinities, he dissociates ichthyosis from psoriasis, on account of a theory he has formed with regard to the formation of the scales. + +Under the head of disorders of the vascular tissue, Mr Wilson has placed vascular n?evi and purpura. Yet what evidence have we that purpura is a disorder of the vascular tissue ? On turning to page 316, we find the following passage? + +" Purpura is a morbid state of the capillary system, characterized by the effusion of blood into the different tissues of the body, this effusion giving rise to the formation of sanguineous patches in considerable numbers, and of various sizes. The capillary vessels of the skin participate in this morbid disposition ; +hence purpura has obtained a place, by courtesy, among cutaneous disorders." +Surely there is nothing here to prove that the capillary system is affected in purpura. We have been in the habit of considering it as dependent on some lesion of the blood, and the observations of Liston, Lane, and others would seem to countenance this opinion. + +Of this we. are quite satisfied, that the capillary vessels have never been shown to be diseased, and hence another discrepancy in the pathological arrangement of our author. + +But it is unnecessary to criticise the classification of Mr Wilson further. We think enough has been said to show that his arrange-ment is not very happy, or consistent with our present knowledge of pathology. Most assuredly, in departing from the principles of arrangement introduced by Willan, he has not given us one that is more simple or correct; but, on the contrary, by the formation of unpathological terms, has thrown increased mystification and confusion on the whole subject. + +The matter of classification is at all times important, and we are well aware of its difficulties. Hence, perhaps, our dislike to the derangement of that which is well established and easily understood* It is curious that writers on dermatology should have acquired their knowledge by following the system of Willan and Bateman, and afterwards endeavoured to supersede it by a natural or pathological arrangement. We observe this in the writings of Alibert, Cazenave, Hebra, and now of Mr Wilson. No doubt that system has its faults, and as our knowledge becomes perfect we naturally endea-vour to arrange it upon a scientific rather than upon an artificial basis. We hope that the efforts which are making in this direction will ultimately be crowned with success. But we must bide our time. Science is not yet sufficiently advanced to solve those difficulties which lie on the threshold of the path, and all the attempts which have been made to form classifications of skin diseases, not by mastering these difficulties, but by putting them aside, have invariably failed, and produced no permanent influence on the minds of the profession. + +Turning from the classification and general pathology of Mr Wilson to the special treatises of which his book is composed, we feel much pleasure in stating that many of its individual parts are carefully compiled, and that some give evidence of considerable research and original investigation. Among the latter, we would especially point out the introductory chapter on the Anatomy and Physiology of the Skin, the section on Diseases of the Sebiparous Glands, and the two concluding chapters on the Itch Insect, and the " Steatozoon" Folliculorum. + +We cannot discover in the work any comprehensive notions respecting the treatment of skin diseases. One of the evidences of an improved pathology will be the introduction of general principles to guide us in our efforts at cure. As it is, we find only in the publication before us the same empirical formula, and reliance upon contradictory individual experience, as in most books on the same subject. + +The following are some of the author's observations on the treatment of chronic eczema:? + +" In the, chronic form of eczema, when the disease is obstinate, and resists our common methods of treatment, it becomes necessary to modify the state of the constitution by various means ; as, for instance, by a course of hydriodate of potash, of mercury, of Donovan's solution, of liquor arsenicalis, or of tincture of cantharides. + +" In employing arsenic and cantharides as therapeutic agents, it will be necessary to watch their effects with care, and bear in mind the serious symp-toms which may result from their abuse. Should any of these symptoms be apparent, namely, nervous disorder, and disorder of the alimentary canal, in the case of arsenic,?or of the urinary system, in the case of cantharides, the medicines must be immediately laid aside, either permanently, or to be resumed after a few days, according to the judgment of the practitioner. Whene^r we put a stop to the exhibition of arsenic, and return to it again, it is necessary to begin with a smaller dose than that at which we left off. Arsenic, when it acts upon the nervous system, performs the part of an alterative, but when its effects are directed upon the digestive system, it appears to me to act, like can-tharides, upon the mucous membrane of the kidney, viz. by counter-irritation, by exciting inflammatory action in the interior, and thus determining from the surface. + +" The best formula for the exhibition of cantharides is one in which it is NEW- SERIES.?NO. IX. MARCH 1847. 3 K +combined wiflh equal parts of compound tincture of camphor, and taken in tincture of cinchona."?Pp. 212, 213. + +We have found that arsenic and cantharides act very well to-gether. Equal parts of Fowler's solution, and the tincture of the latter drug, may be mixed together, of which ten drops form a dose to begin with for an adult. The local remedies available in chronic eczema are, according to Mr Wilson, very numerous. We give them exactly as they are set down by him:? + +" Sulphuret of potash in lotion or ointment, lime water, bichloride of mercury in weak solution, calamine ointment, zinc ointment, sulphate of copper oint-ment, tannin ointment, white precipitate ointment, red precipitate ointment, calomel ointment with watery extract of opium, carbonate of lead ointment, tar ointment, sulphur ointment, and mercurial ointment. + +"To relieve the pain and pruritus which accompany the eruption, the fol-lowing remedies as local applications may be tried, viz. acidulated lotions, alkaline lotions, lotion of super-acetate of lead, emulsion of bitter almonds, or of hydrocyanic acid, lotions of opium or hyoscyamus, camphor mixture, infusion of dulcamara, &c."?P. 213. + +We do not think that much benefit in practice is likely to be obtained by following directions, if such they can be called, like those just given. Certainly chronic eczema is a most rebellious disorder; but, according to our experience, is in no way benefited by irritating applications like red precipitate ointment. We have found alkaline lotions the best local method of treatment, but then the parts should be kept continually moist, by means of saturated cloths, well covered with oil-silk to prevent evaporation. + +The following are the author's observations on the treatment of lepra:? + +" The internal remedies recommended for lepra are, a course of purgatives ; +bichloride of mercury in decoction of sarsaparilla, or dulcamara; hydriodate of potash ; tincture of cantharides ; liquor arsenicalis ; liquor potassae; sulphuric acid ; decoctions of guaiacum, mezereum, and elm bark ; infusion of nettles; milk of sulphur, &c. + +" Of these remedies, that on which I place the greatest reliance is the liquor arsenicalis, or the liquor hydriodatis hydrargyri et arsenici (? 446). The former of these solutions in doses of five drops, and the latter ten to thirty, three times a day, with meals. The latter is the milder remedy of the two, and rarely produces any disagreeable constitutional effects. Of course it is necessary, in using arsenical preparations of every kind, to counsel the avoid-ance of acids, fruits, and vegetables, and to explain to the patient the symptoms which call for the suspension or omission of the remedy. The effect of arsenic on the leprous patches is, in the first instance, to increase their redness, activity, and heat, and subsequently to diminish these symptoms, and render them brownish and dull. When the latter change takes place, the eruptions quickly fade and disappear. The medicine requires to be taken regularly, and to be persisted in for several months."?P. 268. + +" The local remedies are, lotions of sulphuret of potash, alkaline baths, vapour baths and douches, sea-bathing, spirituous solution of bichloride of mercury, zinc ointment, white precipitate ointment, calomel ointment, nitrate of mercury ointment, ointments of acetate and phosphate of mercury, of sul-phate and deutoxide of antimony, ioduret of sulphur ointment, from ten to twenty grains to the ounce, creosote, blisters, nitrate of silver, &c. M. Gibert speaks favourably of an ointment of the ioduret of ammonia, in the proportion of a drachm to an ounce, and also of the ointments of Anthrakokali and Fuli-gokali. In the employment of these applications, care must be taken, in acute cases, to use them only after the reduction of the local excitement, by means of fomentations, emollient baths, &c., and then only of moderate strength. In chronic cases, however, they may be employed from the commencement, and in a more concentrated form, with the vifew of modifying the diseased structures. + +"' My own practice,' says a distinguished author (Rayer) on cutaneous diseases, ' is to begin with the white precipitate ointment, or with that of the protochloride of mercury, unless in those cases where the disease is of very long standing, when I try the ioduret of sulphur in preference.' +" M. Emery of Saint Louis has lately recalled the attention of practitioners to an old but valuable application in leprous affections, namely, tar. Finding, however, that this remedy was objectionable on account of its colour and odour, he had recourse to one of the products of tar, concrete napthaline, which afforded him the most successful results. The preparation which he employs is an ointment composed of Napthaline two to four parts. + +Lard thirty parts. + +This he applies to the diseased skin, on folds of linen, night and morning. The ointment is highly stimulating, and has a powerful smell, which quickly passes away. By means of the napthaline ointment M. Emery succeeded in curing eight patients out of fourteen, in from five weeks to three months."?Pp. 270,271. + +Our own experience is decidedly more in favour of the preparations of pitch as local applications than of any other. Napthaline is a cleanly means of employing it, and deserves a trial. We have seen, in the wards of Dr Hebra at Vienna, scaly diseases success-fully treated by infusions of tar, made with boiling water in the usual manner, of which a pint was drank daily. In this manner, the disorders yielded as rapidly as if the remedy had been applied externally, and, of course, without the unpleasantness resulting from the latter mode of treatment. + +In the treatment of lupus, Mr Wilson has found the best results from a prolonged course of liquor hydriodatis hydrargyri et arsenici. + +He seems to be unacquainted with the extensive experiments lately made at the Hopital St Louis, by M. Emery and others, which have shown the great benefits to be derived from the internal administration of cod-liver oil in these cases. + +The following are the author's observations on the treatment of ring-worm:? + +" The treatment of trichosis offers two indications: firstly, to correct the deranged nutrition by constitutional means: secondly, to excite a new action in the skin by means of moderately stimulating local remedies. + +" In effecting the first of these objects, diet, exercise, clothing, and ventila-tion, are important and powerful agents. And their beneficial influence may be assisted by tonic medicinal remedies, such as the mineral acids, quinine, steel, vegetable bitters, &c., given separately or combined. I have found the syrup of quinine and steel a valuable medicine in this complaint, and one which suits the taste of children better than other remedies. The kind of tonic must, however, be left to the judgment of the practitioner, and be determined by its effect on the economy. + +" In directing the local treatment, I have found it useful to commence with some moderately powerful stimulant, such as acetum cantharidis, or the stronger acetic acid, which I apply once to the diseased spots ; and, after the tenderness has abated, anoint the patches daily with some moderately stimu-lating ointment, such as the unguentum hydrargyri nitratis diluted with ceratum cetacei. Another ointment which I have found of great service, is one composed of a drachm of sulphate of zinc to an ounce of simple cerate. + +When an objection exists to ointments, a lotion of bichloride of mercury or sulphate of zinc will be found useful, correcting the dryness which follows the employment of lotions by means of pomatum or cold cream. It is beneficial to wash the head with soap once a day, and, when dried, to anoint it with pomatum. I regard the keeping the scalp constantly moistened with some oleaginous matter as an important adjuvant to cure. + +" The principle of local treatment being, as I have above stated, moderately stimulant, the remedies applicable to this disease, with advantage, are very numerous, and an extensive choice is left to the practitioner ; so that no excuse exists for using such as are disagreeable in their appearance, odour, or effects. + +The preparations of iodine, for example, are open to objection, on account of the stain which they leave behind, and they are in no wise more useful in their effects than more elegant compounds. A remedy which I frequently use in this disease is liquor ammoniae and olive oil, accommodating the quantity of the alkali to the amount of stimulation which I desire to effect. This preparation has the advantage of being cleanly and easily removed by washing, while it clears the patches more completely than any other that I am acquainted with. Another elegant, as well as useful application, is a pomatum of cantha-ridine, prepared at my request by Mr Savory of Bond Street."?Pp. 419, 420. + +It may be worth while knowing the treatment which has proved very successful in Heriot's Hospital School in Edinburgh,? so' successful, indeed, that the disease is now very rarely seen there. + +A piece of very coarse brown paper is rolled into a cone, and the larger end set on fire. The smoke is directed on the diseased scalp. + +In a little while a drop of empyreumatic oil collects at the extremity of the cone, which should be allowed to fall on the affected part. + +When it separates, a cure is often produced. Should other means fail, it may be thought worth while to try this. + +We could continue making extracts and commentaries, but are now obliged to conclude. This we do, with the remark, that our present mode of treating chronic skin diseases is most unsatisfactory, and often irrational; and that we cannot hope to see greater cer-tainty introduced into our practice, until the pathology of these disorders is further advanced. The microscope has here a fertile field to cultivate. Doubtless, some of the fruits have already been plucked by Kemak, Gruby, Hebra, and our author; but there is still a rich harvest to be gathered in by him who will diligently cultivate the soil. + +1. School for Deaconesses for the Sick and Poor, and other Charitable Institutions at Kaiserswerth on the Rhine. Extract from the Journey of a Tourist. (Rev. C. Major.) Berlin : 1843. + +2. Institution of Protestant Deaconesses or Nursing Sisters at Kaiserswerth, on the Rhine, in Prussia. London : 1846. + +3. Passing Visits to Distant Charitable Institutions. Die Barmberzigen Schwestern. Extracted by permission from Lowe's Edin-burgh Magazine. Edinburgh: 1846. + +In the admirable report given by Dr Balfour, on homoeopathic treatment in Dr Fleischmann's Hospital, Vienna, which appeared in the British and Foreign Medical Review, for October last, a passage occurs well worthy the attention of every medical man, and more especially deserving to be seriously pondered by those who have learnt that the giving of drugs is neither the only nor the most important duty which he has to discharge to his patient. The passage is as follows:? + +" In taking into consideration the adjuvants to treatment, the religious character of the establishment must not be forgotten. The greater part of the patients are Roman Catholics. These find themselves surrounded by all the consolations of religion?by every thing which, in their opinion, tends to insure in the event of death a speedy passage of the soul to the realms of bliss. Their minds, thus set at ease with respect to futurity, are less gloomy and desponding, and, consequently, react less unfavourably upon the body than in opposite circumstances. Nay, looking upon their nurses as self-devoted in the service of Heaven and suffering humanity, they cannot but believe that the blessing of the Almighty will rest upon their labours; and being the object of those labours they naturally enough appropriate a portion of this blessing to them-selves, and imagine that their recovery can hardly fail to be promoted by being the inmates of such an institution. In support of this opinion I may state, that though most of the patients were young, and many dangerously ill, I +never heard one expression indicative of a fear of death, nor one murmur, how-ever slight, unless extorted by the extremest pain, and even then, it was more an aspiration after relief than a grieving at suffering. The severer the disease the more closely do they grasp their rosaries and crucifixes. So long as they are able to read, prayer books are constantly in their hands, and even in the intervals of delirium, nay, often, in the cases of women especially, during delirium itself, the exercises of repeating prayers, or snatches of them, is their occupation. The superiority of attendance is also one great advantage in favour of this hospital, independently of the important fact just stated, that the nurses are spiritual as well as temporal comforters." +This truthful statement of what any one may witness in those hospitals where the patients are nursed by the Sisters of Charity, may well induce us to ask the question, why it is that the profes-sors of a religion which we have been taught to regard as erroneous, should set Protestants such an example in self-denial, and works and labours of love:? + +" Observations of this nature made many Protestants of these countries feel the duty of setting before the eyes of their Roman Catholic neighbours, some examples of charitable works carried on by Protestants without the aid of false doctrine, or monastic vows. The Rev. Mr Fliedner embraced the idea with much fervour?and has been liberally supported in his undertaking by many Christians all over Germany." +This quotation will explain the origin of the interesting institu-tion, the benevolent and successful operations of which it is the intention of the pamphlets at the head of this article to record ; +our object at present, however, is more immediately to show the immense advantage in a medical point of view, which would be conferred on our hospitals, and the sick generally, by the introduction of such an institution into this country. + +The immediate object of the institution will be best explained by another extract from the same pamphlet:? + +" But long after this office had ceased, the Roman Catholics endeavoured to fill the breach by their ' Sisters of Charity,' who have given evidence of their usefulness in our provinces likewise, whilst they are employed in almost every public hospital in France. Owing, however, to their very small number, and particularly to their education in monasteries, the recluse principles, which they often endeavour to spread, these Sisters of Charity of the Romish congre-gation are insufficient to fill this important office under all circumstances, and according to apostolical principles never can be admitted to serve the Protestant Church nor its members. The object of the school for deaconesses in Kaiserswerth is to correct this deficiency, by educating converted female Christians for deaconesses, to be sent out among the poor and sick of all denominations, for usefulness either in public hospitals or private houses ; they may likewise be appointed by Protestant churches, who feel the duty'of renewing this aposto-lical institution in their congregations. + +" It appears that several females, after having been very useful in the hospital of Kaiserswerth, have been sent out to different public hospitals or private families, where they continue to labour with much success in a truly evangelical spirit. Others have been appointed as deaconesses by Presbyterian ministers in their own congregations, and it is to be hoped that these churches will gradually feel the good effect of this very useful institution, which owes its existence to the charity of Mr Fliedner, and the liberal contribution of many Christians all over Germany, among whom I remark the name of the whole Royal family of Prussia, the Duchess of Orleans, and the King of Wir-temberg, whom Mr Fliedner has interested in his benevolent enterprises." +The rules of the institution we shall extract from the second pamphlet on our list:? + +" The rules of the establishment at Kaiserswerth are the following :?The candidates must not be under eighteen years of age, and serve from six months to two years for probation. After this probationary time, those among them who have been found fit individuals for the work of Christ, receive, during divine service, a solemn christian blessing, and then enter upon their duties as deaconesses at the infirmary, which contains from 100 to 110 beds. They engage themselves to serve at least five years, after which time they are allowed to leave, or may renew their engagement. It is understood, that if nearer, personal, or family duties, should make them wish for a change of situation during that period, every reasonable facility is granted to them for that purpose by the direction, vested in a committee. They receive no salary: a very moderate annual sum is paid by the institution or family they serve to the institution at Kaiserswerth, which defrays their personal wants, enables them to keep themselves decent and respectable, and entirely provides for those whose health has suffered in consequence of their hard service.'' +In order that the deaconesses might be properly trained to the duties of their responsible office, Mr Fliedner established near his own house a small infirmary, where the wants of the sick poor could be attended to, while, at the same time, the nurses were acquiring that information, which was to fit them for more exten-sive spheres of duty. During the nine years which have elapsed since the hospital was opened, it has received about 1900 patients, of both sexes, and of all religious denominations. + +We cannot forbear extracting the following truly eloquent and touching passage from the last work on our list, where the scenes enacted in this hospital are painfully contrasted with those which are too frequently witnessed in similar institutions in this country, which takes so large an amount of credit for christian and en-lightened benevolence :? + +" The office of these ' sisters of charity' (in the best sense of the word they are so), which elevates them above the common sick nurse, and engages them in concerns which touch on eternity, is that of reading the Scriptures to the sick and aged, and dropping a word of consolation into the languid ear, while they minister to the bodily wants. This they are authorised and expected to do, so that, instead of doing it by stealth, as a pious sick nurse may do in our hospitals; or, instead of railing on the poor sufferer who cries out in concern for his soul's health, as an impious one has sometimes been known to do, they breathe balm while they turn the pillow, and speak of the way of reconciliation while they endeavour to lull pain. Barmhcrzigen Schwestem, in the true sense of that emphatic title, they soothe the distressed?they speak of sin and the fountain opened, without speaking of penances or confessional, and teach to adore Him who laid down his life for the perishing, without interposing crucifixes, or Mary, or saint, or angel. They are by the bed in the midnight hour, and can seize the moment of coolness and clearness to speak to the afflicted?a moment which neither chaplain, nor medical man, nor friendly visiter, may be so happy as to hit upon ; and, while they are forbidden to be preachers, their living actions, their christian bearing, and their faithful advices, are calculated to drop like balm on the wounded spirit, and have, in many cases, accomplished good which we may justly call incalculable, for its consequences are eternal." +Hard as the labour is, and irksome as the duty must often be found, we rejoice to be enabled to state that numbers have come forward willing to undertake it, and many of these from a rank of life, which shows that they could not have been actuated by any selfish considerations :? + +" The deaconesses are not only of the lower and middle classes, but several also of the higher and highest ranks of life. One young Baroness of the Grand Duchy of Mecklenberg has just been educated at Kaiserswerth, and is now destined to be the matron of the large new model hospital at Berlin, lately established by the King of Prussia, in which, at least, thirty deaconesses will find work, and which is to become a great nursery for training deaconesses to serve in the different parts of the monarchy. The institution at Kaiserswerth has been called upon to send, at least, twelve trained deaconesses without delay to that model hospital. Two other ladies of high rank are at present at Kaiserswerth, devoting themselves to the same offices. Some nurses have also been educated at Kaiserswerth for Switzerland, for France, and for Holland, and the claims from many parts of the continent for deaconesses from Kaiserswerth are so numerous, that this establishment cannot satisfy them all. It results from the testimonies of the administration and the medical officers of those public institutions, and is a fact of general notoriety, that wherever those deaconesses have been intrusted with the care of an hospital or of a branch of the same, a visible change for the better takes place in all departments, and the satisfaction, the gratitude, and the blessings of the patients, follow those self-devoted nurses everywhere." +We think the passages we have quoted are amply sufficient to exhibit the nature and design of the institution, and putting its religious character, for the present, out of consideration, we would ask our professional brethren whether such an institution, properly organized and conducted, would not prove to them an inestimable boon. Could we see the patients in our hospitals treated with kindness and consideration, no longer left to the mercies of hire-lings often of the worst characters, and generally selfish to a degree ; +could we be sure that our orders were strictly attended to, and, if neglected, that such neglect were reported to us; had we the comfortable conviction that cheerfulness and universal kindness reigned in our wards, with how much greater satisfaction could we discharge our duties, and how much more frequently should we expect a beneficial result. Nor are the evils we would remedy, confined to hospitals alone. How often is wealth itself unable to procure the luxury of kind and considerate attention; how almost impossible to obtain conscientious and disinterested service. If Mrs Harris be an imaginary character, Mrs Gamp is by no means so, and were such moral nuisances swept away, and their places supplied by active, intelligent, and well educated nurses, the phy-sician would obtain a coadjutor of inestimable value, and the patient an attendant above all price. + +Let medical men proclaim the boon which such an institution would confer, let them press it on the attention of all who have time or talents, means or opportunities to prove it, and we do not despair before long of seeing among us, a school where those who have the necessary gifts may be trained for the best offices of christian charity. Those who are familiar with the working of the Kaiserswerth Hospital, have not been slow to appreciate its advan-tages, and to endeavour to extend its sphere of usefulness. At the first public meeting in London, in 1845, for the purpose of found-ing the German Hospital there, a wish expressed by Chevalier Bunsen, Prussian Ambassador, +" That some of the Protestant deaconesses of Pastor Fliedner's Institution at Kaiserswerth (near Dusseldorf, for the sick and poor), might come from the Rhine to England for the service of that institution, has been carried into effect in these last days. Four of these deaconesses, educated at the mother institution of Kaiserswerth, arrived last week in London, accompanied by the Rev. Mr Fliedner, the founder of that institution, and have entered upon their duties as nursing sisters, one of them in the capacity of matron of the establish-ment. + +" This new hospital, which is going on exceedingly well, and has, since its opening on the 18th October last, already benefited some hundreds of poor sick Germans, can now be expected to become a still greater blessing to the inmates. + +For these christian sisters, in the strength of faith working by love, have undertaken, under the direction of the committee, the management of the hospital, and are ready to show no less humility than self-devotion in its service. Yea, it is to be hoped (to use the expressions of the Rev. Dr M'Caul and the Venerable Archdeacon Robinson at the above-mentioned public meet-ing), that this plan of Protestant nursing sisters will soon be imitated in the English hospitals, where, no less than in other countries, such self-denying sisters are extremely wanted, according to the general testimony of persons of experience." +We cannot conclude without one other extract from the last of our authorities. No one has had better opportunities of observing the general habits of her sex than the accomplished writer?would that her advice were seriously pondered. How many are the complaints, formidable alike to the sufferer and her friends, and exhausting both to body and mind, which might be altogether prevented, or when they did occur, alleviated, if not removed, by suitable and engrossing occupation. Luxury, refinement, and indolence are slaying their thousands, what but self-denial or Christian principles can preserve them:? + +" In this country we lack a little of the German simplicity, and are so nice about distinctions of rank, and what belongs to our supposed station in society, that it may excite strong displeasure if we siy that there are many single women in Scotland, of the excellent of the earth, who are not so useful in the Church as they might be; that the reason of this is their want of proper guidance in selecting their work, and of support in its prosecution ; and that the deaconess' status in society, and the style of character and bearing ex-pected from her, is exactly what is wanted to confer the necessary energy and steadiness." +We rejoice to see, from the article in Lowe's Magazine, that the subject is attracting attention in Edinburgh, and we earnestly trust that the members of our profession will not be behind-hand in pro-moting the formation of such an institution, which might surely be engrafted without great expense on some of our existing medical charities. + +Ueber die Amputation am Fussgelenke. Von Dr Franz Chelius. + +Mit vier Tafeln. Heidelberg, 1846. 4to. + +On Amputation of the Ankle Joint. By Dr Francis Chelius; with Four Plates. Heidelberg, 1846. 4to. + +Dr Chelius, during a short visit he made to Edinburgh in the summer of 1846, saw Professor Syme perform amputation at the ankle joint. He had also an opportunity of examining three other cases in various stages, after the operation had been completed. He tells us that the results of this operation excited his astonishment, and convinced him that it was one of the most brilliant and valuable with which modern surgery had been enriched. On his return to Heidelberg he found a case of diseased tarsus, in the Surgical Clinic, in which amputation of the leg had already been determined on. His father, the well known and distinguished professor of surgery, permitted him to perform Mr Syme's operation, which was done without accident, and with the most perfect success. + +NEW SERIES.?NO. IX. MARCH 1847. 3 L +The present essay constitutes Dr Chelius' inaugural dissertation. + +It is appropriately dedicated to the surgeon, whom he shows, from a learned critical analysis of all that has been written on the subject, has the merit of originating it, viz. Professor Syme. It gives an excellent description of the mode in which it should be performed, divided into five stages, and concludes with the case which was so successful in his hands. It is illustrated with four very well executed lithographic engravings, exhibiting how the incisions should be made, with a copy of the stump in the instance referred to. + +So much has been said in this Journal concerning the operation, that it is unnecessary for us to analyse minutely the excellent essay of Dr Chelius. It is in the highest degree creditable to him, and is exactly that kind of inaugural dissertation we should expect to see in a young surgeon of talent, and one who has been educated under such a distinguished preceptor as its author can boast of. + +With respect to the operation itself, we sincerely join in his con-cluding remark; and trust that, owing to his admirable monograph, it will soon be adopted and firmly rooted in the surgical practice of Germany. + +Hand Book of Human Anatomy, General, Special, and Typographical. + +Translated from the original German of Dr Alfred Yon Behr, and adapted to the use of the English Student, by John Birkett, F.R.C.S. of England, &c. London, 1846. + +The plan of this work is admirably conceived, but is rather too vast for the purposes of a manual. On the whole, however, we can confidently recommend it as an excellent guide to the medical student during the first year of his studies. + +Elements of Chemistry, including the Actual State and Prevalent Doctrines of the Science. By the late Edward Turner, M.D., +F.R.S.L. and E. Eighth Edition. Edited by Baron Liebig, and William Gregory, M.D., F.R.S.E., &c. Part I.?In-organic Chemistry. London, 1847. + +This work is now universally considered as one of the standard books on Chemistry, a reputation which it has maintained through seven editions, and which the eighth, now published, is in every way calculated to confirm. \ No newline at end of file diff --git a/medical/md/PMC5889981.md b/medical/md/PMC5889981.md new file mode 100644 index 0000000000000000000000000000000000000000..d16cf5cd74b4e423a0bbd63b1a85f84e9de50d84 --- /dev/null +++ b/medical/md/PMC5889981.md @@ -0,0 +1,67 @@ +# Current Practices Of Percutaneous + +![0_Image_0.Png](0_Image_0.Png) Coronary Intervention In Korea Between 2011 And 2015 + +## Han Saem Jeong , Md, And Soon Jun Hong **, Md, Phd** + +Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, Seoul, Korea +► See the article "Trends, Characteristics, and Clinical Outcomes of Patients Undergoing Percutaneous Coronary Intervention in Korea between 2011 and 2015" in volume 48 on page 310. + +The number of percutaneous coronary intervention (PCI) procedures for coronary artery disease has dramatically increased as a result of technical and pharmacological advances. As well as PCI procedures in patients with acute coronary syndromes (ACSs), stable angina is one of the most common indications for PCI. In ACS, previous trials have shown that an invasive strategy of early and appropriate revascularization improved clinical outcomes.1) +However, the value of PCI in preventing major adverse cardiac events compared with optimal medical therapy alone in patients with stable angina is controversial.2) The Clinical Outcomes Utilizing Revascularization and Aggressive Drug Evaluation (COURAGE) trial and the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial failed to show a benefit of PCI over optimal medical therapy in patients with stable angina.3) PCI in patients with stable angina has decreased significantly after COURAGE and BARI 2D trials.4) +Moreover, the baseline characteristics of the patients who received PCI have changed over time. Consequently, identifying national trends is important to develop national clinical guidelines and implementation of these guidelines into clinical practice. In Korea, diversity in ethnicity is quite different from either Europeans or Americans. In addition, recent westernization has influenced lifestyle, diet, and disease patterns in Korea. In this aspect, Han et al.5) provided valuable evidence of the national trend for PCI in Korea. The authors5) reported a large nationwide data showing the current practice of PCI in patients with angina pectoris and acute myocardial infarction (AMI) in Korea, and they have well summarized current trends, characteristics, and clinical outcomes of PCI in Korea between 2011 and 2015. This study showed that comorbidities such as hyperlipidemia increased in patients with angina pectoris and AMI from 2011–2012 to 2014–2015. On the other hand, the incidence of hypertension decreased from 2011 to 2015, but other diseases including diabetes and congestive heart failure did not show significant changes in both groups. Proportion of male patients undergoing PCI slightly increased from 2011 to 2015. In comparison to recent Japanese and U.S. nationwide registry,6) prevalance of hypertension, dyslipidemia, and diabetes mellitus in patients undergoing PCI were relatively low in Korea (56.0% in Korea vs. 74.6% in Japan vs. 81.8% in the USA, 37.8% vs. 63.0% vs. 80.0%, and 33.1% vs. 43.3% vs. 35.8%, respectively). However, in-hospital mortality was relatively higher in Korea when compared to Japan and the USA (2.6% vs. 0.9% vs. 1.1%, respectively). Mean age and Editorial + +| Received: Feb 14, 2018 Accepted: Mar 5, 2018 Correspondence to Soon Jun Hong, MD, PhD | +|-----------------------------------------------------------------------------------------| + +Correspondence to Soon Jun Hong, MD, PhD Department of Cardiology, Cardiovascular Center, Korea University Anam Hospital, 73, Inchon-ro, Seongbuk-gu, Seoul 02841, Korea. E-mail: psyche94@gmail.com Copyright © 2018. The Korean Society of Cardiology This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https:// creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ORCID iDs Han Saem Jeong https://orcid.org/0000-0002-6035-4332 Soon Jun Hong https://orcid.org/0000-0003-4832-6678 Conflict of Interest The authors have no financial conflicts of interest. Author Contributions Conceptualization: Hong SJ; Formal analysis: Hong SJ; Investigation: Hong SJ; Methodology: Hong SJ; Supervision: Jeong HS; Validation: Jeong HS; Writing - original draft: Jeong HS, Hong SJ; Writing - review & editing: Jeong HS. The contents of the report are the author's own views and do not necessarily reflect the views of the *Korean Circulation Journal*. + +proportion of male were comparable with U.S. cohort (64.4 vs. 64.8 years, 69.7% vs. 67.2%, + +![1_image_0.png](1_image_0.png) + +respectively). The proportion of patients over 75 years was silmilar with the USA data.4) In UK +registry between 2007 and 2013, comorbities in patients undergoding PCI in UK were different from Korea,7) higher hypercholesterolemia (52.5%) and less diabetes mellitus (17.6%). + +There was a significant 10% increase in the numbers of PCI for angina from 2011–2012 to 2014–2015 in Korea. However, the number of PCI for AMI decreased marginally between 2011–2012 and 2014–2015. After peak volume in 2006 in the USA, PCI volume in patient with stable angina pectoris dramatically dropped in 2011 after COURGAGE trial.4) The PCI +volume for MI was either steady or showed an increase with time in the USA. In Korean data, the prevalence of risk profiles such as hypertension and diabetes in patients with AMI were steady or decreased slightly between 2011 and 2015. The number of PCI procedures in patients with angina pectoris increased between 2011 and 2015. Recent efforts for educating cardiovascular diseases in general population might have been correlated with the early need for PCI procedures in Korea. Moreover, technical medical advances led to perform PCI in those patients with multiple comorbidities with complex coronary morphology.8) +Nonetheless, as the authors pointed out, the use of optimal medical therapy remained suboptimal in Korea. Greater acceptance in both physicians and patients to adopt optimal medical therapy and implementation of appropriateness criteria for PCI in physicians should be emphasized. Recommended pharmacological therapies such as antiplatelet agent, statin, angiotensin converting enzyme (ACE) inhibitors/angiotensin receptor blockers (ARBs), and beta blocker in patients with angina and AMI will improve future prognosis.9) Market share of drug-eluting stents (DESs) in Korea was about 93% throughout 2011 and 2015, in comparison to relatively higher use (about 25% in 2010) of bare-metal stents (BMSs) in the USA and UK data.4)7) Current practice pattern of mostly using DES for PCI in Korea reflected better angiographic and clinical outcomes of newer-generation DESs over BMS.10) The limitation of this study is that unstable angina was not well defined. However, this study provided a comprehensive national trend for PCI in Korea. It is clear from the worldwide available evidence that PCI in patients with either angina or AMI has improved quality of life and clinical outcomes. This study by Han et al.5) will provide evidence for achieving consensus on PCI in the Korean population with angina and AMI, and will provide fundamental references for future PCI trials in Korea. + +## References + +1. Anderson JL, Adams CD, Antman EM, et al. 2012 ACCF/AHA focused update incorporated into the ACCF/ +AHA 2007 guidelines for the management of patients with unstable angina/non-ST-elevation myocardial infarction: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. *J Am Coll Cardiol* 2013;61:e179-347. + +PUBMED | CROSSREF +2. Pursnani S, Korley F, Gopaul R, et al. Percutaneous coronary intervention versus optimal medical therapy in stable coronary artery disease: a systematic review and meta-analysis of randomized clinical trials. Circ Cardiovasc Interv 2012;5:476-90. + +PUBMED | CROSSREF +3. Fihn SD, Blankenship JC, Alexander KP, et al. 2014 ACC/AHA/AATS/PCNA/SCAI/STS focused update of the guideline for the diagnosis and management of patients with stable ischemic heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines, and the American Association for Thoracic Surgery, Preventive Cardiovascular Nurses Association, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons. *J Am Coll* Cardiol 2014;64:1929-49. + +PUBMED | CROSSREF +4. Bangalore S, Gupta N, Genereux P, Guo Y, Pancholy S, Feit F. Trend in percutaneous coronary + +![2_image_0.png](2_image_0.png) + +intervention volume following the COURAGE and BARI-2D trials: insight from over 8.1 million percutaneous coronary interventions. *Int J Cardiol* 2015;183:6-10. + +PUBMED | CROSSREF +5. Han S, Park GM, Kim YG, et al. Trends, characteristics, and clinical outcomes of patients undergoing percutaneous coronary intervention in Korea between 2011 and 2015. *Korean Circ J* 2018;48:310-21. + +CROSSREF +6. Tsai TT, Patel UD, Chang TI, et al. Validated contemporary risk model of acute kidney injury in patients undergoing percutaneous coronary interventions: insights from the National Cardiovascular Data Registry Cath-PCI Registry. *J Am Heart Assoc* 2014;3:e001380. + +PUBMED | CROSSREF +7. O'Neill D, Nicholas O, Gale CP, et al. Total center percutaneous coronary intervention volume and 30-day mortality: a contemporary national cohort study of 427 467 elective, urgent, and emergency cases. *Circ* Cardiovasc Qual Outcomes 2017;10:e003186. + +PUBMED | CROSSREF +8. Singh M, Rihal CS, Gersh BJ, et al. Twenty-five-year trends in in-hospital and long-term outcome after percutaneous coronary intervention: a single-institution experience. *Circulation* 2007;115:2835-41. + +PUBMED | CROSSREF +9. Messerli FH, Mancia G, Conti CR, Pepine CJ. Guidelines on the management of stable angina pectoris: +executive summary: the task force on the management of stable angina pectoris of the European society of cardiology. *Eur Heart J* 2006;27:2902-3. + +PUBMED | CROSSREF +10. Bangalore S, Amoroso N, Fusaro M, Kumar S, Feit F. Outcomes with various drug-eluting or bare metal stents in patients with ST-segment-elevation myocardial infarction: a mixed treatment comparison analysis of trial level data from 34 068 patient-years of follow-up from randomized trials. Circ Cardiovasc Interv 2013;6:378-90. + +PUBMED | CROSSREF \ No newline at end of file diff --git a/medical/md/PMC5895985.md b/medical/md/PMC5895985.md new file mode 100644 index 0000000000000000000000000000000000000000..dd71005e2d32dfc6c2315ad81d05ef2b26083aae --- /dev/null +++ b/medical/md/PMC5895985.md @@ -0,0 +1,189 @@ + +## Evaluation Of The Prognostic Value Of Multimodal Intraoperative Monitoring In Posterior Fossa Surgery Patients With Cerebellopontine Angle Tumors + +Mohsen Dalvandi (1), Ali Nazemi Rafie (1), Alireza Kamali (2), Alireza Jamshidifard (3) +(1) Department of Neurosurgery, Arak University of Medical Sciences, Arak; (2) Department of Anesthesiology, Arak University of Medical Sciences, Arak; (3) Department of Neurology, Arak University of Medical Sciences, Arak Iran. + +This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. + +## Abstract + +The aim of this study was to compare the outcomes of four intraoperative monitoring approaches in order to compare their prognostic value in surgical complications of the facial nerve. This quasi-experimental study was conducted on 25 patients with Cerebellopontine Angle (CPA) tumors, who were hospitalized in the Valiasr Hospital, affiliated to the Arak University of Medical Sciences, Arak, Iran. The degree of the facial nerve paralysis was assessed based on the House-Brackmann (HB) facial nerve grading system. The Orbicularis oris and Oculi muscles were evaluated by electromyography (EMG) before and after the surgery. Monitoring the facial nerve was performed by the EMG, Facial Nerve Antidromic Potentials (FNAPs), Transcranial Electrical Stimulation (TES), and Brainstem Auditory Evoked Potentials (BAEPs) methods. The mean HB score was 0.36.6±5.83, indicating that the function of the facial muscle was abnormal in all the patients pre-surgery. The comparison of the four monitoring approaches six months after surgery showed that the predictive value of facial nerve TES was higher than the other approaches (P<0.005). Furthemroe, 4% of the patients showed normal function in the facial muscle after the surgery. In addition, 8%, 56%, and 32% of the patients had mild abnormalities, moderate to severe paralysis in the facial muscles, and severe facial nerve dysfunction, respectively. In summary, 36% of the patients showed a mild abnormalities in the follow-up period, and full recovery was observed in 28% of the cases. In conclusion, the transcranial motor evoked potential (TCMEP) is a useful method for monitoring the facial nerve during the surgery along with the continuous EMG recordings. + +Key Words: Multimodal IOM, CPA Tumor Resection, Facial Nerve, Acoustic nerve, Predictive Value Eur J Transl Myol 28 (1): 124-131, 2018 One of the main problems of the neurological surgery is the damage to the peripheral or central nerves. Cerebellopontine angle (CPA) tumors are the most common neoplasm in the posterior fossa, which can lead to unilateral hearing loss, speech impediments, disequilibrium, and other loss of motor control.1In some cases such as those with meningioma, the patient's life is seriously at risk; therefore, the therapeutic measures should be performed as soon as possible for these patients.2,3 There are some factors playing an important role in the treatment process of this tumor. These factors include age, tumor size, time of tumor diagnosis, cranial nerve condition before surgery, surgical route, the adhesion of the facial nerve sheath, and previous underlying diseases 4. Damage to the cranial nerve during tumor removal is inevitable in some cases, especially in the tumors bigger than 4 cm. Therefore, the facial nerve damage may be widespread, leading to practical problems and changing the emotional and social factors in the patient's future life. + +The use of the intraoperative neurophysiological monitoring (IOM) is a common approach to monitor the functional integrity of certain neural structures during the neurological surgery. This approach aims to pervent the damage caused by the manipulation of the nervous tissue,5,6 however, it is not very common in Iran compared to other countires. + +Monitoring the facial nerve during the removal of such tumors as vestibular schwannoma and other posterior fossa tumors can help not only reduce the post-surgery facial nerve damage, but also improve the patient's quality of life and decrease the further medical costs. Therefore, it is necessary to perform some studies assessing the exact criteria for monitoring and determining the respective applicable approaches. In this regard, no accurate method has been proposed for estimating the prognostic value of these approaches in facial nerve function after the CPA surgery. EMG is a well-known technique for monitoring the facial nerve during the CPA operation. This method is used for the registration of facial muscle responses to the electrical and mechanical stimulations.7 However, due to the excessive sensitivity of EMG technique in the administration of neuromuscular blockers, the application of additional methods is required. Antidromic facial nerve potentials (FNAPs), transcranial motor evoked potentials (TCMEPs), and brainstem auditory evoked potentials (BAEPs) are other methods for continuous monitoring of the facial nerve.8. All these methods are effective in the prevention of the damage during the surgery and the prediction of the patient's condition post-surgery. Despite the growing trend of IOM in the cervical spine surgery, there is limited data on the sensitivity, specificity, and predictive values of these techniques in detecting new neurological deficits. Although IOM has a long history, its application has only recently become widespread.9 Accordingly, the commercial IOM machine and its educational programs were recently developed in Iran. Although IOM has no impact on the pathology of the patients, it can affect the process of maintaining the anatomical integrity during the surgery. With this background in mind, the present study aimed to compare the functions of the four intraoperative monitoring approaches in order to compare their prognostic values in surgical complications of the facial nerve. + +## Materials And Methods Ethical Approval + +The present study was performed based the Codes of Medical Ethics released by the Ministry of Health and Medical Education, Iran and Declaration of Helsinki. In this study, all approaches were performed based on the international standard protocol. The stages and techniques of this study were clearly explained to the patients, and their informed consent was obtained. Moreover, they were assured about the confidentiality of their information and the possibility to leave the study at any stage of the project. In addition, no money was obtained from the patients until the sixth month of operation. Participants This quasi-experimental study was conducted on 32 patients with CPA tumor, who were hospitalized in the Valiasr hospital affiliated to the Arak University of Medical Sciences, Arak, Iran. Out of the 32 patients selected by the convenience sampling method, 25 cases were candidates for surgery. The exclusion criteria included suffering from congenital deafness, facial paralysis, diabetes mellitus, or addiction, and those undergoing radio or re-surgery six months after surgery. + +## Study Design + +The patients were selected based on the inclusion criteria to minimize the sampling error or bias risk. In the first step, the degree of the facial nerve paralysis was assessed based on the House-Brackmann (HB) facial nerve grading system. Additionally, the Orbicularis oris and Oculi muscles were evaluated by the EMG prior to the surgery. To determine the type and size of tumor, certain para-clinic tests, such as computed tomography scan and magnetic resonance imaging with or without contrast, were performed by neurologist colleagues. Subsequently, the patients were referred for the surgery. In accordance with the monitoring methods, the anesthetic technique was performed using the Los Angeles Anesthesia Friendliness Scale protocol (modified in 5/17/2004). The resection surgery was performed through the retrosigmoid and middle fossa approaches. The facial VII and acoustic VIII nerves were assessed before, during, and six months after the surgery. Due to the lack of data normality, the obtained data of each patient was paired with his/her data in the four stages. To minimize the bias risk of other confounding variables, the temperature of operating room and that of the rooms the patients were kept in before and after operation was fixed at 25°C. Furthermore, all the patients were operated by the same surgeon. During surgery, the antidromic sensory nerve action potentials of the facial nerve in the CPA were repeatedly recorded by the FNAPs method. The electrical stimulation was performed by corkscrew on the Cz, C3, and C4 regions through the transcranial electrical stimulation (TES) approach. The motor evoked potentials (MEP) of the Orbicularis Oris and Oculi muscles were recorded by the EMG. About four to six transcranial stimulations were performed (typically at the intervals of 2-4 ms) with an intensity of 300-500 mA in the pre-specified regions of the skull by the TES approach. The location of the electrodes during the intraoperative electromyographic monitoring of cranial nerves is shown in Table 1. The extended and freerunning EMG of the orbicularis oris and oculi muscles were also recorded during the surgery. The EMG activity in response to direct electrical stimulation of nerve and spontaneous activities were recorded using a monitoring device (Viasys/Nicolet, USA) containing 52 canals. The other invwstigations were performed pre- and postsurgery by the EMG and BAEP evaluation through electrodiagnostic 4-channel system. These devices were placed in the Laboratory of Clinical Neurophysiology at Medical School of Mashhad University of Medical + +| Table 1. Location of the electrodes in intraoperative electromyographic monitoring of cranial Cranial nerve Muscle V Masseter VII Buccalis, Mentalis Frontalis, Zygomaticus, IX Soft palate X Vocal cord XI Trapezius | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Sciences, Mashhad, Iran, and neurosurgical operating rooms of Valiasr Hospital. The stimulation parameters and recording process were controlled by the computers connected to the device. Time and amplitude parameters were listed based on mSe and mV, respectively, recorded to two decimal places on the computer screen. Our devices were equipped with amplifier system and digital converter; therefore, they were automatically calibrated in each registration stage. Prior to surgery, pure tone and absolute threshold of hearing (ATH) were measured. + +Subsequently, the speech discrimination score (SDS) was obtained, which was repeated three weeks and six months after the surgery. The SDS was standardized by Moslehi et al. in 2001. + +10. The BAEPs of both ears were recorded by stimulating the electrodes fixed in the patients' ears before, during, and after the surgery. The auditory status was monitored by the IOM device and the use of installed speakers of ear canal under surgery providing 31 stimulations per second, which was simultaneously recorded by the electrodes placed on the scalp from both ear canals. The electrodes were placed in both ears and vertex (Cz), and the filtration was adjusted within 1503,000 HZ. The mean of each recorded signal of BAEP +was 1,000-4,000 stimulations, which was repeated + +| Table 2. Change of the facial nerve before and after surgery based on House-Brackmann facial nerve grading scale Cases Pre-surgery Post-surgery After 3 weeks After 6 weeks 1 0.875 1 1 2 0.625 0.75 0.875 3 0.75 0.5 1 4 0.625 0.125 1 5 0.375 0.75 0.625 6 0.75 0.125 1 7 0.125 0.25 0.875 8 0.125 0 0.875 9 0.25 0 0.75 10 0.125 0.25 0.875 11 0.25 0.25 0.875 12 0.5 0.875 1 13 0.125 0 0.875 14 0.25 0 0.75 15 0.25 0.125 0.875 16 0.125 0.25 0.625 17 0.5 0.25 0.875 18 0.375 0.875 1 19 0.875 0.5 1 20 0.75 0.5 1 21 0.25 0 0.75 22 0.25 0 0.625 23 0.375 0.25 0.875 24 0.25 0 0.75 25 0.0 0 0.75 | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +almost every 2 min. The presence of signal and wave V +latency were reported. The changes of V waveform and the lack or presence of wave V latency (more than 1 m) +were reported to the surgeon. As a result, the surgeon was recommended to change the position of the retractor on the cerebellum and reduce its stretch as long as the change in signal be compensated. This lasted for a few seconds, and the signal appeared again. In the BAEP technique, more than a millisecond delay for the wave V +latency was considered significant. Moreover, the audiometry, pure-tone average, and SDS were evaluated and recorded for each patient before and after the surgery by an audiologist. Based on the HB scale, the degree of the facial nerve paralysis of the Orbicularis and Oculi muscles was evaluated through the EMG in the third week and sixth month of the surgery. All data were registered in the medical records of the patients, and then analyzed in the EXCEL and SPSS software using the data regression tests. + +## Results + +About 76% of the patients were female. The mean age of the participants was 50.05±10.81 years. Out of the 25 subjects, 23 patients were diagnosed with vestibular schwannoma, 1 with neurofibromatosis, and 1 with recurrent tumor. Furthermore, meningioma was observed in two males. The mean tumor size was 36.64±5.83 mm, ranging within 24-45 mm. About 76% and 24% of the tumors were removed through the retro-sigmoid and middle cranial fossa approaches, respectively. The change of the facial nerve paralysis degree before and after the surgery based on the HB is shown in Table 2. The mean HB score was 0.36.6±5.83, therefore, the function of the facial muscle was abnormal in all the patients prior to the surgery. Before the surgery, the mean values of ATH and SDS were 43.58±15.97 dB and 38.08± 10.43 in the affected side, respectively. Six months after the surgery, the mean ATH was 10.49±31.76 dB, which was increased in the affected side of eight patients and decreased in seventeen patients. Furthermore, the mean of SDS was 59.92±13.96 after six months, showing an increase in the affected side of the patients. The comparison of ATH scores showed that there was a significant difference in the hearing quality before and after the operation (P<0.005). Likewise, the SDS was significantly different before and after the surgery (P<0.005). However, the ATH and SDS scores showed no correlation with the HB score before and after the operation (P>0.005). The comparison of the facial nerve paralysis degree of the patients six months after the surgery demonstrated no significant relationship between the EMG findings and spikes recorded in the free-running EMG. Furthermore, there was no significant relationship between the facial status and latency changes of BAEP wave and the presence or lack of wave V. The changes of audiology test results in both ears before and after the surgery are shown in Table 3. There was a weak correlation between the HB score and amplitude response of FNAP during the surgery before removing the tumor +(r=0.67, P=0.005). The evaluation of the reflexes and evoked responses of Orbicularis Oris and Oculi after the operation and six months after tumor removal revealed a direct weak correlation between the HB score and amplitude response of FNAP (r=0.59, P=0.005). + +Additionally, a positive correlation was observed before removing the tumor and six months after the operation between the HB score and TES amplitude response of facial muscles (r=0.78, P=0.005). Similarly, a high direct correlation was observed between the HB score and TES amplitude response of facial muscles in the affected side and normal side of each patient after tumor removal +(r=0.807, P=0.005). + +The positive prognostic values of the four monitoring approaches were measured using the following formula: The predictive values of anti-dromic facial nerve + +![3_image_0.png](3_image_0.png) stimulation, facial nerve TES, free-running EMG, and BAER were 36.3, 0.87, 0.43, and 0.6, respectively. The comparison of these four approaches six months after the surgery showed that the predictive value of the facial nerve TES was higher than those of the other approaches. In the patients with the HB score of one, a low incidence of facial nerve involvement was reported postsurgery.However, in some cases, the recovery of facial nerve function was observed immediately after the surgery. In addition, all the patients were found to have such recovery six months after the operation, rendering the mean HB of 0.86±0.12 post-surgery. Following the surgery and getting discharged from the hospital, 4% of the patients were observed to have normal function in the facial muscle. In addition, 8%, 56%, and 32% of the patients had mild abnormalities, moderate to severe paralysis in the facial muscles, and severe facial nerve dysfunction, respectively. In summary, 36% of the patients showed mild abnormalities in the follow-up, and full recovery was observed in 28% of the cases. + +Furthermore, two patients one of whom suffering from Ependymoma and the other one having a history of Cerebral Vascular Accident passed away during the follow-up due to bleeding in the brain stem and spontaneous bleeding unrelated to the surgery on the opposite side (in the supratentorial), respectively. + +## Discussion + +As the findings of our study indicate, the transcranial stimulation of the facial nerve is a useful technique to monitor the facial nerve performance during CPA operation. Other similar studies have also confirmed this finding.4,11-13 Our data showed that the hearing quality + +| Table 3. Changes of audiology test results in both ears before and after the surgery Cases Pre-surgery Six months post-surgery PTA audiometry (dB) SDS (%) PTA audiometry (dB) | SDS (%) | | | | | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|--------|----------|--------|----------|--------|------|----| +| Normal | Affected | Normal | Affected | Normal | Affected | Normal | | | +| side | side | side | side | side | side | side | side | | +| 1 | 8.16 | 26.28 | 64 | 52 | 8 | 43 | 64 | 56 | +| 2 | 12.64 | 22.45 | 72 | 64 | 12.21 | 54 | 72 | 72 | +| 3 | 10.58 | 27.34 | 66 | 42 | 10.32 | 40 | 66 | 54 | +| 4 | 5.9 | 24.64 | 60 | 46 | 6 | 37 | 60 | 54 | +| 5 | 8.6 | 38.46 | 52 | 32 | 8 | 62 | 52 | 44 | +| 6 | 11.5 | 38.82 | 68 | 48 | 10 | 35 | 68 | 56 | +| 7 | 5.7 | 36.80 | 76 | 44 | 5.3 | 30 | 76 | 56 | +| 8 | 9.7 | 41.34 | 68 | 32 | 9.5 | 22 | 68 | 48 | +| 9 | 9.4 | 46.62 | 42 | 30 | 9.7 | 26 | 42 | 46 | +| 10 | 12.3 | 40.86 | 54 | 38 | 12.5 | 30 | 54 | 50 | +| 11 | 7.4 | 48.22 | 44 | 28 | 7 | 20 | 44 | 40 | +| 12 | 6.6 | 58.36 | 54 | 36 | 6.2 | 32 | 54 | 52 | +| 13 | 7.9 | 47.93 | 42 | 22 | 8 | 18 | 42 | 44 | +| 14 | 14.3 | 52.65 | 56 | 30 | 14.5 | 30 | 56 | 40 | +| 15 | 5.9 | 70.62 | 60 | 34 | 6 | 34 | 60 | 54 | +| 16 | 10.4 | 48.38 | 64 | 36 | 10 | 32 | 64 | 48 | +| 17 | 11.7 | 39.18 | 70 | 40 | 12 | 36 | 70 | 56 | +| 18 | 9.7 | 30.41 | 78 | 48 | 9.5 | 36 | 78 | 64 | +| 19 | 8.5 | 20.36 | 86 | 56 | 8.5 | 35 | 86 | 64 | +| 20 | 6.8 | 18.38 | 88 | 48 | 7 | 26 | 88 | 80 | +| 21 | 7.8 | 68.92 | 46 | 32 | 8 | 24 | 46 | 50 | +| 22 | 11.6 | 66.38 | 36 | 28 | 11 | 30 | 36 | 36 | +| 23 | 13.4 | 70.36 | 54 | 32 | 13 | 20 | 54 | 40 | +| 24 | 11.9 | 44.86 | 56 | 28 | 12 | 24 | 56 | 52 | +| 25 | 8.5 | 60.88 | 42 | 26 | 8.2 | 18 | 42 | 40 | +| Affected | | | | | | | | | +| PTA: pure-tone average, SDS: speech discrimination score | | | | | | | | | + +and speech discrimination were improved six months after the operation. Furthemore, a high direct correlation was observed between the HB score and TES amplitude response of facial muscles in the affected side and normal side of the patients after tumor removal. In our study, the TCMEP was performed to measure the optimal stimulation parameters of the facial nerve by using multipulse stimulation of 3-5-pulse trains, duration of 0.2-0.5 ms, and interstimulus interval of 4 ms as performed in the pervious studies.4, 11-13. The comparison of the affected and normal sides of facial nerve gives an optimal estimation from the nerve clinical practice. In our study, the highest electromyographic responses between the two sides of facial nerve were observed when comparing the TES responses of facial muscles. Therefore, the post-surgery stimulation of motor cortex of facial nerve results in improved state of descending motor pathways. The TCMEP findings had the highest correlation with the six-month follow-up results than the other approaches. Regarding this, the TCMEP can be introduced as a successful intraoperative neural monitoring in case it is applied by an experienced neuroanesthesiology team. Moreover, the TCMEP data are useful in distinguishing the subjects with initial paresis/paralysis as well as the recovery of their facial function in the long-term. Cosetti et al. evaluated the efficiency of the TCMEP in monitoring the facial nerve. To this aim, they performed TES technique on 52 patients under the CPA surgery. Similar to our study, the facial status was predicted immediately after the surgery using the HP scale and the changes of electromyographic responses of stimulated muscle before and after the tumor removal. The obtained results of the mentioned study showed that the changes of the electromyographic responses of the facial nerve immediately after the surgery should not be reduced to more than 57%, +compared to that of the pre-surgery. Therefore, they concluded that more than 57% changes in the facial nerve may cause problems in the long-term.7 The weakness of the TCMEP approach is its intermittent feature since this approach is performed every 10 min; therefore, the use of the EMG is constantly suggested. The EMG can present a feedback from the environmental damages, which have shown to have a significant correlation with surgery outcomes in terms of the facial nerve.14 The FNMEP is a standard approach confirmed by the National Institutes of Health, USA, as a proper technique to avoid damaging the facial nerve and facilitate long-term prediction of its performance. In our study, this method was used to predict the facial nerve status after the operation. In a study conducted by Fukuda et al., the FNMEP was used, and the corkscrew was installed on the C3, C4, and Cz regions. The motor responses of the orbicularis oris and oculi were recorded, which were caused by180-550 V supramaximal stimulations. In the mentioned study, there was a correlation between the post-surgery facial nerve function and FNMEP ratios. Accordingly, in case the FNMEP ratio remained > 50%, the patient had a satisfactory facial nerve function.13 Our findings demonstrated that the intraoperative FNMEP monitoring could be useful for predicting the facial nerve function post-surgery. This new method is a valuable adjunct to the conventional facial nerve monitoring techniques. Totally, intraoperative monitoring is an indispensible part of the acoustic neuroma surgery that leads to significant improvements in the functional outcomes of CPA surgery and prevents the facial paralysis.15 According to our results, severe facial nerve dysfunction was observed immediately after the surgery in about one third of the patients. However, the facial function was fully recovered in about one third of cases in the followup period; accordingly, these patients obtained HB grade I or II at the follow-up evaluation. Similar to our study, in the study carrieod out by Cosetti et al, complete paralysis and severe facial nerve dysfunction were observed in 32% of the patients immediately after the operation. Furthemore, the facial function was recovered in more than one fifth of the patients.7 Temporary motor nerve conduction block, neuropraxia or axonotmesis, edema, vasoconstriction, and herpes virus reactivation are introduced as the main causes of immediate paresis or paralysis in the patients with CPA tumor; however, the etiology of this problem have not fully discovered yet. Some of these causes (e.g., conduction block) may rapidly improve; nevertheless, in some cases, the paresis or paralysis may persist for months and even, as can be seen in case of neuropraxia or axonometsis, the patient may not achieve the pre-operative function of facial nerve.16 Immediately after surgery, the occurance of facial nerve paralysis or enhancement of HB score is possible; however, the causes are not clear. These conditions may be due to neurapraxia and temporary conduction block of the facial nerve or perioperative trauma, which are usually recovered a day or two after the surgery. Nonetheless, neurapraxia and axonotmesis injuries may continue for a few months.9 Since the CPA +surgery may be associated with main communication problems and ocular complications, the recognition of the patients at risk is very important due to the necessity of the prevention of such complications in these patients. + +The relationship between TCMEP, HB grading, and other variables, such as age, tumor size, diagnosis time, and preoperative symptoms, are not clear yet. In our study, the investigation of the association between the TCMEP and other approaches showed a positive correlation between the HB score and amplitude responses of FNAP and TES. The predictive value of these variables in terms of the facial nerve are confirmed by similar studies16,17 The outcome of the patients with CPA is largely affected by the rate of tumor adhesion to the facial nerve sheath and facial nerve, which was not possible to check in this study. However, the relationship between these variables was assessed in our study. The data on facial nerve function cannot be achieved through continuous EMG or direct electrical stimulation. Nonetheless, the presence of a stimulating probe is necessary to gain information during the surgery, which can be transferred by the TCMEP monitoring. In the present study, the BAEP approach resulted in the improvement of the hearing quality and speech discrimination six months after the operation. This finding was also confirmed in a study conducted by Yamakami et al.18 However, in a study carried out by Rampp et al., the auditory steady-state response was suggested as a more effective tool in the assessment of the hearing quality and speech discrimination.19. One of the advantages of BAEP approach is the low probability of the anesthetics effectiveness on the BAEP responses. + +As the findings of this study revealed, the TCMEP was a useful method for monitoring the facial nerve during the surgery, which could predict the facial nerve complications along with continuous EMG. Therefore, this method cannot be considered as a complete technique for monitoring the facial nerve during the CPA surgery since it should be simultaneously used along with other monitoring approaches. However, the TCMEP +approach is more favorable for monitoring the facial nerve in the posterior fossa surgery. + +## List Of Acronyms + +CPA: Cerebellopontine Angle HB: House-Brackmann EMG: Electromyography FNAPs: Facial Nerve Antidromic Potentials TES: Transcranial Electrical Stimulation BAEPs: Brainstem Auditory Evoked Potentials TCMEP: Transcranial motor evoked potential ATH: Absolute threshold of hearing SDS: Speech discrimination score IOM: Intraoperative neurophysiological monitoring MEP: The motor evoked potentials + +## Author'S Contributions + +Each author contributed in equal part to the manuscript. + +## Acknowledgments And Funding + +Hereby, we extend our gratitude to the Deputy of Research and Technology as well as all the members and experts of the Research Council of Mashhad University of Medical Sciences for approving the project proposal. We also wish to appreciate all the patients participating in this study. Other thanks go to the personnel of operating room and anesthesiology department of the hospital. Additionally, we would like to thank Dr. A.R. Ashtiani, from department of Neurology for his cooperation. Funding: None. + +## Conflict Of Interest + +The authors declare no conflicts of interests derived from the outcomes of this stud. + +## Ethical Publication Statement + +We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. + +## Corresponding Author + +Ali Nazemi Rafie, Department of Neurosurgery, Arak University of Medical Sciences, Arak, Iran. Email: nazemirafie.ali@gmail.com E-mails of co-authors Mohsen Dalvandi: Mdalvandi@yahoo.com Alireza Kamali: Alikamaliir@yahoo.com Alireza Jamshidifard: arjfard@yahoo.com + +## References + +1. Son DW, Choi CH, Cha SH. Epidermoid tumors in the cerebellopontine angle presenting with trigeminal neuralgia. J Korean Neurosurg Soc 2010;47:271-7. doi: 10.3340/jkns.2010.47.4.271. Epub 2010 Apr 30. + +2. Nakamura M. Facial and cochlear nerve function after surgery of cerebellopontine angle meningiomas. Samii's Essentials in Neurosurgery, Springer 2014:321-32. + +3. House JW, Brackmann DE. Facial nerve grading system. Otolaryngol Head Neck Surg.1985;93(2):146-7. + +4. Acioly MA, Liebsch M, Carvalho CH, et al. + +Transcranial electrocortical stimulation to monitor the facial nerve motor function during cerebellopontine angle surgery. Neurosurgery 2010;66:354-62. + +5. Fehlings MG, Brodke DS, Norvell DC, Dettori JR. + +The evidence for intraoperative neurophysiological monitoring in spine surgery: does it make a difference? Spine 2010;35:S37-S46. + +6. Lall RR, Lall RR, Hauptman JS, et al. + +Intraoperative neurophysiological monitoring in spine surgery: indications, efficacy, and role of the preoperative checklist. Neurosurgical focus 2012;33:E10. + +7. Cosetti MK, Xu M, Rivera A, et al. Intraoperative transcranial motor-evoked potential monitoring of the facial nerve during cerebellopontine angle tumor resection. J Neurol Surg B Skull Base. 2012;73:308-15. doi: 10.1055/s-0032-1321507. + +8. Galloway GM, Nuwer MR, Lopez JR, Zamel KM. + +Intraoperative neurophysiologic monitoring: Cambridge University Press, 2010. + +9. Kim S-M, Kim SH, Seo DW, Lee KW. + +Intraoperative neurophysiologic monitoring: basic principles and recent update. J Korean Med Sci 2013;28:1261-9. doi: 10.3346/jkms.2013. 28.9.1261. Epub 2013 Aug 28. + +10 Mosleh M. Development and evaluation of a speech recognition test for Persian speaking adults. Bimonthly Audiology - Tehran University of Medical Sciences. 2001;9:72-6. + +11. Akagami R, Dong CC, Westerberg BD. Localized transcranial electrical motor evoked potentials for monitoring cranial nerves in cranial base surgery. + +Neurosurgery. 2005;57:78-85. discussion 78-85. + +12. Dong CC, MacDonald DB, Akagami R, et al. + +Intraoperative facial motor evoked potential monitoring with transcranial electrical stimulation during skull base surgery. Clin neurophysiol 2005;116:588-96. + +13. Fukuda M, Oishi M, Takao T, et al. Facial nerve motor-evoked potential monitoring during skull base surgery predicts facial nerve outcome. J Neurol Neurosurg Psychiatry 2008;79:1066-70. doi: 10.1136/jnnp.2007. 130500. Epub 2008 Feb 1. + +14. Prell J, Rachinger J, Scheller C, et al. A Real‐Time Monitoring System for the Facial Nerve. Neurosurgery 2010;66:1064-73. + +15. Youssef AS, Downes AE. Intraoperative neurophysiological monitoring in vestibular schwannoma surgery: advances and clinical implications. Neurosurgical focus 2009;27(4):E9. + +16 Bernat I, Grayeli AB, Esquia G, et al. Intraoperative electromyography and surgical observations as predictive factors of facial nerve outcome in vestibular schwannoma surgery. Otol Neurotol 2010;31:306-12. doi: 10.1097/MAO. 0b013e3181be6228. + +17. Esquia-Medina GN, Grayeli AB, Ferrary E, et al. + +Do facial nerve displacement pattern and tumor adhesion influence the facial nerve outcome in vestibular schwannoma surgery? Audiol Neurootol 2013;18:406-13. doi: 10.1159/000 354115. Epub 2013 Oct 23. + +18. Yamakami I, Ushikubo O, Uchino Y, et al. + +Intraoperative monitoring of hearing function in the removal of cerebellopontine angle tumor: auditory brainstem response and cochlear nerve compound action potential. No shinkei geka Neurological surgery 2002;30:275-82. + +19. Rampp S, Rensch L, Simmermacher S, et al. + +Intraoperative auditory steady-state monitoring during surgery in the cerebellopontine angle for estimation of postoperative hearing classes. J Neurosurg 2017;127:559-568. doi: +10.3171/2016.7.JNS16460. Epub 2016 Oct 14. + +Received for publication: January 25, 2018 Revision received: February 5, 2018 Accepted for publication: February 7, 2018 \ No newline at end of file diff --git a/medical/md/PMC5922562.md b/medical/md/PMC5922562.md new file mode 100644 index 0000000000000000000000000000000000000000..972c49c5edcc937b2bafc891a7dd2ab86954fe57 --- /dev/null +++ b/medical/md/PMC5922562.md @@ -0,0 +1,310 @@ +# Risk Prediction In Stable Cardiovascular Disease Using A High-Sensitivity Cardiac Troponin T Single Biomarker Strategy Compared To The **Esc-Score** + +Moritz Biener,1 Evangelos Giannitsis,1 Manuel Kuhner,1 Thomas Zelniker,1 Matthias Mueller-Hennessen,1 Mehrshad Vafaie,1 Kiril M Stoyanov,2 Franz-Josef Neumann,2 Hugo A Katus,1 Willibald Hochholzer,2 Christian Marc Valina2 +► Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ openhrt-2017-000710). + +To cite: Biener M, Giannitsis E, +Kuhner M*, et al*. Risk prediction in stable cardiovascular disease using a high-sensitivity cardiac troponin T single biomarker strategy compared to the ESCSCORE*. Open Heart* 2018;5:e000710. doi:10.1136/ openhrt-2017-000710 Received 2 September 2017 Revised 25 November 2017 Accepted 27 March 2018 1Zentrum für Innere Medizin, Klinik für Kardiologie, Angiologie und Pneumologie, Universitätsklinikum Heidelberg, Heidelberg, Germany 2Klinik für Kardiologie und Angiologie II, UniversitätsHerzzentrum Freiburg Bad Krozingen, Bad Krozingen, Germany Correspondence to Dr Evangelos Giannitsis; evangelos_giannitsis@med.uniheidelberg.de Abstract Objective To evaluate the prognostic performance of high-sensitivity cardiac troponin T (hs-cTnT) compared with the ESC-SCORE. Methods We included low-risk outpatients with stable cardiovascular (CV) disease categorised into need for nonsecondary and secondary prevention. The prognostication of hs-cTnT at index visit was compared with the European Society of Cardiology-Systematic COronary Risk Evaluation (ESC-SCORE) with respect to all-cause mortality (ACM) and two composite endpoints (ACM, acute myocardial infarction (AMI) and stroke and ACM, AMI, stroke and rehospitalisation for acute coronary syndrome (ACS) and decompensated heart failure (DHF)). + +Results Within a median follow-up of 796 days, a total of 16 deaths, 32 composite endpoints of ACM, AMI and stroke and 83 composite endpoints of ACM, AMI, stroke, rehospitalisation for ACS and DHF were observed among 693 stable low-risk outpatients. Using C-statistics, measurement of hs-cTnT alone outperformed the ESCSCORE for the prediction of ACM in the entire study population (Δarea under the curve (AUC) 0.221, p=0.0039) +and both prevention groups (non-secondary: ΔAUC 0.164, p=0.0208; secondary: ΔAUC 0.264, p=0.0134). For the prediction of all other secondary endpoints, hs-cTnT was at least as effective as the ESC-SCORE, both in secondary and non-secondary prevention. Using continuous and categorical net reclassification improvement and integrated discrimination improvement, hs-cTnT significantly improved reclassification regarding all endpoints in the entire population and in the secondary prevention cohort. In non-secondary prevention, hs-cTnT improved reclassification only for ACM. The results were confirmed in an independent external cohort on 2046 patients. Conclusions Hs-cTnT is superior to the multivariable ESC-SCORE for the prediction of ACM and a composite endpoint in stable outpatients with and without relevant CV disease. Trial registration number NCT01954303; Pre-results. + +Introduction Cardiac troponins are recommended as the preferred biomarkers for the diagnosis of + +## Key Questions + +What is already known about this subject? + +► Elevated values of high sensitivity troponins are indicative of adverse outcome irrespective of the underlying reason. + +What does this study add? + +► This study compares for the first time the prognostic value of high sensitivity cardiac troponin T in relation to the European Society of CardiologySystematic COronary Risk Evaluation. + +How might this impact on clinical practice? + +► Multivariable risk scores are rarely used in daily clinical practice due to inconvenience. + +► In contrast, high sensitivity troponins are widely available and provide an easy-to-use risk stratification tool. + +► They might therefore facilitate risk stratification with a better acceptance in daily clinical practice. + +acute myocardial infarction (AMI1). After more sensitive and highly sensitive troponin assays replaced conventional cardiac troponin assays almost 10 years ago, AMI +could be diagnosed earlier with a more accurate discrimination between unstable angina +(UA) and non-ST elevation myocardial infarction.2 3 Furthermore, a higher prevalence of elevated troponins due to myocardial injury for non-coronary and non-cardiac reasons is observed.4 5 Irrespective of the underlying release mechanism, elevated troponin levels were found to be highly predictive for cardiovascular (CV) endpoints including mortality.6–8 A high predictive value of cardiac troponin has also been confirmed in stable coronary artery disease in large trials like the Prevention of Events with ACE +Inhibition (PEACE)9 or the Heart Outcomes Prevention Evaluation (HOPE) study.10 While several multivariable clinical risk scores have been prospectively validated for the risk stratification of patients with an acute coronary syndrome (ACS),11 12 there is no clinical risk score for individualising secondary prevention in patients with coronary artery disease (CAD), CV disease, peripheral artery disease (PAD) or with a history of remote AMI. In contrast, several risk scores have been developed and validated for risk assessment in primary prevention such as the Framingham Score13 and the European Society of Cardiology-Systematic COronary Risk Evaluation (ESC-SCORE)14 which are recommended by European guidelines.15 The ESC-SCORE has been validated in a large cohort of patients, ethnicities and for the endpoint CV mortality.14 Unfortunately, these multivariable scores are rarely used in clinical routine.16 17 Cardiac troponins are widely available in primary care and in hospitalised patients, provide plausible biological information on underlying pathophysiology, are established in the diagnosis of ACS,1 improve risk assessment in ACS,18 atrial fibrillation19 and pulmonary embolism20 and may guide therapeutic management in several acute and chronic diseases. It is tempting to speculate that a single biomarker-based risk prediction tool may be better adopted for prognostication than a complex multivariate score. Therefore, the aim of this study was to compare the performance of high-sensitivity cardiac troponin T +(hs-cTnT) alone against the European guideline recommended ESC-SCORE in a stable outpatient population presenting with or without relevant CV disease, that is, patients who require secondary prevention measures or not. + +## Methods Study Population + +We screened 965 individuals presenting to the outpatient unit of Hugo A Katus at the Department of Cardiology of the University Hospital of Heidelberg between June 2009 and June 2010. These patients were either healthy (almost healthy with minor manifestation of CV +disease) or asymptomatic with or without stable symptoms or signs of CV disease. Patients without obvious CV +disease, those with chronic arrhythmias like atrial fibrillation, pacemaker follow-up visits or arterial hypertension were summarised into the non-secondary prevention group, whereas patients with a history of documented AMI, previous percutaneous coronary intervention (PCI), previous coronary artery bypass graft +(CABG), angiographically confirmed CAD (defined as >50% luminal obstruction), asymptomatic or symptomatic peripheral arterial disease (PAD), previous transient ischemic attack (TIA) or stroke were categorised into secondary prevention. Individuals who were potentially belonging to both groups were classified as secondary prevention. Patients received routinely ECG, carotid scan, blood testing and stress tests. The latter included exercise ECG, stress echocardiography and stress MRI. Imaging tests were performed at the discretion of the attending cardiologist including coronary CT, cardiac MRI, pulmonary function tests and Ankle-Brachial-Index measurements. + +All patients with acute manifestations of CV disease such as recent ACS or acute heart failure were excluded to avoid a confounding effect of acute hs-cTnT elevation. + +The diagnostic workup comprised a 12-lead-ECG, +stress testing (ECG, echocardiography or MRI), carotid ultrasonography, CT coronary angiography, cardiac MRI, pulmonary function testing as well as Holter ECG +and 24hours blood pressure recording at the discretion of the treating physician. Furthermore, laboratory testing including hs-cTnT, blood count, clinical chemistry and coagulation tests were performed. A detailed description of the study population is available elsewhere.21 Left ventricular (LV) function was determined via echocardiography and categorised according to the ejection fraction (EF) into normal (≥55%), mildly reduced (45%– 54%), moderately reduced (30%–44%) and severely reduced (<30%). If available, MRI was used to determine LV function. Pulmonary artery pressure was measured by echocardiography or right heart catheterisation. Aortic valve stenosis was quantified by two-dimensional echocardiography or heart catheterisation. + +All patients of the original cohort were seen by the same physicians (HAK and EG) and treated according to the current guidelines. Inclusion criteria were a stable clinical course, a documented hs-cTnT value on index visit and at least one follow-up visit. Stable disease was defined by the absence of recent ACS, coronary intervention or acute cardiac decompensation since the previous visit. Patients with a history of heart transplantation were excluded. + +Follow-up was accomplished by hospital records, questionnaires, phone calls and death certificates. All scheduled and unscheduled visits or hospital admissions were recorded during follow-up. Due to the retrospective character of this analysis, informed consent was not possible and had not to be obtained according to a statement of the ethics committee of the University of Heidelberg. + +## Excelsior Cohort + +The main findings of the original study cohort were tested in an independent cohort of 2046 stable patients of the Effect of Clopidogrel Loading and Risk of PCI (EXCELSIOR) trial. A detailed description of the methodology and patient characteristics have been published by Hochholzer *et al*. + +22 In this study, subjects were recruited from consecutive patients referred for an elective invasive coronary angiography due to suspected stable CAD. Individuals were then categorised into a secondary and non-secondary prevention category according to the presence or absence of significant coronary artery stenosis >50% (see online supplementary file 1). + +## Outcome Measures + +Three prognostic endpoints were defined: (1) all-cause mortality (ACM); (2) a composite of ACM, AMI and stroke and (3) a composite of ACM, AMI, stroke and rehospitalisation for ACS and decompensated heart failure (DHF). + +ACM was defined as death from any cause including CV +and non-CV conditions. AMI was defined according to the European Society of Cardiology third universal definition of myocardial infaction1 including ST-segment elevation and non-ST-segment elevation myocardial infarction. Stroke was defined according to the Updated definition of stroke for the 21st century of the American Heart Association/American Stroke Association.23 ACS +was defined according to the European Society of Cardiology Guidelines on the management of ACSs in patients presenting without persistent ST-segment elevation24 and the European Society of Cardiology Guidelines for the management of AMI in patients presenting with ST-segment elevation.25 DHF was defined by presentation to the emergency department with symptoms of heart failure including angina or dyspnoea requiring diuretic therapy. + +## Laboratory Measurements + +Only troponin measurements during routine presentations were considered. Hs-cTnT concentrations were tested immediately from fresh blood samples. Cardiac troponin was measured in serum on a COBAS E411 using the hs-cTnT assay by Roche Diagnostics. The limit of blank and limit of detection have been determined to be 3ng/L and 5ng/L.26 The 10% CV was determined at 13ng/L in 100 measurements in the authors' laboratory. The interassay CV was 8% at 10ng/L and 2.5% at 100ng/L. The intra-assay CV was 5% at 10ng/L and 1% at 100ng/L. The hs-cTnT assays were not affected by a lot to lot variation that occurred in 2009 and 2010.27 + +## Esc-Score + +The CV risk was calculated for every patient according to the original publication of the ESC-SCORE14 considering the risk variables at index visit. Individual risk was calculated according to the ESC-SCORE charts expressed by the 10-year risk for a fatal CV event. The recalibrated low risk chart was used in this population as recommended by the guidelines.15 + +## Statistical Analysis + +Due to the pilot character of this observational study with a prespecified recruitment period of 12 months that was defined by the implementation of hs-cTnT as the routine laboratory troponin assay at the University of Heidelberg, a sample size calculation was not performed. + +Variables were tested for normal distribution using the D'Agostino-Pearson test and were presented either as means±SDdeviation, or as medians with 25th and 75th percentiles. Discrete variables were compared using Χ2 or Fisher's exact test. Continuous variables were compared using either Student's t-test for parametric or MannWhitney U test for non-parametric variables. Alternatively, we used analysis of variance (ANOVA) after logarithmic transformation of the data. If the ANOVA test was found positive (p<0.05), then the Student-Newman-Keuls test for pairwise comparison of subgroups was applied. + +We defined three endpoints to assess the prognostic performance: (1) ACM, (2) a composite endpoint of ACM, recurrent AMI and stroke and (3) a composite endpoint of ACM, recurrent AMI, stroke and hospitalisation for ACS or DHF. + +The prognostic performance of the ESC-SCORE and hs-cTnT for the defined endpoints was evaluated by C-statistics plotting and receiver operating characteristic (ROC) curves comparing the differences of the areas under the curve (AUC). The risk was determined by the 10-year risk for a fatal CV event as indicated by the ESC-SCORE charts and the hs-cTnT value on index visit. AUCs were compared using the test of DeLong *et al*. + +28 To determine the additional value of hs-cTnT in the prediction of the defined endpoints, we performed a logistic regression analysis and compared the prognostic performance of the combined variable (hs-cTnT+ESC-SCORE) with the performance of the ESC-SCORE in an ROC analysis. + +In order to evaluate benefits in risk prediction, reclassification analyses were performed to quantify improvements in model performance (in terms of correct reclassification to respective risk categories) after addition of hs-cTnT.29 Therefore, the categorical and the continuous net reclassification improvement (NRI) and the integrated discrimination improvement (IDI) were calculated. Risk categories for the respective endpoint were set as 0%–10% for low risk, 10%–20% for intermediate risk and ≥20% for high risk according to the recommendations of the National Cholesterol Education Programme.30 Statistical analyses were performed with SPSS V.20.0, MedCalc V.11.1 (MedCalc software, Mariakerke, Belgium), R V.3.1.2 (R Core Team, Vienna, Austria), as well as the R package 'PredictABEL'.31 32 All tests were two-tailed and a p value of <0.05 was considered statistically significant. + +## Results Baseline Characteristics + +We screened 965 patients presenting to the outpatient clinic of HAK. A total of 63 subjects were excluded due to missing data (n=47) or an unstable clinical course +(n=16). Of the remaining 902 stable outpatients, 11 individuals were excluded due to a missing hs-cTnT value and 179 patients could not be included because of less than two visits to the outpatient clinic. Finally, 19 patients were not recruited since they had a history of heart transplantation (figure 1). The final study cohort comprised 693 patients, of which 333 were classified as non-secondary prevention and 360 as secondary prevention. + +Follow-up was available in 686 patients (98.9%) with a median time of 796 days (25th; 75th percentile: 631–935 days). A median of three (25th; 75th percentile: 2–4) + +![3_image_0.png](3_image_0.png) + +scheduled ambulatory visits with a median of three (25th; 75th percentile: 2–4) hs-cTnT measurements were documented. hs-cTnT levels exceeding the 99th percentile +(ie, 14ng/L) on index visit were found in 146 patients (21.1%). A history of an ACS was documented in 27% of all patients. Seven out of 693 patients (1%) had an AMI within 6 months before index visit with a median time since last AMI of 62.6 months (95%CI 48.5 to 79.2) before index visit. + +The baseline characteristics split by cardiac troponin below or exceeding the 99th percentile are displayed in table 1. Most baseline characteristics differed significantly. Patients with values >14ng/L were older, were more likely to be male, had more often CV risk factors such as diabetes mellitus or arterial hypertension and were more often smokers. In addition, they had more often a history of CAD, AMI, CABG, cardiomyopathy, heart failure, arrhythmias, valvular heart disease, vascular and pulmonary diseases, renal failure and stroke/TIA. + +Patients with hs-cTnT values exceeding the 99th percentile had a higher CV risk as indicated by the ESC-SCORE. + +Most patients were seen because of a former ACS (25.4%, median time since last ACS: 62.5 months, 95%CI 28.9 to 160.9 months) or stable angiographically confirmed significant CAD with or without a history of elective coronary revascularisation (26.4%). Five patients (0.7%) were seen following a cerebrovascular event (TIA/stroke). Echocardiography (92.6%) and ECG (94.1%) were performed in almost every patient, and an ECG stress test (64.4%) was performed in the majority of patients. + +## General Outcome Characteristics + +In the overall cohort, we observed 16 deaths (ACM), 32 patients with ACM, AMI or stroke and 83 patients with ACM, AMI, stroke and rehospitalisation for ACS or DHF. Total mortality of the entire cohort was 2.03% during follow-up (796 days) corresponding to a total annual mortality rate of 0.93%. + +The prognostic performance of the ESC-SCORE and hs-cTnT in secondary and non-secondary prevention is displayed in table 2. + +The ESC-SCORE performed superior regarding the prediction of ACM in the non-secondary prevention group compared with the secondary prevention group (figure 2A). Regarding the composite endpoint of ACM, AMI and stroke, we also observed a better performance of the ESC-SCORE in non-secondary prevention compared with secondary prevention patients (figure 2B), whereas the prognostic performance for the prediction of the composite endpoint ACM, AMI, stroke, rehospitalisation for ACS or DHF was comparable between both groups (figure 2C). + +Hs-cTnT performed better in the prediction of ACM in the non-secondary prevention group compared with the secondary prevention group, whereas the prognostication of the composite endpoints of ACM, AMI and stroke and ACM, AMI, stroke, rehospitalisation for ACS or DHF was comparable. + +In the prediction of ACM, the hs-cTnT value on index visit (AUC 0.878, 95% CI 0.805 to 0.950) outperformed the ESC-SCORE (AUC 0.657, 95% CI 0.526 to 0.789) in the overall study population (ΔAUC 0.221, p=0.0039, figure 3A). Hs-cTnT was also superior to the ESC-SCORE in the prediction of ACM in non-secondary prevention (hs-cTnT: AUC 0.962, 95% CI 0.924 to 1.000; ESC-SCORE: AUC 0.798, 95% CI 0.664 to 0.931; ΔAUC 0.164, p=0.0208, figure 3B). In secondary prevention, with 0.264 (p=0.0134, figure 3C) the delta between hs-cTnT (AUC 0.836, 95% CI 0.734 to 0.937) and the ESC-SCORE (AUC 0.572, 95% CI 0.389 to 0.755) was even higher. + +Hs-cTnT and the ESC-SCORE had a comparable performance regarding the prediction of the composite endpoint ACM, AMI and stroke in secondary (ΔAUC +hs-cTnT vs ESC-SCORE: 0.110, p=NS, online supplementary figure 1) and non-secondary prevention (ΔAUC hs-cTnT vs ESC-SCORE: 0.119, p=NS, online supplementary figure 2). + +The prognostic performance of hs-cTnT to predict the composite endpoint ACM, AMI, stroke, rehospitalisation for ACS or DHF was higher in the secondary prevention group (ΔAUC hs-cTnT vs ESC-SCORE: 0.136, p=0.02), + +| Coronary artery disease | | | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|--------------------------|--------------------------|--------------| +| Table 1 | Baseline characteristics according to troponin status All (n=693) | Troponin ≤14ng/L (n=547) | Troponin >14ng/L (n=146) | | +| Presentations (quantity) | 3 (3–5) | 3 (3–5) | 4 (3–6)*** | | +| | Outpatient clinic | 3 (2–4) | 3 (2–4) | 3 (2–4) | +| Admission to hospital | 0 (0–1) | 0 (0–1) | 0 (0–1)*** | | +| Admission to emergency department | 0 (0–0) | 0 (0–0) | 0 (0–1)*** | | +| Age (years) | 66.3±12.3 | 64.2±11.9 | 74.3±10.3*** | | +| Male gender, n (%) | 519 (74.9) | 391 (71.5) | 128 (87.7)*** | | +| Risk factors, n (%) Diabetes mellitus | 136 (19.7) | 92 (16.9) | 44 (30.1)*** | | +| Dyslipidaemia | 580 (84.9) | 455 (84.4) | 125 (86.8) | | +| Hypertension | 574 (84.3) | 442 (82.3) | 132 (91.7)** | | +| Smoking | 32 (13.1) | 4 (6.2) | 28 (15.6)*** | | +| | Ex-smoker | 100 (41.0) | 71 (39.7) | 29 (44.6) | +| Family history of coronary artery disease | 258 (52.9) | 224 (55.6) | 34 (40.0)** | | +| Medical history, n (%) Coronary artery disease | 420 (60.6) | 311 (56.9) | 109 (74.7)*** | | +| Acute myocardial infarction | 185 (26.7) | 136 (24.9) | 49 (33.6)* | | +| | Percutaneous coronary intervention | 293 (42.3) | 229 (41.9) | 64 (44.1) | +| | Coronary artery bypass graft | 99 (14.3) | 54 (9.9) | 45 (30.8)*** | +| Cardiomyopathy | 83 (12.0) | 50 (9.1) | 33 (22.6)*** | | +| Chronic heart failure | 208 (30.0) | 156 (28.5) | 52 (35.6) | | +| Dysarhythmia | 200 (28.9) | 140 (25.6) | 60 (41.1)*** | | +| Valvular disease | 472 (68.1) | 358 (65.4) | 114 (78.1)** | | +| Arterial occlusive disease | 128 (18.5) | 71 (13.0) | 57 (39.0)*** | | +| Pulmonary disease | 80 (11.5) | 56 (10.2) | 24 (16.4)* | | +| Kidney disease | 60 (8.7) | 28 (5.1) | 32 (21.9)*** | | +| History of stroke | 45 (6.5) | 29 (5.3) | 16 (11.0)* | | +| Rheumatic disease | 13 (1.9) | 8 (1.5) | 5 (3.4) | | +| Cancer | 78 (11.3) | 57 (10.4) | 21 (14.4) | | +| Other | 229 (33.0) | 211 (38.6) | 18 (12.3)*** | | +| Clinical chemistry High-sensitivity cardiac troponin T (ng/L) | 6 (3–13) | 4 (3–8) | 21 (17–29)*** | | +| N-terminal pro b-type natriuretic peptide (ng/L) | 193 (84–484) | 153 (78–374) | 443 (197–1472)*** | | +| Creatinine (mg/dL) | 1.0 (0.9–1.1) | 0.9 (0.8–1.1) | 1.0 (1.0–1.0)*** | | +| Estimated glomerular filtration rate (mL/min/1.73m2 ) | 82.9±30.0 | 87.3±28.8 | 65.9±28.2*** | | +| Medication, n (%) Antihypertensive therapy | 576 (83.1) | 438 (80.1) | 138 (94.5)*** | | +| Diuretics | 256 (36.9) | 177 (32.4) | 79 (54.1)*** | | +| Acetylsalicylic acid | 395 (57.0) | 324 (59.2) | 71 (48.6)* | | +| Clopidogrel | 103 (14.9) | 76 (13.9) | 27 (18.5) | | +| Statins | 500 (72.2) | 385 (70.4) | 115 (78.8) | | +| Antiarrhythmics | 414 (59.7) | 299 (54.7) | 115 (78.8)*** | | +| Insulin | 19 (2.7) | 7 (1.3) | 12 (8.2)*** | | +| Oral antidiabetics | 78 (11.3) | 52 (9.5) | 26 (17.8)** | | +| Other | 316 (45.6) | 253 (46.3) | 63 (43.2) | | +| European Society of Cardiology-Systematic COronary Risk | 2.7±1.9 | 2.4±2.0 | 3.1±1.7*** | | +| Evaluation (points) *P<0.05 compared with high-sensitivity cardiac troponin T ≤14ng/L, **P<0.01, ***P<0.001. Presentations: quantity of presentations to outpatient clinic, emergency department or hospital admissions during follow-up. | | | | | + +Open Heart + +| Table 2 | Prognostic performance of the ESC-SCORE and high-sensitivity cardiac troponin T in non-secondary and secondary | | | | | | | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------|----------------------|----------------|-------|--------|----------------|----------|------| +| prevention | Non-secondary prevention | Secondary prevention | | | | | | | +| AUC | SE | 95%CI | AUC | SE | 95%CI | ∆AUC | P values | | +| All-cause mortality ESC-SCORE | 0.798 | 0.0681 | 0.664 to 0.931 | 0.572 | 0.0933 | 0.389 to 0.755 | 0.226 | 0.05 | +| High-sensitivity troponin T | 0.967 | 0.0161 | 0.935 to 0.999 | 0.830 | 0.0516 | 0.729 to 0.931 | 0.137 | 0.01 | +| All-cause mortality, acute myocardial infarction, stroke (endpoint 2) ESC-SCORE 0.794 0.0732 0.650 to 0.937 | 0.590 | 0.0689 | 0.455 to 0.725 | 0.204 | 0.04 | | | | +| High-sensitivity troponin T | 0.675 | 0.1350 | 0.412 to 0.939 | 0.696 | 0.0521 | 0.594 to 0.798 | 0.021 | NS | +| All-cause mortality, acute myocardial infarction, stroke, rehospitalisation for acute coronary syndrome or decompensated heart failure (endpoint 3) ESC-SCORE 0.591 0.0699 0.454 to 0.728 0.525 0.0428 0.442 to 0.609 0.066 NS High-sensitivity troponin T 0.668 0.0757 0.519 to 0.816 0.661 0.0372 0.588 to 0.734 0.001 NS AUC, area under the curve; ESC-SCORE, European Society of Cardiology-Systematic COronary Risk Evaluation; NS, not significant. | | | | | | | | | + +whereas no difference was observed in the non-secondary prevention group (ΔAUC hs-cTnT vs ESC-SCORE: 0.077, p=NS). + +Reclassification analysis according to hs-cTnT status +(>14ng/L at index visit) is displayed for the entire population as well as the secondary and non-secondary prevention group in the online supplementary table 1. Briefly, hs-cTnT significantly improved reclassification regarding all endpoints in the overall population and in the secondary prevention cohort. In non-secondary prevention, hs-cTnT improved reclassification only for ACM. + +We found no statistically significant difference of hs-cTnT concentrations in men compared with women regarding all endpoints (ACM: men: 49.2±95.1ng/L, women: 26.0±20.3ng/L, p=NS; ACM, AMI, stroke: men: 30.0±65.6, women: 16.8±19.1, p=NS; ACM, AMI, stroke, rehospitalisation for ACS or DHF: men: 23.4±44.7, women: 8.7±7.4, p=NS). + +## Discussion + +Our study provides several important findings and supports the significant predictive role of hs-cTnT for risk stratification of patients with stable CAD. First, hs-cTnT outperformed the multivariable ESC-SCORE +for the prediction of ACM in non-secondary as well as in secondary prevention. Second, hs-cTnT provides reliable prognostic information on composite endpoints that also include soft outcome measures like readmission with ACS or DHF. Third, hs-cTnT provided consistent prognostic information in an external cohort with a different baseline risk. + +We decided to compare the prognostication of hs-cTnT +to the ESC-SCORE because it is validated in a large cohort across different countries for the occurrence of CV mortality as an objective and robust endpoint14 and is therefore recommended by ESC guidelines.15 The high prognostic performance of hs-cTnT in stable CV disease has been reported in many studies including large multicenter trials like the PEACE9 or the HOPE study.10 Our group had recently reported about the strong independent predictive value of hs-cTnT in the same low risk cohort of asymptomatic or stable patients with a broad spectrum of CV diseases visiting an outpatient clinic for routine regular check-ups.21 In another recently published analysis, we reported on the performance of hs-cTnT compared with established multivariable scores that are used in some European regions (Prospective Cardiovascular Münster Study (PROCAM) Score) or in + +![5_image_0.png](5_image_0.png) + +![6_image_0.png](6_image_0.png) + +the US (Framingham Scores) in primary prevention.33 In consistency with the both previous studies, hs-cTnT predicted ACM with an AUC of 0.878 and thus—as expected—significantly better than the ESC-SCORE in secondary prevention. To our surprise, hs-cTnT also performed significantly better in non-secondary prevention, which is the domain of CV risk scores including the ESC-SCORE. Although our findings need to be confirmed in larger prospective cohorts, our results provide a strong hypothesis generating finding that could improve adoption of risk stratification tools both in primary and secondary prevention. For the latter, there is no universally established risk stratification tool that allows risk estimation after the subacute phase of ACS or after diagnosis of a relevant CV or cerebrovascular disease manifestation. + +Although the ESC-SCORE has not been validated for soft endpoints such as rehospitalisation for DHF, AMI +or recurrent ACS, we compared the prognostic information incorporated in a single hs-cTnT value as compared with a multivariable score and consistently confirmed our findings on the role of hs-cTnT to predict ACM. In agreement with the previous findings6 34 35 and our own published work on this low risk cohort,21 the prognostic performance of hs-cTnT to predict a variety of softer endpoints than ACM was only moderate with an AUC of only 0.707 for the composite endpoint of ACM, AMI and stroke, and 0.671 for the combined endpoint of ACM, AMI, stroke and rehospitalisation for DHF or ACS. Our results using traditional C-statistics were substantiated by reclassification analyses including continuous and categorical NRI as well as IDI. + +A general shortcoming of CV risk scores is that their predictive performance strongly depends on the similarity of the population in which they are applied to the original derivation cohort. An impaired performance has been reported particularly when the baseline risk differs from the original population.36–38 European guidelines therefore recommend the use of national recalibrations of the ESC-SCORE that consider the general population's risk.15 Nevertheless, this score has not been evaluated in non-caucasian populations and predicts only the endpoint 10-year CV mortality.14 In contrast, the predictive ability of hs-cTnT is more consistent showing an excellent performance across a broad spectrum of stable or asymptomatic patients including low-risk cohorts,39–41 chronic heart failure,42 asymptomatic high risk patients10 and in the general population.7 8 Since risk scores are only rarely used in daily clinical practice,16 17 the use of hs-cTnT within a single-biomarker strategy may facilitate the prognostication of low-risk patients and increase the acceptance of risk stratification in daily practice. Nevertheless, although higher hs-cTnT values have been associated with an increased risk for CV endpoints, it may not be directly modified by lifestyle changes in contrast to some of the variables of the ESC-SCORE like hypertension or smoking status. + +Our results were validated in a large independent study cohort of 2046 stable patients with documented coronary anatomy from the EXCELSIOR trial.22 In the EXCELSIOR cohort, a lower prognostic performance of hs-cTnT +for the prediction of ACM as compared with our study cohort was found (AUC 0.759, 95% CI 0.740 to 0.777 vs AUC 0.878 95% CI0.805 to 0.950). A reason for this observation might be the higher baseline risk since all individuals of the EXCELSIOR cohort underwent coronary angiography. Furthermore, the annual mortality was more than twice as high compared with our cohort (2.1 vs 0.9%). Irrespective of the higher baseline risk, hs-cTnT also outperformed the ESC-SCORE in the prediction of ACM and non-fatal AMI in the EXCELSIOR trial. The use of hs-cTnT in addition to the ESC-SCORE could significantly improve risk classification of patients. Multiple subgroup analyses of this cohort demonstrated that this finding was consistent throughout the whole population of this study. We therefore assume that our results may also be applied to other cohorts including populations at higher baseline risk. + +In conclusion, our study suggests that hs-cTnT is a reliable and convenient risk predictor with a superior performance compared with the ESC-SCORE in non-secondary and secondary prevention that may be applied in a broad spectrum of patients in primary care. It is thereby easier and more convenient to use in daily clinical practice. + +## Limitations + +The findings of this registry are limited by a relatively small sample size and small event rates, particularly occurrence of few all-cause deaths. However, our findings are consistent with the previous findings across the spectrum of stable CV disease and plausible due to the comprehensive integration of different pathophysiological aspects reflected by elevated cardiac troponin in blood. Our findings were validated in an independent large registry consistently confirming our results. Nevertheless, we believe that our results are hypothesis generating and would benefit from additional external validation in prospective trials or registries. Furthermore, there is large controversy regarding the role of sex-specific cut-offs that are being recommended by the Universal Myocardial Infarction Definition Task Force.1 Currently, sex-specific cut-offs have neither been recommended by ESC guidelines nor have they been validated prospectively. In the absence of convincing data supporting an improvement of diagnostic and prognostic reclassification,43 we preferred to use a single, sex-independent cut-off at the 99th percentile of a healthy reference population.26 Acknowledgements We thank Mrs Heidi Deigentasch, Mrs Melanie Hütter, Mrs Elisabeth Mertz and Mrs Monika Arnold for their valuable support regarding data management and follow-up. Contributors All authors contributed to the manuscript. + +Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Competing interests None declared. Patient consent Not required. Ethics approval Ethics Committee, University Hospital Heidelberg. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement No additional data are available. Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/ licenses/by-nc/4.0/ © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted. + +## References + +1. Thygesen K, Alpert JS, Jaffe AS, *et al*. Third universal definition of myocardial infarction. *Eur Heart J* 2012;33:2551–67. + +2. Keller T, Zeller T, Peetz D, *et al*. Sensitive troponin I assay in early diagnosis of acute myocardial infarction. *N Engl J Med* 2009;361:868–77. + +3. Reichlin T, Hochholzer W, Bassetti S, *et al*. Early diagnosis of myocardial infarction with sensitive cardiac troponin assays. N Engl J Med 2009;361:858–67. + +4. Agewall S, Giannitsis E, Jernberg T, *et al*. Troponin elevation in coronary vs. non-coronary disease. *Eur Heart J* 2011;32:404–11. + +5. Giannitsis E, Katus HA. Cardiac troponin level elevations not related to acute coronary syndromes. *Nat Rev Cardiol* 2013;10:623–34. + +6. Saunders JT, Nambi V, de Lemos JA, *et al*. Cardiac troponin T +measured by a highly sensitive assay predicts coronary heart disease, heart failure, and mortality in the Atherosclerosis Risk in Communities Study. *Circulation* 2011;123:1367–76. + +7. de Lemos JA, Drazner MH, Omland T, *et al*. Association of troponin T detected with a highly sensitive assay and cardiac structure and mortality risk in the general population. *JAMA* 2010;304:2503–12. + +8. deFilippi CR, de Lemos JA, Christenson RH, *et al*. Association of serial measures of cardiac troponin T using a sensitive assay with incident heart failure and cardiovascular mortality in older adults. JAMA 2010;304:2494–502. + +9. Omland T, de Lemos JA, Sabatine MS, *et al*. A sensitive cardiac troponin T assay in stable coronary artery disease. *N Engl J Med* 2009;361:2538–47. + +10. Kavsak PA, McQueen MJ. Sensitive and high sensitivity cardiac troponin I concentrations in the Heart Outcomes Prevention Evaluation (HOPE) study--a high risk population. *Clin Chim Acta* 2010;411:1832. + +11. Fox KA, Dabbous OH, Goldberg RJ, *et al*. Prediction of risk of death and myocardial infarction in the six months after presentation with acute coronary syndrome: prospective multinational observational study (GRACE). BMJ 2006;333:1091. + +12. Antman EM, Cohen M, Bernink PJ, *et al*. The TIMI risk score for unstable angina/non-ST elevation MI: A method for prognostication and therapeutic decision making. *JAMA* 2000;284:835–42. + +13. D'Agostino RB, Vasan RS, Pencina MJ, *et al*. General cardiovascular risk profile for use in primary care: the Framingham Heart Study. Circulation 2008;117:743–53. + +14. Conroy RM, Pyörälä K, Fitzgerald AP, *et al*. Estimation of ten-year risk of fatal cardiovascular disease in Europe: the SCORE project. Eur Heart J 2003;24:987–1003. + +15. Piepoli MF, Hoes AW, Agewall S, *et al*. 2016 European Guidelines on cardiovascular disease prevention in clinical practice: The Sixth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of 10 societies and by invited experts) Developed with the special contribution of the European Association for Cardiovascular Prevention & Rehabilitation (EACPR). Atherosclerosis 2016;252:207–74. + +16. Sposito AC, Ramires JA, Jukema JW, *et al*. Physicians' attitudes and adherence to use of risk scores for primary prevention of cardiovascular disease: cross-sectional survey in three world regions. *Curr Med Res Opin* 2009;25:1171–8. + +17. Müller-Riemenschneider F, Holmberg C, Rieckmann N, *et al*. Barriers to routine risk-score use for healthy primary care patients: survey and qualitative study. *Arch Intern Med* 2010;170:719–24. + +18. Mueller M, Celik S, Biener M, *et al*. Diagnostic and prognostic performance of a novel high-sensitivity cardiac troponin T assay compared to a contemporary sensitive cardiac troponin I assay in patients with acute coronary syndrome. *Clin Res Cardiol* 2012;101:837–45. + +19. Stoyanov KM, Giannitsis E, Biener M, *et al*. Prognostic value of elevated high-sensitivity cardiac troponin T in patients admitted to an emergency department with atrial fibrillation. *Europace* 2017. + +20. Giannitsis E, Katus HA. Biomarkers for clinical decisionmaking in the management of pulmonary embolism. *Clin Chem* 2017;63:91–100. + +21. Biener M, Giannitsis E, Lamerz J, *et al*. Prognostic value of elevated high-sensitivity cardiac troponin T levels in a low risk outpatient population with cardiovascular disease. *Eur Heart J Acute* Cardiovasc Care 2016;5. + +22. Hochholzer W, Valina CM, Stratz C, *et al*. High-sensitivity cardiac troponin for risk prediction in patients with and without coronary heart disease. *Int J Cardiol* 2014;176:444–9. + +23. Sacco RL, Kasner SE, Broderick JP, *et al*. An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 2013;44:2064–89. + +24. Roffi M, Patrono C, Collet JP, *et al*. 2015 ESC guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: task force for the management of acute coronary syndromes in patients presenting without persistent st-segment elevation of the European Society of Cardiology (ESC). *Eur Heart J* 2016;37:267–315. + +25. Ibanez B, James S, Agewall S, *et al*. 2017 ESC guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: the Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC). *Eur Heart J* 2018;39:119–77. + +26. Giannitsis E, Kurz K, Hallermayer K, *et al*. Analytical validation of a high-sensitivity cardiac troponin T assay. *Clin Chem* 2010;56:254–61. + +27. Apple FS, Jaffe AS. Clinical implications of a recent adjustment to the high-sensitivity cardiac troponin T assay: user beware. Clin Chem 2012;58:1599–600. + +28. DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. *Biometrics* 1988;44:837–45. + +29. Pencina MJ, D'Agostino RB, D'Agostino RB, *et al*. Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond. *Stat Med* 2008;27:157–72. discussion 207-12. + +30. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). *JAMA* 2001;285:2486–97. + +31. Team RC. R: A language and environment for statistical computing. + +2014 http://www.R-project.org/. + +32. Kundu S, Aulchenko YS, van Duijn CM, *et al*. PredictABEL: an R package for the assessment of risk prediction models. Eur J Epidemiol 2011;26:261–4. + +33. Biener M, Giannitsis E, Kuhner M, *et al*. Prognostic value of highsensitivity cardiac troponin T compared with risk scores in stable cardiovascular disease. *Am J Med* 2017;130. + +34. Omland T, Pfeffer MA, Solomon SD, *et al*. Prognostic value of cardiac troponin I measured with a highly sensitive assay in patients with stable coronary artery disease. *J Am Coll Cardiol* 2013;61:1240–9. + +35. Eggers KM, Lagerqvist B, Oldgren J, *et al*. Pathophysiologic mechanisms of persistent cardiac troponin I elevation in stabilized patients after an episode of acute coronary syndrome. *Am Heart J* 2008;156:588–94. + +36. Scheltens T, Verschuren WM, Boshuizen HC, *et al*. Estimation of cardiovascular risk: a comparison between the Framingham and the SCORE model in people under 60 years of age. Eur J Cardiovasc Prev Rehabil 2008;15:562–6. + +37. Selvarajah S, Kaur G, Haniff J, *et al*. Comparison of the Framingham Risk Score, SCORE and WHO/ISH cardiovascular risk prediction models in an Asian population. *Int J Cardiol* 2014;176:211–8. + +38. Versteylen MO, Joosen IA, Shaw LJ, *et al*. Comparison of Framingham, PROCAM, SCORE, and Diamond Forrester to predict coronary atherosclerosis and cardiovascular events. *J Nucl Cardiol* 2011;18:904–11. + +39. Beatty AL, Ku IA, Christenson RH, *et al*. High-sensitivity cardiac troponin T levels and secondary events in outpatients with coronary heart disease from the Heart and Soul Study. *JAMA Intern Med* 2013;173:763–9. + +40. Koenig W, Breitling LP, Hahmann H, *et al*. Cardiac troponin T measured by a high-sensitivity assay predicts recurrent cardiovascular events in stable coronary heart disease patients with 8-year follow-up. *Clin Chem* 2012;58:1215–24. + +41. Giannitsis E, Spanuth E, Horsch A, *et al*. High-sensitivity cardiac troponin T and N-terminal pro-B-type natriuretic peptide predict mortality in stable coronary artery disease: results from the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. *Clin* Chem Lab Med 2013;51:2019–28. + +42. Nagarajan V, Hernandez AV, Tang WH. Prognostic value of cardiac troponin in chronic stable heart failure: a systematic review. *Heart* 2012;98:1778–86. + +43. Mueller-Hennessen M, Lindahl B, Giannitsis E, *et al*. Diagnostic and prognostic implications using age- and gender-specific cut-offs for high-sensitivity cardiac troponin T - sub-analysis from the TRAPIDAMI study. *Int J Cardiol* 2016;209:26–33. \ No newline at end of file diff --git a/medical/md/PMC5954647.md b/medical/md/PMC5954647.md new file mode 100644 index 0000000000000000000000000000000000000000..612d17a23b0e6f7bb65751afae7f6eba99a04b61 --- /dev/null +++ b/medical/md/PMC5954647.md @@ -0,0 +1,154 @@ + +## The Relationship Between Perceived Social Support In The First Pregnancy And Fear Of Childbirth Abstract + +Background: Numerous empirical evidences have shown that social and environmental circumstances and social relations have an important impact on pregnancy outcomes, women's ability to cope with stressful situations, and childbirth pain management. The present study was conducted to determine the relationship between perceived social support and fear of childbirth. **Materials and Methods:** The present descriptive correlational study was conducted on 270 nulliparous pregnant women who referred for pregnancy care in 2016. The subjects were selected through convenience sampling method. The data collection tools were a demographic characteristics form, the Multidimensional Scale of Perceived Social Support (MSPSS), and the Wijma Delivery Expectancy/Experience Questionnaire (W‑DEQ). The collected data were analyzed using descriptive and inferential statistics tests. **Results:** The mean perceived social support score of the participants was 77.90 and a significant correlation was observed between the perceived social support score and fear of childbirth score (β = −0.18, p = 0.004). The participants' mean score of fear of childbirth was 36.8. The results of study after the evaluation of the effect of predictive variables on the fear of childbirth are as follows: pregnancy preparation classes: *(β* = 3.50*, p* = 0.220); observation of natural childbirth videos: (β = 5.26, p = 0.040); and use of educational software: *(β* =5.82, p = 0.080). **Conclusions:** In order to reduce the fear of childbirth, women's social support structure during pregnancy should be assessed. Moreover, demographic characteristics form the structure and determine the extent of individuals' social network and, through the evaluation of these characteristics during pregnancy, the rate of effective support can be predicted in individuals. Keywords: *Childbirth, fear, Iran, pregnancy, social support* + +## Introduction + +Childbirth is a social occurrence all around the world and can be considered as the happiest occurrence in any parent's life. Becoming a parent is accompanied by obtaining a high social status, and the majority of parents describe it as a positive experience. However, pregnancy in itself is a traumatic occurrence and the mother who encounters can potentially be stressful.[1] +The birth of the first child, in particular, is categorized as an extremely stressful event in psychosocial stress tables.[2] Concern about the adverse physical–psychological effects of pregnancy and fear of childbirth can be noted as two of the stressors of this period. It has been estimated that 1 out of 5 pregnant women have fear of childbirth. Moreover, approximately 6–13% of pregnant women experience severe and debilitating fear, which, in addition to physical outcomes, can result This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercialShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. + +For reprints contact: reprints@medknow.com in psychological complications such as depression and anxiety, negative mood after childbirth, eating disorders,[3] and even uninformed selection of delivery method. It has been proven that fear of childbirth has an important role in the selection of cesarean section by Iranian women.[4] Based on the statistics reported by the World Health Organization (WHO), the prevalence of cesarean section was 46% in Iran in 2014; however, the recommendation of the WHO for an acceptable prevalence of cesarean section by 2014 was a maximum of 5–15%.[5] +Fear of childbirth is an important issue during pregnancy and during and after childbirth, and psychological and social factors have an effective role in its creation.[6] The results of the study by Fisher et al.[7] illustrated that fear of childbirth has two dimensions: individual and social dimension. The social dimension has received less attention in most studies,[6] +How to cite this article: Azimi M, Fahami F, +Mohamadirizi S. The relationship between perceived social support in the first pregnancy and fear of childbirth. Iranian J Nursing Midwifery Res 2018;23:235-9. + +Received: September, 2016. **Accepted:** September, 2017. + +## Masoomeh Azimi1, Fariba Fahami2, Soheila Mohamadirizi2 + +1Student *Research Center,* +School of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran, 2Nursing and Midwifery Care Research Center, Faculty of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran Address for correspondence: Ms. Fariba Fahami, Nursing and Midwifery Care Research Center, Faculty of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran. E‑mail: *Fahami@nm.mui.ac.ir* + +![0_image_0.png](0_image_0.png) + +although it has been found that the social dimension not only affects women's identity but also their perception of childbirth as an abstract event. Social relations, the unofficial support networks of pregnant women, and strong support by midwives can strengthen women's perception of childbirth as a physiological and controllable process.[7] On the contrary, researches have shown that Iranian women's perception of childbirth is derived from their culture and values and is rooted in the environment in which they live.[8] It must be noted that social support has two main dimensions: received and perceived social support. Received social support is the rate of support received by the individual. Perceived social support is the individual's evaluation of the availability of support when necessary and required and is a qualitative‑mental and measurable concept.[9] It is affected by culture, evolution, personality, and support systems.[7] Theoreticians believe relationships are not a source of social support unless the individual perceives them as a suitable and available source for fulfilling their needs.[10] +Social support is an interpersonal relationship which provides individuals with psychological help when required. Those who provide this help include family members, neighbors, colleagues, relatives, and the treatment team, who provide mothers with psychological support and information.[1] Relatives, especially the spouse, illustrate their high regard for the pregnant woman through their support for her and this gives her hope. Previous studies have shown that social support has a significant relationship with reduction of stress during pregnancy,[11] and the lack of support has a relationship with unwillingness to breastfeed,[12] low IQ in infants,[13] postpartum depression (PPD),[14] depression during pregnancy,[15] and cleft palate incidence.[16] Some researchers have reported contradictory results regarding this issue. Some studies have shown that sometimes the support provided is inappropriate, untimely, and contrary to the mother's wishes.[17] Other studies have illustrated that this support was ineffective. Jirka et al. found no difference in the rate of social support of pregnant and nonpregnant couples; they received the same rate of support.[18] +Due to the importance of the role of perceived social support in individuals' psychological wellbeing, the contradictory results of previous studies, the role of cultural difference in social support patterns,[19] and the important role of fear of childbirth in the selection of cesarean section by Iranian women,[4] the present study was undertaken. The present study was conducted to determine the possible challenges regarding mothers' social support and the role of predictive variables on fear of childbirth. It is hoped that with the presentation of the results to prenatal caregivers plans can be made for the improvement of mothers' social support, and thus, a small step can be taken toward the improvement of mothers' health and their empowerment. + +## Materials And Methods + +The present descriptive correlational study was conducted on 270 nulliparous pregnant women who referred to health and treatment centers in Isfahan, Iran, for pregnancy care in 2016. The division of perceived social support in the population into two groups of adequate social support and lack of adequate social support required at least 135 subjects from each population and a total of 270 individuals to observe, with a 90% probability, a significant difference of 0.4 in mean score of fear of childbirth between the two populations at 5% significance level. The study environment consisted of the health and treatment centers of Isfahan. The centers were selected through cluster‑nonrandom and quota sampling methods after obtaining the approval of the Ethics Committee of Isfahan University of Medical Sciences, Isfahan. These centers were selected due to the possibility of easy access to study subjects. The inclusion criteria consisted of nulliparous pregnancy, Persian nationality, gestational age of 35 weeks and higher, lack of recognized psychological disorders, such as depression and mood disorder (these data were retrieved from the mothers' pregnancy care file), and low‑risk pregnancy such as lack of chronic diseases like heart disease, hypertension, and diabetes. In addition, the subjects were questioned regarding participation in childbirth preparation classes, use of computer training programs, and observation of natural childbirth videos; these variables are effective on the mean score of fear of childbirth and their impact was studied in regression analysis. Data were collected through self‑report method and if necessary through interviews using three questionnaires. The data collection tools were a demographic characteristics form, the Multidimensional Scale of Perceived Social Support (MSPSS),[20] and the Wijma Delivery Expectancy/ Experience Questionnaire (W‑DEQ).[21] The demographic characteristics form assessed the age and occupation of the mother and her spouse, education level, and economic status. The MSPSS is a 12‑item tool that evaluates perceived social support in the three dimensions of family (questions 3, 4, 8, 11), friends (questions 6, 7, 9, 12), and specific individual (questions 1, 2, 5, 10). This tool is scored based on seven‑point Likert scale (completely agree = 7; agree = 6; somewhat agree = 5; I have no opinion = 4; somewhat disagree = 3; disagree = 2; completely disagree = 1). The minimum and maximum obtained scores of the MSPSS are, respectively, 12 and 84, and higher scores represent higher social support.[20] The W‑DEQ consists of 16 items scored based on a four‑point Likert scale (lack of fear = 1; low = 2; average = 3; high = 4). Its total score ranges from 16 to 64 and higher scores represent greater fear.[21] +The validity and reliability of the MSPSS were reported for the first time by Zimet et al. + +[20] (Cronbach's alpha = 0.88). In the study by Gao[22] performed on 236 ¦ ¦ ¦ +68 nulliparous pregnant women in China, the validity of the MSPSS was approved through content validity and its reliability was obtained through the calculation of Cronbach's alpha ( = 0.85). The validity and reliability of the MSPSS have also been approved in Iran.[8] The validity and reliability of the W‑DEQ were obtained for the first time by Wijma et al.[21] (Cronbach's alpha = 0.89). In the study by Carlson on 406 nulliparous pregnant women in Sweden, the validity and reliability of the W‑DEQ were calculated through content validity and Cronbach's alpha (α = 0.92), respectively.[23] The validity and reliability of this questionnaire have also been approved in Iran.[24] +After obtaining permission and an introduction letter, the researcher referred to the intended treatment centers, presented the introduction letter to the authorities of the centers, and explained the goals of the study to the personnel of the centers. The researcher referred to the health and treatment centers every day during working hours[8‑13] and selected women who referred for regular pregnancy care and had the inclusion criteria through simple sampling method. The questionnaires were distributed among the subjects and their completion method was explained to them. Before the completion of the questionnaire, a suitable environment was prepared for the participants. The questionnaires were completed in one stage and through self‑report method and, if necessary, through interviews. After collecting the completed questionnaires, the subjects were thanked and catered for. The collected data were analyzed using descriptive (mean, median, prevalence distribution, and percentage) and analytical statistics (Pearson coefficient, and regression) at 95% confidence interval in Statistical Package for the Social Sciences (SPSS) software (version 16, SPSS Inc., Chicago, IL, USA). + +## Ethical Considerations + +Ethical principles were respected in all stages of the study. The objectives of the study were fully explained to the participants. Mothers who were willing to participate in the study were entered into the study after obtaining informed written consents from them. The subjects were completely free to leave the study at any moment of its execution. Moreover, they were assured of the confidentiality of their information. + +## Results + +In the present study, the mean age of the women was 26 years. The mean age of their spouses was 30 years. The education level of most of the participants was higher than diploma (41.30%) and university degree (41.35%). The majority of women were homemakers (85.92%) and most of their spouses were employed (87.71%). In addition, most of the subjects had an average economic status (93%). Their mean MSPSS score was 77.90%; the scores of support by family, friends, and spouse were 28.11, 22.42, and 27.73, respectively. The mean W‑DEQ score of the subjects was 36.81%. The results of single‑variable linear regression analysis showed that perceived social support, age, and spouse's age were predictors of fear of childbirth; 1 point increase in the perceived social support score, 1 year increase in the mother's age, and 1 year increase in the spouse's age resulted in the significant reduction of the mean score of fear of childbirth by, respectively, 0.23, 0.73, and 0.84 points. Education, mother's occupation, spouse's employment, lack of participation in pregnancy preparation classes, and lack of use of pregnancy education software did not cause a significant difference in the fear of childbirth. Nevertheless, household income significantly reduced the fear of childbirth, but observation of natural childbirth videos significantly increased the fear of childbirth [Table 1]. + +| Table 1: Predictors of fear of giving birth: Results of univariate linear regression | | | | +|-----------------------------------------------------------------------------------------|--------|--------------|---------| +| Predictors of fear of giving | β | 95% CI | p | +| birth Perceived social support | −0.18 | −0.39-−0.07 | 0.004 | +| Age | −0.73 | −1.31-0.16 | 0.013 | +| Age of husband | −0.84 | −1.47-0.21 | 0.009 | +| Education Illiterate | - | - | - | +| High school | 1.43 | −37.64-40.49 | 0.943 | +| College (Diploma) | 3.40 | −35.34-42.15 | 0.863 | +| University | −5.38 | −44.11-33.36 | 0.785 | +| Occupation Teaching related | - | - | - | +| Government staff | −9.58 | −28.92-9.75 | 0.330 | +| Workers | −9.17 | −51.52-33.19 | 0.670 | +| Housewife | 3.92 | −13.58-21.41 | 0.659 | +| Husband occupation Employed | - | - | - | +| Unemployed | 7.81 | −0.23-15.85 | 0.057 | +| Family income US$ 750 | 5.65 | −14.04-25.34 | 0.572 | +| Participating in giving birth education Yes | - | - | - | +| No | 3.49 | −2.10-9.09 | 0.220 | +| Using giving birth program Yes | - | - | - | +| No | 5.82 | −0.77-12.42 | 0.083 | +| Watching natural giving birth video Yes | - | - | - | +| No | 5.26 | 0.22-10.30 | 0.0.041 | + + ¦ ¦ ¦ 237 + +## Discussion + +The rate of social support in the present study was 77.90%, which was in agreement with the results of the studies by Sadeghe,[8] Jamshidimanesh,[25] and Gao.[22] In the study by Sadeghe, the mean perceived social support score was 62.71 and the scores of support by family, friends, and spouse were 22.53, 16.56, and 23.61, respectively.[8] In the study by Gao,[22] the mean perceived social support score was 68%. The similarity of the findings in the study by Gao to the present study may be due to the fact that China is also an eastern country with a traditional culture in which the family is the main unit of society. In eastern societies, women have an important and high status in the family and the majority of families with different cultures and statuses provide pregnant women with suitable support. In the present study, family support was the most important source of social support and being in the family environment was an unconditional membership in a support group.[25] Moreover, the support of the spouse is an important source which creates a sense of respect, self‑esteem, and safety during pregnancy.[8] +The mean score of fear of childbirth in the present study was 36.8%, which was in agreement with the results of the studies by Khorsande[3] and Nasiri.[26] In the study by Khorsande,[3] the mean score of fear of childbirth of nulliparous women was 26%. In the study by Nasiri,[26] +the mean score of fear of childbirth was 33.45. From these results it can be inferred that the W‑DEQ is a valid tool that is effective in determining women with fear of childbirth and planning to control their fear. If the pregnant mother imagines she cannot control the situation and believes herself to be unable to tolerate the pain, the resulting fear and anxiety will cause her to accept cesarean section without medical indication. In addition, she will not be immune to the negative effects of stress on health. There was a negative significant relationship between the perceived social support score of and fear of childbirth. In this respect, the study by Fisher et al. showed that social relations, unofficial support networks of pregnant women, and the strong support by midwives can strengthen women's belief that childbirth is a physiological and controllable process, and thus, result in psychological wellbeing and reduced fear of childbirth.[7] Furthermore, Pasha and Faramarze reported that one‑third of women experienced pregnancy as a stressful event and the most unpleasant experience of pregnancy was related to the third trimester. They also found that social support had a negative significant relationship with pregnancy stress; with increase in social support score, pregnancy stress decreased (p < 0.05).[11] Najafe et al.[27] observed a negative significant relationship between pregnancy preparation classes and fear of childbirth (p < 0.001). However, this relationship was not significant in the present study. The difference between Najafi et al. study and the present study may be due to the fact that the study by Najafi et al. was an interventional study or due to the difference in the quality of pregnancy preparation classes. Today, one of the causes of the increase in the prevalence of cesarean section in the world is the fear of childbirth. Furthermore, the statistics of cesarean section in Iran are higher than global standards.[5] Thus, pregnancy preparation classes centered on pregnant women with fear of childbirth can be an effective measure in the reduction of fear of childbirth and the promotion of natural childbirth. No similar study was found on the relationship between observation of natural childbirth videos and fear of childbirth. However, the results of the present study (conducted for the first time in Iran) can reinforce the belief that common natural childbirth videos have not been prepared with the aim of education and empowerment of women. It can also be stated that these videos can increase tension, and thus, inefficiency and fear. Through the conveyance of this important issue to pregnant mothers by health personnel and informing them, a small step can be taken toward decreasing the fear of childbirth. The results of the study by Hamzekhane[28] +suggest that educational software can be more effective in increasing the awareness and knowledge of individuals compared to traditional education methods. Nevertheless, they did not decrease the fear of childbirth in the present study. The findings of the present research were studied along with its limitations such as cultural factors, beliefs, and traditions which could impact the responses of the subjects and could not be controlled by the researcher. + +## Conclusion + +The results of the present study showed that demographic factors determine the receiving of social support, because these factors form the structure and determine the extent of individuals' social network. It has been proven that lower income, education, economic status, and occupational position are accompanied by a smaller and less diverse social network. Thus, the rate of effective support in pregnant mothers can be predicted through the evaluation of the demographic factors of the support process from the beginning of pregnancy until after childbirth. Furthermore, through informing the treatment team of these results they can be asked to assess clients with characteristics such as low age of mother and spouse, low education, and low income in terms of perceived social support dimensions throughout their pregnancy. The continuous assessment of this support trend from the beginning of pregnancy until after childbirth by the treatment team resulted in the empowerment of women and reduced fear of childbirth which will, in turn, result in increased psychological wellbeing among mothers and reduced prevalence of cesarean section. + +238 ¦ ¦ ¦ +Acknowledgement The article was derived from the Master's thesis with project number 395096, Isfahan University of Medical Sciences, Isfahan, Iran. + +Financial support and sponsorship Isfahan University of Medical Sciences. Conflicts of interest Nothing to declare. + +## References + +1. Nazare S, Salare P, Mazlom R, Mehram F. The comparative study of social support postpartum mothers multiparity in the first and second month after delivery. Public Health Research Khorasa 2014;8:1‑9. + +2. Pournamdarian S, Birashk B, Asgharnejad Farid A. The clarification of contribution of meta‑cognitive beliefs in explaining the symptoms of depression, anxiety and stress in nurses. Knowledge & Research in Applied Psychology. 2012;13:86‑94. + +3. Khorsande M, Vakilian K, Masole M. The reasons natural childbirth. J Fasa Univ of Medi Sci 2014;4:161‑6. + +4. Zahede M, Asemane O, Mahmodyan H. Given the role of medical in assistants in reducing caesarean sections; designed and evaluated the psychometric questionnaire to measure attitudes of residents towards elective caesarean sections and vaginal births. Iranian Journal of Medical Ethics and History 2015;8:59‑71. + +5. Word Report on Knowledge for Better Health, Strengthening Health Systems, World Health Organization 2004; Available form: URL: www.who.int/rpc/meeting/publ/en/. [Last accessed on 2009 Aug 29]. + +6. Taheri Z, Khorsande M, Amire M, Hasanzade A. Investigate the causes of childbirth in pregnant woman in Shahrekord. Univ of Medi Scirafsanjan 2015;14:345‑50. + +7. Fisher C, Hauckb Y, Fenwickb J. How social context impacts on women's fears of childbirth. Soc Sci Med 2006;63:64‑75. + +8. Sadeghe H, Mosave S, Jahde F, et al. The relationship between perceived social support with the consent of the pregnant women referred to health center martyr Akbarabadi Tehran. Journal of Preventive Care in Nursing and Midwifery 2013;4:54‑64. + +9. Gulacti F. The effect of perceived social support on subjective well‑being. Procedia‑Social and Behavioral Sciences 2010;2:3844‑9. + +10. Chadwick KA, Collins PA. Examining the relationship between social support availability urban center size, and self‑perceived mental health of recent immigrants to Canada: A mixed‑methods analysis. Soc Sci Med 2015;128:220‑30. + +11. Pasha H, Faramarze M. The role of social support in predication of stress in pregnancy. Journal Babol Univ of Medi Sci 2015;17:52‑60. + +12. Hildingsson I, Tingvall M, Rubertsson C. Partner support in the childbearing period - A follow up study. Women Birth 2008;21:141‑8. + +13. Theodore RF, Thompson JM, Waldie KE, Bercroft DM, +Robinson E, Wild CJ, et al. Determinants of cognitive ability at 7 years: A longitudinal case control study of children born small‑for‑gestational age at term. Eur J Pediatr 2009;168:1217‑24. + +14. Spoozak L, Gotman N, Smith MV, Belanger K, Yonkers KA. + +Evaluation of a social support measure that may indicate risk of depression during pregnancy. J Affect Disord 2009;114:216‑23. + +15. Nore R, Kareme N, Mohamade M. The relationship Between Prenatal depression with Social Support and satisfaction. Journal Univ Tehran Iran 2013;1:10‑15. + +16. Ma J, Huang YQ, Yao C, Ma SQ, Meng T, Ma M, et al. Parental Health and Social Support in the First Trimester of Pregnancy and the Risk of oral Cleft. Plast Reconstr Surg 2015;135:212‑8. + +17. Garcia MF, Ramirez M, Jariego MI. Social support and locus of control as predictors of psychological well‑being in Moroccan and Peruvian immigrant women in Spain. International Journal of Intercultural Relations 2002;26:287‑310. + +18. Jirka J, Schuett SH, Foxall MJ. Loneliness and Social Support in infertile couples. J Obstet Gynecol Neonatal Nurs 1996;25:55‑60. + +19. Logsdon M. Social and professional support for pregnancy and parenting women. MCN Am J Matern Child Nurs 2003;28:371‑6. + +20. Zimet GD, Dahlem NW, Zimet SG, Farley GK. The multidimensional scale of perceived social support. Journal of Personality Assessment 1998;52:30‑41. + +21. Wijma K, Wijma B, Zar M. A new questionnaire for the measurement of fear of childbirth (Psychometric aspects of the W‑DEQ). J Psychosom Obstet Gynecol 1998;19:84‑97. + +22. Gao L, Sun K, Chan SW. Social support and parenting self‑efficacy among Chinese women in the perinatal period. Midwifery 2014;30;532‑8. + +23. Carlsson M, Ziegert K, Nissen E. The relationship between childbirth self‑efficacy and aspects of well‑being, birth interventions and birth outcomes. Midwifery 2015;31:1000‑7. + +24. Khorsandi M, Ghofranipour F, Heydarnia A, Faghih Zadeh S, +Vafaei M, Rousta F, et al. The effect of childbirth preparation classes on childbirth fear and normal delivery among primiparous women. Arak Medical University Journal 2008;11:9. + +25. Jamshidimanesh M, Astaraki L, Moghadam ZM, Taghizade Z, +Haghane H. Maternal‑fetal attachment and its associated factor. Hayat 2012;18:33‑45. + +26. Nasiri F, Sharifi SH. The relationship between personality type and childbirth in pregnant women. IJOGI 2013;16:18‑25. + +27. Najafe F, Abozar K, Jafarzade F, Rahnama P, Gholame B. + +Corporate communications at antenatal classes with fear and type of delivery. Tehran Nursing and Midwifery School (Hayat) 2015;21:30‑40. + +28. Hamzekhane M, Hamidzade A, Vasegh F. The effect of computerized educational program on self‑efficacy of pregnant women with postpartum adjustment. Journal of Knowledge and Health 2013;9:13‑20. \ No newline at end of file diff --git a/medical/md/PMC6030411.md b/medical/md/PMC6030411.md new file mode 100644 index 0000000000000000000000000000000000000000..fe6e27bde3fc9e59163f90ac1f8060febcd8fbd4 --- /dev/null +++ b/medical/md/PMC6030411.md @@ -0,0 +1,39 @@ +# Radiation Nephritis: 99Mtc Hydroxydiphosphonate Bone Scan, 99Mtc Dimercaptosuccinic Acid Renal Scan, And 18F-Fdg Pet/Ct Findings + +Hye Lim Park1, Ie Ryung Yoo1, Yeon Sil Kim2, Jin-Hyoung Kang3, and Ji Eun Lee3 A 20-year-old male had Ewing's sarcoma of the right paraspinal region +(T12–L2 vertebra level) and was treated with chemotherapy and radiation therapy (RT). 99mTc hydroxydiphosphonate bone scan was performed 4 months after the end of treatment. + +Intense uptake was noted in both kidneys (Fig. 1). On the fluorodeoxyglucose +(FDG) positron emission tomography/ computed tomography (PET/CT) for response evaluation, mild uptake was also seen in both kidneys (Fig. 2A and 2B). On 99mTc dimercaptosuccinic acid +(DMSA) scan, photon defects were noted in both kidneys (Fig. 2C). When we reviewed the RT plan, the medial aspect of both kidneys was included in the radiation field (Fig. 2D and 2E). On contrast-enhanced CT, low-densities were noted in both kidneys (Fig. 2F). + +We thus diagnosed radiation nephritis (RN). On 6-month follow-up bone scan, the previously noted intensity on both kidneys was decreased. + +RN is commonly seen following RT +of intra-abdominal tumors or bony metastasis of spine. The histologic findings are vascular sclerosis with tubular ischemic change. Tubular dysfunction and ischemic change explain the photon defect on DMSA scan and the delayed enhancement and retention of contrast on CT. Delayed excretion of radiopharmaceuticals could + +![0_image_0.png](0_image_0.png) cause increased uptake on bone scan. + +CT and bone scan findings were previously reported, but PET/CT findings were reported in only one case. + +Although the exact mechanism of increased activity on PET/CT remains uncertain, delayed tracer excretion via parenchymal injury could be a major cause. Radiation-induced inflammation can also cause. However, the lack + +IMAGE OF INTEREST +Korean J Intern Med 2018;33:837-838 https://doi.org/10.3904/kjim.2017.145 1Division of Nuclear Medicine, Department of Radiology, 2Department of Radiation Oncology, 3Division of Medical Oncology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea Received: March **21, 2017** Revised : May **14, 2017** Accepted: May **14, 2017** +Figure 1. On 99mTc hydroxydiphosphonate bone scan 4 months after radiotherapy for evaluation of metastasis, intense linear band-like uptake was noted in the medial aspect of both kidneys (arrows). + +Correspondence to Ie Ryung Yoo, M.D. Tel: +82-2-2258-1552 Fax: +82-2-2258-1575 E-mail: iryoo@catholic.ac.kr Copyright © 2018 The Korean Association of Internal Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. + +pISSN 1226-3303 eISSN 2005-6648 http://www.kjim.org + +![1_image_0.png](1_image_0.png) + +of evidence of inflammation in other tissues included in the radiation field and the decreased blood flow to the kidneys on enhanced CT indicate that the inflammation was a minor contributor. + +Focal renal activity on PET/CT could be misinterpreted as malignancy. Differential diagnostic criteria for distinguishing RN from malignancy include a RT history, a linear uptake pattern, and a sharp margin of uptake. + +Lesion locations were the medial or upper portion of the kidney. Clinicians need to exclude RN when linear increases in renal activity are incidentally detected on FDG PET/CT. + +## Conflict Of Interest + +No potential conflict of interest relevant to this article was reported. \ No newline at end of file diff --git a/medical/md/PMC6113417.md b/medical/md/PMC6113417.md new file mode 100644 index 0000000000000000000000000000000000000000..454c6187269a9b65207e71cf36c1ba5b8d7907ec --- /dev/null +++ b/medical/md/PMC6113417.md @@ -0,0 +1,92 @@ +# Metabolic Reprogramming After Left Ventricular Assist Device + +Remodeling Without Recovery of Cardiac Energetics* J. Eduardo Rame, MD, MPHIL, +a Emma J. Birks, MD, PHDb Despite impressive changes at the organ, tissue and cellular level in myocardial reverse remodeling with left ventricular assist device support, reprogramming the presumed "endstage" human heart toward the nonfailing phenotype has not been convincingly demonstrated (1–5). Many have questioned the possibility of achieving sustained myocardial "recovery" using our current technology, leading to a call for further investigation by a recently gathered National Heart, Lung, and Blood Institute task force (6). Metabolic adaptations have been reported in the calorie-restricted, exercising, ischemic, and failing myocardium—and recently, the lipidomic signature of the end-stage failing, nondiabetic human heart has revealed rarefied lipid stores and evidence for a newly discovered shift to ketones as an alternative energy fuel (7–10). + +## See Page 432 + +Enter the highly anticipated work by Diakos et al. + +(11) to fill in the translational knowledge gaps between myocardial recovery and metabolic adaptation after left ventricular assist device (LVAD) support. The authors performed targeted and untargeted quantitation of metabolites with mass spectroscopy to characterize the metabolic phenotype of myocardial reverse remodeling with mechanical unloading in nonfailing donor hearts, as well as paired myocardial samples obtained before LVAD and before transplantation after LVAD support. Their analyses revealed the following signatures: 1) increased glycolytic flux without a commensurate increase in glucose oxidation resulting in increased myocardial pyruvate and shuttled lactate levels; 2) a persistent disruption in the tricarboxylic acid (TCA) cycle characterized by splitting between first-span and secondspan levels of intermediates; 3) a deficit in mitochondrial oxidative capacity with decreased State III +and State IV respiration in the chronically failing heart not improved with mechanical unloading; and 4) decreased density and altered structure of mitochondria in failing hearts pre- and post-mechanical unloading. The authors (11) reason that the decoupling of the utilization of glucose in the failing myocardium—glycolysis from oxidation—is linked with deficits in mitochondrial function, both in the cycling of intermediary metabolites and oxidative phosphorylation. In order to interpret the significance of these findings, we first need to step back and understand the current work in the context of recent advances in our understanding of the metabolic adaptation of the failing heart. Second, we need to ask exactly what are the mechanisms behind this remodeling in the adaptive metabolism of the failing heart that fall well short of recovering the cardiac energetics with mechanical assist device support? + +Muscle, both cardiac and skeletal, has a high degree of plasticity to the changing conditions challenging its environment. In response to a change in loading conditions and loss of contracting mass and function, metabolic adaptations have been demonstrated in the failing mammalian heart including decreased utilization of fatty acids with a shift to the more oxygen-sparing energetic substrate of glucose +(12–14). The work by Diakos et al. (11) convincingly demonstrates decreased levels of glucose-1phosphate and glucose-6-phosphate in the endstage failing myocardium, which is indicative of a decreased utilization of glucose, and is consistent with decreased glycolysis in advanced heart failure. + +The importance of this finding, coupled with the demonstration that with mechanical unloading there is an increase in the levels of glycolytic metabolites is extremely important, insofar as it shows that there is not a uniform shift to increased glucose metabolism across the broad spectrum of heart failure. Instead, these data support the dynamic model of metabolic adaptation across the different stages of heart failure, with specific metabolic signatures apparent in responders versus non-responders. Indeed, although not large enough for analysis, the subgroup of patients with significant myocardial functional improvement after mechanical unloading did show greater mitochondrial functional and structural improvement than the "nonresponders." A study investigating the metabolic phenotype of nonselective adrenergic receptor blockade in chronic human heart failure with 18-fluoro-deoxyglucose positron emission tomography demonstrated an increased uptake of glucose in the patients with the most significant degree of myocardial reverse remodeling and improved left ventricular ejection fraction (15). Thus, an improved myocardial phenotype may shift more towards increased reliance on glucose metabolism. + +The work by Diakos et al. (11) extends the observation of myocardial plasticity in metabolic adaptation to the end-stage failing heart transitioning to mechanical support and provides evidence of increased glucose utilization through glycolysis but without the capacity to couple this with the oxidation of pyruvate and increased oxidative metabolism. + +When considering the impact of these results on future investigation, an important question concerns the mechanism of how glycolytic flux becomes uncoupled from glucose oxidation. This uncoupling results in a bottleneck increasing the myocardial levels of pyruvate and lactate above the nonfailing levels after a period of mechanical unloading. Could the increase in glycolysis be the result of improving myocardial insulin resistance that has been reported after LVAD therapy (16,17), with the bottleneck at pyruvate representing a failure to release the brake on pyruvate dehydrogenase (PDH) caused by the excess circulating fatty acids? In essence, the effects of the Randle Cycle (18)—inhibition of glucose oxidation by fatty acid uptake and utilization—may persist until the complete reversal of the heart failure syndrome with subsequent normalization of the peripheral metabolic milieu. Alternatively, is the increased concentration of glycolytic metabolites the result of a "shift back to glucose" and away from ketone metabolism (7,8), as peripheral insulin signaling improves, effectively decreasing or halting hepatic ketogenesis? If so, there may be a novel link switching cardiac substrate utilization on/off under failing conditions which accompanies the metabolic reprogramming to an earlier stage of heart failure marked by a preferential metabolism of glucose. + +Diakos et al. (11) have correctly identified abnormalities in mitochondrial structure and function in the failing heart that persist after a period of mechanical unloading, invoking this broader mechanism as the reason for the impaired oxidation of glucose and the decoupling from glycolysis. They present very convincing data that the TCA cycle, which was recently demonstrated to be disrupted in advanced heart failure with a decreased ratio of succinyl-CoA +(an early second-span intermediate) to acetyl-CoA (the proximal metabolic point of entry), remains abnormal despite effective mechanical unloading with LVAD therapy (8). A proposal is made by the authors that mechanisms involving anaplerosis and cataplerosis interacting with amino acid metabolism can explain the changes in the intermediate metabolites of the Krebs cycle. However, what remains unexplained is the difference in post-LVAD levels between first-span (remain decreased) and secondspan (restored to non-failing levels) intermediates. + +Precisely why are the metabolites after succinyl-CoA +restored as the end-stage failing heart remodels with mechanical unloading? This may again represent a "switch away from ketones" because succinyl-CoA +is no longer shuttled from the TCA cycle to serve as the CoA donor in the rate-limiting step of ketone oxidation through the enzyme SCOT (succinyl-CoA:3oxoacid CoA transferase) that was demonstrated to be significantly up-regulated in the end-stage failing human heart (8) (Figure 1). Thus, the active oxidation of ketones results in a depletion of succinyl-CoA +while reversing this state through a decrease or halting of ketogenesis restores the myocardial pool of succinyl-CoA and second-span TCA cycle intermediates. Given the importance of succinylation and acetylation in the transcriptional and enzymatic control of important metabolic pathways, the myocardial concentration of succinyl-CoA may play a key role in the regulation of metabolic adaptations across the various stages of heart failure. + +What are the therapeutic implications of this work in the field of myocardial recovery with LVAD therapy? It should be noted that the present study by Diakos et al. (11) focused on the effects of mechanical unloading rather than the metabolic signature of + +![2_image_0.png](2_image_0.png) + +recovery because these were paired samples from patients bridged to transplantation. We are approaching the final follow-up phase of the prospective, multicenter study of myocardial reverse remodeling with LVAD and standard optimized heart failure therapies (ReSTAGE HF [Remission from Stage D Heart Failure]). The ReSTAGE-HF study was designed to evaluate the rate of recovery when centers optimize pump speed for unloading and use optimal heart failure therapy. By including intermittent testing of underlying function at set time points in a common protocol the rate of recovery under "best standard" conditions will be determined. Once this has been established the next phase will be to consider adjunctive therapies, that is, drugs and strategies that can be added, to firstly further promote reverse remodeling and enhance the rate and degree of myocardial reverse remodeling, and secondly and importantly, to enhance its durability. Previously, the Harefield, United Kingdom, group successfully added clenbuterol as an adjunctive second stage of the therapy, which was done to enhance the durability of recovery (1). Adding a period of cardiac reloading before explantation is also likely to be beneficial, and the work by these authors suggests this might have beneficial effects on mitochondrial structure and function. Like exercise, which has been shown to improve mitochondrial function, cardiac reloading may prove to be an important addition to recovery protocols. The current study also suggests that novel drugs targeting mitochondrial function and enhancement of metabolic adaptation might serve as important additions to recovery protocols to improve the myocardial function and durability of the therapeutic approach. Starting with this important contribution by Diakos et al. (11), further understanding of the metabolic phenotypes resulting from the reversal of heart failure is likely to enhance, not only our rate of myocardial recovery, but the durability after explantation that may be the real target in our efforts to reprogram the failing human heart. + +REPRINT REQUESTS AND CORRESPONDENCE: Dr. J. Eduardo Rame, Cardiovascular Institute, University of Pennsylvania, 11th Floor South Tower, 3400 Civic Center Boulevard, Philadelphia, Pennsylvania 19104. + +E-mail: Eduardo.rame@uphs.upenn.edu. + +## References + +1. Birks EJ, Tansley PD, Hardy J, et al. Left ventricular assist device and drug therapy for the reversal of heart failure. N Engl J Med 2006;355: +1873–84. + +2. Dipla K, Mattiello JA, Jeevanandam V, Houser SR, +Margulies KB. Myocyte recovery after mechanical circulatory support in humans with end-stage heart failure. Circulation 1998;97:2316–22. + +3. Drakos SG, Wever-Pinzon O, Selzman CH, et al. Magnitude and time course of changes induced by continuous-flow left ventricular assist device unloading in chronic heart failure: insights into cardiac recovery. J Am Coll Cardiol 2013;61: +1985–94. + +4. Klotz S, Barbone A, Reiken S, et al. Left ventricular assist device support normalizes left and right ventricular beta-adrenergic pathway properties. J Am Coll Cardiol 2005;45:668–76. 5. Klotz S, Foronjy RF, Dickstein ML, et al. Mechanical unloading during left ventricular assist device support increases left ventricular collagen cross-linking and myocardial stiffness. Circulation 2005;112:364–74. + +6. NHLBI Working Group on Advancing the Science of Myocardial Recovery with Mechanical Circulatory Support. 2016. Available at: http:// www.nhlbi.nih.gov/research/reports/nhlbi-working -group-advancing-science-myocardial-recovery-mech anical-circulatory-support. Accessed September 18, 2016. + +7. Aubert G, Martin OJ, Horton JL, et al. The failing heart relies on ketone bodies as a fuel. Circulation 2016;133:698–705. + +8. Bedi KC Jr., Snyder NW, Brandimarto J, et al. Evidence for intramyocardial disruption of lipid metabolism and increased myocardial ketone utilization in advanced human heart failure. Circulation 2016;133:706–16. + +9. Kolwicz SC Jr., Airhart S, Tian R. Ketones step to the plate: a game changer for metabolic remodeling in heart failure? Circulation 2016;133: +689–91. + +10. Taegtmeyer H. Failing heart and starving brain: ketone bodies to the rescue. Circulation 2016;134:265–6. + +11. Diakos NA, Navankasattusas S, Abel ED, et al. Evidence of glycolysis up-regulation and pyruvate mitochondrial oxidation mismatch during mechanical unloading of the failing human heart: +implications for cardiac reloading and conditioning. J Am Coll Cardiol Basic Trans Science 2016;1: +432–44. + +12. Davila-Roman VG, Vedala G, Herrero P, et al. + +Altered myocardial fatty acid and glucose metabolism in idiopathic dilated cardiomyopathy. J Am Coll Cardiol 2002;40:271–7. + +13. Taylor M, Wallhaus TR, Degrado TR, et al. An evaluation ofmyocardial fatty acid and glucose uptake using PET with [18F]fluoro-6-thia-heptadecanoic acid and [18F]FDG in patients with congestive heart failure. J Nucl Med 2001;42:55–62. + +14. Tenenbaum A, Fisman EZ. Impaired glucose metabolism in patients with heart failure: +pathophysiology and possible treatment strategies. Am J Cardiovasc Drugs 2004;4: +269–80. + +15. Wallhaus TR, Taylor M, DeGrado TR, et al. Myocardial free fatty acid and glucose use after carvedilol treatment in patients with congestive heart failure. Circulation 2001;103: +2441–6. + +16. Chokshi A, Drosatos K, Cheema FH, et al. Ventricular assist device implantation corrects myocardial lipotoxicity, reverses insulin resistance, and normalizes cardiac metabolism in patients with advanced heart failure. Circulation 2012;125: +2844–53. + +17. Rame JE. Chronic heart failure: a reversible metabolic syndrome? Circulation 2012;125: +2809–11. + +18. Randle PJ, Garland PB, Hales CN, Newsholme EA. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963;1: +785–9. + +## Key Words Cardiac Conditioning, + +cardiac metabolism, left ventricular assist device \ No newline at end of file diff --git a/medical/md/PMC6256180.md b/medical/md/PMC6256180.md new file mode 100644 index 0000000000000000000000000000000000000000..f7bae41c71229be1fba6f27ca093f56782d53862 --- /dev/null +++ b/medical/md/PMC6256180.md @@ -0,0 +1,160 @@ +# Inhibition Of Osteoblast Differentiation By Ritonavir + +YOSHITAKA WAKABAYASHI1, YUSUKE YOSHINO2, KAZUNORI SEO2, ICHIRO KOGA2, TAKATOSHI KITAZAWA2 and YASUO OTA2 1Department of Infectious Diseases, The University of Tokyo Hospital, Tokyo 113‑8655; 2Department of Internal Medicine, Teikyo University, Tokyo 173‑8606, Japan Received March 30, 2018; Accepted September 26, 2018 + +## Doi: 10.3892/Br.2018.1154 + +Abstract. Osteoporosis is one of the chronic complications seen in human immunodeficiency virus (HIV)‑infected patients, and affects patients at high prevalence. The causes of osteoporosis in HIV‑infected patients are multiple, and include chronic HIV +infection, living habits such as smoking and alcohol consumption, and antiretroviral drug use. Among antiretroviral drugs, protease inhibitors have been reported to be associated with osteoporosis. However, it remains to be determined how anti‑HIV drugs affect osteoblast differentiation. In the current study, MC3T3‑E1 cells, a mouse osteoblastic cell line, were cultured in osteoblast differentiation medium with or without different protease inhibitors (ritonavir, lopinavir, darunavir or atazanavir), and alkaline phosphatase (ALP) activity and the expression of Runt‑related transcription factor 2 (Runx2) were analyzed. The ALP activity in MC3T3‑E1 cells cultured with ritonavir was significantly reduced compared with that in cells in only osteoblast inducer reagent, indicating that ritonavir inhibited osteoblast differentiation. Meanwhile, ALP activity was not reduced in cells cultured with any of the other inhibitors. In addition, ritonavir inhibited the expression of Runx2, a key regulator of osteoblast differentiation, in the early period of osteoblast differentiation. To the best of our knowledge, this is the first study to demonstrate that ritonavir inhibits osteoblast differentiation in *vitro*. The present findings may explain the mechanism of osteopenia induced by combination antiretroviral therapy involving protease inhibitors. + +## Introduction + +The progress of combination antiretroviral therapy (cART) has led to increased life expectancy of patients infected with human immunodeficiency virus (HIV)(1). However, as life expectancy has increased, chronic complications have become key challenges (2). Osteoporosis is among the chronic complications seen in HIV‑infected patients; the prevalence of osteoporosis is reportedly three times higher among HIV‑infected patients than non‑HIV‑infected patients (3). The reported prevalence rate of osteopenia in HIV‑infected cohorts ranges from 22 to 71%, and that of osteoporosis from 3 to 33% (4). The causes of osteoporosis in HIV‑infected patients are multiple, and include chronic HIV infection, living habits such as smoking and alcohol consumption, and antiretroviral drug use (5). Among antiretroviral drugs, protease inhibitors have been reported to be associated with osteoporosis, and antiretroviral regimens containing protease inhibitors can accelerate osteopenia and osteoporosis (6). + +Osteoporosis is caused by an imbalance of bone resorption and formation (7). Osteoclasts have a role in bone forma-tion, and osteoblasts that are derived from mesenchymal stem cells are responsible for bone formation (8). Osteoblast differentiation is regulated by transcription factors including Runt‑related transcription factor 2 (Runx2) (9). Runx2 is a positive regulator of genes associated with bone matrix proteins including collagen type I α 1/2 chain (9), and triggers the expression of major bone matrix genes during the early stages of osteoblast differentiation (9). As such, Runx2 is considered to serve a central role in skeletal development and be associated with osteoporosis (9). However, it remains to be determined specifically how anti‑HIV drugs affect osteoblast differentiation. Thus, in the present study, the influence of anti‑HIV drugs on osteoblast differentiation was examined in *vitro*. + +## Materials And Methods + +Cell line and culture. The clonal mouse osteoblastic cell line, MC3T3‑E1 subclone 14, was purchased from American Type Culture Collection (Manassas, VA, USA). The MC3T3‑E1 cells were cultured at 37˚C in 5% CO2 in Dulbecco's modi-fied Eagle's medium (DMEM; Sigma‑Aldrich; Merck KGaA, +Darmstadt, Germany) with 10% fetal bovine serum (Hyclone; GE Healthcare Life Science, Logan, UT, USA), 100 IU/ml penicillin and 100 µg/ml streptomycin. To induce osteoblast differentiation, MC3T3‑E1 cells were cultured at 37˚C in 5% CO2 in DMEM with osteoblast inducer reagent containing 1% L‑ascorbic acid, 2% β‑glycerophosphate and 0.2% hydrocortisone (Takara Bio, Inc., Otsu, Japan). The medium was changed every other day. + +Anti‑HIV drugs. The HIV protease inhibitors ritonavir, darunavir, atazanavir and lopinavir were purchased from Toronto Research Chemicals, Inc. (Toronto, ON, Canada). These drugs were prepared as stock solutions in methanol. All prepared anti‑HIV drugs were mixed with osteoblast differentiation medium to adjust to the maximum serum concentration +(Cmax) that these drugs achieve when they are administered at treatment doses in adult HIV patients. Cmax values of protease inhibitors were based on those on the University of Liverpool website, www.hiv‑druginteraction.org. The Cmax values were: Ritonavir, 11.20 µg/ml; lopinavir, 9.60 µg/ml; darunavir, 6.50 µg/ml; and atazanavir 3.15 µg/ml. Alkaline phosphatase (ALP) activity. MC3T3‑E1 cells were seeded at a density of 1x104 cells/well in a 96‑well microplate in normal culture medium. To induce differentiation, the medium was replaced by DMEM with osteoblast inducer reagent after 1 day in the presence or absence of each protease inhibitor, respectively. The cells were incubated for 7 and 9 days in four types of medium: i) DMEM; ii) DMEM with osteoblast inducer reagent; iii) DMEM with osteoblast inducer reagent and an anti‑HIV drug; and iv) DMEM with vehicle (methanol). For subsequent treatments with ritonavir, MC3T3‑E1 cells were treated with various concentrations (0.1, 0.5, 1.0, 5.0 and 10.0 µg/ml ritonavir) and for various durations (10.0 µg/ml ritonavir for 9, 11 and 14 days). All cells were cultured at 37˚C in a 5% CO2 atmosphere. ALP activity was evaluated using a TRACP & ALP double‑staining kit (Takara Bio, Inc.) according to the manufacturer's protocol. Briefly, the plates were incubated with kit reagents at 37˚C for 15 min, absorbance was measured at 405 nm, and ALP activity was calculated from a standard value (bone ALP; Takara Bio, Inc.). The total protein concentration of each cell was determined by a DC™ Protein Assay (Bio‑Rad Laboratories, Inc., Hercules, CA, USA), and the activity data were normalized to the total protein concentration. Alizarin Red staining. MC3T3‑E1 cells were seeded at a density of 4x104 cells/well in a 24‑well plate. The cells were incubated with 10.0 µg/ml ritonavir or control mediums as above. Mineralization of MC3T3‑E1 cells was confirmed by using an Alizarin Red S staining kit (Cosmo Bio, Co., Ltd., Tokyo, Japan) after 7, 14, 21 and 28 days of culture. Then, cells were washed three times with phosphate‑buffered saline, with 500 µl of 100% methanol added to each well, and incubated at 4˚C for 20 min. Alizarin Red solution was added to each well and incubated at room temperature for 5 min. Each well was then washed again three times and directly observed. Total RNA extraction and cDNA synthesis. MC3T3‑E1 cells seeded at 1x105 cells/well in a 12‑well plate were incubated with 10.0 µg/ml ritonavir or control mediums as above for 3, 5 and 7 days. Total RNA was extracted from the MC3T3‑E1 cells by adding 350 µl Tripure (Roche, Basel, Switzerland) to each well. RNA was isolated according to the manufacturer's instructions. Total RNA was reverse‑transcribed into cDNA +by using ReverTra Ace® qPCR RT Master Mix (Toyobo Life Science, Osaka, Japan) according to the manufacturer's instructions. + +Quantitative polymerase chain reaction (qPCR). Runx2 gene expression in MC3T3‑E1 cells was examined by qPCR. β‑actin was used as an internal control gene. The qPCR was performed with a Roche LightCycler 480 system using a LightCycler 480 Probe Master kit (Roche Diagnostics, Basel, Switzerland). Reactions were performed in a final total volume of 20 µl containing 1x LightCycler 480 Probe Master mix, 0.1 µM of each forward and reverse primer, 3.8 µl distilled H2O and 5 µl of the cDNA template. Specific primers for mouse Runx2 and β‑actin were designed using the Universal ProbeLibrary (Roche Diagnostics). The primers used in the current study were as follows: For Runx2, forward, 5'‑cgtgtcagcaaagcttctttt‑3' and reverse, 5'‑ggctcacgtcgctcatct‑3'; and for β‑actin, forward, 5'‑tgacaggatgcagaaggaga‑3' and reverse, 5'‑cgctcaggaggagcaatg‑3'. The amplification conditions were as follows: Denaturation at 95˚C for 5 min, 45 cycles of amplification (95˚C for 10 sec, 60˚C for 30 sec and 72˚C for 1 sec), and cooling at 50˚C for 10 sec. Relative quantification of target gene expression was determined using the E‑Method (Efficiency Method) from the LightCycler 480 software (10). Lactate dehydrogenase (LDH) activity assay. LDH activity was measured to determine MC3T3‑E1 cell viability following treatment with ritonavir. MC3T3‑E1 cells were plated in 12‑well plates (1x105cells/well) and incubated in DMEM +with osteoblast inducer reagent in the presence or absence of ritonavir (10 µg/ml). On day 8, culture medium was replenished and cells were cultured again for 24 h. Cell medium and lysate extracted with radioimmunoprecipitation assay buffer (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were collected on day 9, and cell viability was evaluated using Cytotoxicity detection KitPLUS (Roche Diagnostics) according to the manufacturer's protocol. Cells in DMEM only were defined as a control. The absorbance was measured at 490 nm, and cell viability was calculated using the following formula: Cell viability (%) = {1‑[(absorbance of medium ‑ absorbance of control medium)/(absorbance of lysate ‑ absorbance of control lysate)]} x100. Statistical analysis. Significant differences among samples were determined by one‑way analysis of variance with post hoc Tukey's honest significant difference test, with P<0.05 considered to indicate statistical significance. At least three samples were tested in three independent experiments. All results are presented as means ± standard error of the mean. The data analysis was performed using JMP software version 12.2 (SAS Institute, Inc., Cary, NC, USA). + +## Results + +Osteoblast differentiation is inhibited by ritonavir. As documented, osteoblasts are differentiated by a medium that contains L‑ascorbic acid, β‑glycerophosphate and steroid (11). Since ALP activity is enhanced during osteoblast differentiation in *vitro*, ALP may be used as an osteogenic differentiation marker (12). First, the present study observed that ALP activity was enhanced in MC3T3‑E1 cells at 7 days after the addition of DMEM with osteoblast inducer reagent compared with in cells cultured in DMEM only (P<0.05; data not shown). Subsequently, the effect of protease inhibitors on + +![2_image_0.png](2_image_0.png) + +osteoblast differentiation was examined. MC3T3‑E1 cells were cultured in osteogenic differentiation medium in the presence or absence of the protease inhibitors ritonavir, lopinavir, darunavir and atazanavir 3.15 µg/ml at their Cmax for 9 days, and then ALP activity was examined. The ALP activity of MC3T3‑E1 cells cultured with ritonavir was significantly reduced compared with that of the control cells in osteoblast induction medium alone (P<0.05). However, ALP activity was not reduced in cells cultured with the other anti‑HIV drugs (Fig. 1A). These results indicated that the protease inhibitor ritonavir inhibited osteoblast differentiation. + +Next, the relationship between ritonavir concentration and osteoblast differentiation was examined. MC3T3‑E1 cells were treated with various concentrations of ritonavir (0.1, 0.5, 1.0, 5.0 and 10.0 µg/ml). ALP activity was significantly reduced in MC3T3‑E1 cells cultured with 5.0 or 10.0 µg/ml ritonavir compared with the controls in osteoblast induction medium alone (P<0.05); however, lower concentrations of ritonavir did not inhibit ALP activity (Fig. 1B). + +The time course of ALP activity in MC3T3‑E1 cells cultured with ritonavir was also examined. MC3T3‑E1 cells were cultured in osteogenic differentiation medium with or without 10.0 µg/ml ritonavir for 9, 11 and 14 days. ALP activity was significantly reduced following ritonavir treatment at each time point (P<0.05; Fig. 1C). Effect of ritonavir on the expression of Runx2 in MC3T3‑E1 cells. RUNX2 is the key gene responsible for the differentiation of human mesenchymal cells into osteoblasts; it upregulates bone matrix proteins including ALP in the early phase of osteoblast differentiation (13). MC3T3‑1 cells were cultured in osteogenic differentiation medium with or without 10.0 µg/ml ritonavir for 3, 5 and 7 days, and the mRNA expression of Runx2 was measured by reverse transcription‑qPCR. On days 3 and 5, the mRNA levels of Runx2 were significantly reduced in differentiating cells cultured with ritonavir compared with in the differentiating controls (P<0.05); however, no significant differences were observed on day 7 (Fig. 2). These results indicated that ritonavir inhibited expression of Runx2 in the early phase of osteoblast differentiation. Osteoblast mineralization is inhibited by ritonavir. In the process of bone formation, osteoblasts need to differentiate and mineralize. Thus, whether ritonavir inhibited osteoblast mineralization was examined. MC3T3‑E1 cells were cultured in DMEM with osteoblast inducer reagent with or without 10 µg/ml ritonavir for 7, 14, 21 and 28 days. Then, cells were stained with Alizarin Red S to assess mineralization. At 7 and 14 days, cells treated with DMEM or with osteoblast induction medium, with or without ritonavir, exhibited no positive staining indicative of mineralization. At 21 and 28 days, cells treated with osteoblast induction medium with or without vehicle exhibited increasing mineralization, while those treated with DMEM only or osteoblast induction medium with ritonavir were not (Fig. 3). These results indicated that ritonavir suppressed or delayed osteoblast mineralization. Cell viability of MC3T3‑E1 cells treated with ritonavir. The cytotoxicity of ritonavir was assessed via measuring LDH activity. MC3T3‑E1 cells were cultured in osteogenic differentiation medium with or without 10.0 µg/ml ritonavir for 9 days, and the levels of LDH activity were measured. + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +There were no statistically significant differences among the groups (Fig. 4). These results showed that ritonavir did not exert cytotoxic effects in MC3T3‑E1 cells. + +## Discussion + +Osteoporosis is one of the complications seen in patients with HIV. HIV itself suppresses osteogenesis (14). Several in *vivo* studies have reported that osteopenia and osteoporosis are common among patients treated with antiretroviral drugs (15‑18). Bedimo et al (15) reported that cumulative exposure to tenofovir and protease inhibitors, particularly lopinavir/ritonavir, was an independent predictor of increased risk of osteoporotic fracture in HIV patients on cART. Further, it was shown in the Data collection on Adverse events of anti‑HIV Drugs study that protease inhibitor use was one of the risk factors for osteoporosis (16,17). Duvivier et al (18) reported that decreased lumbar spine bone mineral density was more pronounced in patients receiving protease inhibitors (indinavir/ritonavir or lopinavir/ritonavir) compared with other antiretroviral drugs. By contrast, a cross‑sectional study identified decreased bone density in HIV‑positive patients irrespective of treatment with or without protease inhibitors (19). Thus, the association between osteoporosis and protease inhibitors in *vivo* is yet to be confirmed. + +The MC3T3‑E1 cell line is a pre‑osteoblast line derived from mouse calvaria (20). It is established to exhibit a time‑dependent and sequential expression of osteoblast characteristics analogous to in *vivo* bone formation, and is used as a bone differentiation and mineralization model in *vitro* (21). In the present study, ritonavir was indicated to affect the pathway of osteoblast differentiation and the time course of differentiation. There are a number of in *vitro* reports describing the relationship between certain antiretroviral drugs and osteoblast differentiation (21,22). Jain and Lenhard reported that two protease inhibitors, lopinavir and nelfinavir, decreased osteoblast ALP activity and gene expression in human mesenchymal stem cells (21). Another report demonstrated that ALP activity decreased significantly in human osteoblast cultures following exposure to nelfinavir and indinavir (22). Santiago et al has previously reported ritonavir may facilitate osteoclast differentiation (23); however, other findings have suggested that ritonavir could inhibit osteoclast formation and function (24). Thus, the exact effect of RTV on osteoblast cells remained unknown. + +Ritonavir was originally used for its antiviral action, but is now used as a booster of other protease inhibitors. The therapeutic adult dose of ritonavir is 600 mg twice a day. When 600 mg ritonavir is taken twice a day, the mean maximum and minimum serum concentrations (Cmax and Cmin) of ritonavir have been determined as 11.2±3.6 and 3.7±2.6 µg/ml, respectively (25). In the present study, osteoblast differentiation was inhibited by 5.0 and 10.0 µg/ml ritonavir. These results indicated the possibility that administration of therapeutic doses of ritonavir may inhibit osteoblast differentiation in *vivo*. However, as a booster, ritonavir is used at a dose of 100 to 200 mg a day, and the expected ritonavir Cmax is ~1.5 µg/ml, and therefore osteoblast differentiation appears unlikely to be inhibited in *vivo*. However, to our knowledge there are no reports of a pharmacokinetic analysis of ritonavir in bone. Thus, osteoblast differentiation may be suppressed in bone when the booster dose of ritonavir is administered. + +In the present study, ritonavir suppressed the expression of Runx2 mRNA. To the best of our knowledge, this is the first study to report a suppressive effect of ritonavir on the expression of Runx2, and that ritonavir may affect osteoblast differentiation and mineralization in MC3T3‑E1 cells. There are numerous pathways associated with osteoblast differentiation including the bone morphogenetic protein BMP and parathyroid hormone pathways (26). Runx2 is an essential transcription factor required for osteogenesis; Runx2‑knockout mice exhibit a complete absence of mature osteoblasts and ossification (26). In particular, Runx2 is a key regulator of osteoblast differentiation and regulates the expression of several osteoblastic genes, including collagen 1, osteopontin, osteocalcin and bone sialoprotein, to induce differentiation (13). In primary osteoblast cells isolated from the calvariae of rats, study has found ALP activity to be significantly decreased by Runx2 small interfering RNA treatment when cells were treated with osteoblast differentiation medium that included ascorbic acid and β‑glycerophosphate (27), indicating that Runx2 expression is upstream of ALP. In the present study, the expression of Runx2 mRNA was suppressed on days 3 and 5, but not on day 7 of treatment with ritonavir in osteoblast differentiation medium. Prior to day 7, the ALP activities of cells with or without differentiation medium did not change (not shown). But ALP activities were suppressed on day 9 or later of treatment with ritonavir in osteoblast differentiation medium. These results collectively indicate that ritonavir suppressed Runx2 directly or other regulators of osteoblast differentiation upstream of Runx2, and, as a result, ALP activity was suppressed. + +The present results also suggested inhibition of osteoblast mineralization by ritonavir. However, bone formation takes 3 to 4 months (28), and cells in the current study were cultured for only 28 days (4 weeks). Thus, the possibility that ritonavir simply delayed osteoblast mineralization cannot be excluded. + +In conclusion, the present study is seemingly the first to demonstrate that ritonavir may inhibit osteoblast differentiation in *vitro*. Expression of Runx2 was suppressed by ritonavir in MC3T3‑E1 cells. This may explain the mechanism of osteopenia induced by cART involving protease inhibitors. + +## Acknowledgements + +Not applicable. + +## Funding + +No funding was received. + +## Availability Of Data And Materials + +The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. + +## Authors' Contributions + +YW, YY, KS, IK, TK and YO designed the study. YW processed the experimental data, performed the analysis, drafted the manuscript and produced the figures. All authors discussed the results and contributed to the final manuscript. + +## Ethics Approval And Consent To Participate + +Not applicable. Patient consent for publication Not applicable. + +## Competing Interests + +The authors declare that they have no competing interests. + +## References + +1. Lohse N, Hansen AB, Gerstoft J and Obel N: Improved survival in HIV‑infected persons: Consequences and perspectives. J Antimicrob Chemother 60: 461‑463, 2007. + +2. Maartens G, Celum C and Lewin SR: HIV infection: +Epidemiology, pathogenesis, treatment, and prevention. Lancet 384: 258‑271, 2014. + +3. Brown TT and Qaqish RB: Antiretroviral therapy and the prevalence of osteopenia and osteoporosis: A meta‑analytic review. AIDS 20: 2165‑2174, 2006. + +4. Del Carpio‑Cano FE, Dela Cadena RA and Sawaya BE: HIV and bone disease: A perspective of the role of microRNAs in bone biology upon HIV infection. J Osteoporos 2013: 571418, 2013. + +5. McComsey GA, Tebas P, Shane E, Yin MT, Overton ET, Huang JS, +Aldrovandi GM, Cardoso SW, Santana JL and Brown TT: Bone disease in HIV infection: A practical review and recommen- dations for HIV care providers. Clin Infect Dis 51: 937‑946, 2010. + +6. Tebas P, Powderly WG, Claxton S, Marin D, Tantisiriwat W, +Teitelbaum SL and Yarasheski KE: Accelerated bone mineral loss in HIV‑infected patients receiving potent antiretroviral therapy. AIDS 14: F63‑F67, 2000. + +7. Kanis JA: Assessment of fracture risk and its application to screening for postmenopausal osteoporosis: Synopsis of a WHO report. WHO Study Group. Osteoporos Int 4: 368‑381, 1994. + +8. Endo I and Mastumoto T: Bone and stem cells: Regulatory mechanism of mesenchymal stem cell differentiation to osteo- blasts. Clin Calcium 24: 555‑564, 2014 (In Japanese). + +9. Komori T: Regulation of bone development and maintenance by Runx2. Front Biosci 13: 898‑903, 2008. + +10. Tellmann G: The E‑Method: A highly accurate technique for gene‑expression analysis. Nat Methods 3: i‑ii, 2006. + +11. Jaiswal N, Haynesworth SE, Caplan AI and Bruder SP: Osteogenic differentiation of purified, culture‑expanded human mesenchymal stem cells in vitro. J Cell Biochem 64: 295‑312, 1997. + +12. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, +Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 284: 143‑147, 1999. + +13. Xiao ZS, Liu SG, Hinson TK and Quarles LD: Characterization of the upstream mouse Cbfa1/Runx2 promoter. J Cell Biochem 82: 647‑659, 2001. + +14. Cotter EJ, Malizia AP, Chew N, Powderly WG and Doran PP: +HIV proteins regulate bone marker secretion and transcription factor activity in cultured human osteoblasts with consequent potential implications for osteoblast function and development. AIDS Res Hum Retroviruses 23: 1521‑1530, 2007. + +15. Bedimo R, Maalouf NM, Zhang S, Drechsler H and Tebas P: +Osteoporotic fracture risk associated with cumulative exposure to tenofovir and other antiretroviral agents. AIDS 26: 825‑831, 2012. + +16. Friis‑Møller N, Reiss P, Sabin CA, Weber R, Monforte A, +El‑Sadr W, Thiébaut R, De Wit S, Kirk O, Fontas E, et al; DAD Study Group: Class of antiretroviral drugs and the risk of myocardial infarction. N Engl J Med 356: 1723‑1735, 2007. + +17. Flint OP, Noor MA, Hruz PW, Hylemon PB, Yarasheski K, +Kotler DP, Parker RA and Bellamine A: The role of protease inhibitors in the pathogenesis of HIV‑associated lipodystrophy: Cellular mechanisms and clinical implications. Toxicol Pathol 37: 65‑77, 2009. + +18. Duvivier C, Kolta S, Assoumou L, Ghosn J, Rozenberg S, +Murphy RL, Katlama C and Costagliola D; ANRS 121 Hippocampe study group: Greater decrease in bone mineral density with protease inhibitor regimens compared with nonnucleoside reverse transcriptase inhibitor regimens in HIV‑1 infected naive patients. AIDS 23: 817‑824, 2009. + +19. Amiel C, Ostertag A, Slama L, Baudoin C, N'Guyen T, +Lajeunie E, Neit‑Ngeilh L, Rozenbaum W and De Vernejoul MC: +BMD is reduced in HIV‑infected men irrespective of treatment. J Bone Miner Res 19: 402‑409, 2004. + +20. Kodama H, Amagai Y, Sudo H, Kasai S and Yamamoto S: +Establishment of a clonal osteogenic cell linefrom newborn mouse calvaria. Jap J Oral Biol 23: 899‑901, 1981. + +21. Jain RG and Lenhard JM: Select HIV protease inhibitors alter bone and fat metabolism ex vivo. J Biol Chem 277: 19247‑19250, 2002. + +22. Malizia AP, Cotter E, Chew N, Powderly WG and Doran PP: HIV +protease inhibitors selectively induce gene expression alterations associated with reduced calcium deposition in primary human osteoblasts. AIDS Res Hum Retroviruses 23: 243‑250, 2007. + +23. Santiago F, Oguma J, Brown AM and Laurence J: Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir. Biochem Biophys Res Commun 417: 223‑230, 2012. + +24. Wang MW, Wei S, Faccio R, Takeshita S, Tebas P, Powderly WG, +Teitelbaum SL and Ross FP: The HIV protease inhibitor ritonavir blocks osteoclastogenesis and function by impairing RANKL‑induced signaling. J Clin Invest 114: 206‑213, 2004. + +25. Ltd AL: Norvir® Tablets Summary of Product Characteristics. + +2010. + +26. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, +Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, et al: +Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89: 755‑764, 1997. + +27. Lin L, Shen Q, Leng H, Duan X, Fu X and Yu C: Synergistic inhibition of endochondral bone formation by silencing Hif1α and Runx2 in trauma‑induced heterotopic ossification. Mol Ther 19: 1426‑1432, 2011. + +28. Sims NA and Martin TJ: Coupling the activities of bone formation and resorption: A multitude of signals within the basic multicellular unit. Bonekey Rep 3: 481, 2014. + +This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License. \ No newline at end of file diff --git a/medical/md/PMC6827196.md b/medical/md/PMC6827196.md new file mode 100644 index 0000000000000000000000000000000000000000..e9d272ec3ced8e81e1f4dd8288b07bb0a56b3e07 --- /dev/null +++ b/medical/md/PMC6827196.md @@ -0,0 +1,252 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +# Improvement Of Solubility And Yield Of Recombinant Protein Expression In E. Coli **Using A Two-Step System** + +Tahereh Sadeghian-Rizi1,2, Azade Ebrahimi1,2, Fatemeh Moazzen1**, Hesam Yousefian2**, +and Ali Jahanian-Najafabadi1,* +1Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran. 2Student Research Committee, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran. + +## Abstract + +Overexpression of recombinant proteins in *Escherichia coli* results in inclusion body formation, and consequently decreased production yield and increased production cost. Co-expression of chaperon systems accompanied by recombinant protein is a general method to increase the production yield. However, it has not been successful enough due to imposed intense stress to the host cells. The aim of this study was to balance the rate of protein production and the imposed cellular stresses using a two-step expression system. + +For this purpose, in the first step, green fluorescent protein (GFP) was expressed as a recombinant protein model under control of the T7-TetO artificial promoter-operator, accompanied by Dnak/J/GrpE chaperon system. Then, in the next step, TetR repressor was activated automatically under the control of the stress promoter ibpAB and suppressed the GFP production after accumulation of inclusion bodies. Thus in this step incorrect folded proteins and inclusion bodies are refolded causing increased yield and solubility of the recombinant protein and restarting GFP expression again. Total GFP, soluble and insoluble GFP fractions, were measured by Synergy H1 multiple reader. Results showed that expression yield and soluble/insoluble ratio of GFP have been increased 5 and 2.5 times using this system in comparison with the single step process, respectively. The efficiency of this system in increasing solubility and production yield of recombinant proteins was confirmed. The two-step system must be evaluated for expression of various proteins to further confirm its applicability in the field of recombinant protein production. + +Keywords: Chaperons; *E. coli*; GFP; Inclusion bodies; Protein solubility; Recombinant proteins. + +## Introduction + +Recombinant proteins have many applications in the course of molecular biology, food industries, and pharmaceutical companies (1). The universality of the genetic codons permits protein expression in heterologous organisms (2). *Escherichia coli* (*E. coli)* is one of the most frequently used production host due to the extensive information of its physiological and genetics processes, ease of cultivation, low cost of its growth, and achievement of high cell density fermentations (3,4). Overexpression of the recombinant protein in this organism often leads to incorrect folding, aggregation, and inclusion body formation. Limitation +*Corresponding author: A. Jahanian-Najafabadi Tel: +98-3137927056, Fax: +98-3136680011 Email: jahanian@pharm.mui.ac.ir of the host chaperon capacity may be one of + +![0_image_2.png](0_image_2.png) + +the causes of this problem (5). Chaperons help proper folding of the newly synthesized proteins and refolding aggregated and misfolded proteins to the native conformation as protein quality control system in cells (6). + +DnaK and GroEL/ES systems are two main chaperon systems in the *E. coli* cells (7-9). + +General approaches for increasing protein production yields are based on the expression of recombinant proteins accompanied by chaperons whose unbalanced expression cause severe stress in host cells lowering the success of the system (10,11). + +Other researchers designed expression processes to equilibrate protein production and cellular stresses. Previously, a two-step procedure reported by Marco *et al.* showed efficient enhancement of solubility. + +In the first step of this procedure the four chaperon systems were coordinately co-overproduced with recombinant proteins to optimized folding. Then in the next step protein biosynthesis was inhibited by removal of isopropyl β- d-1-thiogalactopyranoside (IPTG) to permit chaperone-mediated refolding of the misfolded and aggregated proteins *in vivo*. This strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold (12). Disadvantage of this method is the need of external intervention for protein expression inhibition which is impractical and time consuming with expensive process in the industrial scale. Dragosits *et al.* presented a novel self-regulatory protein production system that couples the control of recombinant protein production with a stress-induced, negative feedback mechanism. In their study the soluble/insoluble ratio of recombinant protein increased but total protein yield decreased (13). In the present research we investigated the latter system by some modifications in the expression vectors, expression conditions, and in the presence of DnaK/J/GrpE chaperon system for increasing protein solubility without decreasing total protein yield. In this manner, recombinant protein production was suppressed by the precise feedback system in the appropriate time by the cell itself and after refolding of the misfolded and aggregated proteins, expression of recombinant protein was restarted. + + + +## Materials And Methods Bacterial Strains + +E. coli strains used in this study were XL1-blue and BL21 [DE3]. All strains were grown on LB agar plates or in LB broth supplemented with 100 μg/mL ampicillin, 50 μg/mL kanamycin and/or 34 μg/mL +chloramphenicol as necessary. + +## Plasmid Construction + +GFP and TetR plasmids The schematic presentation of the produced recombinant vectors is represented in Fig. 1. + +Briefly, to produce the presented constructs, Tet operator (TetO) sequence was inserted downstream of the T7 promoter sequence and a ribosome binding site sequence of the pET28a vector (13). Then, the GFP coding sequence was codon optimized for expression in *E. coli BL21*(DE3) cells and cloned downstream of the ribosome binding site sequence. Finally, the corresponding DNA +sequence was ordered to be synthesized by Biomatik Company (Canada) and cloned into the BglII and XhoI restriction sites of the pET28a plasmid. + +The TetR repressor sequence was also codon optimized for optimal expression in E. coli BL21 and was cloned under the control of stress-sensitive IbpAB promoter (PIbpAB) and a mutant weak ribosome binding site sequence. Finally, the corresponding DNA +sequence was ordered to be synthesized by Biomatik Company (Canada) and cloned to the *BglI*I and XhoI restriction sites of the pET15-b plasmid. All of the above cloning procedures were authenticated by restriction endonuclease digestion and finally by DNA sequencing (14). The confirmed pET28a derived plasmid was designated as pET28-T7- +TetO-GFP and pET15b as pET15b-IPAB-TetR plasmids. + +Under this circumstance, GFP is expected to be expressed under the control of T7 promoter until formation of inclusion bodies enough for activation of PIbpAB +promoter, which results in the production of TetR protein, and consequently inhibition of GFP production via activation of Tet-O +operator on the pET28-GFP. + +## Chaperon Plasmid + +In order to verify the effect of simultaneous application of the chaperones on the production yield of recombinant GFP protein produced by the mentioned controllable system, commercial vector pKJE7 from Takara Company (Takara, Japan) was used for expression of DnaK/J/GrpE chaperon system under control of L-arabinose promoter. + +![2_image_0.png](2_image_0.png) + +Fig. 1. The schematic presentation of the produced recombinant vectors. A, pET15b-TetR vector and B, pET28a-GFP vector +Protein expression Co-transformation of the plasmids was performed by electroporation using Electro-competent *E. coli BL21 (DE3)* cells. Then, the cells were cultivated in the presence of suitable antibiotics in accordance with the plasmids each cell harbored. For the one-step procedure, a single colony from the cells transformed with the pET28a-GFP was used to inoculate 5 mL of LB medium supplemented with kanamycin (50 µg/mL) and incubated overnight at 37 °C and 180 rpm. The overnight culture (500 µL) was used to inoculate the 50 mL LB medium supplemented with kanamycin (50 µg/mL) and incubated in conditions mentioned above. At an optical density of 0.6 at 600 nm, GFP expression was induced by addition of 1 mM IPTG and cells were further incubated overnight at 20 °C. + +Expression of GFP was also optimized based on tuning of three variables, including temperature (25, 30, and 37 °C), +IPTG concentration (0, 0.5, 1, and 1.5 mM), and post-induction cultivation time (2, 4, 6, 8, and 24 h) (15). For the two-step procedure, a single colony of the cells transformed with the pET28a-GFP, pET15b-TetR, and pKJE7 was cultured in 5 mL of LB medium supplemented with kanamycin (50 µg/mL), ampicillin (100 µg/mL), and chloramphenicol +(25 µg/mL), and incubated overnight as mentioned above. L-arabinose (0.4 g/mL) was added first to induce chaperon system expression, and IPTG (1 mM) was added at an optical density of 0.6 at 600 nm. Then, the cells were further incubated overnight at 20 °C. GFP expression was investigated in various combinations of pET28a-GFP with the two other plasmids +(pET15b-TetR and pKJE7) (12,13). In fact, host cell was transformed with the plasmids in 3 combinations including combination 1, pET28a-GFP and pET15bTetR, for evaluation of the effect of TetR on the GFP expression yield and soluble/insoluble ratio; combination 2, pET28a-GFP and pKJE7, for evaluation of the effect of DnaK/J/GrpE +chaperon system on GFP expression yield and soluble/insoluble ratio; and combination 3, pET28a-GFP, pET15b-TetR, and pKJE7, for investigation of the two-step procedure effect on total GFP expression and soluble/insoluble ratio. + +## Protein Purification And Analysis Of Soluble And Insoluble Protein Ratio + +Preparation of soluble and insoluble GFP fractions were performed according to the method based on single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form (16). Briefly, the cells were harvested at 5000 g for 20 min at 4 °C, +resuspended in 15 mL phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 12H2O, 2 mM KH2PO4, pH 8.0), and then lysed by sonication on ice. + +The lysate was centrifuged at 12,000 g for 20 min at 4 °C. The clarified supernatant was collected as soluble fraction of GFP +and retained for analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis +(SDS-PAGE) and fluorescence measurement. + +The insoluble pellet was resuspended in 20 mL +washing buffer (50 mM tris-Hcl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% Triton™ +X-100, 1M urea) and then analyzed by SDS-PAGE. The pellet was washed with 20 mL washing buffer for three times, then washed with PBS buffer to remove TritonTM X-100 detergent and centrifuged at 12000 g for 20 min at 4 °C. For subsequent solubilization and refolding, the purified inclusion body pellet was resuspended in PBS buffer containing 2 M urea, frozen at -20 °C for overnight, thawed at room temperature, and centrifuged at 12,000 g for 15 min at 4 °C. Supernatant was collected as insoluble fraction of GFP and retained for analysis by SDS-PAGE and fluorescence measurement. + +## Fluorescence Measurements + +![3_Image_0.Png](3_Image_0.Png) + +The total GFP, soluble, and insoluble GFP amounts were measured using Synergy H1 Multiple Reader (BioTek, USA) using the following settings: 37 °C, excitation wavelength 485 nm, emission wavelength 535 nm and gain 75. The GFP fluorescence values were reported as GFP fluorescence per optical density at 600 nm (13). + +Statistical analyses Data are presented as mean ± standard deviation (SD) of at least three experiments. Statistical significance was tested by two-tailed t-test and two-way ANOVA test. Analyses were performed using GraphPad Prism 8.0.2 software. P < 0.05 was considered significant. + +## Results Optimization Of Gfp Expression In One-Step Procedure + +Expression of the GFP protein by the transformed *E. coli* BL21 cells with pET28a-GFP plasmid was verified by fluorescent microscopy (Fig. 2A). Then, the optimized conditions for GFP expression was investigated by evaluating the effects of temperature, IPTG concentration, and post-induction incubation time. As shown in Fig. 2B, the maximal GFP expression was achieved at 25 °C, 1 mM IPTG +and a post-induction incubation time of 16 h (results related to the effect of time are not presented). + +![4_image_0.png](4_image_0.png) + +## Effect Of Two-Step Procedure On Gfp Expression + +Figure 3 shows the effect of TetR +expression on GFP production. We observed GFP expression was increased 2.4 times where the feedback loop, encoded by pET15b-TetR, was induced (Fig. 3A). + +Expression of TetR under control of stress-sensitive IbpAB promoter translated to increased soluble/insoluble ratio of the produced GFP (2.6 times higher than the expression of GFP in the absence of TetR) +(Fig. 3B,3C). As shown in Fig. 3A-3C, +co-expression of DnaK/J/grpE with GFP did not exhibit notable effect on the yield of total GFP expression compared with the two other groups but a significant increase of soluble/insoluble ratio was observed +(3 times). Figure 3A-3C shows that total GFP expression was increased 5 times using the two-step expression procedure in comparison with GFP expression in the absence of TetR and DnaK/J/GrpE. On the other hand, the main fraction of total GFP +was in the soluble form (soluble/insoluble ratio 8.5). + + + +## Discussion + +Bacterial inclusion bodies are insoluble protein aggregates formed in the bacterial cytoplasm and periplasm during the overproduction of recombinant proteins, especially those from mammalian or viral origin (17,18). Factors including high copy number of target gene, strong promoter, as well as high temperature during protein expression, and high inducer concentrations favor high level expression of recombinant protein and increase the possibility of the inclusion body formation (19-22). + +Reducing environment of bacterial cytosol, amino acid sequence of protein with highly hydrophobic proteins, lack of post-translational modification, and eukaryotic chaperones also favor this event (23). + +Inclusion bodies formation imposes a great obstacle in the production and downstream processing of recombinant proteins (24). Solubilization and refolding of inclusion bodies into bioactive form is a challenging task. If an efficient and convenient way of decreasing formation of inclusion bodies without decreasing protein production yield could be developed, it would reduce the need of excess chromatographic purification steps and downstream processing costs (25). + +At the midstream level, co-expression of folding modulators such as chaperones, lowering the transcription rate, and reducing the growth temperature have been thoroughly explored strategies which have been successful for an important number of specific proteins but they do not offer generic solutions to this matter (26-28). Co-expression of molecular chaperones and foldases from accessory plasmids with no further control results in imbalanced expression of these proteins and has detrimental effects on the production process (29,30). Dragosits *et al.* developed a synthetic expression platform that enables the cell to shut down the recombinant protein production mechanism automatically by itself through stress-induced feedback to address challenges such as balancing protein production and cellular stress. This system limited stressful protein expression and increased the total soluble fraction but significantly decreased the protein yield. + +The authors suggested further investigation on promoter and repressor engineering, other recombinant proteins, and different inducer concentrations for avoiding such loss (13). + +In another study, Marco *et al.* used a two-step procedure that shows the strongest enhancement of the solubility. In the first step of this procedure the four chaperon systems were coordinately co-overproduced with recombinant proteins to optimized folding and in the next step protein biosynthesis was inhibited by removal of IPTG to permit chaperone mediated refolding of the misfolded and aggregated proteins *in vivo*. This strategy increased the solubility of 64 different heterologous proteins up to 42 folds without decreasing total protein yield (12). + +Disadvantage of this method is requirement of external intervention to inhibit protein expression and an impractical and expensive process in industrial scale. We combined the two methods with some modifications. We substituted pNf-TetR with pET15b-TetR and pET23b-GFP with pET28a. In this study, both soluble/insoluble ratio and total GFP expression was increased (2.5 and 5 times, respectively), while in the study of Dragosits et al. soluble/insoluble ratio was increased up to 1.5 times but total GFP expression was decreased 1.4 times as explained earlier. This may be due to the use of pET15b vector for cloning of TetR, which retains similar copy number to pET28a as carrier of GFP encoding sequence in comparison with pNF. + +Second reason may be related to the use of DnaK/J/GrpE chaperon system and increasing of chaperon capacity of bacterial cells which resulted in folding of misfolded and aggregated GFP, suppression of stress-sensitive IbpAB promoter and excessive expression of TetR as suppressor of GFP expression and resuming of its expression. It would be interested to test the efficacy of this system with other recombinant proteins. + +## Conclusion + + +Altogether, in the present study, the use of an automatic negative feed-back system, combined with co-expression of major cytosolic chaperon system DnaK/J/GrpE, resulted in an increased solubility and total recombinant protein expression. + +Therefore, this system is very applicable, beneficial, and cost-effective in recombinant protein expression field of biotechnology. + +Further studies on using this system for expression of various recombinant therapeutic proteins are undergoing in our laboratory. + +## Acknowledgments + +This study was conducted by Tahereh Sadeghian, and aslo Azadeh Ebrahimi which was financially supported by Student Research Committee of School of Pharmacy and Pharmaceutical Sciences, and Research Deputy of Isfahan University of Medical Sciences, Isfahan, Iran through the Grants No. + +194105 and 194296, respectively. + +## References + +1. Ben-Nissan G, Vimer S, Warszawski S, Katz A, +Yona M, Unger T, *et al*. Rapid characterization of secreted recombinant proteins by native mass spectrometry. Commun Biol. 2018;1(1):213-224. + +2. Kyratsous CA, Silverstein SJ, DeLong CR, +Panagiotidis CA. Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in *Escherichia coli*. + +Gene. 2009;440(1-2):9-15. + +3. Chen R. Bacterial expression systems for recombinant protein production: *E. coli* and beyond. Biotechnol Adv. 2012;30(5):1102-1107. + +4. Panda AK. Bioprocessing of therapeutic proteins from the inclusion bodies of *Escherichia coli*. + +Adv Biochem Eng Biotechnol. 2003;85:43-93. + +5. Yang Z, Zhang L, Zhang Y, Zhang T, Feng Y, Lu X, +et al. Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-stepdenaturing and refolding method. PLoS One. + +2011;6(7):e22981. + +6. Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol. 2013;14(10):630-642. + +7. Calloni G, Chen T, Schermann SM, Chang HC, +Genevaux P, Agostini F, *et al.* DnaK functions as a central hub in the *E. coli* chaperone network. + +Cell Rep. 2012;1(3):251-264. + +8. Aguilar-Rodríguez J, Sabater-Muñoz B, MontagudMartínez R, Berlanga V, Alvarez-Ponce D, Wagner A, *et al*. The molecular chaperone DnaK is a source of mutational robustness. Genome Biol Evol. 2016;8(9):2979-2991. + +9. Kedzierska S, Staniszewska M, Wegrzyn A, +Taylor A. The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from *Escherichia coli* cells. FEBS Lett. 1999;446 (2-3):331-337. + +10. Gopal GJ, Kumar A. Strategies for the production of recombinant protein in *Escherichia coli*. Protein J. + +2013;32(6):419-425. + +11. Xiao S, Shiloach J, Betenbaugh MJ. Engineering cells to improve protein expression. Curr Opin Struct Biol. 2014;26:32-38. + +12. de Marco A, Deuerling E, Mogk A, Tomoyasu T, +Bukau B. Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol. 2007;7:32-40. + +13. Dragosits M, Nicklas D, Tagkopoulos I. A synthetic biology approach to self-regulatory recombinant protein production in *Escherichia coli*. J Biol Eng. 2012;6(1):2-11. + +14. Jahanian-Najafabadi A, Soleimani M, Azadmanesh K, Mostafavi E, Majidzadeh AK. Molecualr cloning of the capsular antigen F1 of Yersinia pestis in pBAD/gIII plasmid. Res Pharm Sci. 2015;10(1): +84-89. + +15. Long X, Gou Y, Luo M, Zhang S, Zhang H, Bai L, +et al. Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in *E. coli*. + +BMC Biotechnol. 2015;1(15):13-21. + +16. Qi X, Sun Y, Xiong S. A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form. Microb Cell Fact. 2015;14:24-35. + +17. Garcia-Fruitos E. Inclusion bodies: a new concept. + +Microb Cell Fact. 2010;9:80-82. + +18. Rodriguez-Carmona E, Cano-Garrido O, +Seras-Franzoso J, Villaverde A, Garcia-Fruitos E. Isolation of cell-free bacterial inclusion bodies. + +Microb Cell Fact. 2010;9:71-79. + +19. Singh A, Upadhyay V, Upadhyay AK, Singh SM, +Panda AK. Protein recovery from inclusion bodies of *Escherichia coli* using mild solubilization process. Microb Cell Fact. 2015;14:41-50. + +20. Carrio MM, Villaverde A. Localization of chaperones DnaK and GroEL in bacterial inclusion bodies. J Bacteriol. 2005;187:3599-3601. + +21. Chang CC, Song J, Tey BT, Ramanan RN. + +Bioinformatics approaches for improved recombinant protein production in *Escherichia coli*: protein solubility prediction. Brief Bioinform. + +2014;15(6):953-962. + +22. Rosano GL, Morales ES, Ceccarelli EA. + +New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci. 2019;28(8):1412-1422 23. Carrio MM, Cubarsi R, Villaverde A. Fine architecture of bacterial inclusion bodies. + +FEBS Lett. 2000;471(1):7-11. + +24. Rosano GL, Ceccarelli EA. Recombinant protein expression in *Escherichia coli*: advances and challenges. Front Microbiol. 2014;5:172-188. + +25. Salehinia J, Sadeghi HMM, Abedi D, Akbari V. + +Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 + +single-chain antibody fragment inclusion bodies. Res Pharm Sci. 2018;13(6):566-574. + +26. Garcia-Fruitos E, Sabate R, de Groot NS, Villaverde A, Ventura S. Biological role of bacterial inclusion bodies: a model for amyloid aggregation. FEBS J. + +2011;278(14):2419-2427. + +27. Liu L, Yang H, Shin HD, Chen RR, Li J, Du G, et al. How to achieve high-level expression of microbial enzymes: strategies and perspectives. Bioengineered. 2013;4(4):212-223. + +28. Martínez-Alonso M, González-Montalbán N, +García-Fruitós E, Villaverde A. Learning about protein solubility from bacterial inclusion bodies. + +Microb Cell Fact. 2009;8(1):4-8. + +29. Martínez-Alonso M, García-Fruitós E, +Ferrer-Miralles N, Rinas U, Villaverde A. Side effects of chaperone gene co-expression in recombinant protein production. Microb Cell Fact. 2010;9(1): 64-69. + +30. Gasser B, Saloheimo M, Rinas U, Dragosits M, +Rodríguez-Carmona E, Baumann K, *et al.* Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact. 2008;7(1):11-28. \ No newline at end of file diff --git a/medical/md/PMC7252995.md b/medical/md/PMC7252995.md new file mode 100644 index 0000000000000000000000000000000000000000..c429f98ff82a1e2d4dc418bc3600f3057b485cba --- /dev/null +++ b/medical/md/PMC7252995.md @@ -0,0 +1,317 @@ +# Prompt Recognition Of Infectious Pulmonary Tuberculosis Is Critical To Achieving Elimination Goals: A Retrospective Cohort **Study** + +Courtney Heffernan ,1 James Barrie,2 Alexander Doroshenko,3 Mary Lou Egedahl,1 Catherine Paulsen,1 Ambikaipakan Senthilselvan,4 Richard Long1 To cite: Heffernan C, Barrie J, +Doroshenko A, *et al*. Prompt recognition of infectious pulmonary tuberculosis is critical to achieving elimination goals: a retrospective cohort study. *BMJ Open Resp Res* 2020;7:e000521. doi:10.1136/ +bmjresp-2019-000521 ► Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ bmjresp-2019-000521). Received 29 October 2019 Revised 16 April 2020 Accepted 29 April 2020 © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. + +1Department of Medicine; Division of Pulmonary Medicine, University of Alberta, Edmonton, Alberta, Canada 2Department of Radiology and Diagnostic Imaging, University of Alberta, Edmonton, Alberta, Canada 3Department of Medicine; Division of Preventive Medicine, University of Alberta, Edmonton, Alberta, Canada 4School of Public Health, University of Alberta, Edmonton, Alberta, Canada Correspondence to Dr Richard Long; rlong@ualberta.ca + +## Abstract + +Introduction All pulmonary tuberculosis (PTB) cases are presumed to be infectious to some degree. This spectrum of infectiousness is independently described by both the acidfast bacilli smear and radiographic findings. Smear-positive patients with chest radiographic findings that are typical for adult-type PTB are believed to be most infectious. + +Hypothesis Characterisation of the presumed most infectious PTB case is possible by reference to readily available clinical features and laboratory results. + +Methods Retrospective cohort study of adult, culturepositive PTB cases (151 smear-positive; 162 smear-negative) +diagnosed between 1 January 2013 and 30 April 2017 in Canada. We describe cases according to demographic, clinical and laboratory features. We use multivariable multinomial logistic regression to estimate the relative risk ratio (RRR) with 95% CI of features associated with an outcome of smear-positive PTB, characterised by 'typical' chest radiograph findings. + +Results Being Canadian-born, symptomatic, having a subacute duration of symptoms and broad-spectrum antibiotic prescriptions were all more commonly associated with smear-positive than smear-negative disease (36% vs 20%; 95% vs 63%; 88% vs 54%; and 59% vs 28%, +respectively). After combining smear status and radiographic features, we show that smear-positive patients with typical chest radiographs were younger, had a longer duration of symptoms (RRR 2.41; 95% CI 1.01 to 5.74 and 2.93; 95% CI +1.20 to 7.11, respectively) and were less likely to be foreignborn, or have a moderate to high-risk factor for reactivation +(RRR 0.40; 95% CI 0.17 to 0.92 and 0.18; 95% CI 0.04 to 0.71, respectively) compared with smear-negative patients with atypical chest radiograph findings. + +Conclusion A clear picture of the presumed most infectious PTB case emerges from available historical and laboratory information; vigilance for this presentation by front-line providers will support elimination strategies aimed at reducing transmission. + +Introduction Elimination of tuberculosis (TB) requires a dual-pronged approach. Both interrupting transmission by promptly diagnosing and + +## Key Messages + +What is the key question? + +► Can we identify a highly infectious pulmonary tuberculosis (TB) case in high-income, low-TB incidence settings? + +What is the bottom line? + +► A combination of readily available clinical (n=6) and laboratory (n=2) features describes the prototypical infectious case of pulmonary TB. + +Why read on? + +► The timely diagnosis of infection-spreading cases, especially by front-line providers, is critical to interrupting transmission, and thus, to meeting TB elimination targets. + +treating infection-spreading cases, and preventing TB among those already infected but who have not yet developed a disease are necessary. As TB becomes less common in high-income, low-incidence settings, the prompt diagnosis of infection-spreading cases by front-line physicians and nurses becomes increasingly difficult. These cases are mainly bacillary positive (smear-positive, culturepositive and smear-negative, culture-positive) +pulmonary TB (hereafter 'PTB'). Among culture-positive PTB cases, those with smearpositive disease are 5–10 times more likely to generate a secondary case than those who are smear-negative.1–3 In addition, among persons with smear-positive PTB, those with 'typical' findings on chest radiograph (defined below) are 10 times more likely to generate a secondary case than smearpositive cases without such features.4 Thus, both the smear status and the presentation of disease on chest radiograph contribute independently to transmission.4–7 Six features from the medical history and two readily available laboratory tests (the chest radiograph and complete blood count (CBC)) have emerged, both within the literature and clinical practice, as being critical to suspecting PTB rather than disease attributable to other respiratory pathogens, and subsequently ordering the collection of sputum or other specimens for mycobacteriological examination.8 9 The relative predictive value of each variable on an outcome of PTB in a specialised TB clinic has previously been shown.10 What has not been shown is how well the combination of these features differentiates the highly infectious case from the minimally infectious case, among whom the collection of a sputum is imperative for achieving public health goals. + +The objective of this current study is to distinguish smear-positive and smear-negative PTB by describing the relative frequency of the aforementioned features in a well-defined cohort of adult patients. While all patients with PTB are presumed infectious to some degree, it is the smear-positive case with typical chest radiographic findings whose timely diagnosis offers the greatest potential for interrupting transmission, and impacting public health. Meeting this objective could establish a clinical threshold for front-line providers to consider the diagnosis and seek subsequent laboratory confirmation, which is important to TB elimination efforts.11 + +## Methods + +This study was performed in Alberta—population over 4.25million in 2016 (Statistics Canada)—one of four major immigrant-receiving provinces in Canada, the other three being British Columbia, Ontario and Quebec. In Alberta, in 2016, the crude incidence rate of TB was 5.6 per 100000 population. University-based pulmonary and infectious disease physicians staff three dedicated public health clinics that provide TB services to the province.9 Language line (translation) services are available as necessary. Introduction of an assessment form into the two clinics supported by University of Alberta staff occurred in 2013. It records relevant information for each referred case. We identified a cohort of consecutive, adult (age >14 years), culture-positive pulmonary cases diagnosed between 1 January 2013 and 30 April 2017 from these two clinics. We supplemented data collected through the assessment form with findings from the Provincial Laboratory for Public Health, which performs all mycobacteriology in the province. Per standard of care, patients whose initial sputum specimens were smear-negative had three additional specimens collected at the time of reporting of the first positive culture to confirm that they were still smear-negative on the start date of treatment, which, in Alberta, is the date of diagnosis. The initial isolate of each case was genotyped using mycobacterial interspersed repetitive units, and laboratory cross-contamination was excluded in all patients with a single, smear-negative, culture-positive specimen.12 Basic demographic and clinical information is used to describe the cohort of patients in this study. These data included: age at diagnosis (15–64 years, >64 years), sex, population group (Canadian born and foreign born), disease type (new active vs relapse/retreatment) and HIV status (positive, negative). We then examined the following eight explanatory variables, which we hypothesise describe the most infectious case of PTB. + +Clinical characteristics included, as above, six pieces of information related to patient histories: (1) symptoms, (2) duration of symptoms, (3) absence of dyspnoea, (4) broad-spectrum antibiotic prescriptions in the 6months antedating diagnosis, (5) epidemiological risk of exposure to, or infection with, *Mycobacterium tuberculosis*, and +(6) risk factors for the reactivation of latent TB infection +(LTBI). Symptoms, if present, were reported as respiratory (dry cough, productive cough, haemoptysis or chest pain), constitutional (fever, night sweats, weight loss or fatigue) or both.13 The absence of dyspnoea is known to favour a diagnosis of PTB over community-acquired pneumonia; parallel reductions in ventilation and perfusion at sites of PTB disease are known to preserve gas exchange.14–16 We documented antibiotic prescriptions in individual records, and confirmed their prescription for respiratory complaints by cross-referencing the cohort against the provincial (Alberta Health) stakeholder registry, which, in turn, reports physician claims for the reimbursement of services provided. + +Epidemiological risks include: a history of TB, and whether it was adequately treated, a history of overt contact with PTB, and whether it was adequately assessed (eg, tuberculin skin test or interferon gamma release assay) and treated, migration from a high-incidence country of birth, or travel and work to/within a high-incidence country, an occupational history (eg, healthcare worker), +and/or social risk factors such as homelessness, incarceration and alcohol/substance misuse where non-overt exposures might occur. Online supplementary table 1 provides a description of social factors including homelessness, incarceration and alcohol/substance misuse. + +We reported the presence or absence of one or more 'moderate' or 'high risk' factors for the reactivation of LTBI, as described in the Canadian Tuberculosis Standards.17 Finally, we report the results of two routine laboratory tests performed at the time of diagnosis: the CBC and the chest radiograph. The results of the CBC are grouped into four patterns based on the presence or absence of anaemia, and/or leucocytosis.18 19 An experienced chest radiologist, blind to the smear status of the patients, re-read the posterior-anterior and lateral chest images performed at the time of diagnosis. In the context of this study, d frontal images were divided into four quadrants by halving a perpendicular drawn from the apex of the lung to the dome of the hemidiaphragm. Documented were the presence or absence of (1) parenchymal infiltrates and their dominant location—no distinction was made between infiltrates that were airspace, interstitial, nodular or some combination of these; (2) cavities, defined as a gas-filled space within pulmonary consolidation, a mass, or a nodule; (3) +adenopathy—hilar, mediastinal or both; and (4) pleural effusion. + +As previously reported, we categorised patients as having 'typical' versus 'atypical' radiographs based on the characteristics listed above.4 20 For patients with infiltration localised to, or predominantly in the upper long zones, with or without cavitation, but with no discernable intrathoracic adenopathy, the radiograph is 'typical' for adult-type PTB. In patients with (1) no abnormality; (2) intrathoracic adenopathy with or without parenchymal disease; (3) a localised or predominant lower lung zone infiltrate, with or without cavitation; (4) an isolated pleural effusion; and (5) a miliary (diffuse micronodular) pattern, the radiograph is 'atypical' for adult-type PTB. We report extent of disease on chest radiograph as normal, minimal, moderately, far advanced or miliary according to criteria established by the US National Tuberculosis and Respiratory Disease Association.21 + +## Statistical Analysis + +We describe cases by smear status according to each of the aforementioned features. We tested the difference in proportions, by smear status, of each feature between two independent samples using the direct test, Fisher's exact test or χ2 test as appropriate. Thereafter, we generated a combined outcome of smear status and radiographic findings, creating an outcome for multinomial logistic regression that was a nominal variable with four responses: atypical smear-negative, typical smear-negative, atypical smear-positive and typical smear-positive, with atypical smear-negative considered to be the baseline group. The model includes all features and confounders considered clinically relevant to making a timely diagnosis of TB from a review of the literature. The total sample size of 313 in this study achieves 80% power or greater to detect an effect size (W) of 0.19 or greater for comparison of proportions between four groups using 3 df, and χ2 test with a significance level (alpha) of 0.05.22 We performed all statistical analyses using STATA V.14, StataCorp 2015. There were no missing data within this cohort. Patient and public involvement We did not involve patients and the public in this study given its retrospective design, but we anticipate that the findings presented herein will lead to benefits for future cohorts of patients. + +## Results + +There were 313 patients in the cohort, 151 (48%) smearpositive and 162 (52%) smear-negative. Being Canadianborn was associated with a greater likelihood of having smear-positive than smear-negative disease (36% vs 20%); otherwise there were no statistically significant differences in the age, sex, disease type,or HIV status between these two groups (see table 1). The leading countries of birth of foreign-born patients were the Philippines (38%), India (16%), Vietnam (5%) and Somalia +(5%); Bhutan, Ethiopia and China each contributed 3% of foreign-born patients. + +As shown in table 2, being symptomatic with either respiratory, constitutional or both types of symptoms at the time of diagnosis was more likely in smear-positive than smear-negative patients (95% vs 63%). Cough (dry or productive) and haemoptysis were more common in smear-positive than smear-negative patients (83% vs 45%, and 25% vs 6%, respectively). Chest pain was equally uncommon in both groups (11% vs 9%). Dyspnoea was also relatively uncommon in both groups (28% vs 14%). Each of the major constitutional symptoms was more common in smear-positive than smear-negative patients (weight loss, 50% vs 28%; fever, 45% vs 20%; night sweats, 24% vs 10%; and fatigue, 21% vs 12%). Finally, having a combination of respiratory and constitutional symptoms (70% vs 28%) and a duration of symptoms ≥2 weeks (88% vs 54%) were statistically significantly more common in smear-positive than smear-negative patients. Among smear-positive and smear-negative patients with any symptoms, 93% and 86%, respectively, had symptoms for ≥2 weeks (data not shown). + +We dichotomised possible combinations of antibiotic prescription, epidemiological risk and reactivation risk, and describe their associations with an outcome of either smear-positive or smear-negative disease (table 3). A history of broad-spectrum antibiotic prescription for a respiratory complaint in the 6 months preceding the date of diagnosis was more likely among smear-positive patients (57% vs 23%); of those, multiple prescriptions were also more likely among smear-positive patients (49% vs 36%—data not shown). Macrolides, penicillins and fluoroquinolones were, in order of frequency, the three major classes of antibiotics prescribed. If one assumes that antibiotic prescriptions for respiratory complaints, in the months immediately preceding their diagnosis, were due to PTB then there were missed opportunities to diagnose these patients. In absolute numbers, there were 291 antibiotic prescriptions in the 6 months antedating a TB diagnosis; 208 among persons ultimately diagnosed with smear-positive PTB and 83 among persons ultimately diagnosed with smear-negative PTB (data not shown). + +Almost all patients in both groups (95%) had some epidemiological risk. The distribution of social risk factors was similar in smear-positive and smear-negative patients. Notably, however, they were much more common among Canadian-born than among foreign-born patients (see online supplementary table 1). A moderate to high risk of reactivation was similarly uncommon in both smearpositive and smear-negative patients (6% vs 9%). + +Anaemia was more common in smear-positive than smear-negative cases (65% vs 42%). Anaemia was usually of the chronic disease, iron deficiency varieties or both (data not shown). A leucocytosis was uncommon in both groups, but more uncommon in smear-negative cases + +| Table 1 | Demographic and clinical characteristics of patients with pulmonary tuberculosis by smear status Smear status* Positive Negative n (%) n (%) | P value‡ | | | +|-------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------|------------|------------|-----------| +| Assessed, n | 313 | 151 | 162 | | +| Age 15–64 years | 248 | 120 (79.4) | 128 (79.0) | 0.38 | +| | >64 years | 65 | 31 (20.6) | 34 (21.0) | +| Sex Male | 173 | 86 (57.0) | 87 (53.8) | 0.56 | +| | Female | 140 | 65 (43.0) | 75 (46.2) | +| Population group† Foreign-born | 226 | 97 (64.3) | 129 (79.7) | 0.002 | +| | Canadian-born | 87 | 54 (35.7) | 33 (20.3) | +| Disease type New active | 280 | 135 (89.4) | 145 (89.5) | 0.97 | +| | Relapse/retreatment | 33 | 16 (10.6) | 17 (10.5) | +| HIV status Negative | 300 | 147 (97.4) | 153 (94.5) | 0.19 | +| | Positive | 13 | 4 (2.6) | 9 (5.5) | +| Total | | | | | +| Characteristics | n | | | | +| *Smear-positive patients had to have one or more airway secretion specimens, on or before the start date of treatment, with a | | | | | + +*Smear-positive patients had to have one or more airway secretion specimens, on or before the start date of treatment, with a semiquantitative smear size of 1+ or greater. †Canadian-born includes 61 indigenous patients and 26 Canadian-born non-indigenous patients. Among the foreign-born patients, one smear-positive and 28 smear-negative were referred to public health through the immigration medical screening surveillance mechanism. ‡Difference in proportions [(smear-positive)−(smear-negative)]; sample size was adequate to detect observable differences. + +(21% and 7%). The mean and median haemoglobin in smear-positive males and females was 127 and 126g/L, and 112 and 112g/L, respectively. In smear-negative males and females, it was 135 and 142g/L, and 123 and 124g/L, respectively. The mean and median leucocyte count was 8.6×109/L and 7.8×109/L in smear-positive cases, and 7×109/L and 7×109/L in smear-negative cases. The reference range for a normal haemoglobin is 135–175g/L and 120–160g/L for males and females, respectively. The reference range for a normal leucocyte count is 4.0–11.0×109/L. + +Chest radiographic features that are 'typical' for adulttype PTB, bilateral disease, cavitation and moderate or far-advanced disease were all more common in smearpositive than smear-negative patients (78% vs 36%; +56% vs 21%; 62% vs 6%; and 81% vs 22%, respectively). Among smear-positive patients, those with cavitation had a statistically significantly higher semiquantitative smear grade (3+ to 4+) vs (1+ to 2+), 82% vs 46% than those without cavitation (data not shown). Only three patients, one smear positive and two smear negative, had a miliary pattern. + +Table 4 shows the findings of our multinomial logistic regression. An absence of dyspnoea was more likely among smear-negative patients with typical chest radiograph findings than smear-negative patients with atypical chest radiograph findings; lower lung zone disease and dependent effusions—atypical findings on chest radiograph—are presumed to more negatively affect gas exchange (relative risk ratio (RRR) 4.71, 95% CI 1.69 to 13.18). Young age and a subacute or chronic duration of symptoms were more greatly associated with smearpositive patients who have typical chest radiographs than smear-negative patients with atypical chest radiographs (RRR 2.41; 95% CI 1.01 to 5.74, and RRR 2.93; 95% CI 1.20 to 7.11, respectively), while being foreign born and having a moderate to high-risk factor for reactivation were less commonly associated with smear-positive PTB with typical chest radiographs than smear-negative patients with PTB with atypical chest radiographs (RRR 0.40; 95% CI 0.17 to 0.92, and RRR 0.18; 95% CI 0.04 to 0.71, respectively) (see table 4 and online supplementary table 2). All major symptoms, respiratory and constitutional, were more commonly associated with typical presentation, cavitation, bilateral and moderate to advanced disease on chest radiograph (see online supplementary table 3). + +## Discussion + +It has previously been established that of the information readily available to front-line providers, three pieces are key to triggering suspicion of PTB. These include symptoms, especially if both respiratory and constitutional and subacute or chronic, and *typical* chest radiographic features; a public health trigger is an epidemiological + +| Table 2 | Symptoms and their duration in patients with pulmonary tuberculosis by smear status Smear status Total | | | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------|------------|------------|------------|--------| +| Characteristics | n | Positive | Negative | | | +| n (%) | n (%) | P value* | | | | +| Assessed, n | 313 | 151 | 162 | - | | +| Symptomatic Yes | 245 | 143 (94.7) | 102 (63.0) | <0.001 | | +| | No | 68 | 8 (5.3) | 60 (37.0) | | +| Symptoms Respiratory | 217 | 133 (88.1) | 84 (51.8) | <0.001 | | +| | Cough (dry) | 149 | 90 (59.6) | 59 (36.4) | | +| | Cough (productive) | 50 | 36 (23.8) | 14 (8.6) | | +| | Haemoptysis | 46 | 37 (24.5) | 9 (5.5) | | +| | Chest pain | 31 | 17 (11.2) | 14 (8.6) | | +| | Absence of dyspnoea | | | | | +| | Yes | 249 | 109 (72.2) | 140 (86.4) | <0.001 | +| | No | 64 | 42 (27.8) | 22 (13.6) | | +| | Constitutional | 176 | 113 (74.8) | 63 (38.8) | <0.001 | +| | Weight loss | 121 | 76 (50.3) | 45 (27.7) | | +| | Fever | 101 | 68 (45.0) | 33 (20.3) | | +| | Night sweats | 52 | 36 (23.8) | 16 (9.8) | | +| | Fatigue | 51 | 31 (20.5) | 20 (12.3) | | +| | Both† | | | | | +| | Yes | 151 | 105 (69.5) | 46 (28.4) | <0.001 | +| | No | 162 | 46 (30.5) | 116 (71.6) | | +| Duration of symptoms ≥2 weeks | 221 | 133 (88.0) | 88 (54.3) | <0.001 | | +| | <2weeks | 92 | 18 (12.0) | 74 (45.7) | | +| *Difference in proportions [(smear-positive)−(smear-negative)]; sample size was adequate to detect observable differences. †Patients with some combination of both respiratory and constitutional symptoms. | | | | | | + +risk of having or acquiring LTBI (see figure 1). Our study shows that although a public health risk was similarly common in smear-positive and smear-negative cases (95% and 97%, respectively), clinical triggers (symptoms: 95% vs 63%, and typical chest radiographic features: 78% vs 36%) were much more common in smear-positive cases; fortuitous given that smear positivity and typical radiographic features are presently the major determinants of infectiousness, and therefore the public health risk PTB poses. + +1–4 7 Other variables, including broad-spectrum antibiotic prescription history, reactivation risk factors and the CBC findings, are supportive in considering the likelihood of PTB. + +Individually, each of the features identified in the multinomial logistic regression model as distinguishing +(typical) smear-positive cases from (atypical) smearnegative cases has precedent in the literature, including younger age23 24; symptoms and their duration25–27; less likely to be foreign-born14 28; and less likely to have a moderate to high-risk factor for reactivation.29–32 When combined, the features have utility for describing the prototypical smear-positive patient with typical chest radiographic features: a young middle-aged man or woman with an epidemiological risk for LTBI, and a subacute or chronic duration of symptoms, a history of broad-spectrum antibiotic prescriptions, rarely having a risk factor for reactivation, and with an anaemia and a normal or low leucocyte count on CBC. + +In the future, semiquantitative results from the real-time PCR method, Xpert MTB/RIF assay, may replace smear microscopy and its current role as a proximate measure of infectiousness.33 34 There is, however, no expectation that this technology will impact the picture of the prototypical infectious case described herein. By contrast, cough aerosol technology might. For reasons that are only speculative at this time, some patients, whether smear positive or smear negative, are better able to generate an infectious aerosol than others.35 Therefore, once available, this technology is expected to further clarify the picture of the most infectious cases. The earlier the detection of these cases, the lesser the transmission and the smaller the reservoir of LTBI, the seedbed of future cases. + +Table 3 Broad-spectrum antibiotic use, epidemiological risk, reactivation risk and laboratory features in patients with + +pulmonary tuberculosis by smear status + +| pulmonary tuberculosis by smear status | Smear status | | | | | +|----------------------------------------------------------------------------------------------------------------------------|------------------------------------------------|------------|------------|------------|--------| +| Total | | | | | | +| Characteristics | n | Positive | Negative | | | +| n (%) | n (%) | P value* | | | | +| Assessed, n | 313 | 151 | 162 | - | | +| Antibiotic use† Yes | 124 | 86 (56.9) | 38 (23.5) | <0.001 | | +| | No | 189 | 65 (43.1) | 124 (76.5) | | +| Epidemiological risk‡ Yes | 301 | 144 (95.4) | 157 (96.9) | 0.48 | | +| | No | 12 | 7 (4.3) | 5 (3.1) | | +| Reactivation risk§ Yes | 23 | 9 (5.9) | 14 (8.6) | 0.35 | | +| | No | 290 | 142 (94.1) | 148 (91.4) | | +| Complete blood count¶ Normal haemoglobin; leucocytosis** | 8 | 7 (4.6) | 1 (0.6) | 0.03 | | +| | Normal haemoglobin; normal/low leucocyte count | 139 | 46 (30.4) | 93 (57.4) | <0.001 | +| | Anaemia and leucocytosis** | 36 | 25 (16.5) | 11 (6.7) | 0.01 | +| | Anaemia and normal/low leucocyte count | 130 | 73 (48.5) | 57 (35.3) | 0 .01 | +| Chest radiographs Typical appearance†† | 176 | 118 (78.1) | 58 (35.8) | <0.001 | | +| | Bilateral‡‡ | 118 | 84 (55.6) | 34 (21.1) | <0.001 | +| | Cavitary | 103 | 93 (61.5) | 10 (6.1) | <0.001 | +| | Extent: moderate to far-advanced disease§§ | 159 | 123 (81.4) | 36 (22.2) | <0.001 | +| *Difference in proportions [(smear-positive)−(smear-negative)]; sample size was adequate to detect observable differences. | | | | | | + +*Difference in proportions [(smear-positive)−(smear-negative)]; sample size was adequate to detect observable differences. †Refers to broad-spectrum antibiotic use in the 6months preceding the date of diagnosis (the start date of treatment). ‡Epidemiological risk for exposure to *Mycobacterium tuberculosis*. §Moderate to high-risk factors for the reactivation of tuberculosis in those with latent tuberculosis (TB) infection, according to the Canadian Tuberculosis Standards. ¶The reference range for a normal haemoglobin is 135–175g/L and 120–160g/L for males and females, respectively. The reference range for a normal leucocyte count is 4.0–11.0x109/L. Anaemia and leucocytosis among smear-positive cases as compared with smear-negative cases (65% vs 42% and 21% vs 7%) were significant at p≤0.05, using Pearson's χ2 test. **Fisher's exact test and χ2 test used to test for differences. + +††Typical=upper lung zone predominant infiltrate, with or without cavitation, but no discernable adenopathy. ‡‡Of the 176 cases without bilateral disease, 27 had a normal chest X-ray (CXR): 25 smear negative and 2 smear positive. One of the 25 smear-negative cases with a 'normal' CXR had evidence of intrathoracic adenopathy and normal-appearing lung parenchyma. §§Includes three cases with a miliary pattern (see text). + +It is a common misconception that smear-negative disease is early disease, and smear-positive disease is late disease and that the extent of disease on chest radiograph is an indication of its duration, that is, that a minimal lesion is early disease and a moderate or advanced lesion is old or chronic disease. Years ago, Kurt Toman used data from longitudinal studies, and periodic X-ray surveys of large populations to dispel this idea.27 He concluded that both types of disease—advanced smear-positive TB and minimal, only culture-positive TB—usually developed within the same time frame; thus, making a diagnosis at first presentation an important piece of the elimination puzzle. A recent proof-of-concept study supports his findings of different phenotypic expressions of disease.20 The proportion of smear-positive and smear-negative patients in our study that were asymptomatic (5% and 37%, respectively) is remarkably similar to what he reported across diverse ethnic groups many years ago.36–38 The strengths of this study include the completeness of the data, made possible by the use of an assessment form and the centralised nature of the programme, and the validation of key variables, for example, the attribution of smear status, radiographic features and broad-spectrum antibiotic prescriptions. Consecutive patients in the jurisdiction and time period of the study were included. This may be considered both a strength (reflecting programmatic realities and an unfiltered comparison of smearpositive and smear-negative disease) and a weakness +(the pathway to diagnosis in smear-negative cases may be through active vs passive case finding activities such as Table 4 Multivariable multinomial regression showing the associations between demographic and clinical characteristics to a combined smear, and radiographic outcome + +| Age (years) Sex Population group Absence of dyspnoea Duration of symptoms* Antibiotics† Epidemiological risk§ Reactivation risk¶ CBC** Demographic and clinical characteristics | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Atypical (base) Typical Atypical **Typical** n (%) n (%) RRR (95%CI) n (%) RRR (95%CI) n (%) **RRR (95%CI)** + +Assessed, n 51 111 33 118 Age (years) >64 15 (23.1) 19 (29.2) 1.00 14 (21.5) 1.00 17 (26.2) 1.00 15–64 36 (14.5) 92 (37.1) 1.68 (0.71 to 3.92) 19 (7.6) 0.56 (0.20 to 1.55) 101 (40.7) 2.41 (1.01 to 5.74) Sex Female 22 (15.7) 53 (37.9) 1.00 13 (9.3) 1.00 52 (37.1) 1.00 Male 29 (16.8) 58 (33.5) 0.77 (0.38 to 1.56) 20 (11.6) 1.15 (0.45 to 2.93) 66 (38.2) 0.90 (0.44 to 1.83) Population group Canadian born 12 (13.8) 21 (24.1) 1.00 8 (9.2) 1.00 46 (52.9) 1.00 Foreign born 39 (17.3) 90 (40.0) 1.00 (0.42 to 2.37) 25 (11.0) 1.09 (0.36 to 3.32) 72 (31.7) 0.40 (0.17 to 0.92) Absence of dyspnoea No 15 (23.4) 7 (11.0) 1.00 16 (25.0) 1.00 26 (40.6) 1.00 Yes 36 (14.4) 104 (41.8) 4.71 (1.69 to 13.18) 17 (6.8) 0.59 (0.22 to 1.60) 92 (37.0) 1.85 (0.79 to 4.34) Duration of symptoms* <2 weeks 18 (19.5) 56 (60.9) 1.00 3 (3.3) 1.00 15 (16.3) 1.00 ≥2weeks 33 (14.9) 55 (24.9) 0.68 (0.31 to 1.75) 30 (13.6) 3.86 (0.93 to 15.92) 103 (46.6) 2.93 (1.20 to 7.11) Antibiotics† No 35 (18.5) 89 (47.1) 1.00 13 (6.9) 1.00 52 (27.5) 1.00 Yes‡ 16 (12.9) 22 (17.7) 0.74 (0.31 to 1.75) 20 (16.1) 2.14 (0.79 to 5.82) 66 (53.2) 1.76 (0.79 to 3.89) Epidemiological risk§ None/low 24 (16.3) 45 (30.6) 1.00 17 (11.6) 1.00 61 (41.5) 1.00 Moderate to high 27 (16.3) 66 (39.8) 0.89 (0.42 to 1.87) 16 (9.6) 1.20 (0.46 to 3.11) 57 (34.3) 0.77 (0.36 to 1.61) Reactivation risk¶ None/low 44 (15.2) 104 (35.9) 1.00 28 (9.7) 1.00 114 (39.3) 1.00 Moderate to high 7 (30.4) 7 (30.4) 0.43 (0.13 to 1.43) 5 (21.7) 1.24 (0.32 to 4.80) 4 (17.4) 0.18 (0.04 to 0.79) CBC** Low risk 5 (11.4) 7 (15.9) 1.00 5 (11.4) 1.00 27 (61.4) 1.00 High risk 46 (17.1) 104 (38.7) 1.26 (0.36 to 3.28) 28 (10.4) 0.64 (0.15 to 2.61) 91 (33.8) 0.49 (0.16 to 1.48) + +*The presence of 'symptoms' and the 'duration of symptoms' were considered collinear—here we report just on the duration of symptoms. †One or more broad-spectrum antibiotics prescribed in the 6months prior to diagnosis. ‡One or more broad-spectrum antibiotic prescription(s) within six months of the PTB diagnosis with no, or a limited and unsustained + +therapeuticbenefit. + +§Epidemiological risks none/low=no known occupational risk, travel history, migration from endemic nation, place of residence in a high-incidence indigenous community, recent contact with a known TB case plus no history of TB; OR, a history of TB/latent TB infection (LTBI) that has been adequately treated. Moderate to high=occupational risk, travel history, migration from endemic nation, place of residence in a high-incidence + +indigenous community, recent contact with a known tuberculosis (TB) case and/or a positive tuberculin skin test (TST)/interferon gamma release + +assay (IGRA) or a history of TB that was inadequately treated or with no record of treatment. ¶Per the Canadian Tuberculosis Standards, 7th Edition. **Low risk=combined normal haemoglobin; leucocytosis and anaemia; leucocytosis. High risk=combined normal haemoglobin; normal/low + +leucocyte count and anaemia; normal/low leucocyte count. + +| Smear negative | Smear positive | | | | | | | +|---------------------------------------------------------------------------------------------------------------------------------------|------------------|-------------|----------------------|-------------|----------------------|-------------|---------------------| +| Atypical (base) | Typical | Atypical | Typical | | | | | +| n (%) | n (%) | RRR (95%CI) | n (%) | RRR (95%CI) | n (%) | RRR (95%CI) | | +| Assessed, n | 51 | 111 | 33 | 118 | | | | +| >64 | 15 (23.1) | 19 (29.2) | 1.00 | 14 (21.5) | 1.00 | 17 (26.2) | 1.00 | +| 15–64 | 36 (14.5) | 92 (37.1) | 1.68 (0.71 to 3.92) | 19 (7.6) | 0.56 (0.20 to 1.55) | 101 (40.7) | 2.41 (1.01 to 5.74) | +| Female | 22 (15.7) | 53 (37.9) | 1.00 | 13 (9.3) | 1.00 | 52 (37.1) | 1.00 | +| Male | 29 (16.8) | 58 (33.5) | 0.77 (0.38 to 1.56) | 20 (11.6) | 1.15 (0.45 to 2.93) | 66 (38.2) | 0.90 (0.44 to 1.83) | +| Canadian born | 12 (13.8) | 21 (24.1) | 1.00 | 8 (9.2) | 1.00 | 46 (52.9) | 1.00 | +| Foreign born | 39 (17.3) | 90 (40.0) | 1.00 (0.42 to 2.37) | 25 (11.0) | 1.09 (0.36 to 3.32) | 72 (31.7) | 0.40 (0.17 to 0.92) | +| No | 15 (23.4) | 7 (11.0) | 1.00 | 16 (25.0) | 1.00 | 26 (40.6) | 1.00 | +| Yes | 36 (14.4) | 104 (41.8) | 4.71 (1.69 to 13.18) | 17 (6.8) | 0.59 (0.22 to 1.60) | 92 (37.0) | 1.85 (0.79 to 4.34) | +| <2 weeks | 18 (19.5) | 56 (60.9) | 1.00 | 3 (3.3) | 1.00 | 15 (16.3) | 1.00 | +| ≥2weeks | 33 (14.9) | 55 (24.9) | 0.68 (0.31 to 1.75) | 30 (13.6) | 3.86 (0.93 to 15.92) | 103 (46.6) | 2.93 (1.20 to 7.11) | +| No | 35 (18.5) | 89 (47.1) | 1.00 | 13 (6.9) | 1.00 | 52 (27.5) | 1.00 | +| Yes‡ | 16 (12.9) | 22 (17.7) | 0.74 (0.31 to 1.75) | 20 (16.1) | 2.14 (0.79 to 5.82) | 66 (53.2) | 1.76 (0.79 to 3.89) | +| None/low | 24 (16.3) | 45 (30.6) | 1.00 | 17 (11.6) | 1.00 | 61 (41.5) | 1.00 | +| Moderate to high | 27 (16.3) | 66 (39.8) | 0.89 (0.42 to 1.87) | 16 (9.6) | 1.20 (0.46 to 3.11) | 57 (34.3) | 0.77 (0.36 to 1.61) | +| None/low | 44 (15.2) | 104 (35.9) | 1.00 | 28 (9.7) | 1.00 | 114 (39.3) | 1.00 | +| Moderate to high | 7 (30.4) | 7 (30.4) | 0.43 (0.13 to 1.43) | 5 (21.7) | 1.24 (0.32 to 4.80) | 4 (17.4) | 0.18 (0.04 to 0.79) | +| Low risk | 5 (11.4) | 7 (15.9) | 1.00 | 5 (11.4) | 1.00 | 27 (61.4) | 1.00 | +| High risk | 46 (17.1) | 104 (38.7) | 1.26 (0.36 to 3.28) | 28 (10.4) | 0.64 (0.15 to 2.61) | 91 (33.8) | 0.49 (0.16 to 1.48) | +| *The presence of 'symptoms' and the 'duration of symptoms' were considered collinear—here we report just on the duration of symptoms. | | | | | | | | + +immigration medical surveillance).39 A limitation of this study includes the relatively small cohort, which is typical in low-incidence settings. A second limitation includes the retrospective study design, which affects the interpretation of the strength of relationship between clinical features and outcomes, though collection of the data evaluated in this study is by protocol and we are confident in their accuracy. This study describes infectious TB cases in a highincome, low-incidence country where the disease is increasingly uncommon. We anticipate this description will contribute to the prompt submission of specimens for + +![7_image_0.png](7_image_0.png) + +Figure 1 Smear-positive pulmonary tuberculosis (TB) cases with typical chest radiographs. This figure describes a diagnostic loop with two entry points: (1) report of one or more classical pulmonary tuberculosis symptoms, and (2) report of a chest radiograph abnormality consistent with adult-type pulmonary tuberculosis. Proceeding clockwise, the percentages describe the proportion of patients with one or more classical symptoms who had each of the other features shown; proceeding counterclockwise the percentages describe the proportion of patients who had a chest radiograph abnormality consistent with adult-type pulmonary tuberculosis with each of the other features. CXR, chest X-ray. + +confirmation by mycobacteriological testing. The prompt recognition of these cases is possible but takes a collaborative effort that includes front-line providers. Such input is understood to be a high-income, low-incidence country effort to follow the WHO/Stop TB Partnership roadmap to engage all care providers in TB prevention and care.11 Limiting the potential for transmission will ultimately aid TB elimination efforts. Electronic recording of the readily available clinical features proposed in this work along with computer-aided detection of adult-type PTB on chest radiographs could conceivably automate this process. + +Acknowledgements The authors are very grateful to the staff of the Edmonton TB Clinic, the Provincial TB Clinic and the Tuberculosis Program Evaluation and Research Unit for assistance in assembling the data presented in this study. Contributors CH and RL conceived this study. CH analysed and interpreted the data and provided initial drafts. JB interpreted the radiographs and was involved in critical review. AD and AS provided assistance with statistical methods and critical reviews. MLE and CP abstracted and collated relevant clinical data and provided critical reviews. RL interpreted the chest radiographs and provided expert oversight and critical reviews for all the drafts. Funding An Alberta Lung Association/Respiratory Health Strategic Clinical Health Network Studentship, and the University of Alberta Pulmonary Research Group award provided financial support to the lead author for her studies, of which this work represents one part. Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. + +Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available upon reasonable request. All data are available upon reasonable request made to the corresponding author. Subsequent secondary analyses will be subject to appropriate REB approvals as necessary. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iD Courtney Heffernan http://orcid.org/0000-0003-1241-7201 + +## References + +1 Behr MA, Warren SA, Salamon H, *et al*. Transmission of Mycobacterium tuberculosis from patients smear-negative for acidfast bacilli. *Lancet* 1999;353:444–9. + +2 Hernández-Garduño E, Cook V, Kunimoto D, *et al*. Transmission of tuberculosis from smear negative patients: a molecular epidemiology study. *Thorax* 2004;59:286–90. + +3 Tostmann A, Kik SV, Kalisvaart NA, *et al*. Tuberculosis transmission by patients with smear-negative pulmonary tuberculosis in a large cohort in the Netherlands. *Clin Infect Dis* 2008;47:1135–42. + +4 Lau A, Barrie J, Winter C, *et al*. Chest radiographic patterns and the transmission of tuberculosis: implications for automated systems. + +PLoS One 2016;11:e0154032. + +5 Catanzaro A. Nosocomial tuberculosis. *Am Rev Respir Dis* 1982;125:559–62. + +6 Bailey WC, Gerald LB, Kimerling ME, *et al*. Predictive model to identify positive tuberculosis skin test results during contact investigations. *JAMA* 2002;287:996–1002. + +7 Golub JE, Bur S, Cronin WA, *et al*. Delayed tuberculosis diagnosis and tuberculosis transmission. *Int J Tuberc Lung Dis* 2006;10:24–30. + +8 Long R. Making a timely diagnosis of pulmonary tuberculosis. *Can* Respir J 2015;22:317–21. + +9 Long R, Heffernan C, Gao Z, *et al*. Do "virtual" and "outpatient" public health tuberculosis clinics perform Equally Well? A ProgramWide Evaluation in Alberta, Canada. *PLoS One* 2015;10:e0144784. + +10 Heffernan C, Doroshenko A, Egedahl ML, *et al*. Predicting pulmonary tuberculosis in immigrants: a retrospective cohort study. *ERJ Open* Res 2018;4. doi:10.1183/23120541.00170-2017. [Epub ahead of print: 20 Apr 2018]. + +11 World Health Organization and STOP TB Partnership. PublicPrivate mix for TB prevention and care: a roadmap. WHO/CDS/ +TB/2018.32. Geneva, Switzerland: WHO, 2018. http://www.who.int/ tb/publications/2018/ppmroadmap 12 Small PM, McClenny NB, Singh SP, *et al*. Molecular strain typing of *Mycobacterium tuberculosis* to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. *J Clin Microbiol* 1993;31:1677–82. + +13 van't Hoog AH, Meme HK, Laserson KF, *et al*. Screening strategies for tuberculosis prevalence surveys: the value of chest radiography and symptoms. *PLoS One* 2012;7:e38691. + +14 Wisnivesky JP, Kaplan J, Henschke C, *et al*. Evaluation of clinical parameters to predict *Mycobacterium tuberculosis* in inpatients. Arch Intern Med 2000;160:2471–6. + +15 Samb B, Henzel D, Daley CL, *et al*. Methods for diagnosing tuberculosis among in-patients in eastern Africa whose sputum smears are negative. *Int J Tuberc Lung Dis* 1997;1:25–30. + +16 Long R, Maycher B, Dhar A, *et al*. Pulmonary tuberculosis treated with directly observed therapy: serial changes in lung structure and function. *Chest* 1998;113:933–43. + +17 The Canadian Lung Association and the Public Health Agency of Canada. *The Canadian tuberculosis standards*. 7th edn, 2013. http:// +www.publichealth.gc.ca/tuberculosis 18 Morris CD. The radiography, haematology and biochemistry of pulmonary tuberculosis in the aged. *Q J Med* 1989;71:529–3. + +19 Morris CD, Bird AR, Nell H. The haematological and biochemical changes in severe pulmonary tuberculosis. *Q J Med* 1989;73:1151–9. + +20 Long R, Asadi L, Heffernan C, *et al*. Is there a fundamental flaw in Canada's post-arrival immigrant surveillance system for tuberculosis? *PLoS One* 2019;14:e0212706. + +21 Falk A, O'Connor JB, Pratt PC, *et al*. Classification of pulmonary tuberculosis. In: Diagnostic standards and classification of tuberculosis. 12th edn. New York: National Tuberculosis and Respiratory Disease Association, 1969: 68–76. + +22 Cohen J. *Statistical power analysis for the behavioral sciences*. 2nd edn. Hillside, NJ: Lawrence Erlbaum Associates, 1998. + +23 Perez-Guzman C, Torres-Cruz A, Villarreal-Velarde H, *et al*. + +Progressive age-related changes in pulmonary tuberculosis images and the effect of diabetes. *Am J Respir Crit Care Med* 2000;162:1738–40. + +24 Chan CH, Woo J, Or KK, *et al*. The effect of age on the presentation of patients with tuberculosis. *Tuber Lung Dis* 1995;76:290–4. + +25 Cohen R, Muzaffar S, Capellan J, *et al*. The validity of classic symptoms and chest radiographic configuration in predicting pulmonary tuberculosis. *Chest* 1996;109:420–3. + +26 Al-Tawfiq JA, Saadeh BM. Radiographic manifestations of culturepositive pulmonary tuberculosis: cavitary or non-cavitary? Int J +Tuberc Lung Dis 2009;13:367–70. + +27 Toman K. *Tuberculosis: case finding and chemotherapy: questions* and answers. Geneva: World Health Organization, 1979. + +28 Phoa L-L, Teleman MD, Wang Y-T, *et al*. Characteristics of patients with delayed diagnosis of infectious pulmonary tuberculosis. Respirology 2005;10:196–200. + +29 Jones BE, Young SM, Antoniskis D, *et al*. Relationship of the manifestations of tuberculosis to CD4 cell counts in patients with human immunodeficiency virus infection. *Am Rev Respir Dis* 1993;148:1292–7. + +30 Skogberg K, Ruutu P, Tukiainen P, *et al*. Effect of immunosuppressive therapy on the clinical presentation and outcome of tuberculosis. Clin Infect Dis 1993;17:1012–7. + +31 Kiyan E, Kilicaslan Z, Gurgan M, *et al*. Clinical and radiographic features of pulmonary tuberculosis in nonAIDS immunocompromised patients. *Int J Tuberc Lung Dis* 2003;7:764–70. + +32 Wilcke JT, Askgaard DS, Nybo Jensen B, *et al*. Radiographic spectrum of adult pulmonary tuberculosis in a developed country. Respir Med 1998;92:493–7. + +33 Lee H-S, Kee S-J, Shin J-H, *et al*. Xpert MTB/RIF assay as a substitute for smear microscopy in an intermediate-burden setting. + +Am J Respir Crit Care Med 2019;199:784–94. + +34 Van Deun A, Tahseen S, Affolabi D, *et al*. Sputum smear microscopy in the Xpert® MTB/RIF era. *Int J Tuberc Lung Dis* 2019;23:12–18. + +35 Jones-López EC, Namugga O, Mumbowa F, *et al*. Cough aerosols of Mycobacterium tuberculosis predict new infection: a household contact study. *Am J Respir Crit Care Med* 2013;187:1007–15. + +36 Banerji D, Andersen S. A sociological study of awareness of symptoms among persons with pulmonary tuberculosis. Bull World Health Organ 1963;29:665–83. + +37 Styblo K, Dankova D, Drepela J, *et al*. Epidemiological and clinical study of tuberculosis in the district of Kalin, Czechoslovakia. *Bull* WHO 1967;37:819–74. + +38 Shimao T. A study of the mode of detection of newly registered pulmonary tuberculosis patients with special reference to their symptoms. In: Reports on medical research problems of the Japan anti-tuberculosis association. , 1974: 21, 17. + +39 Ward HA, Marciniuk DD, Pahwa P, *et al*. Extent of pulmonary tuberculosis in patients diagnosed by active compared to passive case finding. *Int J Tuberc Lung Dis* 2004;8:593–7. \ No newline at end of file diff --git a/medical/md/PMC7592778.md b/medical/md/PMC7592778.md new file mode 100644 index 0000000000000000000000000000000000000000..efe2ecf4c94bca7f88f8df6c7100dfb1a49bd2cb --- /dev/null +++ b/medical/md/PMC7592778.md @@ -0,0 +1,211 @@ +# Traf4 Enhances Osteosarcoma Cell Proliferation And Invasion By Akt Signaling Pathway Weitao Yao, Xin Wang, Qiqing Cai, Songtao Gao, Jiaqiang Wang, And Peng Zhang + +Department of Bone and Soft Tumor, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou, China TRAF4, or tumor necrosis factor receptor-associated factor 4, is overexpressed in several cancers, suggesting a specific role in cancer progression. However, its functions in osteosarcoma are unclear. This study aimed to explore the expression of TRAF4 in osteosarcoma tissues and cells, the correlation of TRAF4 to clinical pathology of osteosarcoma, as well as the role and mechanism of TRAF4 in osteosarcoma metastasis. The protein expression levels of TRAF4 in osteosarcoma tissues and three osteosarcoma cell lines, MG-63, HOS, and U2OS, were assessed. Constructed TRAF4 overexpression vectors and established TRAF4 overexpression of the U2OS cell line. Cell proliferation, cell invasion, protein levels, and TRAF4 phosphorylations were assessed following TRAF4 transfection, as well as the effects of TRAF4 siRNA on cell proliferation and invasion. The results show that TRAF4 protein levels in osteosarcoma tissues were significantly higher than that in normal bone tissues. Importantly, an obvious upregulation of TRAF4 was found in carcinoma tissues from patients with lung metastasis compared with patients without lung metastasis. Consistently, a similar increase in TRAF4 mRNA and protein was also demonstrated in the osteosarcoma cell lines MG-63, HOS, and U2OS compared to normal bone cells, hFOB1.19. When TRAF4 was overexpressed in U2OS cells, cell proliferation was significantly enhanced, accompanied by an increase in Ki67 expression and colony formation. Compared with the control and vector-treated groups, TRAF4 transfection increased the invasion potential of U2OS cells (p < 0.05). + +Interestingly, TRAF4 transfection significantly enhanced the phosphorylation of Akt. After blocking Akt with its specific siRNA, TRAF4-induced cell proliferation and invasion were dramatically attenuated. In summary, our findings demonstrated that TRAF4 enhances osteosarcoma cell proliferation and invasion partially by the Akt pathway. This work suggests that TRAF4 might be an important target in osteosarcoma. Key words: Osteosarcoma; TRAF4; Cell proliferation; Cell invasion; Akt signaling pathway Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, with a peak incidence at the age of 15–19 years (1,2). Osteosarcoma is strongly invasive and has a high tendency to metastasize early (3). Unfortunately, treating metastatic osteosarcoma is a challenge in oncology and is the major cause of treatment failure and death (4). + +Tumor necrosis factor receptor-associated factors +(TRAFs) were initially discovered as adaptor proteins that regulate cell life and death (5). So far, seven members (TRAF1-7) have been identified, and each member shares a common structural domain at the C-terminus (5,6). TRAFs form a complex network in which each TRAF is able to interact with and influence the function of many signaling molecules, either directly or through interactions with other TRAF family members (6). TRAF4 is a unique member of the TRAF protein family (7); it was initially identified in breast carcinoma (8). TRAF4 is required during embryogenesis in key biological processes including the formation of the trachea, the development of the axial skeleton, and the closure of the neural tube (9). TRAF4 deficiency leads to severe developmental alterations (10). TRAF4 is likely to be involved in signal transduction as it directly or indirectly mediates the downstream signal molecules. For instance, TRAF4 downregulates innate immune responses through its direct binding to NOD2 and inhibits NOD2-induced NF- B activation (11) and restricts IL-17-mediated pathology and signaling processes (12). TRAF4 stimulates MEKK4 kinase activity to regulate JNK activation (13). + +Recently, TRAF4 has been considered as an oncogene as it is overexpressed in a wide range of human malignancies, including breast cancer, lung cancer, colon adenocarcinomas, melanomas, neurogenic tumors, and lymphomas +(14). Accumulating evidence indicates that TRAF4 plays critical role in breast cancer, such as antiapoptotic (15), and promotes cell migration (16). It has been reported that TRAF4 interacts with PIPs and acts as a negative regulator of tight junctions and increases cell migration +(17). Moreover, TRAF4 promotes TGF- receptor signaling and drives breast cancer metastasis (18) and TRAF4 overexpression-induced degradation of p53 leading to poor prognosis in breast cancer patients (19). TRAF4 is also involved in maintaining tumorigenic properties in lung cancer; TRAF4 is required for EGF-induced Akt activation, and its deficiency markedly impairs the activity of Akt signaling and Akt-mediated lung cancer glycolysis (20). However, few studies have focused on the function of TRAF4 in osteosarcoma. + +In the present study, we aimed to assess the expression of TRAF4 in osteosarcoma tissue and cell lines, that is, MG-63, HOS, and H2OS. The role and the underlying mechanism of TRAF4 in osteosarcoma cell proliferation and invasion were also explored. + +MATERIALS AND METHODS + +## Specimens + +Fresh osteosarcoma tissue specimens were collected from 62 patients who underwent osteosarcoma resection operation in our hospital between July 2012 and March 2014. In addition, 22 normal bone tissue specimens were collected. Patients with osteosarcoma were matched for gender, age, and lung metastasis (Table 1). The patients with osteosarcoma included 42 males and 20 females; 44 cases were younger than 23 years old, while 20 patients were older than 23 years old. Without any preoperative treatment, all 36 cases were pathologically diagnosed with osteosarcoma postoperatively. There were 41 cases with lung metastases, while 21 others had no metastases. + +The specimens were preserved in liquid nitrogen immediately for subsequent testing. The specimen collection procedure was performed with informed patient consent and the approval of the Medical Ethics Committee. Cell Lines and Cell Culture + +## The Immortalized Human Osteoblast Cell Line Hfob1.19 And The Human Osteosarcoma Cell Lines Mg-63, Hos, + +and U2OS were purchased from American Type Culture Collection (ATCC). Three osteosarcoma cell lines (MG63, U2OS, and HOS) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin at 37°C in a humidified 5% CO2 incubator. The immortalized human osteoblast cell line (hFOB1.19) was grown in DMEM/Ham's F-12 containing 10% and geneticin (400 mg/ml) at a temperature of 34°C. These cells were maintained in the appropriate medium and passaged every 3 days. Construction of Expression Vectors and Cell Transfection Total RNA from U2OS cells were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), based on the manufacturer's instructions. The cDNA was synthesized by reverse transcription of total RNA, using the PrimeScript® RT reagent kit (Takara, Dalian, China) with oligo-dT primers, according to the manufacturer's protocol. Then, the open reading frame of TRAF4 cDNA was cloned and inserted into the pcDNA3.1 vector (Invitrogen) to construct the recombinant pcDNA3.1–TRAF4 expression vector. Control cells and pcDNA3.1 vector-transfected cells were also prepared. For cell transfection, cells were cultured to 60% confluence, and transfection was performed using the FuGENE HD transfection reagent (Roche, Indianapolis, IN, USA) method, as suggested by the manufacturer. siRNA Transfection Akt siRNA and control siRNA (con-siRNA) were purchased from Cell Signaling (Beverly, MA, USA). For transfection, 5 × 104 cells were seeded in each cell of 24well microplates, grown for 24 h to reach 30–50% confluence, and then incubated with a mixture of siRNA and Lipofectamine 2000 reagent (Invitrogen) in 100 ml of serum-free OPTI-MEM according to the manufacturer's instructions. The transfection efficiency was examined by real-time PCR and Western blotting. Western Blotting The proteins were extracted from tissue and cells using RIPA lysis buffer (Beyotime, Nantong, China). The protein Table 1. Correlation of TRAF4 Expression With Pathologic Parameter of Osteosarcoma + +| Pathologic Parameter | Case | Positive Rate (%) | 2 | p | +|------------------------|--------|---------------------|--------|-------| +| Normal tissues | 22 | 2 (9.10%) | 31.466 | 0.000 | +| Carcinoma tissues | 62 | 48 (77.42%) | | | +| Gender Male | 42 | 32 (76.19%) | 0.010 | 0.919 | +| Female | 20 | 15 (75.00%) | | | +| Age <23 | 44 | 34 (77.27%) | 0.388 | 0.533 | +| >23 | 20 | 14 (70.00%) | | | +| Lung metastasis | 41 | 38 (92.69%) | 11.038 | 0.001 | +| Nonmetastasis | 21 | 12 (57.14%) | | | + +concentration in the lysates was determined using a BCA protein assay kit (Beyotime, Nantong, China). Western blotting was performed according to standard methods. + +Proteins were separated by SDS-PAGE and then transferred onto nitrocellulose membranes (GE Healthcare, Germany) in a semidry transblot apparatus using transfer buffer. Membranes were blocked with 10% defatted milk in PBS at 4°C overnight and incubated with the primary antibodies for 60 min. The primary antibodies are as follows: mouse anti-TRAF4 antibody (Santa Cruz, CA, USA), mouse anti- -actin antibody (KangChen Bio-tech, Shanghai, China), rabbit anti-Akt antibody, and rabbit anti-phospho-Akt (p-Ser473) antibody (Cell Signaling Technology, Danvers, MA, USA). After washing with TBST buffer, the membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (KangChen Bio-tech, Shanghai, China). ECL +reagent (Beyotime) was used for detection. All experiments were performed in triplicate, and the results were normalized according to -actin. Real-Time Quantitative PCR +Total RNA and cDNA was obtained as described above. Real-time quantitative PCR (RT-PCR) reactions were performed on a Bio-Rad iQ5 real-time thermal cycler using SYBR® Premix Ex Taq™ II kit (Takara, Dalian, China). The PCR primers specific for the TRAF4 was designed as previously reported (21): 5 -AGGAG TTCGTCTTTGACACCATC-3 (forward), 5 -CTTTGA ATGGGCAGAGCACC-3 (reverse); those for -actin were 5 -CAACTTGATGTATGAAGGCTTTGGT-3 (forward) and 5 -ACTTTTATTGGTCTCAAGTCAGTGTA +CAG-3 (reverse). These primers were all synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The PCR procedure was as follows: polymerase activation for 30 s at 95°C; 40 cycles of amplification each consisting of 95°C for 5 s and 60°C for 20 s; and one cycle of dissociation consisting of 95°C for 15 s, 60°C for 30 s, and 95°C +for 15 s. All reactions were performed in triplicate, and the results were represented as the relative mRNA expression calculated according to the 2−DDCT method (22). + +MTT Assay Cell proliferation was determined by the MTT assay. + +Cells were seeded into 96-well plates, washed twice with PBS, and 10 µl of MTT was added to each well. Then, the cells were incubated at 37°C for 2 h, and 100 µl DMSO was added to dissolve the formazan crystals. Absorbance was measured at 560 nm with a SpectraMax Paradigm Multi-Mode Reader (Molecular Devices, Austria). Soft Agar Assay + +## The Soft Agar Assay Was Performed According To A Previous Report (23). The Control Group, The Vector-Transfected + +group, and the TRAF4-transfected group cells were evenly seeded into 100-mm petri dishes with soft agar. The cells were allowed to grow for 3 weeks until colonies formed. Colonies were stained with crystal violet and counted under a microscope using OpenCFU 3.8-BETA software (24). Cell Invasion Assay The in vitro invasive properties of cells were evaluated using a Transwell chamber assay. According to the manufacturer's protocol, chambers coated with Matrigel (50 µl per filter) (BD, USA) are incubated at 37°C for 30 min to create a Matrigel membrane, and divide the chamber into upper and lower compartments. Serum-free DMEM was added to the upper chambers, and DMEM containing 10% fetal calf serum was added to the lower chambers. The control, the vector transfected, and the TRAF4-transfected cells were cultured for 48 h and transferred to the upper chambers (1 × 105 cells per Transwell). After 24 h of incubation, cells that had migrated to the lower surface were fixed in 90% alcohol and stained with 0.08% crystal violet. Statistical Analysis The SPSS version 19.0 software was used to analyze the related data with chi-square test or t test. The results were considered statistically significant if p < 0.05. + +RESULTS +The Expression of TRAF4 in Human Osteosarcoma Tissue To investigate whether TRAF4 was highly expressed in osteosarcoma tissue, we analyzed the protein level of TRAF4 in normal and different osteosarcoma patient tissues by Western blotting. The results showed that the TRAF4 expression level in osteosarcoma tissue was more than a 2.5-fold increase compared to normal bone tissue. Moreover, TRAF4 expression in osteosarcoma with lung metastatic tissue was more than twofold higher compared with tissue without lung metastases (Fig. 1). These results indicate that TRAF4 might be critical in osteosarcoma. TRAF4 Is Upregulated in Human Osteosarcoma Cell Lines To further verify the expression of TRAF4 in normal bone and osteosarcoma cells, we used RT-PCR to detect TRAF4 mRNA levels and Western blotting to analyze the protein levels in hFOB1.19, MG-63, HOS, and U2OS cell lines. RT-PCR analysis showed that the mRNA levels of TRAF4 in three osteosarcoma cell lines were higher than that in normal cells (Fig. 2A). Consistent with the results of mRNA levels, the protein levels in different osteosarcoma cells were higher than that in normal cells +(Fig. 2B). It is noteworthy that both the mRNA and protein levels of TRAF4 in U2OS cells were higher than those in MG-63 and HOS cells. + +![3_image_0.png](3_image_0.png) + +## Expression Levels Of Traf4 In Traf4-Transfected + +U2OS Cells On the basis of our observations that TRAF4 is more highly expressed in U2OS cells than other two osteosarcoma cell lines, MG-63 and HOS (Fig. 2), we generated TRAF4-overexpressing U2OS cells to investigate the function of TRAF4 in osteosarcoma cells. After TRAF4 transfection, the mRNA and protein levels of TRAF4 were significantly increased (Fig. 3). Therefore, these results confirm that TRF4-overexpressing cells were successfully established. + +## The Effect Of Traf4 Overexpression On Cell Growth + +In order to investigate the role of TRAF4 in osteosarcoma cell growth, cell proliferation was assessed in TRAF4-transfected U2OS cells. The MTT analysis revealed that TRAF4-transfected cells possessed almost twofold higher cell proliferative ability than the other two groups +(p < 0.05) (Fig. 4A). No differences were detected between the control and vector control cells. Ki67 is a nuclear protein that is expressed in proliferating cells (25) +and may be required for maintaining cell proliferation. It had been used as a marker for cell proliferation in cancers + +![3_image_1.png](3_image_1.png) + +24 + +![4_image_0.png](4_image_0.png) + +including osteosarcoma (26–28). We further analyzed the Ki67 protein expression levels and revealed that TRAF4 overexpression promoted the Ki67 protein expression +(Fig. 4B). Additionally, colony formation was also detected by the soft agar assay. We found that U2OS cells transfected with TRAF4 yielded more colonies than the control and vector-transfected cells ( p < 0.05) (Fig. 4C). + +trol and vector-transfected groups. + +The Effect of TRAF4 Overexpression on Cell Invasion To further assess the function of TRAF4 on osteosarcoma cells, U2OS cell invasion was detected after TRAF4 transfection. As shown in Figure 5, forced + +![5_image_0.png](5_image_0.png) + +TRAF4 expression notably upregulated the number of invading cells from 120 to 197 compared to the control group. No difference was found between the control and vector groups (p < 0.05). Hence, these results indicate that TRAF4 is a positive regulator of osteosarcoma cell invasion. TRAF4 Enhances Osteosarcoma Cell Viability and Invasion Partially by the Akt Signaling Pathway The Akt signaling plays a major role in tumorigenesis by regulating cell proliferation, colony formation, cell cycle, cell survival, cell invasion, and metabolism (23,29–31). The fact that TRAF4 enhanced osteosarcoma cell growth and cell invasion was demonstrated in our previous results. To further investigate the underlying mechanism involved in this process, we investigated Akt signaling. Interestingly, the TRAF4-transfected cells exhibited higher Akt phosphorylation levels than the other two groups (p < 0.05) (Fig. 6A). To further explore the function of Akt signaling in TRAF4-induced osteosarcoma cell proliferation and invasion, Akt siRNA was used to knock down Akt level in U2OS cells. After silencing Akt signaling with its specific siRNA, the effect of TRAF4 on enhanced cell viability (p < 0.05) +(Fig. 6B) and invasion (p < 0.05) (Fig. 6C) was obviously decreased. These results indicate that TRAF4 enhances osteosarcoma cell proliferation and invasion partly through the Akt signaling pathway. + +DISCUSSION +Osteosarcoma is a leading malignant tumor in childhood and adolescence. It is a highly aggressive tumor that metastasizes to the lung (4). TRAF4 was first identified overexpressed in breast cancer in 1995 (8). In the following years, accumulating evidence indicated that TRAF4 is highly expressed in a range of human malignancies and tumors (32). However, its role and mechanism in osteosarcoma remains undefined. In this study, our results + +![5_image_1.png](5_image_1.png) showed that TRAF4 was highly expressed not only in osteosarcoma tissue but also in different osteosarcoma cell lines such as MG-63, HOS, and U2OS, indicating a potential critical role of TRAF4 in the development of osteosarcoma. + +Cell proliferation in cancer is associated with cancer development and prognosis. In order to explore the role of TRAF4 in osteosarcoma cell proliferation, we generated TRAF4-overexpressing U2OS cells and assessed cell proliferation. The results showed that TRAF4 overexpression dramatically promoted cell proliferation. We also detected the expression of protein Ki67, which is a marker for proliferating cells (33). Consistent with speculation, TRAF4 promoted Ki67 protein expression. Moreover, we also found that TRAF4 overexpression promoted colony formation. Our data demonstrate that TRAF4 plays an important role in promoting osteosarcoma cell growth. + +A common feature of osteosarcoma is that it metastasizes primarily to the lung (34). Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis (35). Thus, the invasion ability of tumor cells is closely related to tumor metastasis. Previous studies reported that TRAF4 promotes TGFreceptor signaling and drives breast cancer metastasis +(18); moreover, TRAF4 modulates tight junctions and promotes cell migration (17). Our results reveal that TRAF4 expression was higher in osteosarcoma tissue from a patient with lung metastases than in those without metastases, indicating that TRAF4 is a critical molecule during osteosarcoma metastasis. Further in vitro analysis confirmed that TRAF4 overexpression enhanced cell invasion, providing evidence of the positive role of TRAF4 in osteosarcoma metastasis. + +The serine/threonine kinase Akt is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli (30). As a key molecule of this pathway, Akt is relevant to cancer cell survival, proliferation (increased cell number), and growth (increased cell size) (30,36,37). To further clarify the underlying mechanism involved in TRAF4-induced osteosarcoma cell proliferation and invasion, we detected Akt phosphorylation after TRAF4 transfection. As expected, TRAF4 upregulation promoted the activation of the Akt signaling pathway. When this signaling was blocked with Akt siRNA, cell proliferation induced by TRAF4 was decreased. It has been reported that Akt can promote cell invasion in prostate cancer (38) and by increased motility and metalloproteinase production in the HT1080 cell line (39). + +To further explore the mechanism of TRAF4 in promoting osteosarcoma cell invasion, we analyzed Akt function on TAFR4-induced cell invasion. The results show that Akt siRNA impaired the TAFR4-dependent enhancement of osteosarcoma cell invasion. Together, these results suggest that TRAF4 enhances osteosarcoma cell proliferation and invasion partially by the Akt signaling pathway. + +In summary, TRAF4 was confirmed in this study to be overexpressed in human osteosarcoma. Furthermore, TRAF4 may facilitate osteosarcoma progression by enhancing osteosarcoma cell proliferation and invasion partially through the Akt signaling pathway. Our research suggests that TRAF4 plays a critical role in osteosarcoma and may serve as a potential target against osteosarcoma. + +ACKNOWLEDGMENT: The authors declare no conflicts of interest. + +REFERENCES +1. Vijayamurugan, N.; Bakhshi, S. Review of management issues in relapsed osteosarcoma. Expert Rev. Anticancer 14: 151–161; 2014. + +2. Bielack, S.; Carrle, D.; Hardes, J.; Schuck, A. Paulussen, M. Bone tumors in adolescents and young adults. Curr. Treat. Options Oncol. 9:67–80; 2008. + +3. Harting, M. T.; Blakely, M. L. Management of osteosarcoma pulmonary metastases. Semin. Pediatr. Surg. 15:25–29; 2006. + +4. Zwaga, T.; Bovee, J. V.; Kroon, H. M. Osteosarcoma of the femur with skip, lymph node, and lung metastases. Radiographics 28:277–283; 2008. + +5. Arch, R. H.; Gedrich, R. W.; Thompson, C. B. Tumor necrosis factor receptor-associated factors (TRAFs)—A family of adapter proteins that regulates life and death. Gene Dev. 12:2821–2830; 1998. + +6. Bradley, J. R.; Pober, J. S. Tumor necrosis factor receptorassociated factors (TRAFs). Oncogene 20:6482–6491; 2001. 7. Kedinger, V.; Rio, M. C. TRAF4, the unique family member. Adv. Exp. Med. Biol. 597:60–71; 2007. + +8. Regnier, C. H.; Tomasetto, C.; Moog-Lutz, C.; Chenard, M. + +P.; Wendling, C.; Basset, P.; Rio, M. C. Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma. J. Biol. Chem. 270:25715– 25721; 1995. + +9. Régnier, C. H.; Masson, R.; Kedinger, V.; Textoris, J.; Stoll, I.; Chenard, M.-P.; Dierich, A.; Tomasetto, C.; Rio, M.-C. Impaired neural tube closure, axial skeleton malformations, and tracheal ring disruption in TRAF4-deficient mice. Proc. Natl. Acad. Sci. USA 99:5585–5590; 2002. + +10. Shiels, H.; Li, X.; Schumacker, P. T.; Maltepe, E.; Padrid, P. A.; Sperling, A.; Thompson, C. B.; Lindsten, T. TRAF4 deficiency leads to tracheal malformation with resulting alterations in air flow to the lungs. Am. J. Pathol. 157: 679–688; 2000. + +11. Marinis, J. M.; Homer, C. R.; McDonald, C.; Abbott, D. W. + +A novel motif in the Crohn's disease susceptibility protein, NOD2, allows TRAF4 to down-regulate innate immune responses. J. Biol. Chem. 286:1938–1950; 2011. + +12. Zepp, J. A.; Liu, C.; Qian, W.; Wu, L.; Gulen, M. F.; Kang, Z.; Li, X. Cutting edge: TNF receptor-associated factor 4 restricts IL-17-mediated pathology and signaling processes. J. Immunol. 189:33–37; 2012. + +13. Abell, A. N.; Johnson, G. L. MEKK4 is an effector of the embryonic TRAF4 for JNK activation. J. Biol. Chem. 280: 35793–35796; 2005. + +14. Camilleri-Broet, S.; Cremer, I.; Marmey, B.; Comperat, E.; +Viguie, F.; Audouin, J.; Rio, M. C.; Fridman, W. H.; Sautes-Fridman, C.; Regnier, C. H. TRAF4 overexpression is a common characteristic of human carcinomas. Oncogene 26:142–147; 2006. + +15. Zhang, X.; Wen, Z.; Mi, X. Expression and anti-apoptotic function of TRAF4 in human breast cancer MCF-7 cells. Oncol. Lett. 7:411–414; 2014. + +16. Wang, X.; Jin, C.; Tang, Y.; Tang, L. Y.; Zhang, Y. E. + +Ubiquitination of tumor necrosis factor receptor-associated factor 4 (TRAF4) by smad ubiquitination regulatory factor + +TRAF4 ENHANCES OSTEOSARCOMA CELL PROLIFERATION AND INVASION 27 +1 (Smurf1) regulates motility of breast epithelial and cancer cells. J. Biol. Chem. 288:21784–21792; 2013. + +17. Rousseau, A.; McEwen, A. G.; Poussin-Courmontagne, P.; +Rognan, D.; Nomine, Y.; Rio, M. C.; Tomasetto, C.; Alpy, F. TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration. PLoS +Biol. 11:e1001726; 2013. + +18. Zhang, L.; Zhou, F.; Garcia de Vinuesa, A.; de Kruijf, E. M.; +Mesker, W. E.; Hui, L.; Drabsch, Y.; Li, Y.; Bauer, A.; Rousseau, A.; Sheppard, K. A.; Mickanin, C.; Kuppen, P. J.; Lu, C. X.; Ten Dijke, P. TRAF4 promotes TGF- receptor signaling and drives breast cancer metastasis. Mol. Cell 51: 559–572; 2013. + +19. Yi, P.; Xia, W.; Wu, R.-C.; Lonard, D. M.; Hung, M.-C.; +O'Malley, B. W. SRC-3 coactivator regulates cell resistance to cytotoxic stress via TRAF4-mediated p53 destabilization. Genes Dev. 27:274–287; 2013. + +20. Li, W.; Peng, C.; Lee, M. H.; Lim, D.; Zhu, F.; Fu, Y.; +Yang, G.; Sheng, Y.; Xiao, L.; Dong, X.; Ma, W.; Bode, A. + +M.; Cao, Y.; Dong, Z. TRAF4 is a critical molecule for Akt activation in lung cancer. Cancer Res. 73:6938–6950; 2013. + +21. Shen, J.; Qiao, Y.; Ran, Z.; Wang, T. Different activation of TRAF4 and TRAF6 in inflammatory bowel disease. Mediat. Inflamm. 647936:28; 2013. + +22. Schmittgen, T. D.; Livak, K. J. Analyzing real-time PCR +data by the comparative CT method. Nat. Protoc. 3: 1101–1108; 2008. + +23. Zhang, K.; Tian, F.; Zhang, Y.; Zhu, Q.; Xue, N.; Zhu, H.; +Wang, H.; Guo, X. MACC1 is involved in the regulation of proliferation, colony formation, invasion ability, cell cycle distribution, apoptosis and tumorigenicity by altering Akt signaling pathway in human osteosarcoma. Tumour Biol. 35(3):2537–2548; 2014. + +24. Geissmann, Q. OpenCFU, a new free and open-source software to count cell colonies and other circular objects. PLoS +One 8:e54072; 2013. + +25. Romero, Q.; Bendahl, P. O.; Klintman, M.; Loman, N.; +Ingvar, C.; Ryden, L.; Rose, C.; Grabau, D.; Borgquist, S. Ki67 proliferation in core biopsies versus surgical samples —A model for neo-adjuvant breast cancer studies. BMC Cancer 11:341; 2011. + +26. Hernández-Rodríguez, N. A.; Correa, E.; Sotelo, R.; +Contreras-Paredes, A.; Gomez-Ruiz, C.; Green, L.; Mohar, A. Ki-67: A proliferative marker that may predict pulmonary metastases and mortality of primary osteosarcoma. + +Cancer Detect. Prev. 25:210–215; 2001. + +27. Li, B.; Yang, Y.; Jiang, S.; Ni, B.; Chen, K.; Jiang, L. + +Adenovirus-mediated overexpression of BMP-9 inhibits human osteosarcoma cell growth and migration through downregulation of the PI3K/Akt pathway. Int. J Oncol. 41: 1809–1819; 2012. + +28. Urruticoechea, A.; Smith, I. E.; Dowsett, M. Proliferation marker Ki-67 in early breast cancer. J. Clin. Oncol. 23: 7212–7220; 2005. + +29. Brazil, D. P.; Park, J.; Hemmings, B. A. PKB binding proteins: Getting in on the Akt. Cell 111:293–303; 2002. + +30. Manning, B. D.; Cantley, L. C. Akt/PKB signaling: Navigating downstream. Cell 129:1261–1274; 2007. + +31. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Targeting the phosphoinositide 3-kinase pathway in cancer. Nat. Rev. Drug Discov. 8:627–644; 2009. + +32. Camilleri-Broet, S.; Cremer, I.; Marmey, B.; Comperat, E.; +Viguie, F.; Audouin, J.; Rio, M. C.; Fridman, W. H.; Sautes-Fridman, C.; Regnier, C. H. TRAF4 overexpression is a common characteristic of human carcinomas. Oncogene 26:142–147; 2007. + +33. Santisteban, M.; Reynolds, C.; Barr Fritcher, E. G.; Frost, M. H.; Vierkant, R. A.; Anderson, S. S.; Degnim, A. C.; Visscher, D. W.; Pankratz, V. S.; Hartmann, L. C. Ki67: A time-varying biomarker of risk of breast cancer in atypical hyperplasia. Breast Cancer Res. Tr. 121:431–437; 2010. + +34. Longhi, A.; Errani, C.; De Paolis, M.; Mercuri, M.; Bacci, G. Primary bone osteosarcoma in the pediatric age: State of the art. Cancer Treat. Rev. 32:423–436; 2006. + +35. Yamaguchi, H.; Wyckoff, J.; Condeelis, J. Cell migration in tumors. Curr. Opin. Cell Biol. 17:559–564; 2005. + +36. Vivanco, I.; Sawyers, C. L. The phosphatidylinositol 3-kinase-akt pathway in human cancer. Nat. Rev. Cancer 2: 489–501; 2002. + +37. Zhao, G.; Cai, C.; Yang, T.; Qiu, X.; Liao, B.; Li, W.; Ji, Z.; Zhao, J.; Zhao, H.; Guo, M.; Ma, Q.; Xiao, C.; Fan, Q.; Ma, B. MicroRNA-221 induces cell survival and cisplatin resistance through PI3K/Akt pathway in human osteosarcoma. Plos One 8:e53906; 2013. + +38. Shukla, S.; MacLennan, G. T.; Hartman, D. J.; Fu, P.; +Resnick, M. I.; Gupta, S. Activation of PI3K-Akt signaling pathway promotes prostate cancer cell invasion. Int. J. Cancer 121:1424–1432; 2007. + +39. Kim, D.; Kim, S.; Koh, H.; Yoon, S. O.; Chung, A. S.; +Cho, K. S.; Chung, J. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J. 15:1953–1962; 2001. \ No newline at end of file diff --git a/medical/md/PMC7718532.md b/medical/md/PMC7718532.md new file mode 100644 index 0000000000000000000000000000000000000000..ae2c9f41d5161075b39837df323a77632e3d9add --- /dev/null +++ b/medical/md/PMC7718532.md @@ -0,0 +1,340 @@ +# Scientific Article + +www.advancesradonc.org + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +Patterns of Failure After Adjuvant Stereotactic Body Radiation Therapy for Pancreatic Cancer With Close or Positive Margins Ankur K. Patel, MD,a Joshua L. Rodríguez-López, MD,a Nathan Bahary, MD, PhD,b Amer H. Zureikat, MD,c Steven A. Burton, MD,a Dwight E. Heron, MD, MBA, FACRO, FACR,a and Adam C. Olson, MD, MSa,* +Departments of aRadiation Oncology, bMedicine, Division of Oncology, and cSurgery, Division of GI Surgical Oncology, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Received 4 March 2020; revised 31 July 2020; accepted 19 August 2020 + +## Abstract + +Purpose: There is no consensus on treatment volumes for adjuvant stereotactic body radiation therapy (SBRT) for pancreatic cancer. + +Herein, we report patterns of failure after pancreatic SBRT for close/positive margins, which may inform target volume design. + +Methods and Materials: An institutional review board-approved retrospective review of patients with pancreatic adenocarcinoma treated with adjuvant SBRT for close/positive margins from 2009 to 2018 was conducted. Patterns of failure were defined as local (LF) +within the tumor bed, regional (RF) within lymph nodes or anastomoses, or distant (DF). The cumulative incidence of locoregional failure was calculated using the cumulative incidence function accounting for the competing risk of death. LFs were mapped to the planning target volume (PTV) and classified as in-field (completely within the PTV), marginal (partially within the PTV), or out-of-field +(completely outside the PTV). The location of LFs was compared with the Radiation Therapy Oncology Group 0848 contouring atlas to determine whether standard postoperative radiation therapy volumes would have included the LF. + +Results: Seventy-six patients were treated with adjuvant SBRT for close (51.3%) or positive (48.7%) margins. Most (81.6%) received 36 Gy in 3 fractions, with a median PTV volume of 17.8 cc (interquartile range, 12.1-25.6). With a median follow-up of 17.0 months +(interquartile range, 7.3-28.4), crude rates of first isolated LF, isolated RF, and DF þ/e LF or RF were 9.2%, 6.6%, and 56.6%, +respectively. Two-year cumulative incidences of LF, RF, locoregional failure, and DF were 34.9%, 30.8%, 49.2%, and 60.4%, +respectively. Of 28 reviewable LFs, 21.4% were in-field while the remainder were completely outside (60.7%) or partially outside +(17.9%) the PTV. Most LFs (92.9%) would have been encompassed by the Radiation Therapy Oncology Group consensus target volumes. + +Conclusions: After adjuvant pancreatic SBRT for close/positive margins, the majority of LFs were outside the PTV but within contemporary target volumes for conventional radiation therapy. + + 2020 Published by Elsevier Inc. on behalf of American Society for Radiation Oncology. This is an open access article under the CC +BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +## Introduction + +Pancreatic cancer is a highly lethal malignancy, responsible for the fourth-highest cancer death rate in the United States. Despite advances in management, 5-year overall survival (OS) remains dismal at 9%.1 Resection remains the most important curative modality; however, 80% to 85% of patients are unresectable at presentation.2,3 Of resectable patients, 20% to 30% have positive margins +(R1) and fare worse than patients with negative margins +(R0), even with adjuvant chemotherapy.4-12 Adjuvant radiation therapy (RT) may thus have a role after R1 resection and for other high-risk patients; however, studies examining adjuvant RT have produced conflicting results.9,12-20 The role of adjuvant RT is currently being examined in Radiation Therapy Oncology Group (RTOG) +0848.21 One concern with adjuvant conventionally fractionated RT is the delay to initiating systemic chemotherapy, owing to time needed to deliver RT over 5 to 6 weeks as well as potential interruptions in treatment from treatment-induced toxicities. Micrometastatic spread before adjuvant chemotherapy may offset any locoregional control benefits with adjuvant RT. For these reasons, adjuvant stereotactic body radiation therapy (SBRT) +delivered over a few fractions may be an attractive option for select patients. Definitive pancreatic SBRT provides local control rates of 70% to 80%.22-24 Adjuvant pancreatic SBRT provides similar local control with mild toxicities, though the body of evidence for adjuvant SBRT is limited.25-27 A previous report of a prospective trial from our institution involving 50 patients who received adjuvant pancreatic SBRT for close/positive margins showed 2-year local and regional control of 77% +and 73%, respectively. Acute grade 3þ toxicity was 4.1%, and no late grade 3þ toxicity or changes in patientreported quality of life were evident.25 Although SBRT may provide some advantages over conventionally fractionated RT, 1 disadvantage to SBRT +is the size limit on treatment volumes. Dose constraints of adjacent organs, chiefly the duodenum, limit the ability to dose-escalate in this region. Thus, SBRT target volumes are often limited to areas of highest risk of recurrence (ie, the positive margin or critical vascular interfaces); coverage of regional lymph nodes (LNs) and other areas at risk for harboring microscopic disease are usually omitted. Furthermore, although guidelines exist to standardize treatment volumes with adjuvant conventionally fractionated RT, no such consensus has been reached for pancreatic SBRT volumes. Contouring guidelines for postoperative conventionally fractionated RT were developed based on studies examining patterns of failure (POF), and recommend inclusion of surgical anastomoses and regional LNs (Table 1).28 Limited treatment volumes with SBRT that omit areas adjacent to the tumor or tumor bed may not be adequate in preventing adjacent local or regional recurrences. + +Few studies have reported POF after pancreatic SBRT +to inform appropriate treatment volumes.29,30 No studies, to our knowledge, have examined failure locations relative to treatment volumes after adjuvant pancreatic SBRT. + +Herein we report POF in patients treated with adjuvant pancreatic SBRT for close/positive margins. + +## Methods And Materials Eligibility + +An institutional review board-approved retrospective review of consecutive patients treated between October 2009-February 2018 with adjuvant pancreatic SBRT for close/positive margins was conducted. During this time, many patients were treated in a prospective observational trial at our institution , results of which have been previously reported.25 Patients in this trial were included in this retrospective review if they met inclusion criteria. + +Demographic, clinical, pathologic, and treatment details were collected. Included patients had resectable or borderline resectable pancreatic adenocarcinoma and were treated with resection and adjuvant pancreatic SBRT +for close/positive margins. Resectability definitions were based on National Comprehensive Cancer Network guidelines available at the time of treatment. Generally, resectable patients had no arterial tumor contact and/or +-180 venous tumor contact without vein contour irregularity, while borderline resectable patients had arterial tumor contact -180 and/or venous tumor contact >180 or -180 with contour irregularity that was amenable to vein reconstruction. Patients underwent pancreaticoduodenectomy or distal pancreatectomies for pancreatic tail tumors robotic assistance. Positive surgical margins included viable tumor cells present at any resection margin. Close margins included viable tumor cells present within 2 mm of any resection margin. Patients received adjuvant or neoadjuvant chemotherapy or both per physician discretion and patient tolerance. + +Experimental systemic therapy was allowed in institutional protocols based on eligibility and patient/physician preference. + +## Stereotactic Body Radiation Therapy + +Patients were simulated supine with arms raised and vacuum lock bag immobilization. Axial computed tomography (CT) images with 1.25 mm slice thickness were obtained. Intravenous and/or enteral contrast was administered per physician discretion. Four-dimensional CT data were acquired to evaluate target motion. The clinical target volume (CTV) was delineated in conjunction with the operating surgeon and included the area of close/positive margin, clips/fiducials, and other areas at high risk of microscopic disease based on the pathology report. Abutting vasculature was only included if that specific vessel margin was close/positive and was otherwise not routinely included. Elective regional LNs were not included in the CTV. The planning target volume (PTV) included margins for set-up error and target motion and was altered accordingly to respect critical structure tolerance. All patients were treated with 3 fractions of SBRT, every other day, to a dose of 27 to 36 Gy using daily cone beam CT for image guidance. Patients were typically seen 10 to 12 weeks post-SBRT, then every 3 months for 1 year, with subsequent follow-up per physician discretion. CT scans were typically obtained at each follow-up or if there was a concern for recurrence or progression. + +## Endpoints And Statistical Analysis + +Clinical notes and radiologic images/reports were reviewed to capture recurrences. For patterns of failure analysis, failures were classified as local (LF), regional +(RF), locoregional (LRF), or distant (DF) as deemed by the reading radiologist and/or treating physician in radiology reports and/or clinical notes. LFs were recurrences within the operative bed. RFs were recurrences within the regional LNs (new or enlarging LNs) or anastomosis. + +LRFs were defined as local and/or regional failures. DFs were recurrences in nonregional LNs or distant organs. + +The date of recurrence was backdated to the initial imaging study demonstrating any radiographic evidence of progression. Both the site of first failure and the cumulative incidence of all failure types were collected. All patients were evaluated for LF and RF from SBRT completion to death or loss of follow-up, regardless of progression at other sites. + +SBRT treatment plans were accessed to map LFs relative to the PTV. In-field LFs were LFs completely within the PTV. Marginal failures were LFs partially within the PTV, and out-of-field were LFs completely outside the PTV. The location of LFs and RFs was compared with the RTOG 0848 consensus volumes for postoperative treatment of pancreatic cancer28 to determine whether consensus volumes for conventionally fractionated postoperative RT would have included the LF or RF. Recommended contours of each region of interest and CTV expansions were considered when determining whether an LF/RF would have been encompassed by the RTOG consensus volumes (Table 1). To successfully map LFs and RFs relative to the PTV and the RTOG +consensus volumes, the location of each failure relative to the preoperative tumor location and measured distances between the edge of failures and prominent abdominal anatomy (eg, celiac artery [CA], superior mesenteric artery [SMA], portal vein, anastomoses) were recorded and compared with the location and measured distances of the PTV volumes and RTOG consensus volumes relative to the same anatomic landmarks. Patients without available SBRT plans or imaging evidence of LF/RF were excluded from analysis of failure location. + +Crude failure rates were calculated from the number of each failure type within the cohort. Time-to-event analysis was calculated from the date of SBRT completion to the date of the event. OS was calculated using KaplanMeier methods. The cumulative incidence function was used to calculate the cumulative incidence of LF, in-field LF, marginal LF, out-of-field LF, RF, LRF, and DF þ/e LF or RF from the last day of SBRT treatment to the date of failure, accounting for the competing risk of death.31-33 Differences in time to LF by type of LF were analyzed with the log-rank test. Cox proportional hazards regression was used to test the effect of demographic, clinical, pathologic, and SBRT treatment details on the risk of LF +and in-field LF. Log-minus-log plots were used to confirm the proportional hazards assumption. As a sensitivity analysis, continuous variables were also tested as binary categorical variables stratified by the median value. Variables with P values < .10 on univariate analysis were entered into the multivariate model, which was run with backward stepwise selection. P values < .05 were considered statistically significant. Statistical analysis was conducted with Statistical Package for the Social Sciences +(SPSS) Statistics for Windows version 25.0 (SPSS Inc, Chicago, IL) and R version 3.6.2.34 + +## Results + +Seventy-six patients were treated with adjuvant SBRT +for close/positive margins. Table 2 outlines cohort characteristics. Most patients (56.6%) were male and had pancreatic head tumors (80.3%). Most (59.2%) received neoadjuvant chemotherapy, most commonly gemcitabine/ +nab-paclitaxel (48.9%). The majority (81.6%) received adjuvant chemotherapy, most commonly gemcitabine +(41.9%). + +Three-quarters of patients were resectable and 25.0% +were borderline resectable. Most patients (86.8%) underwent pancreaticoduodenectomy. Close margins were seen in 51.3%; positive margins were seen in 48.7%. The most common positive/close margin was retroperitoneal +(52.6%). Twenty patients (26.3%) had multiple positive/ close margins. More than 3/4 (77.6%) had pathologic T2 disease. Pathologic nodal staging was variable: pN0, pN1, and pN2 was seen in 26.3%, 39.5%, and 34.2%, respectively. + +Adjuvant SBRT was delivered a median 2.2 months after surgery (interquartile range [IQR], 1.7-3.0). Most patients (81.6%) received 36 Gy in 3 fractions. Median PTV was 17.8 cc (IQR, 12.1-25.6). Median equivalent 1200 A.K. Patel et al Advances in Radiation Oncology: NovembereDecember 2020 + +| Table 1 | Radiation Therapy Oncology Group consensus treatment volumes for conventionally fractionated postoperative | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------|--------------------| +| pancreatic radiation therapy.28 Region of interest | RTOG* recommendations for | Expansions for CTV | +| volume delineation | | | +| Postoperative bed | Based on preoperative tumor location and | 0.5-1.0 cm | +| pathology reports; include surgical clips. | | | +| Anastomoses | Pancreaticojejunostomy (PJ), choledochaljejunostomy, or hepaticojejunostomy. Do not include pancreaticogastrostomy if present. | 0.5-1.0 cm | +| Abdominal nodal regions Celiac artery (CA) | Most proximal 1.0-1.5 cm | 1.0-1.5 cm | +| Superior mesenteric artery (SMA) | Most proximal 2.5-3.0 cm | 1.0-1.5 cm | +| (includes peripancreatic nodes) | | | +| Porta hepatic | Portal vein (PV) segment that runs anterior and | 1.0-1.5 cm | +| anteromedial to the inferior vena cava. | 2.5-3.0 cm to the right, 1.0 cm to the left, 2.0-2.5 cm anteriorly, and 0.2 cm posteriorly toward the anterior edge of the vertebral body. | | +| Abbreviations: CTV Z clinical target volume; RTOG Z Radiation Therapy Oncology Group (RTOG). Para-aortic Aorta from most cephalad aspect of CA, PJ, or PV (whichever is most cephalad), to bottom of L2; cover to bottom of L3 if needed to encompass extent of preoperative tumor. | | | + +dose in 2-Gray-per-fraction using alpha:beta Z 10 and biologically effective dose using alpha:beta Z 10 was 66.0 Gy (IQR, 66.0-66.0) and 79.2 Gy (IQR, 79.2-79.2), +respectively. No patient had a significant interruption during SBRT; the median time elapsed during the SBRT +course was 6.0 days (IQR, 5.0-7.0). + +Median follow-up after SBRT completion was 17.0 months (IQR, 7.3-28.4). Median OS was 17.3 months (95% +confidence interval, 11.0-23.5 months). Fifty-seven patients (75.0%) experienced a recurrence at any site during follow-up. Table 3 outlines POF. Upon examination of sites of first failure, isolated DF was predominant (30.2%, n Z +23). LF was a component of first failure in 23 patients. + +Crude rates of isolated LF, isolated RF, and DF þ/e LF or RF at first failure were 9.2%, 6.6%, and 56.6%, respectively. A total of 32 (42.1%), 24 (31.6%), 42 (55.3%), and 50 patients (65.8%) developed a LF, RF, LRF, or DF, respectively, during follow-up. Upon examination of LF or RF, 18 patients (23.7%) had an LF DF but without RF, 10 patients (13.2%) had an RF DF but without LF, and 14 patients (18.4%) had both LF and RF DF. The 2-year cumulative incidence of LF, RF, LRF, or DF, accounting for the competing risk of death, was 34.9%, 30.8%, 49.2%, +and 60.4%, respectively (Fig 1). Univariate Cox regression did not reveal any statistically significant predictors of LF +(Table 4), although a trend was seen for positive pancreatic neck margin (P Z .056). + +Of 32 patients with an LF during follow-up, 3 patients had unavailable imaging of the LF, and 1 patient had an unavailable SBRT plan. Thus, 28 of 32 patients (87.5%) +were analyzed for POF relative to treatment volumes. + +Only 21.4% (n Z 6) of LFs were in-field; the remaining were out-of-field (60.7%, n Z 17) or marginal (17.9%, n Z 5). The 2-year cumulative incidence of in-field, marginal, and out-of-field LFs was 7.3%, 5.8%, and 17.9%, +respectively. There were no differences in time to LF +based on type of LF; median time to failure for in-field, marginal, and out-of-field LFs was 5.0, 8.0, and 11.8 months, respectively (P Z .477). All 6 patients with an in-field LF received 36 Gy in 3 fractions and had larger CTV (median, 15.2 cc; IQR, 12.4-16.7) and PTV (median, 27.5 cc; IQR, 22.0-33.9) volumes relative to the entire cohort. However, no variables correlated with risk of in-field LF (Table 4). + +Because RTOG consensus volumes cover areas at risk for LF and RF, every LF (n Z 28) and RF (n Z 24) with available imaging was examined to assess its location relative to recommended RTOG consensus volumes. + +Most LFs (92.9%, n Z 26) would have been encompassed by the RTOG consensus volumes for postoperative conventional RT. Two LFs (7.1%) were deemed to be outside RTOG consensus volumes. Both were infiltrative masses that spread along and cuffed the distal SMA >5 cm beyond the SMA origination from the aorta, and thus outside the caudal extent of the recommended SMA target volume. + +Upon examination of RFs, 62.5% (n Z 15) would have been encompassed by RTOG consensus volumes. + +Of 9 RFs deemed to be outside RTOG consensus volumes, 5 were in high para-aortic or gastrohepatic LNs located 1 cm above the recommended cephalad border of the para-aortic LN target volume, 2 were in para-aortic LNs below the recommended caudal extent of the paraaortic LN volume, and 2 were in mesenteric LNs in the anterior or lower abdomen along the distal SMA, beyond the recommended SMA nodal target volume. The 2-year cumulative incidence of LRF within RTOG consensus volumes was 39.3%. + +| Table 2 | Demographic, clinical, pathologic, and treatment | +|----------------------------------------------------------|----------------------------------------------------| +| detail characteristics of cohort Characteristic | Value (n Z 76) | +| Age at diagnosis (median, IQR) | 66.0 (57.3-73.0) | +| Sex (n, %) Male | 43 (56.6) | +| Female | 33 (43.4) | +| Tumor location (n, %) Body | 8 (10.5) | +| Head | 61 (80.3) | +| Tail | 2 (2.6) | +| Uncinate process | 4 (5.3) | +| Neck | 1 (1.3) | +| Resectability (n, %) Resectable | 57 (75.0) | +| Borderline resectable | 19 (25.0) | +| Surgery type (n, %) Pancreaticoduodenectomy | 66 (86.8) | +| Distal pancreatectomy | 10 (13.2) | +| Margin status (n, %) Positive | 37 (48.7) | +| Close (1 mm) | 37 (48.7) | +| Close (2 mm) | 2 (2.6) | +| Location of positive/close margin (n, %) Retroperitoneal | 40 (52.6) | +| Vascular groove | 35 (46.1) | +| Pancreatic neck | 11 (14.5) | +| Chemotherapy (n, %) Neoadjuvant | 45 (59.2) | +| Gemcitabine | 2 (4.4) | +| FOLFIRINOX | 6 (13.3) | +| Gemcitabine/nab-paclitaxel | 22 (48.9) | +| Gemcitabine/hydroxychloroquine | 9 (20.0) | +| Other | 6 (13.3) | +| Adjuvant | 62 (81.6) | +| Gemcitabine | 26 (41.9) | +| FOLFIRINOX | 4 (6.5) | +| Gemcitabine/capecitabine | 10 (16.1) | +| Gemcitabine/nab-paclitaxel | 13 (21.0) | +| FOLFOX | 4 (6.5) | +| Other | 5 (8.1) | +| Pathologic T stage (AJCC 8th edition) (n, %) pT1a | 1 (1.3) | +| pT1c | 6 (7.9) | +| pT2 | 59 (77.6) | +| pT3 | 10 (13.2) | +| Pathologic N stage (AJCC 8th edition) (n, %) pN0 | 20 (26.3) | +| pN1 | 30 (39.5) | +| pN2 | 26 (34.2) | +| SBRT regimen (n, %) 36 Gy in 3 fractions | 62 (81.6) | +| 30 Gy in 3 fractions | 13 (17.1) | +| 27 Gy in 3 fractions | 1 (1.3) | +| EQD210 (median, IQR) | 66.0 (66.0-66.0) | +| BED10 (median, IQR) | 79.2 (79.2-79.2) | +| (continued on next page) | | + +| Table 2 (continued) Characteristic | Value (n Z 76) | +|--------------------------------------|---------------------| +| CTV volume (median, IQR) | 9.7 cc (6.8-14.4) | +| PTV volume (median, IQR) | 17.8 cc (12.1-25.6) | +| PTV V100% (median, IQR) | 94.8% (90.3-98.1) | +| Months from surgery to SBRT start | 2.2 (1.6-3.0) | +| (median, IQR) | | +| Days elapsed from SBRT start to end | 6.0 (5.0-7.0) | +| (median, IQR) | | +| Abbreviations: AJCC Z American Joint Committee on Cancer; BED10 Z biologically effective dose using an alpha/beta ratio of 10; CTV Z clinical target volume; EQD210 Z equivalent dose in 2 Gray per fraction using an alpha/beta ratio of 10; IQR Z interquartile range; PTV Z planning target volume; PTV V100% Z percent of PTV that received 100% of the prescription dose; SBRT Z stereotactic body radiation therapy. | | + +## Discussion + +In this single-institution retrospective analysis of adjuvant pancreatic SBRT for close/positive margins, we identified a high rate of LRF outside the SBRT PTV but within contemporary RT target volumes. Although the predominant POF at first failure was isolated DF (30.2%), +42.1% of patients developed an LF during follow-up. The 2-year cumulative incidence of LF was 34.9%. Thus, despite macroscopic resection, targeted RT, and systemic therapy, patients with close/positive margins were at substantial risk for developing an LF. LFs within the PTV were infrequent (6 of 28 patients); the 2-year cumulative incidence of in-field LFs was 7.3%. Most LFs were completely outside the PTV (60.7%). Over 90% of LFs would have been encompassed by RTOG consensus volumes for postoperative conventionally fractionated RT +for pancreatic cancer. + +This study is the first, to our knowledge, that describes POF after adjuvant SBRT. Published studies examining POF with pancreatic SBRT are limited to patients treated in the definitive or neoadjuvant setting, and few studies have detailed examination of failures relative to treatment volumes or abdominal vasculature.29,30,35 A Chinese study of >500 medically inoperable patients treated with pancreatic SBRT revealed that most LFs occurred near the CA, SMA, or splenic artery for pancreatic tail tumors29; 80% and 95% of failures near the CA or SMA +occurred within 11 and 13 mm of the vessel, respectively. + +These failures likely would have been included within the consensus postoperative target volumes for conventionally fractionated RT. In-field failures (>80% of volume + +| Table 3 | Patterns of failure with adjuvant SBRT Crude rates | Cumulative incidence (95% CI) | | | +|------------------------------------------------------------------------------------------|------------------------------------------------------|---------------------------------|-------------------|-------------------| +| n (%) | 6-month | 12-month | 24-month | | +| Location of failure based on clinical documentation (n Z 76) No failure | 19 (25.0) | | | | +| Local failure | 32 (42.1) | 17.2% (9.7-26.5) | 29.3% (19.5-39.8) | 34.9% (24.3-45.8) | +| Regional failure | 24 (31.6) | 13.2% (6.7-21.2) | 23.9% (15.0-34.0) | 30.8% (20.7-41.5) | +| Locoregional | 42 (55.3) | 26.3% (17.0-36.6) | 40.8% (29.7-51.5) | 49.2% (37.4-59.9) | +| Distant failure | 50 (65.8) | 33.1% (22.7-43.4) | 50.5% (38.7-61.2) | 60.4% (48.2-70.5) | +| Location of local failure in patients with available plans and imaging (n Z 28) In-field | 6 (21.4) | 4.2% (1.1-10.8) | 5.7% (1.8-12.9) | 7.3% (2.7-15.2) | +| Marginal | 5 (17.9) | 2.9% (0.1-8.9) | 5.8% (1.9-13.2) | 5.8% (1.9-13.2) | +| Out-of-field | 17 (60.7) | 8.0% (3.3-15.6) | 14.8% (7.8-23.9) | 17.9% (10.1-27.6) | +| Abbreviations: CI Z confidence interval; SBRT Z stereotactic body radiation therapy. | | | | | + +![5_image_0.png](5_image_0.png) + +within the prescription isodose) occurred in 22.9%; this number mirrors the in-field failure rate in this study of 21.4%. The distance from primary tumors to retroperitoneal recurrences was larger than the distance from primary tumors to the CA (11.7 vs 9.0 mm, respectively), +suggesting that asymmetrical expansion of SBRT target volumes to include the local retroperitoneal space may be warranted. Kharofa et al30 reported POF in 18 resectable or borderline resectable patients treated with neoadjuvant pancreatic SBRT (33 Gy in 5 fractions) to the tumor and entire circumference of the abutting vessel. After interim analysis showed 2 of 8 patients failed locally at the vessel at the PTV margin, the remaining patients received 25 Gy in 5 fractions simultaneously to an additional PTV +covering the entire pancreatic head/body, with extension of vessel coverage to the CA origin and SMA. With the limitations of a small sample size, the combined PTV +approach led to longer 1-year PFS (60% vs 25%; P Z +.03), 2-year OS (67% vs 25%; P Z .03), and lower 1-year cumulative incidence of LF (38% vs 70%; P Z .45), +raising the question of whether elective coverage of vessel origins with SBRT reduces the risk of failures at the margin of the primary PTV. + +There are no consensus pancreatic SBRT volumes. + +Results from our study support consideration for expanding treatment volumes if feasible. Indeed, initial results from our institutional prospective trial of adjuvant pancreatic SBRT for close/positive margins found CTV +volume was the only predictor for freedom from LF on multivariate analysis (hazard ratio, 1.095; 95% confidence interval, 1.005-1.193; P Z .038).25 However, the tradeoff between expanding target volumes and irradiating more normal tissue must be carefully evaluated. A study by Dholakia et al36 generated hypothetical adjuvant intensity modulated radiation therapy and SBRT volumes encompassing the majority of LFs in >200 patients after pancreaticoduodenectomy. CTV volumes encompassed either 90% of LFs (CTV90) or 80% of LFs (CTV80) based on specific asymmetrical expansions around CA and SMA +contours. With no further margins for setup error, PTV80 and PTV90 volumes ranged from 123.0 to 139.6 cc and 183.2 to 215.7 cc, respectively. These volumes are significantly larger than those in our study but may represent a practical strategy to expand treatment volumes while minimizing irradiation of normal tissue. Although the authors report critical structure dose constraints were met with 25 Gy in 5 fractions to the PTV90 with a simultaneous integrated boost to 33 Gy to PTV80, no patients were actually treated using the hypothetical plans. + +The aforementioned studies highlight the commonality of LFs along vessels after SBRT and underline the importance of detailed POF examinations. Studies reporting LFs after pancreatic SBRT use different LF +definitions, which may inadvertently include nodal failures e the Chinese and Dholakia studies defined LFs as Advances in Radiation Oncology: NovembereDecember 2020 Patterns of failure after pancreatic SBRT 1203 + +| Table 4 | Univariate Cox regression for predictors of LF in all patients (n Z 76) as deemed by the reading radiologist and/or | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------| +| treating physicians and for predictors of in-field LF in patients with documented LF who had available plans and imaging (n Z 28) Univariate analysis Variable HR for LF (95% CI) P value HR for in-field LF (95% CI) P value Age at diagnosis 1.00 (0.96-1.03) .769 1.06 (0.96-1.17) .259 Sex .565 .441 Male Reference Reference Female 0.81 (0.40-1.66) 1.88 (0.38-9.42) Tumor location .999 .650 Body Reference Reference Head 1.05 (0.36-3.04) 0.33 (0.06-1.81) Tail 1.44 (0.16-13.04) NR Uncinate process 1.05 (0.19-5.80) NR Neck NR NR Resectability .127 .856 Resectable Reference Reference Borderline resectable 1.83 (0.84-3.96) 1.17 (0.21-6.60) Surgery type .600 .501 Pancreaticoduodenectomy Reference Reference Distal pancreatectomy 1.27 (0.52-3.10) 1.79 (0.33-9.82) Margin status .862 .395 Positive Reference Reference Close (1 mm) 0.87 (0.42-1.78) 0.87 (0.14-5.29) Close (2 mm) 0.62 (0.08-4.79) 4.26 (0.37-49.65) Retroperitoneal margin .279 .465 Negative Reference Reference Positive/close 1.48 (0.73-3.03) 1.89 (0.34-10.51) Vascular groove margin .256 .717 Negative Reference Reference Positive/close 0.66 (0.32-1.35) 0.73 (0.13-4.02) Pancreatic neck margin .056 .847 Negative Reference Reference Positive/close 2.30 (0.98-5.42) 0.81 (0.09-7.08) Pathologic tumor size (cm) 1.10 (0.82-1.46) .538 0.39 (0.11-1.43) .155 Neoadjuvant chemotherapy .624 .390 No Reference Reference Yes 0.84 (0.42-1.70) 2.57 (0.30-22.07) Adjuvant chemotherapy .183 No Reference Reference Yes 2.25 (0.68-7.42) NR CTV volume (cc) 1.02 (0.98-1.05) .414 1.01 (0.94-1.08) .760 PTV volume (cc) 1.01 (0.98-1.04) .414 1.03 (0.97-1.09) .334 PTV V100% (%) 1.01 (0.97-1.05) .565 1.04 (0.92-1.16) .589 BED10 1.00 (0.96-1.06) .760 1.17 (0.72-1.92) .562 EQD210 1.01 (0.95-1.07) .760 NR .818 Abbreviations: BED10 Z biologically effective dose using an alpha/beta ratio of 10; CI Z confidence interval; CTV Z clinical target volume; EQD210 Z equivalent dose in 2 Gray per fraction using an alpha/beta ratio of 10; HR Z hazard ratio; LF Z local failure; NR Z not reportable; PTV Z planning target volume; PTV V100% Z percent of PTV that received 100% of the prescription dose. | | + +failures in between the diaphragm and bottom of L3, while Kharofa et al considered LFs as failures within the SBRT volume or within institutional fractionated volumes that electively covered the regional vasculature. In our analysis, because no elective nodal volumes were treated, we constrained the definition of LF to failures within the operative bed based on preoperative tumor location to minimize inclusion of nodal failures. Despite this constrained definition, LFs were identified in 42.1% of patients after SBRT targeted to close/positive margins. + +Thus, true LFs occur in a significant proportion of patients, despite SBRT targeting the areas of highest risk of recurrence. Of 72 patients with available imaging and SBRT plans, only 6 (8.3%) experienced an LF within the treatment volume; the majority of LFs were outside or marginal to the treatment volume. This suggests issues with volume and not dose, and thus expanding treatment volumes to include all portions of the pancreas/pancreatic bed and abutting vasculature may be warranted. Because RTOG consensus volumes encompassed most LFs as well as RFs in this study, adopting a similar philosophy with SBRT treatment volumes may be warranted if feasible. + +We note several limitations of this study, including its retrospective nature, relatively small sample size, and the subjective nature of mapping LFs and RFs to PTV volumes and RTOG consensus volumes. The latter issue was mitigated by systematically noting the location and distance of failures relative to abdominal vasculature, which allowed for more precise mapping. + +## Conclusions + +In patients with resectable or borderline resectable adenocarcinoma of the pancreas who received adjuvant SBRT for close or positive margins, the majority of LFs occurred outside the PTV. Future trials involving adjuvant SBRT or hypofractionated RT should consider expansion of treatment volumes if feasible. + +## References + +1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70:7-30. + +2. Li D, Xie K, Wolff R, Abbruzzese JL. Pancreatic cancer. Lancet. + +2004;363:1049-1057. + +3. Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N +Engl J Med. 2014;371:1039-1049. + +4. Konstantinidis IT, Warshaw AL, Allen JN, et al. Pancreatic ductal adenocarcinoma. Ann Surg. 2013;257:731-736. + +5. Gnerlich JL, Luka SR, Deshpande AD, et al. Microscopic margins and patterns of treatment failure in resected pancreatic adenocarcinoma. Arch Surg. 2012;147:753-760. + +6. Delpero JR, Bachellier P, Regenet N, et al. Pancreaticoduodenectomy for pancreatic ductal adenocarcinoma: A +French multicentre prospective evaluation of resection margins in 150 evaluable specimens. HPB. 2014;16:20-33. + +7. Neoptolemos JP, Palmer DH, Ghaneh P, et al. Comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer (ESPAC-4): A +multicentre, open-label, randomised, phase 3 trial. Lancet. 2017; 389:1011-1024. + +8. Stocken DD, Bü Chler MW, Dervenis C, et al. Meta-analysis of randomised adjuvant therapy trials for pancreatic cancer. Br J +Cancer. 2005;92:1372-1381. + +9. Herman JM, Swartz MJ, Hsu CC, et al. Analysis of fluorouracilbased adjuvant chemotherapy and radiation after pancreaticoduodenectomy for ductal adenocarcinoma of the pancreas: +Results of a large, prospectively collected database at the Johns Hopkins Hospital. J Clin Oncol. 2008;26:3503-3510. + +10. Parikh AA, Maiga A, Bentrem D, et al. Adjuvant therapy in pancreas cancer: Does it influence patterns of recurrence? J Am Coll Surg. 2016;222:448-456. + +11. Ghaneh P, Kleeff J, Halloran CM, et al. The impact of positive resection margins on survival and recurrence following resection and adjuvant chemotherapy for pancreatic ductal adenocarcinoma. + +Ann Surg. 2019;269:520-529. + +12. Morganti AG, Falconi M, Van Stiphout RGPM, et al. Multi-institutional pooled analysis on adjuvant chemoradiation in pancreatic cancer. Int J Radiat Oncol Biol Phys. 2014;90:911-917. + +13. Smeenk HG, Van Eijck CHJ, Hop WC, et al. Long-term survival and metastatic pattern of pancreatic and periampullary cancer after adjuvant chemoradiation or observation: Long-term results of EORTC trial 40891. Ann Surg. 2007;246:734-740. + +14. Kaiser MH, Ellenberg SS. Pancreatic cancer: Adjuvant combined radiation and chemotherapy following curative resection. Arch Surg. + +1985;120:899-903. + +15. Neoptolemos JP, Stocken DD, Friess H, et al. A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. N Engl J Med. 2004;350:1200-1210. + +16. Tesfaye AA, Philip PA. Further evidence of effective adjuvant combined radiation and chemotherapy following curative resection of pancreatic cancer. Gastrointestinal Tumor Study Group. Cancer. 1987;59:2006-2010. + +17. Van Laethem JL, Hammel P, Mornex F, et al. Adjuvant gemcitabine alone versus gemcitabine-based chemoradiotherapy after curative resection for pancreatic cancer: A randomized EORTC-4001322012/FFCD- 9203/GERCOR phase II study. J Clin Oncol. 2010; 28:4450-4456. + +18. Schmidt J, Abel U, Debus J, et al. Open-label, multicenter, randomized phase III trial of adjuvant chemoradiation plus interferon alfa-2b versus fluorouracil and folinic acid for patients with resected pancreatic adenocarcinoma. J Clin Oncol. 2012;30:4077-4083. + +19. Kooby DA, Gillespie TW, Liu Y, et al. Impact of adjuvant radiotherapy on survival after pancreatic cancer resection: An appraisal of data from the national cancer data base. Ann Surg Oncol. 2013;20: +3634-3642. + +20. Shinde A, Verma V, Li R, et al. The role of sequential radiation following adjuvant chemotherapy in resected pancreatic cancer. J Gastrointest Oncol. 2019;10:462-473. + +21. Gemcitabine hydrochloride with or without erlotinib hydrochloride followed by the same chemotherapy regimen with or without radiation therapy and capecitabine or fluorouracil in treating patients with pancreatic cancer that has been removed by surgery. Available at: https://clinicaltrials.gov/ct2/show/NCT01013649. Accessed January 9, 2020. + +22. Chuong MD, Springett GM, Freilich JM, et al. Stereotactic body radiation therapy for locally advanced and borderline resectable pancreatic cancer is effective and well tolerated. Int J Radiat Oncol Biol Phys. 2013;86:516-522. + +23. Mahadevan A, Jain S, Goldstein M, et al. Stereotactic body radiotherapy and gemcitabine for locally advanced pancreatic cancer. Int J Radiat Oncol Biol Phys. 2010;78:735-742. + +24. Herman JM, Chang DT, Goodman KA, et al. Phase 2 multiinstitutional trial evaluating gemcitabine and stereotactic body radiotherapy for patients with locally advanced unresectable pancreatic adenocarcinoma. Cancer. 2015;121:1128-1137. + +25. Bernard ME, Sutera PA, Iarrobino NA, et al. Initial results of a prospective study of adjuvant pancreatic stereotactic body radiation therapy for close or positive margins. Adv Radiat Oncol. 2019;4: 294-301. + +26. Rwigema J-CM, Parikh SD, Heron DE, et al. Stereotactic body radiotherapy in the treatment of advanced adenocarcinoma of the pancreas. Am J Clin Oncol. 2011;34:63-69. + +27. Rwigema JCM, Heron DE, Parikh SD, et al. Adjuvant stereotactic body radiotherapy for resected pancreatic adenocarcinoma with close or positive margins. J Gastro Cancer. 2012;43:70-76. + +28. Goodman KA, Regine WF, Dawson LA, et al. Radiation Therapy Oncology Group consensus panel guidelines for the delineation of the clinical target volume in the postoperative treatment of pancreatic head cancer. Int J Radiat Oncol. 2012; 83:901-908. + +29. Zhu X, Ju X, Cao Y, et al. Patterns of local failure after stereotactic body radiation therapy and sequential chemotherapy as initial + +## Advances In Radiation Oncology: Novemberedecember 2020 Patterns Of Failure After Pancreatic Sbrt 1205 + +treatment for pancreatic cancer: Implications of target volume design. Int J Radiat Oncol Biol Phys. 2019;104:101-110. + +30. Kharofa J, Mierzwa ML, Olowokure OO, et al. Pattern of marginal local failure in a phase II trial of neoadjuvant chemotherapy and stereotactic body radiation therapy for resectable and borderline resectable pancreas cancer. Am J Clin Oncol. 2019;42:247-252. + +482-482. + +31. Austin PC, Lee DS, Fine JP. Introduction to the analysis of survival data in the presence of competing risks. Circulation. 2016;133:601-609. + +32. Scrucca L, Santucci A, Aversa F. Competing risk analysis using R: An easy guide for clinicians. Bone Marrow Transplant. 2007;40:381-387. + +33. Kim HT. Cumulative incidence in competing risks data and competing risks regression analysis. Clin Cancer Res. 2007;13:559-565. + +34. R Core Team. R: A Language and Environment for Statistical Computing. + +Vienna, Austria: R Foundation for Statistical Computing; 2019. + +35. Baine MJ, Sleightholm R, Lin C. Incidence and patterns of locoregional failure after stereotactic body radiation therapy for pancreatic adenocarcinoma. Pract Radiat Oncol. 2019;9:e29-e37. + +36. Dholakia AS, Kumar R, Raman SP, et al. Mapping patterns of local recurrence after pancreaticoduodenectomy for pancreatic adenocarcinoma: A new approach to adjuvant radiation field design. Int J +Radiat Oncol Biol Phys. 2013;87:1007-1015. \ No newline at end of file diff --git a/medical/md/PMC7776371.md b/medical/md/PMC7776371.md new file mode 100644 index 0000000000000000000000000000000000000000..1f76f84341fa3911e1bba034f43cfeaf4731ce82 --- /dev/null +++ b/medical/md/PMC7776371.md @@ -0,0 +1,27 @@ +Conclusion: TNFα and IL-18 in combination with CMV viral load may be useful in predicting likelihood of TI-CMV. This is important in situations where tissue biopsies are not feasible, and adds to our diagnostic capability for TI-CMV in SOT recipients. + +Disclosures: Stuart C. Ray, MD, **miDiagnostics, Inc.** (Board Member, Research Grant or Support) Robin K. Avery, MD, **Aicuris** (Scientific Research Study Investigator)**Astellas** (Scientific Research Study Investigator)**Chimerix** (Scientific Research Study Investigator)**Merck** (Grant/Research Support, Scientific Research Study Investigator)**Oxford Immunotec** (Scientific Research Study Investigator)**Qiagen** (Scientific Research Study Investigator)**Takeda/Shire** (Scientific Research Study Investigator) 687. Iatrogenic Kaposi's Sarcoma Following Treatment with Vedolizumab Susanne O. Ajao, n/a1; HAMID SHAABAN, n/a2; Rajasingam Jayasingam, n/a2; 1St. Michael's Medical Center, Bloomfield, New Jersey 2St. Michael Medical Center, Newark, New Jersey Session: P-27. Diagnostics: Virology Background: Kaposi's sarcoma (KS) is a vascular tumor caused by human herpes virus-8 infection (HHV-8) commonly involving the skin. We report a case of a patient with controlled HIV who developed Kaposi's sarcoma on vedolizumab. + +![0_image_1.png](0_image_1.png) + +(UC), Hodgkin's lymphoma in remission and HIV presented with complaints of abdominal pain and bloody diarrhea. He had new tender lesions on his left foot which was absent at his previous admission a month ago. The lesions started as macules and later progressed to tender lesions. Mesalamine and oral corticosteroids were previously prescribed without symptomatic relief and he was started on vedolizumab 2 months ago. On physical exam he had abdominal tenderness, tender cervical and inguinal lymph nodes, and dark macules on his feet. Digital rectal exam revealed bloody mucoid stool. Laboratory showed white blood cell count of 12,600/mm3, ESR of 132 mm/ +hr and CRP of 4.6 mg/dL. His CD4 T-cell count was 873 cells/mm3 and viral load was 50 copies per milliliter. Cervical lymph node biopsy showed polymorphous population of lymphocytes but was negative for malignant cells. Biopsy of the foot plaques showed atypical intradermal vascular and spindle cell proliferation positive for HHV-8 and for vascular marker CD34. The results were consistent with the diagnosis of KS. Serology was also positive for HHV-8 with high viral titers of 74 copies/mL. Colonoscopy showed severe proctitis with deep ulcerations in a continuous pattern in the rectum with a normal sigmoid colon. Follow up colonoscopy showed improved proctitis and he was started on doxorubicin to treat KS with improvement of the foot lesion a month later following treatment. + +Dark macules on the sole of both feet + +Figure 3a: Spindle cells with irregular small vessel proliferation and red blood cell + +![0_image_0.png](0_image_0.png) + +extravasation between tumor cells Figure 3b: Immunohistochemical stain showing ![0_image_2.png](0_image_2.png) + +of lymphocytes to the inflamed tissue. It is approved for the treatment of IBD and has shown efficacy and safety. The iatrogenic form of Kaposi's sarcoma occurs in patients on immunosuppressive therapy, as this patient. + +Conclusion: Patients with IBD on immunosuppressive drugs should be followed up closely and screened for latent viral infections prior to initiating therapy. As in the patient, HHV-8 should be recognized as a likely underlying opportunistic infection in immunocompromised patients with IBD. + +Disclosures: **All Authors**: No reported disclosures + +## 688. Marginal Structure Models To Estimate The Effect Of Cytomegalovirus Infection On Hospitalization Among Children Undergoing Allogeneic Hematopoietic Cell Transplantation + +Yun Li, PhD1; Arman Oganisian, MS2; Craig L. K. Boge, MPH3; Molly Hayes, PharmD, BCPS3; Alexander Newman, MD4; Brian T. Fisher, DO, MPH, MSCE5; Brian T. Fisher, DO, MPH, MSCE5; 1University of Pennsylvania, Philadelphia, Pennsylvania; 2Department of Biostatistics, Epidemiology and Informatics, philadelphia, Pennsylvania; 3Children's Hospital of Philadelphia, Philadelphia, PA; 4Ann and Robert H Lurie Children's Hospital of Chicago, Chicago, IL; 5Children's Hospital of Philadelphia; Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Session: P-27. Diagnostics: Virology Background: Children receiving an allogeneic hematopoietic cell transplant +(HCT) are at risk for cytomegalovirus (CMV) infection in the post-transplant period, necessitating routine surveillance for CMV. Some patients will not have CMV detected \ No newline at end of file diff --git a/medical/md/PMC8028335.md b/medical/md/PMC8028335.md new file mode 100644 index 0000000000000000000000000000000000000000..28a110f6e436659e42fbff5b8e046753e260779d --- /dev/null +++ b/medical/md/PMC8028335.md @@ -0,0 +1,509 @@ +pubs.acs.org/JPCA Article + +![0_image_0.png](0_image_0.png) + +# Assessment Of The Ab Initio Bethe−Salpeter Equation Approach For The Low-Lying Excitation Energies Of Bacteriochlorophylls And Chlorophylls Zohreh Hashemi And Linn Leppert* + +ABSTRACT: Bacteriochlorophyll and chlorophyll molecules are crucial building blocks of the photosynthetic apparatus in bacteria, algae, and plants. Embedded in transmembrane protein complexes, they are responsible for the primary processes of photosynthesis: +excitation energy and charge transfer. Here, we use ab initio manybody perturbation theory within the GW approximation and Bethe−Salpeter equation (BSE) approach to calculate the electronic structure and optical excitations of bacteriochlorophylls a, b, c, d, and e and chlorophylls a and b. We systematically study the effects of the structure, basis set size, partial self-consistency in GW, and the underlying exchange−correlation approximation and compare our calculations with results from time-dependent density functional theory, multireference RASPT2, and experimental literature results. We find that optical excitations calculated with GW+BSE are in excellent agreement with experimental data, with an average deviation of less than 100 meV for the first three bright excitations of the entire family of (bacterio)chlorophylls. + +Contrary to state-of-the-art time-dependent density functional theory (TDDFT) with an optimally tuned range-separated hybrid functional, this accuracy is achieved in a parameter-free approach. Moreover, GW+BSE predicts the energy differences between the low-energy excitations correctly and eliminates spurious charge transfer states that TDDFT with (semi)local approximations is known to produce. Our study provides accurate reference results and highlights the potential of the GW+BSE approach for the simulation of larger pigment complexes. + +- INTRODUCTION +Electronic excitations form the foundation of some of the most fundamental natural processes. In photosynthesis, plants, algae, and bacteria convert solar energy into chemical energy, utilizing a cascade of coupled energy and charge transfer excitations that are performed by pigment−protein complexes with high quantum efficiency. Bacteriochlorophyll (BCL) and chlorophyll (CL) molecules are among the most important building blocks of these pigment−protein complexes.1 They are responsible for the absorption and transfer of excitation energy and for the charge separation necessary for establishing a proton gradient that eventually drives the synthesis of chemical energy in plants and bacteria.2 Accurately calculating the electronic structure and excitations of these molecules from first principles is the prerequisite for understanding their interactions with each other and with the surrounding proteins and, consequently, energy and charge transfer in natural photosynthesis. + +BCL and CL molecules constitute a family of substituted tetrapyrroles with varying absorption properties depending on conjugation and the number and nature of substitutions. CL a and b are present in plants and green algae, whereas green + +ACCESS Metrics & More Article Recommendations *sı Supporting Information +bacteria mostly rely on BCL c, d, and e for excitation energy transfer and BCL a for concentrating excitations close to the reaction center of the photosynthetic unit.3 BCL a is also the main pigment in purple bacteria, whose light harvesting apparatus and reaction center are among the most thoroughly studied natural light-harvesting systems.4 The optical excitation spectrum of these pigments possesses two characteristic absorption bands: (1) the Q band in the visible part of the spectrum, composed of excitations Qy and Qx with high- and low-oscillator strength, respectively and (2) the B (or Soret) band in the near ultraviolet. + +In the field of finite organic and biological molecular systems, neutral excitations and optical spectra are predominantly calculated using time-dependent density functional theory (TDDFT). In conjunction with model Hamiltonian + +Received: February 10, 2021 + +![0_image_2.png](0_image_2.png) Revised: February 16, 2021 Published: March 3, 2021 + +![0_image_1.png](0_image_1.png) + +approaches, TDDFT has been employed for the simulation of large photosynthetic pigment−protein complexes.5,6 The accuracy of its approximations and implementations has been tested for a variety of biochromophores.7,8,47 However, TDDFT's standard approximations are inadequate for describing long-range charge transfer excitations9 and highenergy Rydberg states10 due to self-interaction errors and an incorrect asymptotic behavior. Exchange−correlation (xc) +functionals that contain long-range exact exchange, such as optimally tuned range-separated hybrid functionals (OTRSH), can be employed as a remedy in such cases,11,12 but the use of such functionals requires a tedious per-system tuning procedure. + +Multireference wavefunction-based methods have scarcely been used for molecules as large as BCL and CL. Vertical excitation energies of CL a, based on ADC(2) and different coupled cluster approaches show a spread of ∼0.4 eV, strongly depending on the method, basis set, and structural model used in these calculations.13 −15 In 2016 and 2019, Anda et al. + +reported multistate RASPT2/RASSCF excitation energies of several BCL units within the light-harvesting system 2 of a purple bacterium.16,17 A RASPT2 approach was also combined with electrostatic embedding of fixed-point charges to simulate the effect of the protein environment on excitation energies of the same system by Segatta et al.18 While these reports constitute important advances in the use of wavefunctionbased methods for complex biological molecules, they were performed with relatively small basis sets and show a dependence on the choice of the restricted active space (RAS). + +The ab initio Bethe−Salpeter equation (BSE) approach, when rigorously based on many-body Green's function theory, is an alternative method for describing neutral excitations of correlated many-electron systems.19 It is based on a framework of charged excitation energies that correspond to electron addition and removal energies and that are most frequently calculated within the GW approximation. The GW+BSE +approach has been shown to be successful in predicting the optical spectra of bulk solids20,21 and low-dimensional materials.22 In recent years, it has also begun to be applied to finite systems, such as small molecules,23−25 and larger molecular complexes,26−28 for which its accuracy has been shown to be comparable to single-reference wavefunction methods for both localized and charge transfer excitations,29 at a substantially reduced computational cost. + +In this article, we assess the accuracy of the ab initio GW ++BSE approach for the Qy, Qx, and the first bright B excitation of several members of the BCL and CL family and the chemically closely related bacteriochlorin (BC) molecule. We compare two different approaches for approximating the electronic self-energy Σ = iGW: (1) G0W0, a one-shot method, in which the zeroth-order Green's function G0 and screened Coulomb interaction W0 are constructed from a DFT +eigensystem and directly used to correct DFT eigenvalues perturbatively and (2) partially self-consistent GW (evGnWn), +in which the corrected eigenvalues are used to iteratively recalculate G and/or W until self-consistency is reached. We compare our results to TDDFT calculations with the local density approximation (LDA), two global hybrids and an OTRSH functional, with RASPT2 literature results,16,17 and with experimental data.30,31 We find that the GW+BSE approach used in a partially selfconsistent manner results in excitation energies in the visible and near-ultraviolet within less than 100 meV from experiment for the entire family of pigments studied here. Our results are almost completely independent of the DFT eigensystem used as an input for the GW+BSE calculations. In fact, even a simple and computationally inexpensive LDA starting point leads to excellent agreement with experiment and eliminates spurious charge transfer excitations between Qy and Qx that TDDFT +with (semi)local functionals produces. Contrary to TDDFT, +GW+BSE also correctly predicts the energy difference between the two Q-band excitations, a crucial prerequisite for understanding the coupling of excitations in systems consisting of more than one pigment. Finally, we show that differences between evGnWn+BSE and state-of-the-art TDDFT calculations using an OT-RSH functional can be explained almost entirely based on differences in how electron−hole interactions are described by the xc kernel of TDDFT and the BSE kernel, respectively. Eigenvalue differences as computed with evGnWn and DFT with an OT-RSH functional are almost identical. + +The remainder of this article is structured as follows: we start by briefly reviewing the GW+BSE approach and report computational details and numerical convergence. We then show the effect of different DFT starting points and partial selfconsistency on the excitation energies of the BC molecule. + +After this, we discuss our results for BCL a, b, c, d, and e and CL a and b, followed by a comparison with literature results and an in-depth discussion of differences between our GW ++BSE and TDDFT results for BCL a. + +- METHODS +GW+BSE Approach. In Green's function-based manybody perturbation theory, the calculation of charged excitations, corresponding to electron removal and addition energies, is based on knowledge of the exact interacting singleparticle Green's function G, which can, in principle, be computed from a set of self-consistent integro-differential equationsHedin's equationslinking G to the electronic self-energy Σ, the screened Coulomb interaction W, the irreducible polarizability χ, and the vertex function Γ. + +32 The lowest-order expansion of Σ with respect to W leads to the GW approximation, in which the electronic self-energy Σ = iGW. + +33 Quasiparticle (QP) eigenvalues can be obtained by solving Ä + +Ç +ÅÅÅÅÅÅÅÅÅ +$$\begin{array}{l}{{-\frac{\hbar^{2}}{2m}\nabla^{2}\,+\,V_{\mathrm{ion}}({\bf r})\,+\,V_{\mathrm{H}}({\bf r})\,\biggr]\!\varphi_{n}^{\mathrm{QP}}({\bf r})}}\\ {{+\,\int\!\mathrm{d}{\bf r}^{\prime}\Sigma({\bf r},\,{\bf r}^{\prime};\,\varepsilon_{n}^{\mathrm{QP}})\varphi_{n}^{\mathrm{QP}}({\bf r}^{\prime})\,=\,\varepsilon_{n}^{\mathrm{QP}}\varphi_{n}^{\mathrm{QP}}({\bf r})}}\end{array}$$ +$\left(1\right)$. +here, Vion is the ionic potential, VH is the Hartree potential, and εn QP and φn QP are QP energies and wavefunctions, respectively. + +To avoid the high computational cost of a self-consistent solution of eq 1, the GW approach is commonly used within a one-shot scheme, in which G0 and W0 are constructed from a +(generalized) Kohn−Sham (gKS) eigensystem obtained from a preceding DFT calculation. We use the notation G0W0@gKS +to refer to G0W0 based on the gKS eigensystem (φn gKS;εn gKS) +computed with the xc functional Exc gKS. In this approach, QP +corrections are calculated to first order in Σ as + +$$(\varepsilon_{n}^{\mathrm{qp}})\,-\,V_{\mathrm{xc}}|\varphi_{n}^{\mathrm{gKS}}\rangle$$ +$$=\varepsilon_{n}^{\mathrm{gKS}}+\,\langle\varphi_{n}^{\mathrm{gKS}}|\Sigma$$ + +n n ( ) V n n n QP gKS gKS QP +xcgKS ε = + ⟨ |Σ − | *ε φε φ* ⟩ (2) +where Vxc is the xc potential, and it is assumed that φn QP ≈ φn gKS. + +While the G0W0 approach has been used with much success, in particular, for the calculation of band gaps and band structures of solids, a well-known and well-documented dependence on the gKS eigensystem used to construct G0 Karlsruhe basis sets def2-SVP, def2-TZVP, and def2-TZVPP + +![2_image_0.png](2_image_0.png) + +and their corresponding auxiliary basis sets.42 Figures 1 and S2 show the convergence of the HOMO− +LUMO gap and Qy and Qx excitation energies as a function of and W0 limits its predictive power.34−36 Partial self-consistency in the QP eigenvalues can often mitigate this problem. In eigenvalue self-consistent GW, the gKS eigenvalues used to construct G and/or W are replaced with those from the output of a prior GW step; the self-energy corrections are then iterated until the QP eigenvalues converge. This approach, which we call evGnWn in the following (n refers to the number of iterations), has been shown to remove much of the starting point dependence for a range of different systems.37,38 The BSE is an equation for the two-particle electron−hole Green's function and allows for the calculation of the polarizability including electron−hole interactions through the screened Coulomb interaction W. In practice, the BSE is usually solved by neglecting the frequency dependence of W. Within this static approximation, it can be written in a form equivalent to Casida's equations of TDDFT + +k +$${\binom{A}{-\ B\ -\ A}}{\binom{X^{s}}{Y^{s}}}=\Omega_{s}{\binom{X^{s}}{Y^{s}}}$$ +− − s = Ω(3) +k +where Ωs represents neutral excitation energies and (Xs, Ys) the corresponding eigenvectors.19 A and −A represent resonant and antiresonant transitions that can be expressed as + +$$A_{i a}^{j b}=(\varepsilon_{a}^{\mathrm{QP}}-\varepsilon_{i}^{\mathrm{QP}})\delta_{i j}\delta_{a b}-2(i a|j b)+W_{i j}^{a b}(\omega=0)$$ +and that are coupled through $B$ and $-B$, defined as . +$$B_{i a}^{j b}=-2(i a l b j)\,+\,W_{i i}^{a b}(\omega=0)$$ +ij +ab =− | + = ω (5) +for singlet excitations. In these expressions, i and j are occupied and a and b are unoccupied states and (ia|bj) stands for + +$$(i a l b j)=\iint\mathrm{d}\mathbf{r}\mathrm{d}\mathbf{r}^{\prime}\varphi_{i}^{\mathrm{QP}}(\mathbf{r})\varphi_{a}^{\mathrm{QP}}(\mathbf{r}){\frac{1}{|\mathbf{r}-\mathbf{r}^{\prime}|}}\varphi_{j}^{\mathrm{QP}}(\mathbf{r}^{\prime})\varphi_{b}^{\mathrm{QP}}(\mathbf{r}^{\prime})$$ + +(6) +Note that φi QP = φi gKS, whenever the G0W0 or evGnWn approaches are used to construct A and B. + +Computational Details. Our calculations of charged and neutral excitations were performed using the GW+BSE and TDDFT implementation in the open-source MOLGW software package (version 2B), which relies on Gaussian basis functions.39 We used the frozen-core approximation throughout, which changes excitation energies by less than 1 meV. We also employed the resolution-of-the-identity (RI) method, in order to reduce the calculation of four-center integrals to two- and three-center integrals. For BCL a, the RI changes the QP highest occupied molecular orbital +(HOMO)−lowest unoccupied molecular orbital (LUMO) +gap by less than 50 meV using a 6-31G basis set and BHLYP as a starting point, but we expect the effect of the RI to be even smaller for the larger basis sets used in the remainder of this article.40 To further reduce the computational cost of the evaluation of the GW polarizability, we use the single-pole approximation (see the Supporting Information for details). + +The Tamm−Dancoff approximation, which corresponds to neglecting the B matrix elements in eq 3 is not used as it consistently increases both GW+BSE and TDDFT results by +∼0.3 eV, in agreement with previous findings.6,27 We tested the influence of the Gaussian basis set size on HOMO−LUMO gaps and Qy and Qx excitations of BCL a +(using a structure from ref 16) with G0W0@BHLYP+BSE, +considering seven different basis sets, namely, the Pople basis sets 6-31G, 6-311G, 6-311++G**, and 6-311++G(2d,2p), +combined with the DeMon auxiliary basis set,41 and the + +$$({\mathfrak{I}})$$ + +$$\left({4}\right)$$ + +the inverse number of basis functions, 1/Nbasis for GW+BSE +and TDDFT, respectively (raw data are presented in Tables S1 and S2). We find that the HOMO−LUMO gap depends significantly more on 1/Nbasis than Qy and Qx excitation energies and that TDDFT results are less sensitive to the choice of the basis set than GW+BSE. Based on these tests, we use the 6-311++G(2d,2p) basis set for all calculations reported in the following. We estimate the error in the GW(+BSE) +HOMO−LUMO gap and the Qy and Qx excitation energies by linearly extrapolating to an infinite basis set. By excluding the very small 6-31G and 6-311G basis sets from these fits, we obtain extrapolated values of 3.57 eV for the HOMO−LUMO +gap, 1.11 eV for Qy, and 1.81 eV for Qx. We conclude that using the 6-311++G(2d,2p) basis set for all further calculations, we likely overestimate GW(+BSE) HOMO− +LUMO gaps and Qy and Qx excitation energies by ∼0.1 eV +with respect to the complete basis set limit. Conversely, the use of the single-pole approximation leads to a similar underestimation of the HOMO−LUMO gap and the Qy and Qx excitations, resulting in a fortuitous cancellation of errors. + +We test the effect of different xc functionals on our TDDFT +and GW+BSE results. We use the LDA, two global hybrid functionals (B3LYP and BHLYP), and the range-separated hybrid (RSH) functional ωPBE. In RSH functionals, the Coulomb repulsion is separated into a short-range part and a long-range part, for numerical convenience expressed as + +$${\frac{1}{r}}={\frac{1\,-\,\operatorname{erf}(a r)}{r}}\,+\,{\frac{\operatorname{erf}(a r)}{r}}$$ + +1 1 erf( ) erf( ) ω ω = − + (7) +where ω is called the range separation parameter. The ωPBE +functional uses PBE exchange in the short range and the exact exchange energy in the long range, allowing for a selfinteraction-free description at large electron−electron distances. We obtain the range separation parameter ω through the tuning procedure outlined in ref 43, in which ω is chosen such that the HOMO eigenvalue is as close as possible to the negative ionization potential both for the neutral and anionic system. Consequently, and by construction, the resulting HOMO−LUMO gap is a very good approximation to the fundamental gap of the neutral molecule. We use the Q-Chem code and a 6-31G(d,p) basis set for the tuning.44 The tuned range separation parameters for all systems discussed in the following can be found in Table S3. + +- RESULTS AND DISCUSSION +Bacteriochlorin. To validate our methodological setup and investigate the starting point dependence of the G0W0 approach and the effect of eigenvalue self-consistency on our calculated HOMO−LUMO gaps and excitation energies, we start by examining the BC molecule, for which GW+BSE +results have been reported in ref 27. We use a BC structure from ref 27 and denote the lowest energy excitations Qx and Qy, according to the direction of their transition dipole moments. Table 1 contains our calculated HOMO−LUMO + +Table 1. HOMO−LUMO Gaps, Qx and Qy Excitation + +Energies (in eV), and the Corresponding Oscillator + +Strengths, Γx and Γy, for BC Calculated with the 6-311+ + ++G(2d,2p) Basis Set + +method xc functional H-L gap Qx Γx Qy Γy + +TDDFT LDA 1.38 2.04 0.18 2.39 0.03 + +B3LYP 2.17 2.06 0.23 2.51 0.04 BHLYP 3.27 1.93 0.28 2.55 0.04 + +ωPBE 4.38 1.87 0.23 2.42 0.05 + +G0W0+BSE LDA 4.15 1.21 0.09 1.67 0.04 + +B3LYP 4.36 1.44 0.14 1.97 0.04 BHLYP 4.56 1.67 0.19 2.23 0.05 + +ωPBE 4.59 1.64 0.19 2.26 0.05 + +evGnW0+BSE LDA 4.31 1.41 0.13 1.97 0.04 + +B3LYP 4.43 1.54 0.16 2.12 0.05 BHLYP 4.56 1.66 0.19 2.24 0.05 + +ωPBE 4.57 1.62 0.18 2.27 0.04 + +evGnWn+BSE LDA 4.42 1.51 0.17 2.21 0.05 + +B3LYP 4.55 1.63 0.19 2.24 0.05 BHLYP 4.60 1.69 0.20 2.27 0.05 + +ωPBE 4.56 1.61 0.18 2.26 0.04 + +Expa 1.60 2.30 + +aData for bacteriopheophorbide from refs.31,27 + +gaps, Qx and Qy excitation energies, and oscillator strengths using TDDFT and several flavors of the GW+BSE approach. We find that, as expected, (generalized) Kohn−Sham HOMO−LUMO gaps show a large dependence on the xc functional, with LDA, B3LYP, and BHLYP leading to a significantly lower HOMO−LUMO gap and ωPBE leading to a HOMO−LUMO gap similar to the HOMO−LUMO gap calculated with G0W0 and eigenvalue-self-consistent evGnWn. In turn, Qy and Qx excitation energies from TDDFT are considerably less dependent on the xc functional than HOMO−LUMO gaps. In agreement with previous studies, we find that TDDFT overestimates the experimental values for The Journal of Physical Chemistry A pubs.acs.org/JPCA Article Qx and Qy by up to ∼0.4 eV, depending on the xc functional.27 TDDFT with the OT-RSH ωPBE is in best agreement with experiment, overestimating it by ∼0.2 eV for both excitations. + +We further find that G0W0@LDA+BSE underestimates Qx by 0.4 eV and Qy by 0.6 eV, whereas the use of a BHLYP and ωPBE starting point results in excitations within 0.1 eV of the experimental results. In accordance with prior studies, we observe that most of the starting point dependence of the G0W0+BSE results is inherited from the starting point dependence of the HOMO−LUMO gaps.25 In order to investigate the effect of eigenvalue selfconsistency in the GW+BSE approach, we tested the effect of updating the eigenvalues in the construction of G only +(evGnW0) and of both G and W (evGnWn). Eigenvalue selfconsistency in G alone only slightly changes the results as compared to G0W0. In contrast, full eigenvalue self-consistency largely eliminates the starting point dependence. In particular, using an LDA starting point results in excitation energies within 0.1 eV from experimentsimilar to the ωPBE starting point, but at considerably reduced computational cost. In Table S4, we report similar results for the more complex pigment BCL a. In the remainder of this article, we therefore focus primarily on eigenvalue self-consistent results based on LDA and ωPBE starting points. + +Excitation Energies of Bacteriochlorophylls and Chlorophylls. Next, we turn to reporting the vertical excitation energies of several members of the BCL and CL family of pigments. All structures were obtained from ref 45 and geometry-optimized using DFT as implemented in the Turbomole code with a def2-TZVP basis set and the B3LYP xc functional.46 Atomic coordinates of all relaxed structures can be found in the Supporting Information. We used both LDA +and ωPBE starting points for our evGnWn+BSE and ωPBE for our TDDFT calculations. Unlike the Qx excitation of BCL a and b, which has significant oscillator strength, the Qx excitation of BCL c−e is dark. Following ref 8, we therefore also compare our calculations with experimental results for the higher-energy B band.31 We report the vertical excitation energies and corresponding oscillator strengths of the first six excitations of all pigments in Tables S6 and S7. In these calculations, we included a total of 20 excitations, in order to ensure that the higher lying excitations are well converged. + +Table 2 demonstrates that evGnWn+BSE is in excellent agreement with experiment for the entire family of BCL and CL molecules. The MAE is about 50 meV for the Qy and Qx and between 100 and 200 meV for the B excitation. Our evGnWn+BSE results also accurately reflect the spectral shifts of the Qy excitation when comparing different BCL pigments with each other. For example, the BCL b molecule differs from BCL +a through an ethyliden side group, which shifts the Qy excitation by 40 meV to the red. This red shift is perfectly reproduced in our GW+BSE calculations. This is the first main result of this study. The second one is that our results are essentially independent of the DFT eigensystem used as an input for the GW+BSE approach: a computationally inexpensive LDA starting point results in the same level of agreement with experiment as the more tedious ωPBE +calculation that involves a system-dependent tuning procedure for the range separation parameter ω. This is in stark contrast to TDDFT. TD-LDA leads to spurious excitations with charge transfer character in between Qy and Qx, as well as slightly above Qx, depending on the structure, as discussed below and in the literature.47 TDDFT with the optimally tuned ωPBE + +| GW@LDA+BSE | GW@ωPBE+BSE | TD-ωPBE | expb | | | | | | | | | | +|----------------------------------------------------------------------------------------------------------------------|---------------|-----------|--------|------|------|------|------|------|------|------|------|------| +| molecule | Qy | Qx | B | Qy | Qx | B | Qy | Qx | B | Qy | Qx | B | +| BCL a | 1.52 | 2.08 | 3.25 | 1.50 | 2.10 | 3.16 | 1.75 | 2.16 | 3.33 | 1.60 | 2.15 | 3.46 | +| BCL b | 1.48 | 2.07 | 2.95 | 1.45 | 2.09 | 3.05 | 1.69 | 2.15 | 3.19 | 1.56 | 2.14 | 3.37 | +| BCL c | 1.85 | 2.05 | 2.94 | 1.84 | 2.11 | 3.02 | 2.05 | 2.21 | 3.21 | 1.88 | 2.89 | | +| BCL d | 1.90 | 2.15 | 2.94 | 1.89 | 2.21 | 3.05 | 2.08 | 2.29 | 3.19 | 1.90 | 2.93 | | +| BCL e | 2.01 | 2.04 | 2.78 | 1.96 | 2.13 | 2.88 | 2.10 | 2.23 | 3.02 | 1.92 | 2.72 | | +| CL a | 1.85 | 2.13 | 2.91 | 1.86 | 2.19 | 3.02 | 2.06 | 2.29 | 3.16 | 1.87 | 2.14 | 2.88 | +| CL b | 1.95 | 2.17 | 2.79 | 1.93 | 2.20 | 2.85 | 2.10 | 2.29 | 2.97 | 1.92 | 2.26 | 2.72 | +| MAE | 0.05 | 0.06 | 0.12 | 0.04 | 0.05 | 0.17 | 0.17 | 0.05 | 0.25 | | | | +| a GW+BSE results are based on eigenvalue self-consistent evGnWn. b Experimental results in diethyl ether from ref 8. | | | | | | | | | | | | | + +![4_image_0.png](4_image_0.png) + +Table 2. Qy, Qx and First B Band Excitation Energy of BCLs and CLs Calculated Using a 6-311++G(2d,2p) Basis Seta results in good agreement with experiment for all three excitations, albeit with slightly higher MAEs of 170, 50, and 250 meV for Qy, Qx, and B, respectively. + +In Figure 2, we plot the difference between our calculated results and experiment, averaged over all three excitations, to further highlight qualitative differences between evGnWn+BSE +and TDDFT. For BCL a and BCL b, evGnWn+BSE, on average, underestimates experiment by ∼100 meV, whereas the average TDDFT deviation is close to zero, because TDDFT +slightly overestimates the Qy and Qx excitations but underestimates the B excitation of these pigments. For all other BCL +and the two CL molecules studied here, we consistently find that the average deviation of evGnWn+BSE is significantly smaller than that of TDDFT. Similar to our results for the BC molecule and to other benchmark studies of complex organic molecules,6 TDDFT tends to overestimate all three excitations by between 200 and 300 meV. evGnWn+BSE is in much closer agreement with experiment for these pigments, on average, overestimating their excitation energies by less than 100 meV. We stress again that these results are independent of the DFT +starting point, whereas our TDDFT results rely on a persystem tuning procedure. + +Our results are in excellent agreement with correlated excited-state methods for those systems for which such studies have been reported, primarily CL a and BCL a. ADC(2) +excitation energies of the first three excitations of CL a reported by Suomivuori et al. are 1.97, 2.11, and 2.95 eV, +within ∼0.1 eV of our evGnWn@LDA+BSE results.13 In another study by the same authors, the ADC(2) Qy excitation energy of histidin-ligated BCL a was reported to be 1.46 eV, +again within ∼0.1 eV of our results, although it should be noted that the structures of ligated and free-standing BCL a slightly differ, leading to excitation energy differences of 10−30 meV at the ADC(2) level.14 Furthermore, Sirohiwal et al. used a pair-natural orbital coupled cluster approach to study CL a and reported Qy and Qx excitation energies of 1.75 and 2.24 eV, respectively, for CL a, also within ∼0.1 eV of our GW+BSE +results for these excitations.15 Not only the absolute energies of Qy and Qx excitations are important for understanding and predicting excitation energy and charge transfer in photosynthetic systems but also their relative energy difference, ΔQx−Qy +, plays a role, in particular, in coupled systems of several pigment units. It is therefore reassuring that evGnWn+BSE predicts ΔQx−Qy in very good agreement with experiment, with a deviation of only 10 meV for BCL a, BCL b, and CL a and 120 meV for CL b for the LDA starting point and a slightly larger deviation of, on average, 60 meV for the ωPBE starting point. TDDFT based on ωPBE tends to underestimate ΔQx−Qy +, by, on average, 110 meV for these four pigments. For BCL a, we also show in Table 3 that ΔQx−Qy strongly depends on the xc functional used in the TDDFT calculations, primarily because of the strong dependence of the Qx excitation on the amount of exact exchange, which can be seen by comparing the results based on the LDA (0% of exact exchange), B3LYP (∼23%), and BHLYP +Table 3. Difference between Qx and Qy Excitation Energies +(in eV) Using TDDFT and evGnWn+BSE for BCL a + +| method | xc functional | ΔQx−Qy | +|------------|-----------------|----------| +| evGnWn+BSE | LDA | 0.57 | +| B3LYP | 0.54 | | +| BHLYP | 0.54 | | +| ωPBE | 0.59 | | +| TDDFT | LDA | 0.25 | +| B3LYP | 0.40 | | +| BHLYP | 0.64 | | +| ωPBE | 0.43 | | +| exp8 | 0.55 | | + +(50%). As before, evGnWn+BSE is in excellent agreement with experiment and almost independent of the underlying xc functional. + +The experimental results reported in Tables 2 and 3 are based on measurements in diethyl ether, whereas our calculations are for gas-phase molecules. To approximately account for the effect of the solvent, we extracted experimental reference values for Qy and Qx excitations from a study by Limantara et al.,30 in which electronic absorption spectroscopy was used to obtain Qy and Qx for a large number of nonpolar and polar solvents at room temperature. This study reports regression lines for Qy and Qx excitations of BCL a as a function of R(n) = n2 − 1/n2 + 2, where n is the refractive index of the solvent. The extrapolated values for n = 1 (vacuum) are 1.68 eV (nonpolar) and 1.67 eV (polar) for the Qy and 2.25 eV (nonpolar) and 2.21 eV (polar) for the Qx excitation. Based on these regression parameters, we estimate that the experimental reference values in Table 2 lie ∼50−70 meV below the gas-phase excitation energies. We also calculated the Qy and Qx excitation energies of BCL a with TDDFT (using ωPBE), approximating solvent effects with the COSMO approach as implemented in Turbomole. We used a dielectric constant of 4.33ε0 corresponding to the value in diethyl ether. COSMO red-shifts the Qy and Qx excitation energies by 70 and 50 meV, respectively, supporting our estimate. We conclude that solvent effects are smallwithin the numerical accuracy of our GW+BSE calculationsand do not change our main conclusions. Note that we also neglect the effects of temperature and the 0−0 vibrational energy contribution in our comparison with experimental results. Exact agreement of our calculated results with experiment is therefore not expected. + +Bacteriochlorophyll a. In the remainder of this paper, we will use the BCL a molecule as a case study to compare to available computational literature results for this pigment, discuss the origin of differences between our evGnWn+BSE and TDDFT results, and comment on the effects of the choice of structure on excitation energies. + +Comparison with RASPT2. For the Qy and Qx excitations of BCL a, we compare our GW+BSE and TDDFT calculations to multistate, second-order perturbation theory (RASPT2) +calculations by Anda et al.16,17 For this comparison, we use the molecular geometry reported in ref 16, which is a BCL a unit from the light-harvesting system LH2 of Rhodoblastus acidophilus. This structure was extracted from an experimental X-ray crystallographic structure of the LH2 complex (unit 302 within the structure 1NKZ in the RCSB Protein Data Bank).48 The phytyl tail was truncated and replaced by a hydrogen atom, and no further geometry optimization was carried out. In the following, we will call this structure "A". Our geometryoptimized version of "A", which we relaxed using DFT as + +![5_image_0.png](5_image_0.png) + +implemented in the Turbomole code with a def2-TZVP basis set and B3LYP,46 will be called "R". A visual comparison between "A" and "R" is shown in Figure 3. The large differences that we observe between these two structures are unsurprising, given that we perform our geometry optimizations without taking into account the protein environment in which BCL a "A" is embedded in vivo. Table 4 shows our GW +Table 4. HOMO−LUMO Gaps and Qy and Qx Excitation Energies (in eV) for BCL a Structure "A" ++BSE and TDDFT results for "A" in comparison with the RASPT2 excitation energies from refs.16,17 We find, as before, that when eigenvalue self-consistency is used in GW, HOMO− +LUMO gaps and Qy and Qx excitation energies differ by a maximum of 0.1 eV. Most notably, however, our GW+BSE +excitation energies substantially differ from those calculated with RASPT2, with Qy 0.4 eV and Qx 0.5 eV lower than the RASPT2 result. + +We find that about half of this difference can be traced back to the use of a smaller basis set (ANO-RCC-vDZP) in ref 16. + +Repeating our evGnWn@LDA+BSE calculation with the same basis, we obtain excitation energies of 1.38 eV for Qy and 2.18 eV for Qx. In line with previous studies, we also find that TDDFT with global hybrid functionals (B3LYP and BHLYP) +results in similar excitation energies to RASPT2 for the Qy excitation.17,49 We hypothesize that this agreement is fortuitous. The optimally tuned RSH functional ωPBE has been shown to better describe singlet excitation energies of a + +| method/basis set | xc | | | | +|----------------------------|------|------|------|------| +| functional | H-L | | | | +| evGnWn+BSE/6-311++G(2d,2p) | LDA | 3.62 | 1.17 | 1.90 | +| evGnWn+BSE/ANO-RCC-vDZP | LDA | 3.76 | 1.38 | 2.18 | +| TDDFT/6-311++G(2d,2p) | LDA | 0.92 | 1.59 | 1.99 | +| RASPT2/ANO-RCC-vDZP | 1.61 | 2.40 | | | + +wide variety of organic compounds as compared to global hybrid functionals50−52 and is more than 0.1 eV lower in energy than the RASPT2 Qy excitation energy. Similar trends have also been shown for CL a, where DFT-based multireference CI, just as TDDFT with global hybrid functionals, tends to overestimate experiment by ∼0.2 eV for the Qy and Qx excitation.53 All in all, given that comparisons with experimental data are complicated for an in vivo structure such as "A", we consider it most likely that our GW+BSE +calculations underestimate the excitation energies of structure +"A" by ∼0.1 eV, similar to our results for gas-phase BCL a +(Table 2). The remaining deviations could be attributed to the multireference character of the Qy excitation16 and the choice of the RAS. + +We also note that our GW+BSE results reproduce the energetic order and relative energy differences of the Qy excitation of other BCL units within the LH2 ring that RASPT2 predicts, when using the ANO-RCC-vDZP basis set. + +However, the use of the significantly larger 6-311++G(2d,2p) +basis leads to substantially larger excitation energy differences between these units (Table S7). Finally, it is worth mentioning that our GW+BSE calculations reproduce the relatively large energy difference ΔQx−Qy ≈ 0.8 eV that RASPT2 predicts, whereas TDDFT excitation energy differences are much less sensitive to details of the structure, with ΔQx−Qy ≈ 0.5 eV (using ωPBE) similar to the gas-phase structure of BCL a. We speculate that a geometry optimization of structure "A" within its protein environment would result in a smaller ΔQx−Qy for both RASPT2 and GW+BSE. + +Role of the Electron−Hole Kernel. We find that the difference between GW+BSE and TDDFT excitation energies can be traced back almost entirely to differences in how electron−hole interactions are described in both schemes. The Qy excitation is primarily (∼90%) a HOMO → LUMO +transition, and the HOMO−LUMO gaps, as calculated with DFT-ωPBE and evGnWn@ωPBE, differ by only 0.02 meV +(Table 3). In fact, the density of states (DOS) in the energy range relevant for both the Qy and the Qx excitations based on evGnWn@ωPBE and DFT-ωPBE eigenvalues are almost identical (see Figure 4). To further test our hypothesis, we construct the statically screened Coulomb interaction Wij ab (see eq 4) and solve the BSE based on a DFT-ωPBE eigensystem +(instead of first computing QP eigenvalues using eq 2). We obtain values for the Qy and Qx excitation that are only 20 meV +higher and 40 meV lower than the full GW+BSE solution, respectively, for structure "A". Similarly, for structure "R", the results are within less than 10 and 50 meV for the Qy and Qx excitation, respectively. This observation confirms that differences between the GW+BSE and TDDFT excitation energies are primarily due to differences in the xc and the BSE kernel. + +Generally, the overestimation of excitation energies that we observe with TDDFT is in line with results for other organic π chromophores such as rhodamine and rosamine54 and of phenothiazine dyes,55 for which it has been linked to an insufficient treatment of differential electron correlation between the ground and excited states by most TDDFT xc kernels.56 Charge Transfer Excitations with TD-LDA and GnWn@LDA ++BSE. Finally, motivated by the excellent performance of evGnWn@LDA+BSE, we compare G0W0@LDA+BSE, evGWLDA+BSE, and TD-LDA results for structures "A" and "R" of BCL a. Figure 5 shows the excitation spectrum calculated at + +The Journal of Physical Chemistry A pubs.acs.org/JPCA Article + +![6_image_0.png](6_image_0.png) + +these levels of theory. TD-LDA's severe underestimation of charge transfer excitations is well known9 and leads to spurious excitations with charge transfer character at energies between Qy and Qx for BCL a. + +47 Our comparison of structures "A" and +"R" shows that while the energy of Qy and Qx is changing only slightly when TD-LDA is used, the relative position of these spurious low-oscillator strength excitations depends strongly on the structure. G0W0@LDA+BSE results in a very different, albeit no more reassuring, picture. For both structures, the first excitation already appears at energies below or around 1 eV +and its oscillator strength is considerably lower than with TDLDA; for structure "A", the oscillator strength of Qy is even lower than that of Qx. For structure "R", excitations 2, 3, and 4 have similar, very low, oscillator strength. However, already at the G0W0@LDA+BSE level, no charge transfer excitations are found between Qy and Qxa consequence of the inherent nonlocality of the BSE kernel. Finally, for both structures, eigenvalue self-consistency pushes all excitations to significantly higher energies and results in a quantitatively correct description of Qy and Qx. + +Inspection of the DOS calculated with DFT-xc, G0W0@xc, and GnWn@xc (xc = LDA, ωPBE) shown in Figure 4 is instructive for understanding the contribution of eigenvalue differences to the TDDFT and GW+BSE excitation energies. + +The G0W0@LDA DOS underestimates the HOMO−LUMO +gap and the energy difference between the HOMO and HOMO − 1. In contrast, there is virtually no difference between the HOMO, HOMO − 1, and LUMO energies as calculated with DFT-ωPBE, G0W0@ωPBE, evGnWn@ωPBE, +and evGnWn@LDA. As expected, the DFT-LDA DOS is markedly different, not only underestimating the HOMO− LUMO gap but also significantly underestimating the energy differences between the HOMO − 1, HOMO − 2, and HOMO − 3. Notably, the spurious dark states between Qy and Qx that TD-LDA predicts have significant contributions from transitions involving these lower occupied states. + +The Journal of Physical Chemistry A pubs.acs.org/JPCA Article + +![7_image_0.png](7_image_0.png) + +- CONCLUSIONS +In this article, we performed a systematic first-principles study of the electronic structure and excitations of seven members of the (bacterio)chlorophyll family, which we validated through comparison with calculated and experimental literature results. + +The GW+BSE approach, when used in a partially selfconsistent fashion, is in excellent agreement with experiment for excitations in the visible and near-ultraviolet part of the spectrum. GW+BSE also correctly predicts the energy difference between the low-energy Qy and Qx excitations of these pigments, relevant for the description of the coupling between pigment complexes, present in the light harvesting units and reaction centers of plants and bacteria and crucial for excitation energy and charge transfer. Most importantly, our results are almost entirely independent of the DFT eigensystem used as an input for the GW+BSE calculations. A computationally inexpensive LDA starting point leads to similar results as a more involved optimally tuned ωPBE +starting point. + +It should be noted that the GW approach, despite its implementation using Gaussian basis functions and the use of the RI approximation in MOLGW and other codes, remains a major bottleneck of these calculations due to its O(N4) scaling with system size. Furthermore, our results highlight that the GW approach, more so than DFT, requires careful convergence with respect to the basis set size. This limits its applicability to systems with a few (B)CL pigments at most, until algorithms with better scaling become more widely available.57−59 Our study joins a growing number of results, demonstrating that the GW+BSE approach can accurately predict neutral excitations of complex molecules without empirical parameters.29 With new approaches for combining GW+BSE with large-scale molecular mechanics simulations28 and polarizable continuum embedding60 emerging, an accurate + +prediction of excitation energy and charge transfer in complex molecular environments is within reach. + +- ASSOCIATED CONTENT +*sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpca.1c01240. + +Atomic coordinates (ZIP) Additional convergence data, tuned range-separation parameters, and excitation energies of all molecules discussed in the main manuscript (PDF) +- AUTHOR INFORMATION +Corresponding Author Linn Leppert − MESA+ Institute for Nanotechnology, University of Twente, 7500 AE Enschede, The Netherlands; Institute of Physics, University of Bayreuth, Bayreuth 95440, Germany; orcid.org/0000-0002-4361-4382; Email: l.leppert@utwente.nl Author Zohreh Hashemi − Institute of Physics, University of Bayreuth, Bayreuth 95440, Germany Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jpca.1c01240 + +## Notes + +The authors declare no competing financial interest. + +- ACKNOWLEDGMENTS +The authors are grateful for helpful discussions with C. Filippi. This work was supported by the Bavarian State Ministry of Science and the Arts through the Collaborative Research Network Solar Technologies go Hybrid (SolTech), the Elite Network Bavaria (ENB), and through computational resources provided by the Bavarian Polymer Institute (BPI). + +- REFERENCES +(1) Blankenship, R. E. Molecular Mechanisms of Photosynthesis, 2nd ed.; Wiley Blackwell: Oxford, 2014. + +(2) Hu, X.; Ritz, T.; Damjanovic, A.; Autenrieth, F.; Schulten, K. ́ +Photosynthetic Apparatus of Purple Bacteria. Q. Rev. Biophys. 2002, 35, 1−62. + +(3) Croce, R.; van Amerongen, H. Natural Strategies for Photosynthetic Light Harvesting. Nat. Chem. Biol. 2014, 10, 492−501. + +(4) Cogdell, R. J.; Gall, A.; Köhler, J. The Architecture and Function of the Light-Harvesting Apparatus of Purple Bacteria: From Single Molecules to In Vivo Membranes. Q. Rev. Biophys. 2006, 39, 227− +324. + +(5) Jang, S.; Mennucci, B. Delocalized Excitons in Natural LightHarvesting Complexes. Rev. Mod. Phys. 2018, 90, 035003. + +(6) Shao, Y.; Mei, Y.; Sundholm, D.; Kaila, V. R. I. Benchmarking the Performance of Time-Dependent Density Functional Theory Methods on Biochromophores. J. Chem. Theory Comput. 2020, 16, 587−600. + +(7) Sundholm, D. A Density-Functional-Theory Study of Bacteriochlorophyll b. Phys. Chem. Chem. Phys. 2003, 5, 4265−4271. + +(8) Vokácová, Z.; Burda, J. V. Computational Study on Spectral ̌ +Properties of the Selected Pigments from Various Photosystems: Structure-Transition Energy Relationship. J. Phys. Chem. A 2007, 111, 5864−5878. + +(9) Dreuw, A.; Head-Gordon, M. Failure of Time-Dependent Density Functional Theory for Long-Range Charge-Transfer Excited States: The Zincbacteriochlorin-Bacteriochlorin and Bacteriochlorophyll-Spheroidene Complexes. J. Am. Chem. Soc. 2004, 126, 4007− +4016. + +(10) Tozer, D. J.; Handy, N. C. Improving Virtual Kohn-Sham Orbitals and Eigenvalues: Application to Excitation Energies and Static Polarizabilities. J. Chem. Phys. 1998, 109, 10180−10189. + +(11) Kuritz, N.; Stein, T.; Baer, R.; Kronik, L. Charge-Transfer-Like π -> π * Excitations in Time-Dependent Density Functional Theory: +A Conundrum and Its Solution. J. Chem. Theory Comput. 2011, 7, 2408−2415. + +(12) Kümmel, S. Charge-Transfer Excitations: A Challenge for Time-Dependent Density Functional Theory That Has Been Met. Adv. Energy Mater. 2017, 7, 1700440. + +(13) Suomivuori, C.-M.; Fliegl, H.; Starikov, E. B.; Balaban, T. S.; +Kaila, V. R. I.; Sundholm, D. Absorption Shifts of Diastereotopically Ligated Chlorophyll Dimers of Photosystem I. Phys. Chem. Chem. + +Phys. 2019, 21, 6851−6858. + +(14) Suomivuori, C.-M.; Winter, N. O. C.; Hättig, C.; Sundholm, D.; +Kaila, V. R. I. Exploring the Light-Capturing Properties of Photosynthetic Chlorophyll Clusters Using Large-Scale Correlated Calculations. J. Chem. Theory Comput. 2016, 12, 2644−2651. + +(15) Sirohiwal, A.; Berraud-Pache, R.; Neese, F.; Izsák, R.; Pantazis, D. A. Accurate Computation of the Absorption Spectrum of Chlorophyll a with Pair Natural Orbital Coupled Cluster Methods. + +J. Phys. Chem. B 2020, 124, 8761−8771. + +(16) Anda, A.; Hansen, T.; De Vico, L. Multireference Excitation Energies for Bacteriochlorophylls A within Light Harvesting System 2. + +J. Chem. Theory Comput. 2016, 12, 1305−1313. + +(17) Anda, A.; Hansen, T.; De Vico, L. Qy and Qx Absorption Bands for Bacteriochlorophyll a Molecules from LH2 and LH3. J. Phys. + +Chem. A 2019, 123, 5283−5292. + +(18) Segatta, F.; Cupellini, L.; Jurinovich, S.; Mukamel, S.; Dapor, M.; Taioli, S.; Garavelli, M.; Mennucci, B. A Quantum Chemical Interpretation of Two-Dimensional Electronic Spectroscopy of LightHarvesting Complexes. J. Am. Chem. Soc. 2017, 139, 7558−7567. + +(19) Onida, G.; Reining, L.; Rubio, A. Electronic Excitations: +Density-Functional versus Many-Body Green's-Function Approaches. + +Rev. Mod. Phys. 2002, 74, 601. + +The Journal of Physical Chemistry A pubs.acs.org/JPCA Article +(20) Albrecht, S.; Reining, L.; Del Sole, R.; Onida, G. Ab Initio Calculation of Excitonic Effects in the Optical Spectra of Semiconductors. Phys. Rev. Lett. 1998, 80, 4510. + +(21) Rohlfing, M.; Louie, S. G. Electron-Hole Excitations and Optical Spectra from First Principles. Phys. Rev. B: Condens. Matter Mater. Phys. 2000, 62, 4927. + +(22) Qiu, D. Y.; Da Jornada, F. H.; Louie, S. G. Optical Spectrum of MoS2: Many-Body Effects and Diversity of Exciton States. Phys. Rev. + +Lett. 2013, 111, 216805. + +(23) Grossman, J. C.; Rohlfing, M.; Mitas, L.; Louie, S. G.; Cohen, M. L. High Accuracy Many-Body Calculational Approaches for Excitations in Molecules. Phys. Rev. Lett. 2001, 86, 472−475. + +(24) Tiago, M. L.; Kent, P. R. C.; Hood, R. Q.; Reboredo, F. A. + +Neutral and Charged Excitations in Carbon Fullerenes from FirstFrinciples Many-Body Theories. J. Chem. Phys. 2008, 129, 084311. + +(25) Bruneval, F.; Hamed, S. M.; Neaton, J. B. A Systematic Benchmark of the Ab Initio Bethe-Salpeter Equation Approach for Low-Lying Optical Excitations of Small Organic Molecules. J. Chem. Phys. 2015, 142, 244101. + +(26) Palummo, M.; Hogan, C.; Sottile, F.; Bagalá, P.; Rubio, A. Ab Initio Electronic and Optical Spectra of Free-Base Porphyrins: The Role of Electronic Correlation. J. Chem. Phys. 2009, 131, 084102. + +(27) Duchemin, I.; Deutsch, T.; Blase, X. Short-Range to LongRange Charge-Transfer Excitations in the ZincbacteriochlorinBacteriochlorin Complex: A Bethe-Salpeter Study. Phys. Rev. Lett. + +2012, 109, 167801. + +(28) Wehner, J.; Brombacher, L.; Brown, J.; Junghans, C.; Çaylak, O.; Khalak, Y.; Madhikar, P.; Tirimbo, G.; Baumeier, B. Electronic ̀ +Excitations in Complex Molecular Environments: Many-Body Green's Functions Theory in VOTCA-XTP. J. Chem. Theory Comput. 2018, 14, 6253−6268. + +(29) Blase, X.; Duchemin, I.; Jacquemin, D. The Bethe-Salpeter Equation in Chemistry: Relations with TD-DFT, Applications and Challenges. Chem. Soc. Rev. 2018, 47, 1022−1043. + +(30) Limantara, L.; Sakamoto, S.; Koyama, Y.; Nagae, H. Effects of Nonpolar and Polar Solvents on the Qx and Qy Energies of Bacteriochlorophyll a and Bacteriopheophytin a. Photochem. Photobiol. + +1997, 65, 330−337. + +(31) Scheer, H.; Inhoffen, H. The Porphyrins; Dolphi, Ed.; Academic Press: New York, 1978; Vol. II. + +(32) Fetter, A.; Walecka, J. Quantum Theory of Many-Particle Systems; MacGraw-Hill: New York, 1971. + +(33) Hedin, L. On Correlation Effects in Electron Spectroscopies and the GW Approximation. J. Phys.: Condens. Matter 1999, 11, R489. + +(34) Jiang, H.; Gomez-Abal, R. I.; Rinke, P.; Scheffler, M. FirstPrinciples Modeling of Localized d States with the GW@LDA+U +Approach. Phys. Rev. B: Condens. Matter Mater. Phys. 2010, 82, 045108. + +(35) Liao, P.; Carter, E. A. Testing Variations of the GW +Approximation on Strongly Correlated Transition Metal Oxides: +Hematite (α-Fe2O3) as a Benchmark. Phys. Chem. Chem. Phys. 2011, 13, 15189−15199. + +(36) Marom, N.; Caruso, F.; Ren, X.; Hofmann, O. T.; Körzdörfer, T.; Chelikowsky, J. R.; Rubio, A.; Scheffler, M.; Rinke, P. Benchmark of GW Methods for Azabenzenes. Phys. Rev. B: Condens. Matter Mater. Phys. 2012, 86, 245127. + +(37) Jacquemin, D.; Duchemin, I.; Blase, X. Benchmarking the Bethe-Salpeter Formalism on a Standard Organic Molecular Set. J. + +Chem. Theory Comput. 2015, 11, 3290−3304. + +(38) Kaplan, F.; Weigend, F.; Evers, F.; Van Setten, M. J. OffDiagonal Self-Energy Terms and Partially Self-Consistency in GW +Calculations for Single Molecules: Efficient Implementation and Quantitative Effects on Ionization Potentials. J. Chem. Theory Comput. + +2015, 11, 5152−5160. + +(39) Bruneval, F.; Rangel, T.; Hamed, S. M.; Shao, M.; Yang, C.; +Neaton, J. B. MOLGW 1: Many-Body Perturbation Theory Software for Atoms, Molecules, and Clusters. Comput. Phys. Commun. 2016, 208, 149−161. + +(58) Vlcek, V.; Rabani, E.; Neuhauser, D.; Baer, R. Stochastic GW ̌ +Calculations for Molecules. J. Chem. Theory Comput. 2017, 13, 4997− +5003. + +(59) Förster, A.; Visscher, L. Low-Order Scaling G0W0 by Pair Atomic Density Fitting. J. Chem. Theory Comput. 2020, 16, 7381− +7399. + +(60) Duchemin, I.; Guido, C. A.; Jacquemin, D.; Blase, X. The Bethe-Salpeter Formalism with Polarisable Continuum Embedding: Reconciling Linear-Response and State-Specific Features. Chem. Sci. + +2018, 9, 4430−4443. + +(40) van Setten, M. J.; Caruso, F.; Sharifzadeh, S.; Ren, X.; Scheffler, M.; Liu, F.; Lischner, J.; Lin, L.; Deslippe, J. R.; Louie, S. G.; Yang, C.; Weigend, F.; Neaton, J. B.; Evers, F.; Rinke, P. GW100: Benchmarking G0W0 for Molecular Systems. J. Chem. Theory Comput. + +2015, 11, 5665−5687. + +(41) Godbout, N.; Salahub, D. R.; Andzelm, J.; Wimmer, E. + +Optimization of Gaussian-Type Basis Sets for Local Spin Density Functional Calculations. Part I. Boron Through Neon, Optimization Technique and Validation. Can. J. Chem. 1992, 70, 560−571. + +(42) Zheng, J.; Xu, X.; Truhlar, D. G. Minimally Augmented Karlsruhe Basis Sets. Theor. Chem. Acc. 2011, 128, 295−305. + +(43) Stein, T.; Eisenberg, H.; Kronik, L.; Baer, R. Fundamental Gaps in Finite Systems from Eigenvalues of a Generalized Kohn-Sham Method. Phys. Rev. Lett. 2010, 105, 266802. + +(44) Shao, Y.; Gan, Z.; Epifanovsky, E.; Gilbert, A. T.; Wormit, M.; +Kussmann, J.; Lange, A. W.; Behn, A.; Deng, J.; Feng, X.; et al. + +Advances in Molecular Quantum Chemistry Contained in the QChem 4 Program Package. Mol. Phys. 2015, 113, 184−215. + +(45) Oviedo, M. B.; Negre, C. F. A.; Sánchez, C. G. Dynamical Simulation of the Optical Response of Photosynthetic Pigments. Phys. + +Chem. Chem. Phys. 2010, 12, 6706−6711. + +(46) Turbomole, V7.4 2019, a development of University of Karlsruhe and Forschungszentrum Karlsruhe GmbH, 2019. www. turbomole.org. + +(47) Schelter, I.; Foerster, J. M.; Gardiner, A. T.; Roszak, A. W.; +Cogdell, R. J.; Ullmann, G. M.; De Queiroz, T. B.; Kümmel, S. + +Assessing Density Functional Theory in Real-Time and Real-Space as a Tool for Studying Bacteriochlorophylls and the Light-Harvesting Complex 2. J. Chem. Phys. 2019, 151, 134114. + +(48) Papiz, M. Z.; Prince, S. M.; Howard, T.; Cogdell, R. J.; Isaacs, N. W. The Structure and Thermal Motion of the B800-850 LH2 Complex from Rps. acidophila at 2.0 Å Resolution and 100 K: New Structural Features and Functionally Relevant Motions. J. Mol. Biol. + +2003, 326, 1523−1538. + +(49) List, N. H.; Curutchet, C.; Knecht, S.; Mennucci, B.; Kongsted, J. Toward Reliable Prediction of the Energy Ladder in Multichromophoric Systems: A Benchmark Study on the FMO LightHarvesting Complex. J. Chem. Theory Comput. 2013, 9, 4928−4938. + +(50) Kronik, L.; Stein, T.; Refaely-Abramson, S.; Baer, R. Excitation Gaps of Finite-Sized Systems from Optimally Tuned Range-Separated Hybrid Functionals. J. Chem. Theory Comput. 2012, 8, 1515−1531. + +(51) Refaely-Abramson, S.; Baer, R.; Kronik, L. Fundamental and Excitation Gaps in Molecules of Relevance for Organic Photovoltaics from an Optimally Tuned Range-Separated Hybrid Functional. Phys. + +Rev. B: Condens. Matter Mater. Phys. 2011, 84, 075144. + +(52) Jacquemin, D.; Moore, B.; Planchat, A.; Adamo, C.; +Autschbach, J. Performance of an Optimally Tuned Range-Separated Hybrid Functional for 0-0 Electronic Excitation Energies. J. Chem. + +Theory Comput. 2014, 10, 1677−1685. + +(53) Parusel, A. B. J.; Grimme, S. A Theoretical Study of the Excited States of Chlorophyllaand Pheophytina. J. Phys. Chem. B 2000, 104, 5395−5398. + +(54) Moore, B.; Schrader, R. L.; Kowalski, K.; Autschbach, J. + +Electronic π-to-π* Excitations of Rhodamine Dyes Exhibit a TimeDependent Kohn-Sham Theory "Cyanine Problem". ChemistryOpen 2017, 6, 385−392. + +(55) De Queiroz, T. B.; De Figueroa, E. R.; Coutinho-Neto, M. D.; +Maciel, C. D.; Tapavicza, E.; Hashemi, Z.; Leppert, L. First Principles Theoretical Spectroscopy of Methylene Blue: Between Limitations of Time-Dependent Density Functional Theory Approximations and its Realistic Description in the Solvent. J. Chem. Phys. 2021, 154, 044106. + +(56) Moore, B.; Autschbach, J. Longest-Wavelength Electronic Excitations of Linear Cyanines: The Role of Electron Delocalization and of Approximations in Time-Dependent Density Functional Theory. J. Chem. Theory Comput. 2013, 9, 4991−5003. + +(57) Neuhauser, D.; Gao, Y.; Arntsen, C.; Karshenas, C.; Rabani, E.; +Baer, R. Breaking the Theoretical Scaling Limit for Predicting Quasiparticle Energies: The Stochastic GW Approach. Phys. Rev. Lett. + +2014, 113, 076402. \ No newline at end of file diff --git a/medical/md/PMC8061605.md b/medical/md/PMC8061605.md new file mode 100644 index 0000000000000000000000000000000000000000..6ab26604e2022a16dfc8bd967b72ec3d19460756 --- /dev/null +++ b/medical/md/PMC8061605.md @@ -0,0 +1,47 @@ + +## Sliding Inguinal Hernia In A Case Of Fournier'S Gangrene: A **Rare Case** + +Abstract Fournier's gangrene and inguinal hernia, both are common diseases. However, inguinal hernia in a case of Fournier's gangrene is rarest of rare cases. Only one such case is reported in literature. We present one such rare case. A 50‑year–old man presented with blackish discoloration of scrotum with inguinoscrotal swelling. It was associated with pus discharge and foul smell. There were noassociated co morbidities. The patient was a chronic smoker from 40 years. Serial debridement was done until the wound became healthy and free of infection. After infection control, the patient was operated and hernioplasty was done. Keywords: *Debridement, Fournier's, gangrene, hernia, inguinoscrotal swelling* + +## Introduction + +Fournier's gangrene is a necrotizing fasciitis characterized by a polymicrobial infection of the soft tissues of the perineum, external genitalia and perianal region.[1] It can arise following minor injuries or procedures in the perineal area, such as a bruise, scratch, or opening of a periurethral abscess. Many patients have concurrent illnesses that diminish their defenses, most notably diabetes mellitus and alcoholism. There is a mixed infection of aerobic and anaerobic bacteria in a fulminating inflammation of the subcutaneous tissues which results in an obliterative arteritis of the arterioles to the scrotal skin resulting in gangrene. The condition can spread rapidly to involve the fascia and skin of the penis, perineum, and abdominal wall. Infection spreads rapidly with small necrotic areas of skin which, if untreated, coalesce to involve the entire scrotal and penile coverings, which may then slough, leaving the testes exposed. There may be crepitus and a foul‑smelling exudate. The patient typically becomes septic in a short period of time. Fournier's gangrene is a surgical emergency. Initial management involves intravenous fluid resuscitation and early use of broad spectrum intravenous antibiotics. Urgent wide surgical excision of the dead and infected tissue is essential and the extent of the internal necrosis is typically much This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. + +For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com greater than the external appearances. Supportive care is essential. + +## Case Report + +A  50‑year‑Gentleman presented with a complaint of blackish discoloration of scrotum from 20 days. The patient initially developed pain and pustule over scrotum which later progressed to involving whole of the scrotum and was associated with foul smell, blackish discoloration, and fever. The patient was diagnosed with Fournier's Gangrene and debridement of scrotum was done [Figure 1a]. The patient had also left inguinal hernia and it was irreducible. The patient was chronic smoker from 40 years. There was no significant past medical or surgical history. On examination, the patient was vitally stable. Irreducible left inguinal hernia with Fournier's gangrene of scrotum was present. Patient's hemoglobin was on the lower side (8.4 g %) and total leukocyte count was raised (31,000/ +mm3). Serial debridement was done and culture and sensitivity of tissue was sent [Figure 1b]. Broad spectrum antibiotic started early and later, the patient was shifted on antibiotic according to sensitivity test. Dressings were done twice a day to manage the Fournier's Gangrene. Patient's clinical picture and blood parameters improved significantly. Patient was operated for irreducible inguinal hernia after scrotal infection was controlled and debridement with reduction of hernial sac with anatomical repair of hernia (hernioplasy) +Meghraj Kundan, Vinaysheel Priyadarshi, Chintamani Department of General Surgery, VMMC and Safdarjung Hospital, New *Delhi, India* Submitted: 09‑Dec‑2019 Revised: 02‑Dec‑2020 Accepted: 03-Jan-2021 Published: 08‑Apr‑2021 Address for correspondence: Dr. Vinaysheel Priyadarshi, Post Graduate Resident, Department of Surgery, VMMC and Safdarjung Hospital, New Delhi. E‑mail: *drmeghrajkundan@* gmail.com + +![0_image_0.png](0_image_0.png) + +How to cite this article: Kundan M, Priyadarshi V, +Chintamani. Sliding inguinal hernia in a case of Fournier's gangrene: A rare case. Int J App Basic Med Res 2021;11:106-7. + +![1_image_0.png](1_image_0.png) + +under general anaesthesia was done. Per-operative findings– Dense adhesions were present between gangrenous testis and caecum. Caecum was the content of the hernial sac. Specimen was sent for histopathological examination [Figure 1c]. The patient improved and discharged. Patient was followed up after 2 weeks and then after 3 months. The patient had no complaints. + +## Discussion + +Fournier's gangrene was first described by Baurienne in 1764 and is named after a French venereologist, Jean Alfred Fournier following five cases he presented in clinical lectures in 1883.[2] Initially, it was considered as rapidly progressing idiopathic necrotizing soft‑tissue infection of scrotum. However, later, it was concluded that it is the infection of perineal and genital region, typically seen in elderly, diabetic, or otherwise immune‑compromised and is known to be frequently due to a symbiotic polymicrobial infection. Females can also acquire this infection but there is a male predominance. Sliding hernia is the hernia in which one of the walls of the hernial sac is formed by a viscous. Approximately 3%–5% of all indirect inguinal hernias are sliding hernias. Sigmoid colon being the most common slided organ. Other common slided organs are retroperitoneal appendix, caecum, and urinary bladder. In 1995, Laor et al. + +[3] developed Fournier's Gangrene Severity Index to predict the prognosis of the patients. The treatment of Fournier's gangrene is radical debridement of the gangrene until no infected tissue is left, supported by broad spectrum antibiotics and followed by skin grafting. The treatment of sliding hernia is surgery. Depending on the setting of treating center, surgeon and the patient, treatment can either be done by open surgeries or by laparoscopic surgeries. + +## Conclusion + +Inguinoscrotal hernia in a case of Fournier's gangrene is extremely rare. No literature states the proper treatment for such condition. Fournier's gangrene should managed first as it is a life‑threatening condition if inguinal hernia is uncomplicated. After control of infection, the patient can be taken for surgery to manage the hernia. It can either be done as open surgery using high inguinal approach or can be done laparoscopically. However, in case of complicated inguinal hernia with Fournier's gangrene, further study is needed. + +## Declaration Of Patient Consent + +The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. + +## References + +1. Yecies T, Lee DJ, Sorbellini M, Ramasamy R. Penile Fournier's gangrene. Urology 2013;82:e31. + +2. Fournier JA. Fournier 1832‑1914. Gangrene foudroyante de la verge (overwhelming gangrene). Sem Med 1883. Dis Colon Rectum 1988;31:984‑8. + +3. Laor E, Palmer LS, Tolia BM, Reid RE, Winter HI. Outcome prediction in patients with Fournier's gangrene. J Urol 1995;154:89‑92. \ No newline at end of file diff --git a/medical/md/PMC8080457.md b/medical/md/PMC8080457.md new file mode 100644 index 0000000000000000000000000000000000000000..bb33f5e729e6773b6c6afe246c9ded205dd4d024 --- /dev/null +++ b/medical/md/PMC8080457.md @@ -0,0 +1,154 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +![0_image_3.png](0_image_3.png) + +![0_image_5.png](0_image_5.png) + +Review + +![0_image_2.png](0_image_2.png) + +Epidural analgesia and related ethical issues among pregnant women: +Literature review Sawsan Abuhammad *, Esraa Alholi School of Nursing, Jordan University of Science and Technology, Irbid, Jordan + +| ARTICLE INFO Keywords: Informed consent Epidural analgesia Pregnant women And antenatal period | +|-------------------------------------------------------------------------------------------------------| + +| ABSTRACT Objective: This study aims to review the literature regarding epidural analgesia and related ethical issues among pregnant women. Method: The authors were searched many electronic databases (CHINAL, Ovid Midline, Pub MED and Google Scholar, Science Direct Database, Biomed) under the following keywords (Informed consent, epidural analgesia, pregnant women, and antenatal period). The several quantitative studies published in English were reviewed and analyzed. The authors were analyzed the studies using a literature review matrix subheading to author, years published, type of study, variables, design and methodology, finding, conclusion, limitations for practices and limitations for studies. Results: The themes that emerged from the literature review were: difference between patient and provider in perceptions of informed consent toward EA during childbirth process; factors affecting the women choice of using EA during childbirth process and awareness regarding using of EA in childbirth among women in antenatal clinics. Conclusion: The process of signing of informed consent for EA in women during childbirth is linked to many ethical responsibilities from women and healthcare providers. The informed consent practice can be enhanced by putting greater emphasis on antenatal information, providing suitable timing of information and developments in recent methods of information release and transfer. | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +## 1. **Introduction** + +Pain through childbirth is main concern of most women during childbirth in the world. Childbirth pain is the most extreme pain that most women may experience during their life [1]. Many women have increased awareness of their right to enhance the quality of care through childbirth by using an appropriate pain relief way [2,3]. Currently, epidural anesthesia [EA] is the most effective way to control pain without any risk to the mother or the fetus [4]. However, EA is still not a completely and acceptable practice for many healthcare providers in developing countries despite several benefits of this method [4]. Normal vaginal delivery is an excruciating event resulting from frequent uterine contractions [5]. National Institute for Clinical Excellence [6] in United Kingdom recommends educating pregnant women about the accessibility of useful analgesia methods during childbirth to ensure that they obtain the most favorable experience through childbirth. The information regarding childbirth pain and way to control it are relatively in intuitive stages in developing countries [7]. Insufficient information, antenatal dialogue, and practical experience are still the major concerns towards the practice of EA among obstetricians and healthcare providers. The main key source of receiving information regarding childbirth analgesia methods is from family and friends [8]. Therefore, it is the responsibility of the decision maker to improve the quality of EA +practicing by providing consistent information about childbirth analgesia. There are many advantages of increasing awareness regarding EA +among women which may prevent women from choosing cesarean section to avoid painful childbirth [8]. Because this intervention of healthcare provider holds risks and benefits, women are put in serious position at a critical time when they are vulnerable, in addition to the time needed to attain a lot of information to maintain and support their choices. Accordingly, increasing emphasis on the significance of the decision-making process to the childbirth process is required to enhance prenatal outcomes [12]. Women need to be informed about the advantages and disadvantages connected with EA through childbirth process +[14]. Ethically, pregnant women have the right to obtain informed consent; there is an obligation to have all information regarding EA that +* Corresponding author. + +E-mail address: Shabuhammad@just.edu.jo (S. Abuhammad). + +https://doi.org/10.1016/j.amsu.2021.102305 Received 23 March 2021; Received in revised form 6 April 2021; Accepted 7 April 2021 Available online 13 April 2021 2049-0801/© 2021 The Authors. Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY-NC-ND license +(http://creativecommons.org/licenses/by-nc-nd/4.0/). + +![0_image_4.png](0_image_4.png) + +journal homepage: www.elsevier.com/locate/amsu include advantage and disadvantage of using this way to control pain [9]. + +In many countries, it is the responsibility of the anesthesiologist to get the informed consent from the women for any surgical procedure. This practice is inadequate because it is affected by many ethical factors related to the client and anesthesiologist if they fail to gain ethical informed consent, and this obligate the anesthesiologist to reassess the understanding of the patient [10–12]. The EA consent form is habitually oral and just obtained prior to insertion; therefore, this study aims to assess the process of obtaining informed consent regarding EA among pregnant women. This study aims to review the literature regarding epidural analgesia and related ethical issues among pregnant women. + +## 2. **Method** 2 2.1. Searching Strategies + +Electronic search using the following databases to collect data about informed consent toward epidurals among pregnant women: science direct, PubMed, Google Scholar, Google advanced search with the following search words: Informed Consent, Epidural Analgesia, Antenatal Period, Pregnant Women, Risk Factors, Benefits, Characteristics, Knowledge, Practice, Nurses, Midwives, and Perception. The authors extended the searching to the reference list of each article they found. The articles were systematic review, randomized controlled trial (RCT), crosssectional study, cohort study and descriptive study. The inclusion criteria were articles written in English language with focusing on the main keywords listed previously. The emerging themes from the literature review were: Differences among Patient and Health Care Providers; Factors Affecting the Women Choice of Using EA During Childbirth Process, and Awareness Regarding Using of EA in Childbirth Process among Pregnant Women in Antenatal Clinics. + +## 3. **Results** + +3.1. Difference between patient and provider in perceptions of informed consent toward EA during childbirth process The first theme that emerged from the literature review was difference between patient and provider in perceptions of informed consent. In a comparative study to determine the variation of informed decisionmaking awareness toward EA during childbirth among patient and provider. They found a considerable difference between provider and patient in defining and recognizing pain relief methods. Providers were expected to support the rights of women toward informed decisions compare to the women who are expected to respect the healthcare providers decisions [15]. In another research study to compare the differences in decision-making among health care providers and women regarding utilizing EA throughout childbirth process. It was obvious that choosing of EA depend on the information that women were received from the healthcare providers [15,16]. + +## 3.2. Factors Affecting The Women Choice Of Using Ea During Childbirth Process + +There were many studies were described factors that affecting choice of using EA during childbirth [6,8,16,17]. For example, a prospective cross-sectional study was conducted to assess factors linked to the refusing of EA among 933 among women. This study was collected information about their parity, literacy level, reasons for seeking or refusing EA, a resource of information and satisfaction. This study demonstrated that universal reasons were misconceptions regarding the nature of EA (65.9%) and lacking information about the choice of EA +(20.5%); familiarity with the side effects of EA (70.5%). For participants used EA during childbirth, their choices for having EA were directed by healthcare providers [8]. In two studies in Nigeria and Norway, to compare the characteristics between women who received EA and who not. They found that the education level was higher with women who had received EA during childbirth compared to none. Moreover, women who received childbirth preparation classes are more likely to use EA during childbirth [22,23]. Another national antenatal survey among 8233 found that refusing to seek EA was greater in poor women such as none employed women or less than bachelor's degree [16]. In comparison, a survey study among 320 participants found that 80% of women receiving an epidural during childbirth. The identified factors for using EA include prior epidural use, partner preference, age, language, type of insurance, duration of childbirth, and education [17]. + +In summary, the main reasons for avoiding EA are lack of knowledge and awareness, misconceptions about EA, and desire to experience vaginal delivery [8]. The recommendation of National Institute for Clinical Excellence to educate pregnant women about availability of effective analgesia in childbirth process and find the factors that impacted their decision to choose EA during childbirth [6]. + +3.3. Awareness regarding using of EA in childbirth among women in antenatal clinics Another theme that emerged from this literature review was awareness regarding EA in childbirth among women in antenatal clinics +[7,8,18,20–24]. A descriptive study among multipara pregnant women found that many misconceptions and worries linked with EA use. Braun, Skene, & Merry [20] anticipated three methods to address the impeding factors for obtaining informed consent from healthcare providers that should described in preadmission clinic. These clinics require to inform women regarding pain-relief options during childbirth. A cross-sectional study among 350 women attending antenatal classes aimed to identify the awareness and utilization of EA during childbirth between April 2016 and July 2016 found that only 43.3% of the participants were aware of using EA during childbirth. However, only 7.5% used EA. The results from this study focus on developing a strategy to enhance performance and quality of EA by information providing during pregnancy +[8]. The improving of antenatal information was required teamwork with sufficient resources [7]. + +A study among 40 pregnant and newly birthing women regarding how media, friends, and family informing decision-making for women for using EA during childbirth. The results of the study show that women define birth based on previous experiences. These experiences impact the women's choices regarding use of EA during childbirth process [23]. + +Yet, it is implicit that women's choices of delivery would be enhanced if women informed correctly and professionally regarding EA. Stewart et al. experimental study found that most women in both standard care and intervention groups would receive the information regarding EA +from anesthesiologist prior to childbirth. They found that women need to obtain both verbal and written information from healthcare providers prior to childbirth to formulate a decision regarding using EA (26). It is the responsibility of healthcare to give an adequate information about pain relief options during childbirth to empower pregnant women to make an informed choice. + +## 3.4. Critique + +There were many limitation and gaps in the literature regarding informed consent for EA during childbirth, these limitations are small sample size or focusing on homogeneous sample that could limit the generalizability of the findings [8,17,19,20]. For example, a study by Harkins et al. [17] regarding the impact of women characteristics on healthcare providers choice of having informed consent regarding EA. This study weakens by using homogenous sample from English-literate, married, and highly educated women. + +Similarly, Sitras et al. [25] utilize an investigation to inspect how women gain knowledge regarding informed consent of EA during childbirth. A limitation is the generalizability of the findings that restricted to Norwegian-speaking women who were Caucasian sample. + +Another possible limitation of this study is not using validated instrument. A study in Northern British Columbia where EA is available and physicians directed most of the clinics that improved the chance of using EA, which limit the generalizability the results to other areas [25]. + +## 3.5. Implications And Recommendation + +The study's results draw attention for the importance of legal and ethical principles toward the childbirth process. Healthcare providers should assist women regarding decisions toward EA. It is vital for healthcare providers to find the best method to improve woman understanding of option during childbirth process. Lastly, it is the responsibility of healthcare providers to guarantee the understanding of EA during informed consent. It is recommended that providers spend more effort in implementing "shared decision-making practices". + +## 4. **Conclusion** + +The process of signing of informed consent for EA in women during childbirth is linked to many ethical responsibilities from women and healthcare providers. The informed consent practice can be enhanced by putting greater emphasis on antenatal information, providing suitable timing of information and developments in recent methods of information release and transfer. Changes in existing practice may well be helpful to women to better adhere to the legal standards of obtaining informed consent. There is a need for future research which may add further information to enhance awareness regarding EA during childbirth. + +## Ethical Approval + +No ethical approval is required. + +Sources of funding No funding Author contribution All authors were participated in all stages of this paper. + +Consent No consent is required. + +Registration of research studies 1. Name of the registry: +2. Unique Identifying number or registration ID: +3. Hyperlink to your specific registration (must be publicly accessible and will be checked): + +## Guarantor + +All authors were participated in all steps of this paper. + +## Declaration Of Competing Interest + +No conflict of interest. + +## References + +[1] L.O. Lawani, J.N. Eze, O.B. Anozie, C.A. Iyoke, N.N. Ekem, Obstetric analgesia for vaginal birth in contemporary obstetrics: a survey of the practice of obstetricians in Nigeria, BMC Pregnancy Childbirth 14 (1) (2014 Dec) 1–6. + +[2] E.O. Ogboli-Nwasor, S.E. Adaji, Between pain and pleasure: pregnant women's knowledge and preferences for pain relief in labor, a pilot study from Zaria, Northern Nigeria, Saudi J. Anaesth. 8 (Suppl 1) (2014 Nov) S20. + +[3] E. Ogboli-Nwasor, S.E. Adaji, S.B. Bature, O.S. Shittu, Pain relief in labor: a survey of awareness, attitude, and practice of health care providers in Zaria, Nigeria, J. Pain Res. 4 (2011) 227. + +[4] R.V. Shidhaye, M.V. Galande, V.B. Bangal, S.S. Joshi, U.R. Shidhaye, Awareness and attitude towards labour analgesia of Indian pregnant women, Anaesth. Pain Intensive Care 16 (2) (2012) 131–136. + +[5] H. Aksoy, B. Yücel, U. Aksoy, G. Acmaz, T. Aydin, M.A. Babayigit, The relationship between expectation, experience and perception of labour pain: an observational study, SpringerPlus 5 (1) (2016 Dec) 1–5. + +[6] NICE Intrapartum care for healthy women and babies, Clin. Guidelines (CG 190) +(2014 Dec) 1–103. + +[7] A.R. Braun, L. Skene, A.F. Merry, Informed consent for anaesthesia in Australia and New Zealand, Anaesth. Intensive Care 38 (5) (2010 Sep) 809–822. + +[8] A. Prakash, A. Yadav, H.M. Karim, S.K. Sahoo, P. Jena, K. Aman, Knowledge, awareness and acceptance of labor analgesia among antenatal women in a remote Island: a questionnaire based study, J. Adv. Med. Med. Res. (2017 May 31) 1–7. + +[9] Authority, Informed consent, NHSLA Risk Alert 4 (2) (2014) 53–58. + +[10] G.N. Jackson, T. Sensky, P. Reide, S.M. Yentis, The capacity to consent to epidural analgesia in labour, Int. J. Obstet. Anesth. 20 (3) (2011 Jul 1) 269–270. + +[11] B.M. Broaddus, S. Chandrasekhar, Informed consent in obstetric anesthesia, Anesth. Analg. 112 (4) (2011 Apr 1) 912–915. + +[12] Sakala C, Corry MP. Evidence-based Maternity Care: what it Is and what it Can Achieve. + +[14] V.A. Entwistle, I.S. Watt, Patient involvement in treatment decision-making: the case for a broader conceptual framework, Patient Educ. Counsel. 63 (3) (2006 Nov 1) 268–278. + +[15] H.B. Goldberg, A. Shorten, Differences between patient and provider perceptions of informed decision making about epidural analgesia use during childbirth, J. Perinat. Educ. 23 (2) (2014 Jan 1) 104–112. + +[16] Fisher KT. reportWomen's Recall of Information Received Relating to Labour Epidural Analgesia at an Academic Hospital: a Pilot Study (Doctoral dissertation). + +[17] J. Harkins, B. Carvalho, A. Evers, S. Mehta, E.T. Riley, Survey of the factors associated with a woman's choice to have an epidural for labor analgesia, Anesthesiol. Res. Pract. (2010 Jan 1) 2010. + +[18] P.O. Ezeonu, O.B. Anozie, F.A. Onu, C.U. Esike, J.E. Mamah, L.O. Lawani, R. + +C. Onoh, E.O. Ndukwe, R.L. Ewah, R.O. Anozie, Perceptions and practice of epidural analgesia among women attending antenatal clinic in FETHA, Int. J. Wom. Health 9 (2017) 905. + +[19] A. Barakzai, G. Haider, F. Yousuf, A. Haider, N. Muhammad, Awareness of women regarding analgesia during labour, J. Ayub Med. Coll. Abbottabad 22 (1) (2010 Mar 1) 73–75. + +[20] L. Ray, F. Goffinet, M. Palot, M. Garel, B. Blondel, 681:'Choice'of delivery without epidural analgesia in women at low risk in France Camille, Am. J. Obstet. Gynecol. 197 (6) (2007 Dec 1) S195. + +[21] C.O. Imarengiaye, B.N. Olagbuji, M.C. Ezeanochie, Akhideno II, Clinical correlates of women requesting labour epidural analgesia in a tertiary hospital in Nigeria, Niger. Postgrad. Med. J. 20 (3) (2013 Sep 1) 214–217. + +[22] S.B. Munro, A. Hui, E.A. Gemmell, N. Torabi, A.S. Johnston, P.A. Janssen, Evaluation of an information pamphlet for women considering epidural analgesia in labour, J. Obstet. Gynaecol. Can. 40 (2) (2018 Feb 1) 171–179. + +[23] C. Malacrida, Always, already-medicalized: women's prenatal knowledge and choice in two Canadian contexts, Curr. Sociol. 63 (5) (2015 Sep) 636–651. + +[24] S.J. Brubaker, H.E. Dillaway, Medicalization, natural childbirth and birthing experiences, Sociol. Compass 3 (1) (2009 Jan) 31–48. + +[25] V. Sitras, J. Saltyt ˇ e ˙ Benth, M. Eberhard-Gran, Obstetric and psychological characteristics of women choosing epidural analgesia during labour: a cohort study, PloS One 12 (10) (2017 Oct 18), e0186564. \ No newline at end of file diff --git a/medical/md/PMC8080539.md b/medical/md/PMC8080539.md new file mode 100644 index 0000000000000000000000000000000000000000..497643259b395d190dc229ce6f1217863bd93726 --- /dev/null +++ b/medical/md/PMC8080539.md @@ -0,0 +1,415 @@ +World Journal of W J S C *Stem Cells* + +![0_image_0.png](0_image_0.png) + +Submit a Manuscript: https://www.f6publishing.com *World J Stem Cells* 2021 April 26; 13(4): 281-303 DOI: 10.4252/wjsc.v13.i4.281 ISSN 1948-0210 (online) +REVIEW +Patient-specific induced pluripotent stem cells as "disease-in-adish" models for inherited cardiomyopathies and channelopathies – + +# 15 Years Of Research + +Miruna Mihaela Micheu, Ana-Maria Rosca + +| ORCID number: Miruna Mihaela Micheu 0000-0001-7201-3132; AnaMaria Rosca 0000-0001-5020-1052. Author contributions: Both authors equally contributed to the conception of the paper, the literature review and analysis, drafting and to critically revising and editing the manuscript. | +|---| + +Conflict-of-interest statement: The authors declare that they have no competing interests. + +| Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: htt p://creativecommons.org/License s/by-nc/4.0/ Manuscript source: Invited manuscript Specialty type: Cardiac and cardiovascular systems | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Miruna Mihaela **Micheu,** Department of Cardiology, Clinical Emergency Hospital of Bucharest, Bucharest 014452, Romania Ana-Maria **Rosca,** Cell and Tissue Engineering Laboratory, Institute of Cellular Biology and Pathology "Nicolae Simionescu", Bucharest 050568, Romania Corresponding **author:** Miruna Mihaela Micheu, MD, PhD, Doctor, Department of Cardiology, Clinical Emergency Hospital of Bucharest, Floreasca Street 8, Bucharest 014452, Romania. mirunamicheu@yahoo.com + +## Abstract + +Among inherited cardiac conditions, a special place is kept by cardiomyopathies +(CMPs) and channelopathies (CNPs), which pose a substantial healthcare burden due to the complexity of the therapeutic management and cause early mortality. Like other inherited cardiac conditions, genetic CMPs and CNPs exhibit incomplete penetrance and variable expressivity even within carriers of the same pathogenic deoxyribonucleic acid variant, challenging our understanding of the underlying pathogenic mechanisms. Until recently, the lack of accurate physiological preclinical models hindered the investigation of fundamental cellular and molecular mechanisms. The advent of induced pluripotent stem cell (iPSC) technology, along with advances in gene editing, offered unprecedented opportunities to explore hereditary CMPs and CNPs. Hallmark features of iPSCs include the ability to differentiate into unlimited numbers of cells from any of the three germ layers, genetic identity with the subject from whom they were derived, and ease of gene editing, all of which were used to generate "disease-in-a-dish" models of monogenic cardiac conditions. Functionally, iPSC-derived cardiomyocytes that faithfully recapitulate the patient-specific phenotype, allowed the study of disease mechanisms in an individual-/allele-specific manner, as well as the customization of therapeutic regimen. This review provides a synopsis of the most important iPSC-based models of CMPs and CNPs and the potential use for modeling disease mechanisms, personalized therapy and deoxyribonucleic acid variant functional annotation. + +Key **Words:** Induced pluripotent stem cells; Cardiomyopathy; Channelopathy; Genes; Mutation; Deoxyribonucleic acid variants + +| Country/Territory of origin: Romania Peer-review report's scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 Received: February 1, 2021 Peer-review started: February 1, 2021 First decision: February 28, 2021 Revised: March 11, 2021 Accepted: March 29, 2021 Article in press: March 29, 2021 Published online: April 26, 2021 P-Reviewer: Thummer RP, Wu HY S-Editor: Zhang L L-Editor: Filipodia P-Editor: Wang LL | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved. + +| Core Tip: Induced pluripotent stem cell (iPSC) technology holds a great potential for medical research. Patient-specific iPSC-derived cardiomyocytes offer a unique framework for various applications, such as cardiotoxicity screening, drug discovery, disease modeling, and cell therapy. In the particular case of inherited cardiomyopathies and channelopathies, iPSC-based models have prompted study of disease mechanisms in an individual-/allele-specific manner, as well as the customization of therapeutic regimens. Herein, we present and critically discuss the current knowledge and key experimental approaches that support patient-specific iPSCs as robust "disease-in-adish" models for genetic cardiomyopathies and channelopathies after 15 years of research. | +|---| + +Citation: Micheu MM, Rosca AM. Patient-specific induced pluripotent stem cells as "diseasein-a-dish" models for inherited cardiomyopathies and channelopathies - 15 years of research. + +World J Stem Cells 2021; 13(4): 281-303 URL: https://www.wjgnet.com/1948-0210/full/v13/i4/281.htm DOI: https://dx.doi.org/10.4252/wjsc.v13.i4.281 + +## Introduction + +Stem cell technology is one of the most dynamic areas in modern research, holding a great potential to alleviate or even cure various diseases. Particularly, induced pluripotent stem cells (iPSCs) are of a great interest given that they share the benefits of embryonic stem cells but lack their downsides. First, similar to embryonic stem cells, iPSCs are able to differentiate into tissues derived from all three germ layers, both in *vitro* and in *vivo.* Secondly, iPSCs-derived cells will be immunologically identical to the host, making the use of immunosuppression unnecessary. Thirdly, there are no bioethical issues with the use of iPSCs. These unique features endorse them an excellent candidate for a wide array of applications such as cardiotoxicity screening, drug discovery, disease modeling, and cell therapy. + +Ever since their first mention in 2006[1], we have witnessed a mounting body of data related to this rapidly growing field. Progress has been made in reprogramming and differentiation methods. Strategies for improving the maturity of iPSC-derived cardiomyocytes (iPSC-CMs) have been tested, and new applications to manage cardiac diseases have been tested. A recent Scientific Statement from the American Heart Association acknowledges disease modeling as possibly the most productive use of iPSCs[2]. Several key characteristics endorse iPSCs as an ideal candidate for generating +"disease-in-a-dish" models, particularly with regard to monogenic conditions. First of all, each iPSC line has a donor-specific genetic profile. Secondly, when collected, iPSCs are devoid of many of the epigenetic modifications caused by environmental and lifestyle factors, thus enabling the study of the genetic contribution to the disease. This aspect is of a particular importance in the case of Mendelian cardiac maladies, which are characterized by variable clinical expression and incomplete penetrance as a consequence of complex interactions between genetic backgrounds and environmental disease modifiers[3]. Thirdly, iPSCs are quite malleable to genetic modification; accordingly, by using appropriate genome editing tools such as TALENs and CRISPRCas9, the deoxyribonucleic acid (DNA) sequence can be altered either by introducing causal DNA mutations into wild-type iPSC lines, or by repairing the causative factor to achieve phenotypic rescue in differentiated cells[2,4]. + +Inherited cardiac conditions (ICCs) include a variety of genetic disorders that primarily affect the heart. Among ICCs, a special place is kept by cardiomyopathies +(CMPs) and arrhythmic diseases (*i.e.* channelopathies), which pose a substantial healthcare burden due to the complexity of therapeutic management and occurrence early mortality. Importantly, sudden cardiac death is frequently the first expression of the disease. Understanding the underlying genetic cause is the centerpiece of a timely diagnosis and targeted treatment[5]. + +CMPs are characterized by both structural and functional abnormalities of the ventricular myocardium that are not explained by flow-limiting coronary artery disease or abnormal loading conditions, each entity having particular characteristics at macroscopic and molecular level[6]. Based on morphology, hereditary CMPs comprise the following types: hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic cardiomyopathy (ACM), and left ventricular noncompaction (LVNC). + +Inherited channelopathies (CNPs) are primary electrical disorders caused by mutations in genes encoding cardiac ion channels or associated proteins. As a result, malfunction of specific ion channels or of intracellular calcium handling occur, leading to electrical instability and predisposition to malignant arrhythmias in the absence of structural heart disease[7,8]. The main cardiac channelopathies associated with increased risk of sudden cardiac death are long QT syndrome (LQTS), short QT syndrome (SQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic ventricular tachycardia (CPVT). As comprehensive reviews of the genetics and clinical presentation of various ICCs have been written by our group[3,9] and other groups[10-12], +we briefly point out the core genes associated with the CMPs and CNPs discussed in the present paper (see Tables 1 and 2) +[12-19]. It is to be noted that there is considerable genetic overlap among different CMPs and CNPs (Figure 1A and B, respectively). + +## Modeling Disease-Specific Mechanisms + +Due to the potential to differentiate into functional cardiomyocytes (CMs) that recapitulate patient-specific phenotypes, human iPSCs provide an excellent in *vitro* platform to decipher the underlying disease-specific mechanisms and efficiently study inherited CMPs and CNPs in an individualized manner. + +## Inherited Cardiomyopathies + +HCM and DCM are the most frequently encountered genetic CMPs in daily clinical practice, therefore, unsurprisingly, they have been the most studied iPSC-CM-based models. In a recent report, Eschenhagen *et al*[19]comparatively analyzed 38 original papers that reported the characteristics of iPSC-CMs obtained from patients with HCM/DCM or generated from iPSC lines in which a HCM or DCM mutation had been genetically introduced[19]. In summary, compared with their respective controls, the main features exhibited by HCM iPSC-CMs were the following: larger cell size, increased nuclear localization of nuclear factor of activated T cells (NFAT, a transcription factor) and increased *MYH7* (or *MYH7/MYH6* ratio) expression[20-24]. The most constant aberration identified in DCM lines was reduced peak force development[25], the molecular mechanisms of which are discussed, and explain the main clinical presentation of the disease. As for similarities between the two considered diseases, three anomalies were the most documented: sarcomere disarray, increased *NPPA/NPPB* gene expression, and arrhythmic behavior[20,24,26,27]. + +Although not constantly associated with alterations in contractile force or kinetics, abnormal calcium handling appears to be a key pathological mechanism observed in iPSC-CM models of HCM[20,21,24,28]. Valuable insights related to the molecular mechanisms of HCM pathogenesis have been provided by Seeger *et al*[29]in a model of patient-derived iPSC-CMs harboring a premature stop codon in the *MYBPC3* gene. When compared with the isogenic mutation-corrected iPSC lines, in addition to aberrant calcium signaling, patient-derived iPSC-CMs displayed molecular dysregulations without haploinsufficiency of the MYBPC3 protein. This observation could challenge the existing dogma of haploinsufficiency as the underlying mechanisms for HCM caused by *MYBPC3* premature termination codon mutations. The specific molecular signature included dysregulation of genes involved in calcium handling (ATP2A2, *ATP2B2*, and *CASQ1*), cardiac hypertrophy (GP130, JAK2, *RRAS,* MEK1, *TWEAKR*, and *NPPB*), stress response (HSPB1, HSPB6, HSPB7, *IGF1*, and *IGF2*) , and structural organization of sarcomeres and mechanosensors (*CSRP3* and *TCAP*). + +Disturbed calcium signaling has been shown to be a central pathological mechanism of diastolic dysfunction in familial HCM lines with variations in the MYH7, *MYBPC3*, +and *TNNT2* genes[30]. Using comprehensive functional imaging analysis (*i.e.* calcium imaging and traction force microscopy), Wu *et al*[30]revealed that diastolic Ca2+ +overload and increased myofilament Ca2+ sensitivity contribute to diastolic dysfunction and demonstrated for the first time that patient-specific iPSC-CMs can recapitulate diastolic dysfunction characteristics at the single-cell level. Furthermore, calcium homeostasis was restored by Ca2+ and late Na+ blockers (verapamil, dilitiazem, ranolazine, and electlazine), which was reflected in diastolic function improvement in HCM iPSC-CMs. + +| Table 1 Main genes associated with inherited cardiomyopathies Condition Genotype | Ref. | | +|------------------------------------------------------------------------------------|---------------------------------------------------------------------|--------------| +| HCM1 | MYBPC3, MYH7, TNNT2, TNNI3, TPM1, ACTC1, MYL2, MYL3 | [13] [14,15] | +| DCM | TTN, LMNA, MYH7, TNNT2, BAG3, RBM20, TNNC1, TNNI3, TPM1, SCN5A, PLN | | +| LVNC | Overlap with HCM and DCM | [14,15] | +| ACM | DES, DSC2, DSG2, DSP, JUP, LMNA, PKP2, PLN, RYR2, SCN5A, TMEM43, | [14,15] | +| TTN | | | +| RCM | TTR, TNNI3, DES. Overlap with HCM and DCM | [14,15] | + +1Only genes having definitive evidence for hypertrophic cardiomyopathy have been depicted. ACM: Arrhythmogenic cardiomyopathy; DCM: Dilated cardiomyopathy; HCM: Hypertrophic cardiomyopathy; LVNC: Left ventricular noncompaction; RCM: Restrictive cardiomyopathy. + +| Table 2 Main genes associated with inherited channelopathies Condition Genotype | Ref. | | +|-----------------------------------------------------------------------------------|---------------------|------| +| LQTS1 | KCNQ1 | [16] | +| LQTS2 | KCNH2 | [16] | +| LQTS3 | SCN5A | [16] | +| LQTS4 | ANK2 | [17] | +| LQTS5 | KCNE1 | [17] | +| LQTS6 | KCNE2 | [17] | +| LQTS7 | KCNJ2 | [17] | +| LQTS8 | CACNA1C | [17] | +| LQTS9 | CAV3 | [17] | +| LQTS10 | SCN4B | [17] | +| LQTS11 | AKAP9 | [17] | +| LQTS12 | SNTA1 | [17] | +| LQTS13 | KCNJ5 | [17] | +| LQTS14 | CALM1 | [17] | +| LQTS15 | CALM2 | [17] | +| JLN1 | KCNQ1 | [17] | +| JLN2 | KCNE1 | [17] | +| CPVT1 | RYR2 | [16] | +| CPVT2 | CASQ2 | [16] | +| SQTS | KCNH2, KCNQ1, KCNJ2 | [16] | +| BrS | SCN5A | [18] | + +BrS: Brugada syndrome; CPVT: Catecholaminergic polymorphic ventricular tachycardia; LQTS: Long QT syndrome; SQTS: Short QT syndrome. + +More recently, it has been shown that molecular signaling differs within HCM iPSCCMs with diverse gene mutations. Isogenic models of HCM revealed differential phenotypes and mechanism-driven possible therapeutic targets in *MYH7* and *ACTC1* cell lines, respectively[31]. In spite of sharing key disease hallmarks, such as intracellular calcium overload and calcium transient arrhythmias, which were common in both models, modifications in contractility were entirely divergent, namely decreased contractility of *MYH7* cells and gain of hypercontractility of *ACTC1* cells. Notably, the expression of Ca2+-binding proteins and hypertrophy-associated transcription factor activation also showed opposing behavior. Accordingly, compared with their + +![4_image_0.png](4_image_0.png) + +respective controls, MYH7-R453C mutants were characterized by upregulation of CASQ2, *CALM1* and *CAMK2D* along with MEF and NFAT nuclear translocation prompted by *IRF8* downregulation. Reversed expression patterns of those genes were found in *ACTC1*-E99K iPSC-CMs. The study offered clinically-relevant data, given that the arrhythmogenic phenotype was rescued in both models following treatment with a mixture of dantrolene and ranolazine (a ryanodine receptor antagonist that inhibits sarcoplasmic Ca2+ release into the cytosol and a late sodium current blocker promoting intracellular Ca2+ efflux). In addition, the enhanced contractility displayed in *ACTC1* iPSC-CMs was rescued by mavacamten (a selective allosteric inhibitor of cardiac myosin ATPase). Of note, an earlier study reported differences in the phenotypic features of iPSC-CMs obtained from a family with ACTC1-E99K mutation. These differences varied depending on the source subject, highlighting the value of isogenic iPSC-CMs in genotype-phenotype correlations. Thus, while arrhythmogenesis was manifested in all *ACTC1*-E99K iPSC-CM lines, it was more common in cells obtained from the father, and less apparent in those derived from the two sons. This suggests that, in addition to the causative mutation, other factors (either genetic or epigenetic) +might contribute to the disease phenotype[32]. + +The contractility consequences seem to be dependent on the specific DNA change rather than the affected gene. In contrast to the hypo-contractile phenotype described in *MYH7*-R453C mutants[31], *MYH7*-R403Q, *MYH7*-V606, and *MYH7*-R719 iPSC-CMs exhibited hyperdynamic contraction resulting from an increased proportion of myosin molecules in a disordered relaxed state conformation[33]. Imbalance of myosin configurations (*i.e.* super and disordered relaxed states) led to the destabilization of interacting-heads motif interactions that were followed, in addition to increased contractility, by impaired relaxation, hypertrophic remodeling, excessive energy consumption, and metabolic stress. Mavacamten treatment restored the physiological super relaxed state/disordered relaxed state ratio and relieved downstream functional abnormalities, suggesting that chronic dysregulation of myosin conformations is a central mechanism of HCM. + +Other reports have used iPSCs to decode genetic DCM physiopathology. The most frequently mutated gene in DCM is TTN, with truncating variants explaining up to 30% of familial cases[34,35]. Other genes involved in DCM etiology are shown in Table 1. + +The most commonly encountered genes responsible for DCM are LMNA, DES, *MYH7*, +MYH6, SCN5A, *MYBPC3*, and *TNNT2*, although at a lower prevalence than TTN[36]. + +Patient-derived iPSC-CMs have characteristics consistent with DCM, including sarcomere disorganization, reduced contractile function, and altered calcium handling[25,37-39]. Hinson *et al*[38]investigated the disease phenotypes of iPSCs engineered from DCM subjects with either truncating or missense mutations in TTN, and showing sarcomere insufficiency, impaired responses to mechanical and βadrenergic stress, and attenuated growth factor and cell signaling activation. + +In a series of studies that focused on *TNNT2*-R173W missense mutation, iPSCCMs lines generated from affected family members exhibited a compromised ability to regulate calcium flux, reduced contraction force, and heterogeneous myofilament organization that were exacerbated by βadrenergic stimulation[25]. In-depth analysis uncovered the epigenetic activation of phosphodiesterase genes *PDE2A* and *PDE3A* as the underlying mechanism responsible for the defective βadrenergic signaling and contractile dysfunction[40]. The latest data indicates that *TNNT2*-R173W hampers molecular interactions between troponin and tropomyosin and restricts the binding of PKA to local sarcomere microdomains, resulting in diminished troponin phosphorylation and misalignment of sarcomeric proteins[41]. An R173W variant also altered the interaction between sarcomere microdomain and cytoskeleton filaments via *MYH7* and AMPK, with consequent disturbance of sarcomere protein alignment and impaired contractility. In terms of phenotype rescue, AMPK activation by small molecules, such as A-769662, improved sarcomere-cytoskeleton attachment and partially recovered sarcomere protein misalignment and subsequent impaired contractility in mutated iPSC-CMs[41]. + +The phenotypes and the causal disease mechanisms linked to *LMNA* variants have also been intensively studied. In iPSC-CMs with either a nonsense or missense mutation in *LMNA*, Siu *et al*[39] noticed enhanced nuclear senescence and augmented electrical stress-induced apoptosis that was significantly attenuated by pharmacological inhibition of the ERK1/2 pathway with the MEK1/2 inhibitors U0126 and selumetinib. Other investigators analyzed iPSC-CMs from three patients with distinctive *LMNA* mutations (R225X, Q354, and T518fs)[42]. Although all three types of diseased cells recapitulated the pathophysiological hallmarks of *LMNA*-related DCM, the positive effect of ataluren treatment on the expression of full-length Lamin A/C, nuclear blebbing, apoptosis, and contractility was detected only in the *LMNA*-R225X +mutant, suggesting that the effect might be codon selective[42]. Other traits expressed by *LMNA*-mutated iPSC-CMs were bradyarrhythmia, beat rate variability, abnormal calcium handling, stress hypersensitivity, disorganized sarcomeres, and increased cell size[43,44]. The incriminating mechanisms were aberrant activation of PDGF-signaling, which was rescued by pharmacological and molecular inhibition of PDGF receptor B +[43], or epigenetic inhibition of *SCN5A* that was rescued by *SCN5A* overexpression[45]. + +Mutations in other DCM-related genes such as DES[37], *MYH7*[46], DMD[47,48], PLN[26], or even compound mutations have been modeled[49]. + +Characterization of iPSC-CMs from family members with LVNC who carried a nonsense variant in cardiac transcription factor *TBX20*, compared with iPSCCMs from unaffected relatives, revealed abnormal activation of transforming growth factor-β signaling leading to decreased cell proliferation capability. Moreover, inhibition of the transforming growth factor-β signaling pathway and correction of *TBX20* alteration by CRISPR-Cas9 technology, successfully rectified the pathological phenotype[50]. + +Impairments in contractility, calcium handling, and metabolic activity have been nominated as key features in another model of iPSC-CMs generated from patients with delayed-onset LVNC caused by a missense mutation in the *GATA4* gene[51]. + +In an iPSC model with a *TPM1*-R178H mutation, it was shown that mislocalization of tropomyosin 1 was a central pathological change, triggering disturbance of the sarcomere structure and impaired calcium handling. Comprehensive analysis found involvement of complex molecular pathways centered on downregulation of the expression of numerous genes controlling heart development and positive regulation of cellular processes, including transcription factors (GATA4, *GATA6*) and sarcomeric proteins (MYBPC3, MYH6, TTN, TNNI3, *TNNT2*), thus linking sarcomeric dysfunction to LVNC[52]. + +ACM is another inherited CMP studied in iPSC models. In a series of reports published in 2013, CMs engineered from subjects having mutations in the *PKP2* gene efficiently recapitulated key disease features, including reduced cell surface localization of desmosomal proteins with altered desmosomal structure and a more adipogenic phenotype[53]. These phenotypical changes were accompanied by upregulation of the pro-adipogenic transcription factor peroxisome proliferatoractivated receptor (PPAR)-γ and enhanced activation of respective signaling pathways[54,55]. Furthermore, lipid droplets accumulation was prevented by administration of a specific inhibitor of glycogen synthase kinase 3β (6-bromoindirubin-3'- +oxime)[55]. Subsequent work revealed novel mechanistic insights in ACM pathogenesis or confirmed those already cited. Wen *et al*[56]reported that coactivation of normal PPAR-α and abnormal PPAR-γ pathways in ACM iPSC-CMs triggered markedly increased lipogenesis, apoptosis, Na+channel downregulation and defective intracellular calcium handling[56]. In another iPSC-based model it was found that RhoA/ROCK signaling at the intercalated disc was essential for cardiomyocyte homeostasis[57]. Using patient-derived iPSC-CMs with impaired cell-cell adhesion due to a *PKP2* frameshift mutation, or disturbed RhoA signaling caused by a nonsense MYH10 mutation, Dorn *et al*[57] elegantly demonstrated that cardiomyocyte identity was lost following disruption of the RhoA/MRTF/SRF-signaling circuit. RhoA recruitment to cell-cell junctions was abridged in diseased cells, prompting increased levels of cytosolic G-actin and successive cytoplasmic sequestration of transcription factors such as MRTF that are involved in myocyte identity, preventing their entry into the nucleus. Finally, when exposed to an adipogenic environment, the mutated cells were poised to switch to a brown/beige adipocyte lineage, providing a possible molecular explanation of the clinical phenotype observed in ACM. Interestingly, a recent study reported lipid accumulation, increased pleomorphism, irregular Z-bands, and increased L-type calcium currents in iPSC-CMs carrying a novel frameshift mutation (L5218fs) in the *OBSCN* gene[58]. The phenotypic alterations were accompanied by activation of adipocytokines and PPAR signaling pathways, diminished expression of the mutant protein and its anchor protein Ank1.5, in addition to downregulation of other desmosomal coding genes (*PKP2, JUP, DSP*) +[58]. + +IPSC-CMs generated from an ACM patient with a *DSG2* mutation exhibited complex ion channel dysfunctions and abnormal cellular electrophysiology as well as increased sensitivity to adrenergic stimulation, indicating involvement of ion channel dysfunctions in arrhythmogenesis, independent of structural abnormalities[59]. + +Subsequent work conducted by the same group established for the first time that enhanced *NDPK-B* expression, via activating SK4 channels, contributed to arrhythmogenesis in *DSG2-*related ACM, suggesting that NDPK-B could be a specific therapeutic target in selected patients[60]. + +Ng *et al*[61]reported for the first time that some desmoplakin missense variants, such as DSP-R451G, are functionally equivalent to truncating alleles by promoting pathological vulnerability to calpain proteolysis and subsequent desmoplakin insufficiency. + +There are few data related to restrictive cardiomyopathy modeling by iPSC. CMs harboring homozygous DES-Y122H mutation were reported to display abnormal desmin cytoplasmic aggregates responsible for the pathological phenotype[62]. + +## Inherited Channelopathies + +A second category of genetic cardiac conditions extensively modeled using iPSC technology is represented by CNPs, for which electrophysiology studies exposed alterations of action potential, field potential, or Ca2+ transients in engineered CMs. + +LQTS, in particular the first three types (LQTS1, LQTS2 and LQTS3), benefit from the most well-characterized iPSC-CMs models. Types LQTS that differ according to the underlying channel or gene mutation are shown in Table 1. + +The first model of an arrhythmic syndrome using patient-specific iPSC-CMs was reported in 2010 by Moretti *et al*[63], who generated iPSCs from two affected members of a family with LQTS1 caused by a missense mutation (R190Q) in the *KCNQ1* gene. Mutant iPSC-CMs effectively reproduced the relevant features of the disease, namely prolongation of the action potential duration into atrial-like and ventricular-like cells, and increased occurrence of arrhythmic events when exposed to β-adrenergic agonists. Voltage clamp analysis revealed a substantial 70% to 80% reduction in the slowly activating delayed rectifier potassium currents (IKs) of LQTS1-iPSC-derived ventricular CMs due to a decreased number of functional *KCNQ1* channels in the sarcolemma compared with the healthy counterpart. Beta-blocker treatment of LQTS1 CMs had a protective effect against catecholamine-induced arrhythmia. Similar findings have been reported in subsequent models of iPSC-CMs from LQTS1 with KCNQ1 missense or frameshift mutations[64,65]. Additionally, Wang *et al*[66]identified abnormalities in Ca2+ handling linked to three distinct *KCNQ1* variants (R190Q, G269S, +and G345E). CMs derived from all three edited iPSC lines displayed a characteristic LQTS phenotype and significant prolongation of the action potential duration compared with isogenic controls, which were corrected by treatment with L-type calcium channel antagonists. Similar results have been reported recently following an increase in the number of iPSC-CMs models of autosomal dominant, recessive, and compound heterozygous LQTS1[67-73], including analysis of models derived from specific populations[67,72]. A plethora of LQTS2 iPSC-CMs models developed from patients harboring missense mutations in *KCNH2* have reproducibly shown prolonged action potential, increased arrhythmogenicity, and reduced rapidly activating delayed rectifier potassium current (IKr), compared with healthy control lines. The first analyzes of iPSC-based LQTS2 models were published in 2011 by two independent groups[74,75]. Itzhaki *et al*[74] generated iPSC lines from a patient with a *KCNH2*-A614V +variant. As expected, the derived-CMs exhibited substantial prolongation of the action potential, diminished IKr, early after depolarizations (EADs), and triggered arrhythmias. Specific pharmacological inhibition of IKr worsened the cellular phenotype, while administration of other pharmacological agents such as Ca2+ channel blockers, or KATP-channel openers alleviated the pathological features[74]. The in *vitro* model developed by the second group effectively replicated the variation in clinical phenotypes of two family members carrying the same *KCNH2*-A561V mutation. Although the action potential duration was increased in the iPSC-CMs derived from both the clinically symptomatic patient and the clinically asymptomatic mother, an increased sensitivity (appearance of EADs) to stress (*i.e.* β-adrenoreceptor stimulation) +was detected only in the symptomatic patient-derived cells[75]. By comparing the electrophysiological properties of spontaneously beating CMs produced from LQTS2 cases and controls, it was suggested that cell-to-cell contacts in the syncytium result in compensatory mechanisms with a tendency to protect the repolarization system from major aberrations of physiological parameters. Although a considerable signal difference was detected between LQTS2 and control iPSC-CMs on single-cell patchclamp recordings (a 66% increase in action potential in LQTS2 cells), the differences were more modest (10%-20%) when using a microelectrode array technique on cell aggregates, similar to the surface electrocardiogram in respective patients[76]. In later studies, the diseased phenotype was rescued either by genetic correction of the KCNH2 mutation[77], or by allele-specific ribonucleic acid (RNA) interference, which selectively destroyed the mutant mRNA while leaving the wild-type mRNA +undamaged[78]. Various pharmacological agents were also shown to correct the electrophysiological anomalies[79-81], although for some molecules the effect was mutation-specific[82]. An interesting observation was noted by Spencer *et al*[83], who established that in iPSC-CMs with a *KCNH2*-A422T mutation, the action potentials and intracellular calcium transients were prolonged in parallel. Furthermore, exposure to a Ca2+ antagonist such as nifedipine, abbreviated the action potentials despite the IKr deficit. + +Although abnormal calcium handling is common in both LQTS1 and LQTS2, there are major differences in this regard. Joutsijoki *et al*[84] used an innovative approach to differentiate the Ca2+ transient statistics between these two LQTS-mutated iPSC-CMs. + +By combining machine learning and iPSC technology, the authors analyzed 90 LQTS1 transient signals from two cell lines and 138 LQTS2 signals from four cell lines, resulting in classification accuracies of up to 100%. The findings support the hypothesis that Ca2+ transients are disease-specific or even mutation-specific. + +Patient-specific iPSC-CMs models harboring gain-of-function mutations in the SCN5A gene efficaciously summarized LQTS3 pathognomonic electrophysiological traits, such as abnormal sodium currents and prolonged APD[85-88]. A study by Malan at al[89]complemented prior findings by also showing a high incidence of EADs, a recognized trigger mechanism for arrhythmia, in disease cells. Treatment with mexiletine, specific sodium channel inhibitors, reduced action and field potential durations in LQT3 iPSC-CMs and alleviated EADs in a dose-dependent manner. Other types of LQTS, including LQTS7[90], LQTS8[91], LQTS14, and LQTS15[92-94] have been successfully investigated with iPSC technology. + +CPVT comprises two main subtypes, CPTV1 (caused by mutations in the *RYR2* gene) and CPTV2 (determined by mutations in the *CASQ2* gene). Both genes are key regulators of CM calcium homeostasis, and dysfunction of either gene prompts abnormal intracellular Ca2+ handling and signaling, and increased arrhythmogenicity under adrenergic stimulation. To date, numerous CPVT models have been developed using the iPSC platform, successfully recapitulating the arrhythmogenic phenotype seen in patients[95-104]. In a study published in 2011, Fatima *et al*[95] analyzed the functional properties of iPSC-CMs from healthy donors and a patient with CPVT1 carrying a novel heterozygous autosomal dominant mutation (RYR2-F2483I). Compared with healthy CMs, the mutated cells displayed arrhythmias and delayed afterdepolarizations (DADs), higher amplitudes and longer duration of spontaneous Ca2+ release events in the basal state, as revealed by patch-clamp recordings and calcium imaging studies. Additionally, in CPVT-iPSC-CMs the Ca2+-induced Ca2+- +release events continued after repolarization and were eliminated by increasing cytosolic cAMP levels with forskolin. In another CPVT1 model of iPSC-CMs harboring the RYR2-M4109R mutation, intracellular electrophysiological recordings evidenced increased development of DADs in CPVT-iPSCs-CMs compared with healthy CMs, which were further exacerbated by β-adrenergic stimulation; and, as opposed to previous findings, by forskolin. In contrast, thapsigargin (a specific inhibitor of the sarcoplasmic reticulum calcium ATPase pump) eradicated all afterdepolarizations in those cells, indicating that internal Ca2+ stores play a central role in the pathogenesis of DADs. Indeed, laser-confocal Ca2+ imaging revealed extensive whole-cell Ca2+ +anomalies (such as frequent local and large-storage Ca2+-release events, broad and double-humped transients, and triggered activity) that were aggravated under catecholaminergic stress and alleviated by beta-blockers. Also, *RYR2*-M4109R +mutations significantly reduced the threshold for store-overload-induced Ca2+ +release[96]. Dantrolene[97], flecainide[105], and S107 (an RYR stabilizer[104]) were other pharmacological agents shown to ameliorate the disease phenotypes. + +More recently, the combined iPSC and CRISPR/Cas9 gene editing technics were used to validate preliminary data and, more importantly, to gain further insight into dysfunction produced by variations in *RYR2* gene. Wei *et al*[106], introduced a point mutation into wild-type *RYR2* iPSCs by CRISPR/Cas9 gene editing. Similar to CMs generated from CPVT1 patient harboring F2483I-RyR2 mutation[101], edited iPSC-CMs carrying the same CPVT1-associated variant showed abnormal intracellular Ca2+ handling, including longer and wandering Ca2+ sparks, elevated diastolic Ca2+ leaks, reduced sarcoplasmic reticulum (SR) Ca2+ content, and increased susceptibility to arrhythmias caused by isoproterenol[106], suggesting that F2483I-RyR2 mutation produces leaky RyR2 channels. The same approach was used by Zhang *et al*[107]to assess aberrant Ca2+ signaling and pharmacological sensitivity to three distinct CPVT1associated mutations. While all three diseased iPSC-CM lines exhibited some abnormalities in calcium handling (*i.e.* irregular, long-lasting, spatially wandering Ca2+ sparks and aberrant Ca2+ releases), enhanced SR Ca2+ leaks and diminished SR Ca2+ +contents were seen only in cells with Q4201R and F2483I, but not R420Q. Moreover, fractional Ca2+ release and calcium-induced calcium release gain were higher in Q4201R than in R420Q and F2483I iPSC-CMs, emphasizing that Ca2+ signaling abnormalities may vary depending on the mutation site. Several potential therapeutic interventions, including flecainide, dantrolene, and JTV519 (a Ca2+-dependent blocker of SERCA) were tested, indicating that drug sensitivities may also be mutation dependent. Using a wide-ranging methodology integrating optogenetics, tissue engineering, lab-on-a-chip technology, gene editing, and iPSC technology, Park *et al*[108] +identified calmodulin-dependent protein kinase II activation as a key molecular pathway underlying exercise-triggered arrhythmia in CPVT patients, suggesting that its inhibition might be an effective therapeutic strategy in selected cases. + +Recent data indicate that *RYR2* screening should not be indicated only in subjects with stress- or exercise-induced symptoms. Using patient-specific iPSC-CMs, it has been shown that *RYR2*-H29D variants elicit alteration of calcium homeostasis and molecular modifications such as aberrant SR Ca2+ leak under physiological pacing, pro-arrhythmic electrical phenotypes, impaired and asynchronous contractile properties, and aberrant RyR2 post-translational modifications that occur only at rest[109]. Furthermore, the authors hypothesized that the uncommon location of *RYR2*- +H29D mutations outside the four hot-spot regions linked to CPVT1, might be responsible for the distinct phenotypic expression. iPSC-based platforms have also been used to explore functional abnormalities in CMs generated from CPVT2 patients[99,102,110,111]. Under beta-adrenergic stimulation, patient-derived iPSC-CMs carrying the *CASQ2*-D307H variant demonstrated disease-specific arrhythmogenic characteristics due to Ca2+-transient anomalies, afterdepolarization, reduced threshold for store overload-induced Ca2+-release, and upsurge of diastolic intracellular calcium concentration[99,102,110]. + +SQTS is a rare inheritable, autosomal dominant cardiac condition characterized by abnormally short QT intervals and an increased risk of atrial and ventricular tachyarrhythmias. The causal ion channel genes are shown in Table 2, variation in KCNH2 being the most frequently observed in genotyped cases[7,112]. The first SQTS +model utilizing the iPSC platform was reported in 2018 by El-Battrawy *et al*[113]. The authors generated iPSC-CMs from a patient with a *KCNH2*-N588K mutation and two healthy control subjects. Mutated cardiac myocytes exhibited enhanced IKr density and shortened APD compared with control cells, along with abnormal calcium transients and arhythmic propensity. Carbachol, an acetylcholine receptor agonist, increased the occurrence of arrhythmic events in diseased iPSC-CMs, while quinidine, and not sotalol or metoprolol, prolonged the APD and alleviated carbachol-prompted arrhythmias. In subsequent studies, patient-specific and gene-corrected iPSC-CMs were used to elucidate the SQTS phenotype either at single-cell[114] or tissue level[115]. + +When compared with healthy control and gene-corrected CMs, *KCNH2*-T618I iPSCCMs were shown to display shortened APDs and increased beat-beat interval variability. Although no significant differences in total *KCNH2* expression in control, gene-corrected, and SQTS iPSC-CMs were seen, membrane expression of *KCNH2* was approximately 8-fold higher in mutated iPSC-CMs than in isogenic cells, suggesting that the aforesaid variant results in enhanced membrane expression of *KCNH2*, which may contribute to the increased IKr density. Moreover, the phenotype was successfully rescued by BmKKx2, a short-peptide scorpion toxin acting as a selective IKr blocker[114]. Shinnawi *et al*[115] examined conduction and arrhythmogenesis in confluent 2-dimensional iPSC-derived cardiac cell monolayers generated from a symptomatic SQTS patient also with *KCNH2*-N588K mutation. SQTS-iPSC-CM monolayers were characterized by abnormal repolarization properties and induced sustained re-entrant arrhythmias, while retaining a normal conduction appearance. + +BrS is another cardiac channelopathy that has been modeled using iPSC technology. + +Various genes encoding either sodium, potassium, or calcium channels have been linked to BrS[116]. Among them, the *SCN5A* gene was found to be most commonly mutated (Table 2). That gene encodes the alpha subunit of the main cardiac sodium channel (Nav1.5); loss of function variants result in reduced availability of functional Nav1.5 channels either through decreased plasma membrane channel expression or through altered channel gating properties[117]. iPSC-CMs generated from BrS patients were shown to reflect the pro-arrhythmic phenotype associated with the disease and caused by blunted inward sodium currents, increased triggered activity, and calcium transient abnormalities. Davis *et al*[118] were the first to describe the molecular mechanisms that underlie BrS by using patient-specific iPSC-CMs harboring *SCN5A* _1795insD mutation, which effectively recapped the INa peak reduction and persistent INa associated with overlapped LQTS3/BrS[118]. Another group investigated sodium currents, action potentials and calcium dynamics in iPSC-CMs derived from patients with type 1 BrS carrying two different *SCN5A* variants and in healthy control subjects[119]. Mutated cardiac cells showed reductions in inward sodium current density, reduced maximal upstroke velocity of the action potential (AP), increased burden of triggered activity, abnormal calcium transients, and beating interval variation compared with control iPSC-CMs from healthy controls. Further analysis revealed markedly reduced expression of *KCNJ2*, which encodes Kir2.1 inwardly rectifying potassium channel, an observation not previously described in BrS. Correction of the causative variant by CRISPR/Cas9-mediated genome editing prompted efficient resolution of triggered activity and abnormal Ca2+ transients. + +Additional data were provided by the work of Ma *et al*[120], who reported that a repolarization deficit was involved in BrS. By comparing electrophysiological properties of iPSC-CMs generated from a patient carrying a compound *SCN5A* mutation (A226V and R1629X) and a healthy sibling control, they observed an over 75% loss of sodium current in diseased cells. The decline in INa was reflected by altered action potential morphology with reduced maximum upstroke velocity and action potential amplitude at normal 1.0 Hz pacing frequency. At slow a slow pacing 0.1 Hz pacing frequency, an increased phase-1 repolarization action potential pattern characterized by a marked reduction of action potential duration and increased resting membrane potential occurred in a fraction of BrS CMs. Furthermore, disparities in the levels of transient outward K+currents (Ito) among the iPSC-CMs from either compound carriers or healthy controls were noticed, with 19% to 23% of the studied cells displaying high Ito densities. Importantly, in patient-derived iPSC-CMs, treatment with 4-Aminopyridine, an Ito blocker, completely reversed the increased phase-1 repolarization and restored the APD, indicating a coordinated role of INa and Ito in the arrhythmogenic mechanism of BrS. In-depth analysis of iPSC-CMs derived from two BrS subjects with an *SCN5A*-S1812X variant revealed reduced INa, amplified Ito, and increased ICaL window current probability along with conduction slowing, demonstrating coexistence of repolarization and depolarization impairment in diseased cells[121]. + +At present, it is widely acknowledged that patient-specific genetic background is a key determinant of the phenotypical manifestation of BrS, as was reported by a team of researchers from Spain and United Kingdom[122]. As expected, iPSC-CMs from a patient with a *SCN5A* variation recapitulated the loss of function of Nav1.5 associated with the syndrome. Also, a shift in both activation and inactivation voltagedependence curves and faster recovery from inactivation were reported. Remarkably, conventional heterologous expression systems (*i.e.* immortalized HEK293 cells coexpressing wild-type and mutant channels) failed to exhibit pro-arrhythmic changes in channel function, showing only a reduction in sodium current density, highlighting once again the need to assess the pathophysiological mechanisms of sodium channel mutations in a cardiac- and patient-specific model. + +IPSC technology has also been used to model BrS caused by mutations in genes other than *SCN5A.* Cerrone *et al*[123] were the first to describe the association of BrS and genetic variation in *PKP2*. They analyzed iPSC-CMs derived from five index cases carrying missense mutations in *PKP2* and perceived reduced sodium channel expression and current. The phenotype was rescued by transfection of wild-type *PKP2,* demonstrating that not only loss of PKP2, but also single amino acid mutations, can interfere with INa. + +In the vast majority of clinically-diagnosed BrS cases (85%), the genetic cause is not known despite extensive use of NGS[124]. Few research groups have used iPSC +technology to uncover disease mechanisms at the cellular level in phenotype-positive genotype-negative patients[125,126]. Notably, no clear cellular electrophysiological differences between the iPSC-CMs obtained from BrS patients without identified pathogenic mutations and control-derived cells were seen. That finding indicated that alternative pathophysiological mechanisms may be involved in those specific cases, such as right ventricular fibrosis or diminished cardiomyocyte coupling through gap junctions. Last but not least, BrS may be a multifactorial disorder, caused by an interaction of common genetic variations and environmental factors[125]. + +## Modeling Patient-Specific Therapeutic Regimens + +The right dose of the right drug for the right patient at the right time is not only the mantra of personalized or precision medicine, but a common challenge faced daily by clinicians all over the world[2,127]. With the advent of iPSC technology to guide therapeutic decisions in a patient-specific manner, tailoring treatment to a patient's genetic background is yet to become a reality. + +Prondzynski *et al*[128] employed patient-specific iPSC-CMs to define disease-related mechanisms and also to guide treatment in an HCM-affected family carrying a novel ACTN2 missense mutation[128]. Apart from previously described hallmarks of HCM, +such as myofibrillar disarray, cell hypertrophy, increased myofilament Ca2+ sensitivity, hypercontractility, and prolonged relaxation, iPSC-CMs demonstrated enhanced Ltype calcium channel current and prolonged action potential duration compared with isogenic controls. Following the beneficial results of improved contractile and electrophysiological in *vitro* phenotype with diltiazem, an L-type Ca2+ channel blocker, the findings were translated into clinical settings where standard-dose diltiazem reverted the LQT phenotype in the son and sister of the index patient. + +Although still in early stages, patient-derived iPSCs have been shown to facilitate optimal treatment in arrhythmic disorders. In a stepwise study, Terrenoire *et al*[88] +established a patient-specific therapeutic regimen in a LQTS child with complex genetics and only partially-controlled arrhythmia with high-dose mexiletine[88]. The index patient had a de *novo* mutation in the sodium channel *SCN5A* and a common polymorphism in the potassium channel *KCNH2*. First, electrophysiological analysis of the iPSC-CMs revealed that the *SCN5A* mutation was responsible for the patient's symptoms. Furthermore, the authors found that mexiletine inhibited the IKr potassium channels in iPSC-CMs from both the father and the proband, irrespective of KCNH2 polymorphism, which explained the limited ability of mexiletine to completely correct the repolarization defect. Hence, alternative strategies to control INaL have been tested on patient-derived iPSC-CMs, such as changes in pacing rate or the addition of a second Na+channel blocker. The experimental data recommended mexiletine alone and an increased pacemaker rate as the best therapeutic option, which was further confirmed by the patient's clinical evolution. In another LQTS3 model, mexiletine rescued the abnormal electrophysiology in iPSC-CMs from a patient harboring a *SCN5A* mutation (p.V1763M)[87]. + +Specific drug screening using patient-derived iPSC models has also been performed in CPVT, where β-blockers are the drugs of choice, but often fail to avoid malignant arrhythmias. In symptomatic CPVT patients under standard β-blocker treatment, it was shown that individual-specific iPSC-CMs had a subadequate antiarrhythmic response to β-blockers, while both patient and iPSC-CMs responded more effectively to flecainide[105,110]. Clearly, the antiarrhythmic efficacy of different drugs is dependent on the underlying genetic variation. By patch-clamp analysis alone or by simultaneous patch-clamp and video imaging, Pölönen *et al*[129,130] assessed the antiarrhythmic effects of carvedilol and flecainide in CPVT patient-specific iPSC-CMs carrying diverse *RYR2* variants. They found mutation-specific differences in arrhythmias and drug responses, suggesting that proper treatment may vary even among subjects with mutations in the same genes[129,130]. Evidence from earlier studies indicated that dantrolene was able to restore normal Ca2+ spark properties and rescue the arrhythmogenic phenotype in a patient-specific iPSC model[97]. Subsequent study revealed that not only that the location of the RYR2 mutation was critical for a favorable effect of dantrolene, but also suggested that the drug effect was dependent on the specific DNA alteration. Specifically, the antiarrhythmic effect was detected only in cases carrying mutations in the NH2-terminal or central regions of RYR2 protein. No effect was seen in subjects carrying mutations in the transmembrane region. Moreover, the effect of dantrolene was only minimal in iPSC-CMs with a *Q4201R* variant despite being located in the central region of RYR2 protein, even if at its end[131]. Indeed, the dantrolene binding site is located in the NH2-terminal region of *RYR2* between amino acid 601 and 620. After specific binding, the drug restores normal channel gating and prevents uncontrolled Ca2+ release by stabilizing interdomain interactions between the NH2-terminal and central regions of RYR2, as previously reported[132,133]. + +## Modeling Variants Of Uncertain Significance + +Sequencing of wide-ranging gene panels by high-throughput techniques on a daily basis has increased the rate of positive genetic testing, and it has also increased the detection of variants of uncertain significance (VUS). Recently, our group reported the yield of DNA testing in a cohort of HCM probands[134,135]. Nearly half (45%) of the rare variants identified in our study were novel, and thus classified as of VUS. All but two were found only once in our cohort. Similar results were obtained in other studies, which reported a prevalence of 35%-40% of new mutations, half of which were unique for a family[136]. The conclusive classification of VUS is encumbered by challenges, particularly in cases of "private" mutations, as it involves computational and population-based studies, not rarely misleading[137,138]. Combined use of recent technologies such as iPSC and gene editing have enabled functional annotation in specific cases. + +Lv *et al*[139] used a dual-integrase cassette exchange platform to rapidly and efficiently generate iPSCs with the TNNT2-E251D variant harbored by a woman with severe HCM and otherwise negative genetic testing. Although the mutation was generally predicted to be pathogenic by in *silico* analysis, the allele frequency of 0.03% in the Exome Aggregation Consortium database was inconsistent with the disease incidence (*i.e.* too high), and the ClinVar archive included conflicting interpretations of clinical significance, but mostly VUS. *TNNT2*-E251D iPSC-CMs had normal responses to isoproterenol, suggesting that the variant might not be pathogenic. To exclude the possibility that the failure in attaining a pathological phenotype was due to lack of a permissive genetic background in the studied cells, the authors introduced an E251D point mutation into an edited iPSC line known to be vulnerable to cardiomyopathy with CRISPR-Cas9. Comprehensive investigation of the E251D iPSC-CMs showed normal responses to isoproterenol and no significant increase in cell size or expression of genes previously reported to be upregulated in HCM iPSC-CMs (e.g., *TNNT2,* MYL2, *MYL4,* and *MYH7*). This approach allowed specific recommendations to be made to relatives, namely not to undergo cascade genetic screening for the *E251D* variant. + +In another study, iPSC-CMs were produced from an asymptomatic subject with a HCM associated mutation in *MYL3,* and reported by the ClinVar database to be likely pathogenic[23]. Extensive assays, including measurement of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling, were performed on isogenic iPSC-CMs that were either corrected or carrying homozygous alleles found that the VUS was benign. + +With the goal of functional prediction of pathogenicity, Pettinato *et al*[140] developed a scalable human cardiomyocyte platform to interrogate *TNNT2* variants previously identified in the human population. Using iPSC-CMs in cardiac microtissue and single-cell assays, they examined 51 *TNNT2* variants, including 30 pathogenic/likely pathogenic variants associated with HCM/DCM, and 21 VUS. Experimental evidence including transcriptomic changes and cardiac microtissue contraction, supported the reclassification of two pathogenic/likely pathogenic variants and two VUSs. These findings are of a great interest given that most *TNNT2* variants identified in the human population are classified as of VUS. therefore definite reclassification would enable specific clinical decision making for individuals harboring these variants. + +In a similar manner, iPSC models were used to decipher the pathogenicity of variants detected in patients with inherited CNPs. By combining patient-specific iPSCs and genome editing, Garg *et al*[141] demonstrated the pathogenicity of a novel VUS in the *KCNH2* gene. Compared with healthy control cells, VUS iPSC-CMs displayed electrophysiological abnormalities consistent with LQTS2 phenotype (prolongation of action potential duration and reduced IKr density), which were rescued by VUS correction by CRISPR/Cas9. Furthermore, the introduction of the homozygous KCNH2-T983I variant in a healthy control line recapitulated the hallmark LQTS phenotype, confirming that the mutation was sufficient to prompt the disease. + +Generation of iPSC lines from every single individual with a VUS in a CMP-/CNPrelated gene, followed by allele correction, and functional assessment is laborious and virtually impossible. Hence alternative approaches exploiting already existing and functionally characterized human iPSC lines has been considered. For example, commercially available human iPSC-CMs were used to screen a *KCNJ2* VUS detected in a LQTS7 proband by whole exome sequencing[142]. VUS overexpression was associated with a substantial prolongation of APD with evidence of arrhythmic activity, emulating the clinical phenotype, and thus supporting causality of the variant. + +Chavali *et al*[143] established a patient-independent human iPSC model as a new tool for rapid determination of genetic variant pathogenicity in LQTS. The authors used CRISPR/Cas9 to introduce a *CACNA1C* VUS from an unrelated healthy volunteer into a previously established iPSC line. Functional changes detected in gene-edited iPSCCMs allowed reclassification of *CACNA1C*-N639T variant to ''likely pathogenic". + +Considering all the available evidence, it can be easily seen that a screening platform based on edited human iPSC lines might be more informative than currently used procedures for variant classification, such as computational and populationbased methods. + +## Conclusion + +Due to various genetic and environmental modulatory factors, Mendelian CMPs and CNPs are characterized by variable expressivity and incomplete penetrance, which often delays the clinical management of such patients. One issue to be addressed by upcoming studies is whether iPSCs can be used to identify genetic modifiers and to unveil the protective or aggravating underlying regulatory mechanisms. As a proof of concept, Chai *et al*[144] used complementary physiological and genomic analyses to identify genetic modifiers explaining the variable expressivity observed in a large LQTS2 family. + +Although the feasibility of this new technology for disease modeling and drug testing has been demonstrated, there are currently some limitations that should be addressed in order to further recommend the use of iPSC-CMs in clinical practice. Thus, the main setbacks in using this approach on a large scale are the reproducibility of results among multiple laboratories and the immature phenotype displayed by these cells. + +The first is due to the use of various methods for inducing pluripotency, chromosomal instability throughout the reprogramming process and in *vitro* manipulation, the purity in myocyte composition, and batch disparities in differentiated CMs[145]. Therefore, implementation of standardized protocols for patient-specific lines is important. Second, most iPSC-CMs have an immature structural and functional phenotype, with fetal gene expression, disorganized sarcomeres, primarily relying on glycolysis, and having contractile features different from those of adult CMs, such as spontaneous beating[146]. Those properties could negatively impact the interpretation of the cellular responses to various drugs and the prediction of the clinical value of the respective compounds. Consequently, it is imperative to develop methods to generate CMs with a more mature phenotype in order to improve the predictive value of in *vitro* studies. Recently, important progress in the maturation of iPSC-CMs has been made by using small molecules[147], environmental manipulation[148] and three-dimensional culture[149]. IPSC-based research is still at an early stage. Nevertheless, one can undoubtedly see its boundless potential for advancing personalized clinical management of individuals with inherited CMPs and CNPs. + +## References + +Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. *Cell* 2006; 126: 663-676 [PMID: 16904174 DOI: +1 2 3 4 6 13 14 15 16 10.1016/j.cell.2006.07.024] +Musunuru K, Sheikh F, Gupta RM, Houser SR, Maher KO, Milan DJ, Terzic A, Wu JC; American Heart Association Council on Functional Genomics and Translational Biology; Council on Cardiovascular Disease in the Young; and Council on Cardiovascular and Stroke Nursing. Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine: A Scientific Statement From the American Heart Association. *Circ Genom Precis Med* 2018; 11: e000043 [PMID: 29874173 DOI: 10.1161/HCG.0000000000000043] Popa-Fotea NM, Micheu MM, Bataila V, Scafa-Udriste A, Dorobantu L, Scarlatescu AI, Zamfir D, Stoian M, Onciul S, Dorobantu M. Exploring the Continuum of Hypertrophic Cardiomyopathy-From DNA to Clinical Expression. *Medicina (Kaunas)* 2019; 55 [PMID: 31234582 DOI: +10.3390/medicina55060299] Musunuru K, Arora P, Cooke JP, Ferguson JF, Hershberger RE, Hickey KT, Lee JM, Lima JAC, Loscalzo J, Pereira NL, Russell MW, Shah SH, Sheikh F, Wang TJ, MacRae CA; American Heart Association Council on Genomic and Precision Medicine; Council on Cardiovascular Disease in the Young; Council on Cardiovascular and Stroke Nursing; Council on Cardiovascular Radiology and Intervention; Council on Peripheral Vascular Disease; Council on Quality of Care and Outcomes Research; and Stroke Council. Interdisciplinary Models for Research and Clinical Endeavors in Genomic Medicine: A Scientific Statement From the American Heart Association. *Circ Genom* Precis Med 2018; 11: e000046 [PMID: 29844141 DOI: 10.1161/HCG.0000000000000046] +Micheu MM, Popescu I, Capatana CO, Barbarii LE, Dorobantu M. Molecular autopsy in sudden cardiac death - Ethical issues and clinical implication for relatives. *Rom J Leg Med* 2016; 24: 157163 [DOI: 10.4323/rjlm.2016.157] +5 Elliott P, Andersson B, Arbustini E, Bilinska Z, Cecchi F, Charron P, Dubourg O, Kühl U, Maisch B, McKenna WJ, Monserrat L, Pankuweit S, Rapezzi C, Seferovic P, Tavazzi L, Keren A. Classification of the cardiomyopathies: a position statement from the European Society Of Cardiology Working Group on Myocardial and Pericardial Diseases. *Eur Heart J* 2008; 29: 270-276 +[PMID: 17916581 DOI: 10.1093/eurheartj/ehm342] Fernández-Falgueras A, Sarquella-Brugada G, Brugada J, Brugada R, Campuzano O. Cardiac Channelopathies and Sudden Death: Recent Clinical and Genetic Advances. *Biology (Basel)* 2017; 6 [PMID: 28146053 DOI: 10.3390/biology6010007] +7 Garcia-Elias A, Benito B. Ion Channel Disorders and Sudden Cardiac Death. *Int J Mol Sci* 2018; 19 [PMID: 29495624 DOI: 10.3390/ijms19030692] +8 Popa-Fotea NM, Cojocaru C, Scafa-Udriste A, Micheu MM, Dorobantu M. The Multifaced Perspectives of Genetic Testing in Pediatric Cardiomyopathies and Channelopathies. *J Clin Med* 2020; 9 [PMID: 32635562 DOI: 10.3390/jcm9072111] +9 Kline J, Costantini O. Inherited Cardiac Arrhythmias and Channelopathies. *Med Clin North Am* 2019; 103: 809-820 [PMID: 31378327 DOI: 10.1016/j.mcna.2019.05.001] +10 Wilde AAM, Nannenberg E, van der Werf C. Cardiogenetics, 25 years a growing subspecialism. Neth Heart J 2020; 28: 39-43 [PMID: 32780330 DOI: 10.1007/s12471-020-01444-8] +11 Yogasundaram H, Alhumaid W, Dzwiniel T, Christian S, Oudit GY. Cardiomyopathies and Genetic Testing in Heart Failure: Role in Defining Phenotype-Targeted Approaches and Management. *Can J Cardiol* 2021 [PMID: 33493662 DOI: 10.1016/j.cjca.2021.01.016] +12 Ingles J, Goldstein J, Thaxton C, Caleshu C, Corty EW, Crowley SB, Dougherty K, Harrison SM, +McGlaughon J, Milko LV, Morales A, Seifert BA, Strande N, Thomson K, Peter van Tintelen J, Wallace K, Walsh R, Wells Q, Whiffin N, Witkowski L, Semsarian C, Ware JS, Hershberger RE, Funke B. Evaluating the Clinical Validity of Hypertrophic Cardiomyopathy Genes. *Circ Genom* Precis Med 2019; 12: e002460 [PMID: 30681346 DOI: 10.1161/CIRCGEN.119.002460] +Hershberger RE, Givertz MM, Ho CY, Judge DP, Kantor PF, McBride KL, Morales A, Taylor MRG, Vatta M, Ware SM. Genetic Evaluation of Cardiomyopathy-A Heart Failure Society of America Practice Guideline. *J Card Fail* 2018; 24: 281-302 [PMID: 29567486 DOI: 10.1016/j.cardfail.2018.03.004] Hershberger RE, Givertz MM, Ho CY, Judge DP, Kantor PF, McBride KL, Morales A, Taylor MRG, Vatta M, Ware SM; ACMG Professional Practice and Guidelines Committee. Genetic evaluation of cardiomyopathy: a clinical practice resource of the American College of Medical Genetics and Genomics (ACMG). *Genet Med* 2018; 20: 899-909 [PMID: 29904160 DOI: 10.1038/s41436-018-0039-z] +Ackerman MJ, Priori SG, Willems S, Berul C, Brugada R, Calkins H, Camm AJ, Ellinor PT, +Gollob M, Hamilton R, Hershberger RE, Judge DP, Le Marec H, McKenna WJ, Schulze-Bahr E, Semsarian C, Towbin JA, Watkins H, Wilde A, Wolpert C, Zipes DP. HRS/EHRA expert consensus statement on the state of genetic testing for the channelopathies and cardiomyopathies this document was developed as a partnership between the Heart Rhythm Society (HRS) and the European Heart Rhythm Association (EHRA). *Heart Rhythm* 2011; 8: 1308-1339 [PMID: 21787999 DOI: 10.1016/j.hrthm.2011.05.020] Schwartz PJ, Ackerman MJ, George AL Jr, Wilde AAM. Impact of genetics on the clinical management of channelopathies. *J Am Coll Cardiol* 2013; 62: 169-180 [PMID: 23684683 DOI: 10.1016/j.jacc.2013.04.044] +17 Hosseini SM, Kim R, Udupa S, Costain G, Jobling R, Liston E, Jamal SM, Szybowska M, Morel CF, Bowdin S, Garcia J, Care M, Sturm AC, Novelli V, Ackerman MJ, Ware JS, Hershberger RE, Wilde AAM, Gollob MH; National Institutes of Health Clinical Genome Resource Consortium. + +18 20 21 22 23 24 25 26 27 28 29 30 31 32 Reappraisal of Reported Genes for Sudden Arrhythmic Death: Evidence-Based Evaluation of Gene Validity for Brugada Syndrome. *Circulation* 2018; 138: 1195-1205 [PMID: 29959160 DOI: +10.1161/CIRCULATIONAHA.118.035070] +Eschenhagen T, Carrier L. Cardiomyopathy phenotypes in human-induced pluripotent stem cellderived cardiomyocytes-a systematic review. *Pflugers Arch* 2019; 471: 755-768 [PMID: 30324321 DOI: 10.1007/s00424-018-2214-0] +19 Lan F, Lee AS, Liang P, Sanchez-Freire V, Nguyen PK, Wang L, Han L, Yen M, Wang Y, Sun N, +Abilez OJ, Hu S, Ebert AD, Navarrete EG, Simmons CS, Wheeler M, Pruitt B, Lewis R, Yamaguchi Y, Ashley EA, Bers DM, Robbins RC, Longaker MT, Wu JC. Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells. *Cell Stem Cell* 2013; 12: 101-113 [PMID: 23290139 DOI: 10.1016/j.stem.2012.10.010] Han L, Li Y, Tchao J, Kaplan AD, Lin B, Mich-Basso J, Lis A, Hassan N, London B, Bett GC, +Tobita K, Rasmusson RL, Yang L. Study familial hypertrophic cardiomyopathy using patientspecific induced pluripotent stem cells. *Cardiovasc Res* 2014; 104: 258-269 [PMID: 25209314 DOI: +10.1093/cvr/cvu205] Prondzynski M, Krämer E, Laufer SD, Shibamiya A, Pless O, Flenner F, Müller OJ, Münch J, Redwood C, Hansen A, Patten M, Eschenhagen T, Mearini G, Carrier L. Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes. *Mol Ther Nucleic Acids* 2017; 7: 475-486 [PMID: 28624223 DOI: +10.1016/j.omtn.2017.05.008] Ma N, Zhang JZ, Itzhaki I, Zhang SL, Chen H, Haddad F, Kitani T, Wilson KD, Tian L, Shrestha R, Wu H, Lam CK, Sayed N, Wu JC. Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells. Circulation 2018; 138: 2666-2681 [PMID: 29914921 DOI: 10.1161/CIRCULATIONAHA.117.032273] Mosqueira D, Mannhardt I, Bhagwan JR, Lis-Slimak K, Katili P, Scott E, Hassan M, Prondzynski M, Harmer SC, Tinker A, Smith JGW, Carrier L, Williams PM, Gaffney D, Eschenhagen T, Hansen A, Denning C. CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy. *Eur Heart J* 2018; 39: 3879-3892 [PMID: 29741611 DOI: 10.1093/eurheartj/ehy249] Sun N, Yazawa M, Liu J, Han L, Sanchez-Freire V, Abilez OJ, Navarrete EG, Hu S, Wang L, Lee A, Pavlovic A, Lin S, Chen R, Hajjar RJ, Snyder MP, Dolmetsch RE, Butte MJ, Ashley EA, Longaker MT, Robbins RC, Wu JC. Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy. *Sci Transl Med* 2012; 4: 130ra47 [PMID: 22517884 DOI: 10.1126/scitranslmed.3003552] +Karakikes I, Stillitano F, Nonnenmacher M, Tzimas C, Sanoudou D, Termglinchan V, Kong CW, +Rushing S, Hansen J, Ceholski D, Kolokathis F, Kremastinos D, Katoulis A, Ren L, Cohen N, Gho JMIH, Tsiapras D, Vink A, Wu JC, Asselbergs FW, Li RA, Hulot JS, Kranias EG, Hajjar RJ. Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy. *Nat Commun* 2015; 6: 6955 [PMID: 25923014 DOI: +10.1038/ncomms7955] +Judge LM, Perez-Bermejo JA, Truong A, Ribeiro AJ, Yoo JC, Jensen CL, Mandegar MA, Huebsch N, Kaake RM, So PL, Srivastava D, Pruitt BL, Krogan NJ, Conklin BR. A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress. *JCI Insight* 2017; 2 [PMID: 28724793 DOI: 10.1172/jci.insight.94623] Wang L, Kim K, Parikh S, Cadar AG, Bersell KR, He H, Pinto JR, Kryshtal DO, Knollmann BC. + +Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and proarrhythmic action potential changes in human iPSC cardiomyocytes. *J Mol Cell Cardiol* 2018; 114: +320-327 [PMID: 29217433 DOI: 10.1016/j.yjmcc.2017.12.002] Seeger T, Shrestha R, Lam CK, Chen C, McKeithan WL, Lau E, Wnorowski A, McMullen G, Greenhaw M, Lee J, Oikonomopoulos A, Lee S, Yang H, Mercola M, Wheeler M, Ashley EA, Yang F, Karakikes I, Wu JC. A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay. *Circulation* 2019; 139: 799811 [PMID: 30586709 DOI: 10.1161/CIRCULATIONAHA.118.034624] Wu H, Yang H, Rhee JW, Zhang JZ, Lam CK, Sallam K, Chang ACY, Ma N, Lee J, Zhang H, Blau HM, Bers DM, Wu JC. Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients. *Eur Heart J* 2019; 40: 3685-3695 [PMID: 31219556 DOI: 10.1093/eurheartj/ehz326] +Bhagwan JR, Mosqueira D, Chairez-Cantu K, Mannhardt I, Bodbin SE, Bakar M, Smith JGW, +Denning C. Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics. *J Mol Cell Cardiol* 2020; 145: 43-53 [PMID: 32531470 DOI: 10.1016/j.yjmcc.2020.06.003] Smith JGW, Owen T, Bhagwan JR, Mosqueira D, Scott E, Mannhardt I, Patel A, Barriales-Villa R, Monserrat L, Hansen A, Eschenhagen T, Harding SE, Marston S, Denning C. Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits. *Stem Cell Reports* 2018; 11: 1226-1243 [PMID: 30392975 DOI: 10.1016/j.stemcr.2018.10.006] +33 **Toepfer CN**, Garfinkel AC, Venturini G, Wakimoto H, Repetti G, Alamo L, Sharma A, Agarwal R, 34 35 37 38 39 40 41 43 44 45 46 47 Ewoldt JF, Cloonan P, Letendre J, Lun M, Olivotto I, Colan S, Ashley E, Jacoby D, Michels M, +Redwood CS, Watkins HC, Day SM, Staples JF, Padrón R, Chopra A, Ho CY, Chen CS, Pereira AC, Seidman JG, Seidman CE. Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy. *Circulation* 2020; 141: 828-842 [PMID: 31983222 DOI: 10.1161/CIRCULATIONAHA.119.042339] +Gerull B, Gramlich M, Atherton J, McNabb M, Trombitás K, Sasse-Klaassen S, Seidman JG, +Seidman C, Granzier H, Labeit S, Frenneaux M, Thierfelder L. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. *Nat Genet* 2002; 30: 201-204 [PMID: 11788824 DOI: 10.1038/ng815] Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, Conner L, DePalma SR, McDonough B, Sparks E, Teodorescu DL, Cirino AL, Banner NR, Pennell DJ, Graw S, Merlo M, Di Lenarda A, Sinagra G, Bos JM, Ackerman MJ, Mitchell RN, Murry CE, Lakdawala NK, Ho CY, Barton PJ, Cook SA, Mestroni L, Seidman JG, Seidman CE. Truncations of titin causing dilated cardiomyopathy. *N Engl J Med* 2012; 366: 619-628 [PMID: 22335739 DOI: 10.1056/NEJMoa1110186] Hershberger RE, Morales A. Dilated Cardiomyopathy Overview. 2007 Jul 27 [updated 2018 Aug 23]. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2021 36 Tse HF, Ho JC, Choi SW, Lee YK, Butler AW, Ng KM, Siu CW, Simpson MA, Lai WH, Chan YC, Au KW, Zhang J, Lay KW, Esteban MA, Nicholls JM, Colman A, Sham PC. Patient-specific induced-pluripotent stem cells-derived cardiomyocytes recapitulate the pathogenic phenotypes of dilated cardiomyopathy due to a novel DES mutation identified by whole exome sequencing. Hum Mol Genet 2013; 22: 1395-1403 [PMID: 23300193 DOI: 10.1093/hmg/dds556] Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S, Gorham J, Yang L, Schafer S, Sheng CC, Haghighi A, Homsy J, Hubner N, Church G, Cook SA, Linke WA, Chen CS, Seidman JG, Seidman CE. HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. *Science* 2015; 349: 982-986 [PMID: 26315439 DOI: 10.1126/science.aaa5458] Siu CW, Lee YK, Ho JC, Lai WH, Chan YC, Ng KM, Wong LY, Au KW, Lau YM, Zhang J, Lay KW, Colman A, Tse HF. Modeling of lamin A/C mutation premature cardiac aging using patient‐ specific induced pluripotent stem cells. *Aging (Albany NY)* 2012; 4: 803-822 [PMID: 23362510 DOI: 10.18632/aging.100503] +Wu H, Lee J, Vincent LG, Wang Q, Gu M, Lan F, Churko JM, Sallam KI, Matsa E, Sharma A, Gold JD, Engler AJ, Xiang YK, Bers DM, Wu JC. Epigenetic Regulation of Phosphodiesterases 2A and 3A Underlies Compromised β-Adrenergic Signaling in an iPSC Model of Dilated Cardiomyopathy. Cell Stem Cell 2015; 17: 89-100 [PMID: 26095046 DOI: 10.1016/j.stem.2015.04.020] +Dai Y, Amenov A, Ignatyeva N, Koschinski A, Xu H, Soong PL, Tiburcy M, Linke WA, Zaccolo M, Hasenfuss G, Zimmermann WH, Ebert A. Troponin destabilization impairs sarcomerecytoskeleton interactions in iPSC-derived cardiomyocytes from dilated cardiomyopathy patients. Sci Rep 2020; 10: 209 [PMID: 31937807 DOI: 10.1038/s41598-019-56597-3] +Lee YK, Lau YM, Cai ZJ, Lai WH, Wong LY, Tse HF, Ng KM, Siu CW. Modeling Treatment Response for Lamin A/C Related Dilated Cardiomyopathy in Human Induced Pluripotent Stem Cells. *J Am Heart Assoc* 2017; 6 [PMID: 28754655 DOI: 10.1161/JAHA.117.005677] +42 Lee J, Termglinchan V, Diecke S, Itzhaki I, Lam CK, Garg P, Lau E, Greenhaw M, Seeger T, Wu H, Zhang JZ, Chen X, Gil IP, Ameen M, Sallam K, Rhee JW, Churko JM, Chaudhary R, Chour T, Wang PJ, Snyder MP, Chang HY, Karakikes I, Wu JC. Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy. *Nature* 2019; 572: 335-340 [PMID: 31316208 DOI: 10.1038/s41586-019-1406-x] +Shah D, Virtanen L, Prajapati C, Kiamehr M, Gullmets J, West G, Kreutzer J, Pekkanen-Mattila M, +Heliö T, Kallio P, Taimen P, Aalto-Setälä K. Modeling of *LMNA*-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells. *Cells* 2019; 8 [PMID: 31208058 DOI: 10.3390/cells8060594] Salvarani N, Crasto S, Miragoli M, Bertero A, Paulis M, Kunderfranco P, Serio S, Forni A, Lucarelli C, Dal Ferro M, Larcher V, Sinagra G, Vezzoni P, Murry CE, Faggian G, Condorelli G, Di Pasquale E. The K219T-Lamin mutation induces conduction defects through epigenetic inhibition of SCN5A in human cardiac laminopathy. *Nat Commun* 2019; 10: 2267 [PMID: 31118417 DOI: 10.1038/s41467-019-09929-w] +Yang KC, Breitbart A, De Lange WJ, Hofsteen P, Futakuchi-Tsuchida A, Xu J, Schopf C, +Razumova MV, Jiao A, Boucek R, Pabon L, Reinecke H, Kim DH, Ralphe JC, Regnier M, Murry CE. Novel Adult-Onset Systolic Cardiomyopathy Due to MYH7 E848G Mutation in Patient-Derived Induced Pluripotent Stem Cells. *JACC Basic Transl Sci* 2018; 3: 728-740 [PMID: 30623132 DOI: 10.1016/j.jacbts.2018.08.008] +Lin B, Li Y, Han L, Kaplan AD, Ao Y, Kalra S, Bett GC, Rasmusson RL, Denning C, Yang L. + +Modeling and study of the mechanism of dilated cardiomyopathy using induced pluripotent stem cells derived from individuals with Duchenne muscular dystrophy. *Dis Model Mech* 2015; 8: 457466 [PMID: 25791035 DOI: 10.1242/dmm.019505] +Dick E, Kalra S, Anderson D, George V, Ritso M, Laval SH, Barresi R, Aartsma-Rus A, Lochmüller H, Denning C. Exon skipping and gene transfer restore dystrophin expression in human induced 48 49 50 51 52 53 54 55 57 58 59 60 61 62 63 pluripotent stem cells-cardiomyocytes harboring DMD mutations. *Stem Cells Dev* 2013; 22: 27142724 [PMID: 23829870 DOI: 10.1089/scd.2013.0135] Deacon DC, Happe CL, Chen C, Tedeschi N, Manso AM, Li T, Dalton ND, Peng Q, Farah EN, Gu Y, Tenerelli KP, Tran VD, Chen J, Peterson KL, Schork NJ, Adler ED, Engler AJ, Ross RS, Chi NC. Combinatorial interactions of genetic variants in human cardiomyopathy. *Nat Biomed Eng* 2019; 3: 147-157 [PMID: 30923642 DOI: 10.1038/s41551-019-0348-9] +Kodo K, Ong SG, Jahanbani F, Termglinchan V, Hirono K, InanlooRahatloo K, Ebert AD, Shukla P, Abilez OJ, Churko JM, Karakikes I, Jung G, Ichida F, Wu SM, Snyder MP, Bernstein D, Wu JC. iPSC-derived cardiomyocytes reveal abnormal TGF-β signalling in left ventricular non-compaction cardiomyopathy. *Nat Cell Biol* 2016; 18: 1031-1042 [PMID: 27642787 DOI: 10.1038/ncb3411] Ang YS, Rivas RN, Ribeiro AJS, Srivas R, Rivera J, Stone NR, Pratt K, Mohamed TMA, Fu JD, Spencer CI, Tippens ND, Li M, Narasimha A, Radzinsky E, Moon-Grady AJ, Yu H, Pruitt BL, Snyder MP, Srivastava D. Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis. *Cell* 2016; 167: 1734-1749. e22 [PMID: 27984724 DOI: 10.1016/j.cell.2016.11.033] Takasaki A, Hirono K, Hata Y, Wang C, Takeda M, Yamashita JK, Chang B, Nakaoka H, Okabe M, Miyao N, Saito K, Ibuki K, Ozawa S, Sekine M, Yoshimura N, Nishida N, Bowles NE, Ichida F. Sarcomere gene variants act as a genetic trigger underlying the development of left ventricular noncompaction. *Pediatr Res* 2018; 84: 733-742 [PMID: 30188508 DOI: 10.1038/s41390-018-0162-1] Ma D, Wei H, Lu J, Ho S, Zhang G, Sun X, Oh Y, Tan SH, Ng ML, Shim W, Wong P, Liew R. Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy. *Eur Heart J* 2013; 34: 1122-1133 [PMID: 22798562 DOI: 10.1093/eurheartj/ehs226] Kim C, Wong J, Wen J, Wang S, Wang C, Spiering S, Kan NG, Forcales S, Puri PL, Leone TC, Marine JE, Calkins H, Kelly DP, Judge DP, Chen HS. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. *Nature* 2013; 494: 105-110 [PMID: 23354045 DOI: 10.1038/nature11799] Caspi O, Huber I, Gepstein A, Arbel G, Maizels L, Boulos M, Gepstein L. Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells. *Circ* Cardiovasc Genet 2013; 6: 557-568 [PMID: 24200905 DOI: +10.1161/CIRCGENETICS.113.000188] Wen JY, Wei CY, Shah K, Wong J, Wang C, Chen HS. Maturation-Based Model of Arrhythmogenic Right Ventricular Dysplasia Using Patient-Specific Induced Pluripotent Stem Cells. + +Circ J 2015; 79: 1402-1408 [PMID: 25971409 DOI: 10.1253/circj.CJ-15-0363] +56 Dorn T, Kornherr J, Parrotta EI, Zawada D, Ayetey H, Santamaria G, Iop L, Mastantuono E, Sinnecker D, Goedel A, Dirschinger RJ, My I, Laue S, Bozoglu T, Baarlink C, Ziegler T, Graf E, Hinkel R, Cuda G, Kääb S, Grace AA, Grosse R, Kupatt C, Meitinger T, Smith AG, Laugwitz KL, Moretti A. Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity. *EMBO J* 2018; 37 [PMID: 29764980 DOI: 10.15252/embj.201798133] Chen P, Xiao Y, Wang Y, Zheng Z, Chen L, Yang X, Li J, Wu W, Zhang S. Intracellular calcium current disorder and disease phenotype in *OBSCN* mutant iPSC-based cardiomyocytes in arrhythmogenic right ventricular cardiomyopathy. *Theranostics* 2020; 10: 11215-11229 [PMID: +33042279 DOI: 10.7150/thno.45172] El-Battrawy I, Zhao Z, Lan H, Cyganek L, Tombers C, Li X, Buljubasic F, Lang S, Tiburcy M, Zimmermann WH, Utikal J, Wieland T, Borggrefe M, Zhou XB, Akin I. Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy. *Europace* 2018; 20: f46-f56 [PMID: 29566126 DOI: 10.1093/europace/euy042] Buljubasic F, El-Battrawy I, Lan H, Lomada SK, Chatterjee A, Zhao Z, Li X, Zhong R, Xu Q, Huang M, Liao Z, Lang S, Cyganek L, Zhou X, Wieland T, Borggrefe M, Akin I. Nucleoside Diphosphate Kinase B Contributes to Arrhythmogenesis in Human-Induced Pluripotent Stem CellDerived Cardiomyocytes from a Patient with Arrhythmogenic Right Ventricular Cardiomyopathy. J +Clin Med 2020; 9 [PMID: 32050722 DOI: 10.3390/jcm9020486] +Ng R, Manring H, Papoutsidakis N, Albertelli T, Tsai N, See CJ, Li X, Park J, Stevens TL, Bobbili PJ, Riaz M, Ren Y, Stoddard CE, Janssen PM, Bunch TJ, Hall SP, Lo YC, Jacoby DL, Qyang Y, Wright N, Ackermann MA, Campbell SG. Patient mutations linked to arrhythmogenic cardiomyopathy enhance calpain-mediated desmoplakin degradation. *JCI Insight* 2019; 5 [PMID: +31194698 DOI: 10.1172/jci.insight.128643] Brodehl A, Pour Hakimi SA, Stanasiuk C, Ratnavadivel S, Hendig D, Gaertner A, Gerull B, Gummert J, Paluszkiewicz L, Milting H. Restrictive Cardiomyopathy is Caused by a Novel Homozygous Desmin (DES) Mutation p.Y122H Leading to a Severe Filament Assembly Defect. Genes (Basel) 2019; 10 [PMID: 31718026 DOI: 10.3390/genes10110918] Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flügel L, Dorn T, Goedel A, Höhnke C, Hofmann F, Seyfarth M, Sinnecker D, Schömig A, Laugwitz KL. Patient-specific induced pluripotent stem-cell models for long-QT syndrome. *N Engl J Med* 2010; 363: 1397-1409 [PMID: +20660394 DOI: 10.1056/NEJMoa0908679] Egashira T, Yuasa S, Suzuki T, Aizawa Y, Yamakawa H, Matsuhashi T, Ohno Y, Tohyama S, Okata S, Seki T, Kuroda Y, Yae K, Hashimoto H, Tanaka T, Hattori F, Sato T, Miyoshi S, Takatsuki 64 65 66 67 68 69 70 71 72 73 76 77 78 S, Murata M, Kurokawa J, Furukawa T, Makita N, Aiba T, Shimizu W, Horie M, Kamiya K, +Kodama I, Ogawa S, Fukuda K. Disease characterization using LQTS-specific induced pluripotent stem cells. *Cardiovasc Res* 2012; 95: 419-429 [PMID: 22739119 DOI: 10.1093/cvr/cvs206] Ma D, Wei H, Lu J, Huang D, Liu Z, Loh LJ, Islam O, Liew R, Shim W, Cook SA. Characterization of a novel KCNQ1 mutation for type 1 Long QT syndrome and assessment of the therapeutic potential of a novel IKs activator using patient-specific induced pluripotent stem cell-derived cardiomyocytes. *Stem Cell Res Ther* 2015; 6: 39 [PMID: 25889101 DOI: +10.1186/s13287-015-0027-z] Wang Y, Liang P, Lan F, Wu H, Lisowski L, Gu M, Hu S, Kay MA, Urnov FD, Shinnawi R, Gold JD, Gepstein L, Wu JC. Genome editing of isogenic human induced pluripotent stem cells recapitulates long QT phenotype for drug testing. *J Am Coll Cardiol* 2014; 64: 451-459 [PMID: 25082577 DOI: 10.1016/j.jacc.2014.04.057] Wang Z, Wang L, Liu W, Hu D, Gao Y, Ge Q, Liu X, Li L, Wang Y, Wang S, Li C. Pathogenic mechanism and gene correction for LQTS-causing double mutations in KCNQ1 using a pluripotent stem cell model. *Stem Cell Res* 2019; 38: 101483 [PMID: 31226583 DOI: 10.1016/j.scr.2019.101483] Ge N, Liu M, Krawczyk J, McInerney V, Galvin J, Shen S, O'Brien T, Prendiville T. Generation of eight human induced pluripotent stem cell (iPSC) lines from familial Long QT Syndrome type 1 +(LQT1) patients carrying KCNQ1 c.1697C>A mutation (NUIGi005-A, NUIGi005-B, NUIGi005-C, +NUIGi006-A, NUIGi006-B, NUIGi006-C, NUIGi007-A, and NUIGi007-B). *Stem Cell Res* 2019; 39: 101502 [PMID: 31415974 DOI: 10.1016/j.scr.2019.101502] +Ge N, Liu M, Ding Y, Krawczyk J, McInerney V, Galvin J, Shen S, Prendiville T, O'Brien T. + +Generation and characterization of twelve human induced pluripotent stem cell (iPSC) lines from four familial long QT syndrome type 1 (LQT1) patients carrying KCNQ1 c.1201dupC mutation. Stem Cell Res 2019; 41: 101650 [PMID: 31765965 DOI: 10.1016/j.scr.2019.101650] +Mura M, Lee YK, Pisano F, Ginevrino M, Boni M, Calabrò F, Crotti L, Valente EM, Schwartz PJ, +Tse HF, Gnecchi M. Generation of the human induced pluripotent stem cell (hiPSC) line PSMi004- A from a carrier of the KCNQ1-R594Q mutation. *Stem Cell Res* 2019; 37: 101431 [PMID: 30974404 DOI: 10.1016/j.scr.2019.101431] +Mura M, Lee YK, Pisano F, Ginevrino M, Boni M, Calabrò F, Crotti L, Valente EM, Schwartz PJ, +Tse HF, Gnecchi M. Generation of the human induced pluripotent stem cell (hiPSC) line PSMi005- A from a patient carrying the KCNQ1-R190W mutation. *Stem Cell Res* 2019; 37: 101437 [PMID: 31009818 DOI: 10.1016/j.scr.2019.101437] Mura M, Pisano F, Stefanello M, Ginevrino M, Boni M, Calabrò F, Crotti L, Valente EM, Schwartz PJ, Brink PA, Gnecchi M. Generation of two human induced pluripotent stem cell (hiPSC) lines from a long QT syndrome South African founder population. *Stem Cell Res* 2019; 39: 101510 [PMID: 31398660 DOI: 10.1016/j.scr.2019.101510] Mura M, Bastaroli F, Corli M, Ginevrino M, Calabrò F, Boni M, Crotti L, Valente EM, Schwartz PJ, Gnecchi M. Generation of the human induced pluripotent stem cell (hiPSC) line PSMi006-A from a patient affected by an autosomal recessive form of long QT syndrome type 1. *Stem Cell Res* 2020; 42: 101658 [PMID: 31785541 DOI: 10.1016/j.scr.2019.101658] +Itzhaki I, Maizels L, Huber I, Zwi-Dantsis L, Caspi O, Winterstern A, Feldman O, Gepstein A, +Arbel G, Hammerman H, Boulos M, Gepstein L. Modelling the long QT syndrome with induced pluripotent stem cells. *Nature* 2011; 471: 225-229 [PMID: 21240260 DOI: 10.1038/nature09747] +74 Matsa E, Rajamohan D, Dick E, Young L, Mellor I, Staniforth A, Denning C. Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a long QT syndrome type 2 mutation. *Eur Heart J* 2011; 32: 952-962 [PMID: 21367833 DOI: 10.1093/eurheartj/ehr073] +75 Lahti AL, Kujala VJ, Chapman H, Koivisto AP, Pekkanen-Mattila M, Kerkelä E, Hyttinen J, +Kontula K, Swan H, Conklin BR, Yamanaka S, Silvennoinen O, Aalto-Setälä K. Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture. Dis Model Mech 2012; 5: 220-230 [PMID: 22052944 DOI: 10.1242/dmm.008409] +Bellin M, Casini S, Davis RP, D'Aniello C, Haas J, Ward-van Oostwaard D, Tertoolen LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL. Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome. *EMBO J* 2013; 32: 31613175 [PMID: 24213244 DOI: 10.1038/emboj.2013.240] Matsa E, Dixon JE, Medway C, Georgiou O, Patel MJ, Morgan K, Kemp PJ, Staniforth A, Mellor I, +Denning C. Allele-specific RNA interference rescues the long-QT syndrome phenotype in humaninduced pluripotency stem cell cardiomyocytes. *Eur Heart J* 2014; 35: 1078-1087 [PMID: 23470493 DOI: 10.1093/eurheartj/eht067] Smith JL, Anderson CL, Burgess DE, Elayi CS, January CT, Delisle BP. Molecular pathogenesis of long QT syndrome type 2. *J Arrhythm* 2016; 32: 373-380 [PMID: 27761161 DOI: 10.1016/j.joa.2015.11.009] +79 Schwartz PJ, Gnecchi M, Dagradi F, Castelletti S, Parati G, Spazzolini C, Sala L, Crotti L. From patient-specific induced pluripotent stem cells to clinical translation in long QT syndrome Type 2. Eur Heart J 2019; 40: 1832-1836 [PMID: 30753398 DOI: 10.1093/eurheartj/ehz023] +80 Perry MD, Ng CA, Mangala MM, Ng TYM, Hines AD, Liang W, Xu MJO, Hill AP, Vandenberg JI. Pharmacological activation of IKr in models of long QT Type 2 risks overcorrection of repolarization. *Cardiovasc Res* 2020; 116: 1434-1445 [PMID: 31628797 DOI: 10.1093/cvr/cvz247] +81 82 **Mehta A**, Ramachandra CJA, Singh P, Chitre A, Lua CH, Mura M, Crotti L, Wong P, Schwartz PJ, 83 85 86 87 88 90 92 93 94 95 96 97 Gnecchi M, Shim W. Identification of a targeted and testable antiarrhythmic therapy for long-QT +syndrome type 2 using a patient-specific cellular model. *Eur Heart J* 2018; 39: 1446-1455 [PMID: +29020304 DOI: 10.1093/eurheartj/ehx394] Spencer CI, Baba S, Nakamura K, Hua EA, Sears MA, Fu CC, Zhang J, Balijepalli S, Tomoda K, Hayashi Y, Lizarraga P, Wojciak J, Scheinman MM, Aalto-Setälä K, Makielski JC, January CT, Healy KE, Kamp TJ, Yamanaka S, Conklin BR. Calcium transients closely reflect prolonged action potentials in iPSC models of inherited cardiac arrhythmia. *Stem Cell Reports* 2014; 3: 269-281 +[PMID: 25254341 DOI: 10.1016/j.stemcr.2014.06.003] +Joutsijoki H, Penttinen K, Juhola M, Aalto-Setälä K. Separation of HCM and LQT Cardiac Diseases with Machine Learning of Ca2+ Transient Profiles. *Methods Inf Med* 2019; 58: 167-178 [PMID: 32079026 DOI: 10.1055/s-0040-1701484] +84 Fatima A, Kaifeng S, Dittmann S, Xu G, Gupta MK, Linke M, Zechner U, Nguemo F, Milting H, Farr M, Hescheler J, Sarić T. The disease-specific phenotype in cardiomyocytes derived from induced pluripotent stem cells of two long QT syndrome type 3 patients. *PLoS One* 2013; 8: e83005 [PMID: 24349418 DOI: 10.1371/journal.pone.0083005] Navarrete EG, Liang P, Lan F, Sanchez-Freire V, Simmons C, Gong T, Sharma A, Burridge PW, +Patlolla B, Lee AS, Wu H, Beygui RE, Wu SM, Robbins RC, Bers DM, Wu JC. Screening druginduced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays. *Circulation* 2013; 128: S3-13 [PMID: 24030418 DOI: 10.1161/CIRCULATIONAHA.112.000570] +Ma D, Wei H, Zhao Y, Lu J, Li G, Sahib NB, Tan TH, Wong KY, Shim W, Wong P, Cook SA, +Liew R. Modeling type 3 Long QT syndrome with cardiomyocytes derived from patient-specific induced pluripotent stem cells. *Int J Cardiol* 2013; 168: 5277-5286 [PMID: 23998552 DOI: 10.1016/j.ijcard.2013.08.015] Terrenoire C, Wang K, Tung KW, Chung WK, Pass RH, Lu JT, Jean JC, Omari A, Sampson KJ, Kotton DN, Keller G, Kass RS. Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics. *J Gen Physiol* 2013; 141: 61-72 [PMID: 23277474 DOI: 10.1085/jgp.201210899] Malan D, Zhang M, Stallmeyer B, Müller J, Fleischmann BK, Schulze-Bahr E, Sasse P, Greber B. Human iPS cell model of type 3 Long QT syndrome recapitulates drug-based phenotype correction. Basic Res Cardiol 2016; 111: 14 [PMID: 26803770 DOI: 10.1007/s00395-016-0530-0] +89 Kuroda Y, Yuasa S, Watanabe Y, Ito S, Egashira T, Seki T, Hattori T, Ohno S, Kodaira M, Suzuki T, Hashimoto H, Okata S, Tanaka A, Aizawa Y, Murata M, Aiba T, Makita N, Furukawa T, Shimizu W, Kodama I, Ogawa S, Kokubun N, Horigome H, Horie M, Kamiya K, Fukuda K. Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes. *Biochem Biophys Rep* 2017; 9: 245-256 [PMID: 28956012 DOI: 10.1016/j.bbrep.2017.01.002] +Yazawa M, Hsueh B, Jia X, Pasca AM, Bernstein JA, Hallmayer J, Dolmetsch RE. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. *Nature* 2011; 471: 230-234 [PMID: 21307850 DOI: 10.1038/nature09855] +91 Limpitikul WB, Dick IE, Tester DJ, Boczek NJ, Limphong P, Yang W, Choi MH, Babich J, DiSilvestre D, Kanter RJ, Tomaselli GF, Ackerman MJ, Yue DT. A Precision Medicine Approach to the Rescue of Function on Malignant Calmodulinopathic Long-QT Syndrome. *Circ Res* 2017; 120: +39-48 [PMID: 27765793 DOI: 10.1161/CIRCRESAHA.116.309283] +Rocchetti M, Sala L, Dreizehnter L, Crotti L, Sinnecker D, Mura M, Pane LS, Altomare C, Torre E, +Mostacciuolo G, Severi S, Porta A, De Ferrari GM, George AL Jr, Schwartz PJ, Gnecchi M, Moretti A, Zaza A. Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes. *Cardiovasc Res* 2017; 113: 531-541 [PMID: 28158429 DOI: 10.1093/cvr/cvx006] Yamamoto Y, Makiyama T, Harita T, Sasaki K, Wuriyanghai Y, Hayano M, Nishiuchi S, Kohjitani H, Hirose S, Chen J, Yokoi F, Ishikawa T, Ohno S, Chonabayashi K, Motomura H, Yoshida Y, Horie M, Makita N, Kimura T. Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation. *Hum Mol Genet* 2017; 26: +1670-1677 [PMID: 28335032 DOI: 10.1093/hmg/ddx073] Fatima A, Xu G, Shao K, Papadopoulos S, Lehmann M, Arnáiz-Cot JJ, Rosa AO, Nguemo F, Matzkies M, Dittmann S, Stone SL, Linke M, Zechner U, Beyer V, Hennies HC, Rosenkranz S, Klauke B, Parwani AS, Haverkamp W, Pfitzer G, Farr M, Cleemann L, Morad M, Milting H, Hescheler J, Saric T. In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells. *Cell Physiol Biochem* 2011; 28: 579-592 [PMID: 22178870 DOI: 10.1159/000335753] +Itzhaki I, Maizels L, Huber I, Gepstein A, Arbel G, Caspi O, Miller L, Belhassen B, Nof E, Glikson M, Gepstein L. Modeling of catecholaminergic polymorphic ventricular tachycardia with patientspecific human-induced pluripotent stem cells. *J Am Coll Cardiol* 2012; 60: 990-1000 [PMID: +22749309 DOI: 10.1016/j.jacc.2012.02.066] +Jung CB, Moretti A, Mederos y Schnitzler M, Iop L, Storch U, Bellin M, Dorn T, Ruppenthal S, +Pfeiffer S, Goedel A, Dirschinger RJ, Seyfarth M, Lam JT, Sinnecker D, Gudermann T, Lipp P, Laugwitz KL. Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia. *EMBO Mol Med* 2012; 4: 180-191 +[PMID: 22174035 DOI: 10.1002/emmm.201100194] +98 99 100 101 102 103 104 105 107 108 109 110 111 113 Kujala K, Paavola J, Lahti A, Larsson K, Pekkanen-Mattila M, Viitasalo M, Lahtinen AM, +Toivonen L, Kontula K, Swan H, Laine M, Silvennoinen O, Aalto-Setälä K. Cell model of catecholaminergic polymorphic ventricular tachycardia reveals early and delayed afterdepolarizations. *PLoS One* 2012; 7: e44660 [PMID: 22962621 DOI: 10.1371/journal.pone.0044660] Novak A, Barad L, Zeevi-Levin N, Shick R, Shtrichman R, Lorber A, Itskovitz-Eldor J, Binah O. + +Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to βadrenergic stimulation. *J Cell Mol Med* 2012; 16: 468-482 [PMID: 22050625 DOI: +10.1111/j.1582-4934.2011.01476.x] Di Pasquale E, Lodola F, Miragoli M, Denegri M, Avelino-Cruz JE, Buonocore M, Nakahama H, Portararo P, Bloise R, Napolitano C, Condorelli G, Priori SG. CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia. *Cell Death Dis* 2013; 4: e843 [PMID: 24113177 DOI: 10.1038/cddis.2013.369] Zhang XH, Haviland S, Wei H, Sarić T, Fatima A, Hescheler J, Cleemann L, Morad M. Ca2+ signaling in human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) from normal and catecholaminergic polymorphic ventricular tachycardia (CPVT)-afflicted subjects. *Cell Calcium* 2013; 54: 57-70 [PMID: 23684427 DOI: 10.1016/j.ceca.2013.04.004] Novak A, Barad L, Lorber A, Gherghiceanu M, Reiter I, Eisen B, Eldor L, Itskovitz-Eldor J, Eldar M, Arad M, Binah O. Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations. *J Cell Mol Med* 2015; 19: 2006-2018 [PMID: 26153920 DOI: 10.1111/jcmm.12581] +Lodola F, Morone D, Denegri M, Bongianino R, Nakahama H, Rutigliano L, Gosetti R, Rizzo G, +Vollero A, Buonocore M, Napolitano C, Condorelli G, Priori SG, Di Pasquale E. Adeno-associated virus-mediated CASQ2 delivery rescues phenotypic alterations in a patient-specific model of recessive catecholaminergic polymorphic ventricular tachycardia. *Cell Death Dis* 2016; 7: e2393 [PMID: 27711080 DOI: 10.1038/cddis.2016.304] Sasaki K, Makiyama T, Yoshida Y, Wuriyanghai Y, Kamakura T, Nishiuchi S, Hayano M, Harita T, Yamamoto Y, Kohjitani H, Hirose S, Chen J, Kawamura M, Ohno S, Itoh H, Takeuchi A, Matsuoka S, Miura M, Sumitomo N, Horie M, Yamanaka S, Kimura T. Patient-Specific Human Induced Pluripotent Stem Cell Model Assessed with Electrical Pacing Validates S107 as a Potential Therapeutic Agent for Catecholaminergic Polymorphic Ventricular Tachycardia. *PLoS One* 2016; 11: e0164795 [PMID: 27764147 DOI: 10.1371/journal.pone.0164795] +Preininger MK, Jha R, Maxwell JT, Wu Q, Singh M, Wang B, Dalal A, Mceachin ZT, Rossoll W, +Hales CM, Fischbach PS, Wagner MB, Xu C. A human pluripotent stem cell model of catecholaminergic polymorphic ventricular tachycardia recapitulates patient-specific drug responses. Dis Model Mech 2016; 9: 927-939 [PMID: 27491078 DOI: 10.1242/dmm.026823] +Wei H, Zhang XH, Clift C, Yamaguchi N, Morad M. CRISPR/Cas9 Gene editing of RyR2 in human stem cell-derived cardiomyocytes provides a novel approach in investigating dysfunctional Ca2+ +signaling. *Cell Calcium* 2018; 73: 104-111 [PMID: 29730419 DOI: 10.1016/j.ceca.2018.04.009] +106 Zhang XH, Wei H, Xia Y, Morad M. Calcium signaling consequences of RyR2 mutations associated with CPVT1 introduced via CRISPR/Cas9 gene editing in human-induced pluripotent stem cell-derived cardiomyocytes: Comparison of RyR2-R420Q, F2483I, and Q4201R. *Heart* Rhythm 2021; 18: 250-260 [PMID: 32931925 DOI: 10.1016/j.hrthm.2020.09.007] +Park SJ, Zhang D, Qi Y, Li Y, Lee KY, Bezzerides VJ, Yang P, Xia S, Kim SL, Liu X, Lu F, +Pasqualini FS, Campbell PH, Geva J, Roberts AE, Kleber AG, Abrams DJ, Pu WT, Parker KK. Insights Into the Pathogenesis of Catecholaminergic Polymorphic Ventricular Tachycardia From Engineered Human Heart Tissue. *Circulation* 2019; 140: 390-404 [PMID: 31311300 DOI: 10.1161/CIRCULATIONAHA.119.039711] +Sleiman Y, Souidi M, Kumar R, Yang E, Jaffré F, Zhou T, Bernardin A, Reiken S, Cazorla O, +Kajava AV, Moreau A, Pasquié JL, Marks AR, Lerman BB, Chen S, Cheung JW, Evans T, +Lacampagne A, Meli AC. Modeling polymorphic ventricular tachycardia at rest using patientspecific induced pluripotent stem cell-derived cardiomyocytes. *EBioMedicine* 2020; 60: 103024 +[PMID: 32980690 DOI: 10.1016/j.ebiom.2020.103024] Maizels L, Huber I, Arbel G, Tijsen AJ, Gepstein A, Khoury A, Gepstein L. Patient-Specific Drug Screening Using a Human Induced Pluripotent Stem Cell Model of Catecholaminergic Polymorphic Ventricular Tachycardia Type 2. *Circ Arrhythm Electrophysiol* 2017; 10 [PMID: 28630169 DOI: +10.1161/CIRCEP.116.004725] Ross S, Holliday M, Lim S, Semsarian C. Characterization of the first induced pluripotent stem cell line generated from a patient with autosomal dominant catecholaminergic polymorphic ventricular tachycardia due to a heterozygous mutation in cardiac calsequestrin-2. *Stem Cell Res* 2019; 37: 101450 [PMID: 31039485 DOI: 10.1016/j.scr.2019.101450] Mazzanti A, Underwood K, Nevelev D, Kofman S, Priori SG. The new kids on the block of arrhythmogenic disorders: Short QT syndrome and early repolarization. *J Cardiovasc Electrophysiol* 2017; 28: 1226-1236 [PMID: 28569435 DOI: 10.1111/jce.13265] +112 El-Battrawy I, Lan H, Cyganek L, Zhao Z, Li X, Buljubasic F, Lang S, Yücel G, Sattler K, +Zimmermann WH, Utikal J, Wieland T, Ravens U, Borggrefe M, Zhou XB, Akin I. Modeling Short QT Syndrome Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes. J Am Heart Assoc 2018; 7 [PMID: 29574456 DOI: 10.1161/JAHA.117.007394] +114 **Guo F**, Sun Y, Wang X, Wang H, Wang J, Gong T, Chen X, Zhang P, Su L, Fu G, Su J, Yang S, Lai 115 118 119 120 121 122 123 125 126 128 129 130 R, Jiang C, Liang P. Patient-Specific and Gene-Corrected Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Short QT Syndrome. *Circ Res* 2019; 124: 66-78 +[PMID: 30582453 DOI: 10.1161/CIRCRESAHA.118.313518] Shinnawi R, Shaheen N, Huber I, Shiti A, Arbel G, Gepstein A, Ballan N, Setter N, Tijsen AJ, Borggrefe M, Gepstein L. Modeling Reentry in the Short QT Syndrome With Human-Induced Pluripotent Stem Cell-Derived Cardiac Cell Sheets. *J Am Coll Cardiol* 2019; 73: 2310-2324 [PMID: +31072576 DOI: 10.1016/j.jacc.2019.02.055] +Tse G, Liu T, Li KH, Laxton V, Chan YW, Keung W, Li RA, Yan BP. Electrophysiological Mechanisms of Brugada Syndrome: Insights from Pre-clinical and Clinical Studies. *Front Physiol* 2016; 7: 467 [PMID: 27803673 DOI: 10.3389/fphys.2016.00467] +116 Remme CA. Cardiac sodium channelopathy associated with SCN5A mutations: electrophysiological, molecular and genetic aspects. *J Physiol* 2013; 591: 4099-4116 [PMID: 23818691 DOI: 10.1113/jphysiol.2013.256461] +117 Davis RP, Casini S, van den Berg CW, Hoekstra M, Remme CA, Dambrot C, Salvatori D, Oostwaard DW, Wilde AA, Bezzina CR, Verkerk AO, Freund C, Mummery CL. Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease. *Circulation* 2012; 125: 3079-3091 [PMID: 22647976 DOI: 10.1161/CIRCULATIONAHA.111.066092] Liang P, Sallam K, Wu H, Li Y, Itzhaki I, Garg P, Zhang Y, Vermglinchan V, Lan F, Gu M, Gong T, Zhuge Y, He C, Ebert AD, Sanchez-Freire V, Churko J, Hu S, Sharma A, Lam CK, Scheinman MM, Bers DM, Wu JC. Patient-Specific and Genome-Edited Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Brugada Syndrome. *J Am Coll Cardiol* 2016; 68: 2086-2096 [PMID: 27810048 DOI: 10.1016/j.jacc.2016.07.779] +Ma D, Liu Z, Loh LJ, Zhao Y, Li G, Liew R, Islam O, Wu J, Chung YY, Teo WS, Ching CK, Tan BY, Chong D, Ho KL, Lim P, Yong RYY, Panama BK, Kaplan AD, Bett GCL, Ware J, Bezzina CR, Verkerk AO, Cook SA, Rasmusson RL, Wei H. Identification of an INa-dependent and Itomediated proarrhythmic mechanism in cardiomyocytes derived from pluripotent stem cells of a Brugada syndrome patient. *Sci Rep* 2018; 8: 11246 [PMID: 30050137 DOI: +10.1038/s41598-018-29574-5] Li W, Stauske M, Luo X, Wagner S, Vollrath M, Mehnert CS, Schubert M, Cyganek L, Chen S, Hasheminasab SM, Wulf G, El-Armouche A, Maier LS, Hasenfuss G, Guan K. Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Lossof-Function SCN5A Mutation. *Front Cell Dev Biol* 2020; 8: 592893 [PMID: 33195263 DOI: +10.3389/fcell.2020.592893] Selga E, Sendfeld F, Martinez-Moreno R, Medine CN, Tura-Ceide O, Wilmut SI, Pérez GJ, Scornik FS, Brugada R, Mills NL. Sodium channel current loss of function in induced pluripotent stem cellderived cardiomyocytes from a Brugada syndrome patient. *J Mol Cell Cardiol* 2018; 114: 10-19 +[PMID: 29024690 DOI: 10.1016/j.yjmcc.2017.10.002] Cerrone M, Lin X, Zhang M, Agullo-Pascual E, Pfenniger A, Chkourko Gusky H, Novelli V, Kim C, Tirasawadichai T, Judge DP, Rothenberg E, Chen HS, Napolitano C, Priori SG, Delmar M. Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype. *Circulation* 2014; 129: 1092-1103 [PMID: 24352520 DOI: 10.1161/CIRCULATIONAHA.113.003077] Monasky MM, Micaglio E, Ciconte G, Pappone C. Brugada Syndrome: Oligogenic or Mendelian Disease? *Int J Mol Sci* 2020; 21 [PMID: 32121523 DOI: 10.3390/ijms21051687] +124 Veerman CC, Mengarelli I, Guan K, Stauske M, Barc J, Tan HL, Wilde AA, Verkerk AO, Bezzina CR. hiPSC-derived cardiomyocytes from Brugada Syndrome patients without identified mutations do not exhibit clear cellular electrophysiological abnormalities. *Sci Rep* 2016; 6: 30967 [PMID: 27485484 DOI: 10.1038/srep30967] Miller DC, Harmer SC, Poliandri A, Nobles M, Edwards EC, Ware JS, Sharp TV, McKay TR, +Dunkel L, Lambiase PD, Tinker A. Ajmaline blocks INa and IKr without eliciting differences between Brugada syndrome patient and control human pluripotent stem cell-derived cardiac clusters. Stem Cell Res 2017; 25: 233-244 [PMID: 29172153 DOI: 10.1016/j.scr.2017.11.003] +Abrahams E. Right drug-right patient-right time: personalized medicine coalition. *Clin Transl Sci* 2008; 1: 11-12 [PMID: 20443813 DOI: 10.1111/j.1752-8062.2008.00003.x] +127 Prondzynski M, Lemoine MD, Zech AT, Horváth A, Di Mauro V, Koivumäki JT, Kresin N, Busch J, Krause T, Krämer E, Schlossarek S, Spohn M, Friedrich FW, Münch J, Laufer SD, Redwood C, Volk AE, Hansen A, Mearini G, Catalucci D, Meyer C, Christ T, Patten M, Eschenhagen T, Carrier L. Disease modeling of a mutation in α-actinin 2 guides clinical therapy in hypertrophic cardiomyopathy. *EMBO Mol Med* 2019; 11: e11115 [PMID: 31680489 DOI: +10.15252/emmm.201911115] Pölönen RP, Penttinen K, Swan H, Aalto-Setälä K. Antiarrhythmic Effects of Carvedilol and Flecainide in Cardiomyocytes Derived from Catecholaminergic Polymorphic Ventricular Tachycardia Patients. *Stem Cells Int* 2018; **2018**: 9109503 [PMID: 29760739 DOI: 10.1155/2018/9109503] +Pölönen RP, Swan H, Aalto-Setälä K. Mutation-specific differences in arrhythmias and drug responses in CPVT patients: simultaneous patch clamp and video imaging of iPSC derived cardiomyocytes. *Mol Biol Rep* 2020; 47: 1067-1077 [PMID: 31786768 DOI: 10.1007/s11033-019-05201-y] +131 135 136 137 138 139 140 142 143 147 Penttinen K, Swan H, Vanninen S, Paavola J, Lahtinen AM, Kontula K, Aalto-Setälä K. + +Antiarrhythmic Effects of Dantrolene in Patients with Catecholaminergic Polymorphic Ventricular Tachycardia and Replication of the Responses Using iPSC Models. *PLoS One* 2015; 10: e0125366 [PMID: 25955245 DOI: 10.1371/journal.pone.0125366] Kobayashi S, Bannister ML, Gangopadhyay JP, Hamada T, Parness J, Ikemoto N. Dantrolene stabilizes domain interactions within the ryanodine receptor. *J Biol Chem* 2005; 280: 6580-6587 +[PMID: 15611117 DOI: 10.1074/jbc.M408375200] +132 Paul-Pletzer K, Yamamoto T, Ikemoto N, Jimenez LS, Morimoto H, Williams PG, Ma J, Parness J. + +Probing a putative dantrolene-binding site on the cardiac ryanodine receptor. *Biochem J* 2005; 387: +905-909 [PMID: 15656791 DOI: 10.1042/BJ20041336] +133 Micheu MM, Popa-Fotea NM, Oprescu N, Dorobantu M, Ratiu AC, Ecovoiu AA. NGS data validated by Sanger sequencing reveal a puzzling small deletion of MYBPC3 gene associated with hypertrophic cardiomyopathy. *Rom Biotechnol Lett* 2019; 24: 91-99 [DOI: 10.25083/rbl/24.1/91.99] +134 Micheu MM, Popa-Fotea NM, Oprescu N, Bogdan S, Dan M, Deaconu A, Dorobantu L, GheorgheFronea O, Greavu M, Iorgulescu C, Scafa-Udriste A, Ticulescu R, Vatasescu RG, Dorobanțu M. + +Yield of Rare Variants Detected by Targeted Next-Generation Sequencing in a Cohort of Romanian Index Patients with Hypertrophic Cardiomyopathy. *Diagnostics (Basel)* 2020; 10 [PMID: 33297573 DOI: 10.3390/diagnostics10121061] Alfares AA, Kelly MA, McDermott G, Funke BH, Lebo MS, Baxter SB, Shen J, McLaughlin HM, Clark EH, Babb LJ, Cox SW, DePalma SR, Ho CY, Seidman JG, Seidman CE, Rehm HL. Results of clinical genetic testing of 2,912 probands with hypertrophic cardiomyopathy: expanded panels offer limited additional sensitivity. *Genet Med* 2015; 17: 880-888 [PMID: 25611685 DOI: 10.1038/gim.2014.205] Walsh R, Mazzarotto F, Whiffin N, Buchan R, Midwinter W, Wilk A, Li N, Felkin L, Ingold N, Govind R, Ahmad M, Mazaika E, Allouba M, Zhang X, de Marvao A, Day SM, Ashley E, Colan SD, Michels M, Pereira AC, Jacoby D, Ho CY, Thomson KL, Watkins H, Barton PJR, Olivotto I, Cook SA, Ware JS. Quantitative approaches to variant classification increase the yield and precision of genetic testing in Mendelian diseases: the case of hypertrophic cardiomyopathy. *Genome Med* 2019; 11: 5 [PMID: 30696458 DOI: 10.1186/s13073-019-0616-z] +Campuzano O, Sarquella-Brugada G, Fernandez-Falgueras A, Coll M, Iglesias A, Ferrer-Costa C, Cesar S, Arbelo E, García-Álvarez A, Jordà P, Toro R, Tiron de Llano C, Grassi S, Oliva A, Brugada J, Brugada R. Reanalysis and reclassification of rare genetic variants associated with inherited arrhythmogenic syndromes. *EBioMedicine* 2020; 54: 102732 [PMID: 32268277 DOI: 10.1016/j.ebiom.2020.102732] Lv W, Qiao L, Petrenko N, Li W, Owens AT, McDermott-Roe C, Musunuru K. Functional Annotation of TNNT2 Variants of Uncertain Significance With Genome-Edited Cardiomyocytes. + +Circulation 2018; 138: 2852-2854 [PMID: 30565988 DOI: +10.1161/CIRCULATIONAHA.118.035028] Pettinato AM, Ladha FA, Mellert DJ, Legere N, Cohn R, Romano R, Thakar K, Chen YS, Hinson JT. Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human *TNNT2* Variants. *Circulation* 2020; 142: 2262-2275 [PMID: 33025817 DOI: 10.1161/CIRCULATIONAHA.120.047999] +Garg P, Oikonomopoulos A, Chen H, Li Y, Lam CK, Sallam K, Perez M, Lux RL, Sanguinetti MC, +Wu JC. Genome Editing of Induced Pluripotent Stem Cells to Decipher Cardiac Channelopathy Variant. *J Am Coll Cardiol* 2018; 72: 62-75 [PMID: 29957233 DOI: 10.1016/j.jacc.2018.04.041] +141 Gélinas R, El Khoury N, Chaix MA, Beauchamp C, Alikashani A, Ethier N, Boucher G, Villeneuve L, Robb L, Latour F, Mondesert B, Rivard L, Goyette P, Talajic M, Fiset C, Rioux JD. Characterization of a Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for the Study of Variant Pathogenicity: Validation of a *KCNJ2* Mutation. *Circ Cardiovasc Genet* 2017; 10 [PMID: 29021306 DOI: 10.1161/CIRCGENETICS.117.001755] Chavali NV, Kryshtal DO, Parikh SS, Wang L, Glazer AM, Blackwell DJ, Kroncke BM, Shoemaker MB, Knollmann BC. Patient-independent human induced pluripotent stem cell model: A new tool for rapid determination of genetic variant pathogenicity in long QT syndrome. Heart Rhythm 2019; 16: 1686-1695 [PMID: 31004778 DOI: 10.1016/j.hrthm.2019.04.031] +Chai S, Wan X, Ramirez-Navarro A, Tesar PJ, Kaufman ES, Ficker E, George AL Jr, Deschênes I. Physiological genomics identifies genetic modifiers of long QT syndrome type 2 severity. *J Clin* Invest 2018; 128: 1043-1056 [PMID: 29431731 DOI: 10.1172/JCI94996] +144 Pang L. Toxicity testing in the era of induced pluripotent stem cells: A perspective regarding the use of patient-specific induced pluripotent stem cell–derived cardiomyocytes for cardiac safety evaluation. *Curr Opin Toxicol* 2020; 23: 50-55 [DOI: 10.1016/j.cotox.2020.04.001] +145 Machiraju P, Greenway SC. Current methods for the maturation of induced pluripotent stem cellderived cardiomyocytes. *World J Stem Cells* 2019; 11: 33-43 [PMID: 30705713 DOI: +10.4252/wjsc.v11.i1.33] +146 Parikh SS, Blackwell DJ, Gomez-Hurtado N, Frisk M, Wang L, Kim K, Dahl CP, Fiane A, Tønnessen T, Kryshtal DO, Louch WE, Knollmann BC. Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes. *Circ Res* 2017; 121: 1323-1330 [PMID: 28974554 DOI: 10.1161/CIRCRESAHA.117.311920] +148 **Ruan JL**, Tulloch NL, Razumova MV, Saiget M, Muskheli V, Pabon L, Reinecke H, Regnier M, + +149 Murry CE. Mechanical Stress Conditioning and Electrical Stimulation Promote Contractility and Force Maturation of Induced Pluripotent Stem Cell-Derived Human Cardiac Tissue. *Circulation* 2016; 134: 1557-1567 [PMID: 27737958 DOI: 10.1161/CIRCULATIONAHA.114.014998] Correia C, Koshkin A, Duarte P, Hu D, Carido M, Sebastião MJ, Gomes-Alves P, Elliott DA, Domian IJ, Teixeira AP, Alves PM, Serra M. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes. *Biotechnol Bioeng* 2018; 115: 630-644 +[PMID: 29178315 DOI: 10.1002/bit.26504] +Published by **Baishideng Publishing Group Inc** + +![23_image_0.png](23_image_0.png) + +![23_image_1.png](23_image_1.png) + +7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA +Telephone: +1-925-3991568 E-mail: bpgoffice@wjgnet.com Help Desk: https://www.f6publishing.com/helpdesk https://www.wjgnet.com \ No newline at end of file diff --git a/medical/md/PMC8129987.md b/medical/md/PMC8129987.md new file mode 100644 index 0000000000000000000000000000000000000000..36c3ccdee8ef466adad1a606a96690bb1dc79362 --- /dev/null +++ b/medical/md/PMC8129987.md @@ -0,0 +1,106 @@ +Contents lists available at ScienceDirect + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +![0_image_4.png](0_image_4.png) + +![0_image_7.png](0_image_7.png) + +# Molecular Genetics And Metabolism Reports + +![0_Image_3.Png](0_Image_3.Png) + +![0_Image_5.Png](0_Image_5.Png) + +![0_Image_6.Png](0_Image_6.Png) + +journal homepage: www.elsevier.com/locate/ymgmr + +![0_image_8.png](0_image_8.png) + +Short Communication + +![0_image_1.png](0_image_1.png) + +Successful pregnancy in a woman with glycogen storage disease type 6 Sarah Catharina Grünert *, Stefanie Rosenbaum-Fabian , Luciana Hannibal , Anke Schumann , +Ute Spiekerkoetter Department of General Paediatrics, Adolescent Medicine and Neonatology, Medical Centre- University of Freiburg, Faculty of Medicine, Mathildenstraße 1, 79106 Freiburg, Germany ARTICLE INFO +ABSTRACT + +| Keywords: Glycogen storage disease type 6 GSD VI Pregnancy Ketotic glycogen storage disease Glycogen phosphorylase deficiency PYGL | +|--------------------------------------------------------------------------------------------------------------------------------------------| + +Glycogen storage disease type VI is caused by biallelic variants in the *PYGL* gene that result in hepatic glycogen phosphorylase deficiency. The disorder is clinically characterized by hepatomegaly and recurrent ketotic hypoglycemia from infancy. Although most patients reach adulthood without major complications, no pregnancies in women with GSD VI have been reported so far. We report on a successful pregnancy in a GSD VI patient that resulted in a healthy offspring and describe the pre- and perinatal management. + +## 1. **Introduction** + +Glycogen storage disease type VI (GSD VI, OMIM \#232700) is a rare inherited disorder of glycogen metabolism caused by mutations in the PYGL gene resulting in deficiency of hepatic glycogen phosphorylase +(PYGL) [1–4]. The incidence is approximately 1:65,000–1:85,000 [1]. + +Patients with GSD VI usually present in childhood with hepatomegaly and recurrent hypoglycemia. Other clinical symptoms comprise impaired growth resulting in short stature, osteopenia, delayed puberty and hepatic fibrosis [1,2]. Typical laboratory findings include hypoglycemia with hyperketonemia, elevated transaminase concentrations, hyperlipidemia, and reduced prealbumin concentration [1]. Most adults with GSD VI are asymptomatic [2]. As GSD VI is typically a rather mild disease, women with GSD VI usually reach the child-bearing age without major complications. Nevertheless, to our knowledge, no pregnancies in patients with GSD VI have been reported so far. + +## 2. **Case Presentation** + +The patient is the first child of non-consanguineous parents. Clinical signs of hypoglycemia including sweating and shakiness were reported from early infancy throughout childhood. During early childhood the patient required feeding every 2 h, also at night. Hepatomegaly and elevated transaminases were first noted at 3 years. At the age of 6, a liver biopsy was performed and the diagnosis of GSD VI was made based on reduced activity of hepatic PYGL. Until age 13, the patient received regular cornstarch doses during the night, and no episodes of severe hypoglycemia were reported in the further course. From puberty until the age of 36, the patient was lost to follow-up. When she first presented to our metabolic centre at the age of 36, mild hepatomegaly was present, but otherwise she was in good metabolic control. She was on a selfchosen low-carb diet to reduce weight (body weight 113 kg, BMI 39.1 kg/m2). Mutation analysis of the PYGL gene was performed and yielded compound heterozygosity for the variants c.475G > C (p.Gly159Arg) +and c.697G > A (p.Gly233Ser). The diet was switched to a protein-rich diet with reduction of simple sugars (protein 30% of total energy). At age 38, the patient became pregnant after fertility treatment. Apart from ovarian hyperstimulation syndrome, the pregnancy was uncomplicated. + +Her maximum fasting time during the night was 11 h with blood glucose levels above 4.2 mmol/L in the morning and ketone levels <0.1 mmol/L +with rare exceptions. The patient took a protein-rich bedtime snack or alternatively 30 g of uncooked corn starch. No severe hypoglycemias occurred throughout pregnancy (lowest documented glucose level 3.7 mmol/L), however, no conctinuos glucose monitoring was performed. In compliance with our recommendation, no oral glucose tolerance test was performed in the second trimester. Weight gain during pregnancy was 25 kg (weight before pregnancy 100 kg, BMI 34.6 kg/m2). In the 37th week of gestation, she spontaneously delivered a healthy baby boy +* Corresponding author at: Department of Paediatrics, Adolescent Medicine and Neonatology, University Medical Center, Mathildenstraße 1, 79106 Freiburg, Germany. + +E-mail addresses: sarah.gruenert@uniklinik-freiburg.de (S.C. Grünert), stefanie.rosenbaum-fabian@uniklinik-freiburg.de (S. Rosenbaum-Fabian), luciana. + +hannibal@uniklinik-freiburg.de (L. Hannibal), anke.schumann@uniklinik-freiburg.de (A. Schumann), ute.spiekerkoetter@uniklinik-freiburg.de (U. Spiekerkoetter). https://doi.org/10.1016/j.ymgmr.2021.100770 Received 10 March 2021; Received in revised form 30 April 2021; Accepted 1 May 2021 2214-4269/© 2021 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +S.C. Grünert et al. + +(weight 3400 g (76th centile), length 52 cm (71th centile), head circumference 32.5 cm (6th centile)). Management in the peripartal period included an intravenous glucose-electrolyte infusion (starting dose 10 g glucose/h), and regular blood glucose and ketone measurements in the mother during labour. Under this regimen, blood glucose remained stable throughout delivery. The boy could be breastfed without any problems and shows normal development at age 5 months. + +With him being a heterozygous carrier of the disease, no special precautions needed to be taken. + +## 3. **Discussion** + +GSD VI is a rare disorder with only about 50 cases reported. Due to the rather benign character and good prognosis of the disease, more and more patients will reach child-bearing age. To our knowledge, this is the first pregnancy of a woman with GSD VI. + +To ensure an adequate supply of nutrients and energy to the unborn foetus, several alterations in maternal physiology occur during pregnancy mainly influenced by placental hormones [5]. While the first half of pregnancy is an anabolic state associated with increased insulin sensitivity and fat deposition in adipose tissue, the second half is characterized by a maternal insulin resistant state in order to preserve maternal glucose for foetal metabolism. Additionally, hepatic glucose production increases to meet the growing demands of both the foetus and the mother [6]. As women with GSD VI may not be able to match glucose production with increasing energy demands, patients with GSD +VI are at risk for metabolic derangements with more pronounced hypoglycemia and hyperketonemia during pregnancy [7]. Careful monitoring and optimal dietetic treatment are therefore necessary throughout pregnancy to maintain maternal blood glucose levels in the normal range for the safety and proper development of the fetus [8]. For GSD type I, it has been shown that good metabolic control before conception and throughout pregnancy is directly related to successful outcomes [8]. In patients with GSD III it has been shown that maternal hypoglycemia may be associated with intrauterine growth restriction and low birth weight [7], and similar relations can be expected in GSD VI. The mainstay of therapy in GSD VI is the prevention of prolonged fasting, a protein-rich diet and supplementation with uncooked corn starch, if necessary. Treatment and monitoring have to be adapted on an individual basis. In the non-GSD population, oral glucose tolerance tests are routinely performed in the second trimester to screen for gestational diabetes. It is important to note that this test is contraindicated in women with GSDs, as fasting prior to the ingestion of 75 g of concentrated glucose is required for proper interpretation [8]. Fasting, however, poses a high risk of hypoglycemia on pregnant GSD women, and a large bolus of concentrated glucose can cause metabolic instability and result in lactic acidosis [8,9]. To prevent hypoglycemia during delivery, peripartum management should include administration of a high glucose-electrolyte infusion and regular monitoring of blood glucose and ketones during labour. Optimal planning and interdisciplinary collaboration between metabolic physicians and gynecologists is necessary to guarantee a safe setting for mother and child. + +## 4. **Conclusions** + +Our case demonstrates that successful pregnancy is possible in GSD +VI. Careful monitoring during pregnancy and delivery is necessary to minimize the risk of recurrent hypoglycemia for both mother and child. + +## Acknowledgement + +We are grateful to the patient for her support of this publication. This work was supported by the Metabolic Division in the University Children's Hospital, which is part of the Freiburg Center for Rare Diseases. + +Several authors of this publication are members of the European Reference Network for Rare Hereditary Metabolic Disorders (MetabERN) - Project ID No 739543. We acknowledge David Weinstein, Terry Derks and Ulrike Steuerwald for their clinical advice with respect to glycogen storage diseases and many fruitful discussions. The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the University of Freiburg in the funding programme Open Access Publishing. + +## References + +[1] E. Labrador, D.A. Weinstein, Glycogen storage disease type VI, in: M.P. Adam, H. + +H. Ardinger, R.A. Pagon, S.E. Wallace, L.J. Bean, K. Stephens (Eds.), GeneReviews®, +University of Washington, Seattle, Seattle (WA), 1993. http://www.ncbi.nlm.nih. gov/books/NBK5941/. Accessed 4 Sep 2020. + +[2] Margaret A. Chen, David A. Weinstein, Glyogen storage diseases: diagnosis, treatment and outcome, Trans. Sci. Rare Dis. 1 (2016) 45–72. + +[3] S. Chang, M.J. Rosenberg, H. Morton, C.A. Francomano, L.G. Biesecker, Identification of a mutation in liver glycogen phosphorylase in glycogen storage disease type VI, Hum. Mol. Genet. 7 (1998) 865–870. + +[4] B. Burwinkel, H.D. Bakker, E. Herschkovitz, S.W. Moses, Y.S. Shin, M.W. Kilimann, Mutations in the liver glycogen phosphorylase gene (PYGL) underlying glycogenosis type VI, Am. J. Hum. Genet. 62 (1998) 785–791. + +[5] N.F. Butte, Carbohydrate and lipid metabolism in pregnancy: normal compared with gestational diabetes mellitus, Am. J. Clin. Nutr. 71 (5 Suppl) (2000), 1256S–61S. + +[6] S.C. Kalhan, L.J. D'Angelo, S.M. Savin, P.A. Adam, Glucose production in pregnant women at term gestation. Sources of glucose for human fetus, J. Clin. Invest. 63 +(1979) 388–394. + +[7] R. Ramachandran, Y. Wedatilake, C. Coats, F. Walker, P. Elliott, P.J. Lee, et al., +Pregnancy and its management in women with GSD type III - a single Centre experience, J. Inherit. Metab. Dis. 35 (2012) 245–251. + +[8] I.A. Ferrecchia, G. Guenette, E.A. Potocik, D.A. Weinstein, Pregnancy in women with glycogen storage disease Ia and Ib, J. Perinat. Neonatal. Nurs. 28 (2014) 26–31. + +[9] J. Fernandes, J.F. Koster, W.F. Grose, N. Sorgedrager, Hepatic phosphorylase deficiency. Its differentiation from other hepatic glycogenoses, Arch. Dis. Child. 49 +(1974) 186–191. + +2 \ No newline at end of file diff --git a/medical/md/PMC8311701.md b/medical/md/PMC8311701.md new file mode 100644 index 0000000000000000000000000000000000000000..8daf197dfda4b5ead070f8cc42d6cbc3e5be8874 --- /dev/null +++ b/medical/md/PMC8311701.md @@ -0,0 +1,61 @@ +# The Gutenberg Revolution In Cardiovascular Medicine + +Personalized 3D Printing in Planning Aortic Valve Surgery* Gianluca Torregrossa, MD, Andrea Amabile, MD +The invention of the printing press had a significant democratizing effect on society, known as The Gutenberg Revolution. The simple yet extraordinary concept of movable types paved the way for the era of mass communication, which permanently altered the structure of society. + +The relatively unrestricted circulation of printed ideas transcended borders and ultimately led to the spread of revolutions such as the Protestant Reformation, the Age of Enlightenment, and the establishment of the industrial society in Europe. By 1620, the English philosopher Francis Bacon wrote in his Instauratio Magna that the 3 inventions that "have altered the face and state of the world" were gunpowder, the nautical compass and the printing press +(1). Indeed, the printing press was a key factor in establishing a community of scientists who could easily disseminate their discoveries through scholarly journals, and eventually helped standardize language, grammar, and spelling in the scientific world. + +However, when Johannes Gutenberg built the first printing press in approximately 1436, he was certainly not aware of the immense revolution his invention would have been responsible for: Gutenberg died penniless and his presses were impounded by his + +![0_image_0.png](0_image_0.png) + +creditors. + +Similarly, 3-dimensional (3D) printing has entered clinical research as a tool with potentials that clinicians have not completely explored yet. The application of 3D-printed patient-specific models in cardiovascular medicine has a potential role in several areas (2,3). This technology represents a powerful teaching tool, which can rapidly convey a complex anatomic arrangement to trainees or which can make patients reach a higher level of awareness about their structural heart disease. + +Also, 3D-printed models represent an outstanding tool for surgical or interventional preoperative planning and simulation; they can also be used as functional models to investigate intracardiac flow, and they have a tangible role in testing and innovating new surgical and transcatheter devices. + +In this issue of JACC: Case Reports, Shearn et al. (4) +from the Bristol Heart Institute reported the use of 3D-printed, computed tomography–derived, patientspecific aortic root models for the preoperative planning of neocuspidization surgery (Ozaki procedure) in 2 patients with bicuspid aortic valve (BAV). Neocuspidization surgery has gained popularity over the past 10 years as a novel technique in which, during surgery, the autologous pericardium of a patient is molded into 3 different leaflets to create a competent aortic valve (5). Potential advantages of such technique are several: first, the absence of a stent allows molding of a neo-aortic valve with excellent hemodynamics made by all biological material, so theoretically it is less prone to develop endocarditis; second, the design of the valve creates a large coapting surface between the leaflets allowing to maintain a competent valve even if the aortic root will grow (children) or dilate in the future. Finally, ISSN 2666-0849 https://doi.org/10.1016/j.jaccas.2020.05.033 the use of autologous pericardium offers a potential superior durability respect of the xenogenic biological material used for commercially available prosthetic valves, making this technique attractive for young patients with aortic valve disease. All these advantages are waiting to be reported and verified by groups other than Ozaki himself who has the largest experience and has published excellent midterm results of this technique. + +Shearn et al. (4) should be complimented for their excellent short-term outcomes in applying 3D printing technology to the surgical planning of the Ozaki procedure in BAV morphology. BAV anatomy represents indeed an extra technical challenge for the neocuspidization technique in which a trileaflet valve needs to be constructed in a previously bicuspid annulus by creating a new commissure within the aortic root: the success of this type of procedure heavily relies on the technical experience of the surgeon, as demonstrated by the midterm outcomes of Prof. Ozaki (6). The possibility given by 3D printing technology to bench test the surgery on a patientspecific anatomy represents a unique opportunity for aiming at high-quality reconstructions. + +Interestingly, the authors claimed that the presized leaflets were larger than those ultimately implanted in vivo in both patients raising some concern about the fidelity of the model created. + +Nevertheless, it would certainly be worth further investigating the efficacy of this 2-step approach in a + +## References + +1. Boruchoff DA. The three greatest inventions of modern times: an idea and its public. In: Hock K, +Mackenthu G, editors. Entangled Knowledge: Scientific Discourses and Cultural Differences. + +Münster and New York: Waxmann, 2012;133–63. + +2. Giannopoulos AA, Mitsouras D, Yoo SJ, et al. + +Applications of 3D printing in cardiovascular diseases. Nat Rev Cardiol 2016;13:701–18. + +3. Vukicevic M, Mosadegh B, Min JK, et al. Cardiac 3D printing and its future directions. J Am Coll Cardiol Img 2017;10:171–84. + +4. Shearn AIU, Ordoñez MV, Rapetto F, et al. Rapid prototyping flexible aortic models aids sizing of valve leaflets and planning the Ozaki repair. J Am Coll Cardiol Case Rep 2020;2:1137–40. + +5. Ozaki S, Kawase I, Yamashita H, et al. Reconstruction of bicuspid aortic valve with autologous pericardium. Circ J 2014;78:1144–51. + +6. Ozaki S, Kawase I, Yamashita H, et al. Midterm outcomes after aortic valve neocuspidization with glutaraldehyde-treated autologous pericardium. + +J Thorac Cardiovasc Surg 2018;155:2379–87. + +7. Arjmand SA, Pourghorban R, Tehrai M, et al. + +Comparison of aortic root dimension changes during cardiac cycle between the patients with and without aortic valve calcification using ECG-gated 64-slice and dual-source 256-slice computed tomography scanners: results of a multicenter study. + +Int J Cardiovasc Imaging 2013;29:1391–400. + +KEY WORDS 3D printing, aortic valve surgery, neocuspidization, Ozaki technique larger series of patients (comparing the observedversus-expected leaflet sizing and evaluating the long-term follow-up), with a standardized computed tomography scan protocol and cycle time acquisition for the 3D modeling (because the aortic root anatomy changes in shape throughout the cardiac cycle [7]). + +Although the tendency in cardiovascular medicine has been focused on investigating different treatments for a specific disease (surgical aortic valve replacement vs. transcatheter aortic valve replacement, repair vs. replacement, mechanical vs. biological, coronary artery bypass grafting vs. percutaneous coronary intervention, surgery vs. percutaneous treatment); the future of medicine is shifting to a clinical practice extremely oriented to a patientspecific, tailored approach, in which bench simulation will guide which device or which technique can better address that patient's specific anatomy and disease. + +The paper by Shearn et al. (4) perfectly represents this paradigm: from bedside to bench, and from bench to bedside again. + +ADDRESS FOR CORRESPONDENCE: Dr. Gianluca Torregrossa, Division of Minimally Invasive and Robotic Cardiac Surgery, Department of Surgery, University of Chicago Medicine, 5758 S. Maryland Avenue, Chicago, Illinois 60637. E-mail: gianluca. torregrossa@gmail.com. \ No newline at end of file diff --git a/medical/md/PMC8506526.md b/medical/md/PMC8506526.md new file mode 100644 index 0000000000000000000000000000000000000000..a35458813de0bd6f939207327e574ef31fbfa6af --- /dev/null +++ b/medical/md/PMC8506526.md @@ -0,0 +1,313 @@ +Original Article + +![0_image_0.png](0_image_0.png) + +# Acy1 Regulating Pten/Pi3K/Akt Signaling In The Promotion Of Non-Small Cell Lung Cancer Progression + +## Hong Chen1#, Wei Wang2#, Caizhi Xiao1, Dongqin Xia1, Fangfei Li1**, Shaoyong Liu1** + +1Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, Chongqing, China; 2Key Laboratory for Biorheological Science and Technology of Ministry of Education (Chongqing University), Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, Chongqing, China Contributions: (I) Conception and design: H Chen, W Wang; (II) Administrative support: H Chen, W Wang; (III) Provision of study materials or patients: C Xiao, D Xia; (IV) Collection and assembly of data: H Chen, W Wang, F Li, S Liu; (V) Data analysis and interpretation: H Chen, W Wang, C Xiao, D Xia; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. + +\#These authors contributed equally to this work. + +Correspondence to: Wei Wang. Key Laboratory for Biorheological Science and Technology of Ministry of Education (Chongqing University), +Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, 181 Hanyu Road, Chongqing 400044, China. Email: wangweixiayanai@163.com. + +Background: Non-small cell lung cancer (NSCLC) has a poor prognosis and is the most common cause of cancer-related deaths worldwide. Aminoacylase 1 (ACY1) plays a promoting role in some cancers, but its role in NSCLC is still unclear. + +Methods: Immunohistochemistry, Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting assays were used to determine ACY1 expression patterns in NSCLC tissues and cell lines. The clinical significance of ACY1 in NSCLC was evaluated by χ 2 test and Kaplan-Meier analysis. MTT, flow cytometry, wound healing, and Transwell assays were performed to assess cell growth, apoptosis, migration, invasion, and tumorigenesis under different treatments. Male athymic BALB/C nude mice were used for xenotransplantation experiments. + +Results: The results showed that ACY1 expression was elevated in NSCLC tissue samples and cells, and high ACY1 expression predicted an advanced clinical process and shorter overall survival in patients with NSCLC. Overexpression of ACY1 significantly increased cell growth, migration, invasion, and tumorigenesis, and reduced cell apoptosis, indicating that ACY1 functions as an oncogene in NSCLC. Moreover, ACY1 decreased phosphatase and tensin homolog (PTEN) expression, increased its ubiquitination, and activated PI3K/AKT signaling. Overexpression of PTEN diminished the effects of ACY1 upregulation on cell tumorigenesis promotion. + +Conclusions: This study reveals that ACY1 may promote the progression of NSCLC via activating PI3K/AKT signaling in a PTEN-dependent manner. Our study may provide a better understanding of the pathogenesis and development of NSCLC. + +Keywords: Aminoacylase 1 (ACY1); phosphatase and tensin homolog (PTEN); PI3K/AKT; non-small cell lung cancer (NSCLC) Submitted May 30, 2021. Accepted for publication Aug 16, 2021. doi: 10.21037/atm-21-3127 View this article at: https://dx.doi.org/10.21037/atm-21-3127 + +## Page 2 Of 13 Introduction + +Non-small cell lung cancer (NSCLC), including adenocarcinoma and squamous cell carcinoma, accounts for 80–85% of all lung cancer cases and is the most common cause of cancer-related deaths globally (1,2). NSCLC has been identified as a highly heterogeneous type of cancer, harboring multiple genetic aberrations within its various subtypes (3). Despite recent advances in clinical and experimental oncology, the prognosis of lung cancer remains poor. Approximately 35% of NSCLC cases are diagnosed at stage IV, resulting in an overall 5-year survival rate of only 15% (1,4). Therefore, further understanding of the mechanisms related to NSCLC +progression is needed to develop novel therapeutic strategies. + +Aminoacylase 1 (ACY1) is a homodimeric zinc-binding metalloenzyme which is widely expressed in mammalian tissues. It catalyzes the hydrolysis of Na-acylated amino acids to L-amino acids and an acyl group (5). Recent studies have reported the role of ACY1 in tumorigenesis in several types of human cancer. For example, Miller et al. (6) found that ACY1 expression was reduced in a significant number of small-cell lung cancer (SCLC) +cell lines. Additionally, Hwa and Balabanov *et al.* (7,8) +reported low ACY1 expression in renal clear cell carcinoma. + +Wei *et al.* (9) revealed that the expression of ACY1 was decreased in hepatocellular carcinoma (HCC) tissues and that it functioned as a tumor suppressor. However, ACY1 was reported to be overexpressed in colorectal cancer (CRC), and knockdown of ACY1 significantly inhibited proliferation and induced cell apoptosis (10,11). It is showed that in neuroblastoma, ACY1 has the effect of inhibiting proliferation and metastasis (12). Nevertheless, the roles of ACY1 in NSCLC remain unclear. + +The PI3K/AKT/mTOR pathway is a well-studied signal pathway which plays a key role in the modulation of many cellular functions, such as cell proliferation, apoptosis, metastasis, and invasion (13,14). The hyperactivation of PI3K/AKT/mTOR can be detected in most cancers including NSCLC and is associated with cancer progression (15). Phosphatase and tensin homolog (PTEN), a tumor suppressor, has been reported to induce the dephosphorylation of the phospholipid products of PI3K, thereby resulting in the repression of PI3K/AKT/ mTOR signaling (15,16). Studies have confirmed that PTEN expression is regulated by number of genes which exert oncogenic or suppressive roles in the development + +## Chen Et Al. Effects Of Acy1 On Nsclc + +of NSCLC, such as long noncoding RNA growth arrestspecific 5 (*GAS5*) (17), transforming growth factor beta (TGF-β) (18), and tumor protein 53 target gene 1 (*TP53TG1*) (19). + +Although the expression pattern of ACY1 has been found to be reduced in SCLC (6), to our knowledge, its function and clinical significance in NSCLC has not been clarified. In this study, we aimed to determine the expression profile of ACY1 in NSCLC, to evaluate the association between ACY1 expression and the clinical process and prognosis of patients with NSCLC, and to investigate whether PTEN/ +PI3K/AKT/mTOR signaling is involved *in vitro* and in vivo. It is hoped that the findings of this study can aid in the development of new methods for the diagnosis and treatment of NSCLC. + +We present the following article in accordance with the ARRIVE reporting checklist (available at https://dx.doi. org/10.21037/atm-21-3127). + +## Methods Tissue Samples + +Twenty paired NSCLC and the adjacent normal tissue samples were obtained from patients with NSCLC who underwent pneumonectomy and did not receive any form of chemoradiotherapy. All tissue samples were stored at +−80 ℃ immediately after resection. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Ethics Committee of Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital (Chongqing, China) and informed consent was taken from all the patients. + +## Immunohistochemistry + +First, the tissues were cut into 4-μm slides. After deparaffinization and treatment to remove the endogenous peroxidase activity, antigen retrieval, and blocking with goat serum, the sections were incubated with the primary antiACY1 antibody (no. AF2900, R&D Systems, Minneapolis, MA, USA) at a 1:100 dilution. The corresponding secondary antibody (Zhongshanqinqiao, Beijing, China) was then added and counterstained with Harris hematoxylin. Cell nuclei were dyed with 3,3'-diaminobenzidine (DAB; Serva, Heidelberg, Germany) at a 1:5,000 dilution. + +| Table 1 Primer sequence Genes Sequence ACY1 sense-5'-CAGCGACAGGAGAGTGAGC-3' ACY1 antisense-5'-CACATCCGTGTGGGAGTTGA-3 PI3K sense-5'-AAATGCTTGGGGTGGAAGGG-3' PI3K antisense-5'-ATGTATTCAGTTCAATTGCAGAAGG-3' AKT sense-5'-GAAGGACGGGAGCAGGC-3' AKT antisense-5'-TGTACTCCCCTCGTTTGTGC-3' mTOR sense-5'-CTTAGAGGACAGCGGGGAAG-3' mTOR antisense-5'-TCCAAGCATCTTGCCCTGAG-3' PTEN sense-5'-CAGCAGCTTCTGCCATCTCT-3' PTEN antisense-5'-TGCTTTGAATCCAAAAACCTTACT-3' GAPDH sense-5'-CCACTAGGCGCTCACTGTTCT-3' GAPDH antisense-5'-GCATCGCCCCACTTGATTTT-3' ACY1, aminoacylase 1; PI3K, phosphatidylinositol 3 kinase; AKT, | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +ACY1, aminoacylase 1; PI3K, phosphatidylinositol 3 kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog deleted on chromosome ten; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. + +## Cells Culture + +Human embryonic lung WI-38 cells, along with NSCLC cell lines HCC2935, A549, and H1975, were obtained from the American Type Culture Collection (ATCC, VA, USA). WI-38 cells were grown in Eagle's Minimum Essential Medium; A549 cells in F-12K medium; and HCC2935 and H1975 cells in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ +streptomycin. All reagents used for cell culture were obtained from Thermo Fisher Scientific (Danvers, MA, USA). + +## Cell Transfection + +The lentivirus vector shRNA-ACY1 used to silence ACY1 expression in NSCLC cells and its negative control (shNC) were purchased from OriGene (Beijing, China; no. + +TL315053). The overexpressing lentivirus vectors used to upregulate PTEN (OE-PTEN) and ACY1 (OE-ACY1) were obtained from GenePharma (Shanghai, China). Firstly, the gene fragments were amplified, and the amplified fragments were connected to the lentivirus and packaged. + +To establish stable sh-ACY1- or OE-ACY1-transfected cell lines, H1975 cells were transfected with sh-ACY1 or OE-ACY1and then cultured with 8 μg/mL puromycin for 2 weeks, with the medium being changed every 2 days. Meanwhile, 100 μg/mL of G418 was used to select cells with OE-PTEN transfection. + +## Real Time Quantitative Polymerase Chain Reaction Assay + +RNA was extracted from cells and tissues with an Oligotex mRNA Mini Lit (Qiagen, Valencia, CA, USA). The RNA was then reverse-transcribed into complement DNA (cDNA) with a PrimerScript RT reagent kit (TransGen Biotech, Beijing, China) in accordance with the instructions. Polymerase chain reaction (PCR) amplification was conducted using a SYBR Premix Ex TaqTM II kit +(TransGen Biotech, Beijing, China) in a ViiATM 7 realtime fluorescence quantitative PCR system. Expression of messenger RNA (mRNA) was normalized to that of GAPDH. The relative expressions of mRNA were analyzed with the 2-∆∆CT method. Primer sequences were showed int *Table 1*. + +## Western Blotting Assay + +Cells in different groups and tissues were lysed with RIPA +buffer (Thermo Scientific) at 4 ℃ for 30 minutes. Proteins were extracted and separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane by electroblotting. Nonfat milk was used to block the PVDF membrane at 4 ℃ overnight. + +The membrane was then incubated with the primary antibodies at 4 ℃ overnight. The antibodies were showed in *Table 2*. The membranes were then incubated at room temperature for 1.5 hours with the secondary antibody (1:2,000, Abcam). GAPDH was used as the internal reference. Target bands of proteins were visualized with an efficient chemiluminescence (ECL) western blotting kit (Millipore, Boston, MA, USA). Relative expression of proteins was assessed with the 2−∆∆CT method. + +## Immunoprecipitation Assay + +NSCLC cells were treated with sh-NC, sh-ACY1, OENC, or OE-ACY1 for 48 hours before being rinsed with cold phosphate-buffered saline (PBS) and lysed in immunoprecipitation (IP) lysis buffer (Thermo Fisher Scientific). The cell lysate containing 200 μg proteins + +## Page 4 Of 13 Chen Et Al. Effects Of Acy1 On Nsclc + +| Table 2 Antibodies Names | Dilution | Item No. | Company | +|------------------------------------------------------------------------------------------------------------------------------|------------|------------|---------------------------------------------| +| ACY1 | 1:500 | AF2900 | R&D Systems, MN, USA | +| PI3K | 1:500 | 4252 | Cell Signaling Technology, Beverly, CA, USA | +| p-PI3K | 1:500 | 4228 | Cell Signaling Technology, Beverly, CA, USA | +| AKT | 1:500 | 9272 | Cell Signaling Technology, Beverly, CA, USA | +| p-AKT | 1:500 | 13038 | Cell Signaling Technology, Beverly, CA, USA | +| mTOR | 1:500 | ab2732 | Abcam, Cambridge, MA, USA | +| PTEN | 1:500 | 9552 | Cell Signaling Technology, Beverly, CA, USA | +| Ub | 1:500 | 3933 | Cell Signaling Technology, Beverly, CA, USA | +| GAPDH | 1:500 | TA-08 | Zhongshanjinqiao, Beijing, China | +| ACY1, aminoacylase 1; PI3K, phosphatidylinositol 3 kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; PTEN, | | | | + +was incubated with Dynabeads protein G for 1 hour, and incubated overnight at 4 ℃ with 2 μg of antibody against PTEN, PI3K, AKT, and mTOR, which was followed by incubation with Dynabeads protein G for 1 hour. The immune complex was then loaded onto gels using Ub antibody. + +## Mtt Assay + +For the MTT assay, 3×103 cells (with 200 μL growth medium) were seeded into 96-well plates overnight. Subsequently, MTT (Merck, Germany) was added and incubated at 37 ℃ for 4 hours. After this, 150 μL of dimethyl sulfoxide (DMSO) was added for 10 minutes. The absorbance was measured at 570 nm. + +## Flow Cytometry Assay + +Cells in different groups were seeded into 6-well plates +(5×105). After 24 hours of treatment, cells were digested with 0.25% trypsin and resuspended in binding buffer. Following this, 5 μL of Annexin V-FITC (Annexin V-FITC Apoptosis Detection kit, BD Biosciences, San Jose, CA, USA) and 5 μL of propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) were added to each sample and incubated at 2–8 ℃ for 5 minutes in the dark. Fluorescence-activated apoptotic cell assays were conducted with a flow cytometer (BD FACSVantage SE, BD Biosciences Co., Franklin Lakes, NJ, USA). The sum of the data of Q2 and Q3 was the apoptosis rate (%). + +## Wound Healing Assay + +NSCLC cells were seeded into 6-well plates at a 2×105 cells/well density and transfected with sh-NC, sh-ACY1, and OE-NC or OE-ACY1. The scratch was made using a sterile 20-μL pipette tip, and the medium was replaced with a serum-free medium until the cells reached a confluent monolayer. Cells were then incubated at 37 ℃ for 24 hours. + +The cells were examined and photographed under phase contrast microscopy at 0 and 24 hours. The rate of cell mobility inhibition (%) was calculated as follows: cell mobility = scratch width (24 hours)/scratch width (0 hour) ×100%. + +## Transwell Chamber With Matrigel Assay + +Cell invasion was detected by transwell chambers (8 μm pore size; BD Biosciences, San Diego, CA, USA) +precoated with Matrigel (BD Biosciences). Next, 1.0×105 cells resuspended in 200 μL of serum-free medium were inoculated into the upper chamber of the transwell. Meanwhile, 600 μL of medium containing 15% FBS was added to the lower chamber. The cells were stained with 1% crystal violet for 20 minutes and then washed with PBS. The invaded cells were counted with a microscope after 48 hours. + +## Xenotransplantation Assay + +A total of 12 four-week-old male athymic BALB/C nude mice (Weitonglihua. Co., Ltd., Beijing, China) were used + +to assess cell tumorigenesis. The animals were kept in a professional laboratory for nude mice in an atmosphere of 24±1 ℃ with 60%±5% relative humidity. A random number table method was used to divide the mice into 4 groups (N=3): control, sh-ACY1, OE-ACY1, and OEACY1+OE-PTEN. In detail, 5×106 H1975 cells with stable control, sh-ACY1, OE-ACY1, or OE-ACY1+OE-PTEN transfection were resuspended in 200 μL PBS and then injected into the armpit area of the nude mice. Twentyeight days posttransplantation, the mice were sacrificed, and the tumors were collected for weighing. During the experiment, care was taken to minimize the pain of the mice without affecting the results of the experiment. No deaths occurred during the experiment. Euthanasia was administered when the diameter of the tumor was greater than 2 cm (not involved in this experiment). Protocols involving animals were approved by the ethical committee of Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, and animal experiment was conducted in compliance with Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital guidelines for the care and use of animals. + +## Statistical Analysis + +Each measurement was obtained in 3 parallel tests. All experimental data are presented as the mean ± SD. Statistical analyses were performed on GraphPad Prism 7 software with one-way analysis of variance followed by the Tukey's test for multiple groups and Student's t-test for 2 groups. χ 2 test and Kaplan-Meier analysis were used to evaluate the clinical significance of ACY1 in NSCLC. + +Pearson Correlation Coefficient was used to verify the correlation between ACY1 and PTEN in NSCLC tissues. A P value <0.05 was considered a statistically significant difference. + +## Results Acy1 Expression Was Increased In Nsclc Tissues And Cells + +We first identified the expression patterns of ACY1 and PTEN in NSCLC tissues and cells. Increased ACY1 expression was observed in NSCLC tissues when compared to the adjacent normal tissues in both protein and mRNA +levels, as determined by immunohistochemistry (*Figure 1A*), +RT-PCR (*Figure 1B*), and western blotting (*Figure 1C*). + +The expression of PTEN protein in NSCLC tissues was significantly lower than that of adjacent normal tissues +(*Figure 1C*). In NSCLC tissues, the trends of PTEN +and ACY1 were opposite. In addition, ACY1 and PTEN +expression was also detected in NSCLC cell lines by realtime PCR (RT-PCR) and western blotting assays. ACY1 expression levels were increased in human NSCLC cell lines HCC2935, A549, and H1975 compared to those in human embryonic lung WI-38 cells, while PTEN mRNA +and protein were decreased (*Figure 1D,1E*). These results suggest that ACY1 was deregulated in NSCLC. + +## High Acy1 Expression Predicted An Advanced Clinical Features And Shorter Overall Survival For Patients With Nsclc + +To investigate the role of ACY1 in NSCLC progression, we then assessed its clinical significance in NSCLC. A total of 91 patients with NSCLC were divided into 2 groups according to the expression levels of ACY1, among whom 60 had high ACY1 expression (high expression group) and 31 had low expression (low expression group). As shown in *Table 3,* there were no significant differences in gender, age, or histological type, but patients with ACY1 high expression had significantly lower differentiation (P=0.011), lymph node metastasis (P=0.002), and advanced tumor-node-metastasis (TNM) stage (P=0.006). Moreover, patients in the high expression group had shorter overall survival than those in the low expression group (*Figure 1F*). + +In addition, the expression of ACY1 and PTEN in NSCLC tissues were negatively correlated (*Figure 1G*). + +These results suggest that ACY1 may have the potential to predict the clinical process and outcome of NSCLC +patients. + +## Overexpression Of Acy1 Facilitated Nsclc Cell Conversion To A Malignant Phenotype + +We investigated the effects of ACY1 in the progression of NSCLC *in vitro* via gain/loss-of-function assays. ACY1 expression was decreased to 20–30% when HCC2935 and H1975 cells were transfected with sh-2, while it was increased to 350–380% in mRNA levels and 200–210% +in protein levels when cells were transfected with OEACY1 (*Figure 2A,2B*). Compared with controls, sh-ACY1 transfection significantly increased cell proliferation, and sh-ACY1 impaired cell proliferation in HCC2935 (*Figure 2C*) +and H1975 cells (*Figure 2D*). Cell apoptosis was decreased + +![5_image_0.png](5_image_0.png) + +when HCC2935 and H1975 cells were transfected with OEACY1, and it was increased when ACY1 was downregulated +(*Figure 2E*). In addition, ACY1 upregulation promoted cell migration and invasion, and knockdown of ACY1 caused the opposite to occur (*Figure 3A-3C*). We also detected the effects of ACY1 on epithelial-mesenchymal transition (EMT)-marked proteins. The results showed that when ACY1 was inhibited, the expression of E-cadherin protein increased, and the expression of N-cadherin and Vimentin protein decreased (*Figure 3D,3E*). Overexpression of ACY1 produced the opposite result. Together, these findings demonstrated that ACY1 functioned as an oncogene in NSCLC. + +## Overexpression Of Acy1 Promoted The Activation Of Pi3K/ Akt Signaling Through Downregulation Of Pten + +Subsequently, we investigated the effects of ACY1 expression on the activation of PI3K/AKT signaling. Compared with the control group, ACY1 upregulation significantly increased the protein expression of p-PI3K, p-AKT, and mTOR, whereas it reduced PTEN expression (*Figure 4A*) +and showed no obvious influence in the mRNA levels of PI3K, AKT, mTOR, or PTEN (*Figure 4B*). To further explore the mechanism of ACY1 in regulating PTEN/PI3K/ AKT signaling, we used an IP assay to assess the interaction between ACY1 and PTEN/PI3K/AKT signaling-related proteins. The results showed that ACY1 could only combine + +| Table 3 Association of ACY1 expression and the clinicopathologic features of patients with NSCLC ACY1 expression Variable P value High Low Gender 0.649 Male 37 21 Female 23 10 Age (y) 0.186 <60 38 15 ≥60 22 16 Histological type 0.512 Squamous cell carcinoma 32 14 Adenocarcinoma 28 17 Differentiation 0.011 Well 12 14 Moderate 22 12 Poor 26 5 Lymph node metastasis 0.002 No 28 25 Yes 32 6 TNM stage 0.006 I 10 11 II 17 14 III 33 6 NSCLC, non-small cell lung cancer; ACY1, aminoacylase 1; TNM, tumor-node-metastasis. | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +with PTEN, and this interaction was weakened when ACY1 was overexpressed in H1975 cells (*Figure 4C*). This might have been due to ACY1 inducing the degradation of PTEN protein. Moreover, ACY1 upregulation promoted the protein degradation of PTEN protein (*Figure 5A*), and increased the ubiquitination of PTEN protein (*Figure 5B*), +and vice versa. Overall, these results demonstrated that ACY1 upregulation could promote PI3K/AKT signaling activation via downregulation of PTEN. + +## Acy1 Enhanced Nsclc Cell Tumorigenesis Via Downregulating Pten + +We further examined the effects of ACY1/PTEN in the tumorigenesis of NSCLC cells. Tumorigenesis was significantly enhanced in H1975 cells during stable upregulation of ACY1, while it was decreased at the time of ACY1 downregulation. Overexpression of PTEN +counteracted the enhancement of H1975 cell tumorigenesis induced by ACY1 upregulation (*Figure 6A,6B*), illustrating that ACY1 promoted cell tumorigenesis via downregulating PTEN in NSCLC. + +## Discussion + +The American Joint Committee on Cancer (AJCC) (20,21) has reported that up to 70% of NSCLC patients are incurable due to the extent of cancer at the time of diagnosis. A standard treatment for NSCLC, platinumbased chemotherapy, also shows low response rates of 17–32% and overall survival of 7.4–11.3 months for NSCLC patients at advanced stages (22). Therefore, it is crucial to further investigate the underlying mechanisms of NSCLC. This study focused on understanding the role and mechanism of ACY1, revealed the high fold change in the protein and mRNA expression levels of ACY1 in NSCLC tissues and cells, and showed that ACY1 has an oncogenic role in NSCLC. To our knowledge, there is a lack of studies on the clinical significance of ACY1 in NSCLC. However, the present study demonstrated that NSCLC patients with ACY1 high expression were prone to show low differentiation (P=0.011), lymph node metastasis (P=0.002), and advanced TNM stage (P=0.006), as well as poor prognosis, which was consistent with previous studies concerning the role of ACY1 in CRC (23). + +ACY1 is commonly found in the kidneys and the brain. + +Its deregulation has been associated with autism and convulsions (24,25). ACY1 has also been reported to play a role in the deacetylation of aflatoxin epoxy derivatives and thereby influence cell detoxification function (26). Moreover, studies have found ACY1 to be strongly implicated in carcinogenesis, with different roles in different types of cancers. For instance, low expression of ACY1 was found in HCC tissues, and ACY1 expression was significantly associated with tumor invasiveness; furthermore, overexpression of ACY1 in HCC BEL7402 cells was shown to significantly inhibit cell viability and invasiveness (9), revealing that ACY1 may function as a tumor suppressor. Conversely, ACY1 was reported to be overexpressed in CRC, and knockdown of ACY1 significantly inhibited cell proliferation and induced cell apoptosis (10,11). In contrast to the ACY1 expression Page 8 of 13 + +![7_image_0.png](7_image_0.png) + +profiles in SCLC (6), we found that ACY1 expression was obviously increased in NSCLC tissues and cells in both protein and mRNA levels. To measure the effects of this increase of ACY1 in NSCLC progression, gain/loss-offunction assays were performed in 2 NSCLC cell lines, H1975 and HCC2935. The results showed that cell growth, migration, invasion, and tumorigenesis were enhanced, while cell apoptosis was decreased when ACY1 was upregulated, indicating that ACY1 has a tumor-promoting role in NSCLC. + +In addition, we investigated the mechanism of ACY1 in promoting the progression of NSCLC. We observed that ACY1 could activate PI3K/AKT signaling through downregulating PTEN. Studies have demonstrated that + +Annals of Translational Medicine, Vol 9, No 17 September 2021 **Page 9 of 13** + +![8_image_0.png](8_image_0.png) + +![9_image_0.png](9_image_0.png) + +PTEN protein expression is significantly reduced in poorly differentiated NSCLC tumors compared to moderately differentiated NSCLC tumors. This is one of the main contributors to PI3K/AKT pathway activation and resistance to tyrosine kinase inhibitors (TKIs), including the most common chemotherapy drugs for NSCLC, such as gefitinib, erlotinib, and afatinib (27,28). Furthermore, NSCLC patients with PTEN low expression level always have lower survival rates than those with PTEN high expression (29). This study, along with previous studies, illustrates the crucial role of PTEN in NSCLC. To further investigate the deregulation of PTEN and clarify the mechanism of ACY1 in NSCLC, we carried out western blotting and IP assays and found that ACY1 could interact with PTEN protein but not mTOR, AKT, or PI3K, and could decrease its expression via accelerating its ubiquitination and degradation. Moreover, we observed that PTEN upregulation weakened the effect of ACY1 on tumorigenesis promotion in NSCLC H1975 cells, suggesting that ACY1 facilitated the progression of NSCLC +through PTEN deletion. Although previous studies have reported that activation of ERK1/2 and expression of TGF-β are implicated in ACY1-induced tumor cell proliferation (9,30), the present study revealed, for the first time, that the PTEN/PI3K/AKT pathway may also be the target signaling of ACY1 in carcinogenesis. Based on this, we speculated that in NSCLC, the upregulation of ACY1 protein might activate the PI3K/AKT/mTOR pathway by inducing degradation of PTEN protein, thereby promoting cell proliferation and metastasis capabilities, and inhibiting apoptosis. However, the molecular mechanism underlying the manner in which ACY1 induces PTEN ubiquitination and degradation still needs to be explored by further indepth analysis. + +In conclusion, this study suggests that ACY1 may function as an oncogene in NSCLC via regulating the PTEN/PI3K/AKT signaling pathway. It also demonstrates the clinical significance of ACY1 in NSCL, specifically, how ACY1 expression may be closely associated with an advanced clinical process and shorter overall survival for + +![10_image_0.png](10_image_0.png) + +![10_image_1.png](10_image_1.png) + +## Page 12 Of 13 + +patients with NSCLC. Our study aims to provide a better understanding of the pathogenesis and development of NSCLC. However, further exploration into the mechanism of ACY1 influence in the occurrence and progression of lung cancer is needed. + +## Acknowledgments + +Funding: The study was funded in part by Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital (No. ZY201701001). + +## Footnote + +Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://dx.doi. org/10.21037/atm-21-3127 Data Sharing Statement: Available at https://dx.doi. + +org/10.21037/atm-21-3127 Conflicts of Interest: All authors have completed the ICMJE +uniform disclosure form (available at https://dx.doi. org/10.21037/atm-21-3127). The authors have no conflicts of interest to declare. + +Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Ethics Committee of Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital (Chongqing, China) and informed consent was taken from all the patients. Protocols involving animals were approved by the ethical committee of Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, and animal experiment was conducted in compliance with Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital guidelines for the care and use of animals. + +Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non- + +## Chen Et Al. Effects Of Acy1 On Nsclc + +commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). + +See: https://creativecommons.org/licenses/by-nc-nd/4.0/. + +## References + +1. Dempke WC. Targeted Therapy for NSCLC--A Doubleedged Sword? Anticancer Res 2015;35:2503-12. + +2. Ettinger DS, Wood DE, Akerley W, et al. Non-Small Cell Lung Cancer, Version 6.2015. J Natl Compr Canc Netw 2015;13:515-24. + +3. Jiao Z, Yu A, He X, et al. Bioinformatics analysis to determine the prognostic value and prospective pathway signaling of miR-126 in non-small cell lung cancer. Ann Transl Med 2020;8:1639. + +4. Wu KL, Tsai MJ, Yang CJ, et al. Liver metastasis predicts poorer prognosis in stage IV lung adenocarcinoma patients receiving first-line gefitinib. Lung Cancer 2015;88:187-94. + +5. Heese D, Löffler HG, Röhm KH. Further characterization of porcine kidney aminoacylase I reveals close similarity to 'renal dipeptidase'. Biol Chem Hoppe Seyler 1988;369:559-66. + +6. Miller YE, Minna JD, Gazdar AF. Lack of expression of aminoacylase-1 in small cell lung cancer. Evidence for inactivation of genes encoded by chromosome 3p. J Clin Invest 1989;83:2120-4. + +7. Balabanov S, Zimmermann U, Protzel C, et al. Tumourrelated enzyme alterations in the clear cell type of human renal cell carcinoma identified by two-dimensional gel electrophoresis. Eur J Biochem 2001;268:5977-80. + +8. Hwa JS, Park HJ, Jung JH, et al. Identification of proteins differentially expressed in the conventional renal cell carcinoma by proteomic analysis. J Korean Med Sci 2005;20:450-5. + +9. Wei X, Li J, Xie H, et al. Proteomics-based identification of the tumor suppressor role of aminoacylase 1 in hepatocellular carcinoma. Cancer Lett 2014;351:117-25. + +10. Shi H, Hayes MT, Kirana C, et al. Overexpression of aminoacylase 1 is associated with colorectal cancer progression. Hum Pathol 2013;44:1089-97. + +11. Shi H, Hood KA, Hayes MT, et al. Proteomic analysis of advanced colorectal cancer by laser capture microdissection and two-dimensional difference gel electrophoresis. J Proteomics 2011;75:339-51. + +12. Chen Z, Gao J, Sun J, et al. Aminoacylase 1 (ACY1) Mediates the Proliferation and Migration of + +Neuroblastoma Cells in Humans Through the ERK/ +Transforming Growth Factor β (TGF-β) Signaling Pathways. Med Sci Monit 2021;27:e928813. + +13. Ersahin T, Tuncbag N, Cetin-Atalay R. The PI3K/AKT/ +mTOR interactive pathway. Mol Biosyst 2015;11:1946-54. + +14. Fan X, Huang H, Ji Z, et al. Long non-coding RNA +MEG3 functions as a competing endogenous RNA of miR-93 to regulate bladder cancer progression via PI3K/ AKT/mTOR pathway. Transl Cancer Res 2020;9:1678-88. + +15. Pérez-Ramírez C, Cañadas-Garre M, Molina MÁ, et al. + +PTEN and PI3K/AKT in non-small-cell lung cancer. Pharmacogenomics 2015;16:1843-62. + +16. Xia M, Tong JH, Ji NN, et al. Tramadol regulates proliferation, migration and invasion via PTEN/PI3K/ AKT signaling in lung adenocarcinoma cells. Eur Rev Med Pharmacol Sci 2016;20:2573-80. + +17. Cao L, Chen J, Ou B, et al. GAS5 knockdown reduces the chemo-sensitivity of non-small cell lung cancer (NSCLC) cell to cisplatin (DDP) through regulating miR-21/PTEN axis. Biomed Pharmacother 2017;93:570-9. + +18. Shen H, Guan D, Shen J, et al. TGF-β1 induces erlotinib resistance in non-small cell lung cancer by downregulating PTEN. Biomed Pharmacother 2016;77:1-6. + +19. Xiao H, Liu Y, Liang P, et al. TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis. Cell Biosci 2018;8:23. + +20. Yu Y, Zhang P, Yao R, et al. Prognostic value of log odds of positive lymph nodes in node-positive lung squamous cell carcinoma patients after surgery: a SEER populationbased study. Transl Lung Cancer Res 2020;9:1285-301. + +21. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 2010;17:1471-4. + +22. Kelly K, Crowley J, Bunn PA Jr, et al. Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine Cite this article as: Chen H, Wang W, Xiao C, Xia D, Li F, Liu S. ACY1 regulating PTEN/PI3K/AKT signaling in the promotion of non-small cell lung cancer progression. Ann Transl Med 2021;9(17):1378. doi: 10.21037/atm-21-3127 plus cisplatin in the treatment of patients with advanced non--small-cell lung cancer: a Southwest Oncology Group trial. J Clin Oncol 2001;19:3210-8. + +23. Yu B, Liu X, Cao X, et al. Study of the expression and function of ACY1 in patients with colorectal cancer. Oncol Lett 2017;13:2459-64. + +24. Smith T, Ghandour MS, Wood PL. Detection of N-acetyl methionine in human and murine brain and neuronal and glial derived cell lines. J Neurochem 2011;118:187-94. + +25. Tylki-Szymanska A, Gradowska W, Sommer A, et al. Aminoacylase 1 deficiency associated with autistic behavior. J Inherit Metab Dis 2010;33 Suppl 3:S211-4. + +26. Stocker P, Brunel JM, de Rezende L, et al. Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxinderived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides. Biochimie 2012;94:1668-75. + +27. Jin G, Kim MJ, Jeon HS, et al. PTEN mutations and relationship to EGFR, ERBB2, KRAS, and TP53 mutations in non-small cell lung cancers. Lung Cancer 2010;69:279-83. + +28. Spoerke JM, O'Brien C, Huw L, et al. Phosphoinositide 3-kinase (PI3K) pathway alterations are associated with histologic subtypes and are predictive of sensitivity to PI3K inhibitors in lung cancer preclinical models. Clin Cancer Res 2012;18:6771-83. + +29. Endoh H, Yatabe Y, Kosaka T, et al. PTEN and PIK3CA +expression is associated with prolonged survival after gefitinib treatment in EGFR-mutated lung cancer patients. J Thorac Oncol 2006;1:629-34. + +30. Yijing L, Liu H, Yuan C, et al. The effects of qindancapsule-containing serum on the TGF-β1/ERK +signaling pathway, matrix metalloproteinase synthesis and cell function in adventitial fibroblasts. Pharm Biol 2013;51:712-21. + +(English Language Editors: G. Stone and J. Gray) \ No newline at end of file diff --git a/medical/md/PMC8690856.md b/medical/md/PMC8690856.md new file mode 100644 index 0000000000000000000000000000000000000000..2b28017f65001bc65e898aa884ea3b56635cac68 --- /dev/null +++ b/medical/md/PMC8690856.md @@ -0,0 +1,237 @@ +# Color Change After Tooth Bleaching With Ozone And 10% Ozonized Carbamide Peroxide For In-Office Use + +Thalyta dos Reis Furlani Zouain Ferreira Neves Dias, Francisco Ubiratan Ferreira de Campos, Cecilia Pedroso Turssi, Flávia Lucisano Botelho do Amaral, Fabiana Mantovani Gomes França, Roberta Tarkany Basting* Faculdade São Leopoldo Mandic, Campinas, São Paulo, Brazil +*Correspondence to: Roberta Tarkany Basting, DDS, PhD, rbasting@yahoo.com. + +orcid: 0000-0002-0888-7342 (Thalyta dos Reis Furlani Zouain Ferreira Neves Dias); 0000-0003-0990-2425 (Francisco Ubiratan Ferreira de Campos); 0000-0002-0078-9895 (Cecilia Pedroso Turssi); 0000-0002-8934-6678 (Flávia Lucisano Botelho do Amaral); 0000-0002-2877-6797 (Fabiana Mantovani Gomes França); 0000-0002-5345-5776 (Roberta Tarkany Basting) + +## Abstract + +There is a constant search for bleaching treatments that can offer greater safety with fewer adverse effects, especially in the techniques performed in the office, which usually employ hydrogen peroxide in high concentrations (35% to 40%) that are not recommended by some international control agencies. This *in vitro* study evaluated the color change after tooth bleaching with the use of ozone and a 10% ozonized carbamide peroxide bleaching treatment for in-office use. Thirty molars were allocated (n = 10): three applications of ozone (1 hour every 3 days); three applications of 10% ozonized carbamide peroxide (1 hour every 3 days); 10% carbamide peroxide agent (8 hours a day for 7 days). The teeth were mounted on a plaster model to simulate the dental arch, and trays made of silicone were used for the application of the bleaching agents and to allow ozone to enter through. The ozone concentration used was 60 μg/mL, with an oxygen flow of 0.25 L/min. The values of color change showed no significant differences among treatments. The variations in the parameters over time, as well as the values of ΔEab, ΔE00, and WID , showed that there was no significant difference among the three treatments. The use of ozone and 10% ozon-ized carbamide peroxide for in-office use was effective for tooth bleaching with clinically perceptible and acceptable color alterations. The study was approved on September 10, 2019 by the São Leopoldo Mandic Ethics Research Committee (CAAE No. 17711719.4.0000.5374). Key words: carbamide peroxide; color; color change; gas; hydrogen peroxide; ozone; ozone therapy; oxygen; tooth bleaching; whitening doi: 10.4103/2045-9912.330693 How to cite this article: Dias TdRFZFN, de Campos FUF, Turssi CP, do Amaral FLB, França FMG, Basting RT. Color change after tooth bleaching with ozone and 10% ozonized carbamide peroxide for in-office use. Med Gas Res. 2022;12(3):100-106. + +## I**Ntroduction** + +Tooth bleaching techniques use agents with different concentrations of hydrogen peroxide or carbamide for in-office or at-home treatments.1 Higher concentrations of hydrogen peroxide (between 35% and 40%) are used for in-office techniques, whereas lower concentrations (up to 10% hydrogen peroxide and between 10% and 20% carbamide peroxide, which releases between 3% and 6% hydrogen peroxide), are used for techniques performed at-home by the use of trays.1 According to the American Dental Association,2 products with hydrogen peroxide concentrations up to 3.5% are considered safe, whereas the Committee for Scientific Consumer Products3 of the European Union approves products with up to 6.0% hydrogen peroxide. Thus, there is a need to develop safe bleaching treatments that can effectively deliver desired dental chromatic changes, especially for techniques that use concentrations higher than those recommended by control agencies. + +In this respect, at-home bleaching technique using 10% +carbamide peroxide can be considered a safe and effective procedure.1,4,5 However, some patients do not adapt well to the use of trays, or they may still require an in-office procedure.4 In case of the recommendation about using agents with low peroxide concentration, it may be useful to increase their oxidative capacity for possible use in-office for achieving effective results within a short duration after application. + +Ozone is an agent that has been studied as a powerful oxidizer alone or in association with high concentrations of hydrogen peroxide in.6-10 It has a short half-life and is unstable and dependent on system conditions such as temperature and pressure; therefore, it easily decomposes into pure oxygen.11 In addition to its use as a medical antimicrobial and therapeutic agent,11 its use in in-office bleaching techniques with agents with high concentrations of hydrogen peroxide, at 37.5%7 and 38%,8-10 has shown significant bleaching effects; additionally, it does not affect the mineral content of the enamel.12 Furthermore, ozone bleaching has been shown to result in reduced dental sensitivity compared with the conventional in-office technique.10 By using ozone with low concentrations of bleaching agents, Manton et al.13 found that applying ozone for 40 seconds did not potentiate the bleaching effect of an 8% carbamide peroxide aqueous solution, possibly because it was not used in gel form; the thickener leads to prolonged contact with the tooth surface, slowing the release of free radicals and reducing the duration of application.14,15 The concentration of hydrogen peroxide released by 10% +carbamide peroxide is 10 times less than that of hydrogen peroxide used in in-office techniques.4 Thus, because of the possible benefit of reduced sensitivity9,10 and the potential for oxidation, the use of ozone with a bleaching gel containing 10% carbamide peroxide for in-office techniques should also be evaluated. + +Therefore, the objective of this study was to assess *in vitro* the effects of color change after ozone and a 10% ozonized carbamide peroxide bleaching treatments for in-office use. The null hypothesis for this study was that there are no differences between the changes in the color of teeth subjected to bleaching treatments using ozone and ozonized carbamide peroxide for in-office use compared to 10% carbamide peroxide for at-home use. + +## Materials And M**Ethods** Ethical Aspects And Tooth Selection + +Thirty healthy human third molars were selected from a dental research biorepository after the project was approved on September 10, 2019 by the São Leopoldo Mandic Ethics Research Committee (CAAE No. 17711719.4.0000.5374) and stored in 0.1% thymol solution for a maximum of 6 months.12 Written informed consent (**Additional file 1**) was obtained from the patients to provide the teeth for the experiment. + +## Bleaching Protocols + +Thirty molars were allocated by randomization into three groups (n = 10): CP group: bleaching with 10% carbamide peroxide agent (Opalescence PF 10%/Ultradent); O group: bleaching with three applications of ozone; OCP group: bleaching with three applications of 10% ozonized carbamide peroxide. The groups and bleaching protocols are presented in **Table 1**. The ozone concentration followed the protocol of Santana et al.7 and Tessier et al.16 adapted for use herein. The pH values of the 10% carbamide peroxide and the 10% ozonized carbamide peroxide bleaching agents were measured at different application times (15, 30, 45, and 60 minutes) using a pH meter (MS Tecnopon Equipamentos Especiais Ltda., Piracicaba, SP, Brazil). + +Table 1: Groups, bleaching protocols, materials, and + +manufacturers + +| manufacturers Bleaching | Bleaching | Commercial name and | | +|---------------------------|------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------| +| Groups | treatment | protocol | manufacturer | +| CP | 10% carbamide peroxide | 8 h/d for 7 d | Opalescence PF 10% - Ultradent (Indaiatuba, SP, Brazil) | +| O | Ozone | 1 h every 3 d, for a total of three applications Medplus MX Philozon® (Balneário Camboriú, SC, Brazil) | | +| OCP | 10% ozonized carbamide peroxide | 1 h every 3 d, for a total of three applications Opalescence PF 10% - Ultradent (Indaiatuba, SP, Brazil) and Medplus MX Philozon® (Balneário Camboriú, SC, Brazil) | | + +## Tooth Preparation For Initial Color Evaluation And Plaster Model Assembly + +The allocation of the teeth to the respective groups was performed based on the initial color evaluation by the Vita Classical criterion using a spectrophotometer (VITA EasyShade Advance 4.0, Vita Zahnfabrik, BadSackingen, Germany) in order to distribute the tooth colors equally among the groups, + ¦ 101 and to avoid the risk of bias. For this, the teeth remained im-mersed in an artificial saliva solution (7 mL for each tooth) (1.5 mM Ca; 50 mM KCl; 0.9 mM PO4 +; 20 mM Tris buffer; pH = +7.0), as recommended by Featherstone et al.17 and modified by Serra and Cury,18 for 7 days at 37°C in a bacteriological oven. They were removed from the solution, dried with gauze, and evaluated using a closed white box to prevent the effects of external luminosity and standardization of light during the readings. The color measurements were obtained in duplicate to ensure accuracy.19 The region for the assessment of color was the middle third of the buccal surface of the teeth. Based on the initial evaluations according to Vita Classical (the selected teeth had colors B3, A3.5, and A4), they were equally distributed among the three groups. Therefore, all the groups presented an equal amount of teeth with shades B3, A 3.5 and A4. At the same time, the color data for L*, a*, and b* were recorded by the CIELa*b* system for each tooth. + +After the initial color analysis, the teeth were mounted on a plaster model to simulate the dental arch for the bleaching techniques. The lower arch of a dental mannequin (Prodens - Pronew, São Gonçalo, RJ, Brazil) without the artificial teeth was duplicated with special silicone (RTV-2 silicone rubber, HB FLEX 5528 - HBQUIMICA Representações, Comércio e Serviços Ltda., Custóias, Portugal). After duplication, the natural teeth were positioned in the empty alveoli. To correctly fit the teeth, the regularization of these alveoli was performed with a carbide drill 060 (Horico, Berlin, Germany). A new duplication with special silicone and casting with a special micro-granulated IV plaster (Durone, Dentsply Sirona, São Paulo, SP, Brazil) was carried out with a vacuum spatula to obtain the models. For this, each group of teeth (respective to each bleaching treatment to be evaluated) was mounted on each cast model, using 7 wax for fixing the entire root of each tooth after sealing along the dental arch of the model. This step was carried out to position and fit the teeth in the models to complete the subsequent steps. + +## Manufacturing The Trays + +Three trays made of silicone (Crystal PVC, Bioart, São Carlos, SP, Brazil) (thickness = 3 mm) were used for the application of the bleaching agents (one tray was used for each bleaching treatment). The trays were obtained after molding the plaster models in which the teeth were fixed with the use of alginate and a tray. The molds were cast with a special type IV dental plaster. For the group that would receive the 10% carbamide peroxide treatment (PC group), the tray was cut 2 mm above the gingival sulcus. For the experimental groups that were treated with ozone (O group) or 10% ozonized carbamide peroxide gel (PCO group), wearing was carried out with a number 6 spherical bur coupled to the micromotor in the end region of the vestibule, both on the outer face and the face inside the respective plaster models. A dense condensation silicone (Speedex - Coltene, Rio de Janeiro, RJ, Brazil) paste was used, and it was manipulated for the application using a thickness of 0.5 mm above the occlusal surface of the cast to make room for ozone circulation inside the tray. After the aforementioned processes, the trays were created in the vacuum plasticizer (P7, Bio-art, São Carlos, SP, Brazil) by cutting each tray at the location where wearing was previously carried out in the backend region of the vestibule. Later, four holes were drilled in the vestibular side and one hole was drilled in the lingual surface of the trays. These holes were made for installing the plastic cannulas (Macrobots Descarpack Luer Slip, San Paulo, SP, Brazil) to allow ozone to enter through the vestibular side of the trays and exit from the lingual face of the respective trays. + +## Bleaching Treatment With 10% Carbamide Peroxide - Cp Group + +A disposable syringe (Biotecmed, São Paulo, SP, Brazil) was used to apply 0.02 mL of the bleaching gel on the vestibular surface of the enamel using trays with 10% carbamide peroxide. The bleaching agent was kept on the enamel surface for 8 hours a day in an artificial saliva solution and a bacteriological oven at 37°C, according to the manufacturer's recommendations. Subsequently, the gel was removed from the enamel surface with deionized water for 10 seconds, and the tooth was dried with gauze. Next, the model was immersed in an artificial saliva solution and kept in a bacteriological oven at 37°C. This cycle was repeated for 7 days. After the end of the applications, the enamel surface was washed with distilled water and stored again in the artificial saliva solution for 12 hours in a bacteriological greenhouse at 37°C until the final color assessments are made. + +## Ozone Gas Treatment - O Group + +An ozone gas generator (Medplus MX Philozon, Camboriú, SC, Brazil) was connected to an oxygen cylinder by a flowmeter standard20 to the experiment. The dose adjustment was applied according to the ozone concentration and the acting time. The ozone concentration used was 60 μg/mL, with an oxygen flow of 0.25 L/min, as per the manufacturer's specifications. The application time was 1 hour every 3 days for a total of three applications. For the ozone application, one of the ends of the cannulas was adapted to the four holes in the vestibular region of the tray, whereas the other end of the cannula was adapted to the ozone generator equipment. For the ozone output of the tray, one of the ends of the cannulas was adapted to the tray in the hole in the lingual region, whereas the other end was adapted to the ozone generator vacuum suction system (**Figure 1**). Between the bleaching sessions, the specimens were kept in a humid environment in a bacteriological oven at 37°C. + +![2_image_0.png](2_image_0.png) + +102 ¦ + +![2_image_1.png](2_image_1.png) + +10% ozonized carbamide peroxide treatment - OCP group A disposable syringe (Biotecmed, São Paulo, SP, Brazil) was used to apply 0.02 mL of the 10% carbamide peroxide bleaching agent over the surface of the vestibular enamel of the teeth, and the tray was positioned with the previously prepared cannulas. The tray with plastic cannulas was connected to the ozone generator, as previously presented. The 10% carbamide peroxide, together with the ozone to be applied, remained on the specimens for 1 hour. This application was repeated every 3 days three times. Between the bleaching sessions, the specimens were kept in a humid environment in a bacteriological oven at 37°C. + +## Final Color Analysis + +The final color evaluations were performed 24 hours after completion of treatments using the Vita Classical scale and CIEL*a*b* system, as described for the initial color evaluation. + +The tooth color obtained using the Vital Classical scale was converted to numerical values (1 to 16) as previously reported.4,19 Lower numerical values represented brighter teeth. The color change (ΔEab) obtained by the CIELab system was calculated using the following formula21: ΔEab* = [(ΔL*)2 + (Δa*)2 ++ (Δb*)2 ]1/2, where ΔL = Lfinal - Linitial; Δa = afinal - ainitial; Δb = +bfinal - binitial. The color change was also assessed by CIEDE2000 +(ΔE00), which is based on hue and chroma values,22 and the Whiteness Index for Dentistry (WID), in which the parameters L*, a*, and b* are used in transmittance analysis according to the following equation: WID = 0.511L* - 2.324a* - 1.100b*.23 + +## Statistical Analysis + +Since the data did not meet the assumptions of the one-way analysis of variance, they were analyzed by generalized linear models, considering the design of repeated measures for L*, a*, b*, and the color values using the Vita Classical scale. + +The data from WID were not adjusted to a known distribu-tion. The non-parametric Kruskal-Wallis tests were used to compare the groups, and the Wilcoxon test was used for time comparison (WID). The data for ΔL, Δa, and Δb were not adjusted to a known distribution, and they were analyzed using the non-parametric Kruskal-Wallis test. Generalized linear models were also used, considering the treatment factor, to analyze ΔEab, ΔE00, and ΔWID. R (R Core Development Team 2020, Vienna, Austria) and SAS (SAS Institute Inc., Cary, NC, USA) were used for the analyses, assuming the level of significance of 5%. + +The values for ΔEab above 2.7 were considered clinically acceptable.24,25 The values for ΔE00 of 0.8 and 1.8 were adopted as thresholds for the perceptibility and acceptability criteria.24 The thresholds for ΔWID were 0.72 for perceptibility and 2.60 for acceptability.26 + +## R**Esults** Ph Values + +The pHs of the bleaching agents evaluated at different times +(**Table 2**) were similar and close to neutrality within 60 minutes. There was a slight decrease in pH values for the 10% +ozonized carbamide peroxide throughout the period evaluation (from 7.43 to 7.21) while the 10% carbamide peroxide remained at 7.43 during this time. + +## Ciel*A*B*, Vita Classical And Wi**D Results** + +For the color variable (**Table 3**), there was no significant association between the groups and the times (P > 0.05); there were also no significant differences among the three groups (P > 0.05). For all treatments, there was a significant increase in the parameters L* (teeth became lighter) and WID (teeth became whiter) and a significant decrease in a* (teeth became less red), b* (teeth became less yellow), and in the Vita Classical scale (an average change of 10 shade colors). + +| Table 2: pH values according to treatments and evaluation times Bleaching treatment Initial 15 min 30 min 45 min | 60 min | | | | | +|----------------------------------------------------------------------------------------------------------------------|----------|------|------|------|------| +| 10% carbamide peroxide (CP group) | 7.43 | 7.43 | 7.43 | 7.43 | 7.43 | +| 10% ozonized carbamide peroxide (OCP group) 7.43 | 7.38 | 7.38 | 7.29 | 7.21 | | +| Note: The analysis of pH was performed in triplicate (n = 3) at 25°C. | | | | | | + +Color change (ΔL*, Δa*, Δb*, ΔEab, ΔE00 and ΔWI**D) results** +The variation of parameters L*, a*, and b* over time (Table 4), as well as the values of ΔEab, ΔE00 and ΔWID show that there was no significant difference between the groups (P > 0.05). + +## D**Iscussion** + +The data from this study shows that all the evaluated treatments delivered effective tooth bleaching, thus accepting the null hypothesis. Carbamide peroxide (10%) in tray is recognized for its bleaching effectiveness due to its potential for color change4,5 and its safety.4 The results showed that it had significant effects on the brightness parameters (L*), the reduction of reddish (a*) and yellowish (b*) hues, an average change of 10 shade colors based on the Vita Classical scale, and a clinically perceptible color change by CIEL*a*b*, CIEDE2000, and WID, even with use for 7 days, which was less than what is usually employed clinically (about 14 days of use1, depending on the initial color of the teeth (longer protocol for darker teeth, and shorter for lighter teeth). The values of ΔEab were > +2.7,24,25 and they were considered clinically perceptible. This was also observed for the values of ΔE00 and WID, which were above the limits of perceptibility and acceptability,23,24 denoting the teeth bleaching effectiveness during 7 days of treatment. Some authors reported that the isolated use of ozone did not promote effective bleaching6,13,27; however, this study showed that ozone promoted tooth bleaching with the same effective- + +| Table 3: Color parameters, Vita Classical scale and Whiteness Index for Dentistry depending on the treatments or groups and the evaluation times Variable Before treatment 24 h after application P-value L* P(group)=0.4113; P(time)=0.0075; P(group × time)=0.7529 10% carbamide peroxide 79.15±4.37 84.77±6.46* Ozone 78.04±4.86 81.15±6.67* 10% ozonized carbamide peroxide 77.67±6.92 81.82±6.15* a* P(group)=0.4892; P(time)=0.0184; P(group × time)=0.3499 10% carbamide peroxide 1.52±1.38 –0.69±0.75* Ozone 1.91±1.30 0.20±0.49* 10% ozonized carbamide peroxide 1.33±1.33 –0.24±0.83* b* P(group)=0.5875; P(time) < 0,0001; P(group × time)=0.5732 10% carbamide peroxide 31.52±4.71 12.34±4.93* Ozone 31.55±4.84 11.02±4.05* 10% ozonized carbamide peroxide 31.47±3.56 13.10±4.63* Vita Classical scale P(group)=0.5290; P(time) < 0.0001; P(group × time)=0.2749 10% carbamide peroxide 11.90±1.20 1.80±0.63* Ozone 12.50±1.78 1.60±0.52* 10% ozonized carbamide peroxide 12.10±1.60 2.10±1.10* Whiteness index for dentistry P(time)=0.0051 10% carbamide peroxide 3.13 (–10.71–12.79) 29.00 (24.61–42.61)* Ozone 0.44 (–9.02 to 13.52) 27.43 (23.23–41.89)* 10% ozonized carbamide peroxide 3.09 (–7.34 to 11.06) 27.82 (20.21–36.19)* Note: Data in color parameters and Vita Classical scale are expressed as mean ± SD, and data in whiteness index for dentistry are expressed mean (minimum-maximum values) (n =10). Generalized linear models applied for L*, a*, b* and Vita Classical Scale analysis, and Kruskal-Wallis and Wilcoxon tests for the Whiteness index for dentistry analysis. *P < 0.05, vs. before treatment. | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +www.medgasres.com + +| Table 4: Variation in color based on the parameters L*, a*, and b* by CIELab, CIEDE 2000, and WID systems according to different treatments or groups Variable Value P-value ΔL P=0.7472 10% carbamide peroxide 5.62±5.62 Ozone 3.11±9.51 10% ozonized carbamide peroxide 4.15±8.42 Δa P=0.6099 10% carbamide peroxide –2.21±1.39 Ozone –1.71±1.58 10% ozonized carbamide peroxide –1.57±1.25 Δb P=0.5226 10% carbamide peroxide –19.18±5.53 Ozone –20.53±6.81 10% ozonized carbamide peroxide –18.37±5.03 ΔEab P=0.7168 10% carbamide peroxide 20.93±5.15 Ozone 22.66±7.16 10% ozonized carbamide peroxide 20.68±4.43 ΔE00 P=0.6439 10% carbamide peroxide 11.00±2.76 Ozone 12.35±4.17 10% ozonized carbamide peroxide 11.39±2.60 ΔWID P=0.6960 10% carbamide peroxide 29.10±7.42 Ozone 28.15±11.13 10% ozonized carbamide peroxide 25.98±5.60 Note: Data are expressed as mean ± SD (n = 10). Kruskal-Wallis tests applied for ΔL, Δa, and Δb analyses, and ggeneralized linear models applied for ΔEab, ΔE00, and ΔWID analyses. | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +ness as 10% carbamide peroxide bleaching treatment in tray. + +The differences in results reported by different studies can be justified by factors that influence the oxidative effect of ozone. Factors such as reduced ozone application time - from only 40 seconds,13 4 minutes in the group where only ozone was applied, and 80 seconds repeated three times in the group where ozone and a bleaching gel were used6 - can justify such results. Another factor that may contribute to the ineffectiveness of ozone in bleaching with the use of trays is the design of the trays used. Adjusting the trays to the dental structures (as a "well-fitted" tray) restricts the free flow of ozone, preventing the direct contact of ozone with the vestibular surface of the anterior teeth.27 Thus, there is the need to ensure internal relief for effective ozone circulation inside the trays, as performed in this study. + +In addition to these factors, the ozone concentration of 140 ppm used in the Ozotop unit in the study by Zanjani et al.6 can be cited; the lack of suction capacity limited the use of a higher concentration of ozone. It was also found that the isolated application of ozone was effective in bleaching tetracyclinestained mouse teeth by using it for 3 and 5 minutes.16 Ozone is produced naturally from oxygen via molecular photodissociation in activated oxygen atoms, which react with other oxygen molecules. This radical transitional anion quickly becomes protonated and generates hydrogen trioxide, which, in turn, decomposes into an even more powerful oxidizer, the hydroxyl radical, giving ozone gas a higher oxidizing potential.28 This effect may justify the minimal reduction in the pH of ozonized carbamide peroxide after 60 minutes (from 7.43 to 7.21). In addition, chromophore groups present in the tooth structure can be broken down by ozone to form smaller molecules, resulting in tooth bleaching via one of the following three mechanisms: a) bonding mechanism, which occurs by the double bonding of ozone to form ozonide in the presence of non-aqueous solvents; however, if water is present, ozone is hydrolyzed to other products by cleaving the double bond that would have occurred; (b) a replacement mechanism, where an atom or functional group is replaced by another; (c) a cleavage mechanism, in which a carbon-carbon link is separated to produce organic composite fragments.11,20 Aqueous ozone facilitates a high level of biocompatibility in oral epithelial human cells, gingival fibroblast cells, and periodontal cells,11,20 while gaseous ozone makes the inhalation toxic to the lungs and other organs28; thus, a suitable system for application of ozone as a bleaching agent is required.20 Administration in gaseous form requires a sealing and suction system to avoid inhalation and adverse effects,29 like the trays that were developed and adapted for this study. In this respect, it was ensured that ozone remained in constant flux on the enamel structure with direct application, which may also have contributed to the significant results of color change. In contrast with other studies that used other types of systems, such as machines, in which the lack of suction capacity limited the use of a higher ozone concentration,6 or the use of a rectangular box with side openings for input and output7 and trays with an unsuitable design that did not allow the continuous circulation of ozone over the teeth.27 Because of the significant difference between the proposed treatments, the use of ozone appears to have advantages for shorter application durations to the dental structure and the absence of cytotoxic effects on the pulp over the use of agents containing peroxide.10 This type of tray could have led to dehydration of the teeth during the application of ozone (which enables a "false bleaching" at the end of the procedure), but the evaluation was performed after damping the teeth in artificial saliva for 24 hours. Clinically, dehydration during in-office bleaching may also be related to sensitivity,30 but some clinical studies report that the application of ozone or its combination with bleaching agents can minimize this effect.9,31 The combination of ozone and 10% carbamide peroxide gel applied in the in-office treatment has proven effective in teeth bleaching, although not better than the results of the other evaluated bleaching treatments. Only ozone may have been effective for bleaching since it was applied using the tray, and it remained in contact with the occlusal and lingual surfaces of the teeth, where the effect of carbamide peroxide applied to the vestibular faces was minor or absent. Some studies have shown that the use of low concentrations of hydrogen peroxide +(between 6% and 20%)32,33 or carbamide (between 15% and 40%)34 for in-office use is unsatisfactory given the borderline or minimal color change, unless they were associated with the use of light.35 On the other hand, the use of low peroxide concentrations has been a safety recommendation for bleaching procedures1; and control agencies have recommended gels in lower concentrations.36 Ozone appeared to satisfactorily facilitate tooth bleaching when combined with 10% carbamide peroxide for in-office application; clinically perceptible values in the ΔEab, ΔE00, and WID parameters were obtained. + +Other studies that combined ozone with bleaching agents did not show the enhancement of bleaching compared with ozonefree bleaching treatments,13 not even after employing different ozone application times (4 or 19 minutes, respectively) in combination with 35%10 or 37.5%11 hydrogen peroxide. Al-Omiri et al.8 suggested that ozone enhanced the bleaching response after application for 60 seconds immediately after the application of 38% hydrogen peroxide, in addition to keeping this mix for another 20 minutes on the teeth. In another study, Al-Omiri et al.9 described this potentiation in their study that applied 38% +hydrogen for 20 minutes and ozone for 60 seconds. + +This study suggests that ozone may have potentialized the 10% carbamide peroxide bleaching because it was applied for a shorter duration (total 3 hours of application) than in case of the conventionally applied carbamide peroxide for the 8 hours every day group (which totaled 56 hours of application). However, the benefits and costs of applying each treatment should be considered. + +According to the pH value analysis, neutrality was maintained within 60 minutes; however, there was a slight decrease in values, which was possibly associated with the free radical release. Although the use of ozone alone or in combination with low concentration carbamide peroxide for in-office dental bleaching following the adopted protocols facilitated favorable changes in color, they must be evaluated in clinical trials to not only prove their effectiveness but also their safety of use.28 It is important to consider the potential for soft tissue irritation, sensitivity of teeth, and acceptance of treatment by patients. + +It can be concluded that ozone and 10% ozonized carbamide peroxide for in-office use could promote tooth bleaching with clinically perceptible and acceptable altered chromaticity. + +Author contributions Study conception and design: RTB; data acquisition: TRFZFND, FUFC, CPT, FLBA, FMGF; data interpretation: TRFZFND, FUFC, RTB; data analysis: CPT, FLBA; manuscript writing: TRFZFND, FUFC, CPT, RTB; manuscript revision: CPT, FLBA, FMGF. All authors approved the final version of the manuscript. Conflicts of interest The authors have no financial, economic, commercial, and/or professional interests related to the topics presented in this manuscript. + +Open access statement This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. ©Article authors (unless otherwise stated in the text of the article) 2022. All rights reserved. No commercial use is permitted unless otherwise expressly granted. Additional file Additional file 1: Informed consent form (template). + +## References + +![5_Image_0.Png](5_Image_0.Png) + +1. de Geus JL, Wambier LM, Kossatz S, Loguercio AD, Reis A. Athome vs in-office bleaching: a systematic review and meta-analysis. *Oper Dent*. 2016;41:341-356. + +2. American Dental Association. Dentist dispensed home-use teeth bleaching products [Internet]. http://www.ada.org/ada/seal/standards/guide_home. Accessed by December 9, 2020. + +3. Committee for Scientific Consumer Products. Public consultation on a preliminary opinion on hydrogen peroxide in tooth whiten-ing products. https://ec.europa.eu/health/scientific_committees/ +consultations/public_consultations/sccp_cons_01_en. Accessed by December 9, 2020. + +4. Basting RT, Amaral FL, França FM, Flório FM. Clinical comparative study of the effectiveness of and tooth sensitivity to 10% and 20% carbamide peroxide home-use and 35% and 38% hydrogen peroxide in-office bleaching materials containing desensitizing agents. *Oper Dent.* 2012;37:464-473. + +5. Almeida AF, Torre Edo N, Selayaran Mdos S, et al. Genotoxic potential of 10% and 16% carbamide peroxide in dental bleaching. + +Braz Oral Res. 2015;29:S1806-83242015000100217. + +6. Zanjani VA, Ghasemi A, Torabzadeh H, Jamali M, Razmavar S, +Baghban AA. Bleaching effect of ozone on pigmented teeth. *Dent* Res J (Isfahan). 2015;12:20-24. + +7. Santana MS, Bridi EC, Navarro RS, et al. Dental bleaching with ozone: effects on color and enamel microhardness. *Acta Odontol* Latinoam. 2016;29:68-75. + +8. Al-Omiri MK, Abul Hassan RS, AlZarea BK, Lynch E. Improved tooth bleaching combining ozone and hydrogen peroxide--A blinded study. *J Dent.* 2016;46:30-35. + +9. Al-Omiri MK, Al Nazeh AA, Kielbassa AM, Lynch E. Randomized controlled clinical trial on bleaching sensitivity and whitening efficacy of hydrogen peroxide versus combinations of hydrogen peroxide and ozone. *Sci Rep.* 2018;8:2407. + +10. Al-Omiri MK, Lamfon HA, Al Nazeh AA, Kielbassa AM, Lynch E. Randomized clinical trial on the comparison of bleaching outcomes using either ozone or hydrogen peroxide. *Quintessence Int.* +2018;49:625-634. + +11. Saini R. Ozone therapy in dentistry: A strategic review. *J Nat Sci* Biol Med. 2011;2:151-153. + +12. Tahmassebi JF, Chrysafi N, Duggal MS. The effect of ozone on progression or regression of artificial caries-like enamel lesions in vitro. *J Dent.* 2014;42:167-174. + +13. Manton DJ, Bhide R, Hopcraft MS, Reynolds EC. Effect of ozone and Tooth Mousse on the efficacy of peroxide bleaching. *Aust Dent* J. 2008;53:128-132. + +14. Públio JD, D'Arce MB, Catelan A, et al. Influence of enamel thick-ness on bleaching efficacy: an in-depth color analysis. Open Dent J. 2016;10:438-445. + +15. do Carmo Públio J, Zeczkowski M, Burga-Sánchez J, et al. Influence of different thickeners in at-home tooth bleaching: a randomized clinical trial study. *Clin Oral Investig.* 2019;23:2187-2198. + +16. Tessier J, Rodriguez PN, Lifshitz F, Friedman SM, Lanata EJ. The use of ozone to lighten teeth. An experimental study. Acta Odontol Latinoam. 2010;23:84-89. + +17. Featherstone JDB, O'Really MM, Shariati M, Brugler S. Enhancement of remineralization in vitro and in vivo. In: Leach AS, ed. + +Factors relating to demineralization and remineralization of the teeth. Oxford: IRL; 1986. + +18. Serra MC, Cury JA. The in vitro effect of glass-ionomer cement restoration on enamel subjected to a demineralization and remineralization model. *Quintessence Int.* 1992;23:143-147. + +![6_image_0.png](6_image_0.png) + +19. Carlos NR, Bridi EC, Amaral F, França F, Turssi CP, Basting RT. + +Efficacy of home-use bleaching agents delivered in customized or prefilled disposable trays: a randomized clinical trial. *Oper Dent.* 2017;42:30-40. + +20. Nogales CG, Ferrari PH, Kantorovich EO, Lage-Marques JL. + +Ozone therapy in medicine and dentistry. *J Contemp Dent Pract.* 2008;9:75-84. + +21. Commission Internationale del Éclairage. Commission Internationale del Éclairage. Vienna, Austria: CIE Central Bureau. 2004. + +22. Sharma G, Wu W, Dalal EN. The CIEDE2000 color-difference formula: Implementation notes, supplementary test data, and mathematical observations. *Color Res Appl*. 2005;30:21-30. + +23. Pérez Mdel M, Ghinea R, Rivas MJ, et al. Development of a customized whiteness index for dentistry based on CIELAB color space. *Dent Mater.* 2016;32:461-467. + +24. Paravina RD, Ghinea R, Herrera LJ, et al. Color difference thresholds in dentistry. *J Esthet Restor Dent.* 2015;27 Suppl 1:S1-9. + +25. International Organization for Standardization. ISO/TR 28642: +2016. Technical Report (E): Dentistry - Guidance on colour measurements. + +26. Pérez MM, Herrera LJ, Carrillo F, et al. Whiteness difference thresholds in dentistry. *Dent Mater.* 2019;35:292-297. + +27. Aykut-Yetkiner A, Ertuğrul F, Eden E, Aladağ A, Ergin E, Özcan M. Color assessment after bleaching with hydrogen peroxide versus ozone: a randomized controlled clinical trial. *Gen Dent.* 2017;65:e12-e17. + +28. Naik SV, K R, Kohli S, Zohabhasan S, Bhatia S. Ozone- a biological therapy in dentistry- reality or myth????? *Open Dent J.* +2016;10:196-206. + +29. Loncar B, Mravak Stipetic M, Matosevic D, Tarle Z. Ozone application in dentistry. *Arch Med Res.* 2009;40:136-137. + +30. Mounika A, Mandava J, Roopesh B, Karri G. Clinical evaluation of color change and tooth sensitivity with in-office and home bleaching treatments. *Indian J Dent Res.* 2018;29:423-427. + +31. Suh Y, Patel S, Kaitlyn R, et al. Clinical utility of ozone therapy in dental and oral medicine. *Med Gas Res*. 2019;9:163-167. + +32. Torres CR, Souza CS, Borges AB, Huhtala MF, Caneppele TM. + +Influence of concentration and activation on hydrogen peroxide diffusion through dental tissues in vitro. ScientificWorldJournal. 2013;2013:193241. + +33. Lilaj B, Dauti R, Agis H, et al. Comparison of bleaching products with up to 6% and with more than 6% hydrogen peroxide: whiten-ing efficacy using BI and WI (D) and side effects - an in vitro study. + +Front Physiol. 2019;10:919. + +34. Perdigão J, Baratieri LN, Arcari GM. Contemporary trends and techniques in tooth whitening: a review. *Pract Proced Aesthet* Dent. 2004;16:185-192; quiz 194. + +35. Loiola AB, Souza-Gabriel AE, Scatolin RS, Corona SA. Impact of hydrogen peroxide activated by lighting-emitting diode/laser system on enamel color and microhardness: an in situ design. *Contemp Clin Dent.* 2016;7:312-316. + +36. American Dental Association Council on Scientific Affairs. Tooth whitening/bleaching: Treatment considerations for dentists and their patients; September 2009, Revised November 2010 [cited 2019 June 30]. Available at: "https://www.ada.org/~/ media/ADA/ +About%20the%20ADA/Files/ada_house_of_ delegates_whiten-ing_report.ashx effectiveness". Accessed by December 9, 2020. + +Date of submission: December 6, 2020 Date of decision: January 5, 2021 Date of acceptance: February 7, 2021 Date of web publication: *November 29,* 2021 \ No newline at end of file diff --git a/medical/md/PMC8820049.md b/medical/md/PMC8820049.md new file mode 100644 index 0000000000000000000000000000000000000000..76a4158117d8c824a1e5048318e7d58b9d1b0c53 --- /dev/null +++ b/medical/md/PMC8820049.md @@ -0,0 +1,264 @@ +Contents lists available at ScienceDirect + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +![0_image_5.png](0_image_5.png) + +North American Spine Society Journal (NASSJ) + +![0_image_3.png](0_image_3.png) + +![0_image_4.png](0_image_4.png) + +![0_image_6.png](0_image_6.png) + +journal homepage: www.elsevier.com/locate/xnsj + +![0_image_7.png](0_image_7.png) + +Clinical Studies + +![0_image_1.png](0_image_1.png) + +Spine coding transition from ICD-9 to ICD-10: Not taking advantage of the specificity of a more granular system✩ +Matthew J. Sabatino∗, Patrick J. Burroughs, Harold G. Moore, Jonathan N. Grauer Department of Orthopaedics and Rehabilitation, Yale School of Medicine, 47 College Street, New Haven, CT 06510, United *States* + +| a r t i c l e | i n f o | a b s t r a c t | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------|-------------------| +| Keywords: Spine Clinical coding International classification of diseases Codes ICD ICD-9-CM ICD-10-CM Claim reporting insurance Health care costs Data collection Data interpretation statistical | care and research possibilities. Kolmogorov-Smirnov test. | | + +Background: The transition from International Classification of Diseases, 9th Edition (ICD-9) to the 10th edition (ICD-10) in 2015 increased the number and specificity of diagnostic codes with the goal of facilitating clinical care and research possibilities. + +Considering the potential to default to less specified ICD-10 codes, the current study evaluated the number of codes utilized for spine-related conditions before versus after the transition to ICD-10. + +Methods: The numbers of patients with an index encounter for a primary spine-related non-deformity diagnosis codes indexed as "dorsopathies" were abstracted from the Humana PearlDiver dataset. As the transition from ICD-9 to ICD-10 occurred in 2015, the current study compared the year prior (ICD-9) to the year after (ICD-10). The number of ICD-9 and ICD-10 codes was assessed, and distribution of utilization was compared using the Kolmogorov-Smirnov test. Results:: In 2014, 848,623 patients were assigned one of the 100 unique ICD-9 dorsopathy codes, of which 17 codes (17% of available codes) were used for more than 1% of the patients. In 2016, 840,310 patients were assigned one of the 504 unique ICD-10 dorsopathy codes, of which 21 (4% of available codes) were used for more than 1% of the patients. The top 20 codes in 2014 (ICD-9) and the top 20 codes in 2016 (ICD-10) both represented the majority of the patient population and were not statistically differently represented (p = 0.819). Further, analysis of ICD-10 codes demonstrated a clear bias toward utilizing less specified codes. + +Conclusions: Despite a five-fold increase in available diagnostic codes for spine conditions in ICD-10, in the year after implementation providers continued to select a small proportion of less specific diagnostic codes when treating spine patients. + +## Introduction + +In 2015, the Centers for Medicare & Medicaid Services (CMS) of the United States Department of Health and Human Services implemented the International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10-CM) as the official diagnostic tool for health care.1,2 This marked a conversion from the previous ICD-9-CM, which had served as the primary diagnostic system since 1979.1 The conversion from ICD-9-CM to ICD-10-CM involved increasing the number and complexity of diagnostic codes to provide health caregivers with a greater degree of detail. While the ICD-9-CM codebook had approximately 14,000 distinct diagnostic codes, ICD-10-CM has more than 69,000 diagnostic codes.1 The Center for Disease Control and Prevention (CDC) highlighted the benefits of more enhanced data collection with the transition to ICD-10-CM. These improvements included more specific data for tracking public health conditions, conducting epidemiological research, as well as the potential for enhancing clinical decision-making and payment/reimbursement systems.1,3 Although the CDC stated the intended advantages of the new classification system, only a few authors have published results assessing the improvements in data specificity and diagnostic coding since the conversion.4-7 In 2017, researchers using Veterans Affairs data analyzed the impact the conversion to ICD-10-CM had on the recording of the overall epidemiology of chronic conditions and mortality statistics. 8 Additionally, one group has evaluated the change in reimbursements and insurance denials associated with the conversion in an ophthalmology practice.5 Both of these studies highlighted a tendency for coding to capture similar prevalence of conditions diagnosed; however, the ophthalmology study found a trend in which providers used a higher frequency of less +✩ Given his role as Editor in Chief, Jonathan Grauer, MD had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Tobias Mattei, MD. + +DOI of original article: 10.1016/j.xnsj.2020.100032 ∗ Corresponding author. + +E-mail *address:* Matthew.Sabatino@yale.edu (M.J. Sabatino). + +https://doi.org/10.1016/j.xnsj.2020.100035 Received 23 July 2020; Received in revised form 24 October 2020; Accepted 24 October 2020 Available online 31 October 2020 2666-5484/Published by Elsevier Ltd on behalf of North American Spine Society. This is an open access article under the CC BY-NC-ND license +(http://creativecommons.org/licenses/by-nc-nd/4.0/) + +| Table 1 Comparison of ICD-9 and ICD-10 diagnoses in study cohort. ICD-9 codes ICD-10 codes Categories 720.XX - 721.XX M45.XXX—M49.XXX Spondylopathies 722.X1 - 723.XX M50.XXX Cervical Disc Disorders 722.X2, 722.X3, M51.XXX Thoracic, Lumbar, Sacral Disc Disorders 724.X2, 722.X3 722.X0 - 724.X0 M53.XXX Other and Unspecified Dorsopathies 724.XX M54.XXX Dorsalgia Cross-mapping of spinal conditions that comprise the category "Dorsopathies"; note that deformity codes are excluded from ICD-9 or ICD-10 groups. ICD - International Classification of Diseases. | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +specific or unspecified codes upon conversion to the ICD-10 coding system. + +Spine-related diagnosis codes are typically captured under a general heading of "dorsopathies". The diagnostic category of "dorsopathies" comprises all manner of pathologies originating from the back or neck. The category includes spondylopathies, deformities, disc disorders, radiculopathies as well as back and neck pain. The usage of different ICD-9 versus ICD-10 dorsopathy codes has not previously been reported in the literature. + +The current study was thus performed to assess the hypothesis that, despite ICD-10 offering more diagnostic coding options/granularity for dorsopathy conditions than ICD-9, providers would continue to use a limited subset of available codes and would tend to default to less specific codes. The large national, administrative Humana dataset available from PearlDiver was used for these analyses. + +## Materials And Methods Data Source + +Data were extracted from the Humana patient subset from PearlDiver Patient Records Database (PearlDiver Inc., Colorado Springs, CO, USA), +which captured diagnostic and reimbursement data from 5,497,415 patients in the years 2014 and 2016 cumulatively. This a national subscription database made available by PearlDiver Inc. for the purpose of academic orthopaedic research. The database is stored on a passwordprotected server maintained by PearlDiver Inc. + +The database is searchable using ICD-9 or ICD-10 diagnoses. Our institutional Investigative Review Board has given exemption to studies utilizing this database. + +## Study Cohorts + +Using the ICD-9-CM diagnostic tables, codes were identified under the category "dorsopathies" containing spine (back or neck) as the primary origin or site of diagnosis. This encompassed every ICD-9 code from 720.XX to 724.XX (Table 1). + +Corresponding ICD-10 codes were identified using the publicly available general equivalency mapping (GEM) files provided by CMS.9 This encompassed codes M45.XXX - M54.XXX (Table 1). + +All deformity codes were excluded from the current study cohort +(ICD-9: 737.XX and ICD-10: M40.XXX - M43.XXX). Because the spine deformity coding has evolved to reflect additional stratifications based on age of onset and underlying disease state, the decision was made to exclude them from this analysis. + +Using the PearlDiver Research Program, assessment was performed to identify patients who had one of the spine dorsopathy codes as the primary diagnosis in a clinical encounter in the respective years. Each diagnostic code analyzed was the primary diagnosis for an individual clinical encounter recorded in the insurance database. If a patient had multiple encounters during the assessed year, the earliest visit for the respective year was used as the encounter of record. + +The study cohorts were also analyzed based on service encounter location. For service encounter location, four distinct locations were assessed as designated in PearlDiver - 1) Hospital & Surgical center, 2) +Outpatient Clinic, 3) Skilled Nursing or Rehab Facility, 4) Emergency Room or Urgent Care. Analysis to determine the percentage of patients seen in each type of encounter location was performed to compare between years 2014 and 2016. + +In 2014, the number of patients with the defined ICD-9 codes was assessed. In 2016, the number of patients with the ICD-10 codes was assessed. Demographic data of the two cohorts are listed in Table 2. + +## Subset Analysis + +A subset analysis was completed to illustrate the use of ICD-10 specificity. From the overall category of "dorsopathy" (M45-M54, 504 codes), +the commonly used subset diagnoses that fall under "dorsalgia" (M54, 38 codes) were selected for in-depth analysis. + +This subset analysis is to see whether the providers used specified codes when available. Diagnostic codes that were "unspecified" (denoting an unspecific entity when more specific codes are available) were compared to "specified" codes (denoting a specific anatomic region or sidedness). For example, the diagnostic code of M54.1 is "radiculopathy, site unspecified", and the diagnostic code of M54.16 is "radiculopathy, lumbar region." In this case, "radiculopathy, site unspecified" would be considered unspecified because there are more specific classifications available that clarify location, whereas "radiculopathy, lumbar region" would be considered specified because it captures the most specific description of radiculopathy diagnosis available. Within the category of +"dorsalgia" there are 10 unspecified codes and 28 specified codes. + +## Statistical Analysis + +The utilization of ICD-9 and ICD-10 codes was determined. First, the overall number of patients with primary spine diagnoses was tallied. The number of diagnostic codes that were used for more than 1% of the total cases was then determined for the respective years. + +Comparisons between the datasets to determine differences in distribution patterns were done using the two-sample Kolmogorov–Smirnov +(K–S) test. This is a test that determines whether there is a statistically significant difference in distribution between the two samples. In comparing the two samples, a K–S Test p-value of < 0.05 would indicate a statistically significant difference in distribution between the samples, where a K–S Test p-value of > 0.05 would indicate no statistically significant difference. + +Comparisons between the 2014 and 2016 cohorts with regards to encounter location were done using a Chi-squared test. + +All summary statistics, distributions and calculations were performed using R, version 2.12.2 (R Development Core Team, Vienna, Austria). + +| Table 2 Cohort demographics. Category 2014 ICD-9 Dorsopathy Cohort | 2016 ICD-10 Dorsopathy Cohort | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------|------------------| +| Total patients | 848,623 | 840,310 | +| % Female | 59.50% (504,932) | 59.94% (503,640) | +| Age Under 45 years | 11.58% (98,271) | 9.68% (81,342) | +| 45–64 years | 29.75% (252,465) | 30.48% (256,126) | +| 65+ years | 58.67% (497,887) | 59.84% (502,842) | +| Race White | 56.50% (479,472) | 61.84% (519,648) | +| Non-White | 11.72% (99,459) | 14.45% (121,425) | +| Unknown | 31.78% (269,692) | 23.71% (199,237) | +| Overview of cohort characteristics as reported in PearlDiver Patient Record Database reported as% (count). ICD - International Classification of Diseases. | | | + +![2_image_0.png](2_image_0.png) + +## Results Study Cohorts + +With ICD-9, there were 100 unique codes for "dorsopathy" (720.XX– +724.XX). Within the 2014 Humana dataset, 848,623 patients had one of these codes as their primary diagnosis. Of the 100 unique ICD-9 codes, 17 codes (17% of available codes) were each utilized for more than 1% +of the primary diagnoses (Fig. 1). + +With ICD-10, there were 504 unique codes for "dorsopathy" +(M45.XXX–M54.XXX). Within the 2016 Humana dataset, 840,310 patients had one of these codes as their primary diagnosis. Of the 504 unique ICD-10 codes, 21 codes (4% of available codes) were each utilized for more than 1% of primary diagnoses (Fig. 1). + +## Code Distributions + +For both ICD-9 and ICD-10 codes the majority of patients were assigned a small percentage of the available codes (Fig. 2). It can be noted in the distributions that the overwhelming majority of diagnoses were captured by a small number of codes. + +To highlight the more commonly used codes, the 20 most-utilized codes from ICD-9 were compared to the 20 most-utilized codes from ICD-10 (Fig. 3). Use of the two- sample Kolmogorov-Smirnov test demonstrated that the distributions of these codes were not statistically different (K-S Test p-value = 0.819). + +Table 3 lists the 20 most-utilized codes from ICD-9 and ICD-10, respectively, with their percentage of use in the overall cohort. + +The service encounter comparison from 2014 to 2016 demonstrated a similar proportion of visits to each of the designated location types. + +Although the Chi-squared test produced a statistically significant difference in each of the four location types from 2014 to 2016, this was likely due to a small difference in a large dataset comparison, and did not represent a meaningful difference between groups (Fig. 4). + +## Subset Analysis + +The most frequent ICD-10 coding prefix (M54.XXX - Dorsalgia) has 38 codes, of which there are 10 unspecified codes and 28 specified codes. The unspecified codes were 26.3% of the dorsalgia codes but were used for 71.8% of the dorsalgia encounters. The specified codes were 73.7% +of the dorsalgia codes but were 28.2% of the dorsalgia encounters. + +## Discussion + +The transition from ICD-9 to ICD-10 gave providers more coding options to facilitate clinical care and research. Specifically, the category + +| Top 20 frequently used diagnostic dorsopathy codes, ICD-9 (2014) vs. ICD-10 (2016). Rank ICD-9 Diagnoses (2014) % of Total ICD-10 Diagnoses (2016) | % of Total | | | | +|------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|--------------------------|--------------------------------------------------------|--------| +| All Dorsopathy (720–724) | 100% | All Dorsopathy (M45-M54) | 100% | | +| 1 | Lumbago (724.2) | 20.35% | Low back pain (M54.5) | 19.58% | +| 2 | Backache, unspecified (724.5) | 9.60% | Cervicalgia (M54.2) | 8.50% | +| 3 | Cervicalgia (723.1) | 9.00% | Radiculopathy, lumbar region (M54.16) | 5.70% | +| 4 | Degeneration of lumbar or lumbosacral disc (722.52) | 7.74% | Other disc degeneration, lumbar region (M51.36) | 5.56% | +| 5 | Thoracic or lumbosacral radiculitis, unspecified | 7.13% | Dorsalgia, unspecified (M54.9) | 5.43% | +| (724.4) | | | | | +| 6 | Lumbosacral spondylosis without myelopathy (721.3) | 6.94% | Spinal stenosis, lumbar region (M48.06) | 4.53% | +| 7 | Displacement of lumbar disc without myelopathy | 4.52% | Spondylosis w/out myelopathy, radiculopathy, lumbar | 4.24% | +| (722.10) | (M47.816) | | | | +| 8 | Spinal stenosis, lumbar, w/out neurogen. claudication | 4.21% | Other disc displacement, lumbar region (M51.26) | 2.77% | +| (724.02) | | | | | +| 9 | Cervical spondylosis without myelopathy (721.0) | 3.41% | Spondylosis w/out myelopathy, radiculopathy, cervical | 2.76% | +| (M47.812) | | | | | +| 10 | Degeneration of cervical intervertebral disc (722.4) | 3.00% | Radiculopathy, cervical region (M54.12) | 2.46% | +| 11 | Sciatica (724.3) | 2.99% | Pain in thoracic spine (M54.6) | 2.17% | +| 12 | Brachial neuritis/radiculitis, unspecified (723.4) | 2.68% | Spondylosis w/out myelopathy, radiculopathy, | 2.03% | +| lumbosacral (M47.817) | | | | | +| 13 | Pain in thoracic spine (724.1) | 2.13% | Radiculopathy, lumbosacral (M54.17) | 1.55% | +| 14 | Displacement of cervical disc w/out myelopathy | 1.54% | Intervertebral disc disorders w/radiculopathy, lumbar | 1.50% | +| (722.0) | (M51.16) | | | | +| 15 | Spinal stenosis in cervical region (723.0) | 1.31% | Spinal stenosis, cervical region (M48.02) | 1.48% | +| 16 | Sacroiliitis, not elsewhere classified (720.2) | 1.27% | Other disc degeneration, lumbosacral (M51.37) | 1.38% | +| 17 | Postlaminectomy syndrome, lumbar (722.83) | 1.22% | Other cervical disc degeneration, unspecified cervical | 1.35% | +| region (M50.30) | | | | | +| 18 | Degeneration of disc, site unspecified (722.6) | 0.80% | Sacroiliitis, not elsewhere classified (M46.1) | 1.27% | +| 19 | Disorders of sacrum (724.6) | 0.80% | Lumbago with sciatica, right side (M54.41) | 1.13% | +| 20 | Spinal stenosis, unspecified region (724.00) | 0.71% | Lumbago with sciatica, left side (M54.42) | 1.06% | +| ICD - International Classification of Diseases. | | | | | + +Fig. 3. ICD-9 vs. ICD-10 Code Utilization (Top 20 Codes). + +![3_image_0.png](3_image_0.png) A comparison plot of the top 20, highest volume diagnostic codes of ICD-9 and ICD-10. Analysis of empirical distribution (Kolmogorov–Smirnov Test) is applied to the two samples, a p-value of 0.819 demonstrates that the distributions are not statistically different. This means the overall pattern of common diagnostic code utilization is similar between ICD-9 and ICD-10 despite the availability of hundreds of more specific codes in ICD-10. ICD- International Classification of *Diseases*. + +of dorsopathies increased in complexity from 100 possible diagnostic codes in ICD-9 to 504 possible diagnostic codes in ICD-10. + +The current study found that the majority of diagnoses for dorsopathy spine conditions were captured by a small percentage of available codes, despite a five-fold increase in coding options with the transition from ICD-9 to ICD-10. In 2014, 17 diagnoses each accounted for utilization of more than 1% of the cumulative ICD-9 coding, while in 2016, 21 diagnoses each accounted for utilization of more than 1% of the cumulative ICD-10 coding. + +Fig. 4. ICD-9 vs. ICD-10 diagnostic code comparison by service encounter location. + +A graphical representation of the percentage of unique patient visits attributed to each of the four designated service encounter locations provided in PearlDiver between 2014 (ICD-9) and 2016 (ICD-10). While the Chi-squared test showed a statistically significant difference between the two groups in each location, the overall proportions were similar and represent no meaningful difference between groups. + +ICD- International Classification of *Diseases*. + +Comparison of the top 20 diagnostic code volumes from ICD-9 and ICD-10 demonstrated two distributions that were not statistically significantly different. This showed that providers tended to use the same few codes with similar frequency both in ICD-9 and after the transition to ICD-10. + +Not only did providers use a small fraction of available codes to account for the vast majority of diagnoses, they also tended to select nonspecific diagnostic codes despite having more complex coding choices available. This pattern highlighted by the example of the highest frequency category of dorsalgia (M54.XXX) codes, in which the 26.3% unspecified codes were used 71.8% of the time. + +The current study is the first to evaluate utilization patterns of spine codes in ICD-10 versus ICD-9. Similar to the currently presented results, in radiology Fleming et al. demonstrated that radiology used a small percentage (less than 3%) of available ICD-10 codes for the overwhelming majority (90%) of radiology claim coding.6 Also, in ophthalmology, Hellman et al. found that in an academic ophthalmology practice, the conversion to ICD-10 was associated with a bias toward unspecified codes.5 Consistent with the study hypothesis, the lack of usage of the full breadth of codes (number of codes and specificity of codes) is clearly a limitation to the objectives of introducing ICD-10. Clinical treatment algorithms cannot take advantages of the granular nature of ICD-10 if the specific codes are not used. Clinical research would have the similar limitations and lack of planned ICD-10 advantages. + +There are several possible reasons that the breadth of codes afforded by ICD-10 was not taken advantage of. First and foremost, clinicians could be expected to default to codes that are similar to what they had used in the past. Although new coding options were made available, it is understandable that providers continue to rely on the same habit of coding they have been using for years of practice. + +Electronic medical record systems may further facilitate physicians choosing pre-selected diagnoses or templates. Taking the time to change templated notes or poring through available codes may serve as barriers to change in the area of coding specificity. Without a targeted training +/ incentive, it is hard to imagine a significantly different pattern of code utilization for spine care going forward. + +One opportunity to incentivize improvement in spine coding specificity exists with the new initiatives on reimbursements and bundled payments for spine care. Multiple studies outline the challenges in designing a bundled payment for spine surgery. 10-12 Kahn et al. note there is significant variation in spine surgeon preference and technique for the same coded procedure. However, this variation in provider preference may not account for as much difference in resource utilization as patient-level effects (comorbidities, demographics). 10 In another study, Malik et al. highlighted the limitations of a bundled payment system demonstrating a wide range of costs for the same cervical fusion procedure. + +In both of these studies, differences in patient diagnoses had a stronger association with cost of care than surgeon technique.11 A bundled payment system that reimbursed based on preoperative diagnostic accuracy would likely result in providers utilizing more specific codes. + +A strength of this study is its evaluation of a large private insurer database with millions of patient encounters. This allows for a robust depiction of diagnostic code usage for spine conditions. The samples used from the PearlDiver dataset are equitable in size and scope and therefore offer a valid comparison between ICD-9 in 2014 and ICD-10 in 2016. + +There are limitations to the current study. First, the study is based on a single insurer (Humana), which may not be fully representative of coding seen by other insurers. Secondly, the study assessed the most recent data available for the current work, but it is possible that code usage has further evolved over the most recent few years. Additionally, as the study only evaluated primary diagnosis codes for billed encounters, it is possible that further granularity of secondary diagnoses was not appreciated. Nonetheless, given that the primary codes showed little change, it could be postulated similar trends occurred for secondary diagnoses. + +## Conclusion + +Despite a five-fold increase in available diagnostic codes for spine conditions in ICD-10, immediately after implementation providers continue to select a small proportion of less specific diagnostic codes when caring for spine patients. + +The ongoing initiatives in data registries and bundled care systems for spine care demonstrate an association between diagnostic accuracy and outcomes. Providers should take the opportunity to code for spine care in a manner that is accurate and specific in order to benefit from the additional clarity and granularity provided by ICD-10. + +## Declaration Of Competing Interest + +The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. + +The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: + +## Funding Disclosure Statement + +No financial support was provided for this study, and there was no study-specific conflict of interest for any of the authors of this paper. + +## Supplementary Materials + +Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.xnsj.2020.100035. + +## References + +[1] Centers for Disease Control and Prevention. International classification of diseases, (ICD-10-CM/PCS) transition - background. 2015; https://www.cdc.gov/ +nchs/icd/icd10cm_pcs_background.htm. Accessed August 14, 2019. + +[2] Centers for Medicare and Medicaid Services. The ICD-10 transition: an introduction. 2014; https://www.cms.gov/Medicare/Coding/ICD10/Downloads/ ICD10Introduction20140819.pdf. Accessed 9/2/2019, 2019. + +[3] Centers for Disease Control and Prevention. The ICD-10 transition and public health surveilance. 2013; https://www.cdc.gov/nchs/icd/data/CDC_ICD-10_ +Transition_FactSheet_12_2013.pdf. Accessed 8/12/2019, 2019. + +[4] Boyd AD, Li JJ, Burton MD, et al. The discriminatory cost of ICD-10-CM transition between clinical specialties: metrics, case study, and mitigating tools. J Am Med Inform Assoc 2013;20(4):708–17. + +[5] Hellman JB, Lim MC, Leung KY, Blount CM, Yiu G. The impact of conversion to international classification of diseases, 10th revision (ICD-10) on an academic ophthalmology practice. Clin Ophthalmol 2018;12:949–56. + +[6] Fleming M, MacFarlane D, Torres WE, Duszak R Jr. Magnitude of impact, overall and on subspecialties, of transitioning in radiology from ICD-9 to ICD-10 codes. J +Am Coll Radiol 2015;12(11):1155–61. + +[7] Li B, Evans D, Faris P, Dean S, Quan H. Risk adjustment performance of Charlson and Elixhauser comorbidities in ICD-9 and ICD-10 administrative databases. BMC +Health Serv Res 2008;8:12. + +[8] Yoon J, Chow A. Comparing chronic condition rates using ICD-9 and ICD-10 in VA +patients FY2014-2016. BMC Health Serv Res 2017;17(1):572. + +[9] Centers for Medicare and Medicaid Services. ICD-10-CM and ICD-10 PCS +and GEMs archive. 2018; https://www.cms.gov/Medicare/Coding/ICD10/ArchiveICD-10-CM-ICD-10-PCS-GEMs.html. Accessed 8/14/2019, 2019. + +[10] Kahn EN, Ellimoottil C, Dupree JM, Park P, Ryan AM. Variation in payments for spine surgery episodes of care: implications for episode-based bundled payment. J +Neurosurg Spine 2018;29(2):214–19. + +[11] Malik AT, Yu E, Kim J, Khan SN. Posterior cervical fusion for fracture vs degenerative cervical spine disease: implications for a bundled payment model. Spine J +2019;19(9):S20. + +[12] Ugiliweneza B, Kong M, Nosova K, et al. Spinal surgery: variations in health care costs and implications for episode-based bundled payments. Spine 2014;39(15):1235–42. \ No newline at end of file diff --git a/medical/md/PMC8913344.md b/medical/md/PMC8913344.md new file mode 100644 index 0000000000000000000000000000000000000000..3fdeb8a0736d139cd785c2b484c3fe36664fa656 --- /dev/null +++ b/medical/md/PMC8913344.md @@ -0,0 +1,417 @@ +Contents lists available at ScienceDirect + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +# Saudi Journal Of Biological Sciences + +journal homepage: www.sciencedirect.com Original article + +![0_image_1.png](0_image_1.png) + +The prevalence and intensity of external parasites in domestic pigeons +(Columba livia domestica) in Egypt with special reference to the role of deltamethrin as insecticidal agent Heba M. Salem a,⇑, Nahed Yehia b, Saad Al-Otaibi c, Ahmed M. El-Shehawi c, Alia A.M.E. Elrys d, Mohamed T. El-Saadony e, Marwa M. Attia f aDepartment of Poultry Diseases, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt b Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Giza 12618, Egypt cDepartment of Biotechnology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia d School of Life Science, Nanjing Normal University, Nanjing, Jiangsu, PR China eDepartment of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt fDepartment of Parasitology, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt article info + +| Article history: Received 23 August 2021 Revised 4 October 2021 Accepted 15 October 2021 Available online 22 October 2021 | +|-----------------------------------------------------------------------------------------------------------------------------| + +Keywords: +Dermanyssus gallinae Deltamethrin, Hippobosca equine Menopon gallinae Knemidocoptes spp., Pseudolynchia canariensis + +## Abstract + +This study aimed to investigate the prevalence and intensity of external parasites in domestic pigeons in Giza, Egypt, from January 2020 to December 2020. A total of 300 domestic pigeons (25 pigeons per month) were examined. The birds were divided into groups based on their age. The oxidative stress parameters; serum zinc concentration, serum malondialdehyde (MDA), and serum Nitric oxide were evaluated in single and mixed external parasitic infestations. The prevalence of external parasites in examined pigeons was 80.3%. The detected parasites were Pseudolynchia canariensis (P. canariensis), Hippobosca equina (H. equina), Columbicola columbae (C. columbae), Menopon gallinae (M. gallinae), Knemidocoptes species (spp.) and Dermanyssus gallinae (D. gallinae); their incidences were 41.6, 26, 7, 5,0.33 and 0.33%, respectively. The highest infestation was recorded in both spring and summer. The incidence of disease was higher in squabs and young birds than in adults. The mixed external parasitic infestation was recorded in this study. The infected birds showed decreased serum zinc concentration and elevated MDA and serum Nitric oxide levels. In conclusion, regular monthly treatment with deltamethrin is recommended as an effective drug in the treatment of the infested birds and succeeded in reducing the incidence of externalparasites in the treated birds; in addition, pigeon management measures must be implemented to reduce the risk of external parasites. + + 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +Abbreviations: MDA, serum malondialdehyde; P. canariensis, Pseudolynchia canariensis; H. equine, Hippobosca equine; C. columbae, Columbicola columbae; M. + +gallinae, Menopon gallinae; D. gallinae, Dermanyssus gallinae; spp., Species. + +⇑ Corresponding author. + +E-mail addresses: dr.hebasalem@cu.edu.eg (H.M. Salem), elshehawi@hotmail. + +com (A.M. El-Shehawi). + +Peer review under responsibility of King Saud University. + +| Production and hosting by Elsevier | +|--------------------------------------| + +## 1. Introduction + +Pigeons coexist with different animal and poultry species as well as humans all over the world (Alkharigy et al., 2018). In Egypt, domestic pigeons are a valuable source of essential protein for people as they gain more weight quickly than other birds, its meat is palatable, easy to breed, and require slight management (ElDakhly et al., 2016). Pigeons can be infected with a wide range of pathogens and serve as a reservoir for parasitic diseases. The proximity of pigeons to other domestic birds heightens the risk of parasitic infestation in poultry (Alkharigy et al., 2018). Pigeons harbor both external and/or internal parasites (Attia and Salem 2021). + +Poultry ectoparasites are abundant in the tropics, where poor husbandry practices and favorable climatic circumstances allow https://doi.org/10.1016/j.sjbs.2021.10.042 + +![0_image_3.png](0_image_3.png) + +1319-562X/ 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. + +This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +them to thrive (Imura et al., 2012; Badparva et al., 2015; Salem and Attia 2021). These parasites induce several direct and indirect effects on the pigeon, including weight loss, irritability, decreased productivity, malnutrition, low growth, decreased egg production and the development of a variety of clinical symptoms as; ruffled broken feathers, feathers loss, itching, pruritis, dermatitis, emaciation, inconvenience, death especially in squabs as well as indirect harm by transmitting other diseases (Radfar et al., 2012, Attia and Salem, 2021). The most common external parasites infesting pigeons are P. canariensis, Argas persicus, D. gallinae, M. gallinae, C. + +columbae, and Menocanthus stramineus (Atkinson et al.,2008). + +Insects can harbor a lot of pathogens and convey them to the host from these pathogens ''blood parasites" as Haemoproteus columbae which transmitted by the bite of ectoparasitic hippoboscid flies (Hussein and Abdelrahim, 2016); Plasmodium relictum, which is one of the most common species of avian malaria (Beadell et al., 2006) and Leucocytozoon marchouxi (Swinnerton et al., 2005, Martinsen et al., 2006, Hussein and Abdelrahim 2016). Deltamethrin (a pyrethroid derivative) has become a popular pesticide in many nations due to its low toxicity and wide safety margin for poultry (Eladl et al., 2018). Deltamethrin affects the nervous system of insects that touch or ingest them, which leads to the rapid paralysis and death of these insects (Elyazar et al., 2011). In the same way, deltamethrin considered as an effective insecticide for the control of insects on pigeons and the surrounding environment (Attia and Salem, 2021). Accordingly, the aim of this study is to understand the biodiversity and determine the prevalence of pigeon external parasitic infestation with evaluation of oxidative stress markers and zinc levels which will aid in the planning of actions to improve the health of these birds in Egypt. + +## 2. Material And Methods 2.1 Vvv. Ethical Approval + +This study was designed according to the Ethical Committee, Faculty of Veterinary Medicine regulations. Birds were humanly handled and safely released after clinical investigations. + +## 2.2. Pigeon Sampling + +During the observation period from January to December 2020, 300 pigeons (25 bird per month) were investigated. Three hundred Birds were collected from different poultry clinics, poultry market and pigeon rearing houses from Giza, Egypt which lies in 29.9870N 31.2118E with a hot desert climate. The average ambient temperature in Giza in winter ranged from 21.1 C (70.0)F: +24.2 C (75.6)F with relative humidity 46:57%, in spring, 28.4 C +(83.1)F : 34.9 C (94.8)F with relative humidity 37:44%, in summer 34.5 C (94.1)F : 41 C (106)F with relative humidity 39:53% and in autumn 25 C (77.7)F : 32.4 C (90.3)F with relative humidity 47: 57%. The examined birds were divided according to age as 80 squabs (day old to one month), 100 young (form one month to 7 months old) and 120 adults (above 7 months of age). Every bird was physically examined to detect the age and presence of external parasites (data were retrieved from the owners). + +## 2.3. History And Clinical Examination Of Pigeons 2.3.1. External Parasites + +The feathers and skin were carefully examined for the presence of any macroscopic external parasites by naked eye then parasites were picked up and/ or skin scraping was adopted to identify the parasites using an Olympus Stereoscopic microscope (SZX16; Japan (Soulsby 1986; Attia and Salem, 2021). + +## 2.4. Identification Of The Parasites + +All the collected parasites from the birds and their premises were preserved in 70% ethyl alcohol. Then, these parasites were identified using the keys recorded by Soulsby (1986); Attia and Salem, (2021). + +## 2.5. Blood Samples + +Blood was collected from jugular vein of externally parasitized birds on plain tubes for sera separation. Sera were kept on 20 C for further investigations according to Attia et al., (2020). + +## 2.6. Biochemical Analysis + +Serum zinc concentration were analysed using ionized coupled plasma by mass spectrometry method as described by Page et al., +(2018); Attia et al., (2020). + +## 2.7. Evaluation Of Oxidative Stress Markers + +The level of serum malondialdehyde (MDA) using the reaction of thiobarbituric acid and then separation occurred on HPLC. The detection was performed using UV at 532 nm. Serum Nitric oxide +(NO) level were analysed as mentioned by Khazaei and Nematbakhsh, 2012; briefly; colorimetric NO assay kit was used +(Calbiochem-Novabiochem Corporation, San Diego, Calif), that measures the total nitrate and nitrite on serum based on Griess reaction and measured using wavelength 450 nm as mentioned by Aktas et al., (2017) and Aytekin and Unubol Aypak (2011). + +2.8. Recommendation for external parasites control Deltamethrin (Butox, 12.5 % solution, 1 mL / 4 L of water for birds & 1 mL /L for surrounding environment), is recommended for monthly spraying. Precautions have been taken during deltamethrin spraying, and the remains have been disposed of in a sanitary manner. + +## 2.9. Statistical Analysis + +Data were statistically analyzed by using SPSS Version 18.0 software (Inc., Chicago, IL, USA). Blood parameters in infested and control non infested group were compared by independent T-test following the normality of data. A p- value consider significant when (p < 0.05). + +## 3. Results 3.1. History And Clinical Investigations + +Investigated pigeons were exhibited indications of illness such as general weakness, decreased egg production in layers, unthriftiness, ruffled feathers, decreased weight gain, emaciation with protrusion of keel bone (Fig. 1A) and in some cases; pigeon flies were observed on birds' body, under/or on the wings or on tail (Fig. 1B) and Fig. 2. + +Most of the examined pigeons were positive with at least with one of external parasitic species. The overall prevalence rate was 80.3%. + +## 3.2. External Parasites + +External parasites usually observed on living birds. In case of lice infestation, eggs were attached with feathers roots, while in + +![2_image_0.png](2_image_0.png) + +![2_image_1.png](2_image_1.png) + +red mite; the red spots were observed surround the bird vent, unilateral arthritis was observed in birds mange with presence of dull raised scales tinged with grayish to brown color exudate on shank of pigeon leg (Fig. 1C). + +Two hundred and forty-one; out of 300 examined pigeons, were positive for external parasites infestations. Six different types of external parasites were detected as follow: P. canariensis, H. equina, C. columbae, M. gallinae, Knemidocoptes spp. and D. gallinae with prevalence 41.6, 26, 7, 5, 0.33 and 0.33%, respectively. The intensity of external parasites on pigeon's body were ranged from (1–6), (1– +4), (8–25) and (5–20) in case of P. canariensis, H. equina, C. columbae, and M. gallinae, respectively. + +The prevalence and intensity of ectoparasites in domestic pigeons based on their age were summarized in (Fig. 3 & Table 1) and Fig. 4. + +Single infestation of pigeons with P. canariensis, H. equina, C. + +columbae, M. gallinae, Knemidocoptes spp. and D. gallinae were recorded also; mixed external parasites infestation between +(P. canariensis and H. equina); (P. canariensis and C. columbae); +(C. columbae and M. gallinae) and (P. canariensis; C. columbae and M. gallinae) were observed and summarized in; Table 2. + +The highest prevalence of external parasites was detected in spring and summer seasons as seen in (Table 3). + +Infested pigeon with H. equina showed significantly higher in Nitric oxide (120.56 ± 4.56, ±95% C.I.) while in mixed infestation with P. canariensis it reaches to 145.89 ± 9.43; the Nitric oxide levels was low in M. gallinae reach to 56.89 ± 3.76. Serum MDA +reach to 100.30 ± 6.48, 95% C.I.; in mixed infestation with P. canariensis, H. equina, while it was low in C. columbae infestation. + +zinc levels were decreased with parasitic infestation as it reaches to low levels in P. canariensis, H. equina mixed infestation; all data were compared to control levels of oxidative stress markers and zinc levels (Table 4). + +Periodical, monthly usage of deltamethrin in the infested flocks revealed a marked decrease in external parasites numbers after two days of treatment with relieve of the clinical signs. + +## 4. Discussion + +Pigeons are a point of anxiety since they can spread zoonoses to humans and are a reservoir for numerous parasite diseases affecting poultry (Sari et al., 2008). Most of examined pigeons showed 1827 + +![3_image_0.png](3_image_0.png) + +| Parasites | Positive cases (%) | Parasite's intensity | Age Squabs | Young | | +|--------------------|----------------------|------------------------|--------------|--------------|----------------| +| (80) | (1 00) | | | | | +| P. canariensis | 125 (41.6) | 1–6 | 50 (62.5%) | 65 (65%) | 10 (8.33%) | +| H. equina | 78 (26) | 1–4 | 12 (15%) | 22 (28%) | 44 (36.6%) | +| C. columbae | 21 (7) | 8–25 | 10 (12.5%) | 8 (8%) | 3 (2.5%) | +| M. gallinae | 15 (5) | 5–20 | 6 (0.75%) | 4 (4%) | 5 (4.16%) | +| D. gallinae | 1 (0.33) | - | 0 | 0 | 1 (0.33%) | +| Knemidocoptes spp. | 1(0.33) | - | 0 | 0 | 1 (0.33%) | +| Total | 241 (80.3%) | - | 78/80(97.5%) | 99/100 (99%) | 63/120 (52.5%) | + +![3_image_1.png](3_image_1.png) + +signs of ruffled feathers, severe emaciation. Similar signs were observed in case of parasitic infestation as recorded by Abd ElRahman et al., (2008); Mohamed et al., (2009) and Abdel Rahman et al., (2019). + +Ectoparasites live on feathers and skin of the host, using as a shelter and food provider, they also have a considerable negative influence on host health and productivity. From our results, the overall prevalence of external parasites was 80.3% (241/300) either single or mixed infestations, six different arthropods species were identified as follow: P. canariensis (41.6%), H. equina (26%), C. + +columbae (7%), M. gallinae (5%), D. gallinae (0.33 %), and Knemidocoptes spp (0.33%). Higher prevalence 89% (89/100) was recorded in Tripoli, Libya by Alkharigy et al., (2018) as they found the prevalence of C. columbae (82%), Goniodes gallinae (18%), M. gallinae (3%) +and P. canariensis (1%). Also, Radfar et al., (2011) recorded higher prevalence of external parasites in pigeon, C. columbae (79.41%), M. gallinae (44.11%) and P. canariensis (63.72%) in south khorasan; Iran while, in Colombia, Pérez-García et al., (2015) found that the prevalence of C. columbae (64%), P. canariensis (52%), M. gallinae +(24%). Borji et al., (2012) reported that the prevalence of C. columbae (42.8%), P. canariensis (16.1%) and M. gallinae (7.1%) in Mashhad city Iran. Also, Ali et al., (2020) recorded two external parasites on +(Harami) pigeons, including the shaft louse M. gallinae and P. + +canariensis with 100 and 88.90% prevalence, respectively in Medina, Saudi Arabia. + +In another study, the incidence of H. equina was 26% while Sokół and Michalski, (2015) noticed the low host specificity of H. equina which could attack cattle, dogs, hares, birds, and humans. + +The prevalence of C. columbae was 21% however, Jahantigh et al., (2016), Dranzoa et al., (1999) and Foronda et al., (2004) +recorded higher prevalence of C. columbae 78.40%, 94.1 and 100% in Iran, Uganda, and Tenerife, respectively. + +From our findings, Knemidocoptes spp. was observed in one case only with incidence of 0.33% during summer season and its lesion was found as unilateral arthritis and presence of severe dermatitis that appeared as raised dull color scales with presence of grayish color exudate between scales. Similar findings were observed in + +| Prevalence of single and mixed infestation of external parasites in domestic pigeons. Parasite Type of infestation | Prevalence (Positive cases/300 total examined birds) No. % | | | +|----------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------|-------|------| +| P. canariensis | Single | 90 | 30 | +| H. equina | 66 | 22 | | +| C. columbae | 9 | 3 | | +| M. gallinae | 3 | 1 | | +| D. gallinae | 1 | 0.33 | | +| Knemidocoptes spp. | 1 | 0.33 | | +| P. canariensis + H. equina | mixed | 22 | 7.33 | +| P. canariensis + C. columbae | 31 | 10.33 | | +| C. columbae + M. gallinae | 12 | 4 | | +| P. canariensis + C. columbae + M. gallinae | 6 | 2 | | +| Total No. of positive pigeons | 241 | 80.3 | | + +| Table.3 Seasonal prevalence of external and internal parasites in domestic pigeons. External Parasite Spring | Summer | Autumn | Winter | | | | | | +|----------------------------------------------------------------------------------------------------------------|----------|----------|----------|-------|----|------|----|------| +| No. | % | No. | % | No. | % | No. | % | | +| Pseudolynchia canariensis | 57 | 76 | 60 | 80 | 5 | 6.66 | 3 | 4 | +| Hippobosca equina | 18 | 24 | 48 | 66.6 | 12 | 16 | 0 | 0 | +| Columbicola columbae | 8 | 10.66 | 10 | 13.33 | 2 | 2.66 | 1 | 1.33 | +| Menopon gallinae | 6 | 8 | 9 | 12 | 0 | 0 | 0 | 0 | +| Dermanyssus gallinae | 0 | 0 | 1 | 1.33 | 0 | 0 | 0 | 0 | +| Knemidocoptes spp. | 0 | 0 | 1 | 1.33 | 0 | 0 | 0 | 0 | +| No. = number of positive cases. | | | | | | | | | + +| Oxidative stress markers in relation to single and mixed infection of external parasites. Parasite Oxidative stress markers Nitric oxide MDA serum Zinc P. canariensis 95.57 ± 3.67 78.38 ± 5.17 54.04 ± 2.85 H. equina 120.56 ± 4.56 88.38 ± 4.49 36.77 ± 1.32 C. columbae 67.98 ± 7.00 30.33 ± 3.35 100.24 ± 1.00 M. gallinae 56.89 ± 3.76 33.56 ± 3.46 110.52 ± 2.96 D. gallinae 79.89 ± 5.99 50.33 ± 1.03 33.57 ± 2.82 Knemidocoptes spp. 80.14 ± 6.33 52.35 ± 1.67 34.66 ± 3.62 P. canariensis + H. equina 145.89 ± 9.43 100.30 ± 6.48 28.89 ± 1.52 P. canariensis + C. columbae 110.00 ± 3.59 98.33 ± 2.45 59.97 ± 3.95 C. columbae + M. gallinae 74.49 ± 9.32 60.38 ± 2.98 100.67 ± 2.96 P. canariensis + C. columbae + M. gallinae Control 49.78 ± 0.95 27.89 ± 0.69 118.00 ± 0.94 | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +a parallel study conducted by Abou-Alsoud and Karrouf, (2016) who detected Knemidocoptes Pilae with prevalence of (11.5 %) in Budgerigars in Mansoura public park, Egypt. + +The mixed infestation was reported as follow: P. canariensis + H. + +equina with prevalence 7.33%. P. canariensis + C. columbae with 10.33 % prevalence; C. columbae + M. gallinae (4%) and P. canariensis + C. columbae + M. gallinae (2%). Also, Alkharigy et al., (2018) +recorded mixed infestation with external parasites in Libya. The mixed infestation of pigeon with external parasites could be contributed to that ectoparasites can live together without causing any hurtful effects on each other (Radfar et al., 2011). + +The occurrence of ectoparasitic infestations in squabs (82.5%) +was higher than young (77%) and lowest prevalence was recorded in adults (18%). Same observations were recorded by da Cunha Amaral et al., (2013) in case of P. canariensis infestation but, Msoffe et al., (2010) and Radfar et al., (2012) noticed that the prevalence of P. canariensis were higher in adult pigeons than squabs. Adult pigeons are predicted to have a lower incidence of ectoparasites since they have a higher level of parasite immunity +(Merila et al.,1995) and adult birds use their claws and the bill to get rid of ectoparasites (Clayton et al., 2010; Waite et al., 2012). + +From our results, the intensity (number of external parasites that are found per bird) of external parasites on pigeon's boy ranged from (1:6), (1:4), (8:25) and (5:20) in case of P. canariensis, H. + +equina, C. columbae and M. gallinae, respectively. This result is agreed with Attia and Salem (2021) as they noticed that the intensity of P. canariensis flies was 1: 6 (2.00 ± 1.0) on pigeons' body in El- Gharbia, Egypt. + +The ambient temperature is the key determinant of the frequency, quantity, and diversity of external parasites allover year +(Al-Barwari and Saeed 2012; Attia et al. 2017). In our study the highest prevalence of external parasites infestations was recorded in both spring and summer. The prevalence of P. canariensis was 76% and 80%, C. columbae was 10.66% and 13.33%, M. gallinae was 8 % and 12% in spring and summer, respectively. D. gallinae only detected in summer season only 1.33%. The lowest prevalence of external parasites infestation in our study was recorded in autumn and winter season, P. canariensis was 6.66% and 4%, C. columbae was 2.66% and 1.33% in autumn and winter, respectively. on the other hand, no positive cases of M. gallinae and D. gallinae were recorded in autumn and winter. The differences in the seasonal prevalence may be contributed to the ambient temperature in Egypt exceed 25 Co in both spring and summer which considered a favorable condition for insects' population. + +Continuous exposure of poultry to ectoparasites that act as intermediate hosts for different parasitic worms increases the potential for the spread of parasitic diseases among birds +(Ashenafi and Eshetu, 2004). So, monthly spray of deltamethrin is recommended and provoke acceptable result and succeeded in limitation of external parasites load. This result is agreed with (Attia and Salem, 2021) as they found deltamethrin is effective in P. canariensis control. Deltamethrin has proven its efficiency in controlling insects and we recommend in the future to load the active substance on biological nanoparticles to increase its efficiency. + +Also, recommend the use of some additives on pigeon food and drink as prebiotics (Abd El-Hack et al., 2021b; Yaqoob et al., 2021) probiotics (Abd El-Hack et al., 2021; Alagawany et al., +2021a; El-Saadony et al., 2021a), bioactive plants compounds (ElSaadony et al., 2021b), bioactive peptides (El-Saadony et al., +2021c,e), green synthesized nanoparticles (El-Saadony et al., 2021d,f), herbal extracts (Reda et al., 2021; Abou-Kassem et al., 2021), phytogenic compounds (Abd El-Hack et al., 2021c; Ashour et al., 2020), and essential oils (Alagawany et al., 2021b; ElTarabily et al., 2021; Abd El-Hack et al., 2020) to strengthen their immune system and increase disease resistance. + +The oxidative stress markers as (Nitric oxide and MDA levels) +were raised in parasitic infestation either in single parasites or in mixed infestation due to rapid erythrocyte destruction and secretion of oxygen radicles (Mousa and Soliman, 2016); as recorded by Nazifi et al., (2011) and Razavi et al., (2011) found that the antioxidant enzyme activity of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase, were raised during parasitic infestation. + +## 5. Conclusion + +When we spoke with pigeon owners, we discovered that most of them were unaware of dealing with a parasitic infestation in domestic pigeons, and some of them do not remedy them at all. + +From our findings, pigeons have a significant prevalence of external parasites, with single or mixed infestation. Oxidative stress markers were significantly elevated during either single or mixed external parasites infestations. Pathogenicity studies, regular monitoring, treatment, and control strategies must be put in place to reduce pigeon parasitic infestation considering that these birds are in close contact with other poultry and human. Deltamethrin could be considered as an effective insecticide for controlling of external parasites in the pigeon and would be recommended for regular use hand by hand with biosecurity measures. + +## Declaration Of Competing Interest + +The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. + +## Acknowledgement + +The current work was funded by Taif University Researchers Supporting Project number (TURSP - 2020/221), Taif University, Taif, Saudi Arabia. + +## References + +Abd El-Hack, M.E., El-Saadony, M.T., Swelum, A.A., Arif, M., Abo Ghanima, M.M., +Shukry, M., El-Tarabily, K.A., 2021c. Curcumin, the active substance of turmeric: its effects on health and ways to improve its bioavailability. J. Sci. Food Agric.in press. + +Abd El-Hack, M.E., Alaidaroos, B.A., Farsi, R.M., Abou-Kassem, D.E., El-Saadony, M.T., +Ashour, E.A., 2021. Impacts of supplementing broiler diets with biological curcumin, zinc nanoparticles and Bacillus licheniformis on growth, carcass traits, blood indices, meat quality and cecal microbial load. Animals 11 (7), +1878. + +Abd El-Hack, M.E., El-Saadony, M.T., Shafi, M.E., Alshahrani, O.A., Saghir, S.A., AlWajeeh, A.S., Al-Shargi, O.Y., Taha, A.E., Mesalam, N.M., Abdel-Moneim, A.-M.-E., +2021b. Prebiotics can restrict Salmonella populations in poultry: a review. Anim. Biotechnol. 19, 1–10. + +Abd El-Hack, M.E., El-Saadony, M.T., Shehata, A.M., Arif, M., Paswan, V.K., Batiha, G.- +E.-S., Khafaga, A.F., Elbestawy, A.R., 2020. Approaches to prevent and control Campylobacter spp. colonization in broiler chickens: a review. Environ. Sci. + +Pollut. Res. 28 (5), 4989–5004. + +Abd El-Rahman, M.A.M., Seddiek, S.A., Soliman, A.S., 2008. Some studies on trichomoniasis of pigeons at Qualiobia governorate. Egypt. J. Comp. Pathol. + + +Clin. Pathol. 21 (2), 123–141 http://erepository.cu.edu.eg/index.php/EJCPCP/ article/view/177. + +Abdel Rahman, M.M.I.A., Tolba, H.M.N., Abdel-Ghany, H.M., 2019. Ultrastructure, morphological differentiation, and pathological changes of Ascaridia species in pigeons. Adv. Anim. Vet. Sci. 7 (2), 66–72. https://doi.org/10.17582/journal. + +aavs/2019/7.2.66.72. + +Abou-Alsoud, M.E., Karrouf, G., 2016. Diagnosis and Management of Knemidocoptes Pilae in Budgerigars (Melopsittacus Undulates): Case Reports in Egypt. M J Vete. 1 (1), 007. + +Abou-Kassem, D.E., Mahrose, K.M., El-Samahy, R.A., Shafi, M.E., El-Saadony, M.T., +Abd El-Hack, M.E., Emam, M., El-Sharnouby, M., Taha, A.E., Ashour, E.A., 2021. Influences of dietary herbal blend and feed restriction on growth, carcass characteristics and gut microbiota of growing rabbits. Ital. J. Anim. Sci. 20, 896– 910. + +Aktas, M.S., Kandemir, F.M., Kirbas, A., Hanedan, B., Aydin, M.A., 2017. Evaluation of oxidative stress in pigeon infected with Psoroptes ovis using total antioxidant capacity, total oxidant status, and malondialdehyde level. Vet. Res. 61(2):197– +201.https://doi. 10.1515/jvetres-2017-0025. + +Alagawany, M., El-Saadony, M., Elnesr, S., Farahat, M., Attia, G., Madkour, M., Reda, F., 2021a. Use of lemongrass essential oil as a feed additive in quail's nutrition: its effect on growth, carcass, blood biochemistry, antioxidant and immunological indices, digestive enzymes and intestinal microbiota. Poult. Sci. 100, (6) 101172. + +Alagawany, M., Madkour, M., El-Saadony, M.T., Reda, F.M., 2021b. Paenibacillus polymyxa (LM31) as a new feed additive: Antioxidant and antimicrobial activity and its effects on growth, blood biochemistry, and intestinal bacterial populations of growing Japanese quail. Anim. Feed Sci. Technol. 276, 114920. + +Al-Barwari, S.H., Saeed, I., 2012. The Parasitic Communities of the rock pigeon Columba livia from Iraq: Component and Importance. Turkiye Parazitol Derg 36, 232–239. https://doi.org/10.5152/tpd.2012. + +Ali, M., Ibrahim, R., Alahmadi, S., Elshazly, H., 2020. Ectoparasites and intestinal helminths of pigeons in Medina, Saudi Arabia. J Parasitol. 12, 106(6):721–729. https://doi.10.1645/20-64. DOI: 10.1645/20-64. + +Alkharigy, F.A., El Naas, A.S., EL Maghrbi, A.A., 2018. Survey of parasites in domestic pigeons (Columba livia) in Tripoli, Libya. Open Vet. J. 8(4): 360–366. http:// dx.doi.org/10.4314/ovj.v8i4. + +Ashenafi, H., Eshutu, Y., 2004. Study on Gastro-intestinal helminths of local chickens in Central Ethiopia. Rev. Med. Vet. 155(10): 504–507. https://www. + +revmedvet.com › RMV155_504_507. + +Ashour, E.A., El-Hack, M.E.A., Shafi, M.E., Alghamdi, W.Y., Taha, A.E., Swelum, A.A., +Tufarelli, V., Mulla, Z.S., El-Ghareeb, W.R., El-Saadony, M.T., 2020. Impacts of green coffee powder supplementation on growth performance, carcass characteristics, blood indices, meat quality and gut microbial load in broilers. + +Agriculture 10, 457. + +Atkinson, C.T., Thomas, N.J., Hunter, D.B., 2008. Parasitic diseases of wild birds. John Wiley & Sons, Inc. ISBN: 978-0-813-82081-1. U.S. Library of Congress. https://doi.10.1002/9780813804620. + +Attia, M.M., El-Gameel, S.M., Ismael, E., 2020. Evaluation of tumor necrosis factoralpha (TNF-a); gamma interferon (IFN-c) genes and oxidative stress in sheep: +immunological responses induced by Oestrus ovis (Diptera: Oestridae) +infestation. J. Parasit. Dis.44(2): 332–337.https://doi.org/10.1007/s12639-02001220-w. + +Attia, M.M., Salem, H.M., 2021. Morphological and molecular characterization of Pseudolynchia canariensis (Diptera: Hippoboscidae) infesting domestic pigeons. Int. J. Trop. Insect Sci. https://doi.org/10.1007/s42690-021-00597-2. + +Attia, M.M., Soliman, S.M., Khalf, M.A., 2017. Hydrophilic nanosilica as a new larvicidal and molluscicidal agent for controlling of major infectious diseases in Egypt. Vet World. 10 (9), 1046–1051. + +Aytekin, I., Unubol Aypak, S., 2011. Levels of selected minerals, nitric oxide, and vitamins in aborted Sakis pigeon raised under semitropical conditions. Trop. + +Anim. Health Prod. 43:511–514.https://doi.10.1007/s11250-010-9724-x. + +Badparva, E., Ezatpour, B., Azami, M., Badparva, M., 2015. First report of bird's infection by intestinal parasites in Khorramabad, west Iran. J. Parasit. Dis. 39 (4), 720–724. https://doi.org/10.1007/s12639-014-0427-5. + +Beadell, J.S., Ishtiaq, F., Covas, R., Melo, M., Warren, B.H., Atkinson, C.T., Bensch, S., +Graves, G.R., Jhala, Y.V., Peirce, M.A., Rahmani, A.R., Fonseca, D.M., Fleischer, R.C., 2006. Global phylogeographic limits of Hawaii's avian malaria. Proc R Soc Lond. + +Series B. 273 (2935–2944). https://doi.org/10.1098/rspb.2006.3671. + +Borji, H., Moghaddas, E., Razmi, G.R., Azad, M., 2012. A survey of ecto- and endoparasites of domestic pigeons (Columba livia) in Mashhad, Iran. Iran. J. Vet. Sci. + +Technol. 4(2):37–42. https://doi: 10.4314/ovj.v8i4.2. + +Clayton, D.H., Koo, J., Harbiso, C., Moye, B., Bush, S., 2010. How birds combat ectoparasites. J. Ornithol. 3: 41–71. https//doi: 10.2174/ 1874453201003010041. + +da Cunha Amarala, H., Bergmannb, F.B., Silveirac, T., Silveira dos Santosd, P.R., +Krügera, R.F., 2013. Pseudolynchia canariensis (Diptera: Hippoboscidae): distribution pattern and phoretic association with skin mites and chewing lice of Columba livia (Aves: Columbidae). J. Nat. Hist. 47 (47–48), 2927–2936. https://doi.org/10.1080/00222933.2013.791939. + +Dranzoa, C., Ocaido, M., Katete, P., 1999. The ecto,gastro-intestinal and haemoparasites of live pigeons (Columba livia) in Kampala, Uganda. Avian Pathol. 28, 119–124. https://doi: 10.1080/03079459994830. + +Eladl, A., Hamed, H.R., El-Shafei, R.A., 2018. Prevalence of mites and their impact on laying hen (Gallus gallus domesticus) farm facilities in Egypt, with an analysis of deltamethrin residues in eggs and tissue. Avian Pathol. 47 (2), 161–171. https:// doi.org/10.1080/03079457.2017.1388500. + + +El-Dakhly, K., Mahrous, L.N., Mabrou, G.A., 2016. Distribution pattern of intestinal helminths in domestic pigeons (Columba livia domestica) and turkeys (Meleagris gallopavo) in Beni-Suef province Egypt. J. Vet. Med. Res. 23(1):112–120. https//doi: 10.21608/JVMR.2016.43226. + +El-Saadony, M.T., Alagawany, M., Patra, A.K., Kar, I., Tiwari, R., Dawood, M.A., AbdelLatif, H.M., 2021a. The functionality of probiotics in aquaculture: An overview. + +Fish Shellfish Immunol. 117, 36–52. + +El-Saadony, M.T., Zabermawi, N.M., Zabermawi, N.M., Burollus, M.A., Shafi, M.E., +Alagawany, M., Abd El-Hack, M.E., 2021b. Nutritional aspects and health benefits of bioactive plant compounds against infectious diseases: A review. Food Rev. Int., 1–23 El-Saadony, M.T., Abd El-Hack, M.E., Swelum, A.A., Al-Sultan, Sa.ad., El-Ghareeb, W. + +R., Hussein, E.O.S., Ba-Awadh, H.A., Akl, B.A., Nader, M.M., 2021c. Enhancing quality and safety of raw buffalo meat using the bioactive peptides of pea and red kidney bean under refrigeration conditions. Ital. J. Anim. Sci. 20 (1), 762– 776. + +El-Saadony, M.T., Alkhatib, F.M., Alzahrani, S.O., Shafi, M.E., Abdel-Hamid, S.E., Taha, T.F., Aboelenin, S.M., Soliman, M.M., Ahmed, N.H., 2021d. Impact of mycogenic zinc nanoparticles on performance, behavior, immune response, and microbial load in Oreochromis niloticus. Saudi J. Biol. Sci. 28 (8), 4592–4604. + +El-Saadony, M.T., Khalil, O.S., Osman, A., Alshilawi, M.S., Taha, A.E., Aboelenin, S.M., +Shukry, M., Saad, A.M., 2021e. Bioactive peptides supplemented raw buffalo milk: biological activity, shelf life and quality properties during cold preservation. Saudi J. Biol. Sci. 28 (8), 4581–4591. + +El-Saadony, M.T., Saad, A.M., Najjar, A.A., Alzahrani, S.O., Alkhatib, F.M., Shafi, M.E., +Selem, E., Desoky, E.-S.M., Fouda, S.E.-S.E.-S., El-Tahan, A.M., 2021f. The use of biological selenium nanoparticles in controlling Triticum aestivum L. crown root and rot diseases induced by Fusarium species and improve yield under drought and heat stress. Saudi. J. Biol. Sci. 28 (8), 4461–4471. + +El-Tarabily, K.A., El-Saadony, M.T., Alagawany, M., Arif, M., Batiha, G.E., Khafaga, A.F., +Elwan, H.A., Elnesr, S.S., Abd El-Hack, M.E., 2021. Using essential oils to overcome bacterial biofilm formation and their antimicrobial resistance. Saudi J. Biol. Sci. 28 (9), 5145–5156. + +Elyazar, I. R.F., Hay, S. I., Baird, J. K., 2011. Chapter 2 - Malaria distribution, prevalence, drug resistance and control in Indonesia. ADV PARASIT .74: 41–175. https://doi.org/10.1016/B978-0-12-385897-9.00002-1. + +Foronda, P., Valladares, B., Rivera-Medina, J.A., 2004. Parasites of Columba livia +(Aves: Columbiformes) in Tenerife (Canary Islands) and their role in the conservation biology of the Laurel pigeons. Parasite. 1: 311-316. https//doi: +10.1051/parasite/2004113311. + +Hussein, N.M., Abdelrahim, E.A., 2016. Haemoproteus Columbae infection and its histopathological effects on pigeons in Qena Governorate, Egypt. IOSR-JPBS. 11 (1): 79-90. https//doi: 10.9790/3008-11117990. + +Imura, T., Suzuki, Y., Ejiri, H., Sato, Y., Ishida, K., Sumiyama, D., Murata, K., Yukawa, M., 2012. Prevalence of avian haematozoa in wild birds in ahigh-altitude forest in Japan. Vet. Parasitol. 183: 244–248. https//doi: 10.1016/j.vetpar.2011.07.027. + +Jahantigh, M., Esmailzade Dizaji, R., Teymoori. Y., 2016. Prevalence of external parasites of pigeon in Zabol, southeast of Iran. J. Parasit. Dis. 40(4):1548-1551. + +https//doi: 10.1007/s12639-015-0725-6. + +Khazaei, M., Nematbakhsh, M., 2012. Effect of experimentally induced metabolic acidosis on aortic endothelial permeability and serum nitric oxide concentration in normal and high-cholesterol fed rabbits. Arch. Med. Sci. 8 (4), 719–723. https://doi.org/10.5114/aoms.2012.30296. + +Martinsen, E.S., Paperna, I. Schall, J., 2006. Morphological versus molecular identification of avian Haemosporidia: an exploration of three species concepts. Parasitol. 13(3):279–288. https//doi: 10.1017/S0031182006000424. + +Merila, J., Bjorklund, M., Bennett, G.F., 1995. Geographic and individual variation in haematozoan infections in the greenfinch, Carduelis chloris. Can. J. Zool. 73:1798–1804. https//doi: 10.1139/z95-212. + +Mohamed, I.E., El-Sakkar, G.H., Moursi, M.M., 2009. Pathological studies on pigeon trichomoniasis with reference to the associated bacteria. J. Comp. Pathol. Clin. + +Pathol. 22 (2), 67–87 http://www.pathologyeg.com/publication2009_2/ Publication3.pdf. + +Mousa, S.A., Soliman, S.M., 2016. Oxidant and Antioxidant Status in Pneumonic Goats with Special Reference to Bacterial Etiology. Int. J. Livestock Res. 6 (5), 15– 23. https://doi.org/10.5455/ijlr.20160417045508. + +Msoffe, P.L.M., Muhairwa, A.P., Chiwanga, G.H., Kassuku, A.A., 2010. A study of ectoand endo-parasites of domestic pigeons in Morogoro unicipality. Tanzania. Afr. + +J. Agric. Res. 5 (3), 264–2672. + +Nazifi, S., Razavi, S.M., Kianiamin, P., Rakhshandehroo, E., 2011. Evaluation of erythrocyte antioxidant mechanisms: antioxidant enzymes, lipid peroxidation and serum trace elements associated with progressive anemia in ovine malignant theileriosis. Parasitol. Res. 109 (275–281). https://doi.org/10.1007/ +s00436-010-2248-5. + +Page, C.M., Murphy, T.W., Van, M.L., Emon, J.G., Bowman, P., Wyffels, S.A., Stewart, W.C., 2018. Blood serum mineral element concentrations of weaned Montana ram lambs and their relationship with water quality characteristics. Prof. Anim. Sci. 34 (5), 410–420. https://doi.org/10.15232/pas.2018-01747. + +Pérez-García, J., Monsalve-Arcila, D., MárquezVillegas, C., 2015. Presencia de parásitos y enterobacterias en palomas ferales (Columba livia) en áreas urbanas en Envigado Colombia. Rev. Fac. Na.c Salud Pública. 33 (3), 370–376. + +Radfar, M.H, Asl, E.N., Seghinsara, H.R., Dehaghi, M.M., Faith, S., 2012. Biodiversity and prevalence of parasites of domestic pigeons (Columba livia domestica) in a selected semiarid zone of South Khorasan, Iran. TROP. ANIM. HEALTH PRO. 44 (2):225–229. https//doi: 10.1007/s11250-011-0002-3. + +Radfar, M.H., Fathi, S., Asl, E.N., Dehaghi, M.M., Seghinsara, H.R., 2011. A survey of parasites of domestic pigeons (Columba livia domestica) in South Khorasan, Iran. J. Vet. Res. 4(1):18–23. https//doi: 10.3923/vr.2011.18.23. + +Razavi, S., Nazifi, S., Bateni, M., Rakhshandehroo, E., 2011. Alterations of erythrocyte antioxidant mechanisms: antioxidant enzymes, lipid peroxidation and serum trace elements associated with anemia in bovine tropical theileriosis. Vet. Parasitol. 180(3):209–214. https//doi: 10.1016/j.vetpar.2011.03.011. + +Reda, F., El-Saadony, M., El-Rayes, T., Farahat, M., Attia, G., Alagawany, M., 2021. + +Dietary effect of licorice (Glycyrrhiza glabra) on quail performance, carcass, blood metabolites and intestinal microbiota. Poult. Sci. 100, (1) 101266. + +Salem, M.H., Attia, M.M., 2021. Accidental intestinal myiasis caused by Musca domestica L. (Diptera: Muscidae) larvae in broiler chickens: a field study. Int. J. + +Trop. Insect. Sci. https://doi.org/10.1007/s42690-021-00492-w. + +Sari, B., Karatepe, B., Karatepe, M., Kara, M., 2008. parasites of domestic (Columba livia domestica) and wild (Columba livia livia) pigeons in nig˘de, turkey. Bull. Vet. Inst. Pulawy. 52 (4), 551–554. + +Sokół, R., Michalski, M.M., 2015. Occurrence of Hippobosca equina in Polish primitive horses during the grazing season. Ann. Parasitol. 61 (2), 118–122. + +Soulsby, E.J.L., 1986. Helminths, Arthropods and Protozoa of Domesticated Animals. + +Bailliere Tindall, London, pp. 167–174. + +Swinnerton, K.J., Peirce, M.A., Greenwood, A., Chapman, R.E., Jones, C.G., 2005. + +Prevalence of Leucocytozoon marchouxi in the endangered Pink Pigeon Columba mayeri. Ibis. 147(4):725–737. https/doi: 10.1111/j.1474-919X.2005. 00454.x Waite, J.L., Henry, A.R., Clayton, D.H., 2012. How effective is preening against mobile ectoparasites? An experimental test with pigeons and hippoboscid flies. Int. J. Parasitol. 42(5):463–467. https/doi: 10.1016/j.ijpara.2012.03.005. + +Yaqoob, M., Abd El-Hack, M., Hassan, F., El-Saadony, M., Khafaga, A., Batiha, G., +Yehia, N., Elnesr, S., Alagawany, M., El-Tarabily, K., 2021. The potential mechanistic insights and future implications for the effect of prebiotics on poultry performance, gut microbiome, and intestinal morphology. Poult. Sci. 100, (7) 101143. \ No newline at end of file diff --git a/medical/md/PMC9005259.md b/medical/md/PMC9005259.md new file mode 100644 index 0000000000000000000000000000000000000000..f706fa9f154cfedd77ed8c5ee8432edb580603c2 --- /dev/null +++ b/medical/md/PMC9005259.md @@ -0,0 +1,421 @@ +# Environmental Epidemiology Original Research Article Does Early Life Phthalate Exposure Mediate Racial Disparities In Children'S Cognitive Abilities? Marisa A. Pattia,*, Melissa Eliota, Nan Lia, Kimberly Yoltonb, Bruce P. Lanphearc,D, Aimin Chene, Joseph M. Brauna + +Background: Early life exposure to phthalates may be associated with reduced cognition. However, it is unknown if disproportionate exposure to phthalates contributes to racial disparities in children's intellectual abilities. + +Methods: We used data from 253 mother-child pairs in Cincinnati, OH (the Health Outcomes and Measures of the Environment study, 2003–2006). We measured urinary concentrations of 11 phthalate metabolites twice during pregnancy and up to six times in childhood. We evaluated children's cognitive abilities at ages 5 and 8 years. Using mediation models, we quantified covariate-adjusted direct and indirect effects of race on children's Full-Scale Intelligence Quotient (IQ) scores for individual phthalate metabolite concentrations during gestation and childhood. + +Results: Average IQ scores among Black children (n = 90) were 7.0 points lower (95% confidence interval [CI] = −12, −1.8) than among White children (n = 145) after adjustment for socioeconomic factors. Urinary monobenzyl phthalate and monoethyl phthalate (MEP) concentrations during gestation and childhood were higher among Black than White children. We did not observe evidence that phthalate concentrations mediated the race-IQ association, with the exception of MEP. Childhood MEP concentrations partially mediated the race-IQ association. For instance, each 10-fold increase in MEP concentrations at age 2 years contributed to a 1.9point disparity in IQ scores between Black and White children (95% CI = −4.7, 0.7). Other phthalate metabolite concentrations during pregnancy or childhood did not mediate the race-IQ association. Conclusions: Despite observing racial disparities in exposure to some phthalates and IQ, we found little evidence that phthalates contribute to IQ disparities. Keywords: Racial disparities; Child intelligence; IQ; Phthalates; Gestational exposure; Childhood exposure a +*Department of Epidemiology, Brown University, Providence, RI; bDepartment of* Pediatrics, Division of General and Community Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH; cChild and Family Research Institute, British Columbia Children's Hospital, Vancouver, British Columbia, Canada; dFaculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia, Canada; and e*Department of Biostatistics,* Epidemiology and Informatics, University of Pennsylvania, Philadelphia, PA. J.M.B. served as an expert witness in litigation related to perfluorooctanonic acid contamination in drinking water in New Hampshire. Any funds he received from this arrangement were/are paid to the Brown University and cannot be used for his direct benefit (e.g., salary/fringe, travel). The other authors have no conflicts to report. The results reported herein correspond to specific aims of grant R01 ES024381 to investigator J.M.B.; R01 ES020349 to investigators A.C., B.P.L., and K.Y.; R01 ES011261 to investigator B.P.L.; and R01 ES014575 to investigators B.P.L. and K.Y. from the National Institutes of Health. + +Data and Code Acquisition: Computing code can be obtained from the corresponding author (M.A.P.) upon request. Data from the Health Outcomes and Measures of the Environment (HOME) study are available upon request. The HOME study principal investigators have actively engaged in collaborative data-sharing projects. We welcome new collaborations with other investigators. Interested investigators in the HOME study data can explore data options at the following location and use the available link to contact the investigators to discuss collaborative opportunities: https:// homestudy.research.cchmc.org/. The Data Sharing Committee meets regularly to review proposed research projects and ensure that they do not overlap with extant projects and are an efficient use of scare resources (e.g., cord blood). + +SDC Supplemental digital content is available through direct URL citations in the HTML and PDF versions of this article (www.environepidem.com). + +*Corresponding Author. Address: Brown University, School of Public Health, 121 S Main Street, Box G-S121-4, Providence, RI 02912. E-mail: marisa_patti@ brown.edu (M.A.P.). Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The Environmental Epidemiology. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Environmental Epidemiology (2022) 6:e205 Received: 5 November 2021; Accepted 1 March 2022 Published online 15 March 2022 DOI: 10.1097/EE9.0000000000000205 + +## Isee Introduction 1 + +Phthalates are a class of chemicals used in personal care and consumer products, including cosmetics, pharmaceuticals, vinyl flooring, medical supplies, food packaging, and plastic toys.1–7 Human exposure can occur through dermal absorption, inhalation, or ingestion.1,4,8 Phthalates are quickly excreted from the body (<24 hours), but exposure is chronic and ubiquitous in the US population, including among vulnerable subpopulations like pregnant women, infants, and children.3,9 Exposure may be higher in infants and children than adults due to differences in physiology, diet, and behaviors.9–14 Phthalates are suspected developmental neurotoxicants.15–23 Some epidemiologic studies24,25 report an inverse association of gestational17–22 or childhood phthalate exposure16,18–20 with cognitive abilities (i.e., Intelligence Quotient [IQ]) in children. While measures of intelligence are imperfect, they are strong predictors of educational achievement and work performance.26–30 Racial disparities in child IQ have been documented in the United States and abroad, such that individuals who identify as Black or have darker skin colors have lower measured IQ relative to their White peers.31–33 Parental cognition and genetics alone do not explain the full heterogeneity in child IQ,34,35 emphasizing + +## What This Study Adds + +Phthalates are neurotoxicants associated with diminished cognitive abilities in children. Racial disparities in children's phthalate exposure and cognitive abilities have been documented previously, but it is unclear if disproportionate exposure to phthalates contributes to disparities in child cognition. Using mediation models, urinary concentrations of monoethyl phthalate but not other phthalates partly mediated racial disparities in child cognitive abilities. While racial disparities in exposure to some phthalates and child cognition exist, we found little evidence to suggest phthalates contribute to disparities in child cognition. + +the need to consider external factors such as environmental exposures and socioeconomic features.36 Further, race is a social construct, not a biological variable. No genetic basis exists to explain racial disparities in IQ, indicating that disparities in cognitive abilities are most likely related to environmental factors.31,33,37 Phthalate exposure is ubiquitous in the United States (US), +but urinary concentrations of some phthalates are consistently higher in the Black population.13,38 Thus, highly exposed populations may be more likely to experience phthalate-related health consequences than those with lower levels of exposure. Moreover, prior work suggests that racial and ethnic disparities in reproductive, neonatal, and cardiometabolic health outcomes may be related to racial and ethnic differences in phthalate exposure.13,38–42 For example, some phthalate-containing products, particularly hair products, are more frequently marketed to and used by the Black communities.43 This highlights how certain subpopulations may be more exposed and thus vulnerable to the adverse effects of phthalates.43 Despite the recognition that racial and ethnic differences exist in phthalate exposure and child IQ, no study has evaluated whether phthalate exposure mediates the relation between race and child IQ. + +To evaluate the potential for phthalate metabolite concentrations during gestation and childhood to mediate the race–cognitive abilities relation, we employed previously defined methods for evaluating how environmental exposures contribute to racial disparities.44 The framework outlined by Bellavia et al utilizes multiple mediation analyses to evaluate the contribution of an environmental exposure of interest on the association between race and health outcomes in cases where there is an unequal distribution of both the exposure and outcome. The goal of this approach is to identify modifiable sources of environmental exposures to reduce (and eliminate) unequal distributions of exposures by developing interventions to reduce exposure-related health disparities. + +Using data from the Health Outcomes and Measures of the Environment (HOME) study, we quantified racial disparities in gestational and childhood phthalate exposure and assessed the extent that phthalates mediate the relation between race and child IQ. We hypothesized that racial disparities in phthalate exposure would partly explain the disparity in cognitive abilities between Black and White children. While this study focused on phthalates, it also serves a proof-of-principle analysis of other environmental toxicants as potential explanatory factors of racial disparities in child IQ. + +## Methods Study Participants + +The HOME study is a general population, prospective pregnancy and birth cohort study. Information regarding recruitment and data collection details have been previously published.45 Briefly, we recruited 468 pregnant women from 1263 eligible women from nine prenatal clinics affiliated with three delivery hospitals in the greater Cincinnati, OH area between 2003 and 2006. + +Inclusion criteria specified women be at least 18 years old, 16±3 weeks of gestation, HIV negative, not taking medications for seizures and/or thyroid disorders, living in homes built before 1978, and not having a diagnosis of diabetes, bipolar disorder, schizophrenia, or cancer the resulted in radiation treatment or chemotherapy. The Institutional Review Board at the Cincinnati Children's Hospital Medical Center and all participating hospitals approved this study (protocol code 01-8-5, 2008-0022 and dates of approval: 29 May 2002, 6 August 2007, and 4 August 2011). All participating mothers provided their written, informed consent for themselves and their children. + +Of 468 women who enrolled, 67 dropped out before delivery. Among the 389 live-born, singleton infants, our analysis included mother-child pairs that had at least one measure of child IQ at ages 5 or 8 years, at least one urinary phthalate concentration during gestation and childhood, and no missing covariate information (n = 7). We further restricted the analytic sample to include children who identified as Black or White. We excluded those children who identified as other races due to small sample size. Our final sample included 253 mother-child pairs (eFigure 1; http://links.lww.com/EE/A183). + +## Urinary Phthalate Metabolite Concentration Assessment + +Study staff collected maternal urine samples at approximately 16 and 26 weeks of gestation in polypropylene specimen cups. We collected child urine samples at ages 1, 2, 3, 4, 5, and 8 years. Among children who were not toilet trained, we collected urine by placing surgical inserts in clean diapers. We lined training potties with inserts to collect urine from children who were in the process of being toilet trained. Urine from inserts was collected with a syringe and expressed into a collection cup in the laboratory. For children who were toilet trained, caregivers assisted in the collection of urine using polypropylene specimen cups. We followed previously described protocols to minimize external contamination of urine samples.46,47 All urine samples were refrigerated for less than 24 hours before processing and stored at ≤−20°C until chemical analysis at the Centers for Disease Control and Prevention (CDC). + +Using a modified method of online solid phase extraction coupled with isotope dilution high-performance liquid chromatography–tandem mass spectrometry, CDC staff measured the urinary phthalate metabolite concentrations.13 We measured urinary concentrations of mono(3-carboxypropyl) phthalate +(MCPP), mono-isobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), mono-n-butyl phthalate (MBP), monocarboxyoctyl phthalate (MCOP), monocarboxynonyl phthalate (MCNP), and monoethyl phthalate (MEP). We used three oxidative di(2-ethylhexyl) phthalate (DEHP) metabolites (mono-2-ethyl-5-hydroxyhexyl phthalate, mono-2-ethyl-5-oxohexyl phthalate, and mono-2-ethyl-5-carboxypentyl phthalate [MECPP]) to create a summary DEHP metabolite measure (i.e., ΣDEHP) for both gestational and childhood samples. To calculate concentrations of ΣDEHP (in ng/mL), we divided each DEHP metabolite concentration by its molar mass, summed the individual concentrations, and then multiplied the molar sum by the molar mass of MECPP.48 We did not measure MCOP or MCNP concentrations in maternal urine samples because the method for these biomarkers had not yet been developed. We could not measure concentrations of MBP, MiBP, or MEHP from child urine samples collected at ages 1 to 3 years, as these metabolites were detected in the diaper inserts used during collection.9 The limits of detection (LODs) for the phthalate metabolites ranged from ~0.1 to ~1ng/mL. Values below the LOD were given a value of the LOD/√2 (eTable 3; http://links.lww.com/EE/A183).49 We accounted for urine dilution using urinary creatinine concentrations as described previously.50 Phthalate metabolite concentrations (ng/mL) were first creatinine standardized and log10 transformed before we conducted measurement error correction. Briefly, we accounted for phthalate exposure measurement error using a previously described regression calibration approach.51 For gestational phthalate concentrations, we used a linear mixed model with a random intercept to estimate subject-specific geometric mean creatinine-standardized urinary phthalate concentrations across the two measurement occasions (i.e., 16 and 26 weeks).52 Our model for phthalate concentrations collected on the ith mother at the jth measurement occasion during gestation: Yij ij = + µ ε i , +where µ µ σ i~ ( N , ) Β +2 and ε σ ij~ ( N 0, ) W +2 . Under this model, the reliability of observed geometric mean phthalate concentration for the ith mother across gestation, yi is given R n i B B +W +i += + σ σ/ ( ) σ 2 2 2 +, +where σ B +2 and σW +2 are the between-subject and within-subject variance components, respectively, and ni = 1–2 is the number of available phthalate measures during gestation. + +While this calibration approach is appropriate when geometric mean phthalate concentrations show no time trends across measurement occasions, this was not the case for childhood concentrations, which varied quadratically with child age.47 Therefore, we used a more complex linear mixed-effects model to account for measurement error in childhood phthalate measures. Thus, we included subject-specific age and agesquared terms for the age of urine sample collection from age 1 to 8 years. We included subject-specific intercepts and linear age slopes, while age squared was fixed. For each subject, we used the estimated best linear unbiased predictor to calculate phthalate metabolite concentrations at each time point and averaged across childhood. The average childhood value was calculated by dividing the area under the estimated trajectories of phthalate metabolite concentrations curve by the length of time (age, 1–8 years). This method accounts for measurement error, individual variation in the number of samples ascertained, and exposure time trends in urinary phthalate metabolite concentrations. For the remainder of the manuscript, we refer to these measurement error corrected values as urinary phthalate metabolite concentrations. + +## Cognitive Outcomes + +Three trained examiners assessed children's cognitive abilities using the Wechsler Preschool and Primary Scale of IntelligenceIII (WPPSI-III) at age 5 years (n = 202) and the Wechsler Intelligence Scale for Children-IV (WISC-IV) at age 8 years (n += 220).53–55 Following initial training and certification on these assessments, quality control checks were unannounced and completed (by K.Y.) every 6 months to ensure IQ measures were properly administered and accurately scored. Both instruments assess children's overall intellectual abilities (Full-Scale Intelligence Quotient [FSIQ]) based on subscales for verbal comprehension, working memory, processing speed, and others. In this analysis, we focused on FSIQ as our primary outcome. The FSIQ scores from the WPPSI-III are comparable to those from the WISC-IV, and thus we averaged them if more than one measurement was available to maximize sample size and accommodate our modeling approach.53,54 We used standardized FSIQ +scores (i.e., mean = 100 and SD = 15) based on the US normative data. Higher scores indicate better cognitive performance. + +## Covariate Assessment + +We used directed acyclic graphs to identify potential confounders that may be associated with both race and IQ, considering how confounders may bias the association between race and child IQ through exposure to phthalates during gestation and childhood (eFigure 2; http://links.lww.com/EE/A183 and eFigure 3; http://links.lww.com/EE/A183). Sociodemographic and maternal factors were collected using standardized questionnaires administered by trained interviewers. Child race was determined based on maternal report and assessed separately from maternal race. Child sex was abstracted from hospital medical charts.56 We derived maternal prepregnancy body mass index (BMI) using self-reported height and weight and imputed weight for mothers with missing data.57 Briefly, a SuperLearner algorithm was used to predict prepregnancy weight based on covariates found to be significantly associated with weight from HOME study participants with nonmissing data.58 These covariates included maternal education, marital status, maternal race, insurance status, parity, income, cotinine concentrations, height, and chart-abstracted weight between 13 and 19 weeks of gestation. We assessed secondhand and active tobacco smoke exposure during pregnancy by averaging cotinine concentrations from serum collected at 16 and 26 weeks of gestation and using previously established thresholds to determine active, secondhand, and nonsmoking (unexposed) status using serum cotinine were ≥0.3, <3.0 to ≥0.015, and <0.015ng/mL, respectively.59 Maternal cognitive abilities were assessed when children were ≥1 year using the Wechsler Abbreviated Scale of Intelligence.60 In primary analyses, we adjusted for maternal age, prepregnancy BMI, household income, log10-transformed cotinine concentration, and maternal FSIQ scores. + +## Statistical Analyses + +We calculated univariate summary statistics and characterized distributions of maternal sociodemographic and reproductive factors by child race. We further explored the distribution of urinary phthalate metabolite concentrations across visits by calculating univariate statistics and creating violin plots.61 We also calculated mean child FSIQ scores by strata of the key covariates stratified by race. + +## Mediation Analyses + +First, we quantified the racial disparities in child FSIQ scores. We used a linear mixed model to estimate unadjusted and covariate-adjusted differences in FSIQ scores between Black and White children. This represents the total effect. Second, we examined racial disparities in average gestational and childhood urinary phthalate metabolite concentrations by examining univariate statistics and percentage differences in concentrations of urinary phthalate metabolites between Black and White children. In cases where we identified racial differences in average child urinary phthalate metabolite concentrations, we investigated if differences in urinary phthalate concentrations were also present at individual time points at ages 1, 2, 3, 4, 5, and 8 years. + +Third, for phthalates that differed by race, we conducted mediation analyses following previously established methods for evaluating health disparities by Bellavia et al.44 We evaluated if associations between child race and average FSIQ score were mediated by gestational or childhood urinary phthalate metabolite concentrations using the R package, Mediation.44,62 This package estimates the average causal indirect effects, average direct effects, and total effects based on previously established methods for linear structural equation modeling.63 We estimated the covariate-adjusted, average direct (association between race and child FSIQ), and average indirect effects (association between race and child FSIQ through average gestational and childhood urinary phthalate concentrations). Note that in models of average phthalate metabolite concentrations for childhood, we only included urinary phthalate concentrations from ages 1, 2, 3, 4, and 5 years, as the outcome of childhood FSIQ score included values at ages 5 and 8 years. + +To assess mediation by average gestational and childhood urinary phthalate concentrations, we focused on the natural indirect effect, which is the effect of child race on FSIQ that operates only by changing the value of urinary phthalate metabolite concentrations, as this provides information on the pathways where disparities in child FSIQ originate. We also present the controlled direct effects, which are the effects of child race on child FSIQ score after fixing values of urinary phthalate concentrations (eFigure 4; http://links.lww.com/EE/A183). + +## Secondary And Sensitivity Analyses + +We conducted mediation analyses to quantify the total, direct, and indirect effects of select phthalate metabolites on the association between child race and child FSIQ using phthalate concentrations at individual time points during childhood. Note that for mediation analyses at age 8 years, child FSIQ at age 8 years was used as the outcome (as opposed to average child FSIQ, which includes FSIQ scores ascertained at ages 5 and 8 years). + +We conducted sensitivity analyses to assess the robustness of our results to various adjustments. In addition to previously specified covariates, we adjusted for child sex, quality, and quantity of the caregiving environment as measured using the Home Observation for Measurement of the Environment (HOME inventory),64 time-varying blood lead concentrations, neighborhood socioeconomic position, maternal educational attainment, and parity. HOME inventory measures were assessed by study staff during home visits when children were 1 year of age. The HOME inventory is designed to measure the quality and quantity of stimulation and support available to children in their home.64 In addition to previously identified covariates, we also adjusted for blood lead concentrations at each time point where the relation between phthalates and FSIQ were assessed. We collected whole blood samples from women at 16 and 26 weeks of gestation and children at ages 1, 2, 3, 4, 5, and 8 years using collection materials prescreened for lead contamination.65 Blood lead concentrations were quantified at the Centers for Disease Control and Prevention laboratories using inductively coupled plasma mass spectrometry.66 Neighborhood socioeconomic position among HOME study participants was previously assessed using data from 2000 US census records.67,68 For each participant's 2000 US census tract at 20 weeks of gestation, neighborhood socioeconomic position scores were calculated based on log median household income, percentage of households with interest, dividend, or rental income, the log median value of housing unit, percent of residents who completed high school or college, and percentage of residents with executive, managerial, or professional occupations. For each measure, Z scores were calculated, summed, and categorized into terciles where lower scores represent lower socioeconomic position. We conducted statistical analyses using R Studio (version 4.0.3; R Development Core Team).69 + +## Results Univariate Statistics: Study Participants And Fsiq Scores + +Among 253 mother-child pairs, 90 (38%) were Black children and 145 (62%) were White children. Compared with White children, Black children were more likely to have mothers who were younger (<25 years at the time of gestation) and had acquired less education, with lower household incomes, who were more likely to smoke during pregnancy, be multiparous, and have higher prepregnancy BMI (Table 1; eTable 1; http://links.lww. com/EE/A183). For both Black and White children, FSIQ scores were positively associated with maternal age, maternal education, and household income (Table 2). + +In unadjusted analyses, FSIQ scores among Black children were 16 points lower (β = −16.0 [95% confidence interval +(CI) = −19.4, −12.7]) than among White Children. After adjusting for covariates, Black children had FSIQ scores 7 points +(β = −7.0 [95% CI, −12.1, −1.8]) lower than White children. + +## Univariate Statistics: Mediators (Phthalate Metabolites) + +Urinary MCPP, MiBP, MBzP, MBP, and ΣDEHP concentrations were lower during gestation relative to childhood (Figure 1; eTable 2; http://links.lww.com/EE/A183). During gestation and childhood, MBzP (gestation percentage difference, 16 [95% CI = 13, 21]; childhood percentage difference, 13 [95% CI = 12, 15]) and MEP (gestation percentage difference, 15 [95% CI = 11, 19]; childhood percentage difference, 21 [95% CI = 19, 24]) concentrations were higher in Black compared with White children (eTable 2; http://links.lww.com/EE/A183). + +During pregnancy, urinary ΣDEHP concentrations were higher + +| 5 and/or 8 years by child race, according to covariates: the HOME study (2003–2006) Variable Full sample n (%) Black n (%) White n (%) Overall 253 (100) 90 (36) 145 (57) Maternal age (years) <25 57 (23) 45 (50) 8 (5) 25 to <35 154 (61) 40 (44) 104 (72) 35+ 42 (17) 5 (6) 33 (23) Maternal education High school or less 62 (25) 51 (57) 7 (5) Some college 71 (28) 31 (34) 37 (26) Completed college 120 (47) 8 (9) 101 (70) Maternal race Non-Hispanic Black 83 (33) 82 (91) 1 (0) Non-Hispanic White 152 (60) 5 (6) 142 (98) Other 18 (7) 3 (3) 2 (1) Annual income <$30,000 82 (32) 70 (78) 9 (6) $30,000–$75,000 77 (30) 17 (19) 55 (38) ≥$75,000 94 (37) 3 (3) 81 (56) Maternal smokinga Unexposed 78 (31) 5 (6) 66 (56) Secondhand smoking 116 (46) 40 (44) 71 (49) Active smoking 59 (23) 45 (50) 8 (6) Parity 0 114 (45) 29 (32) 73 (50) 1 79 (31) 29 (32) 46 (32) 2+ 60 (24) 32 (36) 26 (18) Prepregnancy BMI (kg/m2 ) Normal/underweight: <25 125 (49) 32 (36) 83 (57) Overweight: ≥25 to <30 65 (26) 23 (26) 38 (26) Obese: ≥30 63 (25) 35 (39) 24 (17) Child sex Female 140 (55) 57 (63) 76 (52) Percentages reflect columns (e.g., race) within each study sample characteristic. a Maternal smoking during pregnancy estimated based on maternal serum cotinine concentrations | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +in White than Black mothers. All other phthalate metabolite concentrations were similar during gestation and childhood in Black and White participants (percentage differences for average gestation or average childhood, <13; Figure 2). Thus, for subsequent analyses, we focused on MBzP and MEP concentrations. + +## Mediation Analyses + +Gestational phthalate metabolite concentrations did not independently contribute to racial disparities in child FSIQ (Table 3). Average childhood MEP concentrations showed modest evidence for mediation, but not MBzP. Each 10-fold increase in average childhood MEP concentrations contributed to −1.1 point (95% CI = −3.0, 0.8) difference in the natural indirect effect between race and FSIQ, though the confidence interval includes the null. The direct effect of race on child FSIQ scores, controlling for average childhood MEP +concentration, was modestly diminished (β = −5.8 [95% +CI = −12, −0.4]). + +## Secondary Analyses + +The natural indirect effects of MEP on IQ appeared to be stronger during early childhood (Figure 3). The strongest natural indirect effect of MEP on child FSIQ scores was observed at age 2 years (β = −1.9 [95% CI = −4.7, 0.7]; eTable 4; http://links. + +| (2003–2006) | Non-Hispanic Black | Non-Hispanic White | | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|----------------------|-----|-----------| +| Variable | n | Mean ± SD | n | Mean ± SD | +| Overall | 90 | 91±13 | 145 | 107±12 | +| Maternal age (years) <25 | 45 | 89±15 | 8 | 101±14 | +| 25 to <35 | 40 | 91±11 | 104 | 107±12 | +| 35+ | 5 | 103±13 | 33 | 108±13 | +| Maternal education High school or less | 51 | 88±14 | 7 | 98±17 | +| Some college | 31 | 92±13 | 37 | 103±13 | +| Completed college | 8 | 102±5.9 | 101 | 109±11 | +| Annual income <$30,000 | 70 | 89±14 | 9 | 98±19 | +| $30,000 to $75,000 | 17 | 95±11 | 55 | 104±12 | +| ≥$75,000 | 3 | 103±6.6 | 81 | 110±10 | +| Maternal smokingb Unexposed | 5 | 96±7.2 | 66 | 106±12 | +| Secondhand smoking | 40 | 91±12 | 71 | 107±13 | +| Active smoking | 45 | 91±17 | 8 | 111±17 | +| Parity 0 | 29 | 88±14 | 73 | 109±13 | +| 1 | 29 | 91±13 | 46 | 107±10 | +| 2+ | 32 | 93±13 | 26 | 103±12 | +| Prepregnancy BMI (kg/m2 ) Normal/underweight: <25 | 32 | 88±15 | 83 | 108±12 | +| Overweight: ≥25 to <30 | 23 | 95±11 | 38 | 107±13 | +| Obese: ≥30 | 35 | 90±13 | 24 | 104±14 | +| Child sex Male | 33 | 89±13 | 69 | 105±13 | +| Female | 57 | 92±14 | 76 | 109±12 | +| a Average FSIQ values represent the arithmetic mean of repeated child FSIQ scores. In cases where children only had one FSIQ measure, that value was used to represent the mean. Of the full analytic sample (n = 253), n = 169 children had two measures of FSIQ ascertained at 5 and 8 yrs (67%). | | | | | + +lww.com/EE/A183). While the 95% CI of the indirect effect estimate included the null value, the residual disparity in FSIQ +after controlling for MEP was attenuated to less than 5 points +(β = −4.9 [95% CI = −11.5, 1.6]; Figure 4). + +## Sensitivity Analyses + +We conducted sensitivity analyses for MEP. Additional adjustment for child sex, caregiving environment (HOME scores), or parity did not substantially change the pattern of results (eTable 5; http://links.lww.com/EE/A183). However, the total effect of race diminished once we adjusted for additional socioeconomic and environmental factors. Specifically, adjustment for maternal neighborhood socioeconomic position during gestation reduced the racial disparity in child FSIQ scores from 7.0 points (95% CI = −12.1, −1.8) to 6.1 points (95% CI = −11.7, −0.6). Adjusting for time-varying blood lead concentrations also decreased the disparity in child FSIQ scores, with the strongest indirect effect of MEP on child FSIQ observed at age 1 year from 7.0 points to 6.4 points (95% CI = −12.4, −0.4). Adjusting for maternal educational attainment also reduced the racial disparity in child FSIQ from 7.0 points to 6.3 points (95% CI = −11.5, −1.1). In fully adjusted models, where we considered all primary covariates and those included in sensitivity analyses, the racial disparity in child FSIQ was further attenuated to 3.4 points (95% CI = −10.5, 3.7). + +## Discussion + +By applying mediation methods to a longitudinal birth cohort, we sought to determine if racial disparities in children's cognitive abilities could be explained by early life phthalate exposure. During gestation and early childhood, urinary concentrations of MBzP and MEP were higher among Black than White children. Furthermore, child FSIQ scores were on average 7 points lower among Black children than White children after adjusting for socioeconomic factors. We did not observe compelling evidence that phthalate concentrations mediated racial disparities in child FSIQ, with the exception of MEP. We speculate this could be, in part, due to the disparities in urinary MEP concentrations observed between Black and White children, whereas urinary concentrations of other phthalates did not differ substantially. + +There was some evidence that MEP weakly mediated the association between race and child FSIQ. Other investigators have documented racial disparities in child FSIQ31–33 and differences in phthalate exposure across racial groups.13,38,39,43,70 Moreover, some studies report associations between gestational and early life phthalate exposure with decreased child IQ.16–22,24,25 Direct and indirect effects of all phthalate metabolites showed an inverse association with child cognitive abilities, with the exception of MCPP during childhood (β = 1.5 points +[95% CI = 0.2, 3.2]). This finding was counterintuitive, as we did not expect phthalates to have a protective effect; however, these results could be spurious as most indirect coefficients were centered around the null. Further, MCPP is a nonspecific phthalate metabolite, as it represents a breakdown product of several other higher molecular weight phthalate diesters.71 Consistent with our prior work in the HOME study, higher urinary MEP concentrations during childhood were associated with lower FSIQ.16 The lack of stronger mediating effects might be due to the relatively modest association that MEP and other phthalates have with child cognitive abilities. Note, while prior work in the HOME study16 did not identify strong evidence for sex-specific associations, future work may want to consider this in analyses investigating health disparities. + +MEP is of particular interest for several reasons. First, urinary concentrations of MEP are substantially higher in Black populations than White ones.5,72,73 Independent of socioeconomic position, Black women have higher body burdens of some phthalates and other environmental chemicals than White women, partly due to consumer beauty product use.38,74–77 In addition, Black women who are pregnant reported more use and frequency of use of hair care products during pregnancy than White women.42,78 Second, diethyl phthalate (DEP), the parent compound of MEP, is commonly used in a variety of consumer products such as makeup, nail polish, perfume, deodorant, and various hair products (spray, dyes, mousse, crème rinse, nutrients, and conditioners).5,25–28,72,79 The primary route of exposure for DEP is dermal absorption and suggests that frequent use of DEP containing products may result in higher internal body burdens of MEP compared to phthalates with different routes of exposure.72 In fact, frequent use of hair products during pregnancy, more commonly used among Black than White women, may be associated with decreases in gestational age,42 which is a predictor for child neurodevelopmental deficits.80 Some of these products are heavily marketed to Black communities, highlighting how cosmetics can be an environmental injustice81 but also a potentially modifiable source of exposure.42 This is an important consideration for future work, as consumers generally are often unaware of harmful chemical exposures present in their personal care products.81–85 Differences in exposure + +![5_image_0.png](5_image_0.png) + +the HOME study (2003–2006). The shaded area represents the density function of phthalate metabolite concentrations, and the dot represents the median. + +Repeated maternal urinary phthalate metabolites assessed at 16 and 26 weeks of gestation. Repeated child urinary phthalate metabolites assessed at ages 1, 2, 3, 4, and 5 years. Note, the average value of urinary phthalate concentrations does not include those collected at age 8 years since the primary outcome, average FSIQ consists of scores from ages 5 and 8 years. Concentrations of ΣDEHP (in ng/mL) were calculated using the following formula: ΣDEHP +(ng/mL) = (MECPP [ng/mL]/308.33 g/mol + MEHHP [ng/mL]/294.347 g/mol + MEOHP [ng/mL]/292.331 g/mol) × 308.33 g/mol. + +to phthalate-containing products could, in part, be due to discriminative advertising practices, 82,83 directed to maintain European, White beauty standards (i.e., hair relaxers and skin lighteners). 84,86 Our study has some limitations. First, the sample size was modest, and it varied during follow-up. We retained fewer Black than White families, which may result in selection bias. The reductions in sample size due to loss to follow-up also + +![5_image_1.png](5_image_1.png) + +6 + +| through gestational and childhood urinary phthalate metabolite concentrationsc: the HOME study (2003–2006) Gestationc Childhoodd Metabolite Visit β (95% CI) P β (95% CI) | P | | | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|--------------------|------------------|--------------------|-------| +| MCPP | Direct effect | −6.7 (−11.9, −1.6) | 0.01 | −8.6 (−13.8, −3.3) | <0.01 | +| Indirect effect | −0.2 (−1.7, 1.3) | 0.82 | 1.5 (0.2, 3.2) | 0.02 | | +| MiBP | Direct effect | −7.0 (−12.3, 1.9) | <0.01 | −7.1 (−12.2, −1.9) | 0.01 | +| Indirect effect | 0.1 (−0.3, 0.7) | 0.72 | 0.0 (−0.7, 0.5) | 0.86 | | +| MBzP | Direct effect | −7.2 (−12.6, −1.5) | 0.02 | −7.4 (−12.2, −2.3) | <0.01 | +| Indirect effect | 0.1 (−0.4, 0.7) | 0.77 | 0.5 (−0.3, 1.7) | 0.23 | | +| MBP | Direct effect | −7.0 (−11.5, −1.9) | <0.01 | −6.9 (−11.9, −1.9) | <0.01 | +| Indirect effect | −0.1 (−0.5, 0.3) | 0.81 | −0.1 (−0.9, 0.5) | 0.70 | | +| MCNPe | Direct effect | - | - | −7.1 (−12.5, −1.7) | 0.01 | +| Indirect effect | - | - | 0.0 (−0.7, 0.7) | 0.91 | | +| MCOPe | Direct effect | - | - | −7.1 (−12.2, −1.6) | <0.01 | +| Indirect effect | - | - | 0.1 (−0.6, 1.0) | 0.72 | | +| ΣDEHPf | Direct effect | −6.3 (−11.8, −1.0) | 0.01 | −7.0 (−12.3, −1.7) | 0.01 | +| Indirect effect | −0.6 (−1.9, 0.4) | 0.21 | 0.0 (−0.7, 0.7) | 0.92 | | +| MEP | Direct effect | −7.0 (−12.2, −2.0) | <0.01 | −5.8 (−11.6, −0.4) | 0.03 | +| Indirect effect | 0.1 (−0.4, 0.8) | 0.78 | −1.1 (−3.0, 0.8) | 0.24 | | +| a Adjusted for maternal age (continuous), prepregnancy BMI (continuous), income (continuous), log-10 transformed average gestational cotinine concentration (continuous), and maternal FSIQ scores (continuous). | | | | | | + +reduce statistical power and precision, limiting our ability to observe subtler associations. Second, measures of child cognition, including assessing IQ, are historically controversial.26–30 Despite efforts to reduce racial, ethnic, and socioeconomic biases in tools for assessing intelligence, some biases remain.31,33,37 Third, we were unable to consider multiple mediators in our analysis to consider both sources of phthalate exposure (i.e., daily personal care product use or fast-food consumption) in addition to phthalate biomarkers, as recommended by Bellavia et al.44 While assessing urinary concentrations of phthalate metabolites can provide an assessment of phthalate exposure from multiple sources, it remains difficult to directly translate these findings to personal recommendations or policy.44 For example, it may be more practical to intervene on behavior patterns related to phthalate-containing product use as a modifiable exposure, rather than any one specific phthalate metabolite.1 Future work could consider phthalate exposure sources, such as hair or beauty products, in addition to biomarkers of phthalates when investigating racial disparities in child FSIQ. + +Fourth, while we adjusted for many relevant covariates, residual confounding remains a possibility. For example, adjusting for income as a measure of socioeconomic status may not fully capture the cumulative effects of daily experiences for minority populations, such as racism or chronic stress.33,37 Further, given that adjustment for socioeconomic and environmental factors attenuated the racial differences in IQ by almost half of an SD in IQ scores, additional environmental factors are likely to play a major role in explaining racial disparities in IQ. This is further supported from sensitivity analyses, where additional adjustment for socioeconomic position, blood lead concentrations, and parity attenuated the effect of race on the association between phthalates and FSIQ. In fully adjusted models where we considered all covariates from primary and sensitivity analyses, we observed a large attenuation of racial Figure 3. Adjusted indirect associations between child race and average child FSIQ at ages 5 and/or 8 years mediated through gestational and childhood urinary MEP concentrations: the HOME study (2003–2006). Adjusted for maternal age (continuous), prepregnancy BMI (continuous), income (continuous), log-10 + +![6_image_0.png](6_image_0.png) + +transformed average gestational cotinine concentration (continuous), and maternal FSIQ scores (continuous). Repeated maternal urinary phthalate metabolites assessed at 16 and 26 weeks of gestation. Repeated child urinary phthalate metabolites assessed at ages 1, 2, 3, 4, and 5 years. Note, the average value of urinary phthalate concentrations does not include those collected at age 8 years since the primary outcome, average FSIQ consists of scores from ages 5 and 8 years. + +![7_image_0.png](7_image_0.png) + +differences in IQ, emphasizing the potential for multiple maternal, child, and community-level factors to be responsible for disparities in child IQ scores. It is also important to reiterate that race is a social construct, rather than a biological variable. As such, including race as a variable in our models does not capture the totality of minority and marginalized people's experience that may impact child cognitive development. Further, we were underpowered to examine other race or ethnicities in this study, and given the regional nature of the study location (Cincinnati, OH), our findings may not be generalizable to other major US cities. While we considered blood lead levels as an additional coexposure, we did not consider other known chemicals (i.e., organophosphates or polychlorinated biphenyls).17,87 Future work may consider other exposures, as well as chemical mixtures as potential mediators of the race-cognitive abilities relation.88 Fifth, it is possible our study findings are subject to type I +error (in the case of MEP), as we did not account for multiple comparisons. We attempted to mitigate this by conducting our statistical analyses in a stepwise fashion, as to reduce the overall number of statistical tests. Further, secondary and sensitivity analyses were only conducted for MEP, as this was the only phthalate metabolite that was found to partly mediate the relation between child race and cognitive abilities. + +This study had several strengths. First, we had extensive biomarkers of phthalate exposure and two measures of cognitive ability. Most published studies only had one phthalate biomarker, which is subject to substantial within-person variation due to the episodic nature of phthalate exposure and the short half-life of phthalates.3,9,89,90 Second, we used regression calibration to repeated urinary phthalate concentrations from gestation and early childhood to account for measurement error. + +Third, we applied previously established epidemiological methods to evaluate phthalates as a source of racial disparities in child FSIQ.44 + +## Conclusion + +We observed racial disparities in child cognition and exposure to some phthalates during gestation, infancy, and childhood. + +Overall, we did not find that phthalates mediated racial disparities in child IQ. However, further studies are warranted to determine if efforts to reduce exposure to neurotoxicants may diminish some of the disparities in cognitive abilities observed between Black and White children. + +## References + +1. Braun JM, Just AC, Williams PL, Smith KW, Calafat AM, Hauser R. + +Personal care product use and urinary phthalate metabolite and paraben concentrations during pregnancy among women from a fertility clinic. J Expo Sci Environ Epidemiol. 2014;24:459–466. + +2. Carlstedt F, Jönsson BA, Bornehag CG. PVC flooring is related to human uptake of phthalates in infants. *Indoor Air.* 2013;23:32–39. + +3. Hauser R, Calafat AM. Phthalates and human health. Occup Environ Med. 2005;62:806–818. + +4. Rudel RA, Gray JM, Engel CL, et al. Food packaging and bisphenol A +and bis(2-ethyhexyl) phthalate exposure: findings from a dietary intervention. *Environ Health Perspect.* 2011;119:914–920. + +5. Parlett LE, Calafat AM, Swan SH. Women's exposure to phthalates in relation to use of personal care products. *J Expo Sci Environ Epidemiol.* 2013;23:197–206. + +6. Hernández-Díaz S, Su YC, Mitchell AA, Kelley KE, Calafat AM, Hauser R. Medications as a potential source of exposure to phthalates among women of childbearing age. *Reprod Toxicol.* 2013;37:1–5. + +7. Agency for Toxic Substances and Disease Registry (ATSDR). + +Toxicological profile for Di(2-Ethylhexyl)Phthalate (DEHP). Atlanta GA Agency Toxic Subst Dis Regist. Published online 2004:484. + +8. Bornehag CG, Lundgren B, Weschler CJ, Sigsgaard T, Hagerhed-Engman L, Sundell J. Phthalates in indoor dust and their association with building characteristics. *Environ Health Perspect.* 2005;113:1399–1404. + +9. Watkins DJ, Eliot M, Sathyanarayana S, et al. Variability and predictors of urinary concentrations of phthalate metabolites during early childhood. *Environ Sci Technol.* 2014;48:8881–8890. + +10. Woodruff TJ, Zota AR, Schwartz JM. Environmental chemicals in pregnant women in the United States: NHANES 2003-2004. Environ Health Perspect. 2011;119:878–885. + +11. Koch HM, Preuss R, Drexler H, Angerer J. Exposure of nursery school children and their parents and teachers to di-n-butylphthalate and butylbenzylphthalate. *Int Arch Occup Environ Health.* 2005;78:223–229. + +12. Koch HM, Drexler H, Angerer J. Internal exposure of nursery-school children and their parents and teachers to di(2-ethylhexyl)phthalate (DEHP). *Int J Hyg Environ Health.* 2004;207:15–22. + +13. Silva MJ, Barr DB, Reidy JA, et al. Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999-2000. Environ Health Perspect. 2004;112:331–338. + +14. Sørensen LK. Determination of phthalates in milk and milk products by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2006;20:1135–1143. + +15. Desvergne B, Feige JN, Casals-Casas C. PPAR-mediated activity of phthalates: a link to the obesity epidemic? *Mol Cell Endocrinol.* 2009;304:43–48. + +16. Li N, Papandonatos GD, Calafat AM, et al. Identifying periods of susceptibility to the impact of phthalates on children's cognitive abilities. + +Environ Res. 2019;172:604–614. + +17. Doherty BT, Engel SM, Buckley JP, Silva MJ, Calafat AM, Wolff MS. + +Prenatal phthalate biomarker concentrations and performance on the Bayley Scales of Infant Development-II in a population of young urban children. *Environ Res.* 2017;152:51–58. + +18. Factor-Litvak P, Insel B, Calafat AM, et al. Persistent associations between maternal prenatal exposure to phthalates on child IQ at age 7 years. *PLoS One.* 2014;9:e114003. + +19. Huang HB, Chen HY, Su PH, et al. Fetal and childhood exposure to phthalate diesters and cognitive function in children up to 12 years of age: Taiwanese maternal and infant cohort study. *PLoS One.* 2015;10:e0131910. + +20. Kim JI, Hong YC, Shin CH, Lee YA, Lim YH, Kim BN. The effects of maternal and children phthalate exposure on the neurocognitive function of 6-year-old children. *Environ Res.* 2017;156:519–525. + +21. Kim S, Eom S, Kim HJ, et al. Association between maternal exposure to major phthalates, heavy metals, and persistent organic pollutants, and the neurodevelopmental performances of their children at 1 to 2years of age- CHECK cohort study. *Sci Total Environ.* 2018;624:377–384. + +22. Polanska K, Ligocka D, Sobala W, Hanke W. Phthalate exposure and child development: the Polish Mother and Child Cohort Study. *Early* Hum Dev. 2014;90:477–485. + +23. Engel SM, Patisaul HB, Brody C, et al. Neurotoxicity of ortho-phthalates: recommendations for critical policy reforms to protect brain development in children. *Am J Public Health.* 2021;111:687–695. + +24. Ipapo KN, Factor-Litvak P, Whyatt RM, et al. Maternal prenatal urinary phthalate metabolite concentrations and visual recognition memory among infants at 27 weeks. *Environ Res.* 2017;155:7–14. + +25. Téllez-Rojo MM, Cantoral A, Cantonwine DE, et al. Prenatal urinary phthalate metabolites levels and neurodevelopment in children at two and three years of age. *Sci Total Environ.* 2013;461-462:386–390. + +26. Gottfredson LS. *Social Consequences of Group Differences in Cognitive* Ability. University of Delaware; 2004. + +27. Herrnstein RJ, Murray C. The Bell Curve: Intelligence and Class Structure in American Life. Free Press; 1994. + +28. Coyle TR, Pillow DR. SAT and ACT predict college GPA after removing g. *Intelligence*. 2008;36:719–729. + +29. Schmidt FL, Hunter JE. The validity and utility of selection methods in personnel psychology: practical and theoretical implications of 85 years of research findings. *Psychol Bull*. 1998;124:262–274. + +30. Schmidt FL, Hunter J. General mental ability in the world of work: +occupational attainment and job performance. *J Pers Soc Psychol.* 2004;86:162–173. + +31. Thomas D. Racial IQ differences among transracial adoptees: fact or artifact? *J Intell*. 2017;5:1. + +32. Templer DI, Arikawa H. Association of race and color with mean IQ +across nations. *Psychol Rep.* 2006;99:191–196. + +33. LeWinn KZ, Bush NR, Batra A, Tylavsky F, Rehkopf D. Identification of modifiable social and behavioral factors associated with childhood cognitive performance. *JAMA Pediatr.* 2020;174:1063–1072. + +34. Rushton JP, Jensen AR. Thirty years of research on race differences in cognitive ability. *Psychol Public Policy Law*. 2005;11:235–294. + +35. Lynn R. *Race Differences in Intelligence: An Evolutionary Analysis*. + +Washington Summit Books; 2006. + +36. Nisbett RE, Aronson J, Blair C, et al. Intelligence: new findings and theoretical developments. *Am Psychol.* 2012;67:130–159. + +37. Breslau N, Chilcoat HD, Susser ES, Matte T, Liang KY, Peterson EL. + +Stability and change in children's Intelligence Quotient scores: a comparison of two socioeconomically disparate communities. *Am J Epidemiol.* +2001;154:711–717. + +38. Kobrosly RW, Evans S, Miodovnik A, et al. Prenatal phthalate exposures and neurobehavioral development scores in boys and girls at 6-10 years of age. *Environ Health Perspect.* 2014;122:521–528. + +39. Potischman N, Troisi R, Thadhani R, et al. Pregnancy hormone concentrations across ethnic groups: implications for later cancer risk. Cancer Epidemiol Biomarkers Prev. 2005;14:1514–1520. + +40. Faupel-Badger JM, Wang Y, Karumanchi SA, et al. Associations of pregnancy characteristics with maternal and cord steroid hormones, angiogenic factors, and insulin-like growth factor axis. *Cancer Causes* Control. 2011;22:1587–1595. + +41. Henderson BE, Bernstein L, Ross RK, Depue RH, Judd HL. The early in utero oestrogen and testosterone environment of blacks and whites: potential effects on male offspring. *Br J Cancer.* 1988;57:216–218. + +42. Preston EV, Fruh V, Quinn MR, et al. Endocrine disrupting chemical-associated hair product use during pregnancy and gestational age at delivery: a pilot study. *Environ Health*. 2021;20:86. + +43. Huang T, Saxena AR, Isganaitis E, James-Todd T. Gender and racial/ethnic differences in the associations of urinary phthalate metabolites with markers of diabetes risk: National Health and Nutrition Examination Survey 2001-2008. *Environ Health.* 2014;13:6. + +44. Bellavia A, Zota AR, Valeri L, James-Todd T. Multiple mediators approach to study environmental chemicals as determinants of health disparities. *Environ Epidemiol.* 2018;2:e015. + +45. Braun JM, Kalloo G, Chen A, et al. Cohort profile: the Health Outcomes and Measures of the Environment (HOME) study. *Int J Epidemiol.* 2017;46:24. + +46. Jackson-Browne MS, Papandonatos GD, Chen A, et al. Identifying vulnerable periods of neurotoxicity to triclosan exposure in children. + +Environ Health Perspect. 2018;126:057001. + +47. Stacy SL, Eliot M, Etzel T, et al. Patterns, variability, and predictors of urinary triclosan concentrations during pregnancy and childhood. Environ Sci Technol. 2017;51:6404–6413. + +48. Meeker JD, Ferguson KK. Urinary phthalate metabolites are associated with decreased serum testosterone in men, women, and children from NHANES 2011-2012. *J Clin Endocrinol Metab.* 2014;99:4346–4352. + +49. Hornung RW, Reed LD. Estimation of average concentration in the presence of nondetectable values. *Appl Occup Environ Hyg*. 1990;5:46–51. + +50. Larsen K. Creatinine assay by a reaction-kinetic principle. *Clin Chim* Acta. 1972;41:209–217. + +51. Jackson-Browne MS, Papandonatos GD, Chen A, Yolton K, Lanphear BP, Braun JM. Early-life triclosan exposure and parent-reported behavior problems in 8-year-old children. *Environ Int.* 2019;128:446–456. + +52. Measurement Error in Nonlinear Models *: A Modern Perspective*, +Second Edition. Chapman and Hall/CRC; 2006. + +53. Wechsler D. *Manual for the Weschler Intelligence Scale For ChildrenThird Edition (WISC-III)*. The Psychological Corporation; 1991. + +54. Wechsler D. The Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III). The Psychological Corporation; 2002. + +55. Goldstein S, Naglieri JA (eds). *WISC-IV. In. Encyclopedia of Child* Behavior and Development, Springer; 2011. + +56. Werner EF, Braun JM, Yolton K, Khoury JC, Lanphear BP. The association between maternal urinary phthalate concentrations and blood pressure in pregnancy: the HOME study. *Environ Health.* 2015;14:75. + +57. van der Laan MJ, Polley EC, Hubbard AE. Super learner. *Stat Appl* Genet Mol Biol. 2007;6:Article25. + +58. Braun JM, Kalkbrenner AE, Just AC, et al. Gestational exposure to endocrine-disrupting chemicals and reciprocal social, repetitive, and stereotypic behaviors in 4- and 5-year-old children: the HOME study. Environ Health Perspect. 2014;122:513–520. + +59. Benowitz NL, Bernert JT, Caraballo RS, Holiday DB, Wang J. Optimal serum cotinine levels for distinguishing cigarette smokers and nonsmokers within different racial/ethnic groups in the United States between 1999 and 2004. *Am J Epidemiol.* 2009;169:236–248. + +60. David W. *Wechsler Abbreviated Scale of Intelligence* | Second Edition. The Psychological Corporation; 1999. Accessed June 9, 2021. https://www. + +pearsonassessments.com/store/usassessments/en/Store/ProfessionalAssessments/Cognition-%26-Neuro/Wechsler-Abbreviated-Scale-ofIntelligence-%7C-Second-Edition/p/100000593.html 61. Hintze JL, Nelson RD. Violin plots: a box plot-density trace synergism. + +Am Stat. 1998;52:181. + +62. Tingley D, Yamamoto T, Hirose K, et al. Causal mediation analysis; 2019. https://imai.princeton.edu/projects/mechanisms.html 63. Baron RM, Kenny DA. The moderator-mediator variable distinction in social psychological research: conceptual, strategic, and statistical considerations. *J Pers Soc Psychol.* 1986;51:1173–1182. + +64. Caldwell BM, Bradley RH. The home observation for measurement of the environment revisited. *Child Adolesc Ment Health*. 1978;9:25–35. + +65. Braun JM, Hornung R, Chen A, et al. Effect of residential lead-hazard interventions on childhood blood lead concentrations and neurobehavioral outcomes: a randomized clinical trial. *JAMA Pediatr.* +2018;172:934–942. + +66. Jones DR, Jarrett JM, Tevis DS, et al. Analysis of whole human blood for Pb, Cd, Hg, Se, and Mn by ICP-DRC-MS for biomonitoring and acute exposures. *Talanta.* 2017;162:114–122. + +67. Sears CG, Braun JM, Ryan PH, et al. The association of traffic-related air and noise pollution with maternal blood pressure and hypertensive disorders of pregnancy in the HOME study cohort. *Environ Int.* +2018;121(pt 1):574–581. + +68. Diez Roux AV, Merkin SS, Arnett D, et al. Neighborhood of residence and incidence of coronary heart disease. *N Engl J Med.* 2001;345:99–106. + +69. R Core Team. *R: A Language and Environment for Statistical* Computing. R Foundation for Statistical Computing; 2020. https:// www.R-project.org/. + +70. James-Todd TM, Meeker JD, Huang T, et al. Racial and ethnic variations in phthalate metabolite concentration changes across full-term pregnancies. *J Expo Sci Environ Epidemiol.* 2017;27:160–166. + +71. Calafat AM, Silva MJ, Reidy JA, et al. Mono-(3-carboxypropyl) phthalate, a metabolite of di-n-octyl phthalate. *J Toxicol Environ Health A.* +2006;69:215–227. + +72. Wallner P, Kundi M, Hohenblum P, Scharf S, Hutter HP. Phthalate metabolites, consumer habits and health effects. *Int J Environ Res* Public Health. 2016;13:E717. + +73. Bloom MS, Wenzel AG, Brock JW, et al. Racial disparity in maternal phthalates exposure; association with racial disparity in fetal growth and birth outcomes. *Environ Int.* 2019;127:473–486. + +74. James-Todd TM, Chiu YH, Zota AR. Racial/ethnic disparities in environmental endocrine disrupting chemicals and women's reproductive health outcomes: epidemiological examples across the life course. *Curr* Epidemiol Rep. 2016;3:161–180. + +75. Varshavsky JR, Zota AR, Woodruff TJ. A novel method for calculating potency-weighted cumulative phthalates exposure with implications for identifying racial/ethnic disparities among U.S. reproductive-aged women in NHANES 2001-2012. *Environ Sci Technol.* +2016;50:10616–10624. + +76. Branch F, Woodruff TJ, Mitro SD, Zota AR. Vaginal douching and racial/ethnic disparities in phthalates exposures among reproductive-aged women: National Health and Nutrition Examination Survey 2001-2004. *Environ Health.* 2015;14:57. + +77. James-Todd T, Connolly L, Preston EV, et al. Hormonal activity in commonly used Black hair care products: evaluating hormone disruption as a plausible contribution to health disparities. J Expo Sci Environ Epidemiol. 2021;31:476–486. + +78. Chan M, Mita C, Bellavia A, Parker M, James-Todd T. Racial/ethnic disparities in pregnancy and prenatal exposure to endocrine-disrupting chemicals commonly used in personal care products. *Curr Environ* Health Rep. 2021;8:98–112. + +79. Buckley JP, Palmieri RT, Matuszewski JM, et al. Consumer product exposures associated with urinary phthalate levels in pregnant women. J Expo Sci Environ Epidemiol. 2012;22:468–475. + +80. Twilhaar ES, Wade RM, de Kieviet JF, van Goudoever JB, van Elburg RM, Oosterlaan J. Cognitive outcomes of children born extremely or very preterm since the 1990s and associated risk factors: a meta-analysis and meta-regression. *JAMA Pediatr.* 2018;172:361–367. + +81. Zota AR, Shamasunder B. The environmental injustice of beauty: framing chemical exposures from beauty products as a health disparities concern. *Am J Obstet Gynecol.* 2017;217:418.e1–418.e6. + +82. Krieger N. Discrimination and health inequities. *Int J Health Serv.* +2014;44:643–710. + +83. Bailey ZD, Krieger N, Agénor M, Graves J, Linos N, Bassett MT. + +Structural racism and health inequities in the USA: evidence and interventions. *Lancet.* 2017;389:1453–1463. + +84. Parameswaran R, Cardoza K. Melanin on the margins: advertising and the cultural politics of fair/light/white beauty in India. *J Commun* Monogr. 2009;11:213–274. + +85. Bristor JM, Lee RG, Hunt MR. Race and ideology: African-American images in television advertising. *J Public Policy Marketing*. 1995;14:48–59. + +86. Bristor JM, Lee RG, Hunt MR. *Race and Ideology: African-American* Images in Television Advertising; 1995. Accessed February 22, 2021. https://journals.sagepub.com/doi/abs/10.1177/074391569501400105 87. Vreugdenhil HJ, Lanting CI, Mulder PG, Boersma ER, Weisglas-Kuperus N. Effects of prenatal PCB and dioxin background exposure on cognitive and motor abilities in Dutch children at school age. *J Pediatr.* +2002;140:48–56. + +88. Kalloo G, Wellenius GA, McCandless L, et al. Chemical mixture exposures during pregnancy and cognitive abilities in school-aged children. + +Environ Res. 2021;197:111027. + +89. Adibi JJ, Whyatt RM, Williams PL, et al. Characterization of phthalate exposure among pregnant women assessed by repeat air and urine samples. *Environ Health Perspect.* 2008;116:467–473. + +90. Braun JM, Smith KW, Williams PL, et al. Variability of urinary phthalate metabolite and bisphenol A concentrations before and during pregnancy. *Environ Health Perspect.* 2012;120:739–745. \ No newline at end of file diff --git a/medical/md/PMC9128869.md b/medical/md/PMC9128869.md new file mode 100644 index 0000000000000000000000000000000000000000..66454b93f33530619461e707ad29f4adde408cee --- /dev/null +++ b/medical/md/PMC9128869.md @@ -0,0 +1,288 @@ +# Review Article + +![0_image_0.png](0_image_0.png) + +# Challenges In Clinical Research And Care In Pruritus + +Manuel P. PEREIRA1, Claudia ZEIDLER1, Michael STORCK2, Konstantin AGELOPOULOS1, Wolfgang G. PHILIPP-DORMSTON3, Alexander ZINK4 and Sonja STÄNDER1 1Department of Dermatology and Center for Chronic Pruritus, University Hospital Münster, 2Institute of Medical Informatics, University of Münster, Münster, 3Faculty of Health, University Witten/Herdecke, and 4Department of Dermatology and Allergy, School of Medicine, Technical University of Munich, Munich, Germany Acta DermatoVenereologica Advances in dermatology and venereology Chronic pruritus is a frequent global condition. The pathophysiology, underlying aetiology, clinical manifestation, associated burden and response to therapy of chronic pruritus varies from patient to patient, making clinical research and management of this condition challenging. There are still several unmet needs, such as the need to standardize translational research protocols, diagnostic and therapeutic procedures and to enhance the knowledge of the humanistic and economic burden associated with chronic pruritus. Basic and clinical research is of the utmost importance to target these matters. Clinical research has the potential to identify new relevant mechanisms in affected patients, which may lead to identification of novel therapy targets. This article discusses in depth current shortcomings in the daily care of patients with chronic pruritus and the challenges clinical researchers and physicians treating chronic pruritus face in addressing these matters. + +Key words: itch; patient-reported outcome; guideline; clinical trials; clinical research. Accepted Oct 15, 2019; Published Jan 9, 2020 Acta Derm Venereol 2020; 100: adv00028. + +Corr: Sonja Ständer, Department of Dermatology and Center for Chronic Pruritus, University Hospital Münster, Von-Esmarch-Str. 58, DE-48149 Münster, Germany. E-mail: Sonja.Staender@ukmuenster.de Chronic pruritus (CP), defined as pruritus lasting for 6 weeks or longer, is highly prevalent, affecting approximately one-sixth of the population in Germany +(1). Population-based analyses of the nationwide search volume of pruritus on Google suggest an even larger number of individuals suffering from CP within Germany and the USA (2–4). Those affected often report a substantial burden and a relevant impairment of their quality of life (QoL) (5, 6). The aetiology, clinical manifestation, diagnostic measures and therapeutic response to CP vary from patient to patient, which contributes to the challenging management of CP (7). Although recent years have witnessed a marked increase in basic and clinical research efforts in this field, CP researchers and physicians treating CP currently face several challenges when targeting unmet needs that impact on clinical routine. The aim of this review is to highlight and discuss + +## Significance + +Itch lasting for 6 weeks or more is considered chronic and represents a high burden for those affected. Various aspects of chronic itch, including the underlying origin of the itch, symptoms, skin manifestations, response to therapies and impairment of quality of life vary from patient to patient, constituting a challenge for clinicians and clinical researchers. Unmet needs, such as the standardization of experimental and clinical research protocols, diagnostic procedures and therapeutic regimens, as well as a better understanding of associated burdens and the development of novel effective therapies should be targeted by physicians and researchers dealing with chronic itch. + +methodologies used in CP research, as well as current challenges and unmet needs in clinical care. + +## Chronic Pruritus: A Multidimensional Condition + +CP is a heterogeneous condition in terms of demographics, clinical presentation and underlying origin. Although the prevalence of CP increases with age, patients of all age groups, including children, may be affected +(8). Moreover, patients of both sexes and all ethnic backgrounds may suffer from CP. According to the International Forum for the Study of Itch (IFSI), CP may present on inflamed skin (IFSI I), on normal appearing skin (IFSI II), or accompanied by chronic scratch lesions, such as chronic prurigo or lichen simplex chronicus (IFSI III) (9). As for the underlying aetiology, CP may arise from dermatological, systemic, neurological or psychiatric/ psychosomatic conditions. Some patients show multiple causes for the pruritus (multifactorial CP), while in few cases the origin of the pruritus remains unknown despite extensive diagnostic work-up (9). Recent studies have further suggested a substantial impact of climate and weather on the prevalence of pruritus, especially involving specific localizations on the body, but these issues have yet to be examined in epidemiological studies (2–4). Adding to these factors, patients with CP vary in terms of medical history, comorbidities, co-medication, socioeconomic backgrounds and therapy goals, contributing to the complexity of the management of these patients. + +Acta Derm Venereol 2020; 100: adv00028 This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta Journal Compilation © 2020 Acta Dermato-Venereologica. + +doi: 10.2340/00015555-3348 + +## Unmet Needs In Clinical Research + +Due to the complexity and multidimensional nature of CP, clinical research investigating several aspects of pruritic diseases and its management is needed. In particular, clinical research contributes to a better understanding of pathophysiological mechanisms involved in the development and chronicity of CP in humans. Moreover, clinical researchers should focus on various aspects of clinical management, such as standardizing diagnostic and therapeutic procedures, and assessing the humanistic burden and impact of CP on affected patients. Another important area is the performance of longitudinal translational studies, clinical trials investigating novel agents for the treatment of CP, and psychometric research. It should also be investigated how the care of patients with CP is integrated into the health system in order to identify shortcomings and to adopt innovative strategies, as, for example, digital tools to improve care. The next section of this article discusses the challenges and difficulties with regard to these issues. + +## Challenges In Clinical Research And Care Understanding The Pathophysiology + +Basic and clinical research has led in recent decades to a better understanding of the mechanisms underlying CP. + +In clinical-translational research, different methods investigating specific pathophysiological features that augment data obtained from morphological and molecular biological research have been established and validated. The collaboration with biostatisticians and experts of medical informatics is essential for the interpretation of data and for obtaining a comprehensive understanding of the results. An overview of these methods is given in **Table I**. + +Several methodologies have been developed in order to investigate the functions of peripheral nerves. Skin stimulation with pruritogens (e.g. histamine, capsaicin, cowhage, β-alanine) has led to the characterization of several subpopulations of C-fibres. Histamine activates mechano-insensitive C-fibres (CMi-fibres), while cowhage and β-alanine activate heat and mechano-sensitive C-fibres (CMH-fibres) (10). Hyperknesis related to cowhage-induced pruritus, but not histaminergic pruritus, is present in several types of CP compared with controls, arguing for sensitization of CMH-fibres in CP (11). One limitation of this methodology is the high inter-individual variability, while intra-individual variability is low (12). Another method, transcutaneous electrical stimulation, allows the selective activation of subpopulations of peripheral nerve fibres. For example, using the Neurometer® C, Aδ and Aβ fibres are activated by electrical stimulation at a frequency of 5, 250 and 2,000 Hz, respectively (13). Higher itch intensities could be induced upon stimulation at 5 and 2,000 Hz in patients with CP of different origins compared with healthy individuals, arguing for hyperknesis in patients with CP (14). Of note, patients are often anxious when undergoing experimental procedures with electrical stimulation, which leads to biased ratings of evoked sensory symptoms, thus constituting a limitation of this method. Another functional well-established diagnostic tool that is used for clinical itch research is quantitative sensory testing (QST). It is a battery of psychophysical tests, in which response to graded thermal and mechanical stimuli is assessed (15). This procedure, originally developed for determination of neuropathic pain, informs about gain or loss of function of different peripheral nerve fibre subpopulations and hints at possible signs of central sensitization. In itch research it could be shown that patients with CP of different origins have an altered QST profile, arguing for disordered peripheral neuronal mechanisms +(11). Although QST is widely used in neurological routine care, it has some limitations. It is time-consuming, requires highly qualified personnel, as well as the collaboration of patients. Morphological methodologies for investigation of the anatomy of cutaneous cells and expression of receptors and mediators are essential in clinical itch research. In particular, determination of the intraepidermal nerve fibre density is useful in order to reveal potential epidermal neuroanatomical alterations (16). In this examination, a skin biopsy is obtained and intraepidermal nerves are stained with the axonal marker protein gene product 9.5 (17). This simple method, which is also used in routine care, has some limitations. + +Table I. Clinical research methodologies used in itch research + +| Methodology | Mechanism | +|-------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Cutaneous stimulation with histamine | Chemical-evoked itch via activation of mechano-insensitive C-fibres (CMi-fibres), mast cell degranulation | +| Cutaneous stimulation with cowhage or ß-alanine | Chemical-evoked itch via activation of mechano and heat-sensitive C-fibres (CMH-fibres) as well as A-fibres. | +| Hyperknesis | Increased itch in response to (chemical, electrical) pruritogens or pinprick stimulation | +| Alloknesis | Itch response to non-pruritogenic stimuli: touch/brush strokes. Functional testing of itch sensitization phenomena with punctuate or dynamic mechanical stimulation (von Frey filaments, cotton swab) | +| Transcutaneous electrical stimulation | Electrical-evoked itch via selective activation of peripheral nerve fibre classes | +| Quantitative sensory testing | Psychophysical testing of subpopulations of peripheral nerve fibres (C-fibres, Aδ-fibres, Aβ-fibres) and signs of central sensitization using graded thermal and mechanical stimuli | +| Functional magnetic resonance imaging/positron | Identification of activated and deactivated cerebral centres upon pruritic stimulation | +| emission tomography | | +| Conditioned pruritic modulation | Central descending inhibition of pruritus | +| Intraepidermal nerve fibre density | Morphological determination of epidermal nerve fibre numbers (skin biopsy) | + +Acta DermatoVenereologica The skin probe is sensitive to the fixative and needs to be processed in a timely manner in the laboratory. In addition, reference values are available only for the distal lower leg (18, 19), thus interpretation of values from other body sites is difficult. It is a purely morphological examination, which does not inform on functional alterations of peripheral nerve fibres. + +Central processing of pruritus is very challenging. + +Most current knowledge on spinal transmission of itch was obtained from animal studies (20). Functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) studies have been used to investigate transmission and processing of pruritus in upper centres. Most studies have been performed on healthy volunteers exposed to pruritic stimulation during the scans. Brain regions associated with sensory recognition, cognition, motor response and emotional states are activated in response to pruritus (21). Interestingly, both overlapping and distinct brain areas are activated upon histaminergic and non-histaminergic pruritic stimulation, pointing to differences in cerebral processing of distinct pruritus types (22). Patients with CP also show differences in brain activation upon stimulation with histamine compared with healthy individuals, arguing for central sensitization in CP (23). Also, structural changes in grey matter have been observed in patients with CP, showing the neuroplasticity in chronic states (24). Although functional imaging studies have enhanced our understanding of cerebral processing of pruritus, this methodology has some pitfalls. fMRI and PET are very expensive methodologies, which are available only in specialized centres. + +In addition, patients with claustrophobia, ferromagnetic prostheses or implanted devices (e.g. defibrillator) cannot undergo an MRI examination, while others (e.g. children) have difficulty lying still during the procedure, leading to poor imaging quality. More imaging studies, enrolling patients with CP of different origins, are needed in order to establish similarities and differences in cerebral brain processing of pruritus across distinct CP conditions. + +Upper centres exert a descending inhibitory modulation via a noradrenergic and a serotonergic pathway, as shown by imaging studies (21, 25). The paradigm of conditioned pain modulation (CPM), in which a noxious stimulus inhibits pain elsewhere in the body, can be used experimentally to assess descending inhibition (26). In patients with CP, as in those with chronic pain, CPM is impaired, which may contribute to the chronicity of the pruritus (11). An analogous paradigm of conditioned pruritic modulation, in which a pruritic stimulus inhibits pruritus elsewhere in the body, has also been investigated, with conflicting results regarding its effect (27, 28). + +More studies are needed to enhance the understanding of the mechanisms of descending modulation in CP and its role in the chronicity of the disease. + +## Standardization Of Assessment Instruments + +Since pruritus has a subjective dimension, patientreported outcomes (PRO) play a pivotal role. A plethora of standardized questionnaires and scales have been developed in order to gather information on several aspects of CP, including pruritus characteristics, course of the disease, QoL, reactive disorders, such as anxiety, depression and sleep impairment, and therapy goals. + +Pruritus characteristics (e.g. onset, duration and distribution of pruritus, as well as accompanying sensory symptoms and scratching behaviour) can be assessed using general patient-oriented itch questionnaires (29, 30). Mono-dimensional scales, namely the visual analogue scale, the numerical rating scale and the verbal rating scale, have been validated to assess the intensity of pruritus (31). Intensity scales are also validated for the use in electronic diary applications (32). The interpretation of itch intensity scales is challenging, since scores vary from patient to patient according to external factors, such as sex or ethnic background (1, 33). Some efforts have been put into understanding the minimal clinical relevant difference of itch intensity change, e.g. after initiating a therapy (34). However, many factors (e.g. the aetiology of the pruritus, recall period, socioeconomic background) may influence the minimal clinical relevant difference, making the interpretation of results difficult. + +An overview of other instruments for the assessment of itch is shown in **Table II**. + +As for secondary conditions arising due to CP, standardized questionnaires are used to screen for anxiety and + +| Table II. Examples of instruments for itch assessment Instrument Description Visual analogue scale (VAS) Monodimensional scale for assessment of itch intensity; used in clinical trials and routine care (electronic and paper versions available) Numerical rating scale (NRS) Monodimensional scale for assessment of itch intensity; used in clinical trials and routine care (electronic and paper versions available) Verbal rating scale (VRS) Categorial scale for assessment of itch intensity; used in clinical trials and routine care (electronic and paper versions available) Labelled magnitude scale (LMS) Monodimensional scale for assessment of itch intensity; used in experimental research (paper version) Itch Severity Scale (ISS) Multidimensional scale for assessment of itch intensity; used in clinical trials (paper version) Dynamic Pruritus Score (DPS) Patient global impression of change for assessment of itch improvement; used in clinical trials and routine care (electronic and paper versions available) 5-D itch scale (5 dimensions itch Multidimensional questionnaire for assessment of itch characteristics; used in clinical trials (paper version) scale) ItchyQuant Monodimensional scale for assessment of itch intensity; combination of VAS and cartoon depictions of scratching; developed for populations with cognitive limitations (elderly, children); used in routine care (paper version) | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Acta DermatoVenereologica Advances in dermatology and venereology depression (e.g. Hospital Anxiety and Depression Scale (35)), to assess sleep disorders (36, 37) and to measure the impairment of QoL (e.g. Dermatological Life Quality Index (38) and pruritus-specific ItchyQol (39)). + +PRO measures are usually collected via paper and pencil questionnaires, but electronic PRO systems are emerging. Computerized collection of PRO measures offers a couple of advantages compared with paper questionnaires: reduction of errors emerging through typewriting, reduction of missing data by requiring completion, and reduction of invalid data by implementation of skip patterns (40). Furthermore, electronic assessment can increase the compliance of completing questionnaires at home by up to 70%, using, for example, computerized reminder functionality (40). To overcome the drawbacks of paper questionnaires, an electronic PRO system, enabling the patient to complete the PRO measures via digital survey, may be implemented (41–43). Since most PRO measures have been validated as paper tools, it cannot be assumed that the electronic version of the same PRO measures deliver the same results for a patient encounter. Gwaltney et al. (40) showed that "as long as substantial changes are not made to the item text or response scales, equivalence studies should not be necessary to demonstrate anew the equivalence or validity of a computerized measure". However, in order to ensure the validity of electronic measures, it would be preferable if the concrete implementations were examined using test–retest or alternate-forms reliability. + +Standardization of itch assessment instruments across centres treating patients with CP is of great importance, since it would allow the comparability of data. A first step was already taken by the Task Force Pruritus of the European Academy of Dermatology and Venereology. In a consensus conference, it was agreed that pruritus intensity and QoL should be regarded as the 2 most important parameters to be assessed in routine care. The visual analogue scale and the ItchyQol were regarded as the instruments of choice (44). Another important task is the validation of the assessment tools into various languages in order to be used in clinical trials and routine care across different countries. Future clinical research should focus on showing which instruments have better utility for the clinical practice and which tools are better accepted by patients and physicians. + +## Understanding The Humanistic And Economic Burden + +CP, similarly to chronic pain, can lead to a severe impairment of QoL (45–47) and has negative effects on mood, ability to concentrate, quality of sleep, everyday life and work productivity (48, 49). Some patients have mental health problems, such as depression, in response to CP, and are at significantly higher risk of suicide (50, 51). QoL is a highly subjective construct and is influenced by various factors, such as pruritus intensity, but also duration, frequency and localization of pruritus. Another challenge in understanding the impact of pruritic conditions on affected patients is that cross-cultural factors influence the reported intensity of pruritus and QoL rather than the specific dermatological diagnosis. This could be found in a study in 9 European countries enrolling more than 500 patients with CP due to different dermatoses +(6). Clinical research should take the cultural background of patients with CP into account when investigating the humanistic burden of pruritic conditions. + +In addition to limiting QoL, there are often psychiatric complications. Patients with CP and with a psychosomatic/psychiatric comorbidity suffer more from CP and its consequences. Interestingly, even if their symptoms improve, psychological suffering is higher in these patients (52). In order to cope with these associated disorders, individual multimodal medical care is needed, not only for diagnosing the underlying disease of CP and for symptomatic antipruritic therapy, but also for adjunct therapy against, for example, sleep disorders or mental health conditions. An important challenge for clinical researchers is to detect risk factors for developing mental diseases in patients with CP and thus identify patients at risk. This would aid attending physicians in redirecting vulnerable patients to mental health professionals and ultimately to improve care. + +## Definition Of Treatment Goals + +As a multifactorial chronic disease, CP requires complex and cost-intensive therapy. Knowledge of the treatment goals of patients with CP is therefore of great importance in order to plan an efficient therapy regimen. Therapy goals were recorded and evaluated in 2,474 patients with CP using the Patient Benefit Index - Pruritus (PBI-P), which was validated in 2009 for patients with CP and consists of 27 items, including various aspects, such as physical and mental well-being, professional and everyday performance, social and leisure activities, and QoL +in general. The relevance of these items is calculated on the basis of a 5-step Likert scale (0="not at all important" to 4="very important") (53). It was shown that, in addition to demographic data such as sex and age, the CP +IFSI group, pruritus intensity and QoL had a significant impact on different patient needs. Women considered the reduction in physical and psychological symptoms, such as depressive feeling, nervousness, or burning sensations, as more important (54). For men, an important goal was the improvement in aspects of social life, such as social contacts, partnership, and sex life. Patients with CP on inflamed skin (IFSI I) or pruritus with chronic scratch lesions (IFSI III) considered the healing of the skin lesions as a very important treatment goal (54). Overall, it has been shown that therapeutic goals relate to the diagnosis and medical therapy. Reduction in pruritus or confidence in therapy were considered important or very important Acta DermatoVenereologica Advances in dermatology and venereology by the majority of patients with CP. However, patients with CP focus not only on symptom relief, but also on trustworthy and efficient medical action. Their overall need level seems to be higher than in other dermatological patients as, for example, patients with atopic dermatitis (54). The complexity of patients' needs constitute a challenge for clinical researchers, since patients' needs and goals influence how they perceive their disease and the efficacy of a therapy. Patients' needs should thus be taken into consideration when planning studies involving patients with CP. + +## Standardization Of Diagnostic And Therapeutic Procedures + +Owing to the heterogeneity of CP conditions, it is difficult to achieve a standardized diagnostic and therapeutic approach across specialized centres and physicians treating patients with CP. The first guideline addressing CP was published in Germany in 2006 (55) and constituted an important first step in this regard. Since then, the German guideline has been regularly updated (56), while a European guideline was also developed and recently updated (57). + +There is still a lack of randomized controlled trials investigating anti-pruritic drugs, and therefore many of the recommendations of current guidelines are based on case series, case reports and expert opinion. Another challenge that guidelines face is the heterogeneity of CP aetiologies. For instance, while antihistamines are effective in the treatment of urticaria, they do not reduce pruritus in other diseases, such as atopic dermatitis or systemic pruritic conditions (58). Therefore specific therapeutic recommendations are needed for the various pruritic conditions. Clinical trials targeting different pruritic diseases (e.g. atopic dermatitis, uraemic pruritus, cholestatic pruritus, paraneoplastic pruritus) are of the utmost importance in order to generate quality data on which to base therapeutic recommendations. + +Dermatologists are usually the first specialists consulted for CP and play a key role in the management of CP. + +Their job is not only to treat the CP, but also to assign the patient to local physicians of other specialties for specific diagnostic procedures (e.g. medical imaging). However, due to time constraints, taking a comprehensive medical history in patients with CP is challenging in a dermatological practice. Reactive disorders of CP, such as depression and anxiety, need to be considered, as possible causative factors and skin findings, comorbidities and medication must be well documented. The consultation of patients with CP in specialized pruritus centres can offer an important advantage in the management of CP, especially when refractory to basic therapeutic measures, such as antihistamines, topical steroids and the use of emollients. However, the centres are scarce and more are needed for the current demand (7). Pruritus centres should work on an interdisciplinary basis together with medical specialties other than dermatology, such as internal medicine, neurology, pain medicine, psychosomatics, radiology, and medical informatics (59). In addition to outpatient care, specialized centres should also offer the possibility of inpatient care for highly complex patients who require extensive diagnostic procedures or who have relevant psychosocial factors, such as suicidal ideation or severe sleep impairment (59). + +Guideline-based treatment of patients with CP at specialized centres can significantly reduce costs, both in the inpatient and outpatient sectors. This is the result of a recent study in which data from 300 patients with CP were analysed regarding their QoL, health economic burden and therapeutic benefits. Six months after the start of treatment at a specialized pruritus centre there was not only a significant improvement in the pruritus intensity, QoL and therapeutic benefit (PBI-P), but also a significant reduction in all costs (60). + +## Need For Novel Effective Therapies + +Owing to a better understanding of the mechanisms underlying CP, new promising agents with an anti-pruritic effect have been identified and are being tested in randomized controlled trials. These include monoclonal antibodies (e.g. nemolizumab, tralokinumab), neurokinin-1 receptor antagonists (e.g. serlopitant, aprepitant), opioid modulators (e.g. nalbuphine, nalfurafine), phosphodiesterase-4 inhibitors (e.g. apremilast, crisaborole), janus kinase inhibitors (e.g. tofacitinib) among other novel agents. + +The development of innovative drugs faces important challenges. So far, clinical trials have been performed only for a few indications, especially atopic dermatitis, chronic prurigo, uraemic and cholestatic pruritus. For many other pruritic conditions clinical trials are lacking, and thus no novel drugs are available. + +Since these new agents represent a high cost for the healthcare system, it is important to select the patients who can profit the most from them. Clinical trials and observations from routine care should inform which target population is suitable for each drug. + +At present, innovative drugs being tested in clinical trials are available in only a few centres and thus not all patients have access to them. Licensing of these drugs is needed in order to extend the availability of these promising agents to patients in need. Hence, clinical research should focus on producing high-quality data on the safety and efficacy of novel anti-pruritic drugs, so that regulatory agencies can approve these medications. + +## Conclusion + +Clinical research efforts in CP have increased in recent years, leading to better understanding of this condition, and ultimately to better care. However, the multidimensional nature of this condition and the heterogeneous population affected by CP pose a challenge for clinical researchers and attending physicians. Unmet needs, such as the shortage of knowledge on chronicity mechanisms, insufficient standardization of a diagnostic and therapeutic approach to CP patients, and the development of novel promising drugs for refractory CP, ought to be targeted by researchers and physicians dealing with CP. + +## Acknowledgements + +This article was supported by the German Research Foundation +(DFG, No. FOR2690 to KA and SST) and by the European Academy of Dermatology and Venereology (EADV, No. 2016-012 to MP and SST). The authors have no conflicts of interest to declare. + +## References + +1. Matterne U, Apfelbacher CJ, Loerbroks A, Schwarzer T, +Buttner M, Ofenloch R, et al. Prevalence, correlates and characteristics of chronic pruritus: a population-based crosssectional study. Acta Derm Venereol 2011; 91: 674–679. + +2. Zink A, Schuster B, Ruth M, Pereira MP, Philipp-Dormston WG, Biedermann T, et al. Medical needs and major complaints related to pruritus in Germany: a 4-year retrospective analysis using Google AdWords Keyword Planner. J Eur Acad Dermatol Venereol 2019; 33: 151–156. + +3. Tizek L, Schielein M. Influence of climate on Google internet searches for pruritus across 16 German cities: retrospective analysis. J Med Internet Res 2019; 21: e13739. + +4. Wongvibulsin S, Khanna R, Kwatra SG. Anatomic localization and quantitative analysis of the burden of itch in the United States. J Am Acad Dermatol 2019. pii: S01909622(19)30993-4. + +5. Hay RJ, Johns NE, Williams HC, Bolliger IW, Dellavalle RP, +Margolis DJ, et al. The global burden of skin disease in 2010: an analysis of the prevalence and impact of skin conditions. + +J Invest Dermatol 2014; 134: 1527–1534. + +6. Steinke S, Zeidler C, Riepe C, Bruland P, Soto-Rey I, Storck M, +et al. Humanistic burden of chronic pruritus in patients with inflammatory dermatoses: results of the European Academy of Dermatology and Venereology Network on Assessment of Severity and Burden of Pruritus (PruNet) cross-sectional trial. J Am Acad Dermatol 2018; 79: 457–463.e455. + +7. Pereira MP, Steinke S, Bruland P, Ständer H, Dugas M, Augustin M, et al. Management of chronic pruritus: from the dermatological office to the specialized itch center. a review. Itch 2017; 2: e06. + +8. Matterne U, Apfelbacher CJ, Vogelgsang L, Loerbroks A, +Weisshaar E. Incidence and determinants of chronic pruritus: a population-based cohort study. Acta Derm Venereol 2013; 93: 532–537. + +9. Ständer, Weisshaar E, Mettang T, Szepietowski JC, Carstens E, Ikoma A, et al. Clinical classification of itch: a position paper of the International Forum for the Study of Itch. Acta Derm Venereol 2007; 87: 291–294. + +10. Johanek LM, Meyer RA, Hartke T, Hobelmann JG, Maine DN, +LaMotte RH, et al. Psychophysical and physiological evidence for parallel afferent pathways mediating the sensation of itch. J Neurosci 2007; 27: 7490–7497. + +11. Pogatzki-Zahn EM, Pereira MP, Cremer A, Zeidler C, Dreyer T, +Riepe C, et al. Peripheral sensitisation and loss of descending inhibition is a hallmark of chronic pruritus. J Invest Dermatol 2019; pii: S0022-202X(19)31851-2. + +12. Andersen HH, Sorensen AR, Nielsen GA, Molgaard MS, Stilling P, Boudreau SA, et al. A Test-retest reliability study of human experimental models of histaminergic and non-histaminergic itch. Acta Derm Venereol 2017; 97: 198–207. + +13. AAEM Equipment and Computer Committee AAoEM. Technology review: the Neurometer Current Perception Threshold +(CPT). Muscle Nerve 1999; 22: 523–531. + +14. Pereira MP, Agelopoulos K, Kollner J, Neufang G, Schmelz M, Ständer. Selective nerve fibre activation in patients with generalized chronic pruritus: hint for central sensitization? + +Acta Derm Venereol 2019; 99: 1009–1015. + +15. Rolke R, Baron R, Maier C, Tolle TR, Treede RD, Beyer A, +et al. Quantitative sensory testing in the German Research Network on Neuropathic Pain (DFNS): standardized protocol and reference values. Pain 2006; 123: 231–243. + +16. Lauria G, Hsieh ST, Johansson O, Kennedy WR, Leger JM, +Mellgren SI, et al. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society. Eur J Neurol 2010; 17: 903–912, e944–909. + +17. Lauria G, Cornblath DR, Johansson O, McArthur JC, Mellgren SI, Nolano M, et al. EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy. Eur J Neurol 2005; 12: 747–758. + +18. Lauria G, Bakkers M, Schmitz C, Lombardi R, Penza P, Devigili G, et al. Intraepidermal nerve fiber density at the distal leg: a worldwide normative reference study. J Peripher Nerv Syst 2010; 15: 202–207. + +19. Provitera V, Gibbons CH, Wendelschafer-Crabb G, Donadio V, +Vitale DF, Stancanelli A, et al. A multi-center, multinational age- and gender-adjusted normative dataset for immunofluorescent intraepidermal nerve fiber density at the distal leg. + +Eur J Neurol 2016; 23: 333–338. + +20. Akiyama T, Carstens E. Spinal Coding of Itch and Pain. In: +Carstens E, Akiyama T, editors. Itch: mechanisms and treatment. Boca Raton, FL: CRC Press/Taylor & Francis Group, LLC.; 2014. + +21. Mochizuki H, Kakigi R. Central mechanisms of itch. Clin Neurophysiol 2015; 126: 1650–1660. + +22. Mochizuki H, Papoiu ADP, Yosipovitch G. Brain processing of itch and scratching. In: Carstens E, Akiyama T, editors. Itch: mechanisms and treatment. Boca Raton, FL: CRC Press/ +Taylor & Francis Group, LLC; 2014. + +23. Schneider G, Ständer, Burgmer M, Driesch G, Heuft G, +Weckesser M. Significant differences in central imaging of histamine-induced itch between atopic dermatitis and healthy subjects. Eur J Pain 2008; 12: 834–841. + +24. Papoiu AD, Emerson NM, Patel TS, Kraft RA, Valdes-Rodriguez R, Nattkemper LA, et al. Voxel-based morphometry and arterial spin labeling fMRI reveal neuropathic and neuroplastic features of brain processing of itch in end-stage renal disease. J Neurophysiol 2014; 112: 1729–1738. + +25. Mochizuki H, Tashiro M, Kano M, Sakurada Y, Itoh M, Yanai K. + +Imaging of central itch modulation in the human brain using positron emission tomography. Pain 2003; 105: 339–346. + +26. Talbot JD, Duncan GH, Bushnell MC, Boyer M. Diffuse noxious inhibitory controls (DNICs): psychophysical evidence in man for intersegmental suppression of noxious heat perception by cold pressor pain. Pain 1987; 30: 221–232. + +27. van Laarhoven AI, Kraaimaat FW, Wilder-Smith OH, van de Kerkhof PC, Evers AW. Heterotopic pruritic conditioning and itch - analogous to DNIC in pain? Pain 2010; 149: 332–337. + +28. Andersen HH, van Laarhoven AIM, Elberling J, Arendt-Nielsen L. Modulation of itch by conditioning itch and pain stimulation in healthy humans. J Pain 2017; 18: 1437–1450. + +29. Yosipovitch G, Zucker I, Boner G, Gafter U, Shapira Y, David M. A questionnaire for the assessment of pruritus: validation in uremic patients. Acta Derm Venereol 2001; 81: 108–111. + +30. Weisshaar E, Ständer, Gieler U, Matterne U, Darsow U. Entwicklung eines deutschsprachigen Fragebogens zur Erfassung von chronischen Pruritus (AGP-Fragebogen). Hautarzt 2011; 62: 914–927. + +31. Phan NQ, Blome C, Fritz F, Gerss J, Reich A, Ebata T, et al. + +Assessment of pruritus intensity: prospective study on validity and reliability of the visual analogue scale, numerical rating scale and verbal rating scale in 471 patients with chronic pruritus. Acta Derm Venereol 2012; 92: 502–507. + +32. Gernart M, Tsianakas A, Zeidler C, Riepe C, Osada N, Pihan D, et al. ItchApp(c): an app-based ediary for assessment of chronic pruritus in clinical trials. Acta Derm Venereol 2017; 97: 601–606. + +33. Ständer, Stumpf A, Osada N, Wilp S, Chatzigeorgakidis E, +Pfleiderer B. Gender differences in chronic pruritus: women present different morbidity, more scratch lesions and higher burden. Br J Dermatol 2013; 168: 1273–1280. + +34. Reich A, Riepe C, Anastasiadou Z, Medrek K, Augustin M, +Szepietowski JC, et al. Itch assessment with visual analogue scale and numerical rating scale: determination of minimal clinically important difference in chronic itch. Acta Derm Venereol 2016; 96: 978–980. + +35. Snaith RP. The Hospital Anxiety And Depression Scale. Health Qual Life Outcomes 2003; 1: 29. + +36. Glenville M, Broughton R. Reliability of the Stanford Sleepiness Scale compared to short duration performance tests and the Wilkinson Auditory Vigilance Task. Adv Biosci 1978; 21: 235–244. + +37. Johns MW. A new method for measuring daytime sleepiness: +the Epworth sleepiness scale. Sleep 1991; 14: 540–545. + +38. Finlay AY, Khan GK. Dermatology Life Quality Index (DLQI) - +a simple practical measure for routine clinical use. Clin Exp Dermatol 1994; 19: 210–216. + +39. Desai NS, Poindexter GB, Monthrope YM, Bendeck SE, +Swerlick RA, Chen SC. A pilot quality-of-life instrument for pruritus. J Am Acad Dermatol 2008; 59: 234–244. + +40. Gwaltney CJ, Shields AL, Shiffman S. Equivalence of electronic and paper-and-pencil administration of patient-reported outcome measures: a meta-analytic review. Value Health 2008; 11: 322–333. + +41. Rogausch A, Sigle J, Seibert A, Thuring S, Kochen MM, Himmel W. Feasibility and acceptance of electronic quality of life assessment in general practice: an implementation study. Health Qual Life Outcomes 2009; 7: 51. + +42. Storck M, Zeidler C, Rehr M, Riepe C, Dugas M, Ständer, et al. Validation of pruritus measures gathered with the electronic patient-reported outcome system MoPat. Acta Derm Venereol 2018; 98: 38–43. + +43. Wagner LI, Schink J, Bass M, Patel S, Diaz MV, Rothrock N, +et al. Bringing PROMIS to practice: brief and precise symptom screening in ambulatory cancer care. Cancer 2015; 121: 927–934. + +44. Ständer, Zeidler C, Riepe C, Steinke S, Fritz F, Bruland P, et al. European EADV network on assessment of severity and burden of Pruritus (PruNet): first meeting on outcome tools. J Eur Acad Dermatol Venereol 2016; 30: 1144–1147. + +45. Erturk IE, Arican O, Omurlu IK, Sut N. Effect of the pruritus on the quality of life: a preliminary study. Ann Dermatol 2012; 24: 406–412. + +46. Carr CW, Veledar E, Chen SC. Factors mediating the impact of chronic pruritus on quality of life. JAMA Dermatol 2014; 150: 613–620. + +47. Kini SP, DeLong LK, Veledar E, McKenzie-Brown AM, Schaufele M, Chen SC. The impact of pruritus on quality of life: the skin equivalent of pain. Arch Dermatol 2011; 147: 1153–1156. + +48. Kimball AB, Edson-Heredia E, Zhu B, Guo J, Maeda-Chubachi T, Shen W, et al. Understanding the relationship between pruritus severity and work productivity in patients with moderate-to-severe psoriasis: sleep problems are a mediating factor. J Drugs Dermatol 2016; 15: 183–188. + +49. Shutty BG, West C, Huang KE, Landis E, Dabade T, Browder B, et al. Sleep disturbances in psoriasis. Dermatol Online J 2013; 19: 1. + +50. Konda D, Chandrashekar L, Rajappa M, Kattimani S, Thappa DM, Ananthanarayanan PH. Serotonin and interleukin-6: +Association with pruritus severity, sleep quality and depression severity in Prurigo Nodularis. Asian J Psychiatr 2015; 17: 24–28. + +51. Halvorsen JA, Dalgard F, Thoresen M, Bjertness E, Lien L. + +Itch and pain in adolescents are associated with suicidal ideation: a population-based cross-sectional study. Acta Derm Venereol 2012; 92: 543–546. + +52. Schneider G, Grebe A, Bruland P, Heuft G, Ständer. Chronic pruritus patients with psychiatric and psychosomatic comorbidity are highly burdened: a longitudinal study. J Eur Acad Dermatol Venereol 2019; 33: e288–e291. + +53. Blome C, Augustin M, Siepmann D, Phan NQ, Rustenbach SJ, Ständer. Measuring patient-relevant benefits in pruritus treatment: development and validation of a specific outcomes tool. Br J Dermatol 2009; 161: 1143–1148. + +54. Steinke S, Bruland P, Blome C, Osada N, Dugas M, Fritz F, +et al. Chronic pruritus: evaluation of patient needs and treatment goals with a special regard to differences according to pruritus classification and sex. Br J Dermatol 2017; 176: 363–370. + +55. Ständer, Streit M, Darsow U, Niemeier V, Vogelgsang M, +Ständer H, et al. Diagnostisches und therapeutisches Vorgehen bei chronischem Pruritus. J Dtsch Dermatol Ges 2006; 4: 350–370. + +56. Ständer, Zeidler C, Augustin M, Bayer G, Kremer AE, Legat FJ, et al. S2k-Leitlinie zur Diagnostik und Therapie des chronischen Pruritus - Update - Kurzversion. J Dtsch Dermatol Ges 2017; 15: 860–873. + +57. Weisshaar E, Szepietowski JC, Dalgard FJ, Garcovich S, Gieler U, Gimenez-Arnau AM, et al. European S2k Guideline on Chronic Pruritus. Acta Derm Venereol 2019; 99: 469–506. + +58. Klein PA, Clark RA. An evidence-based review of the efficacy of antihistamines in relieving pruritus in atopic dermatitis. Arch Dermatol 1999; 135: 1522–1525. + +59. Ständer, Ständer HF, Steinke S, Bruland P, Dugas M, Augustin M. Chronischer Pruritus: Versorgung in der Praxis. Hautarzt 2016; 67: 640–647. + +60. Steinke S, Müller S, Gutknecht M, Borowski M, Augustin M, Ständer S, editors. Versorgung von Patienten mit chronischem Pruritus: Kosten und Nutzen der spezialisierten Versorgung in einem Kompetenzzentrum. 50. Tagung der Deutschen Dermatologischen Gesellschaft; Berlin, 2019. + +Advances in dermatology and venereology \ No newline at end of file diff --git a/medical/md/PMC9188956.md b/medical/md/PMC9188956.md new file mode 100644 index 0000000000000000000000000000000000000000..a19387ba13332f81e3a7ee1be0bf669554aaa2eb --- /dev/null +++ b/medical/md/PMC9188956.md @@ -0,0 +1,335 @@ +Contents lists available at ScienceDirect + +![0_image_0.png](0_image_0.png) + +![0_image_3.png](0_image_3.png) + +![0_image_4.png](0_image_4.png) + +![0_image_7.png](0_image_7.png) + +![0_image_8.png](0_image_8.png) + +![0_image_2.png](0_image_2.png) + +![0_image_5.png](0_image_5.png) + +![0_image_6.png](0_image_6.png) + +journal homepage: www.elsevier.com/locate/afjem Original article + +![0_image_1.png](0_image_1.png) + +Severe traumatic brain injuries secondary to motor vehicle crashes in two Namibian regions: A retrospective review Paulus Ambundaa,b,∗, Andrit Lourens c a Motor Vehicle Accident Fund of Namibia, Windhoek, Namibia b Department of Health Sciences, Faculty of Health, Applied Sciences and Natural Resources, Namibia University of Science and Technology, Windhoek, Namibia c Division of Emergency Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South *Africa* a r t i c l e i n f o + +## A B S T R A C T + +| Keywords: Traumatic brain injury MVCs Severe Head injury Head trauma TBI | +|----------------------------------------------------------------------------| + +Introduction: Traumatic Brain Injuries (TBIs) are a leading cause of morbidity and mortality among trauma patients globally, with motor vehicle crashes (MVCs) being a major contributor. Namibia had a World Health Organization (WHO) estimated MVC-related fatality rate of 30.4 per 100 000 population in 2016, higher than that of the African continent, while no epidemiological studies describing the distribution and determinants of TBIs exist in the country. The study aimed to describe the characteristics of adult patients (≥18 years) with severe TBI secondary to MVCs which occurred in two regions of Namibia between the years 2014–2018. + +Methods: A retrospective descriptive observational study was conducted in adult patients who sustained severe TBIs secondary to MVCs in two Namibian regions. The inclusion criteria were patients ≥18 years with a severe +(as described on the Motor Vehicle Accident Fund system) MVC-related TBI who sustained an injury in the Otjozondjupa or Khomas regions between the years 2014–2018. + +Results: A total of 87 patients met the inclusion criteria, 65 (74.7%) from the Khomas region, and 22 (25.3%) +from the Otjozondjupa region. The overall mean age of patients was 34 years (SD 11.79), most were male +(n = 78. 89.7%) and 55.2% (n = 48) of all patients sustained an isolated TBI. The majority of the patients were admitted to a state healthcare facility (n = 52, 59.8%). Pedestrians were the most injured (n = 34, 52.3%) in the Khomas region while vehicle drivers were the most injured (n = 11, 50%) in the Otjozondjupa Region. A total of 34 (39.1%) patients died and 53 (60.9%) were discharged from hospital. Overall, there were no statistically significant relationships between patient outcomes and independent variables. Conclusions: The study was to our knowledge the first to describe the epidemiology of TBIs in Namibia. Young individuals are the main people who sustained TBIs, which may subsequently place a socio-economic burden on the country. There is however limited research in Namibia to guide healthcare planning. + +## African Relevance + +- Provide the first quantitative description of TBI epidemiological characteristics in Namibia +- Develop a broader understanding of the impact of MVAs in the Namibian setting +- Empower healthcare sector planning and possible intervention +- Develop a platform for further research + +## Introduction + +Traumatic Brain Injuries (TBIs) have become a global health and socio-economic concern and a major contributor to morbidity and mortality amongst trauma patients [1]. The estimated incidence of TBIs, globally, was 10 million cases per annum and 150 cases per 100 000 population in the United States of America (USA) in 2007 [1]. About 4.8 million people died worldwide in 2013 secondary to a TBI and many more were left disabled [2]. The annual (direct and indirect) medical costs of TBIs in the USA in the year 2009 (USD 302 million and USD +2.8 billion, respectively) demonstrate the significant financial burden caused by TBIs [3]. + +Motor vehicle crash (MVC) related injuries have increased over the last 30 years with MVCs recognised as the leading cause of TBIs [1,4]. The highest number of MVCs emanates from Africa and South-East Asia +[1]. Males sustain TBIs more frequently than females with the age of those injured ranging mainly between 20-40 years [5] and it is a major causative factor of morbidity and mortality amongst young adults Abbreviations: ECC, Emergency Communications Centre; LMICs, Low- and middle-income countries; MVCs, Motor Vehicle Crashes; TBIs, Trauma Brain Injuries; WHO, World Health Organization. ∗ Corresponding author. + +E-mail *address:* paul.ambunda@gmail.com (P. Ambunda). + +https://doi.org/10.1016/j.afjem.2022.04.009 Received 26 May 2021; Received in revised form 18 April 2022; Accepted 22 April 2022 2211-419X/© 2022 The Authors. Published by Elsevier B.V. on behalf of African Federation for Emergency Medicine. This is an open access article under the CC +BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) +globally [6]. The 2018 World Health Organization (WHO) global status report on road safety states that low- and middle-income countries +(LMICs) have 85% of the world's population and 60% of the world's registered vehicles while 93% (13% and 80%, respectively) of MVC-related deaths occurs here [7]. In 2013, MVC-related injuries were estimated to cost LMICs between 1% and 2% of their gross national product [8]. Although MVC-related injuries have increased over the last 3 decades, the mortality rates in South Africa and a few other African countries may be decreasing and likely attributed to the development of pre-hospital systems in these regions [4]. Severe TBI patients commonly require lengthy hospital admissions, and the survivors may require long-term rehabilitation for conditions secondary to the TBI, some from which patients never fully recover [6]. + +Namibia is a sparsely populated, upper-middle-income country in Southern Africa with vast distances between towns. Tertiary level hospitals are mainly located in the capital city, Windhoek (Khomas Region) +where patients can receive definitive care. These challenges are further compounded by the high rate of MVCs exerting more pressure on the already resource-limited facilities. The WHO (2018) states that MVCrelated death rates are highest in Africa at 26.6 per 100 000 population, however, Namibia had an estimated MVC-related death rate of 30.4 per 100 000 population in 2016 [7]. With no published data on the distribution and determinants of TBIs in Namibia and Namibia identified as a country with a high rate of MVC-related deaths [7], the results of this study will be valuable, allowing the first description of TBI characteristics in Namibia, broadening the understanding of the impact of MVCs, and develop a platform for further research. The study aimed to describe and compare the characteristics of adult patients with severe TBI +secondary to MVCs which occurred in two Namibian regions between 2014 and 2018 as captured by the Motor Vehicle Accident (MVA) Fund of Namibia. + +## Methods Study Design + +A descriptive observational study in the form of a retrospective review was conducted to describe the characteristics of adult patients who sustained a severe TBI secondary to a MVC in 2 Namibian regions (Khomas & Otjozondjupa). + +## Study Setting And Population + +Namibia has a population of about 2.5 million [9] and is divided into 14 regions including the Khomas and Otjozondjupa regions. The Khomas region is centrally located with most of the population residing in Windhoek [10]. Otjozondjupa region, one of the biggest regions, has 4 district hospitals [11] providing emergency care but not definitive care, for which patients require inter-hospital transfers (road or air) to tertiary hospitals in Windhoek. The hospitals are located in Okahandja, Otjiwarongo, Okakarara and Grootfontein. Okahandja is closest to Windhoek at 72km while Grootfontein is the farthest at 458 km. The 2 regions contain four of the five top roads for crash location and severity identified in Namibia and recorded some of the highest injury and fatality rates in the country in the period under study, hence the selection as the study setting [12–17]. + +Established in 1991, the MVA Fund of Namibia is an organization that designs, develops, promotes, and implements MVC injury prevention measures and aid persons injured in road crashes. As part of assisting MVC victims, the MVA Fund operates a national 24-hour emergency communications centre and emergency medical response services with bases in various towns across the country. The main source of data is the MVA Fund Emergency Communications Centre (ECC), where crashes are reported, through the Toll-free Accident Response Number. Information collected and recorded by the ECC is verified with the Namibian Police, Emergency Medical Rescue Services - Paramedics and Health Officials from public and private hospitals throughout the country. Only crashes which result in injuries are recorded by the MVA Fund ECC. Hence, crashes that result in property damage only, are duly excluded [13–17]. + +The study population were adult patients (≥18 years old) who sustained a severe TBI (as classified in the MVA Fund system) secondary to an MVC in the Khomas region and Otjozondjupa region in Namibia between the 1st of January 2014 and the 31st of December 2018. The classification of severe TBI is recorded on the MVA Fund system as made by the clinical evaluation of the treating physician. + +## Inclusion And Exclusion Criteria The Inclusion Criteria Were As Follows: + +- Adult patients (≥18 years) +- Severe TBI (as classified in the MVA Fund database) +- Injury sustained in the Khomas or Otjozondjupa region between 1 January 2014 and 31 December 2018 The exclusion criteria were any patient that did not meet the stated inclusion criteria. Patients that died on scene or enroute to hospital were not included. It was hypothesized that the epidemiological characteristics of adult patients who sustained severe TBI in the Khomas region differ from those who sustained injuries in the Otjozondjupa region in terms of the outcome given the time taken to definitive care by patients from the Otjozondjupa region. + +## Data Collection + +Data was collected from the MVA Fund case management google spreadsheet and SIEBEL system (MVA Fund in-house production system). Patients who sustained a severe TBI were extracted from the case management google spreadsheet and the MVA Fund's SIEBEL system was used to determine the costs, ages, and location of MVCs. Patients whose diagnosis was not clearly stipulated as severe TBI on the case management spreadsheet were excluded as the severity of the injury could not be established. + +## Data Analysis + +Data were analysed using SPSS statistical package, Version 25 [18]. + +Descriptive statistics were used to report counts, percentages, mean and standard deviation and presented in tables. Pearson chi-square test for independence was done to determine whether a relationship existed between the dependent variable and independent variables. A p-value of 0.05 was deemed to be statistically significant. + +## Ethical Approval + +The study received ethical approval from the Ethics Committees of the Namibian University of Science and Technology and the Ministry of Health and Social Services of Namibia. In addition, written permission to conduct research was granted by the MVA Fund. To respect patient confidentiality and anonymity, no patient identifiable data was extracted. + +## Results + +During the study period, a total of 20 095 crashes were recorded by the MVA Fund of Namibia in which 33 099 occupants were injured. In the Khomas region, 7725 (38.4%) crashes occurred with 8869 (26.8%) +injuries while 1848 (9.2%) crashes occurred in the Otjozondjupa region with 4372 (13.2%) injuries. A total of 352 TBIs were recorded. This is however not specific to severe TBIs. + +| Table 1 Patient characteristics per region. Khomas Region (n = 65) | Otjozondjupa Region (n = 22) | Total | | +|----------------------------------------------------------------------|--------------------------------|------------|------------| +| Characteristics | n (%) | n (%) | n (%) | +| Gender: Male | 58 (89.2%) | 20 (90.9%) | 78 (89.7%) | +| Female | 7 (10.8%) | 2 (9.1%) | 9 (10.3%) | +| Age Groups: 18-28 Years | 25 (38.5%) | 7 (31.8%) | 32 (36.8%) | +| 29-39 Years | 20 (30.8%) | 13 (59.1%) | 33 (37.9%) | +| 40-50 Years | 11 (16.9%) | - | 11 (12.6%) | +| 51-61 Years | 8 (12.3%) | 2 (9.1%) | 10 (11.5%) | +| >61 Years | 1 (1.5%) | - | 1 (1.2%) | +| Healthcare Facilities: Public Hospital: Katutura State Hospital | 34 (52.3%) | 5 (22.7%) | 39 (44.8%) | +| Windhoek Central State Hospital | 4 (6.2%) | - | 4 (4.6%) | +| Okahandja District Hospital | - | 5 (22.7%) | 5 (5.7%) | +| Otjiwarongo District Hospital | - | 3 (13.6%) | 3 (3.4%) | +| Grootfontein District Hospital | - | 1 (4.5%) | 1 (1.1%) | +| Total | 38 (58.5%) | 14 (63.6%) | 52 (59.8%) | +| Private Hospital: Mediclinic Windhoek | 16 (24.6%) | 5 (22.7%) | 21 (24.1%) | +| Roman Catholic Hospital | 6 (9.2%) | 2 (9.1%) | 8 (9.2%) | +| Lady Pohamba Private Hospital | 4 (6.2%) | 1 (4.5%) | 5 (5.7%) | +| Rehoboth St. Mary's Hospital | 1 (1.5%) | - | 1 (1.1%) | +| Total | 27 (41.5%) | 8 (36.4%) | 35 (40.2%) | + +## Patient Characteristics + +Eighty-seven patients met the inclusion criteria, 22 (25.3%) from the Otjozondjupa region and 65 (74.7%) from the Khomas region. The mean age of patients in Khomas and Otjozondjupa regions was 34 (SD 12.79) +years and 32 (SD 8.36) years respectively, while the mean overall age was 34 (SD 11.79) years. The age group, 29-39 years (n = 33, 37.9%), +contained the highest proportion of patients while 74.7% (n = 65) of all patients were <40 years. Most patients were male (n = 78, 89.7%) while patients were mainly admitted at state healthcare facilities (n = 52, 59.8%) situated in the two regions (Table 1). + +## Mvc Characteristics + +Most patients in the Khomas region travelled by sedan (n = 42, 64.6%) while in the Otjozondjupa region most travelled by pick-up vehicle (n = 10, 45.5%). In the Khomas region, pedestrians (n = 34, 52.3%) were the most injured while in the Otjozondjupa region, the most common position of an injured occupant was the driver (n = 11, 50%) (Table 2). + +## Patient Outcomes + +The mortality rate among all included patients (n = 87) was 39.1% +(n = 34). Amongst the male patients, the mortality rate was 37.1% (29/78) and 55.6% (5/9) amongst females (Table 3). The three age groups, 18-28 years (40.6%), 40-50 years (45.5%) and 51-61 years +(40.0%), each had a mortality rate of 40.0% or higher while the age group 29-39 years had a 36.4% (12/33) mortality rate. The mortality rate amongst patients with an isolated TBI was 39.6% (19/48) and 38.5% (15/39) in the group of patients with compounding injuries. No statistically significant associations were found between patient outcomes (died or discharged from hospital) and the independent variables +(age group, gender, type of MVC, vehicle type, occupant position) and facility (public versus private) for the two regions as well as for the overall data. + +## Medical Cost + +The total medical costs for managing the included patients from the Otjozondjupa region was USD 550 173.47 (N$ 8 423 155.86) and USD 1 315 077.00 (N$ 20 133 842.22) for included patients from the Khomas region. This totaled to USD 1 865 251.34 (N$ 28 556 998.08). The average cost per patient in the Otjozondjupa region was USD 25 007.89 (N$ +382 870.72) while the average cost per patient in the Khomas region was USD 20 231.95 (N$ 309 751.42). These costs include ambulance costs up to hospital expenses involved directly in the medical management of the patients and were extracted from invoices submitted to the MVA Fund for payment of services rendered by medical providers and ambulance service providers. + +## Discussion + +To our knowledge, this is the first research to describe the characteristics of adult patients who sustained a severe TBI secondary to an MVC +in Namibia. Our study found that in the two Namibian regions, male patients, and patients 40 years or younger more commonly sustained severe TBIs secondary to MVCs. No statistically significant difference were found between the mortality rates of severe TBI patients in the 2 regions. + +## Characteristics Of Tbi Patients + +It is well documented in the literature that males sustain TBIs more commonly than females whether in low or high-resource settings [5,19] +and this has been mainly attributed to behavioral factors, amongst others, such as driving under the influence of alcohol & other illicit substances and reckless driving [5,19]. + +Pedestrians in the African region have been identified as vulnerable road users at high risk of injury and death [20,21]. Similarly, the current study found a high proportion of pedestrians sustaining a severe TBI and this may be indicative of unsafe pedestrian conditions requiring further investigation and interventions to prevent further injury in this class. + +Younger persons (20-40 years), similar as suggested in the current study, are the main population to sustain TBIs. Because this group is the economically active population, TBIs can be expected to place a major socioeconomic burden on countries. Additionally, the transport sector is an integral part of modern economies and MVCs cause a negative impact on a country's economy secondary to capital losses and the loss of the labour force [22]. + +| Table 2 MVC characteristics per region. Characteristics Khomas Region (n = 65) | Otjozondjupa Region (n = 22) | Total | | +|-----------------------------------------------------------------------------------------------------------------|--------------------------------|--------------|---------------| +| n (%) | n (%) | n (%) | | +| Vehicle Type: Pick-up | 15 (23.1%) | 10 (45.5%) | 25 (28.7%) | +| Sedan | 42 (64.6%) | 6 (27.2%) | 48 (55.1%) | +| Bus | 3 (4.6%) | 5 (22.7%) | 8 (9.2%) | +| Truck | 3 (4.6%) | - | 3 (3.4%) | +| Motorcycle | 2 (3.1%) | - | 2 (2.3%) | +| Sport Utility Vehicles (SUV) | - | 1 (4.5%) | 1 (1.2%) | +| Type of MVC (as characterized by the Namibian police): Rollover 18 (27.7%) | 10 (45.4%) | 28 (32.2%) | | +| Pedestrian | 34 (52.3%) | 3 (13.6%) | 37 (42.5%) | +| Head-side | 7 (10.8%) | 3 (13.6%) | 10 (11.5%) | +| With animal | 2 (3.1%) | 1 (0.04%) | 3 (3.4%) | +| With fixed object | 2 (3.1%) | 1 (0.04%) | 3 (3.4%) | +| Motorcycle | 2 (3.1%) | - | 2 (2.3%) | +| Fell from moving vehicle | - | 1 (0.04%) | 1 (1.2%) | +| Head-on | - | 3 (13.6%) | 3 (3.4%) | +| Occupant Position: Driver | 11 (16.9%) | 11 (50%) | 22 (25.3%) | +| Passenger | 17 (26.2%) | 8 (36.4%) | 25 (28.7%) | +| Pedestrian | 34 (52.3%) | 3 (13.6%) | 37 (42.5%) | +| Animal driver | 1 (1.5%) | - | 1 (1.2%) | +| Motorcyclist | 2 (3.1%) | - | 2 (2.3%) | +| Table 3 Mortality rate per age group, gender, and injuries. Khomas Region (n = 65) Otjozondjupa Region (n = 22) | Total | | | +| Died n (%) | Died n (%) | deaths | | +| Age groups: 18-28 years | 10/25 (40%) | 3/7 (42.9%) | 13/32 (40.6%) | +| 29-39 years | 8/20 (40%) | 4/13 (30.8%) | 12/33 (36.4%) | +| 40-50 years | 5/11 (45.5%) | - | 5/11 (45.5%) | +| 51-61 years | 3/8 (37.5%) | 1/2 (50%) | 4/10 (40.0%) | +| >61 years | - | - | 0/1 (0%) | +| Total | 26/65 (40.0%) | 8/22 (36.4%) | 34/87 (39.1%) | +| Gender: Male | 22/58 (37.9%) | 7/20 (35.0%) | 29/78 (37.1%) | +| Female | 4/7 (57.1%) | 1/2 (50%) | 5/9 (55.6%) | +| Total | 26/65 (40.0%) | 8/22 (36.4%) | 34/87 (39.1%) | +| Injuries: Isolated TBI | 15/39 (38.5%) | 4/9 (44.4%) | 19/48 (39.6%) | +| Compounding injuries | 11/26 (42.3%) | 4/13 (30.8%) | 15/39 (38.5%) | +| Total | 26/65 (40.0%) | 8/22 (36.4%) | 34/87 (39.1%) | + +## Mortality + +From the data collected, we were unable to determine the mortality period of the patients and for this reason, patient outcomes were described as discharged from hospital or died. The MVA Fund since 2009 has however adopted the WHO definition of road death which is any death occurring immediately or within 30 days as a result of a road crash. + +Although no differences were found between patients that sustained an isolated TBI (39.6%) versus those with compounding injuries with a severe TBI (38.5%), evidence suggests TBI severity to be the major determining factor to patient outcomes while compounding injuries do not necessarily have a significant impact [23]. + +We expected that mortality in the Otjozondjupa would be higher given the increased time to definitive care. This hypothesis was based on the cornerstone of the management of TBI, which is aimed at preventing secondary TBI, making the initial management vital as it can influence patient outcomes [24]. Pakkanen et al.[25] suggests that better patient survival outcomes and better neurological function post-TBI +can likely be linked overall to effective pre-hospital care. The Namibian pre-hospital care system is relatively new and still under-developed. + +In comparison to the rest of the country, the Khomas region has the most developed pre-hospital care services. Services in the Otjozondjupa region are limited with an absence of ambulances in some areas. Finding no difference between the mortality rates of the two regions was therefore not expected due to the limited resources in the Otjozondjupa region and overall time to definitive care. + +Our findings did not suggest worse outcomes in any particular age group, however, other literature suggests older patients have poorer outcomes after sustaining TBIs. This is due to the physiological changes that occur with age which predisposes them to injury and resultant worse outcomes [26]. + +The patients of the current study were admitted and managed at various facilities, some private facilities and others, public facilities. A cross-analysis found no statistically significant difference in the outcome of the patients in regard to whether they were managed at public or private facilities, however, the study sample was small and not powered to detect these differences. + +It is evident that further in-depth studies are required to fully understand and describe the extent of the TBI and more generally the trauma burden in Namibia. Investigating the impact of distance and time to definitive care as well as the impact of pre-hospital interventions on the outcome of severe TBI patients, may assist in focusing attention on the need for emergency care development in the country. Additionally, trauma registries need to be established in Namibia in order to streamline trauma research and care in the country. + +## Medical Costs + +International studies suggest that the financial burden associated with TBIs in high-resource settings are significant [3,27]. Less is known about the economic burden in Africa, however, the impact of TBIs in low-resource settings is believed to be far exceeding those in highresource settings [1]. Modelling suggests that the estimated total number of TBIs in Africa in 2050 will be almost 6 million cases with the highest occurrence in males between 15 and 34 years and the lowest amount in Southern Africa [28]. Although the medical cost from the current study (USD 1 865 251.34/N$ 28 556 998.08) might seem less significant, it only represents data from two Namibian regions, adults with severe MVC-related TBIs and excludes costs such as rehabilitation, medical benefits, injury grants, etc. Webster et al. [29] reported unpublished data from a 2009 internal audit at Groote Schuur Hospital in Cape Town, South Africa, finding that the direct costs of managing 654 TBI +(moderate-severe) patients at this hospital were USD 1 139 696.67 (N$ +17 448 756.00) [29]. Although the South African figure is from 4 years prior to our study, the direct cost (USD 1 315 077.00/N$ 20 133 842.22) of managing the 65 severe TBI patients from the Khomas region alone exceeded that of managing patients in the South African study. This may be indicative of the high costs involved in managing severe TBI patients in the country. The true cost of TBIs in Namibia is likely much higher, potentially constraining the growth of the country. Dedicated studies evaluating and describing the financial burden and economic impact of TBIs of any cause in Namibia are vital. + +The retrospective nature of the current study may present various potential limitations like selection and information bias, issues with missing data and uncertainty about generalisability. The convenience sampling method employed may have subjected the study findings to selection bias. The study sample was small, affecting the ability to detect a statistically significant difference in patient outcomes even if they existed and raises concerns with generalisability. + +In addition, MVC-related TBIs in younger victims, mild and moderate MVC-related TBIs and TBIs secondary to causes other than MVCs was not described. Patients who sustained severe TBIs but were not captured on the tool used for data collection and those that died on scenes or prior to arrival at hospital were not included in the study, potentially resulting in underreporting. The classification of the TBI severity was at the discretion of the medical doctor and the criteria used is not known further restricting the generalisability of the study findings. Although anecdotal information, local neurosurgeons suggest that the Glasgow Coma Scale (GCS) and the Marshall score are the 2 systems used to classify the TBI severity in Namibia. + +Furthermore, the data was collected only from the MVA Fund thus further limiting the study, however, data capturing systems remain under-developed in the country which greatly hinders the conduction of research. Despite these limitations, we hope that this study highlights the need for further research aimed at describing the epidemiology of injuries in Namibia and aid in the development of more rigorous data capturing systems to facilitate research. + +## Conclusion + +The study, to our knowledge, was the first to describe the epidemiology of TBIs in Namibia. Young individuals who are the economically active population sustain more TBIs with potentially greater resultant socio-economic burdens that ensue post-injury. The two regions studied, carried a huge medical cost for managing these patients which is only a fraction of the total cost of managing TBI patients. + +A larger focus needs to be placed on trauma research in Namibia, particularly related to MVCs to curb the TBI issue and inform healthcare planning. This research is a step forward in understanding severe TBI's from MVC's in Namibia and can inform healthcare planning, particularly in the pre-hospital setting. More research is however required to study the effect of pre-hospital care, on the outcome of TBI patients in Namibia, as the literature suggests that adequate pre-hospital care may lead to improved outcomes in TBI patients. + +## Dissemination Of Results + +Results from this study were submitted and presented to the academic staff of the Namibia University of Science and Technology +(NUST). The research report was also shared with the MVA Fund Chief: +Human Capital. + +## Author'S Contributions + +Authors contributed as follows to the conception or design of the work; the acquisition, analysis, or interpretation of data for the work; and drafting the work or revising it critically for important intellectual content: PA contributed 50% and AL contributed 50%. All authors approved the version to be published and agreed to be accountable for all aspects of the work. + +## Declaration Of Competing Interest + +The corresponding author is an employee of the Motor Vehicle Accident Fund of Namibia. The authors declared no further conflict of interest. + +## Acknowledgements + +I would like to thank the MVA Fund staff for the assistance offered during the data collection phaseand Mr. Etuhole Mwahi, the statistician who assisted in the data analysis. + +## References + +[1] Hyder AA, Wunderlich CA, Puvanachandra P, Gururaj G. The impact of traumatic brain injuries: a global perspective. Neurorehabilitation 2007;22(5):341–53. [Internet]Available from https://pubmed.ncbi.nlm.nih.gov/18162698/. + +[2] Haagsma JA, Graetz N, Bolliger I, Naghavi M, Higashi H, Mullany EC, et al. The global burden of injury: incidence, mortality, disability-adjusted life years and time trends from the global burden of disease study 2013. Inj Prev 2016;22(1):3–18. [Internet]Available from http://dx.doi.org/10.1136/injuryprev-2015-041616. + +[3] Humphreys I, Wood R, Phillips C, Macey S. The costs of traumatic brain injury: a literature review. Clinicoecono Outcomes Res 2013;5:281–7. [Internet]Available from: +https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699059/pdf/ceor-5-281.pdf. + +[4] Adeloye D, Thompson J, Ayo C. The burden of road traffic crashes, injuries and deaths in Africa: a systematic review and meta-analysis. Bull World Health Organ 2016;94(7):510–21. [Internet]Available from http://dx.doi.org/10. + +2471/BLT.15.163121. + +[5] Krebs E, Gerardo CJ, Park LP, Ricardo J, Vissoci N, Byiringiro JC, et al. Mortalityassociated characteristics of patients with traumatic brain injury at the university teaching hospital of Kigali, Rwanda. World Neurosurg 2017;102:571–82. [Internet]Available from: https://doi.org/10.1016/j.wneu.2017.03.001. + +[6] Stocchetti N, Zanier ER. Chronic impact of traumatic brain injury on outcome and quality of life: a narrative review. Crit Care 2016;20(148):1–10. [Internet]Available from: https://doi.org/10.1186/s13054-016-1318-1. + +[7] World Health Organisation. Global status report on road safety 2018. Geneva: +World Health Organisation; 2018. p. 424. Available from https://www.who.int/ +publications/i/item/9789241565684. + +[8] World Health Organisation. Global status report on road safety: supporting a decade of action. Geneva: World Health Organisation;; 2013. p. 318. Available from https://apps.who.int/iris/handle/10665/78256. + +[9] Review W.P. Namibia population by year (historical). [Internet]. 2020. Available from: https://worldpopulationreview.com/countries/namibia-population/. + +[10] Kafidi L. Namibia 2011 population and housing census. Wind. Repub. Namib. + +2011:214. + +[11] MoHSS. Regional directorates [Internet]. 2019. Available from: http://www. + +mhss.gov.na/otjozondjupa +[12] NRSC. Road accidents in Namibia: statistical report 2009. 2009. [13] MVA Fund. Road crash and claims report 2014. Windhoek: MVA Fund; 2014. p. 31. [14] MVA Fund. Road crash and claims report 2015. Windhoek: MVA Fund; 2015. p. 52. + +Available from http://www.mvafund.com.na/index.php/shortcode/accordion/tab. + +[15] MVA Fund. Road crash and claims report 2016. Windhoek: MVA Fund; 2016. p. 52. + +Available from http://www.mvafund.com.na/index.php/shortcode/accordion/tab. + +[16] MVA Fund. Road crash and claims report 2017. Windhoek: MVA Fund; 2017. p. 52. + +Available from http://www.mvafund.com.na/index.php/shortcode/accordion/tab. + +[17] MVA Fund. Road crash and claims report 2018. Windhoek: MVA Fund; 2018. p. 52. + +Available from http://www.mvafund.com.na/index.php/shortcode/accordion/tab. + +[18] IBM SPSS statistics for windows. Armonk, NY: IBM Corp; 2017. + +[19] Peeters S, Blaine C, Vycheth I, Nang S, Vuthy D, Park KB. Epidemiology of traumatic brain injuries at a major government hospital in Cambodia. World Neurosurg 2016;97:580–9. Available from: https://doi.org/10.1016/j.wneu.2016.09.127. + +[20] World Health Organisation. Global status report on road safety: time for action. + +Geneva: World Health Organisation; 2009. p. 301. Available from www.who.int/ +violence_injury_prevention/road_safety_status/2009. + +[21] Onywera VO, Blanchard C. Road accidents: a third burden of disease in subSaharan African. Glob Health Promot 2014;20(4):52–5. [Internet]Available from https://doi.org/10.1177/1757975913502688. + +[22] Enu P. Road traffic accidents and macroeconomic conditions in Ghana. Soc Basic Sci Res Rev 2014;2(9):374–93. [Internet]Available from https://www.researchgate. net/publication/276266936_Road_Traffic_Accidents_and_Macroeconomic_ +Conditions_in_Ghana. + +[23] Watanabe TW, Awai YK, Wamura AI, Aegawa NM, Ukushima HF, Kuchi KO. + +Outcomes after traumatic brain injury with concomitant severe extracranial injuries. Neurol Med Chir 2018;58(9):393–9. [Internet]Available from https://doi. + +org/10.2176/nmc.oa.2018-0116. + +[24] Wells AJ, Hutchinson PJ. The management of traumatic brain injury. Surgery 2018;36(11):613–20. [Internet]Available from https://doi.org/10.1016/j.mpsur. + +2018.09.007. + +P. and A. of 12 +[25] Pakkanen T, Nurmi J, Huhtala H, Silfvast T. Prehospital on-scene anaesthetist treating severe traumatic brain injury patients is associated with lower mortality and better neurological outcome. Scand J Trauma, Resusc Emerg Med 2019;27(9):1–7. [Internet]Available from https://doi.org/10.1186/s13049-019-0590-x. + +[26] Nnanna N, Babatola B, Gbalipre F. Traumatic brain injury in adults: age, etiology, and treatment outcome. Med Res Chron. 2016;3(1):29–39. [Internet]Available from: +https://www.academia.edu/22670864/TRAUMATIC_BRAIN_INJURY_IN_ADULTS_ AGE_ETIOLOGY_AND_TREATMENT_OUTCOME. + +[27] Scholten AC, Polinder S, Panneman MJM, Beeck EFVan, Haagsma JA. Incidence and costs of bicycle-related traumatic brain injuries in the Netherlands. Accid Anal Prev 2015;81:51–60. [Internet]Available from: https://doi.org/ +10.1016/j.aap.2015.04.022. + +[28] Wong JC, Linn KA, Shinohara RT, Mateen FJ. Traumatic brain injury in Africa in 2050: a modeling study. Eur J Neurol 2016;23(2):382–6. [Internet]Available from https://doi.org/10.1111/ene.12877. + +[29] Webster J, Balchin R. Traumatic brain injury, the hidden pandemic: a focused response to family and patient experience and needs. S Afr Med J 2015;105(3):194–8. + +doi:10.7196/samj.9014. \ No newline at end of file diff --git a/medical/md/PMC9380560.md b/medical/md/PMC9380560.md new file mode 100644 index 0000000000000000000000000000000000000000..c20f62264e4d46928f4318816875496e376c698e --- /dev/null +++ b/medical/md/PMC9380560.md @@ -0,0 +1,405 @@ +Cite this: RSC Adv., 2022, 12, 23263 + +# Construction Of A Binary S-Scheme S-G-C3N4/Cozf Heterojunction With Enhanced Spatial Charge + +![0_Image_0.Png](0_Image_0.Png) Separation For Sunlight-Driven Photocatalytic Performance† + +Ali Bahadur,a Shahid Iqbal, *b Mohsin Javed,c Syeda Saba Hassan,c Sohail Nadeem,c Ali Akbar, d Rami M. Alzhrani,e Murefah Mana Al-Anazy, f Eslam B. Elkaeed, g Nasser S. Awwad,h Hala A. Ibrahiumij and Ayesha Mohyuddinc A step-scheme (S-scheme) photocatalyst made of sulfurized graphitic carbon nitride/cobalt doped zinc ferrite (S-g-C3N4/Co-ZF) was constructed using a hydrothermal process because the building of Sscheme systems might increase the lifespan of highly reactive charge carriers. Utilizing cutting-edge methods, the hybrid photocatalyst was evaluated by employing TEM, XPS, XRD, BET, FTIR, transient photo-response, UV-vis, EIS and ESR signals. In order to create a variety of binary nanocomposites (NCs), +nanoparticles (NPs) of 6% cobalt doped zinc ferrite (Co-ZF) were mixed with S-g-C3N4 at various concentrations, ranging from 10 to 80 wt%. For photocatalytic dye removal, a particular binary NC +constructed between S-g-C3N4 and Co-ZF produces a huge amount of catalytic active sites. The findings showed that loading of S-g-C3N4 on 6% Co-ZF NPs serves as a good heterointerface for e/h+ +separation and transportation through the S-scheme S-g-C3N4/Co-ZF heterojunction. By boosting the hybrid system's BET surface area for the photocatalytic process, the addition of 6% Co-ZF improves the system's ability to absorb more sunlight and boosts its photocatalytic activity. The highest photo-removal effectiveness (98%), which is around 2.45 times higher than that of its competitors, was achieved by the hybrid photocatalyst system with an ideal loading of 48% Co-ZF. Furthermore, the trapping studies showed that the primary species involved in the MB aqueous photo-degradation were cOH and h+. + +Received 21st November 2021 Accepted 8th August 2022 DOI: 10.1039/d1ra08525e rsc.li/rsc-advances aDepartment of Chemistry, College of Science and Technology, Wenzhou-Kean University, Wenzhou, China bDepartment of Chemistry, School of Natural Sciences (SNS), National University of Science and Technology (NUST), H-12, Islamabad, 46000, Pakistan. E-mail: shahidiqbal.chem@sns.nust.edu.pk cDepartment of Chemistry, School of Science, University of Management and Technology, Lahore, Pakistan dDepartment of Physics, University of Agriculture Faisalabad (UAF), Faisalabad, Punjab, 38000, Pakistan eDepartment of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia fDepartment of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia gDepartment of Pharmaceutical Sciences, College of Pharmacy, AlMaarefa University, Riyadh 13713, Saudi Arabia hChemistry Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia iBiology Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia jDepartment of Semi Pilot Plant, Nuclear Materials Authority, P.O. Box 530, El Maadi, Egypt +† Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d1ra08525e + +## Introduction + +The need for clean water sources has spurred interest on a global scale as a result of the world population's ongoing rise as well as the quickening pace of industrialization and urbanization.1–3 Several textile businesses release byproducts that include water during this fast growth, especially colors like methyl orange (MO) and methylene blue (MB), both of which are very lethal and toxic.4–6 Mixing dirty water with pure water results in considerable environmental pollution.7–9 Degrading incompatible organic molecules and purifying the water are therefore important to save the ecology.10–12 Organic dye molecules are challenging to spontaneously decay due to their bulky and complicated chemical structure.13–15 Organic pollutant elimination is a well-established potential of photon-initiated oxidation processes, notably photocatalysis.16–20 This method's notable benets include the utilization of inexpensive photons, mild working temperatures, nontoxic photocatalysts, and full mineralization.21–26 Light absorption, photocatalytic redox interactions with reactive radicals, and the separation and transport of the photogenerated electron–hole pair are generally the three key components of the photocatalytic process.27–30 Due to its distinctive qualities, such as environmental friendliness, affordability, a moderate working temperature, and high oxidation power, semiconductor-based photocatalysts have been extensively used to remove environmental contaminants from water.31–33 It is a drawback because TiO2 has a signicant bandgap (3.22 eV), which implies that only 4% of the solar spectrum is captured and the rest is lost. There have lately been several novel photocatalytic semiconducting materials that destroy organic complex compounds by reacting to visible light. Physiochemical characteristics, non-toxicity, and outstanding photocatalytic efficiency of spinel ferrites have made them popular photocatalysts in environmental cleanup.34–36 Zinc ferrite (ZnFe2O4) is a spinel-type (AB2O4) +material that is stable because Zn(II) ions dominate the tetrahedral A-site and Fe(III) ions reside in the octahedral B-sites.34 It has been used in several applications because of its optoelectronic qualities, photochemical stability and narrow bandgap +(1.9 eV), including magnetic materials, gas sensors, pigment, hydrogen production, solar energy active photocatalyst for the photo-removal of organic pollutants and as an anode material in batteries.6,37 There are several ways to make zinc ferrite (ZF), including solvothermal, co-precipitation, hydrothermal, microemulsion, sol–gel, the reverse micelle technique, solid-state processes and combustion.38,39 Due to its simplicity, ability to produce crystals with the required morphology, size, and form, high productivity, and improved solubility, the hydrothermal process is more oen used than other synthetic techniques.40–42 Additionally, a lot of crystals may be produced using this technique without altering the composition, and materials with high vapor pressure close to their melting point can also be formed. + +ZnFe2O4 nanocrystals were created by Fan et al. via a hydrothermal technique, and they were then utilized to degrade acid orange II.43 In addition, Sun et al. and Han et al. examined the photocatalytic efficiency of ZnFe2O4 nanoplates and octahedral ZnFe2O4 produced by hydrothermal techniques against RhB.44,45 However, because of insufficient e and h+ pair separation and substantial recombination of photoinduced e/h+, the photocatalytic efficiency of zinc ferrite is severely constrained. + +As a consequence, many areas are being concentrated on increasing the effectiveness of the photocatalytic procedure, involving expanding the light-harvesting spectrum range and boosting the transfer and separation of photoinduced e and h+ +sets.46–48 The use of 2D layered materials and metal-doping are typical methods for achieving these objectives. Numerous investigations have shown that non-local-conjugated structures in conductive polymers improve charge carrier transport and boost photocatalytic performance. Co-ZnFe2O4, 49 ZnFe2O4/gC3N4, 50 ZnFe2O4/graphene,51 and ZnFe2O4/Fe2O3 (ref. 52) are cited in earlier papers as materials that have shown exceptional performance in the elimination of contaminants. + +The cobalt-doped ZnFe2O4 (Co-ZF) NPs used in this study have varying percentages of cobalt (2, 4, 6, 8, and 10 wt%) and a 6% cobalt-doped ZnFe2O4 composite with varying percentages +(12, 24, 48, 60, and 80 wt%) of S-g-C3N4 photodegraded using these synthetic photocatalysts and a suggested mechanism for MB degradation was made. + +## Experimental Materials + +Ferric nitrate nonahydrate (Fe(NO3)3$9H2O, 99% Alfa Aesar), distilled water, Zn(NO3)3$6H2O, 97% Sinopharm, sodium hydroxide (NaOH, 99% Alfa Aesar), polyethylene glycol (PEG, +97% DAEJUNG), MB (C16H18ClN3S, 99% Simpsons), cobalt nitrate hexahydrate (Co(NO3)2$6H2O, 97% DAEJUNG) and thiourea (CH4N2S, 99% Sinopharm). + +## Synthesis Of Znfe2O4 Nps + +ZnFe2O4 NPs were created using the hydrothermal technique. In a nutshell, 2.98 g of Zn(NO3)3$6H2O and 8.09 g of Fe(NO3)3- +$9H2O were dissolved in 31 mL of distilled water before being combined with 11 mL of polyethylene glycol (PEG) and rapidly agitated for 22 minutes. To keep the mixture's pH at 11, a dropwise addition of a 7 M NaOH solution was made with constant stirring. For hydrothermal treatment at 182 C for 17 hours, this suspension was vacuum sealed in a 100 mL Teon autoclave. The temperature was then raised to the normal range. The item underwent many rounds of ltering and distilled water washings before being dried in the oven for three hours at 85 C. A ne powder was created once the product was nished. + +## Synthesis Of Co-Zf Nps + +By adjusting the cobalt content of ZnFe2O4 (2, 4, 6, 8, and 10 wt%), cobalt doped ZnFe2O4 (Co-ZF) NPs were also produced hydrothermally. To make 2% Co-ZF, 0.08 g of Co(NO3)2$6H2O, +8.09 g of Fe(NO3)3$9H2O and 2.91 g of Zn(NO3)3$6H2O were dispersed in 31 mL of distilled H2O. The mixture was then given a further addition of 11 mL of PEG and aggressively agitated for 22 minutes. When the combination's pH reached 11, a dropwise addition of a 7 M solution of NaOH was made while the mixture was continually stirred. For hydrothermal treatment at 182 C +for 17 hours, this suspension was vacuum sealed in a 100 mL +Teon autoclave. Then, it was let cool to room temperature. The precipitates underwent ltration, washing, and three hours of drying at 85 C. The substance was then milled into a ne powder aer drying. The same process was also used to create Co-ZF in a sequence with wt% of 4, 6, 8, and 10. Table 1 lists the different compositions and testing circumstances. + +## Construction Of S-G-C3N4 + +Utilizing thiourea and the thermal polycondensation technique, S-g-C3N4 was created. This process included placing thiourea in the crucible and heating it for three hours at a temperature of 555 C at a pace of 6 C min1. Aer being milled into a ne powder, the resulting yellow color agglomerates were produced. + +## Designing Of S-G-C3N4/Co-Zf Heterostructures + +By combining different amounts (12, 24, 48, 60, and 80 wt%) of S-g-C3N4 with Co-ZF, a series of 6% S-g-C3N4/Co-ZF NCs were made. Solution A was created by dissolving 8.09 g of + +| Table 1 | Co-ZF NPs' chemical make-up and an evaluation of their photocatalytic potential Co(NO3)2$6H2O Zn(NO3)3$6H2O Fe(NO3)3$9H2O | MB dye degradation | | | | | +|-----------|-----------------------------------------------------------------------------------------------------------------------------|----------------------|------|------|----|-----| +| S. no. | Doping in wt% | (g) | (g) | (g) | pH | (%) | +| 1 | Pure ZF | 0 | 2.99 | 8.09 | 11 | 37 | +| 2 | 2% Co-ZF | 0.08 | 2.91 | 8.09 | 11 | 46 | +| 3 | 4% Co-ZF | 0.16 | 2.83 | 8.09 | 11 | 54 | +| 4 | 6% Co-ZF | 0.24 | 2.75 | 8.09 | 11 | 60 | +| 5 | 8% Co-ZF | 0.32 | 2.67 | 8.09 | 11 | 52 | +| 6 | 10% Co-ZF | 0.40 | 2.59 | 8.09 | 11 | 44 | + +Table 2 The progression of the photocatalytic efficacies of the synthesized heterostructures and their composition Fe(NO3)2$9H2O, 2.75 g of Zn(NO3)2$6H2O and 0.24 g of + +![2_image_0.png](2_image_0.png) + +Co(NO3)2$6H2O in 27 mL of distilled water for the synthesis of 12% NCs of S-g-C3N4 with 6% Co-ZF. To create solution B, 0.217 g of S-g-C3N4 were mixed with 16 mL of distilled water. + +Following that, solution A and solution B were combined, and 11 mL of PEG were added. This process took place while the mixture was vigorously stirred for 22 minutes. The pH was + +| Wt% of | MB dye removal | | | +|-----------------|------------------|------|----| +| 6% Co-ZF in NCs | S-g-C3N4 (g) | (%) | | +| 1 | 12% | 0.26 | 69 | +| 2 | 24% | 0.53 | 86 | +| 3 | 48% | 1.06 | 98 | +| 4 | 60% | 1.32 | 86 | +| 5 | 80% | 1.76 | 75 | + +raised to 11 by adding the 7 M solution of NaOH dropwise while stirring the mixture continuously. In a Teon autoclave, the suspension was moved, and it was heated for 17 hours at 182 C. + +The temperature was then lowered to ambient. The sample underwent three hours of drying at 85 C aer being ltered and many times in distilled water. The item was then crushed into a ne powder when it had dried. The same process was also used to create a series of NCs of S-g-C3N4 with 6% Co-ZF (24, 48, 60, and 80 wt%). For their synthesis, Table 2 lists the experimental conditions and S-g-C3N4 dosage. On the other hand, the ESI† includes the material characterizations and photocatalytic activity data. + +## Results And Discussion + +Fig. 1 shows the crystallinity and structure of S-g-C3N4 NSs, 6% +Co-ZF, ZF, and 48% S-g-C3N4/6% Co-ZF NCs as determined by XRD. The cubic spinal structure of 6% Co-ZF, ZF and 48% S-g- +Table 1 Co-ZF NPs' chemical make-up and an evaluation of their photocatalytic potential C3N4/6% Co-ZF NCs is shown by their XRD patterns. Fig. 1 + +![3_image_0.png](3_image_0.png) + +shows that the crystalline form of spinal ZF is signicantly associated with (JCPDS no. 22.1012) each of the diffraction peaks. The peaks at 2q of 35.3, 30.1, 42.1, 52.1, 62.3 and 56.5 are recognized in the (311), (220), (400), (422), (440) and +(511) crystal facets of zinc ferrite. The XRD of 6% Co-ZF is also shown in Fig. 1 for your viewing pleasure. Given that cobalt has a smaller ionic radius (0.57 A) than zinc, the pattern shows that adding 6% of cobalt to ZF marginally enhances the broadness of the peaks (0.59 A). Therefore, a smaller crystallite results from the replacement of Co in ZF. The rise in peak widening is proof that Co was successfully doped in ZF. The S-g-C3N4 diffraction pattern is also seen in Fig. 1. The 002 facets of the crystal are assigned a separate peak in this pattern at 27.37, and this pattern closely resembles (JCPDS 00-087-1526) the pattern of Sg-C3N4 that has been described. The intense peaks in the XRD +pattern of 48% S-g-C3N4/6% Co-ZF NCs are in strong agreement with the existence of pure ZF. This suggests that adding 48% Sg-C3N4 to 6% Co-ZF cannot cause a phase shi, and the fact that there is one peak of SCN at 27.37 indicates that S-g-C3N4 was successfully loaded into 6% Co-ZF. + +As shown in Fig. 2, TEM images were used to assess the surface morphologies of manufactured photocatalysts in addition to their crystal structures. Curiously, TEM investigation of S-g-C3N4 revealed that the produced S-g-C3N4 exhibit almost translucent and stretchable 2D homogeneous NSs constructions with bendy shapes (Fig. 2a). Spherical NPs are collected in erratic formations in the ZF TEM picture shown in Fig. 2b, with the size of the particles varying from 22 to 38 nm. This morphology is known as a 0D morphology. However, the Co-ZF with dimensions between 27 and 49 nm was also shown to have a 0D-like shape (Fig. 2c). The TEM picture (Fig. 2d) for the 48% +S-g-C3N4/6% Co-ZF NCs demonstrates an equal distribution of S-g-C3N4 across the Co-ZF NPs. Co-ZF is evenly dispersed as NPs throughout the S-g-C3N4 segment, which is really a 2D layered structure. The manufacture of 6% Co-ZF NPs on S-g-C3N4 NSs produced the most clearly characterized heterojunction between these two materials. + +Additionally, XPS was used to identify the valence state and + +![4_image_0.png](4_image_0.png) + +elemental conformation and of NCs that were 48% S-g-C3N4/6% +Co-ZF. According to Fig. S1a,† the Zn 2p1/2 and Zn 2p3/2 particles are responsible for the peaks that occurred at 1044.73 eV and 1021.72 eV in the Zn 2p spectra of S-g-C3N4/6% Co-ZF.53,54 Two unique peaks identied at 529.91 and 531.22 eV that may be linked with Fe–O and Zn–O, correspondingly, are validated by the deconvoluted O 1s observations (Fig. S1b†) of S-g-C3N4/6% +Co-ZF.55 Two major peaks of the measurements of Fe 2p were used to determine the oxidation states of Fe3+ in the synthesized photocatalyst (Fig. S1c†), Fe 2p1/2 (722.13) and Fe 2p3/2 +(708.43).56 As shown in Fig. S1d,† the deconvoluted spectra of Co 2p exhibit two distinct peaks that may be assigned to the Co 2p1/2 and Co 2p3/2 at BEs of 793.94 and 778.93 eV. The acquired ndings are in line with the prior work for the Co-ZF NPs. Three unique peaks seen in the C 1s spectra of S-g-C3N4/6% Co-ZF NCs may be assigned to the C–C, C]O, and C–O, individually. These peaks are seen at 283.19, 287.16, and 284.71 eV.57,58 In Fig. S1e,† +the C 1s spectrum is shown. Similarly, three distinct peaks at 397.89, 400.67, and 399.79 eV in the N 1s high-resolution spectra are attributed to the respective N–C–N, N–H, and C– +(C)3 functions (Fig. S1f†).14 The successful synthesis of hybrid Sg-C3N4/6% Co-ZF NCs was shown by these XPS experimental results. The XPS ndings demonstrated the close contact between Co-ZF and S-g-C3N4, resulting in the formation of the nanoscale hybrid system with a content of 48% S-g-C3N4 and 6% +Co-ZF. + +In order to identify the functional group in prepared samples, FTIR spectroscopy is a crucial method. In the 650– +4000 cm1 range, FTIR spectroscopy was used to study samples of 6% Co-ZF, ZF, S-g-C3N4 and S-g-C3N4/6% Co-ZF. Their FTIR +spectra are shown in Fig. 3a. The tetrahedral site's M–O bond is responsible for the distinctive peaks in all FTIR spectra that are in the 849–898 nm range. Our ability to observe the M–O bond's peaks at octahedral positions is limited by our instrumentation. + +Peaks at 842 and 782 cm1 are caused by triazine units, whereas peaks in the area of 1201–702 cm1 are caused by C–N and C]N +stretching in the spectra for S-g-C3N4 and 48% S-g-C3N4/6% CoZF. + +As shown in Fig. 3b, the isotherms of N2 desorption– +absorption for each of the generated samples of ZF, S-g-C3N4, 6% Co-ZF and a 48% S-g-C3N4/6% Co-ZF heterostructure are all simulated. The isotherm of a 48% S-g-C3N4/6% Co-ZF has been reported to be well suited to mesoporous structures and the standard isotherm pattern of the IUPAC.59 ZF, S-g-C3N4, 6% CoZF, and 48% S-g-C3N4/6% Co-ZF heterojunction all had BET +surface areas computed as 7.98, 29.98, 13.74, and 68.73 m2 g1, correspondingly. In comparison to the ZF, S-g-C3N4, 6% Co-ZF, +and the 48% S-g-C3N4/6% Co-ZF have a greater surface area. + +This is most likely the outcome of the phenomena of arrangement and building of numerous integrated components, which not only promotes the well-dened fabrication but also makes 48% S-g-C3N4/6% Co-ZF more active by generating additional active positions for photocatalysis. High photocatalytic activity is provided by photocatalysts made of 48% S-gC3N4/6% Co-ZF due to the well-dened manufacturing of heterointerface and enhanced surface area. However, the mesoporous 48% S-g-C3N4/6% Co-ZF exhibits an extraordinary capacity to suppress the photogenerated e/h+ pairs recombination processes, which subsequently helps to ne-tune the photocatalytic abilities of the 48% S-g-C3N4/6% Co-ZF +photocatalyst. + +Following that, the light-absorption of the synthesized photocatalysts S-g-C3N4, Co-ZF NPs, ZF NPs and 48% S-g-C3N4/6% +Co-ZF were explored by UV-vis spectra. Fig. 3c shows the aggregate collection of UV-vis evaluations with wavelengths between 255 and 755 nm. When the UV-vis measurements of Co-ZF and ZF NPs are compared, a consistent variation in absorption (redshi) is seen. Associating the 48% S-g-C3N4/6% +Co-ZF light absorption spectrum to all other samples, including ZF, S-g-C3N4 and Co-ZF NPs spectra, it is notable that the light cultivation is enriched from 255 nm to 755 nm. This increase in absorption is mostly the result of the integration of the 6% CoZF with S-g-C3N4, which also contributes to enhancing the photocatalytic efficacies of the heterostructures made up of 48% +S-g-C3N4/6% Co-ZF. Additionally, a signicant improvement has been made in the light-harvesting capacity in the 505–755 nm region, which is important for photocatalytic efficiency. + +By creating the Tauc's plot of the UV-vis measurements, shown in Fig. 3c, the energy bandgap values of these produced photocatalysts were evaluated. The computed bandgap values were found to be 2.02 eV, 1.94 eV, and 2.71 eV for the 6% Co-ZF, +ZF and 48% S-g-C3N4/6% Co-ZF, correspondingly, as shown in Fig. 3c. Because cobalt is doped into the ZF NPs, which most likely leads to the creation of new energy regions below the conduction band, the energy bandgap of 6% Co-ZF may be lowered. The sample with a cobalt concentration of 6% had the lowest energy bandgap out of all the designed samples of Co-ZF (2, 4, 8 and 10%). Similar results were obtained when the energy bandgap of the Co-ZF equated to the S-g-C3N4 and the 48% S-gC3N4/6% Co-ZF fell from 2.71 eV for S-g-C3N4 to 2.23 eV for 48% +S-g-C3N4/6% Co-ZF. The effective surface fusion of both components may be to blame for this drop in bandgap values, which signicantly increases the photocatalytic abilities of the binary photocatalyst. + +Fig. 4 compares the photocatalytic efficiency of a sequence of NCs and ZF with MB. The NCs had Co contents of 2, 4, 6, 8 and 10 wt%. Fig. 4a demonstrates that 6% Co-ZF may degrade MB at a faster rate than other samples, eliminating 60% of it in under 86 min. Later, a nanocomposite of 6% Co-ZF is made with various concentrations of S-g-C3N4 (12, 24, 48, 60 and 80 wt%), +and MB degradation is conducted to further improve the photocatalytic efficacy of the nanocomposite. To examine the + +![5_image_0.png](5_image_0.png) + +![6_image_0.png](6_image_0.png) + +![6_image_1.png](6_image_1.png) + +photocatalytic characteristics of generated NCs with various CoZF (12, 24, 48, 60, and 80 wt%) NPs concentrations, visible light illumination of MB was utilized (Fig. 4b). Our investigation of the photodegradation of organic pollutants over a 48% S-gC3N4/6% Co-ZF revealed that MB degradation may be sped up by raising the Co-ZF content in the 48% S-g-C3N4/6% Co-ZF +(Table 2). The highest photodegradation yield of 98% that was observed with this concentration under visible-light irradiation proves that the optimal amount of Co-ZF NPs is 50 wt%. For example, when S-g-C3N4/Co-ZF with 80 wt% of Co-ZF content were used as photocatalysts for MB degradation, the yield was reduced to 75%, suggesting that the yield would be lower with further increasing the Co-ZF NPs content. Even aer six-hour runs, the S-g-C3N4/Co-ZF exhibits the highest photodegradation efficiency and chemical stability (Fig. 5a). + +Fig. 4c and d illustrate the results of computing the photoremoval rate of dye by photocatalysts using the formula, lnCoC + +¼ Kt; where Co and C represent the dye content at zero and t minutes, correspondingly, and K represents the reaction rate. According to this gure, the photo-removal rate of MB is 8.99 104 min1. The photolysis rate of 6% Co-ZF (2.58 103 min1) is greater than the photocatalysis rate of MB utilizing ZF, which is 1.67 103 min1 then by creating a composite of 6% Co-ZF and 48% S-g-C3N4, the rate was further elevated and reached 0.42 103 min1 as a consequence of the synergistic interaction between Co-ZF and S-g-C3N4. Then, it is shown from estimated results that NCs of 48% S-g-C3N4/6% +Co-ZF showed the greatest 98% photo-removal of dye aer 56 min, whereas with 6% Co-ZF and ZF the degradation of dye was 60% and 37%, correspondingly. Additionally, as demonstrated in Table 3, 48% S-g-C3N4/6% Co-ZF photocatalytic efficiency is practically greater than that of past studies. + +Since it is generally known that the chemical constancy is a key factor in a catalyst's ability to be extensively utilized, the 48% S-g-C3N4/6% Co-ZF heterostructures that were created were assessed for their potential to be employed up to six times in the reaction of dye photo-removal under solar light. The catalyst's impressive chemical stability and availability for routine experimental usage were shown by the fact that there was no visible decline in its efficiency even aer 6 runs (Fig. 5a). + +Photocurrent studies have been performed with all of the available catalysts to dive further into the underlying factors that contribute to the exceptional photocatalytic efficacy of 48% +S-g-C3N4/6% Co-ZF for the MB mineralization. This is accomplished by creating a proportional association of photocurrent responses for 48% S-g-C3N4/6% Co-ZF, along with all other catalysts ZF NPs, S-g-C3N4, and 6% Co-ZF NPs, which provides a useful perspective on the transportation of photogenerated electron/hole pairs. The photocurrent response for 6% Co-ZF, +ZF, S-g-C3N4, and 48% S-g-C3N4/6% Co-ZF was explored in a 0.5 M Na2SO4 solution under chopping radiance (Fig. 5b). The photocurrent response of the 48% S-g-C3N4/6% Co-ZF was by far the greatest when compared to all other produced catalysts, + +S. no. Photocatalyst Dyes Light source + +Irradiation time (min) % degradation Ref. + +1 Zn1xDyxFe2O4 MB Mercury lamp 75 97.3 34 + +2 ZnFe2O4 MB Xenon lamp 141 76.2 60 3 CeO2/ZnFe2O4 MB Sunlight 60 90.48 31 + +4 ZF–RGO Fulvic acid Solar light 220 80.1 61 + +5 rGO/ZnFe2O4 MB Sunlight 180 99.8 62 + +6 SnFe2O4/ZnFe2O4 MB Visible light 120 93.2 63 7 ZnFe2O4/rGO MB Visible light 121 99.99 64 + +8 ZnFe2O4@rGO MB Xenon lamp 142 92.5 60 + +9 ZnFe2O4/Ag/AgBr RhB Visible light 80 88 65 + +10 S-g-C3N4/Co-ZF MB Sunlight 56 98 Present work + +demonstrating once again how effectively charges are transferred and consumed in the binary catalyst. On the other hand, + +![7_image_0.png](7_image_0.png) high photocurrent responses provided well-dened heterointerfaces, excellent electron–hole pair separation, and efficient transfer of electron charge carriers in the very advantageous self-assembled binary 48% S-g-C3N4/6% Co-ZF photocatalytic procedure for dye degradation. + +At the electrode–electrolyte junction, an EIS evaluation was recognized in the dark to assess the heterojunction charge transfer rate. The relationship between a shorter arc radius and lowered electron diffusion resistance, faster interfacial photogenerated charge transport, and better exit prociency is oen seen. According to our experimental results (Fig. 5c), the S-gC3N4/Co-ZF with a 48% S-g-C3N4/6% Co-ZF content had the lowest charge-transmission resistance of all the samples that were produced. According to this research, the heterointerface contact of the binary heterostructure S-g-C3N4/Co-ZF may greatly promote electron transport and increase electron consumption, enhancing photocatalytic performance. The transitory photocurrent responses and the EIS data accord quite well. According to the aforementioned experimental ndings, a well-built S-g-C3N4/Co-ZF might signicantly improve light harvesting, quick heterointerface electron transmission, and efficient separation of photoinduced e–h+ sets. + +Additionally, the 48% S-g-C3N4/6% Co-ZF heterostructures may produce a signicant number of effective oxygen species that are useful for removing MB from the 48% S-g-C3N4/6% CoZF under visible light illumination (Fig. 5d). p-Benzoquinone +(BQ), ethylenediaminetetraacetic acid (EDTA), and isopropanol +(IPA) were each employed to capture cOH, cO2 and h+ in the trapping experiment. This demonstrated that the primary energy species involved in the catalytic dye degradation process are cOH and cO2 +. Examining the EPR spectra of 48% S-g-C3N4/ +6% Co-ZF served as additional proof that functional species cOH +and cO2 were present in the photodegradation process +(Fig. S2a and b†). However, the signals are not discernible in the dark, indicating that both cO2 and cOH are constructed through the photolysis reaction practices. The apparent ESR +signals are associated with DMPO$O2 and DMPO$OH +adducts under sunlight enlightenment. The EPR results not only show that the 48% S-g-C3N4/6% Co-ZF heterojunction is produced, but they also show how effective the self-assembled method of S-g-C3N4/Co-ZF construction is for pollutant degradation. Fig. 6 shows a schematic representation of a photocatalytic process in the S-scheme under visible light. The MB +dye degradation mechanism with the help of 48% S-g-C3N4/6% +Co-ZF photocatalyst is shown in eqn (I)–(IV). + +The adsorption of MB dye on 48% S-g-C3N4/6% Co-ZF photocatalyst surface and the e/h+ pair contribute a signicant role in MB degradation. Firstly, the MB molecule was adsorbed on the 48% S-g-C3N4/6% Co-ZF photocatalyst surface. On the illumination of solar light on the S-g-C3N4/6% Co-ZF photocatalyst generate e/h+ pairs. Band gap alignment of Co-ZF with S-gC3N4 minimizes the e/h+ recombination. The holes in the lowest energy state in the conduction band of Co-ZF oxidized the MB dye directly and generate hydroxyl free radicals by reacting with water molecules which further interact with MB +dye to oxidize into valuable products. Similarly, electrons in the highest energy state in the valence band of S-g-C3N4 reduce the adsorbed MB dye into CO2. The degradation of MB is started by either cOH radicals, cO2 radicals, or direct transfer of holes. A +detailed illustration of the photocatalytic mechanism is written below. + +(I) Formation of electron–hole pair + +Co-ZF + $hv\to h^{+}$ (Co-ZF) + e${}^{-}$ (Co-ZF) +S-g-C${}_{3}$N${}_{4}$ + $hv$ $\rightarrow$ h${}^{+}$ (S-g-C${}_{3}$N${}_{4}$) + e${}^{-}$ (S-g-C${}_{3}$N${}_{4}$) +(II) Formation of cOH radical + + **e${}^{-}$ (S-g-C${}_{3}$N${}_{4}$) + H${}_{2}$O${}_{2}$ - ${}^{\prime}$OH + OH${}^{-}$** + $\text{h}^{+}$ (Co-ZF) + H$_2$O $\rightarrow$ 'OH + H$^{+}$' $\text{h}^{+}$ (Co-ZF) + OH$^{-}$ $\rightarrow$ 'OH +(III) Generation of other reactive species + +$\mathbf{v}$ +e + (S-g-C3N4)+O2 / cO2 + +$\mathbf{a}=\mathbf{a}\cdot\mathbf{a}=\mathbf{a}\cdot\mathbf{a}$. + +${\bf H^{+}+O_{2}-O+OOH}$ ${\bf2H^{+}+2^{+}O_{2}-O+O_{2}+H_{2}O_{2}}$ +(IV) MB degradation mechanism MB + Rc / CO2 + H2O + other degradation products where Rc ¼ cOH, cOOH, h+ (Co-ZF), e (S-g-C3N4), cO2 +, cO2. + +## Conclusion + +In a nutshell, the goal of the current work was to create a better nanocomposite system by integrating S-g-C3N4 with a sunlightsensitive photocatalyst with narrow bandgap, Co-ZF, to enhance the photo-responsive window of the material and, in turn, the photo-removal prociency. The hydrothermal technique was successfully used to create the binary S-g-C3N4/Co-ZF photocatalysts. Additionally, the Co-ZF on S-g-C3N4 were consistently loaded aer their rst manufacture. Both binary and individual photocatalysts were used to break down MB in water. It was discovered that the binary S-g-C3N4/Co-ZF NCs considerably improved the photo-removal of MB and sunshine harvesting. + +The ideal loading is 48 wt% for the binary S-g-C3N4/Co-ZF NCs. + +The photolysis rate of dye is 0.42 103 min1, which is almost 2.45 times more than that of the individual photocatalysts (ZF, +S-g-C3N4 and Co-ZF), and Co-ZF demonstrated the greatest photo-removal efficiency with a 98% efficiency. Additionally, the chemical stability experiment of the sample with a 48% S-gC3N4/6% Co-ZF content showed that the hybrid system is extremely robust and reusable. The current research made a ground-breaking suggestion for a brand-new synthesis method and a distinctive binary photo-catalyst that is active in the sunshine. + +## Author Contributions + +Ali Bahadur: conception, performed photocatalytic experiments, visualization of data, writing reviewing, and editing. + +Shahid Iqbal: design of study, performed major experimental works, writing-original dra preparation. Mohsin Javed: conception, design of study, writing-original dra preparation and critical revision, supervision. Syeda Saba Hassan: analysis and/or interpretation of data, performed FTIR analysis. Sohail Nadeem: material synthesis, visualization of data, writing reviewing, and editing. Ali Akbar: methodology, reviewed original manuscript, and critical revision. Rami M. Alzhrani: +draing the revised manuscript, performed XRD analysis and critical revision. Murefah Mana Al-Anazy: reviewed original manuscript, and critical revision. Eslam B. Elkaeed: visualization of data, reviewed the original manuscript and critical revision. Hala A. Ibrahium: analysis and/or interpretation of data, performed FTIR analysis. Ayesha Mohyuddin: conducted XRD analysis, acquisition of data, writing-original dra preparation. + +## Conflicts Of Interest + +The authors declare no conict of interest. + +## Acknowledgements + +The authors express their appreciation to the Deanship of Scientic Research at King Khalid University, Saudi Arabia, for funding this work through research groups program under grant of number RGP.2/164/43. Rami M. Alzhrani would like to acknowledge Taif University Researchers Supporting Project number (TURSP-2020/209), Taif University, Taif, Saudi Arabia. + +This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number +(PNURSP2022R7), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. + +## References + +1 T.-B. Nguyen, P.-N.-T. Ho, C.-W. Chen, C. P. Huang, R.-a. Doong and C.-D. Dong, Environ. Sci.: Nano, 2022, 9, 229–242. + +2 X. Bai, T. Jia, X. Wang, S. Hou, D. Hao and N. Bingjie, Catal. + +Sci. Technol., 2021, 11, 5432–5447. + +3 H. A. Abubshait, S. Iqbal, S. A. Abubshait, M. T. Alotaibi, N. Alwadai, N. Alfryyan, H. O. Alsaab, N. S. Awwad and H. A. Ibrahium, RSC Adv., 2022, 12, 3274–3286. + +4 H. Mao, Q. Zhang, F. Cheng, Z. Feng, Y. Hua, S. Zuo, A. Cui and C. Yao, Ind. Eng. Chem. Res., 2022, 61, 8895–8907. + +5 S. Iqbal, M. Javed, S. S. Hassan, S. Nadeem, A. Akbar, M. T. Alotaibi, R. M. Alzhrani, N. S. Awwad, H. A. Ibrahium and A. Mohyuddin, Colloids Surf., A, 2022, 636, 128177. + +6 K. Riaz, S. Nadeem, A. Chrouda, S. Iqbal, A. Mohyuddin, S. U. Hassan, M. Javed, A. BaQais, N. Tamam, K. Aroosh, A. Rauf, M. A. S. Abourehab, M. I. Jamil, E. B. Elkaeed, R. M. Alzhrani, N. S. Awwad and H. A. Ibrahium, Colloids Surf., A, 2022, 649, 129332. + +7 S. Nadeem, H. H. Khushi, M. Javed, S. Iqbal, H. O. Alsaab, N. S. Awwad, H. A. Ibrahium, T. Akhter, A. Rauf and H. Raza, J. Mol. Struct., 2022, 1252, 132191. + +8 S. Iqbal, M. Javed, M. A. Qamar, A. Bahadur, M. Fayyaz, A. Akbar, H. O. Alsaab, N. S. Awwad and H. A. Ibrahium, ChemistrySelect, 2022, 7, e202103694. + +9 M. Fan, Y. Cheng, X. Peng, H. Zhang, J. Wu, H. Tang, B. Li and Y. Zong, ACS Sustainable Chem. Eng., 2022, 10, 7664– +7676. + +10 M. Javed, M. A. Qamar, S. Shahid, H. O. Alsaab and S. Asif, RSC Adv., 2021, 11, 37254–37267. + +11 G. Sarp and E. Yilmaz, ACS Omega, 2022, 7, 23223–23233. + +12 M. Chandra, U. Guharoy and D. Pradhan, ACS Appl. Mater. + +Interfaces, 2022, 14, 22122–22137. + +13 A. Bahadur, S. Iqbal, H. O. Alsaab, N. S. Awwad and H. A. Ibrahium, RSC Adv., 2021, 11, 36518–36527. + +14 S. Iqbal, A. Bahadur, M. Javed, O. Hakami, R. M. Irfan, Z. Ahmad, A. AlObaid, M. M. Al-Anazy, H. B. Baghdadi, H. S. M. Abd-Rabboh, T. I. Al-Muhimeed, G. Liu and M. Nawaz, Mater. Sci. Eng., B, 2021, 272, 115320. + +15 S. Iqbal, N. Ahmad, M. Javed, M. A. Qamar, A. Bahadur, S. Ali, Z. Ahmad, R. M. Irfan, G. Liu and M. B. Akbar, J. Environ. Chem. Eng., 2021, 9, 104919. + +16 J. Ding, L. Song, X. Li, L. Chen, X. Li, J. Sun, X. Zhang, Y. Wang and X. Tian, ACS Appl. Energy Mater., 2022, 5, 8800–8811. + +17 C. Wang, W. Shi, K. Zhu, X. Luan and P. Yang, Langmuir, 2022, 38, 5934–5942. + +18 U. Sahoo, S. Pattnayak, S. Choudhury, S. Padhiari, M. Tripathy and G. Hota, Ind. Eng. Chem. Res., 2022, 61, 9703–9716. + +19 F. Chen, T. Ma, T. Zhang, Y. Zhang and H. Huang, Adv. + +Mater., 2021, 33, 2005256. + +20 T. Chen, L. Liu, C. Hu and H. Huang, Chin. J. Catal., 2021, 42, 1413–1438. + +21 W. Liu, D. Zhang, R. Wang, Z. Zhang and S. Qiu, ACS Appl. + +Mater. Interfaces, 2022, 14, 31782–31791. + +22 M. Jafarzadeh, ACS Appl. Mater. Interfaces, 2022, 14, 24993– +25024. + +23 S. Iqbal, A. Bahadur, M. Javed, G. Liu, T. I. Al-Muhimeed, A. A. AlObaid, Z. Ahmad, K. Feng and D. Xiao, J. Alloys Compd., 2022, 892, 162012. + +24 L.-Z. Wu, Acta Phys.-Chim. Sin., 2020, 36, 2004005. + +25 S. Shen, J. Pan, W. Zhou, J. Tang, H. Ding, J. Wang, L. Chen, C.-T. Au and S.-F. Yin, 2020, 36, 1905068. + +26 Z. Jin, Y. Li and X. Hao, Acta Phys.-Chim. Sin., 2021, 37, 1912033. + +27 L. Jia, X. Tan, T. Yu and J. Ye, Energy Fuels, 2022, DOI: +10.1021/acs.energyfuels.2c01137. + +28 C. Coromelci, M. Neamtu, M. Ignat, P. Samoila, M. F. Zaltariov and M. Palamaru, Ceram. Int., 2022, 48, 4829–4840. + +29 A. Aridi, D. Naoufal, H. El-Rassy and R. Awad, Ceram. Int., +2022, DOI: 10.1016/j.ceramint.2022.07.046. + +30 S. Iqbal, Appl. Catal., B, 2020, 274, 119097. + +31 H. A. Al-Shwaiman, C. Akshhayya, A. Syed, A. H. Bahkali, A. M. Elgorban, A. Das, R. S. Varma and S. S. Khan, Mater. + +Chem. Phys., 2022, 279, 125759. + +32 S. Anwer, D. H. Anjum, S. Luo, Y. Abbas, B. Li, S. Iqbal and K. Liao, Chem. Eng. J., 2021, 406, 126827. + +33 C. Akshhayya, M. K. Okla, A. M. Thomas, A. A. Al-ghamdi, M. A. Abdel-Maksoud, B. Almunqedhi, H. AbdElgawad, L. L. Raju and S. S. Khan, Mater. Chem. Phys., 2022, 277, 125464. + +34 P. A. Vinosha, J. V. A. Vinsla, J. Madhavan, S. Devanesan, M. S. AlSalhi, M. Nicoletti and B. Xavier, Environ. Res., 2022, 203, 111913. + +35 R. Rajini and A. C. Ferdinand, Chem. Data Collect., 2022, 38, 100825. + +36 R. Muhammad Irfan, M. Hussain Tahir, M. Maqsood, Y. Lin, T. Bashir, S. Iqbal, J. Zhao, L. Gao and M. Haroon, J. Catal., +2020, 390, 196–205. + +37 K. Patil, K. Jangam, S. Patange, S. Balgude, A. G. Al-Sehemi, H. Pawar and P. More, J. Phys. Chem. Solids, 2022, 167, 110783. + +38 R. Belakehal, K. Atacan, N. Guy, A. Megriche and M. ¨ Ozacar, ¨ +Appl. Surf. Sci., 2022, 154315, DOI: 10.1016/ +j.apsusc.2022.154315. + +39 X. Jiang, Z. Wang, M. Zhang, M. Wang, R. Wu, X. Shi, B. Luo, D. Zhang, X. Pu and H. Li, J. Alloys Compd., 2022, 912, 165185. + +40 H. B. Truong, B. T. Huy, S. K. Ray, G. Gyawali, Y.-I. Lee, J. Cho and J. Hur, Chemosphere, 2022, 299, 134320. + +41 B. Al-Najar, A. Younis, L. Hazeem, S. Sehar, S. Rashdan, M. N. Shaikh, H. Albuasa and N. P. Hankins, Chemosphere, 2022, 288, 132525. + +42 S. Choudhary, D. Hasina, M. Saini, M. Ranjan and S. Mohapatra, J. Alloys Compd., 2022, 895, 162723. + +43 G. Fan, Z. Gu, L. Yang and F. Li, Chem. Eng. J., 2009, 155, 534– +541. + +44 Y. Sun, W. Wang, L. Zhang, S. Sun and E. Gao, Mater. Lett., +2013, 98, 124–127. + +45 L. Han, X. Zhou, L. Wan, Y. Deng and S. Zhan, J. Environ. + +Chem. Eng., 2014, 2, 123–130. + +46 Y. Shi, L. Li, Z. Xu, H. Sun, S. Amin, F. Guo, W. Shi and Y. Li, Mater. Res. Bull., 2022, 150, 111789. + +47 Y. Fang, Q. Liang, Y. Li and H. Luo, Chemosphere, 2022, 302, 134832. + +48 S. Gao, D. Feng, F. Chen, H. Shi and Z. Chen, Colloids Surf., A, +2022, 648, 129282. + +49 H.-Y. He, Y. Yan, J.-F. Huang and J. Lu, Sep. Purif. Technol., +2014, 136, 36–41. + +50 S. Renukadevi and A. P. Jeyakumari, Inorg. Chem. Commun., +2020, 118, 108047. + +51 J. Ai, L. Hu, Z. Zhou, L. Cheng, W. Liu, K. Su, R. Zhang, Z. Chen and W. Li, Ceram. Int., 2020, 46, 11786–11798. + +52 X. Zhang, B. Lin, X. Li, X. Wang, K. Huang and Z. Chen, Chem. Eng. J., 2022, 430, 132728. + +53 S. Khosravi-Gandomani, R. Youse, F. Jamali-Sheini and N. M. Huang, Ceram. Int., 2014, 40, 7957–7963. + +54 Z. Zhu, F. Guo, Z. Xu, X. Di and Q. Zhang, RSC Adv., 2020, 10, 11929–11938. + +55 S.-W. Zhao, M. Zheng, H.-L. Sun, S.-J. Li, Q.-J. Pan and Y.-R. Guo, Dalton Trans., 2020, 49, 3723–3734. + +56 M. F. Abdel Messih, M. A. Ahmed, A. Soltan and S. S. Anis, J. + +Phys. Chem. Solids, 2019, 135, 109086. + +57 S. Iqbal, N. Ahmad, M. Javed, M. A. Qamar, A. Bahadur, S. Ali, Z. Ahmad, R. M. Irfan, G. Liu, M. B. Akbar and M. A. Qayyum, J. Environ. Chem. Eng., 2021, 9, 104919. + +58 S. Iqbal, A. Bahadur, S. Ali, Z. Ahmad, M. Javed, R. M. Irfan, N. Ahmad, M. A. Qamar, G. Liu, M. B. Akbar and M. Nawaz, J. Alloys Compd., 2021, 858, 158338. + +59 F. Khurshid, M. Jeyavelan, M. S. L. Hudson and S. Nagarajan, R. Soc. Open Sci., 2019, 6, 181764. + +60 K. S. Riaz, M. S. Nadeem, V. V. Chrouda, P. A. Iqbal, M. Alsawalha, T. Alomayri and B. Yuan, Colloids Surf., +2021, 125835. + +61 J. Feng, ACS Appl. Mater. Interfaces, 2017, 9, 14103–14111. + +62 Q. Sun, K. Wu, J. Zhang and J. Sheng, Nanotechnology, 2019, 30, 315706. + +63 J. Wang, Q. Zhang, F. Deng, X. Luo and D. D. Dionysiou, Chem. Eng. J., 2020, 379, 122264. + +64 G. J. Rani, K. J. Babu, G. G. Kumar and M. A. J. Rajan, J. Alloys Compd., 2016, 688, 500–512. + +65 M. M. Sabzehmeidani, H. Karimi, M. Ghaedi and V. M. Avargani, Mater. Res. Bull., 2021, 143, 111449. \ No newline at end of file diff --git a/medical/md/PMC9431241.md b/medical/md/PMC9431241.md new file mode 100644 index 0000000000000000000000000000000000000000..dbc7f4e955e44777746297b8d99fe001728ecbb5 --- /dev/null +++ b/medical/md/PMC9431241.md @@ -0,0 +1,53 @@ +# P1572 Impact Of Gut Colonization By Gram Negative Multiresistant Bacteria In Patients With + +![0_Image_0.Png](0_Image_0.Png) + +![0_Image_1.Png](0_Image_1.Png) Acute Leukemia Undergoing Intensive Antineoplastic Treatment + +Topic: 30. Infections in hematology (incl. supportive care/therapy) +Christelle Castañon Fernandez 1, 2, Paula López Ugarriza3, 2, Ainhoa Fernández Moreno3, 2, Nicolás Diaz Varela3, Ana María Fernández Verdugo4, Javier Fernández 4, Walter JavierZambrano1, Laura Escalada1, Paola Carolina Tamayo3, Teresa Bernal del Castillo3, 2 1 Hematology Department, Hospital Universitario San Agustín deAvilés, Oviedo, *Spain;* +2 ISPA, Instituto de Investigación Sanitaria del *Principado* de Asturias, Oviedo, *Spain;* +3 Hematology Department, Hospital Universitario Central deAsturias, Oviedo, *Spain;* +4 Servicio de Microbiología, Hospital Universitario Central deAsturias, Oviedo, *Spain* Background: Gut colonization by Gram negative multiresistant bacteria (GNMRB) occurring during chemotherapy treatment for acute leukemia (AL) reduces overall survival (OS) by increasing early non-relapse mortality (NRM). + +The impact of colonization once patients have finished antineoplastic treatment is unknown. + +Aims: To evaluate long-term consequences of GNMRB colonization on AL surviving patients. Methods: Retrospective study including all consecutive AL patients treated with standard intensive chemotherapy with or without Hematopoietic Stem Cell Transplant (HSCT) who completed first line antineoplastic treatment. A landmark analysis was performed including all patients alive 60 days after last chemotherapy cycle of HSCT. + +Primary end point was OS. Secondary end points were non-relapse mortality (NRM) and acute (a) and chronic (c) +Graft Versus Host Disease (GVHD) in allogeneic transplanted patients. Data are presented as median (interquartile range) or proportion. Cumulative incidence of NRM was calculated accounting for the competing risk of leukemicdeath. Survival curves were built using the Kaplan-Meir method and univariate analysis was performed using the Log-Rank test. Statistical analysis was performed using the R 4.1.1 software package. + +## Results: + +From April 2016 to March 2021, 142 patients from a single center were treated with intensive chemotherapy, of whom 96 were alive 60 days after chemotherapy or HSCT. Median age was 55 (42-52) years with 50% (48/96) of female patients. ECOG performance status at diagnosis of the disease was 0-1 in 98% (94/96) patients. A diagnosis of Acute Myeloid Leukemia (AML), high-risk Myelodysplastic Syndrome and Acute Lymphoblastic Leukemia (ALL) +was present in 85% (82/96), 1% (1/96) and 14 (14/96) patients, respectively. Hematopoietic Transplant Comorbidity Index (HCTCI) was low, intermediate and high in 68% (66/96), 16% (16/96) and 16% (16/96) patients, respectively. Within AML patients, ELN17 genetic risk was favourable, intermediate and adverse in 48% (38/79), 18% (14/79) and 34% (27/79) patients. Prophylactic fluoroquinolones were used in 65% (62/96) patients, significantly more used in AML/MDS than in ALL patients: 69% (57/83) vs 38% (5/13), respectively, P=0.03. Throughout the first line treatment, 48% (46/96) patients were colonized by GNMRB, being the recovered microorganisms: Klebisella sp. 61% +(28/46), Enterobacter sp: 17%, (8/46), Citrobacter sp: 11% (5/46), E. Coli: 9% (4/46), and Pseudomonas aeruginosa 2% (1/46). With a median follow up of 17 (7-34) months, there were no differences in univariate analysis of OS between colonized compared to non-colonized patients: [53 months (95% CI: 26-NR) vs NR (95% CI: 26-NR), P= +0.9], (Figure). On the contrary, age and ELN17 genetic risk were significantly associated with OS (Table). + +Additionally, no differences in aGVHD [34% (10/29) vs 45% (10/22)], P=0.4, or cGVHD [18% (4/22) vs 14% (4/29), +P=0.9] were observed between colonized and non-colonized patients. Regarding NRM, a non-significant increase in non-leukemic deaths were observed in colonized patients compared to non-colonized patients: 15% (7/46) vs 8% (4/46) P= 0.6. + +Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. + +Overall Survival Yes No P +Age>65 yo 24.6 (12-NR) NR (53-NR) 0.01 ELN genetic risk Favorable NR (NR-NR) +Intermediate 0.001 + +![1_image_0.png](1_image_0.png) + +20.6 (6.8-NR) +Adverse 26.6 (17.7-NR) +Colonization 53.2 (26-NR) NR (26-NR) 0.9 + +![1_image_1.png](1_image_1.png) + +![1_image_2.png](1_image_2.png) + +Gram negative multiresistant bacteria gut colonization is not associated with worse long-term outcomes in acute leukemia patients compared to non-colonized patients. + +Copyright Information: (Online) ISSN: 2572-9241 +© 2022 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) which allows third parties to download the articles and share them with others as long as they credit the author and the Abstract Book, but they cannot change the content in any way or use them commercially. + +Abstract Book Citations: Authors, Title, HemaSphere, 2022;6:(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. + +Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. \ No newline at end of file diff --git a/medical/md/PMC9444673.md b/medical/md/PMC9444673.md new file mode 100644 index 0000000000000000000000000000000000000000..214d924d5c4f2b6cc0aa41753d9700fecc16fdf2 --- /dev/null +++ b/medical/md/PMC9444673.md @@ -0,0 +1,311 @@ +Original Article + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +![0_image_1.png](0_image_1.png) + +Non-High-Density Lipoprotein Cholesterol and Risk of Cardiovascular Disease: The Japan Epidemiology Collaboration on Occupational + +# Health Study + +Huan Hu1, 2, Ami Fukunaga2, Toshitaka Yokoya3, Tohru Nakagawa4, Toru Honda4, Shuichiro Yamamoto4, Hiroko Okazaki5, Toshiaki Miyamoto6, Naoko Sasaki7, Takayuki Ogasawara7, Naoki Gonmori8, Kenya Yamamoto9, Ai Hori10, Kentaro Tomita11, Satsue Nagahama12, Maki Konishi2, Nobumi Katayama2, Hisayoshi Morioka1, Isamu Kabe13, Tetsuya Mizoue2 and Seitaro Dohi5 for the Japan Epidemiology Collaboration on Occupational Health Study Group 1Department of Public Health, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan 2Department of Epidemiology and Prevention, National Center for Global Health and Medicine, Tokyo, Japan 3Mitsubishi Heavy Industries, Ltd., Kanagawa, Japan 4Hitachi Health Care Center, Hitachi, Ltd., Ibaraki, Japan 5Mitsui Chemicals, Inc., Tokyo, Japan 6NIPPON STEEL CORPORATION, EAST NIPPON WORKS Kimitsu Area, Chiba, Japan 7Mitsubishi Fuso Truck and Bus Corporation, Kanagawa, Japan 8East Japan Works (Keihin), JFE Steel Corporation, Kanagawa, Japan 9Division of Chemical Information, National Institute of Occupational Safety and Health, Kanagawa, Japan 10Department of Global Public Health, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan 11Healthplant Co., Ltd., Tokyo, Japan 12All Japan Labour Welfare Foundation, Tokyo, Japan 13KUBOTA Corporation, Tokyo, Japan Aims: **We aimed to investigate the association between non-high-density lipoprotein cholesterol (non-HDL-C)** +levels and the risk of cardiovascular disease (CVD) and its subtypes. + +Methods: In this contemporary cohort study, we analyzed the data of 63,814 Japanese employees aged ≥ 30 years, without known CVD in 2012 and who were followed up for up to 8 years. The non-HDL-C level was divided into 5 groups: <110, 110-129, 130-149, 150-169, and ≥ **170 mg/dL. The Cox proportional hazards** +model was used to calculate the hazard ratios (HRs) and the corresponding 95% confidence intervals (CIs) for CVD and its subtypes associated with each non-HDL-C group, considering 130-149 mg/dL as the reference group. + +Results: **During the study period, 271 participants developed CVD, including 78 myocardial infarctions and** +193 strokes (102 ischemic strokes, 89 hemorrhagic strokes, and 2 unknowns). A U-shaped association between non-HDL-C and stroke was observed. In the analysis of stroke subtypes, the multivariable-adjusted HR (95% +CI) for hemorrhagic stroke was 2.61 (1.19–5.72), 2.02 (0.95–4.29), 2.10 (1.01–4.36), and 1.98 (0.96-4.08), +while that for ischemic stroke was 1.54 (0.77-3.07), 0.91 (0.46-1.80), 0.73 (0.38-1.41), and 1.50 (0.87-2.56) in the <110, 110-129, 150-169, and ≥ **170 mg/dL groups, respectively. Individuals with elevated non-HDL-C** +levels had a higher risk of myocardial infarction. + +Conclusions: **High non-HDL-C levels were associated with an increased risk of myocardial infarction.** +Moreover, high and low non-HDL-C levels were associated with a high risk of stroke and its subtypes among Japanese workers. + +See editorial vol. 29: 1271-1272 Key words: **Non-high-density lipoprotein cholesterol, Stroke, Myocardial infarction** + +## Introduction + +Cardiovascular disease (CVD) is a leading cause of mortality, accounting for 31% of all global deaths in 2016 1). In Japan, CVD accounted for approximately 23% of all deaths in 2019 2). Stroke is the most prevalent CVD in Japanese, accounting for +>60% of all CVD cases 3). To reduce the burden of CVD, especially stroke, it is necessary to identify the modifiable risk factors. + +Non-high-density lipoprotein cholesterol (nonHDL-C) is the sum of atherogenic lipoproteins (e.g., +very low-density lipoprotein, intermediate-density lipoprotein, and low-density lipoprotein cholesterol (LDL-C)). High levels of non-HDL-C are assumed to carry a high risk of atherosclerotic diseases. Further, high non-HDL-C levels have been consistently associated with an increased risk of coronary heart disease (CHD) and CVD4-7). + +However, the association between non-HDL-C +levels and the risk of stroke is less clear. Studies on the risk of total stroke associated with non-HDL-C levels are mainly from Asian countries, and reported mixed findings. Cohort studies from China and Korea have reported that high non-HDL-C levels are associated with an increased risk of total stroke 8-10). However, a pooled analysis of 10 Japanese cohort studies did not find such an association11). Further, a recent study has reported a U-shaped association in Japanese men12). + +With regard to stroke subtypes, a meta-analysis of 68 prospective studies (mainly conducted in Europe and North America) reported that higher non-HDL-C levels were associated with higher risk of ischemic stroke, but not hemorrhagic stroke 4). However, the pooled analysis of Japanese cohort studies did not find significant associations between non-HDL-C levels and ischemic stroke or hemorrhagic stroke 11). A +Japanese study has reported an increased risk of intracerebral hemorrhage among men with low nonHDL-C levels (<110 mg/dL), suggesting a need for additional research on the association between low non-HDL-C levels and the risk of stroke 12). + +Previous studies on the association between nonHDL-C levels and stroke in Japan were based on cohorts established in the 1980s and the 1990s, and the participants were mainly middle-aged and older community dwelling adults 11, 12). Population characteristics including circulating lipids (e.g., an increasing trend in high-density lipoprotein cholesterol (HDL-C))13, 14) and morbidity (e.g., a decline in the incidence of stroke) 15, 16) have changed markedly over the past decades. In addition, the productivity losses and economic burden due to CVD +in the Japanese working population are substantial, accounting for more than one in five premature deaths in 2015 17). Thus, evidence from a working population based on recent measurements of lipids is warranted to prevent premature CVD deaths in workers. + +## Aim + +In this study, we examined the association between non-HDL-C levels and the risk of CVD and its subtypes using data from an ongoing large-scale cohort study of Japanese workers. + +## Methods Setting + +This cohort study used data from the Japan Epidemiology Collaboration on Occupational Health +(J-ECOH) Study, which is an ongoing multi-company study of workers in Japan. To date, annual health checkup data between January 2008 and March 2020 have been collected. In the J-ECOH Study, the CVD +registry was established in participating companies in April 2012. Details of the J-ECOH Study and CVD +registration have been described elsewhere18, 19). The study protocol, including the consent procedure, was approved by the Ethics Committee of the National Center for Global Health and Medicine, Japan. + +## Participants + +The baseline population of the present study is comprised of workers in 8 participating companies which provided data on serum lipids including total cholesterol. A total of 79,039 participants aged ≥ 30 years attended either the 2010 or 2011 health checkup. We excluded participants who reported a history of CVD at baseline (n=835); those with missing data on total cholesterol, HDL-C, body mass index (BMI), and smoking status or those with missing data necessary for diagnosis of diabetes and hypertension at baseline (n=7,307). We further excluded participants who neither attended any subsequent health checkups nor had information on CVD, mortality, and long-term sick leave (n=7,083). + +Finally, 63,814 participants, comprising 53,550 men + +## And 10,264 Women, Were Included + +Annual Health Checkup The body height and weight were measured using a scale while the participant wore light clothes and no shoes. BMI was calculated as weight in kilograms divided by the square of height in meters. + +Smoking status was assessed by using a selfadministered questionnaire. Blood pressure (BP) was measured using an automatic BP monitor, with the participants in a sitting position. Total cholesterol, LDL-C, and HDL-C levels were measured using the enzymatic method. Plasma glucose levels were measured using either the enzymatic or glucose oxidase peroxidative electrode method. HbA1c levels were measured using a latex agglutination immunoassay, high-performance liquid chromatography, or the enzymatic method. All the laboratories involved in the health checkups of the participating companies received satisfactory scores (rank A or a score >95 out of 100) from external quality control agencies. + +Exposure We used the 2011 health checkup data for exposure assessment. When 2011 data were not available, we used the 2010 checkup data. NonHDL-C levels were calculated by subtracting HDL-C +levels from total cholesterol levels. Non-HDL-C levels were classified into 5 groups according to the previous study among Japanese general population12) and the Japan Atherosclerosis Society guideline20): <110, 110129, 130-149, 150-169, and ≥ 170 mg/dL. + +Outcome Incident CVD events, including fatal and nonfatal myocardial infarction (MI) and stroke, were ascertained from April 2012 to March 2020. For fatal cases, the cause of death was determined based on available information, including data from death certificates (51%), information obtained from the bereaved family or colleagues (21%), and other sources/missing (13%, source not specified; 15%, +missing). For non-fatal cases, the diagnosis of each CVD event was based on data from medical certificates written by a treating physician and submitted to the company through the worker (85%), +confirmation with the treating physician (2%), selfreport (7%), or missing (6%). + +## Covariates + +The covariates included age, sex, smoking status, BMI, hypertension, diabetes, the HDL-C level, and lipid-lowering treatment. Hypertension was defined as systolic BP ≥ 140 mmHg, diastolic BP ≥ 90 mmHg, or receiving medical treatment for hypertension21). + +Diabetes was defined as HbA1c level ≥ 6.5%, fasting plasma glucose level ≥ 126 mg/dL, random plasma glucose level ≥ 200 mg/dL, or receiving medical treatment for diabetes22). + +## Statistical Analysis + +We calculated age-and sex-adjusted baseline characteristics of the subjects using the analysis of covariance for continuous variables and the marginal structural binomial regression model for categorical variables 23). Person-time was calculated from March 31, 2012 (one day before the beginning of the follow-up period) to the date of first occurrence of a CVD event; date of censoring, which was determined individually based on the available information, including data on annual health checkups, sick leave, retirement, and death; or the end of follow-up (for most companies, this was March 31, 2020). Cox proportional hazards regression models were performed to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) for CVD and its subtypes associated with each non–HDL-C group using the 130-149 mg/dL group as the reference group, considering that a U-shaped association may exist between non-HDL-C levels and the risk of total stroke 12). We first adjusted for age (years, continuous) +and sex in model 1, and then further adjusted for current smoker (yes or no), BMI (kg/m2, continuous), +hypertension (yes or no), and diabetes (yes or no) in model 2. We additionally adjusted for the HDL-C +level and lipid-lowering treatment in model 3. Worksite was treated as strata. We tested the linear trend by treating non-HDL-C as a continuous variable and tested the non-linear trend using the fractional polynomials analysis. To further facilitate comparability with previous studies 5, 11), we also calculated the HRs for the development of MI +associated with a one standard deviation (SD) unit change in the non-HDL-C level. + +Multiple sensitivity analyses were performed by excluding participants who were receiving lipidlowering treatment, fatal CVD cases occurred during the first three years of follow-up, and CVD cases without death/medical certificates. Fractional polynomial analysis was conducted using STATA +version 15.0 (StataCorp, College Station, TX, USA). All other statistical analyses were performed using SAS +version 9.3 (SAS Institute, Cary, NC, USA). A twosided P value of <0.05 was considered statistically significant. + +| Table 1. Age- and sex-adjusted means or frequencies of risk factors for CVD according to non-HDL-C levels at baseline Non-HDL-C (mg/dL) <110 110-129 130-149 150-169 ≥ 170 N 9,839 12,823 14,721 12,530 13,901 Male, %a 75.0 80.4 85.9 88.1 90.6 Age (years)b 44.0 (0.09) 45.7 (0.08) 46.8 (0.07) 47.6 (0.08) 47.5 (0.07) Body mass index (kg/m2 ) 21.9 (0.03) 22.9 (0.03) 23.6 (0.03) 24.2 (0.03) 24.8 (0.03) Current smoker, % 35.7 32.7 33.5 34.0 36.0 Total cholesterol (mg/dL) 160.5 (0.18) 181.4 (0.16) 197.7 (0.14) 214.8 (0.16) 245.1 (0.15) High-density lipoprotein cholesterol (mg/dL) 64.9 (0.14) 61.3 (0.12) 58.3 (0.11) 55.9 (0.12) 53.3 (0.12) Lipid-lowering treatment, % 5.1 6.4 6.9 7.3 9.7 Systolic blood pressure (mmHg) 119.5 (0.15) 120.9 (0.13) 121.7 (0.12) 122.9 (0.13) 124.4 (0.12) Diastolic blood pressure (mmHg) 74.8 (0.10) 75.8 (0.09) 76.5 (0.08) 77.5 (0.09) 78.5 (0.09) Hypertension, % 20.3 21.3 22.4 23.3 24.5 HbA1c (%) 5.50 (0.01) 5.56 (0.01) 5.61 (0.01) 5.66 (0.01) 5.76 (0.01) Blood glucose (mg/dL) 98.1 (0.20) 99.2 (0.18) 100.2 (0.16) 101.1 (0.18) 103.1 (0.17) Diabetes, % 7.8 8.1 8.5 8.8 10.0 a, age adjusted; b, sex adjusted | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +| Table 2. Hazard ratio and 95% confidence interval for CVD according to non-HDL-C levels No. of events Person-years Model 1 Model 2 | Model 3 | | | | | +|--------------------------------------------------------------------------------------------------------------------------------------|----------------|----------------|------------------|------------------|------------------| +| Non-HDL-C (mg/dL) <110 | 34 | 61,439 | 1.37 (0.88-2.15) | 1.53 (0.97-2.40) | 1.67 (1.06-2.63) | +| 110-<130 | 38 | 79,235 | 1.04 (0.68-1.60) | 1.10 (0.71-1.69) | 1.14 (0.74-1.76) | +| 130-<150 | 45 | 89,704 | Reference | Reference | Reference | +| 150-<170 | 59 | 76,073 | 1.45 (0.99-2.14) | 1.38 (0.94-2.04) | 1.35 (0.91-1.99) | +| ≥ 170 | 95 | 84,655 | 2.06 (1.44-2.93) | 1.85 (1.30-2.64) | 1.75 (1.23-2.50) | +| P for linear trend | <0.001 | 0.008 | 0.06 | | | +| P for non-linear trend | Not applicable | Not applicable | <0.001 | | | +| Model 1: adjusted for age and ex | | | | | | + +## Results + +Table 1 shows the age-and sex-adjusted baseline characteristics of the study participants according to their non-HDL-C levels. Participants with higher non-HDL-C levels were more likely to be men and current smokers, and had higher BMI, BP, HbA1c levels, and blood glucose levels. Accordingly, they had a higher prevalence of hypertension and diabetes. + +During 391,106 person-years of follow-up, there were 271 incident CVD cases, including 78 MI cases and 193 stroke cases (102 ischemic stroke cases, 89 hemorrhagic stroke cases, and 2 unknown cases). + +Table 2 shows that low and high non-HDL-C levels were associated with an increased risk of total CVD, with a multivariable-adjusted HR (95% CI) of 1.67 (1.06-2.63), 1.14 (0.74-1.76), 1.35 (0.91-1.99) and 1.75 (1.23-2.50) in the <110, 110-129, 150-169, and ≥ 170 mg/dL groups, respectively, compared to the 130-159 mg/d group. + +Table 3 and **Supplementary Fig.1** show a U-shaped association between non-HDL-C levels and the risk of total stroke (P for nonlinear trend =0.001). + +In the analysis of stroke subtypes, the multivariableadjusted HR for hemorrhagic stroke was 2.61 (1.19– +5.72), 2.02 (0.95–4.29), 2.10 (1.01–4.36), and 1.98 +(0.96-4.08), while that for ischemic stroke was 1.54 +(0.77-3.07), 0.91 (0.46-1.80), 0.73 (0.38-1.41), and 1.50 (0.87-2.56) in the <110, 110-129, 150-169, and ≥ 170 mg/dL groups, respectively. + +The association between non-HDL-C levels and the risk of MI was linear **(Table 4)**. The multivariableadjusted HR for MI was 0.85 (0.27-2.63), 0.69 (0.261.82), 1.58 (0.79-3.15), and 1.90 (1.00-3.61) in the + +| Table 3. Hazard ratio and 95% confidence interval for stroke No. of events Person-years | Model 1 | Model 2 | Model 3 | | | +|--------------------------------------------------------------------------------------------|-----------|-----------|------------------|------------------|------------------| +| Total stroke Non-HDL-C (mg/dL) <110 | 30 | 61,439 | 1.69 (1.03-2.79) | 1.80 (1.09-2.98) | 1.92 (1.16-3.20) | +| 110-<130 | 32 | 79,235 | 1.23 (0.75-2.00) | 1.27 (0.78-2.08) | 1.31 (0.80-2.14) | +| 130-<150 | 32 | 89,704 | Reference | Reference | Reference | +| 150-<170 | 38 | 76,073 | 1.32 (0.83-2.12) | 1.28 (0.80-2.04) | 1.25 (0.78-2.04) | +| ≥ 170 | 61 | 84,655 | 1.87 (1.22-2.87) | 1.74 (1.13-2.67) | 1.67 (1.09-2.58) | +| P for linear trend | 0.13 | 0.44 | 0.72 | | | +| P for non-linear trend | 0.001 | 0.001 | 0.001 | | | +| Hemorrhagic stroke Non-HDL-C (mg/dL) <110 | 16 | 61,439 | 2.53 (1.17-5.48) | 2.58 (1.18-5.63) | 2.61 (1.19-5.72) | +| 110-<130 | 18 | 79,235 | 1.97 (0.93-4.18) | 2.02 (0.95-4.28) | 2.02 (0.95-4.29) | +| 130-<150 | 11 | 89,704 | Reference | Reference | Reference | +| 150-<170 | 21 | 76,073 | 2.15 (1.04-4.47) | 2.10 (1.01-4.37) | 2.10 (1.01-4.36) | +| ≥ 170 | 23 | 84,655 | 2.07 (1.01-4.26) | 1.96 (0.95-4.04) | 1.98 (0.96-4.08) | +| P for linear trend | 0.97 | 0.82 | 0.85 | | | +| P for non-linear trend | 0.97 | 0.98 | 0.98 | | | +| Ischemic stroke Non-HDL-C (mg/dL) <110 | 14 | 61,439 | 1.25 (0.63-2.46) | 1.37 (0.69-2.73) | 1.54 (0.77-3.07) | +| 110-<130 | 14 | 79,235 | 0.83 (0.42-1.63) | 0.87 (0.44-1.71) | 0.91 (0.46-1.80) | +| 130-<150 | 21 | 89,704 | Reference | Reference | Reference | +| 150-<170 | 15 | 76,073 | 0.78 (0.40-1.52) | 0.75 (0.39-1.46) | 0.73 (0.38-1.41) | +| ≥ 170 | 38 | 84,655 | 1.76 (1.03-3.00) | 1.61 (0.94-2.75) | 1.50 (0.87-2.56) | +| P for linear trend | 0.05 | 0.22 | 0.52 | | | +| P for non-linear trend | 0.001 | 0.001 | 0.001 | | | +| Model 1: adjusted for age and sex | | | | | | + +| Table 4. Hazard ratio and 95% confidence interval for myocardial infarction No. of events Person-years Model 1 | Model 2 | Model 3 | | | | +|------------------------------------------------------------------------------------------------------------------|------------------|------------------|------------------|------------------|------------------| +| Non-HDL-C (mg/dL) <110 | 4 | 61,439 | 0.58 (0.19-1.77) | 0.71 (0.23-2.19) | 0.85 (0.27-2.63) | +| 110-<130 | 6 | 79,235 | 0.58 (0.22-1.52) | 0.64 (0.24-1.68) | 0.69 (0.26-1.82) | +| 130-<150 | 13 | 89,704 | Reference | Reference | Reference | +| 150-<170 | 21 | 76,073 | 1.77 (0.89-3.53) | 1.64 (0.82-3.28) | 1.58 (0.79-3.15) | +| ≥ 170 | 34 | 84,655 | 2.47 (1.30-4.69) | 2.10 (1.11-3.99) | 1.90 (1.00-3.61) | +| P for linear trend | <0.001 | <0.001 | 0.004 | | | +| 1-SD increase | 1.55 (1.33-1.80) | 1.35 (1.16-1.57) | 1.26 (1.08-1.48) | | | +| Model 1: adjusted for age and sex | | | | | | + +the sensitivity analyses. The associations between nonHDL-C levels and risk of CVD and its subtypes did not change after excluding participants who were +<110, 110-129, 150-169, and ≥ 170 mg/dL groups, respectively (P for linear trend=0.004). + +The study results were generally supported by receiving lipid-lowering treatment, fatal CVD cases occurred within the first three years, and CVD cases without death/medical certificates **(Supplementary** +Table 1, 2, 3). + +## Discussion + +In this large-scale prospective study of a working population in Japan, we found that low and high nonHDL-C levels were associated with an increased risk of CVD. Regarding the CVD subtypes, we found a U-shaped association between non-HDL-C levels and total stroke. For MI, a positive linear relationship was observed. To our knowledge, this is the first study to investigate the association between non-HDL-C levels and CVD using data from a contemporary cohort in Japan. + +Our finding that low and high non-HDL-C +levels were associated with an increased risk of CVD +was partly in line with a previous study, which showed a U-shaped association between non-fasting triglycerides and CVD mortality in Japanese general population24). To our knowledge, no previous study has reported a U-shaped association between nonHDL-C levels and the risk of total stroke, except one recent Japanese study, which suggested a U-shaped association in men12). However, several studies conducted in China and Korea reported a positive association between non-HDL-C levels and the risk of total stroke 8-10). Our study showed that high and very low non-HDL-C levels were associated with a high risk of hemorrhagic stroke, although the nonlinear association was not statistically significant, due in part to the small number of hemorrhagic strokes. A metaanalysis of mainly Western studies and a pooled analysis of 10 Japanese cohort studies have not shown any increase in the risk of hemorrhagic stroke associated with either low or high non-HDL-C levels 4, 11). The Japan Public Health Center-based Prospective Study and the Rotterdam Study reported an inverse association between non-HDL-C and risk of hemorrhagic stroke 12, 25). We have no plausible reasons for these inconsistent data. One potential explanation may be that low non-HDL-C levels (e.g., <110 mg/ +dL) were included in the reference group in prior studies4, 8-12, 25), which may have limited their ability to detect the risk of hemorrhagic stroke associated with high and low non-HDL-C levels. Our findings on hemorrhagic stroke are partially supported by those of the Women's Health Study, which reported an increased risk of hemorrhagic stroke among those with low levels of LDL-C (<70 mg/dL), a major component of non-HDL-C, and among those with high levels of LDL-C (≥ 160 mg/dL)26). + +The mechanism underlying the observed association between non-HDL-C levels and the risk of hemorrhagic stroke is unclear. The possible explanation for the association between the risk of hemorrhagic stroke and low non-HDL-C levels is that low serum cholesterol levels can cause medial layer smooth muscle cell necrosis in intracerebral arteries and that the impaired endothelium may be more prone to microaneursyms that may subsequently rupture 27). The association between an increased risk of hemorrhagic stroke and high non-HDL-C levels may due to the atherosclerotic effects of lipoproteins such as LDL-C. Some studies have shown that cerebral microbleeds are associated with arterial and aortic stiffness28-30). Given the conflicting evidence, further studies are needed to confirm the association between non-HDL-C levels, especially its low levels, and the risk of hemorrhagic stroke. + +We also observed a higher risk of ischemic stroke in people with high (≥ 170 mg/dL) and low (≤ 110 mg/dL) non-HDL-C levels, but this increase in risk was not statistically significant. Our analysis could be underpowered due to small number of ischemic stroke cases included in our study. Studies from both Western and Asian countries, except Japan, have reported a positive association between non-HDL-C +and ischemic stroke 4, 9). In contrast, previous studies in Japan have consistently reported no association11, 12, 31). + +For example, a pooled analysis of 10 Japanese cohort studies did not show an association between nonHDL-C levels and the risk of ischemic stroke 11). + +Lacunar infarctions account for a larger proportion (> 50%) of ischemic stroke in the Japanese population than in Western or other Asian populations (1030%)32). Previous studies have shown non-HDL-C +levels are associated with atherothrombotic infarction +(another subtype of ischemic stroke) but not with lacunar infarction12, 31). The difference in the subtype distribution of ischemic stroke may account for the lack of association between non-HDL-C and ischemic stroke in Japanese studies, including our study. + +In our study, the risk of MI increased with the increasing non-HDL-C levels, with an adjusted HR +(95% CI) of 1.26 (1.08–1.48) for a one SD unit change in the non-HDL-C level. Concordant with our findings, two meta-analyses that were mainly based on studies on Western populations showed that non-HDL-C was positively associated with CHD4, 5), +with one reporting an adjusted HR of 1.50 (1.391.61) for a one SD unit change in the non-HDL-C +level4). Similarly, the pooled analysis of 10 Japanese cohort studies showed that the incidence rate ratio of acute MI was 1.62 (1.35-1.95) for a one SD unit change in the non-HDL-C level 11). Our study was conducted in a working population, including youngand middle- aged people, and used the most recent data on lipids and CVD (study period 2011-2019). + +The findings from our study and previous studies4, 5, 11) +provide strong evidence that non-HDL-C levels can be used to identify individuals at a high risk of MI. Strengths and Limitations The strength of our study is that we included young-and middle-aged people and used data from a large working population. However, our study has some limitations. First, the lack of data on the subtypes of hemorrhagic stroke and ischemic stroke did not allow us to analyze their associations with non-HDL-C levels. Second, the CVD registry data are mainly based on data from medical certificates written by a physician and submitted to the company by the worker. This registry primarily covers relatively severe cases because the submission of a medical certificate is required when taking long-term (≥ 2 weeks) sick leave. + +On the other hand, patients with milder forms of CVD were not well covered by this registration system because they were not required to submit a medical certificate if they took sick leave for <2 weeks. We also noticed that about 20% did not provide medical certificates (self-reported or missing data). However, the results did not change obviously after excluding cases without death/medical certificates. Third, due to the lack of data on socioeconomic status, family history of CVD, and lifestyle factors other than smoking status (e.g., alcohol consumption, diet, and physical activity), we were unable to control for potential effects of these factors. Fourth, our study is a Japanese occupational cohort. Therefore, our findings may not be generalizable to the general population or other racial/ethnic groups. + +## Conclusion + +Using data from a recent cohort study including Japanese workers, our study showed that high nonHDL-C levels were associated with a high risk of MI. + +Moreover, high and low non-HDL-C levels were associated with a high risk of total stroke, especially hemorrhagic stroke. Further studies are needed to confirm the present finding of a U-shaped association between non-HDL-C levels and the risk of total stroke and its subtypes. + +## Notice Of Grant Support + +This study was supported by the Industrial Health Foundation, Industrial Disease Clinical Research Grants (140202-01, 150903-01, 17030101), the Japan Society for the Promotion of Science KAKENHI (16H05251), and the Grant of National Center for Global Health and Medicine (28-Shi-1206, 30-Shi-2003, 19A1006). + +## Conflict Of Interest + +The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript. + +## References + +1) World Health Organization. https: //www.who.int/en/ +news-room/fact-sheets/detail/cardiovascular-diseases- +(cvds) (Accessed April 16, 2021) +2) Ministry of Health, Labor and Welfare. https: //www. + +mhlw.go.jp/toukei/saikin/hw/jinkou/kakutei19/index. + +html (Accessed April 16, 2021) +3) Mo X, Gai RT, Sawada K, Takahashi Y, Cox SE, +Nakayama T, Mori R. Coronary heart disease and stroke disease burden attributable to fruit and vegetable intake in Japan: projected DALYS to 2060. BMC Public Health, 2019; 19: 707 4) Emerging Risk Factors Collaboration, Di Angelantonio E, +Sarwar N, Perry P, Kaptoge S, Ray KK, Thompson A, +Wood AM, Lewington S, Sattar N, Packard CJ, Collins R, +Thompson SG, Danesh J. Major lipids, apolipoproteins, and risk of vascular disease. JAMA, 2009; 302: 19932000 5) Liu H, Deng X, Peng Y, Zeng Q, Song Z, He W, Zhang L, Gao G, Xiao T, Yu X. Meta-analysis of serum nonhigh-density lipoprotein cholesterol and risk of coronary heart disease in the general population. Clin Chim Acta, 2017; 471: 23-28 6) Sniderman AD, Williams K, Contois JH, Monroe HM, +McQueen MJ, de Graaf J, Furberg CD. A meta-analysis of low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol, and apolipoprotein B as markers of cardiovascular risk. Circ Cardiovasc Qual Outcomes, 2011; 4: 337-345 7) Brunner FJ, Waldeyer C, Ojeda F, Salomaa V, Kee F, Sans S, Thorand B, Giampaoli S, Brambilla P, Tunstall-Pedoe H, Moitry M, Iacoviello L, Veronesi G, Grassi G, +Mathiesen EB, Söderberg S, Linneberg A, Brenner H, +Amouyel P, Ferrières J, Tamosiunas A, Nikitin YP, Drygas W, Melander O, Jöckel KH, Leistner DM, Shaw JE, +Panagiotakos DB, Simons LA, Kavousi M, Vasan RS, +Dullaart RPF, Wannamethee SG, Risérus U, Shea S, de Lemos JA, Omland T, Kuulasmaa K, Landmesser U, +Blankenberg S; Multinational Cardiovascular Risk Consortium. Application of non-HDL cholesterol for population-based cardiovascular risk stratification: results from the Multinational Cardiovascular Risk Consortium. + +Lancet, 2019; 394: 2173-2183 8) Gu X, Yang X, Li Y, Cao J, Li J, Liu X, Chen J, Shen C, +Yu L, Huang J, Gu D. Usefulness of Low-Density Lipoprotein Cholesterol and Non-High-Density Lipoprotein Cholesterol as Predictors of Cardiovascular +Disease in Chinese. Am J Cardiol, 2015; 116: 1063-1070 9) Wu J, Chen S, Zhou Y, Wang C, Wang A, Zhang Q, Gao X, Hu H, Wu S, Zhao X. Non-High-Density Lipoprotein Cholesterol on the Risks of Stroke: A Result from the Kailuan Study. PLoS One, 2013; 8: e74634 10) Kim MK, Han K, Joung HN, Baek KH, Song KH, Kwon HS. Cholesterol levels and development of cardiovascular disease in Koreans with type 2 diabetes mellitus and without pre-existing cardiovascular disease. Cardiovasc Diabetol, 2019; 18: 139 11) Tanabe N, Iso H, Okada K, Nakamura Y, Harada A, +Ohashi Y, Ando T, Ueshima H; Japan Arteriosclerosis Longitudinal Study Group. Serum total and non-highdensity lipoprotein cholesterol and the risk prediction of cardiovascular events: the JALS-ECC. Circ J, 2010; 74: +1346-1356 12) Saito I, Yamagishi K, Kokubo Y, Yatsuya H, Iso H, +Sawada N, Inoue M, Tsugane S. Non-High-Density Lipoprotein Cholesterol and Risk of Stroke Subtypes and Coronary Heart Disease: The Japan Public Health Center-Based Prospective (JPHC) Study. J Atheroscler Thromb, 2020; 27: 363-374 13) Arai H, Yamamoto A, Matsuzawa Y, Saito Y, Yamada N, +Oikawa S, Mabuchi H, Teramoto T, Sasaki J, Nakaya N, +Itakura H, Ishikawa Y, Ouchi Y, Horibe H, Kita T. Serum lipid survey and its recent trend in the general Japanese population in 2000. J Atheroscler Thromb, 2005; 12: +98-106 14) Yokoyama S. Trend of HDL increase among Japanese people continues in National Health and Nutrition Survey. J Atheroscler Thromb, 2018; 25: 554-556 15) Iso H. A Japanese health success story: trends in cardiovascular diseases, their risk factors, and the contribution of public health and personalized approaches. EPMA J, 2011; 2: 49-57 16) Toyoda K. Epidemiology and registry studies of stroke in Japan. J Stroke, 2013; 15: 21-26 17) Ministry of Health, Labor and Welfare. https: //www. + +mhlw.go.jp/english/database/db-hh/1-2.html (Accessed April 16, 2021) +18) Hu H, Mizoue T, Sasaki N, Ogasawara T, Tomita K, +Nagahama S, Hori A, Nishihara A, Imai T, Yamamoto M, +Eguchi M, Kochi T, Miyamoto T, Honda T, Nakagawa T, Yamamoto S, Okazaki H, Uehara A, Shimizu M, Murakami T, Kuwahara K, Nanri A, Konishi M, Kabe I, Dohi S; Japan Epidemiology Collaboration on Occupational Health Study Group. Prediabetes and cardiovascular disease risk: A nested case-control study. + +Atherosclerosis, 2018; 278: 1-6 19) Islam Z, Akter S, Inoue Y, Hu H, Kuwahara K, Nakagawa T, Honda T, Yamamoto S, Okazaki H, Miyamoto T, +Ogasawara T, Sasaki N, Uehara A, Yamamoto M, Kochi T, +Eguchi M, Shirasaka T, Shimizu M, Nagahama S, Hori A, +Imai T, Nishihara A, Tomita K, Sone T, Konishi M, Kabe I, Mizoue T, Dohi S; Japan Epidemiology Collaboration on Occupational Health Study Group. Prediabetes, Diabetes, and the Risk of All-Cause and Cause-Specific Mortality in a Japanese Working Population: Japan Epidemiology Collaboration on Occupational Health Study. Diabetes Care, 2021; 44: 757-764 20) Kinoshita M, Yokote K, Arai H, Iida M, Ishigaki Y, +Ishibashi S, Umemoto S, Egusa G, Ohmura H, Okamura T, Kihara S, Koba S, Saito I, Shoji T, Daida H, +Tsukamoto K, Deguchi J, Dohi S, Dobashi K, +Hamaguchi H, Hara M, Hiro T, Biro S, Fujioka Y, +Maruyama C, Miyamoto Y, Murakami Y, Yokode M, Yoshida H, Rakugi H, Wakatsuki A, Yamashita S; +Committee for Epidemiology and Clinical Management of Atherosclerosis. Japan Atherosclerosis Society (JAS) +Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases 2017. J Atheroscler Thromb, 2018; 25: 846-984 21) American Diabetes Association. American Diabetes Association Standards of medical care in diabetes–2017. Diabetes Care, 2017; 40: S1-135 22) Shimamoto K, Ando K, Fujita T, Hasebe N, Higaki J, +Horiuchi M, Imai Y, Imaizumi T, Ishimitsu T, Ito M, Ito S, Itoh H, Iwao H, Kai H, Kario K, Kashihara N, Kawano Y, Kim-Mitsuyama S, Kimura G, Kohara K, +Komuro I, Kumagai H, Matsuura H, Miura K, Morishita R, Naruse M, Node K, Ohya Y, Rakugi H, Saito I, Saitoh S, Shimada K, Shimosawa T, Suzuki H, Tamura K, +Tanahashi N, Tsuchihashi T, Uchiyama M, Ueda S, +Umemura S; Japanese Society of Hypertension Committee for Guidelines for the Management of Hypertension. The Japanese Society of Hypertension Guidelines for the Management of Hypertension (JSH +2014). Hypertens Res, 2014; 37: 253-390 23) Richardson DB, Kinlaw AC, MacLehose RF, Cole SR. + +Standardized binomial models for risk or prevalence ratios and differences. Int J Epidemiol, 2015; 44: 1660-1672 24) Hirata A, Okamura T, Hirata T, Sugiyama D, Ohkubo T, +Okuda N, Kita Y, Hayakawa T, Kadota A, Kondo K, +Miura K, Okayama A, Ueshima H. Relationship between non-fasting triglycerides and cardiovascular disease mortality in a 20-year follow-up study of a Japanese general population: NIPPON DATA90. J Epidemiol, 2021. doi: 10.2188/jea.JE20200399 25) Wieberdink RG, Poels MM, Vernooij MW, Koudstaal PJ, +Hofman A, van der Lugt A, Breteler MM, Ikram MA. + +Serum lipid levels and the risk of intracerebral hemorrhage: the Rotterdam Study. Arterioscler Thromb Vasc Biol, 2011; 31: 2982-2989 26) Rist PM, Buring JE, Ridker PM, Kase CS, Kurth T, +Rexrode KM. Lipid levels and the risk of hemorrhagic stroke among women. Neurology, 2019; 92: e2286-e2294 27) Ooneda G, Yoshida Y, Suzuki K, Shinkai H, Hori S, +Kobori K, Takayama Y, Sekiguchi M. Smooth muscle cells in the development of plasmatic arterionecrosis, arteriosclerosis, and arterial contraction. Blood Vessels, 1978; 148-156 28) Romero JR, Himali JJ, Beiser A, Pase MP, Davis-Plourde KL, Parva P, Seshadri S, Mitchell GF. Aortic stiffness and cerebral microbleeds: The Framingham Heart Study. Vasc Med, 2021: 1358863X20979740 29) Ding J, Mitchell GF, Bots ML, Sigurdsson S, Harris TB, +Garcia M, Eiriksdottir G, van Buchem MA, Gudnason V, +Launer LJ. Carotid arterial stiffness and risk of incident cerebral microbleeds in older people: the Age, Gene/ +Environment Susceptibility (AGES)-Reykjavik study. Arterioscler Thromb Vasc Biol, 2015: 1889-1895 30) van Sloten TT, Protogerou AD, Henry RM, Schram MT, +Launer LJ, Stehouwer CD. Association between arterial stiffness, cerebral small vessel disease and cognitive impairment: A systematic review and meta-analysis. + +Neurosci Biobehav Rev, 2015: 121-130 31) Imamura T, Doi Y, Ninomiya T, Hata J, Nagata M, Ikeda F, Mukai N, Hirakawa Y, Yoshida D, Fukuhara M, Kitazono T, Kiyohara Y. Non-high-density lipoprotein cholesterol and the development of coronary heart disease and stroke subtypes in a general Japanese population: the Hisayama Study. Atherosclerosis, 2014; 233: 343-348 32) Kim BJ, Kim JS. Ischemic stroke subtype classification: an asian viewpoint. J Stroke, 2014; 16: 8-17 + +![9_image_0.png](9_image_0.png) + +| who were taking lipid-lowering medications) No. of events Person-years Model 1 | Model 2 | Model 3 | | | | +|----------------------------------------------------------------------------------|------------------|------------------|------------------|------------------|------------------| +| Total CVD Non-HDL-C (mg/dL) <110 | 32 | 59,263 | 1.51 (0.94-2.42) | 1.70 (1.06-2.74) | 1.86 (1.15-3.01) | +| 110-<130 | 36 | 75,154 | 1.16 (0.74-1.83) | 1.22 (0.78-1.93) | 1.27 (0.80-2.01) | +| 130-<150 | 38 | 83820 | Reference | Reference | Reference | +| 150-<170 | 52 | 70,479 | 1.52 (1.00-2.31) | 1.44 (0.95-2.19) | 1.40 (0.92-2.13) | +| ≥ 170 | 84 | 75,918 | 2.20 (1.50-3.23) | 1.97 (1.34-2.90) | 1.86 (1.26-2.74) | +| Myocardial Infarction Non-HDL-C (mg/dL) <110 | 3 | 59,263 | 0.55 (0.15-2.00) | 0.68 (0.19-2.49) | 0.83 (0.23-3.05) | +| 110-<130 | 5 | 75,154 | 0.62 (0.21-1.82) | 0.67 (0.23-1.95) | 0.72 (0.25-2.12) | +| 130-<150 | 10 | 83820 | Reference | Reference | Reference | +| 150-<170 | 17 | 70,479 | 1.87 (0.85-4.08) | 1.69 (0.77-3.70) | 1.61 (0.74-3.52) | +| ≥ 170 | 31 | 75,918 | 2.98 (1.46-6.08) | 2.46 (1.20-5.04) | 2.22 (1.08-4.56) | +| 1-SD increase | 1.66 (1.39-1.97) | 1.48 (1.23-1.79) | 1.38 (1.13-1.67) | | | +| Hemorrhagic stroke Non-HDL-C (mg/dL) <110 | 16 | 59,263 | 2.53 (1.17-5.47) | 2.64 (1.21-5.77) | 2.64 (1.20-5.79) | +| 110-<130 | 18 | 75,154 | 1.97 (0.93-4.17) | 2.03 (0.96-4.31) | 2.03 (0.96-4.31) | +| 130-<150 | 11 | 83820 | Reference | Reference | Reference | +| 150-<170 | 19 | 70,479 | 1.95 (0.93-4.10) | 1.90 (0.90-3.99) | 1.90 (0.90-3.99) | +| ≥ 170 | 20 | 75,918 | 1.85 (0.89-3.86) | 1.74 (0.83-3.64) | 1.74 (0.83-3.65) | +| Ischemic stroke Non-HDL-C (mg/dL) <110 | 13 | 59,263 | 1.42 (0.69-2.93) | 1.57 (0.75-3.26) | 1.79 (0.85-3.74) | +| 110-<130 | 13 | 75,154 | 0.95 (0.46-1.95) | 0.99 (0.48-2.04) | 1.05 (0.51-2.15) | +| 130-<150 | 17 | 83820 | Reference | Reference | Reference | +| 150-<170 | 14 | 70,479 | 0.91 (0.45-1.84) | 0.86 (0.43-1.75) | 0.83 (0.41-1.69) | +| ≥ 170 | 33 | 75,918 | 1.92 (1.07-3.45) | 1.76 (0.98-3.17) | 1.62 (0.90-2.93) | +| Model 1: adjusted for age and sex | | | | | | + +| Supplementary Table 2. Hazard ratio and 95% confidence interval for CVD according to non-HDL-C levels (excluded fatal CVD cases in the first 3 years) No. of events Person-years Model 1 Model 2 Model 3 Total CVD Non-HDL-C (mg/dL) <110 29 61,433 1.27 (0.79-2.05) 1.44 (0.89-2.33) 1.56 (0.96-2.54) 110-<130 34 79,230 1.02 (0.65-1.61) 1.08 (0.69-1.71) 1.12 (0.71-1.77) 130-<150 41 89,698 Reference Reference Reference 150-<170 55 70,670 1.49 (0.99-2.23) 1.41 (0.94-2.11) 1.38 (0.92-2.06) ≥ 170 85 84,637 2.02 (1.39-2.93) 1.81 (1.24-2.63) 1.72 (1.18-2.51) Myocardial Infarction Non-HDL-C (mg/dL) <110 2 61,433 0.37 (0.08-1.68) 0.46 (0.10-2.10) 0.54 (0.12-2.49) 110-<130 4 79,230 0.50 (0.16-1.59) 0.55 (0.17-1.77) 0.60 (0.19-1.91) 130-<150 10 89,698 Reference Reference Reference 150-<170 20 70,670 2.20 (1.03-4.69) 2.01 (0.94-4.31) 1.94 (0.91-4.15) ≥ 170 29 84,637 2.73 (1.33-5.60) 2.30 (1.16-4.73) 2.09 (1.02-4.31) 1-SD increase 1.60 (1.38-1.86) 1.39 (1.20-1.61) 1.31 (1.12-1.53) Hemorrhagic stroke Non-HDL-C (mg/dL) <110 13 61,433 2.24 (0.98-5.14) 2.39 (1.03-5.52) 2.37 (1.02-5.51) 110-<130 16 79,230 1.92 (0.87-4.24) 1.99 (0.90-4.40) 1.98 (0.90-4.38) 130-<150 10 89,698 Reference Reference Reference 150-<170 18 70,670 2.03 (0.94-4.41) 1.96 (0.91-4.25) 1.97 (0.91-4.28) ≥ 170 18 84,637 1.79 (0.83-3.89) 1.66 (0.77-3.62) 1.71 (0.78-3.73) Ischemic stroke Non-HDL-C (mg/dL) <110 14 61,433 1.25 (0.63-2.46) 1.37 (0.69-2.72) 1.54 (0.77-3.07) 110-<130 14 79,230 0.83 (0.42-1.63) 0.87 (0.44-1.71) 0.91 (0.46-1.80) 130-<150 21 89,698 Reference Reference Reference 150-<170 15 70,670 0.78 (0.40-1.52) 0.75 (0.39-1.46) 0.73 (0.38-1.41) ≥ 170 38 84,637 1.76 (1.03-3.00) 1.61 (0.94-2.75) 1.50 (0.87-2.56) Model 1: adjusted for age and sex Model 2: further adjusted for smoking, BMI, hypertension, and diabetes Model 3: further adjusted for HDL-C | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +| Supplementary Table 3. Hazard ratio and 95% confidence interval for CVD according to non-HDL-C levels (excluded CVD cases without death/medical certificate) No. of events Person-years Model 1 Model 2 Model 3 Total CVD Non-HDL-C (mg/dL) <110 26 61,428 1.29 (0.78-2.14) 1.46 (0.87-2.43) 1.55 (0.93-2.59) 110-<130 34 79,225 1.16 (0.72-1.85) 1.23 (0.77-1.97) 1.26 (0.79-2.02) 130-<150 36 89,685 Reference Reference Reference 150-<170 47 76,044 1.45 (0.94-2.24) 1.38 (0.89-2.13) 1.35 (0.88-2.09) ≥ 170 79 84,608 2.15 (1.45-3.19) 1.92 (1.30-2.86) 1.86 (1.25-2.76) Myocardial Infarction Non-HDL-C (mg/dL) <110 3 61,428 0.52 (0.14-1.84) 0.63 (0.17-2.27) 0.73 (0.20-2.66) 110-<130 4 79,225 0.46 (0.15-1.43) 0.50 (0.16-1.56) 0.54 (0.17-1.69) 130-<150 11 89,685 Reference Reference Reference 150-<170 17 76,044 1.69 (0.79-3.61) 1.57 (0.73-3.35) 1.52 (0.71-3.24) ≥ 170 25 84,608 2.18 (1.07-4.44) 1.86 (0.91-3.79) 1.70 (0.83-3.47) 1-SD increase 1.58(1.33-1.88) 1.38 (1.16-1.63) 1.29 (1.08-1.55) Hemorrhagic stroke Non-HDL-C (mg/dL) <110 12 61,428 2.19 (0.92-5.24) 2.35 (1.00-5.67) 2.29 (0.95-5.55) 110-<130 17 79,225 2.23 (0.99-5.00) 2.32 (1.03-5.22) 2.29 (1.02-5.16) 130-<150 9 89,685 Reference Reference Reference 150-<170 15 76,044 1.90 (0.83-4.35) 1.83 (0.80-4.19) 1.85 (0.81-4.23) ≥ 170 19 84,608 2.13 (0.96-4.70) 1.95 (0.88-4.33) 1.99 (0.89-4.43) Ischemic stroke Non-HDL-C (mg/dL) <110 11 61,428 1.28 (0.59-2.76) 1.42 (0.65-3.08) 1.54 (0.71-3.36) 110-<130 13 79,225 1.01 (0.49-2.10) 1.06 (0.51-2.22) 1.10 (0.53-2.30) 130-<150 16 89,685 Reference Reference Reference 150-<170 14 76,044 0.96 (0.47-1.97) 0.92 (0.45-1.89) 0.90 (0.44-1.85) ≥ 170 35 84,608 2.12 (1.17-3.83) 1.94 (1.07-3.52) 1.84 (1.01-3.34) Model 1: adjusted for age and sex Model 2: further adjusted for smoking, BMI, hypertension, and diabetes Model 3: further adjusted for HDL-C | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| diff --git a/medical/md/PMC9685632.md b/medical/md/PMC9685632.md new file mode 100644 index 0000000000000000000000000000000000000000..caaf385a6860566f3a7b573260f9b683c7922f3a --- /dev/null +++ b/medical/md/PMC9685632.md @@ -0,0 +1,220 @@ +# World Journal Of W J O Orthopedics + +![0_Image_0.Png](0_Image_0.Png) + +Submit a Manuscript: https://www.f6publishing.com *World J Orthop* 2022 November 18; 13(11): 978-985 ORIGINAL ARTICLE +DOI: 10.5312/wjo.v13.i11.978 ISSN 2218-5836 (online) +Basic Study Anatomic relationship of extensor indicis propius and extensor + +# Digitorum Communis: Implications For Tendon Transfer + +Joanne Zhou, Christopher Frey, Nicole Segovia, Jeffrey Yao + +| Joanne Zhou, Christopher Frey, Nicole Segovia, Jeffrey Yao, Department of Orthopaedic Surgery, Stanford University Medical Center, Stanford University, Redwood city, CA 94063, United States | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Specialty type: Anatomy and morphology Provenance and peer review: Unsolicited article; Externally peer reviewed. | Corresponding author: Joanne Zhou, MD, Staff Physician, Department of Orthopaedic Surgery, Stanford University Medical Center, Stanford University, 450 Broadway Street Pavilion A 2nd Fl MC 6120, Redwood City, CA 94603, United States. zhoujy@stanford.edu | +| Peer-review model: Single blind Peer-review report's scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): D Grade E (Poor): 0 | Abstract BACKGROUND The extensor indicis proprius (EIP) tendon is a frequently used donor for a variety of tendon transfers, most commonly for reconstruction of the extensor pollicis longus (EPL). EIP is known to have frequent anatomic variants including split tendons and variations in tendon arrangement. AIM To characterize the anatomy of the EIP at the level of the extensor retinaculum, where tendon harvest is often performed, and share our preferred technique for EIP to EPL transfer. | +| P-Reviewer: Luo ZW, China; Pace V, Italy Received: June 23, 2022 Peer-review started: June 23, 2022 First decision: August 22, 2022 Revised: September 6, 2022 Accepted: October 19, 2022 Article in press: October 19, 2022 Published online: November 18, 2022 | METHODS Twenty-nine fresh-frozen cadaveric forearms without history of forearm or hand injury or surgery were dissected. Tendon circumference and relationship of the EIP and extensor digitorum communis to the index (EDCI) at the metacarpophalangeal (MCP) joint and the distal extensor retinaculum were recorded. Distance from the distal extensor retinaculum to the EIP myotendinous junction was measured. RESULTS EIP was ulnar to the EDCI in 96.5% of specimens (28/29) at the distal edge of the extensor retinaculum. In the remaining specimen, EIP was volar to EDCI. Tendon circumference at the distal extensor retinaculum averaged (9.3 mm ± 1.7 mm) for EDCI and 11.1 mm (± 2.7 mm) for EIP (P = 0.0010). The tendon circumference at | + +RESULTS EIP was ulnar to the EDCI in 96.5% of specimens (28/29) at the distal edge of the extensor retinaculum. In the remaining specimen, EIP was volar to EDCI. Tendon circumference at the distal extensor retinaculum averaged (9.3 mm ± 1.7 mm) for EDCI and 11.1 mm (± 2.7 mm) for EIP (P = 0.0010). The tendon circumference at the index MCP joint averaged 11.0 mm (± 1.7 mm) for EDCI and 10.6 mm (± 2.1 mm) for EIP (P = 0.33). EIP had a greater circumference in 76% (22/29) of specimens at the distal extensor retinaculum whereas EIP had a greater circumference in only 31% (9/29) of specimens at the MCP joint. + +## Conclusion + +The EIP tendon is frequently ulnar to and greater in circumference than the EDCI at the distal extensor retinaculum, which can be taken into consideration for tendon transfers involving EIP. + +Key **Words:** Surgical anatomy; Tendon transfer; Extensor digitorum communis; Extensor indicis proprius; Tendon harvest; Cadaveric Dissection +©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved. + +Core **Tip:** We found that extensor indicis proprius (EIP) was consistently ulnar to extensor digitorum communis to the index in 96.5% of specimens at the distal extensor retinaculum. The EIP had a greater circumference in 76% (22/29) of specimens at the distal extensor retinaculum. This research contributes to the body of knowledge on extensor tendon anatomy and facilitates the smaller incisions and dissection of EIP needed for tendon transfers. + +Citation: Zhou J, Frey C, Segovia N, Yao J. Anatomic relationship of extensor indicis propius and extensor digitorum communis: Implications for tendon transfer. *World J Orthop* 2022; 13(11): 978-985 URL: https://www.wjgnet.com/2218-5836/full/v13/i11/978.htm DOI: https://dx.doi.org/10.5312/wjo.v13.i11.978 + +## Introduction + +The extensor indicis proprius (EIP) is a redundant extrinsic extensor of the index finger. It is present in 96.5% of the population and is commonly used for tendon transfers[1]. To correctly harvest the EIP and plan procedural incisions, a thorough understanding of the arrangement of the extensor tendons and their junctural connections is required. + +Understanding the variability in anatomic relationships is important when harvesting EIP through a small incision. These variations have been documented in clinical and anatomic studies[2]. The EIP originates from the dorsal aspect of the ulna, the interosseus membrane, and the extensor pollicis longus (EPL) septum[3]. It has the most distal muscle belly of all the extensor tendons, and is the only muscle belly that enters the fourth compartment, under the extensor retinaculum and deep to the extensor digitorum communis (EDC) tendons[1]. Multiple studies have noted that anatomic variants are frequent (10%)[4-7] with regards to arrangement and number of slips. Defining each slip as the presence of independent fascial sheaths at the metacarpal level, the EIP has been cited to have double slips in 16% +and triple slips in 7% of cadaveric studies[5]. Gonzalez et al[6] classified the arrangement of EIP and extensor digitorum communis to the index (EDCI) at the metacarpophalangeal (MCP) joint into six types: (1) Single EIP ulnar to a single EDCI; (2) Single EDCI between two slips of EIP; (3) Two slips of EIP ulnar to a single EDCI; (4) Single EIP ulnar to two slips of EDCI; (5) Single EIP radial to a single EDCI; and (6) Single EIP volar to a single EDCI. The most common arrangement is a single EIP inserting ulnar to a single EDCI on the extensor hood in 81%-98.3% of specimens[1,4,6], volar to the EDCI in 10% to 11%[4,5,7], and radial to EDCI in 3%-8%[5]. In other tendon transfers, tendon circumference may also be used as a distinguishing characteristic. Celik et al[8] examined the width and thickness of EDCI and EIP tendons at the level of mid-diaphyseal metacarpal bones and found similar thickness for EDCI and EIP (1.5 mm ± 0.4 mm and 1.47 mm ± 0.38 mm respectively). To our knowledge, no studies have examined the relationship and anatomy of the EIP and EDCI at the level of tendon harvest. + +## Clinical Relevance + +EPL ruptures occur in 0.7% of non-displaced distal radius fractures and 3% of dorsally displaced distal radius fractures[9,10]. Tendon attrition is attributed to friction and compression in the poorly vascularized third compartment between the extensor retinaculum and displaced bone fragments of the dorsal radial tubercle[11]. EIP to EPL transfer is one of the most common tendon transfers in the upper extremity. In addition to anatomic proximity and its redundancy for index finger extension, EIP is synergistic to EPL: The motion of extending the index finger and the thumb interphalangeal (IP) joint is commonly performed together during tasks, allowing patients to recover thumb extension after transfer without extensive relearning. Many techniques exist for harvesting and performing this tendon transfer. The incision for harvest can be made longitudinally or transversely, at the level of the distal or proximal extensor retinaculum, and transfer can occur with two incisions or three with a pulvertaft distal to the retinaculum[12]. Classically described, this tendon transfer has been performed through three incisions: A transverse incision over the MCP joint to transect the ulnar EIP, a longitudinal incision over the thumb metacarpal to dissect out the ruptured tendon and carry out the transfer, and a longitudinal incision at the extensor retinaculum to identify EIP and perform tendon harvest. However, technique adaptations may be considered that use the anatomic relationship of these tendons. We performed a cadaveric study to evaluate the relationship between EDCI and EIP and share our preferred surgical technique for EIP to EPL tendon transfer. We sought to characterize the circumference and relationship of the EIP and EDCI tendons at the distal extensor retinaculum, which is relevant for tendon identification when performing an EPL transfer. + +## Materials And Methods Specimens + +Twenty-nine skeletally mature fresh frozen cadaveric forearms ages 19 to 52 were obtained. Clinical histories confirmed no previous trauma or surgery to the donor. Tendons were defined as independent bands originating from muscle. Tendons were occasionally divided into one or more separable smaller slips at the retinaculum. Individual slips were defined distal to the muscle belly with independent fascial sheaths. + +## Dissection + +All specimens were dissected with a single dorsal longitudinal incision made just ulnar to Lister's tubercle extending from the index MCP joint to proximal to the extensor retinaculum. Subcutaneous fat and superficial nerves and veins were elevated in a single flap and tenosynovium was dissected off the EDCI and EIP. The extensor retinaculum was identified as oblique transverse fibers with roughly parallel proximal and distal edges. A 4-0 Prolene suture was used to measure the circumference of the two tendons at the distal edge of extensor retinaculum and the MCP joint (Figure 1) with a single simple knot, and dissection scissors were used to cut the knot at the intersection. The remaining Prolene suture was then measured with a ruler to derive circumference of the tendon. Prolene was chosen for its high intrinsic memory and thus decreased risk for error in measurement of small caliber circumference tendons[13]. The distance in millimeters between the distal edge of the extensor retinaculum and the myotendinous junction of EIP was measured as well. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. + +## Statistics + +To assess our sample with a statistical two-tailed significance of 0.05, power of 80%, and effect size r = 0.6, our a priori power analysis yielded a sample size of 25 was required. Paired t-tests were applied to compare the values obtained from the EIP and EDCI tendon measurements at the distal extensor retinaculum and MCP joint. P < 0.05 was considered to be significant. + +## Results + +Twenty-nine fresh frozen human cadaver forearms were dissected. They were from 20 men and 9 women. The right forearm was dissected in 16 cases and the left in 13 cases. None of the specimens had vascular or nerve variations that were apparent upon dissection. None had evidence of prior surgery to the hand, forearm, or wrist, and clinical histories of the specimens confirmed no history of trauma to the area. + +In all but one specimen, the EIP tendon was ulnar to the EDCI tendon (96.5%, 28/29); in the remaining specimen, the EIP was volar to the EDCI. The tendon circumference at the distal edge of the extensor retinaculum averaged 9.3 mm (± 1.7 mm) for the EDCI and 11.1 mm (± 2.7 mm) for the EIP (P = 0.001). The tendon circumference at the index MCP joint was measured to be 11.0 mm (± 1.7 mm) for the EDCI and 10.6 mm (± 2.1 mm) for the EIP (P = 0.331). The EIP had a greater circumference in 76% (22/29) of specimens at the distal extensor retinaculum whereas the EIP had a greater circumference in 31% (9/29) of specimens at the MCP joint. The distance from the distal extensor retinaculum to the myotendinous junction of EIP varied widely but averaged 16.2 mm (± 11.1 mm), which is similar to findings in other studies[11] (Table 1). We did not find significant differences in tendon circumference between specimens of different sex, but overall the tendon circumferences were lower for females as compared to male specimens (Table 2). In addition, the distance between the distal extensor retinaculum to myotendinous junction was lower in female specimens compared to that of males (Table 2); this was also not statistically significant. + +In one specimen, the EIP tendon was split at the extensor retinaculum, with the radial branch joining the EDCI tendon at the MCP. However, both slips of the EIP were still found to be ulnar to the EDCI tendon at the distal extensor retinaculum (Figure 2). + +| Table 1 Specimen demographics and measurements Laterality Age (yr) Sex Circumference at dER Circumference at | EIP distance from dER to | Relationship of EIP | | | | | | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------|----------------------------|---------|------|------|------|----|----------------------| +| (mm) | MCP (mm) | myotendinous junction (mm) | to EDCI | | | | | | +| cEDCI | cEIP | cEDCI | cEIP | EIP | | | | | +| Left | 52 | M | 8 | 15 | 12 | 14 | 29 | EIP ulnar | +| Left | 54 | F | 9 | 10 | 13 | 10 | 15 | EIP ulnar | +| Right | 38 | M | 12 | 13 | 12 | 12 | 16 | EIP ulnar | +| Left | 48 | M | 12 | 15 | 14 | 13 | 15 | EIP ulnar | +| Right | 54 | F | 11 | 11 | 13 | 9 | 14 | EIP ulnar | +| Right | 30 | M | 7 | 8 | 11 | 8 | 20 | EIP ulnar | +| Left | 24 | M | 11 | 12 | 9 | 10 | 18 | EIP ulnar | +| Right | 44 | M | 9 | 14 | 11 | 14 | 23 | EIP ulnar | +| Left | 38 | F | 9 | 8 | 10 | 9 | 27 | EIP ulnar | +| Right | 57 | F | 9 | 12 | 11 | 13 | 18 | EIP ulnar | +| Right | 19 | M | 8 | 9 | 10 | 10 | 2 | EIP ulnar | +| Right | 44 | M | 11 | 13 | 11 | 11 | 37 | EIP ulnar | +| Left | 30 | M | 7 | 5 | 14 | 9 | 22 | EIP ulnar | +| Left | 49 | F | 9 | 13 | 10 | 10 | 4 | EIP ulnar | +| Left | 19 | M | 9 | 9 | 8.5 | 11 | 2 | EIP ulnar | +| Right | 48 | M | 9 | 8 | 9 | 9 | 48 | EIP ulnar | +| Right | 48 | M | 8 | 14 | 12 | 14 | 25 | EIP ulnar | +| Left | 44 | M | 11 | 13 | 12 | 8 | 17 | EIP ulnar | +| Right | 24 | M | 10 | 14 | 13 | 12 | 13 | EIP ulnar | +| Left | 35 | M | 14 | 10 | 14 | 10 | 10 | EIP ulnar | +| Left | 20 | M | 9 | 11 | 10 | 8 | 2 | EIP ulnar | +| Right | 20 | M | 9 | 13 | 8 | 11 | 3 | EIP volar | +| Left | 20 | F | 10 | 7 | 11 | 8 | 10 | EIP ulnar | +| Right | 21 | F | 9 | 10 | 10 | 9 | 3 | EIP ulnar | +| Left | 40 | M | 7 | 11 | 10 | 8 | 24 | 2 slips of EIP ulnar | +| Right | 49 | F | 7 | 8 | 11 | 11 | 4 | EIP ulnar | +| Right | 33 | M | 9 | 12 | 9.5 | 12 | 18 | EIP ulnar | +| Right | 20 | F | 9 | 10 | 9 | 8 | 6 | EIP ulnar | +| Left | 33 | M | 9 | 15 | 11 | 15 | 24 | EIP ulnar | +| Average | 36.4 | 9.3 | 11.1 | 11.0 | 10.6 | 16.2 | | | +| Standard | 12.7 | 1.7 | 2.7 | 1.7 | 2.1 | 11.1 | | | +| deviation dER: Distal extensor retinaculum; MCP: Metacarpophalangeal; EIP: Extensor indicis proprius; EDCI: Extensor digitorum communis to the index; M: Male; | | | | | | | | | + +dER: Distal extensor retinaculum; MCP: Metacarpophalangeal; EIP: Extensor indicis proprius; EDCI: Extensor digitorum communis to the index; M: Male; F: Female. + +## Discussion + +Although EIP has a role in index finger extension, it also possesses utility in tendon transfers. It is most often used in cases of EPL rupture, and many anatomic studies have been performed on extensor tendon variations[1,4,8,14]. The causes of these differences remain obscure, although differences in racial grouping have been suggested. South Asian populations have the highest rate of single-slip EIP and the lowest rate of double-slip EIP when compared to Japanese, Europeans and North Americans[1]. + +| Table 2 Measurements of extensor indicis proprius and extensor digitorum communis to the index tendons, segregated by gender Sex Age (yr) ± Circumference at dER (mm) ± Circumference at MCP (mm) EIP distance from dER to myotendinous junction Std Std ± Std (mm) ± Std cEDCI cEIP cEDCI cEIP EIP M 34.7 ± 11.1 9.5 ± 1.9 11.7±2.8 11.1 ± 1.8 11.0 ± 2.3 18.4 ± 11.8 F 40.2 ±1 5.8 9.1 ± 1.1 9.9 ± 2.0 10.9 ± 1.4 9.7 ± 1.6 11.2 ± 8.0 P value 0.28 0.56 0.09 0.77 0.14 0.11 | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Std: Standard deviation; dER: Distal extensor retinaculum; MCP: Metacarpophalangeal; EIP: Extensor indicis proprius; EDCI: Extensor digitorum + +![4_image_0.png](4_image_0.png) communis to the index; M: Male; F: Female. + +Figure 1 Tendon circumference **measurement.** Tendon circumference was measured utilizing 4-0 prolene at distal extensor retinaculum and at the + +![4_image_1.png](4_image_1.png) + +metacarpophalangeal joint for both the extensor indicis proprius and extensor digitorum communis to the index tendons. Prolene was used to obtain accurate circumference measurements. The distal extensor retinaculum was marked prior to measurements to ensure repeatability and consistent measurement to the myotendinous junction. + +Figure 2 Interesting case of a split extensor indicis proprius tendon that joined the extensor digitorum communis to the index **tendon.** A +specimen in which the EIP consisted of two slips, one of which arose from the extensor indicis proprius and joined the extensor digitorum communis to the index just proximal to the metacarpophalangeal joint. + +We chose to measure EIP and EDCI circumference at the distal edge of the extensor retinaculum because this is where EIP is typically harvested to attain sufficient tendon length for transfer. Given that the EIP has the most distal muscle belly, often in the extensor retinaculum, we hypothesized that the EIP would have a greater circumference than the EDCI at the distal extensor retinaculum. Although the EIP circumference 11.1 mm (± 2.7 mm) was statistically greater than EDCI circumference 9.3 mm (± 1.7 mm) (P = 0.001), this was found to be true in only in 76% (22/29) of specimens. Perhaps this could be attributed to the wide variety in distance between the distal edge of the extensor retinaculum and the muscle belly of EIP, which averaged 16.2 mm (± 11.1 mm), similar to findings in other studies[11]. Although we hypothesized that a smaller distance between the myotendinous junction of EIP and the distal edge of the extensor retinaculum would be associated with a greater difference in circumference between EIP and EDCI, this was not found to be statistically significant (P = 0.510). + +In 28/29 specimens with a single insertion for EIP, the EIP was located ulnar to the EDCI at the extensor retinaculum. It was located directly volar in the other specimen. The specimen with a split EIP had both branches ulnar to EDCI. These findings are consistent with the known literature where the vast majority (approximately 99%) of single slip EIP tendons are ulnar to EDCI[15], even when 7% of EIPs had two slips and of these, half had both tendons ulnar[1]. These findings are useful when identifying tendons for transfer with small transverse incisions at the distal extensor retinaculum. Although there are other reports of minimally invasive single incision EIP to EPL transfer[15] with a 3 cm incision placed midway between the course of the EIP and EPL at the level of mid-diaphyseal metacarpal, we believe the frequent anatomic variations in the EDCI and EIP relationship warrant additional incisions for EIP harvest and identification. + +## Preferred Surgical Technique + +Given the finding of the frequently more ulnar EIP to EDCI tendon at the level of the distal extensor retinaculum, we recommend performing a small transverse as opposed to a longitudinal incision at the extensor retinaculum, allowing for a more cosmetic scar. Here, we share our preferred technique using wide awake local EIP to EPL tendon transfer[16]. + +Begin by injecting 30 mL of 1% lidocaine with 1 : 100000 epinephrine 2 cm beyond the distal extent of the planned incisions. For incisions, we recommend a small 1 cm transverse incision just ulnar to Lister's tubercle at the distal aspect of the extensor retinaculum over the EIP tendon, a 1cm transverse incision over the index metacarpophalangeal joint, and a 2 cm longitudinal incision over the thumb carpometacarpal joint, where the distal stump of the EPL is often located for transfer. At the distal extensor retinaculum, fully flex the wrist and identify the ulnar EIP tendon. Perform a small 1cm transverse incision at the index MCP joint to transect the more ulnar EIP tendon. The extensor indicis is consistently "free" from junctura tendinae[3,8], and after dissecting the EIP tendon from EDCI, a small Ragnell retractor may be used to harvest the EIP tendon through the 1 cm incision at the distal extensor retinaculum. A transverse incision acknowledging the frequent relationship of the EIP being more ulnar or volar to EDCI at the distal extensor retinaculum avoids the longitudinal incision previously used by the authors for dissection to the myotendinous junction, improving cosmesis with an incision hidden along Langer's lines. Use a single 4-0 Fiberwire (Arthrex, Naples, FL) suture to secure any remaining distal EIP tendon stump to EDCI at the MCP joint to prevent the tendon stump from interfering with range of motion of the MCP joint. Transfer the harvested tendon subcutaneously into the longitudinal thumb incision (underneath dorsal radial branch of radial nerve), leaving the tendon in the extensor retinaculum. Use a tendon weaver to make 3 Pulvertaft weaves at 90 degree angles to each other and set the appropriate tension for the tendon to allow full extension of MCP and IP joints, or just shy of full extension to accommodate for loosening of the transfer. Place a single 4-0 Fiberwire suture before asking the patient to move the thumb. Check full flexion and full extension to ensure appropriate range of motion. Place a 4-0 Fiberwire horizontal mattress suture through each subsequent weave. Splint in thumb spica with thumb fully extended for 4 wk and start the retraining process with a hand therapist. In cases of suspected abnormal anatomy, we recommend following the EIP tendon proximally with an additional incision at the myotendinous junction to evaluate for the most distal muscle belly in order to isolate the correct slip(s) for tendon transfer. Should there be a split tendon, consider using a single slip for the tendon transfer. + +## Limitations + +Although a power analysis was implemented to detect significance for an effect size of 0.6 with 2 mm difference in tendon circumference, it is inadequately powered to capture all variations due to the relatively low incidence of these tendon variations. We performed our dissections from a random selection of cadaveric specimens but there is a chance that it is not representative of the population as a whole. Lastly, there is a chance of measurement error given the small values being analyzed. To counteract this, Prolene, a synthetic monofilament composed of isostatic crystalline stereoisomer of polypropylene was chosen for its intrinsic high memory characteristics and thus decreased risk for error in measurement with small caliber tendons[13]. Considering these limitations, we believe that our study is well designed to accurately depict the relationship between the EIP and EDCI at the distal retinaculum and builds on the existing literature characterizing the complex relationship of dorsal hand tendons. + +## Conclusion + +We found that EIP was consistently ulnar to EDCI in 96.5% of specimens at the distal extensor retinaculum. The EIP had a greater circumference in 76% (22/29) of specimens at the distal extensor retinaculum. This research contributes to the body of knowledge on extensor tendon anatomy and + +## Article Highlights Research Objectives + +We sought to characterize the arrangement and circumference of the extensor indicis proprius (EIP) tendon with respect to the extensor digitorum communis tendon to the index finger (EDCI) at the level of the extensor retinaculum, where tendon harvest is often performed, and share our preferred technique for EIP to extensor pollicis longus (EPL) transfer. + +## Research Methods + +Twenty-nine fresh-frozen cadaveric forearms were dissected. Tendon circumference and relationship of the EIP and EDCI at the metacarpophalangeal (MCP) joint and the distal extensor retinaculum were recorded. Distance from the distal extensor retinaculum to the EIP myotendinous junction was measured. + +## Research Results + +EIP was ulnar to the EDCI in 96.5% of specimens (28/29) at the distal edge of the extensor retinaculum. In the remaining specimen, EIP was volar to EDCI. Tendon circumference at the distal extensor retinaculum averaged (9.3 mm ± 1.7 mm) for EDCI and 11.1 mm (± 2.7 mm) for EIP (P = 0.0010). The tendon circumference at the index MCP joint averaged 11.0 mm (± 1.7 mm) for EDCI and 10.6 mm (± 2.1 mm) for EIP (P = 0.33). EIP had a greater circumference in 76% (22/29) of specimens at the distal extensor retinaculum whereas EIP had a greater circumference in only 31% (9/29) of specimens at the MCP joint. + +## Research Conclusions + +The EIP tendon is frequently ulnar to and greater in circumference than the EDCI at the distal extensor retinaculum, suggesting a minimally invasive approach to tendon transfer using transverse incisions hidden in Langer's lines would be effective and safe. + +## Research Perspectives + +This research contributes to the body of knowledge on extensor tendon anatomy and facilitates the smaller incisions and dissection of EIP needed for tendon transfers. + +## Research Motivation + +Understanding the variability in anatomic relationships is important when harvesting EIP through a small incision. To our knowledge, no studies have examined the relationship and anatomy of the EIP +and EDCI at the level of tendon harvest at the distal extensor retinaculum. + +## Research Background + +The EIP is a redundant extrinsic extensor of the index finger, commonly used in tendon transfer for EPL ruptures. Many variations of the course and arrangement of the tendons have been previously described. It is important to understand the arrangement and anatomy of the EIP tendon at the level of tendon harvest. + +## Acknowledgements + +The authors sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their families deserve our highest gratitude. + +## Footnotes + +Author contributions: Zhou J contributed to study design, dissections, statistical analysis, and writing the manuscript; Frey C contributed to study design, dissections, statistical analysis, and writing the manuscript; Segovia N carried out statistical analyses and assisted with manuscript revisions; Yao J contributed to study design and manuscript revisions. + +Institutional review board statement: This study was deemed exempt from Institutional Review Board Approval as only donated cadaveric specimens were used. + +Informed consent statement: This study was deemed exempt from informed consent statement as only donated cadaveric specimens were used. + +Conflict-of-interest statement: The authors have nothing to disclose. + +Data sharing statement: The statistical methods of this study were reviewed by our research support center, and the statistical methods and techniques mentioned are appropriate for the research. + +Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BYNC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is noncommercial. See: https://creativecommons.org/Licenses/by-nc/4.0/ + +## Country/Territory Of Origin: United States + +ORCID number: Joanne Zhou 0000-0002-6226-4701; Christopher Frey 0000-0001-7009-7977. + +S-Editor: Wang DM L-Editor: A +P-Editor: Wang DM + +## References + +Yammine K. The prevalence of the extensor indicis tendon and its variants: a systematic review and meta-analysis. Surg Radiol Anat 2015; 37: 247-254 [PMID: 25096501 DOI: 10.1007/s00276-014-1352-0] +1 Hirai Y, Yoshida K, Yamanaka K, Inoue A, Yamaki K, Yoshizuka M. An anatomic study of the extensor tendons of the human hand. *J Hand Surg Am* 2001; 26: 1009-1015 [PMID: 11721244 DOI: 10.1016/S0363-5023(01)70045-7] +2 Zilber S, Oberlin C. Anatomical variations of the extensor tendons to the fingers over the dorsum of the hand: a study of 50 hands and a review of the literature. *Plast Reconstr Surg* 2004; 113: 214-221 [PMID: 14707639 DOI: +10.1097/01.PRS.0000091163.86851.9C] +3 von Schroeder HP, Botte MJ. Anatomy of the extensor tendons of the fingers: variations and multiplicity. *J Hand Surg Am* 1995; 20: 27-34 [PMID: 7722260 DOI: 10.1016/S0363-5023(05)80053-X] +4 von Schroeder HP, Botte MJ. Anatomy and functional significance of the long extensors to the fingers and thumb. *Clin* Orthop Relat Res 2001; 74-83 [PMID: 11210972 DOI: 10.1097/00003086-200102000-00010] +5 Gonzalez MH, Weinzweig N, Kay T, Grindel S. Anatomy of the extensor tendons to the index finger. *J Hand Surg Am* 1996; 21: 988-991 [PMID: 8969421 DOI: 10.1016/S0363-5023(96)80305-4] +6 Mestdagh H, Bailleul JP, Vilette B, Bocquet F, Depreux R. Organization of the extensor complex of the digits. *Anat Clin* 1985; 7: 49-53 [PMID: 3994853 DOI: 10.1007/BF01654629] +7 Celik S, Bilge O, Pinar Y, Govsa F. The anatomical variations of the extensor tendons to the dorsum of the hand. *Clin Anat* 2008; 21: 652-659 [PMID: 18792963 DOI: 10.1002/ca.20710] +8 Skoff HD. Postfracture extensor pollicis longus tenosynovitis and tendon rupture: a scientific study and personal series. Am J Orthop (Belle Mead NJ) 2003; 32: 245-247 [PMID: 12772876] +9 Heidemann J, Gausepohl T, Pennig D. [Narrowing of the third extensor tendon compartment in minimal displaced distal radius fractures with impending rupture of the EPL tendon]. *Handchir Mikrochir Plast Chir* 2002; 34: 324-327 [PMID: +12494385 DOI: 10.1055/s-2002-36303] +10 Lepage D, Tatu L, Loisel F, Vuillier F, Parratte B. Cadaver study of the topography of the musculotendinous junction of the finger extensor muscles: applicability to tendon rupture following closed wrist trauma. *Surg Radiol Anat* 2015; 37: 853858 [PMID: 25577541 DOI: 10.1007/s00276-015-1417-8] +11 Schneider LH, Rosenstein RG. Restoration of extensor pollicis longus function by tendon transfer. *Plast Reconstr Surg* 1983; 71: 533-537 [PMID: 6828587 DOI: 10.1097/00006534-198304000-00015] +12 Swanson NA, Tromovitch TA. Suture materials, 1980s: properties, uses, and abuses. *Int J Dermatol* 1982; 21: 373-378 [PMID: 6752052 DOI: 10.1111/j.1365-4362.1982.tb03154.x] +13 Ozturk K, Kastamoni Y, Dursun A, Albay S. Prevalence of the extensor digitorum, extensor digiti minimi and extensor indicis tendons and their variations. *Hand Surg Rehabil* 2020; 39: 320-327 [PMID: 32259596 DOI: 10.1016/j.hansur.2020.02.010] +14 Lahiri A. A Simple and Quick Tendon Transfer Technique to Restore Thumb Function OPEN ACCESS Surgical Technique. *World J Surg Surg Res* 2020; 3: 1208 15 Lalonde DH. Wide-awake extensor indicis proprius to extensor pollicis longus tendon transfer. *J Hand Surg Am* 2014; 39: 2297-2299 [PMID: 25283490 DOI: 10.1016/j.jhsa.2014.08.024] +16 Published by **Baishideng Publishing Group Inc** + +![8_image_0.png](8_image_0.png) + +![8_image_1.png](8_image_1.png) + +7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA +Telephone: +1-925-3991568 E-mail: bpgoffice@wjgnet.com Help Desk: https://www.f6publishing.com/helpdesk https://www.wjgnet.com \ No newline at end of file diff --git a/medical/md/PMC9929830.md b/medical/md/PMC9929830.md new file mode 100644 index 0000000000000000000000000000000000000000..337f4d002ea2321776b5aa0186d949ef042bbcf0 --- /dev/null +++ b/medical/md/PMC9929830.md @@ -0,0 +1,383 @@ +Original Article + +![0_image_0.png](0_image_0.png) + +# Network And Pathway-Based Analysis Of Genes Associated With Esophageal Squamous Cell Carcinoma + +Wenwu He1,2\#^, Kun Yuan3\#, Jinlan He1, Chenghao Wang2, Lin Peng2, Yongtao Han2**, Nianyong Chen1** +1Department of Head and Neck Oncology and Department of Radiation Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China; 2Department of Thoracic Surgery, Sichuan Cancer Hospital and Research Institute, Sichuan Cancer Center, Cancer Hospital Affiliated to University of Electronic Science and Technology of China, Chengdu, China; 3Department of Anesthesiology, The First Hospital of China Medical University, Shenyang, China Contributions: (I) Conception and design: W He, N Chen; (II) Administrative support: N Chen, Y Han; (III) Provision of study materials: K Yuan, J He; (IV) Collection and assembly of data: C Wang, K Yuan; (V) Data analysis and interpretation: L Peng, W He; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. + +\#These authors contributed equally to this work. + +Correspondence to: Nianyong Chen. Department of Head and Neck Oncology and Department of Radiation Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, 37 Guoxue Alley, Chengdu 610041, China. Email: n_ychen@hotmail.com. + +Background: Although diagnostic methods and treatments have improved over the last few years, the 5-year survival rate of esophageal squamous cell carcinoma (ESCC) patients remains generally poor. The development of high-throughput technology has facilitated great achievements in localization of ESCCrelated genes. To take a further step toward a thorough understanding of ESCC at a molecular level, the potential pathogenesis of ESCC needs to be deciphered. + +Methods: The interaction of ESCC-related genes was explored by collecting genes associated with ESCC +and then performing gene enrichment assays, pathway enrichment assays, pathway crosstalk analysis, and extraction of ESCC-specific subnetwork to describe the relevant biochemical processes. + +Results: Through Gene Ontology (GO) enrichment analysis, many molecular functions related to response to chemical, cellular response to stimulus, and cell proliferation were found to be significantly enriched in ESCC-related genes. The results of pathway and pathway crosstalk analysis showed that pathways associated with multiple malignant tumors, the immune system, and metabolic processes were significantly enriched in ESCC-related genes. Through the analysis of specific subnetworks, we obtained some novel ESCC-related potential genes, such as MUC13, GSTO1, FIN, GRB2, *CDC25C*, and others. + +Conclusions: The molecular mechanism of ESCC is extremely complex. Some inducing factors change the expression status of many genes. The abnormal expression of genes mediates the biological processes involved in immunity and metabolism, apoptosis, and cell proliferation, leading to the occurrence of tumors. + +The genes MUC13, RYK, and FIN may be potential prognostic indicators of ESCC; *GRB2* and *CDC25C* +may be potential targets of ESCC in proliferation. Our work may provide valuable information for further understanding the molecular mechanisms for the development of ESCC. + +Keywords: Esophageal squamous cell carcinoma (ESCC); oncogene; network analysis; pathway crosstalk analysis Submitted Dec 12, 2022. Accepted for publication Jan 10, 2023. Published online Jan 31, 2023. doi: 10.21037/atm-22-6512 View this article at: https://dx.doi.org/10.21037/atm-22-6512 + +## Introduction + +Esophageal squamous cell carcinoma (ESCC), originating from the esophageal mucosa or gland, is malignant and aggressive with a poor prognosis, and accounts for 90% of all cases of esophageal cancer globally. In some eastern Asian and African countries, the incidence of ESCC is extremely high (1,2). An extensive body of research has demonstrated that alcohol and smoking are major risk factors for ESCC. Meanwhile, environmental factors such as the intake of hot beverages, nutritional deficiencies, and limited intake of fruits and vegetables also play a role in the development of this cancer (3,4). + +ESCC is clinically challenging and requires multidisciplinary approaches to diagnosis and treatment. The early detection of ESCC is difficult, and most patients are diagnosed with advanced ESCC when major symptoms such as progressive dysphagia and pain behind the sternum occur. Most patients with ESCC require extensive treatment, including chemotherapy, chemoradiotherapy, and/or surgical resection. Palliative treatments are adopted by patients with advanced or metastatic ESCC. However, the limited clinical approaches for the early diagnosis and treatment of ESCC lead to a 10% 5-year survival rate for patients (1,5,6). + +Currently, countless genomic analyses have increased + +## Highlight Box + +Key findings - At the molecular functional level, genes related to chemical response, response to stimuli, and cell proliferation were significantly enriched in ESCC. + +- Pathway crosstalk analysis showed that pathways related to multiple malignancies, immune system, and metabolic processes were enriched among ESCC genes. + +What is known and what is new? - The 321 extracted genes, of which aberrant expression mediates biological processes such as immunometabolism, apoptosis, and cell proliferation, are involved in the development and progression of ESCC. + +- By ESCC-specific network analysis, we discovered 39 new genes, most of which have never been linked to ESCC. The genes MUC13, RYK, and FIN may be potential prognostic indicators of ESCC. GRB2 and CDC25C may be potential targets of ESCC in proliferation. + +What is the implication, and what should change now? - The molecular mechanisms underlying the development and progression of ESCC require more extensive and in-depth studies. +the notoriety of these 6 ESCC-implicated genes (*TP53*, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2). TP53 Pro72 allele increases the risk of ESCC. In previous research, CDKN2A/RB1 was not expressed in the esophageal mucosa of patients without risk factors whereas p16/pRb expression increased in patients exposed to risk factors or with ESCC (7,8). Although numerous single genetic studies and genetic pathway studies have provided many important insights of the development and prognosis of ESCC, they have not provided clues from the perspective of systems biology. In this study, we conducted a comprehensive collection of ESCC-associated genes from the current studies on ESCC genetic association. Then, we detected the significant biological themes in these genetic factors by performing functional enrichment analyses. Moreover, to ulteriorly reveal the mechanisms of ESCC in a more effective manner, we analyzed the interactions between these biochemical pathways through pathway crosstalk and examined the topological characteristics of these ESCC-associated genes based on human protein-protein interaction (PPI) network. Besides, the ESCC-specific molecular network was deduced and evaluated using the Steiner minimal tree algorithm. This study should provide clues and directions for understanding the molecular mechanism of ESCC from the perspective of systems biology. We present the following article in accordance with the STREGA reporting checklist (available at https://atm.amegroups.com/article/ view/10.21037/atm-22-6512/rc). + +## Methods Identification Of Escc-**Related Genes** + +Candidate genes related to ESCC (the standardized term found through medical subject headings [MeSH]) were curated by retrieving the genetic association studies in the PubMed database. We conducted literature retrieval related to ESCC with the terms (Esophageal Squamous Cell Carcinoma AND polymorphism) OR (Esophageal Squamous Cell Carcinoma AND genotype) OR (Esophageal Squamous Cell Carcinoma AND alleles). By 15 July 2022, a total of 1,131 publications were retrieved. After reviewing all 1,131 publications, only the genetic association studies were selected. From the selected publications, we narrowed our selection by focusing on those that reported 1 or more genes significantly associated with ESCC. In addition, the associated genes from several genome-wide association studies (GWAS), showing genetic association at a genome- + +wide significance level, were selected. To reduce the number of false-positive findings, we excluded studies that included negative or unrelated associations. We carefully read the full reports of selected studies to ensure that the conclusion was consistent with the content. In this way, we eventually screened out 321 genes from 736 articles. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). + +## Functional Enrichment Analysis Of Escc-**Related Genes** + +Applying the software the Database for Annotation, Visualization and Integrated Discovery 6.8 (DAVID 6.8; https://david.ncifcrf.gov/), we converted the names of 321 ESCC-related genes obtained from the literature into Entrez Gene IDs. To investigate the functional features of ESCC-related genes, Gene Ontology (GO) term analysis and pathway analysis were applied for functional enrichment analysis. The GO resource (http://www. geneontology.org/) is an international standard classification system of gene function. We performed gene enrichment analysis using GOseq in R Studio 3.4.2 (R Foundation for Statistical Computing, Vienna, Austria). Based on the Wallenius non-central hyper-geometric distribution, this method allows the estimation of gene length preferences and allows us to accurately calculate the probability of a GO term being enriched by the host gene. The enriched P values were corrected by the Benjamini-Hochberg (BH), procedure and we retained terms with P values less than 0.05 as significantly enriched terms. We used directed acyclic graphs (DAGs) as a graphical representation of the GO enrichment analysis of protein genes. + +## Kyoto Encyclopedia Of Genes And Genomes (Kegg) Pathway Enrichment Analysis + +Upon analyzing the entirety of the KEGG database, the pathways in which the study genes were involved were identified and a network of these pathways was established with the aid of the KEGG database's pathway topology. A gene network specific to a single pathway was created through pathway topology analysis. This network was then mapped to the reference gene network from KEGG, resulting in the study gene network. The Entrez Genes IDs were uploaded into the software KOBAS 2.1.1 (http:// kobas.cbi.pku.edu.cn/) and were compared with the genes included in each canonical pathway based on the KEGG pathway database. Then, we obtained the corresponding KEGG pathway and P values for each pathway. KEGG pathway analysis was performed using tools in KOBAS, corrected P value [false discovery rate (FDR)] obtained by Fisher's exact test, which were based on hypergeometric distribution, and the enriched P values were corrected by the BH method and a threshold of FDR <0.05 was used to select significantly enriched pathways. + +## Pathway Crosstalk Analysis + +To further observe the interconnections and interactions between pathways, we conducted a pathway crosstalk analysis of the above-mentioned pathways of significant enrichment. Herein, to test for the overlap between any pair of given pathways, we imported two measurements: the +( ) A B Jaccard coefficient JC A B +∩ = ∪ and the ( ) ( ) +A B overlap coefficient min A , B +∩ OC = , +where A and B are the lists of genes included in the two tested pathways. To construct the pathway crosstalk, we implemented the following procedure: +(I) Select the pathways with PBH ≤0.05. Meanwhile, the number of candidate genes contained in each pathway was restricted to be bigger than or equal to 3, because pathways with too few candidate genes may insufficiently reflect the biological information. + +(II) Calculate the number of candidate genes that overlap between any pair of pathways. Delete the pathway pair with less than 3 overlapping genes. + +(III) Calculate the overlap of all the pathway pairs that meet the above conditions and rank them by +( ) Score2 = *JC +OC* . + +(IV) Visualize the selected pathway crosstalk with the software Cytoscape 3.5.1 (9). Indicate the degree of pathways by the size of node, whereby the bigger the degree, the greater the size of the node. Use the thickness of the edge to indicate the score between the pathway pairs, whereby the rougher the edge, the higher the score. + +## Subnetwork Extraction + +We merged human protein interaction data downloaded from the Protein Interaction Network Analysis (PINA) platform (updated 21 May 2014) and protein interaction data reported in the recent literature. After removing redundant relationship and self-paired relationship we obtained the final proteins and relationship pairs (10). With these network relationships as a background, we applied + +Klein-Ravi algorithm in GenRev, a network-based software package to explore functional relevance of genes via the Steiner minimal tree algorithm that uses a greedy search +strategy to merge the smaller trees into larger ones until +only one tree connecting all input seeds is built, to extract a subnetwork from the human PPI network by using the 321 ESCC-related genes as seeds (11). In order to verify that this subnetwork was a non-random network, we used Erdos-Renyi algorithm in the igraph R package to generate 100 random background networks with the same number of nodes and edges, and combined the seed gene with the 100 random background networks to generate subnetworks by analyses. Then, we calculated the average values of the shortest-path distance and clustering coefficient. We calculated the number of the average shortest path in the random subnetwork that was shorter than the ESCC +networks, denoted the number as nL; calculated the +number of the average clustering coefficient in random subnetwork that was higher than the ESCC network, +denoted the number as nC; calculated the values of pL and +C +L +L C +$$:{\frac{\mathbf{a_{L}}}{100}},\ \mathbf{p_{C}}={\frac{1}{1}}$$ + +## P N N +, P = . P 100 100 = Results Identification Of Genes Reported To Be Associated With Escc + +By searching PubMed, we only selected genetic association studies and related genes from several GWAS, which showed a significant degree of genome-wide genetic association. We excluded studies that included either negative or unrelated outcomes to reduce the number of false-positive results. + +Altogether, we screened 321 genes reported to be significantly related with ESCC out of 736 articles, which had a range of diverse biological functions. For example, some genes were related to tumor necrosis factor (TNF) signaling (e.g., MMP9, CASP3, MMP3, CASP8, *AKT1*, +FADD, IL6, LTA, PIK3CA, PIK3CB, *ITCH, FAS, PTGS2*, EDN1, IL1B, TNF, *TNFRSF1A, IL18R1*, and *NFKBIA*), +and some genes were related to nucleotide excision repair +(e.g., GTF2H3, *ERCC2*, XPA, ERCC5, *ERCC4, XPC*, and *ERCC1*), and some genes were related to choline metabolism in cancer (e.g., mTOR, *Akt1, PIK3CA, RHEB*, EGFR, EGF, CHKA, *PIK3CB*, and *WASL*). A detailed list of all genes we found to be associated with ESCC is provided in Table S1. + +## Page 4 Of 17 **He Et Al. Network And Pathway-Based Analysis Of Escc Genes** Go Enrichment Analysis In Escc-**Related Genes** + +The GO database provides a standardized description of the gene products from the function, the participating biological pathway, and cellular localization, which comprises the simple annotation of the gene products. Through GO enrichment analysis, we can gather a rough understanding of the biological functions, pathways, or cellular localization of the differential genes. DAGs of biological process (BP; *Figure 1A*), cellular component (CC; *Figure 1B*), and molecular function (MF; *Figure 1C*) +were used to show the GO annotation results. In the GO DAG, annotation moved from more general to more specific as one moved from parent nodes to child nodes. Consequently, a DAG approach was used to provide a clearer understanding of which GO terms were specifically enriched and how these affected other GO terms through upper hierarchies. The top 10 terms with the lowest P value and their parent terms were shown in a GO DAG, the terms with pale marks were significantly enriched, and those with deeper red marks were more significantly enriched. In the DAG of BP, significant enrichment terms, namely, response to chemical (PBH=5.28E-47), cellular response to stimulus +(PBH=1.56E-43), and cell proliferation (PBH=1.42E-42) were identified. An example of a significantly enriched term in the DAG of CC is the GO term "intracellular organelle part". This GO term was enriched at a very low FDR (1.46E-08), and two GO terms at upper hierarchies: "nuclear part" and "nuclear lumen" were enriched as a result. These two GO terms together enriched the term "nucleoplasm." The results were consistent with the pathophysiological background of ESCC, indicating that candidate genes were relatively reliable for subsequent bioinformatics analysis. In addition, we used a dot plot to visualize the P values enriched in the first 10 terms in BP, CC, and MF as well as the number of genes involved in each term. At the same time, intuitively, we also found that the genes previously found are in the GO term, which more distinctly showed the correlation. For example, in BP's dot plot (*Figure 2A*), the term "response to stimulus" contained the largest number of genes but had the lowest rich factor. In CC's dot plot +(*Figure 2B*), the term "organelle" contained many genes and its p-value was minimal. In the MF's dot plot (*Figure 2C*), +we found that the term "damaged DNA binding" had the highest rich factor. Combining all dot plots, we found that there was a trend that the more genes ascribed to a term, the lower the rich factor. This may be because those terms had too many gene numbers. Moreover, we used the + +![4_image_0.png](4_image_0.png) + +![5_image_0.png](5_image_0.png) + +ggplot2 package in the Goseq software to graph the top 10 GO terms in BP, CC, and MF (*Figure 3*). + +## Kegg Pathway Enrichment Analysis In Escc-Related Genes + +Pathway analysis enables researchers to map the genes, proteins, or molecules onto a particular class of metabolic or regulatory networks, or according to an individuals' molecular set of functions, in order to form their own specific pathway. This is very important for elucidating the molecular mechanism of action and identifying biomarkers. We uploaded the differential genes into KOBAS 2.1.1 software and compared them to the genes contained in the canonical approaches based on the KEGG pathway database, and we enriched 240 pathways. A total of 31 pathways with corrected P value (FDR) +<0.05 were retained as the significantly enriched pathways (Table S2). Most of the pathways were related to pathophysiological process of cancer. Among them, several pathways are closely related to certain types of malignant tumors, including bladder cancer (ranked 2nd), melanoma (ranked 4th), prostate cancer (ranked 5th), colorectal cancer (ranked 9th), non-small cell lung cancer (NSCLC; ranked 11th), endometrial cancer (ranked 14th), and glioma (ranked 20th). Many studies have clarified the role of p53 and TNF in the development of cancer. p53 is a well-known tumor suppressor gene. However, in ESCC, the p53 pathway is inactivated by TP53 mutations, so it has carcinogenic effects (12-14). Numerous studies have shown that TNF has antitumor activity and is also an endogenous tumor promoter (15). TNF is down-regulated in ESCC tissues and multivariate analysis has shown that down-regulation of TNF is independently associated + +![6_image_0.png](6_image_0.png) + +with early local tumor recurrence (16,17). In cellular metabolism, we obtained pathways such as proteoglycans in cancer, endocrine resistance, HIF-1 signaling pathway, and microRNAs in cancer. It was worth mentioning that in the microRNAs in cancer pathway, MYC binding protein (MYCBP) was identified as a direct target of miR-26 (18). + +In drug metabolism, we found pathways such as platinum drug resistance (ranked 1st), metabolism of xenobiotics by cytochrome P450, and drug metabolism-cytochrome P450. Cisplatin is an important part of chemotherapy for esophageal cancer (19). However, in the use of platinum drugs, some challenges such as patients' partial + +![7_image_0.png](7_image_0.png) + +antitumor response, drug resistance development, and tumor recurrence limit the patient's life expectancy (20). Furthermore, immune-associated biological processes such as graft-versus-host disease and allograft rejection were also significantly enriched. + +## Pathway Crosstalk Analysis Among Significantly Enriched Pathways + +To further observe the interconnections and interactions between significantly enriched pathways, we conducted a pathway crosstalk analysis of the 31 significantly enriched pathways mentioned above. The approach was based on the assumption that two pathways were considered to crosstalk if they shared a proportion of ESCC-related genes (21). A total of 30 of the above 31 significantly enriched pathways were in accordance with the crosstalk analysis criteria, namely, each pathway contained 3 or more genes, each of which had at least two genes sharing with 1 or more other pathways. The base excision repair pathway was the only non-compliant pathway due to the number of genes overlapping with other pathways being less than or equal to 2. All paths formed by these approaches were used to construct pathway crosstalk and the level of overlap between the two pathways was measured on the basis of the average scores of the coefficients JC and OC. Based on their crosstalk, pathways could be broadly divided into three main parts, each containing more interactions than other pathways and might involve in the same or similar biological processes (*Figure 4*). The first part consisted of 16 pathways, including 9 of the top 10 significantly enriched pathways of KEGG. Most of these 16 pathways were specific cancer pathways, such as prostate cancer and NSCLC. Three of these pathways shared some genes with the platinum drug resistance pathway, and these three pathways were linked to drug metabolism and chemical carcinogenesis. The second part contained seven pathways, which connected the other two parts of pathways that shared no genes. These seven pathways were mainly involved in the regulation of many kinds of receptors and cytokines in cells. At the same time, they played an important role in the regulation of cell proliferation, apoptosis, inflammation in vivo, infection, and other physiological processes. The third pathway was mainly associated with immunity and inflammation. As indicated above, path crosstalk analysis can provide important clues to the development and prognosis of ESCC and provide insights into the ECSS mechanism. + +![8_image_0.png](8_image_0.png) + +## Subnetwork Extraction + +To refine the ESCC-related interactions, we used the Steiner's minimal tree algorithm to extract the ESCC subnetwork from the human PPI network and recently published protein interaction pairs (232,866 pairs of 17,379 proteins were involved). This approach attempts to connect a maximum number of input nodes with a minimum number of linked nodes (we used 308 nodes). As shown in Figure 5, 347 nodes and 1,339 edges were contained. A total of 308 out of the 321 ESCC-related genes were included in the ESCC-specific network, accounting for about 95.95% of the candidate genes and 88.76% of the genes in the ESCCspecific network, demonstrating a high coverage of ESCC-related genes in the subnetwork. It was noteworthy that we found 39 additional proteins, some of which had been reported as associated with ESCC (*Table 1*). In addition, to verify that the sub-network was a non-random network, we used the Erdos-Renyi model in the igraph R package to generate 100 random sub-networks. We calculated the arithmetic average values of the shortest path distance and clustering coefficients in the random sub-networks. The average shortest path distance value and average clustering coefficient were compared with the corresponding values of the ESCC-specific network. For these random background networks, the average shortest path distance was 2.86, bigger than the shortest path distance of ESCC specific network (shortest path distance was 2.60; PL<0.01). The average clustering coefficient of random sub-networks was 0.19, which was significantly smaller than that of ESCCspecific networks (clustering coefficient was 0.33, PC<0.01). + +Therefore, the ESCC-specific network extracted from the entire PPI network was a non-random network. + +## Discussion + +As a highly malignant and aggressive tumor, ESCC is not sensitive to radiotherapy and chemotherapy. In the past few decades, studies of human participants, animals, or cell models have gained insight into the molecular mechanisms of ESCC. Although increasing numbers of genes/proteins are believed to be associated with this disease, with the development of high-throughput technologies, many genes/ proteins are considered potential targets for detection or treatment. However, the understanding of the biological processes associated with the pathogenesis of ESCC at the molecular level is far from complete. Therefore, there is a need to decode the underlying pathogenesis of ESCC at the level of systems biology. In this study, we explored the interactions of these genes by collecting genes related to ESCC as well as utilizing pathway and network analysis systems. We have provided a comprehensive and systematic framework for mapping related biochemical processes. + +Through biological function enrichment analysis, we obtained specific biological processes caused by ESCC-related genes. The GO enrichment analysis provided us with a rough interpretation of the biological functions, + +![9_image_0.png](9_image_0.png) + +carcinoma. + +Annals of Translational Medicine, Vol 11, No 2 January 2023 **Page 11 of 17** + +| Table 1 39 genes associated with ESCC discovered by ESCC-specific network Gene abbreviations Gene ID Species Gene name RAD51C 5889 Homo sapiens RAD51 paralog C (RAD51C) GRB2 2885 Homo sapiens Growth factor receptor bound protein 2 (GRB2) AP2S1 1175 Homo sapiens Adaptor related protein complex 2 sigma 1 subunit (AP2S1) ABI2 10152 Homo sapiens Abl interactor 2 (ABI2) NFKB2 4791 Homo sapiens Nuclear factor kappa B subunit 2 (NFKB2) ZBTB17 7709 Homo sapiens Zinc finger and BTB domain containing 17 (ZBTB17) ATF1 466 Homo sapiens Activating transcription factor 1 (ATF1) APP 351 Homo sapiens Amyloid beta precursor protein (APP) BAG6 7917 Homo sapiens BCL2 associated athanogene 6 (BAG6) DLG3 1741 Homo sapiens Discs large MAGUK scaffold protein 3 (DLG3) BCL6 604 Homo sapiens B-cell CLL/lymphoma 6 (BCL6) PRKACA 5566 Homo sapiens Protein kinase camp-activated catalytic subunit alpha (PRKACA) OGT 8473 Homo sapiens O-linked N-acetylglucosamine (glcnac) transferase (OGT) GSTO1 9446 Homo sapiens Glutathione S-transferase omega 1 (GSTO1) MUC13 56667 Homo sapiens Mucin 13, cell surface associated (MUC13) FN1 2335 Homo sapiens Fibronectin 1 (FN1) HIST2H3A 333932 Homo sapiens Histone cluster 2 H3 family member a (HIST2H3A) DHX9 1660 Homo sapiens Dexh-box helicase 9 (DHX9) IL2RB 3560 Homo sapiens Interleukin 2 receptor subunit beta (IL2RB) CEBPB 1051 Homo sapiens CCAAT/enhancer binding protein beta (CEBPB) SMAD9 4093 Homo sapiens SMAD family member 9 (SMAD9) ATP4A 495 Homo sapiens ATPase H+/K+ transporting alpha subunit (ATP4A) RYK 6259 Homo sapiens Receptor-like tyrosine kinase (RYK) ELAVL1 1994 Homo sapiens ELAV like RNA binding protein 1 (ELAVL1) CYB5A 1528 Homo sapiens Cytochrome b5 type A (CYB5A) HBA1 3039 Homo sapiens Hemoglobin subunit alpha 1 (HBA1) FZD5 7855 Homo sapiens Frizzled class receptor 5 (FZD5) CDC25C 995 Homo sapiens Cell division cycle 25C (CDC25C) TRAF3IP2 10758 Homo sapiens TRAF3 interacting protein 2 (TRAF3IP2) NRF1 4899 Homo sapiens Nuclear respiratory factor 1 (NRF1) PLA2G4A 5321 Homo sapiens Phospholipase A2 group IVA (PLA2G4A) ZDHHC17 23390 Homo sapiens Zinc finger DHHC-type containing 17 (ZDHHC17) LRP1 4035 Homo sapiens LDL receptor related protein 1 (LRP1) MAPK6 5597 Homo sapiens Mitogen-activated protein kinase 6 (MAPK6) JUN 3725 Homo sapiens Jun proto-oncogene, AP-1 transcription factor subunit (JUN) UBC 7316 Homo sapiens Ubiquitin C (UBC) FPGT 8790 Homo sapiens Fucose-1-phosphate guanylyltransferase (FPGT) CALM2 805 Homo sapiens Calmodulin 2 (CALM2) C1D 10438 Homo sapiens C1D nuclear receptor corepressor (C1D) ESCC, esophageal squamous cell carcinoma. | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +pathways, or cellular localization of the differential genes, convincingly demonstrating that these genes involved in immune response, metabolic regulation, cell proliferation, apoptosis, and drug response. In cluster analysis, GO terms relative to apoptosis, cell metabolism, regulation of cell development, DNA damage repair, angiogenesis, and xenobiotic metabolism turned out high enrichment scores, which was consistent with our perception of ESCC so far. For example, in GO MF, key terms such as catalytic activity, binding, protein binding, and damaged DNA binding, along with other enriched terms delineated that the cancer cell has an increased damage repair function and can fully repair the DNA damage caused by chemotherapeutic drugs, which at least partially provides a reason for the unsatisfactory effect of chemotherapy in the treatment of ESCC. + +The results of pathway analysis showed that genes related to esophageal cancer contain multiple tumor pathways, revealing its commonality with other malignancies. We found that 7 pathways: bladder cancer, prostate cancer, melanoma, NSCLC, endometrial cancer, glioma, and pancreatic cancer, were in the same functional annotation clustering, and these pathways all contain the genes *TP53*, +RB1, *EGFR, EGF*, and *CCND1*, which was highly consistent with our understanding of the mechanisms of cancer development. *TP53* encodes a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. The encoded protein responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. In ESCC, over 90% of *TP53* mutations and inactivation of the p53 pathway are associated with patient prognosis and resistance to radiotherapy and chemotherapy. *CCND1* mutations exceeding 20%, deletions of *CDKN2A, RB1*, and *CDKN2A*, and abnormal methylation impair the cell cycle. *EP300, CREBBP*, and NOTCH (NOTCH1, *NOTCH2*, +NOTCH3) are associated with epigenetic processes +(12,22,23). Moreover, *CCND1, EGFR*, and *ERBB2* are likely to be driver genes for the development of ESCC +(24,25). Previous studies have shown that *RASSF1* is a very promising tumor suppressor gene, and the extent of its transcript expression and methylation is related to tumor progression and survival of ESCC patients (26,27). In the RAS signaling pathway and the RAP1 signaling pathway, RASSF1 interacts with and influences PIK3CA, AKT1, EGF, +and *EGFR*. These genes are involved in the formation, proliferation, migration, and angiogenesis of tumors. + +AKT1 is significantly associated with local recurrence of + +ESCC (28). Tumor inhibitors of the *RASSF* family act as RAS apoptosis and senescence effectors. It is speculated that inactivation of the *RASSFlA* tumor suppressor promotes K-RAS-mediated transformation by uncoupling it with apoptotic pathways such as the Hippo pathway (29,30). In addition, we observed that in the cell cycle, proliferation, the *PI3K-AKT* signaling pathway, allograft rejection reaction, MYC, *COX-2, p53*, and RB1 also showed associations. As indicated by these results, a variety of genes and signaling molecules serve as bridges and connect to each other, join to many signaling pathways and form a complex network of ESCC molecules. + +In crosstalk analysis, we identified three major modules. + +The first module contains 16 pathways, most of which are specific cancer pathways, such as prostate cancer, NSCLC, pancreatic cancer, and the like. Among these 16 pathways, 3 share some genes with the platinum drug resistance pathway. These three pathways are related to drug metabolism and chemical carcinogenesis: drug metabolism—cytochrome *P450*, metabolism of xenobiotics by cytochrome P450, and chemical carcinogenesis. The CYP1B1 mutation was significantly associated with ESCC +risk (31,32). Studies have shown that the most important issue associated with ESCC treatment is the intrinsic resistance of chemotherapeutic drugs, and multidrug resistance is increasingly common in patients with ESCC (33,34). Apoptosis is one of the most critical processes in cell proliferation and a key molecular mechanism for anticancer therapy. The *PI3K/Akt/mTOR* pathway is involved in cell proliferation, differentiation, survival, apoptosis, and metastasis (35-37). Sensitized drugs promote the action of oxaliplatin by significantly inducing apoptosis and modulating the *PI3K/Akt/mTOR* pathway, counteracting resistance to chemotherapy (38,39). The second module contains 7 pathways which connect other pathways that do not share genes. These 7 pathways are mainly involved in the regulation of various receptors and cytokines in cells, and they also play an important role in the regulation of cell proliferation, apoptosis, inflammation, infection, and other physiological processes. By analysis of module II and module III, pathways related to immune response, cell adhesion (i.e., *HTLV-I* infection), allograft rejection, graftversus-host disease, the *FoxO* signaling pathway, shared genes such as IL6, FAS, END1, IL1B, *TNF, TNFRSF1A*, +NFKBIA, HLA-A, *HLA-B*, and HLA-C. In these three pathways (graft-versus-host disease, type I diabetes mellitus, and allograft rejection), we found similar processes and common genes such as the HLA family (*HLA-A, HLA*-B, + +HLA-C, HAL-G, HLA- DQB1, *HLA-DRB1*), FAS, *FASLG*, +and some cytokines (*IL6, IL1B*). By targeting the *FASLG* +gene, miR-21 down-regulates cell growth, invades, and induces apoptosis, and by combining with *FASLG* treatment methods, the efficacy of radiotherapy can be enhanced, and unnecessary angiogenesis-promoting effects can be reduced (40-42). By modulating *HLA-G* expression, miR148a is indicated to be involved in the carcinogenesis of primary ESCC. The current results indicate that miR148a is a potential biomarker for ESCC (43). HLA class I is critical for tumor immunity, and its degree of expression is an independent prognostic factor for relapse-free survival +(44,45). Studies have shown that *HLA-G* is highly expressed in ESCC and can be used as a novel tumor marker +(43,46-48). HLA-II molecules are mainly encoded by DP, +DQ, and DR genes, expressed in immune cells, and are responsible for presenting antigenic peptides to CD4+ T +cells to trigger the expansion and differentiation of these T cells and induce a series of antigen-specific immunity responses. Studies have shown that aberrant methylation of HLA-DQB1 and *HLA-DAB1* at different sites is significantly associated with ESCC differentiation and late stages, and their methylation is conducive to the abnormal expression of HLA-II (49-51). Therefore, abnormal expression of HLA-II may lead to immune response or autoimmunity deficiency in some diseases. Their abnormal expression inhibits the function of immune cells such as natural killer (NK) cells and T cells, which promotes the development of ESCC. These pathways related to the immune response and cell adhesion movement profoundly influence the occurrence and development of ESCC, and they also shed a new light for understanding diseases through molecular mechanisms. + +Remarkably, in the ESCC-specific network, we discovered 39 new genes, most of which have never been linked to ESCC, such as APP, TRAF3IP2, *ZBTB17*, +NRF1, CEBPB, HBA1, *ABI2, CALM2, RAD51C*, and AP2S1 (Table 1). Studies have shown that RYK may be an important predictor of ESCC clinical stage staging and prognosis (52). The combination of *MUC13/MUC20* expression is a potential prognostic indicator of neoadjuvant chemotherapy and postoperative ESCC (53,54). *GSTO1* may also be a potential biomarker for early detection (55). The drug resistance/response mechanism of ESCC is very complicated. A study found that differential genes are also key cancer-promoting genes related to chemotherapy resistance through PPI, such as *BMP1* and *DBF4* (56); +prognosis-related genes, such as TTK and *KIF18A*. These studies can help improve the survival rate of patients (57). + +JUN is a chromosome region of human malignancy translocations and deletions associated with various signal transduction pathways (58,59). In ESCC, *BCL6* is upregulated, and its acylation is controlled by *HDAC* and SIRT-dependent mechanisms (60). Interference with this process may lead to cell cycle arrest and apoptosis. Its biological function may indicate that it can become a new regulator in esophageal cancer cells (61,62). *GRB2* is a prominent node involved in the naturally regulated cell proliferation-related pathways of the tyrosine kinase signaling pathway and *Erk1/Erk2* Mapk signaling pathway. + +UBC participates in cell cycle progression and is upregulated in more than 70% of ESCC tissues (63-65). *UEB2C* directly interferes with the level of cyclin B1 protein and alters the proliferation and cell cycle characteristics of ESCC (66). + +Due to activation of the *p53/p21* pathway, *CDC25C* is thus reduced, leading to cell cycle arrest (67,68). Although the quantity and quality of PPI data have greatly improved in recent years, the human PPI network is far from being completed. In addition, due to current technology limitations, there may be some false positive results in the PPI data. These potential deviations associated with human PPI networks may have affected our results. + +## Conclusions + +The mechanism of occurrence and development of ESCC is extremely complex and involves a variety of factors, such as genetic and environmental factors. In this study, we used a systems biology framework to select candidate genes and performed a variety of analyses on ESCC. By integrating information from tandem analysis of GO, pathways, and pathway crosstalk, we found that ESCC was associated with a variety of cancer pathways, cell proliferation, apoptosis, immune responses, and drug metabolism. In addition, in ESCC-specific network, some of these additional genes have been reported to be associated with ESCC. In order to reveal the molecular mechanism of ESCC, we initially constructed its molecular network. Our research facilitates a more in-depth understanding of the mechanism of ESCC. + +## Acknowledgments + +The primary results of the abstract in this study were presented as a meeting poster (No. PS02.037) in the 2018 International Society for Diseases of the Esophagus. + +Funding: This work was funded by Bethune Charitable Foundation (No. HZB-20190528-19). + +## Footnote + +Reporting Checklist: The authors have completed the STREGA reporting checklist. Available at https://atm. amegroups.com/article/view/10.21037/atm-22-6512/rc Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm. amegroups.com/article/view/10.21037/atm-22-6512/coif). The authors have no conflicts of interest to declare. + +Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). + +Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/. + +## References + +1. Smyth EC, Lagergren J, Fitzgerald RC, et al. Oesophageal cancer. Nat Rev Dis Primers 2017;3:17048. + +2. Tao Y, Chai D, Ma L, et al. Identification of distinct gene expression profiles between esophageal squamous cell carcinoma and adjacent normal epithelial tissues. Tohoku J Exp Med 2012;226:301-11. + +3. Testa U, Castelli G, Pelosi E. Esophageal Cancer: +Genomic and Molecular Characterization, Stem Cell Compartment and Clonal Evolution. Medicines (Basel) 2017;4:67. + +4. Holmes RS, Vaughan TL. Epidemiology and pathogenesis of esophageal cancer. Semin Radiat Oncol 2007;17:2-9. + +5. Lagergren J, Smyth E, Cunningham D, et al. Oesophageal cancer. Lancet 2017;390:2383-96. + +6. Song Y, Li L, Ou Y, et al. Identification of genomic alterations in oesophageal squamous cell cancer. Nature + +2014;509:91-5. + +7. Müller LB, Meurer L, Lopes AB, et al. Stepwise expression of CDKN2A and RB1 proteins in esophageal mucosa from patients at high risk for squamous cell carcinoma. Appl Immunohistochem Mol Morphol 2014;22:669-73. + +8. Ni X, Wu P, Wu J, et al. Hyperthermic intraperitoneal perfusion chemotherapy and response evaluation in patients with gastric cancer and malignant ascites. Oncol Lett 2017;14:1691-6. + +9. Shannon P, Markiel A, Ozier O, et al. Cytoscape: +a software environment for integrated models of biomolecular interaction networks. Genome Res 2003;13:2498-504. + +10. Cowley MJ, Pinese M, Kassahn KS, et al. PINA v2.0: +mining interactome modules. Nucleic Acids Res 2012;40:D862-5. + +11. Zheng S, Zhao Z. GenRev: exploring functional relevance of genes in molecular networks. Genomics 2012;99:183-8. + +12. Kishino T, Niwa T, Yamashita S, et al. Integrated analysis of DNA methylation and mutations in esophageal squamous cell carcinoma. Mol Carcinog 2016;55:2077-88. + +13. Wu Z, Zhou J, Zhang X, et al. Reprogramming of the esophageal squamous carcinoma epigenome by SOX2 promotes ADAR1 dependence. Nat Genet 2021;53:881-94. + +14. Li L, Xu J, Qiu G, et al. Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas. Theranostics 2018;8:61-77. + +15. Scott KA, Moore RJ, Arnott CH, et al. An anti-tumor necrosis factor-alpha antibody inhibits the development of experimental skin tumors. Mol Cancer Ther 2003;2:445-51. + +16. Xie Y, Wang B. Downregulation of TNFAIP2 suppresses proliferation and metastasis in esophageal squamous cell carcinoma through activation of the Wnt/β-catenin signaling pathway. Oncol Rep 2017;37:2920-8. + +17. Chen Y, Wang Y, Song H, et al. Expression profile of apoptosis-related genes potentially explains early recurrence after definitive chemoradiation in esophageal squamous cell carcinoma. Tumour Biol 2014;35:4339-46. + +18. Li J, Liang Y, Lv H, et al. miR-26a and miR-26b inhibit esophageal squamous cancer cell proliferation through suppression of c-MYC pathway. Gene 2017;625:1-9. + +19. Lebwohl D, Canetta R. Clinical development of platinum complexes in cancer therapy: an historical perspective and an update. Eur J Cancer 1998;34:1522-34. + +20. Oberoi HS, Nukolova NV, Kabanov AV, et al. + +Nanocarriers for delivery of platinum anticancer drugs. Adv Drug Deliv Rev 2013;65:1667-85. + +21. Jia P, Kao CF, Kuo PH, et al. A comprehensive network and pathway analysis of candidate genes in major depressive disorder. BMC Syst Biol 2011;5 Suppl 3:S12. + +22. Gao YB, Chen ZL, Li JG, et al. Genetic landscape of esophageal squamous cell carcinoma. Nat Genet 2014;46:1097-102. + +23. Huang YH, Cai K, Xu PP, et al. CREBBP/EP300 mutations promoted tumor progression in diffuse large B-cell lymphoma through altering tumor-associated macrophage polarization via FBXW7-NOTCH-CCL2/ +CSF1 axis. Signal Transduct Target Ther 2021;6:10. + +24. Sawada G, Niida A, Hirata H, et al. An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma. PLoS One 2015;10:e0139808. + +25. Cui D, Zhu Y, Yan D, et al. Dual inhibition of cMET +and EGFR by microRNA-338-5p suppresses metastasis of esophageal squamous cell carcinoma. Carcinogenesis 2021;42:995-1007. + +26. Perez-Janices N, Blanco-Luquin I, Torrea N, et al. + +Differential involvement of RASSF2 hypermethylation in breast cancer subtypes and their prognosis. Oncotarget 2015;6:23944-58. + +27. Haluskova J, Lachvac L, Nagy V. The investigation of GSTP1, APC and RASSF1 gene promoter hypermethylation in urine DNA of prostate-diseased patients. Bratisl Lek Listy 2015;116:79-82. + +28. Tian B, Liu J, Zhang N, et al. Oncogenic SNORD12B +activates the AKT-mTOR-4EBP1 signaling in esophageal squamous cell carcinoma via nucleus partitioning of PP1α. Oncogene 2021;40:3734-47. + +29. Schmidt ML, Hobbing KR, Donninger H, et al. RASSF1A +Deficiency Enhances RAS-Driven Lung Tumorigenesis. Cancer Res 2018;78:2614-23. + +30. Yu W, Chu L, Zhao K, et al. A nomogram based on phosphorylated AKT1 for predicting locoregional recurrence in patients with oesophageal squamous cell carcinoma. J Cancer 2017;8:3755-63. + +31. Hyland PL, Freedman ND, Hu N, et al. Genetic variants in sex hormone metabolic pathway genes and risk of esophageal squamous cell carcinoma. Carcinogenesis 2013;34:1062-8. + +32. Malik MA, Upadhyay R, Mittal RD, et al. Association of xenobiotic metabolizing enzymes genetic polymorphisms with esophageal cancer in Kashmir Valley and influence of environmental factors. Nutr Cancer 2010;62:734-42. + +33. Zhou P, Zhang R, Wang Y, et al. Cepharanthine hydrochloride reverses the mdr1 (P-glycoprotein)- mediated esophageal squamous cell carcinoma cell cisplatin resistance through JNK and p53 signals. Oncotarget 2017;8:111144-60. + +34. Chi Y, Wang F, Zhang Y, et al. Apatinib inhibits tumour progression and promotes antitumour efficacy of cytotoxic drugs in oesophageal squamous cell carcinoma. J Cell Mol Med 2022;26:1905-17. + +35. Li T, Wang G. Computer-aided targeting of the PI3K/ +Akt/mTOR pathway: toxicity reduction and therapeutic opportunities. Int J Mol Sci 2014;15:18856-91. + +36. Furusawa S, Wu J. The effects of biscoclaurine alkaloid cepharanthine on mammalian cells: implications for cancer, shock, and inflammatory diseases. Life Sci 2007;80:1073-9. + +37. Wang L, Zhang Z, Yu X, et al. SOX9/miR-203a axis drives PI3K/AKT signaling to promote esophageal cancer progression. Cancer Lett 2020;468:14-26. + +38. Lin JJ, Su JH, Tsai CC, et al. 11-epi-Sinulariolide acetate reduces cell migration and invasion of human hepatocellular carcinoma by reducing the activation of ERK1/2, p38MAPK and FAK/PI3K/AKT/mTOR signaling pathways. Mar Drugs 2014;12:4783-98. + +39. Zhou J, Jiang YY, Chen H, et al. Tanshinone I +attenuates the malignant biological properties of ovarian cancer by inducing apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway. Cell Prolif 2020;53:e12739. + +40. Blaes J, Thomé CM, Pfenning PN, et al. Inhibition of CD95/CD95L (FAS/FASLG) Signaling with APG101 Prevents Invasion and Enhances Radiation Therapy for Glioblastoma. Mol Cancer Res 2018;16:767-76. + +41. Wang N, Zhang CQ, He JH, et al. MiR-21 downregulation suppresses cell growth, invasion and induces cell apoptosis by targeting FASL, TIMP3, and RECK genes in esophageal carcinoma. Dig Dis Sci 2013;58:1863-70. + +42. Du X, Hong L, Sun L, et al. miR-21 induces endothelial progenitor cells proliferation and angiogenesis via targeting FASLG and is a potential prognostic marker in deep venous thrombosis. J Transl Med 2019;17:270. + +43. Chen Q, Luo G, Zhang X. MiR-148a modulates HLA-G expression and influences tumor apoptosis in esophageal squamous cell carcinoma. Exp Ther Med 2017;14:4448-52. + +44. Ito S, Okano S, Morita M, et al. Expression of PD-L1 and HLA Class I in Esophageal Squamous Cell Carcinoma: +Prognostic Factors for Patient Outcome. Ann Surg Oncol 2016;23:508-15. + +45. Jhunjhunwala S, Hammer C, Delamarre L. Antigen presentation in cancer: insights into tumour immunogenicity and immune evasion. Nat Rev Cancer 2021;21:298-312. + +46. Golyan FF, Abbaszadegan MR, Forghanifard MM. + +TWIST1, MMP-21, and HLAG-1 co-overexpression is associated with ESCC aggressiveness. J Cell Biochem 2019;120:14838-46. + +47. Zheng J, Xu C, Chu D, et al. Human leukocyte antigen G is associated with esophageal squamous cell carcinoma progression and poor prognosis. Immunol Lett 2014;161:13-9. + +48. Hu J, Li L, Liu Y, et al. Overexpression of HLA-G Is positively associated with Kazakh esophageal squamous cell carcinoma in Xinjiang, China. Viral Immunol 2013;26:180-4. + +49. Baccar A, Ferchichi I, Troudi W, et al. CD99 and HLAII immunostaining in breast cancer tissue and their correlation with lymph node metastasis. Dis Markers 2013;34:363-71. + +50. De Lerma Barbaro A, De Ambrosis A, Banelli B, et al. + +Methylation of CIITA promoter IV causes loss of HLAII inducibility by IFN-gamma in promyelocytic cells. Int Immunol 2008;20:1457-66. + +51. Jo YS, Lee JC, Li S, et al. Significance of the expression of major histocompatibility complex class II antigen, HLADR and -DQ, with recurrence of papillary thyroid cancer. + +Int J Cancer 2008;122:785-90. + +52. Lv J, Cao XF, Ji L, et al. Association of Wnt1/beta-catenin with clinical pathological characteristics and prognosis of esophageal squamous cell carcinoma. Genet Test Mol Biomarkers 2010;14:363-9. + +53. Shen LY, Wang H, Dong B, et al. Possible prediction of the response of esophageal squamous cell carcinoma to neoadjuvant chemotherapy based on gene expression profiling. Oncotarget 2016;7:4531-41. + +54. Wang H, Shen L, Lin Y, et al. The expression and prognostic significance of Mucin 13 and Mucin 20 in esophageal squamous cell carcinoma. J Cancer Res Ther 2015;11 Suppl 1:C74-9. + +55. Li Y, Zhang Q, Peng B, et al. Identification of glutathione S-transferase omega 1 (GSTO1) protein as a novel tumor-associated antigen and its autoantibody in human esophageal squamous cell carcinoma. Tumour Biol 2014;35:10871-7. + +56. Xie F, Zhang D, Qian X, et al. Analysis of cancer- + +promoting genes related to chemotherapy resistance in esophageal squamous cell carcinoma. Ann Transl Med 2022;10:92. + +57. Yang Y, Sun Z, Shi Y, et al. Integrated bioinformatics analysis to identify key genes related to the prognosis of esophageal squamous cell carcinoma. Transl Cancer Res 2021;10:1679-91. + +58. Li LY, Jiang H, Xie YM, et al. Macrolide analog F806 suppresses esophageal squamous cell carcinoma +(ESCC) by blocking β1 integrin activation. Oncotarget 2015;6:15940-52. + +59. Luo A, Yu X, Li G, et al. Differentiation-associated genes regulated by c-Jun and decreased in the progression of esophageal squamous cell carcinoma. PLoS One 2014;9:e96610. + +60. Jiang YY, Jiang Y, Li CQ, et al. TP63, SOX2, and KLF5 Establish a Core Regulatory Circuitry That Controls Epigenetic and Transcription Patterns in Esophageal Squamous Cell Carcinoma Cell Lines. Gastroenterology 2020;159:1311-1327.e19. + +61. Ahrens TD, Timme S, Hoeppner J, et al. Selective inhibition of esophageal cancer cells by combination of HDAC inhibitors and Azacytidine. Epigenetics 2015;10:431-45. + +62. Chen J, Guo L, Peiffer DA, et al. Genomic profiling of 766 cancer-related genes in archived esophageal normal and carcinoma tissues. Int J Cancer 2008;122:2249-54. + +63. Li X, Jiang Z, Feng J, et al. 2-Acetylamino-3-[4-(2acetylamino-2-carboxyethylsulfanylcarbonylamino) phenyl carbamoylsulfanyl] propionic acid, a glutathione reductase inhibitor, induces G2/M cell cycle arrest through generation of thiol oxidative stress in human esophageal cancer cells. Oncotarget 2017;8:61846-60. + +64. Li LY, Zhang K, Jiang H, et al. Quantitative proteomics reveals the downregulation of GRB2 as a prominent node of F806-targeted cell proliferation network. J Proteomics 2015;117:145-55. + +65. Lu J, Xue L, Jin M, et al. Expression profiling of metastasis-related microRNAs in early esophageal squamous cell carcinoma. Zhonghua Bing Li Xue Za Zhi 2014;43:313-7. + +66. Palumbo A Jr, Da Costa NM, De Martino M, et al. + +UBE2C is overexpressed in ESCC tissues and its abrogation attenuates the malignant phenotype of ESCC cell lines. Oncotarget 2016;7:65876-87. + +67. Wang J, Zhang Z, Che Y, et al. Rabdocoestin B exhibits antitumor activity by inducing G2/M phase arrest and apoptosis in esophageal squamous cell carcinoma. Cancer + +Chemother Pharmacol 2018;81:469-81. + +68. Yin X, Zhang R, Feng C, et al. Diallyl disulfide induces G2/M arrest and promotes apoptosis through the p53/p21 and MEK-ERK pathways in human esophageal squamous Cite this article as: He W, Yuan K, He J, Wang C, Peng L, Han Y, Chen N. Network and pathway-based analysis of genes associated with esophageal squamous cell carcinoma. Ann Transl Med 2023;11(2):102. doi: 10.21037/atm-22-6512 cell carcinoma. Oncol Rep 2014;32:1748-56. + +(English Language Editor: J. Jones) \ No newline at end of file diff --git a/medical/md_images/PMC10088903/3_image_0.png b/medical/md_images/PMC10088903/3_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..c176ed4d7dfb6a4bf07559964b8c28b8014a2a6d --- /dev/null +++ b/medical/md_images/PMC10088903/3_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:a4bdefa03cb7ca95f35130dd29788bdb219f7df486f08f09a35fd14dd6baef4a +size 63608 diff --git a/medical/md_images/PMC10088903/8_image_0.png b/medical/md_images/PMC10088903/8_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..192e8b2416de0c26f059c8d2b0c96d0b8dd1be36 --- /dev/null +++ b/medical/md_images/PMC10088903/8_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:1c4f3eaf83e3a286b7901f9b99cf74cd97c6c52e9d22ca70b9cae37038ae9cef +size 59481 diff --git a/medical/md_images/PMC10088903/PMC10088903.md b/medical/md_images/PMC10088903/PMC10088903.md new file mode 100644 index 0000000000000000000000000000000000000000..8179c8de0c1ef55451d538acf64d71f6d6ea6710 --- /dev/null +++ b/medical/md_images/PMC10088903/PMC10088903.md @@ -0,0 +1,269 @@ +# Open Access Full Text Article O R I G I N A L R E S E A R C H Relationship Between Psychological Contract Violation And Physicians' Destructive And Constructive Behaviors In Tertiary Public Hospitals: An Empirical Evidence In Beijing + +Lujia Hu1,2,*, Chen Chen1,*, Junli Zhu 1, Yao Zhang3, Yun Chen4,5, Yufei Jia6 1School of Public Health, Capital Medical University & Research Center for Capital Health Management and Policy, Beijing, People's Republic of China; 2Department of Basic Health Care, Tianjin Hedong District Health Commission, Tianjin, People's Republic of China; 3Department of Human Resources, Beijing Tongren Hospital, Capital Medical University, Beijing, People's Republic of China; 4The National Clinical Research Center for Mental Disorders & Beijing Key Laboratory of Mental Disorders, Beijing Anding Hospital, Capital Medical University, Beijing, People's Republic of China; 5Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, People's Republic of China; 6Department of Human Resources, Beijing Shijitan Hospital, Capital Medical University, Beijing, People's Republic of China *These authors contributed equally to this work Correspondence: Junli Zhu, School of Public Health, Capital Medical University & Research Center for Capital Health Management and Policy, No. 10, Xitoutiao, Youanmen Wai, Fengtai District, Beijing, 100069, People's Republic of China, Tel +86 18701515093, Fax +86 01083911578, Email smallying@126.com Background: In China, physicians have long faced long working hours, high stress levels, and tensions between physicians and patients, which can lead to negative behaviors. Understanding physicians' expectations and requirements of the hospital and increasing satisfaction with their psychological contract can help improve physician motivation and stabilize the hospital team. + +Aim: The study aims to analyze the relationship between physicians' psychological contract violations and different behavioral choices, encourage hospitals to conclude a balanced psychological contract with physicians, and provide governance and intervention strategies for hospital human resource management. + +Methods: Stratified cluster sampling was used to select 321 physicians from four public hospitals in Beijing for questionnaire surveys. Descriptive statistical analysis, t-test, ANOVA, correlation analysis, and regression models were performed using Stata 15.0 and SPSS 26.0 to analyze the relationship between psychological contract violations, physicians' EVLN behaviors and organizational justice. + +Results: Psychological contract violation had a positive effect on exit behavior and neglect behavior, and a negative effect on voice behavior and loyalty behavior. Organizational justice plays a mediating role between psychological contract violation and physicians' exit, voice and loyalty behaviors. + +Conclusion: Psychological contract violation can drive negative behavior among physicians, and organizational justice can play a mediating role in this. Public hospitals should establish a healthy psychological contract with physicians and place a premium on organizational justice to promote constructive behaviors and prevent destructive behaviors. This study constructs a more complete theoretical framework to explain physicians' behavior, and further dynamic tracking investigations are necessary because the evolution of physicians' behavior is a dynamic and long-term process. + +Keywords: tertiary public hospitals, physicians, psychological contract violation, EVLN, China, mediation effect + +## Introduction + +Physicians, as the main providers of medical services in hospitals, have decision-making authority for admitting and discharging patients, performing clinical operations, and prescribing medications. Yet Chinese medical staff have long faced long working hours, high levels of stress, under-valued labor, and strained physician-patient relationships, especially violence by patients against physicians.1,2 From July 2001 to July 2018, there were 47 medical injuries resulting in the death of medical personnel, which greatly affected physicians' sense of security and work ethic.3 The +"Physician Career Satisfaction" survey conducted by China Youth Daily and Chunyu Doctor revealed that 39.6% of physicians were dissatisfied with their jobs.4 The results of Chinese medical institutions' physician turnover survey in 2015 showed that the proportion of physicians who had the intention to leave was 45.4%, 58.0% and 63.4% in primary, secondary and tertiary hospitals respectively.5 Public hospitals are the main body of China's medical service system, accounting for almost 89% of hospital beds and 92% of hospital admissions.6 As the capital of China, Beijing has over 100 tertiary hospitals and many national medical centers, and the tertiary public hospitals lead the country in terms of workload.7 The high volume of outpatient and emergency care, the complexity of patient conditions, and the heavy teaching and research tasks often make physicians have a higher stress level, which in turn leads to anti-productivity behaviors such as hidden absenteeism.8 Healthcare staff are the important implementer of public hospital reform.9 For hospital managers, understanding physicians' expectations and requirements of the hospital and enhancing the satisfaction of their psychological contract can help improve physicians' motivation, activate physicians' creativity and stabilize the team. + +The psychological contract refers to individual beliefs, shaped by the organization, regarding terms of an exchange agreement between individual and organization.10 Schein (1980) pointed out that the "self-evident" contract that exists in people's subconscious mind is an important factor that influences employees' attitudes and behaviors toward the organization.11 Previous researchers conclude that psychological contract violation may occur when the organization violates on purpose or for unforeseen reasons, and it may also occur when employees' understanding of organizational commitments is inconsistent with the organization.10,12,13 Argyris (1957) first developed the idea that psychological contracts can have an impact on employees' attitudes and behaviors.14 Then some researchers used psychological contract theory to explore the hospital-physician relationship and found that the fulfillment of the psychological contract can result in positive attitudinal and behavioral responses of physicians, such as job satisfaction and organizational citizenship behavior.15,16 The violation of the psychological contract can lead to negative physician attitudes and behaviors, such as job dissatisfaction and thoughts of quitting.17,18 Collins et al (2020) found that psychological contract violation was negatively associated with physicians' job satisfaction and well-being.19 Hirschman (1970) and Farrell (1983) summarized the employee behaviors that emerge in job dissatisfaction as EVLN +behaviors, which are exit behavior, voice behavior, loyalty behavior, and neglect behavior.20,21 "Exit" is the tendency and behavior of employees to leave the organization through resignation, transfer, or finding a new job. "Voice" refers to the behavior of employees to improve their environment and conditions through active efforts such as discussing work difficulties with their supervisors or colleagues, taking actions to solve the difficulties, or seeking outside help. "Loyalty" refers to the behavior of employees to passively but optimistically wait for improvement of the environment and conditions by giving the organization support and acting as a well-behaved citizen. "Neglect" refers to the behavior of employees temporarily abandoning work by reducing effort, being late or absent for a long time, doing private tasks during working hours, and increasing the error rate. According to the destructive-constructive dimensions, "Exit" and +"Neglect" are destructive behaviors, while "Voice" and "Loyalty" are constructive behavior.21 There have been studies exploring the effect of psychological contract violation on employee behavior based on EVLN theory.22,23 Yet studies applying psychological contract theory to physicians' behavior are limited, especially in China. Unlike the international mainstream that the physicians are freelance practitioners, Chinese physicians are "unit persons" and are affiliated with medical institutions, which hinders the mobility of Chinese physicians. Cultural differences between China and the Western countries may also cause Chinese physicians to behave differently in the face of psychological contract violations. Zhang et al (2020) designed a psychological contract scale for pharmacists and argued that psychological contract theory could be used in hospital pharmacy.24 Accordingly, this study takes physicians in Beijing public hospitals as the subject to explore physicians' behavioral choices in the face of psychological contract violations. EVLN theory provides a framework for employees' responses to psychological contract violation. Physicians respond to psychological contract violation by increasing turnover and negligence, as well as reducing their voice and loyalty; psychological contract fulfillment increase physicians' constructive behaviors because their needs are met.25,26 Therefore, we argue the following: +Hypothesis 1: Psychological contract violation is significantly related to physicians' EVLN behaviors. Hypothesis 1a: Psychological contract violation is negatively related to physicians' constructive behaviors in public hospitals, including "voice" and "loyalty" behaviors. Hypothesis 1b: Psychological contract violation is positively related to physicians' destructive behaviors, including +"exit" and "neglect" behaviors. + +Also of interest is that previous research has found that organizational justice can strongly influence the relationship between psychological contract violations and employee responses. Organizational justice refers to the perceptions of fairness in organizational decision-making and resource allocation.27 Leineweber (2020) found that team turnover was negatively related with changes in interpersonal justice perceptions.28 Zhou, P.(2019) found that organizational justice was an important mediator of the negatively correlation between physicians' psychological contract violation and their willingness to leave, and that physicians were likely to have the intention to leave if salary distribution and development opportunities were unfair.29 It has also been shown that when physicians perceive higher levels of psychological contract and organizational justice fulfillment, they have higher job satisfaction, lower intention to leave, and higher motivation to contribute to the organization.16,17,30 Thus we also tested the mediating role of organizational justice in the relationship between psychological contract violation and physician EVLN behavior. This leads to our hypothesis: Hypothesis 2: Organizational justice plays an intermediary role between physicians' psychological contract violation and EVLN behavior. Hypothesis 2a: Psychological contract violation is negatively related to organizational justice. Hypothesis 2b: Organizational justice is significantly related to physicians' constructive behaviors in public hospitals, including "voice" and "loyalty" behaviors. Hypothesis 2c: Organizational justice is significantly related to physicians' destructive behaviors, including "exit" and "neglect" behaviors. + +## Materials And Methods Sample And Data + +In this study, stratified cluster sampling was used to conduct a questionnaire survey of physicians in four public hospitals in Beijing. Beijing's tertiary public hospitals are mainly concentrated in Dongcheng District, Xicheng District, Haidian District, Fengtai District, and Chaoyang District. First, we randomly selected three districts from the above five districts, namely Dongcheng District, Xicheng District and Haidian District. Secondly, we randomly selected one hospital in each of the three districts, and two general hospitals and one specialty hospital were selected. In order to ensure the representativeness of the hospitals, we added one more specialist hospital to the sample. Two general hospitals and two specialty hospitals were finally identified. Finally, the samples were selected from the formal clinicians of the four hospitals by using proportional sampling according to the hospital size. The specific sampling process as shown in Figure 1. The questionnaire was anonymous, and all respondents voluntarily participated and signed written informed consent. To ensure the quality of the investigation, the investigators were trained in questioning techniques and communication skills. This study passed the ethical review of the Medical Ethics Committee of Capital Medical University. + +The inclusion criteria for survey subjects of this study were as follows: (1) voluntary participation in this study and +(2) physicians without management positions and working for more than one year. The exclusion criteria were the physicians who are temporarily in the hospital for advanced studies, job-training, or internship. A total of 321 questionnaires were distributed, of which 292 were valid, with an effective rate of 91%. The sample sizes of the four selected hospitals were 42, 40, 120 and 90, respectively. + +Hu et al **Dovepress** + +![3_image_0.png](3_image_0.png) + +Figure 1 Flowchart of sampling. + +## Instruments + +The instrument in this study was adapted from the relevant literature, which showed sound levels of reliability and constructs validity. The scale for physician psychological contract violation was referenced from Russeau (1990) and Turnley & Feldman (1999).13,31 There are 12 items, such as "The hospital pays salaries and bonuses based on your performance", "The hospital maintains a harmonious relationship between colleagues", etc. The EVLN behavior scale was referenced from Rusbult (1988) and includes 18 items.32 Organizational justice was referenced from Colquitt's +(2001) organizational justice scale.33 The meta-analysis of Hauenstein, McGonigle, and Flinder (2001) suggested that a simpler view of justice is one that focuses on general perceptions of justice, rather than specific forms of justice.34 Therefore, this study explored the overall justice dimension, named perceptions of organizational justice (JUST). Further define the questionnaire items by following several steps: +First, we translated the English scale into Chinese. Second, two professors and three hospital HR managers assisted in reviewing the Chinese questionnaire for appropriateness. Third, a pilot study was executed for modifying the questionnaire. The pilot study was conducted by recruiting medical students because the demographic characteristics of medical students are highly homogeneous and the influence of demographic characteristics factors can be avoided, followed by the fact that medical students mostly have internship experience and their behavioral choices are close to physicians, which can be used as the study subjects for the pilot study. Finally, a total of 211 medical students participated in the questionnaire. A 5-point Likert scale was used, with scores from 1 to 5 representing "strongly disagree" to "strongly agree". Exploratory factor analysis (EFA) was used to extract behavioral dimensions. The items with loads less than 0.5 on their own factor or greater than 0.3 on other factors were removed from the analysis.35 The results of pilot study showed that the scale had acceptable reliability and validity and could be adopted. + +## Analysis + +The relationship between the variables was: (1) EVLN ¼ β11PCV þ β10; (2) JUSTICE ¼ β21PCV þ β20; (3) EVLN ¼ β31PCV þ β32JUSTICE þ β30: Where EVLN represents the exit, voice, loyalty, and neglect behavior; PCV represents the psychological contract violation; βi1 represents the standardized regression coefficients for psychological contract violation in equation i; β32 represents the standardized regression coefficients for organizational justice on EVLN; βi0 represents the constant of equation i. The significant mediating effect must satisfy: 1. The independent variable in Equations 1 and 2 can predict the dependent variable and the intermediate variable, β11, and β21 are significant; 2. The intermediate variable in Equation 3 can predict the dependent variable, and β32 is significant; 3. After the intermediate variable is introduced in Equation 3, the influence of the independent variable on the dependent variable must be weakened or eliminated, β31 < β11 (partial mediation) or β31 is not significant (complete mediation). + +This study used Stata 15.0 and SPSS 26.0 for descriptive statistics analysis, exploratory factor analysis, and regression modeling. The differences between participant characteristics and EVLN behaviors was explored using the independent t-test and one-way ANOVA. The correlations between variables were assessed via Pearson's correlation coefficients. The hierarchical regression analysis was used to test the mediating effects of organizational justice on the relationship between psychological contract violation and EVLN behavior. The Sobel test was used to explore the mediating effect of organizational justice between psychological contract violation and EVLN behavior.36 Cronbach's alphas and coefficients were used to assess the reliability and validity of the scales. + +## Results Reliability And Validity Of The Scale Psychological Contract Violation Scale + +The KMO value was 0.95 and the Bartlett's sphericity test was significant, which demonstrated that the data were suitable for factor analysis. Principal component analysis was used to extract factors with eigenvalues greater than to 1. There was 1 main factor extracted, and the cumulative variance explained was 66.19%, indicating that the scale is a single dimensional structure. Internal consistency measured through Cronbachs' α was 0.95, indicating a good stability of the scale. + +## Evln Scale + +Three items (two neglected behavior items and one voice behavior item) were deleted to ensure the validity of the scale, and 15 items were retained at the end. The KMO value was 0.87 and the Bartlett's sphericity test was significant, allowing the EFA to be conducted. The EFA showed a four-factor solution as shown in Table 1, with a cumulative contribution of 70.24%. The factor composition was the same as the questionnaire dimensions, named exit behavior, neglect behavior, voice behavior, and loyalty behavior, respectively. The Cronbach's α value for the overall scale was 0.75, and the Cronbach's α values for the exit, voice, loyalty, and neglect behavior dimensions were 0.87, 0.89, 0.80, and 0.84, respectively, indicating a good stability of the scale. + +## Organizational Justice Scale + +The KMO value was 0.94 and the Bartlett's sphericity test was significant, which demonstrated that the data were suitable for factor analysis. Principal component analysis was used to extract factors with eigenvalues greater than to 1. There was 1 main factor extracted, and the cumulative variance explained was 70.50%, indicating that the scale is a single dimensional structure. Internal consistency measured through Cronbachs' α was 0.96, indicating a good stability of the scale. + +| Table 1 Factor Load Matrix After Rotation Item Factor 1 Factor 2 Factor 3 | Factor 4 | +|-----------------------------------------------------------------------------|------------| +| T1 | 0.83 | +| T2 | 0.78 | +| T3 | 0.68 | +| T4 | 0.66 | +| N1 | 0.75 | +| N2 | 0.79 | +| N3 | 0.79 | +| N4 | 0.80 | +| V1 | 0.79 | +| V2 | 0.84 | +| V3 | 0.81 | +| L1 | 0.81 | +| L2 | 0.70 | +| L3 | 0.80 | +| L4 | 0.76 | + +## The Respondents' Characteristics + +The characteristics of the respondents are shown in Table 2. It can be seen that the 292 respondents in this study included 71.92% from general hospitals and 28.08% from specialized hospitals. Among the respondents, 185 (63.36%) were female and 230 (78.77%) were married. 3.77% of respondents were aged less than or equal to 25, 40.41% were 26–35 years, 38.70% were 36–45 years and 17.12% were 46–55 years. Most respondents (84.25%) had gotten master's degree or above. Physicians of key disciplines comprised 51.03%, while physicians of non-key disciplines comprised 48.97%. As for working years, 29.11% had worked for over 15 years, followed by those for 11–15 years and those for 6–10 years + +| Table 2 The Basic Information of Respondents (n=292) Variables n (%) | Exit | Neglect | Voice | Loyalty | | | +|------------------------------------------------------------------------|------------------|-------------|---------|-----------|----------|-----------| +| t/F | t/F | t/F | t/F | | | | +| Hospital category | General hospital | 210(71.92) | −1.163 | −2.876*** | 3.569*** | 3.631*** | +| Specialist hospital | 82(28.08) | | | | | | +| Gender | Male | 107(36.64) | 1.295 | 1.921* | −1.326 | −3.428*** | +| Female | 185(63.36) | | | | | | +| Marriage | Married | 230(78.77) | 1.344 | 0.429 | 0.057 | 0.531 | +| Unmarried | 56(19.18) | | | | | | +| Others | 6(2.05) | (Continued) | | | | | + +| Table 2 (Continued). Variables | n (%) | Exit | Neglect | Voice | Loyalty | | +|-----------------------------------|-----------------|------------|-----------|-----------|-----------|----------| +| t/F | t/F | t/F | t/F | | | | +| Age | Age ≤25 | 11(3.77) | 1.185 | 0.619 | 1.971* | 0.985 | +| Age 26–35 | 118(40.41) | | | | | | +| Age 36–45 | 113(38.70) | | | | | | +| Age 46–55 | 50(17.12) | | | | | | +| Section category | Key discipline | 149(51.03) | −2.845*** | −2.887*** | 1.876* | 3.487*** | +| Non-key discipline | 143 (48.97) | | | | | | +| Professional title | Chief physician | 21(7.29) | 1.223 | 0.786 | 1.876 | 1.810 | +| Associate chief physician | 81(27.74) | | | | | | +| Attending physician | 99(33.90) | | | | | | +| Physician or below | 91(31.07) | | | | | | +| The highest degree | Doctor | 134(45.89) | 1.250 | 1.374 | 1.158 | 0.330 | +| Master | 112(38.36) | | | | | | +| Bachelor | 42(14.38) | | | | | | +| Others | 4(1.37) | | | | | | +| Working years | ≤5 years | 89(30.48) | 1.994 | 0.215 | 0.669 | 0.728 | +| 6–10 years | 75(25.68) | | | | | | +| 11–15 years | 43(14.73) | | | | | | +| ≥16 years | 85(29.11) | | | | | | +| Note: *P<0.1, ***P<0.01. | | | | | | | + +and less than or equal to 5 years accounted for 14.73%, 25.68%, and 30.48% respectively. Respondents who were chief physicians accounted for 7.29%, associate chief physicians were 27.74%, 33.90% were attending physicians, and physicians or below comprised 31.07%. + +## The Differences In Demographic Characteristics Of Evln Behavior + +Table 2 shows that there were statistically significant differences in EVLN behaviors among different participants' characteristics. There were significant differences in exit behaviors among physicians in different section category (t= −2.845, p<0.01); there were significant differences in neglect behavior among physicians in different hospital category (t=−2.876, p<0.01), gender (t=1.921, p<0.1) and section category (t=−2.887, p<0.01); there were significant differences in voice behavior among physicians in different hospital category (t=3.569, p<0.01), age (F=1.971, p<0.1) and section category (t=1.876, p<0.1); and there were significant differences in loyalty behavior among physicians in different hospital category (t=3.631, p<0.01), gender (t=−3.428, p<0.01) and section category (t=3.487, p<0.01). + +## Relationship Between The Variables + +Table 3 shows the mean level of the physician's EVLN behavior choices, perceptions of organizational justice and psychological contract violations, and the correlations between them. The mean scores for exit, neglect, voice, and loyalty behaviors were 2.360, 1.894, 3.182, and 3.403, respectively, indicating higher scores for constructive behaviors than + +| Table 3 Correlation Analysis of Variables (n=292) Mean SD Exit Neglect | Voice | Loyalty | PCV | | | | | +|--------------------------------------------------------------------------------------------------------------------------------------|---------|-----------|----------|----------|----------|----------|----------| +| Exit | 2.360 | 0.862 | | | | | | +| Neglect | 1.894 | 0.832 | 0.623** | | | | | +| Voice | 3.182 | 0.749 | −0.022 | −0.041 | | | | +| Loyalty | 3.403 | 0.835 | −0.376** | −0.315** | 0.504** | | | +| PCV | 3.098 | 0.737 | 0.295** | 0.127* | −0.246** | −0.466** | | +| JUST | 3.093 | 0.878 | −0.390** | −0.191** | 0.240** | 0.462** | −0.535** | +| Note: *P<0.1, **P<0.05. Abbreviations: SD, standard deviation; PCV, psychological contract violation; JUST, organizational justice. | | | | | | | | + +destructive behaviors. The results also showed that psychological contract violation was significantly related to physicians' EVLN behaviors. Psychological contract violation was positively correlated with exit behavior (r=0.295, P<0.05) and neglect behavior (r=0.127, P<0.1), and negatively correlated with voice behavior (r=−0.246, P<0.05) and loyalty behavior (r=−0.466, P<0.05), which means physicians who experience higher levels of psychological contract violation were more likely to be burned out or even leave the organization and to be less enthusiastic and attached to their work environment. + +## Regression Analysis Results + +The variance inflation factor for each variable was not greater than 5, so the multi-collinearity was within the acceptable level. In Table 4, the results of model (1) showed that psychological contract violation positively affects physician exit behaviors (P<0.01) and negatively affects physician voice and loyalty behaviors, which indicates that hypotheses H1a and H1b were accurate. Model (2) showed that psychological contract violation negatively affects organizational justice, which indicates that hypotheses H2a was accurate. Model (3) introduced organizational justice based on model (1), and the effect of psychological contract violation on EVLN behavior changed at this point. Organizational justice positively affects constructive behavior and negatively affects destructive behavior, indicating that hypothesis H2b and H2c is + +| Table 4 The Correlation Between Psychological Contract Violation and EVLN and the Mediating Role of Justice Variables Model (1) Model (2) Model (3) Exit Neglect Voice Loyalty JUST Exit Neglect Voice Loyalty PCV 0.332*** 0.094 −0.192*** −0.460*** −0.608*** 0.144* 0.000 −0.116** −0.307*** JUST −0.309*** −0.154** 0.124** 0.251*** Female −0.117 −0.185* 0.134 0.335*** 0.232** −0.045 −0.149 0.105 0.277** Age 26–35 0.387 0.430* 0.027 −0.225 −0.376 0.271 0.372 −0.074 −0.130 Age 36–45 0.240 0.264 −0.212 −0.125 −0.248 0.164 0.226 −0.181 −0.062 Age 46–55 0.199 0.268 −0.016 0.141 0.476 0.346 0.342 −0.075 0.022 Age ≥56 −0.335 0.148 −1.524* 0.273 0.806 −0.085 0.272 −1.625* 0.070 Working years −0.006 −0.009 0.007 −0.000 −0.035** −0.017 −0.014 0.012 0.008 Title1† 0.098 −0.309 0.402* 0.321 −0.008 −0.095 −0.310 0.402* 0.323* Title2† −0.004 −0.414 0.086 0.190 −0.003 −0.005 −0.414* 0.086 0.191 (Continued) | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +(*Continued*) +Table 4 (Continued). + +| Variables | Model (1) | Model (2) | Model (3) | | | | | | | +|--------------------------------------------------------------------------------------------------------------------------------------------------|-------------|-------------|-------------|-----------|----------|----------|----------|-----------|----------| +| Exit | Neglect | Voice | Loyalty | JUST | Exit | Neglect | Voice | Loyalty | | +| Title3† | −0.187 | −0.547** | 0.139 | 0.335 | −0.119 | −0.224 | −0.565** | 0.154 | 0.365 | +| Specialist | 0.017 | 0.297** | −0.352*** | −0.290** | 0.041 | 0.029 | 0.303** | −0.356*** | −0.301** | +| Non-key | 0.242** | 0.249** | −0.140* | −0.260** | −0.045 | 0.228** | 0.242** | −0.135 | −0.249** | +| Constant | 2.132*** | 1.982*** | 3.078*** | 3.259*** | 0.464*** | 2.276*** | 2.053*** | 3.020*** | 3.143*** | +| F | 3.67*** | 2.54** | 4.21*** | 10.78*** | 12.35*** | 5.38*** | 2.80*** | 4.30*** | 12.16*** | +| R2 | 0.137 | 0.099 | 0.154 | 0.318 | 0.348 | 0.201 | 0.116 | 0.168 | 0.363 | +| Adjusted R2 | 0.100 | 0.060 | 0.117 | 0.288 | 0.320 | 0.164 | 0.075 | 0.129 | 0.333 | +| Sobel test Z | 4.294*** | 2.269** | −2.104** | −4.083*** | | | | | | +| Proportion of mediation effect to total effect | 0.566 | 0.992 | 0.394 | 0.332 | | | | | | +| Notes: *P<0.1, **P<0.05, ***P<0.01. † Title1, 2 and 3 represent deputy chief physician, attending physician and physician or below respectively. | | | | | | | | | | + +supported. The results showed that the regression coefficient of psychological contract violation on exit behavior + +![8_image_0.png](8_image_0.png) decreased (P<0.1), indicating that organizational justice partially mediated the effect between psychological contract violation and exit behavior. Figure 2 illustrated the mediation regression equations in a path diagram. The absolute value of the regression coefficients of psychological contract violations on voice and loyalty behaviors decreased (P<0.05), indicating that organizational justice played a part in the mediating role. Hypothesis H2 was supported. Besides, model (1) showed that the regression coefficient of gender was significant in neglect and loyalty behavior (β=−0.185, p<0.1; β=0.335, p<0.01). Model (2) showed that the regression coefficient of gender in organizational justice was significant +(β=0.232, P<0.05); model (3) showed that the regression coefficient of gender was no longer significant in neglect behavior (β=−0.149, P>0.1) and decreased in loyalty behavior (β=0.277, P<0.05), indicating that organizational justice played a mediating role between gender and neglect and loyalty behavior. Table 4 also showed that the mediating role of organizational justice accounts for more than 33% of the total effect and even up to 56.6% of exit behavior. This suggests that when psychological contract violations occur, physician behavior is affected primarily through physicians' perceptions of organizational justice. + +## Discussion + +Under the background of the new round of medical and health system reform, hospital structures and strategies have been adjusted: the previous departmental, hierarchical, and colleague relationships have changed, which may break physicians' previous psychological contracts. Psychological contract violation is inevitable in organizations. The violation and imbalance of the psychological contract can lead to an increase in physician dissatisfaction and trigger different behaviors. Physicians play a vital role in hospitals. Pay attention to guiding physician behaviors and creating a better physician-hospital relationship is of great significance to the challenges of current society. + +The study found that Chinese physicians showed more constructive than destructive behaviors in the organization. + +Construction behaviors scored higher, "loyalty" behaviors scored highest, while destructive behaviors scored lower and +"neglect" behaviors scored lowest. This may be related to the special status of physicians in public hospitals in China. + +Public hospitals are public institutions in China, and physicians in public hospitals are similar to civil servants, with stable jobs and high social status that are very attractive to young physicians. In recent years, the Chinese government has begun to implement reforms in the personnel system of public hospitals, including the removal of institution staff member, making it even more precious to have this status. At the same time, this study also found that the perceived psychological contract violation of Chinese public hospital physicians are both at an upper-middle level, which is higher than the perception of Flemish physicians (2.86) in the study conducted by Trybou (2016) based on data from 130 physicians in six hospitals.30 This study also found that when physicians perceive that the psychological contract is violated, physicians' building behaviors decrease and disruptive behaviors increase, and these results support previous research findings.13,15–17,30 Psychological contract violation was positively associated with physicians' intention to leave, and high fulfillment of their psychological contract provided more motivation to contribute to the hospital. These results are consistent with social exchange theory and control theory.37–39 When physicians feel that there is a discrepancy between reality and what the hospital promises, from the perspective of the physician, this difference reflects an imbalance in the social exchange relationship between them and the organization. Physicians have the motivation to eliminate or reduce this imbalance, including both constructive and destructive behaviors. Therefore, it is important to focus on the breach of the physician's psychological contract when the physician's behavior changes. In the psychological contract established between hospitals and physicians, hospital responsibilities include providing promotions, offering reasonable remuneration, providing educational and training opportunities, and providing career development opportunities.31 The fulfillment of the hospital's promised obligations will enable physicians to perform their duties, promote positive behavior and deter negative behavior. + +The mediating effects of organizational justice are different for different gender. Before introducing organizational justice as the mediating variable, male's neglect behavior is less and loyalty behavior is more than female's, which seems to be "more positive" than female in work. After introducing organizational justice as mediating variable, there is no gender difference in neglect behavior, and the gender difference in loyalty behavior is also reduced, which reminds us that organizational justice is a factor that female attach great importance to. Studies have shown that female may face more potential gender bias in work, which leads to female's low enthusiasm and pessimism about the organizational environment, with the highest number of female physicians changing their area of practice.40 So, the organization must offer equal rights for male and female, increasing female representation and encouraging equal opportunities through female empowerment. The research also shows that people with professional titles above physicians and in general hospitals have more neglect behavior. It could be partly explained that the working objects and contents of this group are more complex, working intensity is higher, but lack advancement because of the limited job opportunities. So it is easy for physicians to job burnout and even neglective behavior, such as attending to fewer patients or spending less time at the bedside.41 Chênevert (2021) also suggested that stress is a strong precursor of emotional exhaustion as well as a 'loss spiral' ultimately culminating in burnout and health problems.42 Therefore, hospitals need to design multiple career development channels for physicians, such as clinical type, teaching type, and scientific research type to make physicians have confidence in their professional development. Meanwhile, hospitals are advised to pay more attention to the health status of physicians with more stress and more complex work tasks, not only helping physicians relieve tension, but also encouraging healthy lifestyles and working patterns. + +In addition, this study found that organizational justice mediated the relationship between physician psychological contract violation and EVLN behavior. A high level of organizational justice has a negative buffering effect on psychological contract violations and physicians' exit behaviors, and a positive buffering effect between psychological contract violations and physicians' voice and loyalty behaviors. The results suggests that hospitals should pay attention to organizational justice in the development process of hospitals and build a scientific distribution system and evaluation system. In addition to justice in allocation outcomes, attention should be paid to justice in the allocation process. Siun +(2019) argued that care for employees and commitment to equitable treatment are the basis of any allocation process.43 Physicians should be allowed to participate in the development of the allocation system as much as possible, and the appraisal system should be combined with physicians' self needs, self-worth, and sense of honor to ensure that the wishes of the majority of employees are met. + +Besides, the study also found that the mediating role of organizational justice was particularly significant for exit behavior (the mediating role of organizational justice accounted for 56.6% of the total effect). Trybon (2016) also showed that in Belgium the organizational justice negatively affected physicians' intention to leave.30 Pan Y. (2016) believes that organizational justice plays a mediating role between nurses' psychological contract fulfillment and EVLN behavior in China;44 Zhou P. (2019) took Chinese public hospital physicians as subjects and found that justice was an important mediator of hospital responsibility fulfillment and physician resignation.29 Hu (2013) found that organizational justice is essential to the stability of professional employees.45 These results suggest that as a positive factor, organizational justice has a greater power on destructive behavior because people who are prone to destructive behavior are more sensitive to justice. When people feel that the decision-making process is fair, people are less likely to behave negatively.46–48 Therefore, hospitals should establish a scientific and effective monitoring system to ensure fairness. Only when physicians' sense of unfairness is reduced, negative behaviors such as resignation will be fundamentally reduced and risks in human resource integration will be decreased. + +This study is an extension of the research into the behavioral choices of physicians in organizations. The study has three theoretical contributions. Firstly, most previous studies have discussed only one behavior, in particular exit behavior. Whereas other behavior may have emerged or are emerging prior to the exit behavior, such as decreasing loyalty and increasing neglect. This study provides a more comprehensive and systematic of physicians' behavior choices based on EVLN theory. Second, EVLN theory is generally used to examine the behavior of corporate employees, and this study applies it to physicians, which is an enrichment of EVLN behavior theory. Third, this study introduces the organizational justice as a mediating variable, which constructs a more complete theoretical framework to explain physicians' behaviors. The study has two practical contributions. First, physicians in tertiary hospitals are more stressed and more prone to burnout due to society's high expectations. Focusing on tertiary hospitals, this study explores the root causes of physician behavior change from a psychological contract perspective and explores new ideas for physician motivation. Second, psychological contract violations do lead to negative physician behaviors. Therefore, monitoring the level of psychological contract violation among medical staff is an important part of human resource management. + +This study is not without its limitations. First, this study is only conducted on Beijing's tertiary public hospitals. + +Further research on factors such as different grades and geographic areas can be considered in the future, while including more hospitals and physicians in order to draw more general conclusions. Second, the evolution of physicians' behaviors is a dynamic and long-term process. This article only conducted a cross-sectional survey and did not conduct dynamic tracking studies. It would be more meaningful to analyze the changes in physicians' behaviors before and after reforms to evaluate reform policies. Third, considering that it is difficult to obtain evaluation information from physicians by multiple parties, the effect indicators of this study mainly rely on individual selfevaluation. In the future, information may be collected through multiple channels and methods to ensure comprehensiveness and objectivity. What's more, quality interview study could be considered in the future to supplement and reinforce the results of the questionnaire. + +## Conclusion + +This study shows that psychological contract violations can have a negative effect on physicians' constructive behaviors and a positive effect on destructive behaviors. The research also shows that organizational justice has a mediating effect between physicians' psychological contract violation and EVLN behavior, with the most significant mediating effect on exit behavior. Therefore, it is important to pay attention to physicians' psychological contract violations and build a good psychological contract relationship between hospitals and physicians to promote their constructive behaviors and prevent the destructive behaviors. + +## Ethics Approval + +The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Capital Medical University (protocol code Z2020SY131, date 2020-11-16). + +## Acknowledgments + +The authors thank the editor and the anonymous reviewers for their helpful and constructive comments, which greatly improved the paper. + +## Funding + +The research is supported by the National Natural Science Foundation of China (no.71974133, 71573182). + +## Disclosure + +The authors declare no conflict of interest. + +## References + +1. Tang L, Guan M. Rise of health consumerism in China and its effects on physicians' professional identity and the physician–patient relationship and communication. *Health Commun*. 2018;33(5):636–642. doi:10.1080/10410236.2017.1290015 2. Bareeqa SB, Ahmed SI, Samar SS, et al. Prevalence of depression, anxiety and stress in China during COVID-19 pandemic: a systematic review with meta-analysis. *Int J Psychiatry Med*. 2021;56(4):210–227. doi:10.1177/0091217420978005 3. Wen X. What can we do about the high incidence of violence against physicians? Beijing News; 2020. Available from: https://baijiahao.baidu.com/ +s?id=1667541621258060511&wfr=spider&for=pc. Accessed February 1, 2023. + +4. Wang C, Cheng J, Qi Y. Practicing medicine in the cracks: 68.1% of the interviewed doctors bluntly said that the doctor's practice environment is poor. China Youth Daily; 2021. Available from: http://politics.people.com.cn/n/2015/0420/c70731-26869588.html. Accessed August 8, 2021. + +5. Xu Q. Over 60% of physicians in tertiary hospitals want to resign, hospitals should hurry to find a solution. Health Community; 2016. Available from: https://www.cn-healthcare.com/article/20160628/content-483834.html?from=timeline&isappinstalled=0. Accessed February 1, 2023. + +6. Barber SL, Borowitz M, Bekedam H, Ma J. The hospital of the future in *China: china*'s reform of public hospitals and trends from industrialized countries. *Health Policy Plan*. 2013;29(3):367–378. doi:10.1093/heapol/czt023 7. Liu J. *Research on the Impact of Job Stressors on Occupational Burnout for Clinicians of Level a Tertiary Hospitals in Beijing*. University of International Business and Economics; 2020. + +8. Zhu J, Zhang M, Zeng J, Liu Y. Current situation and its influencing factors of perceived stress among doctors in Beijing. *Occup Health*. 2016;32 +(14):1888–1893. doi:10.13329/j.cnki.zyyjk.2016.0546 9. Li C, Song X. Study on implementation strategy of knowledge employee's psychological contract management in public hospital. Chin Health Serv Manag. 2012;29(1):64–66+79. + +10. Rousseau DM. Psychological contracts in organizations: understanding written and unwritten agreements. *Adm Sci Q*. 1995;43(1):184–186. + +doi:10.2307/2393595 11. Schein E. *Organizational Psychology*. New Jersey: Prentice-Hall; 1980. + +12. Morrison EW, Robinson SL. When employees feel betrayed: a model of how psychological contract violation develops. *Acad Manag Rev*. 1997;22 +(1):226–256. doi:10.5465/AMR.1997.9707180265 13. Turnley WH, Feldman DC, Discrepancy A. Model of psychological contract violations. *Hum Resour Manag Rev*. 1999;9(3):367–386. doi:10.1016/ +S1053-4822(99) +14. Argyris C. The individual and the organization: some problems of mutual adjustment. *Adm Sci Q*. 1957;2(1):1–24. doi:10.2307/2390587 15. Bellou V. Matching individuals and organizations: evidence from the Greek public sector. *Empl Relat*. 2009;31(5):455–470. doi:10.1108/ +01425450910979220 16. Trybou J, Gemmel P, Desmidt S, Annemans L. Fulfillment of administrative and professional obligations of hospitals and mission motivation of physicians. *BMC Health Serv Res*. 2017;17(28):1–10. doi:10.1186/s12913-017-1990-0 17. Bunderson JS. How work ideologies shape the psychological contracts of professional employees: doctors' responses to perceived breach. *J Organ* Behav. 2001;22(7):717–741. doi:10.1002/JOB.112 18. Jeroen TMB, Canfyn S, Lieven A, Paul G. Physiciaé hospital exchanges and extra-role behaviour of physicians: the moderating role of the chief medical officer. *Int J Healthc Manag*. 2016;9(4):225–235. doi:10.1179/2047971915Y.0000000022 19. Collins A, Beauregard AT. The effect of breaches of the psychological contract on the job satisfaction and wellbeing of doctors in Ireland: +a quantitative study. *Hum Resour Health*. 2020;18(89):1–8. doi:10.1186/s12960-020-00534-3 20. Hanson RA. Exit, voice, and loyalty: response to decline in firms, organizations, and states. By Albert O. Hirschman, (Cambridge: Harvard University Press, 1970. Pp. 162. $6.95.). *Am Polit Sci Rev*. 1970;64(4):1274–1276. doi:10.1017/S0003055400133611 21. Farrell D. Exit, voice, loyalty, and neglect as responses to job dissatisfaction: a multidimensional scaling study. *Acad Manag J*. 1983;26 +(4):596–607. doi:10.2307/255909 22. Turnley WH, Feldman DC. The impact of psychological contract violations on exit, voice, loyalty, and neglect. *Hum Relat*. 1999;52(7):895–922. + +doi:10.1177/001872679905200703 23. Si SX, Wei F, Li Y. The effect of organizational psychological contract violation on managers' exit, voice, loyalty and neglect in the Chinese context. *Int J Hum Resour Manag*. 2008;19(5):932–944. doi:10.1080/09585190801995849 24. Zhang T, Yin C, Geng Y, Zhou YMJ, Sun S, Tang F. Development and validation of psychological contract scale for hospital pharmacists. + +J Multidiscip Healthc. 2020;13:1433–1442. doi:10.2147/JMDH.S270030 25. Chatman JA. Improving interactional organizational research: a model of person-organization fit. *Acad Manag Rev*. 1989;14(3):333–349. + +doi:10.5465/AMR.1989.4279063 26. Griffeth RW, Hom PW, Gaertner S. A Meta-analysis of antecedents and correlates of employee turnover: update, moderator tests, and research implications for the next millennium. *J Manage*. 2000;26(3):463–488. doi:10.1177/014920630002600305 27. Greenberg J. A taxonomy of organizational justice theories. *Acad Manag Rev*. 1987;12(1):9–22. doi:10.5465/AMR.1987.4306437 28. Leineweber C, Peristera P, Bernhard-Oettel C, Eib C. Is interpersonal justice related to group and organizational turnover? Results from a Swedish panel study. *Soc Sci Med*. 2020;265:113526. doi:10.1016/j.socscimed.2020.113526 29. Zhou P, Shi J, Zhou H. Research on the association mechanism and moderating variables between physician's perceived hospital accountability status and turnover willingness. *China Health Res*. 2019;22(6):465–471. doi:10.13688/j.cnki.chr.2019.19032 30. Trybou J, Gemmel P, Annemans L. The impact of economic and noneconomic exchange on physicianì organizational attitudes: the moderating effects of the chief medical officer. *Health Care Manage Rev*. 2016;41(1):75–85. doi:10.1097/HMR.0000000000000047 31. Rousseau DM. New hire perceptions of their own and their employer's obligations: a study of psychological contracts. *J Organ Behav*. 1990;11 +(5):389–400. doi:10.1002/JOB.4030110506 32. Rusbult C, Farrell D, Rogers G, Mainous AG. Impact of exchange variables on exit, voice, loyalty, and neglect: an integrative model of responses to declining job satisfaction. *Acad Manag J*. 1988;31(3):599–627. doi:10.2307/256461 33. Colquitt JA, Conlon DE, Wesson MJ, Porter C, Ng KY. Justice at the millennium: a meta-analytic review of 25 years of organizational justice research. *J Appl Psychol*. 2001;86(3):425–445. doi:10.1037/0021-9010.86.3.425 34. Hauenstein NMA, McGonigle TP, Flinder SW. A meta-analysis of the relationship between procedural justice and distributive justice: implications for justice research. *Empl Responsib Rights J*. 2001;13(1):39–56. doi:10.1023/A:1014482124497 35. Hair JF. *Multivariate Data Analysis: A Global Perspective*. Upper Saddle River, NJ: Pearson; 2010. 36. Sobel ME. Asymptotic confidence intervals for indirect effects in structural equation models. *Sociol Methodol*. 1982;13:290. 37. Adams JS. Toward an understanding of inequity. *J Abnorm Psychol*. 1963;67(5):422–436. doi:10.1037/h0040968 38. Blau PM. *Exchange and Power in Social Life*. Routledge; 1964. 39. Carver CS, Scheier MF. Control theory: a useful conceptual framework for personality-social, clinical, and health psychology. *Psychol Bull*. + +1982;92(1):111–135. + +40. Lim WH, Wong C, Jain SR, et al. The unspoken reality of gender bias in surgery: a qualitative systematic review. *PLoS One*. 2021;16(2):e0246420. + +doi:10.1371/journal.pone.0246420 41. Leung S, Panda M, McIntosh G, et al. Relationship between physician burnout and patient's perception of bedside time spent by physicians. + +J Patient Cent Res Rev. 2021;8(1):58–63. doi:10.17294/2330-0698.1764 42. Chênevert D, Kilroy S, Johnson K, Fournier PL. The determinants of burnout and professional turnover intentions among Canadian physicians: +application of the job demands-resources model. *BMC Health Serv Res*. 2021;21(1):993. doi:10.1186/s12913-021-06981-5 43. Gallagher S, Little M. Procedural justice and the individual participant in priority setting: doctors' experiences. *Soc Sci Med*. 2019;228:75–84. + +doi:10.1016/j.socscimed.2019.03.012 44. Yan P. *An Empirical Study on the Relationship Between Nurses' Psychological Contract and Their EVLN Behavior*. Zhejiang University of Traditional Chinese Medicine; 2016. + +45. Hu G. *Research on the Relationship Between Organizational Justice and Turnover Intention of Knowledge-Based Employees in B Company*. + +Shanghai: East China University of Science and Technology; 2013. + +46. Brockner J, Wiesenfeld BM. An integrative framework for explaining reactions to decisions: interactive effects of outcomes and procedures. + +Psychol Bull. 1996;120(2):189–208. doi:10.1037/0033-2909.120.2.189 47. Moorman RH. Relationship between organizational justice and organizational citizenship behaviors: do fairness perceptions influence employee citizenship? *J Appl Psychol*. 1991;76(6):845–855. doi:10.1037/0021-9010.76.6.845 48. Skarlicki DP, Folger R. Retaliation in the workplace: the roles of distributive, procedural, and interactional justice. *J Appl Psychol*. 1997;82 +(3):434–443. doi:10.1037/0021-9010.82.3.434 Journal of Multidisciplinary Healthcare Dovepress Publish your work in this journal The Journal of Multidisciplinary Healthcare is an international, peer-reviewed open-access journal that aims to represent and publish research in healthcare areas delivered by practitioners of different disciplines. This includes studies and reviews conducted by multidisciplinary teams as well as research which evaluates the results or conduct of such teams or healthcare processes in general. The journal covers a very wide range of areas and welcomes submissions from practitioners at all levels, from all over the world. The manuscript management system is completely online and includes a very quick and fair peer-review system. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. + +Submit your manuscript here: https://www.dovepress.com/journal-of-inflammation-research-journal \ No newline at end of file diff --git a/medical/md_images/PMC10088903/PMC10088903_meta.json b/medical/md_images/PMC10088903/PMC10088903_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..b3947e58e811c11854b7cb2b816327a0f54ab8af --- /dev/null +++ b/medical/md_images/PMC10088903/PMC10088903_meta.json @@ -0,0 +1,288 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Introduction", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 152.2119903564453, + 0.0 + ] + }, + { + "title": "Materials and Methods", + "level": 0, + "is_closed": true, + "n_kids": 3, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 353.0589904785156, + 0.0 + ] + }, + { + "title": "Sample and Data", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 336.0589904785156, + 0.0 + ] + }, + { + "title": "Instruments", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 305.7510070800781, + 0.0 + ] + }, + { + "title": "Analysis", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 661.4290161132812, + 0.0 + ] + }, + { + "title": "Results", + "level": 0, + "is_closed": true, + "n_kids": 5, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 406.8689880371094, + 0.0 + ] + }, + { + "title": "Reliability and Validity of the Scale", + "level": 1, + "is_closed": true, + "n_kids": 3, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 389.8699951171875, + 0.0 + ] + }, + { + "title": "Psychological Contract Violation Scale", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 372.8699951171875, + 0.0 + ] + }, + { + "title": "EVLN Scale", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 278.30999755859375, + 0.0 + ] + }, + { + "title": "Organizational Justice Scale", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 155.75100708007812, + 0.0 + ] + }, + { + "title": "The Respondents\u2019 Characteristics", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 5, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 414.1499938964844, + 0.0 + ] + }, + { + "title": "The Differences in Demographic Characteristics of EVLN Behavior", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 262.9859924316406, + 0.0 + ] + }, + { + "title": "Relationship Between the Variables", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "XYZ", + "view_pos": [ + 70.9999008178711, + 131.75, + 0.0 + ] + }, + { + "title": "Regression Analysis Results", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 7, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 469.84698486328125, + 0.0 + ] + }, + { + "title": "Discussion", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 9, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 677.7520141601562, + 0.0 + ] + }, + { + "title": "Conclusion", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 705.9409790039062, + 0.0 + ] + }, + { + "title": "Ethics Approval", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 593.89404296875, + 0.0 + ] + }, + { + "title": "Acknowledgments", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 537.8469848632812, + 0.0 + ] + }, + { + "title": "Funding", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 481.79998779296875, + 0.0 + ] + }, + { + "title": "Disclosure", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 54.0, + 439.75201416015625, + 0.0 + ] + } + ], + "pages": 14, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 13, + "code": 0, + "table": 6, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC10267944/0_image_0.png b/medical/md_images/PMC10267944/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..ece0b4a2f74e04e367197c6d3a4a32a78261d8e2 --- /dev/null +++ b/medical/md_images/PMC10267944/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:82426ecc951e2e51e8ca4e24f645208fd257294cd2e4f4adf907659c8226e7e3 +size 372 diff --git a/medical/md_images/PMC10267944/8_image_0.png b/medical/md_images/PMC10267944/8_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..ae1ddd1797c6bb793b4395f065559998c872dc2a --- /dev/null +++ b/medical/md_images/PMC10267944/8_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:187207f122b28b1ca237c4129c3dbdd914b48a95dcb097b4a4f7c6ed44ff64b9 +size 36583 diff --git a/medical/md_images/PMC10336507/4_image_0.png b/medical/md_images/PMC10336507/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..d0cb810752d316584473cbda69b941e8a75b66cb --- /dev/null +++ b/medical/md_images/PMC10336507/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0af3910e9712a9a97f835efdbd197085df54136af7614774666215baa5968eea +size 76186 diff --git a/medical/md_images/PMC10336507/6_image_0.png b/medical/md_images/PMC10336507/6_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..06f0d75bc43cd035b0ce2194a0ab03429d13d819 --- /dev/null +++ b/medical/md_images/PMC10336507/6_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:208170e4755eb3a8bcd5c64fba7c09bb989c5afbc163a25405dcb19bba1899f5 +size 25458 diff --git a/medical/md_images/PMC10388603/1_image_1.png b/medical/md_images/PMC10388603/1_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..7e4c7ffadcedcdbcaa97d73ad30306361b405b0b --- /dev/null +++ b/medical/md_images/PMC10388603/1_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:292417a2a488d5f0afa46986ff4160eaa1ca9a531a0d55c51616d4bcc51b4707 +size 41789 diff --git a/medical/md_images/PMC10388603/2_image_1.png b/medical/md_images/PMC10388603/2_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..4881681438eb68e9a53232bc590599605c08fcdc --- /dev/null +++ b/medical/md_images/PMC10388603/2_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:eca1f77da94d2e7b8eeeb0dd86be0fb1a6e8ed7b75010be7e97a77abbcf803d1 +size 250 diff --git a/medical/md_images/PMC10514721/0_image_0.png b/medical/md_images/PMC10514721/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..b5e6f30216c3ee926e3afdfd79f8451a5e5567d1 --- /dev/null +++ b/medical/md_images/PMC10514721/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f7a54d0c3b8d929aaaccc289b4697f19c7ba5f6f2024c90e837e415f174a2578 +size 7528 diff --git a/medical/md_images/PMC10514721/5_image_0.png b/medical/md_images/PMC10514721/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..44d7a127a1a0a03819631c77af24f44a828b8b4f --- /dev/null +++ b/medical/md_images/PMC10514721/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f75f958934a0748ef2c60c3b4fce507e17336c62c14bd6df9509600c80307e67 +size 64516 diff --git a/medical/md_images/PMC10582389/0_image_3.png b/medical/md_images/PMC10582389/0_image_3.png new file mode 100644 index 0000000000000000000000000000000000000000..ff68012471b066a367928866445ffe1a1a5b5413 --- /dev/null +++ b/medical/md_images/PMC10582389/0_image_3.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:328dc6c33293e50f053a2f65d7c989148efc9e9a74a79ef47bd65a309d72a1a4 +size 318 diff --git a/medical/md_images/PMC3061075/5_image_0.png b/medical/md_images/PMC3061075/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..7356d1a430871e19e2d65782c113b8f243f1ea2c --- /dev/null +++ b/medical/md_images/PMC3061075/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:26e7394da4aae84c6dfc90f67776f05bc92673705a3bf8c48f2f782d3a0d340f +size 118746 diff --git a/medical/md_images/PMC3061075/7_image_1.png b/medical/md_images/PMC3061075/7_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..5f12577dd558b1bfaf62af0d6150b3720984941b --- /dev/null +++ b/medical/md_images/PMC3061075/7_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:525c97919fc85cadc2391ff35b7f9853227a067faf5b7743e78e42ebca41e550 +size 35245 diff --git a/medical/md_images/PMC4142939/3_image_1.png b/medical/md_images/PMC4142939/3_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..753cc989064a207c5da24fd3084c1e7f673971b2 --- /dev/null +++ b/medical/md_images/PMC4142939/3_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:bbb5dc7526522fc1571588cae0596387223f138b5a5c1193c4101c632f9121ae +size 21015 diff --git a/medical/md_images/PMC4278874/3_image_0.png b/medical/md_images/PMC4278874/3_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..75a64b35e51f531085b9b77e6eca9ea47299c342 --- /dev/null +++ b/medical/md_images/PMC4278874/3_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:ece80ddb5393264bf9161c371f21c242f5353830c3d0b0779b38c6d22e80d2d5 +size 26160 diff --git a/medical/md_images/PMC4278874/3_image_1.png b/medical/md_images/PMC4278874/3_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..7d779c7d3045d566a0d34e7551cc3e1c5eed57b5 --- /dev/null +++ b/medical/md_images/PMC4278874/3_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:361421c1feb8d59d39d998e2b321f01c3db70609cab5ee9c1cbb2deb9a285f68 +size 107457 diff --git a/medical/md_images/PMC4278874/4_image_0.png b/medical/md_images/PMC4278874/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..8ecc8918a007d49059bb41528949233bfc07fd80 --- /dev/null +++ b/medical/md_images/PMC4278874/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:47eaf268a518f4a6feeabcf30f71fc9246d7287013f61d2c5eee595cb6a87650 +size 40856 diff --git a/medical/md_images/PMC4386234/0_image_0.png b/medical/md_images/PMC4386234/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..fb282d8d5921d5d0264ffcc6877014b16d5146d8 --- /dev/null +++ b/medical/md_images/PMC4386234/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:daa1090f971fd266dc29d337e4f903b233889e9efe4504bfef09e914080af0ff +size 4159 diff --git a/medical/md_images/PMC4386234/6_image_0.png b/medical/md_images/PMC4386234/6_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..bc8eb661581dcd97f6eed3aaade77d514cf8bc27 --- /dev/null +++ b/medical/md_images/PMC4386234/6_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:5e2530e483737541d4e70039a464ae7f1ea27cfd60aa0e6f1dc3bed0792132ad +size 44136 diff --git a/medical/md_images/PMC4492755/0_image_0.png b/medical/md_images/PMC4492755/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..282257d811f7f92115b1efdfe55824b6e021226f --- /dev/null +++ b/medical/md_images/PMC4492755/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:52c797046d13a775ac99cdcfed6fab172070bd3a24d020a600a35a24e71a6602 +size 2248 diff --git a/medical/md_images/PMC4492755/8_image_0.png b/medical/md_images/PMC4492755/8_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..fbf7be2bb9b8929bd3248b122252690a177fa188 --- /dev/null +++ b/medical/md_images/PMC4492755/8_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:75078f0497481b1a152f14ab01f3ff827c1a893218c8323e59e404d66f941de3 +size 18629 diff --git a/medical/md_images/PMC4492755/8_image_1.png b/medical/md_images/PMC4492755/8_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..3dffa99655bfd6dac68f7002fc8cc4f26f5e5fbb --- /dev/null +++ b/medical/md_images/PMC4492755/8_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:20b18e16b87582eab2067f20f0f6366cc62f141d2d07aea56f6bb62accfdbe38 +size 29001 diff --git a/medical/md_images/PMC4640227/5_image_0.png b/medical/md_images/PMC4640227/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..410cecfad470d46d95a6c4c8f0d81321f44fc644 --- /dev/null +++ b/medical/md_images/PMC4640227/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:d247cd005c400672b32b3da5ba1982840fb7bf264fcd449dcaa7fb3298dc7303 +size 71610 diff --git a/medical/md_images/PMC4640227/6_image_0.png b/medical/md_images/PMC4640227/6_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..f27897ea964d52aeede9ab685d5e7202c259ffc6 --- /dev/null +++ b/medical/md_images/PMC4640227/6_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:782e58f3f6d20e2f7117e1b66a5ea3b5a3cfd8e48bb2c0f60d13721fa1540cc5 +size 15468 diff --git a/medical/md_images/PMC4665231/4_image_0.png b/medical/md_images/PMC4665231/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..25442992491d67392e2e5eee994656c11d88afec --- /dev/null +++ b/medical/md_images/PMC4665231/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:33235e85c16a70164e9b5786dacc2de4974e994f05292f1f44edcb322617a2cd +size 175822 diff --git a/medical/md_images/PMC4674186/4_image_0.png b/medical/md_images/PMC4674186/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..fda04afb542e6b334ee4d9249b86c09fe3cf6b24 --- /dev/null +++ b/medical/md_images/PMC4674186/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:69accdfd7ae024087a99c07ef546a00212f53ce67970cd7d5da40844ca897027 +size 36701 diff --git a/medical/md_images/PMC4674186/5_image_0.png b/medical/md_images/PMC4674186/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..2cc016c81acef917395a6cda372b3c311a1e3107 --- /dev/null +++ b/medical/md_images/PMC4674186/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:093e08233d34f333e9cdd06c66e162db0fccde9c64c0356794108009e71d43ba +size 70725 diff --git a/medical/md_images/PMC4674186/PMC4674186.md b/medical/md_images/PMC4674186/PMC4674186.md new file mode 100644 index 0000000000000000000000000000000000000000..d5b514de6264f13cbb9a1e9094f47eb9d543cd14 --- /dev/null +++ b/medical/md_images/PMC4674186/PMC4674186.md @@ -0,0 +1,476 @@ +# Pharmacokinetic And Genomic Effects Of Arsenite In Drinking Water On Mouse Lung In A 30-Day Exposure + +Jaya Chilakapati1, Kathleen Wallace2, Araceli Hernandez-Zavala3, Tanya Moore2, Hongzu Ren2, and Kirk T. Kitchin2 Dose-Response: An International Journal April-June 2015:1-11 ª The Author(s) 2015 DOI: 10.1177/1559325815592392 dos.sagepub.com + +## Abstract + +The 2 objectives of this subchronic study were to determine the arsenite drinking water exposure dependent increases in female C3H mouse liver and lung tissue arsenicals and to characterize the dose response (to 0, 0.05, 0.25, 1, 10, and 85 ppm arsenite in drinking water for 30 days and a purified AIN-93M diet) for genomic mouse lung expression patterns. Mouse lungs were analyzed for inorganic arsenic, monomethylated, and dimethylated arsenicals by hydride generation atomic absorption spectroscopy. The total lung mean arsenical levels were 1.4, 22.5, 30.1, 50.9, 105.3, and 316.4 ng/g lung tissue after 0, 0.05, 0.25, 1, 10, and 85 ppm, respectively. At 85 ppm, the total mean lung arsenical levels increased 14-fold and 131-fold when compared to either the lowest noncontrol dose (0.05 ppm) or the control dose, respectively. We found that arsenic exposure elicited minimal numbers of differentially expressed genes (DEGs; 77, 38, 90, 87, and 87 DEGs) after 0.05, 0.25, 1, 10, and 85 ppm, respectively, which were associated with cardiovascular disease, development, differentiation, apoptosis, proliferation, and stress response. After 30 days of arsenite exposure, this study showed monotonic increases in mouse lung arsenical (total arsenic and dimethylarsinic acid) concentrations but no clear dose-related increases in DEG numbers. + +## Keywords + +arsenic, mice, drinking water, genomics, dose response + +## Introduction + +Inorganic arsenic causes cancer in skin, bladder, lung, liver, and kidneys. Arsenic causes lung cancer via inhalation and ingestion of drinking water.1 In 2001, the United States Environmental Protection Agency (EPA) set the arsenic drinking water standard of 0.01 ppm2 based on extrapolation of the human cancer data from the high arsenic exposure areas in Taiwan.3 In 2002, the International Agency for Research on Cancer (IARC) classified arsenic in drinking water as a cause of lung, bladder, and skin cancers.3 Much is known about arsenic-induced carcinogenic effects in urinary bladder and skin. Lung cancer is the leading cause of cancer-related mortality in the United States and worldwide. The IARC has classified arsenic as a group 1 carcinogen for lung cancer.3 Humans exposed to inorganic arsenic in their drinking water have demonstrated elevated risks of death from lung cancer in Taiwan,4,5 Chile,6 and Argentina.7 Epidemiologic evidence supports the presence of a causal association between exposure to elevated arsenic concentrations in drinking water and the risk of developing or dying from lung cancer. + +Beginning in the 1990s, epidemiological studies in Chile showed that the rate of lung and bladder cancer in arsenicpolluted areas was considerably higher than mean cancer rates of nearby areas.6,8 Less is known about the risk of lung cancer at lower exposure levels than achieved in Taiwan, Chile, or Argentina. Arsenic also causes noncancer pulmonary health effects such as chronic obstructive pulmonary disease and bronchiectasis.6,9 For these reasons, we conducted our studies 1 Alcon Labs (R9-7), Fort Worth, TX, USA +2 Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Durham, NC, USA +3 Seccio´ n de Investigacio´ n y Posgrado, Escuela Superior de Medicina del Instituto Politecnico Nacional, Mexico City, Mexico ´ +Corresponding Author: Kirk T. Kitchin, US Environmental Protection Agency, Integrated Systems Toxicology Division, 109 TW Alexander Drive (B105-03), Durham, NC 27711. Email: kitchin.kirk@epa.gov Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License +(http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (http://www.uk.sagepub.com/aboutus/openaccess.htm). + +| Table 1. Some Arsenic-Induced Tumor-Related Health Effects in Mice. Time of Mouse Strain Arsenic Treatment Exposure | Organ | Findings | Reference 23 | | | +|-----------------------------------------------------------------------------------------------------------------------|-------------------------------------------|----------------------------------------|---------------------|-------------------------------------------------|-------| +| A/J mice | DMAV 400 ppm | 50 weeks | Lung | Hyperplasia, adenoma, and adenocarcinoma Tumors | 24,25 | +| Female C3H | AsIII 85 ppm | Gestational days Liver, lung, adrenal, | | | | +| mice | 8-18 in utero | and uterus | | | | +| Ogg1-deficient | DMAV 200 ppm | 72 weeks | Lung | Tumors | 26 | +| mice | | | | | | +| Male ddY mice | DMAV 200 or 400 ppm | 25 weeks | Lung | Adenocarcinomas and | 27 | +| þ 4-nitroquinoline 1-oxide | adenosquamous carcinomas | | | | | +| K6/ODC | DMAV 10 or 100 ppm, AsIII 10 ppm 5 months | Skin | Squamous papillomas | 28 | | +| K6/ODC | MMAIII 10, 50, or 75 ppm | 26 weeks | Skin | Squamous papillomas | 29 | +| Tg.AC | AsIII 200 ppm þ TPA | 22 weeks | Skin | Tumors | 30 | +| Hairless mice | DMAV 1000 ppm þ UVB light | 25 weeks | Skin | Tumors | 31 | +| p53 þ/– | DMAV 50 ppm | 80 weeks | Not organ specific | Tumors | 32 | +| Abbreviations: As, inorganic arsenic; MMA, monomethylarsonic acid; DMA, dimethylarsinic acid; UVB, ultraviolet beta. | | | | | | + +primarily in the lung, a lesser studied organ that still is a large contributor to arsenic cancer-induced death in humans. + +Inorganic arsenic ingested through drinking water is quickly absorbed into the bloodstream and is transported to the liver where it is metabolized through methylation processes to generate monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) metabolites.10 Most of the arsenic metabolites are excreted in the urine; however, some are deposited in lung, liver, kidney, nails, and hair.11 In earlier toxicological thinking, arsenic was sometimes considered to be a promoter and not an initiator of carcinogenesis, as no mutagenic potential was shown in early experimental models.12 However, there has been growing evidence that MMA and DMA are cytotoxic and genotoxic in cell lines.13-17 Arsenic-induced carcinogenesis may occur through several biological mechanisms, particularly via the highly biologically active MMA and DMA metabolites.18 These mechanisms include chromosomal abnormalities, oxidative stress, altered DNA repair, altered DNA methylation patterns, altered growth factors, enhanced cell proliferation, promotion/progression, gene amplification, and suppression of p53.18-21 Thus, several mechanisms for carcinogenesis with ingested arsenic exposure have been suggested, but the percentage of causality of each pathway and the particulars on how and when these pathways operate to cause lung cancer are not well understood. + +Humans are the best model to study arsenic carcinogenesis. + +However, since it is not possible to study the mechanisms of carcinogenesis in humans (particularly with interior organs such as the lungs), animal models of carcinogenesis are frequently used. Unfortunately, with respect to exterior dose and organ susceptibility, rats and mice are not as sensitive as humans to arsenic-induced toxicity and carcinogenicity. In a recent study, whole-genome DNA methylation and gene expression changes were evaluated in lungs from female C57BL/6 mice exposed for 90 days to 50 ppm arsenate (As) +in drinking water.22 This study reported a subset of 17 genes showing differential methylation that included a substantial number that has been demonstrated to play a functional role in cancer-related processes.22 Of the available arsenic rodent models (Table 1), we chose a sensitive mouse model to study the mechanisms, that is, female C3H mouse, primarily because of positive carcinogenicity data in a gestational arsenic exposure from the Waalkes group.24,33 The C3H mouse strain is known to have a high spontaneous incidence of tumors.34 Arsenic can elicit many diverse effects in humans as well as in animal models. It is now well accepted that more than 1 causal mechanism may be involved in its carcinogenic potential (eg, oxidative stress, sulfhydryl group occupancy, DNA +methylation, etc). With greater use of appropriate animal models, particularly with various chronic or subchronic in vivo exposures using arsenic, a better understanding of the adverse effect can be achieved and an easier extrapolation to human exposures be made. A major question that remains to be answered is the level of risk of cancer with human arsenic exposure, particularly in the low-exposure range of 0 to approximately 200 ppb arsenic. In the United States, some past arsenic exposures might have reached 0.05 to 0.2 ppm in certain areas, but in the future, major regulated American water systems should be at 0.01 ppm or lower.2 The objective of this subchronic study was to characterize both the dose–response relationship for tissue arsenical accumulation in mouse lung and liver (a well-studied organ for arsenical speciation) and the gene expression patterns involved in arsenical-induced lung effects in female C3H mice. Mice were administered sodium arsenite via drinking water at 0 +(control), 0.05, 0.25, 1, 10, and 85 ppm as either elemental arsenic (lowest 2 concentrations) or as arsenite (top 3 concentrations; Table 2). A purified diet (AIN-93M) was used to reduce arsenicals present in the diet and thereby lower the mouse tissue arsenic background levels. As a result, the effects of low arsenite concentrations in drinking water could be seen. Mice were exposed to sodium arsenite in their drinking water for 30 days, and the lung samples from each treatment group were analyzed for gene expression and pathway involvement to identify transcription changes that may be linked to arsenic carcinogenesis. + +Table 2. Mouse Liver Concentrations of Arsenicals After 30-Day Exposure to a Wide Range of Drinking Water Arsenite Concentrations. + +| Arsenite Drinking Water | Arsenite Drinking Water Concentration, ppm as elemental arsenic | iAs, ng/ga | MMA, ng/ga | DMA, ng/ga | Total As, ng/ga | +|-----------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|---------------|---------------|--------------|-------------------| +| Concentration, ppm as arsenite 0 (controls) | 0 (controls) | Not detectedb | 5.54 + 1.47 | 11.5 + 4.6 | 17.0 + 5.8 | +| 0.087c | 0.05c | 17.1 + 3.0d | 1.33 + 1.19 | 2.3 + 1.1 | 20.7 + 2.0 | +| 0.434c | 0.25c | 19.8 + 0.8d | Not detectedb | 1.3 + 0.16 | 21.1 + 0.7 | +| 1 | 0.58 | 16.8 + 1.4d | 2.04 + 0.17 | 2.4 + 0.3 | 21.2 + 1.9 | +| 10 | 5.77 | 27.2 + 1.6d | 19.4 + 2.28d | 27.5 + 5.3 | 74.0 + 8.6d | +| 85 | 49.0 | 25.9 + 5.3d | 21.6 + 45.4d | 183. + 32.7d | 65.8 + 138d | +| Abbreviations: As, inorganic arsenic; EPA, United States Environmental Protection Agency; iAs, inorganic arsenic; MMA, monomethylarsonic acid; DMA, | | | | | | + +Abbreviations: As, inorganic arsenic; EPA, United States Environmental Protection Agency; iAs, inorganic arsenic; MMA, monomethylarsonic acid; DMA, dimethylarsinic acid; SEM, standard error of the mean; MCL, maximum contaminant level. + +aValues are the mean + SEM for 5 or 6 samples. The experimental animals were adult female C3H mice. Data are expressed as ng of the arsenical, for example, arsenate, MMA(V), and DMA(V), and not as elemental arsenic. The total As is the sum of arsenate, MMA(V), and DMA(V). To decrease their background tissue arsenical concentrations, mice were fed a purified diet (AIN 93M) for 14 days prior to the study and also during the 30 days of drinking water arsenite exposure. bThe limit of detection is about 0.2 ng/g tissue for all analytes. Arsenic drinking water concentrations are expressed as ppm sodium arsenite and as ppm elemental arsenic in 2 different columns. + +cThe lower concentrations of 0.087 and 0.434 (as arsenite) were used and not 0.05 and 0.25 ppm to make them match elemental arsenic in the lower groups, asthe EPA MCL is expressed as elemental arsenic. The higher groups at 1 to 85 are ppm of the whole compound and not as elemental arsenic. Adapted from Chilakapati et al.35 dStatistically significant versus control at P < .001 via a Kruskal-Wallis 1-way analysis of variance on ranks followed by individual t tests to determine P values. + +## Materials And Methods Chemicals + +Sodium arsenite (82.9%—arsenite and 17.1%—As) was purchased from Sigma-Aldrich (St Louis, Missouri). This arsenite–As ratio hydride generation data were obtained after the animal exposure was completed. All other chemicals used were the highest grade commercially available. + +## Animals + +Female C3H mice (52- to 55-day old) were obtained from Charles River Laboratories (Raleigh, North Carolina). The animals were housed in an AAALAC-approved animal facility at the EPA, Research Triangle Park, North Carolina, and maintained according to the National Institutes of Health ''Guideline on the Care and Use of Laboratory Animals.'' The animal room was kept on a 12-hour light/12-hour dark cycle at a temperature of 22C + 1-C and humidity of 50% + 10%. The animals were group housed 5 per cage in clear polycarbonate cages with Alpha Dri (Shepherd Specialty Papers, Inc., Portage, MI) used as bedding. For gene expression analysis, 4 mice per dose group were utilized. The animal experimentation protocol was approved by an Institutional Animal Care and Use Committee. + +High levels of dietary arsenic are found in standard laboratory chow.36 A diet with high and variable background levels of the toxicant under investigation is expected to skew or mask experimental results (both pharmacokinetic and pharmacodynamics). Laboratory diet can also considerably confound the analysis of gene expression studies. The As level of purified diets (AIN-93G and AIN-93M) with an ultra-trace mineral mix was analyzed for As and found to have 20 ppb.37 In our study, mice were acclimated on a purified diet, AIN-93M diet (Harlan-Tekland, Madison, Wisconsin), for 2 weeks prior to initiation of the arsenite study to allow for washout of residual arsenic in their system. Mice were provided with carbonpurified water at pH 3.3 to 3.8 as a vehicle for administration because mice consume more water at this pH range.38 Mice also consume less deionized or distilled water when compared to carbon-purified water at all pH ranges.38 + +## Experimental Design + +Body weights, food, and drinking water consumption were monitored weekly. The water bottles were changed 2 times a week. + +Female C3H mice were exposed to increasing concentrations of arsenite (0, 0.087, 0.434, 1, 10, and 85 ppm as arsenite or 0, 0.05, 0.25, 0.58, 5.77, and 49 expressed as elemental arsenic ppm) in their drinking water (pH 3.3-3.8) for 30 days. + +The EPA regulates drinking water arsenic as elemental arsenic. Therefore, the lower concentrations were selected to be 0.05 and 0.25 ppm as elemental arsenic (which is 0.087 and 0.434 ppm when expressed as arsenite). Thus, the 2 lowest concentrations come out even as elemental arsenic (0.05 and 0.25), while the 3 higher groups (1, 10, and 85) have even numbers when expressed as ppm of arsenite. In much of this article, these 5 water arsenic concentrations are simply referred to as 0.05, 0.25, 1, 10, and 85 ppm. + +On day 31, the animals were euthanized using carbon dioxide. Livers were flash frozen with liquid nitrogen and stored at 80C. The left lungs of 4 selected mice per dose group were instilled with formaldehyde for pathology. All other lungs were removed and flash frozen in liquid nitrogen for later use. Histological examination of the lungs was done by light microscopy of hematoxylin and eosin-stained slides in an unblind manner.39 A standard lesion scoring system was used for lung histology. + +## Lung And Liver Analysis + +The determination of arsenical species (inorganic arsenic, MMA, and DMA) in tissue samples was performed using a Perkin Elmer 5100 atomic absorption spectrophotometer +(Perkin-Elmer, Norwalk, Connecticut). Tissue samples +(0.02-0.1 g, N ¼ 5 or more per each treatment group) were digested using 2 mol/L phosphoric acid.40 This method generates pentavalent arsenicals from both tri- and pentavalent arsenicals in tissues. Thus, we were unable to distinguish whether the arsenicals in the tissue samples were either in the tri- or in the pentavalent oxidation state. + +## Gene Expression Analysis + +Frozen mouse lungs (4 mice/group) were homogenized in TRIzol. Total RNA was isolated from mouse lungs according to the TRIzol RNA extraction protocol (Invitrogen, Carlsbad, California) followed by RNeasy Mini Kit (RNeasy; Qiagen, Valencia, California). RNA quality was ascertained spectrophotometrically (ratio of A260-A280) using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware) +and also by checking ratio of 18S to 28S RNA using the RNA +Nano Chip on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California). Total RNA (3 mg) was labeled using the Affymetrix One-Cycle complementary DNA Synthesis protocol and hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips (containing 39 000 probesets; all Affymetrix products from Affymetrix, Inc., Santa Clara, California) as described by the manufacturer. Biotin-labeled complementary RNA (cRNA) +was then quantified using NanoDrop and evaluated for quality after fragmentation on a 2100 Bioanalyzer. Following overnight hybridization at 45-C to Affymetrix Mouse Genome 430A 2.0 GeneChips (for 0-85 ppm doses) in an Affymetrix Model 640 GeneChip hybridization oven, the arrays were washed and stained using an Affymetrix 450 fluidics station as recommended by the manufacturer and scanned on an Affymetrix Model 3000 scanner. After scanning, raw data (Affymetrix .cel files) were obtained using Affymetrix GeneChip Operating Software (version 1.4). Data (.cel files) were analyzed and statistically filtered using Rosetta Resolver version 6.0 software +(RosettaBiosoftware, Seattle, Washington). Differentially expressed genes (DEGs) were determined using a 1-way analysis of variance (P .05) by Rosetta Resolver (Microsoft, Inc. + +Redmond, WA), a Benjamini-Hochberg false discovery rate +(FDR) multiple testing correction (<.05), followed by a Scheffe's post hoc test and a +1.5-fold change cutoff. The DEGs were evaluated for canonical pathways and biological function using Ingenuity Pathway Analysis (Agilent Technologies, Santa Clara, California). A data set containing gene identifiers and their corresponding expression values of fold-changes was uploaded as a tab-delimited text file. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. A fold-change cutoff of 1.5-fold and P .05 was set to identify genes whose expression was differentially regulated. These genes were then used as the starting point for generating biological networks. + +## Results Liver Arsenic Accumulation + +As the mice were on a purified AIN-93M diet for over 6 weeks, the control animals accumulated very low arsenic tissue levels. + +In the liver arsenical level data, biological variation occurred in the exposure range of 0 to 1 ppm arsenite without any clear treatment effects. Mice start accumulating higher concentrations of total arsenic in liver at 10 and 85 ppm (4.3- and 38.7-fold, respectively) as shown in Table 2. Interestingly, at 0.05, 0.25, and 1 ppm concentrations, the MMA and DMA +concentrations found were considerably lower than the concentrations of iAs (inorganic arsenic) in the liver. + +## Lung Arsenic Accumulation + +The control animals accumulated almost undetectable arsenic levels in their lungs. With pulmonary iAs, DMA, and total As levels, there were clear elevations observed in the 0.05, 0.25, and 1.0 ppm treatment groups. Interestingly, there was no detectable MMA in the 0.25 ppm group, which also had the lowest number of differential genes expressed. Then, similar to the liver accumulation data, mice continued to accumulate more pulmonary arsenic at 10 and 85 ppm as shown in Table 3. + +However, the pulmonary inorganic arsenic accumulation started declining at 10 and 85 ppm when compared to 0.05 to 1 ppm treatment groups. Mouse pulmonary arsenic was elevated, that is, 16.3-, 21.8-, 76.1-, and 229-fold in the 0.05, 0.25, 1.0, and 85 ppm treatment groups, respectively. Although the 85 ppm group has an unusually low pulmonary iAs concentration of only 2 ng/g, the pulmonary concentrations of MMA (44.8 ng/g) and DMA (27.0 ng/g) were much higher. + +## Pathology + +By light microscopy of hematoxylin and eosin-stained sections, no significant changes in mean lesion scores occurred in the treated lung tissue (data not shown). The treated lungs resembled those of the control animals. + +## Dose Response In Number Of Genes + +There is no clear dose–response relationship in gene expression for arsenite in drinking water (Figure 1). Exposure to 0.05, 0.25, 1.0, 10, and 85 ppm resulted in 77, 38, 90, 87, and 87 DEGs, respectively. + +## Venn Analysis + +A +1.5-fold change cutoff was applied to the DEGs to conduct Venn analysis. Overall, there was not much overlap between adjacent doses. The top 3 exposures (85, 10, and 1 ppm) and bottom 3 exposures (1, 0.25, and 0.05 ppm) had 1 common gene, respectively (Figures 2 and 3). + +## Pathway Analysis + +To identify the unique and common responses to iAs by mouse lung, changes at the pathway and functional level were analyzed. Table 4 shows the results of an Ingenuity Pathway Analysis of the biological process level of the genes across all dose groups. + +Cellular functions such as adhesion, cell cycle control, growth and differentiation, proteolysis, and phosphorylation + +| Table 3. Mouse Lung Concentrations of Arsenicals After 30-Day Exposure to a Wide Range of Drinking Water Arsenite Concentrations.a Arsenite Drinking Water Concentration, ppm as arsenite iAs, ng/ga MMA, ng/ga DMA, ng/ga Total As, ng/ga 0 (controls) Not detectedb Not detectedb 1.28 + 0.43 1.38 + 0.5 0.087c 21.1 + 2.0d 0.72 + 0.64 0.67 + 0.22 22.5 + 2.4d 0.434c 26.2 + 3.2d Not detectedb 3.96 + 0.70e 30.1 + 3.3d 1 22.4 + 1.8d 1.25 + 0.52e 7.38 + 1.05d 50.9 + 6.9d 10 16.8 + 3.0d 7.95 + 0.95d 80.6 + 12.0d 105. + 14.5d 85 2.0 + 2.0 44.8 + 5.6d 270. + 41.7d 316. + 51.3d Abbreviations: As, inorganic arsenic; EPA, United States Environmental Protection Agency; MMA, monomethylarsonic acid; DMA, dimethylarsinic acid; SEM, standard error of the mean; MCL, maximum contaminant level. | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Abbreviations: As, inorganic arsenic; EPA, United States Environmental Protection Agency; MMA, monomethylarsonic acid; DMA, dimethylarsinic acid; SEM, + +![4_image_0.png](4_image_0.png) + +standard error of the mean; MCL, maximum contaminant level. + +aValues are the mean + SEM for 5 or 6 samples. The experimental animals were adult female C3H mice. Data are expressed as ng of the arsenical, for example, arsenate, MMA(V), DMA(V), and not as elemental arsenic. The total As is the sum of arsenate, MMA(V), and DMA(V). To decrease their background tissue arsenical concentrations, mice were fed a purified diet (AIN 93M) for 14 days prior to the study and also during the 30 days of drinking water arsenite exposure. bThe limit of detection is about 0.2 ng/g tissue for all analytes. Arsenic drinking water concentrations are expressed as ppm sodium arsenite and as ppm elemental arsenic in 2 different columns. + +cThe lower concentrations of 0.087 and 0.434 (as arsenite) were used and not 0.05 and 0.25 ppm to make them match elemental arsenic in the lower groups as the EPA +MCL is expressed as elemental arsenic. The higher groups at 1 to 85 are ppm of the whole compound and not as elemental arsenic. Adapted from Chilakapati et al.35 dStatistically significant versus control at P < .001 via a Kruskal-Wallis 1-way analysis of variance on ranks followed by individual t tests to determine P values. eStatistically significant versus control at P < .01. + +were modulated by drinking water arsenic exposure (Table 4). This study has shown genomic changes in the pathways of cell death (0.05 and 0.25 ppm), cell cycle (0.05 and 1 ppm), +and cell growth and proliferation (0.25 ppm). All 3 pathways could be causally connected to carcinogenesis both in this mouse lung system and in other species and target organs. + +These 3 pathways were always statistically different at all 5 exposure levels: cell death (P values of 6.9 E-07 to 4.9 E-02), cell cycle (2.4 E-05 to 4.9 E-02), and cellular growth and proliferation (8.0 E-04 to 4.9 E-02; Table 4). Differentially expressed genes were mapped using Ingenuity Pathway Analysis canonical pathways (data not presented). Most biological phenomena occur through interactions of multiple genes, via signaling pathways or other functional relationships. We could identify common pathways in the dose range used, though they were not found in each and every dose used. Interleukin 6 and acute-phase response signaling were the only 2 that showed up at 0.25 ppm dose. Ras homolog gene family, member A (RhoA) signaling, calcium signaling, actin cytoskeleton signaling, protein kinase A signaling, and acute-phase response signaling have been noted as major pathways that overlap somewhat among the different doses. + +Five signaling pathways that have been significantly impacted in at least 2 dose levels of arsenic exposure (Table 5 shows a partial list of genes exhibiting differential expression in these pathways) are now sequentially presented. + +## Ras Homolog Gene Family, Member A Signaling (0.05, 1, And 10 Ppm) + +Ras homolog gene family, member A is a critical signaling molecule regulating a variety of cellular processes, such as + +![5_image_0.png](5_image_0.png) + +cytoskeletal organization, adhesion, and apoptosis. It is recently considered responsive to reactive oxygen species (ROS).41 The RhoA is regulated by several proteins and particularly by RhoGDI proteins, which maintain RhoA in its inactive GDP-bound form.42 DNA microarrays have revealed that arsenite can downregulate messenger RNA levels of RhoGDI +proteins.43,44 Rho-activating effect of MMAIII has been reported recently.45 + +## Calcium Signaling (0.05 And 1 Ppm) + +Studies in cells show that the calcium stores and calcium receptors are a primary target of arsenic-induced deregulation, while the disturbance of [Ca2þ]i homeostasis is related to reduced cell viability, DNA damage, and apoptosis.46-48 But, to introduce toxic effects, arsenic compounds do not necessarily need to be taken up and biotransformed, since they could trigger death mechanisms by interacting with the cell membrane or simply by small amounts of arsenic passing through the membrane. Such instances could be sufficient to modify other physiological processes such as calcium homeostasis.48-50 + +## Actin Cytoskeleton Signaling (0.05, 1, And 10 Ppm) + +Arsenite-caused mitochondrial damage has been shown to play a crucial role in the accumulation of ROS leading to arsenic mutagenicity in vitro.51 This increase in mitochondria-dependent ROS production by arsenite can be partly attributed to its interference in the dynamics of the actin cytoskeleton.52,53 Actin carbonylation induced by H2O2 results in an increase in the lag phase and a decrease in both the polymerization rate and the polymerization extent.54 Protein carbonylation may actually interfere with the actin bundling activity and disturb the integrity of the actin cytoskeleton under arsenite exposure.55 + +## Protein Kinase A Signaling (0.05 And 1 Ppm) + +Protein kinase A (PKA) regulates processes as diverse as growth, development, memory, and metabolism. The PKA represents a major regulator in cyclic adenosine monophosphate +(cAMP) signal transduction pathway. The PKA is a multimeric protein consisting of 2 catalytic subunits complexed with a regulatory subunit dimer.56 One study demonstrated the existence of synergism between arsenic and cAMP that triggers maturation pathway for acute promyelocytic leukemia (APL) + +| Table 4. Ingenuity Analysis of Biological Processes in Significant Differentially Expressed Genes Across All Doses. Name 0.05 ppm 0.25 ppm 1 ppm 10 ppm | 85 ppm | | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|----------------------|----------------------|----------------------|----------------------| +| Cell death | 169 | 21 | 96 | 11 | 11 | +| P value range | 6.87E-07 to 2.80E-02 | 2.31E-03 to 4.81E-02 | 3.24E-03 to 2.83E-02 | 1.41E-02 to 2.89E-02 | 4.4E-03 to 4.85E-02 | +| Gene expression | 140 | 15 | 89 | 16 | 30 | +| P value range | 3.73E-06 to 2.55E-02 | 5.89E-03 to 4.33E-02 | 5.28E-04 to 2.83E-02 | 1.76E-04 to 2.89E-02 | 7.2E-03 to 4.85E-02 | +| Cell morphology | 90 | 21 | 64 | 34 | 34 | +| P value range | 6.38E-06 to 3.01E-02 | 2.92E-03 to 4.32E-02 | 2.16E-04 to 2.83E-02 | 7.61E-04 to 4.85E-02 | 7.61E-04 to 4.85E-02 | +| Cellular development | 118 | 15 | 63 | 12 | 34 | +| P value range | 6.38E-06 to 3.01E-02 | 6.14E-03 to 4.53E-02 | 3.69E-04 to 2.83E-02 | 6.88E-03 to 2.89E-02 | 7.61E-04 to 4.85E-02 | +| Cell cycle | 82 | 15 | 33 | 12 | 13 | +| P value range | 2.42E-05 to 2.92E-02 | 1.07E-02 to 4.66E-02 | 1.48E-04 to 2.83E-02 | 7.33E-03 to 2.89E-02 | 1E-02 to 4.85E-02 | +| Protein synthesis | 22 | 6 | 7 | 8 | - | +| P value range | 2.51E-02 to 2.51E-02 | 2.82E-03 to 4.22E-02 | 1.84E-04 to 1.84E-04 | 5.58E-03 to 1.46E-02 | - | +| Carbohydrate | 12 | 12 | 8 | 17 | 15 | +| metabolism | | | | | | +| P value range | 3.94E-03 to 2.54E-02 | 3.06E-03 to 4.85E-02 | 4.62E-03 to 2.83E-02 | 2.4E-04 to 2.49E-02 | 7.61E-04 to 2.76E-02 | +| Cellular growth and | 154 | 24 | 15 | 7 | 7 | +| proliferation | | | | | | +| P value range | 1.50E-03 to 2.90-02 | 3.06E-03 to 4.22E-02 | 7.99E-04 to 2.83E-02 | 1.46E-02 to 2.89E-02 | 2.24E-03 to 4.85E-02 | +| Cellular function and | 40 | 12 | 31 | 13 | 21 | +| maintenance | | | | | | +| P value range | 2.98E-03 to 2.54E-02 | 9.54E-03 to 4.97E-02 | 2.87E-05 to 2.83E-02 | 4.23E-03 to 2.89E-02 | 2.82E-03 to 3.55E-02 | +| Cellular assembly and | 90 | 18 | 73 | 21 | 42 | +| organization | | | | | | +| P value range | 4.46E-05 to 2.64E-02 | 9.54E-03 to 4.99E-02 | 1.29E-04 to 2.83E-02 | 2.11E-03 to 2.89E-02 | 2.24E-03 to 4.85E-02 | +| Cellular compromise | 23 | 5 | 24 | 5 | 10 | +| P value range | 2.82E-03 to 2.54E-02 | 1.07E-02 to 4.22E-02 | 1.84E-04 to 2.83E-02 | 7.1E-03 to 2.89E-02 | 2.76E-02 to 3.07E-02 | +| Posttranslational | - | - | 7 | 4 | 8 | +| modification | | | | | | +| P value range | - | - | 1.84E-04 to 1.84E-04 | 1.46E-02 to 2.21E-02 | 6.94E-03 to 3.75E-02 | +| Lipid metabolism | 44 | 11 | 14 | 24 | 25 | +| P value range | 2.26E-04 to 2.54E-02 | 4.78E-03 to 4.22E-02 | 6.49E-03 to 2.83E-02 | 2.92E-06 to 2.89E-02 | 8.01E-04 to 4.86E-02 | +| Small molecule | 57 | 18 | 23 | 30 | 38 | +| biochemistry | | | | | | +| P value range | 2.26E-04 to 2.54E-02 | 4.78E-03 to 4.85E-02 | 4.62E-03 to 2.83E-02 | 2.92E-06 to 2.89E-02 | 7.61E-04 to 4.86E-02 | +| Molecular transport | 22 | 11 | 18 | 21 | 23 | +| P value range | 1.91E-03 to 2.54E-02 | 4.78E-03 to 4.22E-02 | 4.62E-03 to 2.83E-02 | 2.11E-05 to 2.77E-02 | 4.4E-03 to 4.51E-02 | +| Cellular movement | 81 | 11 | 17 | 29 | 34 | +| P value range | 6.58E-04 to 2.54E-02 | 7.02E-03 to 4.85E-02 | 1.25E-03 to 2.83E-02 | 1.42E-04 to 2.46E-02 | 1.95E-03 to 4.85E-02 | +| RNA Posttranscriptional modification | 14 | - | 1 | 5 | 14 | +| P value range | 5.25E-04 to 1.05E-02 | - | 2.83E-02 to 2.83E-02 | 1.78E-03 to 2.47E-02 | 4.23E-05 to 3.55E-02 | + +cells. Although neither arsenic nor cAMP is sufficient alone in promoting terminal differentiation, these 2 signals cooperated to induce the maturation of APL cells.57 + +## Acute-Phase Response Signaling (0.25 And 10 Ppm) + +The acute-phase response is a rapid inflammatory response that provides protection against microorganisms using nonspecific defense mechanisms. In addition to infection, it can also be triggered by tissue injury, trauma or surgery, neoplastic growth, or immunological disorders. Typically, it consists of fever, an increase in inflammatory factors (such as proinflammatory cytokines), and a change in concentration of several plasma proteins (the acute-phase proteins) due largely to an altered hepatic metabolism. Arsenic exposure is known to increase the production of ROS.58-60 Alterations in intracellular oxidation– +reduction (redox) reactions have been shown to activate signal cascades that regulate early response genes. This can also cause the activation of transcription factors (eg AP-1 and nuclear factor kB) and produce oversecretion of proinflammatory and growth-promoting cytokines,17,61 resulting in increased cell proliferation and finally carcinogenesis.62,63 + +## Discussion + +A lot of past research effort has been directed toward the role of arsenic on carcinogenesis in either skin or urinary bladder with little emphasis on lung as the target organ. This article is + +| Table 5. Sodium Arsenite Dose–Response Altered Gene Expression in 5 Common Significantly Impacted Pathways. Pathway/Gene Gene Symbol Gene ID 0.05 ppm 0.25 ppm 1 ppm | 10 ppm | 85 ppm | | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------|----------|----|----| +| RhoA signaling | ACTA1 | 11459 | 1.61 | | +| Calcium signaling | CREB5 | 231991 | 1.60 | | +| MYH8 | 17885 | 2.32 | | | +| Actin cytoskeleton signaling | ACTA1 | 11459 | 2.07 | 1.61 | +| MYH8 | 17885 | 2.32 | | | +| PXN | 19303 | | | | +| Protein kinase A signaling | CREB5 | 231991 | 1.60 | | +| GRK1 | 24013 | 3.23 | | | +| PDE3A | 54611 | 1.53 | | | +| PXN | 19303 | 2.07 | | | +| Acute-phase response signaling | ALB | 11657 | 6.29 | | +| APOA1 | 11806 | 3.50 | | | +| IKBKE | 56489 | 2.22 | | | +| SERPINA1 | 20703 | 14.1 | | | +| TTR | 22139 | 25.3 | | | +| a Genes that participate in multiple pathways may not be represented under all pathways. The numbers in the dose columns are fold-change values. | | | | | + +directed toward the effects of drinking water arsenic exposure on mouse lung incorporation of arsenicals and genomics. In general, mice are relatively resistant to arsenic toxicity due to their excellent metabolism and/or excretion of arsenic. However, it would be interesting to try longer periods of exposure +(Table 1) and then study changes at the genomic level. + +This study is unique in terms of the wide dose range studied +(as low as 0.087 to as high as 85 ppb, a 977-fold range in exposure). These exterior drinking water arsenite concentrations were found to drive a 39-fold elevation in mouse hepatic total arsenic concentration (Table 2), while in the lung an even higher 229-fold elevation in pulmonary total arsenic (Table 3) was observed. These doses reflect low to high drinking water levels in humans as well as doses required for animal carcinogenesis studies.24 In this study, we found that female C3H mice do not show any evidence of arsenic-induced major clinical– biological changes at the doses and time of exposure used in this experiment but do show changes at the genomic level. We also did not find any histological evidence of toxicity in the lungs by light microscopy, although we would expect to find histological changes if longer exposure time points were used. + +For example in 1 time course study,64 mice showed major redox changes in liver at the 6- to 15-month time points, but few changes were observed at only 3 months after the start of arsenic exposure. Lungs are exposed to the highest oxygen tensions in the body; thus, ROS could preferentially form in pulmonary tissues. + +This study is one of the few studies that has used a purified low arsenic diet. Thus, in control mice, we did not detect iAs in liver or lung, and very low total As was found in mouse lungs. + +Using a purified diet, increases in mouse tissue arsenic levels could be detected with arsenite exposures as low as 0.05 ppm in the case of iAs in both mouse liver and lung. At the intermediate exposure concentrations of 0.25 and 1 ppm, hepatic iAs was elevated along with iAs, DMA, and total As in mouse lungs. At 10 and 85 ppm arsenite exposures, almost all tissue arsenicals were elevated often to a fairly large degree in the 5- to 225-fold range (Tables 2 and 3). This combined pharmacokinetic–pharmacodynamic data set can be useful in better linking exterior drinking water exposure concentrations, interior speciated arsenical tissue concentration, and subsequent adverse biological effects. + +This 5 exposure-level dose–response study has resulted in pulmonary and hepatic iAs, MMA, and DMA data over the unusually broad range from 0.05 to 85 ppm. These data sets could be of utility to pharmacokinetic modelers interested in +(1) low exposure concentrations applicable to current human exposures and risk assessments and (b) extrapolation from animal to human exposures. + +A similar study was previously conducted in K6/ODC mice administered 0, 0.05, 0.25, 1.0, and 10 ppm sodium arsenite in their drinking water for 4 weeks65 to identify potential biomarkers and modes of action for skin tumorigenicity. K6/ODC +transgenic mice are hairless mice, especially useful to detect the skin carcinogenicity of mutagenic chemicals administered at low doses within months of exposure.28 With respect to the mouse skin study, (1) low exposures to arsenite caused an unexpectedly large number of DEGs (437-511) in the 3 lowest exposure groups and (2) the highest exposure group had the most DEGs (1227), fitting simple dose–response theories.65 With respect to the present lung study from animals eating a purified AIN-93M diet, (1) the lowest exposure group responded very well (77 DEGs), (2) surprisingly, 0.25 resulted in lower number of DEGs when compared to 0.05 ppm, and (3) +at higher arsenic exposures of 10 and 85 ppm which greatly increased total, MMA, and DMA lung arsenical concentrations (Table 3), there was not a dramatic increase in DEGs found (87 DEGS at both dose levels). This lack of responsiveness to tissue total arsenicals of 76 and 229 times control levels is quite unexpected and hard to explain. If the pulmonary responses were largely driven by the arsenite concentration alone, the observed pattern makes more sense. In pulmonary iAs concentration, there is a big jump from not detected to 21 ng/g iAs when compared to the control and 0.05 ppm groups. Increasing the exposure level to 10 ppm did not increase lung iAs concentrations. At 85 ppm arsenite exposure, the pulmonary iAs was actually decreased to 2 ng/g only about 10% of what it was at lower exposures. Venn analysis of the top and bottom 3 doses showed that the effects are fairly different across this wide dose range and that arsenic can be acting differently in different regions of the exposure range, more dependent on the dose than similar based on the same exposure compound. In both of these studies (and the current study),65 arsenic-induced genomic effects often appear only over certain dose windows and are not simple monotonic (always increasing or decreasing) biological effects. The large dose range used and the large amount of spacing between the drinking water doses in our studies have produced a genomic data set with many nonmonotonic effects, just as in a prior arsenic study in K6/ODC mice.65 In comparing these 2 similar dose–response studies with arsenite, one is struck with several overall facts: (1) lower arsenite concentrations are responding much more than anticipated or easily explained, (2) simple monotonic dose–response models are not well matching the experimental results, and (3) +the lowest arsenite exposures of 50 ppb (the former American maximum contaminant level [MCL]) showed both observable effects and a higher number of DEGs than did at least 1 higher exposure concentrations. From this, we conclude that genomic dose–response studies of arsenicals in vivo after longer exposure times are still in their infancy and we are not close to understanding the types of genomics results that are being obtained. If arsenic operates via multiple mechanisms in multiple dose windows, then one would expect a genomics pattern as complex as the one actually observed. Nevertheless, such low-level subchronic exposures are the key to understanding important chronic diseases such as arsenic-induced carcinogenesis and cardiovascular diseases. + +In the attempt to link mouse lung arsenical concentrations +(Table 3) with genomic effects (Tables 4 and 5), one can see where the larger mouse lung arsenicals increases are (e. g. + +0.087 ppm for iAs, 10 ppm for DMA) as the exterior arsenite dose is increased. Based on Table 3 data, from control to 0.087 ppm arsenite, the lung iAs appears to be the likely driver of genomic effects. Increasing to 0.434 ppm arsenite, the large increase from 0.67 to 3.96 ng/g lung DMA is notable. At 1, 10, and 85 ppm arsenite, both mouse lung MMA and DMA concentrations rose dramatically, without major increases in lung iAs concentration (Table 3). Thus, depending where you are on the dose–response curve, the more likely genomic drivers are iAs (0.087 ppm), DMA (0.434 ppm), or MMA and DMA together (1, 10, and 85 ppm). + +Genes identified via microarray in screening of tissues exposed to arsenic are a valuable tool in determining the mechanism of arsenic toxicity and cancer formation. Gene expression changes can act as sensitive indicators of molecular processes at doses where standard toxicological end points are not responsive and these genomic changes could provide insights into the precursor events that might eventually lead to serious disease outcomes.66 Moreover, genomic tools have the potential to uncover adverse events at treatment levels below the limits of detection of traditional measures of toxicity, at much earlier times, and provide a wealth of mechanistic information.66 The list of candidate genes and processes identified in this study will need further investigation to confirm their role in events that occur at low exposures to arsenic and that could potentially lead to carcinogenicity following chronic exposure of iAs to mice. + +In summary, this study demonstrates that exposure to drinking water arsenic at levels that have been found in the past in US drinking water (0.05 ppm) is associated with altered genomic expression profiles in the C3H mouse lung. A definitive understanding of the various mechanisms of arsenic action in experimental animal models will both provide useful data and assist in model selection and development in arsenic risk assessment. With further validation, some of these genes may be useful as intermediate biomarkers in studies investigating arsenic-associated chronic diseases. Future investigation of the effects of arsenic on the function of these pathways is warranted. These data identify pathways that will help guide investigations into mechanisms of arsenic's health effects and help clarify the threshold for biologic effects and potential disease risk. These data not only facilitate understanding the mechanism of lung carcinogenicity of iAs but also provide useful information for establishing an appropriate animal model for pulmonary arsenic risk assessment. + +## Acknowledgments + +We thank Gail Nelson and Janice S. Lee for reviewing this manuscript as part of EPA clearance procedures. + +## Author'S Note + +Although the research described in this article has been supported by the US Environmental Protection Agency through an EPA postdoctoral trainee award to Jaya Chilakapati, it has not been subjected to official Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. This article has been approved for publication by the US EPA +NHEERL clearance procedure. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. This manuscript does not necessarily reflect the views of US EPA. + +## Declaration Of Conflicting Interests + +The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. + +## Funding + +The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the United States Protection Agency. This work was supported by UNC/EPA Cooperative Agreement EPA CR 833237. + +## References + +1. Smith AH, Ercumen A, Yuan Y, Steinmaus CM. Increased lung cancer risks are similar whether arsenic is ingested or inhaled. + +J Expo Sci Environ Epidemiol. 2009;19(4):343-348. + +2. US Environmental Protection Agency, 2008. Web site. http:// +water.epa.gov/lawsregs/rulesregs/sdwa/arsenic/index.cfm. +Accessed June 1, 2013. Updated April 27, 2015. Published September 17, 2013. + +3. International Agency for Research on Cancer, 2004. Some Drinking Water Disinfectants and Contaminants, Including Arsenic, Vol 84. Lyon, France: IARC Press; 97-156. + +4. Guo HR, Chiang HS, Hu H, Lipsitz SR, Monson RR. Arsenic in drinking water and incidence of urinary cancers. Epidemiology. + +1997;8(5):545-550. + +5. Wu MM, Kuo TL, Hwang YH, Chen CJ. Dose response relation between arsenic concentration in well water and mortality from cancers and vascular diseases. Am J Epidemiol. 1989;130(6): +1123-1132. + +6. Smith AH, Goycolea M, Haque R, Biggs ML. Marked increase in bladder and lung cancer mortality in a region of Northern Chile due to arsenic in drinking water. Am J Epidemiol. 1998;147(7): +660-669. + +7. Hopenhayn-Rich C, Biggs ML, Smith AH. Lung and kidney cancer mortality associated with arsenic in drinking water in Cordoba, Argentina. Int J Epidemiol. 1998;27(4):561-569. + +8. Sancha AM, O'Ryan R. Managing hazardous pollutants in Chile: +arsenic. Rev Environ Contam Toxicol. 2008;196:123-146. + +9. Smith AH, Marshall G, Yuan Y, et al. Increased mortality from lung cancer and bronchiectasis in young adults after exposure to arsenic in utero and in early childhood. Environ Health Perspect. + +2006;114(8):1293-1296. + +10. Aposhian HV. Enzymatic methylation of arsenic species and other new approaches to arsenic toxicity. Annu Rev Pharmacol Toxicol. 1997;37:397-419. + +11. Chiou HY, Hsueh YM, Liaw KF, et al. Incidence of internal cancers and ingested inorganic arsenic: a seven-year follow-up study in Taiwan. Cancer Res. 1995;55(6):1296-1300. + +12. Goering PL, Aposhian HV, Mass MJ, Cebrian M, Beck BD, +Waalkes MP. The enigma of arsenic carcinogenesis: role of metabolism. Toxicol Sci. 1999;49(1):5-14. + +13. Ahmad S, Kitchin KT, Cullen WR. Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin. Toxicol Lett. 2002;133(1):47-57. + +14. Kligerman AD, Doerr CL, Tennant AH, et al. Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: +Induction of chromosomal mutations but not gene mutations. + +Environ Mol Mutagen. 2003;42(3):192-205. + +15. Andrewes P, Kitchin KT, Wallace K. Dimethylarsine and trimethylarsine are potent genotoxins in vitro. Chem Res Toxicol. + +2003;16(8):994-1003. + +16. Styblo M, Del Razo LM, Vega L, et al. Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Arch Toxicol. 2000;74(6):289-299. + +17. Vega L, Styblo M, Patterson R, Cullen W, Wang C, Germolec D. + +Differential effects of trivalent and pentavalent arsenicals on cell proliferation and cytokine secretion in normal human epidermal keratinocytes. Toxicol Appl Pharmacol. 2001;172(3):225-232. + +18. Kitchin KT. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol Appl Pharmacol. 2001;172(3):249-261. + +19. Kitchin KT, Conolly R. Arsenic induced carcinogenesis - oxidative stress as a possible mode of action and future research needs for more biologically based risk assessment. Chem Res Toxicol. 2010;23(2):327-335. + +20. Kitchin KT, Wallace K. The role of protein binding of trivalent arsenicals in arsenic carcinogenesis and toxicity. J Inorg Biochem. 2008;102(3):532-539. + +21. Tapio S, Grosche B. Arsenic in the aetiology of cancer. Mutat Res. 2006;612(3):215-246. + +22. Boellmann F, Zhang L, Clewell HJ, et al. Genome-wide analysis of DNA methylation and gene expression changes in the mouse lung following subchronic arsenate exposure. Toxicol Sci. 2010; 117(2):404-417. + +23. Hayashi H, Kanisawa M, Yamanaka K, et al. Dimethylarsenic acid, a main metabolite of inorganic arsenics, has tumorigenicity and progression effects in the pulmonary tumors of A/J mice. Cancer Lett. 1998;125(1-2):83-88. + +24. Waalkes MP, Liu J, Ward JM, Diwan BA. Animal models for arsenic carcinogenesis: inorganic arsenic is a transplacental carcinogen in mice. Toxicol Appl Pharmacol. 2004;198(3):377-384. + +25. Waalkes MP, Liu J, Ward JM, Diwan BA. Enhanced urinary bladder and liver carcinogenesis in male CD1 mice exposed to transplacental inorganic arsenic and postnatal diethylstilbestrol or tamoxifen. Toxicol Appl Pharmacol. 2006;215(3):295-305. + +26. Kinoshita A, Wanibuchi H, Morimura K, et al. Carcinogenicity of dimethylarsinic acid in Ogg1-deficient mice. Cancer Sci. 2007; 98(6):803-814. + +27. Yamanaka K, Ohtsubo K, Hasegawa A, et al. Exposure to dimethylarsinic acid, a main metabolite of inorganic arsenics, strongly promotes tumorigenesis initiated by 4-nitroquinoline 1-oxide in the lungs of mice. Carcinogenesis. 1996;17(4):767-770. + +28. Chen Y, Megosh LC, Gilmour SK, Sawicki JA, O'Brien TG. K6/ +ODC transgenic mice as a sensitive model for carcinogen identification. Toxicol Lett. 2000;116(1-2):27-35. + +29. Chen Y, O'Brien TG, Del Razo LM, Thomas DJ, Kitchin KT. + +Tissue levels of arsenicals and skin tumor response following administration of monomethylarsonous acid and arsenite to K6/ +ODC mice. J Environ Pathol Toxicol Oncol. 2008;27(1):43-52. + +30. Germolec DR, Spalding J, Yu HS, et al. Arsenic enhancement of skin neoplasia by chronic stimulation of growth factors. Am J +Pathol. 1998;153(6):1775-1785. + +31. Yamanaka K, Katsumata K, Ikuma A, Hasegawa A, Nakano M, +Okada S. The role of orally administered dimethylarsinic acid, a main metabolite of inorganic arsenics, in the promotion and progression of UVB-induced skin tumorigenesis in hairless mice. + +Cancer Lett. 2000;152(1):79-85. + +32. Salim EI, Wanibuchi H, Morimura K, et al. Carcinogenicity of dimethylarsinic acid in p53 heterozygous knockout and wild-type C57BL/6 J mice. Carcinogenesis. 2003;24(2):335-342. + +33. Waalkes MP, Liu J, Ward JM, and Diwan BA. Enhanced urinary bladder and liver carcinogenesis in male CD1 mice exposed to transplacental inorganic arsenic and postnatal diethylstilbestrol or tamoxifen. Toxicol Appl Pharmacol. 2006;215(3):295-305. + +34. Dragani TA, Manenti G, Pierotti MA. Genetics of murine lung tumors. Adv Cancer Res. 1995;67:83-112. + +35. Chilakapati J, Wallace K, Thomas DJ, et al. Effects of arsenite in drinking water on mouse lung in a 30 day exposure (Abstract number 1587). Paper presented at: Society of Toxicology Meeting; March 2009. + +36. Kozul CD, Nomikos AP, Hampton TH, et al. Laboratory diet profoundly alters gene expression and confounds genomic analysis in mouse liver and lung. Chem Biol Interact. 2008;173(2): +129-140. + +37. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN76A rodent diet. J Nutr. 1993;123(11):1939-1951. + +38. Ahlborn GJ, Nelson GM, Grindstaff RD, et al. Impact of life stage and duration of exposure on arsenic-induced proliferative lesions and neoplasia in C3 H mice. Toxicology. 2009;262(2): +106-113. + +39. Crissman JW, Goodman DG, Hildebrandt PK, et al. Best practices guideline: toxicologic histopathology. Toxicol Pathol. 2004; 32(1):126-131. + +40. Hughes MF, Devesa V, Adair BM, Styblo M, Kenyon EM, +Thomas DJ. Tissue dosimetry, metabolism and excretion of pentavalent and trivalent monomethylated arsenic in mice after oral administration. Toxicol Appl Pharmacol. 2005;208(2): +186-197. + +41. Cai J, Niu X, Chen Y, et al. Emodin-induced generation of reactive oxygen species inhibits RhoA activation to sensitize gastric carcinoma cells to anoikis. Neoplasia. 2008;10(1):41-51. + +42. Olofsson B. Rho guanine dissociation inhibitors: pivotal molecules in cellular signaling. Cell Signal. 1999;11(8):545-554. + +43. Andrew AS, Warren AJ, Barchowsky A, et al. Genomic and proteomic profiling of responses to toxic metals in human lung cells. Environ Health Perspect. 2003;111(6):825-835. + +44. Rea MA, Gregg JP, Qin Q, Phillips MA, Rice RH. Global alteration of gene expression in human keratinocytes by inorganic arsenic. Carcinogenesis. 2003;24(4):747-756. + +45. Lim KM, Shin YS, Kang S, et al. Potentiation of vasoconstriction and pressor response by low concentration of monomethylarsonous acid (MMA(III)). Toxicol Lett. 2011;205(3):250-256. + +46. Florea AM, Bu¨sselberg D. Arsenic trioxide in environmentally and clinically relevant concentrations interacts with calcium homeostasis and induces cell type specific cell death in tumor and non-tumor cells. Toxicol Lett. 2008;179(1):34-42. + +47. Florea AM, Splettstoesser F, Bu¨sselberg D. Arsenic trioxide +(As2O3) induced calcium signals and cytotoxicity in two human cell lines: SY-5Y neuroblastoma and 293 embryonic kidney +(HEK). Toxicol Appl Pharmacol. 2007;220(3):292-301. + +48. Florea AM, Bu¨sselberg D. Anti-cancer drugs interfere with intracellular calcium signaling. Neurotoxicology. 2009;30(5): +803-810. + +49. Florea AM, Yamoah EN, Dopp E. Intracellular calcium disturbances induced by arsenic and its methylated derivatives in relation to genomic damage and apoptosis induction. Environ Health Perspect. 2005;113(6):659-664. + +50. Florea AM, Bu¨sselberg D. Occurrence, use and potential toxic effects of metals and metal compounds. Biometals. 2006;19(4): +419-427. + +51. Liu SX, Davidson MM, Tang X, et al. Mitochondrial damage mediates genotoxicity of arsenic in mammalian cells. Cancer Res. 2005;65(8):3236-3242. + +52. Gourlay CW, Carpp LN, Timpson P, Winder SJ, Ayscough KR. A +role for the actin cytoskeleton in cell death and aging in yeast. + +J Cell Biol. 2004;164(6):803-809. + +53. Breitenbach M, Laun P, Gimona M. The actin cytoskeleton. RAScAMP signaling and mitochondrial ROS in yeast apoptosis. + +Trends Cell Biol. 2005;15(12):637-639. + +54. Dalle-Donne I, Milzani A, Colombo R. H2O2-treated actin: +assembly and polymer interactions with cross-linking proteins. Biophys J. 1995;69(6):2710-2719. + +55. Lii CK, Lin AH, Lee SL, Chen HW, Wang TS. Oxidative modifications of proteins by sodium arsenite in human umbilical vein endothelial cells. Environ Toxicol. 2011;26(5):459-471. + +56. Skalhegg BS, Tasken K. Specificity in the cAMP/PKA signaling pathway: differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci. 2000;5:D678-D693. + +57. Zhu Q, Zhang JW, Zhu HQ, et al. Synergic effects of arsenic trioxide and cAMP during acute promyelocytic leukemia cell maturation subtends a novel signaling cross-talk. Blood. 2002; 99:1014-1022. + +58. Barchowsky A, Klei LR, Dudek EJ, Swartz HM, James PE. Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite. Free Radic Biol Med. 1999;27(11-12):1405-1412. + +59. Barchowsky A, Roussel RR, Klei LR, et al. Low levels of arsenic trioxide stimulate proliferative signals in primary vascular cells without activating stress effector pathways Toxicol Appl Pharmacol. 1999;159(1):65-75. + +60. Liu SX, Athar M, Lippai I, Waldren C, Hei TK. Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity. Proc Natl Acad Sci U S A. 2001;98(4):1643-1648. + +61. Germolec DR, Yoshida T, Gaido K, et al. Arsenic induces overexpression of growth factors in human keratinocytes. Toxicol Appl Pharmacol. 1996;141(1):308-318. + +62. Simeonova PP, Luster MI. Mechanisms of arsenic carcinogenicity: genetic or epigenetic mechanisms. J Environ Pathol Toxicol Oncol. 2000;19(3):281-286. + +63. Simeonova PP, Wang S, Toriuma W, et al. Arsenic mediates cell proliferation and gene expression in the bladder epithelium: association with activating protein-1 transactivation. Cancer Res. + +2000;60(13):3445-3453. + +64. Santra A, Maiti A, Das S, Lahiri S, Charkaborty SK, Mazumder DN. Hepatic damage caused by chronic arsenic toxicity in experimental animals. J Toxicol Clin Toxicol. 2000;38(4):395-405. + +65. Ahlborn GJ, Nelson GM, Ward WO, et al. Dose response evaluation of gene expression profiles in the skin of K6/ODC mice exposed to sodium arsenite. Toxicol Appl Pharmacol. 2008; 227(3):400-416. + +66. Irwin RD, Boorman GA, Cunningham ML, Heinloth AN, Malarkey DE, Paules RS. Application of toxicogenomics to toxicology: +basic concepts in the analysis of microarray data. Toxicol Pathol. + +2004;32(suppl 1):72-83. \ No newline at end of file diff --git a/medical/md_images/PMC4674186/PMC4674186_meta.json b/medical/md_images/PMC4674186/PMC4674186_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..faefef68942f6988415f12973b4e25e5af6923c4 --- /dev/null +++ b/medical/md_images/PMC4674186/PMC4674186_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 11, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 0, + "code": 0, + "table": 5, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC4934132/0_image_2.png b/medical/md_images/PMC4934132/0_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..91d1b6f91191f3a57e98371227892aea4212a8e0 --- /dev/null +++ b/medical/md_images/PMC4934132/0_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:528a2f812ebb689d5f4f8c005913e189284c345b3a8675b9718cdcc74aa48576 +size 14163 diff --git a/medical/md_images/PMC5159658/2_image_0.png b/medical/md_images/PMC5159658/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..6a2a6e360a427c54c9180c9f64410ffb7206c07f --- /dev/null +++ b/medical/md_images/PMC5159658/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e43fce2d7785cceef49e0b1d41985b3a33239628c08531df0b1054bc38a5f21e +size 26334 diff --git a/medical/md_images/PMC5274876/PMC5274876.md b/medical/md_images/PMC5274876/PMC5274876.md new file mode 100644 index 0000000000000000000000000000000000000000..5ab99ecc39a10ab479738676a36bf86449bae3c9 --- /dev/null +++ b/medical/md_images/PMC5274876/PMC5274876.md @@ -0,0 +1,630 @@ + +## Abdominal And Pelvic Surgery. + +^utiieiiford Morison, F.E.C.S., Surgeon to the Roycd Infirmary, and Consulting Surgeon to the Dental Hospital, Newcastlc-on-Tyne. + +operations The term " abdominal surgery is, tty^^terior abdominal wa^ which open the peritoneum throug i operations Pecu, t to and the term " pelvic surgery 0 ^mited applicatlon\ anC\ +women. I intend to make no such contents in males include all the abdominal and pel remarks. . + +females, and the walls of these cavi 1 > hospital prac i I also intend to give the statistics of my^ ^ by ^yseU 1903. These will include every case p ^ Infirmary an during nine months of work, both n ^ limited, as a private hospital here ; and the one who has possible, to experiences based upon g struck with t > +looked over Hospital Reports can a ^ forth in ^?in' 0st namely, that the results of opera 10' ? g series of ?-ficant a very different showing to the s< rtned with an insig difficult and dangerous operations, pei medical literatui ? + +mortality, which are published in ie5Ults have been is apt to be forgotten that these remarkably They have their published because they are excep ion ^ operators, w 10 ^ +uses; they show what can be done by ihiMP^ a st]rouluS to a careful selection of cases, ant and danger improved results. It is, however, ?Ses, and to buiy the iudge of general results by these while the latter occ P +former in oblivion, as is apt to be c on , _ +foremost position. . ?orV anc\ their patents, ^ +, Medical men, not practising surg^y- d They may ha be misled by statistics of an excePtl0^taken with imp"nlty'likely learn that operations cannot be un ^ that the u recovery is not always "uneventful , +an q{ hf It i{ +sometimes happens in surgery as 1to the know g ^ +fetter that such facts should not be br o sonal expei be profession, and of the public, by bitten P ^ and then for this is likely to produce a bad imp labilities than P +work. We require rather to indicate pioDa ^ +Chieflyfor this reason, I think ifc +^sults of the whole of ^^riodTentioned, my work for ais0 of its failure complete record, not only of its ?ucc ,' mUCh. _ "Royal and its disappointments, which ?fte^ ueen done in t ^ +T The major part of the work has been building Infirmary, a busy general hospital, anc vhich would more com ^ +a century old, and without a single featuie it to the modern operating surgeon. Indeed, the surgical arrange' ments in the operating theatre, and in the wards, are inferior to those of the old Eoyal Infirmary of Edinburgh, as it was when 1 held the post of a house surgeon in 1875. It is a matter of equal interest and importance to know how far surgical results are influenced by the elaborate and often costly arrangements of modern hospitals ; and this is an additional reason for my record of the year's work. If this question is not fully answered sooner, it can be so here when the New Infirmary, at present in course of , +erection, has been completed. + +The relative paucity of emergency cases is explained by the fact that most of those admitted to my department at the infirmary are operated upon by my assistants. The large pi'0' portion of operations for diseases of women is due to the fact that all surgical cases come under the care of the surgeons, and into the surgical wards. + +Diagnosis.?A careful study of the history, a careful con-sideration of the symptoms, and a skilful interpretation of the physical signs, will lead, in the great majority of cases, to a correct diagnosis. The difficulties and uncertainties of diagnosis have been overrated, and this to such an extent as to discourage the practitioner in his efforts to arrive at a definite opinion before proceeding to operation. + +There are certain lines of inquiry to be followed in every case-The general appearance of the patient should be the first con' sideration, for it may teach a most useful lesson. It is impossible to describe the gait and bearing of diseased persons ; these must be learned by repeated and patient observation. The most important lesson, however, is soon and easily learned; it is to answer the question, Does the patient appear to be seriously ill> +or not ? Even in the absence of signs of serious organic disease, if the general appearance of the patient suggests illness, it is wise to hesitate before offering a very hopeful prognosis. In other words, the diagnosis and prognosis must not be based simply upon local physical signs, but must take into consideration a wider field. + +The yellow complexion of jaundice, the waxy anoemia of long' continued haemorrhage, the muddy anaemia of syphilis and of cancer?these may convey a useful hint; so may the shape of the abdomen, the way the patient walks, or sometimes the presence of an odour. Then, with a view to ascertaining the possible source of mischief, it is well to ask the question, Where is the chief trouble ? After this, in difficult cases, the history of the illness should be carefully inquired into; and it is usually best to let patients tell the story in their own way, asking questions now and again on points requiring more definite information. It is import-ant to know when the illness commenced. A tumour, for example, which seems to be the probable cause of illness, although ABDOMINAL and pelvic surgery. 23 +^^uantTf6^sl'ons ?f Malignancy, is not likely to be years. keen recognisable for more than two or three SlQ +?n which srfD* ?YMPT0MS-?There are certain signs and symptoms Under th? f if . s^ess must be laid. They may be summarised AS.!_Tiowl,ngheadin?s:- are ittmorf- f6 aracter ?f this, and its position and distribution, abdomiml ? P?*nts- nominal ] Nearly all acute instances of serious ? ml acute instances ui senuua severity wh ?e^10ns are ushered in by a pain of overwhelming roughly ']np 1 llsually> at the very commencement of it, can be however fl +^ * +? ^le neighbourhood of the lesion. Very soon, again bppn16 ^ain becomes diffuse, while at a later stage it may asain kp Aumuse, wnue at a later stage it may c^aracter Ttv ^ie situation of disease. The deadly abdominai +? + +t-mus^ be iusisted upon. When a serious Probable tfC^ as^roP^e bas occurred?one so serious as to make it hope of re +& no^nng but immediate operation offers a reasonable +'locations!?^~t5e ?ai? is alwa>'s one of the most important +^reetino- indeed, it may be the only one. A common dying j'' +0m worst cases is?" Oh ! doctor, I am dying! I am traction1 of^^011^ ?a*n great severity suggests the forcible con- +?bstructi0 Un?fcriated muscle. It occurs in cases of intestinal ki?tt of ^ st?neJ calculi passing down the ureter, and reten-pregna11116' an^.^ occlirs physiologically as the termination does not *s a m?re severe pain than any other, but +^cute ?nve^ ^ie message of impending death. + +chiefly inv lrmous pain occurs in inflammatory affections, tuinou +? V111? .^e Pei'itoneum, chronic continuous pain in cases importance' efPe^a^y ?f a malignant character. It is of some abdomina] l ?.w ^bat pain never attends certain organic bfemorrhoi t Pe^v^c diseases, such as, for example, uncomplicated So that th'1CS' slmP^e tumours, and enlargement of the prostate; Tender S^n?P^om P?ir>ts to complications, or to malignancy. + +aild the r\CS'S\-- 1S c^aracteristic of inflammatory complications; ^iscoverv <"iS 10n ^le lesion may often be determined by the different A ,rou^1 careful steady pressure of the finger tip over +?^'Burnev'ar S ? ^le abdominal wall, of the most sensitive point. ^0l1; oftp S ^01,n^ *n appendicitis, is a remarkably useful illustra-a Point or>n n? SS c^aracteristic, in disease of the gall-bladder, is the rectus '?S1H ^le ^P the ninth rib and at the outer edge of point niav Ir)USC duodenal or intestinal or gastric ulcer, this aily reasn ^ a Sllide in localisation. It is difficult to offer Hiqidjf1 ? exPianation of this point tenderness. + +sign of ^ ^Le abdominal muscles.?Rigidity is the earliest Pertance a11 Unc^rtying peritonitis, and one of the greatest im-sP?t of S,-lfn ai(^ diagnosis and prognosis. Over an inflamed hardness- 11 i?n-6urn ^ie muscles become board-like from their j and if the peritonitis spreads, so does the muscular rigidity over the spreading area of disease. In cases of general peritonitis, the whole abdominal wall is so hard and fixed as to stop all respiratory abdominal movements. + +Rigidity, like tenderness, is not only possessed of value as a diagnostic sign, but may also be a good guide to the localisation of the lesion. For example, localised rigidity in the right iliac fossa (and it is surprising how restricted in area it may be) is often the chief diagnostic localising point in cases of appendicitis. + +A similar condition in the right hypochondrium is equally characteristic of cholecystitis. + +Vomiting is a common symptom in all acute abdominal lesions, and one o.f value in diagnosis and prognosis. Eepeated vomiting alone, in an acute case, suggests a serious lesion. The quantity and character of the vomited matter may be the most important aids to diagnosis. A large quantity of watery bile-stained vomit suggests intestinal obstruction, which may be the result of acute peritonitis, or may be of purely mechanical origin. If this be followed by liquid having the appearance of egg beaten up in beef-tea, or by thick pea-soupy liquid with a faecal odour, there can be no doubt that the contents of the small intestine are regurgitating into the stomach. Indeed, faecal vomit, for practical purposes, is considered as pathognomonic of intestinal obstruction; and so it is, but occasional exceptions occur. On one occasion, on the strength of this sign, I operated and found a malignant growth adherent to the transverse colon. Post-mortem examination showed a malignant ulcer of the stomach invading the transverse colon, and a fistulous communication between them. On a second occa-sion, I was guided to a correct diagnosis in a similar case by the fact that part of a turpentine enema was vomited during the act of its administration. + +A large quantity of yeasty, evil-smelling matter vomited in the evening, containing perhaps recognisable portions of food partaken of in the morning (orange is easily recognised) is characteristic of stomach dilatation due to stricture of the pylorus, for it occurs in no other condition. Hsematemesis in young females is often the only warning of the presence of gastric ulceration. Hsematemesis and melsena in young males of duodenal ulcer, and in older persons it may be the first evidence of cirrhosis of the liver. Coffee ground vomiting after an abdominal or pelvic operation (except in the early hours, when it may be due to abdominal shock) is always regarded as of very evil omen, for it is associated with the petechial haemorrhages of the gastric mucous membrane always met with post-mortem in general septic peritonitis. + +To know the force with which vomited matter is ejected may be useful in diagnosis. The forceful ejection of a large quantity in cases of intestinal obstruction, and the frequent quiet regurgita-tion of mouthfuls in general peritonitis, are suggestive points. + +ABDOMINAL and pelvic surgery. + +?Of the two ordinary contents of r>a n\,a ^nowlecifE nfS?1 i! t}l.e mosfc useful hints are obtained P s flatus per rectus -?i behaviour ?f the former. + +Inability to te ^arge bowel IrJlf. + +? Pathogn0mo +? nJ Yu painful rumbling and belching, e passage of fjqt-presence of intestinal obstruction; the +. e most certain n en% foundV, J1161"-. + +Tt,e faot tllat abSndant pus is +\Vo?Wri ^hat at som1SpeCt;* +fi Pe^ature hoc i ?7 +" UP in the abdomen without any rise in f,,QS associatpr7 ? -fx Per,10(^ +? ^tborneter iq nf6C S01Tle authors to express the opinion that the j-L +riay be mifilpofj; no Practical use in abdominal diagnosis. + +e ^emperaturp +? ?n^ a ^ew observations are made, and if 1S Pons^ered) is quite certain; for blood in temnernf,,,,1 ^s?*n ruPtured ectopic gestation, may cause a normal on +^ &A f +^ PVS *n large quantity may be found e- After a serious operation, moreover, a rise of a r. Peritoneal ca temperature, even up to 102?, may be a good sign, especially if the pulse does not show any excessive speed, for it indicates reaction. + +Nevertheless, after allowing for the chances of error occasioned in these exceptional circumstances, it is still possible to find the temperature an important aid in abdominal diagnosis. + +The tongue.?A dirty furred tongue always suggests the need for care in abdominal diagnosis. After an operation it means that the patient is not yet out of danger. A moist brown tongue usually means that the bowels require attention and are not being properly evacuated. A dry brown tongue is of worse omen, for it is often associated with failing circulation; that is, if no other cause is known to exist, such as an insufficiently emptied bladder or damaged bowel after strangulated hernia. In old people, dryness of the tongue, especially in the morning, is not of such serious import as in younger and more vigorous persons. A +scarlet-red moist tongue is suggestive of the presence of suppura-tion. A dry red tongue indicates feeble vitality and a want of reparative power. + +Menstruation.?In women the menstrual history may be of the greatest value. Excessive and irregular flow are always the result of a less or more serious organic cause which requires thorough investigation. A young healthy adult woman, previously regular, who has missed a period over a week, is, in all probability, pregnant. It is a curious fact that, though women may under certain circumstances be full of anxiety to deceive themselves and others into the belief that pregnancy is impossible, I have never known one to give a misleading menstrual history. If a diagnosis that the abdominal or pelvic swelling is a tumour be never made, in the presence of amenorrhcea, till pregnancy has been positively excluded, many painful and damaging mistakes will be avoided. + +Facial expression.?The patient's expression may convey an unmistakable message. Who that has ever seen the grey pinched face of general septic peritonitis is likely to forget it? The presence of jaundice following an acute attack of abdominal pain is characteristic of gall stones. Pigmentation of the skin of the forehead suggests a possible pregnancy. Dilated pupils with alternate flushing and pallor of the face and neck are seen only in highly nervous, emotional subjects. Drooping, heavy upper eyelids in young women indicate a tendency to the grave forms of hysteria which are sometimes associated with abdominal conditions simulating tumour. A dilated condition of the capillaries of the cheeks and nose may aid in the diagnosis of a cirrhotic liver or enlarged spleen. The waxy anaemia of recurrent haemorrhage suggests inquiry as to the presence of haemorrhoids or metrorrhagia-The deadly pallor of a ruptured extra-uterine gestation has been known to afford the greatest aid to diagnosis in an acute abdominal emergency. + +Examination of the Abdomen and Pelvis. Inspection- +^ ^ -?The patient should lie in bed with the si ^ ahdomen fully raised, with the chest and exposed legs covere U1 >1 to view. + +. . + +i.pfi Fixity during ie- Any peculiarity of the parietes is n ? + +j^ocalised oedema spiration occurs in all inflammatory state - +enlargement suggests the presence of suppuration un +^ie deep venous of the superfical veins, some obstructor pregnancy circulation; either a thick, dark pigmented linea.alb . P?d wrinkling of in progress or recently passed; marked c +_ . + +the parietes, a recent confinement. + +protuberant. If ist The abdomen may either be flat 01 pi Exp0SUre of flat it may be possible to note movem . .1^ the stomach its surface to the cool air stimulates p ? + +tajsis is only visi e and intestinal canal, and though norma 1 ovement of hyper through the thinnest parietes, the lncreas ^ ^ diagnosis, trophied stomach or intestine may be a_ , a wave of vigorous It is possible to watch, in the dilateds. ? 'te at the stricture peristalsis pass from left to right, an outline of 1 Pylorus. In cases of intestinal obstructs3 bypertrophied intestinal coils tQ make a may be to iocate the site5 diagnosis certain, and it.may also ?-i^?mttern assumed by obstruction by * noting the ladder-like^ p caecum, and small intestine, the right iliac distensionol ^ A sn;all appearance of the transverse colon as +^ ?vioric one, when tumour may also be seen, especia, y a ^ m0bility or xi J +abdomen is flat and the patient thi , +1 ments. , +may be observed during the respiratory nt may be loca If the abdomen is enlarged, the en and the +, +rounded, etc., 0 character of its surface, e.g., whether n ' +eff0rts or clian?, distinguished. Its mobility during respm y ncy, th in position, may also be noted; win e m the case Movements of a foetus may be clearly se ? + +y discovei _ +If the enlargement is general, ^stTnd Hmbs are wasted whilst the abdomen is enlarged the +_ or ^ ^ay show this is certain evidence of organic is ? + +neral obesi y, the abdominal enlargement is associa +^ +^ +which the abdominal swelling may e abdomen may Shape of the abdomen.?The shape a ]arge tumour help in diagnosis. The forward c0*ve^tyffeneral enlargement tinguishes it from the more latera an free fluid 0 or of fat. + +Palpation.?This is ^n_t important method o certainly the examining the ??y to know how to <3 abdomen; hence it is ne it to the greatest advantage. Both the patient and the examiner's hand should be thoroughly warm, otherwise the difficulties of the + +![7_image_0.png](7_image_0.png) + +Fig. 1.?Showing regions of tumours?(a) Ovarian, uterine, vesicle ; (b) ileo- csecal flexure valve, of crecum, appendix, right half of the pelvis ; (c) sigmoid colon, left half of the pelvis ; (d) kidney ; (e) liver, gall- bladder ; (/) spleen, splenic flexure of colon; (g) gastric, pancreatic; (h) small intestine, omentum. + +Fig. 1.?Showing regions of tumours?(a) Ovarian, uterine, vesicle ; (b) ileo-cecal valve, caecum, appendix, right half of the pelvis ; (c) sigmoid flexure of colon, left half of the pelvis ; (d) kidney ; (e) liver, gall- bladder ; (/) spleen, splenic flexure of colon; (g) gastric, pancreatic; +(h) small intestine, omentum. + +examiner's Bat band is gently applied and moved easily fiom Pla to place over the whole. + +j rigidity of the Tenderness and rigidity.?Tendeme^ ' tenderness and parietes are first sought for. A 1Jcallsec 1 ' iesi0n underneath-rigidity affords evidence of an inflamina ) +distinctly feel In cases of intestinal obstruction, the a arrest of the forcible peristaltic movements, and loca iss ,stomach peristaltic wave, the probable site of o produce the eases, manipulations over the stomac, may induce a splashing sounds characteristic of dila >e however, so far as peristaltic wave strong enough to be te , 'ista|sis sometimes I have observed, not so strong as P ? + +palpation is to observed in intestinal cases. The greate? + +tumour is present enable the observer to decide as to W1 can ^0 it, or so not; and this may be so easy that any? + +muscUlar or fat difficult that no one can. Except in stable methods and abdomens, it should always be possible, by suitaD especially ^ 0 bv the. air! + +in ,.*lllanual exam-5??n' \? sa3'. + +' whether a +^?up is present or +^.'a^ in the great it qI S of lnstances +*Tfoealsopos- rpn. t0. f?i'm a cor-cv)Qt estimate of the Paracter and rela3?n*of the tumour. + +aJL??6 of an an- +Fig. '2. + +"=?uietic in the most difficult cases 0[t1el^. ?? n 0f the parietal f?r> in the absence of sensation, the re c. ^ without muscles allows of more thorough PalPat1^' . be resorted to dangers of its own; this method shou c ^ quite small but seldom, and then with the grea^ ca ' and with a finger tumour can be detected in the flat, lax a j.]ie'abdomen (Fig-^)' +the rectum or the vagina and a hand o lpated. All of the whole contents of the normal pelvis can P ient \[e m a these manipulations may be aided by a o the greates position such as to relax the abdomma back, this is to be extent; and when the patient is lying on e ^ea(j and keep-found by raising the shoulders and suppoi ? ? which alter-lng the thighs flexed. A small elongai intussusception, lately hardens and contracts, is charac v 0f a tumour by If the abdomen is enlarged, the is difficult. The mos inspection and palpation may stnl be easy exalnination. 1 definite evidence is to be obtained by i ^ ^be presence of 18 scarcely possible to form a wrong opinion a tumour, even in tlie fattest or most muscular abdomen, if it can be felt between the two hands, either one on each side of the abdomen (Fig. 3), or one in front and the other behind (Fig. 4). + +If a definite swelling is certainly present, it is well to re-member that in the case of male patients, nine out of ten such growths are malignant; and in women, nine out of ten are the pregnant uterus. Before going any further, therefore, the possibility of either of these conditions should be fully considered. It is also important to have the bladder undistended, because it may be and frequently is mistaken for a cyst; and to have the colon empty, for solid faeces in it resemble so closely a malig-nant growth as to have often misled skilled observers. + +When a tumour has been discovered, the next step is to try to ascertain whether its contents are fluid or solid. + +A fluid under tension, as in the distended gall-bladder, may produce so hard a tumour that it feels like anything but fluid. A soft solid tumour, on the contrary, such as a lipoma, a soft sarcoma, or an cedematous fibroid, yields a sensation of elasticity so closely resembling fluctuation as occasionally to deceive the most wary. Tor my own guidance, I have made, for cases of abdominal tumour, the following rule: that, if I +cannot feel fluctuation, or cannot detect a fluid thrill in the tumour, it is pro-bably solid ; it is to be remembered, how-ever, that the gall-bladder forms an exception to this rule. In the pelvis it is more likely to be correct to regard as a cyst every doubtful tumourwhich is in the least elastic, and which is not very definitely hard. + +Next, it is well to discover, if possible, the relation of the tumour to the abdominal organs; for example, a gall-bladder tumour in the right bypochondrium re-sembles, and is often mistaken for, a mov-able kidney. This error can be certainly avoided by feeling the kidney behind and separate from the tumour. + +Then, the tumour may be mobile or fixed. If mobile, its movements may be independent as in the case of an omental growth or of any pedunculated tumour; or, though movable, it may in its wanderings carry with it some organ to which it is Fig. 3. + +![9_image_0.png](9_image_0.png) + +Fig. 4. + +ABDOMINAL and pelvic surgery. 31 attached. ^11 t ^fluenced by th urnoufs above the umbilicus are more or less +\l0se that are n +? lesP'rat?ry movements of the diaphragm; and +^nejs, spleen ?un1ec^ec^ with the viscera in contact with it, liver, +?l0ve dunno. stomach, unless they are fixed by adhesions, lrispiration and S^llatl^nj' they move freely downwards on forced u?oien, niovemp11^3^ ?n exPiration. In the lower abdomen of +^oiniriunicate fh +" +.S ? a u'er*ne or ovarian tumour are liable to 6 case of ovir'611 niovements, more in the case of uterine, less in fl Mobility no lan'i^le ceryix of the uterus felt per vaginam. + +two examin-a i +? ^ested by moving the tumour between it frnr~ examming hands, passing ?0 uuuuo, u'om one to the other; or by taking the patient change posi-tion, turning on to the side, or standing up, or resting on knees aud elbows. The latter position ls useful in testing whether a tumour lying over the aorta and pulsating is attached to the vessel or not. + +The surface of the tumour may next be palpated. It may be rounded and smooth, as in the case +?f unilocular cysts, or craggy and Modular, as in many malignant growths; or with rounded bosses upon it, as in some multilocular cysts (kidney or ovarian) and uterine fibroids. + +Its edge may be sharp, as in the case of a dislocated liver, Notched, as occurs cliaracteristic-aUy in an enlarged spleen; hard aild nodulated, as in a malignant growth. + +Succussion or gurgling over the tumour may show it to be over- +*apped by stomach or intestine. + +Fig. 5. + +Fig. 5. + +? ur intestine. . nrnours overl\in? + +-Pulsation is perceptible in most sma , disappears +!e aorta. It is not of an expansile cliaracte , ractel.s +*!>en the tnmonr is lifted off the aorta; by both ot tl.es +^ay be distinguished from that of an aneu ) Qn palpation A cyst or other collection of fluid 81V^S \ ^ +HQiiid on oiip ^ 4-1-^ - *- +v,x "IhprWFig^ &)? + +- *??ui on one side of the cyst, a fljP 0D Sections in which it is n?tmg the quality of the thrill and the d multilocUlar cyst, o lri?8t easily transmitted, the diagnosis 0ne with thick contents, is possible. in fluid, ballot* +- +inent may be felt. It suggests pregnancy or cancerous peritonitis; for other conditions causing it (ruptured ovarian cyst, uterine fibroid, and free fluid) are very rare. + +Enormous distension of the abdomen, without corresponding swelling of the legs, suggests a very large, slowly-growing ovarian cyst. Enormous abdominal distension, with corresponding swelling of the legs, suggests ascites. Distension which disappears after the night's rest and gradually increases up to the evening, suggests tympanitis. + +Percussion.?Though of less value than inspection or palpation, percussion may be a valuable aid to diagnosis. Over the majority of abdominal tumours of any size, there is a dull area correspond-ing roughly to the size of the tumour, and this area is dull on both light and strong percussion. + +If palpation has detected a definite tumour, and if this lias a tympanitic patch or strip on it, adhesions to the stomach or intestine are suggested; this is to be expected in renal tumours in front of which the dolon lies, and in other retroperitoneal growths, such as pancreatic cysts, aneurysms, and solid tumours, lying behind the stomach or intestines. The tympanitic note, in these instances, may only be discoverable on light percussion. + +The typical dulness of free fluid with a patient lying on the back is, of course, to be looked for at the dependent parts. + +appears first in the flanks, and, as the quantity increases, forms a gradually increasing band above the pubis. A large quantity oi fluid can be discovered by any one; it is a knowledge of how smalls quantities may be found that is of importance. Within a fe^' +hours of their onset, all acute abdominal lesions, which are about to be followed by general septic peritonitis, develop a sufficient amount of peritoneal fluid to be discoverable by careful percussion-Each flank, in its most dependent part, will be found to be dull on light percussion, and the upper limit of this dulness must be care-fully noted. On turning the patient over, the upper flank becomes resonant, and the dull area 011 the dependent side reaches a higher limit. This evidence, if conclusive, is of great significance. I*1 young women, this sign occurring together with profound sudden anaemia may be the only available evidence of ruptured ectopia gestation, with a dangerous escape of blood into the peritoneal cavity. The distended intestines of intestinal obstruction may cause dulness in one or both flanks, but a change of posture is not likely to increase the dulness of the dependent side, so that this point is as important to note as the fact of dulness itself. + +The tympanitic corona of an ovarian cyst (Fig. 6) is evidence of very great value in favour of a cyst, as distinguished from an encysted collection of fluid (Fig. 7). I have never found this sig11 in anything but tumour, and in its absence I have many times declined, never with any cause for regret, to accept the view that a tumour was present, when everything else appeared to be abdominal and pelvic fir as 1 have ?bse J +favour of this belief. This sign is resemble ova^npo\iec- to be found in the cases which so , -caj the encys. f^e and in which mistakes have become j'n this c011t \1 sjgns tion of fluid due to tuberculous 'he flanks, and th?. Jon shape of the abdomen, the rf??anc?1"11 Mislead; but of au encysted collection of fluid, m y jess Y-shaped of the upper abdomen shows a m -whereas, in a cy resonance, the point of the V "np^rds. convexity of the resonant area points up cases of enormous distension, the a Jsenpe of dulness and presence of a tympanitic note all over are the best evidence that the swelling is due to excessive gas 111 ^e intestines. + +Auscultation is of less value in abdom-lllal diagnosis than any of the other methods Mentioned, but it cannot be neglected with +"npunity. One of the most certain evidences of chronic intestinal obstruction is le presence of borborygmi, which may be +?ud enough to be heard by any one in the Same room as the patient. Another sound ni?re conclusive still, is produced by the passase nf -- j +* Sf^0f intestinal contents through a contracted poition +> which may be heard with the ear close to the abdomma^ +wall or with the stethoscope. A souna like that produced by milk squirting into the milking-pail is also reliable evidence of intestinal obstruction, and patients will frequently tell themselves of this sound attending the forcible rumblings. It is said by reliable authorities that auscultation offers a useful means of distinguishing between cases of acute intestinal obstruction and acute peritonitis; that in the first, peristaltic movements are heard, whilst in the second, they are absent. I have not found this to be so true as to be Fig. 6.?Ovarian cyst?shape of dull area. + +Fig. 6.?Ovarian cyst?shape of dull area. + +lily? + +\ +\ + +``` + st." ?ncysted ascites? + Pe of dull area. +Fig 7 + sW En^sted ascites? + pe of dull area. + +``` + +helpful. . ,nnce of auscultation There is one condition in which the imp cages 0f suspec e Can scarcely be exaggerated, and tins . t^e foetal hear Pregnancy. After four months, the sou ^ heard. ^ +^ perceptible, after the fifth month 1 . eVjclence of P1' o exception, there is no absolute} c ^ ,ef0und either rea nancy. Every other sign and symptom ratX, grave issues ban^ +Sluiulated, hence the importance of this. hoUid be the rule ( +?n the diagnosis of pregnancy, I positive opinion un e ride which I always act upon) to offer n P +o ED. MED. 533?NEW SER.?VOL. XV.?I- +the foetal heart can be heard. More professional reputations have been wrecked upon this rock than upon any other I know of, and it is only by adhering strictly to the rule I have named that mistakes can always be avoided, as mistakes should be, when reputations and lives may be at stake. Lest there should be an}' +misunderstanding as to my meaning, it is necessary to explain that in ordinary cases, and under the usual conditions, i.e., as a practical guide to action, the symptoms and signs of pregnancy are so straightforward and definite that careful examination, any time after the sixth week, will enable a skilled observer to offer an opinion, which is seldom wrong, as to the existence or otherwise of pregnancy. As a matter of scientific evidence, the foetal heartsounds only are to be thought of. + +The statement is often made that gall stones have been heard and felt grating against each other in the gall-bladder, but I have not personally heard or felt them do so, though I have often tried. + +The pathological condition of a gall-bladder filled by stones adds to my doubt of this possibility; and the frequency with which the ninth costal cartilage can be so manipulated as to produce rough friction, suggests a possible source of error. + +Auscultatory percussion is a valuable aid in marking the outline of a dilated stomach. A bimanual stethoscope with the ear piece over the stomach can distinguish by the loudness of the sound when a flicking finger, starting at a distance from and gradually approaching, has reached the stomach. By marking this spot and others in the same way, the area occupied by the stomach may be clearly mapped out. + +A systolic murmur may be heard over many tumours near the abdominal aorta which are not aneurysms; in some abdominal aneurysms no murmur is to be heard, so that no conclusion must be based on the presence of this sign alone. + +Instrumental aids.?The use of special instruments as aids to diagnosis is, in the great majority of cases, unnecessary; and fortunately so, because some of them require considerable practice and opportunity and skill for their satisfactory handling; and their application may not be devoid of risk. Nevertheless, Rontgen rays, the cystoscope, stomach tubes, and the various specula, are essentials for the diagnosis and proper treatment of some abdominal and pelvic diseases. + +Examination of the blood.?Recent developments in the ex-amination of the blood and the urine promise to be of value in the diagnosis and prognosis of abdominal disease, and to afford guidance as to the propriety, or otherwise, of undertaking serious operations. + +The most useful and definite information we have so fa1' derived from these examinations is, that leucocytosis is strong evidence in favour of the presence of pus. + +Aspiration.?I have so often had to deplore disasters following ABDOMINAL AND PELVIC SU +?..tors and trocars, &u the use of exploring syringes,^a&^ condemnation ^rgery) who almost share the strong e, a^ies on abdomi +? ^ately Pre" expressed by many recent au "Except as 1 ,, a g??c would entirely prohibit theii ^ +? + +g Would pro? 1 +) a sUspected liminary to a possible opera i. ? + +puncture incision rule; but in the pelvis especially;^ec * ^tor {rom pus collection through the & ugeleSS opera may give valuable aid, and may l cases where a being carried out. PXT)lore in al1 defect in the Exploratory incision.--- -R undoubtedly -^g of the diagnosis cannot at once be m > k +> insufficient import- present system, because it f.^Vless there ib one diagnostic powers, and 1 +. Vdiate +' +diagnosis an g ^een ex ant reason demanding imm ^ nS 0f diagn?sl harmless should not explore till othei is n0t alway happen m hausted. An exploratory lapa AccideUbs tionas *?r procedure that is_ generally supp g 0{ the op ^besions may the aseptic technique and m :s^ing adhesion , +1 who ar "4 in the separation. of ?? and ?or the form'subsequently and give g are often parried out a suffering from debilitating 1 ocedure can , ^ cancer is exploration, unless some remedial^ ^ inop*^ The the same time. Tor example, P expjoratory t 0f general decidedly not benefited by ' a certain am uit 0f the operation must lead in any ca g^ partly as , bance, Part ^ +disturbance, varying in diitcren intestinal di conBequence, anaesthetic, partly as the result o the ^ A a co as the ^ +result of the confinemen geri()Usly alteied +^ steadilj the patient's condition may he wbo has be ? tQ live, this aud, especially in the case of P ^ 011\y a slior t0 a con-downhill, is very emaciated, an shorten i _ n}ay be a very serious matter, a against siderable extent. , ..uy"ht that I am 1111 +" be empl?ye I do not wish it to be t1 ^ink they ough tine. Ther exploratory operations, but mere matter o . essenti with great judgment, and not as a^. is doubtful,itof are many cases in which, if th in cases, an earl) +to make it certain as soon aS P. important 0 i-ca\ operat1011' suspected malignant disease, i ^ all early r T operation diagnosis from the point o regards explo . T^e a reasonah rule that I should lay do^j* ken where thei> l h ls. that caTrjed they should only he unc ^ure can he r0wths, t Probability that some remedial p ^ ^ the removal |nagtoInosis, ?ut,?SUcbi procedures, for m f +' +aI1Ge of mtes sbould n separation of adhesions, the pe i ratory laPiUrm than down for its operative treatment. + +JGGU ^?? ?^en assumed that the abdominal +?fa?na% whicli t?o patient is necessarily associated with any com 7 bas> I thin7- 'T* +discovered. An ovarian cyst the size Plain ibai1 one nor.?6' ^ ^10m adhesions and tenderness, has, on abi]rf made bvw ,ed of abdomin T?n'i ?0611 exclsed, because the patient comanv f!l0vvledge I.)o J1 ' I. V1'c> and other pains; yet such a cyst to or p l0uble. a i exist for years without growing or causing +^ore ?er*ne orano-IlaJ +abrlf/6.11 ]ess disnlo ne^' 00se enough to be grasped by the hands, 8a^nal and I'?! + +than this, has been fixed for obscure +,ue^ stomach n ^mplaints; tucks have been sewn in the Uter, the m'riW +6 . ated liver and spleen have been 6S' a(*hesions of stomach and of ovaries and cases out of ten no real that- resu^ed. \rn : f?11. + +1e Patient, and some discredit to surgery been don?^' and in Koori 1-l ave bepn o and ai1. 0rganic pn,1(f . Sent practitioner or physician will allow abd.51' less tbnf8 1S at bottom of every abdominal trouble, w>a] in', and SurSeiT is_ the only treatment for every 8Uro0lla^e evi'deripp' ?m ^dyancing claims without strong and e$tpL ?Qs and thpiv 111, eir favour, there is real danger that 01 ^bey now +.W01'k may not continue to be held in the high I have already indicated some of the difficulties in selection of the best time to do an operation in acute cases i the same anxiety arises in chronic ones. My view is, that pallid' ive operations entailing discomfort and a departure from normal exterior, should be as long as possible postponed. Souie years ago, following text-book teaching, I was in the habit ?j recommending early gastrostomy in oesophageal obstruction, aflL +early colostomy in cancer of the rectum. A few submissive anc intelligent persons may be pleased with the results, but i average patient has serious objections to being fed through a h?*e in the stomach as long as he can swallow, or to defecating oVel ? + +the iliac fossa, as long as fseces can come through the rectum ; +in my experience, he does not hesitate to express disgust at the result of these operations. If, however, the oesophageal obstruc-tion is complete, and the patient hungry, or if a rectal cancel is blocking the bowel, and symptoms of obstruction are evidence, no operations will be more appreciated. The dels) +is not good for operation statistics, but surgery was made f?1' ? + +man, not man for surgery, and it is well to remember this 111 these cases. On the other hand, palliative operations, when ^ +apparent defect is in evidence, such as gastroenterostomy aD( +intestinal anastomosis, cannot be too soon undertaken, when ^ +need for their performance has been once recognised. + +The form of operation to be employed is a subject for gra^ +consideration. The natural desire of surgeons is to do a comply and radical operation, and it is scarcely necessary to emphasi' the importance of doing this when it is possible with a prospeC +of success. In many cases of intestinal obstruction, howeve1' those especially in which the small intestine is distended, ' +radical operation offers so small a chance of recovery that t'1 attempt to do one should not be entertained. Drainage of distended intestine, for a time, offers a chance of temporary, 01 even in some rare cases, of permanent recovery; and at a latel date an attempt may be made to remove the cause with a bette hope of success. + +The complete removal of pancreatic cysts, unless these ^ +exceptionally favourably circumstanced, has been attended b)r high rate of mortality; the suture of these to the pariet^ +followed by prolonged drainage and careful after-treatment, ^ +been followed by a high percentage of recovery. A hopele?' +looking case of pyonephrosis may be improved by drainage to su->i?l ?Pc^a^on.?The patients are, as a rule, ad-c!?n js made of n V? yS }jefore operation. A careful examina-Pecial attention ^ ^enera^ condition and of the chest and heart, uitiou to the ? 1Si ?aid ^le examination of the urine. In +? ^e following Ua +. + +^ath on admission, they are submitted Pufgative n +? J?1.ePara^011- On the day before operation, a dose l0rning a siit)7,]nClne' usua^7 castor-oil, is administered, and next +,1011 area is r)?3T SoaP ail(^ water enema. The skin of the opera- lair is shaved nft i i?n Preceding the operation. All +' +?aP (made hv +;U s^n washed with water and spirit-ethylate(j smrin11X'ng toSetj|ier equal parts of soft soap and len ^lethylatpd ? ? s^n *s further cleansed with turpentine, afery solution SPiriti and fina% corrosive sublimate (1 in 1000 n +.covered bv .11(1 hot water li'0* a^er thorough cleaning with soft soap +^truments snf? a ^ ^ carbolic lotion for an hour before use. + +.,Ul>se who has an<' ^ie are handed to the surgeon by a 6 instruments -18 7 ^r ^er so^e duty. The carbolic lotion covering in lotions 1S i1 ^ in 60 before the operation begins. + +'000 aqnPnilQ USl i are, corrosive sublimate 1 in 1000 and 1 in v Tjle site of S?lutlon> and carbolic acid, 1 in 20. + +]VlUngoutin (") ^Pc'rataon is isolated and surrounded by towels +,JePlaced durincfk on' ^ ^0, on which instruments may lands of the siiv 16 ?I)ei]ati011 without fear of contamination. The +?aP) turpentijip^6011/ assistants, and nurses, are washed with spirit-nail brusli id i ^ i llle^ylated spirit, and finally scrubbed with +. The patipnf' ln, .00 corrosive sublimate. + +i in lf)nnnS n *s washed first with 1 in 1000 and then +]? ^ring thl corrosive sublimate. + +lx^ate is -J f?coinse of the operation 1 in 10,000 corrosive subp'ts and the n,01 hands of those engaged in it. The assist- +?r Slops Pq?,USes wear rubber gloves throughout the operation. + +liniate ion Pfevi?usly boiled and stored in 1 in 1000 corrosive +,?f hot 1 nwLT.ph^ Bffore use, the mops are wrung +^0r speeinl corrosive solution. + +P?Ugesj W], +? + +i cases nothing has been found so satisfactory as 7?^unft o?7 ape1kePfc in 1 in 20 carbolic lotion. These are jjwhich ),.lv 'i ni 10,000 corrosive solution just before use. + +r/?r?ugh wash'6 0611 C011tact with septic cases are purified by 1 Ul'e carbolic Ujf an<^ ^ien steeping for twenty-four hours in p,8,6^ The pK '01- sutures and ligatures catgut is nearly always +}li ier, and th(M +1 ^micJse^ gut is placed for twenty-four hours in ethylated snv^/18^ a solution of corrosive sublimate in Pint (1 in 1000) for a week. During the operation it is used direct from methylated spirit in which it is kept-For muscles and for the deep sutures of hernia operations, thick catgut is employed, size No. 8, corresponding to No. 19 standard wire guage, and for the skin a finer size, No. 2, standard wire guage No. 25. + +Thick through and through silk sutures are used for the abdominal wall in malignant cases, as there is in these a special tendency to rupture of the wound, and in cases for which it is desired to speedily terminate the operation. + +Silkworm gut is used to suture the skin in septic cases. + +For dressings, boiled gauze kept in a solution of corrosive sublimate in methylated spirits (1 in 1000) until use, is employed. + +This is wrung out and covered by pads of corrosive wood-wool, and fixed by bandages. + +The anaesthetic.?The choice of an anaesthetic is left to a skilled anaesthetist. When I personally am responsible, my pre-ference is for chloroform until the laryngeal reflex is abolished, then ether for the remainder of the operation, unless the respira-tory movements become too vigorous, when a return to chloroform is suggested. + +The proportion of nurses to patients in the surgical wards is one nurse to eight patients in the male, and one nurse to six patients in the female wards. + +After-treatment.?The after-treatment of abdominal opera-tion cases may be considered under the following headings :? + +Shock.?A certain amount of shock follows all abdominal operations. Of late years it has been minimised by the methods adopted for its prevention, the most important of which still is to keep the patient warm. Nevertheless, a serious amount of shock may follow severe operations, or operations done upon debilitated patients. + +A warm bed is of the greatest importance. The entire bed should be heated before the patient is placed in it, and the body-heat should be maintained by carefully-applied hot bottles and abundant clothing. It is necessary to lay stress upon the careful application of hot bottles, for many and grievous burns have been inflicted upon unconscious patients in the endeavour to restore their body-heat by the too assiduous application of hot bottles; and so easily is this done, that any nurse who has not been specially warned of the danger, and taught in a most emphatic way how it is to be avoided, namely, by abundant woollen wrapping round the bottles, will almost certainly sooner or later come to grief. In ordinary cases, this treatment, namely, the application of warmth and quiet, are sufficient. For worse cases, or for debilitated people, the foot of the bed is raised; the face is rubbed with a dry hot towel; the mouth and pharynx are swabbed with a sponge on forceps, wrung out of brandy; and an enema of 10 oz. of coffee and a teaspoonful of coarse brown sugar is ordered, with gr. of strychnine hypod +^ockeC^ \ pint of next degree in which the patient is seno +^ temperature normal saline (1 drm. of salt to 1 pint o w , ^ ^ jjigginson's +?f 105?,is injected with an aspirator need e c < +si10Ck has assumed syringe, under each pectoral muscle;, an +^as been loss of dangerous proportions, and especially i ? + +^ from 1 to o Wood, then, in addition to the measures ' +usuaiiy the pints of normal saline are transfusec in pT)eated. + +Median cephalic, and the dose of strychnin P +ctjon namely, Three symptoms follow every abdominal sickness, pain, thirst. + +. Regarding these, ^QVQi rules which are I have certain Se +-g treated by followed, except in special instances. + +prom twenty-abstinence from mouth-feeding in ordinary , +_o.- give the f?nr to forty-eight hours after opera ion, , D? m flVe to ten patients small (1 gr.) doses of caloine , reP +passage of flatus, times, one every hour. This, if retained, a ds ^^"relieve the and satisfies the natural craving tor somes tie ^ the patient discomfort present, a feeling not to 1P nalomel dosing, and ls comfortable at the termination of the well aione; if especially if some flatus has been passed)we?i ordered> After n?t, a soap and water enema, about if P ' . + +tg n enema ol the colon has been cleaned, if sickness s P ^ every two +^ ?z. of milk, 2 oz. of beef-tea, anc o . ^ intermittent +?nrs, is given through a, tube and +_ +un ? + +teg tjie rectum of Pumping action of the Higgmson synng ickness, though the s<>me patients.) Up to this time, moderate Vomiting, which Patient has vomited, need cause no ansety-s ^ ^ th rd Persists after this period, especially i i , conSiderabie +(la)T, and if the vomited matter is gre?n ^ of the early quantity, is an anxious symptom; toil . . which, though now xndications of commencing septic pen a tumblerful rare as a sequel of operation, is stil a P +small teaspoonful +?f hot water, as hot as it can be drunk, description for an +?* soda bicarbonate in it, is my favourite p have not +?bstinate but not serious vomiting, oi, ? . j 0f the soda. Foi +^oved with the enema, a seidlitz pow er avail, and that is, Serious vomiting, only one remedy is surprising to an) +^ashing the stomach out through a u . + +^ re^ef may follow +?ne unacquainted with this metho r it in favourable the thorough cleansing of the stomac'h e +^ t^e tube, and the +"Wee. Some patients, however, are satisfactory; and to hashing out is therefore incomplete and tiiuul good. A +persevere in these cases means doing n +{our hours a large fair substitute may be found by ordering eve y ^ ^ ^ or drink such as previously suggestec, oi ? + +for it is strong, whisky and potash water; or even, i the ?third day, some a glass of beer may be allowed. Attei nervous patients continue to retch and bring up nioutlifuls of what they have swallowed. A glass of champagne with a little food, such as a small raw meat sandwich, will sometimes put an end to the trouble; if not, a small hypodermic injection ?* +morphia (? gr.), if the bowels have been well moved, or, if u?t> +1 oz. of castor-oil may be administered. + +Thirst.?The most distressing symptom after the majority ?* +abdominal operations is thirst, for it is the most difficult to relieve. No explanation of it, so far as I know, is satisfactory; and none of the many treatments proposed prevent or cure it-It may be relieved by rectal enemata of warm water, by trans-fusion or infusion of normal saline, or by leaving saline solution in the peritoneal cavity; but it is never entirely prevented 01 completely relieved. Water, as hot as can be borne, in teaspoonfnl doses, frequently repeated, is allowed from the first in all my casesIce makes the tongue sore and dry, increases the flatulence already present, and, melting in the stomach, dissolves and promotes vomiting. Nothing is so useful in relieving the thirst as a dose of morphia, the administration of which requires so much careful consideration, that I shall deal with it specially after the con-sideration of the third symptom mentioned. + +Pain.?It may be taken for granted that the pain following abdominal section is greater than is experienced in the ordinary 01 in most of the extraordinary illnesses of life; and, in my opinion, it is more than patients should be required to bear, unless the strongest possible reasons can be offered to the contrary. Follow-ing the lead given by Lawson Tait for several years, I refused morphia, only giving it in the worst cases. Now my rule is ? g1'- +of morphia hypodermically the first night, but never after. + +Morphia.?There can be 110 doubt that patients who have been treated for a few days by repeated doses of morphia are in ever)" +respect damaged, and great difficulty will be found in estimating the extent of the damage, or in forming a reasonable opinion as to their progress. The " sudden bad turn +" is most frequently seen m them. A more definite and grave objection to the repeated use of morphia is, that a free action of the bowels is difficult to obtain when the patient is under its influence; and it requires 110 long experience in abdominal work to convince any one of the para-mount importance of this. A single strong dose the first night, however, secures rest and relieves all symptoms, and, so far as 1 can judge after several years' trial of it, its advantages exceed an)' +possible disadvantages from its use. + +Condition of the bowels.?The pain complained of is chiefly of a griping character, and is much relieved by the free passage of flatus. It depends, I believe, upon severe but irregular and therefore inefficient contraction of the muscular coats of the intestinesAt an early hour a rectal tube is introduced to aid the passage of flatus; and this the calomel prescribed generally secures before ABDOMINAL and pelvic surgery. 43 aild Water enm ??1C011<^ c^a7 after the operation. If not, a soap rnay be niadp +^ 1S ^ }? produce the desired result, and this UsUal preserin/'11016- e^"ec^ve bj the addition of turpentine. My Pended in a t101\ administer first .1 oz. of turpentine, sus-Uater. This n +^ Parley water, then 1 pint of soap and Passed. Ho\vla? e.rePeated every two hours till flatus is freely lneasiires nit; ?\ei *reety the bowels have acted after these +??k depresspf]011 S 1n:sua^7 complain of some abdominal discomfort, +,a^ Quitp Jn1' ave no appetite for food until after the fourth le}r have harl n?ni ^iey ?et iac^ these symptoms till after castor-oij. Aft le usi.ia^ dose 011 the third or fourth day of 1 oz. of +..^nd that t 611 ever7thing less nauseous known to me, Infective id Vn a Wa^s return to this. Nothing has proved so +?Uows the ln0V'ng eYeiT discomfort. Occasionally diarrhoea lll0derate (levr^fh-011 " eat^ ?f the usual constipation. In a the stools?n Ge' ? -lla^ beneficial and requires no treatment. + +^re of oniuiri G" ? eils*ve' ioz- ?f castor-oil with 5 minims of tinc- +*Pecac. co ~ +1 1S +nY- rou^ne prescription; if not, 5 grs. of pulv. + +Creta. + +' ? ?ls~ bismuth trisnit., and 1 gr. of hydrarg. c. + +V the 1U^6 *S a^ow nothing but sips of hot water hours or f]ln 1 j us ^las '3een passed, i.e. during the first thirty- +~ ?2- of mil It oeieab?ts- Weakly patients get during this time 111 the same ?% 1 ^ea' and ^ oz~ whisky per rectum, and +^ of hot w,/} ? 1 wa^er swallowed is not retained, a tumbler-a Cup of teT w^fi11 an(^ moriliug- soon as fiatus has passed, Slilall and' crJ111 c.1.eai}1 and sugar if desired, and following that +;vater are mZ +** +7 increasing allowances of milk and bar ley eild of a vvpo]611 1 .U1'ing the first four days fluid diet, and by the +? W:i! ?ry food is the rule-ley are all'm 1 Patients can pass urine unaided, as a general rule, 1J?ay be harmf l +? {'? S0' c catheter is then unnecessary, and 1%ht, ft jg If, however, everything does not seem to be u?a!Hine thfi "!ake iDi?y as to urination, and to ladder mav 1^ P0?as^um> for it is astonishing how distended a lt; ls only mrf\iiV1^thout causing definite complaint, in cases where aild of women +^ emP^ed> and this is especially true of old people +^dominal r!f ~^ei^n nurses have a "knack" of doing well with +&et hold of ses' and both patient and surgeon are lucky if they +*kilfmiv wi>]UC *?n11 one' be raised comfortably and packed up sitting nosh ! ^ vs a^ the back and under the knees into a half-k? most nati!w 01 aL^m.e' eyen 021 the second day, is a great relief s^e> then' f 011y iS* ^?^nS over occasionally from the back to one 8reat trouhl? /.le ?^er an^ back again, if it can be done without most naHm.f ^as can be by a skilful nurse), is a change that Empathy SiaiG ^rate^1^ f?r- ^ little drink, a little well-timed n encouragement, even perhaps a gentle reproof on occasion, are things certain to make the difference between a happy and an unhappy patient, and I, for my part, believe that in the worst cases the skilled attentions of an intelligent sympathetic nurse can and do account for the continuance of life and for escape from death. + +It is safe to say in the great majority of cases, that the prognosis may be given before the patient leaves the operation table, but it is still more safe to say that seldom anything goes wrong with a patient who has got over the fourth day without untoward symptoms. The fourth day is the critical one after abdominal operations. Sepsis, haemorrhage, and shock are at the present day dangers of the operation rather than complications after it. + +Complications following Abdominal Operations.?Per itonitis. + +?Though antiseptics and increased skill in the technique of operations have almost abolished septic peritonitis, they have not entirely done so. A septic focus which is already present, and which has successfully resisted attempts to deal with it, may be the centre of a spreading infection and the cause of general peritonitis. In such instances, from the first it may be possible to see that the patient is unlikely to do well, but the more usual course is that nothing extraordinary is noted till the end of the second or third day. The pulse steadily increases in rapidity, a sign of the greatest value in prognosis; sickness and mental de-pression are present, and some distention of the abdomen, first noticed in the epigastrium, with inability to pass flatus, are the first warnings of the much dreaded peritonitis. From definite general septic peritonitis following abdominal operations, recovery is rare whatever is left undone or whatever may be done. My views as to what should be left undone are decided and may be useful to some one, as they are based upon a not inconsiderable experience of such cases. At one time I held very strongly, on pathological grounds, the view that no one could recover from such a condition except by the help of surgery, and that some might recover if operation could be carried far enough. The consequence of this belief was that I left nothing in the way of cleansing and irrigation and drainage undone; but the result invariably was that, the more completely the operation was performed, the sooner the patient died. I believe now that the question of operation should not be entertained in these cases, and that a small percent-age of them do get better if gently helped to do it. The measures I suggest are in the main, treatment of the symptoms, and so long as the abdomen, even though not distended, is not tense, I enter-tain some hope. A tense distended abdomen is wholly unfavourable. + +The first step is, to stop all nourishment and drinks by the mouth, and to cease the administration of purgatives. The next, to wash out the stomach with abundance of hot water, and then v of to administer a small Morphia. hypodermic injection (sa} oth a j is to be re-Nutriment is to be given by the rectum. ^ continuous lieved by venous transfusion of n?im' ' +. + +^ restlessness hypodermic infusion of the same, and mar ^e p ? + +far as by small repeated doses of morphia. The body h?t^ of ^ possible, to be maintained by artificial hea , ^ every six or heart by strychnine, given hypodermicaliy bo o +? + +itonitis eight hours). Every day that the victim +^ r)ur;ncr the whole lives increases the chance of ultimate ieco"v ei) ? , , Q|, out for later part of the illness, it is needful to keep a < +t-on ^ese localised collections of is pus in the abdomen, o to be strongly recommended. + +. . , ,. f anfesthetics, Pneumonia. ? The prolonged admmistratic>1 ^ and the especially of ether, necessary for abdomin p abdominal inability to cough afterwards, owing to the nan of the att to wound, predispose to collections of mucus in he l"ngs,.^ and Pneumonia. One type of this commences eievated tempera- +?n the following day the patient will ha\ +difficulty in Jure, a quick pulse, more or less cyanos > breathing, tment 0f this with moist rales all over the chest. + +a sudden condition, if vigorous and undertaken a on , +bed, end to the whole trouble. The patient must be laise ^ ^ +^d held in this position by some one support g on whilst the abdominal wound is suppor et > +attempt must either side, an honest and, if necessary, pro on^d^ttemp he made by coughing to get rid of the accum coug]1 with In the more ordinary type, a lug en P +pn'eumonia, are rusty sputum, and the found physical chest-signs P usually during the first week a? e . + +win(y operation in My impression is, that pneumonic a ejgew]iere (it Newcastle Infirmary is much more com ^ far as I can occurs in about 10 per cent, of my cases)' +'ur overcrowded judge, it is the only serious illness depen e from it is and insanitary building. Fortunately, the healing very low, and is almost limited to stomac ca , ^ interfered +<* the wounds of persons attacked appears not to with by it. + +, . too rustom to pass Cystitis.?Several years ago, when 1 ^ +was 0ne of the catheters after every abdominal operation, nnrurred in a con-dreaded complications of after-treatmen , 01 j only after a siderable percentage of cases, and of e_n the iast five years Painful and prolonged convalescence. + +in a hospital I cannot recall a single instance of cathe ei things: first, Patient. This change is attributable 1 tninK +requency, and to the fact that catheters are used with much, 16-12 weeks) leaks.1,2 Ultimately, once a leak has matured and contains a fibrous septum, a septotomy may be necessary for definitive treatment. We present our approach to a refractory gastric sleeve leak managed with various endoscopic modalities. + +A 48-year-old woman was transferred to our hospital 2 weeks after sleeve gastrectomy with fever, tachycardia, and hypotension. She was found to have pneumoperitoneum and underwent an exploratory laparotomy with the concomitant placement of percutaneous drains into an abscess cavity. Follow-up imaging demonstrated a largevolume leak from the proximal aspect of her gastric sleeve +(Fig. 1A and B). Upper endoscopy confirmed a 3-cm defect along the proximal staple line in communication with a perigastric abscess cavity (Fig. 2; Video 1, available online at www.VideoGIE.org). Severe stenosis of the distal lumen was also noted. Given the thick tenacious material within the leak cavity, which did not readily pass through the percutaneous drains, the decision was made to place 10F 3-cm double-pigtail stents to provide internal drainage of the cavity through the gastric lumen. Pneumatic dilation with a 30-mm diameter balloon to 20 pounds per square inch was also performed distal to the leak site to decrease the pressure gradient and promote healing. + +Despite these interventions, the leak persisted with ongoing drainage through the laparotomy incision, resulting in skin breakdown at the abdominal wall. Repeated endoscopy 6 weeks after the index surgery was performed, at which point the abscess cavity was debrided and a covered self-expandable metal stent was placed. Twelve weeks after the index surgery, the stent was removed, and a chronic fibrous septum was found (Fig. 3). Owing to the patient's inability to tolerate oral intake, the decision was made to perform a septotomy. + +The septotomy was performed with an insulated cutting knife (Fig. 4). The septum was divided, with care being taken to avoid injury to the leak cavity wall. Foreign material was removed to promote healing, and bleeding was treated with the insulated cutting knife and argon plasma coagulation (APC). + +The patient was discharged the following day. Subsequent imaging demonstrated resolution of her fluid collection (Fig. 5). She was able to advance her diet without further symptoms. + +Sleeve gastrectomy has rapidly become a popular bariatric surgery option, representing 59% of all bariatric procedures performed in the United States in 2017.3 Although this procedure is less morbid than other bariatric surgery options, staple line leaks are estimated to occur in 1% to 7% of patients after sleeve gastrectomy.2 The proposed mechanism includes mechanical disruption of the staple line, relative ischemia resulting from + +![0_image_0.png](0_image_0.png) + +![1_image_0.png](1_image_0.png) + +![1_image_3.png](1_image_3.png) + +![1_image_1.png](1_image_1.png) + +![1_image_2.png](1_image_2.png) + +Figure 3. Repeated endoscopy demonstrating a fibrous septum separating the leak cavity and the gastric lumen that extended 3 cm distally. + +the takedown of the short gastric arteries, and increased intraluminal pressure.4 Simultaneous distal sleeve stenosis may be present.4 Septotomy with needle-knife, APC, or both has been reported for the successful treatment of late or chronic leaks to completely expose the lumen of the leak cavity and to equalize pressures between the gastric lumen and perigastric collection.2,5-7 Available published reports have described a mean of 2.3 to 5 septotomy procedures to completely divide the septum.2,7 The current case demonstrates the novel use of an insulated cutting knife with APC to safely perform the septotomy in a single session. Removal of foreign body material and pneumatic dilation of the distal sleeve remain important adjunctive strategies for the successful endoscopic management of chronic sleeve leaks. + +## Disclosure + +Dr Schulman is a consultant for Apollo Endosurgery, Boston Scientific, and Microtech. The other author disclosed no financial relationships relevant to this publication. + +Abbreviation: APC, argon plasma coagulation. + +## References + +1. Christophorou D, Valats JC, Funakoshi N, et al. Endoscopic treatment of fistula after sleeve gastrectomy: results of a multicenter retrospective study. Endoscopy 2015;47:988-96. + +2. Mahadev S, Kumbhari V, Campos JM, et al. Endoscopic septotomy: an effective approach for internal drainage of sleeve gastrectomyassociated collections. Endoscopy 2017;49:504-8. + +3. Estimate of Bariatric Surgery Numbers, 2011-2017. ASMBS Professional Resource Center Volume 2019. Gainesville, FL: ASMBS; 2018. + +4. Kumbhari V, Cai JX, Schweitzer MA. Endoscopic management of bariatric surgical complications. Curr Opin Gastroenterol 2015;31: +359-67. + +5. Ortega CB, Guerron AD, Portenier D. Endoscopic abscess septotomy: a less invasive approach for the treatment of sleeve gastrectomy leaks. J Laparoendosc Adv Surg Tech A 2018;28:859-63. + +6. Campos JM, Ferreira FC, Teixeira AF, et al. Septotomy and balloon dilation to treat chronic leak after sleeve gastrectomy: technical principles. + +Obes Surg 2016;26:1992-3. + +7. Shnell M, Gluck N, Abu-Abeid S, et al. Use of endoscopic septotomy for the treatment of late staple-line leaks after laparoscopic sleeve gastrectomy. Endoscopy 2017;49:59-63. + +Division of Gastroenterology and Hepatology, University of Michigan, Ann Arbor, Michigan, USA. Copyright ª 2019 American Society for Gastrointestinal Endoscopy. + +Published by Elsevier Inc. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +https://doi.org/10.1016/j.vgie.2019.04.006 + +## Read Articles In Press Online Today! Visit Www.Videogie.Org + +VideoGIE posts in-press articles online in advance of their appearance in a monthly edition of the journal. These articles are available on the *VideoGIE* website by clicking on the "Articles in Press" tab. Articles in Press represent the final edited text of articles that are accepted for publication but not yet scheduled to appear in a specific issue. They are considered officially published as of the date of Web publication, which means readers can access the information and authors can cite the research months prior to its availability in an issue. To cite Articles in Press, include the journal title, year, and the article's Digital Object Identifier (DOI), located in the article footnote. Visit the website today to stay current on the latest research in the field of gastrointestinal endoscopy. \ No newline at end of file diff --git a/medical/md_images/PMC6831913/0_image_0.png b/medical/md_images/PMC6831913/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..3ce81896b19c85637d09afc37e7553cb3d9f9c2d --- /dev/null +++ b/medical/md_images/PMC6831913/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:19f9333f4fdf5b426e410ebf715840e89c1bf3ae21b33390f7aae7d1153a5afe +size 229933 diff --git a/medical/md_images/PMC6831913/1_image_0.png b/medical/md_images/PMC6831913/1_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..4f42a38428d6cdde1dba7c490d41ba3806070a54 --- /dev/null +++ b/medical/md_images/PMC6831913/1_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:bc5dc2274aa5f341b0491a92cc37ecd84ad8c7006cfff42a6ef528445bb50f70 +size 261358 diff --git a/medical/md_images/PMC6831913/PMC6831913.md b/medical/md_images/PMC6831913/PMC6831913.md new file mode 100644 index 0000000000000000000000000000000000000000..b47a54b4bcac752ec5d7c2ce5e006cb3aae47d1d --- /dev/null +++ b/medical/md_images/PMC6831913/PMC6831913.md @@ -0,0 +1,59 @@ +# Tools And Techniques Step-By-Step Approach To Endoscopic Gastroplasty By A Novel Single-Channel Endoscopic Suturing System Gontrand Lopez-Nava, Md, Phd,1 Ravishankar Asokkumar, Mbbs, Mrcp1,2 + +Endoscopic gastroplasty (EG) reduces gastric volume by + +![0_image_0.png](0_image_0.png) + +use of a series of full-thickness internal plications and mimics the surgical sleeve gastrectomy.1 EG with use of the Overstitch device (Apollo Endosurgery, Austin, Tex, USA) is shown to be safe and effective in achieving weight loss.2,3 However, the device has certain limitations: +(1) it requires a dual-channel endoscope, which is not widely available; (2) the endoscope is stiff and less maneuverable; (3) the field of vision gets impaired when the device is mounted; and (4) the suction ability decreases with the accessories in the working channel. Recently, a novel single-channel endoscopic suturing device was designed to overcome these limitations and ease the performance of full-thickness suturing. We describe the technique of EG with the novel single-channel suturing system, and we provide tips for successful gastroplasty (Video 1, available online at www.VideoGIE.org). + +The single-channel endoscopic suturing system (Overstitch Sx; Apollo Endosurgery) can be mounted on most flexible endoscopes with a diameter ranging from 8.8 to 9.8 mm (Fig. 1). It has an external catheter sheath, which has 2 separate working channels independent of the scope channel and can be used to operate the overstitch accessories (anchor exchange, tissue helix, suture cinch). + +The needle-driver assembly is fixed at the side of the distal scope end and does not interfere with the field of view. + +Unlike the conventional device, the tissue helix, when introduced through the working channel, appears at the central axis of the needle-driver assembly, which enables easy alignment of the tissue for suturing without the need for complex endoscope maneuvering. The longer distal length of the suturing system allows capturing a large amount of tissue with the full-thickness bites. + +For successful EG, here are some tips and tricks. We perform the procedure with the patient in the left lateral position and under general anesthesia. We inspect the stomach for the presence of adverse pathologic conditions before performing gastroplasty.4 We administer antibiotics before the procedure and use CO2 insufflation and waterjet function. + +![1_image_0.png](1_image_0.png) + +We insert an overtube and advance the endoscope holding the external sheath. We start at the distal body and perform a U-shaped suture pattern composed of 8 to 10 full-thickness bites. We use the external working channel for accessories and spare the scope channel. We do not premark with argon plasma coagulation because the uneffaced view of the single-channel system allows us to locate the site for suturing precisely. We approach the target site from a distance, and under direct visualization we position the helix device over the tissue. We apply gentle pressure and rotate the helix clockwise 3 times to anchor the tissue. + +Excessive pressure and rotation may penetrate the abdominal cavity, causing a leak, pneumoperitoneum, or the trapping of adjacent structures. We pull the tissue inside the distal assembly of the suturing device, which additionally displaces the adjacent structures from the path of the needle track. Whenever resistance is encountered during traction, we rotate the helix anticlockwise once or twice to reduce the tissue bulk or remove it and choose a different site for suturing. + +To achieve full-thickness bites consistently, we place the tissue captured by the helix perpendicular to the needle driver. We always start at the anterior wall, 1 cm proximal to the incisura, continue to traverse through the greater curvature as far as the posterior wall, and repeat the pattern in reverse before we cinch the suture to form a plication. The flexibility of the endoscope and the smooth edges of the distal cap allow us to maneuver the device with ease without causing mucosal injury and bleeding. + +We ensure that the suture travels in 1 direction without creating loops because this prevents incomplete tissue approximation and suture breakage. We advocate securing the cinching catheter with the left hand and applying intermittent and incremental tension to tighten the suture, because this will bring the walls of the stomach together to form a sleeve. Any overzealous and forceful traction of the suture will result in thinning and premature suture disruption before cinching. We follow the above steps and perform 4 to 5 suture plications in the proximal part of the gastric body, sparing the fundus (Fig. 2). We postulate that this technique decreases the anteroposterior and craniocaudal diameters of the stomach. We do not routinely advance the scope to the pylorus or duodenum after EG because overdistention and inadvertent manipulation may loosen the sutures. + +We close the needle driver, ensure it is free of tissue and suture, and withdraw it inside the overtube and safely remove them together. + +In conclusion, EG may be safely performed using the novel single-channel suturing system. The design of the device facilitates easier performance of gastroplasty, and its compatibility with most single-channel endoscopes from different platforms increases accessibility to full-thickness suturing in clinical practice. + +## Disclosure + +Dr Lopez-Nava is a consultant for Apollo Endosurgery and USGI Medical. All other authors disclosed no financial relationships relevant to this publication. + +## Acknowledgment + +Supported by a Robert W. Summers grant from the American Society for Gastrointestinal Endoscopy, United States. + +Abbreviation: EG, endoscopic gastroplasty. + +## References + +1. Abu Dayyeh BK, Rajan E, Gostout CJ. Endoscopic sleeve gastroplasty: a potential endoscopic alternative to surgical sleeve gastrectomy for treatment of obesity. Gastrointest Endosc 2013;78:530-5. + +2. Lopez-Nava G, Sharaiha RZ, Vargas EJ, et al. Endoscopic sleeve gastroplasty for obesity: a multicenter study of 248 patients with 24 months follow-up. Obes Surg 2017;27:2649-55. + +3. Sharaiha RZ, Kumta NA, Saumoy M, et al. Endoscopic sleeve gastroplasty significantly reduces body mass index and metabolic complications in obese patients. Clin Gastroenterol Hepatol 2017;15:504-10. + +4. Lopez-Nava G, Galvao MP, Bautista-Castano I, et al. Endoscopic sleeve gastroplasty: how I do it. Obes Surg 2015;25:1534-8. + +Bariatric Endoscopy Unit, HM Sanchinarro University Hospital, Madrid, Spain (1); Department of Gastroenterology and Hepatology, Singapore General Hospital, Singapore (2). Copyright ª 2019 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. This is an open access article under the CC +BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +https://doi.org/10.1016/j.vgie.2019.06.008 + +## Videogie **Quiz** + +Think you can outsmart *VideoGIE*? Try your luck with our new Quiz series. Using cases published in *VideoGIE*, we post questions designed to improve the education of our fellows. Go to http://www.videogie.org to submit your answer, and keep checking back for more quizzes! \ No newline at end of file diff --git a/medical/md_images/PMC6831913/PMC6831913_meta.json b/medical/md_images/PMC6831913/PMC6831913_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..eea89d0f2e1fd4f553ce6961a0ad6090661137dc --- /dev/null +++ b/medical/md_images/PMC6831913/PMC6831913_meta.json @@ -0,0 +1,70 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Step-by-step approach to endoscopic gastroplasty by a novel single-channel endoscopic suturing system", + "level": 0, + "is_closed": false, + "n_kids": 2, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 585.0, + 783.0 + ] + }, + { + "title": "Disclosure", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 585.0, + 783.0 + ] + }, + { + "title": "References", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 585.0, + 783.0 + ] + } + ], + "pages": 3, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 13, + "code": 0, + "table": 0, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC6912138/PMC6912138.md b/medical/md_images/PMC6912138/PMC6912138.md new file mode 100644 index 0000000000000000000000000000000000000000..e3db9fa39d15a3393d767bd98fd145f5cd323a9d --- /dev/null +++ b/medical/md_images/PMC6912138/PMC6912138.md @@ -0,0 +1,264 @@ + +## Effect Of An Italian Propolis On The Growth Of **Listeria** Monocytogenes, Staphylococcus Aureus And Bacillus Cereus **In Milk And** Whey Cheese + +Francesca Pedonese, Giada Verani, Beatrice Torracca, Barbara Turchi, Antonio Felicioli, Roberta Nuvoloni Department of Veterinary Sciences, University of Pisa, Italy + +## Abstract + +Propolis antimicrobial activity has been limitedly studied in food, particularly in dairy products. We studied the antimicrobial activity of an alcoholic extract of an Italian propolis in sterile skim milk, pasteurized cow's milk, and cow's and goat's whey cheese (ricotta). Following the determination of the minimal inhibitory concentration on Gram+ and Gram- bacteria, the extract was employed at 2 and 5% (P2, P5), +using controls with the same ethanol concentrations (E2, E5) and without any addition. In milk trials, *Listeria monocytogenes*, +Staphylococcus aureus, Bacillus cereus, and *Pseudomonas fluorescens* were tested. P2 and P5 samples registered significant decreases of Gram+ bacteria in skim milk. The same was true for P5 in cows' milk, but only with *S. aureus* for P2. Ricotta was inoculated with *L. monocytogenes*, S. aureus and *B. cereus* and stored at 8.5°C. In cow's milk ricotta, *L. monocytogenes* counts in P5 were always lower than control during the storage time, significantly so from the 14th day. In goat's ricotta, *L. monocytogenes* counts in P5 were at least one logarithm lower than E5, whereas the extract didn't show a significant effect on S. + +aureus and *B. cereus*. The antimicrobial activity of propolis, particularly on L. + +monocytogenes, could be employed in ready-to-eat refrigerated dairy products. + +## Introduction + +Among beehive products, propolis is a complex material, collected by honeybees from plant buds and exudates and enriched by beeswax and bee secretions (Zabaiou et al., 2017). It has been used in folk medicine for centuries and its beneficial effects are well known (Sforcin and Bankova, 2011) +and attributed to a variety of active compounds, including flavonoids (Huang *et al.*, + +## 2014). + +A promising area of propolis use involves its application as a preservative in different foods, especially fruit juices, fruits and vegetables, due to its antimicrobial and antioxidative properties (Bankova *et al.*, 2016). Though the antimicrobial effect of propolis has been extensively studied (Banskota *et al.*, 2001; De Vecchi and Drago, 2007), knowledge about its antimicrobial activity in food of animal origin is quite limited and mostly focused on meat and fish products, where beneficial antimicrobial effects (mainly decrease of mesophilic and psychrotrophic counts) are reported in beef patties, sausages, filleted and minced fish meat (Pobiega *et al.*, 2019). + +There are only few studies about milk and/or dairy products and they are focused on some specific applications of the propolis antimicrobial properties (shelf-life of cheese: Metwalli, 2011; *L. monocytogenes* in refrigerated milk: Thamnopoulos *et al.*, +2018). With the aim to increase knowledge about practical effects of propolis in milk and dairy products, we evaluated the antimicrobial activity of an Italian propolis against food microorganisms in milk at optimum growth temperature and investigated the antimicrobial effects on *L. monocytogenes, Staphylococcus aureus* and Bacillus cereus in a refrigerated ready-toeat dairy product (ricotta), commonly consumed and potentially exposed to thermal abuse. + +## Materials And Methods Propolis And Ethanolic Extract Preparation + +Propolis was collected in Val di Cecina +(Tuscany, 50-450 m above sea level) in a single harvesting season. Pollen analysis of propolis was carried out following Ricciardelli D'Albore (1979). Raw propolis was stored at -20°C, finely ground, extracted with 70% ethanol (10 g in 100 ml) and repeatedly submitted to refrigerated centrifugation (9000 rpm, 15 min) and filtration phases, after being frozen at -20°C, to remove insoluble waxes. On the obtained ethanolic extract of propolis (EEP), flavonoids quantification and determination of dry residue content were performed according to Popova *et al.* (2004) and Baldini *et al.* (1996), respectively. + +## Bacterial Cultures + +Overall, 7 different microorganisms of relevance for food safety and quality were studied, 4 Gram+ (*L. monocytogenes* ATCC +7644, *S. aureus* ATCC 25923, *S. aureus* Correspondence: Francesca Pedonese, Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy. Tel.: +39.050-2216707 - Fax: +39-050-2210654 E-mail: francesca.pedonese@unipi.it. Key words: Propolis, *Listeria monocytogenes,* Staphylococcus aureus, Bacillus cereus, Dairy. Conflict of interest: The authors declare no potential conflict of interest. Funding: this research was funded by Pisa University grants (Fondi di Ateneo). Received for publication: 17 January 2019. Revision received: 6 August 2019. Accepted for publication: 28 August 2019. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). + +©Copyright: the Author(s), 2019 Licensee PAGEPress, Italy Italian Journal of Food Safety 2019; 8:8036 doi:10.4081/ijfs.2019.8036 ATCC 35556, high biofilm-producer, B. + +cereus DSV12, a wild strain of food origin) and 3 Gram- (*Salmonella enterica* serovar Typhimurium ATCC 14028, Escherichia coli ATCC 25922, *Pseudomonas fluorescens* ATCC 13525). For milk and whey cheese trials, those showing the highest in vitro susceptibility to EEP in preliminary tests were chosen. + +## Antimicrobial Disk Susceptibility Test + +The test was made according to CLSI +(2013), using Tryptone Soy Agar (TSA, +Oxoid, Basingstoke, UK), a bacterial *inoculum* of 0.5 McFarland turbidity and sterile filter paper disks with 10 µL each of EEP, 70% ethanol and dimethyl sulfoxide (DMSO); the inhibition zone diameters were determined after 24 hours of incubation at 25°C (pseudomonads) and 37°C +(other bacteria). + +## Minimal Inhibitory Concentration And Minimal Bactericidal Concentration Determination Assay + +MIC values (the lowest concentration that inhibits visible microbial growth) were determined for EEP and 70% ethanol following Wiegand *et al.* (2008) with minor modifications. The assay was performed in microtiter plates using 10 μL of bacterial inoculum and 190 μL of each dilution. EEP +was diluted in DMSO (1:3) and two-fold dilutions from 1/8 to 1/16,384 were prepared in Tryptone Soy Broth (Oxoid). For the bacterial *inoculum* an overnight broth culture of each microorganism, spectrophotometrically adjusted at about 1.5×108 cfu/mL, was used. The microplates were incubated at 25°C (pseudomonads) and 37°C (other bacteria) for 24 hours. For MBC assay, a loopful from MIC and higher dilutions wells was inoculated onto TSA, with the same incubation, and the lowest concentration with no growth was considered as the MBC value. MIC/MBC assays were made in triplicate. + +## Quantification Of Eep And Ethanol Effect On Bacterial Growth In Milk + +Bacterial growth was tested in sterilized skim milk (Skim Milk Powder, Oxoid) and in cow's milk, pasteurized at 63°C for 30 minutes. Total bacterial counts were determined in Plate Count Agar (Oxoid) at 30°C +for 72 hours before and after milk pasteurization. An overnight culture of each microorganism (the Gram+ ones and *P. fluorescens*) was inoculated at 1% in each type of milk with 2% EEP (P2), 5% EEP (P5), 2% ethanol (E2), 5% ethanol (E5) and in milk alone (control). After 24 hours at 37°C (25°C for *P. fluorescens*), bacterial counts were determined on the following media (Oxoid): Listeria Selective Agar Base with Oxford Supplement for *L. monocytogenes* (Heo *et al.*, 2014), Baird Parker Agar with Egg Yolk-Tellurite for coagulase-positive staphylococci (UNI, 2004), Mannitol Egg Yolk Polymixin Agar for *B. cereus* (FDA BAM, 2017), Pseudomonas Agar Base with CFC Supplement for *P. fluorescens* (Chiesa et al., 2014). + +## Quantification Of Eep And Ethanol Effect On Bacterial Growth In Whey Cheese + +Bacterial growth was tested in an industrial cow's whey cheese and in an artisanal goat's one, purchased at the beginning of shelf-life, at retail level and directly from Article the producer, respectively, and immediately inoculated. P2, P5, E2, E5 and control samples were prepared and inoculated with a pool *inoculum* of 4 chosen microorganisms +(the Gram+ ones). Before mixing, the concentration of each microorganism was spectrophotometrically adjusted in saline solution at about 1.5×108 cfu/mL. The final inoculum was used at 1% in ricotta. Control samples without *inocula* were tested to exclude the presence of *L. monocytogenes*, +S. aureus and *B. cereus*. Ricotta samples were stored at 8.5±0.5°C for 28 and 14 days, for cow's and goat's product, respec- + +| the tested microorganisms. | MIC | MBC | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------|---------|-------|---------| +| | EEP | Ethanol | EEP | Ethanol | +| L.m. 7644 | 1.78 | 13.91 | 7.11 | 55.63 | +| S.a. 25923 | 0.89 | 13.91 | 3.55 | 55.63 | +| S.a. 35556 | 1.78 | 13.91 | 3.55 | 55.63 | +| B.c. DSV12 | 0.89 | 13.91 | 3.55 | 55.63 | +| S.T. 14028 | 3.55 | 13.91 | 28.44 | 55.63 | +| E.c. 25922 | 3.55 | 13.91 | 7.11 | 55.63 | +| P.f. 13525 | 1.78 | 13.91 | 28.44 | 55.63 | +| EEP: ethanolic extract of propolis. L.m.: Listeria monocytogenes; S.a.: Staphylococcus aureus; B.c.: Bacillus cereus; S.T.: Salmonella enterica serovar Typhimurium; E.c.: Escherichia coli; P.f.: Pseudomonas fluorescens. Results are the mode of three independent trials. | | | | | + +| Table 2. Growth of the tested microorganisms in milk with different percentages of propolis ethanolic extract and ethanol. P2 P5 E2 E5 C Skim milk L.m. 7644 6.40±0.48b 3.92±0.14c 8.04±0.19a 7.50±0.34aa 8.27±0.07a (-1.87) (-4.35) (-0.23) (-0.77) S.a. 25923 5.98±0.50b 5.31±0.26b 7.84±0.16a 7.46±0.07a 7.96±0.08a (-1.98) (-2.65) (-0.12) (-0.50) S.a. 35556 5.74±0.33b 5.33±0.42b 8.19±0.05a 8.09±0.13a 8.37±0.08a (-2.63) (-3.04) (-0.18) (-0.28) B.c. DSV12 3.31±0.35b 2.94±0.32b 6.57±0.92a 6.86±0.24a 6.03±0.44a (-2.72) (-3.09) (+0.54) (+0.83) P.f. 13525 6.78±0.11c 6.55±0.07c 7.45±0.41ab 6.95±0.20bc 7.87±0.18a (-1.09) (-1.32) (-0.42) (-0.92) Pasteurized cow's milk L.m. 7644 5.59±0.31a 4.66±0.22b 5.79±0.45a 5.82±0.14a 6.06±0.31a (-0.47) (-1.40) (-0.27) (-0.24) S.a. 25923 6.30±0.16c 5.09±0.13d 7.10±0.31ab 6.62±0.27bc 7.42±0.21a (-1.12) (-2.33) (-0.32) (-0.80) S.a. 35556 7.04±0.35a 4.49±0.09b 7.44±0.43a 7.13±0.36a 7.71±0.32a (-0.67) (-3.22) (-0.27) (-0.58) B.c. DSV12 6.21±0.27aa 3.12±0.56b 5.75±0.28aa 6.25±0.21aa 5.97±0.47a (0.24) (-2.85) (-0.22) (0.28) P.f. 13525 7.32±0.20 6.98±0.24 7.18±0.36 6.89±0.42 7.15±0.16 (0.17) (-0.17) (0.03) (-0.26) P2: milk with 2% propolis ethanolic extract; P5: milk with 5% propolis ethanolic extract; E2: milk with 2% ethanol (70%); E5: milk with 5% ethanol (70%); C: control (milk). L.m.: Listeria monocytogenes; S.a.: Staphylococcus aureus; B.c.: Bacillus cereus; P.f.: Pseudomonas fluorescens. Results are mean values of three independent trials ± standard deviation. In brackets: difference in bacterial counts in comparison with the corresponding control. Values are expressed in log cfu/mL. abcDifferent letters in the same row denote significant differences (P<0.05). | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + + Article tively, based on their shelf-life and analyzed immediately after inoculation (t0) and weekly (t7, t14, t21, t28) to quantify the different microorganisms, as described for milk tests. + +## Statistical Analysis + +For each microorganism and substrate +(milk and whey cheeses) one-way ANOVA +and Tukey HSD test for *post-hoc* comparisons were performed (R software v. 3.5.0, R Foundation for Statistical Computing, Vienna, Austria), to evaluate the differences in bacterial counts at each time, considering the sample type (P2, P5, E2, E5, C) as factor. Differences were considered statistically significant with P value <0.05. + +| and ethanol during storage. | P2 | P5 | E2 | E5 | C | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------|-------------|------------|-------------|-------------|------------| +| Cow's whey cheese | | | | | | | +| L.m. 7644 | t0 | 5.48±0.43 | 5.58±0.28 | 5.59±0.34 | 5.50±0.32 | 5.59±0.27 | +| | (-0.11) | (-0.01) | (0.00) | (-0.09) | | | +| | t7 | 6.53±1.66 | 5.38±0.36 | 6.06±0.40 | 5.39±0.92 | 7.20±0.89 | +| | (-0.67) | (-1.82) | (-1.14) | (-1.81) | | | +| | t14 | 7.30±0.74a | 5.02±0.24b | 7.32±0.02a | 5.79±0.12ab | 7.30±0.74a | +| | (0.00) | (-2.28) | (0.02) | (-1.51) | | | +| | t21 | 7.12±0.91ab | 4.87±0.21b | 7.67±0.19a | 6.45±1.06ab | 8.13±0.10a | +| | (-1.01) | (-3.26) | (-0.46) | (-1.68) | | | +| | t28 | 7.71±0.08a | 4.53±0.48b | 7.99±0.17a | 6.86±0.96a | 8.22±0.18a | +| | (-0.51) | (-3.69) | (-0.23) | (-1.36) | | | +| S.a. 25923/35556 | t0 | 5.41±0.08 | 5.36±0.04 | 5.43±0.01 | 5.25±0.10 | 5.37±0.10 | +| | (0.04) | (-0.01) | (0.06) | (-0.12) | | | +| | t7 | 4.75±0.20 | 4.56±0.45 | 4.97±0.38 | 4.88±0.21 | 4.85±0.16 | +| | (-0.10) | (-0.29) | (0.12) | (0.03) | | | +| | t14 | 4.38±0.51 | 3.81±1.11 | 4.81±0.32 | 4.58±0.03 | 4.38±0.35 | +| | (0.00) | (-0.57) | (0.43) | (0.20) | | | +| | t21 | 4.27±0.65 | 3.52±1.10 | 4.73±0.12 | 4.38±0.03 | 4.59±0.15 | +| | (-0.32) | (-1.07) | (0.14) | (-0.21) | | | +| | t28 | 4.29±0.91 | 3.34±0.80 | 4.63±0.21 | 4.12±0.08 | 4.11±0.38 | +| | (0.18) | (-0.77) | (0.52) | (0.01) | | | +| B.c. DSV12 | t0 | 3.71±0.70 | 3.68±0.68 | 3.73±0.64 | 3.61±0.73 | 3.75±0.65 | +| | (-0.04) | (-0.07) | (-0.02) | (-0.14) | | | +| | t7 | 1.57±0.98 | 1.33±0.58 | 3.37±0.58 | 2.71±0.98 | 3.23±0.46 | +| | (-1.66) | (-1.90) | (0.14) | (-0.52) | | | +| | t14 | 1.35±0.49 | 1.35±0.49 | 2.44±1.05 | 2.00±0.43 | 2.30±0.85 | +| | (-0.95) | (-0.95) | (0.14) | (-0.30) | | | +| | t21 | 1.00±0.00 | 1.00±0.00 | 1.35 ± 0.49 | 1.00±0.00 | 1.85±0.21 | +| | (-0.85) | (-0.85) | (-0.50) | (-0.85) | | | +| | t28 | 1.00±0.00 | 1.00±0.00 | 1.23±0.40 | 1.57±0.51 | 1.57±0.51 | +| | (-0.57) | (-0.57) | (-0.33) | (0.00) | | | +| Goat's whey cheese | | | | | | | +| L.m. 7644 | t0 | 5.37±0.18 | 5.40±0.21 | 5.41±0.25 | 5.45±0.22 | 5.46±0.29 | +| | (-0.09) | (-0.06) | (-0.05) | (-0.01) | | | +| | t7 | 7.00±0.11a | 5.44±0.28b | 6.90±0.24a | 6.86±0.31a | 7.10±0.08a | +| | (-0.10) | (-1.66) | (-0.21) | (-0.24) | | | +| | t14 | 7.01±0.15ab | 5.70±0.72b | 7.06±0.14ab | 6.97±0.18ab | 7.16±0.14a | +| | (-0.15) | (-1.46) | (-0.10) | (-0.19) | | | +| S.a. 25923/35556 | t0 | 4.98±0.28 | 5.00±0.28 | 5.07±0.39 | 4.96±0.30 | 4.92±0.23 | +| | (0.06) | (0.08) | (0.15) | (0.04) | | | +| | t7 | 5.10±0.19 | 4.95±0.30 | 5.25±0.31 | 5.11±0.10 | 5.18±0.03 | +| | (-0.08) | (-0.23) | (0.07) | (-0.07) | | | +| | t14 | 5.08±0.08 | 4.87±0.24 | 5.16±0.23 | 5.18±0.07 | 5.03±0.02 | +| | (0.05) | (-0.16) | (0.13) | (0.15) | | | +| B.c. DSV12 | t0 | 3.86±0.25 | 3.91±0.24 | 3.83±0.32 | 3.65±0.36 | 3.84±0.26 | +| | (0.02) | (0.07) | (-0.01) | (-0.19) | | | +| | t7 | 3.04±0.80 | 2.27±0.38 | 3.07±0.75 | 3.12±0.81 | 3.01±0.38 | +| | (0.03) | (-0.74) | (0.06) | (0.11) | | | +| | t14 | 1.00±0.00 | 1.00±0.00 | 1.35±0.49 | 1.00±0.00 | 1.35±0.49 | +| | (-0.35) | (-0.35) | (0.00) | (-0.35) | | | +| P2: milk with 2% propolis ethanolic extract; P5: milk with 5% propolis ethanolic extract; E2: milk with 2% ethanol (70%); E5: milk with 5% ethanol (70%); C: control (milk). L.m.: Listeria monocytogenes; S.a.: Staphylococcus aureus; B.c.: Bacillus cereus. Results are mean values of two independent trials ± standard deviation. In brackets: difference in bacterial counts in comparison with the corresponding control. Values are expressed in log cfu/g. abcDifferent letters in the same row denote significant differences (P<0.05). | | | | | | | + +## Results Characterization Of Propolis And Eep + +Pollen analysis of propolis revealed a wide variety of pollen types. They were identified, according to Louveaux *et al.* (1978), as belonging to *Castanea* (27% of total pollen), *Hedera* (10%), and Coronilla/Hippocrepis, *Olea* f., *Pinus* f., +Trifolium gr., *Quercus* gr., all in percentages of 4.5-7%, with various *Compositae*, Umbelliferae and *Graminaceae* in lower quantities. A dry residue content of 8.44% and a flavonoids content of 2.3% (w/w) was found in EEP. + +## Antimicrobial Activity Of Eep + +Gram+ bacteria showed EEP inhibition zone diameters of 12 mm (*L. monocytogenes*), 13 mm (*B. cereus*) and 19-20 mm +(*S. aureus*), higher than those of Gram- (1011 mm). Ethanol and DMSO did not inhibit any of the strains, except for a slight inhibition recorded for ethanol against *P. fluorescens* (7 mm) and *E. coli* (8 mm). As for EEP MIC (Table 1), values of 0.89-1.78 mg/mL were obtained for Gram+ bacteria, and in the range 1.78 (*P. fluorescens*)-3.55 mg/mL for Gram-; ethanol values were ever 13.91 mg/mL, showing that the predominant effect was exerted by propolis compounds. EEP MBC values for Gram+ were 3.55-7.11 mg/mL, and 7.11-28.44 mg/mL for Gram-, with ethanol values of 55.63 mg/mL. + +## Effect Of Eep And Ethanol On Bacterial Growth In Milk + +As shown in Table 2, P2 and P5 determined in skim milk significant differences in counts in comparison with control for all microorganisms. However, for *P. fluorescens*, a lower EEP effect was noted, together with a significant inhibiting effect of E5. As for the pasteurized cow's milk, total bacterial count was 3.80 log cfu/mL in raw milk and below 2.18 log cfu/mL after the pasteurization. Its results were similar to skim milk, although counts significantly decreased only in P5 for all microorganisms, with the exception of *P. fluorescens*, +and in P2 just in the case of *S. aureus* 25923; apart from *P. fluorescens*, E5 effect was lower of, at least, 1.16 log cfu/mL in comparison with P5. + +## Effect Of Eep And Ethanol On Bacterial Growth In Whey Cheese + +Results are shown in Table 3. In cow's product, in P5 *L. monocytogenes* counts were lower than in control at each time during storage, with differences progressively higher and statistically significant from t14. In comparison with E5, differences in counts, negligible at t7, increased in time, showing an inhibiting effect of propolis rather than of ethanol. As for *S. aureus*, the 2 strains were enumerated together, but the 2 types of colonies were different enough to confirm that the strains were balanced in their growth. EEP didn't obtain significant antibacterial effects; this was probably influenced by the fact that, unsurprisingly, staphylococci didn't grow at 8.5°C and even decreased over time, including in control. Anyway, P5 was the most active, with differences of 0.86 (t21) and 0.76 log cfu/g (t28) between P5 and E5. Overall, *B. cereus* showed lower counts than the other bacteria in milk and whey cheese controls. In cow's whey cheese, as for *S. aureus*, counts decreased already in control, with too low values to correctly evaluate the effect of EEP and ethanol. Anyway, at t7 P5 gave rise to 1.90 and 1.38 log cfu/g differences versus control and versus E5, respectively. In goat's product, as for *L. monocytogenes*, significant differences versus control were present at t7 and t14. Significant differences between P5 and E5 were obtained at t7, but not at t14, due to high standard deviations; differences were in both cases higher than 1 log cfu/g. + +## Discussion + +The chemical composition of propolis strictly depends from the plant sources and is a result of geographical location, climate conditions and environmental factors (Bankova *et al.*, 2016). The pollen types found in our propolis were related to the type of vegetation of the geographical environment, with woodland and rural areas. + +Particularly, chestnut groves were situated within the production area and were responsible for the high content in *Castanea* pollen. Dry residue of EEP was analogous to that of Gutiérrez-Cortés and SuarezMahecha (2014), and in the range recorded by Cveck *et al.* (2007) and Barbeira *et al.* (2013). Flavonoids content was in the range determined by Marghitas *et al.* (2010) in ethanolic extracts of Romanian propolis. The same authors used EEP to determine the antimicrobial activity with a disk diffusion test on *L. monocytogenes, S. aureus*, B. + +cereus, E. coli and *P. aeruginosa* and obtained inhibition values comparable to ours. Considering EEP MIC values, various authors found results similar to ours: Miorin et al. (2003) obtained values of 0.36-3.65 mg/mL for *S. aureus* strains, slightly higher than those of Freitas Santana *et al.* (2012). More recently, Ristivojević *et al.* (2016) +found MIC of 0.1-1.9 mg/mL for *L. monocytogenes* and 0.4-13.7 mg/mL for S. + +aureus. Finally, Mascheroni *et al.* (2014) +using chitosan-propolis beads found MIC values of 0.8-1 mg/mL for *S. aureus*, L. innocua and *B. cereus*. Our study focused on milk and whey cheese, and, at the best of our knowledge, trials in milk at *optimum* bacterial temperature and in whey cheese during refrigerated storage were not performed before. Results showed some differences in bacterial behavior in the different growth substrates, but, noteworthily, in all tested matrices, EEP revealed a not negligible antimicrobial effect, higher than that determined by ethanol, particularly against L. monocytogenes. + +## Conclusions + +The noteworthy antimicrobial activity of propolis, particularly on *L. monocytogenes*, together with its beneficial properties, could be advantageously exploited, especially in ready-to-eat dairy products to be stored in refrigerated conditions. The development of non-alcoholic formulations (Jansen-Alves *et al.,* 2018) could make it suitable for all consumers. + +## References + +Baldini M, Fabietti F, Giammarioli S, Onori R, Orefice L, Stacchini A, 1996. Metodi di analisi utilizzati per il controllo chimico degli alimenti. Rapporti ISTISAN +96/34, Istituto Superiore di Sanità, Roma, Italy. ISSN 1123-3117. + +Bankova V, Popova M, Trusheva B, 2016. + +New emerging fields of application of propolis. Maced J Chem Chem Eng 35:1-11. + +Banskota AH, Tezuka Y, Kadota S, 2001. + +Recent progress in pharmacological research of propolis. Phytoter Res 15:561-71. + +Barbeira PJS, Paganotti RSN, Ássimos AA, +2013. Development of a multivariate calibration model for the determination of dry extract content in Brazilian commercial bee propolis extracts through UV-Vis spectroscopy. Spectrochim Acta A Mol Biomol Spectrosc 114:441-8. + +Chiesa F, Lomonaco F, Nucera D, Garoglio D, Dalmasso A, Civera T, 2014. Distribution of *Pseudomonas* species in a dairy plant affected by occasional blue discoloration. Ital J Food Saf 9:1722. + +Clinical and Laboratory Standards Institute +(CLSI), 2013. Performance standards for antimicrobial susceptibility testing; twenty-third informational supplement. CLSI document M100-S23. Wayne, PA, USA. + +Cveck J, Medić-Sarić M, Jasprica I, Zubčić S, Vitali D, Mornar A, VedrinaDragojević, Tomić S, 2007. + +Optimisation of an extraction procedure and chemical characterisation of Croatian propolis tinctures. Phytochem Anal 18:451-9. + +De Vecchi E, Drago L, 2007. Propolis antimicrobial activity: what's new? Infez Med 15:7-15. + +FDA BAM (U. S. Food and Drug Administration Bacteriological Analytical Manual). *Bacillus cereus*. + +Available from: +https://www.fda.gov/food/laboratorymethods-food/bam-bacillus-cereus. + +Accessed July 28th 2019. + +Freitas Santana H, Teixeira Barbosa AA, +Ferreira SO, Cuquetto Mantovani H, 2012. Bactericidal activity of ethanolic extracts of propolis against Staphylococcus aureus isolated from mastitic cows. World J Microbiol Biotechnol 28:485-91. + +Gutiérrez-Cortés C, Suarez Mahecha H, +2014. Antimicrobial activity of propolis and its effect on the physicochemical and sensorial characteristics in sausages. Vitae Columbia 21:90-6. + +Heo EJ, Song BR, Park HJ, Kim YJ, Moon JS, Wee SH, Kim JS, Yoon Y, 2014. + +Rapid detection of *Listeria monocytogenes* by Real time PCR in processed meat and dairy products. J Food Prot 77:453-8. + +Huang S, Zhang C-P, Wang K, Li GQ, Hu F-L, 2014. Recent advances in the chemical composition of propolis. Molecules 19:19610-32. + +Jansen-Alves C, Fernandes KF, CrizelCardoso MM, Krumreich FD, Borges CD, Zambiazi RC, 2018. + +Microencapsulation of propolis in protein matrix using spray drying for application in food systems. Food Bioprocess Tech 11:1422-36. + +Louveaux J, Maurizio A, Vorwohl G, 1978. + +Methods of melissopalynology. Bee World 59:139-57. + +Marghitas LA, Mihai CM, Chirila F, +Dezmirean DS, Fit N, 2010. The study of the antimicrobial activity of Transylvanian (Romanian) propolis. Not Bot Horti Agrobo 38:40-4. + +Mascheroni E, Figoli A, Musatti A, Limbo S, Drioli E, Suevo R, Talarico S, Rollini M, 2014. An alternative encapsulation approach for production of active chitosan-propolis beads. Int J Food Sci Tech 49:1401-7. + +Metwalli SAH, 2011. Extended shelf-life of Kareish cheese by natural preservatives. Egypt. J Agric Res 89:639-49. + +Miorin PL, Levy Junior NC, Custodio AR, +Bretz WA, Marcucci MC, 2003. Antibacterial activity of honey and propolis from *Apis mellifera* and Tetragonisca angustula against Staphylococcus aureus. J Appl Microbiol 95:913-20. + +Pobiega K, Kraśniewska, K, Gniewosz M, +2019. Application of propolis in antimicrobial and antioxidative protection of food quality - A review. Trends Food Sci Technol 83:53-62. + +Popova M, Bankova V, Butovska D, Petkov V, Nikolova-Damianova B, Sabatini AG, Marcazzan GL, Bogdanov S, 2004. Validated methods for the quantification of biologically active constituents of poplar-type propolis. Phytochem Anal 15:235-40. + +Ricciardelli D'Albore G, 1979. L'origine géographique de la propolis. Apidologie 10:241-67. + +Ristivojević P, Dimkić I, Trifković J, Berić T, Vovk I, Milojković-Opsenica D, +Stanković S, 2016. Antimicrobial activity of Serbian propolis evaluated by means of MIC, HPTLC, bioautography and chemometrics. PLoS One 11:e0157097. + +Sforcin JM, Bankova V, 2011. Propolis: is there a potential for the development of new drugs? J Ethnopharmacol 133:25360. + +Thamnopoulos I-A, Michailidis GF, +Fletouris DJ, Badeka A, Kontominas MG, Angelidis AS, 2018. Inhibitory activity of propolis against Listeria monocytogenes in milk stored under refrigeration. Food Microbiol 73:16876. + +UNI, 2004. UNI EN ISO 6888-1: 2004. + +[Microbiologia di alimenti e mangimi per animali - Metodo orizzontale per la conta di stafilococchi coagulasi- positivi (*Staphylococcus aureus* e altre specie) - Tecnica che utilizza il terreno agar Baird-Parker]. [Regulation in Italian]. Italian Unification Institute ed., Milan, Italy. + +Wiegand I, Hilpert K, Hancock RE, 2008. + +Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163-75. + +Zabaiou N, Fouache A, Trousson A, Baron S, Zellagui A, Lahouel M, Lobaccaro JMA, 2017. Biological properties of propolis extracts; something new from an ancient product. Chem Phys Lipids 207: 214-22. \ No newline at end of file diff --git a/medical/md_images/PMC7044854/3_image_0.png b/medical/md_images/PMC7044854/3_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..e742b581096ca98959a2764aeed1ae2ea2b74838 --- /dev/null +++ b/medical/md_images/PMC7044854/3_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e6d74fe023ee06fd18fa86fb8e7bf3f6a0fd631cc7cffc57ed4d0a68d09f16db +size 33485 diff --git a/medical/md_images/PMC7075768/0_image_0.png b/medical/md_images/PMC7075768/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..f86efc913662363056f6caa42656012b5d6e1101 --- /dev/null +++ b/medical/md_images/PMC7075768/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:04efcf76ff606e38c29af1b4adc9d9e08e0c73c4c6527e87dfb7c9b43e1ca8cf +size 14312 diff --git a/medical/md_images/PMC7075768/0_image_1.png b/medical/md_images/PMC7075768/0_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..64df58830331540df4c0f8413273bdd1cf484738 --- /dev/null +++ b/medical/md_images/PMC7075768/0_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:6a5d4acf96b28eb91d88e460723ced36d39fe339682a60c419b82a0991a532f7 +size 3056 diff --git a/medical/md_images/PMC7075768/0_image_3.png b/medical/md_images/PMC7075768/0_image_3.png new file mode 100644 index 0000000000000000000000000000000000000000..339f93faf47acbc9c984c23748dbe83f39bf7a9c --- /dev/null +++ b/medical/md_images/PMC7075768/0_image_3.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e8797030d6ddbf5ebe784bf8906eaaa13326da7255c7be60b9f6d304f0b3d702 +size 3199 diff --git a/medical/md_images/PMC7075768/0_image_5.png b/medical/md_images/PMC7075768/0_image_5.png new file mode 100644 index 0000000000000000000000000000000000000000..464338192d6355f24312363f4d8a9566b0595c58 --- /dev/null +++ b/medical/md_images/PMC7075768/0_image_5.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:d3280d22665462c8748b32e1eda50da7fcea0716a417978e48cbbeb5eff6179e +size 1865 diff --git a/medical/md_images/PMC7075768/2_image_0.png b/medical/md_images/PMC7075768/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..da1fd704e02c42fec0ed9a700f848ea73a043ce4 --- /dev/null +++ b/medical/md_images/PMC7075768/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:deafbf2a34bebe39443010b2933801f17d71fe7efd0436556d2df27941c36d77 +size 124790 diff --git a/medical/md_images/PMC7075768/2_image_2.png b/medical/md_images/PMC7075768/2_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..5b499ee6b21ab37fbefcf42e70331ac27a72372a --- /dev/null +++ b/medical/md_images/PMC7075768/2_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:bff88dcab265f87342fb81810a06dae4b067ebab749e58401b2749d5433ab969 +size 88099 diff --git a/medical/md_images/PMC7075768/PMC7075768_meta.json b/medical/md_images/PMC7075768/PMC7075768_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..0df28b3aec02201b35d7d7d8343dc5e6618f4881 --- /dev/null +++ b/medical/md_images/PMC7075768/PMC7075768_meta.json @@ -0,0 +1,137 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Cubital tunnel syndrome: a surgical modification to in situ decompression to improve results", + "level": 0, + "is_closed": false, + "n_kids": 7, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Materials and methods", + "level": 1, + "is_closed": false, + "n_kids": 1, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Operative procedure", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Results", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 794.0, + 596.0, + 794.0 + ] + }, + { + "title": "Discussion", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 794.0, + 596.0, + 794.0 + ] + }, + { + "title": "Conclusion", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 794.0, + 596.0, + 794.0 + ] + }, + { + "title": "Disclaimer", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 794.0, + 596.0, + 794.0 + ] + }, + { + "title": "References", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 794.0, + 596.0, + 794.0 + ] + } + ], + "pages": 6, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 4, + "code": 0, + "table": 3, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC7448026/25_image_0.png b/medical/md_images/PMC7448026/25_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..49bb9c7785e01e3a8ac8422128f01d2125f1f148 --- /dev/null +++ b/medical/md_images/PMC7448026/25_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:9c3192a3e7d54d8ca26c2e9cc13ea61d2d733e57b9092cc4c4369e8efc199d4c +size 15312 diff --git a/medical/md_images/PMC7448026/37_image_0.png b/medical/md_images/PMC7448026/37_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..e0bd015b400800d77ca0e3040ec69bb9de33ba1a --- /dev/null +++ b/medical/md_images/PMC7448026/37_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8b6dd0f3146da40b3d11979753c364c5ccafaea4eab206594509d66c79984ccf +size 15252 diff --git a/medical/md_images/PMC7448143/0_image_0.png b/medical/md_images/PMC7448143/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..e4923b55c74a9c725201780c2f0455758cb30f97 --- /dev/null +++ b/medical/md_images/PMC7448143/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:d3a77d6c8ce184416b1d9c5b2fe803add77afb46adf5c26a11bfa253310de349 +size 7354 diff --git a/medical/md_images/PMC7448143/5_image_0.png b/medical/md_images/PMC7448143/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..a5fd70a1da64b300051ee1656c48c35c4de511dc --- /dev/null +++ b/medical/md_images/PMC7448143/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:6ee26174a1f292564cc5e12de486fdcb2d20e5889a8adfad3f34297954696599 +size 45315 diff --git a/medical/md_images/PMC7707702/1_image_0.png b/medical/md_images/PMC7707702/1_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..18d5d1193a037c3d515f2bc7c44fac9207cc3789 --- /dev/null +++ b/medical/md_images/PMC7707702/1_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:9d51516420aca4997de83531fac866af8af34ca787c88976860f3c1d988ca754 +size 481856 diff --git a/medical/md_images/PMC7723529/0_image_0.png b/medical/md_images/PMC7723529/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..9eea925cdb5f43b0c6c1a3df1aa998230461f492 --- /dev/null +++ b/medical/md_images/PMC7723529/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:3e5aaa6822067c99f6051ec1fded85019cd8ddabc6701309fd2ddd172fc81586 +size 1582 diff --git a/medical/md_images/PMC7723529/2_image_0.png b/medical/md_images/PMC7723529/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..618d69dfb590fd02c8dab15be9dc11db752a9295 --- /dev/null +++ b/medical/md_images/PMC7723529/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:1954f7ea2f44395b71c0de1627db147948402b1d56d1dd58aeedec55284e0d6c +size 73948 diff --git a/medical/md_images/PMC7919521/0_image_1.png b/medical/md_images/PMC7919521/0_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..12ebb64d5afb74c78d7aeb3b9e8cdc51c0648f2e --- /dev/null +++ b/medical/md_images/PMC7919521/0_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0af3aab7229589cf109b2a88978ccf9477d60e62d1ca552b3aa7f34e7bad0b6d +size 5558 diff --git a/medical/md_images/PMC7919521/1_image_0.png b/medical/md_images/PMC7919521/1_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..4f7eb028dd8ab41f31ef297bbc9ba50bfe6367a7 --- /dev/null +++ b/medical/md_images/PMC7919521/1_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:66eeb4e125b5aca9ee06ef903028ce8f2a0f78e960d74092c4ae4409dea8940b +size 152597 diff --git a/medical/md_images/PMC8050878/1_image_0.png b/medical/md_images/PMC8050878/1_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..587ff9469445321e27da301c8a47a20832c45fb1 --- /dev/null +++ b/medical/md_images/PMC8050878/1_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:fc61734e4bbf56ce5dba770985d89f9a425332e870e438186080593fc240609b +size 40517 diff --git a/medical/md_images/PMC8050878/2_image_0.png b/medical/md_images/PMC8050878/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..5163d48db32a27ebc3af775899993585260ae5df --- /dev/null +++ b/medical/md_images/PMC8050878/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:23bcc8807834a44b3bbbb962f45914cedccaa85eb7c620990e8ae6d14b535171 +size 42095 diff --git a/medical/md_images/PMC8050878/PMC8050878.md b/medical/md_images/PMC8050878/PMC8050878.md new file mode 100644 index 0000000000000000000000000000000000000000..79179302df359dab8507a362bc1b889ae98a05c8 --- /dev/null +++ b/medical/md_images/PMC8050878/PMC8050878.md @@ -0,0 +1,148 @@ +# Developing The Covid-19 Intensive Care Medical Equipment Distribution Platform: Outcomes And Lessons **Learned** + +Emmanuel Adeoluwa Akinluyi ,1,2 David Stell,1 Nayanee Perera,1 Christopher Sibley-Allen1 + +## Abstract + +During the first wave of the coronavirus pandemic, the UK government took the decision to centralise the procurement, allocation and distribution of mission-critical intensive care unit (ICU) medical equipment. Establishing new supply chains in the context of global shortages presented significant challenges. This report describes the development of an innovative platform developed rapidly and voluntarily by clinical engineers, to mobilise the UK's shared medical equipment inventory, in order to match ICU capacity to dynamically evolving clinical demand. The 'Coronavirus ICU Medical Equipment Distribution' platform was developed to optimise ICU equipment allocation, distribution, collection, redeployment and traceability across the National Health Service. Although feedback on the platform has largely been very positive, the platform was built for a scenario that did not fully materialise in the UK and this affected the implementation approach. As such, it was not used to its full potential. Nonetheless, the platform and the insights derived and disseminated in its development have been extremely valuable. It provides a prototype for not only optimising system capacity in future pandemic scenarios but also a means for maximally exploiting the large amount of new equipment in the UK health system, as a result of the coronavirus pandemic. This early stage innovation has demonstrated that a system-wide pooled information resource can benefit the operations of individual organisations. It has also generated numerous lessons to be borne in mind in innovation projects. + +To cite: Akinluyi EA, Stell D, +Perera N, *et al*. Developing the COVID-19 intensive care medical equipment distribution platform: outcomes and lessons learned. *BMJ Open Quality* 2021;10:e001383. doi:10.1136/ bmjoq-2021-001383 Received 31 January 2021 Revised 27 February 2021 Accepted 12 March 2021 © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. + +1Medical Physics, Guy's and St Thomas' NHS Foundation Trust, London, UK +2School of Biomedical Engineering & Imaging Sciences, Faculty of Life Sciences and Medicine, King's College London, London, UK Correspondence to Dr Emmanuel Adeoluwa Akinluyi; Emmanuel.Akinluyi@kcl.ac.uk + +## Introduction Background + +During the Coronavirus pandemic's first wave, there was a need to rapidly expand the critical care capacity of the National Health Service (NHS). The UK government had taken the early decision to centralise the procurement, allocation and distribution of mission-critical intensive care unit (ICU) +medical equipment to meet this challenge.1 This system-level approach had the potential advantage of leveraging economies of scale in procurement, instead of local market forces determining outcomes. However, rapidly establishing new supply chains in the context of global shortages presented significant challenges. + +Clinical engineers are key specialists whose remit includes 'developing, managing, assessing, installing and maintaining (health) +technologies for their safe and cost-effective use'.2 National and regional networks of clinical engineers formed in the early phase of the UK's pandemic response with the support of NHS England. These networks created communication channels between NHS clinical engineers and the central national teams, procuring and allocating ICU equipmentand they facilitated numerous initiatives.3 As clinical demand fluctuated across the UK, the network escalated the need for responsive and efficient allocation and redistribution of equipment. This would mean overcoming integration and communication challenges, to enable robust and effective ICU capacity where it was needed most. + +Here we describe an innovative platform developed rapidly and voluntarily by clinical engineers, to mobilise the UK's shared medical equipment inventory, in order to match ICU capacity to dynamically evolving clinical demand. It involved ICU equipment allocation, distribution, collection, redeployment and traceability across the NHS. + +## Objectives + +The team hoped to address several challenges: +► *Provide early sight of incoming unfamiliar* equipment models to clinical engineering teams. + +► Allow organisations to express preference for equipment models: under normal circumstances, and especially when clinical staff are newly trained and stressed, standardised inventory is safety critical4 5—these communications enable this. + +► *Signal demand*: allowing organisations to communicate their current and forecasted shortfall, on the basis of local arrangements. + +## + +► *Enable optimal allocation*: incorporating the above into decision-support for allocators. + +► *Assure efficiency and robustness*: in equipment receipt and deployment6 ; cutting out costly delays by engaging the correct stakeholders in each organisation. + +► *Enable transfers between organisations*: enabling direct-'swapping' between organisations to achieve standardisation. + +► *Improve traceability* of centrally owned equipment assets + +## Methods + +Early in the pandemic, the designers recognised the need for centralised equipment distribution teams to be able to offer equipment to healthcare providers and for healthcare providers to be able to accept or reject offers based on local need. The Coronavirus ICU Medical Equipment Distribution (CIMED) platform was conceived to address this. + +The CIMED platform requirements (in the form of +'user stories'7) were captured from key stakeholders including regional and national equipment distributors, NHS Trust Clinical Engineering leads and logistics providers. The final list of requirements was related to redistribution and return of equipment and the need for nationwide traceability. + +Initially the designers envisaged a database with web interfaces for equipment distributors and for hospitalbased equipment management staff. The distributors' interface would allow them to offer equipment to hospitals, which they could then accept or reject using their own interface. Existing products were surveyed, but no suitable option was identified. A commercial web developer was approached with the concept; however, the offered development timeframe was prohibitive. The designers, therefore, opted to develop their own solution. + +To minimise development time, the designers used a pre-existing web-based office suite (Google docs) as a development platform. This suite includes spreadsheet software (used for data storage and the user interface), an email client (used to send notifications to users and to the logistics provider) and a scripting platform (used to create workflows and relational database functionality). + +In the absence of upfront resourcing, this was a voluntary effort with no guaranteed buy-in. + +## Results What Was Delivered? + +A system of interconnected tables was created (see figure 1). These were used for capture, storage, transfer and reporting of data relevant to equipment allocation and distribution. Workflows were email driven. + +The system included a reference data set that held a complete inventory of all centrally procured equipment, including location information, together with contact and delivery details for every acute healthcare provider in the UK. + +'Region profiles' were created to allow regional equipment allocators to view a complete list of the providers in + +![1_image_0.png](1_image_0.png) + +their region, a list of all the equipment available for allocation. They would have the ability to allocate equipment efficiently (more so, using optimisation-driven decision support) and to offer equipment to individual providers with a few mouse clicks, instead of via time-consuming phone and email conversations. + +'Organisation profiles' were created for all healthcare providers. Identifying the relevant contacts was a significant challenge. Users within these organisations could use their profile to enter contact and delivery details, together with information about local equipment needs and preferences. The profiles contained a list of equipment located with that provider, drawn from the reference data set. Users could arrange equipment return to central loan stock and accept or reject equipment offers from regional allocators with a few mouse clicks. The profile also gave users early sight of the models available for allocation with a complete list of all models available within the system. + +A 'transfer report', viewable by the national logistics provider, contained summary information about all equipment deliveries and collections arranged using CIMED. This information was sent by email every time a delivery or collection was requested by any user of the system. Barcode-scannable collection slips with equipment decontamination prompts and declarations were generated and emailed out. + +## How Was It Used? + +CIMED was used by the national logistics provider. A relatively small number of healthcare providers used the system directly to return superfluous equipment and to reconcile central inventory records with their local inventory data. Others used a more manual process, which was partly defined by the CIMED team. + +## Discussion Performance + +CIMED worked and has been used; feedback has been very positive. The platform was robust and stable with + +![2_image_0.png](2_image_0.png) + +users reporting no problems related to its core functionality. Users at some institutions had difficulty accessing the platform due to local firewall settings—however most engineers were able to resolve this. Two users commented that some elements of the system were slow to update and respond. The designers agree that these are important limitations. + +These limitations could be overcome by changing the system architecture. This would require use of a custom web application and SQL database. This option had been rejected in the early stages due to the development time required. With hindsight, given the limitations of the office suite and the functionality required in the finished product, development of a custom web application may have been preferable. However, this could not have been foreseen with the information available at the beginning of the project. + +CIMED relied on equipment location data gathered from central equipment distributors. This data had been recorded in different ways and to varying quality standards. The majority of queries from health organisations that adopted CIMED were concerning errors in this underlying data. Correction of these errors required direct user engagement, which was labour intensive although curating the inventory data were an unexpected benefit of the system. + +## Extent Of System-Wide Adoption + +Central distributors initially saw CIMED as a platform for coordinating return of medical equipment from healthcare providers to central loan stock. Contacts at over 100 healthcare organisations were given access to CIMED. Eighty-two transfers were issued via CIMED *directly* in the first wave. Many opted to use the manual equivalent, which used CIMED templates for email-based transfer requests to be handled by a dedicated central team. + +The user access mechanism was a significant limitation. Users were issued personal links, rather than using more familiar, navigate-to and login processes. In the period between the first and second waves, most organisations naturally continued to hold equipment. This was a natural response to uncertainty, while system-wide plans for scaling up ICU capacity were being clarified. Similar behaviour is observed in local 'medical equipment library' services.8 As a result, the traffic of medical equipment transfers as a whole, corresponded to increases in ICU activity as illustrated in figure 2. As each user engaged with the platform intermittently, retrieving/recalling their personal link proved harder than initiating the manual process for transfer requests. + +## Extent Of Adoption By Central Decision-Makers + +While CIMED became a tool for mobilising national ICU equipment capacity via a returns process, features that would facilitate the (re)allocation of equipment and interorganisational transfers were not adopted. This platform was developed voluntarily by one particular service rather than being centrally commissioned. Hence, the team simultaneously built and promoted the solution. In an unfamiliar, highly sensitive environment, with sporadic demand levels, reluctance from would-be adopters was understandable. Although feedback was positive, the required promotion alongside supporting local pandemic commitments exceeded our capacity as volunteers. + +## Conclusions Impact + +CIMED had limited direct impact, as it was built for a scenario that did not fully materialise in the UK. Its development, however, drove engagement between operational teams and decision-makers, informing various other logistic processes in the pandemic. + +## Key Lessons + +The CIMED platform shows that a system-wide pooled information resource can benefit the operations of individual organisations. Philanthropic efforts can rapidly develop innovative solutions, but rapid adoption is complex and should not be underestimated when planning. Clinical engineers and other professional groups can play an important role. + +## Managing Expectations + +At the outset, the risk of nonadoption was fully understood by the development team. Partial adoption and limited use, while disappointing crucially represented part of the nation's pandemic readiness. It was important to reflect on and appreciate this, to motivate an alreadystretched workforce. + +## Further Work + +CIMED can yet be useful, and the redistribution process it supports is vital for the resettling of services in-between periods of high intensive care demand. Similarly the system remains available for the scenario of scarce devices and mismatch of supply and demand for ICU medical equipment. In a period of low activity, the London regional Clinical Engineering Network identified that CIMED could allow different providers to exchange heterogeneous medical equipment inventories in a mutually supported movement towards standardisation. + +The ambition to build regional partnerships to support healthcare delivery is at the forefront of the NHS long-term plan. The learning from this model of pan-organisational medical equipment asset management could inform the emerging integrated care systems structure within the NHS. Equipment services on a larger population footprint could improve economies of scale for purchasing, training and maintenance. It could also achieve better capture of data around inequalities in provision and safety and improve standardisation of equipment assets to best serve regional communities and organisations. The team is open to discussions on partnerships for reimplementing CIMED using a different architecture, for future pandemics or general use. More broadly, these experiences will inform subsequent efforts to explore, understand and improve technology adoption processes, for the benefit of patients and the public.9 Acknowledgements The authors would like to thank Ruth Thomsen—Regional Scientific Director, NHSE&I Medical Directorate. She and her team supported and informed the development of this platform. They were instrumental in gathering and engaging its users across the UK. The authors would also like to thank Professor Steve Keevil, Head of the Medical Physics Department, Guy's & St Thomas' NHS Foundation Trust, who recognised the need to support the wider system during the pandemic. + +Contributors EAA initiated the development. DS and EAA jointly developed the platform with support from NP and CS-A, and with DS leading on the implementation. CS-A facilitated the dissemination of the platform. All authors contributed to the final version of this paper. EAA is primarily responsible for the overall content. + +Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. + +Competing interests None declared. + +Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. + +Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data may be obtained from a third party and are not publicly available Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iD Emmanuel Adeoluwa Akinluyi http://orcid.org/0000-0002-4215-5559 + +## References + +1 National Audit Office. Investigation into how government increased the number of ventilators available to the NHS in response to COVID-19. + +The Stationery Office, 2020. Available: https://www.nao.org.uk/report/ increasing-ventilator-capacity-in-response-to-covid-19/ +2 World Health Organization. *Human resources for medical devices, the* role of biomedical engineers. Vancouver: World Health Organization, 2017: 33. + +3 Institute for Physics & Engineering in Medicine. UK contribution to global CE day 2020, 2020. Available: https://www.ipem.ac.uk/ AboutIPEM/PublicEngagement/GlobalClinicalEngineeringDay.aspx 4 Gibbins M, Kelly FE, Cook TM. Airway management equipment and practice: time to optimise institutional, team, and personal preparedness. *Br J Anaesth* 2020;125:221–4. + +5 Care Quality Commission. Supporting note: standardisation. Available: +https://www.cqc.org.uk/sites/default/files/documents/20110506_ supporting_note_-_standardisation_updated_for_external_publication. pdf [Accessed 24 Dec 2020]. + +6 Medicines and Healthcare products Regulatory Agency. *Managing* medical devices: guidance for healthcare and social services organisations, 2015: 22–6. + +7 Lucassen G, Dalpiaz F, Werf vander. The use and effectiveness of user stories in practice. In: *International working conference on* requirements engineering: foundation for software quality. Cham: Springer, 2021: 205–22. + +8 Werth J, Furniss D. Medical equipment library design: revealing issues and best practice using DiCoT. In: *Proceedings of the 2nd ACM* SIGHIT International Health Informatics Symposium, 2012: 583–92. + +9 Institution of Mechanical Engineers, Meese H. Healthcare solutions: +improving technology adoption. information and library service, institution of mechanical engineers, 2020. Available: https://www. + +imeche.org/docs/default-source/1-oscar/reports-policy-statementsand-documents/imeche-healthcare-solutions.pdf?sfvrsn=68e29c12_2 \ No newline at end of file diff --git a/medical/md_images/PMC8050878/PMC8050878_meta.json b/medical/md_images/PMC8050878/PMC8050878_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..b18dfdec9172ec98aab4a01944b4aa33b48bc0ab --- /dev/null +++ b/medical/md_images/PMC8050878/PMC8050878_meta.json @@ -0,0 +1,199 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Developing the COVID-19 intensive care medical equipment distribution platform: outcomes and lessons\u00a0learned", + "level": 0, + "is_closed": false, + "n_kids": 18, + "page_index": 0, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffAbstract\ufeff", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffIntroduction\ufeff", + "level": 1, + "is_closed": false, + "n_kids": 2, + "page_index": 0, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffBackground\ufeff", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffObjectives\ufeff", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffMethods\ufeff", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffResults\ufeff", + "level": 1, + "is_closed": false, + "n_kids": 2, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "What was delivered?", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "How was it used?", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeff\ufeffDiscussion\ufeff\ufeff", + "level": 1, + "is_closed": false, + "n_kids": 3, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffPerformance\ufeff", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Extent of system-wide adoption", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Extent of adoption by central decision-makers", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeff\ufeffConclusions\ufeff\ufeff", + "level": 1, + "is_closed": false, + "n_kids": 4, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffImpact\ufeff", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Key lessons", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Managing expectations", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Further work", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "\ufeffReferences\ufeff", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "Fit", + "view_pos": [] + } + ], + "pages": 4, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 16, + "code": 0, + "table": 0, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC8183208/1_image_0.png b/medical/md_images/PMC8183208/1_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..ac69fae776f9c5aedcd3a2ad5fef92ea8ba9810b --- /dev/null +++ b/medical/md_images/PMC8183208/1_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:23d03a3f5b7291ef70e224f38a35a3e344892ca2abd58998b250180a45985c62 +size 118088 diff --git a/medical/md_images/PMC8183208/2_image_2.png b/medical/md_images/PMC8183208/2_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..fc445a3b7d8fd73feec91ca3f378cd1ca124960f --- /dev/null +++ b/medical/md_images/PMC8183208/2_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:bbde90e87d34fc59055e83cf148f7312053476e07b16efe444e033e7f051c0d5 +size 104904 diff --git a/medical/md_images/PMC8643537/0_image_2.png b/medical/md_images/PMC8643537/0_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..c14e6d79e8b17d2ca8813f95a3b255b2cba76565 --- /dev/null +++ b/medical/md_images/PMC8643537/0_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:fbf6a307a48d40c5c2130e02b7f868bd654ecdb5ccd37d2474b48cc08f8c9fb3 +size 4032 diff --git a/medical/md_images/PMC8857593/0_image_0.png b/medical/md_images/PMC8857593/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..6b320bea604fcb1eb886badd8f56edf962c652c1 --- /dev/null +++ b/medical/md_images/PMC8857593/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:11aa7dac0185a833bd45e2938a505d1cf6a7ae874cca86012a56a8df2f09b020 +size 5194 diff --git a/medical/md_images/PMC8857593/0_image_2.png b/medical/md_images/PMC8857593/0_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..18bdbf2e3637fef4719824d51b551ae78b69b92f --- /dev/null +++ b/medical/md_images/PMC8857593/0_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:fad094b6638716de3d19e400ee62d2f36b4e1abb9d243936c710e92f461d7869 +size 14500 diff --git a/medical/md_images/PMC8857593/0_image_4.png b/medical/md_images/PMC8857593/0_image_4.png new file mode 100644 index 0000000000000000000000000000000000000000..589a8e4ce974c8a7f1cdbd6fef4769ca55894ffe --- /dev/null +++ b/medical/md_images/PMC8857593/0_image_4.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:7b8d820d3f918de1cdd1fd844b452bb2b8e4687d680665ebff41e49dfea2234b +size 4017 diff --git a/medical/md_images/PMC8857593/0_image_6.png b/medical/md_images/PMC8857593/0_image_6.png new file mode 100644 index 0000000000000000000000000000000000000000..b7da94e56b2a8855b264821bf08dcc29d979a933 --- /dev/null +++ b/medical/md_images/PMC8857593/0_image_6.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:20476d237b38933f1cebdcfcd7f92dde3eb20987d3fda7f7e503ca1951c02c09 +size 897 diff --git a/medical/md_images/PMC8857593/PMC8857593.md b/medical/md_images/PMC8857593/PMC8857593.md new file mode 100644 index 0000000000000000000000000000000000000000..3914f055fcbbf0e420f26ff1ae9569b951876ff4 --- /dev/null +++ b/medical/md_images/PMC8857593/PMC8857593.md @@ -0,0 +1,276 @@ + + +![0_image_3.png](0_image_3.png) + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +![0_image_4.png](0_image_4.png) + +![0_image_5.png](0_image_5.png) + +![0_image_7.png](0_image_7.png) + +journal homepage: http://www.arthroplastytoday.org/ + +![0_image_6.png](0_image_6.png) + +Original research Causes and Clinical Outcomes of Patellar Post Impingement Takenori Tomite, MD, PhD a, *, Hidetomo Saito, MD, PhD b, Hiroaki Kijima, MD, PhD b, Noriyuki Ishikawa, MD, PhD a, Yuji Hatakeyama, MD, PhD a, Hiroshi Tazawa, MD, PhD a, Naohisa Miyakoshi, MD, PhD b a Department of Orthopedic Surgery, Akita Red Cross Hospital, Akita, Japan b Department of Orthopedic Surgery, Akita University Graduate School of Medicine, Akita, Japan + +| article | info | +|-----------|--------| + +| Article history: Received 28 July 2021 Received in revised form 25 November 2021 Accepted 19 December 2021 Available online xxx | +|-----------------------------------------------------------------------------------------------------------------------------------| + +Keywords: +Patellar post impingement Posterior stabilized total knee arthroplasty bi-cruciate stabilized total knee arthroplasty New Knee Society Score Anterior knee pain + +## Abstract + +https://doi.org/10.1016/j.artd.2021.12.008 + +![0_image_2.png](0_image_2.png) + +2352-3441/© 2021 The Authors. Published by Elsevier Inc. on behalf of The American Association of Hip and Knee Surgeons. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +Arthroplasty Today 14 (2022) 48e52 Background: Patellar post impingement (PPI), which occurs when the post impinges on the patella, may reportedly cause poor total knee arthroplasty (TKA) outcomes. The causes of PPI and its effect on clinical outcomes and symptoms were investigated. + +Material and methods: The study subjects were 100 patients who underwent TKA (65 posterior stabilized TKA, 35 bi-cruciate stabilized [BCS] TKA). Whether PPI occurred during surgery was investigated; the patients were then classified into a PPIþ group and a PPIe group, and whether the implant or patellar shape was related to the occurrence of PPI was examined. The measurement parameters included patellar shape, joint range of motion (ROM), and clinical outcome using the new Knee Society Score. + +Results: There were 38 knees in the PPIþ group and 62 knees in the PPIe group. There was no difference in patellar shape between the 2 groups, but PPI was more frequent in patients with short patellar tendon and those with low patellar height. In terms of implant type, the PPIþ group included 12 patients (18.5%) +who underwent posterior stabilized TKA and 26 (74.3%) who underwent BCS TKA. There was no difference between the 2 groups in either joint ROM or new Knee Society Score. These results suggest that the position of the patella and implant shape that causes the post to be positioned anteriorly may contribute to PPI. + +Conclusion: PPI occurred more frequently in knees with low patellar height and in patients who had undergone BCS TKA. PPI had no effect on joint ROM or clinical outcome. + +© 2021 The Authors. Published by Elsevier Inc. on behalf of The American Association of Hip and Knee Surgeons. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ +licenses/by-nc-nd/4.0/). + +## Introduction + +Total knee arthroplasty (TKA) is the established method of surgery for end-stage osteoarthritis of the knee [1]. Posterior stabilized (PS) TKA has been shown to have good long-term outcomes +[2,3]. One advantage of PS TKA is that the post-cam structure artificially induces rollback in a highly reproducible manner. + +However, several problems with the post-cam structure have been reported, and this structure could be said to be one of the weak points of PS TKA. These post-cam issues include fracture and wear of the post itself [4,5], as well as characteristic problems such as patellar clunk syndrome [6]. Another problem is the occurrence of anterior tibial post impingement, which occurs when the knee is extended or lightly flexed [7,8], and recent studies using motion analysis, such as KneeSIM (LifeModeler, Inc, San Clemente, CA), have shown that posterior inclination of the tibia is associated with anterior tibial post impingement [9]. Implant shape and positioning are, thus, also associated with problems with the post-cam structure, and this is, thus, an area that merits in-depth investigation. + +Patellar post impingement (PPI) has been identified as an issue for implants capable of deep flexion that have been developed in the past few years, and it occurs when the anterior part of the tibial post impinges on the patella when the knee is deeply flexed (Fig. 1). + +PPI has been reported as causing anterior knee pain, increased patellofemoral pressure, wear on the polyethylene of the patellar component and the tibial post, and decreased range of motion (ROM) [10]. The radiolucency of both the patellar component and the tibial post means that PPI is difficult to assess postoperatively, and much about this issue remains unclear [11]. This may be the +* Corresponding author. Akita Red Cross Hospital, Kamikitate Sarutaazanaeshirosawa 222-1, Akita City, Akita 018-1495, Japan. Tel.: þ81 18 829 5000. + +E-mail address: takenoritomite@yahoo.co.jp + +![1_image_0.png](1_image_0.png) + +Figure 1. The arrow shows where PPI is occurring. +reason that, although some studies have addressed PPI, there have + +![1_image_1.png](1_image_1.png) been few reports of its association with actual clinical outcomes and clinical symptoms. We hypothesized that differences in implant type would affect the incidence of PPI and that PPI would reduce ROM and result in poorer clinical outcomes. The first objective of this study was, therefore, to ascertain whether PPI had actually occurred during surgery in patients who underwent either PS TKA or bi-cruciate stabilized (BCS) TKA and to investigate whether patellar shape, height, or the type of implant was associated with any difference in its incidence. A further objective was to classify these patients into 2 groups depending on the occurrence of PPI and investigate whether there were any differences between these groups in ROM, clinical outcomes, or clinical symptoms. + +## Material And Methods + +The study subjects were 100 patients who had undergone unilateral TKA. The implant used for PS TKA was a Persona (Zimmer Biomet, Warsaw, IN), and that used for BCS TKA was a Journey II +(Smith & Nephew, Memphis, TN). PS TKA was conducted for 65 knees, with BCS TKA for 35. The patients included 20 men and 80 women, with a mean age of 76 ± 6 years. All underwent TKA for osteoarthritis of the knee for the first time. Patients who had previously undergone knee joint surgery such as osteotomy, meniscectomy/suturing, or anterior cruciate ligament reconstruction and those with rheumatoid arthritis were excluded. All operations were performed by the same surgeon. In all cases, the bone was exposed by the medial parapatellar approach, the modified gap technique was used, the implant was anchored with cement, and patellar resurfacing was performed. We gained informed consent of all patients. + +Whether PPI occurred intraoperatively after the insertions of the femoral component, tibial component, and patellar component and all insertions were completed was investigated, and if PPI did occur, the angle was also measured. The patients were classified accordingly into a PPIþ group, in which PPI occurred, and a PPIe group, in which no PPI occurred. The parameters evaluated were (a) patellar length, (b) patellar width, (c) patellar thickness, (d) patellar tendon length, (e) patellar height (Insall-Salvati ratio), (f) patella articularnonarticular angle, (g) patella inferior pole angle, and (h) patella Figure 2. Parameters evaluated on radiographs. + +![1_image_2.png](1_image_2.png) + +![1_image_3.png](1_image_3.png) + +![1_image_4.png](1_image_4.png) + +50 + +| Table 1 Patients' characteristics. Characteristics | PPIþ group | PPI group | P value | +|------------------------------------------------------------------------------------------------------------------------------|------------------|------------------|-----------| +| Number of patients | 38 | 62 | | +| Age (y) | 75.9 (71.0-82.0) | 76.2 (70.0-81.0) | n.s | +| Sex (male/female) | 7/31 | 13/49 | n.s | +| Body mass index (kg/m2 ) | 26.1 (20.5-36.5) | 26.8 (21.1-36.9) | n.s | +| Number of legs (right/left) | 20/18 | 31/31 | n.s | +| There were no significant differences between the 2 groups in age, sex, body mass index, or laterality of the affected side. | | | | + +facet angle, which were all measured on radiographs (Fig. 2). Whether the use of different implants affected the incidence of PPI +was also investigated. Joint ROM and new Knee Society Score (new KSS), a patient-based assessment tool, were evaluated preoperatively and at 1 year postoperatively, and patients were also asked if they had anterior knee pain and crepitation. + +The study was approved by the local ethics committee of the Akita Red Cross Hospital. Informed consent was obtained from all patients. + +## Statistical Analysis + +All data were analyzed using the Statistical Package for the Social Sciences (SPSS) ver. 23.0 (IBM Corp., Armonk, NY). An unpaired t-test was used to evaluate patients' characteristics, X-ray measurements, joint ROM, and new KSS assessments, with P < .05 considered significant. + +## Results + +In the 100 patients, PPI actually occurred intraoperatively in 38 knees (38%, PPIþ group) and did not occur in 62 (62%, PPIe group). + +There were no significant differences between the 2 groups in age, sex, body mass index, or laterality of the affected side (Table 1). PPI +occurred at a mean angle of 127.3-. At the 1-year postoperative follow-up visit, no patients had required implant replacement because of PPI. In terms of implant type, the PPIþ group included 12 of 65 knees (18.5%) treated by PS TKA and 26 of 35 (74.3%) treated by BCS TKA, with PPI more than 4 times as common when BCS TKA was performed than when PS TKA was performed. + +Patellar length, patellar width, patellar thickness, patellar tendon length, patellar height (Insall-Salvati ratio), patella articularnonarticular angle, patella inferior pole angle, and patella facet angle were measured on preoperative radiographs. Postoperatively, because the patellar component is radiolucent, patellar thickness, + +| Results of X-ray measurements. Measurement | PPIþ group | PPI group | P value | +|--------------------------------------------------------------------------------------------------------------|--------------|-------|-----------| +| Preoperative Patellar length (cm) | 45.3 | 44.4 | n.s | +| Patellar width (cm) | 51.2 | 53.8 | n.s | +| Patellar thickness (cm) | 20.2 | 20.8 | n.s | +| Patellar tendon length (cm) | 40.3 | 44.6 | <.05 | +| Patellar height (Insall-Salvati) | 0.88 | 1.04 | <.05 | +| Patellar articular-non articular angle () | 117.4 | 113.2 | n.s | +| Patellar inferior pole angle () | 49.6 | 53.2 | n.s | +| Patellar facet angle () | 139.9 | 140.6 | n.s | +| Postoperative Patellar length (cm) | 44.4 | 43.9 | n.s | +| Patellar width (cm) | 50.7 | 53.2 | n.s | +| Patellar tendon length (cm) | 39.2 | 44.6 | <.05 | +| Patellar height (Insall-Salvati) | 0.87 | 1.03 | <.05 | +| PPI was significantly more likely to occur in patients with a short patellar tendon and low patellar height. | | | | + +patellar articular-nonarticular angle, patellar inferior pole angle, and patellar facet angle were not measured. Intraoperatively, however, the patellar thickness was measured using calipers. Patellar osteotomy was conducted to remove a section of bone of the same thickness as that of the type of patellar component used, and it was confirmed that the patellar thickness after patellar component placement was the same as its preoperative value. Because the preoperative and postoperative patellar thicknesses were the same, there was no great difference in the patellar facet angle compared with its preoperative value. There were significant differences between the PPIþ and PPIe groups in patellar tendon length and patellar height (Table 2). These results showed that, although the shape of the patella itself had no effect on the occurrence of PPI +compared with its preoperative shape, it was more likely to occur in patients with a short patellar tendon and low patellar height. + +In terms of joint ROM, there was no difference between the PPIþ and PPIe groups in either extension or flexion. Although there was a greater improvement in flexion in the PPIe group, there was no significant difference between the 2 groups in either extension or flexion (Table 3). + +Preoperatively, there were no significant differences between the PPIþ and PPIe groups in the new KSS results for symptoms, satisfaction, expectation, or functional activity. Although the preoperative scores for symptoms, satisfaction, and functional activity were often somewhat higher in the PPIe group, the difference between the 2 groups was not significant for any of these parameters (Table 4). + +When the 38 patients with PPI were asked if they had anterior knee pain and crepitation, 3 of 38 (7.9%) complained of anterior knee pain and 1 of 38 (2.6%) of crepitation. Two of the 3 patients who complained of anterior knee pain had undergone BCS TKA, and 1 had undergone PS TKA; the patient who complained of crepitation had undergone BCS TKA. + +## Discussion + +| Table 3 Preoperative and postoperative joint ROM. Range of motion PPIþ group | PPI group | P value | | +|--------------------------------------------------------------------------------|-------|-----------|-----| +| Preoperative extension () | 9.3 | 8.4 | n.s | +| Preoperative flexion () | 118.3 | 116.5 | n.s | +| Postoperative extension () | 4.3 | 4.1 | n.s | +| Postoperative flexion () | 124.9 | 126.8 | n.s | +| The joint ROM was significantly improved postoperatively compared with preoperatively in both the PPIþ and PPIe groups. There was no significant difference | | | | + +The most important information obtained from this study is that PPI occurs more frequently in patients with a shorter patellar tendon and a lower patellar height, a result obtained from actual + +| Table 4 Preoperative and postoperative new KSS results. KSS domain PPIþ group | PPI group | P value | | +|------------------------------------------------------------------------------------------------------------------------------------|------|-----------|-----| +| Preoperative Symptoms | 7.8 | 7.5 | n.s | +| Satisfaction | 13.0 | 13.3 | n.s | +| Expectation | 12.3 | 12.4 | n.s | +| Functional activity | 36.3 | 36.5 | n.s | +| Postoperative Symptoms | 20.2 | 21.2 | n.s | +| Satisfaction | 24.3 | 25.4 | n.s | +| Expectation | 10.3 | 10.2 | n.s | +| Functional activity | 60.4 | 61.6 | n.s | +| There was no significant difference between the PPIþ and PPIe groups in either their preoperative or postoperative new KSS scores. | | | | + +![3_image_0.png](3_image_0.png) + +intraoperative findings in living knees, and that it is more common in BCS TKA. The radiolucency of both the patellar component and the tibial post means that PPI is difficult to assess postoperatively [11], and few studies have addressed the problem. Minoda et al. defined the distance between the lower edge of the patellar component and the cut surface of the tibia as the tibial-patellar clearance (TPC), +which represents the length of the patellar tendon, and they reported that shorter TPCs resulted in smaller PPI angles, and longer TPCs resulted in larger PPI angles [12]. They also stated that the design of the tibial post affects the occurrence of PPI [11,12], but those studies were conducted using Sawbones model bones. Verborgt and Victor reported that a raised joint line, patella infera, tooanterior placement of the tibial component, and smaller femoral component size are associated with the occurrence of PPI [10]. Taking the results of these previous reports in conjunction with those of the present study, the important causes of PPI may be (1) the positions of the patella, such as patella infera, a small TPC, or a raised joint line and (2) an implant shape that places the post in a more anterior position. In light of the study by Verborgt and Victor [10] on the causes of PPI, it may be that, if the joint line is raised, the relatively lower position of the patellar makes the occurrence of PPI +more likely. In the present study, the patients' original joint line was difficult to define and was, therefore, not evaluated. However, no additional osteotomy of the distal femur was carried out during surgery in patients who underwent either BCS TKA or PS TKA, with only the amount of bone designated by the osteotomy guide being cut away in either case. Thus, the osteotomies performed in patients who underwent BCS TKA or PS TKA were of similar size. The position of the tibial component was evaluated on radiographs, and no patient who underwent either BCS TKA or PS TKA showed an obvious anterior overhang. Placing the post in a more anterior position that results in too-anterior placement of the tibial component may also make the occurrence of PPI more likely. BCS is designed specifically to provide postoperative anterior-posterior knee stability, and its AP +position places the femur more anteriorly relative to the tibia than conventional PS, in which their positional relationship is close to that of the normal knee [13]. In terms of the shapes of the Persona and Journey II implants used for PS TKA and BCS TKA, respectively, the post is positioned more anteriorly in BCS TKA than in PS TKA, as can be seen in Figure 3 and Table 5. PPI may, thus, occur more frequently in BCS TKA because more anterior post placement makes it more likely to occur when the knee is flexed. A few cases of low-riding patella after TKA have been reported [14,15], but other studies have found no change in patellar height in the short-term postoperative period [16,17]. The present results are short term, covering 1 year postoperatively, but there was no difference in patellar height between before and after surgery. + +The occurrence of PPI has been reported to cause decreased ROM, anterior knee pain, increased patellofemoral pressure, and polyethylene wear of the patellar component and tibial post [10]. In the present study, ROM and anterior knee pain were investigated, but there was no significant difference in ROM between the PPIþ and PPIe groups. Only 3 of 38 (7.9%) patients who developed PPI +complained of anterior knee pain, and only 1 of 38 (2.6%) complained of crepitation. These percentages are not much higher than those reported by Waters and Bentley, who found that 25.1% of + +| implant to the base of the post. Persona A (mm) | B (mm) | Journey II | A (mm) | B (mm) | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|--------------|-----------|----------|----| +| (size: femur/tibia) | (size: tibia) | | | | | +| Size: 3-5/C-D | 42 | 14 | Size: 1-2 | 42 | 10 | +| Size :6-9/C-D | 42 | 13 | | | | +| Size: 3-5/E-F | 46 | 17 | Size: 3-4 | 48 | 12 | +| Size: 6-9/E-F | 46 | 16 | Size: 5-6 | 53 | 14 | +| Anterior-posterior length (A) and distance from the anterior surface of the implant to the base of the post (B) in frequently used sizes of the Persona and Journey II | | | | | | + +Size: 3-5/C-D 42 14 Size: 1-2 42 10 + +Size :6-9/C-D 42 13 Size: 3-5/E-F 46 17 Size: 3-4 48 12 + +Size: 6-9/E-F 46 16 Size: 5-6 53 14 + +Anterior-posterior length (A) and distance from the anterior surface of the implant + +to the base of the post (B) in frequently used sizes of the Persona and Journey II + +implants used for PS TKA and BCS TKA, respectively. In the Persona, the distance to + +the base of the post varies depending on the size of the femur, and the "Size" entries + +show the femoral and tibial implant sizes. The Persona tibia C-D size is approximately equivalent to the Journey II tibia 1-2 size, and the anterior-posterior lengths + +of the E-F and 3-4 sizes are about the same, but the distance to the post (B) is shorter in the Journey II, meaning that the post is positioned more anteriorly in the Journey II than in the Persona. + +52 post-TKA patients complained of anterior knee pain if patellar resurfacing was not conducted and 5.3% if it was [18]. Anterior knee pain is usually discussed in the context of whether to resurface the patella, and some studies have found that omitting patellar resurfacing may cause anterior knee pain [19,20]. Other studies, however, have found that there is no difference in the occurrence of anterior knee pain regardless of whether the patella is resurfaced +[21,22]. Guman et al. conducted a systematic review and metaanalysis of risk factors for anterior knee pain, and they reported that patients who did not undergo patellar resurfacing and those who underwent infrapatellar fat pad excision tended to experience anterior knee pain [23]. In the present study, as many factors that might cause anterior knee pain as possible were eliminated by performing patellar resurfacing in all cases and excising the infrapatellar fat pad to the extent to which it did not interfere with the field of view. In terms of patellofemoral pressure, Sawaguchi et al. + +compared patellofemoral contact stress in mobile-bearing to that in fixed-bearing TKA, and they found that contact stress is lower in mobile-bearing TKA than in fixed-bearing TKA [24], but to the best of our knowledge, no study has measured the patellofemoral pressure using other types of devices such as BCS TKA and PS TKA. In addition, no study has yet reported that the occurrence of PPI +increases wear of the patellar component or the tibial post. + +As a method of dealing with PPI when it occurs, Jegyun et al. + +reported that resecting the lower portion of the patella increases the space between the patella and the tibial post, preventing PPI from occurring [25]. However, if the lower portion of the patella is resected, it may not be possible to carry out appropriate patellar resurfacing. In terms of surgical procedure, what is important is not to raise the joint line unnecessarily, as reported by Verborgt and Victor [10]. In patients with a low patellar tendon and when carrying out BCS TKA, it may be possible to adapt the procedure by means such as resecting a slightly greater amount of the patella if it is of sufficient thickness. + +The most important limitation of this study was that it was only possible to investigate 2 types of implants. Minoda et al. investigated PPI in a variety of different implants [11], and they found that TPC of 20 mm for PS TKA with a Persona implant and 22 mm for BCS +TKA with a Journey II implant is required to achieve flexion 130- to prevent PPI from occurring. They showed that PPI is more likely to occur in BCS TKA, unless the position of the patella is higher. However, that study used Sawbones models, and it would be quite difficult to insert a range of different implants into living knees for investigation. A second limitation is the short follow-up period of 1 year. No study has yet examined the long-term postoperative outcomes of PPI. In such a short study period of 1 year, PPI +did not cause any serious symptoms that caused implant failure or required revision, but implant failure and patient symptoms are among the issues that must be monitored long term in the future. + +Third, the small sample size should be mentioned. As noted previously, the mean angle at which PPI occurred was 127.3, but in 21 +(55%) of the 38 patients who developed PPI, the ROM did not reach the angle at which PPI occurred, and it improved to greater than the angle at which PPI occurred in 17 (45%) of these patients. In view of the small number of patients in whom ROM improved to above the angle at which PPI actually occurred, and taken in conjunction with the second limitation described previously, studies with a larger sample size and longer follow-up time might show a drop in the KSS score, diminished ROM, or larger numbers of patients complaining of anterior knee pain or crepitations. + +## Conclusion + +PPI occurred more frequently in knees with a shorter patellar tendon, which is approximately equivalent to a low patellar height, and in patients who had undergone BCS TKA, suggesting that the occurrence of PPI may be determined by the position of the patella and implant shape. PPI had no effect on joint ROM or clinical outcome at 1 year postoperatively, and the postoperative complaint rate was also low. + +## Conflicts Of Interest + +The authors declare that there are no conflicts of interest. + +## References + +[1] Jones CA, Beaupre LA, Johnston DW, Suarez-Almazor ME. Total joint arthroplasties: current concepts of patient outcomes after surgery. Rheum Dis Clin North Am 2007;33(1):77. + +[2] Michael A, Clarke HD. Long-term results of posterior cruciate-substituting total knee arthroplasty. Clin Orthop Relat Res 2002;404:51. + +[3] Stern SH, Insall JN. Posterior stabilized prosthesis. Results after follow-up of nine to twelve years. J Bone Joint Surg Am 1992;74:980. + +[4] Callaghan JJ, O'Rourke MR, Goetz DD, et al. Tibial post impingement in posterior-stabilized total knee arthroplasty. Clin Orthop Relat Res 2002;404: 83. + +[5] Mestha P, Shenava Y, D'Arcy JC. Fracture of the polyethylene tibial post in posterior stabilized (Insall Burstein II) total knee arthroplasty. J Arthroplasty 2000;15(6):814. + +[6] Hozack WJ, Rothman RH, Booth Jr RE, Balderston RA. The patellar clunk syndrome. A complication of posterior stabilized total knee arthroplasty. Clin Orthop Relat Res 1989;241:203. + +[7] Hamai S, Miura H, Higaki H, et al. Evaluation of impingement of the anterior tibial post during gait in a posteriorly-stabilised total knee replacement. + +J Bone Joint Surg Br 2008;90(9):1180. + +[8] Tamami M, Tomita T, Yamazaki T, Yoshikawa H, Sugamoto K. Factors in high-flex posterior stabilized fixed-bearing total knee arthroplasty affecting in vivo kinematics and anterior tibial post impingement during gait. J Arthroplasty 2013;28(10):1722. + +[9] Okamoto S, Mizu-uchi H, Okazaki K, et al. Effect of tibial posterior slope on knee kinematics, quadriceps force, and patellofemoral contact force after posterior-stabilized total knee arthroplasty. J Arthroplasty 2015;30(8):1439. + +[10] Verborgt O, Victor J. Post impingement in posterior stabilised total knee arthroplasty. Acta Orthop Belg 2004;70:46. + +[11] Minoda Y, Sugama R, Ohta Y, Ikebuchi M, Nakamura H. Impingement of the patellar component against the tibial post depends on the design of the postcam mechanism: comparison between 12 posterior stabilized total knee prostheses. Knee 2018;25:676. + +[12] Minoda Y, Sugama R, Ohta Y, Nakamura H. Impingement of the patellar component against the tibial post after posterior stabilized and constrained TKA: a pilot study. Arch Orthop Trauma Surg 2017;137:861. + +[13] Victor J, Bellemans J. Physiologic kinematics as a concept for better flexion in TKA. Clin Orthop Relat Res 2006;452:53. + +[14] Koshino T, Ejima M, Okamoto R, Mori T. Gradual low riding of the patella during postoperative course after total knee arthroplasty in osteoarthritis and rheumatoid arthritis. J Arthroplasty 1990;5:323. + +[15] Meneghini RM, Ritter MA, Pierson JL, et al. The effect of the Insall-Salvati ratio on outcome after total knee arthroplasty. J Arthroplasty 2006;21:116. + +[16] Filipe C, Berbardo SP, Rui P, Joao T. Patellar height after total knee arthroplasty: comparison of 3 methods. J Arthroplasty 2017;32:552. + +[17] Jawhar A, Sohoni S, Shah V, Scharf HP. Alteration of the patellar height following total knee arthroplasy. Arch Orthop Trauma Surg 2014;134:91. + +[18] Waters TS, Bentley G. Patellar resurfacing in total knee arthroplasty: a prospective, randomized study. J Bone Joint Surg Am 2003;85:212. + +[19] Pakos EE, Ntzani EE, Trikalinos TA. Patellar resurfacing in total knee arthroplasty: a meta-analysis. J Bone Joint Surg Am 2005;87:1438. + +[20] Parvizi J, Rapuri VR, Seleh KJ, et al. Failure to resurface the patella during total knee arthroplasty may result in more knee pain and secondary surgery. Clin Orthop Relat Res 2005;438:191. + +[21] Barrack RL, Bertot AJ, Wolfe MW, et al. Patellar resurfacing in total knee arthroplasty: a prospective, randomized, double-blind study with five to seven years of follow-up. J Bone Joint Surg Am 2001;83:1376. + +[22] Campbell DG, Duncan WW, Ashworth M, et al. Patellar resurfacing in total knee replacement: a ten-year randomized prospective trial. J Bone Joint Surg Br 2006;88:734. + +[23] Guman D, Chang L, Weiwei L, et al. Different factors conduct anterior knee pain following primary total knee arthroplasty: a systematic review and meta-analysis. J Arthroplasty 2018;33(6):1962. + +[24] Sawaguchi N, Majima T, Ishigaki T, et al. Mobile-bearing total knee arthroplasty improves patellar tracking and patellofemoral contact stress: in Vivo measurements in the same patients. J Arthroplasty 2009;25(6):920. + +[25] Jegyun C, Bongju L, Sangyeop S, Gunil J, Taehyeon J. Analysis of impingement between patella bone and bearing post in cruciate-substituting high eflexion total knee arthroplasty. Clin Orthop Surg 2016;8(2):157. \ No newline at end of file diff --git a/medical/md_images/PMC8857593/PMC8857593_meta.json b/medical/md_images/PMC8857593/PMC8857593_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..5bdd3fecb0536619df85c3986c908c59e5d044b7 --- /dev/null +++ b/medical/md_images/PMC8857593/PMC8857593_meta.json @@ -0,0 +1,145 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Causes and Clinical Outcomes of Patellar Post Impingement", + "level": 0, + "is_closed": false, + "n_kids": 8, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Introduction", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Material and methods", + "level": 1, + "is_closed": false, + "n_kids": 1, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Statistical analysis", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Results", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Discussion", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Conclusion", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "Conflicts of interest", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + }, + { + "title": "References", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 794.0, + 0.0 + ] + } + ], + "pages": 5, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 4, + "code": 0, + "table": 7, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC9011202/13_image_1.png b/medical/md_images/PMC9011202/13_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..ea4e88db800d6e3da79713281423da516266d431 --- /dev/null +++ b/medical/md_images/PMC9011202/13_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:1ff484cd5fa4d43e535fc0547993e9805ab38d2d75f5ba06b22099b99fb6562f +size 13019 diff --git a/medical/md_images/PMC9488732/PMC9488732.md b/medical/md_images/PMC9488732/PMC9488732.md new file mode 100644 index 0000000000000000000000000000000000000000..69f63d84cb274d3a20b3ae4d23043d30fa64044e --- /dev/null +++ b/medical/md_images/PMC9488732/PMC9488732.md @@ -0,0 +1,534 @@ +# Systematic Review On Long-Term Adverse Effects Of Inhaled + +![0_Image_0.Png](0_Image_0.Png) Corticosteroids In The Treatment Of Copd + +Marc Miravitlles 1,6, Ariadna Auladell-Rispau2,6, Mònica Monteagudo3,4, Juan Carlos Vázquez-Niebla2, Jibril Mohammed5, Alexa Nuñez1 and Gerard Urrútia2 1Pneumology Dept, Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Barcelona Hospital Campus, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, Spain. 2Institut d'Investigació Biomèdica Sant Pau (IIB Sant Pau), CIBER de Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain. 3Primary Care University Research Institute Jordi Gol (IDIAP Jordi Gol), Barcelona, Spain. 4Medicine Dept, Universitat Autònoma de Barcelona (UAB), Bellaterra (Cerdanyola del Vallès), Barcelona, Spain. 5Dept of Physiotherapy, Bayero University, Kano, Nigeria. 6Both authors contributed equally and are considered first authors. + +| Corresponding author: Marc Miravitlles (marcm@separ.es) | | +|-----------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Shareable abstract (@ERSpublications) Long-term use of inhaled corticosteroids in COPD is associated with a significantly increased risk of side-effects, especially oral candidiasis, dysphonia, pneumonia, mycobacterial disease, diabetes and fractures https://bit.ly/3t0AGfO Cite this article as: Miravitlles M, Auladell-Rispau A, Monteagudo M, et al. Systematic review on longterm adverse effects of inhaled corticosteroids in the treatment of COPD. Eur Respir Rev 2021; 30: 210075 [DOI: 10.1183/16000617.0075-2021]. Abstract Inhaled corticosteroids (ICSs) are indicated for the prevention of exacerbations in COPD; however, a significant proportion of patients at low risk of exacerbations are treated with ICSs. We conducted a systematic review including a diversity of types of study designs and safety outcomes with the objective of describing the risk of adverse effects associated with the long-term use of ICSs in patients with COPD. A total of 90 references corresponding to 83 studies were included, including 26 randomised clinical trials (RCTs), 33 cohort studies, and 24 nested case–control (NCC) studies. Analysis of 19 RCTs showed that exposure to ICSs for ⩾1 year increased the risk of pneumonia by 41% (risk ratio 1.41, 95% CI 1.23–1.61). Additionally, cohort and NCC studies showed an association between ICSs and risk of tuberculosis and mycobacterial disease. There was a strong association between ICS use and local disorders such as oral candidiasis and dysphonia. The association between ICSs and the risk of diabetes and fractures was less clear and appeared significant only at high doses of ICSs. Since most patients with COPD are elderly and with frequent comorbidities, an adequate risk–benefit balance is crucial for the indication of ICSs. | | +| Copyright ©The authors 2021 This version is distributed under the terms of the Creative Commons Attribution NonCommercial Licence 4.0. For commercial reproduction rights and permissions contact permissions@ersnet.org This article has supplementary material available from err.ersjournals.com Received: 12 Jan 2021 Accepted: 26 March 2021 | Introduction Inhaled corticosteroids (ICSs) combined with long-acting bronchodilators are indicated for patients with COPD who experience exacerbations despite treatment with long-acting β2-agonists (LABAs) and long-acting antimuscarinic agents (LAMAs) [1]. Furthermore, recent studies have shown that the efficacy of ICSs in the prevention of exacerbations is higher in patients with high concentrations of blood eosinophils, but very limited in patients with blood eosinophil levels below 100 cells·μL–1 [2, 3]. Despite these indications, studies in different countries have demonstrated excessive and inadequate use of ICS in patients with mild or moderate COPD who are at low risk of exacerbations [4–6]. The development of inhaled forms of corticosteroids for respiratory diseases, initially asthma and later COPD, represented a great advance, because they opened up the possibility of adequately treating these diseases with lower doses of corticosteroids and with significant reductions in systemic exposure to the | + +Corresponding author: Marc Miravitlles (marcm@separ.es) +The development of inhaled forms of corticosteroids for respiratory diseases, initially asthma and later + +![0_image_1.png](0_image_1.png) COPD, represented a great advance, because they opened up the possibility of adequately treating these diseases with lower doses of corticosteroids and with significant reductions in systemic exposure to the drug. However, there is some systemic absorption of ICSs [7], and due to the prolonged life expectancy of patients with COPD, patients can be exposed to the drug for decades, with possible risks of long-term corticosteroid-related side-effects. This is particularly important in COPD, because the population affected is usually older individuals with frequent comorbidities, such as type 2 diabetes mellitus and mycobacterial infections, among others, which may be susceptible to being aggravated by corticosteroids [8]. The large randomised clinical trials (RCTs) conducted at the beginning of the century revealed an increased risk of nonfatal pneumonia associated with the use of ICSs in COPD [9]. In addition, several studies observed a relationship between ICSs and local disorders such as oral candidiasis and dysphonia [10]; however, other less frequent side-effects may require larger populations exposed for longer periods of time to be identified. We undertook the following analysis with the objectives of identifying and describing studies reporting the risks of adverse events associated with long-term use of ICSs in adult patients with COPD and estimating these risks. + +## Methods Design And Selection Criteria + +We conducted a systematic review with a broad focus, including a diversity of types of study designs and safety outcomes. Studies were included that met the following criteria for study design, population, intervention and outcomes: 1) RCTs, observational controlled studies ( prospective or retrospective cohort studies, nested case–control studies (NCCs) and single cohort studies (if the sample was >500 participants); 2) adult patients diagnosed with COPD (if the study included a mixed population, specific data for patients with COPD should be available); 3) studies reporting data on safety outcomes in patients exposed to long-term use (defined as at least a 1-year period of continuous treatment) of ICSs alone or in combination; 4) studies reporting the rate (or the odds for case–control studies) of at least one of the following safety outcome measures: pneumonia, mycobacterium infections (tuberculosis (TB) and nontuberculous mycobacteria (NTM)), sepsis, osteoporosis, bone fractures, diabetes mellitus (new-onset, progression and/or switch from oral medication to insulin), glaucoma, cataract, hypertension, adrenal insufficiency, dysphonia and oral candidiasis. + +## The Study Was Registered In Prospero (Crd42020168023) In April 2020. + +Search strategy and study selection For the electronic search, we designed a specific algorithm for MEDLINE and EMBASE (supplementary table S1). In addition, we performed a search for other reviews, either systematic or narrative, on the safety of ICSs and manually reviewed all the references included. The Covidence® software was used to manage the bibliography retrieved from the search across all the review process. Three authors (A. AuladellRispau, J. Mohammed and G. Urrútia), working in pairs, screened the search results based on the title and abstract independently. A full-text copy of each eligible reference was retrieved and the same researchers independently confirmed eligibility based on the inclusion criteria. Disagreements were discussed and solved by consensus. + +## Data Extraction + +The same authors extracted all relevant data on the main characteristics (design and methods, study population and intervention) and results of the selected studies. + +## Data Analysis + +Since the review includes several study designs that cannot be combined in a meta-analysis, the results are presented separately for each type of study design. Whenever possible, a meta-analysis was performed separately for RCTs and cohort studies, providing that these studies reported a measure of comparative effects such as hazard ratios (HR) or risk ratios (RR). Data are displayed using forest plots. We examined between-study heterogeneity using the I2 statistic. The quality of the clinical trials included in the meta-analyses was evaluated with the Cochrane risk of bias assessment tool [11]. When studies performed comparisons using ICSs in both study groups, results (rates) were reported narratively. Likewise, in NCC studies, in which the measure of association was the odds ratio (OR), the results are presented narratively. + +## Results + +Our search in MEDLINE through PubMed yielded a total of 2711 bibliographic references, and the search in EMBASE provided 4287 references; both searches included references published up to 20 October 2020. An additional search was performed to identify other reviews related to this topic and included a total of 240 references. The search results, as well as the decisions made during the eligibility process, are shown in a PRISMA flowchart (supplementary figure S1). A total of 90 references from 83 studies were included in the review, corresponding to 26 RCTs [9, 12–43], 33 cohort studies [44–76] and 24 NCC studies [77–100]. A complete description of these studies is provided in supplementary table S2. Safety outcomes have been grouped into four main categories according to the nature of the events: infectious, metabolic, local and other. + +## Infectious Adverse Events + +Pneumonia In total, 47 studies reported this outcome (19 RCTs, 20 cohort studies and eight NCC studies). The pooled analysis of 19 RCTs with 66 485 participants showed that exposure to ICSs for ⩾1 year increased the risk of pneumonia by 41% (RR 1.41, 95% CI 1.23–1.61; p<0.00001; I2=55%) [9, 12–14, 16, 19, 22, 23, 25, 26, 29, 30, 33–39] (figure 1). An interaction was found between the risk of pneumonia and the type of ICS used, with the highest risk being associated with fluticasone (10 studies with 45 870 participants) [9, 12–14, 16, 23, 25, 26, 30, 39]. A similar trend was observed with beclometasone (two studies with 4221 participants) [33, 38] and mometasone (one study with 911 participants) [22], but the low number of available studies limited the statistical power of the analysis and, therefore, resulted in a low-precision estimate. Conversely, exposure to budesonide (six studies with 13 479 participants) [19, 29, 34–37] was not associated with an increased risk of pneumonia, although a high heterogeneity was observed due to a recent large study which observed an increased risk [34]. Of the 21 cohort studies that reported this outcome, 16 described the effects in terms of HR and could be meta-analysed [46, 47, 49, 50, 53, 54, 57– +59, 61, 66–69, 73, 74]. Exposure to ICSs for ⩾1 year increased the risk of pneumonia by 26% (HR 1.26, 95% CI 1.14–1.38, p=0.0001; I2=93%) (supplementary figure S2). + +The remaining studies which were not suitable for the meta-analysis are summarised in table 1. Of these, only one study compared ICS use with no ICS use [71], while the remaining studies used ICSs in both study arms and, therefore, only provided descriptive frequency data. Incidence rates were highly variable among studies, and the risk of pneumonia in patients receiving fluticasone seemed to be higher than in patients receiving budesonide [52, 72], although one other study did not show this association [55]. According to one study (that was included in the meta-analysis), the risk of pneumonia was associated with higher doses of ICSs and the effects of ICSs were only significant in patients with forced expiratory volume in 1 s (FEV1) ⩾50% [53]. + +The results of eight NCC studies are summarised in table 2 [80, 81, 88, 92, 96, 97, 99, 100]. Overall, they confirm the association between ICS exposure and the risk of pneumonia. Higher doses of ICSs, current use of ICSs and exposure to fluticasone were also found to be associated with a higher risk of pneumonia. + +## Tb And Ntm + +Nine studies reported this outcome: five cohort studies [56, 65, 71, 75, 76] and four NCC studies [77–79, 90]. None of these studies were suitable for a meta-analysis. Overall, the cohort studies found that the use of ICSs was associated with the risk of developing TB, especially for patients receiving high ICS doses and with a history of previous pulmonary TB [56, 65, 75]. Only one study did not find this association [71]. According to one study, long-term treatment with budesonide was associated with rates of TB similar to those with fluticasone [76]. Two NCC studies observed an association between ICS exposure and pulmonary TB [78, 90] and two others associated ICSs with NTM [77, 79] (tables 1 and 2). Sepsis Only one cohort [70] and one NCC study [82] related to the development of sepsis were identified. The cohort study compared the effect of budesonide and fluticasone on the risk of sepsis in patients with COPD and found incidence rates of sepsis of 4.99 and 5.74 per 100 person-years, respectively. Fluticasone was associated with a higher risk of sepsis (adjusted HR (aHR) 1.15, 95% CI 1.07–1.24) and septic shock (aHR 1.14, 95% CI 1.01–1.29) compared with budesonide [70]. + +The NCC study included 163 514 patients treated for COPD, 1704 of whom were hospitalised or died with sepsis during follow-up (incidence rate 1.94 per 1000 per year). The risk of sepsis was not increased with ICS, even at high doses, while the risk was increased with oral corticosteroids [82]. + +## Metabolic Adverse Events + +Osteoporosis In total, 12 studies reported this outcome (eight RCTs, two cohort studies and one case–control study). + +The reporting methods for bone mineral density (BMD) data in the RCTs precluded pooling results in a meta-analysis [9, 21, 28, 29, 31, 32, 35, 42]. In one study, BMDs of the lumbar spine ( p=0.007) and the femoral neck ( p<0.001) were significantly lower in the ICS group (triamcinolone) versus placebo after 3 years [42]. Another study showed small but statistically significant differences in changes from baseline + +| ICS | | | | | | | | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------|--------------------------------------------------------------------------------------------------------------------------|--------------------------------|-----------------------------------------------------|-------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|----|----|----| +| Events | No ICS | | | | | | | | | | +| Events | Risk of bias | | | | | | | | | | +| Study or subgroup [ref.] | Total | Total | Weight % Risk ratio (95% CI) | Risk ratio (95% CI) | A | B | C | D | E | F | +| Fluticasone AARON [12] ANZUENTO [13] CALVERLEY [9] CALVERLEY [23] DRANSFIELD [25] FERGUSON [26] LIPSON [14] VESTBO [14] VESTBO [16] WEDZICHA [12] Subtotal Total events | 1 26 587 50 154 29 609 94 465 80 | 145 394 3098 658 2437 394 8285 1396 8297 1680 26 784 | 1 10 395 24 27 15 97 83 377 53 | 304 403 3086 665 818 388 2070 1403 8271 1678 19 086 | 0.2 2.8 12.7 5.1 6.2 3.6 10.5 8.6 12.3 7.4 69.4 | 2.10 (0.13–33.28) 2.66 (1.30–5.44) 1.48 (1.32–1.66) 2.11 (1.31–3.38) 1.91 (1.28–2.86) 1.90 (1.04–3.49) 1.57 (1.27–1.93) 1.14 (0.86–1.51) 1.23 (1.08–1.40) 1.51 (1.07–2.12) 1.49 (1.31–1.69) | | | | | +| 2095 | 1082 | | | | | | | | | | +| Heterogeneity: Tau2=0.01; Chi2=17.03, df=9 (p=0.05); I2=47% Test for overall effect: Z=6.18 (p<0.00001) Budesonide CALVERLEY [19] KERWIN [29] RABE [34] RENNARD [35] SHARAFKHANEH [36] VESTBO [37] Subtotal Total events Heterogeneity: Tau2=0.29; Chi2=20.07, df=5 (p=0.001); I2=75% Test for overall effect: Z=0.06 (p=0.95) 13 6 261 30 7 16 511 282 6404 988 815 145 9145 9 8 48 40 0 24 511 174 2125 976 403 145 4334 | 2.2 1.5 8.2 5.2 0.2 3.8 21.0 | 1.44 (0.62–3.35) 0.46 (0.16–1.31) 1.80 (1.33–2.44) 0.74 (0.47–1.18) 7.43 (0.43–129.71) 0.67 (0.37–1.20) 1.02 (0.59–1.75) | | | | | | | | | +| 333 | 129 | | | | | | | | | | +| Beclometasone PAPI [33] VESTBO [38] Subtotal Total events Heterogeneity: Tau2=0.00; Chi2=0.40, df=1 (p=0.52); I2=0% Test for overall effect: Z=1.06 (p=0.29) 18 40 764 17 1614 19 2378 768 | 3.2 | 1.06 (0.55–2.05) | | | | | | | | | +| 1075 | 4.3 | 1.40 (0.82–2.41) | | | | | | | | | +| 1843 | 7.5 | 1.25 (0.83–1.90) | | | | | | | | | +| 58 | 36 | | | | | | | | | | +| Mometasone CALVERLEY [22] Subtotal Total events Heterogeneity: Not applicable Test for overall effect: Z=1.54 (p=0.12) 25 616 | 6 | | | | | | | | | | +| 616 | 295 | 2.0 | 2.00 (0.83–4.81) | | | | | | | | +| 295 | 2.0 | 2.00 (0.83–4.81) | | | | | | | | | +| 25 | 6 | | | | | | | | | | +| Total Total events Heterogeneity: Tau2=0.03; Chi2=40.19, df=18 (p=0.002); I2=55% Test for overall effect: Z=5.01 (p<0.00001) Test for subgroup differences: Chi2=2.80, df=3 (p=0.42); I2=0% Risk of bias legend A Random sequence generation (selection bias) B Allocation concealment (selection bias) C Blinding of participants and personnel (performance bias) D Blinding of outcome assessment (detection bias) E Incomplete outcome data (attrition bias) F Selective reporting (reporting bias) 38 923 25 558 | 100.0 | 1.41 (1.23–1.61) | | | | | | | | | +| 2511 | 1253 | 0.05 | 5 | 20 | | | | | | | +| 0.2 | 1 | | | | | | | | | | +| Favours ICS | Favours no ICS | | | | | | | | | | +| FIGURE 1 Pooled risk ratios for pneumonia (Mantel-Haenszel, random-effects model). ICS: inhaled corticosteroid. | | | | | | | | | | | + +for budesonide 320 μg compared with all other treatments for total lumbar spine BMD ( p⩽0.037) and for budesonide 320 μg versus formoterol for total hip BMD ( p=0.012) [35]. However, the remaining six RCTs did not find such an effect [9, 21, 28, 29, 31, 32]. In addition, two cohort studies reported a significant BMD decline with ICS use [60], and an evident dose–response relationship with increased risk of osteoporosis at mean ICS exposures of 500 μg·day–1 or greater [62]. Finally, one NCC found that long-term use of ICSs was associated with osteoporosis in multivariate regression analysis (HR 0.70, 95% CI 0.59–0.83; p<0.0001) [91]. + +| TABLE 1 Outcomes from cohort studies (not suitable for meta-analysis) First author Results | Conclusions | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| [ref.] | | | +| Infectious complications: pneumonia Cohort studies with no ICS use in control group WU [71] Pneumonia event rate with ICS use: 703/8813 (8.0%) | The aim of the study was to explore the association between | | +| Pneumonia event rate with no ICS use: 1654/35 252 (4.7%) | post-ICS pulmonary infections (pneumonia and TB) and lung cancer. The incidence observed in those exposed to ICSs was substantially higher than in those not exposed. | | +| Cohort studies with ICS use in both study arms YANG [72] Pneumonia: Event rate for fluticasone/salmeterol: 12.11 per 100 person-years Event rate for budesonide/formoterol: 10.65 per 100 person-years aHR 1.13 (95% CI 1.08–1.20) | Patients receiving fluticasone/salmeterol had a higher incidence rate and a higher risk of pneumonia than patients receiving budesonide/formoterol (13% increase of risk). | | +| Pneumonia requiring mechanical ventilation: Event rate for fluticasone/salmeterol: 3.94 per 100 person-years Event rate for budesonide/formoterol: 3.47 per 100 person-years aHR 1.14 (95% CI 1.05–1.24) | Patients receiving fluticasone/salmeterol had a higher incidence rate and a higher risk of pneumonia requiring mechanical ventilation than patients receiving budesonide/formoterol (14% increase of risk). | | +| JANSON [52] | Pneumonia event rate for fluticasone/salmeterol: 11.0 per 100 patient-years Pneumonia event rate for budesonide/formoterol: 6.4 per 100 patient-years RR 1.73 (95% CI 1.57–1.90; p<0.001) | The observed risk with fluticasone/salmeterol was greater than with budesonide/formoterol (73% increase of risk). | +| KERN [55] | Pneumonia event rate for fluticasone/salmeterol: 702/3697 (19.0%) | The proportion of patients diagnosed with pneumonia was similar in each group. No evidence of an association was | +| Pneumonia event rate for budesonide/formoterol: 634/3697 | observed. | | +| (17.2%) | | | +| OR 0.92 (95% CI 0.81–1.04) | | | +| JANSON [53] | The highest risk of pneumonia was associated with a high- vs | ICS use increased the risk of pneumonia in patients with COPD. | +| low-dose of ICSs (HR 1.41, 95% CI 1.23–1.62) | Asthma was an independent risk factor for pneumonia in the | | +| The risk of pneumonia was significantly increased with ICSs in | COPD population. Multivariate analysis identified independent | | +| patients with FEV1 ⩾50% but not in those with FEV1 <50%, with | predictors of pneumonia in the overall population. The | | +| both low- and high-dose ICS use: | highest risk of pneumonia was associated with high-dose ICS. | | +| Low-dose ICS vs no ICS: if FEV1 <50%, HR 1.06 (95% CI 0.91–1.25), and if FEV1 ⩾50%, HR 1.20 (95% CI 1.05–1.38) High-dose ICS vs no use: if FEV1 <50%, HR 0.98 (95% CI 0.81–1.17), and if FEV1 ⩾50, HR 1.31 (95% CI 1.10–1.56) | | | +| Infectious complications: TB and NTM lung disease KIM [56] The study assessed the risk of TB in patients with COPD depending on ICS use and the presence of a TB scar at baseline: ICS use with TB scar: HR 26.9 (95% CI 3.36–215.75; p=0.002) ICS use without TB scar: HR 9.88 (95% CI 1.11–87.93; p=0.04) No ICSs with TB scar: HR 3.85 (95% CI 0.35–42.55; p=0.272) No ICSs without TB scar: reference group | The use of ICSs was strongly associated with the risk of developing TB. The highest risk was observed in patients who were ICS users and had previous TB lesions. | | +| SHU [65] | This prospective cohort study assessed the risk of developing active TB during follow-up according to the use and dose of ICS: High-dose ICS: 5/50 Medium-dose ICS: 2/72 No ICS: 3/238 (p=0.010) | The use of high-dose ICSs and prior pulmonary TB were associated with an increased risk of pulmonary TB in patients with COPD. | +| LEE [75] | Univariate time-dependent Cox regression analysis in 23 594 patients with COPD revealed that ICS dose-dependently increased the risk of pulmonary TB (HR 1.01, 95% CI 1.00–1.02); in multivariate analysis, the effect of ICSs became obscured; adding ICSs into the multivariate model produced a minimal effect on the other co-variables (HR 1.01, 95% CI 0.99–1.03) | Although ICS therapy has been shown to predispose patients with COPD to pneumonia in large RCTs, it does not increase the risk of TB in real-world practice. Continued | + +| TABLE 1 Continued First author Results | Conclusions | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| [ref.] | | | +| Wu [71] | TB event rate for ICS use: 182/8813 (2.1%) | The aim of the study was to explore the association between | +| TB event rate for no ICS use: 678/35 252 (1.9%) | post-ICS pulmonary infections (pneumonia and TB) and lung cancer. The incidence of TB observed in those exposed to ICSs was similar to that in those not exposed. | | +| HUANG [76] | Fluticasone/salmeterol cohort: incidence rate 0.15/100 person-years Budesonide/formoterol cohort: incidence rate 0.17/100 person-years aHR 0.900 (95% CI 0.565–1.435) | Long-term treatment with budesonide/formoterol was associated with rates of TB similar to those of fluticasone/ salmeterol in patients with COPD. | +| Metabolic complications: fractures CHO [46] In patients with severe COPD: ICSs alone: 22/169 (13%); rate 53.8 per 1000 patient-years ICS/LABAs: 28/638 (4.4%); rate 24.1 per 1000 patient-years In patients with non-severe COPD: ICSs alone: 38/277 (13.7%); rate 25.1 per 1000 patient-years ICS/LABAs: 70/911 (7.7%); rate 12.4 per 1000 patient-years Cumulative hazards during study period and 5-year cumulative hazards of fracture: New ICS users: 0.150 and 0.084 New ICS/LABA users: 0.120 and 0.045 (p<0.001) | Among newly diagnosed patients with COPD and new users of ICSs or ICS/LABA, use of ICS/LABA in a single inhaler was associated with delayed first hospitalisation for fracture, as compared with use of ICSs alone. | | +| GONNELLI [51] Prevalence of vertebral fractures (radiology): No treatment (n=509): 32.3% ICS (n=1664): 42.6% Other treatments (n=449): 41.8% Prevalence of vertebral fractures (radiology) in patients with ICSs (n=1664) by dose: ICS ⩽750 g: 39.9% ICS 750–1500 μg: 41.9% ICS >1500 μg: 46% Fractures vs no fractures: ICS >1500 μg·day–1 : OR 1.40 (95% CI 1.04–1.89; p 0.03) ICS 750–1500 μg·day–1 : OR 1.36 (95% CI 0.93–1.72; NS) ICS <750 μg·day–1 : OR 1.26 (95% CI 0.98–1.89; NS) No treatment: OR 1.00 (reference group) | The prevalence rates of vertebral fractures increased in patients taking higher daily doses of ICS. The risk of vertebral fractures was significantly increased in patients taking the highest dose (>1500 ug) of ICS. The use of ICSs at doses ranging from 751–1500 ug was associated with a positive, but not significant, increase in vertebral fracture risk. | | +| Metabolic complications: diabetes CAUGHEY [45] Diabetes-related hospitalisations: After 5 years, 19.8% of ICS users had a diabetes-related hospitalisation vs 16.2% in non-users (p=0.18) When stratified by ICS dose, patients who received a total DDD of ICSs ⩾0.83/day had a 94% increased likelihood of diabetes-related hospitalisation (HR 1.94, 95% CI 1.14–3.28; p=0.014) compared with patients not receiving ICS; lower ICS doses (<0.83 DDD/day) not associated with increased risks of diabetes-related hospitalisations | In patients with diabetes and COPD, an increased risk of diabetes-related hospitalisations was only evident with the use of high doses of ICS. A dose-dependent increase in risk associated with ICS use was observed. | | +| FLYNN [49] | Worsening of existing diabetes: ICS use: 326.1 per 1000 person-years No ICS use: 318.4 per 1000 person-years HR 0.57 (95% CI 0.25–1.30) | ICS use was not associated with new-onset diabetes nor worsening of existing diabetes. | +| PRICE [63] | Change in HbA1c: ICS use: median 0.18% (IQR −0.23–0.6%) No ICS use: median 0.03% (IQR −0.33–0.5%) Adjusted difference 0.16% (95% CI 0.05–0.27%) Increase in rate of hospitalisation for diabetes mellitus: ICS use: 41 (6.0%) No ICS use: 31 (4.5%) Progression to insulin: ICS use: 28 (4.7%) No ICS use: 12 (2%) | For patients with COPD and comorbid type 2 diabetes mellitus, ICS therapy may have a negative impact on diabetes control. Patients prescribed higher cumulative doses of ICSs may be at greater risk of diabetes progression. Continued | + +| TABLE 1 Continued First author Results | Conclusions | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| [ref.] | | | +| PRICE [62] | Diabetes progression: Patients initiating ICS: mean rate 33.3 per 100 patient-years Control (LABD): mean rate 37.2 per 100 patient-years HR 1.04 (95% CI 0.87–1.25) Diabetes onset: Patients initiating ICS: mean rate 1.25 diagnoses per 100 patient-years Control (LABD): mean rate 1.05 diagnoses per 100 patient-years aHR 1.27 (95% CI 1.07–1.50; p=0.006) | For patients prescribed ICS, compared with LABD, the risk of diabetes onset was significantly increased with overall no increase in risk of diabetes progression. The risk of diabetes progression also showed a clear dose–response relationship with mean daily ICS exposure for all patients and for the GOLD A/B and GOLD C/D subgroups. | +| AJMERA [44] | New-onset diabetes: ICS use: 7.4% No ICS use: 1.39% aOR 1.23 (95% CI 1.07–1.47) | ICS use was associated with an increased risk of new-onset diabetes. | +| Local complications: eye disorders FLYNN [49] Cataract-related outcomes: cumulative exposure univariate analysis HR 1.43 (95% CI 1.11–1.83); multivariate analysis HR 1.42 (95% CI 1.07–1.88) | There was a significant association between ICS use and increased cataract-related admissions. | | +| KENDZERSKA | Cataract surgery: adjusted association, HR 0.93 (95% CI 0.88–0.98) | No association between ICS use and cataract surgery. | +| [54] | | | +| Local complications: oral candidiasis DEKHUIJZEN Oral candidiasis: [48] ICS/LABA: 5.5% No ICS: 2.7% aOR 2.18 (95% CI 1.84–2.59) Comparison between type of ICS: Budesonide: 5.7% Fluticasone: 7.0% aOR 0.77 (95% CI 0.63–0.94) After adjusting for intended ICS daily dose: fully aOR 1.04 (95% CI 0.54–2.00) | ICS use increases the incidence of oral thrush in COPD, and this effect is dose-dependent for fluticasone/salmeterol therapies. Significantly fewer patients prescribed budesonide developed oral thrush compared with patients prescribed fluticasone. However, after adjusting for intended ICS daily dose, no significant differences were found in the incidence of oral thrush between budesonide and fluticasone study arms. A pattern of increasing odds for oral thrush with increasing ICS dose was observed. | | +| ICS: inhaled corticosteroid; TB: tuberculosis; aHR: adjusted hazard ratio; RR: risk ratio; OR: odds ratio; HR: hazard ratio; FEV1: forced expiratory | | | + +ICS: inhaled corticosteroid; TB: tuberculosis; aHR: adjusted hazard ratio; RR: risk ratio; OR: odds ratio; HR: hazard ratio; FEV1: forced expiratory volume in 1 s; NTM: nontuberculous mycobacteria; RCT: randomised clinical trial; LABA: long-acting β2-agonist; NS: nonsignificant; DDD: defined daily dose; GOLD: Global Initiative for Chronic Obstructive Lung Disease; aOR: adjusted odds ratio; IQR: interquartile range; LABD: long-acting bronchodilators. + +Fractures In total, 19 studies reported this outcome: nine RCTs, five cohort studies and five NCC studies. The pooled analysis of 10 RCTs with 41 342 participants did not observe a higher risk of fracture with exposure to ICSs (RR 1.10, 95% CI 0.97–1.25; p=0.13; I2=0%) [9, 14, 16, 18, 24, 25, 27, 31, 34, 43] +(figure 2). No differences were observed depending on the type of ICS used; however, this analysis is not very informative since all but two studies used fluticasone [27, 34]. + +Five cohort studies reported this outcome [46, 49–51, 54]; of these, only three compared ICSs versus no ICSs and reported the effects in terms of HRs. These studies did not find an increased risk of fractures in patients exposed to ICSs for ⩾1 year (HR 1.00, 95% CI 0.97–1.03, p=0.79; I2=0%) [49, 50, 54] +(supplementary figure S3). Two additional cohort studies reported on fractures, but as they lacked a control group without ICS use, the results are presented in a descriptive way (rates) in table 1 [46, 51]. Four out of the five NCC studies showed an increased risk for fractures with ICS use [84, 89, 93, 95], with two showing an increasing risk with increased doses of ICSs [84, 95] and one found an increased risk only with fluticasone [93]. The remaining study only evaluated ICS exposure for up to 1 year and found no increased risk of nonvertebral fracture, but it could not evaluate long-term ICS use, very high ICS doses or vertebral fractures [87] (table 2). + +## Diabetes + +The pooled analysis of four RCTs with 31 151 participants showed that long-term exposure to ICSs was not associated with an increased risk of diabetes (RR 1.10, 95% CI 0.96–1.26; p=0.15; I2=0%) [14, 16, 25, 34] +(figure 3). However, four additional cohort studies found that exposure to ICSs for ⩾1 year marginally + +| TABLE 2 Outcomes from nested case–control studies First author [ref.] Results | Conclusions | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Infectious complications: pneumonia CASCINI [80] Incidence rate for current ICS use: 87/1000 patient-years | ICS use was associated with an excess risk of pneumonia. The | | +| Incidence rate for past ICS use: 32/1000 patient-years | effect was greatest for higher doses and elderly patients. | | +| aRR (current ICS use vs no use): 2.29 (95% CI 1.99–2.63) aRR (past ICS use vs no use): 1.23 (95% CI 1.07–1.42) In current ICS users, a dose-related trend was tested | | | +| ERNST [81] | aRR of hospitalisation for pneumonia (current ICS use vs | The use of ICSs is associated with an excess risk of pneumonia | +| no use): 1.70 (95% CI 1.63–1.77) | hospitalisation and of pneumonia hospitalisation followed by | | +| aRR of pneumonia hospitalisation followed by death within | death within 30 days among elderly patients with COPD. | | +| 30 days (current ICS use vs no use): 1.53 (95% CI 1.30– 1.80) | | | +| RR of hospitalisation for pneumonia (highest doses of ICSs vs non-users): 2.25 (95% CI 2.07–2.44) The risk of pneumonia increased with higher ICS doses | | | +| JOO [88] | aOR of pneumonia hospitalisation (current ICS users vs | The use of ICSs among patients with newly diagnosed COPD is | +| non-users): 1.38 (95% CI 1.31–1.45) | associated with an increased risk of hospitalisation for pneumonia. | | +| MAPEL [92] | Relative to patients using SABDs, the control group, the only treatment associated with a nonsignificant increased risk of pneumonia was ICSs used alone (OR 1.29, 95% CI 0.96–1.73; p=0.09) | Patients with COPD using ICSs alone had a slightly increased risk of pneumonia that did not reach statistical significance (adjusted risk 26–29%). Those who used FSC or other combinations of ICSs with LABA did not have an increased | +| Users of FSC had no increased risk for pneumonia relative | risk for pneumonia. | | +| to SABDs (OR 1.03, 95% CI 0.74–1.42) | | | +| SUISSA [96] | RR of serious pneumonia (current ICS use vs no use): 1.69 | ICS use by patients with COPD increases the risk of serious | +| (95% CI 1.63–1.75) | pneumonia. The risk is particularly elevated and dose-related | | +| RR of serious pneumonia with fluticasone: 2.01 (95% CI | to fluticasone. | | +| 1.93–2.10) | | | +| RR of serious pneumonia with budesonide: 1.17 (95% CI 1.09–1.26) The risk of serious pneumonia was sustained with long-term ICS use and declined gradually after stopping ICS use, disappearing after 6 months The rate of serious pneumonia was higher with fluticasone (RR 2.01, 95% CI 1.93–2.10), but was much lower with budesonide (RR 1.17, 95% CI 1.09–1.26) | | | +| SUISSA [97] | Incidence rate of serious pneumonia: 2.8/100 person-years | The study assessed the effect of ICSs discontinuation in COPD on the incidence of serious pneumonia. Discontinuation of ICS use in COPD is associated with a reduction in the elevated risk of serious pneumonia, particularly with fluticasone. | +| aRR of serious pneumonia (discontinued use vs current use): 0.63 (0.60–0.66) Discontinuation of ICSs was associated with a decrease in the rate of serious pneumonia; the risk reduction was rapidly evident, increasing from 20% in the first month to 50% by the fourth month after discontinuation | | | +| THORNTON SNIDER | Pneumonia (current ICS users vs non-users): OR 1.26 (95% | ICS use, particularly current use and high-dose use, is | +| [99] | CI 1.16–1.36) | associated with increased pneumonia risk. | +| Pneumonia (current high-dose users vs non-users): OR 1.55 (95% CI 1.25–1.92) The risk increased with higher ICS doses | | | +| WANG [100] | Pneumonia (current use of ICSs vs no use): aOR 1.25 (95% | ICSs are significantly associated with an increased risk of | +| CI 1.20–1.30) | pneumonia in patients with COPD. The effect is prominent for | | +| Pneumonia (past use of ICSs vs no use): aOR 1.21 (95% CI | fluticasone-containing ICSs but not for budesonide-containing | | +| 1.16–1.26) | ICSs. | | +| There was an increase in the OR with an increase in the average daily dosage Fluticasone users were more likely to be at a higher risk of pneumonia; in contrast, there were no statistically significant associations between the risk of pneumonia and the use of budesonide | Continued | | + +| TABLE 2 Continued First author [ref.] Results | Conclusions | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Infectious complications: TB and NTM lung disease ANDRÉJAK [77] NTM: Patients with COPD vs no COPD: aOR 13.1 (95% CI 7.4– 23.3) COPD on current ICS therapy vs no COPD: aOR 29.1 (95% CI 13.3–63.8) COPD and former ICS use (> 6 months) vs no COPD: aOR 3.8 (95% CI 0.9–16.8) | Chronic respiratory disease, particularly COPD treated with ICS therapy, is a strong risk factor for NTM-PD. The risk is highly reduced when ICSs are withdrawn. The risk of NTM disease is higher with higher doses of ICS. | | +| BRODE [79] | NTM-PD: Current ICS use vs non-ICS use: aOR 1.86 (95% CI 1.60– 2.15) aOR for current fluticasone use: 2.09 (95% CI 1.80–2.43) aOR for current budesonide use: 1.19 (95% CI 0.97–1.45) There was a strong dose–response relationship between incident NTM-PD and cumulative ICS dose over 1 year | This study suggests that ICS use is associated with an increased risk of NTM-PD but was not significant for TB. | +| TB: Current ICS use vs non-ICS use: aOR 1.43 (95% CI 0.95–2.16) | | | +| BRASSARD [78] | TB: Any ICS use: RR 1.27 (95% CI 1.05–1.53) Current ICS use: RR 1.33 (95% CI 1.04–1.71) | Exposure to ICSs is not associated with risk of TB in the presence of OCSs but is associated with increased TB risk in non-users of OCSs. | +| LEE [90] | TB: aOR for ICS use: 1.20 (95% CI 1.08–1.34) The association was dose-dependent (p for trend <0.001) | ICS use increases the risk of TB. A subgroup analysis revealed that ICS use increased the risk of TB development among non-users of OCSs but not among OCS users. | +| Metabolic complications: fractures GONZALEZ [84] Fracture RR (any use of ICSs vs no use): 1.00 (95% CI 0.97– | Long-term ICS use at high doses is associated with a modest | | +| 1.03) | increase in the risk of hip and upper extremity fractures in patients with COPD. This dose-duration risk increase does not | | +| The fracture rate was increased with >4 years of ICS use at | appear to be higher for women. | | +| daily doses ⩾1000 μg in fluticasone equivalents (RR 1.10, 95% CI 1.02–1.19) | | | +| JOHANNES [87] | ORs for exposure in the preceding 30 days were 1.05 (95% CI 0.89–1.24), 1.13 (95% CI 0.90–1.40), and 0.97 (95% CI 0.78–1.21) for all ICS, fluticasone propionate and other ICS, respectively No dose–response effect was present | No increased nonvertebral fracture risk with ICS exposure as a class or with fluticasone propionate alone was detected. This study could not evaluate very high-dose ICS, long-term ICS exposure or vertebral fracture risk. | +| LEE [89] | Exposure to ICSs at any time during follow-up was not associated with an increased fracture risk (aOR 0.97, 95% CI 0.84–1.11); however, current high-dose ICS users (⩾700 µg per day) had an increased risk of fractures compared with patients with no exposure (aOR 1.68, 95% CI 1.10–2.57) | In patients with COPD, current use of high-dose ICS was associated with an increased risk of nonvertebral fractures. | +| MILLER [93] | Use of FSC in the year prior to the index date was associated with a statistically significant increase in the odds of nonvertebral fractures (aOR 1.25, 95% CI 1.07– 1.47) | Use of FSC was associated with an elevation in the risk of nonvertebral fractures. No increased risk was observed for other ICS use. | +| PUJADES-RODRÍGUEZ | Risk of fracture increased with increasing mean daily doses | | +| [95] | of inhaled corticosteroid (p for trend 0.007) and was most marked in those whose daily dose was >1600 µg (OR 1.80, 95% CI 1.04–3.11) | Use of ICSs is associated with a significant increase in fracture risk, particularly at higher doses. | +| Metabolic complications: diabetes GAYLE [83] Type 2 diabetes mellitus: The aOR for patients receiving ICSs (higher doses) significantly increased compared with patients receiving no ICS therapy (OR 1.73, 95% CI 1.65–1.82) Patients with a high number of ICS prescriptions were more likely to develop diabetes (OR 1.83, 95% CI 1.48–2.26) for 16–20 prescriptions and 1.61 (95% CI 1.53–1.69) for 1–5 prescriptions | Incidence of type 2 diabetes mellitus among patients with COPD is high and exposure to ICSs and frequent exacerbations are associated with a higher risk of type 2 diabetes mellitus among patients with COPD. Continued | | + +| TABLE 2 Continued First author [ref.] Results | Conclusions | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| SUISSA [98] | Current use of ICSs vs no ICS use was associated with a 34% increase in the rate of new-onset diabetes (RR 1.34, 95% CI 1.29–1.39) and diabetes progression (RR 1.34, 95% CI 1.17–1.53) | In patients with respiratory disease, ICS use is associated with modest increases in the risks of diabetes onset and diabetes progression. The risks are more pronounced at the higher doses currently prescribed in the treatment of COPD. | +| The risk increases were greatest with the highest ICSs doses, equivalent to fluticasone 1000 g per day or more (RR 1.64, 95% CI 1.52–1.76 and RR 1.54, 95% CI 1.18– 2.02, respectively) | | | +| Local complications: eye disorders JICK [86] After adjusting for age and gender, incidence rates of cataract development in the ICS subjects were slightly higher than in the nonexposed cohort (RR 1.3, 95% CI 1.1–1.5) | Age appears to modify the association between use of ICSs and the risk of cataract development with an increased effect among the oldest patients at the highest dosages of ICS. This finding may be owing to a differential effect of the drug at | | +| Among individuals ⩾40 years of age, the RR increased with | different ages or to the influence of diagnostic bias and | | +| use of increasing numbers of ICS prescriptions; this | uncontrolled confounding among older patients. | | +| trend was not evident in those under age 40 | | | +| GONZALEZ [85] | The aRR for glaucoma was 1.05 (95% CI 0.91–1.20) with ICS | Current use and continuous use of high-dose ICSs did not result | +| use in the preceding 30 days | in an increased risk of glaucoma or raised intra-ocular | | +| There was no dose-related effect of ICSs on the risk of | pressure requiring treatment. | | +| glaucoma or raised intra-ocular pressure requiring treatment; continuous use of high-dose ICSs for ⩾3 months was not associated with an increased risk of glaucoma | | | +| MILLER [94] | 2941 incident cataract cases and 327 incident glaucoma | FSC or other ICS exposure was not associated with an increased | +| cases were identified in the COPD cohort (n=53 191) | odds of cataracts or glaucoma, nor was a dose–response | | +| Fluticasone (as fixed-dose combination) or ICS | relationship observed. | | +| prescriptions were not associated with risk of incident cataracts or glaucoma for any exposure (dose) category, after adjusting for confounders | | | +| A trend was observed according to the duration and the number of prescriptions in the last year for cataracts, but not for glaucoma | | | +| ICS: inhaled corticosteroid; aRR: adjusted risk ratio; RR: risk ratio; aOR: adjusted odds ratio; SABD: short-acting bronchodilator; OR: odds ratio; FSC: fluticasone propionate/salmeterol fixed-dose combination; LABA: long-acting β2-agonist; TB: tuberculosis: NTM: nontuberculous mycobacteria; NTM-PD: nontuberculous mycobacteria pulmonary disease; OCS: oral corticosteroid. | | | + +increased the risk of new-onset diabetes (HR 1.06, 95% CI 1.00–1.13, p=0.06; I2=79%) [49, 54, 62, 64] +(figure 4). This analysis included the most conservative estimate (risk associated with low-dose ICS) of one study [64], that showed a dose–response relationship with the HR ranging from 1.076–1.150 from low- to high-dose ICSs (all p<0.0001). Among the five cohort studies, two found an association between ICSs and risk of diabetes onset [44, 63], one found an increased risk to progression to insulin treatment with high ICS doses [62] and another found an association between high ICS doses and increased risk of diabetes-related hospitalisations [45]. Only one study did not find an association between ICS use and diabetes [49] (table 1). Two NCCs also found a positive association between exposure to ICSs and an increased risk in diabetes-related outcomes, in particular with high doses of ICSs [83, 98] (table 2). + +## Local Complications + +Eye disorders 13 studies reported this outcome (nine RCTs, two cohort studies and three NCC studies). The pooled analysis of nine RCTs with 40 981 participants showed that exposure to ICSs was associated with a trend to a higher risk of an eye disorder, but the increase was not statistically significant (RR 1.08, 95% CI +0.93–1.25; p=0.32; I2=0%) [9, 14, 16, 18, 24, 25, 29, 34, 40] (supplementary figure S4). The results did not change when evaluating cataracts or other eye disorders separately (supplementary figure S5). Two additional cohort studies assessed this outcome, with conflicting results. While one study [49] observed an increased risk of cataract development associated with cumulative ICS exposure (HR 1.43, Risk of bias A B C D E F + +![10_image_0.png](10_image_0.png) + +| ICS | | | | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------|---------------------| +| Events | No ICS | | | | +| Events | | | | | +| Study or subgroup [ref.] | Total | Total | Weight % Risk ratio (95% CI) | Risk ratio (95% CI) | +| Fluticasone BURGE [18] CALVERLEY [9] DRANSFIELD [25] MALTAIS [31] VESTBO [14] VESTBO [16] WEDZICHA [24] WOUTERS [43] Subtotal Total events Heterogeneity: Tau2=0.01; Chi2=8.02, df=7 (p=0.33); I2=13% Test for overall effect: Z=1.41 (p=0.16) 9 143 47 12 45 175 17 5 376 3098 2437 141 1396 8297 658 189 16592 17 118 8 7 45 166 12 5 375 3086 818 142 1403 8271 665 184 14944 | 2.5 28.0 2.9 2.0 9.7 36.1 3.0 1.1 85.3 | 0.53 (0.24–1.17) 1.21 (0.95–1.53) 1.97 (0.94–4.16) 1.73 (0.70–4.26) 1.01 (0.67–1.51) 1.05 (0.85–1.30) 1.43 (0.69–2.97) 0.97 (0.29–3.31) 1.12 (0.96–1.32) | | | +| 453 | 378 | | | | +| Budesonide PAWELS [27] RABE [34] Subtotal Total events Heterogeneity: Tau2=0.00; Chi2=0.48, df=1 (p=0.49); I2=0% Test for overall effect: Z=0.05 (p=0.96) 5 130 643 3 6404 44 7047 634 | 0.8 | 1.64 (0.39–6.85) | | | +| 2125 | 13.9 | 0.98 (0.70–1.38) | | | +| 2759 | 14.7 | 1.01 (0.73–1.40) | | | +| 135 | 47 | | | | +| Total Total events Heterogeneity: Tau2=0.00; Chi2=8.83, df=9 (p=0.45); I2=0% Test for overall effect: Z=1.50 (p=0.13) Test for subgroup differences: Chi2=0.34, df=1 (p=0.56); I2=0% 23639 17 703 | 100.0 | 1.10 (0.97–1.25) | | | +| 588 | 425 | 0.02 | 10 | 50 | +| 0.1 | 1 | | | | +| Favours ICS | Favours no ICS | | | | +| Risk of bias legend A Random sequence generation (selection bias) B Allocation concealment (selection bias) C Blinding of participants and personnel (performance bias) D Blinding of outcome assessment (detection bias) E Incomplete outcome data (attrition bias) | | | | | + +FIGURE 2 Pooled risk ratios for fractures (Mantel-Haenszel, random-effects model). ICS: inhaled corticosteroid. + +| ICS | | | | | | | | | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------|--------------------------------------------------------------------------------------|-------------------------------|---------------------|----|----|----|----|----|----| +| Events | No ICS | | | | | | | | | | +| Events | Risk of bias | | | | | | | | | | +| Study or subgroup [ref.] | Total | Total | Weight % Risk ratio (95% CI) | Risk ratio (95% CI) | A | B | C | D | E | F | +| DRANSFIELD [25] RABE [34] VESTBO [14] VESTBO [16] Total Total events Heterogeneity: Tau2=0.00; Chi2=2.27, df=3 (p=0.52); I2=0% Test for overall effect: Z=1.43 (p=0.15) 55 198 23 301 2437 6404 1396 8297 18534 14 53 16 290 818 2125 1403 8271 12617 | 5.2 19.8 4.4 70.6 100.0 | 1.32 (0.74–2.36) 1.24 (0.92–1.67) 1.44 (0.77–2.72) 1.03 (0.88–1.21) 1.10 (0.96–1.26) | | | | | | | | | +| 577 | 373 | 0.02 | 10 | 50 | | | | | | | +| 0.1 | 1 | | | | | | | | | | +| Favours ICS | Favours no ICS | | | | | | | | | | +| Risk of bias legend A Random sequence generation (selection bias) B Allocation concealment (selection bias) | | | | | | | | | | | + +95% CI 1.11–1.84), another study [54] reporting on patients undergoing cataract surgery did not observe this association (HR 0.93, 95% CI 0.88–0.98). Only one of the three NCC studies found a significant association between ICS use and cataract development [86], but no association with glaucoma was described [85, 93, 96] (table 2). + +Risk of bias legend A Random sequence generation (selection bias) B Allocation concealment (selection bias) C Blinding of participants and personnel (performance bias) D Blinding of outcome assessment (detection bias) E Incomplete outcome data (attrition bias) F Selective reporting (reporting bias) +FIGURE 3 Pooled risk ratios for diabetes (Mantel-Haenszel, random-effects model) in randomised clinical trials. VESTBO [14] and DRANSFIELD [25] measured effects on glucose levels; VESTBO [16] and RABE [34] measured hyperglycaemia/new-onset diabetes mellitus. ICS: inhaled corticosteroid. + +| Hazard ratio | Hazard ratio | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------|----|----------| +| (95% CI) | (95% CI) | | | +| Study or subgroup [ref.] | log[Hazard ratio] | SE | Weight % | +| FLYNN [49] KENDZERSKA [54] PRICE [62] SAEED [64] Total Heterogeneity: Tau2=0.00; Chi2=13.99, df=3 (p=0.003); I2=79% Test for overall effect: Z=1.90 (p=0.06) –0.3425 0.0198 0.239 0.0733 0.2215 0.0204 0.0874 0.0005 2.0 39.9 10.5 47.6 100.0 | 0.71 (0.46–1.10) 1.02 (0.98–1.06) 1.27 (1.07–1.51) 1.08 (1.07–1.08) 1.06 (1.00–1.13) 0.2 | 2 | | +| 0.5 | 1 | 5 | | +| Favours ICS | Favours no ICS | | | + +FIGURE 4 Pooled hazard ratios for diabetes (inverse variance method, random-effects model) in cohort studies. Data from SAEED [64] refer exclusively to patients treated with low-dose inhaled corticosteroids (ICS). + +## Oral Candidiasis + +The pooled analysis of 16 RCTs with 33 725 participants showed that exposure to ICSs almost tripled the risk of oral candidiasis (RR 2.89, 95% CI 2.36–3.55; p<0.00001; I2=32%) [9, 12, 18–20, 22, 24, 26, 27, 33–35, 38–40, 43] (supplementary figure S6). The results were quite consistent across studies and also according to the ICS used. Only one cohort study assessed this outcome [48]. The rate of oral candidiasis in patients exposed to ICSs was 5.5%, with a significant increased risk compared with non-ICS users. No significant difference was observed between patients exposed to budesonide and fluticasone. A pattern of increasing odds for oral candidiasis with increasing ICS dose was observed [48]. + +## Dysphonia + +The pooled analysis of nine RCTs with 22 841 participants showed that exposure to ICSs increased the risk of dysphonia by 277% (RR 3.77, 95% CI 2.81–5.05; p<0.00001; I2=0%) [9, 12, 13, 18, 22, 29, 34, 35, 38] (supplementary figure S7). The results were highly consistent across studies and also according to the ICS used. + +## Other Adverse Events + +Hypertension The pooled analysis of eight RCTs with 48 207 participants showed that exposure to ICSs was not significantly associated with an increased risk of hypertension overall (RR 1.05, 95% CI 0.89–1.24; p=0.57; I2=39%) [9, 14, 17, 22, 30, 33, 34, 36] (supplementary figure S8). + +## Adrenal Suppression + +Two RCTs assessed this outcome. One case of adrenal insufficiency in patients assigned to fluticasone (n=4135) was observed in one study [16]. The other trial found no evidence that long-term use of a moderate dose of inhaled triamcinolone suppresses either basal or stimulated adrenal function in older patients with COPD (n=221) [41]. + +## Discussion + +Treatment of respiratory diseases with ICSs has significantly reduced the risk of side-effects associated with the chronic use of oral corticosteroids; however, the use of ICSs is not free from possible complications. In this systematic review we observed a significantly increased risk of local side-effects, such as oral candidiasis and dysphonia; and an increased risk of some systemic effects such as respiratory infections ( pneumonia and mycobacterial disease). Other systemic side-effects such as diabetes-related outcomes and bone fractures were only significantly associated with ICSs at high doses and with prolonged exposures in some studies, especially cohort and NCC studies. Finally, we could not find clear evidence of increased risk of sepsis, eye disorders, hypertension or adrenal suppression. In this analysis we included not only RCTs, but also cohort and NCC studies, because most RCTs have a duration of 1 year, and this duration may not be long enough to investigate the risk associated with ICSs treatment. It should be taken into account that patients with COPD could be treated with ICSs for decades, and these patients are usually elderly with frequent comorbidities [8], making them more susceptible to the development of corticosteroid-related side-effects. The quality of the RCTs included in the meta-analyses was, in general, high. Only observational studies were included in the narrative review (with the exception of a few cohort studies) and these studies were not used to estimate the magnitude of the associations between ICSs and side-effects; therefore, no quality analysis was conducted. In general, the quality of observational studies is low (or at best moderate) because: 1) control of confounding variables is not adequate; 2) there is no blinding of the interventions to prevent measurement errors; and 3) they are often retrospective and collect information from registries of medical records from administrative databases. For these reasons, they have only been included in our review as complementary information. The descriptive data in supplementary table S2 provide complete details about the characteristics of these studies that will help the interested reader to interpret their results. An increased susceptibility to infections is a recognised side-effect of the use of systemic corticosteroids. Among other properties, corticosteroids, including ICS, decrease the macrophage production of cytokines that is required for antibacterial immunity [101]. Our meta-analysis of data from 19 RCTs demonstrated a 41% increased risk of pneumonia with ICS, and this significant increase was also observed in cohort and NCC studies. The increased risk of pneumonia was well identified in the 3-year TORCH study with fluticasone propionate [9]. Since then, several studies and reviews have confirmed this association. In 2014, a Cochrane review concluded that the use of ICSs in COPD was associated with an increased risk of serious adverse pneumonia events, but it did not significantly affect mortality [102]. The authors could not find significant differences in risk between fluticasone propionate and budesonide. In contrast, we observed a reduced risk of pneumonia with budesonide compared with fluticasone, as suggested by other authors [52]. A recent systematic review of evidence from direct-comparison studies showed a significantly increased risk of 13.5% and 14.4% for pneumonia and severe pneumonia, respectively, among fluticasone compared with budesonide users [103]. The risk associated with the use of ICSs may be more relevant in patients with other risk factors for pneumonia. In two replicate, 1-year studies, the risk factors associated with at least a two-fold increase in the risk of pneumonia with fluticasone furoate treatment were being a current smoker, having prior pneumonia, a body mass index <25 kg·m−2, and severe airflow limitation [104]. Another factor to consider is the blood eosinophil count, since an increased risk of pneumonia has been observed when ICSs are prescribed to COPD patients with low blood eosinophil counts [105], and particularly if they have chronic bronchial infection or associated bronchiectasis [106]. The immunosuppressive effect of ICSs has been related to an increased risk of TB and nontuberculous mycobacteria pulmonary disease (NTM-PD); however, this effect has not been observed in RCTs due to the low incidence of cases of mycobacterial disease. A recent study observed an incidence rate of TB ranging from 0.15 to 0.17 per 100 person-years in fluticasone or budesonide users, respectively [75], which is approximately 100 times lower than the incidence of pneumonia in ICS users with COPD [52, 72]. Therefore, the impact of ICSs on pulmonary mycobacterial diseases should be explored in large observational studies. The majority of cohort and NCC studies indicated a significant increase in the risk of TB and NTM-PD associated with the use of higher ICS doses, without a clear difference between fluticasone and budesonide [56, 65, 75–78, 90, 92]. Interestingly, one of the most important risk factors for the development of TB in ICS users is a previous history of TB [56, 92]; therefore, the incidence of TB in patients with COPD treated with ICSs is very different in different geographic areas depending on the prevalence of TB in the area. Another interesting observation is that the use of oral corticosteroids is more strongly associated with the development of TB, and consequently, the increase in risk of TB associated with the use of ICSs is not observed in patients using oral corticosteroids simultaneously [76, 78]. The number of studies dedicated to the risk of NTM-PD is very limited, but they consistently show an increased risk with the use of ICS. ANDRÉJAK et al. [77] observed a significantly increased risk of NTM-PD +in patients with COPD compared with controls, which was significantly higher with the current use of ICS, with higher doses of ICSs and with an increased number of previous hospital admissions. The adjusted OR +for NTM-PD for COPD patients with current ICS use versus controls was 29.1 (95% CI 13.3–63.8). + +In a large RCT, the use of triamcinolone was associated with a significant reduction of BMD [42], which was not observed in shorter, 1-year RCTs. Additionally, cohort studies show a relationship of ICS use with significant BMD decline and a dose–response relationship, with a significant effect at ICS doses of 500 μg·day–1 or greater [46, 51]. It is unclear whether and how these effects translate to fractures. The NCC studies showed a similar effect of ICSs in terms of an increase in risk of fractures with a dose– +response relationship and only significant at high doses [84, 87, 89, 93, 95]. However, results from RCTs and cohort studies did not show a significant risk of fractures associated with ICS use. The lack of association observed in RCTs may be due to the shorter duration of observation and the selection of participants of a younger age and with fewer comorbidities. From the evaluation of the results, it appears that the risk of fractures is only significant when ICSs are used at high doses during prolonged periods of time and in particular in patients with other risk factors. One of the largest studies [84] found a 10% +increased risk of fractures with the use of fluticasone at doses ⩾1000 μg·day–1 for >4 years and an 11% increased risk with budesonide at doses ⩾1600 μg·day–1 for >4 years. However, there are some uncertainties about this modest but significant increase in the risk of fractures. In fact, in a recent narrative review of the evidence, CARAMORI et al. [107] concluded that the exact relationship between long-term ICS +use and bone fracture incidence in patients with COPD remains unclear, primarily due to the lack of information about baseline BMD and other risk factors for fractures such as smoking or physical activity as well as a lack of a standardised definition of fractures. Systemic corticosteroids are known to increase the risk of diabetes, but the risk associated with ICSs is less clear. This is particularly important, because in most studies diabetes appears as one of the most frequent comorbidities in patients with COPD [8]. The meta-analysis of RCTs did not show a significant effect of ICSs on the risk of diabetes; however, the cohort and the NCC studies consistently showed an increased risk of diabetes-related outcomes, in particular when high doses of ICSs are used. A large study in Canada showed a 34% increase in the rate of diabetes and another 34% increase in the risk of diabetes progression, with these risks increasing to 64% and 54%, respectively, with the use of high doses of fluticasone of +⩾1000 μg·day–1 [98]. + +We could not conclusively demonstrate an increased risk of eye disorders associated with ICS use in COPD. The pooled analysis of data from eight RCTs did not show a significant effect of ICSs on different eye problems. No evidence of an increased risk of glaucoma was observed in the cohort and NCC studies either; however, results for the associated risk of cataracts were more conflicting. One cohort and one NCC study found a significantly increased risk of cataracts associated with the cumulative ICS exposure [49, 85], while two other studies did not find any significant associations [54, 93]. In contrast to eye disorders, the results observed with other local side-effects such as oral candidiasis and dysphonia were much clearer. The use of ICSs was associated with an almost three-fold higher risk of oral candidiasis and almost 3.5-fold higher risk of dysphonia compared with non-users, without significant differences between different drugs. The high frequency of these disorders has been well documented in RCTs and also in observational studies and, although not severe, may result in discontinuation of treatment in some cases. Since the long-term use of systemic corticosteroids may increase the risk of hypertension and adrenal suppression, we also wanted to investigate the evidence of a possible increase in the risk of these complications with the use of ICS. The pooled analysis of eight RCTs did not observe an increased risk of hypertension, and one RCT found only one case of adrenal insufficiency among >4000 patients treated with fluticasone [16]; consequently, no reliable conclusions could be drawn about the risk of adrenal suppression associated with the use of ICS. Our study has attempted to provide a comprehensive overview of the risks associated with the use of ICSs in patients with COPD. However, it has the limitation that our search was restricted to studies on patients with COPD, and therefore, we may have missed some information from studies that analysed the adverse effects of ICSs in respiratory patients in general, without a clear identification of those with COPD. In conclusion, we observed that the use of ICSs in patients with COPD is associated with an increased risk of several side-effects. The highest risk corresponded to local disorders, such as oral candidiasis and dysphonia, followed by infectious complications such as pneumonia and mycobacterial diseases, and diabetes-related outcomes, although with lower frequency. The risks of osteoporosis, bone fractures and eye disorders are less clear. For most of these complications a dose–response relationship was observed, indicating that lower doses of ICS should be used in patients with COPD whenever possible. This information should help clinicians to make informed decisions about the prescription of ICSs in patients with COPD. + +Provenance: Submitted article, peer reviewed. This study is registered on www.crd.york.ac.uk/prospero with identifier CRD42020168023. Conflict of interest: M. Miravitlles reports speakers fees from AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, Menarini, Rovi, Bial, Zambon, Sandoz, CSL Behring, Grifols and Novartis; consultancy fees from AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Bial, Gebro Pharma, CSL Behring, Laboratorios Esteve, Ferrer, Mereo Biopharma, Verona Pharma, Kamada, TEVA, Sanofi, pH Pharma, Novartis and Grifols; and research grants from GlaxoSmithKline and Grifols, outside the submitted work. A. Auladell-Rispau has nothing to disclose. M. Monteagudo has nothing to disclose. J.C. Vázquez-Niebla has nothing to disclose. J. Mohammed reports a grant from the European Respiratory Society (ERS Fellowship in Guidelines Methodology), outside the submitted work. A. Nuñez has nothing to disclose. G. Urrútia has nothing to disclose. Support statement: This study was funded by an unrestricted grant from Boehringer Ingelheim. Funding information for this article has been deposited with the Crossref Funder Registry. + +References 1 Nici L, Mammen MJ, Charbek E, et al. Pharmacologic management of chronic obstructive pulmonary disease. + +an official American Thoracic Society Clinical Practice Guideline. Am J Respir Crit Care Med 2020; 201: e56–e69. + +2 Pascoe S, Barnes N, Brusselle G, et al. Blood eosinophils and treatment response with triple and dual combination therapy in chronic obstructive pulmonary disease: analysis of the IMPACT trial. Lancet Respir Med 2019; 7: 745–756. + +3 Singh D, Bafadhel M, Brightling CE, et al. Blood eosinophil counts in clinical trials for chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2020; 202: 660–671. + +4 Barrecheguren M, Monteagudo M, Ferrer J, et al. Treatment patterns in COPD patients newly diagnosed in primary care. A population-based study. Respir Med 2016; 111: 47–53. + +5 Koblizek V, Milenkovic B, Barczyk A, et al. Phenotypes of COPD patients with smoking history in central and Eastern Europe - the POPE Study. Eur Respir J 2017; 49: 1601446. + +6 Casas A, Montes de OCA M, Menezes AM, et al. Respiratory medication used in COPD patients from seven Latin American countries: the LASSYC Study. Int J Chron Obstruct Pulmon Dis 2018; 13: 1545–1556. + +7 Brutsche MH, Brutsche IC, Munawar M, et al. Comparison of pharmacokinetics and systemic effects of inhaled fluticasone propionate in patients with asthma and healthy volunteers: a randomised crossover study. Lancet 2000; 356: 556–561. + +8 Divo MJ, Casanova C, Marin JM, et al. COPD comorbidities network. Eur Respir J 2015; 46: 640–650. + +9 Calverley PM, Anderson JA, Celli B, et al. Salmeterol and fluticasone propionate and survival in chronic obstructive pulmonary disease. N Engl J Med 2007; 356: 775–789. + +10 Roland NJ, Bhalla RK, Earis J. The local side effects of inhaled corticosteroids: current understanding and review of the literature. Chest 2004; 126: 213–219. + +11 Higgins JP, Altman DG, Gøtzsche PC, et al. The Cochrane Collaboration's tool for assessing risk of bias in randomised trials. BMJ 2011; 343: d5928. + +12 Aaron SD, Vandemheen KL, Fergusson D, et al. Tiotropium in combination with placebo, salmeterol, or fluticasone-salmeterol for treatment of chronic obstructive pulmonary disease: a randomised trial. Ann Intern Med 2007; 146: 545–555. + +13 Anzueto A, Ferguson GT, Feldman G, et al. Effect of fluticasone propionate/salmeterol (250/50) on COPD +exacerbations and impact on patient outcomes. COPD 2009; 6: 320–329. + +14 Vestbo J, Leather D, Diar Bakerly N, et al. Effectiveness of fluticasone furoate-vilanterol for COPD in clinical practice. N Engl J Med 2016; 375: 1253–1260. + +15 Bakerly ND, Woodcock A, Collier S, et al. Benefit and safety of fluticasone furoate/vilanterol in the Salford Lung Study in chronic obstructive pulmonary disease (SLS COPD) according to baseline patient characteristics and treatment subgroups. Respir Med 2019; 147: 58–65. + +16 Vestbo J, Anderson JA, Brook RD, et al. Fluticasone furoate and vilanterol and survival in chronic obstructive pulmonary disease with heightened cardiovascular risk (SUMMIT): a double-blind randomised controlled trial. Lancet 2016; 387: 1817–1826. + +17 Brook RD, Anderson JA, Calverley PM, et al. Cardiovascular outcomes with an inhaled beta2-agonist/ +corticosteroid in patients with COPD at high cardiovascular risk. Heart 2017; 103: 1536–1542. + +18 Burge PS, Calverley PM, Jones PW, et al. Randomised, double blind, placebo controlled study of fluticasone propionate in patients with moderate to severe chronic obstructive pulmonary disease: the ISOLDE trial. + +BMJ 2000; 320: 1297–1303. + +19 Calverley PM, Boonsawat W, Cseke Z, et al. Maintenance therapy with budesonide and formoterol in chronic obstructive pulmonary disease. Eur Respir J 2003; 22: 912–919. + +20 Calverley P, Pauwels R, Vestbo J, et al. Combined salmeterol and fluticasone in the treatment of chronic obstructive pulmonary disease: a randomised controlled trial. Lancet 2003; 361: 449–456. + +21 Ferguson GT, Calverley PMA, Anderson JA, et al. Prevalence and progression of osteoporosis in patients with COPD: results from the TOwards a Revolution in COPD Health study. Chest 2009; 136: 1456–1465. + +22 Calverley PM, Rennard S, Nelson HS, et al. One-year treatment with mometasone furoate in chronic obstructive pulmonary disease. Respir Res 2008; 9: 73. + +23 Calverley PMA, Stockley RA, Seemungal TAR, et al. Reported pneumonia in patients with COPD: findings from the INSPIRE study. Chest 2011; 139: 505–512. + +24 Wedzicha JA, Calverley PM, Seemungal TA, et al. The prevention of chronic obstructive pulmonary disease exacerbations by salmeterol/fluticasone propionate or tiotropium bromide. Am J Respir Crit Care Med 2008; 177: 19–26. + +25 Dransfield MT, Bourbeau J, Jones PW, et al. Once-daily inhaled fluticasone furoate and vilanterol versus vilanterol only for prevention of exacerbations of COPD: two replicate double-blind, parallel-group, randomised controlled trials. Lancet Respir Med 2013; 1: 210–223. + +26 Ferguson GT, Anzueto A, Fei R, et al. Effect of fluticasone propionate/salmeterol (250/50 microg) or salmeterol (50 microg) on COPD exacerbations. Respir Med 2008; 102: 1099–1108. + +27 Pauwels RA, Löfdahl CG, Laitinen LA, et al. Long-term treatment with inhaled budesonide in persons with mild chronic obstructive pulmonary disease who continue smoking. European Respiratory Society Study on Chronic Obstructive Pulmonary Disease. N Engl J Med 1999; 340: 1948–1953. + +28 Johnell O, Pauwels R, Löfdahl CG, et al. Bone mineral density in patients with chronic obstructive pulmonary disease treated with budesonide Turbuhaler. Eur Respir J 2002; 19: 1058–1063. + +29 Kerwin EM, Ferguson GT, Mo M, et al. Bone and ocular safety of budesonide/glycopyrrolate/formoterol fumarate metered dose inhaler in COPD: a 52-week randomized study. Respir Res 2019; 20: 167. + +30 Lipson DA, Barnhart F, Brealey N, et al. Once-daily single-inhaler triple versus dual therapy in patients with COPD. N Engl J Med 2018; 378: 1671–1680. + +31 Maltais F, Schenkenberger I, Wielders PLML, et al. Effect of once-daily fluticasone furoate/vilanterol versus vilanterol alone on bone mineral density in patients with COPD: a randomised, controlled trial. Ther Adv Respir Dis 2020; 14: 1753466620965145. + +32 Mathioudakis AG, Amanetopoulou SG, Gialmanidis IP, et al. Impact of long-term treatment with low-dose inhaled corticosteroids on the bone mineral density of chronic obstructive pulmonary disease patients: +aggravating or beneficial? Respirology 2013; 18: 147–153. + +33 Papi A, Vestbo J, Fabbri L, et al. Extrafine inhaled triple therapy versus dual bronchodilator therapy in chronic obstructive pulmonary disease (TRIBUTE): a double-blind, parallel group, randomised controlled trial. Lancet 2018; 391: 1076–1084. + +34 Rabe KF, Martinez FJ, Ferguson GT, et al. Triple inhaled therapy at two glucocorticoid doses in moderate-to-very-severe COPD. N Engl J Med 2020; 383: 35–48. + +35 Rennard SI, Tashkin DP, McElhattan J, et al. Efficacy and tolerability of budesonide/formoterol in one hydrofluoroalkane pressurised metered-dose inhaler in patients with chronic obstructive pulmonary disease: +results from a 1-year randomized controlled clinical trial. Drugs 2009; 69: 549–565. + +36 Sharafkhaneh A, Southard JG, Goldman M, et al. Effect of budesonide/formoterol pMDI on COPD +exacerbations: a double-blind, randomised study. Respir Med 2012; 106: 257–268. + +37 Vestbo J, Sørensen T, Lange P, et al. Long-term effect of inhaled budesonide in mild and moderate chronic obstructive pulmonary disease: a randomised controlled trial. Lancet 1999; 353: 1819–1823. + +38 Vestbo J, Papi A, Corradi M, et al. Single inhaler extrafine triple therapy versus long-acting muscarinic antagonist therapy for chronic obstructive pulmonary disease (TRINITY): a double-blind, parallel group, randomised controlled trial. Lancet 2017; 389: 1919–1929. + +39 Wedzicha JA, Banerji D, Chapman KR, et al. Indacaterol-glycopyrronium versus salmeterol-fluticasone for COPD. N Engl J Med 2016; 374: 2222–2234. + +40 Lung Health Study Research Group, Wise R, Connett J, Weinmann G, et al. Effect of inhaled triamcinolone on the decline in pulmonary function in chronic obstructive pulmonary disease. N Engl J Med 2000; 343: +1902–1909. + +41 Eichenhorn MS, Wise RA, Madhok TC, et al. Lack of long-term adverse adrenal effects from inhaled triamcinolone: Lung Health Study II. Chest 2003; 124: 57–62. + +42 Scanlon PD, Connett JE, Wise RA, et al. Loss of bone density with inhaled triamcinolone in Lung Health Study II. Am J Respir Crit Care Med 2004; 170: 1302–1309. + +43 Wouters EF, Postma DS, Fokkens B, et al. Withdrawal of fluticasone propionate from combined salmeterol/ +fluticasone treatment in patients with COPD causes immediate and sustained disease deterioration: a randomised controlled trial. Thorax 2005; 60: 480–487. + +44 Ajmera M, Shen C, Sambamoorthi U. Concomitant medication use and new-onset diabetes among medicaid beneficiaries with chronic obstructive pulmonary disease. Popul Health Manag 2017; 20: 224–232. + +45 Caughey GE, Preiss AK, Vitry AI, et al. Comorbid diabetes and COPD: impact of corticosteroid use on diabetes complications. Diabetes Care 2013; 36: 3009–3014. + +46 Cho KH, Kim YS, Linton JA, et al. Effects of inhaled corticosteroids /long-acting agonists in a single inhaler versus inhaled corticosteroids alone on all-cause mortality, pneumonia, and fracture in chronic obstructive pulmonary disease: a nationwide cohort study 2002–2013. Respir Med 2017; 130: 75–84. + +47 Crim C, Calverley PM, Anderson JA, et al. Pneumonia risk in COPD patients receiving inhaled corticosteroids alone or in combination: TORCH study results. Eur Respir J 2009; 34: 641–647. + +48 Dekhuijzen PNR, Batsiou M, Bjermer L, et al. Incidence of oral thrush in patients with COPD prescribed inhaled corticosteroids: effect of drug, dose, and device. Respir Med 2016; 120: 54–63. + +49 Flynn RW, MacDonald TM, Hapca A, et al. Quantifying the real life risk profile of inhaled corticosteroids in COPD by record linkage analysis. Respir Res 2014; 15: 141. + +50 Gershon AS, Campitelli MA, Croxford R, et al. Combination long-acting β-agonists and inhaled corticosteroids compared with long-acting β-agonists alone in older adults with chronic obstructive pulmonary disease. JAMA 2014; 312: 1114–1121. + +51 Gonnelli S, Caffarelli C, Maggi S, et al. Effect of inhaled glucocorticoids and β2 agonists on vertebral fracture risk in COPD patients: the EOLO study. Calcif Tissue Int 2010; 87: 137–143. + +52 Janson C, Larsson K, Lisspers KH, et al. Pneumonia and pneumonia related mortality in patients with COPD +treated with fixed combinations of inhaled corticosteroid and long acting β2 agonist: observational matched cohort study (PATHOS). BMJ 2013; 346: f3306. + +53 Janson C, Johansson G, Ställberg B, et al. Identifying the associated risks of pneumonia in COPD patients: +ARCTIC an observational study. Respir Res 2018; 19: 172. + +54 Kendzerska T, Aaron SD, To T, et al. Effectiveness and safety of inhaled corticosteroids in older individuals with chronic obstructive pulmonary disease and/or asthma. A population study. Ann Am Thorac Soc 2019; 16: 1252–1262. + +55 Kern DM, Davis J, Williams SA, et al. Comparative effectiveness of budesonide/formoterol combination and fluticasone/salmeterol combination among chronic obstructive pulmonary disease patients new to controller treatment: a US administrative claims database study. Respir Res 2015; 16: 52. + +56 Kim JH, Park JS, Kim KH, et al. Inhaled corticosteroid is associated with an increased risk of TB in patients with COPD. Chest 2013; 143: 1018–1024. + +57 Lee MC, Lee CH, Chien SC, et al. Inhaled corticosteroids increase the risk of pneumonia in patients with chronic obstructive pulmonary disease: a nationwide cohort study. Medicine (Baltimore) 2015; 94: e1723. + +58 Lin SH, Ji BC, Shih YM, et al. Comorbid pulmonary disease and risk of community-acquired pneumonia in COPD patients. Int J Tuberc Lung Dis 2013; 17: 1638–1644. + +59 Lin SH, Perng DW, Chen CP, et al. Increased risk of community-acquired pneumonia in COPD patients with comorbid cardiovascular disease. Int J Chron Obstruct Pulmon Dis 2016; 11: 3051–3058. + +60 Liu SF, Kuo HC, Liu GH, et al. Inhaled corticosteroids can reduce osteoporosis in female patients with COPD. + +Int J Chron Obstruct Pulmon Dis 2016; 11: 1607–1614. + +61 Morros R, Vedia C, Giner-Soriano M, et al. Neumonías adquiridas en la comunidad en pacientes con enfermedad pulmonar obstructiva crónica tratados con corticoides inhalados u otros broncodilatadores. Estudio PNEUMOCORT [Community-acquired pneumonia in patients with chronic obstructive pulmonary disease treated with inhaled corticosteroids or other bronchodilators. Study PNEUMOCORT]. Aten Primaria 2019; 51: 333–340. + +62 Price DB, Voorham J, Brusselle G, et al. Inhaled corticosteroids in COPD and onset of type 2 diabetes and osteoporosis: matched cohort study. NPJ Prim Care Respir Med 2019; 29: 38. + +63 Price DB, Russell R, Mares R, et al. Metabolic effects associated with ICS in patients with COPD and comorbid type 2 diabetes: a historical matched cohort study. PLoS One 2016; 11: e0162903. + +64 Saeed MI, Eklöf J, Achir I, et al. Use of inhaled corticosteroids and the risk of developing type 2 diabetes in patients with chronic obstructive pulmonary disease. Diabetes Obes Metab 2020; 22: 1348–1356. + +65 Shu CC, Wu HD, Yu MC, et al. Use of high-dose inhaled corticosteroids is associated with pulmonary tuberculosis in patients with chronic obstructive pulmonary disease. Medicine 2020; 89: 53–61. + +66 Suissa S, Dell'Aniello S, Ernst P. Comparative effectiveness of LABA-ICS versus LAMA as initial treatment in COPD targeted by blood eosinophils: a population-based cohort study. Lancet Respir Med 2018; 6: 855–862. + +67 Suissa S, Dell'Aniello S, Ernst P. Comparative effectiveness and safety of LABA-LAMA vs LABA-ICS treatment of COPD in real-world clinical practice. Chest 2019; 155: 1158–1165. + +68 Suissa S, Dell'Aniello S, Ernst P. Comparative effects of LAMA-LABA-ICS vs LAMA-LABA for COPD: cohort study in real-world clinical practice. Chest 2020; 157: 846–855. + +69 Tashkin DP, Miravitlles M, Celli BR, et al. Concomitant inhaled corticosteroid use and the risk of pneumonia in COPD: a matched-subgroup post hoc analysis of the UPLIFT® trial. Respir Res 2018; 19: 196. + +70 Wang CY, Lin YS, Wang YH, et al. Risk of sepsis among patients with COPD treated with fixed combinations of inhaled corticosteroids and long-acting beta2 agonists. Aging (Albany NY) 2019; 11: 6863–6871. + +71 Wu MF, Jian ZH, Huang JY, et al. Post-inhaled corticosteroid pulmonary tuberculosis and pneumonia increases lung cancer in patients with COPD. BMC Cancer 2016; 16: 778. + +72 Yang HH, Lai CC, Wang YH, et al. Severe exacerbation and pneumonia in COPD patients treated with fixed combinations of inhaled corticosteroid and long-acting beta2 agonist. Int J Chron Obstruct Pulmon Dis 2017; 12: 2477–2485. + +73 Yawn BP, Li Y, Tian H, et al. Inhaled corticosteroid use in patients with chronic obstructive pulmonary disease and the risk of pneumonia: a retrospective claims data analysis. Int J Chron Obstruct Pulmon Dis 2013; 8: 295–304. + +74 Yeh JJ, Lin CL, Kao CH. Associations among chronic obstructive pulmonary disease with asthma, pneumonia, and corticosteroid use in the general population. PLoS One 2020; 15: e0229484. + +75 Lee CH, Lee MC, Shu CC, et al. Risk factors for pulmonary tuberculosis in patients with chronic obstructive airway disease in Taiwan: a nationwide cohort study. BMC Infect Dis 2013; 13: 194. + +76 Huang TM, Kuo KC, Wang YH, et al. Risk of active tuberculosis among COPD patients treated with fixed combinations of long-acting beta2 agonists and inhaled corticosteroids. BMC Infect Dis 2020; 20: 706. + +77 Andréjak C, Nielsen R, Thomsen VØ, et al. Chronic respiratory disease, inhaled corticosteroids and risk of non-tuberculous mycobacteriosis. Thorax 2013; 68: 256–262. + +78 Brassard P, Suissa S, Kezouh A, et al. Inhaled corticosteroids and risk of tuberculosis in patients with respiratory diseases. Am J Respir Crit Care Med 2011; 183: 675–678. + +79 Brode SK, Campitelli MA, Kwong JC, et al. The risk of mycobacterial infections associated with inhaled corticosteroid use. Eur Respir J 2017; 50: 1700037. + +80 Cascini S, Kirchmayer U, Belleudi V, et al. Inhaled corticosteroid use in chronic obstructive pulmonary disease and risk of pneumonia: a nested case-control population-based study in Lazio (Italy) - The OUTPUL Study. COPD 2017; 14: 311–317. + +81 Ernst P, Gonzalez AV, Brassard P, et al. Inhaled corticosteroid use in chronic obstructive pulmonary disease and the risk of hospitalisation for pneumonia. Am J Respir Crit Care Med 2007; 176: 162–166. + +82 Ernst P, Coulombe J, Brassard P, et al. The risk of sepsis with inhaled and oral corticosteroids in patients with COPD. COPD 2017; 14: 137–142. + +83 Gayle A, Dickinson S, Poole C, et al. Incidence of type II diabetes in chronic obstructive pulmonary disease: +a nested case-control study. NPJ Prim Care Respir Med 2019; 29: 28. + +84 Gonzalez AV, Coulombe J, Ernst P, et al. Long-term use of inhaled corticosteroids in COPD and the risk of fracture. Chest 2018; 153: 321–328. + +85 Gonzalez AV, Li G, Suissa S, et al. Risk of glaucoma in elderly patients treated with inhaled corticosteroids for chronic airflow obstruction. Pulm Pharmacol Ther 2010; 23: 65–70. + +86 Jick SS, Vasilakis-Scaramozza C, Maier WC. The risk of cataract among users of inhaled steroids. + +Epidemiology 2001; 12: 229–234. + +87 Johannes CB, Schneider GA, Dube TJ, et al. The risk of nonvertebral fracture related to inhaled corticosteroid exposure among adults with chronic respiratory disease. Chest 2005; 127: 89–97. + +88 Joo MJ, Au DH, Fitzgibbon ML, et al. Inhaled corticosteroids and risk of pneumonia in newly diagnosed COPD. Respir Med 2010; 104: 246–252. + +89 Lee TA, Weiss KB. Fracture risk associated with inhaled corticosteroid use in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2004; 169: 855–859. + +90 Lee CH, Kim K, Hyun MK, et al. Use of inhaled corticosteroids and the risk of tuberculosis. Thorax 2013; 68: +1105–1113. + +91 Lu PC, Yang YH, Guo SE, et al. Factors associated with osteoporosis in patients with chronic obstructive pulmonary disease - a nationwide retrospective study. Osteoporos Int 2017; 28: 359–367. + +92 Mapel D, Schum M, Yood M, et al. Pneumonia among COPD patients using inhaled corticosteroids and long-acting bronchodilators. Prim Care Respir J 2010; 19: 109–117. + +93 Miller DP, Watkins SE, Sampson T, et al. Long-term use of fluticasone propionate/salmeterol fixed-dose combination and incidence of nonvertebral fractures among patients with COPD in the UK General Practice Research Database. Phys Sportsmed 2010; 38: 19–27. + +94 Miller DP, Watkins SE, Sampson T, et al. Long-term use of fluticasone propionate/salmeterol fixed-dose combination and incidence of cataracts and glaucoma among chronic obstructive pulmonary disease patients in the UK General Practice Research Database. Int J Chron Obstruct Pulmon Dis 2011; 6: 467–476. + +95 Pujades-Rodríguez M, Smith CJ, Hubbard RB. Inhaled corticosteroids and the risk of fracture in chronic obstructive pulmonary disease. QJM 2007; 100: 509–517. + +96 Suissa S, Patenaude V, Lapi F, et al. Inhaled corticosteroids in COPD and the risk of serious pneumonia. + +Thorax 2013; 68: 1029–1036. + +97 Suissa S, Coulombe J, Ernst P. Discontinuation of inhaled corticosteroids in COPD and the risk reduction of pneumonia. Chest 2015; 148: 1177–1183. + +98 Suissa S, Kezouh A, Ernst P. Inhaled corticosteroids and the risks of diabetes onset and progression. Am J +Med 2010; 123: 1001–1006. + +99 Thornton Snider J, Luna Y, Wong KS, et al. Inhaled corticosteroids and the risk of pneumonia in Medicare patients with COPD. Curr Med Res Opin 2012; 28: 1959–1967. + +100 Wang CY, Lai CC, Yang WC, et al. The association between inhaled corticosteroid and pneumonia in COPD +patients: the improvement of patients' life quality with COPD in Taiwan (IMPACT) study. Int J Chron Obstruct Pulmon Dis 2016; 11: 2775–2783. + +101 Sabroe I, Postma D, Heijink I, et al. The yin and the yang of immunosuppression with inhaled corticosteroids. Thorax 2013; 68: 1085–1087. + +102 Kew KW, Seniukovich A. Inhaled steroids and risk of pneumonia for chronic obstructive pulmonary disease. + +Cochrane Database Syst Rev 2014; 10: CD010115. + +103 Lodise TP, Li J, Gandhi HN, et al. Intraclass difference in pneumonia risk with fluticasone and budesonide in COPD: a systematic review of evidence from direct-comparison studies. Int J Chron Obstruct Pulmon Dis 2020; 15: 2889–2900. + +104 Crim C, Dransfield MT, Bourbeau J, et al. Pneumonia risk with inhaled fluticasone furoate and vilanterol compared with vilanterol alone in patients with COPD. Ann Am Thorac Soc 2015; 12: 27–34. + +105 Pavord ID, Lettis S, Anzueto A, et al. Blood eosinophil count and pneumonia risk in patients with chronic obstructive pulmonary disease: a patient-level meta-analysis. Lancet Respir Med 2016; 4: 731–741. + +106 Martinez-Garcia MA, Faner R, Oscullo G, et al. Inhaled steroids, circulating eosinophils, chronic airway infection, and pneumonia risk in chronic obstructive pulmonary disease. A network analysis. Am J Respir Crit Care Med 2020; 201: 1078–1085. + +107 Caramori G, Ruggeri P, Arpinelli F, et al. Long-term use of inhaled glucocorticoids in patients with stable chronic obstructive pulmonary disease and risk of bone fractures: a narrative review of the literature. Int J +Chron Obstruct Pulmon Dis 2019; 14: 1085–1097. \ No newline at end of file diff --git a/medical/md_images/PMC9488732/PMC9488732_meta.json b/medical/md_images/PMC9488732/PMC9488732_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..de9725e0c0b6c5b17c4455db7444832bc03666e4 --- /dev/null +++ b/medical/md_images/PMC9488732/PMC9488732_meta.json @@ -0,0 +1,367 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Systematic review on long-term adverse effects of inhaled corticosteroids in the treatment of COPD", + "level": 0, + "is_closed": true, + "n_kids": 6, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 794.0, + 596.0, + 0.0 + ] + }, + { + "title": "Abstract", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Introduction", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Methods", + "level": 1, + "is_closed": true, + "n_kids": 4, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Design and selection criteria", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Search strategy and study selection", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Data extraction", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Data analysis", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Results", + "level": 1, + "is_closed": true, + "n_kids": 4, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Infectious adverse events", + "level": 2, + "is_closed": true, + "n_kids": 3, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Pneumonia", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "TB and NTM", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Sepsis", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Metabolic adverse events", + "level": 2, + "is_closed": true, + "n_kids": 3, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Osteoporosis", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Fractures", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Diabetes", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Local complications", + "level": 2, + "is_closed": true, + "n_kids": 3, + "page_index": 9, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Eye disorders", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 9, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Oral candidiasis", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Dysphonia", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Other adverse events", + "level": 2, + "is_closed": true, + "n_kids": 2, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Hypertension", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Adrenal suppression", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "Discussion", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 11, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + }, + { + "title": "References", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 14, + "view_mode": "XYZ", + "view_pos": [ + 0.0, + 0.0, + 0.0 + ] + } + ], + "pages": 19, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 54, + "code": 0, + "table": 11, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC9685680/PMC9685680.md b/medical/md_images/PMC9685680/PMC9685680.md new file mode 100644 index 0000000000000000000000000000000000000000..e8e62c5a57e5b42e0b7f6136e5176f65454e48cc --- /dev/null +++ b/medical/md_images/PMC9685680/PMC9685680.md @@ -0,0 +1,453 @@ +World Journal of W J C P *Clinical Pediatrics* + +![0_image_0.png](0_image_0.png) + +Submit a Manuscript: https://www.f6publishing.com *World J Clin Pediatr* 2022 November 9; 11(6): 463-484 SYSTEMATIC REVIEWS + +## Insulin Pumps In Children - A Systematic Review + +DOI: 10.5409/wjcp.v11.i6.463 ISSN 2219-2808 (online) +Mohammed Al-Beltagi, Nermin Kamal Saeed, Adel Salah Bediwy, Reem Elbeltagi + +| Mohammed Al-Beltagi, Department of Pediatrics, Faculty of Medicine, Tanta University, Tanta 31511, Algharbia, Egypt | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Specialty type: Endocrinology and metabolism Provenance and peer review: Invited article; Externally peer reviewed. | Mohammed Al-Beltagi, Department of Pediatrics, University Medical Center, King Abdulla Medical City, Arabian Gulf University, Manama 26671, Manama, Bahrain Mohammed Al-Beltagi, Department of Pediatrics, University Medical Center, Dr. Sulaiman Al Habib Medical Group, Manama, Bahrain, Manama 26671, Manama, Bahrain | +| Peer-review model: Single blind Peer-review report's scientific quality classification Grade A (Excellent): A Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 | Nermin Kamal Saeed, Medical Microbiology Section, Department of Pathology, Salmaniya Medical Complex, Ministry of Health, Kingdom of Bahrain, Manama 12, Manama, Bahrain Nermin Kamal Saeed, Department of Microbiology, Irish Royal College of Surgeon, Bahrain, Busaiteen 15503, Muharraq, Bahrain Adel Salah Bediwy, Department of Chest Disease, Faculty of Medicine, Tanta University, Tanta 31527, Alghrabia, Egypt | +| P-Reviewer: Liao JX, China; Olsen MT, North Zealand | Adel Salah Bediwy, Department of Chest Disease, University Medical Center, King Abdulla Medical City, Arabian Gulf University, Dr. Sulaiman Al Habib Medical Group, Manama 26671, Manama, Bahrain | +| Received: July 8, 2022 Peer-review started: July 8, 2022 First decision: August 1, 2022 Revised: August 2, 2022 Accepted: September 21, 2022 Article in press: September 21, 2022 Published online: November 9, 2022 | Reem Elbeltagi, Department of Medicine, The Royal College of Surgeons in Ireland - Bahrain, Busiateen 15503, Muharraq, Bahrain Corresponding author: Mohammed Al-Beltagi, MBChB, MD, MSc, PhD, Chairman, Professor, Department of Pediatrics, Faculty of Medicine, Tanta University, Al Bahr Street, Tanta 31511, Algharbia, Egypt. mbelrem@hotmail.com Abstract BACKGROUND Insulin pump therapy is a real breakthrough in managing diabetes Mellitus, particularly in children. It can deliver a tiny amount of insulin and decreases the need for frequent needle injections. It also helps to maintain adequate and optimal | + +BACKGROUND Insulin pump therapy is a real breakthrough in managing diabetes Mellitus, particularly in children. It can deliver a tiny amount of insulin and decreases the need for frequent needle injections. It also helps to maintain adequate and optimal glycemic control to reduce the risk of metabolic derangements in different tissues. Children are suitable candidates for pump therapy as they need a more freestyle and proper metabolic control to ensure adequate growth and development. Therefore, children and their caregivers should have proper education and training and understand the proper use of insulin pumps to achieve successful pump therapy. The pump therapy continuously improves to enhance its performance and increase its simulation of the human pancreas. Nonetheless, there is yet a long way to reach the desired goal. + +## Aim + +To review discusses the history of pump development, its indications, types, proper use, special conditions that may enface the children and their families while using the pump, its general care, and its advantages and disadvantages. + +## Methods + +We conducted comprehensive literature searches of electronic databases until June 30, 2022, related to pump therapy in children and published in the English language. RESULTS We included 118 articles concerned with insulin pumps, 61 were reviews, systemic reviews, and meta-analyses, 47 were primary research studies with strong design, and ten were guidelines. + +## Conclusion + +The insulin pump provides fewer needles and can provide very tiny insulin doses, a convenient and more flexible way to modify the needed insulin physiologically, like the human pancreas, and can offer adequate and optimal glycemic control to reduce the risk of metabolic derangements in different tissues. + +Key **Words:** Insulin pump; Children; Diabetes mellitus; Keto-acidosis; Continuous glucose monitoring; Insulin therapy ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved. + +Core **Tip:** The insulin pump is a significant step in proper diabetes management in the way to simulate the human pancreas. Insulin pumps undergo significant daily improvement every day to enhance their performance. The insulin pump is beneficial for children with type I diabetes mellitus. However, proper training and education of the children and their families are mandatory for the appropriate function of the insulin pumps. They should know how to use it properly and overcome the difficulties they may encounter and the different scenarios they may meet. The way still long to achieve our goals. + +Citation: Al-Beltagi M, Saeed NK, Bediwy AS, Elbeltagi R. Insulin pumps in children - a systematic review. World J Clin Pediatr 2022; 11(6): 463-484 URL: https://www.wjgnet.com/2219-2808/full/v11/i6/463.htm DOI: https://dx.doi.org/10.5409/wjcp.v11.i6.463 + +## Introduction + +The insulin pump is a giant breakthrough in diabetes mellitus (DM) treatment. Treating diabetes with an insulin pump is the method most similar to the normal physiologic function of the pancreas. The pump delivers insulin in 2 different ways simulating the human pancreas. It delivers a continuous small insulin quantity as a "background insulin" to maintain the basal metabolic rate and bolus insulin doses when needed to metabolize the ingested food. The insulin pump is particularly needed in the management of type I DM. The insulin pump is continuously undergoing massive improvement. Nonetheless, there is yet a long way to reach the desired goal[1]. + +## Materials And Methods + +This review aims to discuss the history of pump development, its indications, types, proper use, special conditions that may enface the children and their families while using the pump, its general care, and its advantages and disadvantages. In this narrative review, we conducted comprehensive literature searches of electronic databases, PubMed, Embase, Cochrane Library, Reference Citation Analysis (RCA), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Web of Science, Scopus, Library and Information Science Abstracts (LISA), and the National Library of Medicine (NLM) catalog up until June 30, 2022, related to pump therapy in children. Reference lists were inspected, and citation searches were done on the included studies. We included open access papers for English-language studies concerned with pump therapy and diabetes mellitus in children. We also reviewed many review articles concerned with pump therapy in children. We also reviewed the articles that are + +## Results + +Figure 1 showed the flow chart of the reviewed articles. We included 118 articles concerned with insulin pumps, 61 were reviews, systemic reviews, and meta-analyses, 47 were primary research studies with strong design, and ten were guidelines. + +## Discussion + +For a long time, there is a need for proper management of children with diabetes. The result of this review showed that despite the many challenges that enface the use of insulin pumps in children, the advantages are far more than the challenges. + +## Historical Background + +Diabetes has been described in the Papyrus Ebers (c. 1550 BC), Ayurvedic medicine (5th/6th century BC), Greek medicine (1st century BC), and medieval Islamic medicine by Avicenna for a long time[2]. Oskar Minkowski and Joseph von Mering first linked diabetes to pancreatic illness in 1889. In 1910, Sir Edward Albert Sharpey-Shafer proposed that the missing pancreatic component be named insulin, after the Latin word Insula (island). Frederick Grant Banting and his colleagues discovered and refined insulin for clinical use in Toronto, Canada, between 1920 and 1922[3]. + +Leonard Thompson, a fourteen-year-old boy, was the first human to receive an insulin injection, controlling his high blood sugar within 24 h. Shortly after, Eli Lilly's medical company started mass insulin production from cattle and pigs to supply the needs of North America. After about 14 years, Harold Percival Himsworth differentiated type I Diabetes Mellitus from type II in 1936. In the same year, Novo Nordisk Pharmaceuticals developed long-acting insulin. By 1978, genetic engineering enabled the human being to have the first synthetic "human" insulin produced by E. coli bacteria. After four years, Eli Lilly made the human biosynthetic insulin commercially available for the first time with the brand name Humulin in 1982[4,5]. + +The first insulin pump was invented in 1963 by Arnold Kadish, who developed a prototype backpack pump to deliver insulin and glucagon. However, Dean Kamen developed the first wearable insulin infusion pump in 1973, which started to be produced commercially by AutoSyringe Inc in 1976[6]. In the same year, continuous subcutaneous insulin infusion was started successfully. The glucose-controlled insulin infusion system was developed by BioStar company to be the first step in the development of artificial pancreas development in the 1980s. During the same period, the first blood glucose homebased monitors were available to accurately monitor blood glucose levels at home. A few years later, the prefilled syringe-insulin pen delivery systems appeared, providing a safe and convenient insulin delivery method with accurate dosing[7]. In 1983, MiniMed released the first small-sized programmable insulin pump, followed by the soft-set infusion set in 1987, using a soft, flexible cannula to ensure customer comfort. In 1992, MiniMed redesigned the pump to be programmed to include meal bolus memory and daily insulin totals. Continuous upgrading of the pump continued to prolong blood glucose recording to 3 days in 1999. The same year showed constant upgrading of the pump to enable remote programming abilities to control, administer, and stop insulin delivery and to program multiple delivery patterns and different alert types, including vibration mode and child-block[8]. + +By 2000, the concept of implantable insulin pumps emerged to help patients with significantly uncontrolled diabetes. In 2003, the wireless "intelligent" insulin pump was introduced, which can automatically transmit a blood glucose value from a glucose meter to the insulin pump, which calculates the proper insulin dosages. In 2004, the pump was upgraded to notify and alarm diabetes patients of potentially severe high or low glucose fluctuations. In 2005, the system was upgraded to display updated real-time blood glucose values every five minutes, plus the alarming system for severe blood glucose fluctuations, which provided significant help to people with diabetes who need better glucose control[9]. In 2006, a real-time insulin pump system with a continuous glucose monitoring system became available, which was a significant step in developing a "closed-loop" insulin delivery system or what is called "artificial pancreas" trying to mimic some human pancreas functions. Updating the pump system continued over the years, so by 2012, the concept of next-generation artificial pancreas systems was elaborated. In 2018, Bekiari et al[11] showed that the artificial pancreas is "effective and safe" in treating people with type 1 diabetes[10,11]. + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +## Indications Of Insulin Pump + +The Key target of diabetes management is to maintain adequate and optimal glycemic control to reduce the risk of metabolic derangements in different tissues. Although it may be a simple goal, it is not always easy to achieve in practice. There are currently two ways to deliver insulin: Multiple daily subcutaneous injections (conventional insulin therapy) or continuous subcutaneous insulin infusion, also identified as insulin pump therapy[12]. There are many difficulties with conventional insulin therapy, including the variable glycemic control with frequent occurrence of hypoglycemia, abnormal weight gain related to the insulin therapy, augmented by improper dosage calculation related to human error, and the lack of adherence to insulin therapy, especially with multidose regimens[13]. Using an insulin pump to manage DM depends on many factors, including the patient's desire, daily life routine, and knowledge and experience with the disease. More than 25% of patients with type I DM are currently using insulin pump therapy. It is especially indicated in the presence of high hemoglobin A1C, poor glycemic control with problematic hypoglycemia such as nocturnal hypoglycemia, recurrent hypoglycemia, activity-induced hypoglycemia, recurrent diabetic ketoacidosis, frequent hospitalization, large total daily dose, presence of progressive complications such as gastroparesis, inability to selfadminister insulin (such as in pre-school or grade-school children), the need for more meal time flexibility, or the inability to predict food or meal intake (such as in infants or toddlers)[14]. Baretić et al +[15] showed that nocturnal hypoglycemia is the main indication for insulin pumps in adults with type I DM, especially with limited affordability. Patients with type 2 Diabetes who fail to have adequate glycemic control with multidose insulin therapy may have better control with pump therapy, improving HbA1c, and limiting weight gain[16]. So, the insulin pump can be sued for type I and II DM in adults and children, especially for those who want more flexibility and proper mealtime adjustment. About 1/1000 of patients with DM are currently using insulin pumps, and their number is increasing; 90% have type I DM, while only 10% have type II. About 6% of adult patients with type I DM use it, while it reaches about 19% in children with type I DM[17]. + +## Types Of Insulin Pumps + +There are two main types of pumps; the first type is a 'tethered' pump that uses a fine tube connecting the pump with a cannula (Figure 2A). The patient can wear the pump in a pocket or fasten it + +![4_image_0.png](4_image_0.png) + +![4_image_1.png](4_image_1.png) + +to a belt and should change the tubing every 2-3 d. The patch pump (micro pump) is another type without tubing or may have a very short flexible plastic tube (cannula) inserted under the skin (Figure 2B). The pump usually adheres to the skin with an adhesive patch and is wirelessly controlled with a handheld controller unit[18]. The insulin pump is generally formed of the central pump unit connected to an insulin reservoir which usually holds between 176-300 units of short-acting insulin. Another new version of the insulin pump has a built-in Continuous Glucose Monitor (CGM). The pump is supplied with an alarm system activated when the blood glucose reaches a predetermined low or high level[19]. A SmartGuard Technology can be added to CGM to stop insulin supply for two hours if the user's blood glucose reaches a predetermined low level pre-settled without activating the alarm system. Some pumps use a hybrid closed-loop technology by using the SmartGuard technology to permit the users to select from increasing the levels of automation that best suit their needs. The auto mode enables automatic adjustment of the basal insulin delivery according to the glucose reading of the user's CGM sensor and recent insulin delivery[20]. + +There is also a bolus calculator, able to automatically calculate the doses and inform the person if they are too closely set together. However, in this mode, the user should enter the details of carbohydrate intake, confirm the mealtimes, and adjust the correction boluses. Some insulin pumps have an Insulin on board (IOB) feature so that the pump can calculate how much insulin remains active in the patient's body from the previous insulin bolus dose[21]. Some pumps are also waterproof to tolerate up to 12 feet underwater for about 24 h. This feature enables patients with diabetes to enjoy swimming with minimal risk of hypoglycemia. The vast advances in insulin pumps are related to significant software and artificial intelligence progress. For example, some pumps allow choosing an exercise set to change the person's glucose target[22] automatically. Most of the new versions of insulin pumps are compatible via Bluetooth with smartphones with different applications such as the Carelink Connect app, which allows family members or caregivers to access the patient's information and show all readings and permits notifications and alarms. People also can deliver insulin remotely using a smartphone-like Personal Diabetes Manager device. The suitable age for the pump use differs according to the pump type and version, in which the manufacturers determine the appropriate age of use according to each pump's features[23]. + +## Use Of The Insulin Pump + +The children and their caregivers should understand the proper use of insulin pumps to achieve successful pump therapy. The children and their caregivers should have a comprehensive education program about diabetes management and insulin pump use before starting it. They should be able to adjust the blood glucose levels using multiple daily insulin injections and monitor blood glucose frequency at least four times daily during the last two months before the insulin pump therapy[24]. They should also learn to test ketones when the blood glucose level is more than 13.3 mmol/L (240 mg/dL) and recognize symptoms of ketoacidosis when present. Before using the pump, the children or their caregivers should know the suitable age for using it as predetermined by the manufacturers[25]. They should know whether the pump is waterproof, watertight, water-repellent, water-resistant, or not. If the pump is not, they should remove it before showering or swimming. If the pump is waterproof, they should know how many feet in-depth and how long. The patient also should know the maximum time the pump can be removed without affecting its performance, *e.g.*, one hour for the Aviva Combo insulin pump[26]. The child and his/her caregiver should know the number of insulin units the insulin cartridge can hold. However, they should know that the half-life of the insulin pods may not depend on how much is used. The the pump uses is fast-acting because of its rapid onset and offset. Even fast-acting takes some time for its action onset and offset. We should also note the presence of significant individual variations in the onset, duration, and offset of the same type of insulin [27]. + +The patients and their caregivers should be aware of the lowest basal delivery rate the pump can deliver. They should understand the available basal (background) insulin patterns such as daily, weekend, exercise, and night shifts. They also should be aware of the insulin-to-carbohydrate ratio program feature. This mode helps the patient calculate an estimated insulin bolus dose to cover any carbohydrates the patient eats or drinks or improve high blood glucose[20]. The most important thing to properly using the insulin pump is calculating the total daily insulin dose, which equals the basal and the bolus insulin doses delivered each day. Total daily basal insulin is a continuous insulin infusion needed to metabolize hepatic glucose production over 24 h, while the total daily bolus insulin is the insulin units given on needs to control the meal-related glucose peaks over 24 h[28]. + +The pump's total daily insulin dose is 25% less than the current total daily insulin injection dose. To calculate the pump's total daily insulin dose, we usually take the average of the 25%-reduced current total daily insulin injection dose and the weight-based insulin daily dose, which equals the body weight multiplied by 0.5. The pump's total daily insulin dose is less than the current total daily insulin injection dose as the pump delivers the insulin more efficiently, and the insulin is more regularly absorbed than the intermittent daily injections[26]. 40%-50% of the pump's total daily insulin dose is delivered as a total daily basal insulin, while the other 50%-to-60% will be delivered as bolus doses related to the meal intake (Figure 3). The total daily basal insulin is delivered at an hourly rate. However, it can be programmed at different rates throughout the day according to daily activities and circumstances[29]. + +Meanwhile, the percentage of the total daily basal insulin from the pump's total daily insulin dose differs according to the age of the patients; 20% to 40% from the pump's total daily insulin dose in prepubertal and pubertal subjects, 30%-40% from puberty to adulthood, and 40% to 50% in adults. The rest of the pump's total daily insulin dose is divided into bolus doses according to the meals. The ratio of basal to bolus insulin is also dependent on the amount of carbohydrate diet, decreasing with a high carbohydrate diet[30]. + +Basal insulin is the insulin level required to balance gluconeogenesis and peripheral glucose utilization. Basal insulin keeps the blood sugar relatively constant without food intake. The pump delivers the basal insulin in a tiny quantity per hour to control the blood glucose levels over a predetermined period[31]. If a desired change in the basal insulin delivery is required, the adjustment should begin two to three hours before the expected change in the blood glucose to prevent significant fluctuations in its levels; so that the blood glucose level fluctuations are less than 30 mg/dL up or down, either during the day or at night. In the flat basal profile, the total daily basal insulin is divided equally over the day hours (divided by 24)[32]. However, the basal requirements show significant variation within the same person day-on-day, based on circadian rhythm, meal and activity levels, exercise, illness and stress, and the potential changes in the insulin absorption from the cannula. There are various types (4-6) of modified basal profiles. The patient usually starts with a flat basal profile; then, the profile is modified according to the patient's activity[33]. Some patients may need to increase the basal rate in the early morning to neutralize the Dawn phenomenon. Conversely, we may need to reduce the basal rate between mid-morning and mid-afternoon. Meanwhile, some patients may require an increase in the basal rate in the evening (dusk phenomenon) due to a reduction in physical activity later in the day (Figure 4)[34]. + +Suppose the patient has a fixed period of activity that occurs daily at the same time, such as walking or swimming, when there is a risk of hypoglycemia. In that case, the basal rate can be modified to accommodate these activities by reducing the basal rate 60-90 min before the expected activity. In a situation with an increased likelihood of hyperglycemia, such as during illness, the patients may increase the basal rate 30%-50% above the flat basal rate[35]. It should be noted that Blood insulin levels need 2-3 h to reach a steady state after a basal rate change. Consequently, the changes in the basal rate usually occur in blocks of hours. Most patients need to have multiple basal rates throughout the daytime. Basal rates modified to match the patient's activity are associated with improved outcomes [36]. Some insulin pumps can be programmed to suspend/stop insulin delivery from the pump for a particular time before, during exercise, or in hypoglycemia. To reach the proper basal profile, the patients need to check their blood sugar as frequently as eight times daily during the pump's first month. After getting the target profile, the frequency of checking is about four times daily. However, extra checks may be needed during times of exercise, traveling, illness, or the beeping of the pump alarm[17]. + +Bolus insulin is an intermittent insulin bolus infusion intended to parallel the rises in blood glucose related to food intake. It depends on personalized carbohydrate-insulin ratios, which are the amount of insulin needed to match the ingested carbohydrate in grams without causing hypo/hyperglycemia[37]. It is calculated by dividing 500 in adults or 300 in children by the total insulin daily dose. For example, if a child needs 45 insulin units as a daily dose, his carbohydrate-insulin ratio is 300/45 = 6.6. This means that the child needs one Insulin unit for every 6.6-gram carbohydrate. For a meal that contains 120 gm of carbohydrates, the child needs 18 insulin units to maintain the blood glucose levels within the normal range[38]. The insulin pump can deliver various forms of insulin bolus. There are three main types of bolus insulin; the typical bolus, the square-wave bolus, and the dual-wave bolus (Figure 5)[39]. In the typical bolus, the pump delivers the immediately on top of the basal rate for three minutes. The + +![6_image_0.png](6_image_0.png) + +![6_image_1.png](6_image_1.png) + +Figure 4 Circadian changes in the basal insulin **profile.** An example of the circadian changes in the basal insulin profile. Between 12 midnight to 4:0 am, the basal rate is reduced by 20% to increase 20% between 4:0 am to 7:0 am, then will be 100% between 7:0 am to 10:0 am to be reduced by 10% between 10:0 am to 10:0 pm, then it increases again by 10% between 10:0 pm to 12 midnight. + +extended bolus delivers the insulin over a longer period than the typical bolus (the insulin dose is not delivered at once). The square wave bolus is an extended bolus that delivers the insulin over a fixed extended duration (2-4 h)[40]. We usually need it when the food is ingested over a longer time, such as during social events, or delayed digestion, such as in gastroparesis. The dual-wave bolus delivers the insulin in two or more waves (dual or multi-waves], 30%-70% of the insulin bolus is delivered as the typical bolus, and the rest of the bolus is delivered over a predetermined fixed duration, such as 2-4 h (biphasic, dual, or multi-wave). The dual/multi-wave bolus is helpful with meals with high protein or fat content (*e.g.*, pizza, pasta, cheese, or fish) as protein and fat usually take longer to increase blood glucose levels than carbohydrates, which immediately affect blood glucose[41]. + +In the super bolus (Figure 6), the pump delivers the basal insulin for the next 1-4 h is added to the bolus, followed by temporarily suspending the basal insulin delivery for those hours (basal rate is 0% for the next 1-4 h) without stopping the pump. This super bolus is needed with a diet high in carbohydrates and low in protein and fat, such as white toast, jam, white rice, sugary drinks, jelly sweets, *etc.* However, if the post-meal blood sugar persists to be high after a super-bolus insulin dose, we should revise the insulin: carbohydrate ratio. If the post-meal blood sugar fluctuates, we should revise carbohydrate counting as it often shows a wide range of variations[42]. A comparison between the different types of insulin boluses is shown in Table 1. + +| Table 1 Comparison between the different types of insulin boluses Bolus type Normal Extended | Dual wave | Super bolus | | | +|------------------------------------------------------------------------------------------------|-------------------------------------------|------------------------------------|-------------------------------------------|-----------------------------------------| +| Indication | With most diets, 10- | Rarely needed: With lengthy | With low carb & high protein & | With high carbohydrate meal | +| 20 min before the | mealtime & gastroparesis | fat (> 25 gm fat) meals | | | +| meal | | | | | +| Method of | Deliver insulin | Deliver all the insulin in an | | | +| insulin | immediately | extended fashion over a chosen | | | +| delivery | duration which may be the meal length. | 60%-70% of the bolus is | The basal insulin units of the next 1-4 h | | +| delivered immediately, while | are added to the bolus, followed by a | | | | +| 30%-40% is delivered over 2-4 h | temporary 0% of basal rate for that 1–4 h | | | | +| When to test | Before bolus and | Before bolus, after the end of the | Before bolus, after the end of the | Before bolus, after one & 4 h after the | +| blood glucose | after 4 h | bolus, & after 4 h | bolus, & after 4 h | bolus | + +![7_image_0.png](7_image_0.png) + +![7_image_1.png](7_image_1.png) + +Correction bolus is the number of insulin units delivered by the pump when the blood glucose level is above the target range. It depends on the individual insulin sensitivity factor (ISF), which is the amount of decrease in blood glucose level for every one unit of rapid insulin. It calculates the correction bolus dose, which is the number of insulin units the patient needs to bring the blood glucose into the target range without inducing hypoglycemia[43]. To calculate ISF, we divide 100 by the average total daily insulin dosage over three or four days (TDD) to get the result in mmol/L (100 Rule) or divide 1800 by the average TDD to get the results in mg/dL (1800 Rule). So, when the average TDD is 75 Insulin Units, ISF will be 1.33. Thus, one unit of rapid-acting would reduce the blood sugar by 1.33 mmol/L (24 mg/dL) across the next 2-4 h[43]. The following formula can calculate the correction dose: [(Current Blood Glucose - Target Blood Glucose)/ISF]. However, the Association of British Clinical Diabetologists advises routinely starting with the rule of 130 rather than the 100 rule [ISF = 130/TDD] to achieve the optimal ISF and estimate how much one unit of insulin will decrease the blood glucose level in mmol/L[44]. We should ensure the adequacy of ISF by testing the blood glucose after 4-5 h after the last insulin bolus dose. Carbohydrate was consumed more than three hours ago, and the patient can wait for 4-5 h until the next meal[43]. + +The caregivers and their child should adequately adjust the pump setting for the IOB and the active insulin time, which is the duration that an insulin dose continues to work after its delivery. Most pumps set this time to be four hours; however, it can be shortened or prolonged in the presence of hypo/hyperglycemia[26]. With short active insulin time, the pump includes the effects of the more recent insulin doses, omitting the impact of the insulin from the previous doses, thus increasing the risk of hypoglycemia[28]. On the other hand, long active insulin time causes overestimation of the insulin on board, and the pump gives smaller boluses which can cause hyperglycemia. Longer active insulin time than four hours is needed in cases with impaired renal functions, as the kidneys clear about 80% of the insulin. Those needing TDD less than or equal to 40 Units/day should reduce active insulin time. In comparison, those receiving a bolus dose of more than 10 Units or TDD of 60 Units/d or more should consider increasing active insulin time[45]. + +The caregivers and their child should recognize the accepted target blood glucose level, which is individualized according to the child's status and is comfortable with it. Some insulin pumps correct the blood glucose level to a single target level but give the correction bolus when a predefined threshold is reached[46]. Other pumps correct to a higher figure in a predefined target range, while others correct to the mid-target range. The target range usually lies between 4.5-5.5 mmol/L (80-100 mg/dL) (± one mmol/L). However, the target level is modified according to the presence of different factors. Those with high HbA1c need a higher target as the setpoint for hypoglycemic symptoms is higher than for more well-regulated patients with diabetes. They frequently have episodes of pseudohypoglycemia until they are used to lower glucose values[47]. Those with HbA1c > 10% should start with high target blood glucose between 9-10 mmol/L (160-180 mg/dL), then reduce the target level monthly until we reach the normal target blood glucose levels. This gradual decrease of the target is particularly indicated in the presence of diabetic retinopathy, which may deteriorate with the rapid lowering of blood glucose levels. To maintain the target glucose blood level, the caregivers and the children need to frequently test the blood glucose levels, especially in the presence of hypo/hyperglycemia, and to know which insulin doses need to be modified, the basal rate, or the bolus insulin[48]. + +When there is blood glucose variation (hypo/hyperglycemia) during the night, before breakfast, or when a meal is skipped or delayed, the basal rate of insulin needs to be adjusted. However, the insulinto-carbohydrate ratio and ISF should be revised when the blood glucose variation (hypo/hyperglycemia) occurs within four hours of the bolus or meal or after giving a correction insulin bolus. To test the basal rate, we should eliminate the influence of other factors that could affect it by dividing the day into windows, testing the basal rate for each window on different days, and ensuring that the tested person follows his/her usual routine without strenuous activity before the test by 24 h and during the test period[49,50]. Hypoglycemia should not present during the last 12 h before testing, and the patients should be fasting for the tested period except for water. We use the standard basal rate during testing, stop testing with hypoglycemia (< 4 mmol/L or 72 mg/dL), and give a corrective bolus with hyperglycemia of more than 14 mmol/L (252 mg/dL). We should record blood glucose levels, basal rate, and carbohydrate intake. There are four main periods to test the basal rate: Morning testing from 7:00 am to 12:00 noon, after noon testing from 12:00 noon to 18:00, evening basal from 18:00 to 22:00, and overnight testing from 21:00 to 7:00 am[51]. Overnight testing is usually done first, followed by the other periods, each on different daily bases. First, we correct hypoglycemia; then, we correct hyperglycemia with a 10%-20% decrease or increase, *i.e.*, 0.025-0.05 unit/ hour one to hours before the expected change, to keep the blood glucose variation less than 1.5 mmol/L (28 mg/dL)[52]. + +For bolus adjustment, we try to keep the two-hours post-meals to be 1.6-3.2 mmol/L (29-58 mg/dL) +higher than the pre-meal blood glucose levels. If the two-hours post-meal blood glucose level increases to less than 1.6 mmol/L (29 mg/dL), decrease the total bolus by 10%-20%. If the two-hour post-meal blood glucose level increases to more than 3.2 mmol/L (58 mg/dL), increase the total bolus by 10%-20%. Slattery et al[53] suggested that insulin administration 15-20 min before the meal will provide optimum postprandial glucose control. To adjust the correction bolus dose, adjust the insulin sensitivity factor by a 10%-20% increase or decrease to make the two-hours post-meal blood glucose level halfway to the target and reach the target by four-hours post-meal. If the patient uses continuous glucose monitoring (CGM), a retrospective analysis of the time associated with blood sugar fluctuations can help modify the basal rate[54]. Some pumps have a wizard to change the basal rate according to the blood sugar levels. However, CGM should be associated with an event diary to document the meal timing and the carbohydrate intake for accurate assessment. CGM is indicated with frequent or severe hypoglycemia, hypoglycemia unawareness such as in young children, suboptimal glycemic control, or monitoring of young children or beloved ones by family members or friends[55,56]. + +Suppose the patient needs to change the insulin pump therapy to subcutaneous insulin for any reason. In that case, we need to calculate the total daily basal insulin given by the pump and increase it by 20% for the first few days and by 30% in the following days to calculate how much long-acting basal subcutaneous insulin is needed. This dose can be administered as a single or divided dose depending on the type of long-acting insulin used[57,58]. The bolus pre-meal insulin and the corrective insulin doses are given as usual. The pump should continue working for two hours after starting the first long-acting insulin dose. Doing this shifting in the morning is recommended to avoid troubleshooting at night[59]. + +Alternatively, if we need to resume the pump therapy after subcutaneous therapy, we resume it two hours before the next long-acting insulin dose. Again, we need to reprogram the pump setting and temporarily reduce the basal insulin rate by about 30% for the next 24 h. Blood glucose levels should be checked one to two hours after starting pump therapy[18]. So this shift is better to be in the morning for easy monitoring. Suppose we want to shift the insulin therapy from continuous intravenous infusion to pump therapy. In that case, we can shift without waiting for the coming meal and discontinue the intravenous insulin infusion only 60 min after starting the pump therapy[60]. + +## Pump Therapy In Special Situations Sports And Exercise + +Exercise has a long-term beneficial effect on blood glucose regulation regardless of the type of exercise. However, it has a short-term modification on the blood glucose level to be considered during the pump therapy—two significant difficulties enface children with type I diabetes who are willing to exercise regularly. The first problem is controlling the blood glucose level, and the second is the possible occurrence of hypoglycemia during or following exercise[61]. Aerobic exercise reduces blood glucose levels and enhances insulin sensitivity for 16 h following the exercise. On the other side, high-intensity aerobic exercise or that associated with high adrenaline could cause hyperglycemia. Resistance training or anaerobic exercise increases blood glucose levels and insulin resistance, which persist 6-8 h after exercise. Brief periods of anaerobic exercise activity (*e.g.*, short sprints or weight lifting) during moderate-intensity aerobic exercise may decrease the risk of hypoglycemia[62]. However, all types of physical exercises may increase the risk of nighttime and even the next day hypoglycemia, mainly when performed in the afternoon, independent of sex[63]. + +We can prevent the development of exercise-induced hypoglycemia by reducing the bolus or basal insulin, increasing carbohydrate intake, or adjusting the exercise regimen. The basal insulin can temporarily be reduced one to two hours before and throughout the exercise. The degree of basal rate reduction depends on the intensity of exercise, 30% in low-intensity, 50% in moderate-intensity, and up to 100% reduction in high-intensity exercise. If modification of the basal insulin is not feasible, a fastacting carbohydrate (15-20 gm without bolus insulin dose) can be given immediately before the short, intense exercise. For more extended, moderately intense exercise, a solid snack with slow-release carbohydrates can be given. If an exercise is planned within two hours after a meal, the premeal-bolus insulin dose can be reduced by 25%-75% according to the exercise intensity[64]. If we are anticipating anaerobic exercise-induced hyperglycemia, we can give a 50% extra bolus dose 30-60 min before the exercise to antagonize the expected increase in the blood sugar. Alternatively, we can increase the basal rate by 10%-20%, 30-90 min before the exercise, and continue at this rate when post-exercise hypoglycemia persists. In the case of combined aerobic and anaerobic exercise, hypoglycemia is more frequent but less than pure aerobic exercise. Hypoglycemia can be prevented with a reduction of basal insulin by up to 50%. If these changes are impossible, we should consider the artificial pancreas. The artificial pancreas system appeared to get efficient and secure control of blood glucose levels during exercise and four hours later[65]. + +## Troubleshooting Hypoglycemia + +If severe hypoglycemia is present, the patient should not stop the pump but keep it running and check blood glucose levels. When confirmed, the patient is advised to take 15 gm of fast-acting carbohydrates and to recheck blood glucose after 10-15 min. If hypoglycemia persists, another dose of fast-acting carbohydrates is given, and the basal insulin could be temporarily reduced by 10%-20%. As the insulin type used in pump therapy is short-acting, long-acting carbohydrate is not a valid option to treat hypoglycemia with pump therapy. Patients with type I DM on pump therapy should be well trained to recognize and manage the episodes of hypoglycemia they may encounter. The patients should not rely on the hypoglycemia symptoms. Pseudohypoglycemia may occur in patients with diabetes with the typical symptoms of hypoglycemia with blood glucose levels of more than 3.9 mmol/L (70 mg/dL)[66]. + +Disabling hypoglycemia occurs when repeated episodes of hypoglycemia result in persistent anxiety and impaired quality of life. It usually occurs with tight DM control and with an increasing duration of diabetes. Severe hypoglycemia is that associated with the patient's inability to self-treat with marked impairment of cognitive functions, contrary to non-severe hypoglycemia in which the patient can selftreat and mild cognitive function impairment. Therefore, all children and adolescents with type I DM +should have annual screening for impaired hypoglycemia awareness using a valid screening tool such as Gold Score or Clarke Score[67]. These tools can identify the increased risk of severe hypoglycemic events. CGM can detect when more than 10% of the reading is less than four mmol/L (72 mg/dL) or when there are more than three readings with less than three mmol/L (54 mg/dL) per week, which increases the risk of severe hypoglycemic events[68]. It is crucial to investigate the underlying cause of disabling hypoglycemia, such as cortisol, a Growth Hormone, Coeliac disease, insulin Antibodies, *etc.* If no reason is detected, we should start an intensive education program for the family and the child. Then we can update the pump to sensor augment pump {SAP} without low glucose suspend (LGS). If not improved, we can shift to SAP with LGS. Islet cell or pancreas transplant could be the last option[69]. The child should be reassessed every 3-6 mo. + +## Sick Child + +Children with diabetes and adequate metabolic control should not encounter more infections or illnesses than children without diabetes. However, when a child with type I DM gets sick, we should rule out the presence of uncontrolled or symptomatic hyperglycemia, ketoacidosis, dehydration, and hypoglycemia[70]. The most crucial point to be emphasized is never to stop insulin; it is a prevalent mistake that leads to the development of ketoacidosis. When a child with diabetes has vomiting, we should consider insulin deficiency till proven otherwise. Consequently, the insulin dose may need to be increased or decreased according to the blood glucose level and the presence or absence of ketosis. The blood glucose should be monitored more frequently, at least every 3–4 h, particularly during the night and occasionally every 1–2 h. we also need to monitor for ketosis[70]. We should not depend on urinary ketones' presence to diagnose ketoacidosis as there is a long time lag between the pump stoppage and appearance of the ketone bodies in the urine and the lack of association between the ketones in the urine and the ketones in the blood. Blood beta-hydroxybutyrate monitoring is beneficial in too young children or when urine collection is hard to get[71]. + +In the presence of hyperglycemia, we need to check for ketosis. If the urine ketones are absent or small or the blood ketones are less than 0.6 mmol/L (11 mg/dL), we give a pump correction bolus by drinking extra low-carbohydrate fluids and hourly checking of the blood glucose. If the blood glucose starts to decrease, then continue monitoring and accordingly decide to give another bolus dose or not. If the blood glucose fails to decrease after one hour from the corrective dose, then we supplement with a syringe or pen injection. Suppose the urine ketones are moderately or severely increased, or the blood ketones are equal to or more than 0.6 mmol/L (11 mg/dL). In that case, we should check for a pump delivery failure or a significant medical problem, *e.g.*, severe infection. Accordingly, we can shift to insulin therapy by pen or syringe according to the severity of hyperglycemia and degree of ketonuria/ketonemia[70]. The pump should be checked for mechanical or catheter difficulties by replacing the insulin in the pump, the infusion set, and the cannula while continuing to give bolus doses with a pen or syringe until the hyperglycemia is controlled. Then, we must resume the pump therapy with a temporary basal rate of 120%, hourly blood glucose monitoring, and extra low-carbohydrate diet fluids[72]. + +If the child exhibits signs of diabetic ketoacidosis (DKA), the pump therapy should be discontinued due to the altered tissue perfusion in DKA, which impairs insulin absorption and affects the reliability of the pump therapy. The pump should be temporarily discontinued; the cannula should be removed, then follow the standard protocol to manage DKA. Once DKA is fixed, pump therapy is resumed at the patient's standard basal rate, but the patient should maintain intravenous insulin infusion till he receives a meal bolus[73]. Wang et al[74] showed that using nano-insulin pumps in children with DKA can quickly correct children's blood glucose and ketone body levels. If there is ketosis with low blood glucose, the child should drink extra high-carbohydrate fluids with continuous blood glucose monitoring. In case of persistent vomiting or failure of the previous attempt to control the disorder, the child should be hospitalized[75]. + +## Insulin Pump Use In A Hospitalized Child + +When hospitalized with a severe medical condition, children with type I DM need aggressive management of diabetes. Adequate and meticulous control of diabetes is associated with significant morbidity and mortality improvement in children and adults with diabetes[76]. However, intensive insulin therapy is associated with an increased hypoglycemia rate and complications without a significant mortality reduction. Therefore, continuous monitoring of the blood glucose level is crucial to prevent hypo/hyperglycemia[77]. Non-critically ill children hospitalized for elective and non-acute hospitalizations who can operate their insulin pumps are allowed to continue using them. Fasting prior to elective surgical procedure is allowed with proper adjustment of the basal insulin rate to prevent the development of hypoglycemia. However, if the children cannot operate their pump, they can transition to the basal-bolus regimen[78]. If the child is expected to go into a minor procedure with general anesthesia for less than two hours and is expected to eat or drink within 2-3 h, check the blood glucose levels hourly and adjust the insulin pump accordingly (Figure 7)[79]. + +It is acceptable that hospitalized patients continue to self-control their DM using an insulin pump except if they are unconscious, confused, or have severe pain, undergo major surgery or procedure under general anesthesia lasting more than two hours, or develop DKA. However, if children are hospitalized due to a critically ill condition or surgery that needs anesthesia for more than two hours + +![11_image_0.png](11_image_0.png) + +![11_image_1.png](11_image_1.png) + +and will miss more than one meal, they need to shift to intravenous insulin therapy. The patients should remove the insulin pump and keep it safe to be resumed once the procedure finishes unless being critically ill. Frequent blood glucose monitoring (or CGM) offers safe insulin delivery control[80]. Care should be taken while doing specific procedures in the hospital to avoid damage to the insulin pump. The insulin pump should be covered by a lead apron while doing an X-ray, computerized tomography scan, cardiac catheterization, or insertion of a pacemaker/automatic implantable defibrillator. Ultrasonography can be done when needed, but the operator should avoid direct pointing the transducer to the pump. On the other hand, the pump should be removed while doing magnetic resonance imaging. Upper or lower gastrointestinal endoscopies do not require the removal of the pump[81]. + +## Use Of Corticosteroid With Pump Therapy + +As DM type I is an autoimmune disease, it is frequently associated with other autoimmune disorders, such as autoimmune thyroid disease[82]. Corticosteroids are frequently used in high doses to treat these autoimmune disorders such as asthma and inflammatory bowel diseases. Steroids usually increase insulin resistance, insulin requirement, and the risk of hyperglycemia. Hyperglycemia is predicted to occur approximately four to eight hours following oral steroids administration and sooner following intravenous steroids[83]. On the contrary, blood glucose levels may return to pre-steroid levels 24 h following intravenous steroids discontinuation. If oral steroids are withdrawn gradually over many weeks, glucose levels may decrease dose-dependently[84,85]. + +Interestingly, the daily timing of steroid-induced hyperglycemia depends on the type of steroid use. + +For example, prednisolone, usually given in the morning, induces hyperglycemia in the late morning and persists into the evening with a gradual fall of blood glucose back overnight, often reaching baseline levels the following morning[86]. Therefore, children on pump therapy need to increase the basal insulin rate to 130%-150% during the expected times of maximal hyperglycemia. However, we may occasionally need to increase the basal insulin rate to 200%-300% of the standard rate. Other management options are adjusting ICR or giving corrective insulin bolus when needed. Frequent blood glucose monitoring every 4 h should be done. Once steroid therapy is reduced or stopped, the insulin requirement can be decreased back to pre-steroid levels; therefore, readjusting insulin rates may be needed[87]. + +## Improving Insulin Absorption At The Insertion Sites + +Warming the injection site increases the blood flow, increasing the subcutaneous insulin absorption rate. Warming can be done by massaging or using special warming devices, which is particularly helpful in managing postprandial hyperglycemia[88,89]. Local application of recombinant human hyaluronidase increases the subcutaneous insulin dispersion and absorption rate at the injection site, conferring both ultrafast absorption and action[90]. Using silicon microneedles integrated within the pump trans-dermally delivers efficiently, safely, and painlessly. It also helps decrease the + +## Insulin Pump And School Days + +School life occupies an integral part of children's daily life. Consequently, children using an insulin pump should be able to manage it and have a good education about the proper use of the pump and the management of different problems that they may enface. This awareness is crucial for young children who may need continuous supervision of the pump data and settings, especially for carbohydrate intake, blood glucose monitoring, and insulin administration. The responsible teachers or the school healthcare worker should be well trained in the different scenarios the child using an insulin pump may encounter to allow rapid interference when needed. A clear school health plan to manage students with Type I DM should be available and practiced in recognizing and managing specific acute emergencies such as hypo/hyperglycemia with/without ketonemia[87]. + +## Puberty In Children Using An Insulin Pump + +Puberty is associated with increased sex hormone levels which increase the degree of insulin resistance and, consequently, increase the insulin requirement during puberty. Puberty is associated with higher hemoglobin A1c levels and rates of diabetes complications[92]. Adolescence is also associated with an increased risk of alcohol consumption and considerably increases the diabetes risk in young adulthood and makes diabetes control more difficult. Alcohol consumption in the evening increases the risk of hypoglycemia, especially at night and after the next breakfast, which needs frequent blood glucose monitoring, particularly at night and early morning[93,94]. + +## Menstrual Cycle In An Adolescent Female With Type I Dm + +Menses is associated with high fluctuations of the glycaemic control with an increased risk of hyperglycemia and increased insulin resistance during periovulation and early luteal phases (premenstrual period), possibly due to the rising oestradiol levels that occur before ovulation, which may need a temporary increase in the basal insulin rate[95]. On the contrary, when menses start, the progesterone level begins to drop, causing a sharp decline in the insulin requirement, which may increase the hypoglycemia risk steeply[96]. If the adolescent girl has a disturbed menstrual cycle and needs to use contraceptive pills to regulate the cycle, care should be taken while using them as an increase in the basal insulin requirement may occur, especially with high-dose contraceptive pills or with long-acting progesterone injections[97]. + +## Traveling With An Insulin Pump + +Despite traveling being a welcome retreat from our everyday life difficulties, it is associated with an extraordinary increase in daily activity, which could increase the risk of hypoglycemia and becomes a real challenge in patients with diabetes. It is particularly challenging in the presence of time zone differences > 3 h, significant variations in the basal rates, such as the marked Dawn phenomenon, recurrent or severe episodes of hypoglycemia, or reduced hypoglycemia awareness. The pump basal rate is reduced by 10%-20% during traveling for the first 24 h and gradually changes the pump timing setting to the new local time upon arrival at the destination by 2-3 h each day. This change should be associated with frequent blood glucose monitoring. Disconnecting the pump during take-off and landing is also essential to avoid pressure effects on the pump delivery mechanism[98]. + +## General Care For Children With Dm Who Require Insulin Pump Therapy Skin Care And Prevention Of Insertion Site Infection + +Skin changes occur in one-third of patients with diabetes. Children with high glucose levels tend to have dry skin[99]. Therefore, they need adequate blood sugar control and proper skin hydration with specific lotions for diabetes following site change, especially in pump care. Itching is frequent at the insertion site; the child is advised to gently rub and avoid scratching the affected area to avoid skin breakage [100]. The insertion set should be applied to dry, unwet, non-broken skin. The insertion site should be rotated ideally every three days, over 6-10 sites, to long-term preserve skin integrity. Certain areas are advised more than others due to their superior insulin-absorptive capabilities, more comfort, and better convenience. The most common sites suitable for insertion are the abdomen (two-centimetre radius around and away from the navel), upper buttocks, upper thigh (medial and lateral areas, at least 5 cm away from hip or knee joints), upper buttocks (flank), upper arm, and rarely forearm (Figure 8A). The area with the least hair should be chosen as the hair decreases the longevity of the insertion site. Every site should take at least one week for recovery and should be away from the next site by at least one to two inches. Different patterns of insertion site rotations are demonstrated in Figure 8B[101]. + +## Vaccination + +Patients with DM, especially those requiring pump therapy, need intense vaccination programs. + +![13_image_0.png](13_image_0.png) + +Figure 8 Approved insertion sites and different types of insertion site **rotation.** A: There are four approved sites for the subcutaneous injection of insulin: Insulin Absorption Rate Fluctuation Depending on Injection Site; B: shows different types of insertion site rotation. Note that all the rotation patterns keep away from the umbilicus by at least one inch. + +They should receive an annual flu vaccination. They also should consider having Streptococcus pneumoniae vaccines, and the coronavirus disease 2019 (COVID-19) vaccination is strongly recommended[102]. + +The use of insulin pumps in special situations such as traveling, swimming, bathing, imaging, and other procedures is summarized in Table 2. + +## Advantages Of Insulin Pump Therapy + +The insulin pump provides fewer needle injections as there is no need to inject insulin every time needed. It also can provide small insulin doses down to ten times less (0.05-0.1 Units) than an insulin pen or syringe, which is particularly useful in insulin-sensitive young children. Meanwhile, the insulin pump provides a convenient and more flexible way to modify the needed insulin more physiologically, relatively similar to the human pancreas[103]. Hence basal insulin can be programmed to match the person's activity, the changing daily requirement, hormonal changes, pubertal spurts, stress, illness, traveling, and any other situations. At the same time, insulin bolus can be delivered in different ways considering various conditions such as gastroparesis, malabsorption, or even to match the ingested foods. On the other hand, insulin delivery can be temporarily reduced or suspended in certain situations, such as hypoglycemia[32]. + +This incredible flexibility in the insulin delivery system and the marked less in blood glucose fluctuations allow a better quality of life. Using rapid-acting insulin delivered in a low volume tailored to the individual needs allows the insulin pump to overcome the variation in insulin absorption that is usually observed with long-acting insulin, resulting in more consistent and reliable insulin absorption and consequently less fluctuation of both insulin profile and blood glucose level[104]. This feature also helps to decrease the need for snacks, especially before exercise, consequently decreasing the rate of weight gain[105]. However, Boucher-Berry et al[106] found that lower bolus to basal insulin ratios increases the risk of excess weight gain. Hence, proper bolus and basal insulin dose adjustments are mandatory during insulin therapy. Pump therapy also improves the patient experience and satisfaction due to technology motivation and improved self-management. Pumps can also integrate easily with the new technology, so they can link easily with blood glucose measuring technology, bolus advisors, and wizards for diabetes management, forming a closed-loop system similar to an artificial pancreas which significantly improves the patient's quality of life[107]. + +## Disadvantages Of Insulin Pump Therapy + +There are some disadvantages to insulin pump therapy that may limit its use. Being worn all the time (24 h a day/7 days a week), even during sleep, showering, and sports, with continuous reminders of being diabetic, can influence body image and self-confidence. Luckily, numerous accessories are accessible to make wearing the pump hidden and convenient[108]. Because insulin pumps use only rapid-acting insulin without a long-acting insulin depot, there is a high risk of rapid development of DKA with technical failure or interruption of insulin flow such as air in the tubing, a damaged infusion set, or infection at the insertion site. Therefore, the patient should monitor blood glucose levels more frequently, at least four times daily, and a specialized well-trained interdisciplinary care team should be accessible in case of emergency. The pump also should not be disconnected for a long time (short-acting + +| Al-Beltagi M et al. Insulin pumps in children | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Table 2 Special situations with insulin pumps Situation Management Air travel The patient should have a note from the physician about the need for the pump before traveling The patient should inform the security about the pump before passing through the security checkpoint with an X-ray detector All the suppliers and the accessories should be carried in a separate bag for easy inspection The metal detectors and whole-body scanners do not damage the device Shower and bathing A warm shower is associated with more rapid insulin absorption than usual, increasing the risk of hypoglycemia A warm bath (e.g., hot tub or sauna) can expose insulin to high temperatures, which could be spoiled when exposed for an extended period High temperatures can also damage the pump, so it is crucial to check the owner manual for temperature specifications about ideal operating temperatures for the pump Knowing the type of the pump as waterproof, watertight, water-repellent, and water-resistant is essential If the insulin pump is not water waterproof, it is better to disconnect the pump and keep it in a dry place Swimming Knowing the type of the pump as waterproof, watertight, water-repellent, and water-resistant is essential If the pump is waterproof, they should know how many feet in-depth and how long The patient also should know the maximum time the pump can be removed without affecting its performance, e.g., one hour for the Aviva Combo insulin pump Some pumps can be worn while swimming. It will be adjusted to deliver a specific basal rate throughout the swimming If the pump is not compatible with swimming according to the manufacturer's guidelines, the pump can be removed, leaving the cannula attached in place and covered with a dressing and a cap If the pump is removed for more than one hour, testing the blood glucose using a glucometer is advised with the recommended amount of carbohydrates and insulin intake, and continue swimming All types of sports, including martial arts and those with possible body contact, are permitted if there are no other systemic contraindications Contact sports may increase the risk of dislodgement of the pump cannula Ensure adequate hydration during any exercise The pump can be removed for up to one hour Contact sports After one hour, testing the blood sugar using a glucometer is advised with the recommended amount of carbohydrates and insulin intake, and continue sports X-ray Radiation can provoke electrical currents in the electric circuit, impairing the pump function The pump should be removed when possible Imaging Safe to keep on an insulin pump in position if the x-ray beam is less than 3 seconds at a time and if a lead apron protects the pump Dental X-ray The patient should ensure that the pump is covered by the lead apron he wears Ultrasound The ultrasound beam should not directly point at the pump or the insertion site The pump should be removed when possible If unable to remove the pump, relocate it to another area away from the anatomic examination site and cover it with a lead apron Switch off the pump during the examination and set a reminder timer to reoperate the pump just after finishing the radiological procedure CT-scan and Fluoroscopy After finishing the procedure, the pump should be checked for any possible malfunction with frequent blood glucose monitoring The MRI magnetic field is strong enough to magnetize the pump's motor and thus damage it The pump should be removed and kept outside the MRI room whenever possible If it is impossible to remove the pump and the patient uses a metal cannula or an Omnipod, he/she must remove it before entering the room and insert a new cannula after finishing the test MRI If the pump is accidentally exposed to an MRI field, it should be stopped and disconnected immediately and checked by the maintenance team for any malfunction before resuming its use | + +| Insulin pump renewal | The pump is renewed if there is evidence of clinical benefits over the past four years The pump is renewed after warranty expiration to obtain the safest result. However, it can still be used but with an increased risk of malfunction The child pump users can shift to MDT between 12-18 yr of age. However, they can continue using the pump as an adult They need at least three monthly follow-up appointments to ensure the adequacy of MDT with a smooth transition to the adult clinic Pediatric pump users can take a holiday off from the pump therapy. During this holiday, they can receive MDT as a training period and are not considered as a pump therapy failure | +|------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| The transition from Pediatric to the adult pump user | Strong cooperation between the pediatric and the adult pump therapy clinic is needed to ensure a smooth transition of the adolescent to an adult pump or MDT user | + +CT: Computed tomography; MDT: Multidose therapy; MRI: Magnetic resonance imaging. + +insulin). This limitation could affect the child's daily activities, such as swimming. However, many pump types are waterproof and fit for swimming[109]. + +Insulin pump setup occasionally is complicated and difficult to proceed with compared to pen or syringe use. Therefore, adequate training and education are mandatory for both the child and the caregiver. It also needs a high level of motivation, understanding, and education to achieve the best benefit of the pump and avoid complications. In addition, the insertion set, including the infusion set and cannula, requires to be replaced every 2-3 d. In addition, the infusion set may be prone to incorrect priming, air bubbles, tubing breakdown, and kinking or slippage of the cannula, which can interrupt insulin delivery[110]. + +Infection of the insertion site occurs in 17% of patients on insulin pumps over a period of three years, especially when the infusion set is left for longer than it should[111]. Risk factors for insertion site infections include large insulin doses, poor patient selection, inadequate patient education, lack of hygienic measures, infrequent changes of the infusion set as requested by the manufacturer, and incorrect cannula insertion[112]. Staphylococcal or streptococcal bacterial infections are the most common infection at the insertion site, followed by Rhizomucor *pusillus* fungal cellulitis[113]. Occasionally the infection may progress to cellulitis or collect into an abscess requiring surgical drainage. Primary tuberculous infection of the insulin injection site is rare but reported complications of insulin therapy[114]. Changing the insertion set should be done according to the manufacturer's guidelines or pump educator to diminish the chance of infection. It is also imperative that the patient follow proper hygienic measures, especially washing the hands and periodically changing the site to decrease infection chances. The cost of the pump is not only limited to the device but also running costs for accessories, cartridge syringes, batteries, skin preparation items, cannulas, and infusion sets, which are significantly higher than the regular insulin pens and syringes. Because insulin is a lifesaving essential medicine, every effort should be made to minimize the cost to the patient and his family[115]. + +## Discontinuation Of Insulin Pump Therapy + +About 3% of the pump user discontinue pump therapy within one year. The most frequent causes, according to Wong et al[116], include difficulties in pump insertion/adhesive (60%), interference with sports activities (42%), discomfort with wearing the pump (38%), interference with intimacy (34%), +pump dysfunction (28%), and problematic hyperglycemia (28%). Beato-Víbora et al[117] 2015 showed that pump therapy improves glycaemic control and hypoglycemia awareness, decreases hypoglycemia frequency, and can be sustained for many years. However, 5% of the users discontinued the pump therapy due to a lack of clinical advantage, technical problems, safety issues, or user choice. + +The pump therapy should be discontinued for recurrent DKA as it affects the local absorption of insulin due to altered tissue perfusion. It should also be discontinued when there is a pump failure or mismanagement, inadequate blood glucose monitoring (less than four times/day), recurrent injection site infection, intentional overdosing, failure to meet the objective of pump therapy, or with the parents' or the child's wishes. Pump therapy should be discontinued if the patient becomes unconscious, confused, and unable to self-management, such as severe pain or illness. It should also be discontinued if the patient goes for major surgery with general anesthesia for more than two hours[118]. Limitation of the study: some of the included articles were in favor of using pumps which may carry the risk of bias. However, we tried to include most of the available studies to minimize the risk of bias. + +## Conclusion + +The insulin pump is a giant breakthrough in DM management, especially in the pediatric age. The insulin pump provides fewer needles and can provide very tiny insulin doses. It provides a convenient and more flexible way to modify the needed insulin physiologically, like the human pancreas. It can offer adequate and optimal glycemic control to reduce the risk of metabolic derangements in different tissues. However, there are some disadvantages to insulin pump therapy that do not necessarily prevent its use. It should be discontinued with recurrent diabetic ketoacidosis. + +## Article Highlights Research Background + +The insulin pump is a giant breakthrough in diabetes mellitus (DM) treatment. Treating diabetes with an insulin pump is the method most similar to the normal physiologic function of the pancreas. + +## Research Motivation + +We are motivated to write this manuscript to decrease the gap in understanding of insulin pump use among children health care professionals, parents, and children with diabetes mellitus who need intensive insulin therapy. + +## Research Objectives + +To identify all the existing evidence-based research for the proper use of insulin pumps among children with diabetes Mellitus and to increase awareness among these patients and their families. + +## Research Methods + +We conducted comprehensive literature searches of electronic databases until June 30, 2022, related to pump therapy in children and published in the English language. The selected articles were subsequently explored to identify the most recent evidence-based research and existing guidelines for the proper use of insulin pumps in children. + +## Research Results + +We identified 118 articles concerned with insulin pumps, 61 were reviews, systemic reviews, and metaanalyses, 47 were primary research studies with strong design, and ten were guidelines. These articles covered the different aspects of insulin pump use in children with diabetes mellitus. + +## Research Conclusions + +The insulin pump is a giant breakthrough in pediatric DM management. It provides fewer needles and can provide very tiny insulin doses with a convenient and flexible way to modify the needed insulin physiologically, like the human pancreas. It can offer adequate and optimal glycemic control to reduce the risk of metabolic derangements in different tissues. + +## Research Perspectives + +Continuous modification and upgrading of the insulin pump are expected to proceed. These modifications will probably help to make insulin pumps more physiologic and similar to the human pancreas. + +## Acknowledgements + +We thank the anonymous referees for their valuable suggestions. + +## Footnotes + +Author contributions: Al-Biltagi M, Saeed NK, Bediwy AS, and Elbeltagi R collected the data and wrote and revised the manuscript. + +Conflict-of-interest statement: All authors declare no conflict of interest. + +PRISMA 2009 Checklist statement: The authors have read the PRISMA 2009 Checklist, and the manuscript was prepared and revised according to the PRISMA 2009 Checklist. + +Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BYNC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is noncommercial. See: https://creativecommons.org/Licenses/by-nc/4.0/ + +## Country/Territory Of Origin: Bahrain + +ORCID number: Mohammed Al-Beltagi 0000-0002-7761-9536; Nermin Kamal Saeed 0000-0001-7875-8207; Adel Salah Bediwy 0000-0002-0281-0010. + +S-Editor: Liu JH +L-Editor: A +P-Editor: Liu JH + +## References + +McAdams BH, Rizvi AA. An Overview of Insulin Pumps and Glucose Sensors for the Generalist. *J Clin Med* 2016; 5 [PMID: 26742082 DOI: 10.3390/jcm5010005] +1 Karamanou M, Protogerou A, Tsoucalas G, Androutsos G, Poulakou-Rebelakou E. Milestones in the history of diabetes mellitus: The main contributors. *World J Diabetes* 2016; 7: 1-7 [PMID: 26788261 DOI: 10.4239/wjd.v7.i1.1] +2 Lee J, Sanders DPM. From Thebes to Toronto and the 21st Century: An Incredible Journey. *Diabetes Spectr* 2002; 15: 56-60 [DOI: 10.2337/diaspect.15.1.56] +3 Lewis GF, Brubaker PL. The discovery of insulin revisited: lessons for the modern era. *J Clin Invest* 2021; 131 [PMID: 33393501 DOI: 10.1172/JCI142239] +4 Fralick M, Zinman B. The discovery of insulin in Toronto: beginning a 100 year journey of research and clinical achievement. *Diabetologia* 2021; 64: 947-953 [PMID: 33492422 DOI: 10.1007/s00125-020-05371-6] +5 Thomas LJ, Bessman SP. Prototype for an implantable insulin delivery pump. *Proc West Pharmacol Soc* 1975; 18: 393398 [PMID: 1178703] +6 Riley WJ, Silverstein JH, Rosenbloom AL, Spillar R, McCallum MH. Ambulatory diabetes management with pulsed subcutaneous insulin using a portable pump. *Clin Pediatr (Phila)* 1980; 19: 609-614 [PMID: 6996894 DOI: 10.1177/000992288001900907] +7 Kaufman FR, Halvorson M, Miller D, Mackenzie M, Fisher LK, Pitukcheewanont P. Insulin pump therapy in type 1 pediatric patients: now and into the year 2000. *Diabetes Metab Res Rev* 1999; 15: 338-352 [PMID: 10585620 DOI: 10.1002/(sici)1520-7560(199909/10)15:5<338::aid-dmrr57>3.0.co;2-y] +8 9 **Marcus A**. Diabetes care - insulin delivery in a changing world. *Medscape J Med* 2008; 10: 120 [PMID: 18596953] +Kesavadev J, Saboo B, Krishna MB, Krishnan G. Evolution of Insulin Delivery Devices: From Syringes, Pens, and Pumps to DIY Artificial Pancreas. *Diabetes Ther* 2020; 11: 1251-1269 [PMID: 32410184 DOI: 10.1007/s13300-020-00831-z] +10 Bekiari E, Kitsios K, Thabit H, Tauschmann M, Athanasiadou E, Karagiannis T, Haidich AB, Hovorka R, Tsapas A. + +Artificial pancreas treatment for outpatients with type 1 diabetes: systematic review and meta-analysis. BMJ 2018; 361: +k1310 [PMID: 29669716 DOI: 10.1136/bmj.k1310] +11 Sherr J, Tamborlane WV. Past, present, and future of insulin pump therapy: better shot at diabetes control. Mt Sinai J +Med 2008; 75: 352-361 [PMID: 18729180 DOI: 10.1002/msj.20055] +12 Pickup JC. Insulin-pump therapy for type 1 diabetes mellitus. *N Engl J Med* 2012; 366: 1616-1624 [PMID: 22533577 DOI: 10.1056/NEJMct1113948] +13 Mbundu Ilunga R, Camponovo C, Le Dizès O, Wojtusciszyn A. [Insulin pump treatment: For whom and how to set it up on an outpatient? *Rev Med Suisse* 2020; 16: 1191-1196 [PMID: 32520457] +14 Baretić M, Kraljević I, Renar IP. NOCTURNAL HYPOGLYCEMIA--THE MAIN INDICATION FOR INSULIN PUMP THERAPY IN ADULTHOOD. *Acta Clin Croat* 2016; 55: 93-99 [PMID: 27333724 DOI: 10.20471/acc.2016.55.01.14] +15 Cengiz E, Bode B, Van Name M, Tamborlane WV. Moving toward the ideal insulin for insulin pumps. *Expert Rev Med* Devices 2016; 13: 57-69 [PMID: 26560137 DOI: 10.1586/17434440.2016.1109442] +16 Berget C, Messer LH, Forlenza GP. A Clinical Overview of Insulin Pump Therapy for the Management of Diabetes: Past, Present, and Future of Intensive Therapy. *Diabetes Spectr* 2019; 32: 194-204 [PMID: 31462873 DOI: 10.2337/ds18-0091] +17 Yen PM, Young AS. Review of Modern Insulin Pumps and the Perioperative Management of the Type 1 Diabetic Patient for Ambulatory Dental Surgery. *Anesth Prog* 2021; 68: 180-187 [PMID: 34606570 DOI: 10.2344/anpr-68-03-16] +18 Paul N, Kohno T, Klonoff DC. A review of the security of insulin pump infusion systems. *J Diabetes Sci Technol* 2011; 5: 1557-1562 [PMID: 22226278 DOI: 10.1177/193229681100500632] +19 20 Biester T, Kordonouri O, Holder M, Remus K, Kieninger-Baum D, Wadien T, Danne T. "Let the Algorithm Do the Work": Reduction of Hypoglycemia Using Sensor-Augmented Pump Therapy with Predictive Insulin Suspension (SmartGuard) in Pediatric Type 1 Diabetes Patients. *Diabetes Technol Ther* 2017; 19: 173-182 [PMID: 28099035 DOI: 10.1089/dia.2016.0349] Sussman A, Taylor EJ, Patel M, Ward J, Alva S, Lawrence A, Ng R. Performance of a glucose meter with a built-in automated bolus calculator versus manual bolus calculation in insulin-using subjects. *J Diabetes Sci Technol* 2012; 6: 339344 [PMID: 22538144 DOI: 10.1177/193229681200600218] +21 22 **Vettoretti M**, Cappon G, Facchinetti A, Sparacino G. Advanced Diabetes Management Using Artificial Intelligence and Continuous Glucose Monitoring Sensors. *Sensors (Basel)* 2020; 20 [PMID: 32664432 DOI: 10.3390/s20143870] +Doyle-Delgado K, Chamberlain JJ. Use of Diabetes-Related Applications and Digital Health Tools by People With Diabetes and Their Health Care Providers. *Clin Diabetes* 2020; 38: 449-461 [PMID: 33384470 DOI: 10.2337/cd20-0046] +23 Kesavadev J, Das AK, Unnikrishnan R 1st, Joshi SR, Ramachandran A, Shamsudeen J, Krishnan G, Jothydev S, Mohan V. Use of insulin pumps in India: suggested guidelines based on experience and cultural differences. *Diabetes Technol* Ther 2010; 12: 823-831 [PMID: 20807118 DOI: 10.1089/dia.2010.0027] +24 Dhatariya K. Blood Ketones: Measurement, Interpretation, Limitations, and Utility in the Management of Diabetic Ketoacidosis. *Rev Diabet Stud* 2016; 13: 217-225 [PMID: 28278308 DOI: 10.1900/RDS.2016.13.217] +25 Paldus B, Lee MH, O'Neal DN. Insulin pumps in general practice. *Aust Prescr* 2018; 41: 186-190 [PMID: 30670886 DOI: 10.18773/austprescr.2018.056] +26 Zisser H, Jovanovic L. OmniPod Insulin Management System: patient perceptions, preference, and glycemic control. Diabetes Care 2006; 29: 2175 [PMID: 16936173 DOI: 10.2337/dc06-0986] +27 King AB. Continuous glucose monitoring-guided insulin dosing in pump-treated patients with type 1 diabetes: a clinical guide. *J Diabetes Sci Technol* 2012; 6: 191-203 [PMID: 22401339 DOI: 10.1177/193229681200600124] +28 Kuroda A, Kaneto H, Yasuda T, Matsuhisa M, Miyashita K, Fujiki N, Fujisawa K, Yamamoto T, Takahara M, Sakamoto F, Matsuoka TA, Shimomura I. Basal insulin requirement is ~30% of the total daily insulin dose in type 1 diabetic patients who use the insulin pump. *Diabetes Care* 2011; 34: 1089-1090 [PMID: 21430086 DOI: 10.2337/dc10-2149] +29 Demir G, Atik Altınok Y, Özen S, Darcan Ş, Gökşen D. Initial Basal and Bolus Rates and Basal Rate Variability During Pump Treatment in Children and Adolescents. *J Clin Res Pediatr Endocrinol* 2021; 13: 198-203 [PMID: 33374094 DOI: 10.4274/jcrpe.galenos.2020.2020.0171] +30 Silver B, Ramaiya K, Andrew SB, Fredrick O, Bajaj S, Kalra S, Charlotte BM, Claudine K, Makhoba A. EADSG +Guidelines: Insulin Therapy in Diabetes. *Diabetes Ther* 2018; 9: 449-492 [PMID: 29508275 DOI: 10.1007/s13300-018-0384-6] +31 McCall AL, Farhy LS. Treating type 1 diabetes: from strategies for insulin delivery to dual hormonal control. *Minerva* Endocrinol 2013; 38: 145-163 [PMID: 23732369] +32 Heden TD, Kanaley JA. Syncing Exercise With Meals and Circadian Clocks. *Exerc Sport Sci Rev* 2019; 47: 22-28 +[PMID: 30334851 DOI: 10.1249/JES.0000000000000172] +33 Chawla R, Madhu SV, Makkar BM, Ghosh S, Saboo B, Kalra S; RSSDI-ESI Consensus Group. RSSDI-ESI Clinical Practice Recommendations for the Management of Type 2 Diabetes Mellitus 2020. *Indian J Endocrinol Metab* 2020; 24: 1-122 [PMID: 32699774 DOI: 10.4103/ijem.IJEM_225_20] +34 Chetty T, Shetty V, Fournier PA, Adolfsson P, Jones TW, Davis EA. Exercise Management for Young People With Type 1 Diabetes: A Structured Approach to the Exercise Consultation. *Front Endocrinol (Lausanne)* 2019; 10: 326 [PMID: 31258513 DOI: 10.3389/fendo.2019.00326] +35 36 Elleri D, Allen JM, Kumareswaran K, Leelarathna L, Nodale M, Caldwell K, Cheng P, Kollman C, Haidar A, Murphy HR, Wilinska ME, Acerini CL, Dunger DB, Hovorka R. Closed-loop basal insulin delivery over 36 hours in adolescents with type 1 diabetes: randomized clinical trial. *Diabetes Care* 2013; 36: 838-844 [PMID: 23193217 DOI: 10.2337/dc12-0816] +Howard JY, Watts SA. Bolus Insulin Prescribing Recommendations for Patients With Type 2 Diabetes Mellitus. Fed Pract 2017; 34: S26-S31 [PMID: 30766313] +37 King AB, Kuroda A, Matsuhisa M, Hobbs T. A Review of Insulin-Dosing Formulas for Continuous Subcutaneous Insulin Infusion (CSII) for Adults with Type 1 Diabetes. *Curr Diab Rep* 2016; 16: 83 [PMID: 27457238 DOI: 10.1007/s11892-016-0772-0] +38 Heinemann L. Insulin pump therapy: what is the evidence for using different types of boluses for coverage of prandial insulin requirements? *J Diabetes Sci Technol* 2009; 3: 1490-1500 [PMID: 20144405 DOI: 10.1177/193229680900300631] +39 Lukka M, Tillmann V, Peet A. Decreased Need for Correction Boluses with Universal Utilisation of Dual-Wave Boluses in Children with Type 1 Diabetes. *J Clin Med* 2022; 11 [PMID: 35330014 DOI: 10.3390/jcm11061689] +40 Lee SW, Cao M, Sajid S, Hayes M, Choi L, Rother C, de León R. The dual-wave bolus feature in continuous subcutaneous insulin infusion pumps controls prolonged post-prandial hyperglycaemia better than standard bolus in Type 1 diabetes. *Diabetes Nutr Metab* 2004; 17: 211-216 [PMID: 15575341] +41 Kowalczyk E, Dżygało K, Szypowska A. Super Bolus: a remedy for a high glycemic index meal in children with type 1 diabetes on insulin pump therapy? *Trials* 2022; 23: 240 [PMID: 35351180 DOI: 10.1186/s13063-022-06173-4] +42 Hanas R, Adolfsson P. Bolus Calculator Settings in Well-Controlled Prepubertal Children Using Insulin Pumps Are Characterized by Low Insulin to Carbohydrate Ratios and Short Duration of Insulin Action Time. *J Diabetes Sci Technol* 2017; 11: 247-252 [PMID: 27470666 DOI: 10.1177/1932296816661348] +43 Davidson PC, Hebblewhite HR, Steed RD, Bode BW. Analysis of guidelines for basal-bolus insulin dosing: basal insulin, correction factor, and carbohydrate-to-insulin ratio. *Endocr Pract* 2008; 14: 1095-1101 [PMID: 19158048 DOI: 10.4158/EP.14.9.1095] +44 Berián J, Bravo I, Gardel-Vicente A, Lázaro-Galilea JL, Rigla M. Dynamic Insulin Basal Needs Estimation and Parameters Adjustment in Type 1 Diabetes. *Sensors (Basel)* 2021; 21 [PMID: 34372462 DOI: 10.3390/s21155226] +45 Silverstein J, Klingensmith G, Copeland K, Plotnick L, Kaufman F, Laffel L, Deeb L, Grey M, Anderson B, Holzmeister LA, Clark N; American Diabetes Association. Care of children and adolescents with type 1 diabetes: a statement of the American Diabetes Association. *Diabetes Care* 2005; 28: 186-212 [PMID: 15616254 DOI: 10.2337/diacare.28.1.186] +46 Pozzilli P, Battelino T, Danne T, Hovorka R, Jarosz-Chobot P, Renard E. Continuous subcutaneous insulin infusion in diabetes: patient populations, safety, efficacy, and pharmacoeconomics. *Diabetes Metab Res Rev* 2016; 32: 21-39 [PMID: 25865292 DOI: 10.1002/dmrr.2653] +47 Ceriello A, Prattichizzo F, Phillip M, Hirsch IB, Mathieu C, Battelino T. Glycaemic management in diabetes: old and new approaches. *Lancet Diabetes Endocrinol* 2022; 10: 75-84 [PMID: 34793722 DOI: 10.1016/S2213-8587(21)00245-X] +48 49 **Boronat M**, Sánchez-Hernández RM, Rodríguez-Cordero J, Jiménez-Ortega A, Nóvoa FJ. Suspension of basal insulin to avoid hypoglycemia in type 1 diabetes treated with insulin pump. *Endocrinol Diabetes Metab Case Rep* 2015; **2015**: +140081 [PMID: 25614824 DOI: 10.1530/EDM-14-0081] Ogiso K, Koriyama N, Obo T, Tokito A, Nishio Y. Basal insulin ameliorates post-breakfast hyperglycemia via suppression of post-breakfast proinsulin/C-peptide ratio and fasting serum free fatty acid levels in patients with type 2 diabetes. *Diabetol Int* 2021; 12: 161-170 [PMID: 33786271 DOI: 10.1007/s13340-020-00457-3] +50 Nauck MA, Lindmeyer AM, Mathieu C, Meier JJ. Twenty-Four Hour Fasting (Basal Rate) Tests to Achieve CustomTailored, Hour-by-Hour Basal Insulin Infusion Rates in Patients With Type 1 Diabetes Using Insulin Pumps (CSII). J +Diabetes Sci Technol 2021; 15: 360-370 [PMID: 31633384 DOI: 10.1177/1932296819882752] +51 Trief PM, Cibula D, Rodriguez E, Akel B, Weinstock RS. Incorrect Insulin Administration: A Problem That Warrants Attention. *Clin Diabetes* 2016; 34: 25-33 [PMID: 26807006 DOI: 10.2337/diaclin.34.1.25] +52 Slattery D, Amiel SA, Choudhary P. Optimal prandial timing of bolus insulin in diabetes management: a review. *Diabet* Med 2018; 35: 306-316 [PMID: 29044708 DOI: 10.1111/dme.13525] +53 King AB, Armstrong DU. A prospective evaluation of insulin dosing recommendations in patients with type 1 diabetes at near normal glucose control: bolus dosing. *J Diabetes Sci Technol* 2007; 1: 42-46 [PMID: 19888378 DOI: 10.1177/193229680700100107] +54 De Ridder F, den Brinker M, De Block C. The road from intermittently scanned glucose monitoring to hybrid closed-loop systems: Part A. Keys to success: subject profiles, choice of systems, education. *Ther Adv Endocrinol Metab* 2019; 10: 2042018819865399 [PMID: 31384420 DOI: 10.1177/2042018819865399] +55 Aye T, Block J, Buckingham B. Toward closing the loop: an update on insulin pumps and continuous glucose monitoring systems. *Endocrinol Metab Clin North Am* 2010; 39: 609-624 [PMID: 20723823 DOI: 10.1016/j.ecl.2010.05.005] +56 Nicolajsen T, Samuelsson A, Hanas R. Insulin doses before and one year after pump start: children have a reversed dawn phenomenon. *J Diabetes Sci Technol* 2012; 6: 589-594 [PMID: 22768890 DOI: 10.1177/193229681200600314] +57 Cope JU, Samuels-Reid JH, Morrison AE. Pediatric use of insulin pump technology: a retrospective study of adverse events in children ages 1-12 years. *J Diabetes Sci Technol* 2012; 6: 1053-1059 [PMID: 23063031 DOI: 10.1177/193229681200600509] +58 Davidson MB. Insulin Therapy: A Personal Approach. *Clin Diabetes* 2015; 33: 123-135 [PMID: 26203205 DOI: +10.2337/diaclin.33.3.123] +59 Kelly JL. Continuous Insulin Infusion: When, Where, and How? *Diabetes Spectr* 2014; 27: 218-223 [PMID: 26246783 DOI: 10.2337/diaspect.27.3.218] +60 Codella R, Terruzzi I, Luzi L. Why should people with type 1 diabetes exercise regularly? *Acta Diabetol* 2017; 54: 615630 [PMID: 28289908 DOI: 10.1007/s00592-017-0978-x] +61 Yardley JE, Sigal RJ, Perkins BA, Riddell MC, Kenny GP. Resistance exercise in type 1 diabetes. *Can J Diabetes* 2013; 37: 420-426 [PMID: 24321724 DOI: 10.1016/j.jcjd.2013.07.020] +62 Metcalf KM, Singhvi A, Tsalikian E, Tansey MJ, Zimmerman MB, Esliger DW, Janz KF. Effects of moderate-tovigorous intensity physical activity on overnight and next-day hypoglycemia in active adolescents with type 1 diabetes. + +Diabetes Care 2014; 37: 1272-1278 [PMID: 24574352 DOI: 10.2337/dc13-1973] +63 64 Buoite Stella A, Assaloni R, Tonutti L, Manca E, Tortul C, Candido R, Francescato MP. Strategies used by Patients with Type 1 Diabetes to Avoid Hypoglycemia in a 24×1-Hour Marathon: Comparison with the Amounts of Carbohydrates Estimated by a Customizable Algorithm. *Can J Diabetes* 2017; 41: 184-189 [PMID: 27939876 DOI: 10.1016/j.jcjd.2016.09.007] Quirós C, Bertachi A, Giménez M, Biagi L, Viaplana J, Viñals C, Vehí J, Conget I, Bondia J. Blood glucose monitoring during aerobic and anaerobic physical exercise using a new artificial pancreas system. *Endocrinol Diabetes Nutr (Engl Ed)* +2018; 65: 342-347 [PMID: 29483036 DOI: 10.1016/j.endinu.2017.12.012] +65 66 Seaquist ER, Anderson J, Childs B, Cryer P, Dagogo-Jack S, Fish L, Heller SR, Rodriguez H, Rosenzweig J, Vigersky R; American Diabetes Association; Endocrine Society. Hypoglycemia and diabetes: a report of a workgroup of the American Diabetes Association and the Endocrine Society. *J Clin Endocrinol Metab* 2013; 98: 1845-1859 [PMID: 23589524 DOI: +10.1210/jc.2012-4127] Xie X, Guo J, Bremner KE, Wang M, Shah BR, Volodin A. Review and estimation of disutility for joint health states of severe and nonsevere hypoglycemic events in diabetes. *J Comp Eff Res* 2021; 10: 961-974 [PMID: 34287017 DOI: 10.2217/cer-2021-0059] +67 Farrell CM, McCrimmon RJ. Clinical approaches to treat impaired awareness of hypoglycaemia. *Ther Adv Endocrinol* Metab 2021; 12: 20420188211000248 [PMID: 33796253 DOI: 10.1177/20420188211000248] +68 Choudhary P, Olsen BS, Conget I, Welsh JB, Vorrink L, Shin JJ. Hypoglycemia Prevention and User Acceptance of an Insulin Pump System with Predictive Low Glucose Management. *Diabetes Technol Ther* 2016; 18: 288-291 [PMID: 26907513 DOI: 10.1089/dia.2015.0324] +69 Brink S, Joel D, Laffel L, Lee WW, Olsen B, Phelan H, Hanas R; International Society for Pediatric and Adolescent Diabetes. ISPAD Clinical Practice Consensus Guidelines 2014. Sick day management in children and adolescents with diabetes. *Pediatr Diabetes* 2014; 15 Suppl 20: 193-202 [PMID: 25182314 DOI: 10.1111/pedi.12193] +70 Laffel L. Sick-day management in type 1 diabetes. *Endocrinol Metab Clin North Am* 2000; 29: 707-723 [PMID: +11149158 DOI: 10.1016/s0889-8529(05)70160-2] +71 Cescon M, DeSalvo DJ, Ly TT, Maahs DM, Messer LH, Buckingham BA, Doyle FJ 3rd, Dassau E. Early Detection of Infusion Set Failure During Insulin Pump Therapy in Type 1 Diabetes. *J Diabetes Sci Technol* 2016; 10: 1268-1276 [PMID: 27621142 DOI: 10.1177/1932296816663962] +72 Evans K. Diabetic ketoacidosis: update on management. *Clin Med (Lond)* 2019; 19: 396-398 [PMID: 31530688 DOI: 10.7861/clinmed.2019-0284] +73 Wang J, Zhang L, Wang X, Dong J, Chen X, Yang S. Application of Nano-Insulin Pump in Children with Diabetic Ketoacidosis. *J Nanosci Nanotechnol* 2021; 21: 5051-5056 [PMID: 33875090 DOI: 10.1166/jnn.2021.19356] +74 Choudhary A. Sick Day Management in Children and Adolescents with Type 1 Diabetes. *J Ark Med Soc* 2016; 112: 284286 [PMID: 27434984] +75 102 van den Berghe G, Wouters P, Weekers F, Verwaest C, Bruyninckx F, Schetz M, Vlasselaers D, Ferdinande P, Lauwers P, Bouillon R. Intensive insulin therapy in critically ill patients. *N Engl J Med* 2001; 345: 1359-1367 [PMID: 11794168 DOI: 10.1056/NEJMoa011300] +76 Griesdale DE, de Souza RJ, van Dam RM, Heyland DK, Cook DJ, Malhotra A, Dhaliwal R, Henderson WR, Chittock DR, Finfer S, Talmor D. Intensive insulin therapy and mortality among critically ill patients: a meta-analysis including NICE-SUGAR study data. *CMAJ* 2009; 180: 821-827 [PMID: 19318387 DOI: 10.1503/cmaj.090206] +77 Partridge H, Perkins B, Mathieu S, Nicholls A, Adeniji K. Clinical recommendations in the management of the patient with type 1 diabetes on insulin pump therapy in the perioperative period: a primer for the anaesthetist. *Br J Anaesth* 2016; 116: 18-26 [PMID: 26675948 DOI: 10.1093/bja/aev347] +78 79 **Chase HP**. Don't take away my pump! *Diabetes Forecast* 2005; 58: 55-56 [PMID: 16187438] +Pasquel FJ, Lansang MC, Dhatariya K, Umpierrez GE. Management of diabetes and hyperglycaemia in the hospital. Lancet Diabetes Endocrinol 2021; 9: 174-188 [PMID: 33515493 DOI: 10.1016/S2213-8587(20)30381-8] +80 Umpierrez GE, Klonoff DC. Diabetes Technology Update: Use of Insulin Pumps and Continuous Glucose Monitoring in the Hospital. *Diabetes Care* 2018; 41: 1579-1589 [PMID: 29936424 DOI: 10.2337/dci18-0002] +81 Kawasaki E. Type 1 diabetes and autoimmunity. *Clin Pediatr Endocrinol* 2014; 23: 99-105 [PMID: 25374439 DOI: 10.1297/cpe.23.99] +82 Amusan O, Armeni E, Raichura H. Audit of the identification and management of steroid-induced hyperglycaemia and steroidinduced diabetes. *Journal of Diabetes Nursing* 2019; 23: JDN093, 1-7 83 Beaupere C, Liboz A, Fève B, Blondeau B, Guillemain G. Molecular Mechanisms of Glucocorticoid-Induced Insulin Resistance. *Int J Mol Sci* 2021; 22 [PMID: 33435513 DOI: 10.3390/ijms22020623] +84 Tamez-Pérez HE, Quintanilla-Flores DL, Rodríguez-Gutiérrez R, González-González JG, Tamez-Peña AL. Steroid hyperglycemia: Prevalence, early detection and therapeutic recommendations: A narrative review. *World J Diabetes* 2015; 6: 1073-1081 [PMID: 26240704 DOI: 10.4239/wjd.v6.i8.1073] +85 86 Shah P, Kalra S, Yadav Y, Deka N, Lathia T, Jacob JJ, Kota SK, Bhattacharya S, Gadve SS, Subramanium KAV, George J, Iyer V, Chandratreya S, Aggrawal PK, Singh SK, Joshi A, Selvan C, Priya G, Dhingra A, Das S. Management of Glucocorticoid-Induced Hyperglycemia. *Diabetes Metab Syndr Obes* 2022; 15: 1577-1588 [PMID: 35637859 DOI: 10.2147/DMSO.S330253] Maahs DM, Horton LA, Chase HP. The use of insulin pumps in youth with type 1 diabetes. *Diabetes Technol Ther* 2010; 12 Suppl 1: S59-S65 [PMID: 20515309 DOI: 10.1089/dia.2009.0161] +87 Freckmann G, Pleus S, Haug C, Bitton G, Nagar R. Increasing local blood flow by warming the application site: +beneficial effects on postprandial glycemic excursions. *J Diabetes Sci Technol* 2012; 6: 780-785 [PMID: 22920802 DOI: 10.1177/193229681200600407] +88 Cengiz E, Weinzimer SA, Sherr JL, Tichy EM, Carria L, Cappiello D, Steffen A, Tamborlane WV. Faster in and faster out: accelerating insulin absorption and action by insulin infusion site warming. *Diabetes Technol Ther* 2014; 16: 20-25 +[PMID: 24367934 DOI: 10.1089/dia.2013.0187] +89 Muchmore DB, Vaughn DE. Accelerating and improving the consistency of rapid-acting analog insulin absorption and action for both subcutaneous injection and continuous subcutaneous infusion using recombinant human hyaluronidase. J +Diabetes Sci Technol 2012; 6: 764-772 [PMID: 22920800 DOI: 10.1177/193229681200600405] +90 Hultström M, Roxhed N, Nordquist L. Intradermal insulin delivery: a promising future for diabetes management. J Diabetes Sci Technol 2014; 8: 453-457 [PMID: 24876605 DOI: 10.1177/1932296814530060] +91 Cho YH, Craig ME, Donaghue KC. Puberty as an accelerator for diabetes complications. *Pediatr Diabetes* 2014; 15: 1826 [PMID: 24443957 DOI: 10.1111/pedi.12112] +92 Liang W, Chikritzhs T. Alcohol consumption during adolescence and risk of diabetes in young adulthood. *Biomed Res Int* 2014; **2014**: 795741 [PMID: 24757678 DOI: 10.1155/2014/795741] +93 Turner BC, Jenkins E, Kerr D, Sherwin RS, Cavan DA. The effect of evening alcohol consumption on next-morning glucose control in type 1 diabetes. *Diabetes Care* 2001; 24: 1888-1893 [PMID: 11679452 DOI: 10.2337/diacare.24.11.1888] +94 Brown SA, Jiang B, McElwee-Malloy M, Wakeman C, Breton MD. Fluctuations of Hyperglycemia and Insulin Sensitivity Are Linked to Menstrual Cycle Phases in Women With T1D. *J Diabetes Sci Technol* 2015; 9: 1192-1199 [PMID: 26468135 DOI: 10.1177/1932296815608400] +95 Trout KK, Rickels MR, Schutta MH, Petrova M, Freeman EW, Tkacs NC, Teff KL. Menstrual cycle effects on insulin sensitivity in women with type 1 diabetes: a pilot study. *Diabetes Technol Ther* 2007; 9: 176-182 [PMID: 17425444 DOI: 10.1089/dia.2006.0004] +96 Cursino K, Sider M, Pavin EJ, dos Santos Pde N, Bahamondes L, Zantut-Wittmann DE, Fernandes A. Insulin resistance parameters in users of the injectable contraceptive depot medroxyprogesterone acetate during one year of use. *Eur J* Contracept Reprod Health Care 2016; 21: 22-29 [PMID: 26140543 DOI: 10.3109/13625187.2015.1059415] +97 MacNeill G, Fredericks C. Vacation ease: travelling with an insulin pump. *Can J Diabetes* 2015; 39: 178-182 [PMID: 26004904 DOI: 10.1016/j.jcjd.2015.02.004] +98 Pathania YS, Budania A. Diabetic Stiff Hand Syndrome in a Child. *J Paediatr Child Health* 2021; 57: 1347 [PMID: 34169601 DOI: 10.1111/jpc.15629] +99 Heinemann L, Kamann S. Adhesives Used for Diabetes Medical Devices: A Neglected Risk With Serious Consequences? *J Diabetes Sci Technol* 2016; 10: 1211-1215 [PMID: 27566734 DOI: 10.1177/1932296816662949] +100 Messer LH, Berget C, Beatson C, Polsky S, Forlenza GP. Preserving Skin Integrity with Chronic Device Use in Diabetes. Diabetes Technol Ther 2018; 20: S254-S264 [PMID: 29916740 DOI: 10.1089/dia.2018.0080] +101 Hingorani A, LaMuraglia GM, Henke P, Meissner MH, Loretz L, Zinszer KM, Driver VR, Frykberg R, Carman TL, Marston W, Mills JL Sr, Murad MH. The management of diabetic foot: A clinical practice guideline by the Society for Vascular Surgery in collaboration with the American Podiatric Medical Association and the Society for Vascular Medicine. *J Vasc Surg* 2016; 63: 3S-21S [PMID: 26804367 DOI: 10.1016/j.jvs.2015.10.003] +103 **Pisano M**. Overview of insulin and non-insulin delivery devices in the treatment of diabetes. P T 2014; 39: 866-876 104 107 110 116 +[PMID: 25516696] +Bruttomesso D, Crazzolara D, Maran A, Costa S, Dal Pos M, Girelli A, Lepore G, Aragona M, Iori E, Valentini U, Del Prato S, Tiengo A, Buhr A, Trevisan R, Baritussio A. In Type 1 diabetic patients with good glycaemic control, blood glucose variability is lower during continuous subcutaneous insulin infusion than during multiple daily injections with insulin glargine. *Diabet Med* 2008; 25: 326-332 [PMID: 18307459 DOI: 10.1111/j.1464-5491.2007.02365.x] +Andersen HU, Hangaard S, Hommel E, Ridderstråle M. Six-Year Follow-Up After Insulin Pump Initiation: HbA1c Is Significantly Reduced Without Weight Gain. *J Diabetes Sci Technol* 2018; 12: 535-536 [PMID: 28927286 DOI: +10.1177/1932296817731424] +105 Boucher-Berry C, Parton EA, Alemzadeh R. Excess weight gain during insulin pump therapy is associated with higher basal insulin doses. *J Diabetes Metab Disord* 2016; 15: 47 [PMID: 27777901 DOI: 10.1186/s40200-016-0271-5] +106 Breton MD, Kanapka LG, Beck RW, Ekhlaspour L, Forlenza GP, Cengiz E, Schoelwer M, Ruedy KJ, Jost E, Carria L, Emory E, Hsu LJ, Oliveri M, Kollman CC, Dokken BB, Weinzimer SA, DeBoer MD, Buckingham BA, Cherñavvsky D, Wadwa RP; iDCL Trial Research Group. A Randomized Trial of Closed-Loop Control in Children with Type 1 Diabetes. + +N Engl J Med 2020; 383: 836-845 [PMID: 32846062 DOI: 10.1056/NEJMoa2004736] +Ghazanfar H, Rizvi SW, Khurram A, Orooj F, Qaiser I. Impact of insulin pump on quality of life of diabetic patients. Indian J Endocrinol Metab 2016; 20: 506-511 [PMID: 27366717 DOI: 10.4103/2230-8210.183472] +108 Alshami A, Purewal T, Douedi S, Alazzawi M, Hossain MA, Ong R, Sen S, Cheng J, Patel S. Effect of Insulin Pump Use on Diabetic Ketoacidosis in Type 1 Diabetes Mellitus: A Matched Cohort Study. *J Clin Med* 2021; 10 [PMID: 33668749 DOI: 10.3390/jcm10050898] +109 AbdulAziz YH, Al-Sallami HS, Wiltshire E, Rayns J, Willis J, McClintock J, Medlicott N, Wheeler BJ; Paediatric Society of New Zealand Diabetes Clinical Network. Insulin pump initiation and education for children and adolescents - a qualitative study of current practice in New Zealand. *J Diabetes Metab Disord* 2019; 18: 59-64 [PMID: 31275875 DOI: 10.1007/s40200-019-00390-6] Pickup JC, Yemane N, Brackenridge A, Pender S. Nonmetabolic complications of continuous subcutaneous insulin infusion: a patient survey. *Diabetes Technol Ther* 2014; 16: 145-149 [PMID: 24180294 DOI: 10.1089/dia.2013.0192] +111 Patel B, Priefer R. Infections associated with diabetic-care devices. *Diabetes Metab Syndr* 2021; 15: 519-524 [PMID: +33668001 DOI: 10.1016/j.dsx.2021.02.023] +112 Wickline CL, Cornitius TG, Butler T. Cellulitis caused by Rhizomucor pusillus in a diabetic patient receiving continuous insulin infusion pump therapy. *South Med J* 1989; 82: 1432-1434 [PMID: 2814631 DOI: 10.1097/00007611-198911000-00024] +113 Chakraborty PP, Chakraborty M, Dasgupta S. Primary Mycobacterium tuberculosis infection over insulin injection site. + +BMJ Case Rep 2016; **2016** [PMID: 27873753 DOI: 10.1136/bcr-2016-218054] +114 Brown-Georgi J, Chhabra H, Vigersky RA. The Rising Cost of Insulin for Pump Users: How Policy Drives Prices. J +Diabetes Sci Technol 2021; 15: 1177-1180 [PMID: 32757774 DOI: 10.1177/1932296820947100] +115 Wong JC, Boyle C, DiMeglio LA, Mastrandrea LD, Abel KL, Cengiz E, Cemeroglu PA, Aleppo G, Largay JF, Foster NC, Beck RW, Adi S; T1D Exchange Clinic Network. Evaluation of Pump Discontinuation and Associated Factors in the T1D Exchange Clinic Registry. *J Diabetes Sci Technol* 2017; 11: 224-232 [PMID: 27595711 DOI: 10.1177/1932296816663963] +Beato-Víbora P, Yeoh E, Rogers H, Hopkins D, Amiel SA, Choudhary P. Sustained benefit of continuous subcutaneous insulin infusion on glycaemic control and hypoglycaemia in adults with Type 1 diabetes. *Diabet Med* 2015; 32: 1453-1459 [PMID: 26213236 DOI: 10.1111/dme.12869] +117 Jarosz-Chobot P. [The use of continuous subcutaneous insulin infusion (CSII) with personal insulin pumps in the treatment of children and adolescents with diabetes type 1]. *Wiad Lek* 2004; 57: 263-266 [PMID: 15518073] +118 Published by **Baishideng Publishing Group Inc** + +![22_image_0.png](22_image_0.png) + +![22_image_1.png](22_image_1.png) + +7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA +Telephone: +1-925-3991568 E-mail: bpgoffice@wjgnet.com Help Desk: https://www.f6publishing.com/helpdesk https://www.wjgnet.com \ No newline at end of file diff --git a/medical/md_images/PMC9685680/PMC9685680_meta.json b/medical/md_images/PMC9685680/PMC9685680_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..6d506eaa30cbaf69eca7ef25f58892a977e64fdf --- /dev/null +++ b/medical/md_images/PMC9685680/PMC9685680_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 23, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 165, + "code": 0, + "table": 4, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC9713283/3_image_0.png b/medical/md_images/PMC9713283/3_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..f91771810298421f0a4ecd396c27a5f32f7be359 --- /dev/null +++ b/medical/md_images/PMC9713283/3_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:dd115b39aac002894c210934a2e4d06668f69f4b0c96aad092fb9b45a7546cbe +size 85628 diff --git a/medical/md_images/PMC9713283/4_image_0.png b/medical/md_images/PMC9713283/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..5ed97d31138d0e2e1859d704a8e1fb5575e11e7e --- /dev/null +++ b/medical/md_images/PMC9713283/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e4ded37056762a92661783a65ed89785b5d52228d6f912f981e760cb9f7db61a +size 22208 diff --git a/medical/md_images/PMC9713283/5_image_0.png b/medical/md_images/PMC9713283/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..cf250fec7197f8eb860c5764a4afd76c21128934 --- /dev/null +++ b/medical/md_images/PMC9713283/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:b2f3da3d42c761fb2f971dceeb10f8281e2730a94069e28d51def4577e474f76 +size 561126 diff --git a/medical/md_images/PMC9826941/8_image_0.png b/medical/md_images/PMC9826941/8_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..3fa3f832784d0737673823cc131218163c1e34f1 --- /dev/null +++ b/medical/md_images/PMC9826941/8_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:7757e45ddb27bc531e2f569ed1f29b68c4449adbcc2defcb295afbf0d92d90d4 +size 34601 diff --git a/medical/md_images/PMC9860146/11_image_0.png b/medical/md_images/PMC9860146/11_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..ac150fc6d38bd485365b6415a7d0c960a539e4e8 --- /dev/null +++ b/medical/md_images/PMC9860146/11_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:5cf293bc9b3278364eb07b79a5b5bf28be9e4faf51f5145d7ac2b82e29655d3c +size 4102 diff --git a/medical/md_images/PMC9860146/15_image_0.png b/medical/md_images/PMC9860146/15_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..80d56171ebf49fcf08322a16b8cc79d1acb5f430 --- /dev/null +++ b/medical/md_images/PMC9860146/15_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:4c9a1c5df36273db778fc81893433bbcb6179f0f4c7a852cd505a45c9da60d34 +size 4319 diff --git a/medical/md_images/PMC9860146/8_image_0.png b/medical/md_images/PMC9860146/8_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..01a0525ad8ae8dbe448468941e2cb88897d002de --- /dev/null +++ b/medical/md_images/PMC9860146/8_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:174bfc099c62e87b1fcc6b8aaa46feb39b92f558c93a8eae9412221f21a983d7 +size 1538 diff --git a/medical/md_images/PMC9860146/9_image_0.png b/medical/md_images/PMC9860146/9_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..1987685b2c3ee13e680c3303a839c4f48eb0dc10 --- /dev/null +++ b/medical/md_images/PMC9860146/9_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:3e351bb34ff7302121a44babe9c07dc50ce0be1bf2dda9bd58f21eddca5397fa +size 4088 diff --git a/medical/md_images/PMC9980335/PMC9980335.md b/medical/md_images/PMC9980335/PMC9980335.md new file mode 100644 index 0000000000000000000000000000000000000000..ba60fd9607e1f19547f3fbf9959df217daa3085a --- /dev/null +++ b/medical/md_images/PMC9980335/PMC9980335.md @@ -0,0 +1,336 @@ +Prevalence of self-reported chronic conditions and poor health among older adults with and without vision impairment in China: a nationally representative cross-sectional **survey** +Yiran Gu,1 Haozhe Cheng,2,3 Xiaoyun Liu,3 Xiaodong Dong,2,3 Nathan Congdon,4,5 Xiaochen Ma 3 To cite: Gu Y, Cheng H, Liu X, +et al. Prevalence of selfreported chronic conditions and poor health among older adults with and without vision impairment in China: a nationally representative cross-sectional survey. BMJ Open Ophthalmology 2023;8:e001211. doi:10.1136/ bmjophth-2022-001211 +► Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi. org/10.1136/bmjophth-2022001211). Received 24 November 2022 Accepted 7 February 2023 © Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. For numbered affiliations see end of article. Correspondence to Dr Xiaochen Ma; xma@hsc.pku. edu.cn + +## Abstract + +Objective To examine the self-reported prevalence of 13 chronic conditions and poor health among Chinese adults aged 45 years and older with and without self-reported vision impairment. + +Design Cross-sectional study from the China Health and Retirement Longitudinal Study 2018, a nationally representative survey of Chinese adults aged 45 years and older involving 19 374 participants. + +Methods We used logistic regression to assess the association between vision impairment and 13 common chronic conditions and between vision impairment and poor health for those with any of these chronic conditions. + +Results Older people with self-reported vision impairment were significantly more likely to report all 13 chronic conditions (all p<0·05). After controlling for age, gender, education, residential status (rural vs urban), smoking and BMI, the highest adjusted odds were for hearing impairment (OR=4.00 (95% CI 3·60 to 4·44]) and depression (OR=2.28 (95% CI 2.06 to 2.51)). The lowest risk, though still significant, was for diabetes (OR=1·33 (95% CI 1.11 to 2.05)) and hypertension (OR=1.20 (95% CI 1.04 to 1.38)). After controlling for these potential confounding factors, among older people with chronic conditions, those with vision impairment were 2.20 to 4.04 times more likely to have poor health, compared with those without vision impairment (all p<0.001), with the exception of cancer (p=0.595). + +Conclusions Higher prevalence of chronic conditions is strongly associated with vision impairment among older Chinese adults and poor health is strongly associated with vision impairment among people with chronic conditions. + +## Introduction + +Management of the rising prevalence of chronic disease, especially in low-resource settings, is the main challenge facing healthcare systems worldwide and is, thus, a global development imperative.1 As global life expectancies rise, the proportion of the population affected by chronic conditions climbs as well, as does the prevalence of + +## What Is Already Known On This Topic + +⇒ Vision impairment (VI) is associated with higher prevalence of chronic conditions. However, little is known about the impact of VI on specific chronic conditions among older adults in lower and middleincome countries such as China. + +## What This Study Adds + +⇒ Self-reported prevalence of all 13 chronic conditions assessed in this nationally representative sample of 19 374 Chinese adults aged 45 years or older was higher among persons with VI (all p<0·05). Among people with chronic conditions, those with VI were 2·20 to 4·04 times more likely to have poor health, compared with those without VI (all p<0·001), with the exception of cancer (p=0·595). + +## How This Research Might Affect Research, Practices And Policy + +⇒ These nationally representative data provide strong evidence that VI is associated with increased risk of a range of common chronic diseases in low or middle-income country such as China. This provides further impetus for aggressive policies to address prevalent causes of VI, many of which are treatable at low cost, and also suggests that investment in such policies might be a way to reduce the burden of chronic disease in this setting. + +multimorbidity, the cooccurrence of two or more chronic conditions in a single person.2 Chronic conditions, and, in particular, multimorbidity, are associated with a broad range of functional consequences, poor quality of life and well-being and are also a major contributor to health inequalities and constitute an important economic burden.2–5 Vision impairment (VI) is among the most disabling of chronic conditions and increasingly research attention has focused on the coexistence of VI and other chronic conditions.6 7 However, the majority of existing studies have focused on the association between VI and single chronic disease. A number of studies have reported that persons with VI experience a variety of chronic conditions at higher rates than those with normal vision, including diabetes,8 stroke,9 10 depression11–13 and cognitive decline.14 15 Studies describing these associations in low or middle-income countries (LMICs) are relatively rare, highlighting the need for more such data.6 As the most populous and rapidly ageing LMIC,16 China had 59.3million people with moderate to severe VI or blindness in 2019, nearly equivalent to the entire population of the United Kingdom.17 Like many other countries, China has witness a considerable disease burden shifting from communicable, maternal, neonatal and nutritional conditions to non-communicable diseases (NCDs).18 19 The national data show that among population aged 15 years or older, the prevalence of NCDs has risen from 15.7% in 2008 to 34.3% in 2018.20 The economic losses associated with these diseases over the period 2010–2030 are (measured in real US dollar with the base year 2010) +estimated to be US$7.7trillion for China.21 In addition, 70% of middle-aged or elderly Chinese with chronic conditions have multimorbidity, resulting in an estimated number of 240million population.4 The current study aims to address the relative lack of data on VI and chronic disease risk in LMICs by analysing a nationally representative cross-sectional sample of older Chinese adults, with data on 13 common chronic conditions. We aim to provide updated and robust national prevalence estimates for these 13 chronic conditions among older adults with and without VI. Furthermore, we will examine how health status differs between people with and without VI among those with chronic conditions. We hypothesise that VI is associated with higher prevalence of chronic conditions, and also with higher prevalence of poor health among those with such conditions. + +## Methods + +This study explores the cross-sectional association between VI and other chronic conditions and health status. We use data from the nationally representative China Health and Retirement Longitudinal Study (CHARLS). The detailed description of sampling strategy and cohort profile has been reported elsewhere.22 Briefly, CHARLS collects a nationally representative sample of Chinese residents aged 45 years and older, selected from 150 county-level administrative units in 28 provinces of China, using multistage stratified probability-proportionate-to-size sampling. CHARLS examines social, economic and health information among community-dwelling persons aged 45 years or older in China. Trained at Peking University, CHARLS staff members conducted one-to-one interviews in respondents' homes with the use of face-toface computer-assisted personal interview technology.22 23 Our study reports data from the latest wave of CHARLS, +conducted in 2018. Among 19816 participants, 19752 (99.7%) self-reported their vision function and 19717 +(99.5%) reported on chronic diseases/conditions and health status. Our final analytic database includes 19374 participants (97.8%) with complete vision and health data. Even though this non-response rate (2.20%) is low, we used survey weights from CHARLS to address potential non-response bias.23 Vision impairment (self-reported distance vision) Self-reported poor distance vision was used to measure VI. It was ascertained using the question: 'How good is your eyesight for seeing things at a distance, like recognising a friend from across the street (with glasses or corrective lenses if you wear them)? Would you say your eyesight for seeing things at a distance is excellent, very good, good, fair, or poor?' Responses of 'don't know' were excluded, and respondents answering 'poor' were classified as having vision impairment, while those responding 'excellent' to 'fair' were considered to have no vision impairment. + +## Chronic Conditions + +Following previous studies,4 5 we sought to include chronic conditions that are prevalent among elderly Chinese, which resulted in a list of 13 common chronic conditions. Among these conditions, 11 (hypertension, heart disease, dyslipidaemia, stroke, arthritis, asthma, chronic lung disease, cancer, kidney disease, diabetes and liver disease) were assessed based on self-reported diagnosis by a doctor. Participants were asked 'have you been diagnosed by with (conditions listed below, read by interviewers on by one) by a doctor?'. Similar to the assessment of VI, hearing impairment was defined as having responded 'poor' to the question 'Is your hearing excellent, very good, good, fair or poor (with a hearing aid if you normally use it and without if you normally don't)?'. + +In addition, depression was assessed with the 10-item Center for Epidemiologic Studies Depression Scale (CES-D 10), which detects depression risk in general populations, including older adults.24 25 Participants were asked 'how often' 10 depressive symptoms they have experienced during the past week on a 4-point scale, ranging from 0 (less than 1day) to 3 (5–7days). The CES-D 10 scores were summed to derive scores ranging 0–30. Participants with CES-D 10 scores larger or equal to 10 were classified as having clinically significant depressive symptoms.26 The CESD-10 was highly validated for use in general populations in China and other settings.27 28 + +## Health Status + +Self-rated health status was evaluated by asking this question: 'Would you say your health is very good, good, fair, poor, or very poor?' and treated as a dichotomous variable.29 30 Poor health status was defined as having responded 'poor' or 'very poor,' while good health status was defined by any of the following answers: 'very good', 'good' or 'fair'. + +Other covariates Demographic, socioeconomic and health-related behavioural covariates included self-reported age group (45–54 years, 55–64 years, 65–74 years, 75 years and above), gender (male or female), education (illiterate, some primary school, completed primary school, completed junior high school and above) and ever smoking (yes or no). Body mass index (BMI) was categorised as normal (BMI <24kg/m2), overweight (BMI from 24 kg/m2 to 28kg/m2), and obese (BMI> 28kg/m2). + +A participant's urban versus rural residence was determined based on the administrative classification of the participant's basic residing community (*hukou*, the Chinese household registration system).31 Every Chinese citizen is required to register in one and only one place of residence, which determines access to numerous social resources, such as education, healthcare and work.32 Statistical analysis Frequencies (percentages) are reported to summarise the distributions of characteristics for participants with and without VI. Weighted prevalence of various chronic diseases was calculated separately for those with and without VI using the survey weight. Then, the weighted prevalence of self-rated poor health among people reporting any of each chronic disease was calculated for persons with and without VI. Multivaraite logistic regressions were applied to analyse the association between VI and chronic conditions and the association between VI and poor health among participants with chronic diseases. All models are based on the 19374 participants with complete data for both VI and chronic disease status. All analyses were conducted using Stata V.16.0 (StataCorp LP, College Station, Texas). + +## Results + +The mean age of 19374 respondents was 62.0 years, 52.4% were women, 34.4% and had junior school or higher education level (table 1). Among these, 4006 (20.6%) reported VI. Compared with those without VI (p<0.001 for all), these respondents had a higher mean age (65.2 vs 61.0), were more likely to be women (61.3% vs 50.1%), illiterate (35.4% vs 19.3%), rural-dwelling + +| Table 1 | Characteristics of study participants drawn from the China Health and Retirement Longitudinal Study (CHARLS) | | | | | | +|------------------------|----------------------------------------------------------------------------------------------------------------|-------------------|--------------|--------------|---------------|--------------| +| 2018 survey | Total | Vision impairment | | | | | +| Total (N=19374) | Yes (N=4006) | No (N=15368) | | | | | +| n (%) | 95%CI | n (%) | 95%CI | n (%) | 95%CI | P value | +| Age, years | < 0.001 | | | | | | +| 45–54 | 5409 (28.2%) | 27.6 to 28.9 | 694 (17.7%) | 16.6 to 19.0 | 4698 (31.0%) | 30.3 to 31.8 | +| 55–64 | 6327 (33.0%) | 32.3 to 33.7 | 1107 (28.3%) | 26.9 to 29.7 | 5194 (34.3%) | 33.5 to 35.0 | +| 65–74 | 5040 (26.3%) | 25.7 to 26.9 | 1309 (33.4%) | 32.0 to 34.9 | 3713 (24.5%) | 23.8 to 25.2 | +| ≥75 | 2395 (12.5%) | 12.0 to 13.0 | 806 (20.6%) | 19.3 to 21.9 | 1544 (10.2%) | 9.7 to 10.7 | +| Gender | < 0.001 | | | | | | +| Male | 9267 (47.6%) | 46.9 to 48.3 | 1552 (38.7%) | 37.2 to 40.3 | 7664 (49.9%) | 49.1 to 50.7 | +| Female | 10221 (52.4%) | 51.7 to 53.1 | 2454 (61.3%) | 59.7 to 62.8 | 7704 (50.1%) | 49.3 to 50.9 | +| Education | < 0.001 | | | | | | +| Illiterate | 4439 (22.8%) | 22.2 to 23.4 | 1417 (35.4%) | 33.9 to 36.9 | 2968 (19.3%) | 18.7 to 19.9 | +| Some primary school | 4011 (20.6%) | 20.0 to 21.2 | 891 (22.2%) | 21.0 to 23.6 | 3094 (20.1%) | 19.5 to 20.8 | +| Primary school | 4326 (22.2%) | 21.6 to 22.8 | 834 (20.8%) | 19.6 to 22.1 | 3481 (22.7%) | 22.0 to 23.3 | +| Junior high school and | 6712 (34.4%) | 33.8 to 35.1 | 864 (21.6%) | 20.3 to 22.9 | 5825 (37.9%) | 37.1 to 38.7 | +| above | | | | | | | +| Residence | < 0.001 | | | | | | +| Urban | 3693 (21.4%) | 20.8 to 22.0 | 567 (15.8%) | 14.6 to 17.0 | 3110 (22.9%) | 22.2 to 23.7 | +| Rural | 13543 (78.6%) | 78.0 to 79.2 | 3022 (84.2%) | 83.0 to 85.4 | 10446 (77.1%) | 76.3 to 77.8 | +| Ever smoking | < 0.001 | | | | | | +| No | 10199 (57.2%) | 56.5 to 57.9 | 2336 (61.8%) | 60.3 to 63.4 | 7808 (56.0%) | 55.1 to 56.8 | +| Yes | 7629 (42.8%) | 42.1 to 43.5 | 1442 (38.2%) | 36.6 to 39.7 | 6146 (44.0%) | 43.2 to 44.9 | +| BMI | < 0.001 | | | | | | +| Normal | 7647 (53.4%) | 52.6 to 54.2 | 1775 (56.5%) | 54.8 to 58.2 | 5831 (52.5%) | 51.5 to 53.4 | +| Overweight | 4773 (33.3%) | 32.6 to 34.1 | 982 (31.3%) | 29.7 to 32.9 | 3774 (34.0%) | 33.1 to 34.8 | +| Obese | 1905 (13.3%) | 12.8 to 13.9 | 384 (12.2%) | 11.1 to 13.4 | 1511 (13.6%) | 13.0 to 14.2 | +| BMI, body mass index. | | | | | | | + +| Table 2 | Prevalence of different chronic conditions among participants with and without self-reported vision impairment Total Vision impairment (n=19374) Yes (n=4006) No (n=15368) Prevalence (%) 95%CI Prevalence (%) 95%CI Prevalence (%) 95%CI P value | | | | | | | +|----------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|------|--------------|------|--------------|--------| +| Hypertension | 13.9 | 12.9 to 14.9 | 18.3 | 16.5 to 20.2 | 12.9 | 11.8 to 14.1 | <0.001 | +| Heart attack | 7.3 | 6.7 to 8.0 | 11.4 | 9.7 to 13.0 | 6.4 | 5.7 to 7.0 | <0.001 | +| Dyslipidaemia | 11.1 | 10.2 to 12.1 | 13.4 | 11.7 to 15.1 | 10.6 | 9.6 to 11.6 | <0.001 | +| Stroke | 5.1 | 4.6 to 5.6 | 8.1 | 7.0 to 9.3 | 4.4 | 3.9 to 4.9 | <0.001 | +| Arthritis | 10.0 | 9.2 to 10.7 | 14.4 | 12.4 to 16.3 | 9.2 | 8.3 to 10.0 | <0.001 | +| Asthma | 2.1 | 1.8 to 2.4 | 4.0 | 3.1 to 4.8 | 1.6 | 1.4 to 1.9 | <0.001 | +| Chronic lung disease | 5.1 | 4.7 to 5.6 | 8.3 | 7.2 to 9.5 | 4.4 | 3.9 to 4.9 | <0.001 | +| Cancer | 1.3 | 1.1 to 1.5 | 1.9 | 1.3 to 2.5 | 1.1 | 0.9 to 1.4 | 0.0224 | +| Kidney disease | 4.2 | 3.5 to 4.9 | 5.9 | 4.7 to 7.0 | 3.8 | 3.0 to 4.5 | <0.001 | +| Diabetes | 5.4 | 4.9 to 5.9 | 6.3 | 5.3 to 7.2 | 5.2 | 4.6 to 5.8 | 0.0065 | +| Liver disease | 3.4 | 2.8 to 4.0 | 4.4 | 3.6 to 5.3 | 3.1 | 2.4 to 3.8 | <0.001 | +| Hearing impairment | 14.1 | 13.2 to 15.0 | 33.3 | 31.3 to 35.2 | 9.1 | 8.5 to 9.8 | <0.001 | +| Depression | 41.4 | 39.7 to 43.0 | 60.5 | 58.1 to 63.0 | 37.0 | 35.2 to 38.8 | <0.001 | +| Condition | | | | | | | | + +(84.2% vs 77.1%), have never smoked (61.8% vs 56.0%) and have a normal BMI (56.5% vs 52.5%). + +The weighted prevalence of all 13 chronic conditions was significantly higher among people with VI compared with those without (p<0.001 for all, table 2). Depression (60.5% (95% CI 58.1, 63.0)) was the most prevalent chronic condition among participants with VI, compared with 37.0% (95% CI 35.2 to 38.8) of people without VI. + +The prevalence of hearing impairment also differed strikingly between those with and without VI (33.3% (95% CI +31.3 to 35.2) vs 9.10% (95% CI 8.50 to 9.80)). + +Participants with VI reported a significantly higher weighted prevalence of poor health than did those without (p<0.001 for all except cancer, p=0.542); table 3). + +For participants with VI, the prevalence of self-reported poor health status exceeded 50% among those selfreporting all of the 13 chronic diseases. People with VI +and diagnosed asthma reported the highest prevalence of poor health of 72.7% (95% CI 63.6 to 81.9). + +After controlling for potential confounding factors, VI was significantly associated with greater odds of selfreporting all 13 chronic diseases (all p<0.05) among participants in the current study (table 4). The highest adjusted ORs were for hearing impairment (OR=4.00 (95% CI 3.60, 4.44)) and depression (OR=2.28 (95% CI 2.06 to 2.51)), while diabetes (OR=1.33 (95% CI 1.11 to 2.05)) and hypertension (OR=1.20 (95% CI 1.04 to 1.38)) had the lowest. + +Table 3 Prevalence of self-rated poor health among participants with and without self-reported vision impairment reporting + +different chronic diseases + +| different chronic diseases Total | Vision impairment | No vision impairment | | | | | | +|------------------------------------|------------------------------------------------------------------------------|------------------------|-------|--------------|-------|--------------|--------| +| Prevalence of selfreported poor health | Prevalence of selfreported poor health | Prevalence of selfreported poor health | | | | | | +| (%) | 95%CI | (%) | 95%CI | (%) | 95%CI | P value | | +| Hypertension | 34.8 | 30.3 to 39.3 | 58.2 | 53.1 to 63.3 | 27.6 | 21.8 to 33.5 | <0.001 | +| Heart attack | 43.0 | 39.4 to 46.6 | 63.6 | 56.8 to 70.5 | 34.6 | 30.5 to 38.8 | <0.001 | +| Dyslipidaemia | 37.9 | 33.0 to 42.7 | 63.6 | 58.0 to 69.3 | 29.9 | 23.8 to 36.0 | <0.001 | +| Stroke | 51.8 | 47.5 to 56.2 | 68.5 | 62.1 to 74.9 | 44.6 | 39.5 to 49.6 | <0.001 | +| Arthritis | 32.6 | 29.0 to 36.2 | 54.9 | 48.3 to 61.5 | 26.1 | 22.6 to 29.6 | <0.001 | +| Asthma | 57.5 | 51.5 to 63.4 | 72.7 | 63.6 to 81.9 | 48.5 | 40.9 to 56.1 | <0.001 | +| Chronic lung disease | 44.7 | 40.5 to 48.8 | 64.0 | 58.0 to 70.0 | 36.2 | 31.4 to 40.9 | <0.001 | +| Cancer | 55.8 | 47.1 to 64.6 | 60.2 | 43.9 to 76.4 | 54.2 | 43.4 to 64.9 | 0.5422 | +| Kidney disease | 53.2 | 45.4 to 61.0 | 70.6 | 62.8 to 78.4 | 46.5 | 35.3 to 57.7 | <0.001 | +| Diabetes | 34.4 | 30.2 to 38.6 | 57.0 | 48.8 to 65.3 | 27.6 | 23.0 to 32.2 | <0.001 | +| Liver disease | 40.6 | 32.2 to 49.1 | 66.6 | 57.3 to 75.8 | 30.9 | 22.3 to 39.5 | <0.001 | +| Hearing impairment | 48.9 | 46.0 to 51.8 | 61.2 | 57.8 to 64.7 | 37.0 | 33.3 to 40.7 | <0.001 | +| Depression | 38.6 | 36.5 to 40.8 | 58.3 | 55.3 to 61.4 | 31.3 | 28.9 to 33.6 | <0.001 | +| Condition 4 | Gu Y, et al. BMJ Open Ophth 2023;8:e001211. doi:10.1136/bmjophth-2022-001211 | | | | | | | + +| Table 4 | Odds of different chronic diseases among | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------| +| participants with versus without self-reported vision impairment Condition OR 95%CI P value Hypertension 1.197 1.037 to 1.381 0.014 Heart attack 1.659 1.417 to 1.941 <0.001 Dyslipidaemia 1.415 1.228 to 1.630 <0.001 Stroke 1.544 1.294 to 1.844 <0.001 Arthritis 1.635 1.385 to 1.930 <0.001 Asthma 1.716 1.318 to 2.234 <0.001 Chronic lung disease 1.610 1.343 to 1.929 <0.001 Cancer 1.440 1.014 to 2.044 0.041 Kidney disease 1.552 1.266 to 1.903 0.002 Diabetes 1.333 1.111 to 1.599 <0.001 Liver disease 1.641 1.314 to 2.049 <0.001 Hearing impairment 3.998 3.600 to 4.439 <0.001 Depression 2.277 2.064 to 2.512 <0.001 | | + +After controlling for potential confounding factors, VI was associated with greater odds of self-reported poor health among persons self-reporting 12 of the 13 chronic conditions (p<0.001 for all, table 5), the exception being cancer (p=0.595). The highest adjusted odds were for hypertension (OR=4.04 (95% CI 3.04 to 5.36)) and dyslipidaemia (OR=4.02 (95% CI 3.03 to 5.33)). + +## Discussion + +VI is an important public health problem in China.33–38 Due to population growth and ageing, the prevalence of eye disease has steadily increased over the past decades.39 Data from the Global Burden of Diseases, Injuries and Risk Factors Study (GBD) 2019 show that the age-standardised prevalence of moderate and severe VI in China increased more rapidly than in any other + +Table 5 Odds of self-rated poor health among participants + +reporting different chronic diseases with versus without selfreported vision impairment + +Condition OR 95%CI **P value** + +Hypertension 4.037 3.040 to 5.360 <0.001 Heart attack 2.366 1.714 to 3.264 <0.001 Dyslipidaemia 4.022 3.033 to 5.332 <0.001 Stroke 2.200 1.509 to 3.208 <0.001 Arthritis 2.849 2.037 to 3.986 <0.001 Asthma 3.334 1.835 to 6.057 <0.001 Chronic lung disease 3.364 2.292 to 4.937 <0.001 Cancer 1.241 0.560 to 2.749 0.595 Kidney disease 3.392 2.170 to 5.301 <0.001 Diabetes 3.796 2.602 to 5.538 <0.001 Liver disease 2.320 1.460 to 3.688 <0.001 Hearing impairment 2.621 2.148 to 3.198 <0.001 Depression 2.900 2.546 to 3.303 <0.001 + +et al. *BMJ Open Ophth*8 5 G20 countries from 1990 to 2019.17 Our analysis using nationally-representative data revealed that people with VI had significantly elevated prevalence of self-reported chronic conditions. We also found that VI was significantly associated with higher weighted prevalence of poor health among those reporting chronic conditions. + +We conducted a systematic literature search for published papers addressing VI and chronic conditions among Chinese populations in PubMed and Web of Science for English publications and China National Knowledge Infrastructure for Chinese publications on 20 January 2023. We used the search terms ''vision disorders'', ''visual impairment'', ''vision impairment'', ''vision disorder'', ''vision loss'', ''loss of vision'', "eye disease" and ''eye disorder'' to identify vision impairment. To identify chronic conditions, we use the search terms "chronic conditions", "chronic diseases", "multimorbidity", +"comorbidity", "cardiovascular disease or CVD", "cardiovascular metabolic disease, CMD" as well as each of 13 common chronic conditions included in our analysis. We limited our search to studies published in English in 2010 or later, using data on adults collected in China. + +We found no studies examining the prevalence and effects of VI across a broad range of chronic conditions among older Chinese adults. We did encounter publications confirming the association between VI and a number of individual chronic condition in a dyadic analytic fashion. Such analyses have been limited to a few conditions, most notably cardiometabolic disease and its risk factors: diabetes,40 stroke,41 42 hypertension43 and dyslipidaemia.35 VI is also found to coexist frequently alongside hearing loss in elderly populations, and this dual sensory impairment is reported to be associated with negative impacts on function, cognitive abilities, mental health and well-being.12–14 Although rigorous casual evidence is limited, a number of potential pathways have been hypothesised to explain the observed association between VI and specific conditions. For example, in the area of cognitive decline and dementia, some common causes (eg, neurodegenerative or microvascular disease) of both VI and poor cognition might explain the correlation.44 45 Several indirect pathways have also been proposed. Poor vision is known to increase cognitive load,46 which causes sensory stress and takes a negative impact on cognitive function that increases dementia risk. VIs may also be associated with risk factors for cognitive impairment, such as depression, social isolation, and lack of physical activity.47 48 Sensory deprivation hypothesis suggests that a prolonged lack of adequate sensory input may directly result in cognitive deterioration and result in direct alteration of brain structure and function.49 In addition, there is another body of literature specifically studying multimorbidity, the coexistence of two or more chronic conditions; however, vision has rarely been included in such analyses. A few studies have examined visual impairment in conjunction with other chronic diseases,4 5 but these analyses have focused on the cumulative number of conditions, including VI, rather than assessing VI as a risk factor for other chronic conditions. + +Our analysis using CHARLS 2018 data is, according to the above review, the most up-to-date, nationally representative estimate of the prevalence and impact of VI +across a broad range of chronic conditions among older Chinese adults. Our analysis extended on previous work by including a broad inventory of 13 chronic conditions highly prevalent among older Chinese adults. Our analysis closely mirrors that of a cross-sectional US population study from the National Health Interview Survey +(2010–2014).50 Both studies used a similar self-reported definition of VI and included a similar list of 13 chronic conditions, controlling for similar confounders. In both studies, participants with VI were more likely to report chronic conditions than those without. Among those with chronic conditions, participants reporting VI were more likely also to report poor health status than were those without VI, although the magnitude of that associated was larger in the Chinses setting (adjusted ORs for the 13 conditions: 2.20 to 4.04 in China vs 1.66 to 2.70 in the USA) +The demographic characteristics, social-economic development, risk profiles of chronic diseases and the healthcare systems in the USA and China differ in many ways. Nevertheless, the patterns of the prevalence and impact of VI across a broad range of chronic conditions among older adults in China parallel those found in their counterparts in the USA. This reflects the global epidemiological transition in low and middle-income countries, as previously seen in high-income countries. China, like many other countries in the world, is undergoing rapid epidemiological transition and population ageing with a rising burden of NCDs. Consequently, the prevalence of multimorbidity is increasing.4 5 The coexisting of VI and other chronic conditions warrants further investigation and contextually relevant evidence to inform polices. + +In addition, the larger magnitude of the association between VI and poor health among people with chronic conditions in our study highlights the challenge China and many other LMICs are facing regarding management of NCDs. Hypertension is a case in point. Among the US adults with hypertension, 81.6% are aware, 73.1% are on treatment, and 48.6% have well-controlled blood pressure. + +51 By contrast, the corresponding rates are only 44.7%, 30.1%, and 7.2% in China.52 Many factors contribute to these differences, a crucial one being the suboptimal quality of primary healthcare (PHC) +in China.53 Shortfalls in financing, service integration, education and training still exist in China that undermine its PHC system's ability to deliver services for the prevention and management of chronic conditions in equitable fashion.53 Our results have profound public health implications for eye health services in China and other LMICs: the fact that VI is linked with the prevalence of a broad list of chronic conditions underscores the importance of prevention and treatment of VI as public health priorities. Due to traditional beliefs,14 older Chinese adults often view age-related eye disease as an inevitable part of ageing and may be reluctant to receive formal diagnosis or treatment. A recent cross-sectional study in rural areas across nine Chinese provinces shows that mild and moderate-to-severe visual impairment are mostly caused by uncorrected refractive error and cataract, both highly treatable.34 Our results suggest that public awareness should be raised and VI among the elderly treated more actively. The additional burden of poor health among people with chronic conditions suffering from VI highlights the need for more frequent vision assessment,54 particularly among those with hypertension and dyslipidaemia. As the global burden of NCDs increases, PHC is emerging as the focus of both prevention and life-long management of chronic disease.55 In the era of increasing disease and economic burden of NCDs and multimorbidity,4 18 19 56 health system must shift from a single disease focus to a person-centred approach.57 The GBD 2019 China Study17 shows that age-standardised prevalence of all common blinding eye diseases dropped over the past three decades in China, except for diabetic retinopathy. Their analysis found that the increase was attributed more to age-specific prevalence than to population ageing, largely due to the substantial changes in the lifestyle of Chinese people. Within the context of universal health coverage (UHC), it is of particular importance to integrate the prevention and control of chronic eye diseases into China's national management of chronic diseases. As China advances its UHC in the Healthy China 2030 policy,58 this also illustrates the need for an Integrated People-centred Eye Care strategy59 in China.60 The causal relationship between VI and general health is complex, recently published reviews have summarised a number of potential pathways (illustrated in online supplemental figure S1)6 61: (1) direct pathways through the effect of VI on systemic health: increased risk of chronic disease, decreased functional status, frailty; (2) indirect pathways through activity and participation: +reduced access to healthcare, increased injuries, limitations in physical activity, increased risk of social isolation, leading to depression and dementia; (3) shared common risk factors (individual-level traits, environmental and health system characteristics) including smoking, alcohol consumption, socioeconomic status and conditions with both ocular and systemic manifestations (eg, cardiovascular disease, diabetes, hypertension, stroke). However, most of the existing studies are conducted from, future studies based on representative data from LMICs are warranted to generate context-relevant evidence for countries in the developing world. + +Strengths of the present study included the large, nationally representative sample, and the detailed information regarding demographic, socioeconomic and health-related behavioural characteristics in addition to self-reported data on vision, chronic conditions and health status collected in CHARLS. Compared with previous studies,12 14 40–43 the wider array of 13 chronic conditions allows us to explore more fully the impact of VI on the health of older Chinese adults. + +Despite these strengths, our study has several limitations. First, the data we used were cross-sectional; we, therefore, cannot interpret the associations found as causal. Irrespective of their direction, these associations underscore the need for more attention to VI in the current health system. Second, our measurement of VI +was non-objective. As a large-scale, nationally representative survey, it was only practical for CHARLS to acquire self-reported vision measures. Similar definition of selfreported poor vision was also used based on the English Longitudinal Study of Ageing62 as well as the National Health Interview Survey50 conducted among older adults in England and the USA. Although previous studies have shown a strong correlation between self-reported vision and objective measurements,63 some discrepancies may exist.64 Last but not least, we used participant-reported diagnosis by a physician for 12 of the 13 chronic illnesses studied (with the exception of depression). Despite China's recent health system reform, which extended essential healthcare and insurance coverage to the entire population, high-quality care for prevention and management of chronic conditions is still less accessible in rural settings, particularly at the PHC level.53 As a result, the actual prevalence of the included chronic conditions might be higher than the current estimate, due to underdiagnosis. Hence, the actual association between VI and prevalence of chronic conditions might be even stronger than the current estimate in our analysis. + +A higher prevalence of a broad range of chronic conditions is significantly associated with VI among older Chinese adults. Among those with chronic conditions, the burden of poor health is significantly greater among persons with VI compared with those without. These findings from a nationally representative cohort suggest the need for further attention among both policymakers and researchers to the coexistence of VI and chronic conditions, both to better understand the problem and to inform contextually relevant strategies to promote healthy ageing among older persons in China and other LMICs. + +Author affiliations 1School of Public Health, University of California Berkeley, Berkeley, California, USA +2School of Public Health, Peking University, Beijing, China 3China Center for Health Development Studies, Peking University, Beijing, China 4Centre for Public Health, Queen's University Belfast, Belfast, UK +5Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, Guangdong, China Acknowledgements We thank Mr. LIU Zhifang (Health Bureau of Yongyang District, Handan) for providing insightful comments. + +Contributors XM has full access to all data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design: XM. Data analysis: YG, XM. Interpretation of data: XM, NC. Drafting of the manuscript: YG, HC, XD, XM. Critical revision of the manuscript for important intellectual content: XM, NC. Supervision: XM, XL. XM is the guarantor. + +Funding National Natural Science Foundation of China (Number 71703002; Number 72174009) and China Medical Board Grants (Number 17-267). + +Competing interests None declared. Patient and public involvement Patients and/or the public were involved in the design, or conduct, or reporting, or dissemination plans of this research. Refer to the Methods section for further details. Patient consent for publication Consent obtained directly from patient(s) Ethics approval This study involves human participants. All participants provided written informed consent, and survey protocols were approved by the Peking University Ethics Review Board (IRB approval number: IRB00001052-11015). Participants gave informed consent to participate in the study before taking part. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available in a public, open access repository. Data are available upon reasonable request. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iD Xiaochen Ma http://orcid.org/0000-0002-8251-3373 REFERENCES +1 Nugent R, Bertram MY, Jan S, *et al*. Investing in non-communicable disease prevention and management to advance the sustainable development goals. *Lancet* 2018;391:2029–35. + +2 Barnett K, Mercer SW, Norbury M, *et al*. Epidemiology of multimorbidity and implications for health care, research, and medical education: a cross-sectional study. The Lancet 2012;380:37–43. + +3 The Lancet. Making more of multimorbidity: an emerging priority. + +The Lancet 2018;391:1637. + +4 Ma X, He Y, Xu J. Urban–rural disparity in prevalence of multimorbidity in China: a cross-sectional nationally representative study. *BMJ Open* 2020;10:e038404. + +5 Zhao Y, Atun R, Oldenburg B, *et al*. Physical multimorbidity, health service use, and catastrophic health expenditure by socioeconomic groups in China: an analysis of population-based panel data. The Lancet Global Health 2020;8:e840–9. + +6 Burton MJ, Ramke J, Marques AP, *et al*. The lancet global health commission on global eye health: vision beyond 2020. *Lancet Glob* Health 2021;9:e489–551. + +7 Zheng DD, Christ SL, Lam BL, *et al*. Patterns of chronic conditions and their association with visual impairment and health care use. + +JAMA Ophthalmol 2020;138:387–94. + +8 Klein R, Klein BE, Moss SE, *et al*. The Beaver dam eye study. + +retinopathy in adults with newly discovered and previously diagnosed diabetes mellitus. *Ophthalmology* 1992;99:58–62. + +9 Sand KM, Midelfart A, Thomassen L, *et al*. Visual impairment in stroke patients--a review. *Acta Neurol Scand Suppl* 2013:52–6. + +10 Sand KM, Wilhelmsen G, Naess H, *et al*. Vision problems in ischaemic stroke patients: effects on life quality and disability. Eur J Neurol 2016;23 Suppl 1:1–7. + +11 Jones GC, Rovner BW, Crews JE, *et al*. Effects of depressive symptoms on health behavior practices among older adults with vision loss. *Rehabil Psychol* 2009;54:164–72. + +12 Xie T, Liu D, Guo J, *et al*. The longitudinal effect of sensory loss on depression among chinese older adults. *J Affect Disord* 2021;283:216–22. + +13 Fenwick EK, Ong PG, Man REK, *et al*. Vision impairment and major eye diseases reduce vision-specific emotional well-being in a Chinese population. *Br J Ophthalmol* 2017;101:686–90. + +14 Ma X, Wei J, Congdon N, *et al*. Longitudinal association between self-reported sensory impairments and episodic memory among older adults in China: a prospective cohort study. J Geriatr Psychiatry Neurol 2022;35:382–91. + +15 Uhlmann RF, Larson EB, Koepsell TD, *et al*. Visual impairment and cognitive dysfunction in Alzheimer's disease. *J Gen Intern Med* 1991;6:126–32. + +16 World Bank. Population ages 65 and above in china. 2022. Available: +https://data.worldbank.org/indicator/SP.POP.65UP.TO.ZS?view= chart&locations=CN [Accessed 14 Jul 2022]. + +17 Xu T, Wang B, Liu H, *et al*. Prevalence and causes of vision loss in china from 1990 to 2019: findings from the global burden of disease study 2019. *Lancet Public Health* 2020;5:e682–91. + +18 Yang G, Wang Y, Zeng Y, *et al*. Rapid health transition in china, 19902010: findings from the global burden of disease study 2010. *Lancet* 2013;381:1987–2015. + +19 Zhou M, Wang H, Zeng X, *et al*. Mortality, morbidity, and risk factors in china and its provinces, 1990-2017: a systematic analysis for the global burden of disease study 2017. *Lancet* 2019;394:1145–58. + +20 National Health Commission. China's health statistics yearbook 2021. Peking Union Medical College Press, 2020. + +21 Bloom DE, Chen S, Kuhn M, *et al*. The economic burden of chronic diseases: estimates and projections for China, Japan, and South Korea. *The Journal of the Economics of Ageing* 2020;17:100163. + +22 Zhao Y, Hu Y, Smith JP, *et al*. Cohort profile: the China health and retirement longitudinal study (CHARLS). *Int J Epidemiol* 2014;43:61–8. + +23 Zhao YS, Chen X, *et al. China health and retirement longitudinal* study wave 4 user's guide: national school of development. Peking University, 2020. + +24 Beekman AT, Deeg DJ, Van Limbeek J, *et al*. Criterion validity of the center for epidemiologic studies depression scale (CES-D): results from a community-based sample of older subjects in the Netherlands. *Psychol Med* 1997;27:231–5. + +25 Turvey CL, Wallace RB, Herzog R. A revised CES-D measure of depressive symptoms and a DSM-based measure of major depressive episodes in the elderly. *Int Psychogeriatr* 1999;11:139–48. + +26 Zhou L, Ma X, Wang W. Relationship between cognitive performance and depressive symptoms in chinese older adults: the china health and retirement longitudinal study (CHARLS). *J Affect Disord* 2021;281:454–8. + +27 Chen H, Mui AC. Factorial validity of the center for epidemiologic studies depression scale short form in older population in China. *Int* Psychogeriatr 2014;26:49–57. + +28 Ruiz M, Hu Y, Martikainen P, *et al*. Life course socioeconomic position and incidence of mid-late life depression in China and England: a comparative analysis of CHARLS and ELSA. J Epidemiol Community Health 2019;73:817–24. + +29 Yu T, Jiang Y, Gamber M, *et al*. Socioeconomic status and self-rated health in China: findings from a cross-sectional study. *Medicine* (Baltimore) 2019;98:e14904. + +30 Haseli-Mashhadi N, Pan A, Ye X, *et al*. Self-Rated health in middle-aged and elderly Chinese: distribution, determinants and associations with cardio-metabolic risk factors. *BMC Public Health* 2009;9:368. + +31 Yep R, Wang J, Johnson T. *Handbook on urban development in* china. Edward Elgar Publishing, 2019. + +32 Liu Z. Institution and inequality: the hukou system in China. Journal of Comparative Economics 2005;33:133–57. + +33 Zhao J, Ellwein LB, Cui H, *et al*. Prevalence of vision impairment in older adults in rural China: the China nine-province survey. Ophthalmology 2010;117:16.e1:409–16, . + +34 Zhao J, Xu X, Ellwein LB, *et al*. Causes of visual impairment and blindness in the 2006 and 2014 nine-province surveys in rural china. Am J Ophthalmol 2019;197:80–7. + +35 Fu Z, Chen CT, Cagnone G, *et al*. Dyslipidemia in retinal metabolic disorders. *EMBO Mol Med* 2019;11:e10473. + +36 Luo Y, He P, Guo C, *et al*. Association between sensory impairment and dementia in older adults: evidence from China. *J Am Geriatr Soc* 2018;66:480–6. + +37 Wang J, Xiao LD, Wang K, *et al*. Gender differences in cognitive impairment among rural elderly in China. *Int J Environ Res Public* Health 2020;17:3724. + +38 Wang L, Zhu Z, Scheetz J, *et al*. Visual impairment and ten-year mortality: the liwan eye study. *Eye (Lond*) 2021;35:2173–9. + +39 Wang B, Congdon N, Bourne R, *et al*. Burden of vision loss associated with eye disease in China 1990-2020: findings from the global burden of disease study 2015. *Br J Ophthalmol* 2018;102:220–4. + +40 Song P, Yu J, Chan KY, *et al*. Prevalence, risk factors and burden of diabetic retinopathy in china: a systematic review and meta-analysis. J Glob Health 2018;8:010803. + +41 Siong KH, Woo GC, Chan DY-L, *et al*. Prevalence of visual problems among stroke survivors in Hong Kong Chinese. *Clin Exp Optom* 2014;97:433–41. + +42 Gangwani RA, Lian JX, McGhee SM, *et al*. Diabetic retinopathy screening: global and local perspective. *Hong Kong Med J* +2016;22:486–95. + +43 Liu L, Quang ND, Banu R, *et al*. Hypertension, blood pressure control and diabetic retinopathy in a large population-based study. PLoS One 2020;15. + +44 Chan VTT, Sun Z, Tang S, *et al*. Spectral-domain OCT measurements in alzheimer's disease: a systematic review and meta-analysis. Ophthalmology 2019;126:497–510. + +45 Wayne RV, Johnsrude IS. A review of causal mechanisms underlying the link between age-related hearing loss and cognitive decline. Ageing Res Rev 2015;23(Pt B):154–66. + +46 Pigeon C, Li T, Moreau F, *et al*. Cognitive load of walking in people who are blind: subjective and objective measures for assessment. Gait Posture 2019;67:43–9. + +47 McDonnall MC. Risk factors for depression among older adults with dual sensory loss. *Aging Ment Health* 2009;13:569–76. + +48 Cosh S, Naël V, Carrière I, *et al*. Bidirectional associations of vision and hearing loss with anxiety: prospective findings from the ThreeCity study. *Age Ageing* 2018;47:582–9. + +49 Whitson HE, Cronin-Golomb A, Cruickshanks KJ, *et al*. American geriatrics Society and National Institute on aging bench-to-bedside conference: sensory impairment and cognitive decline in older adults. *J Am Geriatr Soc* 2018;66:2052–8. + +50 Crews JE, Chou C-F, Sekar S, *et al*. The prevalence of chronic conditions and poor health among people with and without vision impairment, aged ≥65 years, 2010-2014. *Am J Ophthalmol* 2017;182:18–30. + +51 Aggarwal R, Chiu N, Wadhera RK, *et al*. Racial/Ethnic disparities in hypertension prevalence, awareness, treatment, and control in the United States, 2013 to 2018. *Hypertension* 2021;78:1719–26. + +52 Lu J, Lu Y, Wang X, *et al*. Prevalence, awareness, treatment, and control of hypertension in china: data from 1·7 million adults in a population-based screening study (china peace million persons project). *Lancet* 2017;390:2549–58. + +53 Li X, Krumholz HM, Yip W, *et al*. Quality of primary health care in china: challenges and recommendations. *Lancet* 2020;395:1802–12. + +54 Park SJ, Ahn S, Woo SJ, *et al*. Extent of exacerbation of chronic health conditions by visual impairment in terms of health-related quality of life. *JAMA Ophthalmol* 2015;133:1267–75. + +55 Hanson K, Brikci N, Erlangga D, *et al*. The lancet global health commission on financing primary health care: putting people at the centre. *Lancet Glob Health* 2022;10:e715–72. + +56 LY YANGJ, XY CHEN, *et al*. Economic burden among middleaged and elderly patients with chronic diseases. Chinese Health Economics 2019;05:71–3. + +57 Pearson-Stuttard J, Ezzati M, Gregg EW. Multimorbidity-a defining challenge for health systems. *Lancet Public Health* 2019;4:e599–600. + +58 The State Council. Opinions of the state council on the implementation of the healthy china action. 2019. Available: http:// www.gov.cn/zhengce/content/2019-07/15/content_5409492.htm [Accessed 1 Feb 2023]. + +59 WHO. World report on vision. 2019. Available: https://www.who.int/ +publications/i/item/9789241516570 [Accessed 29 Jan 2023]. + +60 Wang NC. Without eye health , there is no great health—the contribution and prospect of cataract blindness prevention and treatment to national health. *China Medicine* 2020;15:1817–9. + +61 Ehrlich JR, Ramke J, Macleod D, *et al*. Association between vision impairment and mortality: a systematic review and meta-analysis. Lancet Glob Health 2021;9:e418–30. + +62 Liljas AEM, Carvalho LA, Papachristou E, *et al*. Self-Reported vision impairment and incident prefrailty and frailty in English communitydwelling older adults: findings from a 4-year follow-up study. J +Epidemiol Community Health 2017;71:1053–8. + +63 Kenny Gibson W, Cronin H, Kenny RA, *et al*. Validation of the selfreported hearing questions in the Irish longitudinal study on ageing against the whispered voice test. *BMC Res Notes* 2014;7:361. + +64 Kim SY, Kim H-J, Kim M-S, *et al*. Discrepancy between selfassessed hearing status and measured audiometric evaluation. + +PLoS One 2017;12:e0182718. \ No newline at end of file diff --git a/medical/md_images/PMC9980335/PMC9980335_meta.json b/medical/md_images/PMC9980335/PMC9980335_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..fbadb6f631f7dafdbe0333077c6f408c053215fb --- /dev/null +++ b/medical/md_images/PMC9980335/PMC9980335_meta.json @@ -0,0 +1,136 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Prevalence of self-\u00adreported chronic conditions and poor health among older adults with and without vision impairment in China: a nationally representative cross-\u00adsectional\u00a0survey", + "level": 0, + "is_closed": false, + "n_kids": 11, + "page_index": 0, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Abstract", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Introduction\ufeff\ufeff", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Methods", + "level": 1, + "is_closed": false, + "n_kids": 5, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Vision impairment (self-reported distance vision)", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Chronic conditions", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Health status", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Other covariates", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Statistical analysis", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Results", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "Discussion", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "Fit", + "view_pos": [] + }, + { + "title": "References", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "Fit", + "view_pos": [] + } + ], + "pages": 8, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 31, + "code": 0, + "table": 4, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file