diff --git a/medical/md/PMC10074947.md b/medical/md/PMC10074947.md new file mode 100644 index 0000000000000000000000000000000000000000..21461081fad507b68097acc900ea3b2789e24e0d --- /dev/null +++ b/medical/md/PMC10074947.md @@ -0,0 +1,243 @@ +World Journal of + +![0_image_0.png](0_image_0.png) + +Gastrointestinal W J G P *Pathophysiology* +Submit a Manuscript: https://www.f6publishing.com *World J Gastrointest Pathophysiol* 2023 March 22; 14(2): 34-45 DOI: 10.4291/wjgp.v14.i2.34 ISSN 2150-5330 (online) +ORIGINAL ARTICLE +Observational Study Novel CABIN score outperforms other prognostic models in predicting in-hospital mortality after salvage transjugular intrahepatic portosystemic shunting Jake Krige, Eduard Jonas, Chanel Robinson, Steve Beningfield, Urda Kotze, Marc Bernon, Sean Burmeister, Christo Kloppers + +| Jake Krige, Eduard Jonas, Chanel Robinson, Urda Kotze, Marc Bernon, Sean Burmeister, Department of Surgical Gastroenterology, University of Cape Town Health Sciences Faculty, Cape Town 7925, Western Cape, South Africa | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Specialty type: Gastroenterology and hepatology Provenance and peer review: Unsolicited article; Externally peer reviewed. | Steve Beningfield, Department of Radiology, University of Cape Town Health Sciences Faculty, Cape Town 7925, Western Cape, South Africa | +| Peer-review model: Single blind | Christo Kloppers, Department of Surgical Gastroenterology, University of Cape Town, Faculty of Health Sciences, Cape Town 7925, Western Cape, South Africa | +| Peer-review report's scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 | Corresponding author: Jake Krige, FACS, FRCS (Ed), MD, MSc, PhD, Full Professor, Department of Surgical Gastroenterology, University of Cape Town Health Sciences Faculty, Anzio Road, Observatory, Cape Town 7925, Western Cape, South Africa. jej.krige@uct.ac.za Abstract BACKGROUND Transjugular intrahepatic portosystemic shunt (TIPS) is now established as the salvage procedure of choice in patients who have uncontrolled or severe recurrent variceal bleeding despite optimal medical and endoscopic treatment. | +| P-Reviewer: Aseni P, Italy Received: November 29, 2022 Peer-review started: November 29, 2022 First decision: January 31, 2023 Revised: February 23, 2023 Accepted: March 10, 2023 Article in press: March 10, 2023 Published online: March 22, 2023 | AIM To analysis compared the performance of eight risk scores to predict in-hospital mortality after salvage TIPS (sTIPS) placement in patients with uncontrolled variceal bleeding after failed medical treatment and endoscopic intervention. METHODS Baseline risk scores for the Acute Physiology and Chronic Health Evaluation (APACHE) II, Bonn TIPS early mortality (BOTEM), Child-Pugh, Emory, FIPS, model for end-stage liver disease (MELD), MELD-Na, and a novel 5 category CABIN score incorporating Creatinine, Albumin, Bilirubin, INR and Na, were calculated before sTIPS. Concordance (C) statistics for predictive accuracy of inhospital mortality of the eight scores were compared using area under the receiver operating characteristic curve (AUROC) analysis. | + +RESULTS +Thirty-four patients (29 men, 5 women), median age 52 years (range 31-80) received sTIPS for uncontrolled (11) or refractory (23) bleeding between August 1991 and November 2020. Salvage TIPS controlled bleeding in 32 (94%) patients with recurrence in one. Ten (29%) patients died in hospital. All scoring systems had a significant association with in-hospital mortality (P < 0.05) on multivariate analysis. Based on in-hospital survival AUROC, the CABIN (0.967), APACHE II (0.948) and Emory (0.942) scores had the best capability predicting mortality compared to FIPS (0.892), BOTEM (0.877), MELD Na (0.865), Child-Pugh (0.802) and MELD (0.792). + +## Conclusion + +The novel CABIN score had the best prediction capability with statistical superiority over seven other risk scores. Despite sTIPS, hospital mortality remains high and can be predicted by CABIN category B or C or CABIN scores > 10. Survival was 100% in CABIN A patients while mortality was 75% for CABIN B, 87.5% for CABIN C, and 83% for CABIN scores > 10. + +Key **Words:** Transjugular intrahepatic portosystemic shunt; Risk score; Portal hypertension; Variceal bleeding; Mortality ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. + +Core **Tip:** This study compared the performance of a new CABIN score with seven existing risk scores to predict in-hospital mortality after salvage transjugular intrahepatic portosystemic shunt (TIPS) placement in 34 patients with uncontrolled variceal bleeding after failed medical treatment and endoscopic intervention. Using concordance statistics for predictive accuracy of in-hospital mortality the novel 5 category CABIN score incorporating Creatinine, Albumin, Bilirubin, INR and Na outperformed the APACHE II, BOTEM, Child-Pugh, Emory, FIPS, MELD and MELD-Na scores when compared by area under the receiver operating characteristic curve (AUROC) analysis. Survival was 100% in CABIN A patients while mortality was 75% for CABIN B, 87.5% for CABIN C, and 83% for CABIN scores > 10. + +Citation: Krige J, Jonas E, Robinson C, Beningfield S, Kotze U, Bernon M, Burmeister S, Kloppers C. Novel CABIN score outperforms other prognostic models in predicting in-hospital mortality after salvage transjugular intrahepatic portosystemic shunting. *World J Gastrointest Pathophysiol* 2023; 14(2): 34-45 URL: https://www.wjgnet.com/2150-5330/full/v14/i2/34.htm DOI: https://dx.doi.org/10.4291/wjgp.v14.i2.34 + +## Introduction + +Transjugular intrahepatic portosystemic shunt (TIPS) is now established as the salvage procedure of choice in patients who have uncontrolled or severe recurrent variceal bleeding despite optimal medical and endoscopic treatment[1]. Key clinical distinctions exist in the spectrum of patients undergoing TIPS, ranging from high-risk cirrhotic patients with liver decompensation and uncontrolled variceal bleeding necessitating an emergent salvage TIPS (sTIPS) to those with well-preserved liver function undergoing an elective TIPS for refractory bleeding. Current risk stratification of patients who have refractory variceal bleeding and require sTIPS is however imperfect. Although TIPS is a minimally invasive procedure, appropriate patient selection is crucial to identify patients who would benefit from the procedure, considering the substantial risks of hepatic encephalopathy, liver failure and increased overall morbidity and mortality in high-risk individuals[2,3]. + +Several prognostic and risk scores have been developed to identify patients at risk for a poor clinical outcome after sTIPS. These include the Acute Physiology and Chronic Health Evaluation (APACHE) II [4], Bonn TIPS early mortality (BOTEM)[5], Child-Pugh (C-P)[6], Emory[7], Freiburg index of post-TIPS survival (FIPS)[8], model for end-stage liver disease (MELD)[9], and Model for End-Stage Liver Disease sodium (MELD-Na)[10] scores. In this study the accuracy of a novel CABIN score, which was developed to overcome limitations of existing scoring systems, was compared to established risk scores for the prediction of in-hospital mortality following sTIPS. + +## Materials And Methods + +In this retrospective observational analysis, eight risk scores were evaluated in a cohort which included all adult patients who underwent sTIPS for uncontrollable or life-threatening refractory variceal bleeding in the Surgical Gastroenterology Unit at Groote Schuur Hospital and the University of Cape Town Private Academic Hospital between August 1991 and November 2020. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for reporting observational studies[11]. Baseline demographic, clinical and endoscopic data and biochemical variables were collected on admission. The anonymized and de-identified information were retrieved from a prospectively maintained ethics approved registry for patients treated for esophageal varices (Table 1). + +Details of the acute bleeding management protocol and the endoscopic interventional techniques used in our unit have been published previously[12-15]. In patients who had endoscopically uncontrolled bleeding a Minnesota balloon tube or a Danis esophageal stent (Ella-CS, Hradec Kralove, Czech Republic) was inserted to tamponade variceal bleeding and endotracheal intubation was used for airway protection when indicated[16]. In this high-risk group with uncontrolled variceal bleeding and those with refractory life-threatening bleeding despite endoscopic intervention and somatostatin infusion, sTIPS was performed as an emergency procedure under general anaesthesia with placement of an expandable uncovered 10 mm Wallstent (Boston Scientific, Marlborough, MA, United States)[15]. + +The study protocol followed the Baveno recommendations and defined uncontrolled or persistent variceal bleeding as the need for a transfusion of 4 units of blood or more within 6 h and the inability to achieve an increase in systolic blood pressure to 70 mmHg or more or a pulse reduction to less than 100/min. Contraindications to sTIPS in our unit were severe pulmonary hypertension, severe tricuspid regurgitation, congestive heart failure, fibropolycystic liver disease, uncontrolled systemic sepsis and unrelieved biliary obstruction. Relative contraindications were congenital hepatic fibrosis, portal vein thrombosis, obstruction of all hepatic veins and severe coagulopathy (INR > 5). + +Details of the newly developed five component CABIN score are given in Supplementary Table 1. + +Each CABIN variable is scored from one to five and the cumulative total is calculated by adding the individual values of the five biochemical components (Creatinine, Albumin, Bilirubin, INR (international normalized ratio) and Na (sodium). The best total CABIN score computes at 5 points and the worst at 25 points. Four CABIN categories (A-D) were established (A: 5-10 points, B: 11-15, C: 16-20, D: 21-25). + +The CABIN score and seven previously described scoring systems, APACHE II, BOTEM, Child-Pugh, Emory, FIPS, MELD, and MELD-Na scores were calculated based on clinical evaluation and laboratory values obtained before the sTIPS procedure. The primary study outcome measure was prediction of inhospital mortality after sTIPS and compared the relative performances of the seven established scoring models and the new CABIN score. + +## Statistical Analysis + +All clinical data and variables were collected and managed using the REDCap electronic data capturing software licensed to the University of Cape Town[17]. Statistical computations were made using IBM SPSS statistics (version 26.0, IBM, United States). Statistical significance was set at P < 0.05. Continuous data were reported as mean ± SD or medians and range and discrete data as percentages. To evaluate the performance of the various scoring systems to predict in-hospital mortality the concordance Cstatistic [area under the curve (AUC) of the receiver operating characteristic (ROC) curves] was used. + +## Ethical Considerations + +The study protocol was approved by the Human Research Ethics Committee (HREC Ref No. 120/2019) of the University of Cape Town and the research was conducted in accordance with the Declaration of Helsinki. + +## Results + +A total of 564 patients with variceal bleeding were treated during the study period. In 530 patients (94%), bleeding was controlled by endoscopic intervention and medication. In 34 patients (6%) who constitute the study population and underwent sTIPS, bleeding was either uncontrollable ab *initio* (n = 11) or life-threatening refractory (n = 23) despite optimal endoscopic and pharmacological management. + +The demographic and clinical data of the patients are summarized in Table 1. No patients had a concomitant HCC or portal vein thrombosis at the time of TIPS insertion. Before sTIPS 19 patients had a median of three (1-9) injection sclerotherapy treatment (IST) sessions and 20 had a median of two (1-6) endoscopic variceal ligation (EVL) sessions with a median of 10 bands placed per session. Five patients had both IST and EVL. Median units of blood transfused before sTIPS was six (3-12), and 14 patients required either Minnesota balloon tamponade (n = 12) or placement of a Danis stent (n = 2) for temporarily control of bleeding before the sTIPS procedure. Eleven patients required endotracheal intubation and mechanical ventilation and nine required inotropic support. + +Technical success for sTIPS was 100% and therapeutic success (control of bleeding) was achieved in 31 of 34 (91%) patients. Bleeding persisted in two patients (6%) despite a patent sTIPS on repeat USdoppler examination and one patient developed recurrent bleeding in hospital during the index + +| Table 1 Demographic, clinical characteristics and risk prediction scores of 34 patients undergoing salvage transjugular intrahepatic portosystemic shunt, n % Variable Total cohort (n = 34) Survived (n = 24) In-hospital death (n = 10) P value Demographics Age (mean ± SD) 52 ± 11.6 50 ± 10.5 57 ± 12.9 0.107 Sex Male 29 (85) 22 (92) 7 (70) 0.104 Female 5 (15) 2 (8) 3 (30) Cause of cirrhosis Alcohol related 22 (65) 15 (63) 7 (70) 0.938 Non-alcohol related 12 (35) 9 (37) 3 (30) Child-Pugh grade A 3 (9) 3 (12) 0 0.022 B 19 (56) 16 (67) 3 (30) C 12 (35) 5 (20) 7 (70) Risk prediction scores APACHE II (mean ± SD) 13.4 ± 4.7 11.4 ± 3.3 18.3 ± 3.8 0.196 BOTEM (mean ± SD) 5.4 ± 1.1 5.0 ± 0.9 6.3 ± 0.7 0.964 CABIN (mean ± SD) 10.9 ± 5.0 8.3 ± 1.8 17.0 ± 3.8 0.133 CHILD-PUGH (mean ± SD) 8.9 ± 1.8 8.2 ± 1.8 10.6 ± 2.0 0.001 EMORY (mean ± SD) 3.2 ± 0.9 2.8 ± 0.7 4.3 ± 0.5 0.497 FIPS (mean ± SD) -0.3 ± 0.9 -0.6 ± 0.9 0.5 ± 0.5 0.205 MELD (mean ± SD) 15.0 ± 6.2 13 ± 4.8 19.8 ± 6.7 0.007 MELD-Na (mean ± SD) 16.9 ± 7.4 14 ± 5.3 23.9 ± 7.1 < 0.001 | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +SD: Standard deviation; APACHE II: Acute physiology and chronic health evaluation II; BOTEM: Bonn TIPS early mortality; CABIN: Creatinine, Albumin, Bilirubin, INR, Sodium score; FIPS: Freiburg index of post-TIPS survival; MELD: Model for end-stage liver disease; MELD-Na: Model for end-stage liver disease sodium. + +admission after initial control of bleeding by sTIPS. + +Ten patients (29.4%) died in hospital at a median of 5 d following the procedure (range 1-10 d) of progressive liver failure (n = 4), MOF (2), alcoholic cardiomyopathy (n = 2) or uncontrolled variceal bleeding (n = 2). Mortality in C-P grade A patients was 0%, in C-P grade B patients 16% and C-P grade C patients 58%. In patients who died the median C-P score was 11, (range 7-13), median MELD score was 18 (range 11-29) and median MELD Na score was 25 (range 11-33). Nine of the 12 (75%) patients who required pre-sTIPS balloon tamponade died, while all nine (100%) patients who were hypotensive (systolic blood pressure < 70 mmHg) and with the combination of > 8 unit blood transfusion, inotropic support, balloon tamponade and mechanical ventilation died. + +The two patients with persistent bleeding after TIPS underwent repeat endoscopy and ultrasoundguided Histoacryl and coil injection of residual gastric varices with resolution. The patient with recurrent bleeding in hospital underwent a gastric devascularization for control of gastric varices. + +Figure 1 shows the graphic representation of the comparative performances of the eight risk scores in predicting in-hospital death following sTIPS. The CABIN score (AUROC 0.967) had the highest discriminative ability in predicting in-hospital death compared to the APACHE II (AUROC 0.948), BOTEM +(AUROC 0.877), C-P (AUROC 0.802), EMORY (AUROC 0.942), FIPS (AUROC 0.892), MELD (AUROC 0.792), and MELD-Na (AUROC 0.865) scores as detailed in Table 2. The median CABIN score in the 24 in-hospital TIPS survivors was 8 (range 5-18) compared to a median of 17 (range 11-22) in the 10 deaths. CABIN A patients had a 100% survival, compared to 25% and 12.5% survival in CABIN B and CABIN C category patients respectively. CABIN points of 11 or more provided a clear survival cut-off. No patients with CABIN scores < 10 died while 83% of patients with CABIN scores of > 11 died. + +| Table 2 Performance of various risk prediction scores in predicting in-hospital death following salvage transjugular intrahepatic portosystemic shunt In-hospital deaths AUC STD error P value 95% confidence interval APACHE II 0.948 0.035 0 0.879–1.000 BOTEM 0.877 0.059 0.001 0.762–0.992 CABIN 0.967 0.028 0 0.912–1.000 CHILD-PUGH 0.802 0.084 0.006 0.638–0.967 EMORY 0.942 0.038 0 0.868–1.000 FIPS 0.892 0.055 0 0.783–1.000 MELD 0.792 0.082 0.008 0.631–0.952 MELD Na 0.865 0.077 0.001 0.713–1.000 | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +AUC: Area under the curve; SD: standard deviation; APACHE II: Acute physiology and chronic health evaluation II; BOTEM: Bonn TIPS early mortality; + +![4_image_0.png](4_image_0.png) CABIN: Creatinine albumin bilirubin INR sodium score; FIPS: Freiburg index of post-TIPS survival; MELD: Model for end-stage liver disease; MELD-Na: Model for end-stage liver disease sodium. + +Figure 1 Performance of various risk prediction scores in predicting in-hospital death following salvage transjugular **intrahepatic** portosystemic **shunt.** APACHE II: Acute physiology and chronic health evaluation II; BOTEM: Bonn TIPS early mortality; CABIN: Creatinine, Albumin, Bilirubin, INR, Sodium score; FIPS: Freiburg index of post-TIPS survival; MELD: Model for end-stage liver disease; MELD-Na: Model for end-stage liver disease sodium; TIPS: Transjugular intrahepatic portosystemic shunt; ROC: Receiver operating characteristic. + +## Discussion + +The unique safety profile and minimally invasive characteristics conferred by TIPS provide an effective reduction in portal pressure and make the procedure the ideal rescue intervention for variceal bleeding not controlled by endoscopic intervention and pharmacological therapy[18]. In this study we compared the relative performances of eight scoring models, including the novel CABIN score, in predicting inhospital mortality in a high-risk cohort of patients who underwent sTIPS placement. Although sTIPS +controlled variceal bleeding in 94% of patients, over-all in-hospital mortality was 29.4% and increased exponentially in those who required > 8 unit blood transfusion, inotropic support, esophageal balloon tamponade and mechanical ventilation. Log-rank comparisons of survival curves showed that of the eight scores evaluated, the CABIN, APACHE II and Emory scores had the highest AUROC values and the best discriminatory ability with C-statistic values all exceeding 0.9. Of these three top contenders, the CABIN score (0.967) had the best discriminatory and predictive capability. As a collorary, this study also demonstrates the predictive ability of the CABIN score with 100% survival observed in patients in the CABIN A category (< 10 points) after sTIPS. + +The reported mortality rate after TIPS placement varies widely due to differing inclusion criteria, timing of TIPS placement, the spectrum and severity of the underlying liver disease and inclusion in some reports of patients with active bleeding during urgent TIPS as well as stable patients undergoing elective TIPS[19-21]. In the 22 studies exclusively reporting salvage or rescue TIPS in patients with uncontrolled life-threatening or endoscopically unmanageable variceal bleeding, as in this study, inhospital mortality rates range from 17% to 56% which are significantly higher than for elective TIPS[2243] (Table 3). Accurate prediction of outcome following sTIPS is thus a crucial element of management and the optimal prognostic score should ideally be able to distinguish two groups, patients with a better prognosis and likely to survive and those with a high or prohibitive risk of death. + +Most of the current prognostic scores used in sTIPS patients have intrinsic limitations due to the selection and weighting of the constituent components. The MELD score, which was initially created to predict survival following elective placement of TIPS, is currently the most widely used liver-related prognostic score both in clinical practice and research and especially as a tool for organ allocation[9]. + +Although the MELD score was a prospectively developed and validated indicator of the severity of endstage liver disease that utilizes quantitative and objective measures, including bilirubin, creatinine and INR values, the score has potential limitations. A further caveat is the maximum assigned value of serum creatinine which is capped at four even when the measured serum level is higher. Modifications to overcome MELD shortcomings have included reweighting the model's coefficients, altering the laboratory components and the addition of new variables including serum sodium ('MELD-Na'), +albumin [termed '5-variable MELD' (5vMELD)][44] and female gender (MELD 3.0)[45]. These modifications are more discriminative than either MELD or MELD-Na in transplant assessment and use similar elements as the CABIN score. + +The inclusion of subjective clinical components in other proposed prognostic models may also limit precision and reproducibility of score assignments. The C-P, Emory and BOTEM scores all have at least one component that may be perceived as subjective while the APACHE II and BOTEM scores lack specificity for liver disease which limits their capacity to predict outcomes after liver interventions such a sTIPS. In addition the C-P, Emory, and BOTEM scores are limited by a ceiling effect in which laboratory values above a particular cut-off level are not distinguished from one another in terms of higher scoring[4-7,9,10]. The FIPS overcomes some of these limitations by using four objective components, age, bilirubin, albumin, and creatinine levels[8]. + +In a meta-analysis, which included 11 studies and 2037 patients Zhou et al[46] found that MELD was superior to the C-P score in predicting 3-mo survival after TIPS but not 1-mo, 6-mo or 12-mo survival. + +Zhang et al[47] found that C-P grade C and MELD > 10 but not the Emory, BOTEM or SB/PLT scores were predictors of survival in Chinese cirrhotic patients treated with TIPS. Gaba et al[48] reported that MELD and MELD-Na scores had the best capability to predict early mortality in an American population compared with bilirubin and the C-P, Emory, PI, APACHE II, and BOTEM scores. In a comparison of the MELD, C-P and Emory scores Schepke et al[49] found that all three models predicted 3-mo survival with similar accuracy, but the MELD score was marginally superior to the C-P score for both 12- and 36-mo survival. In patients with refractory variceal bleeding Rubin et al[39] found that survival was inversely proportional to C-P class and APACHE II scores. The single determinant most closely associated with decreased survival in the first month following TIPS was the APACHE II score, with a score of 18 stratifying patients into low and high mortality risk groups (Table 3). Only one of 13 patients with C-P class C cirrhosis and an APACHE II score exceeding 18 survived > 30 d[39]. In the Hermie study early mortality was associated with a MELD score of at least 19 and hemodynamic instability at the time of admission[32] (Table 3). If hemodynamic instability was combined with a high MELD score, the 6-week mortality peaked at 77.8%[32]. In a multicentre French study Walter et al[50] reported that sTIPS mortality was > 90% in patients who had lactate levels ≥ 12 mmol/L and/or a MELD score ≥ 30. + +In view of these differing outcomes, the development of a prognostic model to accurately stratify the risk profile of patients undergoing sTIPS may be invaluable in guiding treatment. The novel CABIN score used in this study was developed as a point-based tool to improve prognostic prediction specifically for patients undergoing emergent sTIPS and circumvents the complex computations of the MELD and other scores. This new score avoids subjective elements and can be calculated at the bedside providing a refined, granular grading system from a minimal laboratory dataset with scores ranging from 5 to 25. The CABIN score achieved significant prognostic discrimination reflected by in-hospital survival of 100% in patients in the CABIN A category (5-10 points), while patients in the CABIN B category (11-15) score had a 25% and those in the CABIN C category (16-20) a 12.5% survival. Our model predicted in-hospital mortality with high accuracy and showed statistical superiority over the other seven contenders, including MELD and C-P scores. Moreover, of all the examined models, only the CABIN, APACHE II and Emory scores exceeded a C-statistic value of 0.9. + +There are inevitable and specific limitations to our study. Firstly, this investigation is limited by its small sample size, retrospective design, and lack of a control group. Secondly, the study has a clear selection bias which restricts universal applicability as these patients were treated in a single, wellresourced tertiary care referral center with round the clock skilled endoscopic and TIPS access. Thirdly, because patients were accrued over three decades, technical differences in TIPS placement and improvements in medical care during the study period would have contributed to differences in clinical + +| Krige J et al. Risk scores for salvage TIPS Table 3 Published series of salvage transjugular intrahepatic portosystemic shunt for uncontrolled variceal bleeding Initial control of bleeding % 30-d mortality % Ref. Country No. of C-P grade patients A/B/C Persistent/Recurrent Survival % Prognostic factors rebleeding Azoulay et al France 58 3/8/47 90 29 17 51.7 (12 Sepsis, vasoactive [22], 2001 mo) drugs, balloon tamponade Bañares et al Spain 56 11/22/23 95 28 22 (1 mo) 72 (30 d) Ascites, HE, albumin [23], 1998 Barange et al France 32 3/14/15 90 25 14 75 (30 d) ND [24], 1999 Bizollon et al France 28 0/11/17 96 25 (40 d) 18 52 (2 yr) ↑Creatinine, ↑bilirubin [25], 2001 Casadaban et al 101 2/46/52 89 31 21 44 (12 mo) ↑Bilirubin, ↑creatinine, United [26], 2015 States ↑INR, non-alcoholic liver disease Chau et al[27], England 84 4/17/63 98 34 30 (30 d) 66 (30 d) ND 1998 Encarnacion et United 64 2/32/31 98 19 29 (6 mo) 56 (12 mo) Haemodynamic al[28], 1995 States instability Gazzera et al Italy 82 ND 94 25.6 13.4 74.4 (30d) Child-Pugh C, ↑ [29], 2012 creatinine, ↑PT Gerbes et al Germany 11 91 27 27 73 (12 mo) ND [30], 1998 Helton et al United 23 0/15/18 74 56 (in 39 ND Emergency TIPS, [31], 1993 States hospital) active bleeding Hermie et al Belgium 32 ND/ND/14 97 31 0 69 MELD > 19, [32], 2018 Haemodynamic instability Jabbour et al United 25 ND/ND/8 96 44 ND 56 (30 d) Child-Pugh C, urgent [33], 1996 States TIPS Jalan et al[34], Scotland 19 3/3/13 100 42 15.6 58 (30 d) Liver failure, sepsis 1995 Maimone et al England 144 11/55/78 ND 36 (6 wk) 29 64 (6 wk) ↑MELD, ↑Child-Pugh [36], 2019 score Le Moine et al Belgium 24 3/13/9 96 17 25 29 (5 mo) ND [35], 1994 McCormick et England 20 1/7/12 100 60 (40 d) 40 30 ND al[37], 1994 Patch et al[38], England 54 5/20/29 91 48 (6 wk) 11 53 (6 mo) Ventilation, ↑WBC, 1998 platelets, ↑creatinine Rubin et al[39], United 49 3/23/23 84 40% 16 ND C-P grade C, APACHE 1995 States II > 18 Sanyal et al United 30 1/7/22 100 37 7 60 (6 wk) > 70 yr, bilirubin >6 [40], 1996 States mg/dL, creatinine > 3 mg/dL, HE, ARDS Tyburski et al United 33 0/5/28 ND 27 15 58 (12 mo) Albumin < 2.5 g/dL, [41], 1997 States bilirubin > 3 mg/dL, PT > 15 s Tzeng et al Taiwan 107 ND ND 28 ND 50 (12 mo) C-P score > 11, MELD [42], 2009 > 20 Zhu et al[43], China 58 5/36/7 91.2 12.3 (6 wk) 10.5 (6 wk) 81.8 (12 Ventilation, ICU 2019 mo) APACHE: Acute Physiology and Chronic Health Evaluation; ARDS: Adult Respiratory Distress Syndrome; C-P: Child-Pugh; INR: International | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +APACHE: Acute Physiology and Chronic Health Evaluation; ARDS: Adult Respiratory Distress Syndrome; C-P: Child-Pugh; INR: International normalized ratio; TIPS: Transjugular intrahepatic portosystemic shunt; MELD: Model for end-stage liver disease; HE: Hepatic encephalopathy; ND: No data; ICU: Intensive care unit; WBC: White blood cells. + +outcomes over time. Fourthly, this new score has been developed using a derivation dataset and requires confirmation and external validation in a similar sTIPS patient group. The robustness of this study is enhanced by the prospective data collection, supervision by the same investigators during the study period, restriction of subjects to a well-defined cohort of cirrhotic patients with uncontrolled exsanguinating bleeding and complete follow-up. The use of all-cause mortality as the primary outcome provided a consistent and objective end point. + +## Conclusion + +In conclusion, the novel CABIN prognostic score, which is objective, quantitative, and reproducible, combines five easily obtained laboratory test results and provides improved statistical power predicting in-hospital mortality in patients with uncontrolled variceal bleeding undergoing sTIPS. The CABIN score identified high-risk patients and outperformed other scoring systems in predicting in-hospital mortality. Despite the fact that mortality was 75% for CABIN B, 87.5% for CABIN C, and 83% for CABIN scores > 10 in this study, this high-risk category should not be denied consideration for an emergency TIPS and should be assessed on a case-by-case basis especially in units where there is prompt access to liver transplantation after sTIPS. This study was based on a small defined cohort of predominantly alcoholic decompensated cirrhotic patients undergoing emergent TIPS and this newly developed derivative CABIN score will need further prospective external validation before being considered for general clinical application. + +## Article Highlights Research Background + +Transjugular intrahepatic portosystemic shunt (TIPS) is now established as the salvage procedure of choice in patients who have uncontrolled or severe recurrent variceal bleeding despite optimal medical and endoscopic treatment. + +## Research Motivation + +Although TIPS is a minimally invasive procedure, appropriate patient selection is crucial to identify patients who would benefit from the procedure, considering the substantial risks of hepatic encephalopathy, liver failure and increased overall morbidity and mortality in high-risk individuals. + +## Research Objectives + +In this study the accuracy of a novel CABIN score, which was developed to overcome limitations of existing scoring systems, was compared to established risk scores for the prediction of in-hospital mortality following sTIPS. + +## Research Methods + +Eight risk scores were evaluated in a cohort which included all adult patients who underwent sTIPS for uncontrollable or life-threatening refractory variceal bleeding. A new five component CABIN score was devised in which each CABIN variable was scored from one to five and the cumulative total is calculated by adding the individual values of the five biochemical components (Creatinine, Albumin, Bilirubin, INR (international normalized ratio) and Na (sodium). The best total CABIN score computes at 5 points and the worst at 25 points. Four CABIN categories (A-D) were established (A: 5-10 points, B: 11-15, C: 16-20, D: 21-25). The CABIN score and seven previously described scoring systems, Acute Physiology and Chronic Health Evaluation (APACHE) II, Bonn TIPS early mortality (BOTEM), ChildPugh, Emory, FIPS, model for end-stage liver disease (MELD), and MELD-Na scores were calculated based on clinical evaluation and laboratory values obtained before the sTIPS procedure. The primary study outcome measure was prediction of in-hospital mortality after sTIPS and compared the relative performances of the seven established scoring models and the new CABIN score. + +## Research Results + +In 34 patients (6%) who underwent sTIPS, bleeding was either uncontrollable ab *initio* (n = 11) or lifethreatening refractory (n = 23) despite optimal endoscopic and pharmacological management. Ten patients (29.4%) died in hospital at a median of 5 d following the procedure (range 1-10 d). Nine of the 12 (75%) patients who required pre-sTIPS balloon tamponade died, while all nine (100%) patients who were hypotensive (systolic blood pressure < 70 mmHg) and with the combination of > 8 unit blood transfusion, inotropic support, balloon tamponade and mechanical ventilation died. The CABIN score [area under the receiver operating characteristic curve (AUROC) 0.967] had the highest discriminative ability in predicting in-hospital death compared to the APACHE II (AUROC 0.948), BOTEM (AUROC 0.877), C-P (AUROC 0.802), EMORY (AUROC 0.942), FIPS (AUROC 0.892), MELD (AUROC 0.792), and MELD-Na (AUROC 0.865) scores. The median CABIN score in the 24 in-hospital TIPS survivors was 8 (range 5-18) compared to a median of 17 (range 11-22) in the 10 deaths. CABIN A patients had a 100% survival, compared to 25% and 12.5% survival in CABIN B and CABIN C category patients respectively. CABIN points of 11 or more provided a clear survival cut-off. No patients with CABIN scores < 10 died while 83% of patients with CABIN scores of > 11 died. + +## Research Conclusions + +The novel CABIN prognostic score, which is objective, quantitative, and reproducible, combines five easily obtained laboratory test results and provides improved statistical power predicting in-hospital mortality in patients with uncontrolled variceal bleeding undergoing sTIPS. The CABIN score identified high-risk patients and outperformed other scoring systems in predicting in-hospital mortality. Despite the fact that mortality was 75% for CABIN B, 87.5% for CABIN C, and 83% for CABIN scores > 10 in this study, this high-risk category should not be denied consideration for an emergency TIPS and be assessed on a case by case basis especially in units where there is prompt access to liver transplantation after sTIPS. + +## Research Perspectives + +This study was based on a small defined cohort of predominantly alcoholic decompensated cirrhotic patients undergoing emergent TIPS and this newly developed derivative CABIN score will need further prospective external validation before being considered for general clinical application. + +## Footnotes + +Author contributions: Krige J, Jonas E and Robinson C designed the research study; Krige J, Jonas E, Robinson C and Kotze U collected the data and performed the research; Krige J, Jonas E, Robinson C, Kotze U, Beningfield S, Bernon M, Burmeister S, and Kloppers C analyzed the data and wrote the manuscript; All authors have read and approve the final manuscript. + +Institutional review board statement: The study protocol was approved by the Human Research Ethics Committee +(HREC Ref No. 120/2019) of the University of Cape Town and the research was conducted in accordance with the Declaration of Helsinki. + +Informed consent statement: Since this was a retrospective observational study using existing anonymized data, the requirement for informed consent from the study participants was waived by the Institutional Review Board. + +Conflict-of-interest statement: All the authors declare no conflict of interest. + +Data sharing statement: Dataset available from the corresponding author at jej.krige@uct.ac.za. + +STROBE statement: The authors have read the STROBE Statement—checklist of items, and the manuscript was prepared and revised according to the STROBE Statement—checklist of items. + +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: South Africa + +ORCID number: Jake Krige 0000-0002-7057-9156; Eduard Jonas 0000-0003-0123-256X; Chanel Robinson 0000-0002-93852709; Steve Beningfield 0000-0003-0727-2650; Urda Kotze 0000-0003-1405-474X; Marc Bernon 0000-0002-7967-8548; Sean Burmeister 0000-0003-0888-7606; Christo Kloppers 0000-0003-2438-6879. + +S-Editor: Liu JH +L-Editor: A +P-Editor: Liu JH + +## References + +Rajesh S, George T, Philips CA, Ahamed R, Kumbar S, Mohan N, Mohanan M, Augustine P. 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Hepatology 2021; 74: 2085-2101 [PMID: 34018627 DOI: 10.1002/hep.31913] +Published by **Baishideng Publishing Group Inc** + +![12_image_0.png](12_image_0.png) + +![12_image_1.png](12_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/PMC10078391.md b/medical/md/PMC10078391.md new file mode 100644 index 0000000000000000000000000000000000000000..806f408297540bc46b6788a203d9d6be15e5389d --- /dev/null +++ b/medical/md/PMC10078391.md @@ -0,0 +1,181 @@ +DOI: 10.1111/dth.15911 ORIGINAL ARTICLE +Gradually increasing the dosing interval of Secukinumab for moderate to severe plaque psoriasis: A single-center, uncontrolled, prospective study in 36 weeks Xi-Bei Chen | Yu-Xin Zheng | Li-Ran Ye | Xue-Yan Chen | Xiao-Yong Man Department of Dermatology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Correspondence Xiao-Yong Man, Department of Dermatology, Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jiefang Road, Hangzhou, China. Email: manxy@zju.edu.cn Funding information National Natural Science Foundation of China (NSFC), Grant/Award Numbers: 81630082, 81930089 + +## Abstract + +Secukinumab is a recombinant, fully human monoclonal anti-IL-17A antibody approved to treat moderate-to-severe psoriasis and psoriatic arthritis. Its effectiveness and safety have been confirmed, but a gradual increase in the secukinumab dosing interval has not been investigated. To assess the feasibility, efficacy, and safety of gradually increasing the secukinumab dosing interval; the interval duration was determined by changes in the Psoriasis Area and Severity Index scores. Patients with moderate-to-severe plaque psoriasis received secukinumab 300 mg subcutaneously at baseline and weeks 0, 1, 2, and 3. At week 4, the improvement from baseline PASI guided the next injection time until week 36. In total, 83 patients were recruited. PASI 75 was achieved by 80%, 96%, and 95% of patients at weeks 4, 12, and 36, respectively. PASI 90 was achieved by 54%, 95%, and 84% of patients at weeks 4, 12, and 36, respectively. PASI 100 was achieved by 28%, 89%, and 68% of patients at weeks 4, 12, and 36, respectively. The average PASI score (1.05 ± 1.83) was significantly lower at week 36 than at baseline. Most patients reached PASI 75 at week 36 in our modified study. This study may provide information for future biotherapies. + +KEYWORDS +feasibility, gradually increasing dose interval, PASI, Secukinumab + +## 1 | Introduction + +Psoriasis is a chronic inflammatory disease with a prevalence of approximately 2% among the world population.1 IL-17A, produced by Th17 cells, is considered an essential proinflammatory cytokine that mediates the development of psoriasis.2 Secukinumab is a recombinant, fully human monoclonal anti-IL-17A antibody and was the first biologic to target IL-17A to be approved by the US Food and Drug Administration to treat moderate-to-severe psoriasis and psoriatic arthritis in 2015.3 Secukinumab has been established as efficacious and safe in both long-term clinical trials and real-world management of moderate to severe plaque psoriasis.4–9 The results of several realworld evidence studies were better than those of phase II and III clinical trials. A total of 80%–83.5% of the treatment group achieved PASI +75 at week 12, while 78.5%–92.1% reached PASI 75 at week 52.10,11 Psoriasis is considered a disabling disease with no cure, and biologics have enormously enhanced the ability to treat this condition in recent years.12 The overall costs of these medications and the uncertainty relating to the necessary length of therapy place an economic Xi-Bei Chen and Yu-Xin Zheng equally contributed to the presented paper burden on the healthcare system. In addition, patients have many 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. Dermatologic Therapy published by Wiley Periodicals LLC. + +questions about the emerging biologics that we have not been able to answer, such as whether monthly injection regimen or lifelong treatment is necessary. + +Regarding the pharmacokinetic profile of secukinumab, the peak concentrations is reached 5–6 days after the first subcutaneous injection. The mean elimination half-life is approximately 27 days,2 and the blood level is stable at 20 days; therefore, the recommended dose of secukinumab for plaque psoriasis is 300 mg subcutaneously every week from baseline to week 4 and once every 4 weeks thereafter. Most phase II, phase III, and real-world studies were conducted around this recommended dose, but some studies adjusted the duration of treatment. A GAIN study by Reich et al. revealed the potential benefit of the Q2W dosing in some suboptimal responders but did not reach statistical significance compared with the Q4W dose.13 Another therapeutic administered without the initial weekly loading dose was inferior to secukinumab at the labeled dose.14 The feasibility of gradually increasing the dosing interval of secukinumab for treatment is still unknown. Herein, we designed a modified therapy that gradually increases the dosing interval of secukinumab based on the response of the PASI score. This study aims to evaluate the feasibility, safety, and efficacy of modified dosing of secukinumab compared with the recommended and FDA-approved dose regimen. + +## 2 | Methods 2.1 | Patients + +This was a single-center, open-label, uncontrolled, prospective study to investigate the feasibility, efficacy, and safety of a tapering dose of secukinumab in real-world settings from January 2020 to March 2022. The inclusion criteria included patients aged >18 years with moderate-to-severe plaque-type psoriasis for at least half a year, with or without psoriatic arthritis. Prior treatments, including topical, systemic, phototherapy, or biologic therapy alone or in combination, were also included. Patients with previous biologics experience at least a one-month washout period. Moderate-to-severe plaque-type psoriasis was defined at enrollment by PASI score ≥ 10 or BSA (body surface area; %) ≥ 10%. The study was conducted according to the principles of the Declaration of Helsinki and approved by the Human Research Ethics Board of Second Affiliated Hospital, Zhejiang University School of Medicine (approval number 2020–354) and. Informed consent was obtained from all the participants in the study. + +## 2.2 | Study Design + +The patients were treated with 300 mg of secukinumab during a screening period of 0–3 weeks (subcutaneous injection four times). Then, at the beginning of week 4, the improvement from baseline PASI guided the next injection time. The PASI 75 response was used as a measure of effective treatment. In our study, patients achieving PASI 75 and less than PASI 90 accepted the recommended dose every 4 weeks from week 4. At the initial consultation, patients with PASI 90 or better skipped the dose and were given four additional weeks. If the patients had sustainably achieved PASI 90 or better at the follow-up visit, an additional 4 weeks was added but with a restriction of 20 weeks. If patients did not achieve a PASI 75 response at the latest follow-up due to prolonged treatment, they resumed 1 month of treatment. Changing therapy should be considered if patients were unable to reach PASI 75 more than 5 times. The detailed design is shown in Figure 1. + +## 2.3 | Statistical Analysis + +The demographic and clinical characteristics of this study population are expressed as percentages (%), means, and standard deviations (SDs). Clinical effectiveness included the proportion of patients achieving a PASI reduction score (PASI 75, PASI 90, PASI 100) at weeks 4, 8, 12, 24, and 36. The reduction score from baseline to the endpoints at weeks 4, 8, 12, 24, and 36. If patients discontinued therapy because of adverse events or loss to follow up, they were removed from the study. Data from resistant patients were preserved in the statistical analysis until termination of treatment. The significance of differences in mean values from each point was assessed by the Kruskal–Wallis test (multiple comparisons), where p values <0.05 were considered to be statistically significant. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA). + +## 3 | Results 3.1 | Patient Profiles And Baseline Characteristics + +The group included 83 patients; 3 patients were censored, two due to absenteeism and another due to inflammatory bowel disease at week 16. Furthermore, six patients switched to biologic therapy due to continuous therapeutic resistance. Two of them terminated therapy at week 12, one patient stopped at week 24, and another three-ended therapies at week 36. The other 74 patients had a 36-week follow-up visit, but two patients did not strictly comply with the prescribed time with a 1–2 month delay. Our results still included these two patients, as they did not prolong for more than 5 months. The mean time since the first diagnosis of psoriasis was 24.8 (±10.76) years, and the mean duration of psoriasis was 14.84 (±9.08) years. A mean baseline PASI score of 21.53 (±9.81) and a mean baseline BSA score of 47.76 (±22.84) indicated the severity of the disease. Comorbidities were reported in 17.5% of the group. The most common comorbidities were hyperlipidemia (8.75%), diabetes mellitus (7.5%), hypertension (6.25%), cardiovascular disease (6.25%), and obesity (6.25%). In addition, eight patients (10%) had concurrent psoriatic arthritis, and eight patients had hepatitis B virus. Smoking was recorded in 15 (18.75%) patients. A total of 11.25% of the group had previously received one biologic, only one of the patients accepted two biologics, and none + +![2_image_0.png](2_image_0.png) + +were treated with three biologics or more. The demographics and clinical characteristics of the patients are summarized in Table 1. + +## 3.2 | Efficacy And Feasibility + +A positive effect on skin lesions could be observed in our modified therapy group. A 80% of patients reached PASI 75, while 54% of patients reached PASI 90, and 28% even reached PASI 100 at week 4. At week 12, the PASI score improved, with 96% of patients reaching PASI 75,88% reaching PASI 90 and 76% clearing of disease. At week 24, 96% of patients received a PASI 75 score, 86% received a PASI 90 score, and 76% achieved PASI 100. At the endpoint (week 36), the same percentage received PASI 75, 84% received PASI 90 and 68% obtained clearing of disease (Figure 2). The average PASI score for patients significantly decreased from 21.53 (±9.81) at baseline to 3.71 (±4.24) at week 4 and a lowest point of 0.59 (±1.94) at week 12 but recovered to 1.047 (± 1.83) at week 24 and remained stable at 1.38 (± 2.2) until week 36 during our observation period (Figure 3). Each assessment point compared with baseline had a significant reduction with p < 0.0001. + +## 3.3 | Safety + +A total of 28 subjects experienced adverse events during the 36-week period, including 10 infections (nasopharyngitis, fungal infection, conjunctivitis, otitis externa), five cases of pruritus, four cases of eczema, one case of headache, and one case of IBD. The case confirmed the diagnosis of IBD, which was moderate, and discontinued therapy. All the others were of mild severity, easily manageable, and did not lead to drug discontinuation. None of the hepatitis B virus reactivation or tuberculosis infection cases were reported. No new or unexpected safety signals were noted. The percentage of AEs was similar to or less than that in other clinical trials4,15 (Table 2). + +## 3.4 | Social Perspective + +Patients needed a total of twelve injections based on the recommended regimen compared with 7.96 (±1.14) times in our modified study (Table 1). This means that our study can reduce the costs of secukinumab treatment, resulting in $2141 savings per patient during the 36 weeks of treatment. + +## 4 | Discussion + +Earlier studies of dose adjustment have typically increased dosages or shortened treatment time, but our study aimed to guide subsequent treatments based on response to treatments. Previous studies have reported that biologic agents lose efficacy over time, but secukinumab is believed to have improved long-term effectiveness.4,16–20 Our modified study outcomes illustrate a similar efficacy as previous studies. + +Research from Galluzzo et al.11 revealed that 92% of patients naive to biological therapies achieved PASI 75, while 84% reached PASI 100 at + +## 4 Of 6 Chen + +| TABLE 1 | Baseline demographics and characteristics Secukinumab initiators (n = 80), | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------|-------------------------------------------------| +| Characteristic | n (%) or mean (SD) | | +| Male/female (n) | 43(53.75) | | +| Age (years) | 39.43 ± 12.42 | | +| BMI, (kg/m2 ) | 23.62 ± 4.167 | | +| Normal/underweight (<25 kg/m2 ) | 55(68.75) | | +| Overweight (25≤ to <30 kg/m2 ) | 23(28.75) | | +| Obese (>30 kg/m2 ) | 5(6.25) | | +| Age at disease onset (years) | 24.80 ± 10.76 | | +| Duration of psoriasis (years) | 14.84 ± 9.08 | | +| PASI at baseline | 21.53 ± 9.81 | | +| BSA at baseline | 47.76 ± 22.84 | | +| Current smoker | 15(18.75) | | +| Psoriatic arthritis | 8(10) | | +| Baseline comorbidities (n [%]) Hypertension | 5(6.25) | | +| Hyperlipidemia | 7(8.75) | | +| Cardiovascular disease | 5(6.25) | | +| Diabetes mellitus | 6(7.5) | | +| Thyroid cancer | 2(2.5) | | +| Treatment history (n [%]) | 74(92.5) | | +| Biologic naive | 71(88.75) | | +| Biologic experienced | 9(11.25) | | +| No. of prior biologics (n [%]) 1 | 8(10) | | +| 2 | 1(1.25) | | +| ≥3 | 0(0) | | +| Average times of subcutaneous | 7.96(±1.14) | | +| injections at 36 weeks | FIGURE 3 | Mean change of PASI score from baseline to week | +| 4, 8, 12, 24, 36. PASI, Psoriasis Area and Severity Index; n, number of patients; ****, p < 0.0001. TABLE 2 Summary of adverse events Patients (n = 81) Any AEs (n [%]) 28(34.6) Death (n [%]) 0 Nasopharyngitis 6(7.4) Pruritus 5(6.2) Eczema 4(4.9) Urticaria 3(3.7) Diarrhea 3(3.7) Upper respiratory tract infection 2(2.5) Fungal infection 1(1.2) | | | +| Note: Data are expressed as mean ± SD or percentage, unless indicated otherwise. Abbreviations: BMI, body mass index; BSA, body surface area; PASI, psoriasis area and severity index; SD, standard deviation. | Otitis externa | 1(1.2) | +| Headache | 1(1.2) | | +| Conjunctivitis | 1(1.2) | | +| SAEs (n [%]) IBD | 1(1.2) | | +| Abbreviations: AE, adverse event; IBD, inflammatory bowel disease; SAE, | | | + +Abbreviations: AE, adverse event; IBD, inflammatory bowel disease; SAE, serious adverse event. + +![3_image_0.png](3_image_0.png) + +week 36. Our modified treatments revealed a close percentage of PASI 75 at the end of 36 weeks and a slightly reduced rate of PASI 100. Dose interval lengthening from week 4 did not significantly reduce the PASI 75 response. Achieving PASI <3 has become one of the critical considerations in long-term disease management.13 Here, we obtained an average PASI of 1.38 (±2.2) at the endpoint, this parameter indicates that minimal disease activity was maintained. However, the PASI 75 response rate in our study was 96% at week 24 and was superior to other real-world settings in Italy, Germany, and Spain, as they had an 82.11%–89% of PASI 75 at weeks 24.7,14,15 + +## Acknowledgments + +![4_Image_0.Png](4_Image_0.Png) Conflict Of Interest Data Availability Statement Ethics Statement Orcid References Author Contributions + +The resulting disparities may be due to the fundamental level of BMI, baseline PASI score and ethnic population. + +Previous studies have found that patients with a complex history of biologic treatment are resistant to new biologic therapies.9,11 Notably, in our study, four of the six patients who were resistant to treatment had a history of prior biologic treatment. The relatively small number of patients in our study who had experience with biologic treatment may also lead to good results. + +A pooled analysis evaluated the long-term safety of secukinumab and indicated that the most common AE was nasopharyngitis, followed by headache, upper respiratory tract infection, pruritus, diarrhea, and hypertension.21 Nasopharyngitis (7.5%), pruritus (6.25%), +and eczema (5%) were the top three incidences in our study, followed by urticaria and diarrhea. Other AEs can be relieved spontaneously or with topical medications. Only one serious AE occurred during our observation period. In recent years, inflammatory bowel disease has been found at a higher prevalence in psoriasis patients and is considered a comorbidity.5,22,23 One young male patient discontinued the drug due to IBD at week 16. The patient was never diagnosed with Crohn's disease or ulcerative colitis before the treatment. The comorbidities may be exacerbated during therapeutic interventions for psoriasis. + +Some limitations of this modified study, such as wide-ranging inclusion criteria, not randomized, open-label, and a lack of a control group, should be considered when evaluating these results. Patients treated with biologic agents, as well as psoriatic arthritis patients, were also included, which may influence the effectiveness of treatment. The open-label trial may have created selection, maintenance, and measurement biases. Our study lacked a control group, and the difference in treatment between the screening and stabilization phases may also cause biases. We used PASI scores as evaluation criteria, while other indicators, such as DLQI (Dermatology Life Quality Index) or IGA (Investigator's Global Assessment), were excluded, leading to incomplete conclusions. Furthermore, the PASI response is an indicator used to assess the efficacy of secukinumab. + +Overall, the safety and efficacy of our tapering dose regimen were consistent with those of the previously recommended dose. The recommended regimen requires 12 injections, costing a total of $6360 compared with 7.96(±1.14) injections in our modified regimen used in this study.24 From a societal perspective, we saved $2141 per patient in the 9 months of treatment. This study demonstrates the feasibility of gradually increasing the dosing interval of secukinumab and may serve as a reference for biologic treatment experience, although a longer follow-up time and expanded sample sizes are necessary to investigate this possibility. + +This work was supported by grants from the National Natural Science Foundation of China (NSFC) (Nos. 81930089, 81630082). There is no conflict of interest to disclose. Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. The study was approved by the Human Research Ethics Board of Second Affiliated Hospital, Zhejiang University School of Medicine (approval number 2020-354) and was conducted according to the principles of the Declaration of Helsinki. + +Xiao-Yong Man https://orcid.org/0000-0003-3331-5538 1. Menter A, Gelfand JM, Connor C, et al. Joint American Academy of Dermatology-National Psoriasis Foundation guidelines of care for the management of psoriasis with systemic nonbiologic therapies. J Am Acad Dermatol. 2020;82(6):1445-1486. + +2. Frieder J, Kivelevitch D, Menter A. Secukinumab: a review of the antiIL-17A biologic for the treatment of psoriasis. Ther Adv Chronic Dis. + +2018;9(1):5-21. + +3. Sanford M, McKeage K. Secukinumab: first global approval. Drugs. + +2015;75(3):329-338. + +4. Lacour JP, Paul C, Jazayeri S, et al. Secukinumab administration by autoinjector maintains reduction of plaque psoriasis severity over 52 weeks: results of the randomized controlled JUNCTURE trial. J Eur Acad Dermatol Venereol. 2017;31(5):847-856. + +5. Deodhar A, Mease PJ, McInnes IB, et al. Long-term safety of secukinumab in patients with moderate-to-severe plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis: integrated pooled clinical trial and post-marketing surveillance data. Arthritis Res Ther. 2019; 21(1):111. + +6. Kishimoto M, Taniguchi A, Fujishige A, et al. Efficacy and safety of secukinumab in Japanese patients with active ankylosing spondylitis: 24-week results from an open-label phase 3 study (MEASURE 2-J). Mod Rheumatol. 2020;30(1):132-140. + +7. Thaci D, Korber A, von Kiedrowski R, et al. Secukinumab is effective in treatment of moderate-to-severe plaque psoriasis: real-life effectiveness and safety from the PROSPECT study. J Eur Acad Dermatol Venereol. 2020;34(2):310-318. + +8. Papp KA, Gooderham M, Beecker J, et al. Rationale, objectives and design of PURE, a prospective registry of patients with moderate to severe chronic plaque psoriasis in Canada and Latin America. BMC Dermatol. 2019;19(1):9. + +9. Megna M, Di Costanzo L, Argenziano G, et al. Effectiveness and safety of secukinumab in Italian patients with psoriasis: an 84 week, multicenter, retrospective real-world study. Expert Opin Biol Ther. 2019;19(8):855-861. + +10. Caldarola G, Mariani M, Pirro F, et al. Comparison of short- and longterm effectiveness of ixekizumab and secukinumab in real-world practice. Expert Opin Biol Ther. 2021;21(2):279-286. + +11. Galluzzo M, Talamonti M, De Simone C, et al. Secukinumab in moderate-to-severe plaque psoriasis: a multi-center, retrospective, Xiao-Yong Man, Xi-Bei Chen; Methodology: Xiao-Yong Man, Xi-Bei Chen; Formal analysis and investigation: Xi-Bei Chen, Yu-Xin Zheng, Li-Ran Ye; Writing - original draft preparation: Xi-Bei Chen, Yu-Xin Zheng; Writing - review and editing: Xi-Bei Chen, Yu-Xin Zheng, Li- Ran Ye, Xue-Yan Chen; Funding acquisition: Xiao-Yong Man; Supervision: Xiao-Yong Man. + +real-life study up to 52 weeks observation. Expert Opin Biol Ther. + +2018;18(7):727-735. + +12. Ceccarelli M, Venanzi Rullo E, Berretta M, et al. New generation biologics for the treatment of psoriasis and psoriatic arthritis. State of the art and considerations about the risk of infection. Dermatol Ther. 2021;34(1):e14660. + +13. Reich K, Korber A, Mrowietz U, et al. Secukinumab 2-weekly vs. 4-weekly dosing in patients with plaque-type psoriasis: results from the randomized GAIN study. Br J Dermatol. 2021;184(5): 849-856. + +14. Gisondi P, Rovaris M, Piaserico S, Girolomoni G. Efficacy of secukinumab without the initial weekly loading dose in patients with chronic plaque psoriasis. Br J Dermatol. 2020;182(1):175-179. + +15. Michelsen B, Georgiadis S, Di Giuseppe D, et al. Real-world 6 and 12-month drug retention, remission and response rates of Secukinumab in 2,017 psoriatic arthritis patients in 13 European countries. + +Arthritis Care Res (Hoboken). 2021;74:1205-1218. + +16. Bodemer C, Kaszuba A, Kingo K, et al. Secukinumab demonstrates high efficacy and a favorable safety profile in pediatric patients with severe chronic plaque psoriasis: 52-week results from a phase 3 double-blind randomized, controlled trial. J Eur Acad Dermatol Venereol. + +2021;35(4):938-947. + +17. Rich P, Sigurgeirsson B, Thaci D, et al. Secukinumab induction and maintenance therapy in moderate-to-severe plaque psoriasis: a randomized, double-blind, placebo-controlled, phase II regimen-finding study. Br J Dermatol. 2013;168(2):402-411. + +18. Galluzzo M, D'Adamio S, Silvaggio D, Lombardo P, Bianchi L, +Talamonti M. In which patients the best efficacy of secukinumab? + +Update of a real-life analysis after 136 weeks of treatment with secukinumab in moderate-to-severe plaque psoriasis. Expert Opin Biol Ther. 2020;20(2):173-182. + +19. Augustin M, Jullien D, Martin A, Peralta C. Real-world evidence of secukinumab in psoriasis treatment–a meta-analysis of 43 studies. + +J Eur Acad Dermatol Venereol. 2020;34(6):1174-1185. + +20. Yiu ZZN, Mason KJ, Hampton PJ, et al. Drug survival of adalimumab, ustekinumab and secukinumab in patients with psoriasis: a prospective cohort study from the British Association of Dermatologists biologics and Immunomodulators register (BADBIR). Br J Dermatol. 2020; 183(2):294-302. + +21. van de Kerkhof PC, Griffiths CE, Reich K, et al. Secukinumab longterm safety experience: a pooled analysis of 10 phase II and III clinical studies in patients with moderate to severe plaque psoriasis. J Am Acad Dermatol. 2016;75(1):83-98 e84. + +22. Ni C, Chiu MW. Psoriasis and comorbidities: links and risks. Clin Cosmet Investig Dermatol. 2014;7:119-132. 23. Blair HA. Secukinumab: a review in moderate to severe pediatric plaque psoriasis. Paediatr Drugs. 2021;23(6):601-608. 24. Cosentyx. Prescribing information. Switzerland: Novartis Pharmaceutical Corporation, https://www.pharma.us.novartis.com/sites/www. + +pharma.us.novartis.com/files/cosentyx.pdf (2016, accessed 28 Jun 2017). + +How to cite this article: Chen X-B, Zheng Y-X, Ye L-R, Chen X-Y, Man X-Y. Gradually increasing the dosing interval of Secukinumab for moderate to severe plaque psoriasis: A single-center, uncontrolled, prospective study in 36 weeks. Dermatologic Therapy. 2022;35(12):e15911. doi:10.1111/dth. 15911 \ No newline at end of file diff --git a/medical/md/PMC10078477.md b/medical/md/PMC10078477.md new file mode 100644 index 0000000000000000000000000000000000000000..64934f1c37e0b8d06f8cbecbf596115aa75b7c31 --- /dev/null +++ b/medical/md/PMC10078477.md @@ -0,0 +1,557 @@ +DOI: 10.1111/desc.13264 REGISTERED REPORT + +# Coping With Dialects From Birth: Role Of Variability On Infants' Early Language Development. Insights From Norwegian Dialects + +Natalia Kartushina1,2 **Julien Mayor2** +1Center for Multilingualism in Society across the Lifespan, Department of Linguistics and Scandinavian Studies, Faculty of Humanities, University of Oslo, Henrik Wergelands hus, Oslo, Norway 2Department of Psychology, Faculty of Social Sciences, University of Oslo, Oslo, Norway Correspondence Natalia Kartushina, Center for Multilingualism in Society across the Lifespan, Faculty of Humanities, University of Oslo, Henrik Wergelands hus, Niels Henrik Abels vei 36, 0313 Oslo, Norway. + +Email: Natalia.Kartushina@iln.uio.no Funding information Norges Forskningsråd, Grant/Award Number: +223265; HUP (Helse, Utviklings og Personlighetspsykologi) funding, University of Oslo, Norway + +## Abstract + +![0_Image_0.Png](0_Image_0.Png) + +Previous research suggests that exposure to accent variability can affect toddlers' familiar word recognition and word comprehension. The current preregistered study addressed the gap in knowledge on early language development in infants exposed to two dialects from birth and assessed the role of dialect similarity in infants' word recognition and comprehension. A 12-month-old Norwegian-learning infants, exposed to native Norwegian parents speaking the same or two Norwegian dialects, took part in two eye-tracking tasks, assessing familiar word form recognition and word comprehension. Their parents' speech was assessed for similarity by native Norwegian speakers. First, in contrast to previous research, our results revealed no listening preference for words over nonwords in both monodialectal and bidialectal infants, suggesting potential language-specific differences in the onset of word recognition. Second, the results showed evidence for word comprehension in monodialectal infants, but not in bidialectal infants, suggesting that exposure to dialectal variability impacts early word acquisition. Third, perceptual similarity between parental dialects tendentially facilitated bidialectal infants' word recognition and comprehension. Forth, the results revealed a strong correlation between the raters and parents' assessment of similarity between dialects, indicating that parental estimations can be reliably used to assess infants' speech variability at home. Finally, our results revealed a strong relationship between word recognition and comprehension in monodialectal infants and the absence of such a relationship in bidialectal infants, suggesting that either these two skills do not necessarily align in infants exposed to more variable input, or that the alignment might occur at a later stage. + +KEYWORDS +dialect, infant, language development, speech variability, word comprehension, word recognition This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. + +© 2022 The Authors. *Developmental Science* published by John Wiley & Sons Ltd. + +## 1 Introduction 1.1 Word Representations In Monodialectal Infants + +Human speech is an acoustic signal composed of bursts, hisses, vocalizations and voice modulations, that a child will learn to perceive as a sequence of meaningful units, for example, words (Gervain & Werker, 2008). This is a challenging task. First, in fluent speech, there are no systematic pauses between words to segment them. Second, and most important, words are produced differently, that is, have physically different acoustic signals, across speakers (Jaeger & Weatherholtz, 2016; Peterson & Barney, 1952), and contexts (Steinlen, 2005), meaning that there is no robust mapping between an acoustic signal and a word. In order to learn and recognize words efficiently, the child has to discover which acoustic detail allows to distinguish meaning in her language, that is, *lexically relevant cues* (e.g., changes in voice onset time to distinguish the English "bed" and "pet"), and which are not (e.g., genderrelated changes in pitch or affect-related changes in pitch amplitude). + +Adapting to speech variability requires considerable experience and language exposure. While 6- to 8-month-old infants recognize words produced by familiar speakers (Bergelson & Swingley, 2012, 2013; Tincoff & Jusczyk, 2012), they fail to recognize a newly learnt word, if it is produced in a different affect (Singh et al., 2004) or voice (Houston & +Jusczyk, 2000) from what was used during learning. These studies suggest that young infants stock in memory all acoustic details, between and within-talker, present in variable speech, even if they are lexically irrelevant. It is only by the end of the first year that infants start processing speech variability in adult-like way: they understand words across speakers (Houston & Jusczyk, 2000) and affects (Singh et al., 2004), and show reliable speaker-independent recognition of familiar words over nonsense words (Hallé & Boysson-Bardies, 1994). + +Yet, effective language use entails the ability to understand speakers across accents (e.g., dialects and foreign accented speech), who might differ in phonetic detail from the familiar (native) accent, but preserve the phonological structure of a word. For instance, the word "bat" is produced /bæt/ in South England but /bαt/ in England's East midlands regions. Adaptation to dialectal variation relies on phonological constancy, that is, the ability to disregard the phonetic (accented) variations and to capitalize on the phonological structure of the word in order to recognize it (Best et al., 2009). This ability has been shown to develop between 19 and 22 months of age (Best et al., 2009; van Heugten & Johnson, 2014), that is, almost 1 year after that infants start mastering word recognition in their native (familiar) accent, suggesting that adaptation to dialectal variability requires more robust and/or mature phonological representations of words. + +However, infants might show phonological constancy even earlier, at the age of 15 months, if they had been familiarized with the accent/dialect prior to the test (van Heugten & Johnson, 2014). Fifteenmonth old Canadian English-learning infants who heard a familiar story in unfamiliar Australian English accent showed listening preference for the known words as compared to the nonsense words, suggesting that they had recognized words produced in unfamiliar Research Highlights +∙ We examined the role of dialectal exposure from birth on early word comprehension and phonological word form recognition in 12-month-old Norwegian infants. + +∙ Unlike English and French peers, neither mono- nor bidialectal 12-month-old Norwegian infants recognized familiar words, suggesting cross-linguistic differences in the onset of word recognition. + +∙ Monodialectal, but not bidialectal infants showed evidence of word comprehension, suggesting that early exposure to dialects might affect word comprehension in bidialectal infants. + +∙ Performance in the word recognition and word comprehension tasks were highly correlated in monodialectal infants, but not in bidialectal infants. + +accent. Therefore, brief exposure to unfamiliar accented speech promotes the mappings between the sounds and the words in unfamiliar accent and in the native accent, and boosts infants' word recognition across accents (phonological constancy). Other studies have shown that speaker variability augments phonological processing in word learning in 14-month-old infants (Rost &McMurray, 2009). Given these results, one might expect that infants exposed to dialectal variability from birth should show reliable word recognition in familiar dialects. Yet, recent studies on multidialectal infants suggest that variability does not benefit phonological processing, but, on the other hand, hinders the establishment of stable word representations (Buckler et al., 2017; Durrant et al., 2015; Floccia et al., 2012; van Heugten & Johnson, 2017). + +## 1.2 Word Representations In Bidialectal Infants + +So far, by the time we submit the final paper, six studies1 − performed by two research groups − have examined early word knowledge in infants and toddlers naturally exposed to one versus two dialects (Buckler et al., 2017; Durrant et al., 2015; Floccia et al., 2012; van der Feest & Johnson, 2016; van Heugten & Johnson, 2017). The youngest age group examined so far refers to infants of 12.5-months of age. Van Heugten and Johnson (2017) assessed mono-dialectal and multidialectal infants' preference for familiar over nonsense words, using the Headturn Preference paradigm. The results revealed that 12.5- to 14.5-month-old infants exposed to multiple dialects (∼33% of exposure to Canadian English and ∼65% to other variants of English, which included foreign-accented English2) failed to show preference for familiar over nonsense words produced in the dominant regional accent (Canadian English), whereas infants receiving uniform input succeed in this task. The authors concluded that exposure to multiple accents impacts the precision/quality of early word representations, which are less constrained in 12.5- to 14.5-month-old multidialectal infants, as compared to infants receiving uniformly accented speech. + +Word comprehension studies in older bidialectal infants/toddlers, on the other hand, suggest that infants' word representations are dialect-sensitive and reflect the properties of the incoming speech +(Durrant et al., 2015; van der Feest & Johnson, 2016). At 20 months of age, bidialectal infants detect only those mispronunciations that concern the specific details that vary across dialects, as, for example, vowel mispronunciations for infants exposed to South and East England dialects, yet they disregard consonant mispronunciation (Durrant et al., 2015). At 24 months of age, infants show dialect-sensitive accurate knowledge of the phonological categories in both Dutch dialects: +multidialectal infants adapt their phonological expectations based on the phonetic contrastive cues relevant for a speaker's dialect (van der Feest & Johnson, 2016). So, the same type of mispronunciation, for example voicing of a devoiced fricative consonant, is detected as "error" in the dialect that maintains voicing contrasts, but is disregarded in the dialect that devoices fricatives (although see Floccia et al., 2012 for infants' preference for community dominant accent). These results concord with previous research in monodialectal and bilingual infants showing that infants are sensitive to fine-grained acoustic features of parental input (Cristia, 2011) and use them to build their phonemic representations (Bosch & Ramon-Casas, 2009, 2011). + +In sum, the results of a handful of available studies in bidialectal (English or Dutch learning) infants suggest that exposure to two dialects hinders the establishment of stable word form representations in young 12.5- to 14.5-month-old infants (one study only), but benefits later word processing, as 24-month-old bidialectal toddlers demonstrate dialect-specific sensitivity to the incoming speech and efficient word recognition (van der Feest et al., 2022).3 However, due to differences in task (word form recognition in 12.5- to 14.5-month-old infants and word comprehension and mispronunciation sensitivity in 20- to 24-month-old infants) and accent type (native vs. foreign) between studies, the developmental trajectory of word form recognition and word comprehension in bidialectal infants remains unclear. + +## 1.3 Factors Boosting Accent Adaptation + +Studies in monodialectal infants have shown that, under specific conditions, infants can show adaptation to an unfamiliar accent (including foreign) even before the critical 19 months of age (Best et al., +2009; Mulak et al. 2013; van Heugten et al., 2018). For example, 12and 15-month-old infants can recognize familiar words in unfamiliaraccented speech, if they have been familiar with this unfamiliar accent prior to the test (van Heugten & Johnson, 2014) or when the unfamiliar accent variant was very close to their native accent (Schmale et al., +2010), respectively. Is it possible that dialectal similarity (accent distance between dialects) and dialect (accent) exposure, factors facilitating accent adaptation in monodialectal infants, influence early language development in bidialectal infants who are naturally exposed to two dialects? + + AND **3 of 19** +Schmale et al. (2012) suggested that the amount of accent variability in language input can modulate infants' adaptation strategy when faced with unfamiliar dialects. The authors distinguished two adaptation strategies, lexical specific and general. Infants exposed to little inconsistency in dialectal input are likely to use lexical top-down strategy to accommodate accents (see also White & Aslin, 2011). Lexical strategy relies on clear evidence of differences in pronunciation between dialects (e.g., /bæt/ in South England but /bαt/ in England's East midlands regions) and is facilitated by the presence of visual cues (e.g., pictures of objects as in the word comprehension task). In line with this hypothesis, 12-month-old monodialectal infants have been shown to recognize familiar words produced in an unfamiliar accent minimally different from the native one (Schmale et al., 2010). In contrast, infants exposed to highly inconsistent input, when dialects differ along a number of dimensions, including segmental and suprasegmental cues, are likely to use a general expansion strategy, which may require an expansion (or relaxation) of phonemic categories in order to accommodate deviating examples (Schmale et al., 2012). The general expansion strategy might have negative consequences, as infants can erroneously accept close speech sounds as being members of the same category, for example, /sek/ and /sæk/ can be perceived as good candidates of +/sak/ "sock" (White & Aslin, 2011). In line with this hypothesis, 24month-old Canadian-English infants, exposed to at least one foreignaccented caregiver, showed delayed and less efficient word comprehension, as compared to infants exposed to uniform input (Buckler et al., 2017). These results align with previous research showing that inconsistencies in parental speech are inversely related to the accuracy of early phonemic representations in infancy and childhood (Bosch +& Ramon-Casas, 2009, 2011; Mayr & Montanari, 2015). Note that, while 18-month-old monodialectal infants tolerate some deviations in foreign-accented speech and use lexical strategy to recognize familiar word forms, they fail to recognize mispronounced versions of words produced in foreign-accented speech, suggesting that monodialectal infants do not accommodate (severe) phonological deviations (at least in the absence of a referent, as shown in van Heugten et al., 2018). In sum, although previous research suggests that the degree of dissimilarity in pronunciation between parental accents might modulate the quality of infants' early word representations, with highly dissimilar variants leading to coarser word representations, yet, to the best of our knowledge, no study, so far, has examined this question empirically. + +Studies in young monodialectal infants have shown that accent exposure can boost infants' adaptation to (unfamiliar) regional and foreign accents. For instance, as little as 2 min of exposure can enable monodialectal infants to segment and recognize words produced in unfamiliar accents (Schmale et al., 2012; van der Feest & Johnson, 2016; van Heugten & Johnson, 2014). In general, research in monodialectal infants has shown a linear positive relationship between the quantity of language input/exposure and infant's vocabulary size (Legacy et al., 2018; Rowe, 2012). Similarly, research in bilingual toddlers revealed a strong relationship between a child's early language development (vocabulary size and grammatical complexity) and the amount of input she receives in that language (Hoff et al., 2012). + +Interestingly, there was no relationship between these skills across 4 of 19 AND +languages (Hoff et al., 2018), suggesting that input contribution is language-specific.4 Yet, studies in bidialectal infants showed either no relationship between the amount of dialectal exposure and infants' ability to recognize (van Heugten & Johnson, 2017) and understand words in this dialect (Buckler et al., 2017; Durrant et al., 2015), or infants' indifference to the frequency of accent exposure, with better word recognition in a community dominant accent (Floccia et al., 2012). + +Therefore, more research is needed to understand the role of dialect exposure in bidialectal infants' word form recognition and word comprehension abilities. + +## 1.4 The Current Study + +The current study fills a gap in bidialectal developmental literature and examines word recognition and word comprehension in 12-monthold Norwegian-learning infants exposed to two Norwegian dialects spoken by native speakers of Norwegian. To the best of our knowledge, this is the first study that examines language development in Norwegian infants exposed to dialects, which is very common in Norway. The first aim of our study is to replicate, conceptually, in Norwegian infants exposed to bidialectal Norwegian speech, the results of van Heugten and Johnson (2017) study with multidialectal Canadian– +English infants (one third of them were exposed to foreign-accented English). The second aim of our study is to extend our knowledge on early language development in multidialectal infants by directly examining their comprehension of familiar words (including the temporal dynamics analysis), using an eye-tracker and an intermodal preferential looking (IPL) task, and comparing it to their word recognition skills. + +Although both tasks share a recognition component, they do not tap into the same processes.While word recognition informs about infants' ability to recognize familiar word forms over similar-sounding unfamiliar ones (infants' development of sensitivity to phonological distinctiveness, Best et al., 2009), word comprehension task provides insights into infants' development of word-object mappings (lexico-semantical knowledge). Therefore, successful word recognition (preference for familiar words) might operate in an absence of an established mapping between this word and its referent; likewise, successful word comprehension might operate in an absence of accurate word phonological representation, as infants can use semantical/contextual (Bergelson & Aslin, 2017a, 2017b), perceptual (Arias-Trejo & Plunkett, 2010), and frequency (Kartushina & Mayor, 2019) cues to disambiguate between objects and show word comprehension even when items are mispronounced (Mani & Plunkett, 2010). To the best of our knowledge, the current study would be the first to examine the relationship between word recognition and word comprehension abilities in 12-month-old infants. Importantly, the third aim of the study is to assess, for the first time, the role of dialectal similarity and dialectal exposure on infants' ability to recognize and understand words. + +In Oslo and its surroundings, it is not uncommon that Norwegian parents speak different dialects to their infants (around 50% of families who took part in previous research), providing thus rich but often inconsistent linguistic input as compared to infants growing up in monodialectal families. For instance, a child growing up in a bidialectal family has to learn, similarly to monodialectal Norwegian infants, that some differences in sound production between parents are languagespecific and lexically relevant (e.g., changes in vowel height /y/-/ø/, as in lys "light" vs. løs "loose" and changes in vowel length /ɑ/-/ɑ:/, as in *takk* +"thanks" vs. tak "roof"). However, in addition, they are to discover that some of these differences are dialect-specific and lexically irrelevant +(e.g., trill or tap /ɾ/ in Eastern dialect (Oslo region) vs. uvular /ʁ/ inWestern dialect [e.g., Bergen], so /ɡoːɾ/ vs /ɡoːʁ/ refer to the same word gård "farm"). Bearing in mind that there are also differences in the use of lexical and phrasal tones between some Norwegian dialects, retrieving lexically relevant cues and building representations for words might become a very challenging task for a bidialectal Norwegian child. + +In the current study, we examined word recognition and word comprehension in two groups of Norwegian infants: monodialectal infants receiving similar input from both parents speaking the Oslo (Eastern) dialect, and bidialectal infants exposed to the Oslo dialect and to a different type of Norwegian dialect (that can belong to one of the remaining three group-types of dialects: Western, Central, and Northern). All four types of dialects are mutually intelligible, but clearly recognizable even by an untrained ear for their differences at segmental and suprasegmental levels, that is, the phonetic realization of a number of sounds (or their omission) and the use of lexical pitch accents5 (Johnsen, 2012; Kerswill, 2016; Mæhlum & Røyneland, 2012; Røyneland, 2009), but also for their differences at morpho-syntactic (in particular related to differences in gender attribution for words) and lexical levels. Word recognition was tested using the Visual Fixation paradigm (as in Frank et al., 2020), where infants heard, in a random order, eight lists of familiar and nonsense words. Longer listening times to word lists were interpreted as evidence for listening preference driven by the recognition of familiar word forms. Word comprehension was tested using the IPL task (as in Bergelson & Aslin, 2017b; Bergelson & Swingley, 2012), that has recently been adapted for Norwegian and tested with 6- to 9-month-old infants (Kartushina & Mayor, 2019). On each trial, infants saw two pictures of familiar objects on the screen and heard a sentence "Look at the 'target'!," where the target was the label of one of the objects. Longer looking times at the target as compared to the distractor were interpreted as evidence for word comprehension. + +The following hypotheses were considered for monodialectal infants. Although, to date and to the best of our knowledge, no study has examined word recognition and word comprehension in Norwegian 12-month-old infants, data from parental reports suggest that having more lexically relevant cues (as compared to English, Norwegian has additional vowel lengthening and pitch modulation) does not delay word acquisition in Norwegian infants. A comparison of the median number of words understood by 12-month old Norwegianand American–English-speaking infants, as reported by their parents using Communicative Development Inventories (CDIs; retrieved from wordbank.stanford.edu, see Frank et al., 2017; Kristoffersen & Simonsen, 2012; Simonsen et al., 2014), reveals comparable scores: 64 words for Norwegian and 77 words for English. Therefore, we expected that monodialectal Norwegian infants, similarly to British English +(Vihman et al., 2004) and French (Hallé & Boysson-Bardies, 1994) +infants, would show reliable listening preference for words over nonsense words and would show word comprehension for familiar words used in the IPL task, similarly to English-learning 12-month-old infants (Bergelson & Swingley, 2015; van Heugten & Johnson, 2017). + +The following hypotheses were considered for bidialectal infants' performance in word recognition and word comprehension tasks. First, if exposure to accent variability hinders the establishment of accurate +(phonological) word representations, as reported in van Heugten and Johnson (2017) study with multiaccented infants, then bidialectal Norwegian infants, exposed to two dialects spoken by native Norwegian speakers, would show no preference for words over nonsense words +(H0).6 However, if we do observe word recognition in our bidialectal infants (H1), then we would conclude that exposure to accent variability, present in families speaking two Norwegian dialects natively, does not delay infants' early word representations (development of phonological constancy). In addition, following Schmale et al. (2012) +hypothesis on the role of accent similarity in monodialectal infants' accent adaptation, we predicted that bidialectal infants exposed to similar dialects would show better word recognition as compared to bidialectal infants exposed to dissimilar dialects. This result would suggest that large inconsistencies between parental dialects lead to broader or coarser phonological representations for words. Finally, we also predicted that the amount of exposure to the target (Oslo) dialect would modulate infants' word recognition accuracy, with more exposure leading to better word recognition. + +Second, we hypothesized that, in the word comprehension task, bidialectal infants would orient their gaze to the target object (H1) +after hearing its name, demonstrating that bidialectal infants' word representations were precise enough to disambiguate between two familiar, phonologically dissimilar words (e.g., "foot" and "banana"), when visual cues are present (top-down lexical facilitation). However, if bidialectal 12-month-old infants fail to display word comprehension (H0), we would conclude that regular exposure to accent variation in parental speech (i.e., inconsistent word pronunciations between speakers' dialects) weakens/delays the establishment of word - object associations required for successful word comprehension in naturalistic listening conditions. In addition, we expected that the degree of perceptual similarity between parental dialects would not affect infants' word comprehension, given that visual cues facilitate object disambiguation (even when the accented pronunciation of a word deviates from the target, van Heugten et al., 2018), yet, we predicted that infants exposed to the target dialect more frequently would show better word comprehension. + +Finally, given the lack of studies on the relationship between word recognition and comprehension in 12-month-old infants, two possible outcomes were considered in the current study. First, word comprehension and word recognition might follow distinct developmental patterns. This possibility stems from research showing that infants can map a word to its referent from as early as 6 months of age (at least for some familiar items as, e.g., bottle, banana, hand, see Bergelson & Aslin, 2017b; Bergelson & Swingley, 2012; Tincoff & Jusczyk, 1999), whereas they fail to recognize familiar word forms before the age of 11 months (Carbajal & Peperkamp, 2017; Hallé & Boysson- + AND **5 of 19** +Bardies, 1994; Vihman et al., 2004, 2007). These studies suggest that infants can disambiguate between two familiar items (likely by relying on other available cues, as, e.g., semantic, conceptual, perceptual, and frequency cues, see Kartushina & Mayor, 2019) despite the lack of a phonologically detailed word form representation (required to succeed in the recognition task). Yet, an alternative outcome can also be expected. A recent study suggests that infants' word knowledge contributes to their development of the phonetic categories (Swingley & +Alarcon, 2018), implying a tight relationship between lexico-semantical and phonological abilities in infants (see also Swingley, 2009). Similarly, Van Heugten and Johnson (2017) revealed a positive relationship between early lexico-semantical and phonological abilities in 18month-old bidialectal infants by showing that the vocabulary size (as revealed by parental reports in the CDI) predicted their performance in the recognition task. In line with these studies, we expected a positive correlation between word comprehension and word recognition abilities, with word comprehension performance being superior or equal to word recognition. + +## 2 Methods 2.1 Participants + +Word recognition at 11–12 months of age tested with eye-tracking paradigms has an effect size of Cohen's d ranging between 0.59 and 0.777 (Bergmann et al., 2018); word comprehension at 12 months of age assessed with IPL paradigms has an effect size of Cohen's d = 0.64 +(Bergelson & Aslin, 2017a). To detect the smallest effect size (0.59) with a power of 0.80, we needed twenty-five participants in each group (Faul et al., 2009). However, in order to make sure that the design was powerful enough to detect a potential interaction between the trial type and infant group, we recalculated the sample size based on the interaction effect size ηp 2 = 0.106 reported in van Heugten and Johnson's (2017) +study. To reach 80% power, we needed seventy participants (n = 35 in each group). + +Infants were recruited from the National Registry (Folkeregister). + +The following criteria were used to include infants: (1) the child was born full term (gestational weeks > 37); (2) the child is exposed to 90% +Norwegian or more at home; (3) both parents speak Norwegian to the child; and (4) the child has no developmental delays and no history of chronic ear infections. Total 111 parents expressed their interest in taking part in the study and filled in the consent form. All children were born full term, with no history of hearing and/or language disorders. + +Among those, 23 participants were excluded from the study: 16 parents canceled their appointment, due to a child's sickness or a Covid19related quarantine; one parent did not fill in participant background questionnaire; four babies cried during the experiment and were not able to provide data for both tasks, so the experiment was stopped, and two babies had less than 90% of exposure to Norwegian. + +Among the remaining 88 participants who completed both tasks, 16 were excluded in the word recognition task and 12 were excluded in the word comprehension task, as they did not validate at least half TABLE 1 Participants who have successfully completed both tasks, final sample + +| Receptive vocabulary mean (sd) | | | | | +|----------------------------------|--------|------------|-----------|---------| +| Monodialectal | 35 | 18/17 | 367 (7.0) | 72 (67) | +| Bidialectal | 35 | 13/22 | 368 (6.5) | 73 (55) | +| Age in days | | | | | +| Group | Number | Boys/girls | mean (sd) | | + +of the trials in each task, after that we applied by-trial exclusion criteria (cf. Section 2). Among the remaining 72 participants in the word recognition task and 76 participants in the word comprehension task, 70 participants, which was our required sample size, successfully completed both tasks (see Table 1 for details). There were no differences in receptive vocabulary size (p = 0.9), as measured by the Norwegian version of the CDI for 8- to 16-month-old infants (Simonsen et al., 2014), +or age (p = 0.6) between the two groups. Half of them (n = 35) were selected from monodialectal families where both parents speak Oslo (Eastern) dialect, whereas the other half from bidialectal Norwegian families, where parents speak two different dialects among four large groups of Norwegian dialects: Eastern, Western, Central, or Northern Norwegian. + +The inclusion criteria in the bidialectal group was a minimum 30% +of exposure to each dialect8; that was assessed by the language background questionnaire filled in by parents before their visit to the lab (see Section 2.3 for details). The study has been approved by the Norwegian Centre for Research Data (NSD). + +## 2.2 Stimuli 2.2.1 Word Recognition Task + +The stimuli were prepared closely following the methods used in van Heugten and Johnson studies (2014, 2017).We have selected 12 familiar words (ball, bade, bil, drikke, pappa, bleie, hund, klemme, katt, takk, mamma, vann; ball, bath, car, drink, father, diaper, dog, hug, cat, thanks, mother, water) and have created twelve nonsense words strictly matched in phonemes, onset, rhyme, and the number of phonemes +(*brall, made, dil, klakke, tippa, keie, ban, blamme, patt, hokk, vumma, benn*). + +On average, 70% of 12-month-old Norwegian infants know the 12 selected familiar words (Simonsen et al., 2014). Note that all familiar words used in the study have a very similar segmental pronunciation across the four Norwegian dialects, with the exception of *drikke* that has a guttural "r" in the Western dialect. + +A native Norwegian female speaker, who was born and grew up in Oslo (Eastern dialect), was recorded while reading the stimuli in a child-directed speech style using the following recording parameters: +44 kHz, 16 bits, two channels. Known and nonsense words were read by pairs in order to match the intonation pattern between them. The recordings were checked for auditory quality and saved to individual wav files. Some residual noise in the recordings was removed in Praat (Boersma & Weenink, 2020) using the *remove noise* function (window length 0.025 s, filter 80–10 kHz, smoothing bandwidth 40 Hz, and spectral subtraction noise reduction method). Known and nonsense words were closely matched for average length (723 and 748 ms, p = 0.39), +average pitch of the first vowel (208 and 206 Hz, p = 0.76) and average pitch of the second vowel (253 Hz and 247 Hz, p = 0.41). All twentyfour word types were equated for amplitude: the mean amplitude was set to 70 dB. We created eight word lists, four for each word type. + +Within each list, each word was repeated twice, resulting in 24 words; their order differed between lists. Within each list, words were interspersed with 740–750 ms silences; all lists last 35.09 s. The word lists +(four known and four nonsense word lists) were combined pseudorandomly to create four presentation orders, with the restriction that lists of the same word type could not occur more than twice in a row (see https://osf.io/6btq2/). + +## 2.2.2 Word Comprehension Task + +The stimuli for the word comprehension task were similar to those used in a recent study (Kartushina & Mayor, 2019). Sixteen pictures, depicting familiar objects were used in this task (for detailed description refer to Kartushina & Mayor, 2019), see https://osf.io/6btq2/. Pictures were assembled in eight picture pairs (see Figure 1). The objects within each pair were edited so that their relative brightness and size were approximately the same. The eight picture-pairs were laid out on a light-gray background with the size matching the experimental screen (as shown in Figure 1). An additional set of eight picture-pairs was created by switching the sides of the objects within each pair to counterbalance the side of object presentation, resulting in 16 picture pairs, in total. + +Sixteen audio files (described in Kartushina & Mayor, 2019) were used to prompt infants' looks at the target. A native female speaker of Eastern Norwegian dialect (different from the one used in the word recognition task) was recorded while reading at a slow speed and in a child-directed fashion four types of sentences: "Can you find the < target > ?," "Where is the < target > ?," "Do you see the < target > ?" +and "Look at the < target > !." The target words were the 16 labels of the items depicted on the pictures. The same sentence-frame was used for the two words within a pair (e.g., "Look at the apple!" and "Look at the foot!"). Therefore, each type of sentence was paired with two picture pairs. The following parameters were used for recordings: 16 bits, 2 channels, 44 kHz. Similar to word recognition task, the stimuli average amplitude was equalized and was set to 70 dB. In line with previous research (as in Bergelson & Swingley, 2012), a 1.5 s silence was added before the sentence onset. Trials ended 3.5 s after the target word onset. The length of the sentences varied from 5.4 to 6.2 s (m = 5.7 s). + +In order to fully counterbalance the side of picture presentation, audio files (n = 16) and picture pairs (n = 16) were combined to create 32 video files (to run in Tobii TX300 eye-tracker). That is, infants heard each target word twice: once when the target picture is on the left side and once when the target picture is on the right side of the screen. Four presentation order lists were created, with the restriction that there are at least two different picture pairs between two similar picture AND **7 of 19** + +![6_image_0.png](6_image_0.png) + +pairs (e.g., "apple-foot," "hair-banana," "bread-leg," "foot-apple"). Note that the target words used in the study have a very similar segmental pronunciation across the four Norwegian dialects, with the exception of *brillener* glasses*, brød* bread *and hår* hear that have a guttural "r" in the Western dialect (trill in other dialects) and *bein* leg that is produced with an [e] in Western dialect ([a] in other dialects). + +## 2.2.3 Language Background Questionnaire + +Infants' exposure to each dialect was collected from the Language Background questionnaire that parents filled in prior to their lab visit (see https://osf.io/6btq2/). To measure dialectal exposure, we asked parents to indicate, how much time (in %) the child heard her mother's and her father's speech. The sum should equal 100%. In addition, parents were asked to fill in a word knowledge questionnaire. This questionnaire asked, for each of the twenty words used in the word comprehension and in the word recognition tasks (three of them are shared between the two tasks), how frequently parents would have used it (on a scale from 0-never to 5-very frequently) while interacting with the child or in her presence, since their baby was born. + +## 2.2.4 Perceived Similarity Between Parental Dialects + +As far as the dialectal similarity is concerned, to the best of our knowledge, there is no objective (global) measure allowing to quantify relative perceptual difference/distance between two (Norwegian) +dialects. However, Norwegian speakers are quite aware of the differences between dialects: after a brief exposure to an unfamiliar speaker, they can easily name her/his accent and indicate the region of Norway where the speaker grew up. + +Dialectal similarity in parental speech was assessed by an independent group of 40 raters (all native Norwegian speakers). To record natural (spontaneous) parental speech, at the end of the language background web-questionnaire, parents were prompted to click on a link that redirected them to an experimental platform e-Babylab (Lo et al., submitted)—a web application developed based on the framework provided in Frank and colleagues study (Frank et al., 2016)—where each parent was shown five simple pictures (selected from the CELF battery used to assess language production and comprehension, Wiig et al., +2013) and was asked to describe9 each of them by answering one question (Hva gjør jenta/gutten? - "What is the girl/boy doing?"). The parents were instructed to give a subject-verb-object type answers (e.g., "the girl opens the door," "the boy reads a book," etc. see "Pictures to record parental speech" on https://osf.io/6btq2/). Audio recordings from both parents were combined into pairs, such that each family contributed to five recording pairs. Native Norwegian speakers were instructed to rate the overall/holistic similarity between the two dialects in a pair on a scale from 1-very similar to 10-very distinct (similar to the scale that parents used to rate dialect similarity in the family) by pressing a corresponding key touch on a key board. On each trial, raters were asked to listen to five audio recordings, each of them containing the same sentence produced by both of the child's parents (e.g., Gutten leser en bok - "The boy is reading a book"). Participants could listen to the recordings as many times as they wanted, yet they were asked to make an assessment of dialectal similarity based on their first holistic impression. The experiment was run on a university platform via e-Babylab (Lo et al., 2021), raters' recruitment was implemented on https://www.prolific.co/. Raters were paid around 18 euros for that task. To facilitate the assessment, we provided an example of two very distinct dialects (Stavanger and Tromsø, who are dissimilar in a number of phonological, i.e., segmental, prosodic and suprasegmental, and lexical cues) and two very similar dialects (Oslo and Hamar). + +## 2.3 Procedure + +Data collection were performed in one session at the BabyLing laboratory, at the University of Oslo, equipped with an Eye-link 1000 eyetracker.10 Prior to the lab visit, parents received an information letter, briefly presenting the aims of the study, and filled in three questionnaires (see https://osf.io/6btq2/), that is, a language background and parenting attitudes questionnaire, and the Norwegian version of the McArthur Communicative Development Inventory (Simonsen et al., 2014), using online forms provided for the academic use by the University of Oslo, https://nettskjema.uio.no/ (similar to Qualtrics). A female 8 of 19 AND + +## 2.4 Eye-Tracking Data Processing 2.4.1 Exclusion Criteria + +native speaker of Norwegian received parent(s) with their child in the reception room of the lab and briefly explained the tasks that the child would perform. Then, the parent signed the consent form and the experimenter accompanied her to the eye-tracking room with her child. + +Both tasks were performed on an Eye-link 1000 eye-tracker, 500 Hz sampling rate (monocular), infant mode calibration and a 1280 × 1024 pixels screen resolution. The child was sitting on her parents' lap facing the experimental computer screen fitted with an eye-tracker base. Parents wore sound-attenuating headphones through which they heard masking music (a custom blend of instrumental music and a pastiche of randomly timed and random amplitude stimulus materials from Frank et al., 2020). The parents were asked not to talk to the child, point to the screen or shift their bodies. The experimenter was sitting in the same room, behind the parent, so neither the child nor the parents saw her. The experimenter was wearing noise-attenuating headphones presenting the same masking music, as for the parents. The stimuli were presented at the average amplitude of 65 dB through two speakers, positioned at the left and right sides of the screen. The experimenter was able to monitor infants' looking behavior via the control screen, situated in front of the experimenter. The test started with an automated 5-point calibration procedure (slow version), which was followed by the tasks. The order of the tasks was counterbalanced across participants. All infants were tested in both word comprehension and word recognition tasks, with a break between them, when required. For both tasks, infants were assigned, randomly, to either of the four presentation order lists. Both tasks were gaze-contingent automatically monitored by the eye-tracker. + +In the word recognition task, at the beginning of each trial (eight in total), a spinning circle (accompanied by a tinkle) appeared, on a black background, in the middle of the screen. As soon as the infant fixated the circle for 500 ms, the program initiated the first word list in the presentation order, together with an image of a colorful checkerboard on the screen. The list was played until the infant looked away for 2 s or until the maximum trial length of 35.09 s (stimulus duration) was reached (van Heugten & Johnson, 2017). Then, next word list from the presentation order was initiated. The task stopped after all eight lists had been presented to the child. + +In the word comprehension task, at the beginning of each trial (32 in total), a spinning flower (accompanied by a bird tweeting) appeared, on a gray background, in the middle of the screen and was replaced by the test stimuli after that the child fixated the flower for 500 ms. On each test trial, infants saw two pictures displayed on the right and left sides of the screen and heard, after 1.5 s, a target sentence prompting them to look at either of the two pictures. The pictures remained on the screen for 3.5 s after the target-word onset. The trials were triggered automatically by the eye-tracker. The task stopped after that all 32 trials were presented to the child. + +At the end of the experiment, parents were able to choose a small gift for their infant (e.g., a toy) and were reimbursed for travel costs. The experimental protocols and the materials can be found on the Open Science Framework depository https://osf.io/6btq2/. + +The following criteria (as in Kartushina & Mayor, 2019) were used to exclude infants based on their behavior in both tasks: (a) failed calibration of the eye-tracker; (b) software problem (e.g., technical reasons: software stops displaying images or playing sounds for more than 50% of the trials); (c) the child did not contribute to at least 50% of the experimental data (Bergelson & Aslin, 2017a; Frank et al., 2020). + +The following criteria were used to exclude single trials in the word recognition task: trials with no continuous looking at the screen for at least 2 s during the test trial (Frank et al., 2020). The following criteria were used in the word comprehension task: first, no continuous looking at either image for at least 0.5 s in the postnaming period and, second, no looking was recorded in the prenaming period, due either to the child not looking at the pictures or to the eye-tracker's failure to track the child's gaze (Bergelson & Aslin, 2017a, 2017b). In addition, similar to Tincoff and Jusczyk (2012) study, individual item trials were removed from individual child data if parents reported in the word knowledge questionnaire that they did not use the produced word with the child (or in her presence), since the child was born. Finally, for both tasks, we excluded trials in which the experimenter would have reported that the parent interfered (e.g., pointed to the screen, shifted his/her body, or moved his/her chair), or the trial was interrupted by a third person or due to a technical error. If the experimenter heard audible crying then she terminated the experiment. + +## 2.4.2 Dependent Measures Word Recognition Task + +Our dependent measure was looking time (LT), defined as time spent fixating the screen (the area of the checkerboard) during the test trials, excluding the time when the child looked away from the screen even though the looks away were below the threshold for terminating a trial (Frank et al., 2020). Given that looking times were not normally distributed, we log transformed them prior to the analyses (Csibra et al., 2016). + +## Word Comprehension Task (1) Mixed-Effects Regression Analysis + +Similar to previous research (Bergelson & Swingley, 2012), on each trial, we identified two naming windows: prenaming (from the start of the trial to the target word onset) and postnaming (367–3500 ms after target onset). The target and the distractor areas of interest were limited to an invisible 800 × 680 pixel rectangle around each object. + +Our dependent measure was a baseline-corrected proportion of target looking (*Prop_target*). Similar to previous research (e.g., Bergelson +& Aslin, 2017a), it was computed by subtracting the proportion of time that infants looked at the target at prenaming window from the proportion of looking time at the target during the postnaming period (postnaming_T/(postnaming_T + postnaming_D) - (prenaming_T/(prenaming_T + prenaming_D)), T = Target, D = Distractor). + +Therefore, the dependent measure Prop_target varied between –1 and 1; the chance level was 0. + +(2) Cluster permutation analysis To provide insights into the differences (if any) in dynamics of word comprehension in bidialectal as compared to mono-dialectal infants, we performed cluster-permutation analyses. The dependent measure, the target proportion looking metric *Prop_target_bin*, was computed, in 10 ms time bins, as the proportion of target looking +[target/(target+distractor)] from the beginning of the trial to its end. + +## 2.5 Dialectal Similarity And Language Exposure + +In total, raters assessed the similarity between parental dialects in 76 family dyads (the highest number of participants successfully completing at least one task). There were 80 trials (note that for three parents, recordings were very noisy, thus impossible to assess perceptually), divided into two lists with 40 trials in each, plus four control trials +(recorded stimuli by our research assistant presenting clear-cut dialectal differences in each of them).11 It took around 40 min to complete one list. Similarity scores for five sentences from the rating task were averaged across raters to obtain one similarity score between parental dialects for each infant. The *similarity* score varied between 1: very similar and 10: very distinct. For the statistical analyses, the similarity score was recoded (1 = 0, 10 = 9). There was one similarity measure per infant. Bidialectal infants' *exposure* to Oslo dialect was collected from the language background questionnaire; it varied between 0% and 70%. + +## 3 Results + +All analyses were performed in R (R Core Team, 2020) using the lme4 package (Bates et al., 2015); p-values were computed using the lmerTest package (Kuznetsova et al., 2017). Lsmeans (Lenth, 2016) function in R was used to perform follow-up tests from the interactions, if needed. The package *sjPlot* was used to visualize the output of the regression models (Lüdecke, 2021). + +## 3.1 Assessment Of Perceived Similarity Between Parental Dialects + +Forty native Norwegian speakers (20 for each list) rated the perceived similarity between parents' dialects. Similarity scores for each family (n = 70 in the final sample) were averaged across 20 participants +(mean = 4.0, range between 1.25 and 8.4) and compared to the perceived similarity reported by parents (mean = 3.9, range between 1 and 10). A correlation analysis revealed a significant positive relationship between the assessment of dialect similarity reported by the parents and by the raters (r = 0.83, t = 12.25, df = 67, p < 0.001). + +TABLE 2 Summary of the mixed-effect regression model run on + +fixation time for the word recognition task + +| fixation time for the word recognition task log(Fixation) Predictors Estimates | CI | p | | +|----------------------------------------------------------------------------------|------------|----------------|--------| +| (Intercept) | 9.88 | 9.69 to 10.06 | <0.001 | +| Trial | −0.09 | −0.13 to −0.05 | <0.001 | +| Type [nonwords] | −0.09 | −0.26 to 0.09 | 0.331 | +| Group [Bidialectal] | −0.07 | −0.30 to 0.16 | 0.544 | +| Trial * Type [nonwords] | 0.04 | −0.01 to 0.09 | 0.155 | +| Trial * Group [Bidialectal] | 0.03 | −0.02 to 0.08 | 0.201 | +| (Trial * Type [nonwords]) * | −0.03 | −0.08 to 0.02 | 0.298 | +| Group [Bidialectal] | | | | +| Random effects σ2 | 0.30 | | | +| τ00 ChildID | 0.10 | | | +| τ00 List | 0.00 | | | +| NChildID | 70 | | | +| NList | 8 | | | +| Observations | 474 | | | +| Marginal R2/ | 0.074 / NA | | | +| Conditional R2 | | | | + +## 3.2 Word Recognition + +Following preregistration, we performed mixed-effects regression analyses that included the main effects of trial type (known vs. nonsense word), group (mono- vs. bidialectal), and trial number, capturing the basic effects of each on looking time (we expected longer looking times for known words, possibly longer looking times in monodialectal group and shorter looking times on later trials). In addition, we included two-way interactions of trial type with trial number (to model faster habituation to nonsense words) and of trial type with group (to model possible longer looking times for known words in mono-dialectal infants as compared to bidialectal), and one three-way interaction between trial type, group, and trial number (to model possible faster habituation to nonsense words in bidialectal infants). The random structure included by-subject intercept (a model with a slope did not converge) and by-item (list) intercept. Trial number was recoded to range from 0 to 7 (instead of 1 to 8). Trial type was dummy-coded, with real word trials as the reference level; monolingual infants were set as the reference level for group. + +As can be seen in Table 2, there was a significant effect of trial, indicating that infants looked to the screen less by the end of the study +(cf. Figure 2). Other factors were not significant. Hence, the results of this model indicate that: (1) 12-month-old Norwegian infants did not show preference for listening familiar words as compared to nonsense nonwords (Bayes Factor t-test analysis confirmed that our data presented no evidence in favor of word recognition in Norwegian 12month-old infants, BF = 0.1), (2) overall, there were no differences in looking behavior between monodialectal and bidialectal infants; and, + +![9_image_0.png](9_image_0.png) + +importantly, (3) there were no difference in looking behavior between monodialectal and bidialectal infants for either words or nonwords, that is, nonsignificant trial × group interaction. + +As preregistered, we performed mixed-effects regression analyses to examine the role of dialectal similarity and exposure to the dominant (Oslo) dialect in bidialectal infants' early word recognition. The model was similar to the one used in the above analyses on word recognition, with the following differences: the factor *group* was omitted from the model while *perceived similarity*, as rated by native Norwegian speakers (which was recoded to range from 0 to 9), the categorical variable exposure12 - and their interaction with trial type (to model possible worse word recognition in infants exposed to very dissimilar dialects and only little to the Oslo dialect) were included in the model. The random structure was identical to the original model (i.e., by-child and -list intercepts). The results are summarized in Table 3. Similar to previous analyses, there was a significant effect of trial. In addition, there was a marginally significant effect of exposure, suggesting that bidialectal infants with no exposure to the Oslo dialect at home listened to word lists less than those who were exposed to the Oslo dialect at home. A +marginally significant effect of rated similarity suggests that bidialectal infants exposed to more dissimilar dialects at home displayed less interest in listening to words. + +## 3.3 Word Comprehension + +First, before running the analyses, we removed individual trials where the target word was reported as never used in a child's presence by the parents. Then, we fitted, as preregistered, mixed-effect regression analyses on the dependent measure Diff_prop_looking (cf. Section 2 for details), with the fixed factors group (monodialectal and bidialectal) and trial number, mean-centered (modeling possible shorter looking times on last trials); the random structure included by-subject and by-item (picture) intercepts. The results of the model are summarized in Table 4. + +As can be seen, there were no differences between the two groups of infants and the effect of trial was not significant either. However, the intercept of the model approached significance, suggesting that monodialectal infants had larger proportion of looks to the target as compared to the distractor, at the reference level of trial (mean), hence, displaying evidence of word comprehension (cf. Figure 3). Follow-up analyses showed a significant difference from zero in monodialectal infants (m = 0.026, t = 2.26, df = 34, p-value = 0.015) and no difference in bidialectal infants (m = 0.003, t = 0.25, df = 34, p-value = 0.40. + +In sum, the results show that while 12-month-old Norwegian infants exposed to one dialect at home looked significantly above chance at the target when hearing the matching label as compared to when hearing the name of a distractor (evidence of word comprehension), bidialectal infants, on the other hand, did not show evidence of word comprehension. Yet, the difference between monodialectal and bidialectal infants was not significant. The intercept only model (as per preregistration) +was not significant (β = 0.015,se = 0.009, df = 19.6, t = 1.52, p = 0.145). + +Similar to the analyses of word recognition, and as preregistered, mixed-effects regression analyses were performed to examine the role of dialectal similarity and exposure on bidialectal infants' early word comprehension. The model was similar to the one used in the above analyses of word comprehension, with the following, preregistered, differences: we removed the factor *group* and added a continuous variable *similarity* (from 0 to 9) and a categorical variable *exposure*, and their interaction. The results revealed marginally significant intercept + +TABLE 3 Summary of the mixed-effect regression model run on + +fixation time in the word recognition task, for bidialectal infants + +| fixation time in the word recognition task, for bidialectal infants log (Fixation) Predictors Estimates CI p (Intercept) 10.32 9.78 to 10.87 <0.001 Trial −0.06 −0.10 to −0.02 0.002 Type [nonwords] −0.14 −0.65 to 0.36 0.572 Rated_sim_zero −0.11 −0.24 to 0.01 0.070 Exposure_binom [No] −0.88 −1.80 to 0.03 0.059 Trial * Type [nonwords] 0.01 −0.05 to 0.07 0.706 Type [nonwords] * Rated_sim_zero −0.01 −0.12 to 0.10 0.904 Rated_sim_zero * Exposure_binom [No] 0.19 −0.03 to 0.40 0.097 Type [nonwords] * 0.51 −0.30 to 1.33 0.217 Exposure_binom [No] (Type [nonwords] * Rated_sim_zero) * Exposure_binom [No] −0.07 −0.27 to 0.12 0.460 Random effects σ2 0.27 τ00 ChildID 0.12 τ00 List 0.00 NChildID 35 NList 8 Observations 238 Marginal R2/ 0.138/NA Conditional R2 | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +TABLE 4 Summary of the mixed-effect regression model run on + +difference in looking proportion for the word comprehension task + +| difference in looking proportion for the word comprehension task Diff_prop_looking Predictors Estimates CI p (Intercept) 0.02 −0.00 to 0.05 0.053 Group [Bidialectal] −0.02 −0.05 to 0.01 0.235 Trial_c 0.00 −0.00 to 0.00 0.138 Random effects σ2 0.10 τ00 ChildID 0.00 τ00 trial 0.00 ICC 0.02 NChildID 70 Ntrial 16 Observations 1956 Marginal R2/ 0.002/0.018 Conditional R2 | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +## And 11 Of 19 + +and effect of rated similarity, suggesting that both bidialectal infants exposed to the dominant accent at the similarity reference level of zero, that is, very similar, and infants exposed to similar dialects showed marginal word comprehension (see Table 5). + +An exploratory analysis with the fixed factors similarity, groups and their interactions, and a random structure identical to the above analyses, examined the role of dialect perceived similarity on infants' word comprehension in both groups (Table 6). As expected and revealed by a significant group by similarity interaction, given that monodialectal infants are raised in a homogeneous dialectal home environment, monodialectal infants' word recognition was not affected by similarity between parents' dialects, Spearman r = 0.20, p = 0.12, whereas bidialectal infants' performance was negatively affected by an increase in dissimilarity between parental dialects, Spearman r = −0.28, p = 0.05 +(see Figure 4 for results in perceived similarity between parents' dialects, as reported by native Norwegian speakers and parents themselves). + +## 3.4 Time-Course Analysis Of Looking Behavior In The Word Comprehension Task + +As per registration, we performed three cluster permutation analyses on looking preference to examine the dynamic of word recognition in monodialectal and bidialectal infants and reveal their potential differences. For that, we compared the average proportion of looks at the target object to 50% (the chance) (1) in monodialectal infants, (2) +in bidialectal infants, and (3) between monodialectal and bidialectal infants. Similar to previous research (Dautriche et al., 2018), averaged Prop_target_bin scores were transformed via the arcsin square function to align with the t-test assumptions. Time bins with significant effects (t > 1.69 for one-tailed test with 35 subjects, p < 0.05) over a window from −350 to 3500 ms were grouped into a cluster. Then, we computed the size of the cluster, as the sum of all t-values within this cluster and evaluated, by simulation, the probability of observing a cluster of the same size by chance. For that, we ran 1000 simulations, where the type of word, target versus chance (for the analyses [1] and [2]) and monodialectal versus bidialectal (for the analysis [3]) were assigned randomly for each infant. Then, for each simulation, we computed the size of the biggest cluster using the same procedure as the one that was used to compute the size for the real data. If the probability of observing a cluster of this size, or bigger, was smaller than 5% (p < 0.05) in the simulated dataset, we would then conclude that the differences (target vs. chance in [1] and [2]; and monodialectal vs. bidialectal in [3]) were significant. + +The results of the cluster permutation analysis for monolingual infants revealed the biggest cluster t = 35.54, occurring between 890 and 1060 ms after word onset, was not significant (p = 0.19). No cluster was identified for bidialectal infants, and no cluster was significant in the comparison between monodialectal and bidialectal infants, with the biggest cluster of t = 7.3, occurring between 860 and 890 ms +(Figure 5). + +![11_image_0.png](11_image_0.png) + +TABLE 5 Summary of the mixed-effect regression model run on difference in looking proportion in bidialectal infants for the word comprehension task + +| comprehension task | Diff_prop_looking | | | +|----------------------|---------------------|---------------|-------| +| Predictors | Estimates | CI | p | +| (Intercept) | 0.09 | −0.01 to 0.20 | 0.067 | +| Rated_sim_zero | −0.02 | −0.04 to 0.00 | 0.077 | +| Exposure_binom [No] | −0.08 | −0.25 to 0.08 | 0.322 | +| Rated_sim_zero * | 0.01 | −0.02 to 0.05 | 0.388 | +| Exposure_binom [No] | | | | +| Random effects σ2 | 0.09 | | | +| τ00 ChildID | 0.00 | | | +| τ00 trial | 0.00 | | | +| ICC | 0.03 | | | +| NChildID | 35 | | | +| Ntrial | 16 | | | +| Observations | 958 | | | +| Marginal R2/ | 0.005/0.038 | | | +| Conditional R2 | | | | + +TABLE 6 Summary of the mixed-effect regression model on the role of perceived similarity between parents' dialects on difference in looking proportion in bidialectal and monodialectal infants for the word comprehension task + +| word comprehension task | Diff_prop_looking | | | +|---------------------------|---------------------|---------------|-------| +| Predictors | Estimates | CI | p | +| (Intercept) | 0.00 | −0.03 to 0.04 | 0.842 | +| Rated_sim_zero | 0.02 | −0.01 to 0.06 | 0.159 | +| Group [Bidialectal] | 0.06 | −0.02 to 0.14 | 0.161 | +| Rated_sim_zero * Group | −0.04 | −0.07 to 0.00 | 0.051 | +| [Bidialectal] | | | | +| Random Effects σ2 | 0.10 | | | +| τ00 ChildID | 0.00 | | | +| τ00 trial | 0.00 | | | +| ICC | 0.02 | | | +| NChildID | 69 | | | +| Ntrial | 16 | | | +| Observations | 1924 | | | +| Marginal R2/ | 0.004/0.020 | | | +| Conditional R2 | | | | + +## 3.5 Relationship Between Word Recognition And Word Comprehension Tasks + +Finally, and as preregistered, we examined the relationship between word recognition and word comprehension tasks. For that, for each infant, in both monodialectal and bidialectal group, we computed an average word recognition/preference score, as the difference between average fixation times on known and nonsense trial types, and an average word comprehension score. A Pearson correlation analysis revealed that while monodialectal infants showed a strong relationship between these two variables (r = 0.53, t = 3.58, df = 33, p = 0.001), +bidialectal infants showed no relationship at all (r = −0.02, t = −0.11, + +![12_image_0.png](12_image_0.png) + +![12_image_1.png](12_image_1.png) + +14 of 19 AND + +![13_image_0.png](13_image_0.png) + +df = 33, p = 0.91). An additional linear regression analysis on word preference as a function of difference in looking proportion and group confirmed this significant interaction between group and word preference, β = −65296.0,se = 27359.3, t = −2.39, p = 0.019 (see Figure 6). + +## 4 Discussion + +The main aims of the current preregistered study were to examine word recognition and word comprehension in Norwegian 12-monthold infants and to assess whether exposure to two dialects at home (as frequently encountered in Norway and Oslo, in particular) modulates their performance. Seventy monolingual Norwegian infants exposed to native Norwegian speakers at home, were assessed, in an eye-tracking paradigm on listening preference for familiar words and word comprehension with an IPL paradigm. Half of them were exposed to two different Norwegian dialects at home, whereas the other half were exposed to one dialect (i.e., both parents spoke the same dialect). In brief, our results revealed (1) no listening preference for words, as compared to nonwords, in Norwegian 12-month-old infants; (2) evidence of word comprehension in monodialectal infants, but not in bidialectal infants; +(3) a strong relationship between word recognition and comprehension in monodialectal infants and the absence of such a relationship in bidialectal infants. It what follows we will discuss these results together with the results on the role of accent exposure and similarity between parental dialects. + +First, our results revealed that Norwegian 12-month-old infants +(n = 70) did not show word recognition, as indicated by an absence of listening preference for Norwegian familiar words as compared to nonwords, composed of the same sounds. These results are at odds with previous research in English (Vihman et al., 2007), French (Hallé & Boysson-Bardies, 1994), and Japanese (Tamekawa et al., 1997) 10 to 12-month-old infants, who display a modest effect size (Hedge's) of d = 0.53 in word recognition. We offer three possible interpretations of our result. + +According to the first interpretation, Norwegian infants do not recognize familiar words without their referents, because they have not yet established stable phonological forms for Norwegian words, suggesting cross-linguistic differences in the onset of phonological word (form) recognition. Norwegian language is a phonologically complex language, for instance, to differentiate vowel minimal word pars, it uses vowel lengthening, formants and voice pitch. Given that words were embedded in lists, we cannot tell how many words need to be acquired to trigger word recognition, however, a recent meta-analysis reported larger effect sizes with higher word familiarity (Carbajal et al., 2021). In line with this result, our analyses revealed a marginally significant effect of exposure to the Oslo dialect at home in bidialectal infants, suggesting that familiarity with the accent promotes word recognition +(although note that words used in the task had very similar segmental pronunciation across Norwegian dialects, they might slightly differ in terms of suprasegmental features, i.e., tones in disyllabic words). + +It is, therefore, possible that Norwegian infants require more time to establish stable phonological word representations and to recognize familiar words in a list of word forms. Other studies have similarly reported cross-language differences in word recognition: for instance, Vihman et al. (2007) revealed no word recognition in 12-month-old +(British) English and Welsh infants, and Carbajal and colleagues, in a recent meta-analysis on word recognition, reported larger effect sizes in Romance languages and in "older" infants (Carbajal et al., 2021), suggesting that the absence of word recognition in our study can be accounted by cross-linguistic differences in infants' onset of phonological word form recognition. + +According to the second interpretation, the lack of word recognition can be due to methodological differences (e.g., paradigms, statistical analyses) between the current study and previous research. For instance, the head-turn preference paradigm, mainly used in previous research, can lead to larger effect sizes than a central fixation design +(Frank et al., 2020) used in the current study, which might have prevented us from detecting an effect. In addition, although the study has been preregistered and had a sample size three times more than typically used in word recognition studies, it is not impossible that the power analysis estimates for the current study were too optimistic given the larger effect sizes in central fixation designs and overall modest sample sizes used in previous research. As far as the statistical analysis is concerned, Carbajal and Peperkamp (2017) have shown that mean looking times, as traditionally used in word recognition research, disregard time dependencies and might not describe infants' attention adequately; in their work with a central fixation paradigm (as in the current study), while an analysis of mean looking times revealed word recognition in 11-month-old French infants, a trial-by-trial analysis (as in the current study) revealed no effect of word type when controlling for effect of trial order and infant and list variability. More research is needed to investigate the roles of the method, analyses and language on the onset of word form recognition in infants, by using, eventually, a large cohort of infants' ages (e.g., from 11 to 15 months) and the same study design (e.g., stimuli, procedure, sampling, etc.) across different languages. A recent ManyBabies Consortium initiative (Frank et al., +2020) examined infants' preference for infant-directed speech across 67 labs and dozens of countries using the same stimuli and centrally performed analyses, and reported that the effect sizes were substantially lower than in the meta-analysis of previous literature, and that they varied considerably across methods used to collect infants' preference, suggesting, first, that the effect sizes retrieved from published studies might be overestimated and, second, that they depend crucially on the experimental method used. + +According to the third interpretation, Norwegian infants did recognize familiar words; yet, they did not prefer listening to them more than to pseudowords, due to a likely overall large variability in overheard speech to which they can be regularly exposed in Oslo. Although half of our sample were exposed to one Norwegian dialect at home, it is still very likely that infants encountered other dialects elsewhere, for example, in the playgrounds, social/family gatherings, or on child TV, as regional dialects are used by people in all social strata, and their use is promoted across different domains of language use (Dragojevic et al., 2021). So, a mispronounced word (a pseudoword) in the Oslo dialect could be still entertained by young children as being potentially a meaningful word in a different Norwegian dialect. This interpretation is supported by a marginally significant effect of rated perceptual similarity between parental dialects in bidialectal infants and suggests AND **15 of 19** +that bidialectal infants exposed to more dissimilar dialects at home displayed less preference for listening familiar words. To summarize, a recent study has shown that already at 12.5 months of age infants exposed to dialect/accent variability at home display differences in language skills when compared to infants exposed to uniformly accented speech, by failing to show listening preference for familiar words (van Heugten & Johnson, 2017). In the current study, we failed to reveal any differences in familiar word recognition between monodialectal and bidialectal Norwegian 12-month-old infants. Future research needs to examine the onset of word recognition in Norwegian and other languages where regional dialect variability is prevalent. + +Our second important result revealed that monodialectal infants looked significantly above chance at the target when hearing the matching label as compared to when hearing the name of a distractor (evidence of word comprehension), whereas bidialectal infants, on the other hand, did not show evidence for word comprehension. Our results are in line with previous research in older bidialectal infants (Buckler et al., 2017; Durrant et al., 2015; Floccia et al., 2012), suggesting that variability in speech affects early word comprehension (see below). The results of the cluster-permutation analyses of the looking gaze during the trial, however, did not reveal significant time windows in either group, due to likely a considerable variability across children and also across trials, which are not taken into account when averaging the data for each 10 ms bins across all trials (unlike in mixed-effect analyses of the difference in proportion data). + +In the current study, we examined, in addition, the role of perceived similarity between parental dialects, as assessed by native Norwegian listeners, and exposure to the dominant Oslo dialect on bidialectal infants' performance in word recognition and word comprehension eye-tracking tasks. First of all, we noticed a strong relationship between perceived similarity between parents' dialects, as reported by parents and by an independent group of native Norwegian speakers, suggesting that parental evaluations (although slightly stricter, i.e., +overall higher rating ranges) can be used reliably to assess infants' speech variability at home. To the best of our knowledge, this is the first study that assessed the role of similarity of parental dialects on early language development. Our results revealed that bidialectal infants exposed to similar dialects or to the Oslo dominant accent at home showed marginally significant word comprehension and listening preference for words (although not word recognition), suggesting that dialect familiarity and similarity between parents' dialects facilitate word comprehension (see van Heugten & Johnson, 2014 for similar results on the role of familiarity on accent adaptation in monodialectal infants) and might facilitate word recognition. Although at odds with previous studies in bidialectal infants, showing no relationship between the amount of dialectal exposure and infants' ability to recognize (van Heugten & Johnson, 2017) and understand words in this dialect (Buckler et al., 2017; Durrant et al., 2015), this result on the role of dialect exposure confirms our hypothesis and is in line with research in monodialectal infants exposed to unfamiliar accents. + +A positive effect of perceived similarity between parental dialects on word comprehension is at odds with our original hypothesis, where we predicted that visual cues would facilitate object disambiguation 16 of 19 AND +(see van Heugten et al., 2018); yet, this result corroborates the hypothesis that infants exposed to little inconsistency in dialectal input are likely to use lexical top-down strategy to accommodate accents, which facilitates word recognition/comprehension (Schmale et al., 2010, 2012; White & Aslin, 2011). These results, together with the results in the word recognition task, suggest that Norwegian infants exposed to dialects that differ substantially along a number of acoustico-phonetic dimensions, including segmental and suprasegmental cues, are likely to use a general expansion strategy, which may require a "general" expansion (or relaxation) of phonemic categories (Schmale et al., 2012) to accommodate for deviating examples that can span phonemic boundaries. The general expansion strategy is opposed to top-down lexically guided strategy; it can be beneficial when exposed to unfamiliar speakers with various language or accent backgrounds, as even considerable deviations from the norms would trigger word comprehension; yet, general expansion can also lead to larger processing costs, due to less restricted lexical access (see Schmale et al., 2012). This strategy seems to affect, particularly, word comprehension, as infants exposed to perceptually highly dissimilar dialects showed impaired word comprehension. Similar findings were reported in slightly older 20-month-old bidialectal British English infants (Durrant et al., 2015), who showed similar looking times for mispronounced and for accurately pronounced words referring to the target picture. Although the lack of word comprehension in Norwegian 12-month-old bidialectal infants is in line with previous research in older bidialectal infants learning other languages and indicates that differences in language skills between monodialectal and bidialectal infants can be observed as early as at 12 months of age, this result needs to be taken with caution, due to the lack of difference between monodialectal and bidialectal Norwegian infants, and should be confirmed with older infants, as word comprehension even in monolingual Norwegian infants is not yet robust at 12 months of age, as compared, for instance, to same-age American English peers (Bergelson & +Aslin, 2017a; Bergelson & Swingley, 2013; although word comprehension is more firmly established after 14 months of age, see Bergelson, 2020). A previous study has already revealed that Norwegian monodialectal infants might be lagging behind their American–English peers, who reportedly display evidence of word comprehension from as early as 6 months of age, whereas Norwegian infants' word comprehension only emerges by 8–9 months of age, provided that infants can exploit additional disambiguation cues (Kartushina & Mayor, 2019). Research with older Norwegian infants is needed to assess the onset of word comprehension in bidialectal infants. + +Finally, our results revealed a significant correlation between two direct measures of early language development - word comprehension and familiar word recognition - in monodialectal infants, but not in bidialectal infants. These results suggest that monodialectal infants who recognize familiar words without their referents have established word-object mappings for some frequently used words and indicate that successful word comprehension aligns with or implies listening preference for words. The lack of such a relationship in bidialectal infants suggests either that these two skills do not necessarily align in infants exposed to more variable input, or that the alignment might occur at a later stage, when bidialectal infants start showing word comprehension. To the best of our knowledge, this is the first time when a relationship between these two skills is assessed in young infants. + +Infants in many countries are exposed to dialectal variability, yet very little is still known about whether such variability in early language input impacts language development and, if so, how different levels of variability differentially impact language acquisition. The results of the current study, together with the results of previous research, suggest that exposure to dialect/accent variability impacts very early stages of language acquisition. In particular, our results for familiar word recognition and word comprehension suggest that early word representations in bidialectal infants are likely phonologically less specified (and/or broader) and vary in specificity/broadness as a function of dialect similarity in parental/family speech. However, already by 2 years of age, bidialectal infants show dialect-sensitive accurate knowledge of the phonological categories in both dialects: +they adapt their phonological expectations based on the phonetic contrastive cues relevant for a speaker's dialect (van der Feest & Johnson, 2016), and flexibly adapt to variable input (van der Feest et al., 2022); +by 2.5 years of age, bidialectal infants show an advantage in word learning in accented speech (Kartushina et al., 2021), and by roughly 3 years of age bidialectal infants show similar processing times as their peers, suggesting that difficulties related to processing inconsistent speech input might be overcome by 3 years of age (Buckler et al., 2017). Yet, more research is needed to examine the role of degree of dialect/accent similarity on language acquisition, as revealed in the current study. + +Overall, although bidialectal infants seem to essentially catch up with their monodialectal peers by 3 years of age, an impact on their speech processing seems to persist when the task demands are high (Buckler +& Johnson, 2020), in line with the assumption that early exposure to accent variation continues to impact language processing into adulthood (Chen et al., 2017; Kirk et al., 2018), in addition to the beneficial effects of adapting better to regional dialects in children (Levy et al., 2019) and of better word learning from multidialectal input in toddlers (Kartushina et al., 2021). + +## Acknowledgment + +We would like to thank Ada Koleini for her help with data collection. We thank parents and their infants for taking part in the study. NK was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme [project number 223265]. + +## Conflict Of Interest + +All authors declare that they have no conflicts of interest to disclose. + +## Data Ava Ilab Il Ity Statement + +The data that supports the findings of this study (including stimuli, questionnaires, the consent form, results and analyses) is openly available in OSF at https://osf.io/6btq2/. + +## Orcid + +Natalia Kartushina https://orcid.org/0000-0003-4650-5832 Julien Mayor https://orcid.org/0000-0001-9827-5421 + +## Endnotes + +1Note that in Buckler et al.'s (2017) study, infants were exposed to both Canadian English and a non-native variant of English (cf. footnote 2). + +2Note that foreign-accented speech is qualitatively different from regional-accented speech produced by native speakers. The pattern of +(mis)pronunciations in foreign-accented speech is unstable and unpredictable; the alternations are not limited to sounds, but include also prosodical, supra-segmental (co-articulation) and, importantly, segmental +'errors. + +3Yet, 24-month-old infants exposed to bi-accented input, where one of the accents is spoken by a non-native speaker (e.g., non-native variants of English), showed less efficient word comprehension as compared to 24month-old infants who received unimodal input, suggesting that regular exposure to foreign-accented speech might affect early word comprehension (Buckler et al., 2017). + +4Although note that in this study bilingual infants were exposed to two quite distinct languages, English and Spanish, which might contribute to differences in the effect. + +5 For instance, Oslo dialect is a low pitch dialect, whereas Bergen is a high pitch dialect, meaning that pitch accents follow opposite patterns in Oslo and Bergen. + +6 It is also possible that bidialectal infants - who are used to hear inconsistent word production - are interested to the same degree in familiar and nonsense word forms, which might contribute to the null result in the word form recognition task. This alternative interpretation of the null result has been previously introduced in van Heugten and Johnson +(2017), but challenged by the same authors, because (1) in their study, bidialectal infants did show word preference, only at a later age (at 18 months) and (2) bilingual infants (who can be expected to behave similarly in this design) show word recognition by the age of 11 months (Vihman et al., 2007). + +7Note that in van Heugten and Johnson (2017) study, that used the HPP +procedure, the effect size of Cohen's d = 0.71. In a paired two-sided t-test, 80% power to detect this effect requires 18 participants in each group, as revealed by the *pwr.t.test* function (Champely et al., 2018) in R (R Core Team, 2020). + +8One child in the bidialectal group, who successfully completed both tasks had 20% of exposure to the Northern dialect. Parents made an error in the questionnaire and we kept this child after that we corrected the number. + +9We selected a question-answer task rather than a reading task for the following reasons: first, we aimed to collect natural spontaneous speech (as typically addressed to a child), second, we wanted to capture phonological and lexico-semantical and morpho-syntactic (to a lesser extend) specificities of each dialect, and third, we wanted to avoid written language bias on speech production (as there is no standard writing system in Norwegian - words and morphosyntax vary across dialects - using any writing system, e.g., Bokmål for Oslo dialect or Nynorsk for Bergen dialect, would threaten the authenticity of dialectal production). + +10 In the original study, we planned to use Tobii TX300 eye-tracker; in the meantime, the lab acquired an Eye-link 1000 eye-tracker, so we adapted the task procedures to the new eye-tracker. + +11These trials were used to check the quality of participants' ratings. None of the participants made errors on the control trials. + +12Given that the variable "exposure" was bimodally distributed, that is, either 0 or 50%–80%, we dichotomized it into a categorical variable "no +(exposure)" and "yes." + +## References + +Arias-Trejo, N., & Plunkett, K. (2010). The effects of perceptual similarity and category membership on early word-referent identification. Journal of Experimental Child Psychology, 105(1–2), 63–80. https://doi.org/10.1016/ j.jecp.2009.10.002 Bates, D., Mächler, M., Bolker, B., & Walker, S. (2015). Fitting linear mixedeffects models using lme4. *Journal of Statistical Software,* 67(1), 1–48. + +https://doi.org/10.18637/jss.v067.i01 AND 17 of 19 Bergelson, E. (2020). The comprehension boost in early word learning: +Older infants are better learners. *Child Development Perspectives,* 14(3), +142–149. https://doi.org/10.1111/cdep.12373 Bergelson, E., & Aslin, R. N. (2017a). 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Insights from Norwegian dialects. + +Developmental Science, 26, e13264. + +https://doi.org/10.1111/desc.13264 \ No newline at end of file diff --git a/medical/md/PMC10282595.md b/medical/md/PMC10282595.md new file mode 100644 index 0000000000000000000000000000000000000000..a61fcf990ecae8e0cc825c53c2e363a23cc49b97 --- /dev/null +++ b/medical/md/PMC10282595.md @@ -0,0 +1,100 @@ +DOI: 10.1002/rcr2.1181 CASE REPORT +Pathological complete response in an advance stage IIIB non-small cell lung cancer secondary to neoadjuvant osimertinib targeted therapy: A case report and review of literature Chun Ian Soo1 | Diana Bee-Lan Ong 2 | Ka Kiat Chin1 | Leng Cheng Sia 1 | Vijayan Munusamy 1 | Nur Husna Ibrahim1 | Thian Chee Loh1 | Jiunn Liang Tan1 | Mau Ern Poh1 | Chee Kuan Wong 1 | Yong Kek Pang 1 | Chong Kin Liam1 1Division of Respiratory Medicine, Department of Medicine, University Malaya, Kuala Lumpur, Malaysia 2Department of Pathology, University Malaya, Kuala Lumpur, Malaysia Correspondence Chun Ian Soo, Division of respiratory medicine, department of medicine, University of Malaya, Lembah Pantai, 59100 Kuala Lumpur, Malaysia. Email: scianray@gmail.com Associate Editor: David Lam + +## Abstract + +![0_Image_0.Png](0_Image_0.Png) + +Neoadjuvant chemotherapy is a therapeutic option for potentially resectable non-small cell lung cancer (NSCLC). The role of neoadjuvant targeted therapy +(NTT) remains less explored. This case highlights the use of neoadjuvant osimertinib in a case of advanced NSCLC. A 67-year-old woman had a left lower lobe lung mass measuring 5.0 5.1 7.0 cm with an enlarged subcarinal lymph node (LN) on her positron emission tomography scan. Following biopsy, a diagnosis of stage IIIB N2 (cT3N2M0) EGFR exon 19 deletion mutation-positive lung adenocarcinoma was established. NTT using osimertinib 80 mg once daily was commenced. Subsequent re-imaging at 3 months (ycT2bN2M0), 6 months (ycT1cN2M0) and 9 months showed tumour downstaging and resolution of the subcarinal LN (ycT1cN0M0). She underwent left lower lobectomy with systematic nodal dissection. All surgical specimens demonstrated no evidence of malignant cells (ypT0N0). Osimertinib could be the preferred NTT for potentially resectable NSCLC. + +KEYWORDS neoadjuvant targeted therapy, non-small cell lung cancer, Osimertinib, pathological complete response + +## Introduction + +Osimertinib, a third-generation Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor (TKI), is a recognized first-line treatment of metastatic EGFR-mutant non-small cell lung cancer (NSCLC).1 However, the role of neoadjuvant EGFR-TKI in NSCLC remains uncertain. We report a case of pathological complete response (pCR) +defined as 'the absence of residual viable tumour cells in all the pathological samples of the resected primary lung tumour and lymph nodes following neoadjuvant therapy +(ypT0N0)', with osimertinib in a patient with stage IIIB N2 EGFR-mutant NSCLC. + +## Case Report + +A 67-year-old lady with bronchial asthma complained of worsening cough and mild wheezing for 4 months. Prior to this, her asthma had been well controlled on one puff of Budesonide/Formeterol (160 mcg/4.5 mcg) inhalation taken twice daily. There was no improvement despite optimizing her treatment. She has no other medical illness. She does not smoke and has no family history of malignancy. Physical examination of her respiratory system revealed no significant findings apart from occasional soft expiratory rhonchi heard over the left lower zone. + +This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. + +© 2023 The Authors. Respirology Case Reports published by John Wiley & Sons Australia, Ltd on behalf of The Asian Pacific Society of Respirology. + +![1_image_0.png](1_image_0.png) + +A computed tomography (CT) Thorax confirmed a left lower lobe lung lesion measuring 5.0 5.1 7.0 cm with an enlarged subcarinal lymph node (LN) (Figure 1A, B). A +diagnosis of stage IIIB N2 (cT3N2Mx) EGFR exon 19 deletion mutation-positive lung adenocarcinoma was established following endobronchial ultrasound-guided fine needle + +NEOADJUVANT THERAPY WITH OSIMERTINIB 3 of 5 + +![2_image_0.png](2_image_0.png) + +aspiration (FNA) of the subcarinal LN and endoscopicendobronchial ultrasound-guided FNA sampling of the left lower lobe mass (Figure 2A, B). The tumour cells also expressed 90% programmed death-ligand 1(PD-L1). Other molecular biomarker such as anaplastic lymphoma kinase +(ALK) was negative. The patient's Eastern Cooperative Oncology Group (ECOG) performance status was 0 and she opted for curative surgical resection. A full body positron emission tomography-computed tomography (PET-CT) and a magnetic resonance imaging of the brain demonstrated absence of distant metastasis (cT3N2M0). Following discussion at the lung tumour board meeting, she was commenced on neoadjuvant targeted therapy (NTT) using osimertinib 80 mg once daily. Re-imaging at 3 months (ycT2bN2M0) +(Figure 1C, D) and 6 months demonstrated tumour downstaging (ycT1cN2M0) (stage IIIA) (Figure 1E, F). However, surgical resection was not possible because of the absence of a demarcation plane between the mass and descending aorta. At 9 months, the primary tumour had further shrunken in size to 2.0 2.2 1.8 cm and there was a resolution of the subcarinal LN (ycT1cN0M0) (Figure 1G, H). + +She underwent left lower lobectomy with systematic lymph node dissection (stations 11L, 4L, 7, 8 and 9 LNs). There was no evidence of residual malignant cells on histopathological examination of all surgical specimens (ypT0N0) +(Figure 2C–F). Throughout her NTT, 6- and 12-months postoperative period, she tolerated osimertinib well with only grade 1 skin reaction (dry skin) based on U.S. National Cancer Institute's Common Terminology Criteria for Adverse Events.2 A 6- and 12-months post resection PETCT demonstrated no recurrence. The patient was continued on osimertinib 80 mg once daily for 3 years post-surgery. + +## Discussion + +Neoadjuvant chemotherapy is a therapeutic option for cases of potentially resectable NSCLC. Recently, EGFR-TKI-NTT +has also been explored because targeted therapy with EGFRTKIs results in better treatment outcomes and lower treatment-related toxicity compared to platinum-doublet chemotherapy in patients with metastatic NSCLC.3 Although there is still a paucity of data on EGFR-TKI-NTT, +some early reports supported its role.3–6 In a case series of 13 patients, Hu et al has reported a 69.2% objective response rate, 100% complete resection rate and 100% pathologic downstaging rate with the use of osimertinib-NTT.7 When compared to conventional neoadjuvant chemotherapy, EGFR-TKI-NTT demonstrated higher objective response rate and median progression-free survival.4 However, in a pooled analysis of these early studies, NTT only resulted in a 14% downstaging and 0% pCR rates.3 On the other hand, retrospective studies have reported pCR following neoadjuvant chemotherapy is associated with improved survival +(HR for survival, 0.49%; 95% CI, 0.43–0.56).8 But, the median rate of pCR following neoadjuvant chemotherapy is low (4%) (range, 0%–16%).9 Interestingly, although previous neoadjuvant studies only involved patients up to stage IIIA disease, the phase II NEOS trial recruited patients with stage IIIb disease.10 The preliminary results from the interim analysis of this study revealed that in a cohort of 28 patients, osimertinib is a potential agent for NTT with a 95% complete resection rate. + +In our patient, osimertinib was used as NTT for stage IIIB (cT3N2M0) NSCLC with EGFR exon 19 deletion mutation. Generally, there is no consensus on the optimal duration of NTT, but it rarely exceeds 60 days based on previous studies.4 This patient had an extended duration of NTT up to 9 months because complete surgical resection was not possible at 3 and 6 months, respectively, despite achieving a reasonable downstaging of the disease. Histopathological examination of the resected left lower lobe and the dissected LNs showed no evidence of malignant cells. Hence, confirming a pCR. Throughout the entire duration of her NTT and up to present, the patient only experienced mild side-effect from osimertinib. This reinforces the better tolerated toxicity profile of EGFR-TKI-NTT compared to conventional cytotoxic chemotherapy. Nevertheless, although uncommon, it is important to monitor for potential serious effects of osimertinib when used for the long term, such as vision changes, interstitial lung disease, and arrythmias. Future studies are required to demonstrate the safety of osimertinib when used as EGFR-TKI-NTT. + +In conclusion, this case provides some early insights on the efficacy and benefit of osimertinib-NTT in pathologically downstaging and potentially achieving pCR in stage IIIB N2 NSCLC. The findings of this case report need to be confirmed by the outcome of the highly anticipated ongoing NeoADAURA (NCT04351555) clinical trial comparing three cycles (9 weeks) of neoadjuvant osimertinib with or without chemotherapy versus chemotherapy alone for EGFR-mutated resectable (stage II-IIIB N2) non-squamous NSCLC.11 + +## Author Contributions + +Chun Ian Soo and Chong Kin Liam provided conceptualization and contributed equally for the case report. All authors have contributed substantially to the data acquisition and drafting of the original and revised manuscript and have read and approved the final version. + +## Conflict Of Interest Statement None Declared. Data Availability Statement + +The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. + +## Ethics Statement + +The authors declare that appropriate written informed consent was obtained for the publication of this manuscript and accompanying images. + +## Orcid + +Chun Ian Soo https://orcid.org/0000-0001-8560-7888 Leng Cheng Sia https://orcid.org/0000-0001-6927-2571 Thian Chee Loh https://orcid.org/0000-0002-5399-7030 Chee Kuan Wong https://orcid.org/0000-0002-8678-4664 + +## References + +1. Ettinger DS, Wood DE, Aisner DL, Akerley W, Bauman JR, Bharat A, +et al. Non–small cell lung cancer, version 3.2022, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2022;20(5):497–530. + +2. United States, Department of Health and Human Services, National Institutes of Health, National Cancer Institute (nci). Common Terminology Criteria for Adverse Events (CTCAE). Ver. 5.0. Bethesda, MD. + +[cited 2023 Mar 1]. Available from: https://ctep.cancer.gov/ protocoldevelopment/electronic_applications/docs/ctcae_v5_quick_ reference_8.5x11.pdf 3. Sun L, Guo YJ, Song J, Wang YR, Zhang SL, Huang LT, et al. Neoadjuvant EGFR-TKI therapy for EGFR-mutant NSCLC: a systematic review and pooled analysis of five prospective clinical trials. Front Oncol. 2021;10:586596. + +4. Zhong WZ, Chen KN, Chen C, Gu CD, Wang J, Yang XN, et al. Erlotinib versus gemcitabine plus cisplatin as neoadjuvant treatment of stage IIIA-N2 EGFR-mutant non-small-cell lung cancer (EMERGING-CTONG 1103): a randomized phase II study. J Clin Oncol. 2019;37(25):2235–45. + +5. Belluomini L, Riva ST, Simbolo M, Nocini R, Trestini I, Avancini A, +et al. Anticipating EGFR targeting in early stages of lung cancer: leave no stone unturned. Cell. 2021;10(10):2685. + +6. Liu X, Zheng Y, Mai S, Tong Y, Yang L, Huang M, et al. Case report: +an initially unresectable stage III pulmonary sarcomatoid carcinoma qith EGFR mutation achieving pathological complete response following neoadjuvant therapy with osimertinib plus chemotherapy. Front Oncol. 2022;12:12. + +7. Hu Y, Ren S, Yang L, Tong Z, Wang R, Han W, et al. Osimertinib as neoadjuvant therapy for resectable non-small cell lung cancer: a case series. Front Pharmacol. 2022;13:1620. + +8. Waser NA, Adam A, Schweikert B, Vo L, McKenna M, +Breckenridge M, et al. 1243P pathologic response as early endpoint for survival following neoadjuvant therapy (NEO-AT) in resectable non-small cell lung cancer (rNSCLC): systematic literature review and meta-analysis. Ann Oncol. 2020;31:S806. + +9. Hellmann MD, Chaft JE, William WN Jr, Rusch V, Pisters KM, Kalhor N, +et al. Pathological response after neoadjuvant chemotherapy in resectable non-small-cell lung cancers: proposal for the use of major pathological response as a surrogate endpoint. Lancet Oncol. 2014;15(1):e42–50. + +10. Lyu C, Fang W, Ma H, Wang J, Jiao W, Wang R, et al. Osimertinib as neoadjuvant treatment for resectable stage II-IIIB EGFR mutant lung adenocarcinoma (NEOS). J Clin Oncol. 2021;39(suppl 15):8524. + +11. Tsuboi M, Weder W, Escriu C, Blakely C, He J, Dacic S, et al. Neoadjuvant osimertinib with/without chemotherapy versus chemotherapy alone for EGFR-mutated resectable non-small-cell lung cancer: +NeoADAURA. Future Oncol. 2021;17:4045–55. + +How to cite this article: Soo CI, Ong DB-L, Chin KK, Sia LC, Munusamy V, Ibrahim NH, et al. Pathological complete response in an advance stage IIIB non-small cell lung cancer secondary to neoadjuvant osimertinib targeted therapy: A case report and review of literature. Respirology Case Reports. 2023;11:e01181. https://doi.org/10.1002/ rcr2.1181 \ No newline at end of file diff --git a/medical/md/PMC10700950.md b/medical/md/PMC10700950.md new file mode 100644 index 0000000000000000000000000000000000000000..58282f5b81949245210d48223e2fa52d0f4e470e --- /dev/null +++ b/medical/md/PMC10700950.md @@ -0,0 +1,439 @@ + +## Review Article + +AHBPS + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +![0_image_2.png](0_image_2.png) + +Annals of Hepato-Biliary-Pancreatic Surgery +# Vascular Tumors Of The Liver: A Brief Review + +Sujata Sarangi1,Balamurugan Thirunavukkarasu2, Sudeep Khera2, Selvakumar B3, Taruna Yadav4 1Department of Pathology, Grant Govt. Medical College, Mumbai, India, 2Department of Pathology and Laboratory Medicine, All India Institute of Medical Sciences, Jodhpur, India, 3Department of Surgical Gastroenterology, All India Institute of Medical Sciences, Jodhpur, India, 4Department of Diagnostic and Interventional Radiology, All India Institute of Medical Sciences, Jodhpur, India Vascular tumors of the liver are mesenchymal lesions from endothelial cells. They range from common benign lesions such as haemangioma, intermediate tumors like Kaposi sarcoma, and perivascular epithelioid cell tumor to malignant tumors such as hepatic epithelioid hemangioendothelioma and hepatic angiosarcoma in adults. Pediatric vascular tumors of the liver also include benign, locally aggressive, borderline, and malignant masses with haemangiomas being the most common benign tumors and epithelioid hemangioendothelioma being an uncommon pediatric malignancy. The list of these lesions is completed by nodular regenerative hyperplasia, solitary fibrous tumour, and hepatic small vessel neoplasms (HSVN). Some of these tumors are uncommon and rare. This review article aimed to enumerate hepatic vascular tumors along with their imaging, histopathology, molecular findings for accurate diagnosis that can result in better management. + +Key Words: Liver, Vascular tumour, Diagnostic + +## Introduction + +The liver parenchyma comprises of epithelial components and mesenchymal components such as blood vessels and adipose tissues. Vascular tumors can be formed from endothelial cells of vascular channels. They range from common benign tumors such as haemangiomas and intermediate entities such as Kaposi sarcoma (KS) and PEComas to malignant tumors such as hepatic epithelioid hemangioendothelioma (HEHE) and hepatic angiosarcoma [1,2]. They also include tumors like solitary fibrous tumour (SFT) and hepatic small vessel neoplasms (HSVN). Haemangiomas are the most common pediatric vascular tumors of the liver. They are different from adult hepatic haemangiomas [3]. + +As most of these tumors are uncommon, their diagnostic ap- +Received: March 30, 2023, **Revised:** September 8, 2023, Accepted: September 18, 2023, **Published online:** November 13, 2023 Corresponding author: Sudeep Khera, MD Department of Pathology and Laboratory Medicine, All India Institute of Medical Sciences, Jodhpur 342005, India Tel: +91-7726859981, E-mail: drsudeepkhera@gmail.com ORCID: https://orcid.org/0000-0002-2413-5749 + +Copyright Ⓒ The Korean Association of Hepato-Biliary-Pancreatic Surgery + +![0_image_3.png](0_image_3.png) + +![0_image_4.png](0_image_4.png) + +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. + +proach and treatment modalities are ambiguous and non-standardised [4,5]. Early and accurate diagnosis will help us better manage these rare tumors. + +## Benign Masses And Tumors Adult Hepatic Haemangioma + +Hepatic haemangiomas in adults and pediatric patients are different entities. Hepatic haemangiomas in adults are slowflow venous malformations. They are not actual neoplasms/ +tumors according to the International Society for Study of Vascular Anomalies (ISSVA) classification [6]. They are the most common liver lesions with a prevalence of 3%–20%. They might contribute up to 70% of all benign liver tumors. They can be single or multiple. They can present at any age with a strong female predilection (female-to-male ratio: 4:1 to 5:1) [7,8]. + +They are usually asymptomatic. They are detected as incidental findings on imaging. Among patients who develop symptoms, vague abdominal pain is the most common symptom. Haemangiomas of more than 5 cm in size are called giant haemangiomas [1]. They rarely present as an abdominal lump. They can cause compression of surrounding structures depending on their location. They might cause gastric compression and early satiety when present in the left lobe of liver, compression of inferior vena cava and limb edema when + +![1_image_0.png](1_image_0.png) +Fig. 1. Multiphasic contrast-enhanced computed tomography (CT) in a 52-yearold male patient showing a giant hepatic haemangioma (arrow in A). Axial images in (A) arterial, (B) portal venous, (C) hepatic venous, and (D) 3-minute delayed phases show typical peripheral discontinuous nodular enhancement with progressive centripetal filling of contrast. Note that the density of nodular contrast in the lesion matches with the aorta (blood pool). + +present in the right or caudate lobe, and biliary or portal vein compression leading to jaundice or portal hypertension when present in a central location. Haemangiomas might have intra-lesional thrombosis with subsequent inflammation, hyalinization, and fibrosis that can increase pain and constitutional symptoms such as fever, weight loss, and increased erythrocyte sedimentation rate. However, they usually have a normal leucocyte count. Giant haemangiomas may rarely present as a generalized consumptive coagulopathy syndrome (Kasabach-Merrit syndrome) due to intra-lesional thrombus and fibrinolysis leading to disseminated intravascular coagulation and thrombocytopenia [8]. + +On ultrasonography (USG), haemangiomas appear as sharp homogeneous hyperechoic masses. On contrast-enhanced ultrasound (CEUS), these lesions may show peripheral enhancement in arterial phase and become isoechoic in later phases. + +On contrast-enhanced computed tomography (CECT), they appear as lesions with peripheral, globular enhancement in arterial phase with progressive central enhancement and retention of contrast in delayed phases. On magnetic resonance imaging (MRI), they appear as hypointense on T1 and very hyperintense on T2 weighted images. On contrast-enhanced MRI (CEMRI), a progressive enhancement pattern is similar to CECT. A few haemangiomas might show atypical features on imaging. Giant haemangiomas may show atypical features on imaging due to thrombosis, hyalinization, fibrosis, cystic degeneration, or calcifications. A subset of small haemangiomas may be rapidly-filling completely on arterial phase, mimicking + +![1_image_1.png](1_image_1.png) hepatocellular carcinoma (HCC). However, they usually retain contrast on delayed phases. Haemangiomas in background of cirrhosis might have more fibrosis. They might be difficult to differentiate from HCC. Haemangioma is usually confidently diagnosed by imaging with ultrasound/multiphasic CT/MRI. Very rarely, a biopsy may be needed to confirm the diagnosis of atypical haemangiomas [8-10]. A giant hemangiomas and multiple hemngiomas are shown in Fig. 1 and 2. + +Microscopically, they can be typified into cavernous, capillary, and sclerosing [7,11]. Cavernous haemangioma are large dilated spaces lined by endothelial cells. They are the most common type. Giant haemangiomas differ only in size by being more than 5 cm with the same histopathology [1]. Capillary haemangiomas comprise of small caliber capillaries. Sclerosing haemangiomas are fibrosed and sclerosed with degenerative changes. Cavernous haemangiomas also regress to sclerosing haemangiomas if they are asymptomatic for a long period [12]. + +Diffuse haemangiomas in liver (haemangiomatosis) might be associated with some syndromes such as Osler-Weber-Rendu disease [13] and medications such as estrogen [14] and metoclopramide [15] with a female predilection. These may present with abdominal pain, distension, weight loss, fever, night sweats, and jaundice. These lesions are ill- defined. They may present with complications such as disseminated intravascular coagulation (Kasabach-Merritt syndrome) [16]. Major differential diagnoses on histopathology are HEHE and hepatic hemangiosarcoma (HHS) [17]. On histopathology, haemangiomatosis will present as an ill-defined mass of capillaries while HEHE will show cords and strands of cells in a myxohyaline stroma and HHS will show highly pleomorphic cells without any vasoformation [2]. + +As most haemangiomas are asymptomatic, they need no treatment or follow-up imaging. When they produce symptoms or complications, surgical resection or enucleation is the treatment of choice. A pre-operative angioembolization of the feeder vessels may help reduce intra-operative bleeding. When the location is not feasible for surgical treatment or if the patient is not a good operative candidate, angioembolization may be offered. Haemangioma rupture spontaneously or after blunt trauma is exceptionally rare. It has a high mortality [8]. + +## Pediatric Hepatic Haemangiomas + +Haemangiomas are the most common benign tumors in the pediatric age group. They are benign proliferative vascular tumors originating from endothelial cells. They are the most common benign vascular tumors of infancy, accounting for 12% of all childhood hepatic tumors with a male-to-female ratio of 1:2 [18-20]. Cutaneous haemangiomas are the most common, followed by liver as the second most common site. Liver haemangioma can be congenital haemangioma (CH) +which is present at birth. It starts to develop in fetal life or infantile haemangioma (IH) which is rarely present at birth. CH +usually presents as a single lesion. It is completely formed at birth. It generally does not grow after birth. It can be further subdivided into three types: 1) rapidly involuting congenital haemangioma (RICH), the most common subtype, which involutes before the age of 14 months; 2) non-involuting congenital haemangioma (NICH), which does not regress and may grow with growth of child; 3) partially involuting congenital haemangioma (PICH). CH is glucose transporter-1 (GLUT1) negative while IH is GLUT1 positive [18,21]. + +Infantile hepatic haemangiomas (IHH) are the commonest tumors of infancy, more common than CH [18]. IHH can be further subdivided into focal, multifocal, and diffuse types with nearly half of all cases being associated with cutaneous haemangiomas. Focal type is typically solitary. It is a heterogeneous mass with dilated vascular channels with or without calcifications. Multifocal and diffuse types may grow to a large size and cause peripheral arteriovenous (AV) shunting. + +The classical triad for clinical diagnosis of IHH includes palpable liver, cardiac failure, and cutaneous haemangiomas in an infant. Most of the IHH lesions grow during the first year of life and then spontaneously regress by 5–7 years [9,18,19]. These patients with multifocal and diffuse types may present with cardiac failure, liver failure, consumption coagulopathy (Kasabach-Merritt syndrome), hypothyroidism, or abdominal compartment syndrome. + +On USG, IHH may present as a complex liver mass, single or multiple, ranging from hypoechoic to hyperechoic with large draining hepatic veins due to intra-lesional AV shunting and high blood flow. On multiphasic CT/MRI, early nodular peripheral enhancement and delayed progression to the center of the lesion, similar to adult haemangiomas, are seen. In larger tumors, there is central hypodensity due to either fibrosis, hemorrhage, or necrosis. The aorta typically has a decreased + +Fig. 3. Ultrasound images in an infant with congenital hepatic haemangioma. (A) Colour doppler image in a 19-day-old neonate shows a large hyperechoic mass with internal cystic areas and vascularity. Lesion is delineated by red arrows. Mass is causing splaying of hepatic veins. (B) Followup ultrasound at the age of 15 months shows a significant decrease in the size of the mass lesion with increased hyperechoic fibrous component. Mass is delineated by yellow arrows. + +![2_image_0.png](2_image_0.png) + +Fig. 4. Contrast- enhanced computed tomography (CT) images in a 28-day-old female neonate showing diffuse infantile hepatic haemangiomas who presented with abdominal distension. Axial images in (A) portal venous, (B) 3-minute delayed phases show typical peripheral nodular enhancement with progressive centripetal filling of contrast in delayed phase. Intervening normal liver parenchyma is seen between lesions. + +![3_image_0.png](3_image_0.png) + +caliber distal to the origin of the hepatic mass, indicating large blood supply to the tumour. On MRI, IHHs are hypointense on T1-weighted images and hyperintense on T2-weighted images. Lesions with significant AV shunting and blood pooling may show flow voids on T2-weighted images [9,10]. Cases of pediatric hemengioma have been depicted in Fig. 3 and 4. + +Microscopically, they can be divided into two types: type 1 and type 2. Type 1 is the most common type. It shows proliferation of small, irregular capillary-like vascular spaces lined by bland or plump endothelial cells separated by variable connective tissue. Extramedullary hematopoiesis and entrapped hepatocytes might be seen in the periphery of the lesion. Type 2 lesion is more uncommon. It is aggressive, comprising of irregular branching vascular structures lined by pleomorphic, hyperchromatic endothelial cells, frequent mitotic activity. Although Type 2 is pleomorphic, it has better prognosis. HEHE +differs from IHH in having intracellular vascular lumina with luminal red blood cells (RBCs) [2]. IHH is immunopositive for factor VIII related antigen, CD31, CD34, and GLUT1 [22]. + +Treatment of asymptomatic IHH is conservative as most will involute and regress spontaneously. Symptomatic IHH are treated with propranolol and medical therapy of cardiac failure if present. If medical therapy fails, then angioembolization of the IHH, surgical ligation of hepatic artery, and surgical resection of the lesion might be done [18,19]. + +![3_image_1.png](3_image_1.png) + +Fig. 5. Multiphasic contrast-enhanced computed tomography (CT) in a 45-year-old male patient showing a large regenerative nodule in the background of Budd-Chiari syndrome. Axial images in (A) non-contrast, (B) ar terial, (C) por tal venous, and (D) hepatic venous phases show a well-defined nodule in segment II (red arrow). Nodule is iso-hypoattenuating in non-contrast CT (A), showing arterial enhancement (B) which homogeneously progresses in portal venous phase and becomes isoattenuating in hepatic venous phase (D) with the background parenchyma. Hepatomegaly, heterogeneous reticular enhancement of liver, splenomegaly, prominent azygous system, and ascites are features of BuddChiari syndrome seen in this case due to supra hepatic inferior vena cava (IVC) stenosis. + +## Nodular Regenerative Hyperplasia + +Nodular regenerative hyperplasia (NRH) is a rare condition of the liver that can cause portal hypertension. It comprises 0.7% to 2.6% of hepatic lesions with a mean age of presentation at 40 years [1]. These vasculopathies are among causes of non-cirrhotic portal hypertension. Common etiologies of NRH are myelo- and lympho-proliferative diseases, autoimmune diseases, inflammatory conditions, immunodeficiency disorders, primary biliary cholangitis, Budd-Chiari syndrome, collagen-vascular diseases, and congenital and acquired hepatic macrovascular abnormalities (Rendu-Osler-Weber, Abernethy malformation). These may also occur after chemotherapy such as highly active anti-retroviral therapy (HAART), +platinum-based chemotherapy, thiopurines, azathioprine, and thioguanine [23]. NRH develops due to obstructive vasculopathy or secondary damage to sinusoids. They are usually asymptomatic until the lesion becomes large enough to cause portal hypertension. They present as small tan white nodules without perinodular fibrosis. A case has been depicted in case Fig. 5. On microscopic examination, these lesions comprise of diffuse nodules of hyperplastic hepatocytes around the portal tract with centrilobular atrophy of hepatocytes. Due to compression of the central veins, sinusoids become congested. Regenerative hepatocytes have clear, vacuolated cytoplasm along with cholestasis. They are 2–3 plates thick while adjacent hepatocytes are usually one plate thick. There is no evidence of fibrosis or extramedullary haematopoiesis in the liver parenchyma [24]. Liver transplant may be one of the treatment options when significant complications develop secondary to portal hypertension [1]. + +## Locally Aggressive Or Borderline Malignant Tumors Kaposi Sarcoma + +KS is a low-grade mesenchymal tumour of blood vessels. It is lymphatic. It primarily involves skin. It can be disseminated to other organs. It has four variants, namely classic KS, endemic (African) KS, iatrogenic (organ transplant-related) KS, and acquired immunodeficiency syndrome (AIDS)-related KS. Hepatic KS is most commonly associated with AIDS or organ transplantation. It is usually detected on autopsy. Its clinical features such as nausea, abdominal bloating, lethargy, and fever are usually non-specific and related to systemic diseases. Hepatic KS appears as hepatomegaly with hyperechoic bands and nodules along peripheral branches of portal veinson ultrasound and hypodense nodules in hilar, periportal or capsular in location on CT with delayed enhancement. On MRI, they display high signal on in-phase T1-weighted images and low signal on out-of-phase images due to intracellular fat. They are isointense on T2-weighted images [25,26]. + +On histopathology, these tumors are typically perivascular with main concentration around peripheral portal branches. + +There is presence of nodules with macrovesicular steatosis and the perinodular area showing presence of small vessels. Nodules are comprised of spindle shaped cells with intervening slit like or sieve-like blood vessels [2]. Intracytoplasmic hyaline globules, extravasated RBCs and hemosiderin laden macrophages are common findings. These tumors are typically immunopositive for HHV-8, showing a stippled nuclear staining pattern. They are also positive for ERG, CD34, CD31, and D240 [2]. However, they are negative for myoid, melanocytic, and epithelial markers [2]. + +## Perivascular Epithelioid Cell Tumour (Pecoma) + +Perivascular epithelioid cell tumors (PEComas) are a family of mesenchymal tumors composed of distinctive perivascular epithelioid cells (PECs). They are rare tumors with more frequency in females and a mean age of presentation at 45 years. They can present as painless masses at a wide range of anatomical sites. Most of them are asymptomatic, but may present with non-specific symptoms such as nausea, abdominal bloating, indigestion, and loss of appetite. + +In AML, macroscopic fat (may be more than 50% of the tumour) can be detected on CT or MRI. In rest of PEComas, radiological findings are non-specific with absence of macroscopic fat [27,28]. Hypervascularity and arterio-venous connections or dysmorphic vessels in or surrounding lesions are hallmarks of PEComas on imaging [27,29]. + +These are rare tumors arising from perivascular endothelial cells. They show myoid and melanocytic differentiation [30]. They comprise of angiomyolipoma (AML), epithelioid AML, and lymphangioleiomyomatosis [2]. Hepatic AML are very uncommon. They often present in association with Tuberous Sclerosis resulting from loss of heterozygosity in TSC2 locus +[2]. They are also seen in association with TFE3 gene rearrangement [2]. Grossly, these are well circumscribed with a fibrous or fleshy cut surface. Microscopically, these tumors are comprised of nests, trabeculae, or sheet like architecture of cells that are epithelioid to spindle with granular eosinophilic to clear cytoplasm [2]. They are perivascular in orientation. They are immunopositive for HMB45, Melan A, MITF, SMA, Desmin, and h-Caldesmon [30]. These tumors have uncertain malignant potential with clinically malignant tumors metastasizing to liver, lymph nodes, and bone [2]. + +## Solitary Fibrous Tumour + +SFT is a rare soft tissue tumour. Its incidence in the liver is less than 1% [1]. It is seen in the 5th to 6th decade of life as a solitary, well-defined lesion often with haemorrhage and cystic degeneration [31]. + +SFT was previously known as hemangiopericytoma. It is a tumour arising from pericytes which lie near endothelial cells of blood vessels. Liver involvement might be primary or metastatic. Primary hepatic SFTs are very rare with only a few case reports. They are usually seen in elderly females. They are mostly benign with progressive growth. However, a few cases of malignant potential have also been reported [32]. These patients may remain asymptomatic or may present with acute abdomen in case the tumour ruptures. Their clinical features are generally nonspecific, such as vomiting, weight loss, post prandial fullness, abdominal distension, anorexia, and fatigue. A few cases of hypoglycemia due to secondary secretion of insulin-like growth factor-2 (IGF-2) by tumour cells have been reported [32,33]. + +SFTs are well-defined hypoto hyperechoic masses on USG +and hypodense on non-contrast CT. They show minimal to heterogenous enhancement on CECT. MRI is the imaging modality of choice that shows a hypointense mass on T1-weighted and an iso-hypointense mass on T2-weighted images due to a high content of fibrous collagen tissue and hypocellularity. Hyperintensity of T2-weighted images can be due to cystic or myxoid degeneration, vascular structures, and hypercellular areas. On CE-MRI, there is a typical intense heterogeneous enhancement which persists into the late phase [34]. + +Microscopic examination reveals that SFT has hypocellular and hypercellular areas with cellular areas being comprised of spindle cells in a patternless arrangement interspersed with staghorn vessels. It has been found that 50% of SFTs show atypia and increased mitotic activity as features of malignancy [31]. + +On immune-histochemistry, these tumors are positive for CD 34 and STAT-6. They show molecular alterations in the form of somatic mutations between NAB2 and STAT6 genes [2,31]. Aggressive surgery is the treatment of choice [35]. + +## Malignant Tumors + +Hepatic epithelioid hemangioendothelioma HEHE is a rare malignant and locally aggressive hepatic neoplasm that develops mainly in adults. It comprises of less than + +![5_image_0.png](5_image_0.png) + +1% of all vascular tumors [1]. It presents commonly between 30 and 50 years of age with a male-to-female ratio of 1:2 [9]. It has shared features of haemangioma and angiosarcoma. It has metastatic potential. However, it is less aggressive than primary angiosarcoma [36]. HEHE is usually incidentally detected, but may present with pain abdomen, jaundice, weight loss, and liver failure in advanced cases due to parenchymal replacement by tumour [9,19]. + +HEHEs may appear on imaging as solitary nodule or multiple nodules or diffuse confluent masses involving both lobes +[36,37]. Solitary nodules are small, usually in a subcapsular location. Multiple nodules may be large in subcapsularor deep locations. They are usually associated with capsular retraction [9]. Multiple nodules then grow to coalesce and form large confluent masses. The active infiltrative tumour is usually at the edge of the mass. There might be fibrosis, hyalinisation, or calcification in the central part [20,38]. + +They are usually hypoechoic on ultrasound. On plain CT, +they are hypodense to normal. Upon contrast administration, they become isodense. Hence, plain CT may be more reliable to estimate the extent of lesion. Depending on the extent of hyalinization, fibrosis, and so on within the tumour, the contrast enhancement may also be heterogeneous. On MRI, they are hypointense on T1- and hyperintense on T2-weighted images. A few signs such as "Target sign" and "Lollipop sign" have been described on imaging, more discernible on MRI. On CE-MRI, peripheral enhancement with delayed central enhancement may be seen. These tumors commonly show hepatic vein or portal vein invasion [9,38,39]. These may metastasize to lymph nodes and lungs [2,40]. Fig. 6 shows a case of HEHE. + +According to recent WHO classification of soft tissue tumors, they can be classified into two groups: WWTR1-CAMTA1 rearranged tumors and YAP1-TFE3 rearranged tumors +[2]. Microscopically, WWTR1-CAMTA1 rearranged tumors show cords, strands, or small nests of large endothelial cells with abundant eosinophilic cytoplasm embedded in a myxohyaline stroma with cells having vesicular, round to oval sometimes indented nuclei. A few tumour cells may even show intracytoplasmic vacuoles with erythrocytes [2]. YAP-TFE3 rearranged tumors have solid nests or pseudo alveolar arrangement of epithelioid cells enmeshed in a fibrous stroma with tumour cells having abundant, densely eosinophilic cytoplasm, although intracytoplasmic vacuoles are rare [2]. These tumors are immunopositive for ERG, CD31, podoplanin (D2-40), FLI1, von Willebrand factor, CAMTA1 in WWTR1-CAMTA1 rearranged tumors, and TFE3 in YAP-TFE3 rearranged [4143]. They are negative for S100P, SOX10, Desmin, and EMA [40]. Angiosarcoma is the major differential diagnosis. It can be differentiated on the basis of ill-formed vasoformation with pleomorphic cells. It lacks the above-mentioned mutations. HEHEs, although extremely rare, can also present in pediatric population at around 12 years of age. They share similar histopathological features as those of adults [3]. + +Surgical management, either resection or liver transplantation, is the treatment of choice and the only modality that can lead to a 5-year survival rate of 75%–80%. Only 10% lesions are diagnosed early. They are amenable to resection. Majority undergo liver transplantation. Extrahepatic disease may be present in one-third of patients at presentation. However, surgical management still gives good results and prolongs survival +[9,19]. There is no role of chemotherapy or radiotherapy. + +## Hepatic Angiosarcoma + +Hepatic angiosarcoma or hemangiosarcoma is a rare aggressive malignant tumour arising from endothelial cells. It is the most common primary sarcoma of the liver, contributing to 1% of all liver tumors in adults. It is more common in elderly men, with a male-to-female ratio of 3:1 [9,20]. History of previous exposure to Thorotrast, vinyl chloride, arsenicals, and androgenous steroids has been found in 25%–40% cases [20,44]. Clinically, it presents with generalized weakness, weight loss, abdominal pain, anemia, or hepatomegaly. About half of patients may present with liver failure with jaundice, splenomegaly, hepatic encephalopathy, or ascites. Thrombocytopenia (Kasabach-Merrit syndrome) and rupture with acute hemoperitoneum are rare presentations. + +If it is associated with previous thorotrast exposure, metallic density in 'network fashion' in upper abdominal masses +(liver, spleen or mesenteric lymph nodes) is noted on plain radiographs or CT [45]. On USG, it presents as single or multiple hyperechoic lesions with variable echogenicity due to hemorrhages. On CT, the reticular pattern of thorotrast deposition in liver and spleen with its characteristic circumferential deposition in the periphery of the nodule is noted. In cases other than thorotrast exposure, on plain CT, it usually presents as single or multiple hypodense masses (hyperdense if hemorrhagic) which show arterial enhancement on contrast injection that persists during portal and delayed phases. The pattern of contrast enhancement may be centripetal similar to a haemangioma or a reverse haemangioma pattern. On MRI, it is hypointense on T1-weighted images and predominantly heterogeneously hyperintense on T2-weighted images [46]. The inhomogeneity on T2-weighted images differentiates angiosarcoma from haemangiomas. As thorotrast does not produce any signal on MRI, its presence can easily be missed. On CEMRI, enhancement patterns are similar to CECT. As imaging findings of angiosarcoma overlap with other vascular tumors of liver, the only reliable finding to diagnose it on imaging is its rapid growth on serial imaging studies. Percutaneous trucut biopsy is contraindicated due to the risk of bleeding. Fine needle aspiration cytology may be done for diagnosis [9]. Angiosarcomas are the most common malignant mesenchymal tumors of the liver comprising 2% of primary hepatic tumors with a predominantly male occurrence. They present commonly in the 6th to 7th decade [47-49]. On histopathology, these tumors have features ranging from bland cells having + +| Table 1. Summary of hepatic vascular lesions Entities Inc (%) Age Sex | Presentation | Gross | Microscopic examination | IHC | Molecular | Mx | Articles | | | +|-------------------------------------------------------------------------|---------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------|---------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|--------------------|-----------------------|-----------------------| +| Hemangioma | 1–6 | Children to adults | Common in females | If large or long standing Thrombosis, post traumatic bleeding or pressure related symptoms | CD 31, CD 34, | Nothing specific | Resection if large | Lerut and Iesari [1], | | +| ERG, FLI-1, | Thampy et al. [11], | | | | | | | | | +| Factor VIII | Miyaki et al. [12], Kristidis et al. [21], Yoon et al. [58], Brouwers et al. [59] | | | | | | | | | +| Hemangiomatosis | Rare | Children to adult | Common in females | Kasabach-Merritt syndrome Ill-defined | Similar to | Similar to | Nothing specific | Liver transplant | Lerut and Iesari [1], | +| hemangiomas | hemangiomas | Thampy et al. [11], Maeda et al. [16], Lazăr et al. [31] | | | | | | | | +| HEHE | < 1 | 30–50 years | Common in females | Mostly | Asymptomatic | | | | | +| asymptomatic | masses but can coalesce into large masses having whitish firm surfaces Well circumscribed; > 5 cm: Giant hemangioma | Cavernous hemangioma: Most common; large dilated spaces lined by endothelial cells. WWTR1-CAMTA1 rearranged tumors: cords, strands or small nests of large endothelial cells with abundant eosinophilic cytoplasm embedded in a myxohyaline stroma with cells having vesicular, round to oval sometimes indented nuclei. YAP-TFE3 rearranged tumors: Solid nests or pseudo alveolar arrangement of epithelioid cells enmeshed in a fibrous stroma with tumor cells have abundant, densely eosinophilic cytoplasm. ERG, CD31, podoplanin (D2-40), FLI1, von Willebrand factor, CAMTA1 in WWTR1- CAMTA1 rearranged tumors and TFE3 in YAP-TFE3 rearranged. Negative for S100P, SOX10, Desmin and EMA. | WWTR1-CAMTA1 rearrangemnet; YAP-TFE3 rearrangement | Wide local excision | Lerut and Iesari [1], Bannoura and Putra [3], Miller et al. [36], Da Ines et al. [37], Lau et al. [40], Flucke et al. [41], Lazăr et al. [31] | | | | | +| Angiosarcoma | 2 | 60–70 years | Common in males | Portal | Ill-defined, multifocal | Features ranging | | | | +| hypertension/ | from bland | | | | | | | | | +| liver failure | cells having vasoformation to sheets and cords of pleomorphic cell without any vasoformation.2 Tumour cells are plump, pleomorphic and mitotically active. Can be spindle shaped, polygonal, epithelioid and primitive round cells, forming papillae or solid nests within vascular lumina. Positive: CD31, CD34, ERG, FLI1, VEGF and factor VIII. Negative: HHV-8 | Upregulation of | Surgical | | | | | | | +| TIE1, KDR, TEK | resectionand | | | | | | | | | +| and FLT | adjuvant chemotherapy | WHO [2], Lazăr et al. [31], Yasir and Torbenson [47], Mehrabi et al. [48], Li et al. [49], Omiyale and Carton [50], Huang et al. [51] | | | | | | | | + +| Table 1. Continued 1 Entities Inc (%) | Age | Sex | Presentation | Gross | Microscopic examination | IHC | Molecular | Mx | Articles | +|-----------------------------------------|-----------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------|----------------------------------------------------------------------------------------|----------------------------|------------------------------------------------------------------------|-------------------------------------------------------------------------------|---------------------------------------|------------| +| Kaposi sarcoma | Predominantly | Predominantly | Predominantly | Indolent; | Multinodular | Typically | | | | +| HIV | HIV | HIV | Locally | perivascular | | | | | | +| associated | associated | associated | aggressive | with main concentration around peripheral portal branches. Presence of nodules with macrovesicular steatosis and the perinodular area showing presence of small vessels. Nodules are comprised of spindle shaped cells with intervening slit like or sieve like blood vessels. Typically immunopositive for HHV-8 showing stippled nuclear staining pattern | Nothing specific | Palliative | WHO [2] | | | +| PEComa | Rare | Common in females | Mean age 45 years | Commonly associated with tuberous sclerosis; May range from benign to malignant | Well-circumscribed with a fibrous or fleshy cut surface | Comprised of nests, trabeculae or sheet like architecture of cells which are epithelioid to spindle with granular eosinophilic to clear cytoplasm Immunopositive for HMB45, Melan A, MITF, SMA, Desmin and h-Caldesmon. Loss of heterozygosity in TSC2 locus. TFE3 gene rearrangement | Benign: Resection Malignant: Chemotherapy including mTOR inhibitors [42] | WHO [2], Folpe and Kwiatkowski [30] | | +| HIHE | 20 of pediatric | Common in females | Mean age 6 months | Congestive | | | | | | +| hepatic | cardiac failure, | | | | | | | | | +| tumors | hepatomegaly and failure to thrive Solitary to multiple well circumscribed masses with red tan, soft to spongy cut surface with coexisting cavernous hemangiomas | Type 1: | Factor VIII | | | | | | | +| proliferation of | related | | | | | | | | | +| small, | antigen, | | | | | | | | | +| irregular | CD31, CD34 | | | | | | | | | +| capillary-like | and GLUT1 | | | | | | | | | +| vascular spaces lined by bland or plump endothelial cells separated by variable connective tissue. Type 2: irregular branching vascular structures lined by pleomorphic, hyperchromatic endothelial cells, frequent mitotic activity. | Can be associated | May regress; | Lerut and Iesari [1], | | | | | | | +| with Wilms | Solitary: | WHO [2], | | | | | | | | +| tumour | resection | Christison-Lagay | | | | | | | | +| Steroids, | et al. [60], | | | | | | | | | +| Embolization | Mo et al. [22] | | | | | | | | | +| and transplant can be tried [43]. | | | | | | | | | | +| HSVN | Recent entity | Common in males [22] | Mean age: 54 years [22] | Mostly | Infiltrative borders | Irregular vessels | | | | +| asymptomatic | lined by flat to oval endothelial cells showing hobnailing. | CD31, CD34 and FLI-1 | GNAQ, GNA11, | Resection and follow-up | Lazăr et al. [31], | | | | | +| or GNA14 | Walcott-Sapp | | | | | | | | | +| mutations | et al. [56], Joseph et al. [57] | | | | | | | | | +| SFT | Less than 1 | Not specific | 50–60 years | Asymptomatic | Solitary, | Hypocellular | | | | +| to acute | well- defined | and | | | | | | | | +| abdomen | lesion | hypercellular areas with the cellular areas being comprised spindle cells in a patternless arrangement interspersed with staghorn vessels. | CD 34 and STAT-6 | Somatic mutations between NAB2 and STAT6 genes. | Aggressive surgery | Lerut and Iesari [1], WHO [2], Lazăr et al. [31], Adams et al. [35] | | | | + +| Table 1. Continued 2 Entities Inc (%) | Age | Sex | Presentation | Gross | Microscopic examination | IHC | Molecular | Mx | Articles | +|-----------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------|---------------------|---------|----------------------------|-----------------|-------------|------|------------| +| NRH | 0.7 to 2.6 | Not specific | Mean age: 40 years | Portal hypertension | Small nodules | Diffuse nodules | | | | +| without | of hyperplastic | | | | | | | | | +| cirrhosis | hepatocytes around the portal tract with centrilobular atrophy of hepatocytes. Sinusoids become congested with compression of the central veins. The regenerative hepatocytes are have clear, vacuolated cytoplasm along with cholestasis and are 2–3 plates thick as compared to the adjacent hepatocytes which are usually one plate thick. No evidence of fibrosis or extramedullary hematopoiesis in the liver parenchyma. | Lerut and Iesari [1], Shastri et al. [23], WHO [2] | | | | | | | | +| HEHE, hepatic epithelioid hemangioendothelioma; PEComa, perivascular epithelioid cell tumour; HIHE, hepatic epithelioid hemangioendothelioma; | | | | | | | | | | + +HEHE, hepatic epithelioid hemangioendothelioma; PEComa, perivascular epithelioid cell tumour; HIHE, hepatic epithelioid hemangioendothelioma; HSVN, hepatic small vessel neoplasms; NRH, nodular regenerative hyperplasia; IHC, immunohistochemistry. + +vasoformation to sheets and cords of pleomorphic cells without any vasoformation [2]. These tumour cells are plump, pleomorphic, and mitotically active. They can be spindle-shaped, polygonal, epithelioid, and primitive round cells, forming papillae or solid nests within vascular lumina [2]. Intratumoural haemorrhage can also be seen. These tumors are immunopositive for CD31, CD34, ERG, FLI1, VEGF, and factor VIII but negative for HHV8 ruling out KS [2]. Pediatric angiosarcomas are very rare hepatic tumors having similar biopsy features as those of adults, presenting more commonly in females at around 40 months of age with abdominal distention and pain [3]. These tumors show upregulation of vascular specific receptor tyrosine kinases such as TIE1, KDR, TEK, and FLT [50]. Surgical management and adjuvant chemotherapy are treatments of choice for a better survival [51]. Antiangiogenic therapy, recombinant therapy, and immunotherapy have been tried with or without chemotherapy, showing highly debatable results [31]. + +Angiosarcoma is an aggressive tumour with an extremely + +| Table 2. Benign and intermediate hepatic vascular tumors: a comparison among literature Hepatic hemangiomas Kaposi Sarcoma | HSVN | | | | | | +|------------------------------------------------------------------------------------------------------------------------------|------------------|--------------------|---------------------|-------------------|------------------------|-----------------------| +| Entities | Gill et al. [53] | | | | | | +| Bajenaru et al. [61] | Jia et al. [62] | Van Leer-Greenberg | Arora and Goldberg | (described first; | Mulholland et al. [65] | | +| et al. [63] | [64] | 17 cases) | | | | | +| Incidence | 0.4%–20% | 0.4%–7.3% | 12-24; 34% with HIV | > 50% in GIT | Rare | 22–23 cases till date | +| Age | 30–50 yr | 30–50 yr | 20–40 yr | Patients with HIV | 24–83 yr | | +| M:F | 1:4.5 to 1: 5 | 1:1.2 to 1:6 | Common in males; | Males | Common in males | | +| Africa | | | | | | | +| Treatment | Resection if | Resection if | HAART and | HAART | Resection | | +| symptomatic | symptomatic | chemotherapy | | | | | +| GIT, gastrointestinal tract; HAART, highly active anti-retroviral therapy; HSVN, hepatic small vessel neoplasms. | | | | | | | + +| Table 3. Malignant hepatic vascular lesions: a comparison among literature Hepatic angiosarcomas | Hepatic hemangioendothelioma | | | +|----------------------------------------------------------------------------------------------------|-------------------------------------------------|----------------------------|----------------------------------------| +| Entities | Kumar et al. [66] | Chen et al. [67] | Kou et al. [68], Virarkar et al. [69] | +| Incidence (%) | 0.1–2 | 0.1–2 | 1–2/million | +| Age (yr) | 60–70 | 60–70 | 30–50 | +| M:F | 3:1 to 4:1 | 3:1 | 2:3 | +| Treatment | Complete hepatic resection or radical resection | Complete hepatic resection | Surgical | +| Median survival | 6 mon | 6 mon | 5 yr survival: 75% | + +poor prognosis. Most patients present with advanced tumors involving both lobes of liver. They usually die within six months of diagnosis. Up to 50% may also have extrahepatic metastases to lung, bone, or spleen [20]. Rarely, if the lesion is localised to one lobe, surgical resection may be done. Palliative angioembolisation may be done for symptomatic relief or in cases of tumour rupture with hemoperitoneum. Liver transplantation is contraindicated due to its aggressive nature and early recurrence post-transplant [9,19]. + +## Hepatic Small Vessel Neoplasm + +HSVN is a recently identified rare vascular tumour of liver that shows features of both angiosarcoma and cavernous haemangioma. It has an infiltrative growth pattern with minimal cytological atypia and mitotic activity [52]. It occurs mainly in men, with a mean age of 54 years and a size of 2.1 cm [53]. It is usually asymptomatic. It can cause epigastric fullness when large. Its imaging features are non-specific, showing strong enhancement on arterial phase with the highest enhancement on portal phase and equivocal findings on delayed phases on CT +and diffusion-weighted MRI [54,55].The final diagnosis is usually made on immunohistochemistry and Ki-67 proliferation index. + +HSVNs are different from haemangiomas in having infiltrative borders. They are not as malignant as angiosarcomas as they do not have atypical cells or high mitotic activity [56]. + +Joseph et al. [57] have suggested that these tumors may harbour GNAQ, GNA11, or GNA14 mutations. These tumors have uncertain malignant potential. They present as poorly defined haemorrhagic tumors. Microscopically, they comprise of infiltrative neoplasm having irregular vessels lined by flat to oval endothelial cells showing hobnailing [23]. These tumors are immunopositive for CD31, CD34, and FLI-1 [31,56]. Their major differential diagnosis is angiosarcoma. Both entities can be diagnosed and differentiated on the basis of GLUT-1, p53, Ki-67, and c-myc expression. HSVN has higher Ki-67 than haemangiomas. However, if it is more than 10%, then angiosarcomas should be suspected [31]. Immunopositivity for GLUT1, p53, and c-mycgoes in favour of angiosarcoma [31]. Surgical resection is curative. No recurrence has been reported [8,53]. + +## Conclusion + +There are not many vascular tumors of the liver. They are definitely uncommon and difficult to discern other than typical haemangiomas. A correct diagnosis is essential to decide about the management and prognosis. All vascular tumors of liver that can be diagnosed on histopathology and treated accordingly are summarized in Table 1-3 [1-3,11,12,16,2123,30,31,35-37,40,41,47-51,53,56-69]. + +## Funding None. Conflict Of Interest + +No potential conflict of interest relevant to this article was reported. + +## Orcid + +Sujata Sarangi,https://orcid.org/0000-0002-9169-6933 Balamurugan Thirunavukkarasu, https://orcid.org/0000-0002-5583-1720 Sudeep Khera, https://orcid.org/0000-0002-2413-5749 Selvakumar B, https://orcid.org/0000-0002-2280-3862 Taruna Yadav, https://orcid.org/0000-0002-3644-6093 + +## Author Contributions + +Conceptualization: SS. Data curation: SS, BT, TY, SB. Methodology: SS. Writing - original draft: all authors. 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World J Gastrointest Oncol 2020;12:248-266. \ No newline at end of file diff --git a/medical/md/PMC10848733.md b/medical/md/PMC10848733.md new file mode 100644 index 0000000000000000000000000000000000000000..013094d8ee0b827ae2690b39b8d64c5c20130768 --- /dev/null +++ b/medical/md/PMC10848733.md @@ -0,0 +1,452 @@ +GEOLO GY + +# Lithospheric Thickness Records Tectonic **Evolution** By Controlling Metamorphic **Conditions** + +Zhen-Jie Zhang1,2,3, Guo-Xiong Chen4, Timothy Kusky4, Jie Yang2,3, Qiu-Ming **Cheng2,3,5*** +The lithosphere, as the outermost solid layer of our planet, preserves a progressively more fragmentary record of geological events and processes from Earth's history the further back in time one looks. Thus, the **evolution** of lithospheric thickness and its cascading impacts in Earth's tectonic system are presently unknown. **Here,** we track the lithospheric thickness history using machine learning based on global lithogeochemical data of basalt. Our results demonstrate that four marked lithospheric thinning events occurred during the Paleoarchean, early Paleoproterozoic, Neoproterozoic, and Phanerozoic with intermediate thickening scenarios. These events respectively correspond to supercontinent/supercraton breakup and assembly periods. Causality investigation further indicates that crustal metamorphic and deformation styles are the feedback of lithospheric thickness. Cross-correlation between lithospheric thickness and metamorphic thermal gradients records the transition from intraoceanic subduction systems to continental margin and intraoceanic in the Paleoarchean and Mesoarchean and a progressive emergence of large thick continents that allow supercontinent growth, which promoted assembly of the first supercontinent during the **Neoarchean.** +Copyright © 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). + +## Introduction + +How Earth evolved from its initial hot accretion to the present-day active-lid plate tectonic/mantle convection system is poorly known and hotly debated. Some models suggest that early Earth had a stagnant lid, with no plate tectonics but with numerous plumes pumping magmas from the deep onto a thick crust that dripped back into the interior (1–3). Other models suggest that the early hot magma ocean evolved quickly into a primitive form of plate tectonics with small shallow subduction systems on a mafic-dominated lithosphere, which gradually formed felsic crusts and continents, and that the stagnant lid regime is still billions of years in the future (*4–10*). + +Asthenospheric cooling (*11–14*), which controlled the density differences between the oceanic lithosphere and underlying asthenosphere, has been considered the critical factor that induced and maintained the plate tectonics system (1–3, 15, 16). Asthenospheric temperature reflects the balance between heat gain by natural radioactive decay of Earth's interior and conductive heating at the core-mantle boundary and heat loss by convective and conductive cooling through Earth's lithosphere (17). + +However, the asthenospheric temperature has changed markedly throughout Earth's history (15, 17). Given the smooth secular decay of primordial accretional heat, this marked change of asthenospheric temperature is likely caused by heat release processes controlled by the lithosphere. However, on the other hand, asthenospheric temperature also affects the lithospheric thickness by controlling the partial melting of the upper mantle because the temperature is the most important factor controlling lithospheric rheology (18). Changes in the lithospheric thickness, induced, for instance, by tectonic processes, affect not only the thermal state of the crust but also the heat emissions from Earth's interior, thus retroactively affecting the temperature of the upper mantle in a feedback mechanism, influencing tectonic regimes (19). Therefore, as the rigid outer layer of Earth, the lithosphere records interactions with the underlying asthenospheric mantle, becoming the longterm archive of Earth's evolution (20). + +Because of the temperature dependence of silicate rheology, rapid changes in mantle temperature are strongly inhibited (21). Thus, most Earth scientists assume that the global lithosphere volume has grown slowly but steadily over Earth's history as the result of the secular slow cooling of the asthenosphere since the Archaean (1, 22). Others suggest that most of the continental crust volume, as an important part of lithosphere, formed very early in the Hadean and has been constantly recycled since (23, 24). Recently, Tang et al. (25) used Eu anomalies in detrital zircons as a crustal thickness proxy and suggested that the mean thickness of continental crust changed quasi-sinusoidally over Earth's history. Even some cratons, which were thought to have remained stable over billions of years, have experienced substantial lithospheric thinning and even destruction, for example, the North China, Dharwar, Amazonian, São Francisco, and North Siberian Cratons (*26–29*). Therefore, the mean thickness of global lithosphere is not carved in stone and does not necessarily remain thick once thickened by tectonic or magmatic processes. This concept is supported by recent numerical models and isotopic data showing that continents can be recycled to the deep mantle and then return to relaminate the bases of other lithospheric plate in massive regional relamination events, complicating ideas of slow lithospheric and crustal growth through time (30). Therefore, as the interaction product of multilayers of Earth, the lithosphere continues to emerge, rework, and disappear, and its thickness should markedly change and record the evolution of tectonic regimes during Earth's history. + +Mantle-derived xenoliths as probes of Earth's deep interior can directly reveal the compositional and thermal evolution of the lithosphere (31–34). However, deciphering the evolution of lithospheric thickness is challenged by the difficulties in accessing rock records directly from the deep crust and mantle (20). Basalts and basaltic rocks are the most widespread and abundant igneous rocks on Earth. Since basalts are solidified melts ultimately derived from the asthenosphere, they are expected to contain information about the mantle conditions from which they were derived and thus have been used to infer the geophysical and geochemical heterogeneity of the mantle source (35). However, the incompatible trace elements of the basaltic primary melts are highly sensitive to the composition of the mantle source and re-equilibration with the surrounding mantle during their ascent (36). Experimental petrology has shown that the oxides of the basalt primary melts in all tectonic settings on Earth are sensitive to the pressure of melting +(Fig. 1) and less sensitive to variations in mantle compositions, hence being a good choice to estimate the lithosphere thickness +(*37–41*). Clinopyroxene barometry results for megacrysts in basalts, e.g., the Cenozoic alkali basalts from eastern continental China (42), also indicate that the lithospheric thickness has substantial controls on the oxides compositional variations of these basalts. + +This makes it possible to estimate the lithospheric thickness using the oxides of basalts. + +Benefiting from the vast and ever-expanding global geo-databases (e.g., EarthChem, GEOROC, etc.), we can now obtain the lithogeochemical big data of global basalts (Fig. 2 shows the distribution of samples used in this study). However, it is difficult to find relations between oxides and lithosphere thickness in high-dimensional space. Machine learning (ML) succeeds in regression problems by exploring a larger function space that can connect data to some target or label (43, 44). Random forest (RF) is an outstanding supervised ML algorithm, which can provide the best performance in many geoscience applications (44, 45). Here, we compiled a lithogeochemical database of global basalts spanning the past 3.8 billion years (Ga) (Fig. 2 and see data S1 for detailed database) to reconstruct global lithospheric thickness (Fig. 3) using the RF method, gaining insight into the initiation and development of plate tectonics with the aim to resolve some of the uncertainties evident from the varying conclusions in the cited works above. + +## Results And **Discussion** Global Lithospheric Thickness **Reconstruction** + +We used 1392 experimental basaltic liquid compositions in equilibrium with olivine and orthopyroxene (data S2), compiled from the LEPR (Library of Experimental Phase Relations) database and published literature, as the training and testing data to train an RF regression model to reconstruct their average final pressure (PF) +values. The lithospheric thickness then can be estimated by the PF +of basaltic melt extraction from the mantle (37), i.e., lithospheric thickness = PF/(gρ), where ρ is the density and g is the acceleration due to gravity. After the conditional average treatment effect (CATE) estimation by the causal forest (CF) (46) ML method, we found that the PF is almost irrelevant to the final temperature +(TF) (figs. S1 and S2; see Materials and Methods for details). Therefore, in the lithospheric thickness estimation, which is proportional to the PF, the TF variable can be ignored. The input features of the model include major oxide compositions, i.e., SiO2, TiO2, Al2O3, total FeO, MnO, MgO, CaO, Na2O, and K2O. We next compiled a lithogeochemical database of ~25,000 global basalts (Figs. 2 and 3A and data S1) to reconstruct global lithospheric thickness since et al., Sci. *Adv.* 9, 15 2023 2 of 13 +~3.8 Ga ago using the trained RF regression model (figs. S3 and S4; see Materials and Methods for details). The initial chronological and oxide data were obtained from the EarthChem data repository (http://portal.earthchem.org/). Samples were filtered to include only those basalts with MgO of 7 to 17 weight % (wt %) to minimize the extent of fractional crystallization (15). Then, we manually removed the samples with total oxides below 95 wt % or loss on ignition above 3 wt %, as samples with these properties may have been weathered or secondary altered. To reduce the influence of extreme values, we eliminate the suspect outlier data that have at least one oxide plotted outside the three interquartile range (3IQR; see Materials and Methods for details) box based on box plots for each oxide during each 100 million years. Considering that the alteration of basalt inevitably leadsto the depletion of key elementssuch as Na, K, and Ca, the utilization of the 3IQR technique enables the elimination of any samples exhibiting anomalous behavior. The final 24,194 chronological and oxide analyses of basaltic rocks are retained in the compiled databases (Fig. 2 and data S1; see Materials and Methods for details). + +We used a randomized split of the dataset into training (70%) +and testing (30%) datasets, and ensured that the construction of decision trees in the training process was also randomized. To assess the uncertainty of the predictions generated by our established RF regression model, we iterated this process, building 100 models and obtaining 100 prediction sets (data S1). The average of these 100 prediction sets was used as the calculated result for each individual sample, while the variance was used to evaluate the uncertainty of the predictions. All performance of our RF regression models yields a high prediction accuracy on the testing datasets (>0.86; Fig. 4, A and B, and data S1), which ensures the accuracy of the results of lithospheric thickness reconstruction. The average variance of the PF values across the 100 individual predictions for all samples was 0.36 GPa, corresponding to a lithospheric thickness uncertainty of 11.4 km (Fig. 4C and data S1). Overall, the prediction results exhibited a relatively low level of uncertainty. + +Our approach calculates the thickness of all magmatically active lithosphere, which we call the active lithosphere similar to the definition method of the active crust in Tang et al. (25). Most of this continental lithosphere forms at active zones including convergent plate margins (47), continental rifts, and intraplate hotspots (fig. + +S5), whereas oceanic lithosphere forms at mid-ocean ridges, backarc basins, or extensional fore-arcs (48, 49). Our results indicate that the active lithospheric thickness fluctuated markedly throughout Earth's history (Fig. 3B) but has remained in the range of 60 to 140 km for the past 4 Ga and shows no apparent gradual secular change. We note that we define "active lithosphere" but do not differentiate between active continental and active oceanic lithospheres. Although the thickness of active lithosphere is not the real average lithospheric thickness, which surely should contain the inactive lithosphere, the evolved tendencies are the same under this strict and same sampling strategy and thus can be used to constrain global evolution. + +Our results are plotted in Fig. 3, which shows that obvious lithospheric thickening occurred in early Earth (>3.5 Ga), whereas Earth experienced its first documentable lithospheric thinning process during the Paleoarchean (3.5 to 3.2 Ga). Later, slow and long-term lithospheric thickening occurred during the Mesoarchean to Neoarchean (3.2 to 2.5 Ga), and then another marked thinning occurred during the early Paleoproterozoic (2.5 to 2.0 Ga). + +![2_image_0.png](2_image_0.png) + +After this, progressive thickening during late Paleoproterozoic to Mesoproterozoic (2.0 to 1.0 Ga) reached the peak average thickness of ~140 km at the late Mesoproterozoic (~1.3 Ga). Another more marked thinning episode then occurred throughout the early Neoproterozoic (1.0 to 0.8 Ga), reducing the average thickness to ~73 km and remaining stable until the terminal Proterozoic. During the Paleozoic (0.5 to 0.3 Ga), the latest thinning episode arose, declining the average thickness to ~60 km. Then, the lithospheric thickness increased to ~73 km at present, which is consistent with the observation of modern active lithospheric thickness (~71 km) +obtained by LITHO1.0 (50) (fig. S5). We also validate our RF regression model by reconstructed Cenozoic lithospheric thickness in eastern China (Fig. 5 and data S4), which shows consistency with the modern lithosphere-asthenosphere boundary depth (39). + +We have tested whether the model we trained might exhibit limited performance in high-pressure environments, given the abundance of basalt production in low-pressure tectonic settings such as mid-ocean ridges and ocean islands (Fig. 4). However, despite slightly higher uncertainty, our RF regression model still demonstrated good performance in estimating samples from high-pressure tectonic settings, as validated by the Cenozoic lithospheric thickness in eastern China (Fig. 5). We further developed an RF regression model by training it exclusively on samples with pressures below 3 GPa, excluding high-pressure samples due to their limited availability. The application of this newly devised model yielded predicted results wherein the pressure values appeared relatively smaller. However, these values still exhibited a consistent relative trend with the original model (fig. S7). Notably, discrepancies between the two models only surfaced during the early Archean epoch, potentially attributed to a scarcity of available samples from this particular period. Thus, the model we trained exhibits a high level of robustness, as evidenced by its consistent performance in predicting pressure values by two different models. Meanwhile, we observed a strong correlation between the experimental and predicted pressures (Fig. 4), indicating that PF plays a pivotal role in shaping the composition of basalt. The presence of outliers surrounding the systematic trends (Fig. 4) could potentially be attributed to compositional variations in the mantle source resulting from either melt reactions or contamination from the crust. Consequently, we assume that the variation in mantle source signifies a secondary influence on the compositional characteristics of basalt as it is overshadowed by the lid effect (PF) (38, 39). + +## Secular Change In Crustal Metamorphism **Driven** By Lithospheric **Thickness** + +The preserved pressure-temperature (P-T ) conditions, i.e., metamorphic thermal gradients represented by the thermobaric ratio +(T/P), of crustal metamorphism changed substantially during Earth's history (Fig. 3C), which has been used to infer a secular evolution of Earth's tectonic processes (51–55). The metamorphic rocks recording these conditions originated from the reworking of crustal rocks through dehydration and melting at lithospheric depths (56). To compare the secular record of lithospheric thickness with the evolution of metamorphic thermal gradients, we calculate the global Pearson correlation coefficient (PCC) (Fig. 6A), i.e., every individual global PCC at time t is calculated by the two time series from time = t to present (time = 0), and show the plot of local PCCs, calculated by the two time series in a particular time window for the PF and T/P ratio (Fig. 6B). This analysis reveals a global PCC value + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +of 0.37, exhibiting high statistical significance (P = 5 × 10−14), +between lithospheric thickness and crustal metamorphism time series since 3.8 Ga ago. There is a substantial increase in the global PCC values at 2.3 Ga (>0.5) and reaching 0.7 after 1.6 Ga (Fig. 6A), indicating increasing coupling between lithospheric thickness and crustal metamorphic thermal gradients from 3.8 Ga. + +Further continuous wavelet transform based on Morlet wavelets +(see Materials and Methods for details) is used to detect the periodic patterns for the PF and T/P ratio and their persistence through time. + +Three statistically significant peaks at approximately 0.3, 0.6, and 1.2 Ga were observed for both spectra (Fig. 6, C and D). They correspond to the periodic signals of the Wilson, supercontinent, and superocean cycles (57). Further wavelet coherence analyses show strong consistency and indicate substantial in-phase coherence for 3 cycles, especially 0.6 Ga of the supercontinent cycle, between the two series throughout Earth's history (Fig. 6E). Therefore, we used 0.6 and 1.2 Ga as the time window size to calculate the local PCCs (Fig. 6B), tracking the synergy between lithospheric thickness and crustal metamorphic thermal gradients for supercontinent and superocean cycles. + +To test their correlations, we show the scatter plot of the metamorphic thermal gradient after Neogene [0.023 Ga to present (53)] +versus present lithospheric thickness obtained by LITHO1.0 (50) (Fig. 7 and data S5). The results show a strong positive correlation. To quantify the strength of synchrony and causality association between PF and T/P, we plotted the time-lagged cross-correlation coefficients (TLCCs) and windowed TLCCs (see Materials and Methods for details) between these two nonstationary time series +(Fig. 8) (58, 59). The maximum PCC appears at the positive coordinates (T/P leads part), and right-skewed PCC curves indicate that when T/P is offset by 0.2 Ga, the correlation is the highest (Fig. 8A). The windowed TLCC shows an obvious and steady 0.2-Ga offset for T/P ratio series throughout Earth's history (Fig. 8B). The lagged series could be interpreted as the effect if a significant cross-correlation is found between the two offset time series. That is to say, the lithospheric thickness can determine the crustal metamorphic conditions after 0.2 Ga. + +## Link To Geodynamic **Processes** + +Conditions of the lithosphere control the thermal state of the crust and buffer the temperature of the upper mantle, thus affecting + +![4_image_0.png](4_image_0.png) + +![4_image_1.png](4_image_1.png) + +tectonic regimes. Lower strength vertical damage and higher strength horizontal shortening of the lithosphere due to subduction would thicken the lithosphere and maintain its thermal blanket effect (19, 53), thus preventing mantle heat loss. Ball et al. (60) have revealed the consistency between mantle potential temperature and lithospheric thickness during intraplate volcanism in the Neogene-Quaternary. The gradually accumulated radiation heat increases the geothermal gradient, conducive to the generation of metamorphism with high T/P ratios. Therefore, the lithospheric thickness and thermal gradient should be positively correlated during plate subduction. The local PCC curve is coupled with the multiple supercontinent cycles, when the age window size is 0.6 Ga, which is referred to asthe periodicity of a supercontinent cycle (57). Supercontinent assembly periods show an increase in PCC values, whereasthe PCC values declined during the breakups (Fig. 6B). The systematic assessment of the basic geology, geochronology, structure, petrology, sedimentology, and tectonic facies analysis has shown that plate subduction is dominant during the last few supercontinent assembly periods (1–3, 10). This is consistent with our hypothesis that the geodynamic process of plate subduction results in a high positive correlation between the lithospheric thickness and metamorphic thermal gradient. + +However, the mechanism of decoupling between the lithospheric thickness and metamorphic thermal gradient is less clear. The decoupling mainly occurs during the last few supercontinent breakup periods (Fig. 6B). This reason can be attributed to hot mantle upwellings that destroy and thin the lithosphere (61, 62) but increase metamorphic thermal gradients during the supercontinent breakup periods, thus decoupling the lithospheric thickness and metamorphic thermal gradients. However, the decoupling also occurs before the Mesoarchean (Fig. 6B). The tectonic regimes during this period are still highly controversial (1–3, 10). Therefore, the decoupling before the Mesoarchean can also be induced by abnormally hot mantle upwellings or plumes. However, intraoceanic subductionaccretionary regime can also lead to decoupling. On early Earth, the continents were on their way to formation, and if plate subduction existed at that time, then it was dominated by juvenile oceanic arcs, with accretionary orogens forming at sites of subduction (10). + +Under this regime, although horizontal shortening of the lithosphere due to subduction would thicken the lithosphere, the lithosphere was fragmented. The asthenospheric cooling could not be prevented because of the addition of cold crustal materials by subduction and the absence of a lithospheric thermal blanket. Therefore, the decoupling between the lithospheric thickness and metamorphic thermal gradient can also appear in the intraoceanic subduction-accretionary regime. + +## Implications For Subduction **Initiation** + +We infer that some form of dynamic lid (i.e., plate tectonics) was operating on the early Earth because of the high positive global PCC values between lithospheric thickness and crustal + +![5_image_0.png](5_image_0.png) + +![6_image_0.png](6_image_0.png) + +metamorphism from 3.8 Ga to the present (Fig. 6A). Since the asthenospheric temperature was relatively high and stable during early Earth, the rapid convection of hot mantle (63) would likely make many arc systems that would collide, thicken the crust, and gradually form continents (10). Thus, active plate subduction systems have been dominant since early Earth, not gradually expanding from the weak zones of a stagnant or sluggish lid (8, 10). This hypothesis is also supported by the lithospheric thickness fluctuating between 120 and 80 km before Mesoarchean (>2.8 Ga), just as its extreme fluctuation in post-Archean times (Fig. 3B). Hot mantle upwellings or plumes under the stagnant or sluggish lid regime will elevate the geotherm and thus can also change the lithospheric thickness (64). However, the change should be more unidirectional and less volatile because of the rapid convection of hot mantle (63). + +There is a tipping point recorded by our local PCCs curve, which reveals an obvious transformation at ~2.8 Ga when the age window size is 1.2 Ga of a global superocean cycle (Fig. 6B). Before ~2.8 Ga, there is a negative correlation (r < 0) between the metamorphic conditions and lithospheric thickness. However, a shift occurs after +~2.8 Ga, leading to a strong positive coupling between PF and T/P +(r > 0). This result indicates that the subduction styles of plate tectonics, which have been extant on Earth from the very beginning, have extraordinarily changed from the Neoarchean. Progressive lithospheric thickening and coupling evolution between the lithospheric thickness and metamorphic thermal gradients after ~2.8 Ga reveal that the Wilson cycle or supercontinents start to dominate asthe continents grew larger (Figs. 3B and 6B). Thisturning point is consistent with the widespread distribution of Neoarchean-Paleoproterozoic collisional orogens (10). The evident intermediate increasing scenario of local PCCs from negative to positive between +~3.6 and 2.8 Ga indicates the secular transitions between the modern Wilson cycle and intraoceanic accretionary tectonic cycles from the early nearly pure intraoceanic subduction-accretionary tectonic regime (10, 47, 56). It provides evidence that the onset of plate tectonics might have been before the Paleoarchean, as early as the Eoarchean (10) or Hadean (65). The increasing global PCC values (Fig. 6A) since Eoarchean also supports this point. + +If the high PCC intervals in the supercontinent cycle scale (age window size is 0.6 Ga) after the Proterozoic, then this may represent several supercontinent assemblies caused by modern Wilson cycle plate subduction. The first high PCC interval between 2.8 and 2.3 Ga indicates that the modern Wilson cycle plate subduction has become the dominant tectonic regime that promoted the assembly of the earliest supercontinent during the Archaean. + +The lithospheric thickness during the Paleoarchean records the first nonlinear thinning process. This marked thinning means a large-scale lithospheric collapse, which carries a large amount of cold material into the hot asthenosphere. Reduced asthenosphere temperatures would inhibit the melting of the high-density oceanic mantle lithosphere (66) and hinder the generation of lowdensity oceanic crust (1, 3), thereby retaining the oceanic lithospheric density. The cooler asthenosphere would widen the disparities in density between the overlying continental lithosphere and subducting oceanic lithosphere, finally prompting the global modern Wilson cycle of continental rifting, plate subduction, and collision events (15, 67). This could be an early record of lithospheric thinning, where the continental roots sink to the asthenosphere, + +![6_image_1.png](6_image_1.png) + +and, eventually, when the thermal buoyancy exceeds the chemical negative buoyancy, returns to underplate and thicken the lithosphere again (30), or it could relate to continental margin collisions that thicken lithosphere (47). Therefore, the initiation of modern Wilson cycle plate tectonic appears before the Paleoarchean, and it becomes the dominant tectonic regime after ~2.8 Ga. + +## Implications For Plate Tectonic **Evolution** + +The geological record shows evidence of intraoceanic subduction and arc magmatism as far back as the oldest preserved rocks (68). These primitive intraoceanic arc systems would collide, thicken the lithosphere, and gradually form continents (10). However, large supercontinents are apparently absent until the Neoarchean, after the completion of secular transitions from the early nearly pure intraoceanic subduction-accretionary orogens to the modern Wilson cycle tectonic regimes during 3.5 to 2.8 Ga (Fig. 6B). The assembly of the earliest proposed supercontinent of Kenorland, at 2.7 to 2.5 Ga (69), broke up after ~2.3 Ga, and the thick lithosphere is markedly thinned again (Fig. 3B). Then, subduction forces the assembly of continental blocks to form the Columbia supercontinent (with Nuna as its core) from 2.0 to 1.8 Ga (70). Our result indicates that the lithosphere consecutively thickened during late Paleoproterozoic to Mesoproterozoic (2.0 to 1.0 Ga), during collisions related to assembly of the Nuna and Rodinia [assembled between 1.3 and 0.9 Ga (61)] supercontinents (71). Thickened lithosphere reduced heat emissions from the interior of Earth and finally formed the thermal blanketing effect (19), preventing asthenospheric cooling (53). Subsequently, superplume events, which were possibly enhanced by the insulation of the thermal blanketing effect (61), invigorated massive magmatism, inducing the following breakup of Rodinia and another marked lithospheric thinning event. The supercontinent breakup and thinning of the lithosphere finally broke the thermal blanketing, facilitating heat emissions from Earth's interior. Progressive subduction further promoted asthenospheric cooling, thereby developing the self-sustaining dynamics of modern plate tectonics, which are characterized by paired low and intermediate T/P metamorphism (Fig. 3C) (3, 56) with distinctive blueschists (67). Earth was eventually primed for the arrival of a colorful Phanerozoic with the Cambrian explosion of metazoans. + +Because of the thermal blanketing effect caused by long-term thickened lithosphere, the Wilson cycle plate tectonics developed before the Neoproterozoic were accompanied by more abundant ductile and warm intraoceanic subduction zones, which were represented by paired metamorphism with high and intermediate T/P +ratios (Fig. 3C) and first documented back to the Neoarchean (55). + +Until the late Mesoproterozoic, accompanied by stronger metamorphism at the bottom of the lithosphere and making it denser, the asthenosphere is finally unable to sustain its buoyancy to the extremely thick lithosphere, resulting in the catastrophic collapse of the lithosphere. The reduced asthenosphere temperatures, caused by the accelerated cold lithosphere addition and heat dissipation, could not sustain the ductile and warm subduction. Therefore, after substantial lithospheric thinning, the Wilson cycle plate tectonics active in the Neoproterozoic were characterized by large continents and dominance of rigid and cold subduction zones, which were represented by paired metamorphism with intermediate and low T/P ratios, and the lithosphere will never be able to reach its former peak thickness (~140 km at the late Mesoproterozoic) +(Fig. 3C). Notably, plate tectonics with a preponderance of cold subduction might have flared up as early as the Paleoproterozoic, recorded by low T/P metamorphism (72–75) or Neoarchean (55, 76) (Fig. 3C). However, these data are limited, and there is no way currently resolved to discriminate between a dominant thermal regime and a preservational effect. Likewise, the extant plate tectonic regime has both hot and cold subduction systems, and it is uncertain, which will be preserved in the future geological record, billions of years from now. Although there are only a few metamorphic thermal gradient records before 2.8 Ga in our study, marked thinning during Paleoarchean recordsthe global lithospheric collapse and thus can still reveal that plate subduction was in operation, in some form, in the Paleoarchean, and there is no justification from the geological record or calculated lithospheric thicknesses to argue for an early stagnant lid Earth. + +## Materials And **Methods** Chronological And Oxide Data Compilation For **Global** Natural Basaltic **Rocks** + +Our compiled global basaltic rocks database (see data S1) contains 24,194 chronological and oxide analyses of whole rock, aged between 3.8 Ga and the present. The chronological and oxide data were obtained from the EarthChem data repository (http:// portal.earthchem.org/). + +We identified the erroneous age data according to the methodology presented by Doucet et al. (77) and proactively rectified inaccurate age, primarily resulting from erroneous age units such as thousand years and million years, to ensure the utmost accuracy and reliability of our analyses. Because rocks older than Eoarchean in age (>3.8 Ga) have rarely been collected, we first selected the basaltic samples with type = volcanic, age < 3.8 Ga, SiO2 (43 to 55 wt +%), and content values > 0 wt % for MgO, TiO2, Al2O3, FeO (or Fe2O3), CaO, MnO, K2O, and Na2O. Samples were filtered to include only those basalts with MgO of 7 to 17 wt % to minimize the extent of fractional crystallization (15). Then, we manually removed the samples with total oxides below 95 wt % or loss on ignition above 3 wt %, as samples with these properties could have been weathered or secondary altered. Iron oxides content was recalculated to the total FeO. To reduce the influence of extreme values, we eliminate the suspect outlier data that plot outside the 3IQR box based on box plots for each oxide during each 100 million years bin. Only basaltic samples with all their oxide analyses plotted within their own 3IQR boxes are eligible for inclusion in the compiled databases. + +The final 24,194 chronological and oxide analyses of basaltic rocks in the compiled databases exhibit a relatively uniform spatial distribution (Fig. 2). On the basis of our total 24,194 samples, it is necessary to have at least 68 or more samples for each bin to achieve a confidence level of 90% that the real value falls within ±10% of the surveyed value. Although their temporal distributions were not uniform, a minimum of 66 samples were collected within each bin (Fig. 3A). This number almost meetsthe statistical criteria necessary for conducting temporal analyses. Secular compositional evolution of global basaltic rocks and the reconstructed lithospheric thickness evolution are shown in fig. S8. + +## Experimental Data Compilation For Basaltic **Liquid** Compositions + +Our compiled experimental basaltic database (see data S2) contains 1392 experimental basaltic liquid compositions in equilibrium with olivine and orthopyroxene, compiled from the LEPR database (https://lepr.earthchem.org/) and published literature. Samples were filtered to include only those basalts with SiO2 (43 to 55 wt +%) and MgO (7 to 17 wt %). We also manually removed the samples with total oxides below 95 wt % and missing pressure and temperature. + +## Litho1.0 + +The LITHO1.0 (50) global lithospheric model, with a 1 × 1 arc-deg spatial resolution, is a tessellated model of the crust and uppermost mantle of Earth, extending into the upper mantle to include the lithospheric lid and underlying asthenosphere. The model is parameterized laterally by tessellated nodes and vertically as a series of geophysically identified layers, such as water, ice, (upper, middle, and lower) sediments, (upper, middle, and lower) crystalline crust, lithospheric lid, and asthenosphere (https://igppweb.ucsd. edu/~gabi/litho1.0.html). + +## Data Processing **Flow** + +The data processing flow in this study can be followed by referring to fig. S9. Eight major data processing steps were conducted in this study: +1) Determine input variables for calculating the estimation of final pressure (PF) (or lithospheric thickness). We performed causality analysis using CF and determined that the temperature variable could be disregarded in the estimation (fig. S2). + +2) Training RF regression model. An RF model wastrained using experimental basalt data (data S2). The performance of the model was assessed and illustrated in Fig. 4. + +3) RF prediction of the PF values for new global data. The trained RF regression model was applied to calculate the PF values for global basalts (data S1). + +We repeated steps 2 and 3 100 times, which resulted in obtaining the average PF value and its SD for each basalt sample. + +4) RF regression model validation. The reconstructed Cenozoic lithospheric thickness in eastern China obtained using the RF regression model was compared with the lithospheric thickness obtained by the LITHO1.0 model. This step aimed to validate the performance and accuracy of the RF regression model (Fig. 5). + +5) Reconstructed PF and T/P series. The calculated PF values +(data S1) and compiled T/P (data S3) data were discrete points. + +To obtain smooth fitting curves for the PF and T/P serials since 3.8 Ga ago, we used the locally weighted scatterplot smoothing [locally weighted scatterplot smoothing (LOWESS)] method. This approach allowed us to visualize the trend and variations in the PF and T/P time series (Fig. 6A and data S6). + +6) Identifying coupling between PF and T/P time serials. We calculated global and local PCCs between the PF and T/P time series to assess their coupling (Fig. 6, A and B, and data S6). Global PCC provided insights into the overall coupling between the two evolution curves, while local PCC allowed for the assessment of the coupling at specific moments or time intervals. + +7) Identifying periodicity and in-phase coherence for the PF and T/P time serials. Continuous wavelet transform was used to detect the periodic patterns persistence in the PF and T/P ratio through et al., Sci. *Adv.* 9, 15 2023 9 of 13 time (Fig. 6, C and D). Wavelet coherence analyses were used to determine the consistency and in-phase coherence between the two series (Fig. 6E). + +8) Examining causal relationships between the PF and T/P time series. To examine potential causal relationships between the PF and T/P time series, the TLCCs and windowed TLCC were calculated (Fig. 8 and data S6). TLCC analysis allowed for the assessment of correlations at different time lags and within specific time windows, providing insights into temporal dynamics and potential causal connections. + +The methods mentioned above will be detailed below. All data and computational source codes are available. + +## Outlier Detection By The Iqr + +The IQR represents the range of the middle half of dataset (from the first to the third quartile). We can use the IQR to create "fences" +(limits) around the data and then define outliers as any values that fall outside those fences. The outliers are any values greater than the upper fence or less than the lower fence. + +We can obtain the outliers by following steps: sort data from low to high; identify the first quartile (Q1) and the third quartile (Q3); +calculate IQR = Q3 − Q1; calculate the upper and lower fences by Q3 + (a × IQR) and Q1 - (a × IQR); and use the fences to highlight any outliers, all values that fall outside the fences. When a = 3, it is the 3IQR method. + +## Causal **Forests** + +Randomized controlled experiments provide a means to assess the impact of an action or intervention on subsequent outcomes. The quantification of this impact is achieved by comparing the average difference between samples that underwent the intervention and those similar to them but did not (referred to as CATE). + +An approach known as CF (46) uses causal trees to estimate the CATE by adapting the RF model. While RF constructs an ensemble of decision trees to minimize prediction error, CF constructs an ensemble of causal trees to capture low-dimensional heterogeneity in treatment effects, leveraging the extensive feature space inherent in big data. In RF, data partitioning is performed iteratively to minimize the prediction error of an outcome variable. Similarly, CF partitions the data but with the aim of maximizing the divergence in the relationship between the outcome variable and the "treatment" +(action or intervention) variable. This approach aims to unveil variations in treatment effects across the sample. When treatment effects exhibit heterogeneity, it becomes possible to condition the calculation of the CATE on a covariate CATE ¼ EfYðT ¼ 1Þ YðT ¼ 0ÞjX ¼ xg where X, Y, and T are the covariates, outcome, and treatment variables, respectively. + +We used the EconML Python library from Microsoft (www. + +microsoft.com/en-us/research/project/econml/)to build the forest double ML estimator (also known as CF), i.e., estimators with T = +1 or T = 0, to estimate CATE. We use the synthetic minority oversampling technique (SMOTE) function (78) for oversampling before the CF training. Please refer to fig. S1 for detailed CATE calculation workflows. The oxide compositions of basalts as a response to the pressuretemperature (P-T ) conditions, as the thermodynamical phase equilibria govern elemental behaviors (79). Typically, the final temperature (TF) associated with basaltic melt extraction from the mantle undergoes collective variations alongside the PF. The two parameters together provide insights into the physical-chemical conditions governing basaltic melting or melt-solid equilibration at the bottom of the lithosphere. However, the interplay between the two factors remains uncertain. Is it imperative to incorporate TF as a variable in lithospheric thickness estimations? Addressing this question necessitates that the assessment of causality through the calculation of heterogeneous treatment effects, specifically the CATE, can help with the final variable selection (80). CATE quantifies the extent to which an outcome variable (Y ) is altered by an interventional change in treatment variable (T), such as the inclusion or exclusion of TF in PF estimations. + +We built two CF double ML estimators to value the causality between the PF and TF (figs. S1 and S2, A and B). Because of the skewed nature of the PF and TF variables, with a majority of samples falling within a specific range, we used SMOTE to create a balanced dataset (78). Subsequently, this dataset was randomly split into training (80%) and testing (20%) datasets. The CF models were trained using the training data, followed by estimating the CATE using the testing set. + +Our findings reveal a CATE value of 0.013 GPa when using TF as the treatment variable for PF regression, corresponding to a lithospheric thickness estimation of 0.43 km (fig. S2D). Conversely, when using PF as the treatment variable for TF regression, the CATE value is 44°C (fig. S2C). Although direct comparison is not feasible, the CATE values are three and two orders of magnitude smaller than the respective PF and TF values. This implies that PF has minimal influence on TF regression, while TF is considerably affected by PF. Consequently, in the lithospheric thickness estimation, which is directly proportional to PF, the TF variable can be disregarded. Our conclusions from big data and CF ML are consistent with the experimental observations (38, 39, 42). + +## Rf **Regression** + +RF (81) is a powerful and versatile supervised ML algorithm that grows and combines multiple decision trees to create a "forest." It can be used for both classification and regression problems. It offers a variety of advantages from accuracy and efficiency to relative ease of use. The principle of RF regressor is briefly described as follows: +The RF train multiple decision trees, e.g., the CART (classification and regression tree) used in this study, by different subtraining sets obtained by bootstrap sampling. The final prediction result was reached by the average prediction results of multiple decision trees (fig. S3). + +When training every single CART, the weighted summary of the node Gini impurities can be calculated by + +$$G(x_{i},v_{i j})={\frac{n_{\mathrm{left}}}{N_{\mathrm{s}}}}H(X_{\mathrm{left}})+{\frac{n_{\mathrm{right}}}{N_{\mathrm{s}}}}H(X_{\mathrm{right}})$$ + +where xi is a splitting variable; vij is a splitting value; nleft, nright, and Ns are the number of training samples belonging to left node, right node, and all nodes after splitting; Xleft and Xright are samples belonging to left node and right node; H(X ) is the impurity function/criterion, and the impure function used in this study is the mean squared error criterion + +$$H(X)={\frac{1}{n}}\sum_{i=1}^{n}(y_{i}-{\overline{{y}}}_{i})^{2}$$ + +where yi isthe actual value, yi isthe predicted value, and n isthe total number of data points. + +Every time a split of a node is made on variable k, the Gini impurity criterion for the two descendent nodes is less than the parent node, and then we define the total reduction of the criterion at node k nk ¼ wk � Gk wleft � Gleft wright � Gright where wk, wleft, and wright is the weight of the number of training samples of node k and its left and right subnodes to the total number of training samples; Gk, Gleft, and Gright represent the Gini impurity of node k and its left and right subnodes after branching. + +The RF regressor library used in this study is from scikit-learn in Python (https://scikit-learn.org/). Our dataset is unbalanced between every 100-million-year bins, so we use the SMOTE function (78) for oversampling. Randomized search and cross-validation (RandomizedSearchCV) hyperparameter optimization technique (82) is applied to find the optimal parameters for RF +model. Please refer to fig. S4 for detailed lithosphere thickness calculation workflow and the performance of the used RF regression model. + +## Reconstructed Pf And T/P **Series** + +The PF and T/P ratio series used for synchrony and causality are calculated by the averages for each 10 million years, with an overlap of 100 million years on both sides. The time-series line obtained using this overlap bin size provides a comprehensive representation of the evolutionary process, offering detailed information while preserving statistical significance. Then, the two time series were smoothed by the LOWESS method with frac = 0.05, i.e., 5% of the data used when estimating each y value. For the final normalized reconstructed PF and T/P series, please see data S6. The LOWESS method used in this study is from statsmodels module in Python (www. statsmodels.org/). + +## Pearson Correlation **Coefficient** + +PCC is a representative way to the measure similarity between two variables. It indicatesthat as one variable changes in value, the other variable tends to change in a specific direction. Understanding the correlation relationship is useful becausewe can use the value of one variable to predict the value of the other variable. Similar to other correlation coefficients, PCC varies between −1 and +1 with 0 implying no correlation. Correlations of −1 or +1 imply an exact linear relationship. PCC (r) between X and Y variables can be calculated by following formula + +$$r={\frac{1}{n-1}}\sum_{i=1}^{n}\left({\frac{x_{i}-{\overline{{x}}}}{\sigma_{X}}}\right)\left({\frac{y_{i}-{\overline{{y}}}}{\sigma_{Y}}}\right)$$ + +where σX and σY are SDs of the X and Y variables and x and y are their average values. + +## Time-Lagged Cross-Correlation **Coefficients** + +TLCC can identify directionality between two signals such as a leader-follower relationship in which the leader initiates a response that is repeated by the follower. TLCC method continuously calculates the correlation coefficients between two time series by shifting one time series in a certain direction with a selected step size. The TLCC values with k lagged between two time series (x and y) can be calculated by + +$$\operatorname{TLCC}_{k}(x,y)={\frac{\sum_{i=0}^{N-1-k}(x_{i}-{\overline{{x}}})(y_{i+k}-{\overline{{y}}})}{{\sqrt{\left(x_{i}-{\overline{{x}}}\right)^{2}}}{\sqrt{\left(y_{i+k}-{\overline{{y}}}\right)^{2}}}}}$$ + +where k is lagged steps; xi and *yi+k* are the discrete signal values in the two time series at i and i + k time, respectively; N is the total number of discrete signal values in the two time series; and x and y are the average signal values for the two time series, respectively. Windowed TLCC produces a matrix of correlation values calculated within a time window of a specified length. + +## Wavelet **Analysis** + +Wavelet transform (83) is the most widely used method for determining the periodicities of time-series signals. The wavelet transform of a time series *x(ti*) of length N with sample spacing Δt can be expressed as follows + +$$W(s)={\frac{\Delta t}{\sqrt{s}}}\sum_{j=0}^{N-1}x(t_{i})\psi[\Delta t(j-i)/s]$$ + +where ψ is the mother wavelet, s is the scale expansion factor, and i represents the position shifting. The Morlet is the most popular wavelets used for time-frequency analysis because it can achieve a better balance between time and frequency. It consists of a plane wave modulated by a Gaussian + +$$\psi(t)=\pi^{-1/4}e^{i w t}e^{-t^{2}/2}$$ + +where ω is nondimensional frequency. + +Wavelet cross-correlation analysis (84) is a method for quantifying the localized correlation between two nonstationary series in time and frequency space by using wavelet transform. The wavelet coherence, i.e., cross-correlation, between time series X and Y can be given by + +$$R^{2}(s)={\frac{|S[W_{i}^{X Y}(s)]/s|^{2}}{|S[W_{i}^{X}(s)]/s|\cdot|S[W_{i}^{Y}(s)]/s|}}$$ + +where S is a smoothing operator defined by the wavelet type used; WXY +i is the cross-wavelet transform and can be defined as: +WXY +i ðsÞ ¼ WXi ðsÞ � WYi ðsÞ. R2 takes a value between 0 and 1, which resembles the implication of traditional correlation coefficient. + +In this study, wavelet analysis is used to detect the periodic patterns for the PF and T/P serials and their persistence through time. + +The wavelet toolbox used in this study is from www.glaciology.net/ wavelet-coherence. + +## Supplementary **Materials** This Pdf File **Includes:** Figs. S1 To S9 Legends For Data S1 To S6 + +Other Supplementary Material for this manuscript includes the **following:** +Data S1 to S6 REFERENCES AND **NOTES** 1. R. J. Stern, The evolution of plate tectonics. Philos. Trans. A Math Phys. 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Methodology: Z.-J.Z. and G.-X.C. Investigation: Z.-J.Z. + +Visualization: Z.-J.Z. Supervision: Z.-J.Z. and Q.-M.C. Writing—original draft: Z.-J.Z. Writingreview and editing: Z.-J.Z., G.-X.C., T.K., J.Y., and Q.-M.C. Competing **interests:** The authors declare that they have no competing interests. Data and materials **availability:** All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. All datasets and computational source codes used in this paper are available on Dryad (https://doi.org/10.5061/dryad.3bk3j9kr2) or MyDEE (https://mydde.deeptime.org/org-portal/MyDDE/project/654c6d05afe0567cf0c286af). + +Submitted 12 April 2023 Accepted 16 November 2023 Published 15 December 2023 10.1126/sciadv.adi2134 \ No newline at end of file diff --git a/medical/md/PMC2816841.md b/medical/md/PMC2816841.md new file mode 100644 index 0000000000000000000000000000000000000000..82aeed96b6c46f9f1091ec335a7b0f0b188ecaab --- /dev/null +++ b/medical/md/PMC2816841.md @@ -0,0 +1,161 @@ +# Alternative Therapy And Abnormal Liver Function During Adjuvant Chemotherapy In Breast Cancer Patients + +Although hepatotoxicity has been rarely reported during adjuvant chemotherapy in breast cancer patients, we observed a high frequency in our patients who were also taking alternative agents. We therefore sought to determine the association between hepatotoxicity and alternative agents during adjuvant chemotherapy in breast cancer patients. All breast cancer patients were treated with the same chemotherapeutic regimen and had normal baseline liver function test (LFT). LFT was checked repeatedly during each cycle of chemotherapy. Patients showing LFT abnormalities were asked about use of alternative agents, and, after the end of chemotherapy, a questionnaire was administered to each patient on their use of alternative agents. Of 178 patients, 65 (36.5%) admitted using alternative therapy, and significantly more patients in this group developed LFT abnormalities (37/65, 56.9%) than those who denied taking alternative therapy (25/113, 22.1%, p=0.001). Although LFT abnormalities were mild to moderate and normalized in most patients after cessation of alternative agents, it remained a serious problem in one patient. In conclusion, alternative therapy may be one of the etiologies for abnormal LFT in breast cancer patients receiving adjuvant chemotherapy. + +Key Words : *Breast Neoplasms; Complementary Therapies; Drug Therapy; Hepatitis, Toxic* + +## Introduction + +The use of alternative therapy in cancer patients has become increasingly popular, although this approach has not been scientifically tested and has the potential to interact with conventional treatment (1-6). To date, there have been no studies prospectively investigating the use of alternative therapies in breast cancer patients receiving adjuvant chemotherapy, or determining the risk of abnormal liver function in concomitantly treated patients. Also, there are few published reports focusing on hepatotoxicity from chemotherapy with adriamycin or the CMF (cytoxan+methotrexate+5-fluorouracil) regimen in adjuvant chemotherapy trial (7, 8). We previously found that 34% of patients who had received adjuvant adriamycin followed by CMF chemotherapy for breast cancer developed abnormal liver functions (9). Since many of our patients were also taking various kinds of herbal medicines, a majority of which are sold as ''health food'' without evidence of proven efficacy or safety, we thought that the association between these alternative therapies and abnormal liver function warranted further study. We designed this study to determine the frequency of hepatotoxicity during adjuvant chemother- + +## Jin-Hee Ahn, Sung-Bae Kim, Mi Ra Yun, Jung-Shin Lee, Yoon-Koo Kang, Woo Kun Kim + +Division of Hematology-Oncology, Department of Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea Received : 28 November 2003 Accepted : 11 February 2004 Address for correspondence Woo Kun Kim, M.D. Division of Hematology-Oncology, Department of Medicine, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap-dong, Songpa-gu, Seoul 138-736, Korea Tel : +82.2.3010-3216, Fax : +82.2-3010-6961 E-mail : wkkim@amc.seoul.kr +*This study was supported by grants from the Clinical Performance Improvement Project of ASAN Medical Center, Korea. + +*This study was presented at the 6th international conference of the Asian Clinical Oncology Society (ACOS), 17 November 2003. + +apy in breast cancer patients, and we compared results between patients who had and had not taken alternative agents. + +## Materials And Methods + +This study was conducted at an outpatient oncology clinic on breast cancer patients who received adjuvant chemotherapy from April through December 2002 with AC (adriamycin 60 mg/m2+cyclophosphamide 600 mg/m2, every 4 weeks) +after curative surgery and had normal liver functions prior to chemotherapy. Each patient had a baseline history, a physical examination, complete blood count (CBC) and liver function test (LFT). The latter included assays for serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin, as well as the hepatitis B virus (HBV) markers, HBsAg and anti-HBs, anti-HCV antibody, and a baseline liver ultrasonography within 2-4 weeks before chemotherapy. LFT was repeated before each cycle of chemotherapy, one month after the last cycle and at every 3 month follow-up visit after chemotherapy. Abnormal LFT was defined as the elevation of aminotransferase (AST and/ +or ALT>40 IU/L) or bilirubin (>1.5 mg/dL) concentration. + +Alternative agents were defined as any product, not approved for use in conventional medicine, including herbs, other botanical agents or any biological preparations sold as ''health food.'' Any patients found to have abnormal LFT during the chemotherapy period were asked about the use of any alternativeagent and requested to cease its use. If the liver function did not return to normal after these agents were stopped, ultrasonography of the liver, and hepatitis panels for HBV and HCV were repeated at the attending physician's discretion. + +Also, HBV DNA probe and HBeAg were assayed if the patient was an HBV carrier. After the end of chemotherapy, all patients were interviewed by an oncology nurse specialist and questionnaires were filled in for the history of use of any alternative agents. We used SPSS (version 11.0) statistical software package for data entry and analysis. Chi-square test was used for comparing the categorical variables between patients who admitted taking alternative therapy and those who did not. Statistical significance was defined as a p-value less than 0.05. + +The protocol was reviewed and approved by the Clinical Performance Improvement Project of our center. All patients provided verbal informed consent before entering onto the study. + +## Results Patient Characteristics + +During the study period, there were 203 patients with breast cancer who had undergone curative surgery and received adjuvant AC chemotherapy. Of these, 25 patients were excluded because of abnormal LFT prior to chemotherapy. Thus, a total of 178 breast cancer patients (median age, 46 yr; range, 2972), were included in this study. Six patients were HBsAg Table 1. Alternative agents taken by our patients + +| Table 1. Alternative agents taken by our patients Alternative therapy Examples Herbs (70) Panax ginseng (ginseng steamed red 35), Daucus carota (carrot juice 15), other kinds of ginseng (6), Ulmus davidiana (Slippery elm 6), Angeliaca keiskei (2), Acer pictum (1), Yeast (1), Elentherococcus sessiliflorus (root bark of various araliaceous shrubs 1), Euonymus hamiltonianus (1), Morus alba (Mulberry leaves 1), Mistletoe (1) Diets (19) Various uncooked grains and raw vegetables (12), ''Pumpkin juice'', ingredients unknown (3), Vegetarian diet (2), ''Pear juice'', ingredients unknown (1), ''Grapefruit juice'', ingredients unknown (1) Mushroom (13) Phelinus linteus (8), Ganoderma lucidum (5) Others (7) Agaricus (3), Crucian juice (2), Royal jelly (1), Shark cartilage (1) *Numbers in the parenthesis are the numbers of patients. | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +carriers and one patient was positive in an anti-HCV antibody test. Of the 150 patients for whom we had results of baseline liver ultrasonography, 37 had underlying fatty liver disease. + +Five patients received red blood cell transfusions during hospitalization for breast surgery, but none had transfusions during the chemotherapy period. + +## Alternative Therapy + +At the first follow-up visit after the end of 4 cycles of chemotherapy, 65 (36.5%) patients admitted they had taken some type of alternative agent(s) during chemotherapy period, whereas 113 patients (63.5%) denied taking any alternative agents. Duration of alternative therapy varied from 1 week to 3 months. Steamed red Ginseng (*Panax ginseng*) was the most common herbal therapy (35/65, 53.8%), and many patients had taken several different agents (Table 1). Baseline characteristics were similar between patients who admitted using alternative therapies and those who did not (Table 2). + +## Abnormal Liver Function + +We found that 62 of 178 patients (34.8%) developed abnormalities in LFT (Table 3). According to NCI common toxicity criteria (Table 4), 43 patients (69.4%) had grade 1 toxicity, 16 (25.8%) had grade 2, 2 (3.2%) had grade 3, and 1 (1.6%) had grade 4. Of the 65 patients who took alternative therapy, 37 (56.9%) developed an LFT abnormality, compared with 25/114 (22.1%) who denied taking alternative therapy (p= 0.001). Development of hepatotoxicity of grade 2 or higher was significantly more common in the alternative therapy group (13/65, 20.0%) than in the other group (6/113, 5.3%; p=0.002). All patients who admitted using alternative agents had started taking them one or two months before the LFT + +Table 2. Patient characteristics according to the use of alternative + +therapy + +| therapy | With | Without | +|-------------------------------------------------------------------------------------------------------------------------------|----------------------|----------------------| +| alternative therapy | alternative therapy | | +| Total number of patients | 65 (36.5%) | 113 (63.5%) | +| Age (yr), median | 44 (29-72) | 46 (30-72) | +| BMI (kg/m2 )* | 23.4 (18.9-28.6) | 23.2 (17.5-35.7) | +| Hemoglobin, median | 12.0 g/dL (9.0-14.1) | 12.0 g/dL (9.0-14.4) | +| RBC transfusion | 1 patient | 4 patients | +| AST* | 20 IU/L(12-37) | 20 IU/L (10-37) | +| ALT* | 15 IU/L (6-40) | 14 IU/L (3-40) | +| ALP* | 50 IU/L (30-162) | 52 IU/L (14-128) | +| Total bilirubin | 0.7 mg/dL (0.2-1.3) | 0.7 mg/dL (0.1-1.4) | +| HBV carrier | 1 patient | 5 patients | +| HCV Ab (+) | 0 | 1 patient | +| Fatty liver disease | 12/54 (22.2%) | 25/97 (25.8%) | +| *Numbers are median value of BMI and liver function test. BMI, body mass index; AST, aspartate aminotransferase; ALT, alanine | | | + +*Numbers are median value of BMI and liver function test. + +BMI, body mass index; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; HBV, hepatitis B virus; HCV Ab, hepatitic C virus antibody. + +| therapy Abnormal liver | With alternative therapy | Without alternative | +|---------------------------|----------------------------|---------------------------| +| function test (LFT) | (No. of patients) | therapy (No. of patients) | +| Incidence* | 37/65 (56.9%) | 25/113 (22.1%) | +| Grade 1/2/3/4 | 24/11/1/1 | 19/5/1/0 | +| Grade ≥2* | 13/65 (20.0%) | 6/113 (5.3%) | +| Time to normalization | median 1.9 months | median 2.9 months | +| of liver function | (range 1-8 months) | (range 1-10 months) | +| *p-value< 0.05. | | | + +abnormality was detected. After discontinuing the agents, however, the liver function abnormality was normalized within a median 1.9 months (range, 1-8 months). In contrast, normalization of liver function abnormalities took a median 2.9 months (range, 1-10 months) in the other group. + +The patient with grade 4 hepatotoxicity was an HBV carrier who had used *Panax ginseng*and *Ulmus davidiana* (Slippery elm) for 3 months from the beginning of adjuvant chemotherapy. After the third cycle of chemotherapy, AST and ALT were increased to 134 IU/L and 164 IU/L, respectively, and the patient was asked to stop taking the alternative agents. After the fourth cycle of chemotherapy, the patient presented with further increases in AST and ALT to 748 IU/L and 732 IU/L, respectively, and her bilirubin was 3.6 mg/dL. This resulted in a delay in the administration of tamoxifen, and hospitalization was required. No evidence for the replication of HBV was observed, judging by the negative results of HBeAg and HBV DNA probe, and liver ultrasonography showed a pattern of non-specific chronic liver disease. It took more than 6 months for the increased aminotransferases and bilirubin to return to their normal ranges. + +Significantly more patients who had underlying fatty liver disease (18/37, 48.6%) developed abnormalities in LFT than patients who did not (35/113, 30.7%; p=0.047). The frequency of underlying fatty liver disease was not different between the patients who took alternative agents (22.2%) and those who did not (25.8%). Of the 6 HBV carriers, 2 developed abnormal LFT during chemotherapy, but there was no evidence of viral replication. + +## Discussion + +Since data regarding hepatotoxicity during adjuvant chemotherapy in breast cancer patients is scarce, it is difficult to determine how often it occurs and what agents are involved. Mild liver function abnormalities have been reported in 7% of breast cancer patients after adjuvant CMF chemotherapy (7), whereas the frequency of mild abnormal LFT within one year after the cessation of CAF (cytoxan+adriamycin+5-fluorouracil) chemotherapy was found to be 77%, suggesting that LFT abnormalities were manifestations of drug toxicity (8). Of these patients, however, 33% (33/97) had abnormal liver functions Table 4. Hepatotoxicity according to NCI common toxicity criteria + +| Grade | 1 | 2 | 3 | 4 | +|-----------------------------------------------------------------------|---------|-----------|------------|----------| +| AST, ALT* | ≤2.5×N� | 2.6-5.0×N | 5.1-20.0×N | > 20.0×N | +| (IU/L) | (≤100) | (100-200) | (200-800) | (>800) | +| Total bilirubin | <1.5×N | 1.5-3.0×N | >3.0×N | | +| (mg/dL) | (<1.8) | (1.8-3.6) | (>3.6) | | +| *AST, aspartate aminotransferase; ALT, alanine aminotransferase; � N, | | | | | + +*AST, aspartate aminotransferase; ALT, alanine aminotransferase; �N, + +upper normal limit. + +prior to the start of chemotherapy, an unusually high percentage. In contrast, only 25 of our 203 patients (12.3%) had abnormal LFT before chemotherapy. + +In our study, 36.5% of breast cancer patients admitted using alternative therapy during the adjuvant chemotherapy period. The development of abnormal LFT was significantly more frequent in these patients than in those who denied taking alternative agents (56.9% vs. 22.1%, p=0.001). In most cases, LFT abnormalities were mild to moderate, rarely led to interruption of chemotherapy, and normalized after the cessation of alternative agents or spontaneously within several months, even in patients who denied using alternative agents. These results strongly indicate that alternative agents could cause liver toxicity during adjuvant chemotherapy. + +In our study, two of six HBV carriers showed elevation of aminotransferases during adjuvant chemotherapy, but there was no evidence of HBV replication. The reactivation of HBV infection in subjects receiving cytotoxic treatment has been well documented in patients with hematologic malignancies receiving steroid-containing high-dose chemotherapy (10-12). Although the numbers of HBV carriers in our study is too small to draw a firm conclusion, HBV carrier status alone does not seem an additional risk factor for abnormal LFT. + +Interests in alternative therapies have been increasing. In previous population-based surveys, 48% and 72% of breast cancer patients admitted using at least one type of alternative therapy (4, 13). Among these alternative therapies were dietary therapies, herbal remedies, spiritual healing, and psychological methods, with the prevalence of each type dependent on ethnicity (4). In our study, we considered only orally ingested alternative therapies, perhaps explaining why the percentage of our patients taking alternative treatment was lower than those observed elsewhere. + +Some herbal and botanical agents possess complex biological activities that could affect many aspects of carcinogenesis and treatment (14, 15). Despite an extensive series of laboratory studies, few clinical trials have tested the efficacy and safety of these herbal and botanical agents. Heavy-metal contamination is not uncommon in Asian herbal or botanical agents, and these agents can act through a variety of mechanisms to alter the pharmacokinetic profile of concomitantly administered drugs (16, 17). Since several herbal remedies are associated with hepatotoxicity (18, 19), it was not surprising that our patients who took alternative agents had a higher rate of developing abnormal liver function during the chemotherapy. + +The alternative agents taken by our patients varied widely, such that we could not sort out the agents responsible for hepatotoxicity. The determination of patients who ingested alternative agents was based totally on their own statements, and it is our practice to discourage our patients from taking alternative agents. Thus, we cannot completely rule out the possibility that our patients under-reported usage of alternative agents. In addition, we did not perform serial liver ultrasonography on all patients who developed LFT abnormalities. Therefore, we cannot determine precisely whether individual LFT +abnormalities are due to the use of alternative therapies, to hepatotoxicity induced by chemotherapeutic agents, or to aggravation of underlying fatty liver disease, either by chemotherapy or alternative agents. + +In conclusion, our results indicate that the use of alternative agents can be one of the etiologies for the development of abnormal LFT while on AC adjuvant chemotherapy in breast cancer patients. Our findings suggest the need for more prospective studies regarding the relative contribution to hepatotoxicity by adjuvant chemotherapy and by alternative agents. + +## References + +1. Ernst E, Cassileth BR. *The prevalence of complementary/alternative* medicine in cancer. A systemic review. Cancer 1998; 83: 777-82. + +2. Kelly KM, Jacobson JS, Kennedy DD, Braudt SM, Mallick M, Weiner MA. *Use of unconventional therapies by children with cancer at* an urban medical center. J Pediatr Hematol Oncol 2000; 22: 412-6. + +3. Richardson MA, Sanders T, Palmer JL, Greisinger A, Singletary SE. + +Complementary/alternative medicine use in a comprehensive cancer center and the implications for oncology. J Clin Oncol 2000; 18: 250514. + +4. Lee MM, Lin SS, Wrensch MR, Adler SR, Eisenberg D. *Alternative* therapies used by women with breast cancer in four ethnic populations. J Natl Cancer Inst 2000; 92: 42-7. + +5. Ang-Lee MK, Moss J, Yuan CS. *Herbal medicines and perioperative* care. JAMA 2001; 286: 208-16. + +6. Labriola D, Livingston R. Possible interactions between dietary antioxidants *and chemotherapy. Oncology 1999; 13: 1003-8.* +7. Hirvikoski PP, Kumpulainen EJ, Johansson RT. *Hepatic toxicity* caused by adjuvant CMF/CNF in breast cancer patients and reversal by tamoxifen. Breast Cancer Res Treat 1997; 44: 269-74. + +8. Larroquette CA, Hortobagyi GN, Buzdar AU, Holmes FA. *Subclinical hepatic toxicity during combination chemotherapy for breast cancer. JAMA 1986; 256: 2988-90.* +9. Ahn JH, Bahng H, Kim JG, Kim SB, Ahn SH, Chang H, Lee JS, Kim SH, Kim WK. Retrospective analysis of the results of adjuvant chemotherapy in breast cancer patients with 10 or more positive nodes: Nonrandomized comparison of adriamycin-containing regimens. Cancer Res Treatm 2002; 34: 84-90. + +10. Stroffolini T, Andriani A, Bibas M, Barlattani A. Successful treatment with lamivudine for reactivated hepatitis B infection following chemotherapy for non-Hodgkin's lymphoma. Ann Hematol 2002; 81: 48-9. + +11. Sekine R, Taketazu F, Kuroki M, Takagi S, Imawari M, Kanazawa Y, Kawakami M. Fatal hepatic failure caused by chemotherapyinduced reactivation of hepatitis B virus in a patient with hematologic malignancy. Int J Hematol 2000; 71: 256-8. + +12. Clark FL, Drummond MW, Chambers S, Chapman BA, Patton WN. + +Successful treatment with lamivudine for fulminant reactivated hepatitis B infection following intensive therapy for high-grade non-Hodgkin's lymphoma. Ann Oncol 1998; 9: 385-7. + +13. Adler SR. *Complementary and alternative medicine use among women* with breast cancer. Med Anthropol Q 1999; 13: 214-22. + +14. Cohen I, Tagliaferri M, Tripathy D. *Traditional Chinese medicine* in the treatment of breast cancer. Semin Oncol 2002; 29: 563-74. + +15. Rose DP, Connolly JM, Rayburn J, Coleman M. Influence of diets containing eicosapentaenoic or docosahexaenoic acid on growth and metastasis of breast cancer cells in nude mice. J Natl Cancer Inst 1995; 87: 587-92. + +16. Marcus DM, Grollman AP. *Botanical medicines: The need for new* regulations. N Engl J Med 2002; 347: 2073-6. + +17. Fugh-Berman A. *Herb-drug interactions. Lancet 2000; 355: 134-8.* 18. Ernst E. *Harmless herbs? A review of the recent literature. Am J Med* 1998; 104: 170-8. + +19. De Smet PA. *Herbal remedies. N Engl J Med 2002; 347: 2046-56.* \ No newline at end of file diff --git a/medical/md/PMC2816938.md b/medical/md/PMC2816938.md new file mode 100644 index 0000000000000000000000000000000000000000..42f44443ed9128a7e8133853baccb1c83216297b --- /dev/null +++ b/medical/md/PMC2816938.md @@ -0,0 +1,158 @@ + +## Video Article Primary Culture And Plasmid Electroporation Of The Murine Organ Of Corti. + +Mark Parker1,2,3, Aurore Brugeaud1,2, Albert S. B. Edge1,2,4 1Department of Otology and Laryngology, Harvard Medical School 2Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary 3Department of Communication Sciences and Disorders, Emerson College 4Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard Correspondence to: Mark Parker at Mark_Parker@meei.harvard.edu URL: http://www.jove.com/video/1685 DOI: doi:10.3791/1685 Keywords: Neuroscience, Issue 36, hearing, mice, cochlea, organ of Corti, organotypic, culture, hair cell, stem cell, gene expression, in vitro Date Published: 2/4/2010 Citation: Parker, M., Brugeaud, A., Edge, A.S.B. Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.. J. Vis. *Exp.* (36), e1685, doi:10.3791/1685 (2010). + +## Abstract + +In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells. + +An increasingly essential tool in auditory research is the isolation and *in vitro* culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants. This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured *in vitro* in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti. + +## Video Link + +The video component of this article can be found at http://www.jove.com/video/1685/ + +## Protocol Day 1. Sterilization And Coating Of Glass Coverslips. + +1. Dry sterilize **glass microscope coverslips** in an autoclave. + +2. Place the sterilized coverslips into two wells of a pre-sterilized **four-well cell culture dish**. 3. Coat the coverslips with 1:1 **poly-L-ornithine** and **laminin** supplemented with 20% **Fetal Bovine Serum (FBS)** overnight at 4 °C. + +a. 400 μL poly-L-ornithine (0.01% solution stored at 4 °C) b. 400 μL laminin (50 μg/mL stock solution stored in aliquots at -20 °C) c. 200 μL FBS (stored in aliquots at -20 °C) +4. Heat-sterilize the **dissection tools** overnight in a 150°C incubator. + +5. Make culture medium containing 10% serum and 10 mg /mL ampicillin. + +a. 90 mL **Dulbecco's Modified Eagle Medium** +b. 5 mL FBS (stored in aliquots at -20 °C) +c. 5 mL **horse serum** (stored in aliquots at -20 °C) +d. 10 μL **ampicillin** (10 mg/mL stock solution stored at 4 °C ) + +## Day 2. Isolation Of The Organ Of Corti. + +1. Sterilize the **positive flow dissection hood**. + +a. turn on UV light for 20 min b. spray all surfaces with **70% ethanol** and wait 5 min before use. + +2. Decapitate mouse pup (P4) at the base of the foramen magnum using **operating scissors**. + +3. Briefly rinse the head in 10 cm dish containing 70% ethanol. 4. Remove the epidermis using a **scalpel blade**. + +5. Open the cranium along the sagittal suture using a scalpel blade and then bisect the forebrain. Retain the caudal forebrain for further dissection. + +6. Remove the forebrain, cerebellum and brainstem using blunt dissection. 7. Remove the temporal bones (fig 2A), dip them briefly in 70% ethanol, and transfer them to a 3 mm dish containing sterile HBSS. + +8. Using **forceps**, remove the bulla and surrounding tissue from the petrous portion of the temporal bone. + +9. Locate the conch shaped cochlea (fig 2B) and separate it from the vestibular system using forceps. 10. At this stage of development, the bony labyrinth is not completely calcified and is easily dissected using forceps. Remove the bony labyrinth of the cochlea by careful separation starting at the basal end and moving apically using forceps. + +11. The spiral ligament and attached organ of Corti is coiled along the spiral of the modiolus (fig 2C). Carefully remove the organ of Corti by securing the spiral ligament at the hook region of the base using forceps and unwinding it as you move apically. + +12. Starting at the base, remove the spiral ligament from the organ of Corti using **\#55 fine forceps** (fig 2D). + +Micro-isolation of the organ of Corti sensory epithelium (Optional). + +13. Remove the organ of Corti hook region of the base using two **½ cc Insulin Syringes** with permanently attached U-100 28G½ needles as forceps. + +14. Starting at the apex, remove the spiral limbus from the row of inner hair cells and proceed basally (fig 2E-F). + +Plating the organ of Corti explant 15. Remove the poly-L-ornithine/laminin/FBS solution from the culture wells and add 130 μl of culture medium. 16. Transfer the dissected organ of Corti to the coated glass coverslip in the culture well and orient the explant so that the cilia of the hair cells are pointing up. + +17. Remove the medium in the culture well using a 200 μL pipette. Make sure that the basilar membrane makes solid contact with the coated glass coverslip. + +18. Carefully add 130 μL of culture medium to the organ of Corti using a 200 mL pipette. Apply two drops onto the surface of the organ of Corti and then slowly add the remaining volume to the side of the coverslip. Make sure that the organ of Corti does not float in the culture media, but remains affixed to the coverslip. + +19. Incubate overnight at 37 °C in the presence of 5% CO2. + +## Day 3. Electroporation Of The Reporter Gene Into Cultured Organ Of Corti. + +1. Remove the culture medium from the organ of Corti culture. + +2. Add 130 μL H2O for 1 min and then remove using a 200 μL pipette. + +3. Add 30 μL of DsRed reporter plasmid (2mg /mL H2O stored at -20 °C). + +4. Advance the electrodes of the **electroporator** using a micromanipulator so that the anode and cathode are on either side of the culture. + +5. Generate a pulse (27V, 30 ms duration, 10 pulse trains) to electroporate the reporter gene into the organ of Corti explant culture. + +a. Optional: reverse the polarity of the pulse to ensure electroporation of the transgene on both the modiolar and spiral ligament sides of the explant. + +6. Wait 5 min. + +7. Add 130 μL of the **Fugene 6: DNA solution** (3 parts Fugene to 2 parts DNA). This solution should be prepared prior to the start of the electroporation procedure (step 20) using a 3 mL round bottom **polystyrene test tube** in a **laminar flow hood**. To make this solution: +a. Add 2.4 μL of **Opti-MEM** (stored at 4 °C) to the test tube. + +b. Add 0.6 μL of Fugene 6 reagent (stored at 4 °C) to the test tube. Make sure to add the Fugene directly to the Opti-MEM and avoid direct contact with the sides of the test tube. + +c. Vortex for 1 second. d. Incubate 5 min at room temperature. + +e. Add 2.0 μL **DsRed reporter plasmid** double stranded DNA (stored at -20 °C at 100 μg DNA/mL H2O aliquots). + +f. Vortex for 1 second. g. Incubate 15 min at room temperature. h. Add 200 μL of culture medium. i. Vortex for 1 second. + +8. Incubate overnight at 37 °C in 5% CO2. + +9. Add 2 mL culture medium to the culture well and incubate for 37 °C for up to 10 days. + +## Representative Results + +We present a method for the isolation of the organ of Corti from a perinatal mouse. The procedure can be used for mice as young as embryonic day 16 up to about postnatal day 6, at which point the bony labyrinth becomes sufficiently calcified to render the dissection cumbersome. Once the organ of Corti is dissected, it may be plated and cultured either in its entirety (fig 3) or as micro-isolated sensory epithelium (fig 4). We have further presented a technique to express exogenous genes in the cultured organ of Corti. The organotypic culture is useful for many other types of study, such as the analysis of organ of Corti gene expression using RT-PCR or in situ hybridization, organ of Corti co-culture with spiral + +![2_image_0.png](2_image_0.png) + +ganglion cells or exogenous stem cells using the micro-isolate3, or *in vitro* analysis of hair cell death and regeneration. + +Figure 1. Cross section of the P4 murine organ of Corti. (A) A cross section from the basal turn of a cryosectioned cochlea obtained from a P4 mouse illustrates the general structures of the murine cochlea described in this protocol. The scala media is bordered by the spiral ligament and stria vascularis laterally, the Reissner s membrane superiorly, the spiral limbus medially, and the basilar membrane inferiorly. The box indicated the region expanded in B. (B) The organ of Corti is located on the superior side of the basilar membrane and includes one row of inner hair cells, three rows of outer hair cells, and their respective supporting cells. Dashed line 1 indicates the location along the basilar membrane that is removed during this organ of Corti dissection. Dashed line 2 indicates the location along the basilar membrane that is removed during the micro-isolation procedure. Green indicates immunohistochemical labeling of calbindin, which labels interdental cells of the spiral limbus, cochlear hair cells, spiral ganglion neurons, as well as cells of the spiral ligament and stria vascularis 7. DAPI labeling of nuclei is in blue. To see a larger version of this image please click here. + +![3_image_0.png](3_image_0.png) + +Figure 2. Organ of Corti dissection. Images from the accompanying video of the organ of Corti dissection highlight A) the cochlea and + +![3_image_1.png](3_image_1.png) + +vestibular system located within the isolated temporal bone (red), B) the bony labyrinth of the cochlea, C) the spiral ligament and attached organ of Corti after removal of the bony labyrinth, D) removal of the spiral ligament and stria vascularis (red) from the organ of Corti, E) micro-isolation of the sensory epithelium from the spiral limbus (red), and F) the isolated spiral limbus (left) and sensory epithelium (right). To see a larger version of this image please click here. + +Figure 3. Cultured organ of Corti explant. DIC image showing the organ of Corti of a P4 Atoh1-nGFP mouse that was isolated, plated, and cultured for five days as described. This mouse has been genetically engineered so that cells that express the pro-hair cell gene Atonal homolog 1 (Atoh1 aka Math1) exhibit a green fluorescent protein that is localized to the nucleus8. The organs of Corti from these mice exhibit a nuclear GFP label in all hair cell nuclei and therefore allow for easy visualization of the sensory epithelium using an epifluorescent microscope. The relatively large spiral limbus can be seen lateral to the sensory epithelium. Mesenchymal cells that have originated from the organ of Corti have migrated away from the explant. Blue nuclear label is DAPI. To see a larger version of this image please click here. + +![4_image_0.png](4_image_0.png) + +Figure 4. Micro-isolated sensory epithelium. Epifluorescent image obtained from the sensory epithelium that has been isolated from a + +![4_image_1.png](4_image_1.png) + +P4 murine organ of Corti as described and cultured overnight. The micro-isolate was then fixed in 4% paraformaldehyde and processed for immunolabeling of Myosin 7a which labels cochlear hair cells. Note the absence of the comparatively larger spiral limbus from the figure 3. Blue nuclear label is DAPI. To see a larger version of this image please click here. + +Figure 5. Electroporation of DsRed reporter gene into the cultured organ of Corti. Whole organs of Corti from P4 Atoh1-nGFP mouse pups were isolated, plated, and then electroporated with the DsRed reporter gene as described and then viewed under an epifluorescent microscope. Cells of the organ of Corti explant that expressed the transgenic DsRed reporter protein exhibit a red fluorescence and endogenous cells of the sensory epithelium exhibit a green nuclear fluorescence. Transgenic cells can be seen throughout the spiral limbus and sensory epithelium. To see a larger version of this image please click here. + +## Discussion + +There are several details that are critical for the success of this procedure. The shorter the time from temporal bone isolation to organ of Corti incubation, the greater the chance that the organs will attach to the coverslip and result in viable organ cultures. Therefore, it is important to limit the amount of time between dissection and placing the organs in the incubator. The choice of antibiotic is also crucial, since many aminoglycoside antibiotics are ototoxic and will result in hair cell death. Although it is preferable to forgo the use of antibiotics altogether, this leaves open the possibility for contamination. Therefore, we suggest the use of 10 μg /mL ampicillin as a general rule to overcome potential contamination problems. The most troublesome aspect of this, and other procedures for the primary culture of the organ of Corti, is tendency of the organs to float off the plates during incubation. Although floating organ cultures can remain viable for 5-7 days, there are drawbacks of culturing floating organs. For instance, floating organ cultures often fold onto themselves after 4-5 days rendering microscopy problematic. Subsequently, the structural integrity of the organ can become compromised when compared to organs that have been affixed to the coverslip. We have found that the following techniques help to ensure that the organ of Corti does not float in the culture media, but remains affixed to the coverslip. First, coat the glass coverslips in 1:1 polyornithine/laminin supplemented with 20% FBS as described. The overnight incubation called for in this protocol is the minimum time that the plate should be coated. In our laboratory, we often coat all of the plates that we need for one week and keep them at 4 °C until the day prior to use when we move them to the incubator for an overnight incubation. Second, after the organs are transported to the coated coverslips, orient the explant so that the cilia of the hair cells face up. This orientation will facilitate the adherence of the basilar membrane to the culture dish. Third, remove the media within the well to affix the explant to the coated dish. This will ensure contact between the culture dish and the basilar membrane and enhance the ability of the explants to adhere to the glass. This will also assist in maintaining the structural integrity of the rows of hair cells. Lastly, carefully drip 2 drops of culture medium onto the surface of the organ of Corti using a 200 μL capacity pipette and then slowly fill the well by dripping the remaining volume (of the total 130 μL) on the side of the coverslip. It is important to work quickly to assure the attachment of the organ of Corti to the coated coverslip. From the initiation of the dissection to the incubation of the explants, it typically takes 10 minutes for a practiced operator to complete this organ of Corti isolation procedure. In this protocol, we also present a method for the micro-isolation of the sensory epithelium from the spiral limbus of the organ of Corti. In this procedure, the spiral limbus is dissected away from the sensory epithelium using 28G½ insulin needles as dissection tools. The resulting microisolate consists of the rows of hair cells and their corresponding supporting cells (fig 3). The isolated sensory epithelium can then be cultured as described in this protocol. This micro-isolation procedure should be compared to the enzymatic separation of the sensory epithelia from surrounding tissue4. In mammals, as well as non-mammalian species such as chickens, thermolysin digestion of isolated vestibular organs results in the isolation of sensory epithelia from the basement mesenchymal cells4. In the rat cochlea, thermolysin digestion results in the separation of the greater epithelial ridge, lesser epithelial ridge and accompanying sensory epithelia from the basement membrane5. However, it is unclear whether the cochlear sensory epithelium can attach to the coated plates without the accompanying mesenchymal cells. While both the mechanical micro-isolation method and enzymatic digestion method result in the separation of the sensory epithelia from the spiral limbus, advantages of the micro-isolate dissection over the thermolysin digestion include a relatively shorter protocol, cheaper reagents, and potentially less stress to the explant due to enzymatic effects of the digestion. Additionally, the basement membrane is left intact in this approach, which may enhance attachment of the sensory epithelium to the culture plate. Disadvantages of this method include the need to develop the skills for this delicate dissection and potential mechanical damage to the sensory epithelium resulting from the micro dissection. As an example of the utility of this procedure, we also present one example of the use of the organ of Corti cultures; the electroporation of exogenous genes into the explant culture. The electroporation procedure described above is based on previous methods of organ of Corti electroporation. Notably, Zheng and Gao (2000) describe the electroporation of isolated rat organs of Corti where the explants are held in place for electroporation by a molded groove of agarose and then plated on collagen coated 8-well LabTek slide in serum-free medium 2. An advantage of their approach is that the organs are oriented so that the top surfaces of the explants face the cathode, which should theoretically result in an even distribution of electroporated cells across the explant. In our hands however, the method that we describe improved on this procedure because the organ of Corti remained affixed to the coverslip throughout the procedure thereby reducing the manipulation of the explant after electroporation. Additionally, a higher percentage of organs of Corti remained attached to the coverslips using this presented method. Our method is taken from Jones, *et al.* (2006) which uses the addition of the Fugene 6 reagent to increase the efficiency of gene expression after the electroporation. In the Jones *et al.* (2006) protocol, the organs are electroporated, incubated for 5 minutes with 100 μL of Fugene 6 transfection reagent, and plated 6. Our method differs in the use of a 3:2 ratio of Fugene 6 reagent to plasmid DNA, which we found empirically to provide optimal transgenic expression with minimum organ toxicity. We do not use undiluted Fugene 6 reagent, which can result in toxicity to the cultures. The electrode configuration in our protocol, as well as Jones *et al.* (2006), results in primary gene expression in either the spiral limbus or sensory epithelium depending upon the position of the cathode. Although there are DsRed positive cells on the side of the culture distant to the cathode, there is a higher concentration of transgenic cells closer to the cathode. To ensure a complete expression of the transgene in both sides of the organ of Corti explant, the current can be reversed for a second pulse train by simply inverting the leads. The presented protocol results in robust expression of the transgene throughout the organ of Corti (fig 5). + +## Acknowledgements + +The authors would like to thank Demêmes Danielle and Douglas Cotanche and for their efforts in teaching us the methods for the isolation of the organ of Corti. Additionally, we would like to thank Ishmael Stefanov-Wagner for engineering the electroporator electrodes; Sherry Lin for her contributing artwork to the video animation; and Matthew Chana, Jason Meeker, and Kendra Marshall (www.goodfightproductions.com) for producing the video. This work was funded by grants (R03DC010065-Parker; RO1DC007174-Edge; P30DC05209- MEEI Core Support for Hearing Research) from the NIDCD. + +## References + +1. Sobkowicz, H. M., Bereman, B., and Rose, J. E. Organotypic Development of the Organ of Corti in Culture. *Journal of Neurocytology* 4 (5), +543 (1975); Kelley, M. et al., *Development* 119 (4), 1041 (1993). + +2. Zheng, J. L., and Gao, W. Q. Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. *Nature* Neuroscience 3 (6), 580 (2000). + +3. Martinez-Monedero, R. et al. THE POTENTIAL ROLE OF ENDOGENOUS STEM CELLS IN REGENERATION OF THE INNER EAR. J +Neurobiol 66 (4), 319 (2006). + +4. Saffer, L. D., Gu, R., and Corwin, J. T. An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles .*Hearing Research* 94 (1-2), 14 (1996). + +5. Zhang, Y., et al. Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge. Journal of Neuroscience Methods 164 (2), 271 (2007). + +6. Jones, J. M., et al. Inhibitors of Differentiation and DNA Binding (Ids) Regulate Math1 and Hair Cell Formation during the Development of the Organ of Corti. *J. Neurosci.* 26 (2), 550 (2006). + +7. Daniela, B., and Josef, S. Calbindin and S100 protein expression in the developing inner ear in mice. *The Journal of Comparative Neurology* 513 (5), 469 (2009). + +8. Helms, A. W., et al. Autoregulation and multiple enhancers control Math1 expression in the developing nervous system. *Development* 127 (6), +1185 (2000). + +9. Zheng, J. L. and Wei-Qiang, G. Differential Damage to Auditory Neurons and Hair Cells by Ototoxins and Neuroprotection by Specific Neurotrophins in Rat Cochlear Organotypic Cultures. *European Journal of Neuroscience* 8 (9), 1897 (1996). \ No newline at end of file diff --git a/medical/md/PMC2864425.md b/medical/md/PMC2864425.md new file mode 100644 index 0000000000000000000000000000000000000000..def6df4c457c924a3601ddffee39059d3be838cb --- /dev/null +++ b/medical/md/PMC2864425.md @@ -0,0 +1,42 @@ +# Editorial How Can We Target Pulmonary Inflammation In Cystic Fibrosis? + +![0_image_0.png](0_image_0.png) + +With each year our enhanced understanding of the pathogenesis of cystic fibrosis (CF) is challenged by new discoveries related to the increasingly complex nature of this disorder. Originally described as a genetic disorder principally due to defective chloride ion conductance at epithelial surfaces and a predisposition to bacterial colonization have been moved into the category of an inflammatory disorder because of increasing research efforts. As our knowledge of the molecular and cellular events associated with CF has burgeoned, we now have a greater appreciation of its protean inflammatory manifestations. This MiniHot Topic on "Mechanisms of Pulmonary Inflammation in Cystic Fibrosis" presents a series of comprehensive review articles in which the authors describe novel mechanisms regulating inflammation in the CF lung and propose and dissect the potential benefits and side-effects of diverse treatments aimed at ameliorating the inflammatory consequences of CF lung disease. + +CF is the most common serious inherited metabolic disorder among Caucasians. It is a recessive condition with a known gene defect, which is invariably fatal. CF affects the liver, pancreas and intestinal tract but the major causes of morbidity and mortality are the lung disease. This generally begins in childhood with the production of thick mucus, chronic airway infections and inflammation leading to progressive decline in lung function and death from respiratory failure. Although gene therapy heralded great promise for the treatment of CF, this approach, whilst likely to repair the chloride ion secretion defect in CF, has no effect on the chronic pulmonary inflammatory manifestations of the disorder. + +Current treatments for CF are broad ranging. In addition to exercise regimes and nutritional supplementation strategies, a number of CF therapies targeting mucus overproduction, inflammation and infection, along with gene therapeutics, protein assist/repair and salt transport restoration drugs are either fully developed or in Phase I, II or III trials. Continuous cycles of infection lead to increased morbidity and mortality in the setting of CF. Prompt antibiotic therapy is associated with reduced decline in lung function and improved patient outcomes. Symptomatically, inhaled/ nebulised bronchodilator therapy provides relief from breathlessness, and mucolytic therapy can be very useful in enabling patients to expectorate sputum. Both are associated with improvements in lung function. Now there is an increasing focus on the therapies targeting the underlying pathogenesis, and in particular underlying inflammation which is central to the disease process in CF. In this respect non steroidal anti-inflammatory medications (NSAIDS), steroids and azithromycin have proved to be useful but their chronic use is associated with long term sequelae including osteoporosis, peptic ulcer disease and cataracts. + +Recently, a number of novel mechanisms regulating the pulmonary inflammatory manifestations of CF have been reported. + +Teichgraber *et al*. described an age-dependent relationship between ceramide generation and lung inflammation, cell death and infection in CF knockout mice [1]. Their study highlighted acid sphingomyelinase as a potential therapeutic target in CF. JE +Blalock's group identified the new collagen-derived proinflammatory tripeptide molecule, proline-glycine-proline (PGP), +detectable in CF sputum that is chemotactic for neutrophils [2]. They also described a role for high mobility group box protein1 (HMGB1) in the ENaC overexpressing Scnn1b+ mouse model [3]. Taggart *et al*. have proposed secretory leucoprotease inhibitor (SLPI) as a multifunctional therapeutic for chronic inflammatory lung disease characterised by a high protease burden +[4-6]. Their studies showed how SLPI acts not only as an antiprotease but also as an anti-inflammatory by directly inhibiting toll-like receptor (TLR) signalling and NFB activation in macrophages. Their thesis promotes the idea that antiprotease therapies are likely to have significant benefit for CF. + +Protease-mediated lung destruction is a hallmark feature of CF lung disease. In their article Quinn *et al*. outline the major protease families present in the CF lung and the various antiproteases that have been proposed as their possible therapeutic inhibitors. Therapies targeting the serine protease neutrophil elastase (NE) are described however, as NE is not the only culprit causing the lung damage in CF, other major proteases in the CF lung including metalloproteases (MMPs), cysteinyl cathepsins and bacterial-derived proteases are also addressed as potential targets. Antiprotease strategies to dampen the pulmonary inflammation in CF and halt further damage to the airways are certainly an attractive therapeutic option and Quinn *et al*. + +expound the use of antiproteases themselves rather than small synthetic molecule protease inhibitor drugs, due to their additional therapeutic properties. For example the antibacterial and anti-inflammatory properties of SLPI are discussed. Finally the feasibility of using appropriate cocktails combining inhibitors of classes of proteases, which may form part of the future of personalised medicine programmes, is also considered. + +In their review Gaggar *et al*. examine the potential impact of two new small molecule inflammatory mediators in CF lung disease, PGP and HMGB1. These novel molecules are described as playing an important and heretofore underappreciated role in pulmonary inflammation in CF. PGP, a collagen-derived peptide generated via prolyl endopeptidase, MMP-8 and MMP-9 activity, although by no means the only chemotactic molecule in the CF lung, according to Gaggar *et al*. may fulfil a role as significant as IL-8. HMGB1, possibly more familiar as a late mediator of inflammation in sepsis, is a DNA-binding protein which once released from its intracellular location can activate the receptor for advanced glycation end products (RAGE) and TLRs 2 and 4. Various strategies are proposed that have the potential to antagonise HMGB1 and PGP in the CF lung. + +Becker *et al*. believe that ceramide plays an important role in the pathogenesis of CF and that pharmacological or genetic inhibition of acid sphingomyelinase, the enzyme responsible for generating ceramide, has therapeutic potential for the treatment of CF. Their mouse models have generated convincing data that ceramide is important in promoting inflammation and pathological changes in the CF lung yet they freely admit that its role in the development of fibrosis remains to be determined. Furthermore they are quick to point out that in addition to its undesirable proinflammatory effects ceramide is actually required for the effective removal of bacteria from the lung during acute infection. Accordingly their favoured approach is to use aerosolised agents such as amitryptyline to partially inhibit ceramide production thereby restoring normal pulmonary ceramide concentrations. The usefulness of any particular therapeutic for the pulmonary inflammatory manifestation of CF must be suited to the context of CF lung disease. Any particular treatment must have a clearly defined use - should it be given at the onset of an exacerbation or is it more suited for long-term administration to quell chronic inflammation? These are considerations which should be addressed in the evaluation of the novel therapeutic strategies described here. Notwithstanding the veracity with which each of the authors expound the importance of their chosen therapeutic targets and their respective roles in the pathogenesis of CF lung disease, it is clear that this disease is dependent not only on the factors described in this series of articles but on additional known, and no doubt yet to be discovered, processes. Some of the major determinants of CF lung disease include a decreased airway surface liquid volume, increased mucus viscosity, chronic microbial colonisation, an impaired protease-antiprotease balance and increased pulmonary inflammation mediated in part by proinflammatory lipid- or collagen-derived molecules such as ceramide and PGP. However, other idiosyncratic factors including genotype, gender, colonisation status, co-morbidities, other organ involvement, treatment regimens and exacerbation history all play their part too. With respect to the novel targets discussed here I look forward to the rapid development and proven success of therapies directed at these targets in our ongoing challenge to enhance the health and quality of life for individuals with CF. + +## References + +[1] Teichgräber V, Ulrich M, Endlich N, Riethmüller J, Wilker B, De Oliveira-Munding CC. Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat Med 2008; 14(4): 382-91. + +[2] Gaggar A, Jackson PL, Noerager BD, O'Reilly PJ, McQuaid DB, Rowe SM. A novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation. J Immunol 2008; 180(8): 5662-9. + +[3] Rowe SM, Jackson PL, Liu G, *et al.* Potential role of high-mobility group box 1 in cystic fibrosis airway disease. Am J Respir Crit Care Med 2008; 178(8): 822-31. + +[4] Taggart CC, Greene CM, McElvaney NG, O'Neill S. Secretory leucoprotease inhibitor prevents lipopolysaccharide-induced IkappaBalpha degradation without affecting phosphorylation or ubiquitination. J Biol Chem 2002; 277(37): 33648-53. + +[5] Greene CM, McElvaney NG, O'Neill SJ, Taggart CC. Secretory leucoprotease inhibitor impairs Toll-like receptor 2- and 4-mediated responses in monocytic cells. Infect Immun 2004; 72(6): 3684-7. + +[6] Taggart CC, Cryan SA, Weldon S, Gibbons A, Greene CM, Kelly E. Secretory leucoprotease inhibitor binds to NF-kappaB binding sites in monocytes and inhibits p65 binding. J Exp Med 2005; 202(12): 1659-68. + +## Catherine M. Greene (Guest Editor) + +Respiratory Research Division Department of Medicine Royal College of Surgeons in Ireland Education and Research Centre Beaumont Hospital Dublin 9 Ireland E-mail: cmgreene@rcsi.ie \ No newline at end of file diff --git a/medical/md/PMC3245411.md b/medical/md/PMC3245411.md new file mode 100644 index 0000000000000000000000000000000000000000..e414e871ecb4688e59685e755d940eb53d7a523b --- /dev/null +++ b/medical/md/PMC3245411.md @@ -0,0 +1,141 @@ + +## Characterization Of Alternanthera Mosaic Virus **And Its Coat Protein** + +Anna A. Mukhamedzhanova*,1, Alexander A. Smirnov1, Marina V. Arkhipenko1, Peter A.Ivanov1, Sergey N. Chirkov1, Nina P. Rodionova1,†, Olga V. Karpova1 and Joseph G. Atabekov1,2 1*Department of Virology, Moscow State University, Moscow 119991, Russia* 2*A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991, Russia* Abstract: A new isolate of Alternanthera *mosaic virus* (AltMV-MU) was purified from Portulaca *grandiflora* plants. It has been shown that the AltMV-MU coat protein (CP) can be efficiently reassembled *in vitro* under different conditions into helical RNA-free virus-like particles (VLPs) antigenically related to native virus. The AltMV-MU and VLPs were examined by atomic force and transmission electron microscopies. The encapsidated AltMV-MU RNA is nontranslatable in vitro. However, it can be translationally activated by CP phosphorylation or by binding to the TGB1protein from the virus-coded movement triple gene block. + +Keywords: *Alternanthera mosaic virus*, coat protein, potexviruses, translation activation, triple gene block protein 1, virus-like particles. + + Flexible filamentous virions of *Alternanthera mosaic* virus (AltMV) are typical for the family *Flexiviridae* (genus Potexvirus) [1]. AltMV was purified from *Alternanthera* pungenes (*Amaranthaceae*) in Australia by Geering and Thomas [2] who showed that AltMV was closely related to Papaya mosaic virus (PapMV). A potexvirus from symptomless portulaca plants (AltMV-Po) has been also isolated and characterized in Italy [3]. Furthermore, the AltMV isolates from phlox (AltMV-PA) and portulaca were identified in the United States [4]. Recently, the complete nucleotide sequence of the genome of another AltMV isolate +(AltMV-MU) has been determined by our group and deposited in GenBank (accession number FJ822136) [5]. + +The RNA sequence analysis allowed to characterize AltMVMU as a separate strain with a new genotype. In the present work some properties of the virions and the coat protein (CP) +of AltMV-MU that caused symptomless infection in Portulaca grandiflora plants were investigated. + +## Transmission Electronic And Atomic Force Microscopy Of Virions And Viruslike Particles Formed By Altmv-Mu Coat Protein + + The AltMV-MU has been propagated in *P. grandiflora* and purified from systemically infected leaves by the method developed for *potato virus X* (PVX) purification [6] using a modified (50mM Tris-HCl, 10mM EDTA pH 8.0) extraction buffer. Virus concentration was estimated using an average extinction coefficient of 2.84 for potexviruses [2]. Purified virus had an A260/A280 ratio of 1.44 and yield was of 0.34 mg/g of leaves harvested at 21-25 days after inoculation. + + Viral RNA was isolated from the purified viral particles by phenol deproteinization. The method of salt deproteinization with 2M LiCl described for PVX CP [7] +was used to obtain the AltMV CP preparation. The extinction coefficient of 0.7 for AltMV CP was calculated +(http://us.expasy.org/tools/protparam.html). + + A single protein band with apparent size of 22 kDa was observed when dissociated virions and purified CP +preparations were analyzed by 8-20% SDS-PAGE and Western blot (Fig. 1). The Mr of the protein deduced from the amino acid sequence was 22, 1 x 103 which was close to the value determined by SDS-PAGE. + +![0_image_0.png](0_image_0.png) + +Fig. (1). 1 μg of purified AltMV-MU virions and CP preparation were analysed in 8-20% SDS-PAGE. Lane M shows molecular weight markers (Fermentas). Gel was stained with Coomassie G250. Western blot (WB): proteins were transferred to PVDF +membrane and incubated with mouse AS to AltMV-MU virions +(dilution 1:10000) and anti-mouse antibodies conjugated with horseradish peroxidase (Promega); the reaction was visualized by the ECL system (Amersham Biosciences). + +![1_image_0.png](1_image_0.png) + + For transmission electron microscopy (TEM) on copper grids [8], the purified viral particles (60 g/ml) and CP +aggregates (60 g/ml) were negatively stained with 2% (w/v) +uranyl acetate. Particles lengths were measured manually from digital prints by *Image J* program. Atomic force microscopy (AFM) measurement was carried out using a NanoScope IIIa multimode-scanning probe microscope +(Digital Instruments) in tapping mode in air, as described previously [8, 9]. + + According to the TEM data, the mean length and diameter of AltMV-MU virion were 570 nm and 13 nm, respectively (Fig. 2a). Based on AFM images, the virions average height was 6-8 nm (Fig. 2b). + + The ability of PapMV CP to assemble VLPs *in vitro* at pH 4.0 in the absence of genomic RNA is known since seventies [10]. By contrast, assembly of PapMV CP at pH +8.0 was limited by production of "small 13-33S aggregates" +[10]. Here, we examined the ability of RNA-free CP AltMVMU to form VLPs under those conditions. + + We found that similarly to PapMV CP, AltMV-MU CP +could be assembled *in vitro* into VLPs at pH 4.0 and low ionic strength. In 0.01M citrate buffer, pH 4.0 a considerable part of AltMV-MU VLPs stuck together producing long bundles (Fig. 2c, d). However, in contrast to PapMV, TEM +and AFM analyses showed that AltMV-MU CP formed filamentous VLPs at pH 8.0 as well (Fig. 2e, f). In 0.01M +Tris-HCl buffer at pH 8.0, AltMV-MU CP formed VLPs, comparable in size to virus particles under the same condition (0.01M Tris-HCl buffer, pH 8.0) or even exceeding them in length (Fig. 2a, b, e, f). In agreement with the AFM data the length of VLPs formed by AltMV-MU CP +at pH 8.0 ranged from 60 nm to 2000 nm, the height was of 6-7 nm and the width of about 20 nm (Fig. 2f). + + Apparently, the assembly of AltMV-MU CP into VLPs was less dependent on pH than that of PapMV. Therefore, the conditions of assembly and the size of the products formed from AltMV-MU and PapMV CPs were considerably different [10]. + +## Immunological Properties Of Virions And Virus-Like Particles + + Antigenic specificity of AltMV-MU virions was compared with that of RNA-free VLPs assembled from AltMV-MU CP at pH 4.0. Antisera against AltMV-MU +virions and VLPs have been obtained using a purified native virus preparation and a preparation of VLPs assembled at the pH 4.0 as antigens. Antigenic properties of AltMV-MU and VLPs were compared by indirect ELISA [11]. Purified preparations of native AltMV-MU and VLPs, both diluted with PBS pH 7.4 up to the concentration of 5 g/ml, were applied to the wells of MaxiSorp microplate (Nunc) and adsorbed at 4°C overnight. Mouse antisera to AltMV-MU and VLPs as well as non-immune mouse serum used as a negative control were titrated against each antigen. + +Horseradish peroxidase-labelled anti-mouse IgG W402 +(Promega) was used as detection antibodies. Mouse antiserum to native AltMV-MU recognized the homologous antigen and some VLPs antigenic determinants which presumably are common for both structures but differently exposed on the VLPs and the native virus (Fig. + +3a). Similarly, mouse antiserum to VLPs strongly reacted with VLPs and also with native AltMV-MU even though the reaction with the virus was clearly weaker (Fig. 3b). These data indicate that native AltMV-MU and VLPs are antigenically related, but not identical. This was not surprisingly because native virus particles (Fig. 2b) and VLPs assembled at the pH 4.0 in the absence of genomic RNA (Fig. 2d) looked quite different morphologically suggesting some differences in their antigenic properties. In particular, some CP epitopes exposed at the surface of native virus particle, could be found hidden or disrupted at the VLPs assembly. On the other side, the neotope appearance cannot be ruled out at the VLPs formation. + + The AltMV-MU, PVX and *Potato aucuba mosaic virus* +(PAMV), members of *Potexvirus* group were analyzed by Western blotting. Blots were developed with polyclonal antisera against PVX diluted 1:20000, AltMV-MU - 1:10000 and PAMV - 1:3000 (Fig. 3c). Both antisera to PVX and PAMV have been found to react with the AltMV-MU (Fig. + +3c, lanes 1, 6). At the same time, the antiserum to AltMVMU reacted neither with PVX nor with PAMV (Fig. 3c, lanes 4, 5). Phylogenetic analysis of 26 coat protein sequences from genus *Potexviru*s was performed previously [5]. It shows that all three proteins are distanly located from each other within the appropriate tree that might explain the absence of cross-reactivity in case of antiserum to AltMVMU. + +## Translational Activation Of Altmv-Mu Virions By Tgb1 Protein + + Recently we have shown that, contrary to *Tobacco* mosaic virus (TMV) and some other viruses, encapsidated PVX RNA was completely nontranslatable *in vitro.* The translational activation of PVX virions may be reached by two ways: either phosphorylation of PVX CP or TGB1 +(triple gene block 1) protein binding to terminal subunits of the polar PVX helix, TGB1 is one of three PVX movement proteins [6, 12]. It is believed that binding of TGB1 to one end of PVX particles induces a structural transition +(remodeling) of the PVX CP into a metastable form. As a result of this transition, the 5' end of viral RNA becomes accessible for ribosomes [13]. Here, we report that similarly to virion PVX RNA, AltMV-MU encapsidated RNA was also untranslatable *in vitro* (Fig. 4a, lane 2) but could be activated by CP phosphorylation of the AltMV-MU virions by protein kinase C (PKC) (Fig. 4a, lane 3). + + A similar effect could be reached as a result of interaction of virions with AltMV-MU TGB1. The AltMVMU TGB1 with N-terminal thioredoxin (Trx-tag), S-tag and His-tag was expressed in *E. coli* BL21 (DE3) strain using the pET 32(a)+ vector according to a Novagen protocol +(Novagen pET System Manual, 11th Edition). The fusion protein with His-tag was purified using Ni-NTA affinity resin (Qiagen) and then specifically digested with enterokinase (Novagen, Tag-off rEK Cleavage/Capture Kit) +to remove the Trx-tag, S-tag and His-tag. The data presented at Fig. (4a) lane 4 showed that putative TGB1-CP AltMVMU interaction resulted in translation of encapsidated genome RNA *in vitro*. This interaction was confirmed by Far-western blot analysis (Fig. 4b). It could be presumed that two ways of translational activation of encapsidated RNA +are peculiar features of some other members of genus Potexvirus. It is known that the PapMV VLPs have been used successfully as an epitope presentation system. Moreover, PapMV VLP platforms have the advantage of triggering both arms of the immune responses, namely, the humoral and CTL responses, a characteristic that is unique for vaccine platforms [14, 15]. As mentioned above, AltMV-MU is closely related to *papaya mosaic virus* (PapMV) [5]. We believe that AltMV-MU VLPs can be regarded as good candidates for presentation of foreign epitopes as well. + +Characterization of AltMV-MU Coat Protein The Open Virology Journal, 2011, Volume 5 **139** + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +Fig. (4). (a) Translational activation of encapsidated AltMV-MU +RNA *in vitro*. SDS-PAGE in 8-20% polyacrylamide gel of 35Slabeled translation products in the wheat germ extract. Gel was dried and autoradiographed. Translation of purified AltMV-MU +RNA (lane 1); AltMV-MU virions (lane 2); AltMV-MU virions after phosphorylation with PKC (lane 3), AltMV-MU preparations incubated with AltMV-MU TGB1 (lane 4). AltMV-MU RNA +concentration in the translation sample was 40 μg/ml. Arrows indicate the positions of molecular weight markers and the bold arrow indicates the position of the protein encoded by the 5'- +proximal AltMV-MU replicase gene (174 kDa). (b) Far-western blot (Far-WB) analysis of interaction of different TGB1 proteins with CP AltMV-MU at pH 8.0. Purified TGB1 PAMV (1 μg, lane 1, negative control) and TGB1 AltMV-MU (1 μg, lane 2) proteins were separated by 8-20% SDS-PAGE. TGB1s were transferred to PVDF membrane, incubated with CP AltMV-MU (5μg/ml), mouse AS to CP AltMV-MU (dilution 1:10000) and anti-mouse antibodies conjugated with horseradish peroxidase (Promega); reaction was visualized by the ECL system (Amersham Biosciences). + +## Acknowledgements + +![3_Image_2.Png](3_Image_2.Png) + + We are grateful to Dr. Stephan Winter (German Collection of Microorganisms and Cell Cultures, Braunschweig) for providing the initial portulaca isolate and Dr Sergey Saunin and Mikhail Savvateev (AIST-NT +Company) for Atomic force microscopy assays. This work was funded in part by the Russian Foundation for Basic Research (Grant No. 10-04-000-89-a) and the Federal Agency of Science and Innovation (FASI) (contracts No 16.512.11.2184). + +## References + +[1] Martelli GP, Adams MJ, Kreuze JF, Dolja VV. Family Flexiviridae: A case study in virion and genome plasticity. Annu Rev Phytopathol 2007; 45:73-100. + +[2] Geering AD, Thomas, JE. Characterisation of a virus from Australia that is closely related to papaya mosaic potexvirus. Arch Virol 1999; 144: 577-92. + +[3] Ciuffo M, Turina M. A potexvirus related to Papaya mosaic virus isolated from moss rose (Portulaca grandiflora) in Italy. Plant Pathol 2004; 53: 515. + +[4] Hammond J, Reinsel MD, Maroon-Lango CJ. Identification and full sequence of an isolate of Alternanthera mosaic potexvirus infecting Phlox stolonifera. Arch Virol 2006; 151: 477-93. + +[5] Ivanov PA, Mukhamedzhanova AA, Smirnov AA, Rodionova NP, +Karpova OV, Atabekov JG. The complete nucleotide sequence of Alternanthera mosaic virus infecting Portulaca grandiflora represents a new strain distinct from phlox isolates. Virus Genes 2011; 42: 268-71. + +[6] Atabekov JG, Rodionova NP, Karpova OV, Kozlovsky SV, +Novikov VK, Arkhipenko MV. Translational activation of encapsidated potato virus X RNA by coat protein phosphorylation. + +Virology 2001; 286: 466-74. + +[7] Homer RB, Goodman RM. Circular dichroism and fluorescence studies on potato virus X and its structural components. Biochim Biophys Acta 1975; 378: 296-304. + +140 *The Open Virology Journal, 2011, Volume 5 Mukhamedzhanova et al.* + +[8] Karpova OV, Zayakina OV, Arkhipenko MV, *et al.* Potato virus X +RNA-mediated assembly of single tailed ternary "coat proteinRNA-movement protein" complexes. J Gen Virol 2006; 87: 273140. + +[9] Kiselyova OI, Yaminsky IV, Karpova OV, *et al.* AFM study of potato virus X disassembly induced by movement protein. J Mol Biol 2003; 332: 321-5. + +[10] Erickson JW, Bancroft JB, Horne RW. The assembly of papaya mosaic virus protein. Virology 1976; 72: 514-7. + +[11] Clark MF, Adams AN. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 1977; 34: 475-83. + +[12] Atabekov JG, Rodionova NP, Karpova OV, Kozlovsky SV, +Poljakov VY. The movement protein-triggered *in situ* conversion of potato virus X virion RNA from a nontranslatable into a translatable form. Virology 2000; 271: 259-63. + +[13] Rodionova NP, Karpova OV, Kozlovsky SV, Zayakina OV, +Arkhipenko MV, Atabekov JG. Linear remodeling of helical virus by movement protein binding. J Mol Biol 2003; 333: 565-72. + +[14] Denis J, Majeau N, Acosta-Ramirez E, *et al.* Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization. + +Virology 2007; 363: 59-68. + +[15] Leclerc D, Beauseigle D, Denis J, *et al.* Proteasome-independent major histocompatibility complex class I cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human T cells. J Virol 2007; 81: 1319-26. +Received: July 5, 2011 Revised: September 9, 2011 Accepted: September 21, 2011 © Mukhamedzhanova *et al*.; Licensee *Bentham Open*. + +This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http: //creativecommons.org/licenses/by-nc/ 3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. \ No newline at end of file diff --git a/medical/md/PMC3377154.md b/medical/md/PMC3377154.md new file mode 100644 index 0000000000000000000000000000000000000000..d8088459813350d530688da2370fd08a40f71aa5 --- /dev/null +++ b/medical/md/PMC3377154.md @@ -0,0 +1,26 @@ +# Gorham'S Disease Of Femur - A Response + +Sir, We read with great interest the manuscript entitled "Gorham's disease of femur," by Baba et al. + +1 There is still no clear consensus about the treatment of Gorham's disease as it shows variable response to medical and surgical treatment. In 2009, we published our report on treatment of diaphyseal Gorham's disease with radiotherapy and fibular graft.2 We had similar belief as that of the authors that radiotherapy converts the proliferating blood vessels to fibrous tissue. Subsequent fibular graft transplantation into the defect results in good incorporation. In our previously published article, we reported about the successful transplantation of a fibular graft after radiation therapy in a case of radial diaphysis Gorham's disease in a 15‑year‑old girl who had a followup of 2 years at that time.2 But unfortunately, we noticed the recurrence of the disease after 5 years of surgery and the fibular graft in her case showed resorption. It is quite difficult to predict when the disease process actually ceases its activity in Gorham's disease. In our case whether it was a recurrence or it was because of residual disease pathology at that site is quite difficult to say. Whatever may be the cause of resorption of the fibular graft in our case, we would like to say that the followup of the patient (2 years) in the article of Baba et al. is quite short to predict the outcome. We, from our own experience, would like to suggest the authors that they should follow their patient for a long time to have definite conclusion about the outcome of radiation treatment in Gorham's disease. We still believe that the response to treatment is quite variable and unpredictable in Gorham's disease. Long term outcome of all patients treated till date with different procedures must be reported to have better understanding about the disease and its treatment. + +## Sujit Kumar Tripathy, Ramesh Kumar Sen1**, Tarun Goyal2** + +Department of Orthopaedics, Friarage Hospital, Northallerton, UK, 1Department of Orthopaedics, Postgraduate Institute of Medical Education and Research, Chandigarh, India, 2*Department of* Orthopaedics, Freeman Hospital, Newcastle upon Tyne, UK +Address for correspondence: Dr. Sujit Kumar Tripathy, Clinical Fellow, Department of Orthopaedics, Northallerton, DL6 1JG, United Kingdom. E‑mail: sujitortho@yahoo.co.in + +## References + +1. Baba AN, Bhat YJ, Palijor SD, Nazir A, Khan NA. Gorham's disease of femur. Indian J Orthop 2011;45:565‑8. + +2. Tripathy SK, Sen RK, Agarwal A. Fibular grafting in diaphyseal Gorham's disease: Report of two cases and review of literature. Eur J Orthop Surg Traumatol 2009;19:485‑90. + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +## Authors' Reply + +Sir, We appreciate Tripathy et al. + +1 for the interest shown in our paper2 and thank them for sharing their experience on the long term followup of Gorham's disease managed with radiotherapy and bone grafting. Gorham's disease is a rare disorder of the musculoskeletal system. The etiology and pathophysiology of the disease remains undetermined. Being uncommon with an unpredictable course, the management of the disease has not been standardized. A number of treatment methods including bisphosphonates, estrogens, magnesium, calcium, vitamin D, vitamin B12, fluoride, calcitonin, cisplatin, actinomycin D, thalidomide, somatotrophin, interferons, amino acids, placental extracts, and transfusions of placental blood have been used, but none has been found to be effective in majority of cases. Surgery and radiation therapy remain the principal treatment modalities of the condition. Surgical treatment usually has involved prosthetic replacement of the affected bone (especially in proximal femur) or amputation in severe cases, with most of the studies showing good long term results.3 Bone grafting has been tried in a few cases, but it has usually yielded poor results as the graft is usually found to resorb, generally within the first year, or after a number of years, as stated by Tripathy et al. Radiotherapy has shown promise in the treatment of Gorham's disease and halting the disease process, probably due to the radiosensitive nature of the proliferating capillary‑like or lymphatic‑like vessels which play a role in the pathogenesis of the disease. Most of the studies showing lack of effect of radiotherapy have used low doses, generally less than 20 Gy. Definitive radiation therapy in moderate doses (40–45 Gy) appears to result in a good outcome and few long term complications.4 Radiotherapy appears to be the best bet in managing this condition. This view is supported by a recent literature review wherein radiotherapy was seen to prevent the disease progression in 77.3% patients of Gorham's disease.5 \ No newline at end of file diff --git a/medical/md/PMC3421985.md b/medical/md/PMC3421985.md new file mode 100644 index 0000000000000000000000000000000000000000..31afa60256471a70a381cc76040c5006a846e379 --- /dev/null +++ b/medical/md/PMC3421985.md @@ -0,0 +1,300 @@ + + +![0_image_0.png](0_image_0.png) + +# In Posidonia Oceanica Cadmium Induces Changes In Dna Methylation And Chromatin Patterning A Putative Positive Feedback Regulation Mechanism In Csacs2 Expression Suggests A Modified Model For Sex Determination In Cucumber (Cucumis Sativus L.) Maria Greco, Adriana Chiappetta, Leonardo Bruno And Maria Beatrice Bitonti* Zheng Li1,2,*, Shu Wang3,*, Qianyi Tao1,2, Junsong Pan2, Longting Si4, Zhenhui Gong1 And Run Cai2,† + +Department of Ecology, University of Calabria, Laboratory of Plant Cyto-physiology, Ponte Pietro Bucci, I-87036 Arcavacata di Rende, Cosenza, Italy * To whom correspondence should be addressed. E-mail: b.bitonti@unical.it 1 College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China 2 School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China 3 College of Horticulture and Landscape Architecture, Southwest Forest University, Kunming, Yunnan 650224, China 4 College of Horticulture, Shenyang Agriculture University, Shenyang, Liaoning 110161, China Received 29 May 2011; Revised 8 July 2011; Accepted 18 August 2011 +* These authors contributed equally to this work. + +y To whom correspondence should be addressed. E-mail: cairun@sjtu.edu.cn Abstract Received 13 December 2011; Revised 27 March 2012; Accepted 4 April 2012 In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants. It is well established that the plant hormone ethylene plays a key role in cucumber sex determination. Since the unisexual control gene M was cloned and shown to encode an ethylene synthase, instead of an ethylene receptor, the 'one-hormone hypothesis', which was used to explain the cucumber sex phenotype, has been challenged. Here, the physiological function of CsACS2 (the gene encoded by the M locus) was studied using the transgenic tobacco system. The results indicated that overexpression of CsACS2 increased ethylene production in the tobacco plant, and the native cucumber promoter had no activity in transgenic tobacco (PM). However, when PM plants were treated with exogenous ethylene, CsACS2 expression could be detected. In cucumber, ethylene treatment could also induce transcription of CsACS2, while inhibition of ethylene action reduced the expression level. These findings suggest a positive feedback regulation mechanism for CsACS2, and a modified 'one-hormone hypothesis' for sex determination in cucumber is proposed. + +Key words: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE, DNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile. + +Key words: CsACS2, Cucumis sativus L., ethylene induction, one-hormone hypothesis, positive feedback regulation, sex determination Introduction In the Mediterranean coastal ecosystem, the endemic seagrass Posidonia oceanica (L.) Delile plays a relevant role by ensuring primary production, water oxygenation and provides niches for some animals, besides counteracting coastal erosion through its widespread meadows (Ott, 1980; Piazzi et al., 1999; Alcoverro et al., 2001). There is also considerable evidence that P. oceanica plants are able to absorb and accumulate metals from sediments (Sanchiz et al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus influencing metal bioavailability in the marine ecosystem. + +For this reason, this seagrass is widely considered to be a metal bioindicator species (Maserti et al., 1988; Pergent et al., 1995; Lafabrie et al., 2007). Cd is one of most widespread heavy metals in both terrestrial and marine environments. + +In angiosperms, the mechanisms of sex determination, which have been extensively studied in recent years, involve a number of different genetic and epigenetic factors +(Tanurdzic and Banks, 2004). As a result of its diversity in sex types, cucumber (Cucumis sativus L.) has been researched as a model for more than half a century +(Atsmon, 1968; Tsao, 1988; Perl-Treves, 1999). Cucumber plants generally produce male and female flowers separately on the same individual (monoecious); however, certain lines (hermaphrodite and andromonoecious) also produce bisexual flowers (Galun, 1961; Shifriss, 1961; Kubicki, 1969). Morphologically, all floral buds are initially hermaphroditic with staminate and pistillate primordia Although not essential for plant growth, in terrestrial plants, Cd is readily absorbed by roots and translocated into aerial organs while, in acquatic plants, it is directly taken up by leaves. In plants, Cd absorption induces complex changes at the genetic, biochemical and physiological levels which ultimately account for its toxicity (Valle and Ulmer, 1972; Sanitz di Toppi and Gabrielli, 1999; Benavides et al., 2005; Weber et al., 2006; Liu et al., 2008). The most obvious symptom of Cd toxicity is a reduction in plant growth due to an inhibition of photosynthesis, respiration, and nitrogen metabolism, as well as a reduction in water and mineral uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., 2000; Shukla et al., 2003; Sobkowiak and Deckert, 2003). + +At the genetic level, in both animals and plants, Cd can induce chromosomal aberrations, abnormalities in (Kubicki, 1969). A systematic morphogenetic analysis of male and female flower development revealed that the origin of floral reproductive organ differentiation and sex determination occurs after the carpel primordia initiation stage (Bai et al., 2004). The selective arrest of either the staminate or pistillate organs results in female or male flowers, respectively. Furthermore, if the arrest does not happen, hermaphroditic flowers are formed (Atsmon and Galun, 1960; Goffinet, 1990; Perl-Treves, 1999). + +Early genetic studies indicated that three major genes are responsible for sex expression and segregation in cucumber: F/f, M/m, and A/a. The F gene promotes femaleness, while the recessive m gene regulates the appearance of bisexual ª 2011 The Author(s). + +This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. + +ª 2012 The Author(s). + +This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. + +flowers on the plant. Furthermore, in combination with the homozygous recessive f gene, the recessive a gene can intensify the androecious nature in cucumber plants (Galun, 1961; Kubicki, 1969; Robinson et al., 1976). Additionally, sex expression can also be regulated by many environmental factors such as photoperiod, temperature, and plant hormones such as ethylene, indole-3-acetic acid, and gibberellic acid (Atsmon, 1968; Takahashi et al. 1983; Perl-Treves, 1999; Yamasaki et al., 2005). + +Preliminary physiological investigations revealed that a strong correlation exists between the plant hormone ethylene and the sex phenotype in cucumber. Elevated endogenous levels of ethylene can promote femaleness, whereas the inhibitors of ethylene biosynthesis or the ethylene response suppress the development of female reproductive organs (Atsmon and Tabbak, 1979; Takahashi and Jaffe, 1984). The arresting action of ethylene on stamen development can also be found in some other plant species. Overexpression of a cucumber ethylene synthesis gene, CsACO2 (Cucumis sativus ACC oxidase gene 2), can restrain Arabidopsis stamen development, mimicking female cucumber flowers (Duan et al., 2008). + +The 'one-hormone hypothesis' posits that the F locus, which controls the gynoecious phenotype, regulates ethylene levels, promoting the development of the pistillate primordial state; and the dominant M locus, which is important in determining the monoecious condition, mediates the inhibition of stamen development by ethylene. The ethylene signal is not transmitted if the genotype is homozygous for m; as a result, stamen development is not inhibited, and the plant produces hermaphroditic flowers (Yin and Quinn, 1995; Yamasaki et al., 2001). According to this hypothesis, the F locus encodes a gene for ethylene production, and the M locus encodes an ethylene-response factor, which might be a receptor of ethylene. + +Recently, the F locus has been cloned and shown to encode 1-aminocyclopropane-1-carboxylic acid synthase +(ACS) (Trebitsh et al., 1997; Mibus et al., 2004), which has been verified as being involved in ethylene biosynthesis (Li et al., 2009). Furthermore, an ethylene receptor gene, CsETR1 (Cucumis sativus ethylene receptor 1), has been observed to play a key role in stamen arrest (Wang et al., 2010). A cucumber nuclease-encoding gene, CsCaN +(Cucumis sativus calcium-dependent nuclease), has been shown to respond to the ethylene signal and is involved in the anther-specific DNA damage in the primordia of developing female flowers (Gu et al., 2011). However, the cloning of the M locus, which was previously thought to encode an ethylene receptor, reveals that it is not part of the ethylene signalling pathway, but rather encodes an enzyme involved in ethylene biosynthesis. This gene, CsACS2, also encodes an ACS (Boualem et al., 2009; Li et al., 2009). So, the response relationship between the CsACS2 gene and ethylene needs to be clarified. + +Previous work indicated that the CsACS2 gene product is enzymatically active in Escherichia coli and can synthesize ACC (1-aminocyclopropane-1-carboxylic acid), which is a precursor of the ethylene molecule (Li et al., 2009). In this study, the function of this gene was studied using the transgenic tobacco system. The results indicate that the overexpression of CsACS2 increases ethylene production in tobacco, and these transgenic plants (P35) display short internodes and delayed flowering. Moreover, the original cucumber promoter of CsACS2 was not active in transgenic tobacco (PM), and did not promote transcription of the gene. However, when the PM plants were treated with exogenous ethylene, expression of CsACS2 could be detected. In cucumber plants, a certain amount of ethylene treatment could induce the transcription of CsACS2, while inhibition of ethylene action could reduce expression levels to varying degrees. These findings suggest a positive feedback regulation mechanism for CsACS2, which could be involved in flower development in cucumber. + +## Plant Materials + +Sterile-grown seedlings of tobacco Nicotiana tabacum pv. + +Bairihong, which were provided by Professor Lingxia Zhao +(Shanghai Jiaotong University, Shanghai), were used for Agrobacterium-mediated transformation. A gynoecious cucumber inbred line G06 (FFMM) was used for isolation of the wild-type CsACS2 gene and its promoter. A monoecious cucumber line M06 (ffMM), which was the near-isogenic line (NIL) with G06 for the F +locus, was used to study the exogenous ethylene-inducing expression of CsACS2 in cucumber. A second gynoecious line, G34 (FFMM), +together with the gynoecious line G06, was used to analyse the level of transcription of the CsACS2 gene during inhibition of ethylene action. A hermaphroditic cucumber line H34 (FFmm), together with its NIL G34 for the M locus, was used to study the promoters sequence and compare the relative expression of CsACS2 in near genotypic backgrounds, except for M/m. A traditional andromonoecious line 'Lemon' (ffmm) was used to analyse the gene expression and response to the ethylene-related chemical treatments. All cucumber lines were maintained by inbreeding at the Plant Department of Shanghai Jiaotong University and the College of Horticulture at Northwest A&F University. Conditions for germination and growth were according to Li et al. (2009). + +Cloning of CsACS2 and its promoter Cucumber apical shoots without any immature leaves were excised from the shoot apices with an Olympus SZ61 stereomicroscope (Olympus, Japan). As described previously (Li et al., 2009), apical shoot total RNA was isolated from G06 cucumber plants at the four-leaf stage (;20 d after sowing). Reagents for RNA extraction were purchased from TIANGEN (China). DNase I (TaKaRa, China) was used to remove contaminating DNA, and first-strand cDNA was synthesized with the RevertAid First Strand cDNA +Synthesis Kit (Fermentas). The CDS (coding sequence) of the CsACS2 gene was amplified using a Long-Distance PCR Kit +(Takara, China) with the appropriate primers (Supplementary Table S1 available at JXB online). + +To isolate the CsACS2 promoter, genomic DNA was extracted as described previously (Li et al., 2008). The promoter fragment of the CsACS2 gene from G06 was amplified using a Long-Distance PCR Kit (Takara, China). The promoter regions from the NILs G34 and H34 were also amplified to compare the homologous cisacting elements. Putative cis-acting elements in the CsACS2 promoter were identified by searching the PLACE database (http://www.dna.affrc.go.jp/PLACE/) (Higo et al., 1999). + +All the DNA fragments were subcloned into the T/A-cloning vector pUCm-T (Fermentas) and sequenced as previously described (Li et al., 2008). + +Generation of transgenic tobacco plants The CDS of CsACS2 was inserted into the pBI121 plasmid in the sense orientation under the control of the Cauliflower mosaic virus (CaMV) 35S promoter to give the pBI121-P35S-CsACS2 construct (Fig. 1A), which resulted in the transgenic tobacco plants designed P35. Using the restriction sites (ClaI and BamHI) flanking the CaMV 35S promoter, the cucumber promoter fragment was inserted to produce the pBI121-PCsACS2-CsACS2 plasmid +(Fig. 1A), which resulted in the transgenic tobacco plants designed PM. The two expression cassettes were introduced into tobacco N. tabacum pv. Bairihong by Agrobacterium-mediated transformation (Hoekema et al., 1983). DNA was isolated from leaves of kanamycin-resistant regenerated plants to assay for positive transformation events by PCR +using gene-specific primers (Supplementary Table S1 at JXB oline). PCR-positive transgenic plants were subjected to dot blotting analysis. Five P35 and three PM transgenic tobacco plants were chosen and self-pollinated to obtain homozygous T2 offspring. + +Dot blotting and quantitative RT-PCR Genomic DNA and total RNA of transgenic, wild-type tobacco and cucumber plants were extracted using the method described previously (Li et al., 2008, 2009). + +Dot blotting: Tobacco genomic DNA samples (40 lg) were blotted onto Hybond-N+ nylon membranes with the Minifold I DotBlot System (Amersham, China). Hybridizations were performed using the protocol provided by the manufacturer (Gene Images random priming labeling module and Gene Images CDP-Star detection module from Amersham, China). + +Semi-quantitative RT-PCR: First-strand cDNA was synthesized according to a method described previously(Li et al., 2009). Using the appropriate gene-specific primers (Supplementary Table S1 at JXB online), PCR was performed for 30 cycles (94 C, 30 s; 55 C, 30 s; 72 C, 30 s). The products were separated on 1% agarose gels stained with ethidium bromide. The results were analysed using Gene Tools software from Syngene (CA, USA). The reverse transcription-PCRs (RT-PCRs) for two housekeeping genes (tobacco Actin gene AY189972 and cucumber Actin3 gene DQ115883) were performed under the same conditions as described above to estimate whether equal amounts of RNA had been used in the RT-PCRs. Quantitative RT-PCR: Cucumber RNA extraction and first-strand cDNA synthesis were described previously. PCR was performed in a 96-well plate using an ABI 7500 Fast Real-Time PCR System +(Applied Biosystems, USA), with SYBR Green Realtime PCR +Master Mix (TaKaRa, China). The amplification was initiated by heating to 94 C for 10 min, followed by 40 cycles at 94 C for 5 s and 62 C for 34 s. The amplification specificity was tested by a dissociation curve (65–90 C). To compare results from different reactions and samples, CsACS2 amplification was normalized to CsActin3 using the CT values. Ethylene production in transgenic tobacco and cucumber cultivars The time course of ethylene evolution was examined in transgenic tobacco and cucumber cultivars according to Yamasaki et al. (2001). To examine ethylene production from the two transgenic tobacco lines, 10 g samples of leaf tissue from each of three P35 plants (T3-8, T3-22, and T3-26) and one PM plant (TM-3) were cut into 1 cm2 pieces and enclosed individually in 5 ml vessels sealed with rubber stoppers. After incubation at 25 C for 1 h, 1 ml of head gas was withdrawn from each vessel using a gas-tight syringe. The gas samples were collected once every hour during a 3 h incubation period. To examine ethylene production from the five cucumber cultivars G06, M06, G34, H34, and 'Lemon', shoot apices were excised at the four-leaf stage. Head gas was sampled after incubation at 25 C for 16 h. All samples were injected into a gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and an activated alumina column. The instrument was calibrated with standard ethylene gas. + +Each result was derived from three independent biological replicates. + +Chemical treatments To investigate the effect of ethylene on CsACS2 expression, ethephon (an ethylene-releasing agent) was applied to transgenic tobacco and cucumber plants (M06) at various concentrations. + +AgNO3 (ethylene action inhibitor) and aminoethoxyvinyl glycine +(AVG, ethylene biosynthesis inhibitor) were also applied to two cucumber lines, G06 and G34. Ethephon and AgNO3 were purchased from Sigma (China). AVG was provided by Professor Qiguang Wen (IPPE, CAS, Shanghai). + +Ethephon treatment of transgenic plants: Briefly, 50 ll aliquots of 0.5 mM and 1.0 mM ethephon in water containing 0.1% (v/v) Tween-20 were applied to the apex of flower buds of the PM transgenic plant line TM-3 using a micropipette once a day for 3 d. The same treatment with water containing 0.1% (v/v) Tween-20 was the control (and was also used in the experiment below). The apex buds, leaves adjacent to the buds, and untreated lower leaves were then excised for RNA extraction. + +Ethephon treatment of cucumber plants: As the expression of CsACS2 was not detected in apical shoot, the monoecious cucumber line M06 was selected to analyse the effect of ethylene on CsACS2 transcription. The apical shoots of four-leaf-stage M06 seedling were soaked in water containing 0.1, 0.5, and 1.0 mM ethephon with 0.1% (v/v) Tween-20 for 2, 4, and 6 h, respectively. The apices were then excised for RNA extraction. + +AgNO3 and AVG treatment of cucumber plant: The cucumber lines G06 and G34, which both express CsACS2 at high levels, were selected to analyse the inhibitory effect of ethylene on CsACS2 expression. Aliquots of 50 ll of 1.0 mM AgNO3 and 1.0 mM AVG +in H2O containing 0.1% (v/v) Tween-20 were applied to the apices of the two cucumber lines at the four-leaf stage using a micropipette once a day for 3 d. The apices were then excised for RNA +extraction. + +To study the effects of chemical application on the sexual expression of cucumber plants, several of the treated plants were allowed to grow to maturity. These included G06 and G34 treated with AgNO3 (1.0 mM), M06 treated with ethephon (0.5 mM and 1.0 mM), H34 treated with AgNO3 (1.0 mM) and ethephon +(0.5 mM and 1.0 mM), and 'Lemon' treated with AgNO3 +(1.0 mM) and ethephon (0.5 mM and 1.0 mM). The application method was as given above for AgNO3 and AVG treatment of cucumber plants. Every treatment employed five plants, and the number of male, female, and bisexual flowers was recorded up to the 30th node on the main stem. + +Overexpression of CsACS2 in transgenic tobacco plants Transgenic tobacco plants were generated to analyse the biological function of under the control of two + +![3_image_0.png](3_image_0.png) + +different promoters. After identification by PCR and dot blotting (Fig. 1B), five P35 and three PM transgenic tobacco plants were chosen for self-pollination to produce the homozygous T2 offspring. + +RT-PCR revealed a high level of expression of CsACS2 in the P35 transgenic lines, T3-6, T3-8, T3-22, and T3-26 +(Fig. 1C), and the plants showed delayed flowering compared with wild-type tobacco (Fig. 1D). The overexpression lines also had a shorter internode distance and increased leaf number before flowering (Fig. 1D). These phenotypes might have a relationship with an endogenous hormone balance disorder. + +The P35 plants also showed higher ethylene production. + +Using gas chromatography analysis, it was found that leaves of three overexpression lines, T3-8, T3-22, and T3-26, produced more ethylene than did the wild-type +(Fig. 2). This result indicated that the CsACS2 gene is involved in ethylene biosynthesis in planta, and it could lead to more ethylene production in the transgenic tobacco system. + +## Using Rt-Pcr, It Was Not Possible To Detect Any Expression + +of CsACS2 in the PM lines; and the right-hand part of Fig. 1B shows this result in root, stem, leaf, and flower bud from the TM3 line. This is consistent with the fact that there was no visible change in phenotype of transgenic PM +lines (Fig. 1D), and indicated that the native CsACS2 promoter from cucumber had no activity in tobacco. + +Moreover, ethylene production in TM3 was not significantly difference from that in wild-type tobacco (Fig. 2). + +However, after ethephon treatment, some differences in the expression level could be detected in the flower bud and its adjacent leaf (Fig. 3). It was supposed that treatment with 1.0 mM ethephon could induce more accumulation of CsACS2-specific mRNA due to transcription than 0.5 mM +ethephon treatment, and that induction in flower bud was more sensitive than in leaf. + +![4_image_1.png](4_image_1.png) + +![4_image_0.png](4_image_0.png) + +To analyse the effects of ethylene on CsACS2 expression in cucumber, the basal level of transcription needed to be determined initially. As a result of the different expression patterns of CsACS2 (Fig. 4), the cucumber line M06 was selected for the ethylene induction experiments, while lines G34 and G06 were used to study the inhibitory effect of ethylene on CsACS2 expression. + +In M06, after 2 h treatment with 1.0 mM ethephon, the expression of CsACS2 could be detected; the transcription level peaked at 4 h after treatment, and then declined rapidly at 6 h. With 0.1 mM and 0.5 mM ethephon treatment, transcription was first detected at 4 h, and reached the highest level at 6 h (Fig. 5). This result indicated that certain concentrations of exogenous ethylene can induce the expression of CsACS2 in cucumber, and that higher concentrations result in more rapid induction and accumulation of CsACS2specific mRNA. Possible causes for the observed decline in CsACS2 expression after treatment with 1.0 mM ethephon for 6 h will be discussed below. + +Reduced expression of CsACS2 in cucumber in response to chemical treatment After 3 d of treatment with AgNO3 and AVG, a decline in CsACS2 expression was observed in cucumber shoot apices. + +![5_image_0.png](5_image_0.png) + +![5_image_1.png](5_image_1.png) + +For the AgNO3 treatment, expression decreased by 42% +and 18% in G34 and G06, respectively. However, treatment with AVG had a more pronounced inhibitory effect than was observed for AgNO3; CsACS2 expression decreased by 60% and 95% in G34 and G06, respectively (Fig. 6). Therefore, the inhibitory effects of the endogenous ethylene response and biosynthesis negatively controlled CsACS2 expression. + +Effects of chemical treatment on sex determination in cucumber Besides the effects on the expression of CsACS2, ethylene has a significant correlation with sex determination in cucumber plants, and it can induce femaleness. Table 1 shows the effect of ethylene-related treatments on the gynoecious (G06 and G34), monoecious (M06), hermaphrodite (H34), and andromonoecious ('Lemon') cucumber lines. AgNO3 is known to interfere with ethylene action, and its application promoted the conversion of female/ hermaphrodite flowers into male flowers, which increased male flower number in all four cucumber cultivars tested +(G06, from 0.5 to 13.5; G34, from 0 to 10.4; H34, from 0 to 13.5; and 'Lemon', from 16.6 to 28.4). Moreover, ethephon could induce development of female flowers in M06, with female flowers produced on 5.6 and 7.4 nodes after treatment with 0.5 mM and 1.0 mM ethephon, respectively. In bisexual lines, 0.5 mM ethephon treatment resulted in more bisexual flowers (16.5) in 'Lemon'. Unexpectedly, when the concentration was increased to 1.0 mM, some female flowers (8.9) were produced, along with a further reduction of both male and bisexual flowers. A similar phenomenon was found with line H34, in which female flowers were first produced at 0.5 mM ethephon (3.2 nodes), and increased to 11.3 nodes at 1.0 mM. The number of bisexual flowers on H34 plants decreased to 24.3 and 12.6 nodes, respectively, at 0.5 mM and 1.0 mM ethephon. These results confirm the action of ethylene in arresting stamen development, which was expressed as the conversion of male/hermaphrodite into female flowers in cucumber plants with ethephon treatment, and suggest that there is a significant correlation between ethylene content and sex expression in cucumber. + +CsACS2 promoter sequence analysis The 1958 bp promoter fragments derived from G34 and H34 genomic DNA were sequenced, and no DNA polymorphisms were identified between them (GenBank accession no. FJ529216). Additionally, analysis of the conserved CsACS2 promoter sequence indicated that there were two EREs (ethylene-responsive elements; AWTTCAAA) at positions –154 to –147 and –1334 to –1327, respectively +(Table 2). The EREs were identified as an ethyleneresponsive enhancer element in Solanum lycopersicum +(Montgomery et al., 1993), Dianthus caryophillus (Itzhaki et al., 1994), and Lycopersicon chilense (Tapia et al., 2005). These two cis-acting elements might take part in the ethylene-regulated expression of CsACS2. Moreover, one cytokinin (CTK) response element and one LEAFY element were also found in the CsACS2 promoter. These findings could be in agreement with the spatial expression pattern of CsACS2 in the cucumber shoot apical meristem (Saito et al., 2007). There is a long history of research into the mechanisms of sex determination in cucumber, and extensive physiological studies indicate that the plant hormone ethylene is the key point in the regulatory pathways. Based on the studies of Yin and Quinn (1995) and Yamasaki et al. (2001), the 'onehormone hypothesis' was proposed, in which the core view + +![6_image_0.png](6_image_0.png) + +Cultivar and genotype Treatment No. of flowers per plantz + +| Male flowers | Bisexual flowers | Female flowers | Abortion | | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------|------------------|------------|-------------|-----------| +| G06 (FFMM) | Control | 0.560.3 a | 0.0 a | 28.861.0 b | 0.860.8 a | +| AgNO3 (1.0 mM) | 13.562.1 b | 0.0 a | 15.562.2 a | 1.060.9 a | | +| G34 (FFMM) | Control | 0.0 a | 0.0a | 28.560.8 b | 1.160.4 a | +| AgNO3 (1.0 mM) | 10.461.7 b | 0.0 a | 18.261.8 a | 0.960.8 a | | +| M06 (ffMM) | Control | 28.860.7 b | 0.0 a | 0.660.5 a | 0.660.5 a | +| Ethephon (0.5 mM) | 18.262.8 a | 0.0 a | 5.661.2 b | 3.661.8 a,b | | +| Ethephon (1.0 mM) | 15.765.6 a | 0.0 a | 7.463.2 b | 6.963.2 b | | +| H34 (FFmm) | Control | 0.0 a | 29.660.5 d | 0.0 a | 0.560.4 a | +| AgNO3 (1.0 mM) | 13.561.0 b | 16.361.0 b | 0.0 a | 0.460.2 a | | +| Ethephon (0.5 mM) | 0.0 a | 24.361.2 c | 3.260.5 b | 2.461.3 a | | +| Ethephon (1.0 mM) | 0.0 a | 12.662.0 a | 11.360.8 c | 6.162.1 b | | +| 'Lemon' (ffmm) | Control | 16.661.7 b | 13.162.0 c | 0.0 a | 0.560.3 a | +| AgNO3 (1.0 mM) | 28.461.7 c | 1.361.5 a | 0.0 a | 0.560.3 a | | +| Ethephon (0.5 mM) | 12.363.0 a | 16.562.5 c | 0.0 a | 1.860.5 a | | +| Ethephon (1.0 mM) | 10.362.0 a | 8.461.3 b | 8.962.0 b | 2.463.0 a | | +| AgNO3 and ethephon were applied to the shoot apices of cucumber plants at the four-leaf stage. The number of male, female, and bisexual flowers was recorded up to the 30th node on the main stem. Nodes with no flowers were defined as 'abortion'. z Mean values of five independent trials. Means followed by different letters are significantly different at the 5% level by LSD test. | | | | | | + +Table 2. Specific putative cis-acting elements in the promoter of CsACS2 (negative values indicate the first nucleotide position of the cis-elements with respect to the transcription initiation site located at position +1) + +Response cis-element Sequence + +(5' to 3') + +Position + +is that ethylene regulates expression of the three cucumber flower phenotypes (female, male, and hermaphrodite). According to this hypothesis, the F locus is involved in ethylene synthesis, and the M locus is part of the ethylene + +| (5' to 3') | | | | +|-----------------------------------------|---------------------|----------|------------| +| CTK response | CPBCSPOR [TATTAG] | TATTAG | –1407a | +| Ethylene response | ERE [A(A/T)TTCAAA] | AATTCAAA | –154,–1334 | +| LEAFY response | LEAFYATAG [CCAATGT] | CCAATGT | –1715a | +| a Sequence of the complementary strand. | | | | + +signalling pathway. It was previously shown that the products of both CsACS1G (the F locus gene) and CsACS2 (the M locus gene) possess ACS enzyme activity, and that they might both be involved in ethylene biosynthesis (Li et al., 2009). In the present study, overexpression of CsACS2 in transgenic tobacco resulted in production of increased levels of ethylene gas, thus confirming that CsACS2 encodes an ethylene synthesis-related enzyme. + +Therefore, the regulation pattern and the response relationship between CsACS2 and ethylene needs to be clarified. + +The present results demonstrated that the native CsACS2 promoter from cucumber could be induced by ethephon. This effect was detected in both transgenic tobacco and cucumber. These results suggest that the ethylene-responsive elements (possibly the EREs) in the cucumber CsACS2 promoter had a conserved function in the two plants. + +Unexpectedly, in cucumber plant, it was found that when the ethephon concentration was increased to 1.0 mM, and the treatment extended to 6 h, the expression of declined significantly. This is in agreement with the results of the study of Wang et al. (2010), in which treatment with ethephon at 1.0 mM for 6 h caused DNA damage in cucumber protoplasts. + +The results of experiments in which chemical treatments interfered with endogenous ethylene action also confirmed this conclusion. To summarize, it was demonstrated that CsACS2 could cause increased ethylene production, and that expression of the CsACS2 gene can be induced by ethylene. Thus, a positive feedback mechanism could exist in the regulation of the CsACS2 gene. In a previous study, CsACS2 expression analysis indicated significant differences (>9-fold) between NILs for the M locus (Li et al., 2009). + +The experiments were repeated with more accurate shoot apex materials, and it was confirmed that the transcription level was much higher in the gynoecious line, G34 (FFMM), than in its hermaphrodite NIL partner, H34 (FFmm) +(Fig. 4). Considering the very close genetic relationship, the difference in expression probably derives from the CsACS2 (M locus gene) itself. In this study, the allelic gene promoter fragments isolated from G34 and H34 did not show any sequence polymorphism. In previous work, it was also demonstrated that the recessive m allele, which is mutated at a single nucleotide, encoded an inactive product (Li et al., +2009). Thus, the putative positive feedback mechanism suggested here could explain this difference between the NILs. It is proposed that the ethylene derived from the wild-type M locus (CsACS2) induced self-expression, which was absent in the mm genotype because of the nonfunctional mutant. + +The involvement of ethylene in the development of the gynoecium has been reported in hermaphrodite plants such as Arabidopsis and tobacco (De Martinis and Mariani, 1999; Tsuchisaka and Theologis, 2004). Moreover, ethylene production can directly disrupt stamen development in Arabidopsis (Duan et al., 2008). However, in the present study, relatively high concentrations of ethylene did not have an obvious arresting effect on tobacco stamen development (Supplementary Fig. S1 at JXB online), and ethephon treatments also did not cause any significant changes in filament length in PM transgenic plants (unpublished data). This leads to the proposal that stamen development in tobacco is less sensitive to ethylene than in cucumber and Arabidopsis. + +The action of ethylene in arresting stamen development in cucumber was confirmed in the present study. The classical chemical application tests were repeated, and it was found that AgNO3 could induce stamen development, which led to an increase in the number of male flowers in all four cucumber genotypes. It was also shown that ethylene could induce pistil development and stamen arrest in three genetic backgrounds. + +As observed by Yamasaki et al. (2001), treatment with a low concentration of ethephon (0.5 mM in the present study) could induce the production of female flowers in the monoecious line (M06), but was not able to convert hermaphrodite into female flowers in the andromonoecious line ('Lemon'). Unexpectedly, it was found that relatively high concentrations of ethephon could induce the change from bisexual flowers to female flowers in the hermaphrodite (H34 at 0.5 mM and 1.0 mM) and andromonoecious +('Lemon' at 1.0 mM) lines. As the M locus encodes an ethylene synthase and caused ethylene production in planta, it is proposed that there could be a local ethylene threshold to control the action of ethylene in arresting stamen development in cucumber. Under natural conditions, the ethylene derived from the continuous expression of CsACS2, which is induced by CsACS1G and maintained by positive feedback regulation, stays above the threshold and arrests the stamen development from stage 5 to stage 12 shown by Bai et al. (2004) and Satio et al. (2007), leading to female flowers in cucumber. In the mm genotype (hermaphrodite and andromonoecious lines), the ethylene threshold is not maintained, and stamen development does not arrest. In this case, the appropriate concentration of exogenous ethylene could compensate for this threshold and arrest stamen development, which was revealed when the lines H34 and 'Lemon' were treated with 1.0 mM ethephon. + +Moreover, in the H34 line, 0.5 mM ethephon induced female flowers, but this concentration was ineffective in +'Lemon'. This finding was consistent with higher endogenous levels of ethylene in the H34 line than in 'Lemon' +(Supplementary Fig. S2 at JXB online). + +Saito et al. (2007) performed a comprehensive study on the expression pattern of CsACS2. Their results showed that the CsACS2 mRNA initially accumulated just beneath the pistil primordia of flower buds at the bisexual stage, and this coincided with the sex determination stage (stage 6) in cucumbers (Bai et al., 2004). It was noticed in the study that the continuous accumulation of CsACS2 mRNA was correlated with the final establishment of female flowers. These results imply the relationship between the expression of CsACS2 and the continuous arrest of stamen development. As has been proposed from this study, the positive feedback mechanism which leads to a constant level of transcription of CsACS2 could supply continuous ethylene, and could therefore constantly arrest stamen development. + +Combining all the previous data for F and M, the following modified model for sex determination in cucumber is proposed (Fig. 7). + +(i) Both F (CsACS1G) and M (CsACS2) encode ACC +synthases, and their cooperation produces female, male, or bisexual flowers. + +(ii) In the primordial buds that will develop into female flowers, F may be the first specific gene activated, and the eventual product, ethylene, promotes pistil primordia development. At the same time, ethylene activates M, which then begins its positive feedback expression. The continuous accumulation of ethylene beneath the region of the pistil primordia by translation of the M gene mRNA constantly arrests stamen primordia development, resulting in female flowers. + +![8_image_0.png](8_image_0.png) synthases. F is the first specific gene activated and its eventual product, ethylene, promotes pistil primordia development. Concomitantly, ethylene activates M, and CsACS2 then begins its positive feedback activating expression. The continuous accumulation of ethylene constantly arrests the development of stamen primordia, resulting in the production of female flowers. + +(iii) In the pre-determined stamen primordial buds, F will not be activated, and there will not be enough ethylene to start pistil primordia development and promote the expression of M. As a result, the pistil primordia do not develop and the arrest of stamen development does not happen. + +Thus, male flowers develop. + +(iv) In the F-mm genotype, F will be activated normally, and pistil primordia develop without resistance. However, there will not be enough ethylene accumulation to eliminate the development of stamen primordia because of the inactive ACC synthase mutant in the mm genotype. Thus, bisexual flowers develop. + +The detailed mechanism of how ethylene arrests stamen development is still unknown in cucumber, and the ethylene threshold proposed here is speculative. The hypothesis stated above is probably not the final one, and it can only explain the current research results in cucumber. Identifying more mutations in the sex expression pathway and cloning the a gene in cucumber may help to reveal more complete information in this area. Supplementary data are available at JXB online. + +Figure S1. Flower phenotypes in transgenic tobacco plants. + +Figure S2. Ethylene production from shoot apices of the five cucumber cultivars. + +Table S1. Primers used in this study The authors wish to thank Professor Lingxia Zhao (Shanghai Jiaotong University, Shanghai) for the tobacco Nicotiana tabacum pv. Bairihong; Professor Qiguang Wen +(Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai) for the chemical reagent AVG; +and Dr Shoujun Wu (Northwest A&F University, Yangling) for his critical reading of this work. This work was supported by the National Natural Science Foundation of China (no. 31101540), Research Fund for the Doctoral Program of Higher Education of China (no. + +20110204120004), and Starting Research Fund from the Northwest A&F University (no. 2010BSJJ060). + +Atsmon D. 1968. The interaction of genetic, environmental, and hormonal factors in stem elongation and floral development of cucumber plants. Annals of Botany 32, 877–882. + +Atsmon D, Galun E. 1960. 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Genetics 182, 1381-1385. + +Li Z, Pan JS, Guan Y, Tao QY, He HL, Si LT, Cai R. 2008. + +Development and fine mapping of three co-dominant SCAR markers linked to the M/m gene in the cucumber plant ( Cucumis sativus L. ). + +Theoretical and Applied Genetics 117, 1253-1260. + +Mibus H, Tatlioglu T. 2004. Molecular characterization and isolation of the F/f gene of femaleness in cucumber ( Cucumis sativus L.). Theoretical and Applied Genetics 109, 1669-1676. Montgomery J, Goldman S, Deikman J, Margossian L, +Fischer RL. 1993. Identification of an ethylene-responsive region in the promoter of a fruit ripening gene. Proceedings of the National Academy of Sciences, USA 90, 5939–5943. + +Perl-Treves R. 1999. Male to female conversion along the cucumber shoot: approaches to studying sex genes and floral development in Cucumis sativus . In: Ainsworth CC, ed. Sex determination in plants . Oxford: BIOS Scientific Publishers, 189-286. Robinson RW, Munger HM, Whitaker TW, Bohn GM. 1976. Genes of Cucubitaceae. HortScience 11, 554-568. Saito S, Fujii N, Miyazawa Y, Yamasaki S, Matsuura S, Mizusawa H, Fujita Y, Takahashi H. 2007. Correlation between development of female flower buds and expression of the CS-ACS2 gene in cucumber plants. Journal of Experimental Botany 58, 297-2907. Shifriss O. 1961. Sex control in cucumber. Journal of Heredity 52, 5-12. + +Takahashi H, Jaffe MJ. 1984. Further studies of auxin and ACC induced feminization in the cucumber plant using ethylene inhibitors. + +Phyton 44, 81–86. + +Takahashi H, Saito T, Suge H. 1983. Separation of effects of photoperiod and hormones on sex expression in cucumber. Plant and Cell Physiology 24, 142 - 154. + +Tanurdzic M, Banks JA. 2004. Sex-determining mechanisms in land plants. The Plant Cell 16, S61–S71. + +Tapia G, Verdugo I, Yanez M, Ahumada I, Theoduloz C, +Cordero C, Poblete F, Gonzalez E, Ruiz-Lara S. 2005. + +Involvement of ethylene in stress-induced expression of the TLC1.1 retrotransposon from Lycopersicon chilense Dun. Plant Physiology 138, 2075–2086. Trebitsh T, Staub JE, O'Neill SD. 1997. Identification of a 1-aminocyclopropane-1-carboxylic acid synthase gene linked to the Female (F) locus that enhances female sex expression in cucumber. + +Plant Physiology 113, 987–995. + +Tsao TH. 1988. Sex expression in flowering. Acta Phytophysiological Sinica 14, 203–207. + +Tsuchisaka A, Theologis A. 2004. Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1carboxylate synthase gene family members. Plant Physiology 136, 2982-3000. + +Wang DH, Li F, Duan QH, Han T, Xu ZH, Bai SN. 2010. Ethylene perception is involved in female cucumber flower development. The Plant Journal 61, 862–872. + +Yamasaki S, Fujii N, Takahashi H. 2005. Hormonal regulation of sex expression in plants. Vitamins and Hormones 72, 79–110. Yamasaki S, Fujii N, Matsuura S, Mizusawa H, Takahashi H. 2001. The M locus and ethylene-controlled sex determination in andromonoecious cucumber plants. Plant and Cell Physiology 42, 608-619. + +Yin T, Quinn JA. 1995. Tests of a mechanistic model of one hormone regulating both sexes in Cucumis sativus (Cucurbitaceae). American Journal of Botany 82, 1537-1546. \ No newline at end of file diff --git a/medical/md/PMC3510905.md b/medical/md/PMC3510905.md new file mode 100644 index 0000000000000000000000000000000000000000..c41214f146d541a2cb137bfe3b05a6cf89477636 --- /dev/null +++ b/medical/md/PMC3510905.md @@ -0,0 +1,219 @@ +# Nardostachys Jatamansi **Extract Prevents Chronic** Restraint Stress-Induced Learning And Memory Deficits In A Radial Arm Maze Task + +Gloria Karkada, K. B. Shenoy, Harsha Halahalli1, K. S. Karanth2 Departments of Applied Zoology, Mangalore University, 1Physiology, 2Pharmacology, K. S. Hegde Medical Academy, Mangalore, Karnataka, India Address for correspondence: Dr. K. S. Karanth, Emeritus Professor, Department of Pharmacology, K. S. Hegde Academy of Medical Sciences, Nitte University, Nithyanandanagar, Mangalore 575 018, Karnataka, India. E-mail: sudhakarkaranth01@gmail.com + +## Abstract + +Nardostachys jatamansi is traditionally used in alternative medicine for treatment of neuropsychiatric disorders. We investigated the potential of *N. jatamansi* extract (NJE) in protecting against chronic stress-induced impairments in spatial learning and memory. The rats were exposed to 21 days of chronic restraint stress and simultaneously received 100 mg or 200 mg/kg body weight of NJE following which acquisition and retention of hippocampus-dependent spatial memory were tested in a partially-baited eight arm radial maze. Animals treated with 200 mg/kg body weight NJE had learning curves comparable to control unstressed animals, made significantly more correct choices (38%, P < 0.001), and fewer reference memory errors (53%, P < 0.01) on the eighth day of training compared to untreated stressed animals as well as stressed animals which received vehicle or a lower dose (100 mg/kg) of NJE. NJE-treated animals also made significantly higher correct choices (31%, P < 0.001) than untreated animals in a retention test 10 days after the training period. We propose that NJE has a protective effect of stress-induced impairments in hippocampus-dependent learning and memory behavior in rats. + +Key words: Chronic stress, hippocampus, learning, memory, *Nardostachys jatamansi* + +## Introduction + +Nardostachys jatamansi (commonly called as *Jatamansi*) +belonging to the family *Valerianaceae* is a medicinal herb found in parts of India, Tibet, and China. It is often used in Ayurvedic and Unani traditions of alternative medicine as a neuroprotective agent in the treatment of epilepsy, hysteria, insomnia, and convulsions.[1] The rhizomal extract of N. + +jatamansi (NJE) has been particularly studied for its effect on the central nervous system. It has been shown to possess anticonvulsant activity,[2] antidepressant activity,[3] and + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +neuroprotective effects in a rat cerebral ischemia model.[4] Although *N. jatamansi* is also recommended as a nootropic agent in Ayurveda, not many studies have investigated this aspect systematically. NJE has been reported to improve performance in elevated plus maze and passive avoidance tasks in young mice and also to ameliorate aging-related deficits in these tasks. Further, it has also been reported to reverse diazepam and scopolamine-induced amnesia. + +[5] The effect of NJE on hippocampus-dependent spatial memory has received scant attention. A reduction in the hippocampal volume and cell numbers has been reported in animal models of aging, depression, and alcoholismconditions that have all been associated with memory loss in humans.[6,7] Chronic stress is associated with hippocampusdependent learning and memory impairments have been reported in animals subjected to 6 h of physical restraint each day for 21 days.[8] Behavioral deficits in animal models of chronic stress have been associated with loss of hippocampal neurons, dendritic atrophy, and increase in dendritic spines and excrescences.[9-11] Such models therefore provide an useful approach to study the pharmacological potential of agents in preventing/reversing stress-related cognitive impairments. Pretreatment with NJE has been reported to reduce markers of stress such as elevation of plasma corticosterone, gastric ulceration, and increase in adrenal and spleen weights in rats subjected to stress.[12,13] This antistress effect prompted us to explore the effects of NJE on stress-induced deficits in learning and memory. We investigated the potential of NJE in protecting against chronic restraint stressinduced impairments in spatial learning and memory in a partially baited eight arm radial arm maze (RAM) task. This model has been successfully used in the past to study changes in hippocampus-dependent cognitive tasks.[14,15] +We hypothesized that NJE reduces the impact of chronic restraint stress on performance in this task. + +## Materials And Methods Subjects + +Adult male Wistar rats aged 2–2.5 months (200–250 g body weight) obtained from the Central Animal House of K. S. Hegde Medical Academy, Mangalore, were used in the study. The Institutional Animal Ethics Committee for the use of animals in research approved the experimental protocols (Approval letter No. KSHEMA/AEC/044/2006, dated 18-09-2006) . All efforts were made to minimize both the suffering and the number of animals used. The rats were reared in a light-controlled (12-h light dark) environment with *ad libitum* access to water and food except during RS and RAM training periods. + +## Experimental Groups + +The animals were divided into five groups each of ten animals. (1) Normal control (NC) did not undergo any stress procedure, (2) Restraint stress (RS) subjected to a 21 day chronic restraint stress protocol (as explained below), (3) RS + JM1: received 100 mg of *N. jatamansi* per kg body weight for 21 days along with chronic stress, (4) RS + JM2: +rats received 200 mg of *N. jatamansi* per kg body weight for 21 days along with chronic stress, and (5) VC: received vehicle (Alcohol + Tween 80 + water—1:2:5) along with chronic stress for 21 days. In addition to these animals used in the behavioral experiments, an additional group of six animals was used for the evaluation of the effectiveness of the chronic restraint stress model by estimating serum corticosterone levels during the stress protocol (see below for details). + +Chronic restraint stress Chronic restraint stress involved placing the rats in a closefitting wire mesh rodent restrainer for 6 h per day for 21 days. This form of stress has been reported to elevate corticosterone levels, increase adrenal and spleen weights, cause gastric ulcers, and to induce deficits in spatial learning and memory.[8,14] + +## Corticosterone Analysis + +Stress is associated with alterations in the corticosterone level. The serum corticosterone level can therefore be used to assess the effectiveness of experimental stress protocols. In order to establish the effectiveness of the chronic restraint stress model employed in this study, four blood samples were obtained from an additional group of six animals on Days 0, 3, 10, and 21 (Day 1 indicates the start of the stress procedure). Collection tubes containing sodium citrate as anticoagulant were used to sample from the retro-orbital venous plexus. Samples were centrifuged at 4° C for 15 min at 3000 rpm. Serum aliquots were aspirated and stored in sealable polypropylene microcentrifuge tubes at −70° C until assayed for serum corticosterone concentrations using enzymeimmunoassay (EIA) kit (Cayman chemicals Inc., Lewis, USA). + +Preparation and administration of the N. jatamansi extract Botanically identified roots and rhizomes of *N. jatamansi* were purchased from a recognized and licensed supplier (Amsar Private Ltd, Indore, India). An ethanolic extract was prepared following standardized methods described earlier. + +[4] They were cut into small pieces, powdered and refluxed with 95% ethanol in a Soxhlet extractor for 6–8 h. The extract was evaporated to dryness under reduced pressure and temperature using a rotary vacuum evaporator, and the dried residue was stored at 4° C. The yield of the dry extract from the crude powder was about 10%. Previous studies investigating neurological actions of NJE have suggested 200 mg/kg body weight as an effective dose in mediating its antidepressant and memory-enhancing actions.[3,5] Accordingly, doses of 200 mg/kg body weight and 100 mg/kg were administered orally for 21 days. + +Assessment of spatial memory using eight arm radial maze The animals were assessed for spatial memory in a partially baited RAM. Learning and memory in the RAM were assessed in a separate set of animals as described earlier.[14] +The eight arm radial maze consisted of eight equally spaced arms (42 × 12 × 12 cm3) radiating from an octagonal central platform. Prior to the training, the animals were kept on a restricted diet and body weight was maintained at 85% of their free feeding weight, with water available *ad libitum*. The animals were provided two sessions of acclimatization on two consecutive days prior to beginning of training. During these sessions, they were allowed us to explore the baited arms of the maze for 10 min. + +During the training period, the rats were given two acquisition trials per day until they achieved learning criteria as specified below. At the beginning of each trial, the maze was thoroughly cleaned with 70% ethanol and four of the arms (2, 3, 5, and 7) were baited with food reinforcement (Kellogg's Planets and Stars™, Kellogg India Ltd., Mumbai, India). The rat was placed in the center of the octagon and was allowed a free choice. An arm choice was recorded when a rat ate a bait or reached the end of an arm. The maze arms were not rebaited, and only the first entry into the baited arm was recorded as a correct choice. An entry into an unbaited arm was considered a reference memory error (RME) and any re-entry was considered as a working memory error (WME). The trial continued until the rat entered all the four baited arms or 5 min had elapsed. At the end of the trial, the rat was returned to the home cage for a period of 1 h before running the second trial. Training was continued until the rats attained the criteria of 80% correct choice (at least four correct entries out of five). Data from the two trials for each day were averaged and entered into further analysis. Ten days after acquisition, rats were evaluated for retention of the task. Rats were given two trials, and the average of the scores percentage correct choice, reference, and WME was taken for analysis. + +## Results + +Effectiveness of the chronic stress procedure Serum corticosterone levels were elevated by chronic restraint stress A repeated easures ANOVA performed on the corticosterone levels measured on Days 0 (baseline), 3, 10, and 21 of chronic stress revealed a significant difference within-subjects [F(3, 12) = 22.547, P < 0.001)] [Figure 1]. + +Pairwise comparisons (adjusted for multiple comparisons by the Bonferroni method) revealed that Days 3 and 21 corticosterone levels were significantly higher than Day 0 (baseline) levels (P = 0.001 and P <0.05, respectively). Mean corticosterone levels on Day 10 was higher than Day 0, but this difference did not reach statistical significance in the post hoc pairwise tests. By Day 21 corticosterone levels were significantly lower than Day 3 levels (P = 0.001). + +## Acquisition % Correct Choices During Learning Phase + +A two -way repeated measures ANOVA (RM-ANOVA) was performed with training day (Day) as within-subjects factor and experimental group (Group) as between-subjects factor. A significant main effect was found for Day [P < 0.001, F (7, 315) = 24.37], group [P 0< 0.001, F = (4, 45) = 6.42] as well as the Day by Group interaction [P +< 0.001, F = (28, 315) = 2.86] [Figure 2]. This suggests that the performance of the animals changed with days of training and that this change in performance differed between the groups. + +Performance on Day 8 was further analyzed within the RM-ANOVA framework to assess the group differences in learning. A significant simple main effect was found for groups at this time point [P < 0.001, F(4, 45) = 8.29]. + +Pairwise comparisons, adjusted for multiple comparisons using the Bonferroni method revealed a significantly higher %CC for NC animals on Day 8 compared to RS animals (P < 0.001). The RS + JM2 group showed significantly higher %CC compared to the RS and the VC groups (P < 0.001). The VC and the RS + JM1 groups did not differ significantly from the RS group [Figure 3]. A trend analysis performed to more precisely characterize the interaction between Day and Group revealed that the linear effect of Days by Group interaction was significant [P < 0.001, F(4, 45) = 6.31] with no such significant effects at higher-order nonlinear trends. A one-way ANOVA test was used to compare the slopes of the linear regression of learning curves. This showed significant between groups difference in the slopes of the learning curves [P < 0.001, F(4, 45) = 108.2]. *Post hoc* Tukey's tests revealed that the slope of the learning curve for the RS group was significantly lower than that of the NC group and that the slope for the RS+JM2 group was significantly higher than both the RS and VC groups but did not differ from the slope of the NC group [Table 1]. + +## Reference Memory Errors During The Learning Phase + +![2_Image_0.Png](2_Image_0.Png) + +| maze acquisition | NC | RS | RS+J1 | RS+J2 | VC | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------|-------|---------|---------|------| +| Mean | 4.21 | 1.70 | 1.34 | 3.85 | 0.85 | +| SD | 0.40 | 0.39 | 0.46 | 0.58 | 0.47 | +| MD | Q | | | | | +| NC vs. RS | 2.506 | 17.01 | <0.001 | | | +| NC vs. RS + J1 | 2.868 | 19.47 | <0.001 | | | +| NC vs. RS + J2 | 0.36 | 2.444 | >0.05 | | | +| NC vs. VC | 3.363 | 22.83 | <0.001 | | | +| RS vs. RS + J1 | 0.362 | 2.458 | >0.05 | | | +| RS vs. RS + J2 | −2.146 | 14.57 | <0.001 | | | +| RS vs. VC | 0.8566 | 5.815 | <0.01 | | | +| RS + J1 vs. RS + J2 | −2.508 | 17.03 | <0.001 | | | +| RS + J1 vs. VC | 0.4946 | 3.358 | >0.05 | | | +| RS + J2 vs. VC | 3.003 | 20.38 | <0.001 | | | +| NC, Normal control; RS, Restraint stress; VC, Vehicle control; JM1, Jatamansi extract 100 mg; JM2, Jatamansi extract 200 mg (n = 10 for each group), One-way ANOVA: P < 0.001, F(4, 45) = 108.2, MD, Mean difference; q: Post hoc Tukey's test statistic | | | | | | + +![3_image_0.png](3_image_0.png) + +A two-way repeated measures ANOVA (RM-ANOVA) was performed with training day (Day) as within-subjects factor and the experimental group (Group) as between-subjects factor. A significant main effect was found for Day [P < 0.001, F (7, 315) = 10.23], Group [P < 0.001, F = (4, 45) = 4.33] as well as the Day by Group interaction [P < 0.001, F = (28, 315) = 3.10] [Figure 4]. This suggests that the RME rates changed with days of training and that these changes differed between the groups. + +Reference memory errors on Day 8 were further analyzed within the RM-ANOVA framework to assess the group differences in learning. A significant simple main effect was found for groups at this time point [P < 0.001, F(4, 45) = 9.61]. Pairwise comparisons, adjusted for multiple comparisons using the Bonferroni method revealed a significantly fewer RME for NC animals on Day 8 compared to RS animals (P < 0.01). The RS+JM2 group made significantly less RME compared to the RS and VC +groups (P < 0.01). The VC and the RS+JM1 groups did not differ significantly from the RS group [Figure 5]. Trend analysis performed to characterize the interaction between Day and Group revealed that the *linear* effect of Days × Group interaction was significant [P < 0.001, F(4, 45) = 6.79] with no such significant effects at higher-order nonlinear trends. The RS and VC groups had slopes which did not deviate significantly from zero, i.e., showed no change in RME rates over time. However, the NC and the JM treated, stressed groups had negative slopes which were significantly non-zero suggesting that the RME decreased linearly with number of training days. A one-way ANOVA test was used to compare the slopes of the linear regression of RME over days curves. This showed significant between groups difference in the slopes of the RME curves [P < 0.001, F(4, 45) = 111.90]. *Post hoc* Tukey's tests revealed that the slope of the RME curve for the NC group was significantly lower than that of the RS group and that the slope for the RS+JM2 group was significantly lower than both the RS and VC [Table 2]. + +## Working Memory Errors During The Learning Phase + +![4_Image_2.Png](4_Image_2.Png) + +A two-way repeated measures ANOVA (RM-ANOVA) was performed with the training day (Day) as within-subjects factor and the experimental group (Group) as betweensubjects factor. A significant main effect was found for Day +[P < 0.001, F(7, 315) = 18.72], Group [P < 0.001, F = (4, 45) += 1.86] as well as the Day by Group interaction [P < 0.001, F = (28, 315) = 2.71] [Figure 3]. Exploration of the simple main effects of Group within the different time points (Days) suggested significant effects on Day 1 [P < 0.001, F(4, 45) = 6.22], Day 6 [P < 0.049, F(4, 45) = 2.60], and Day 7 [P < 0.045, F(4, 45) = 2.65]. Pairwise comparison of WME between the groups at these time points with Bonferroni adjustment for multiple comparisons was performed. On Day 1, the NC group had significantly higher WME +compared to each of the other groups (P < 0.01). On Day 6, the only significant difference was between NC and VC +(P < 0.05). No other significant between-group differences were found with *post hoc* pairwise comparisons [Figure 6]. + +The differences on Day 1 are possibly due to high variability + +![4_image_0.png](4_image_0.png) + +![4_image_1.png](4_image_1.png) in WME and the lack of significant differences in later days of training suggests that the groups did not differ in the WME during the acquisition of the partially baited RAM task. + +![4_image_3.png](4_image_3.png) + +| radial arm maze acquisition NC | RS | RS+JM1 | RS+JM2 | VC | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------|----------|----------|-------|------| +| Mean | −0.20 | 0.03 | 0.10 | −0.13 | 0.04 | +| SD | 0.04 | 0.03 | 0.04 | 0.05 | 0.04 | +| MD | q | | | | | +| NC vs. RS | −0.23 | 18.79 | <0.001 | | | +| NC vs. RS+JM1 | −0.30 | 25.02 | <0.001 | | | +| NC vs. RS+JM2 | −0.07 | 5.60 | <0.01 | | | +| NC vs. VC | −0.24 | 20.06 | <0.001 | | | +| RS vs. RS+JM1 | −0.08 | 6.23 | <0.001 | | | +| RS vs. RS+JM2 | 0.16 | 13.19 | <0.001 | | | +| RS vs. VC | −0.02 | 1.28 | >0.05 | | | +| RS+J1 vs. RS+JM2 | 0.24 | 19.42 | <0.001 | | | +| RS+J1 vs. VC | 0.06 | 4.96 | <0.01 | | | +| RS+J2 vs. VC | −0.18 | 14.47 | <0.001 | | | +| NC, Normal control; RS, Restraint stress; VC, Vehicle control; JM1, Jatamanasi extract 100 mg; JM2, Jatamanasi extract 200 mg (n = 10 for each group), One-way ANOVA: P < 0.001, F(4, 45) = 111.90, MD, Mean difference; q, Post hoc Tukey's test statistic | | | | | | + +![5_image_0.png](5_image_0.png) + +## Retention % Correct Choice (%Cc) During Retention Testing + +A one-way ANOVA was used to analyze the % correct choices during the retention testing trial performed on the tenth day following completion of the partially baited RAM task. A significant group difference was found [P < 0.01, F(4, 45) = 5.90)] which was further explored with *post hoc* Tukey's pairwise comparisons. This revealed significantly lower % CC by the RS group compared to the NC group. The RS + JM2 group showed significantly higher %CC +compared to the RS and VC groups (P < 0.001). The VC +and the RS+JM1 groups did not differ significantly from the RS group [Figure 7]. + +## Reference Memory Errors During Retention Testing + +A one-way ANOVA was used to analyze the RME during the retention testing trial performed on the tenth day following completion of the partially baited RAM task. A significant group difference was found [P < 0.01, F(4, 45) = 4.37)] which was further explored with *post hoc* Tukey's pairwise comparisons. This revealed significantly higher RME by the VC and RS+JM1 groups compared to the NC group. The RS+JM2 group showed a trend for lower RME compared to the RS and VC groups, but this was not statistically significant [Figure 8]. + +## Discussion + +The current study assessed the effects of concurrent treatment with NJE on hippocampus-dependent spatial learning and memory in rats subjected to 21 days of chronic RS. The salient findings in the current study are that chronic restraint stress impairs learning of the eight-arm radial maze task and that this impairment is ameliorated by concurrent treatment with NJE. Animals treated with 200 mg/kg body weight NJE had learning curves comparable to control unstressed animals, made significantly more correct choices and fewer reference memory errors on the eighth day of training compared to untreated stressed animals as well as stressed animals which received vehicle or a lower dose (100 mg/kg) of NJE. Compared to untreated animals, NJE-treated animals also made significantly higher correct choices and fewer RME in a retention test 10 days after the training period. To the best of our knowledge, this is the first report of the protective effects of NJE on cognitive functions in chronically stressed animals. The stress model used in our study was effective as evidenced by the changes in plasma corticosterone during the stress period [Figure 1]. The chronic RS procedure resulted in impairments in the acquisition and retention of the partially baited radial maze task has been reported in earlier studies using this model.[15-17] +NJE has been demonstrated to protect against acute restraint and cold stress induced elevation in plasma corticosterone, increase in adrenal and spleen weights and gastric ulceration.[18] These findings were associated with a reversal of the stress-induced elevation of lipid peroxidase and NO levels and decrease in catalase activity in the brain and therefore the anti-stress effect of NJE was attributed to its anti-oxidant properties. This study provides preliminary evidence for the protective effect of NJE in chronic stress. NJE has been reported in an earlier study to improve performance in passive avoidance tasks and to reverse drug-induced impairments in this task.[5] Findings from this study indicate that NJE has a protective effect on hippocampus-dependent spatial memory and learning as well. Chronic restraint stress has been shown to result in impaired learning and retention in spatial memory tasks like the RAM task[15,17] and the water maze task.[19] Such impairment has been associated with increase in circulating corticosteroids,[20] alterations in the hippocampal morphology-like retraction of the apical dendrites in the CA3 region of the hippocampus,[21] changes in the hippocampal dendritic spine number and shape,[22] loss of cells,[10] reduced neurogenesis in the dentate gyrus,[23] reduced BDNF expression,[24] impaired long-term potentiation at hippocampal synapses,[25] and alterations in neurotransmitter levels.[26] The protective effect of NJE on stress-induced deficits in the RAM task may be mediated through modulatory influences on one or more of these multiple factors. In this context, we have recently demonstrated that NJE treatment was associated with an elevation in AChE activity levels in the frontal cortex of both chronically stressed animals as well as unstressed controls.[27] Acute administration of NJE has been reported to enhance GABA levels in the rat brain while subchronic administration has been associated with increase in monoamines like NE, DA, 5-HT, 5-HIAA as well.[28] The modulatory effects of NJE on neurotransmitter systems may therefore be important in mediating its protective effects on hippopcampal function. Further studies are required to investigate these possibilities. A few other studies have investigated the possible mechanisms of the action of NJE on the central nervous system in general. Studies in a mouse model of depression have suggested altered monoamine oxidase +(MAO-A and MAO-B) activities[3] and changes in the levels of neurotransmitters such as norepinephrine, dopamine, serotonin, 5-hydroxyindoleacetic acid, and γ-aminobutyric acid.[28] Enhanced anti-oxidant defenses, restoration of dopamine and its metabolites, enhanced striatal dopaminergic D2 receptor binding, and tyrosine hydroxylase expression have been correlated with the antiparkinsonism activity of NJE.[29] It has also been proposed that GABAergic neuronal stimulation by NJE may antagonize the effect of glutamate and protect the neurons from cerebral ischemia. Some of these mechanisms may also play a role in mediating the effects of NJE on stress-induced cognitive impairment demonstrated in this study. To summarize, we demonstrate the ameliorative effects of NJE administration on RAM learning and memory in a chronic restraint stress model. This protective effect was seen at an oral dose of 200 mg/kg, but not at 100 mg/kg. We propose that these effects may be mediated through modulatory influences of NJE on hippocampal function. + +## References + +1. Uniyal MR, Issar RK. Commercially and traditionally important medicinal plants of Mandakini Valley of UttaraKhand, Himalayas. J Res Indian Med 1969;4:83. + +2. Rao VS, Rao A, Karanth KS. Anticonvulsant and neurotoxicity profile of *Nardostachys jatamansi* in rats. J Ethnopharmacol 2005;102:351-6. + +3. Dhingra D, Goyal PK. Inhibition of MAO and GABA: Probable mechanisms for antidepressant-like activity of *Nardostachys jatamansi* DC in mice. Indian J Exp Biol 2008;46:212-8. + +4. Salim S, Ahmad M, Khan SZ, Ahmad SA, Islam F. Protective effect of Nardostachys jatamansi in rat cerebral ischemia. Pharmacol Biochem Behav 2003;74:481-6. + +5. Joshi H, Parle M. *Nardostachys jatamansi* improves learning and memory in mice. J Med Food 2006;9:113-8. + +6. Heine VM, Maslam S, Joel M, Lucassen PJ. Prominent decline of newborn cell proliferation, differentiation, and apoptosis in the aging dentate gyrus, in absence of an age-related hypothalamuspituitary-adrenal axis activation. Neurobiol Aging 2004;25:361-75. + +7. Herrera DG, Yague AG, Johnsen–Soriano S. Selective impairment of hippocampal neurogenesis by chronic alcoholism: Protective effects of an antioxidant. Proc Natl Acad Sci U S A 2003;100:7919-24. + +8. Sunanda, Rao BS, Raju TR. Chronic restraint stress impairs acquisition and retention of spatial memory task in rats. Curr Sci 2000;17:1581-4. + +9. Sunanda, Rao MS, Raju TR. Effect of chronic restraint stress on dendritic spines and excrescences of hippocampal CA3 pyramidal neurons--a quantitative study. Brain Res 1995;694:312-7. + +10. Uno H, Tarara R, Else JG, Suleman MA, Sapolsky RM. Hippocampal damage associated with prolonged and fatal stress in primates. J Neurosci 1989;9:1705-11. + +11. Watanabe Y, Gould E, McEwen BS. Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res 1992;588:341-5. + +12. Glavin GB, Murison R, Overmier JB, Pare WP, Bakke HK, Henke PG, +et al. The neurobiology of stress ulcers. Brain Res Rev 1991;16:301-43. + +13. Welch BL, Welch A. Sustained effects of brief daily stress (fighting) +upon brain and adrenal catecholamines and adrenal, spleen, and heart weights of mice. Physiology 1969;64:100-7. + +14. Srikumar BN, Raju TR, Rao BS. The involvement of cholinergic and noradrenergic systems in behavioral recovery following oxotremorine treatment to chronically stressed rats. Neuroscience 2006;143:679-88. + +15. Titus AD, Rao BS, Harsha HN, Ramkumar K, Srikumar BN, Singh SB, +et al. Hypobaric hypoxia-induced dendritic atrophy of hippocampal neurons is associated with cognitive impairment in adult rats. Neuroscience 2007;145:265-78. + +16. Bindu B, Rekha J, Kutty BM. Postinsult enriched housing improves the 8-arm radial maze performance but not the Morris water maze task in ventral subicular lesioned rats. Brain Res 2005;1063:121-31. + +17. Srikumar BN, Raju TR, Shankaranarayana Rao BS. Contrasting effects of bromocriptine on learning of a partially baited radial arm maze task in the presence and absence of restraint stress. Psychopharmacology 2007;193:363-74. + +18. Lyle N, Bhattacharyya D, Sur TK, Munshi S, Paul S, Chatterjee S, et al. Stress modulating antioxidant effect of *Nardostachys jatamansi*. Ind J Biochem Biophy 2009;46:93-8. + +19. Wright RL, Conrad CD. Enriched environment prevents chronic stress-induced spatial learning and memory deficits. Behav Brain Res 2008;187:41-7. + +20. Coburn-Litvak PS, Pothakos K, Tata DA, McCloskey DP, Anderson BJ. Chronic administration of corticosterone impairs spatial reference memory before spatial working memory in rats. Neurobiol Learn Mem 2003;80:11-23. + +21. Conrad CD, LeDoux JE, Magarinos AM, McEwen BS. Repeated restraint stress facilitates fear conditioning independently of causing hippocampal CA3 dendritic atrophy. Behav Neurosci 1999;113: 902-13. + +22. Donohue HS, Gabbott PL, Davies HA, Rodriguez JJ, Cordero MI, Sandi C, *et al*. Chronic restraint stress induces changes in synapse morphology in stratum lacunosum-moleculare CA1 rat hippocampus: A stereological and three-dimensional ultrastructural study. Neuroscience 2006;140:597-606. + +23. Pham K, Nacher J, Hof PR, McEwen BS. Repeated restraint stress suppresses neurogenesis and induces biphasic PSA-NCAM expression in the adult rat dentate gyrus. Eur J Neurosci 2003;17: 879-86. + +24. Murakami S, Imbe H, Morikawa Y, Kubo C, Senba E. Chronic stress, as well as acute stress, reduces BDNF mRNA expression in the rat hippocampus but less robustly. Neurosci Res 2005;53:129-39. + +25. Jay TM, Rocher C, Hotte M, Naudon L, Gurden H, Spedding M. + +Plasticity at hippocampal to prefrontal cortex synapses is impaired by loss of dopamine and stress: Importance for psychiatric diseases. Neurotox Res 2004;6:233-44. + +26. Sunanda, Rao BS, Raju TR. Restraint stress-induced alterations in the levels of biogenic amines, amino acids, and AChE activity in the hippocampus. Neurochem Res 2000;25:1547-52. + +27. Karkada G, Shenoy KB, Halahalli H, Karanth KS. Differential effect of *Nardostachys jatamansi* rhizome extract on acetylcholinesterase in different regions of brain in rats under chronic stress. Biomedicine 2011;31:13-21. + +28. Prabhu V, Karanth KS, Rao A. Effects of *Nardostachys jatamansi* on biogenic amines and inhibitory amino acids in the rat brain. Planta Med 1994;60:114-7. + +29. Ahmad M, Yousuf S, Khan MB, Hoda MN, Ahmad AS, Ansari MS, +et al. Attenuation by *Nardostachys jatamansi* of 6-hydroxydopamine-induced parkinsonism in rats: behavioral, neurochemical, and immunohistochemical studies. Pharmacol Biochem Behav 2006;83:150-60. + +How to cite this article: Karkada G, Shenoy KB, Halahalli H, Karanth KS. *Nardostachys jatamansi* extract prevents chronic restraint stressinduced learning and memory deficits in a radial arm maze task. J Nat Sc Biol Med 2012;3:125-32.. + +Source of Support: Nil. **Conflict of Interest:** None declared. + +## Dispatch And Return Notification By E-Mail + +The journal now sends email notification to its members on dispatch of a print issue. The notification is sent to those members who have provided their email address to the association/journal office. The email alerts you about an outdated address and return of issue due to incomplete/incorrect address. If you wish to receive such email notification, please send your email along with the membership number and full mailing address to the editorial office by email. \ No newline at end of file diff --git a/medical/md/PMC3546278.md b/medical/md/PMC3546278.md new file mode 100644 index 0000000000000000000000000000000000000000..982e6b8feebc2bfb0ff4cf5e11ed48b9b5f0af58 --- /dev/null +++ b/medical/md/PMC3546278.md @@ -0,0 +1,27 @@ +Letter to the **Editor** +Open **Access** + +# Reply To Commentary On "A Case Of Erdheim-Chester Disease **With** Asymptomatic Renal **Involvement"** + +Hyun Jung **Lee,** MD, Tae Min **Kim,** MD, PhD +Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, *Korea* We express our thanks to Dr. Cavoli for interest in our case [1] and shared experience of your case. A 60-year-old female with ErdheimChester disease (ECD)suffered from azotemia with bilateral hydronephrosis within 2 years of initial diagnosis.Author regarded a metforminassociated lactic acidosis combined with azotemia asthe cause of her presentations.Although nearly 10% of cases with metformin-associated lactic acidosis were met for all three criteria (i.e., arterial pH<7.35, blood lactate>5 mmol/L, and detectable plasma metformin concentration) +[2], plasma metformin concentration was not given in this patient. In addition, fever, abdominal pain, and leukocytosis raised a possibility of urinary tract infection accompanied by azotemia. + +Regardless of causality, a wait-and-see policy was possible after the correction of azotemia in this patient. Similarly, our patient did not any specific treatment for ECD until August 2012 (more than 4 years from initial detection of left renal mass) and did not any genito-urinary symptoms [3]. Therefore, a wait-and-see policy might be acceptable for asymptomatic ECD patients. Considering that interferon-α appeared to be effective against bilateral hydronephrosis [4], its response might be important to design a treatment strategy in this patient. Recently, 13 (54%) of 24 ECD patients harbored *BRAF* V600E mutation [5] and the identification of *BRAF* V600E mutation would provide a theoretical rationale for the use of *BRAF* inhibitors in patients with *BRAF* V600E-positive ECD with any symptoms or disease progression. + +## R E F E R E N C E S + +1. Li Cavoli G. Commentary on "A case of Erdheim-Chester disease with asymptomatic renal involvement". Cancer Res Treat. 2012;44:279. + +2. Kajbaf F, Lalau JD. The criteria for metformin-associated lactic acidosis: the quality of reporting in a large pharmacovigilance database. Diabet Med. 2012 Sep 21 [Epub]. + +http://dx.doi.org/10.1111/dme.12017. + +3. Lee HJ, Lee KY, Shin DY, Lee YG, Choi SY, Moon KC, et al. A case of Erdheim-Chester disease with asymptomatic renal involvement. Cancer Res Treat. 2012;44:146-50. + +4. HarocheJ, Amoura Z, Trad SG, Wechsler B, Cluzel P, Grenier PA,etal. Variabilityin theefficacy of interferon-alphain Erdheim-Chester disease by patientand site of involvement: + +results in eight patients. Arthritis Rheum. 2006;54:3330-6. + +5. Haroche J, Charlotte F, Arnaud L, von Deimling A, Helias-Rodzewicz Z, Hervier B, et al. High prevalence of BRAF V600E mutations in Erdheim-Chester disease but not in other non-Langerhans cell histiocytoses. Blood. 2012;120:2700-3. + diff --git a/medical/md/PMC3964284.md b/medical/md/PMC3964284.md new file mode 100644 index 0000000000000000000000000000000000000000..6de34c7fdd560c4a31f587aff14c0f2afd1e8e92 --- /dev/null +++ b/medical/md/PMC3964284.md @@ -0,0 +1,310 @@ +Stem Cell Reports + +![0_image_1.png](0_image_1.png) + +![0_image_2.png](0_image_2.png) + +![0_image_3.png](0_image_3.png) + +Repor t +# Engineered Biomaterials Control Differentiation And Proliferation Of Humanembryonic-Stem-Cell-Derived Cardiomyocytes Via Timed Notch Activation + +Jason C. Tung,1 Sharon L. Paige,2,4 Buddy D. Ratner,1 Charles E. Murry,1,2,3,4,5,* and Cecilia M. Giachelli1,5,* 1Department of Bioengineering, University of Washington, Seattle, WA 98195, USA +2Department of Pathology, University of Washington, Seattle, WA 98195, USA +3Department of Medicine/Cardiology, University of Washington, Seattle, WA 98195, USA +4Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109, USA +5These authors contributed equally to this work +*Correspondence: murry@uw.edu (C.E.M.), ceci@u.washington.edu (C.M.G.) +http://dx.doi.org/10.1016/j.stemcr.2014.01.011 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Summary + +For cell-based treatments of myocardial infarction, a better understanding of key developmental signaling pathways and more robust techniques for producing cardiomyocytes are required. Manipulation of Notch signaling has promise as it plays an important role during cardiovascular development, but previous studies presented conflicting results that Notch activation both positively and negatively regulates cardiogenesis. We developed surface- and microparticle-based Notch-signaling biomaterials that function in a time-specific activation-tunable manner, enabling precise investigation of Notch activation at specific developmental stages. Using our technologies, a biphasic effect of Notch activation on cardiac differentiation was found: early activation in undifferentiated human embryonic stem cells (hESCs) promotes ectodermal differentiation, activation in specified cardiovascular progenitor cells increases cardiac differentiation. + +Signaling also induces cardiomyocyte proliferation, and repeated doses of Notch-signaling microparticles further enhance cardiomyocyte population size. These results highlight the diverse effects of Notch activation during cardiac development and provide approaches for generating large quantities of cardiomyocytes. + +## Introduction + +Specific control of cellular fate by biological surface modification has garnered recent attention for the ability to create biomimetic microenvironments (Lutolf and Hubbell, 2005). Normally, the body contains stem cell niches composed of complex, spatially and temporally controlled mixtures of soluble chemokines, insoluble extracellular matrix molecules, and cells expressing transmembrane receptor ligands that direct cell fate. Much focus has been given to modifying surfaces to mimic these stem cell niche microenvironments in order to control cellular fate (Lutolf and Hubbell, 2005; Keselowsky et al., 2005; Hoffman and Hubbell, 2004). In these studies, molecular immobilization is proposed to have a critical role by increasing protein stability, promoting persistent signaling, and inducing receptor clustering (Irvine et al., +2002). Despite the attention given to mimicking stem cell niches via surface modifications, few studies have utilized cell-cell surface-ligand-receptor interactions for controlling cellular fate. + +One particularly promising cell-surface pathway is the Notch pathway, which has been shown to play an important role in development and normal cell function, regulating such events as cell growth, proliferation, survival, migration, and differentiation (Artavanis-Tsakonas et al., +1999). The Notch pathway is initiated upon binding of a cell-surface-bound Notch ligand with a Notch receptor + +![0_image_0.png](0_image_0.png) + +![0_image_4.png](0_image_4.png) + +on a second cell, triggering two proteolytic cleavages that release the Notch intracellular domain (NICD) from the plasma membrane. Once released, the NICD translocates to the nucleus where it binds to and converts the CSL transcription factor from a transcriptional repressor to an activator, allowing for Notch target-gene transcription (Bray, 2006; Mumm and Kopan, 2000). Activation of the pathway contributes to numerous cell-fate decisions including maintenance of hematopoietic stem cells in an undifferentiated state (Varnum-Finney et al., 2000b), induction of endothelial-to-mesenchymal transformation +(Noseda et al., 2004), expansion of neural precursors (Oishi et al., 2004), and inhibition of differentiation toward an osteoblastic phenotype (Sciaudone et al., +2003). During cardiac morphogenesis, the Notch signaling pathway is crucial as Notch perturbation has been implicated in the pathogenesis of various human cardiovascular diseases (Nemir and Pedrazzini, 2008; Joutel and Tournier-Lasserve, 1998). However, past studies have presented conflicting conclusions, stating that Notch activation can both promote and inhibit cardiac differentiation (Schroeder et al., 2003; Nemir et al., 2006; Noggle et al., 2006; Jang et al., 2008; Lowell et al., 2006; Chen et al., 2008; Fox et al., 2008; Yu et al., 2008). Thus, we hypothesized that Notch signaling plays multiple roles in cardiac development from human embryonic stem cells, with the precise effect on cellular fate being highly + +![1_image_0.png](1_image_0.png) context-dependent. + +Because the Notch pathway is a cell-cell signaling pathway, unique approaches must be taken to successfully activate signaling. Common approaches include in vitro coculture with Notch-ligand-presenting cells (Neves et al., 2006) and transfection with constitutively active forms of the NICD. Unfortunately, these approaches possess several disadvantages. Coculture systems result in unrelated cell-to-cell interactions, and heterogeneity between cell lines and cell-culture conditions may induce varying levels of ligand expression (Sokolova and Epple, 2008). Overexpression of the NICD results in the pathway being permanently activated, when often only transient activation is desired. Gene transfection also results in heterogeneous conditions, whereas transfection efficiency and cytotoxicity may compromise cell viability and normal gene expression. In addition, because of the ability of Notch ligands to bind with multiple Notch receptors, genetic modifications that serve to overactivate single Notch receptors may fail to properly address the complexity of Notch activation. The use of genetically modified Notch receptors can also result in the expression of Notch receptors at nonphysiologic levels. Notch-activating surface modifications avoid these issues through the engineering of homogenous surfaces that allow for precise activation of the Notch signaling pathway. As previously published by our group, compared to soluble treatment and nonspecific immobilization of a Notch ligand, oriented ligand immobilization results in a more potent induction of Notch-mediated epithelial differentiation in vitro and in a neonatal foreskin-rafted organ culture model (Beckstead et al., 2006, 2009). + +Compared to use of a soluble ligand that can act as a Notch agonist or antagonist depending on the context +(Artavanis-Tsakonas et al., 1999; Hicks et al., 2002; Varnum-Finney et al., 2000a; Small et al., 2001), these Notch-signaling surfaces provide an approach for precisely and reliably controlling Notch activation for use in guiding cellular fate. + +In the present study, we have engineered oriented Notch-signaling biomaterials capable of precisely controlling stem cell fate. In order to determine the context-dependent effect of Notch activation on cardiac development, Notch activation was precisely targeted at several developmental stages. The effect of oriented Jagged-1 surfaces on cardiomyocyte differentiation at the pluripotent embryonic stem cell and cardiovascular progenitor cell developmental stage were assessed, and changes in terminally differentiated human embryonic stem cell (hESC)-derived cardiomyocyte proliferation following Notch activation were also determined. + +## Results + +Oriented Jagged-1 Immobilization on Surfaces and Microparticles Activates Notch Signaling in a Controllable Manner An indirect immobilization scheme was used to control Notch ligand surface orientation and increase Notchsignaling activity. 125I radioactivity experiments demonstrate that Jagged-1 can be indirectly immobilized on a tissue culture polystyrene (TCPS) surface (Figure 1A). By increasing precoat concentration, we observed dosedependent increases in Jagged-1 surface concentration, eventually reaching a maximum surface concentration of 120 ng/cm2, an amount that corresponds to levels expected for a theoretical Jagged-1 protein monolayer. Activation of Notch signaling through the CSL-dependent pathway in human embryonic kidney, neonatal (HEKn) cells plated on our oriented Notch ligand surfaces was analyzed via a CBF-1 luciferase assay. Oriented Jagged-1 surfaces incubated in at least 7 nM Jagged-1 precoat solutions resulted in a 3-fold or greater increase in Notch/CBF-1 signaling at 24 hr postplating compared to TCPS control +(Figure 1B). By further increasing the Jagged-1 incubation solution concentration, a greater than 4-fold increase could be observed. + +In order to repeatedly activate Notch signaling without the need to replate cells, an oriented Jagged-1-modified fibrin microparticle signaling platform was developed. + +Using an aqueous in oil stirred emulsion approach, fibrin microparticles with an approximate diameter of 40 mm were synthesized (Figure 1C). As was done with our surfaces, Jagged-1 was bound to fibrin microparticles via an indirect immobilization approach. Utilizing free terminal carboxyl groups on the fibrin microparticles, 1-ethyl3-[3-dimethylaminopropyl]carbodiimide hydrochloride/ +N-hydroxysulfosuccinimide (EDC/NHS) chemistry was used to create amine-reactive sulfo-NHS esters, allowing covalent attachment of antipolyhistidine molecules via their free primary amines and subsequent formation of amide bonds. Subsequent incubation in Jagged-1 solutions resulted in the affinity immobilization of Jagged-1 on antipolyhistidine-modified fibrin microparticles. 125I radiolabeled Jagged-1 served to confirm immobilization to fibrin microparticles (Figure 1D). Additionally, the quantity of Jagged-1 immobilized to fibrin microparticles could be fine-tuned in a dose-dependent manner by varying Jagged-1 precoat solution concentration. The ability to activate Notch signaling through the CSL-dependent pathway was again determined via a HEKn CBF-1 luciferase assay. Cells treated with oriented Jagged-1-modified fibrin microparticles showed a dose-dependent activation of Notch/CBF-1 signaling (Figure 1E), whereas no Notch activation was observed in the unmodified fibrin microparticle + +![2_image_1.png](2_image_1.png) + +Jagged-1-Mediated Notch Activation in Undifferentiated hESCs Increases Ectodermal Gene Expression Because we believed the role of Notch activation in cardiogenesis to be time-specific, we began by investigating the role of Notch activation in the differentiation of pluripotent embryonic stem cells (ESCs). To determine this, undifferentiated hESCs maintained in mouse-embryonicfibroblast-conditioned media were replated in serumcontaining media on either control surfaces or oriented Jagged-1 surfaces. As displayed in our control samples without Notch activation, these conditions initiated spontaneous differentiation and departure of ESCs from the pluripotent state. In hESCs plated on oriented Jagged-1 surfaces, expression of the Notch target gene, HES1, was significantly increased compared to control surfaces, demonstrating Notch-signaling activation (Figure 2A). All Figure 1. Characterization of Oriented + +![2_image_0.png](2_image_0.png) Jagged-1 Biomaterials +(A) 125I radiolabeling shows dose-dependent increases in surface concentration of Jagged-1 indirectly immobilized on a TCPS +surface. A maximum surface concentration corresponding to levels expected for a theoretical Jagged-1 protein monolayer was achieved. (B) CBF-1 luciferase assays measuring Notch/CSL signaling in HEKn cells plated on oriented Jagged-1 surfaces show a dosedependent increase in signal activation. + +(C) A stirred emulsion reaction mixture generates 40 mm diameter fibrin microparticles. + +(D) 125I radiolabeling showing the dosedependent indirect immobilization of Jagged-1 on fibrin microparticles. (E) CBF-1 luciferase assay results measuring Notch/CSL signaling in HEKn cells treated with oriented Jagged-1-modified fibrin microparticles at various ratios, unmodified fibrin microparticles, or oriented Jagged-1positive control surface. Cells treated with oriented Jagged-1-modified fibrin microparticles showed a dose-dependent activation of Notch/CBF-1 signaling. Results are representative of three independent experiments and are mean ± SD, n = 3. Points marked by a given symbol denote statistical significance (p < 0.05 compared to points that are unmarked or marked by a different symbol). + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +Figure 2. The Role of Notch Signaling Activation in the Differentiation of Human Pluripotent Embryonic Stem Cells Undifferentiated human embryonic stem cells were plated on control or oriented Jagged-1 surfaces, and gene expression was analyzed via quantitative PCR (qPCR) up to 8 days after initial plating. Notch gene: HES1; pluripotency gene: OCT4; mesodermal gene: BRACHYURYT; endodermal gene: FOXA2; ectodermal gene: SOX1. (A) hESCs plated on oriented Jagged-1 surfaces display increased expression of the Notch target gene, HES1, peaking 1 day after plating, indicating successful activation of the Notch pathway via oriented Jagged-1 surfaces. (B–E) Activation of Notch signaling does not alter pluripotency, mesoderm, or endoderm gene expression compared to controls but does increase ectodermal gene expression. Results are representative of three independent experiments and are mean ± SD, n = 3; symbols denote p < 0.05 compared to controls. + +experimental groups downregulated expression of the OCT4 pluripotency gene and upregulated markers of mesoderm formation (BRACHYURYT) and endoderm formation +(FOXA2) in a comparable manner (Figures 2B–2D). In contrast, a sustained increase (>2-fold) expression of the ectodermal gene, SOX1, was observed on oriented Jagged-1 surfaces compared to control surfaces (Figure 2E). Thus, Jagged-1-mediated Notch activation encourages ectodermal gene expression in undifferentiated hESCs, demonstrating a relationship between Jagged-1-mediated Notch activation and ectoderm formation at this early developmental time point. + +Oriented Jagged-1 Surfaces Promote Cardiomyocyte Differentiation of KDR+ Progenitor Cells Next, we sought to determine the effect of Notch activation at a later multipotent cardiac progenitor developmental stage. KDR+ cardiovascular progenitor cells are a multipotent cell population with the capacity to differentiate into endothelial, smooth muscle, and cardiac cell types (Yang et al., 2008). For our experiments, we generated populations enriched for KDR+/platelet-derived-growth-factorreceptor-a-positive progenitor cells (>90%). This cell population actively expresses mRNA transcripts of the Notch ligand, JAGGED-1, and the Notch target gene, HES1, compared to an undifferentiated hESC population +(Figure 3A), suggesting that Jagged-1-mediated Notch activation can occur in this cell population. + +To determine whether Jagged-1-mediated Notch activation influences their differentiation, KDR+ progenitors +(>90% purity) were plated on oriented Jagged-1 surfaces. In culture, beating loci were observed on oriented Jagged-1 surfaces 8 days after plating, whereas cells on control surfaces did not exhibit any contractions until 12 days after plating. At 12 days after plating, fluorescence-activated cell sorting (FACS) analysis demonstrated that Jagged-1induced Notch activation in KDR+ progenitor cells resulted in a >20-fold increase in the percent of cells expressing cardiac troponin T (TNNT2) (from -3% to -70%), roughly a 50% decrease in the population of smooth muscle actin+ +cells (from 14% to 7%), and no significant change in CD31+ endothelial cells (Figures 3C–3F). This Notch-mediated increase in cardiac differentiation resulted in an 8-fold increase in cardiac differentiation efficiency (Figure 3G), as every KDR+ progenitor cell plated on an oriented Jagged-1 surface generated roughly two TNNT2+ cardiomyocytes, whereas control surfaces required more than four KDR+ progenitors to generate a single TNNT2+ cardiomyocyte. + +Additionally, cardiomyocytes expressed the cardiac transcription factor NKX2.5 and the cytoskeletal protein a-actinin, with a-actinin staining displaying the striations characteristic of sarcomeric formation (Figure 3B). Perhaps the most striking difference in these differentiated cardiomyocytes is observed in culture, as these differentiated cardiomyocytes form sheets of spontaneously beating cells, with synchronous waves of contraction propagating throughout the entire culture (Movie S1 available online). These results demonstrate that Jagged-1-mediated Notch activation through oriented Jagged-1 surfaces provides a nongenetic approach capable of inducing highly efficient cardiac differentiation in human KDR+ progenitor cells. + +Combined with the results from our undifferentiated hESC studies, these findings demonstrate a biphasic effect of Notch activation during cardiac differentiation and highlight the temporally dependent nature of Notch signaling. + +## Oriented Jagged-1 Surfaces Induce Hesc-Derived + +Cardiomyocyte Proliferation Upon determining context-specific effects of Notch activation on cardiac differentiation, we sought to determine the role of Notch activation in guiding the development of terminally differentiated cardiomyocytes. hESC-derived MYH7+ cardiomyocytes exhibit significant increases in mRNA levels of the Notch ligand, JAGGED-1, and the Notch signaling target gene, HES1, compared to an undifferentiated hESC population (Figure 4A). These results demonstrate JAGGED-1 expression and HES1 transcription in hESC-derived MYH7+ cardiomyocytes, suggesting that, + +![4_image_0.png](4_image_0.png) like the KDR+ progenitor cells, Jagged-1-mediated Notch activation can occur in definitive cardiomyocytes. + +hESC-derived MYH7+ cardiomyocytes were plated on several surfaces to determine whether Notch-signaling activation could promote cardiac proliferation. We found that 55% of cardiomyocytes plated on oriented Jagged-1 surfaces exhibited positive staining for bromodeoxyuridine +(BrdU) incorporation, a significant increase compared to 42% of cardiomyocytes plated on control surfaces (Figures 4B and S1). This is the most robust mitogen we have found to date for hESC-derived cardiomyocytes, significantly surpassing the 47% BrdU incorporation rates observed with insulin growth factor 1 (IGF-1) (McDevitt et al., 2005). + +Additionally, ɣ-secretase inhibitor treatment, which blocks the proteolytic activation of the Notch intracellular domain, inhibited this increase in proliferation, significantly decreasing the proliferative cardiomyocyte population to 38%, and verifying that the proliferative effects of oriented Jagged-1 surfaces are indeed Notch mediated. + +Interestingly, ɣ-secretase inhibitor treatment of cardiomyocytes plated on gelatin control surfaces served to further decrease the proliferative population to 33%, suggesting that basal hESC-derived cardiomyocyte proliferation is partially a result of endogenous levels of Notch signaling. + +Lastly, unoriented Jagged-1 surfaces synthesized by nonspecific protein adsorption displayed proliferative levels +(43%) significantly lower than oriented Jagged-1 surfaces and more analogous to control surfaces, demonstrating that the Jagged-1 immobilization technique influences the capacity of Notch-signaling surfaces to induce proliferation in hESC-derived cardiomyocytes. These results highlight the ability of oriented Jagged-1 surfaces to induce Notch-mediated proliferation of hESC-derived cardiomyocytes, highlighting yet another distinct developmental stage at which Notch signaling displays potent effects. + +## Oriented Jagged-1-Modified Fibrin Microparticles Induce The Proliferation Of Hesc-Derived + +Cardiomyocytes While effective for inducing Notch signaling at a singular time point, oriented Jagged-1 surfaces only result in an initial spike in Notch activation. To achieve greater versatility of Notch activation, we developed a microparticlebased Notch-signaling platform, which through repeated addition of modified microparticles allows for sustained signaling activation or activation at multiple distinct time points. hESC-derived MYH7+ cardiomyocytes were treated with oriented Jagged-1-modified fibrin microparticles to determine if this technology could successfully induce proliferation in differentiated cardiomyocytes. We found that 44% of cardiomyocytes treated with oriented Jagged-1modified fibrin microparticles at a 1:1 cell:particle ratio + +![5_image_0.png](5_image_0.png) + +![5_image_1.png](5_image_1.png) + +incorporated BrdU, a statistically significant increase from the 32% incorporation rate observed in controls exposed to particles containing only the antipolyhistidine modification (Figure 4C). In addition, this increase in cardiomyocyte proliferation matched that of cardiomyocytes plated on oriented Jagged-1 surfaces, indicating that our microparticle-based signaling platform is an equally effective activator of Notch signaling. Importantly, no statistically significant difference was observed in cardiomyocytes treated with antipolyhistidine-modified fibrin microparticles compared to cells plated on a gelatin surface, demonstrating that the increase in cardiomyocyte proliferation is specifically due to the immobilization of Jagged-1 on fibrin microparticles and not due to a nonspecific effect of the particles. Cells treated with unoriented Jagged-1modified fibrin microparticles at a 1:1 cell:particle ratio exhibited only 32% BrdU incorporation, indicating that unoriented Jagged-1-modified fibrin microparticles are unable to induce cardiomyocyte proliferation. A doseresponse curve showed that cardiomyocytes treated with one particle per two cells had significantly increased proliferation compared to controls (38% versus 32%), and still higher proliferation rates were observed with a 1:1 particle:cell ratio (44%). These studies demonstrate that oriented Jagged-1-modified fibrin microparticles are capable of inducing proliferation in hESC-derived cardiomyocytes in a dose-dependent manner. + +As our Notch-signaling microparticles possess the ability to repeatedly activate Notch signaling, we sought to demonstrate the utility of this technology by attempting to further heighten cardiomyocyte population growth via the administration of multiple treatments. hESC-derived cardiomyocytes were subjected to multiple doses of modified microparticles, and cell population growth was measured over a 5-day time span. Compared to oriented Jagged-1 surfaces, which served to increase cell numbers by about 4-fold after 5 days (3-fold increase in controls), +cardiomyocytes receiving two 1:1 cell:particle doses of oriented Jagged-1-modified fibrin microparticles 2 days and 4 days after plating displayed >6-fold increase in cell + +![6_image_0.png](6_image_0.png) number (Figure 4D). These results demonstrate that repeated dosing with Notch-signaling microparticles has potential for the large-scale generation of hESC-derived cardiomyocytes. + +## Discussion + +In the present study, we describe the development of several oriented Notch-signaling biomaterials and their use in generating hESC-derived cardiomyocytes. Our results indicate that Notch activation has a biphasic effect over the course of human cardiac differentiation, favoring ectodermal differentiation at an undifferentiated pluripotent stem cell stage and increasing cardiac differentiation at the KDR+ cardiovascular progenitor stage. Additionally, Notch activation induces proliferation in the differentiated cardiomyocyte cell population. Importantly, by taking a physiologically relevant approach and triggering Notch activation through the presentation of a Notch ligand and not through the use of constitutively active or druginducible Notch receptors, these findings demonstrate the natural potential of Notch activation to influence multiple stages of human cardiac development. These results highlight the temporally dependent potent effects of Notch activation and suggest that Notch-signaling technologies may be of value for generating differentiated cardiomyocytes for clinical applications. + +Recent studies have begun to investigate the role of Notch signaling in guiding cardiogenic fate decisions. Several studies have demonstrated that Notch inhibition through either expression of defective RBP-Jk (Schroeder et al., 2003), Notch1-deficient cells (Nemir et al., 2006), +or treatment with the gamma secretase inhibitors (Noggle et al., 2006; Jang et al., 2008) can both promote or inhibit cardiomyocyte differentiation, whereas Notch activation in cells as observed through reporter gene activation +(Nemir et al., 2006) or activation through DLL1-expressing + +Figure 4. Effects of Oriented Jagged-1 + +![7_image_0.png](7_image_0.png) + +![7_image_1.png](7_image_1.png) Biomaterial-Mediated Notch-Signaling Activation on the Induction of Cardiomyocyte Proliferation After attachment, differentiated MYH7+ +cardiomyocytes were stimulated as described for 48 hr in serum-free media and labeled with BrdU during the last 24 hr of treatment, at which point induction of proliferation was determined via immunostaining. + +(A) qPCR analysis of mRNA gene expression demonstrates transcription of the Notch ligand, JAGGED-1, and the Notch-signaling target gene, HES1, in hESC-derived MYH7+ +cardiomyocytes. + +(B) BrdU proliferation assays demonstrate induction of hESC-derived cardiomyocyte proliferation. MYH7+ cardiomyocytes plated on oriented Jagged-1 surfaces display statistically significant greater levels of proliferation compared to controls. ɣ-secretase inhibitor treatment reverses the proliferative effects of oriented Jagged-1 surfaces, verifying that oriented Jagged-1 surface bioactivity is Notch specific. The mode of Jagged-1 immobilization also plays a considerable role in the effectiveness of Jagged-1 surfaces to induce proliferation, as randomly oriented Jagged-1 surfaces are unable to induce cardiomyocyte proliferation. + +(C) Treatment with oriented Jagged-1modified fibrin microparticles results in a significant increase in cardiomyocyte proliferation as measured by BrdU incorporation. Method of Jagged-1 immobilization influences the microparticle bioactivity as oriented Jagged-1-modified fibrin microparticles successfully induce cardiomyocyte proliferation, whereas cardiomyocytes treated with unoriented Jagged-1-modified fibrin microparticles display proliferation levels similar to negative controls. The degree of cardiomyocyte proliferation is further controlled in a dose-dependent manner by oriented Jagged-1modified fibrin microparticles through adjustment of the particle:cell ratio. (D) Oriented Jagged-1-modified fibrin microparticles can repeatedly induce cardiomyocyte proliferation to further heighten population growth. Oriented Jagged-1 surfaces accelerate cardiomyocyte population growth compared to controls, whereas repeated treatment with oriented Jagged-1-modified fibrin microparticles further increases the rate of cardiomyocyte population growth. Results are representative of three independent experiments and are mean ± SD, n = 7 (A–C) or 3 (D). Conditions marked by a given symbol denote statistical significance (p < 0.05 compared to experimental groups that are unmarked or marked by a different symbol). +OP9 feeder layers (Lowell et al., 2006) leads to reduced cardiac differentiation in favor of neuroectoderm formation. + +At a more specified developmental stage, timed activation of Notch signaling in cardiac mesoderm promotes cardiac development, whereas Notch activation in mouse-derived hemangioblasts respecifies them to a cardiac cell fate (Chen et al., 2008). In addition, the Notch signaling pathway has been shown to be active in proliferating neonatal rat cardiomyocytes, with exogenous Notch activation serving to stimulate cardiomyocyte proliferation and cell-cycle re-entry (Collesi et al., 2008; Campa et al., 2008). Our results help to resolve some of the discrepancies between these studies, showing that not only does Notch signaling play a significant role in the differentiation and proliferation of human cardiomyocytes, but the timing of Notch activation is also critical, as pathway activation can have positive or negative effects on the generation of human-derived cardiomyocytes depending on the developmental stage at which it is triggered. + +In this study, we have shown that oriented Jagged-1 biomaterials, immobilized on either a solid surface or microparticles, are able to precisely activate the Notch signaling pathway for use in controlling cellular fate. In guiding cardiomyocyte development, we have demonstrated multiple temporally distinct effects, where Notch activation at the pluripotent state acts in an anticardiogenic manner by favoring ectodermal differentiation and is procardiogenic at later stages, serving to increase cardiac differentiation in KDR+ progenitors and induce proliferation in differentiated cardiomyocytes. Whereas the exact mechanism by which the Notch signaling pathway exerts these effects on differentiation in our system is unclear, reports have suggested significant interaction between Notch and other signaling pathways, including the transforming growth factor b (TGF-b)/bone morphogenetic protein (BMP) (Guo and Wang, 2009) and Wnt pathways (Hayward et al., +2008). Interestingly, our results appear to coincide with previous work that suggested that the Notch and TGF-b/ +BMP pathways work synergistically, whereas Notch inhibits the Wnt pathway during the process of cardiomyocyte-directed differentiation (Chen et al., 2008). These results demonstrate the importance of timing Notch activation, as pathway activation can have positive or negative effects on cardiomyocyte differentiation depending on the developmental stage at which signaling is activated. + +Our studies utilized the Notch ligand Jagged-1 for inducing Notch activation. Previous studies have highlighted the contrasting effects stemming from activating the Notch signaling pathway via the Delta versus the Jagged ligand family in various organs and tissues, including the developing nervous and immune systems +(Amsen et al., 2004; Brooker et al., 2006). In addition, a family of glycosyltransferases called Fringe proteins are known to modify O-fucose on Notch receptors through the addition of N-acetylglucosamine, a modification that serves to restrict activation of Notch receptors to only Delta ligands, inhibiting Jagged-mediated Notch activation (Haines and Irvine, 2003). In future studies, investigating the effects of Fringe proteins or Delta-mediated Notch activation in our systems would serve to provide even further clarity on the precise role of Notch signaling in guiding cellular fate decisions at these developmental stages. + +Our data suggest that surfaces remain active primarily over the course of 24 hr. Decreased activity after this time period may result from either inadequate amounts of ligand remaining on the surfaces or self-regulation of the Notch pathway, as most Notch-mediated processes require only a transient pulse of activity that in some cases lasts + +![8_image_0.png](8_image_0.png) only a fraction of the cell cycle (Ambros, 1999). We were able to surpass the effects of oriented Jagged-1 surfaces through treatment with multiple doses of Notch-signaling microparticles, demonstrating the promise of using this technology to repeatedly activate or maintain Notch signaling activation. Though useful, this technology may possess several limitations. Previous studies have demonstrated that, during neonatal rat cardiomyocyte maturation, expression levels of Notch-signaling components are actually downregulated over time (Collesi et al., 2008), serving to limit the proliferation of the maturing cardiomyocyte population. As both of our Notch-signaling platforms require expression of endogenous Notch receptors in our cells of interest in order to achieve successful Notch activation, the aging of hESC-derived cardiomyocytes in culture may limit their responsiveness to our Notch-signaling technologies. In addition, as neonatal rat cardiomyocytes mature and decrease Notch pathway activity, forced activation of the pathway results in pronounced activation of DNA damage checkpoint, eventually leading to mitotic arrest and cell apoptosis (Campa et al., 2008). + +These findings again highlight the important role our engineered signaling platforms can play in activating Notch signaling in a physiologically relevant manner. + +In conclusion, we have expanded on previous work by demonstrating the potential application of Notchsignaling surface modifications for use in precisely controlling cell-fate decisions at several developmental stages of human cardiomyocyte development. Our findings suggest several approaches that allow for more efficient hESCderived cardiomyocyte generation. Furthermore, because the Notch signaling pathway plays a key context-dependent role in numerous developmental processes, these Notch-signaling biomaterials have the potential for widespread application in directing cell-fate decisions in a variety of cell systems. + +## Experimental Procedures + +Preparation of Oriented JAG1 Biomaterials Oriented JAG1 surfaces were fabricated using an indirect immobilization scheme, and for antipolyhistidine control surfaces and unoriented JAG1 surfaces, proteins were attached using a direct immobilization scheme (Beckstead et al., 2006, 2009). Fibrin microparticles were fabricated using a stirred-emulsion approach and JAG1 indirectly immobilized using antipolyhistidine and EDC/NHS chemistry. + +Human Embryonic Stem Cell Differentiation Assay Undifferentiated hESCs were grown as described by Xu et al. (2001) +and seeded onto oriented JAG1 surfaces, antipolyhistidine, or Matrigel with differentiation analyzed via quantitative RT-PCR. + +Cardiovascular Progenitor Cell Differentiation Assay + +![9_image_0.png](9_image_0.png) + +KDR+ cardiovascular progenitor cells were generated via an embryoid-body-based differentiation system (Yang et al., 2008) and plated on either Matrigel, antipolyhistidine, or oriented JAG1 surfaces. Differentiation was characterized via FACS and immunofluorescent staining. + +## Human-Embryonic-Stem-Cell-Derived + +Cardiomyocyte Proliferation Assay hESC-derived MYH7+ cardiomyocytes were generated from KDR+ progenitors as described previously using oriented JAG1 surfaces. + +Dissociated cardiomyocytes were replated in chamber slides and treated as described. Proliferation was characterized via AlamarBlue and immunostaining. + +## Statistical Analysis + +Data were analyzed for significance using a one-way ANOVA and a Tukey's post hoc test for pairwise comparison. The software used was SPSS for Windows and significance set at 95% confidence. + +Detailed methods can be found in the Supplemental Information. + +## Supplemental Information + +Supplemental Information includes Supplemental Experimental Procedures, one figure, one table, and one movie and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr.2014. 01.011. + +## Acknowledgments + +This work was supported by P01 HL094374, R01 HL084642, P01 GM081719, U01 HL100405 (to C.E.M.), R01 HL64387 (to C.E.M. + +and B.D.R.), T32 EB001650 (to J.C.T.), and unrestricted research funds (to C.M.G.). Received: August 16, 2013 Revised: January 20, 2014 Accepted: January 23, 2014 Published: February 27, 2014 + +## References + +Ambros, V. (1999). Cell cycle-dependent sequencing of cell fate decisions in Caenorhabditis elegans vulva precursor cells. Development 126, 1947–1956. + +Amsen, D., Blander, J.M., Lee, G.R., Tanigaki, K., Honjo, T., and Flavell, R.A. (2004). 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Cell Stem Cell 2, 461–471. \ No newline at end of file diff --git a/medical/md/PMC4506991.md b/medical/md/PMC4506991.md new file mode 100644 index 0000000000000000000000000000000000000000..36c1a91f21236aec12eb06918a5da827169c5781 --- /dev/null +++ b/medical/md/PMC4506991.md @@ -0,0 +1,391 @@ + + +![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_4.png](0_image_4.png) + +![0_image_6.png](0_image_6.png) + +journal homepage: www.elsevier.com/locate/redox + +![0_image_5.png](0_image_5.png) + +## Abstract 1. Introduction + +| Article history: Received 14 June 2015 Accepted 15 June 2015 Available online 29 June 2015 Keywords: Aryl hydrocarbon receptor (AhR) Bilirubin Hypoxia-inducible factor (HIF) NADPH oxidase Nitric oxide synthase Toll-like receptor 4 (TLR4) Superoxide | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Research Paper + +![0_image_2.png](0_image_2.png) + +Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase Gila Idelman, Darcey L.H. Smith, Stephen D. Zucker n Division of Digestive Diseases, University of Cincinnati, Cincinnati, OH 45267-0595, USA +It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW +264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide (O2 +−) production, respectively. The generation of both nitrate and O2 +− in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated O2 +− production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-β, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-β release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin. + +& 2015 Published by Elsevier B.V. + +iNOS inhibitors prevent cardiovascular collapse and abrogate liver, lung, renal, and gastrointestinal injury [3], supporting that NO +mediates many of the harmful consequences of endotoxemia [4]. + +We [5] and others [6] have demonstrated that the administration of bilirubin, an endogenous product of heme catabolism, to LPStreated rats ameliorates tissue injury, reduces serum nitrate concentrations, and attenuates the expression of iNOS message in the liver. Physiologically relevant concentrations of bilirubin +(r50 mME3 mg/dL) also have been shown to suppress LPS-stimulated iNOS up-regulation and nitrate production by murine macrophages in vitro [5,6]. However, the mechanism(s) by which bilirubin exerts these effects remains poorly delineated. + +LPS binding to TLR4 triggers two distinct intracellular signaling pathways [7]. The adaptor protein myeloid differentiation factor +(MyD88)-dependent pathway culminates in the early activation of MAPK and nuclear factor-κB (NF-κB), leading to increased expression of inflammatory cytokines, such as tumor necrosis factor-α (TNFα) +and interleukin-6 (IL-6) [8]. The MyD88-independent Toll/IL-1 receptor (TIR) domain-containing adaptor (TRIF)-dependent pathway Expression of the inducible isoform of nitric oxide synthase +(iNOS), which generates nitric oxide (NO) from the catalyzed conversion of L-arginine to L-citrulline, is triggered by the binding of LPS to Toll-Like Receptor 4 (TLR4) [1,2]. In rodent models of sepsis involving the intravenous administration of LPS, selective Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; Cyp1A1, cytochrome P450, family 1, subfamily A, polypeptide 1; HE, hydroethidine; HIF-1α, hypoxia inducible factor-1α; iNOS, inducible nitric oxide synthase; IL-6, interleukin-6; Inf-β, interferon-β; IRF3, interferon regulatory factor 3; ISRE, interferon-sensitive response element; MyD88, myeloid differentiation factor-88; Nox, NADPH oxidase; PMA, phorbol myristate acetate; ROS, +reactive oxygen species; SOD, superoxide dismutase; TEMPOL, 4-hydroxy-2,2,6,6tetramethylpiperidine-N-oxyl; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TLR4, toll-like receptor-4; TNFα, tumor necrosis factor-α n Correspondence to: Division of Digestive Diseases, University of Cincinnati, 231 Albert B. Sabin Way, MSB 7262B, Cincinnati, OH, 45267-0595 USA. + +Fax: þ1 513 558 1744. + +E-mail address: zuckersd@ucmail.uc.edu (S.D. Zucker). + +http://dx.doi.org/10.1016/j.redox.2015.06.008 2213-2317/& 2015 Published by Elsevier B.V. + +article info is characterized by the late-phase activation of NF-κB, up-regulation of iNOS and interferon-β (Inf-β), and downstream modulation of numerous interferon-inducible genes [7,9]. In previous studies, bilirubin has not been found to influence the early activation of NF-κB +or MAPK in response to LPS [5,6], suggesting that it does not exert an effect on the MyD88-dependent TLR4 pathway. On the other hand, the ability of bilirubin to modulate TRIF-dependent TLR4 signaling has not previously been investigated. + +It is postulated that NADPH oxidases (Nox), which generate superoxide anion (O2 +−) from molecular oxygen, play an important role in LPS-stimulated iNOS up-regulation. Support for this conclusion is derived from the observation that the increased expression of iNOS in response to LPS is abolished by Nox inhibitors, such as apocynin [6,10,11]. It has further been shown that treatment of macrophages with LPS stimulates the cellular production of reactive oxygen species (ROS) [12,13], a process that also is ef-ficiently abrogated by Nox inhibitors [6,14,15]. Specific evidence that superoxide mediates TLR4 signaling is provided by the demonstrated co-localization of TLR4 with the Nox4 isoform, and by the fact that knockdown of Nox4 prevents both LPS-induced ROS +generation and NF-κB activation [16]. It is notable that bilirubin is able to efficiently scavenge Nox-derived ROS [6,17], likely due to its potent antioxidant properties [18] and its ability to undergo redox cycling within the cell [19]. To assess whether bilirubin suppresses LPS-stimulated up-regulation of iNOS by inhibiting Nox activity, we investigated the effect of bilirubin on TLR4-mediated iNOS expression in RAW 264.7 murine macrophages. Our findings indicate that bilirubin disrupts TRIF-dependent TLR4 signaling and modulates iNOS expression by scavenging NADPH oxidase-derived superoxide, an effect that is mediated by the hypoxia-inducible transcription factor-1α (HIF-1α). + +## 2. Materials And Methods 2.1. Materials + +Bilirubin (bilirubin IXα) was obtained from Porphyrin Products +(Logan, UT) and further purified according to the method of McDonagh and Assisi [20]. Hydroethidine (dihydroethidium) and Accutase were purchased from Life Technologies. Apocynin was obtained from Cayman Chemical. Superoxide dismutase (SOD; +bovine erythrocyte, BioUltra, Z97% SDS-PAGE), catalase (bovine liver), catalase-polyethylene glycol (PEG-catalase), (7)-α-tocopherol (synthetic, Z96% HPLC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the TLR-4 inhibitors cinnamaldehyde, mastoparan, and resveratrol were purchased from Sigma-Aldrich. + +## 2.2. Cell Culture + +RAW 264.7 murine macrophages and MOVAS, an SV-40 immortalized murine aortic smooth muscle cell line, were obtained from ATCC (Rockville, MD). Cells were grown in Dulbecco's Modified Eagle Medium containing 4 mM L-glutamine, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10% FBS at 37 °C in 5% CO2. Bilirubin stock solutions were prepared freshly in 50 mM potassium phosphate (pH 12), as described previously [5]. The addition of an aliquot (r1% v:v) of bilirubin solubilized in this manner had no appreciable effect on the pH of the medium, and neither bilirubin nor the potassium phosphate vehicle altered cell viability. + +## 2.3. Assays For Cellular Nitrate And Cytokine Production + +RAW cells (2 106) were seeded and grown in 12-well plates containing 1 mL media. Nitrate accumulation in the cell supernatant was determined 24 h following the addition of the indicated treatments using a Nitrate/Nitrite Colorimetric Assay kit +(Cayman Chemical, Ann Arbor, MI), as previously described [5]. + +Briefly, nitrite is produced from the catalyzed conversion of nitrate and then quantified colorimetrically using Griess reagent, based on a standard curve. IL-6, Inf-β, and TNFα levels in the cell culture medium were quantified using commercially available mouse ELISA kits (Thermo Scientific, Rockford, IL). Data are normalized to cellular protein, as determined by Bio-Rad protein assay, which trivially impacted results. + +## 2.4. Measurement Of Cellular Superoxide Production + +Cells were grown on 6 well-plates, lifted with Accutase, washed, and suspended in 5 mL of phenol red-free RPMI at a concentration of 2 106 cells/mL. Cell suspensions were loaded with 10 mM hydroethidine (HE) at 37 °C for 5 min and then maintained on ice until 5 min prior to the initiation of the experiments (when the indicated treatments are added), which were performed at 37 °C. Fetal bovine serum (10%) was added to the cell suspension in order to maintain bilirubin solubility. Cells were activated by the addition of 16 mM phorbol myristate acetate (PMA) or 1 mg/mL LPS, +along with various inhibitor compounds, as described. Superoxide production was quantified by the time-dependent increase in mean fluorescence intensity (ex: 488 nm, em: 610 nm long pass filter) of the cell population, as measured by flow cytometry. + +## 2.5. Determination Of Cellular Hydrogen Peroxide Production + +Cellular hydrogen peroxide was quantified using an Amplex Red assay kit (Molecular Probes), according to the manufacturer's instructions. Briefly, RAW 264.7 cells were seeded in 96-well plates (30,000 cells per well) and grown in phenol red-free RPMI. Cells were activated by the addition of PMA or LPS, along with 50 mM Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) +and 0.1 U/mL HRP at a total volume of 100 mL. Hydrogen peroxide production was quantified from the time-dependent changes in absorbance at 560 nm, using a standard curve. + +## 2.6. Quantitative Pcr Analysis + +Total cellular RNA was extracted and RT-PCR was performed using an Mx3000P system (Stratagene, Cedar Creek, TX). Primers for murine iNOS (sense: 5′-acatgcagaatgagtaccgg-3′; antisense: 5′- +tcaacatctcctggtggaac-3′), cytochrome P450, family 1, subfamily A, polypeptide 1 (Cyp1A1; sense: 5′-cgggatccttacagcccaagcagc-3′, 5′- +ggggtacccagagcactcttcaggag-3′) and GAPDH (sense: 5′-tcaacagcaactcccactcttcca-3′; antisense: 5′-accctgttgctgtagccgtattca-3′) +were employed, with the latter used to control for amplification. + +## 2.7. Determination Of Hif-1Α Activation + +HIF-1α DNA binding activity was measured using an ELISAbased HIF-1α Transcription Factor Assay Kit (Cayman Chemical), +according to the manufacturer's instructions. Briefly, 1 107 cells were grown in a 100 mm culture disk and, following the indicated treatments and incubation times, nuclear proteins were isolated using a Cayman Nuclear Extraction Kit. The nuclear extracts were incubated with HIF-1α response elements immobilized on a 96well plate, and the HIF transcription factor complex detected using an HIF-1α-specific primary antibody and HRP-conjugated secondary antibody. + +2.8. Plasmid preparation and luciferase reporter assay A 1192-bp fragment from the 5′-flanking region of the wildtype (WT) murine iNOS gene (kindly provided by R. Lyons, University of New Mexico Health Science Center, Albuquerque, NM) was inserted into a pGL3-basic vector containing a luciferase reporter (Promega). Plasmid HRE-del, possessing a deletion of the 6-bp hypoxia response element (HRE) sequence (226 to 221), +and HRE-TT, containing a 2-bp exchange (TACGTG to TATTTG) within the HRE of the iNOS promoter, also were created. RAW +264.7 cells were transiently transfected with the indicated reporter constructs using Effectene Transfection Reagent (Qiagen, Inc.). Promoter activity was quantified utilizing a dual Luciferase Reporter Assay System (Promega) and employing a co-transfected Renilla reporter vector to control for transfection efficiency. + +## 2.9. Statistical Methods + +Data were analyzed using a statistical program (SSI SigmaStat, San Jose, CA). Mean values were compared by ANOVA with t-test to assess for significance. For data that were not normally distributed, a Kruskal–Wallis analysis of variance on ranks was performed. + +## 3. Results + +3.1. Role of NADPH oxidases and reactive oxygen species in mediating TLR4 signaling To evaluate the contribution of NADPH oxidase-derived superoxide to TLR4 signaling, we employed a fluorescent assay using hydroethidine, a redox-sensitive probe with specificity for O2 +− [21]. RAW 264.7 cells were pre-loaded with HE and superoxide production was quantified by measuring the time-dependent changes in cellular fluorescence intensity using FACS. We first validated the assay using the classical Nox activator phorbol myristate acetate (PMA). As expected, incubation of cells in the presence of PMA induced a sharp increase in HE fluorescence that was markedly attenuated by simultaneous treatment with a maximally effective dose (500 U) of SOD (Fig. 1A), which catalyzes the conversion of superoxide to H2O2 and O2. As shown in Fig. 1B, +treatment of RAW 264.7 cells with LPS also increases cellular superoxide generation, albeit at substantially lower levels than PMA. + +Notably, apocynin, failed to suppress superoxide production by RAW 264.7 cells in response to stimulation with either PMA or LPS +(Fig. 1B), indicating that this compound did not inhibit Nox activity. While surprising, these findings are consistent with previous reports that apocynin does not impede the function of NADPH oxidase in cells lacking significant myeloperoxidase activity, which is required for apocynin activation [22,23]. + +Concordant with our observations in PMA-treated cells, incubation of RAW 264.7 cells with SOD abrogated LPS-stimulated superoxide production (Fig. 1C). Similarly, 50 mM bilirubin was found to efficiently suppress superoxide production in both LPSactivated and in non-stimulated cells (Fig. 1C), in the absence of any alteration in cell viability (data not shown). We speculate that bilirubin appears to be more effective than SOD in this assay + +![2_image_0.png](2_image_0.png) + +![3_image_0.png](3_image_0.png) +system because it also scavenges hydrogen peroxide, which is able to react with HE to a limited extent [21]. Additionally, we show that cinnamaldehyde, which inhibits TLR4 signaling by preventing receptor oligomerization [24], completely suppresses LPS-stimulated O2 +− production (Fig. 1D), supporting that TLR4 mediates the increase in Nox activity induced by LPS. + +Since superoxide undergoes both catalyzed and spontaneous dismutation to form hydrogen peroxide, we further assessed cellular H2O2 by Amplex Red colorimetric assay. In a similar manner to superoxide, both PMA and LPS were observed to induce a timedependent increase in hydrogen peroxide production by RAW +264.7 cells, with PMA producing a more robust response (Fig. 2A). + +However, in contrast with its marked inhibitory effect on superoxide production (Fig. 1A and C), SOD did not significantly alter cellular ROS levels (Fig. 2B), most likely because it generates H2O2 from O2 +−. In support of this proposition, the SOD mimetics [25] +Tiron (disodium 4,5-dihydroxy-1,3-benzenedisulfonate) and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) also did not alter cellular hydrogen peroxide generation in response to PMA (Fig. 2B). Notably, treatment with the cell permeant polyethylene-glycolated form of catalase (PEG-catalase) [26], an enzyme that degrades hydrogen peroxide to form water and oxygen, significantly reduced PMA-stimulated H2O2 formation, while the non-PEG form of the enzyme had minimal effect, suggesting that PMA-stimulated H2O2 is primarily intracellular. As anticipated, bilirubin, which rapidly and spontaneously traverses cellular membranes [27], also reduced cellular H2O2 production in response to PMA (Fig. 2B) and to LPS (C), providing further support that it acts as a potent scavenger of Nox-derived ROS. In contrast, despite a lack of effect on PMA- or LPS-stimulated superoxide production, apocynin caused a marked reduction in cellular hydrogen peroxide (Fig. 2B and C) supporting, as previously proposed by Heumüller et al. [22], that it acts as a H2O2 scavenger. + +3.2. Mechanism of LPS-stimulated nitrate production by murine macrophages When RAW 264.7 macrophages were incubated in the presence of LPS, a marked increase in cellular nitrate production (indicative of iNOS activity) was observed (Fig. 3A). Consistent with previous reports [5,6], this effect was dose-dependently attenuated by simultaneous treatment with bilirubin or apocynin (Fig. 3A). To determine whether the activation of iNOS by LPS is mediated through TLR4, cells were incubated in the presence of the TLR4 inhibitors cinnamaldehyde [24], mastoparan [28], or resveratrol [29]. Each of these agents effectively inhibited cellular nitrate production in response to LPS (Fig. 1B), validating an essential role for TLR4 in mediating LPS-induced iNOS activity [30,31]. Since treatment with LPS previously has been shown to activate NADPH +oxidase (Nox) and generate reactive oxygen species in RAW 264.7 macrophages [13,32], we sought to determine whether LPS-stimulated iNOS activation is ROS-dependent. We found that treatment with PEG-catalase, but not SOD, effectively blocked cellular nitrate production (Fig. 3C), supporting that hydrogen peroxide +(rather than superoxide) is the primary ROS mediator of LPS-induced iNOS activity. Consistent with these findings, the antioxidant α-tocopherol also was found to exhibit an, albeit modest, dose-dependent inhibitory effect on nitrate release (Fig. 3C). The observation that nitrate production is similar between cells stimulated with 16 mM PMA or 1 mg/mL LPS (Fig. 3C) suggests that iNOS expression is regulated by relatively low-levels (1 mM) of ROS (Fig. 2C). + +## 3.3. Effect Of Bilirubin On The Activation Of Hif-1Α By Lps + +We have validated that LPS stimulates iNOS activity in RAW +264.7 cells through a mechanism that involves TLR4-dependent ROS production. As there exists a growing body of evidence that Nox-derived ROS modulate the activity of hypoxia inducible factor-1α [33] and, as activation of HIF-1α induces iNOS expression through binding to a hypoxia response element (HRE) on the iNOS promoter [34,35], we postulated that bilirubin may inhibit LPS- + +![4_image_0.png](4_image_0.png) + +![4_image_1.png](4_image_1.png) +stimulated iNOS up-regulation by scavenging Nox-generated ROS +and thereby prevent activation of HIF-1α. To test this hypothesis, we created a luciferase reporter construct containing a 1.2-kb fragment of the wild-type murine iNOS promoter, as well as two constructs in which the 6-bp HRE was either deleted (HRE-del) or disrupted by a 2-bp (CG to TT) exchange (HRE-TT) (Fig. 4A). + +We first examined the effect of bilirubin on LPS-stimulated HIF- +1α nuclear binding activity in RAW 264.7 cells. As shown in Fig. 4B, treatment with bilirubin abolishes the LPS-induced increase in HIF-1α nuclear binding, supporting that bilirubin prevents activation of HIF-1α. Consistent with the known inhibitory effect of bilirubin on iNOS expression [5,6], we found that treatment of transiently transfected RAW 264.7 cells with bilirubin caused a significant reduction in the LPS-stimulated increase in luciferase activity of the wild-type iNOS promoter construct (Fig. 4B). The responses of both the HRE-TT (Fig. 4C) and HRE-del (D) reporter constructs to LPS also were blunted as compared with that of the wild-type construct, confirming a contribution of HIF1α to TLR4-mediated iNOS up-regulation. Deletion of the HRE also abolished the inhibitory effect of bilirubin on the iNOS promoter response to LPS (Fig. 4D), consistent with the proposition that bilirubin suppresses iNOS up-regulation by preventing HIF-1α activation. + +3.4. Role of the aryl hydrocarbon receptor in modulating iNOS expression HIF-1α binds to the hypoxia response element as a heterodimeric complex with the transcription cofactor aryl hydrocarbon nuclear translocator (ARNT), a constitutively expressed protein that resides within the cell nucleus and mediates several signal transduction pathways. In addition to HIF-1α, ARNT also binds to the aryl hydrocarbon receptor (AhR), which facilitates its association with xenobiotic-responsive elements to mediate biological effects of halogenated aromatic hydrocarbons. A functional interference between the hypoxia (HIF-1α-dependent) and AhR pathways, through competition for ARNT binding, has previously been proposed [36,37]. Since it has been reported that bilirubin serves as an activator ligand for AhR [38,39], we speculated that bilirubin might modulate iNOS expression by activating AhR which, in turn, would compete with HIF-1α for binding to ARNT. + +To assess whether activation of AhR could cause reciprocal inhibition of HIF-1α-mediated iNOS expression, we examined the effect of TCDD, a prototypic AhR activator, on LPS-stimulated nitrate production in RAW 264.7 cells. As shown in Fig. 5A, treatment with TCDD suppressed nitrate production in a dose-dependent manner, supporting that activation of AhR can lead to reciprocal inhibition of LPS-mediated (HIF-1α-dependent) iNOS upregulation. Because of an inability to detect expression of either of the classic AhR inducible genes, Cyp1A1 or Cyp1B1, in RAW 264.7 cells, we utilized murine smooth muscle cells, which previously have been shown to express AhR, Cyp1A1, and iNOS [40,41]. + +Treatment of MOVAS with LPS was found to up-regulate the expression of iNOS mRNA, while co-incubation with TCDD significantly attenuated this response (Fig. 5B), consistent with the proposed reciprocal inhibition of HIF-1α by AhR. In a parallel manner, the TCDD-stimulated expression of Cyp1A1 was inhibited by simultaneous treatment of MOVAS with LPS (Fig. 5C). On the other hand, bilirubin, even at pathophysiological concentrations +(100 mM), exhibited a rather modest ability to induce Cyp1A1 expression in comparison with a half-maximal dose of TCDD +(Fig. 5D). + +## 3.5. Influence Of Bilirubin On Tlr-4 Signaling Pathways + +The binding of LPS to TLR4 activates two principal downstream signaling pathways [7]. The MyD88-dependent pathway leads to activation of NF-κB and the production of pro-inflammatory cytokines such as IL-6 and TNFα, while the TRIF-dependent pathway induces interferon-β through activation of interferon regulatory factor 3 (IRF3). To determine the effect of bilirubin on these two TLR4 pathways, we treated RAW 264.7 cells with LPS and measured the production of IL-6, TNFα, and Inf-β (Fig. 6). Co- + +![5_image_0.png](5_image_0.png) +incubation of cells in the presence of bilirubin resulted in a marked reduction in Inf-β production (Fig. 6B), in the absence of an effect on IL-6 (A) or TNFα (data not shown), suggesting that bilirubin specifically inhibits TRIF-dependent TLR4 signaling. In contrast, apocynin inhibited both IL-6 and Inf-β production (Fig. 6A and B), +supporting a distinct mechanism of action. The induction of IL-6 and Inf-β also was effectively abolished by treatment with cinnamaldehyde (Fig. 6C and D), mastoparan, or resveratrol (data not shown), validating that LPS-induced cytokine production is mediated through TLR4. While treatment of cells with SOD, PEGcatalase, or α-tocopherol did not significantly alter LPS-stimulated cytokine release (Fig. 6E and F), the combination of SOD plus PEGcatalase was found to exert a modest inhibitory effect on Inf-β production. These data suggest a limited role for ROS in modulating the TLR4-dependent induction of cytokines. + +## 4. Conclusions + +It was first speculated over 75 years ago that bilirubin exerts anti-inflammatory effects when it was observed that patients with rheumatoid arthritis experienced a remission of symptoms after developing jaundice from superimposed liver disease [42]. More recently, bilirubin has been shown to prevent tissue injury by inhibiting the up-regulation of iNOS by LPS [5,6]. In the present studies, we attempt to elucidate the molecular mechanism +(s) underlying this effect. Employing RAW 264.7 murine macrophages, we validated that physiological concentrations of bilirubin significantly inhibit iNOS expression and cellular nitrate production in response to LPS treatment. Our finding that TLR4 receptor antagonists effectively abrogate nitrate production (Fig. 3B), are consistent with prior reports that cells [30] and animals [31] de-ficient in TLR4 fail to up-regulate iNOS in response to LPS. We further show that TLR4 inhibitors suppress LPS-mediated superoxide production (Fig. 1D), also in line with previous investigations demonstrating that TLR4 interacts directly with NADPH oxidase +[16] and that TLR4 activation causes an increase in intracellular ROS [43,44]. We found that both PEG-catalase and α-tocopherol, but not SOD, suppress LPS-stimulated nitrate production (Fig. 3C), +suggesting that hydrogen peroxide (rather than superoxide) serves as the primary ROS signaling intermediate involved in the regulation TLR4-mediated iNOS expression. Finally, we demonstrate + +![6_image_0.png](6_image_0.png) + +that bilirubin, a potent chain-breaking antioxidant that undergoes intracellular redox cycling [18,19], is an efficient scavenger of LPSinduced O2 +− and H2O2 (Figs. 1C and 2C). Taken together, these data support that bilirubin suppresses TLR4-mediated up-regulation of iNOS by scavenging Nox-derived reactive oxygen species. + +As LPS activates HIF-1α through an ROS-dependent mechanism +[45] and, since the iNOS promoter contains a hypoxia responsive element [34,35], we speculated that bilirubin inhibits the LPSstimulated induction of iNOS by suppressing ROS production, and thereby preventing HIF-1α activation. This hypothesis is supported by our finding that bilirubin blocks LPS-stimulated nuclear binding of HIF-1α and that the induction of iNOS by LPS is significantly blunted in cells transfected with promoter constructs where the hypoxia response element has been modified or deleted (Fig. 4D +and E). The observation that bilirubin exerts no additional effect on iNOS expression beyond that which occurs when the HRE is deleted from the promoter, further supports that HIF-1α is the predominant mechanism through which bilirubin acts to suppress LPS-mediated iNOS induction. + +Because bilirubin has been postulated to serve as an endogenous ligand for the aryl hydrocarbon receptor [38,39], we speculated that it may also regulate iNOS expression by activating AhR, which could compete with HIF-1α for ARNT binding. While we were able to demonstrate that the simultaneous activation of HIF-1α with LPS and AhR with TCDD resulted in the reciprocal regulation of message for iNOS and Cyp1A1, bilirubin was noted to exert a quite modest effect on Cyp1A1 expression at all but supraphysiologic concentrations (Fig. 5D). These findings are reasonably concordant with those of Sinal and Bend [38], who demonstrated a dose-dependent increase in Cyp1A1 expression in Hepa1c1c7 cells exposed to 100 µM bilirubin, reaching approximately one-third the level achieved with TCDD. Phelan et al. reported a more robust effect of 50 mM bilirubin on Cyp1A1 induction in liver and intestinal cell lines, approaching that of TCDD +[39]; however, the absence of albumin in the culture medium in these studies could have resulted in supraphysiologic free bilirubin concentrations. Given its relatively modest influence on AhR activation, we suspect that bilirubin is unlikely to regulate iNOS +expression through ARNT-dependent reciprocal inhibition of HIF1α, at least under physiological conditions. A proposed schema via which bilirubin may modulate LPS-dependent iNOS up-regulation is depicted in Fig. 7. + +Although apocynin is commonly utilized as a specific inhibitor of NADPH oxidase, it is well-established that this effect is dependent upon activation by cellular myeloperoxidases [46]. In cells lacking myeloperoxidase activity, apocynin not only fails to block the generation of O2 +−, but has variably been shown to scavenge +[22] or even augment [23] the production of H2O2. Consistent with prior studies in HEK293 cells [22] and vascular fibroblasts [23], we found that apocynin had no effect on either PMA or LPS-stimulated superoxide production as measured by hydroethidine fluorescence. These findings suggest that apocynin does not inhibit Nox activity in RAW 264.7 cells, at least over the 45 min incubation period employed. Despite its lack of effect on superoxide production, apocynin effectively blocked cellular hydrogen peroxide generation (as assessed by Amplex Red), supporting the + +![7_image_0.png](7_image_0.png) + +![7_image_1.png](7_image_1.png) + +hypothesis originally put forth by Heumüller et al. that apocynin acts as a scavenger for peroxide-dependent ROS [22] . These data highlight the caution that is required when employing apocynin as a Nox inhibitor. + +One notable observation is that treatment of RAW 264.7 cells with bilirubin results in a marked reduction in LPS-stimulated interferon-β production, in the absence of an effect on cellular IL-6 release. These data suggest that bilirubin exerts a specific inhibitory effect on the TRIF-dependent TLR4 signaling pathway, which also is believed to modulate iNOS up-regulation [47] . + +However, unlike iNOS, it appears unlikely that the influence of bilirubin on Inf- β is mediated through HIF- 1 α , since the murine Inf-β promoter does not possess a hypoxia response element [48] . Seleme et al. have shown that TLR3-mediated induction of Inf-β is abolished in macrophages derived from mice possessing a spontaneous mutation in the Ncf1 gene encoding the p47 pʰox subunit of Nox, while production of IL-6 remained unaltered [49] . These investigators further reported a reduction in TRIF message and + +![8_image_0.png](8_image_0.png) + +protein levels in TLR3-stimulated macrophages derived from these superoxide-deficient animals, supporting that Nox is required for activation of the TRIF-dependent pathway, at least with respect to TLR3. However, our finding that incubation of RAW 264.7 cells in the presence of SOD had no effect on LPS-mediated Inf-β production suggests that this mechanism may not be relevant to TLR4-mediated processes. Nevertheless, our data clearly show that bilirubin disrupts TLR4-dependent Nox activation and interferon-β production, which likely represents the molecular mechanism +(s) underlying its inhibitory effect on iNOS expression in response to LPS. As Inf-β previously has been shown to act in a paracrine manner to augment LPS-stimulated iNOS expression by murine macrophages [50,51], the ability of bilirubin suppress interferon-β production (in conjunction with its influence on HIF-1α activation) +could contribute to its observed inhibitory effect on iNOS upregulation. Bilirubin is produced as part of the normal physiological degradation of heme via the sequential activity of heme oxygenase +(HO) and biliverdin reductase. Although markedly elevated serum bilirubin concentrations (420 mg/dLE340 mM) can cause neurologic injury (kernicterus) in newborns, toxicity in adults is negligible, and bilirubin has been administered intravenously to patients without sequellae [52], reaching serum levels as high as 22 mg/dL (normalr1.2 mg/dLE20 mM). More modest (o3-fold) +elevations in serum bilirubin, at the levels employed in the present studies, are commonly encountered in individuals with Gilbert's syndrome, a benign condition resulting from polymorphisms in the gene encoding UGT1A1, the principal bilirubin conjugating enzyme. Indeed, we have shown that oral [53] or intraperitoneal +[17] administration of bilirubin to rodents produces significant physiological effects at serum levels that are 3- to 4-fold baseline +(r0.4 mg/dL) [5]. Nitric oxide generated by the activity iNOS has been implicated in the pathogenesis of a number of clinical disorders, including inflammatory bowel disease, cardiovascular disease, rheumatoid arthritis, and cancer [54]. In animal models of sepsis and intestinal malignancy, treatment with bilirubin has been shown to ameliorate tissue injury and suppress iNOS expression in the liver, intestine, kidney, heart, and aortic tissues +[5,6,53]. Although human macrophages, even when stimulated, produce little to no nitric oxide [55] as a result of epigenetic modifications that effectively silence the iNOS gene [56], the fact that epidemiological studies support an inverse association between serum bilirubin levels and cardiovascular disease [57,58], +inflammatory bowel disease [59], colorectal cancer [60], and rheumatoid arthritis [61], raise the possibility of a potential therapeutic benefit of bilirubin through its ability to inhibit iNOS upregulation. + +## Acknowledgments + +The authors wish to thank Megan Vogel for her critical reading of the manuscript. This study was supported by National Institutes of Health research Grant DK063954 (SDZ). No financial disclosures are necessary for any of the authors. + +## References + +[4] F.S. Laroux, K.P. Pavlick, I.N. Hines, S. Kawachi, H. Harada, S. Bharwani, J. + +M. Hoffman, M.B. 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Res. 2 (6) (2010) 256–260, http://dx.doi.org/ 10.4021/jocmr444w 22043258. \ No newline at end of file diff --git a/medical/md/PMC4654091.md b/medical/md/PMC4654091.md new file mode 100644 index 0000000000000000000000000000000000000000..d509586b4e11f26812035c5a27d5e953b559da26 --- /dev/null +++ b/medical/md/PMC4654091.md @@ -0,0 +1,117 @@ +Ann Rehabil Med 2015;39(5):833-837 pISSN: 2234-0645 - eISSN: 2234-0653 http://dx.doi.org/10.5535/arm.2015.39.5.833 + +![0_image_0.png](0_image_0.png) + +# Multiple Lower Extremity Mononeuropathies By Segmental Schwannomatosis: A Case Report Na Yeon Kwon, Md, Hyun-Mi Oh, Md, Young Jin Ko, Md, Phd + +Department of Rehabilitation Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea Schwannoma is an encapsulated nerve sheath tumor that is distinct from neurofibromatosis. It is defined as the occurrence of multiple schwannomas without any bilateral vestibular schwannomas. A 46-year-old man with multiple schwannomas involving peripheral nerves of the ipsilateral lower extremity presented with neurologic symptoms. Electrodiagnostic studies revealed multiple mononeuropathies involving the left sciatic, common peroneal, tibial, femoral and superior gluteal nerves. Histologic findings confirmed the diagnosis of schwannoma. We reported this rare case of segmental schwannomatosis that presented with neurologic symptoms including motor weakness, which was confirmed as multiple mononeuropathies by electrodiagnostic studies. + +Keywords Schwannomatosis, Mononeuropathies, Electromyography + +## Introduction + +Schwannoma is mostly a solitary lesion as reported by Park et al. [1]. However, it can occur as multiple lesions in patients with neurofibromatosis or schwannomatosis [2]. These are characterized by the multiple schwannomas in the absence of bilateral vestibular schwannomas [3]. Pain is reported in several cases with schwannomatosis along the peripheral nerves [4,5]. However, motor weakness by multiple mononeuropathies has rarely been reported. We presented this rare case of segmental schwannomatosis with electrodiagnostic confirmation of multiple mononeuropathies involving the left lower extremities. + +## Case Report + +A 46-year-old man presented with a 5-year history of tingling sensation in his left foot and weakness in his left great toe extension. He had visited other clinics for further evaluation, and a follow-up lower extremity magnetic resonance imaging (MRI) revealed multiple nodules along the course of sciatic, tibial and peroneal nerves (Fig. 1). He was referred to the orthopedic department in our hospital. Under the clinical impression of schwannomatosis, excisions were done at multiple distinct locations including superior and inferior parts of the posterior tibia, middle portion of posterior thigh, popliteal fossa, distal leg above medial ankle joint, distal and proximal portion of plantar aspect and around the common peroneal nerve. The lesions were grossly oval-shaped, multilobulated masses of 3 cm in the greatest dimension, and histologic findings confirmed schwannoma. + +After 2 years, the patient was referred to our rehabilitation clinic with worsening of weakness and palpating nodules at the calf and medial portion of posterior thigh. Lower extremity MRI revealed well-defined masses along the neurovascular bundles. A T2-weighted image showed multiple hyperintense tumors along the course of tibial + +![1_image_0.png](1_image_0.png) + +and peroneal nerves with heterogeneous enhancement (Figs. 2, 3). + +Medical Research Council scale for muscular weakness showed the following grades: left lower extremities with hip flexor 5/4 (right/left), hip abductor 5/4, hip extensor 5/4, knee flexor 5/4, knee extensor 5/4, ankle dorsiflexor 5/3, hallucis extensor 5/2, and ankle plantar flexor 5/3. The patient complained of tingling sensation below the left malleolus and left calf hyperalgesia. Deep tendon reflexes at left lower extremity were decreased. + +Electrodiagnostic motor nerve conduction studies showed prolonged distal latencies, reduced compound muscle action potential amplitudes in the left tibial nerve and the left femoral nerve. The tibial nerve conduction velocities were decreased. In sensory nerve conduction studies, sensory nerve action potentials (SNAP) of the left sural nerve were not evoked. The SNAP amplitudes were reduced in the left superficial peroneal and saphenous nerves. H-wave study showed no response in the left tibial nerve. Needle electromyography revealed abnormal spontaneous activities in the left gluteus medius, biceps femoris (long head), peroneus longus, tibialis posterior, tibialis anterior and gastrocnemius muscles with substantially reduced interference patterns. Peroneal innervated muscles including left biceps femoris (short head) and peroneus longus showed large amplitudes and prolonged durations of motor unit action potentials (Tables 1, 2). + +![1_image_1.png](1_image_1.png) + +Fig. 2. (A) Sagittal T2-weighted image shows multiple hyperintense tumors along the course of the tibial nerve. (B) Proximal lesion has a similar appearance with a bright signal. + +![2_image_1.png](2_image_1.png) + +![2_image_0.png](2_image_0.png) + +Fig. 3. (A) Axial T2-weighted image demonstrates the well-circumscribed lesions along the course of the tibial nerve. (B) Distal lesions are well-circumscribed and extend into the soleus muscle. + +| Table 1. Nerve conduction findings | Right | Left | | | | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|---------------|-------------|-------|---------------|-------------|-----|----| +| Nerve | Stimulation | Latency (ms) | Amplitudea) | CV | Latency (ms) | Amplitudea) | CV | | +| (m/s) | (m/s) | | | | | | | | +| Motor Femoral | Inguinal area | 4.75 | 8.8 | - | 4.50 | 0.1 | - | | +| | Peroneal EDB | Ankle | 3.70 | 5.8 | - | NR | NR | - | +| Fibular head | 11.45 | 4.7 | 42.6 | NR | NR | - | | | +| Knee | 13.20 | 4.6 | 51.4 | NR | NR | - | | | +| | Peroneal TA | Fibular head | 2.75 | 4.2 | - | 2.80 | 4.1 | - | +| Knee | 4.50 | 3.3 | 51.4 | 4.70 | 3.2 | 47.4 | | | +| | Tibial | Ankle | 4.70 | 16.8 | - | 7.85 | 0.3 | - | +| Popliteal fossa | 13.95 | 12.2 | 41.1 | 24.10 | 0.1 | 23.4 | | | +| Sensory Sural | Calf | 3.55 | 12.5 | - | NE | NE | - | | +| | Superficial peroneal | Lateral leg | 3.25 | 9.3 | - | 3.45 | 3.2 | - | +| | Saphenous | Medial leg | 2.55 | 6.8 | - | 2.50 | 3.1 | - | +| CV, conduction velocity; EDB, extensor digitorum brevis; TA, tibialis anterior; NE, not evoked; NR, no response. a)Amplitudes are measures in millivolt (mV, motor) and in microvolt (μV, sensory). | | | | | | | | | + +These results indicated that neurologic symptom was associated with multiple mononeuropathies involving left sciatic, common peroneal, tibial, femoral and superior gluteal nerves. The patient refused our recommendation of lumbosacral plexus MRI for possible plexopathy. He received conservative therapies that required continuous follow-up evaluations for any signs of recurrence. + +## Discussion + +Schwannomas are benign nerve sheath tumors that can arise along the course of nerves or spinal roots [2]. About 30% of schwannomatosis affect a single limb or one half of the body, as in our case [4,6]. Schwannomatosis limited to a single limb is defined as segmental schwannomatosis [3]. Patients complain of neurologic changes although these symptoms are uncommon unless the size of tumor becomes large [7]. Chronic pain, either local or + +## Na Yeon Kwon, Et Al. + +| Table 2. Findings of needle electromyography in left lower extremity Muscle IA ASA | MUAP | Interferential pattern | | | +|--------------------------------------------------------------------------------------------------|--------|--------------------------|------------------------|----------| +| Both paraspinalis (L3-S1) | Normal | None | | | +| Gluteus maximus | Normal | None | Normal | Full | +| Gluteus medius | Normal | ++ | Normal | Reduced | +| Adductor magnus | Normal | None | Normal | Reduced | +| Iliopsoas | Normal | None | Normal | Reduced | +| Biceps femoris (short) | Normal | None | Large amplitude (6 mV) | Discrete | +| Biceps femoris (long) | Normal | ++ | Normal | Discrete | +| Tibialis anterior | Normal | + | Normal | Reduced | +| Tibialis posterior | Normal | + | Normal | Reduced | +| Gastrocnemius (medial) | Normal | + | Normal | Reduced | +| Peroneus longus | Normal | ++ | Large amplitude (6 mV) | Discrete | +| IA, insertional activity; ASA, abnormal spontaneous activity; MUAP, motor unit action potential. | | | | | + +diffuse, is the most common symptom in schwannomatosis patients [8]. Moreover, Gonzalvo et al. [9] described pain as the initial symptom in most patients. MacCollin et al. [6] also presented several cases with schwannomatosis along the course of peripheral nerves. + +However, motor weakness by peripheral neuropathies due to schwannomatosis has rarely been reported. Ogose et al. [5] reported a case of incomplete peroneal neuropathy among patients with the extraspinal lesion. Park et al. [1] presented a case of schwannoma involving sciatic nerve, which showed weakness of the gastrocnemius muscle and a posterior tibial neuropathy confirmed by electrodiagnostic studies. + +Schwannomatosis is clinically distinguished from neurofibromatosis based on lack of café-au-lait spots and neurofibromas [7]. On MRI finding, fusiform shape and target sign can be seen in either lesions, but several findings can be helpful to differentiate between the two lesions. Eccentrically positioned lesions with different anatomical locations can be schwannoma, whereas centrally located masses can suggest neurofibroma. After gadolinium injection, a small schwannoma has a tendency to be enhanced homogeneously, while large schwannomas can be heterogeneously enhanced [7]. In the present case, biopsy was done from the left leg and schwannoma was confirmed by histology. + +Clinicians always need to make a decision to select sufficient image studies based on cost-effectiveness, but sometimes they are insufficient. Therefore, electromyography can be a useful tool to make a correct diagnosis, and localize the disorder. Diagnostic considerations for electromyography could include not only lesions but also proximal and distal nerve involvement along the whole extremities Although the recurrence of schwannomatosis is rare, there are several reports on recurrence [6,10] as in this case, and the probability of malignant transformation is still unknown. + +Surgery is a treatment of choice for solitary schwannoma. However, repeated surgeries are not recommended because of the possibility of iatrogenic nerve injuries and worsening pain. Therefore, management should be focused on prevention of disease progression and strict surveillance [6,10]. + +In summary, we reported a rare case of segmental schwannomatosis, presenting with motor weakness, with electrodiagnostic confirmation of multiple mononeuropathies. Finally, clinicians should be aware that schwannomatosis could cause variable symptoms including not only pain, but also motor weakness. Strict surveillance and regular follow-up are essential to prevent possible neurological deficits. + +## Conflict Of Interest + +No potential conflict of interest relevant to this article was reported. + +## References + +1. Park NK, Lee JH, Kim DS, You JH, Cho IH. Posterior tibial neuropathy by schwannoma in midthigh. J Korean EMG Electrodiagn Med 2005;7:139-42. + +2. Goldblum JR, Folpe AL, Weiss SW. Enzinger and Weiss's soft tissue tumors. 6th ed. Philadelphia: Elsevier Saunders; 2014. + +3. Ferner RE. Neurofibromatosis 1 and neurofibromatosis 2: a twenty first century perspective. Lancet Neurol 2007;6:340-51. + +4. Ellis SJ, Gordin S, Edgar MA, Morris CD. Multiple ipsilateral lower extremity masses in a 46-yearold man. Schwannomatosis. Clin Orthop Relat Res 2007;456:268-74. + +5. Ogose A, Hotta T, Morita T, Otsuka H, Hirata Y. Multiple schwannomas in the peripheral nerves. J Bone Joint Surg Br 1998;80:657-61. + +6. MacCollin M, Woodfin W, Kronn D, Short MP. Schwannomatosis: a clinical and pathologic study. Neurology 1996;46:1072-9. + +7. Pilavaki M, Chourmouzi D, Kiziridou A, Skordalaki A, +Zarampoukas T, Drevelengas A. Imaging of peripheral nerve sheath tumors with pathologic correlation: pictorial review. Eur J Radiol 2004;52:229-39. + +8. Merker VL, Esparza S, Smith MJ, Stemmer-Rachamimov A, Plotkin SR. Clinical features of schwannomatosis: a retrospective analysis of 87 patients. Oncologist 2012;17:1317-22. + +9. Gonzalvo A, Fowler A, Cook RJ, Little NS, Wheeler H, McDonald KL, et al. Schwannomatosis, sporadic schwannomatosis, and familial schwannomatosis: a surgical series with long-term follow-up. J Neurosurg 2011;114:756-62. + +10.Molina AR, Chatterton BD, Kalson NS, Fallowfield ME, +Khandwala AR. Multiple schwannomas of the upper limb related exclusively to the ulnar nerve in a patient with segmental schwannomatosis. J Plast Reconstr Aesthet Surg 2013;66:e376-9. \ No newline at end of file diff --git a/medical/md/PMC4762793.md b/medical/md/PMC4762793.md new file mode 100644 index 0000000000000000000000000000000000000000..9e6928d358fc3f1d7cadd7047f3a7636928f7291 --- /dev/null +++ b/medical/md/PMC4762793.md @@ -0,0 +1,74 @@ +Case Report + +![0_image_2.png](0_image_2.png) + +Parinaud's oculoglandular syndrome associated with Sporothrix schenckii Cassio Porto Ferreira a,b,c,d,*, Jose´ Augusto daCosta Nery a,b,c,d, Ana Cristina Oliveira de Almeida c,d, Luiz Cla´udio Ferreira d, Suzana Corte-Real c,d, Fa´tima Conceic¸a˜o-Silva c,d a Souza Arau´jo Outpatient Clinic, Brazil b Leprosy Laboratory-Lahan, Brazil cOswaldoCruz Institute-IOC, Brazil dOswaldoCruz Foundation-Fiocruz, Brazil A R T I C L E I N F O +A B S T R A C T +Article history: +Received 23 April 2014 Received in revised form 4 May 2014 Accepted 7 May 2014 Keywords: +Syndrome Eye Fungi Sporothrix schenckii Sporotrichosis Epidemic Zoonotic + +## Introduction + +Parinaud oculoglandular syndrome is a rare eye disease caused by different etiologic agents, including bacteria, viruses and fungi. + +It is characterized by a granulomatous conjunctivitis, accompanied by adjacent preauricular lymphadenopathy which is almost always caused by local trauma and can bring consequences if not treated promptly. We present a case caused by the dimorphic fungus Sporothrix schenckii from the sporotrichosis epidemic occurred in Rio de Janeiro, Brazil. + +## Case Report + +Male, 21 years-old, single, student, resident in Seropedica city/ +Rio de Janeirostate/Brazil.Refers to appearance of afinger injury two months after a sick cat scratch with subsequent involvement of the +* Corresponding author at: Souza Arau´ jo Outpatient Clinic-OswaldoCruz Institute - IOC/ Oswaldo CruzFoundation-Fiocruz Brazil. + +Tel.: +55 21 2598 4242; fax: +55 21 2768 8008. + +E-mail address: drcassioferreira@yahoo.com.br (C.P. Ferreira). + +http://dx.doi.org/10.1016/j.idcr.2014.05.001 2214-2509/- 2014 Published by Elsevier Ltd. + +Open access under CC BY-NC-ND license. + +![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) + +The Parinaud oculoglandular syndrome is a rare eye disease caused by different etiologic agents, including bacteria, viruses and fungi. It is characterized by a granulomatous conjunctivitis, accompanied by adjacent preauricular lymphadenopathy and can bring consequences if not treated promptly. We present a case of Parinaud oculoglandular syndrome caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form occurring in Rio de Janeiro, Brazil. + +- 2014 Published by Elsevier Ltd. + +Open access under CC BY-NC-ND license. + +eye. The clinical examination presents erythematous crusted lesion of 0.3 cm in size, located on the index finger (Fig. 1A) and in the right eye was observed a small rounded painful lesion with sessile base and soft consistency, located in the lower tarsal conjunctiva (Fig. 1B) +and preauricular lymphadenopathy. A fungal culture was realized with isolation of the fungus S. schenckii (Fig. 1C and D). Itraconazole 100 mg day was administered and the patient is in regular monitoring in Dermatology and Ophthalmology Departments. + +## Discussion + +Parinaud's oculoglandular syndrome is a curious medical conditionand ithas beenassociated withseveral differentinfectious diseases, especially to cat scratch disease (Bartonella henselae), but sometimes it has been reported in granulomatous chronic infectious like tuberculosis, atypical mycobacteria and syphilis [1] and even some viruses (herpes simplex virus) [2]. Fungal infections also have been associated to this syndrome especially sporotrichosis (S. schenckii) [3], blastomycosis (Blastomyces dermatitidis) [4] and coccidioidomycosis (Coccidioides immitis) [5]. Sporotrichosis is a subacute or chronic mycosis that affect all ages, currently distributed throughout the world, especially in tropical and + +![1_image_0.png](1_image_0.png) + +subtropical zone [6]. Infection almost always occurs by traumatic inoculation of soil, plants and organic matter contaminated with fungus [6] and may be localized in the conjunctiva as a primary infection or may be secondary involving of the eyelid and face [7]. + +Numerous soft, yellow, granulomatous nodules, which may ulcerate, develop in the palpebral or bulbar conjunctiva of the involved eye [7]. The preauricular and submandibular lymph nodes are grossly enlarged and may suppurate [7]. There is no evidence how the infectious agent has been disseminated throughout the Rio de Janeiro municipality and its outskirts, but it is beyond reasonable doubt that the close interaction with cats represents a key form of transmission of the fungus [8–10]. Felines have very close contact withcontaminatedsoil andorganicmatter andconstitute a reservoir of this agent [11]. The gold standard for sporotrichosis diagnosis is culture [6] and drug of choice to treat these patients has been oral itraconazole [6]. Ophthalmologists and dermatologists must be aware of its occurrence especially in endemic regions in order to avoid diagnostic pitfalls. + +## Authors' Contributions + +Cassio Porto Ferreira and Jose´Augusto da Costa Nery: clinical care and paper preparation. Ana Cristina Oliveira de Almeida: laboratorydiagnosis, paper preparation and English review. Luiz Cla´udio Ferreira: technical support. Suzana Corte-Real and Fa´tima Conceic¸a˜o-Silva: laboratory diagnosis and paper preparation. + +## Conflict Of Interest Statement + +None. + +## Role Of Funding Source Ethical Approval + +The authors declare that a written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. Ethics committee approval: CEP 14/2001 Instituto Oswaldo Cruz– CPqHEC/FIOCRUZ. + +## Acknowledgement + +The authors thank Dr Euzenir Nunes Sarno, PhD, for the support and valuable tips. + +## References diff --git a/medical/md/PMC4764964.md b/medical/md/PMC4764964.md new file mode 100644 index 0000000000000000000000000000000000000000..0b4376f7a4cb37b0ee84452a33a62deebcda27bb --- /dev/null +++ b/medical/md/PMC4764964.md @@ -0,0 +1,143 @@ +# Effectiveness Of Spiritual Group **Therapy On** Quality Of Life And Spiritual Well-Being Among Patients With Breast Cancer + +Sakineh Zamaniyan1, MA; Jafar Bolhari2, MD; +Ghasem Naziri3, PhD; Majid Akrami4, MD; +Sara Hosseini4, MA 1Department of Psychology, Islamic Azad University, Karaj Branch, Karaj, Iran; 2School of Behavioral Sciences and Mental Health, Iran University of Medical Sciences, Tehran, Iran; 3Fars Sciences and Research Branch, Department of Psychology, Islamic Azad University, Shiraz, Iran; 4Breast Diseases Research Center, Department of Surgical Oncology, Shiraz University of Medical Sciences, Shiraz, Iran Correspondence: Sakineh Zamaniyan, MA; Breast Clinic, Motahari Clinic, Nemazee Sq., Shiraz, Iran Tel: +98 917 7188435 Fax: +98 71 36474673 Email: zamaniyan91@yahoo.com Received: 19 April 2014 Revised: 20 July 2014 Accepted: 27 July 2014 + +## What'S Known + +• Spirituality is a human dimension, the consideration of which can be therapeutic. • Spiritual needs often go unaddressed on the part of health care professionals. • Many patients with cancer seek recourse to religion to cope with their disease and enrich their quality of life. + +What's New +• In the present study, spiritual group therapy improved the spiritual well-being and quality of life of patients with breast cancer. We consolidated the techniques of spiritual group therapy in the form of a treatment package. + +Abstract Cancer is deemed the century's major health problem, and its increasing growth during the last decades has made experts concerned more than ever. Of all types of cancer, breast cancer is regarded as the second most common disease among women. The aim of this study was to determine the effectiveness of spiritual group therapy on quality of life and spiritual well-being among patients suffering from breast cancer. The present research was carried out between March and June 2011. The sample consisted of 24 participants randomly assigned to 2 groups: an experimental group (n, 12) and a control group (n, 12). All the subjects completed questionnaires on quality of life and spiritual well-being in pretest and posttest. The experimental group received 12 sessions of spiritual group therapy. The results demonstrated improvement in quality of life and spiritual wellbeing in the experimental group. In conclusion, spiritual group therapy can be used to improve quality of life and spiritual wellbeing (religious health and existential health) among patients with breast cancer. + +Please cite this article as: Zamaniyan S, Bolhari J, Naziri G, Akrami M, Hosseini S. + +Effectiveness of Spiritual Group Therapy on Quality of Life and Spiritual WellBeing among Patients with Breast Cancer. Iran J Med Sci. 2016;41(2):140-144. + +Keywords - Breast neoplasms - Spiritual therapies - Quality of life - Psychotherapy - Group - Existentialism + +## Introduction + +Cancer is universally recognized as the century's major health concern, and its mounting escalation during the past few decades and detrimental impact on all physical, emotional, spiritual, social, and economic aspects of human life have rendered experts concerned more than ever.1 Of all types of cancer, breast cancer is viewed as the second most common disease among women. Breasts are symbols of femininity and as such the majority of women find the prospect of losing them unthinkable. A woman's reaction to any kind of actual or suspected disease may include fear of deformity, loss of attraction, and death; consequently, breast cancer is indubitably horrifying to any woman.2 Breast cancer treatments such as radiotherapy, chemotherapy, and surgery could cause serious physical and psychological side effects, making treatment programs and rules more difficult to follow. Patients may, therefore, find it harder to adhere to the treatment protocol and inadvertently undermine the efficacy of the treatment, which could negatively impact their own life expectancy.3 At this stage, patients suffering from breast cancer are liable to complain of various kinds of problems such as sleeping disorders, high levels of anxiety, and reduced quality of life sometimes even 2 years after the initial diagnosis. Treatment seems to come to its desirable end once such difficulties have been overcome.4 Since modern treatment methods have turned cancer on many occasions into an acute and often curable disease out of an incurable one, various aspects of cancer psychiatry such as reaction toward diagnosis and also treatment have become increasingly prominent.5 One dimension of human life is spirituality, which enables individuals to communicate and integrate with the universe. Communication and integration endow hope and meaning to human life and elevate it beyond the confines of time and place. Religious/spiritual group therapy is a form of psychotherapy drawing upon special principles and religious/spiritual techniques to empower patients to attain a nonmaterial understanding of self, universe, incidents and phenomena, and ultimately health and growth.6 The results of a research carried out by Meraviglia7 under the title of "Effects of Spirituality in Breast Cancer survivors" on 84 women aged between 34 and 80 years suffering from breast cancer whose disease had been diagnosed 5 years earlier, along with his another study conducted on 60 patients aged between 33 and 83 years suffering from lung cancer, showed that patients should be encouraged to seek spirituality as an effective tool in dealing with physical and psychological responses to cancer. + +Given the reach and influence of religious culture in Iranian society, drawing upon religion as an important source of compatibility is anticipated. Due to the lack of research evidence in this domain in our country, the present study was conducted to investigate the efficiency of spiritual group therapy in enhancing patients' well-being and spiritual health. + +## Patients And Methods + +The present research was a quasi-experimental project of pretest-posttest type with a control group carried out between March and June 2011, in collaboration with Shiraz University of Medical Sciences. The study population was selected from among patients referring to Amir Hospital and Omid Hospital. For the purpose of this research, among a large number of patients suffering from breast cancer and aged between 18 to 65 years, 24 individuals were selected after clinical interviews through the available sampling method. The participants were then randomly assigned to two 12-member control and test groups. All the 24 participants were + +tested using the research tools, comprising structured clinical interview for DSM-IV (SCID-I), quality-of-life questionnaire (WHOQOL-26), and spiritual health scale (SWB-20). The test group members thereafter received 12 sessions of group spiritual treatment (Table 1),8 whereas the control group members did not receive any kind of treatment until the test group members were fully treated and the second group of questionnaires was collected. + +The inclusion criteria for the present research were as follows: 1. Having elementary education and literacy level 2. Being at least 18 and at most 60 years of age 3. Not having the diagnostic criteria for clear psychiatric disorders such as psychosis, major depression, obsessive-compulsive disorder, and personality disorders based on the SCID-I conducted by the researcher Subsequently, all the participants under test completed the research questionnaire in 2 rounds. The test group participated in the 12 group-treatment sessions in the form of a 120-minute session in a week. + +## Results + +From the 24 participants, 43% were 50 and 57% under 50 years old, while 21% were single and 79% were married. Apropos education, 37.5% of the study population were below the high school diploma, 50% had a high school diploma, + +| Table 1: Treatment sessions in brief Treatment session Session title First session Members becoming familiar with and talking to each other about the concept of spirituality and religion and the impact it has on a person's life Second session Self-consciousness and communication with oneself and listening to the inner voice Third session Self-concept Fourth session The word of God, communication with God or with any superior power the patient believes in, prayer and conversation with God Fifth session Altruism (doing something spiritual collectively) Sixth session Relationship with holy sites Seventh session Resentment and lack of forgiveness, self-forgiveness, and feeling guilty Eighth session Forgiveness Ninth session Death and fear of death and toil Tenth session Faith and trust in god Eleventh session Gratitude and thanksgiving Twelfth session Final session | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +and 12.5% had college education. Also, 65% of the participants in each group underwent chemotherapy or had recently undergone it. + +As is shown in Table 2, there were significant differences in quality of life and some of its dimensions (psychological and social dimensions) between the 2 groups following the spirituality group-therapy sessions among the experimental subjects. However, as regards the physical dimension, no significant difference was observed between the 2 groups after the therapy sessions. + +The eta-squared in this table shows that 39%, +5%, 26%, and 2% of the changes in the scores of quality of life, physical, psychological, and social aspects were created by the implementation of our treatment method. + +With respect to the spiritual health dimension, there were significant differences between the control and test groups. Additionally, 36%, 2%, and 38% of the changes in these variables were caused by the implementation of our treatment method. + +As is demonstrated in Table 3, there was no significant difference in the social aspect of quality of life between the 2 groups. + +## Discussion + +In parallel with the findings of the present research, it has been previously observed that religiosity and spirituality can play a significant role in enhancing quality of life among patients with cancer.9 Abedi et al.10 suggested that prayers and religious practices could affect not only emotional moods but also physical quality and that such practices could sometimes improve a patient with physical disease in a few moments or in a few days. Quing, McCola, and Larson11 also reported that many of their patients acknowledged the positive effects of religion on their mental and physical health. + +Rahmati et al.12 concluded that spiritual/ +religious group therapy could generally be effective in mental improvement in patients with schizophrenia. Rocha and Falek13 concluded that spirituality and religion could have no impact on the social dimensions of quality of life. + +Prayer reduces anxiety, promotes spirituality, and is an appropriate method for coping with diseases. In a research conducted by Hojjati et al.,10 a direct relationship was reported between prayer and well-being: More prayer was correlated with better health.One study probed into well-being, religious adaptation, and quality of life among African-American women who had undergone treatment for breast cancer and reported that the women who probed into well-being and religious adaptation had more positive adaptation and that there was a meaningful relationship between spiritual well-being, physical and emotional aspects of quality of life, and performance health.14 Askari et al.15 posited that religious beliefs and optimism were predictors of spiritual health. + +Spiritual and religious beliefs and practices are effective in promoting adaption to cancer by + +| Table 2: Comparison of the results of the statistical analysis of covariance at pretest and posttest in the experimental and control groups Research variables Resources Eta-squared df Mean F Sum of P value changes squares squares Quality of life (total) Posttest 0.39 1 362.8 13.26 362.8 0.002 Pretest 0.15 1 105.08 3.84 105.08 0.063 Physical dimension Posttest 0.5 1 3.44 1.69 3.44 0.293 Pretest 0.164 1 12.12 4.11 12.12 0.055 Psychological dimension Posttest 0.26 1 35.27 7.5 32.27 0.012 Pretest 0.012 1 1.17 0.25 1.17 0.62 Social dimension Posttest 0.2 1 53.64 2.12 53.64 0.034 Pretest 0.108 1 26.6 2.54 26.6 0.126 Spiritual health Posttest 0.36 1 721.72 11.62 721.72 0.003 Pretest 0.0 1 0.354 0.0006 0.354 0.94 Religious health Posttest 0.2 1 94.46 5.12 94.46 0.034 Pretest 0.07 1 28.72 1.56 28.72 0.225 Existential health Posttest 0.38 1 323.31 12.99 323.31 0.002 Pretest 0.06 1 36.26 1.45 36.27 0.241Table 3: Results of t-tests on the differential scores of the social dimension Groups Mean±SD F Level of significance t df P value Control 2.08±3.62 1.94 0.17 1.42 22 0.169 Experimental 4.5±4.64 | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +affecting existential concerns such as the search for the meaning of life and hope. + +Today, faith and spirituality are regarded as some of the most significant sources of physical health and quality of life. Evidence shows that spiritual interventions could be helpful in preventing or improving an extensive range of physical problems and coping with acute pains, diseases, and death.6 Various studies have confirmed a meaningful relationship between spirituality and religion and quality of life, spiritual well-being, and meaning of life.16 Our results revealed that spiritual group therapy enhanced quality of life and its psychological and social dimensions as well as spiritual well-being and its religious and existential aspects. Furthermore, there is a great deal of evidence indicating that religion is a protective factor against depression and that it facilitates recovery.17 Prominent among the salient points of spiritual group therapy is that it can improve the patient's attitude toward life or disease. The importance of stressors is determined through cognitive evaluations under the influence of beliefs and personal values such as self-control and existential and spiritual beliefs. Individuals make use of available resources and various coping strategies in order to manage their stress. Based on this standpoint, it can be argued that values affect significant cognitive evaluations in the process of dealing with a problem; thus, spirituality can help individuals assess negative events in different ways. Hence, spirituality offers a stronger sense of control and herewith leads to more psychological compatibility.18 The second factor that should be taken into account is the coordination of therapy sessions in groups. Groups help improve patients' communicative skills and provide them with an effective supportive system. In addition, when surrounded by others afflicted with the same problem, the individual does not consider the problem as something exclusive and tends to become more hopeful.19 The third factor of note is the particular characteristics of patients suffering from cancer and some national and cultural features of our society. Conflicts, diseases, and severe traumas usually drive individuals away from their routine course of life and make them aware of temporary values and goals. Individuals in such situations need some tool to seek more long-lasting goals and values. Holding such group therapies probably confers such required opportunities. Experts believe that spirituality is described and formed by acceptable actions and beliefs in a certain culture.20 The fourth noteworthy factor in regard to spiritual group therapy is related to some procedures and techniques and also the impact of prayer, benediction, and communication with God. Essa Zadegan21 reported that therapeutic metaphor could be an innovative method whereby patients utilize their power of imagination to achieve better insight. + +There are some limitations in the current study. The therapist had inadequate experience in treating patients suffering from cancer via group therapy and spiritual therapy. Moreover, the small sample size of this study precludes generalization of the results. Furthermore, the number of group therapy sessions was limited +(12 sessions). It seems that more time is needed for the treatment to become complete. + +## Conclusion + +According to our patients' statements as well as the results of our statistical analyses, spiritual group therapy could be deemed a suitable method for treating disorders such as depression and promoting the quality of life as well as the religious and existential dimensions of spiritual health in patients suffering from breast cancer. + +## Acknowledgment + +The author would like to thank Mrs. Eslami, an employee of Omid Charity, and Mr. Mohamadreza Yabandeh and Mrs. Farideh Gholami for their valuable help as therapist assistants. + +## Conflict Of Interest: None Declared. References + +1. Kamangar F, Dores GM, Anderson WF. + +Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol. 2006;24:2137-50. doi: 10.1200/JCO.2005.05.2308. PubMed PMID: 16682732. + +2. Kaplan JL, Porter RS. Information on medical care for women. Translated by Zolanvary M. Tehran: Meyar-e-elm; 2006. Persian. + +3. Spira JL, Reed GM. Group psychotherapy for women with breast cancer: 1st ed. + +Washington: American Psychological Association; 2003. + +4. Kayser K, Scott JL. Helping couples cope with women's cancers: an evidence-based approach for practitioners. 1st ed. New York: +Springer Science & Business Media; 2008. + +5. Sadock BJ, Sadock VA. Kaplan and Sadock's synopsis of psychiatry: Behavioral sciences/clinical psychiatry. Translated by Arjmand M. Tehran: Arjmand Press; 2008 6. Richards PS, Bergin AE. A spiritual strategy for counseling and psychotherapy. 2nd ed. + +Washington: American Psychological Association; 2005. + +7. Meraviglia M. Effects of spirituality in breast cancer survivors. Oncol Nurs Forum. 2006;33:E1-7. doi: 10.1188/06.ONF.E1-E7. PubMed PMID: 16470229. + +8. Hartz GW. Spirituality and mental health: +clinical application. Translated by Kamgar A & Jaafar E. Mashhad: Ravan publication; 2008. Persian. + +9. Lim JW, Yi J. The effects of religiosity, spirituality, and social support on quality of life: a comparison between Korean American and Korean breast and gynecologic cancer survivors. Oncol Nurs Forum. 2009;36:699 708. doi: 10.1188/09.ONF.699708. PubMed PMID: 19887358. + +10. Hojjati H, Motlagh M, Nuri F, Sharifnia H, +Mohammadnejad E, Heydari B. Relationship between different dimensions of prayer and spiritual health in hemodialysis patients. Iranian Journal of Critical Care Nursing. 2009;2:149-52. + +11. Snoep L. Religiousness and happiness in three nations: A research note. Journal of Happiness Studies. 2008;9:207-11. doi: 10.1007/s10902-007-9045-6. + +12. Rahmati M, Falahi Km, Rahgouy M, +Rahgozar M. The effect of spiritualreligious group therapy on mental status of schizophrenic inpatients. Nursing Research. 2010;5:13-20. Persian. + +13. Rocha NSd, Fleck MPdA. Evaluation of quality of life and importance given to spirituality/religiousness/personal beliefs (SRPB) in adults with and without chronic health conditions. Revista de Psiquiatria Clínica. 2011;38:19-23. + +14. Holt CL, Clark EM, Kreuter MW, Rubio DM. + +Spiritual health locus of control and breast cancer beliefs among urban African American women. Health Psychology. 2003;22:294299. doi: 10.1037/0278-6133.22.3.294. + +15. Askari P, Ruoshani Kh, Mehri Aderyani M. + +Relationship between religious reliefs and optimism with spiritual health in Ahvaz Islamic Azad university Students. Journal of Yaftehaye nou dar Ravanshenasi. 2010;27 40. Persian. + +16. Barbeito S, Vega P, Ruiz de Azua S, +Balanza-Martinez V, Colom F, Lorente E, et al. Integrated treatment of first episode psychosis with online training (e-learning): +study protocol for a randomised controlled trial. Trials. 2014;15:416. doi: 10.1186/17456215-15-416. PubMed PMID: 25348346; PubMed Central PMCID: PMC4219047. + +17. Aukst-Margetic B, Jakovljevic M, Margetic B, +Biscan M, Samija M. Religiosity, depression and pain in patients with breast cancer. Gen Hosp Psychiatry. 2005;27:250-5. doi: 10.1016/j.genhosppsych.2005.04.004. PubMed PMID: 15993256. + +18. Simoni JM, Martone MG, Kerwin JF. + +Spirituality and psychological adaptation among women with HIV/AIDS: implications for counseling. Journal of Counseling Psychology. 2002;49:139-47. doi: 10.1037/0022-0167.49.2.139. + +19. Corey G. Group counseling methods and theories. Translated by Askari F, Khodabakhshi M, Darini M, Askari F. Tehran: Shabnam Danesh; 2008. Persian. + +20. Miller G. Incorporating Spirituality in Counseling and Psychotherapy: Theory and Technique. 1st ed. New Jersey: Willey & +Sons; 2003. + +21. Essa Zadegan A. The use of metaphors in counseling and psychotherapy. Novel Research and Consulting. 2008;26:105-75. Persian. \ No newline at end of file diff --git a/medical/md/PMC4959331.md b/medical/md/PMC4959331.md new file mode 100644 index 0000000000000000000000000000000000000000..5d1d9ae3ba39a05a25a7b8acbf7f7eb92ab0931c --- /dev/null +++ b/medical/md/PMC4959331.md @@ -0,0 +1,19 @@ + + +![0_image_0.png](0_image_0.png) + +## Living Your Yoga + +Author: Dr. Judith Hanson Lasater, Ph. D., P.T. + +Year: 2015 Publisher: Rodmell Press, Berkeley, California The author of the book Dr. Judith Hanson Lasater is a Yoga teacher, with more than four decades of experience from California. She is also the author of several Yoga books pertaining to lifestyle adaptations through yogic principles and practices. This book addresses yogic practices to be cultivated for personal growth to live in harmony with nature catering to healthy internal and external environment. The book is in three parts. The author discusses the qualities to be cultivated under each part with reference to Patanjali's Yoga Sutras and *Bhagavad‑Gita*, quoting the relevant verses. The ways to cultivate the qualities (such as love and compassion) and relevant mantras for daily living are given for each quality. She has shared her personal experience, some stories, and illustrations under each quality. The first part speaks concerning awareness of internal disciplines and qualities to be cultivated for bringing in such disciplines. These practices make our actions conducive to personal growth. To abide in our own self is to be present with a sense of fullness and to create a loving environment without self‑judgment. As a means to abide in our own true nature, we must commit to develop awareness by observing our lives, our thoughts, our words, and our actions. This is in line with Patanjali's aphorism tada drashtuh svarupe avasthanam (Yoga Sutra 1.3) implying when we come to realize our true nature we are free from miseries. To observe our bodily sensations and the breath means to consciously relax our body and slow down our breath. When we are relaxed we see things as they are. When we are in relaxation, it gives room for expansion, coinciding with the infinite. This helps us to love others and ourselves without any expectations. This state makes us witness our own thoughts and not be carried away by them. It helps to develop faith and a willingness to accept reality. Elaborating *Patanjali* and *Bhagavad‑Gita*, the author explains abhyasa and vairagya. Abhyasa is doing everything intimately present in each and every moment, + +![0_image_1.png](0_image_1.png) not performing actions mechanically without inner transformations. Vairagya is letting go which is an act of surrender. This, as per the author, is the key to living in yoga. It is possible to extend this thinking bringing in the importance of practice. Instead of just witnessing our thoughts, we identify the mind and its modifications. We become one with the flow of thoughts. Hence, abhyasa is important which is constant inner practice and vairagya, the act of detachment. The second part speaks about remaining truly open and aware of intimate relationships. The first quality presented is compassion that is awareness of others' suffering. We should let go the desire to control what we see around us. The author observes that the urge to control comes from the fear of death and unhappiness. These refer to the five klesas that the sutras talk about. Inner peace brings us freedom and wholeness, which binds us all with universal love. Further, the author suggests that greed and anger springs from fear. However, there is another dimension to this; namely, fear comes from inordinate involvement to the material. Hence, Bhartrhari (a famous poet) says that vairagya or dispassion makes a person fearless. Clinging to a particular preference whether it is from attachment or aversion creates suffering. Hence, Lord Krishna gives the solution of being in moderation and maintaining equanimity. Recognizing one's preferences to attachment and aversion is half the solution. We suffer when we identify ourselves with pain. We have choice to either suffer or not to suffer! Only change is enduring and certain in all things - change of seasons, day, changes in the crust of the earth, inhalation, exhalation, etc., Quantum physics also teaches about the uncertainty and impermanent nature of the material world around us. Accepting the changes also makes us fearless. We may also say the main reason of annoyance is our uncontrolled responses to pleasant or unpleasant conditions in which we get involved. It is good to remind oneself the attitudes that Sage Patanjali recommend for cultivating pleasant disposition. The mind of a person is clarified through cultivating attitudes of friendliness, compassion, cheerfulness, and nonjudgmental mindset toward happiness, misery, virtue, and vice, respectively (Sutra 1.33). With practice of these right attitudes, we achieve pleasant disposition in various kinds of situations that may arise in one's relationship with people around. The third part is to transcend ordinary way of thinking. First, the author goes on to remark that the answer to greed is aparigraha or nonhoarding. A balance between your needs and desire as suggested by Sage Patanjali should be implemented. It may be said that the follower of the law of love cannot hoard anything for oneself. There are two aspects that face us in our life: They are impermanent material nature and our material cravings that never satisfy our desires. The author discusses the concept of Karma Yoga, to work without attachment to the outcome as Lord Krishna also says. The author mentions that when we can live with deep faith in our connections, it makes us fearless, want‑less, and needless. We should develop the wisdom to see the interconnectedness which is the thread connecting us all. Sharing this connectedness is love. Love comes when one does not react. Success in life is living with an open, loving heart, which connects us with the Divine within and in others. We should be grounded in truthfulness from which the universe springs. This makes action and fruition depend on the supreme. The author concludes that our actions and practice must be an offering to the universal principle with an awareness of gratitude. In conclusion, the reviewer feels that the prescribed ten values of Patanjali under the title, "yama and niyama" are essential and includes all that is experienced and listed by the author under various headings. These characteristics help in developing a personality, which has obtained through external and internal purity. Such a person has a joyful disposition, develops mastery over his/her senses, and has moderation in enjoyment. Yama and niyamas should be practiced regularly and with consistency. In this age of yoga practice, the focus is only on asanas and pranayama, with little emphasis or even information on yama and niyama. Thus, the importance of this book in introducing these components of yoga cannot be overemphasized. + +Karuna Nagarajan Directorate of Distance Education, S-VYASA University, Bengaluru, Karnataka, India. + +E-mail: karuna.nag6@gmail.com This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. + +![1_image_0.png](1_image_0.png) + +How to cite this article: Nagarajan K. Living your yoga. Int J Yoga 2016;9:178-9. \ No newline at end of file diff --git a/medical/md/PMC4989436.md b/medical/md/PMC4989436.md new file mode 100644 index 0000000000000000000000000000000000000000..2297e720d34b318c60105a8505df270730a74e0e --- /dev/null +++ b/medical/md/PMC4989436.md @@ -0,0 +1,49 @@ +# Three Cases Of Idiopathic Superior Laryngeal Neuralgia Treated By Superior Laryngeal Nerve Block Under Ultrasound Guidance + +Jian-Ping Wu1, Hui Liu1, Jian-Xiong An1, Doris K Cope2, John P **Williams2** +1 Department of Anesthesiology, Pain Medicine and Critical Care Medicine, Aviation General Hospital of China Medical University and Beijing Institute of Translational Medicine, Chinese Academy of Sciences, Beijing 100012, China 2 Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh 15213, USA +Key words: Laryngeal Pain; Superior Laryngeal Nerve Neuralgia; Ultrasound‑guided Laryngeal Nerve Block In treating superior laryngeal neuralgia (SLN), superior laryngeal nerve blocks (SLNBs) can provide immediate relief of persistent pain. Although SLNB was effective, the procedure can increase the risks due to the proximity to the carotid artery, external vein, and other vessels. To avoid complications such as advertent intravascular injection, local anesthetic toxicity from intravascular absorption, and intratracheal injection, we successfully blocked the superior laryngeal nerve under ultrasound guidance in three patients suffering from SLN. The written informed consent before treatment was obtained for each patient participating in this study. A 58‑year‑old male was admitted to our hospital; he had a complaint of a fish bone that had stuck to his throat 6 months ago, and the bone was immediately removed (case 1). Subsequently, he experienced paroxysmal burning and stabbing pain in his left throat. Pain was associated with deep inspiration and coughing. Tenderness over the left thyrohyoid membrane was elicited by light pressure. Neurological examination was negative. The left SLNBs under ultrasound guidance using 3 ml of 2% lidocaine were done for four times, which was completed for every 2 days. The patient experienced no pain when we followed up 6 months following the procedures. A 70‑year‑old female who developed left‑side SLN for 2 years was admitted to our hospital (case 2). She had had a cough for a week just before the onset of the neuralgia. She experienced paroxysmal stabbing pain in the left side of the throat, triggered by swallowing and aspiration. There was tenderness to pressure over the left thyrohyoid membrane. + +![0_image_0.png](0_image_0.png) + +10.4103/0366-6999.187859 +Chinese Medical Journal ¦ August 20, 2016 ¦ Volume 129 ¦ Issue 16 **2007** +Neurological examination was negative, and 10 trials of SLNB were done. Although the paroxysms occurred once a year for a few months after the last block therapy, she was able to deal with the pain with carbamazepine alone. A 57‑year‑old male with complaints of right‑side throat pain for the previous 5 months was admitted to our hospital (case 3). He had a history of severe smoking for about 20 years. The paroxysmal stabbing pain occurred almost every day. Neurological examination was negative. After 10 trials of SLNB, pain was alleviated. He remained asymptomatic for about 1 year after the last SLNB. A portable ultrasound machine, Venue 50, (GE Healthcare, Chalfont St. Giles, UK) was brought to the operating room, with the patient lying in the supine position with head turned away from the affected side and a pillow placed under the patient's neck. After preparation of the skin with antiseptic solution, ultrasound examination is performed using a high‑resolution linear array transducer. + +The transducer is applied transversely to the middle aspect of the neck to obtain a short axis view of the thyrohyoid Address for correspondence: Prof. Jian‑Xiong An, Department of Anesthesiology, Pain Medicine and Critical Care Medicine, Aviation General Hospital of China Medical University and Beijing Institute of Translational Medicine, Chinese Academy of Sciences, Beijing 100012, China E‑Mail: anjianxiong@yeah.net This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms. + +For reprints contact: reprints@medknow.com +© 2016 Chinese Medical Journal ¦ Produced by Wolters Kluwer ‑ Medknow Received: 27‑04‑2016 **Edited by:** Qiang Shi How to cite this article: Wu JP, Liu H, An JX, Cope DK, Williams JP. Three Cases of Idiopathic Superior Laryngeal Neuralgia Treated by Superior Laryngeal Nerve Block under Ultrasound Guidance. Chin Med J 2016;129:2007-8. + +membrane. One can easily identify the thyroid cartilage and the common carotid artery. The superior laryngeal nerve is usually found medial to the artery between the hyoid bone and thyroid cartilage, and a block needle is inserted in the plane of the ultrasound beam in a medial to lateral orientation. It is often easy to visualize the needle. The needle was inserted with 35° to the skin and the depth was approximately 2 cm. When the needle located medial to the common carotid artery, with no blood or air of the aspirated, the test dose of 2 ml of 2% lidocaine was injected [Figure 1]. The procedure is completed for every 2 days until the pain disappeared. The superior laryngeal nerve arises from the vagus nerve. It runs inferiorly and anteriorly behind the carotid artery to pass the lateral extent of the hyoid bone.[1] It divides into internal and external branches. The internal branch passes immediately inferior to the greater horn of the hyoid bone and approaches the thyrohyoid membrane accompanied by the superior laryngeal artery, a branch of the superior thyroid artery. The superior laryngeal nerve is so located that any regional inflammation or further local trauma,[2] +aggravated by normal laryngeal movements, can produce localized neuritis at the thyrohyoid membrane and cause the throat pain. Aydin et al. + +[3] described a case of SLN after acute laryngitis and treatment with a single injection of a local anesthetic. The patient received a single nerve block near the right hypothyroid membrane and the symptoms decreased dramatically in a few minutes. He had no pain for 1 year following the procedure. Neuropathic pain is derived from abnormal afferent inputs caused by axonal damage, myelin disruption, and inflammatory processes. Local anesthetics can interrupt or alleviate pain and have been shown to suppress ectopic neural discharges from nerve fibers. It was posited that SLNB may be useful in the diagnosis and treatment of SLN.[4] +Ultrasound has been used to image the human body for over half a century. The images can be acquired in "real time," thus providing instantaneous visual guidance for many interventional procedures including those for regional anesthesia and pain management.[5] +In these three cases, the use of ultrasound made it possible to perform SLNB in patients suffering from SLN. All of the three patients recovered from SLB without any Figure 1: Transverse sonographic view of the patient's neck at the level + +![1_image_0.png](1_image_0.png) + +of the thyrohyoid membrane. An in‑plane method was used to block the superior laryngeal nerve. Arrow indicates the entry of the needle from the 10 o'clock direction. Triangle indicates the thyrohyoid membrane, blue indicates the carotid artery, and red indicates the external carotid vein. L: Left, R: Right. + +complications. These cases illustrate a potential clinical application for ultrasound assistance in pain management. + +## Financial Support And Sponsorship Nil. Conflicts Of Interest + +There are no conflicts of interest. + +## References + +1. Furlan JC. Anatomical study applied to anesthetic block technique of the superior laryngeal nerve. Acta Anaesthesiol Scand 2002;46:199‑202. doi: 10.1034/j.1399‑6576.2002.460214.x. + +2. Bruyn GW. Superior laryngeal neuralgia. Cephalalgia 1983;3:235‑40. + +doi: 10.1046/j.1468‑2982.1983.0304235.x. + +3. Aydin O, Ozturk M, Anik Y. Superior laryngeal neuralgia after acute laryngitis and treatment with a single injection of a local anesthetic. Arch Otolaryngol Head Neck Surg 2007;133:934‑5. doi: 10.1001/ archotol.133.9.934. + +4. Takahashi Sato K, SuzukiM, IzuhaA, Hayashi S, IsosuT, MurakawaM. + +Two cases of idiopathic superior laryngeal neuralgia treated by superior laryngeal nerve block with a high concentration of lidocaine. J Clin Anesth 2007;19:237‑8. doi: 10.1016/j.jclinane.2006.09.003. + +5. Singh M, Chin KJ, Chan VW, Wong DT, Prasad GA, Yu E. Use of sonography for airway assessment: An observational study. J Ultrasound Med 2010;29:79‑85. \ No newline at end of file diff --git a/medical/md/PMC5105473.md b/medical/md/PMC5105473.md new file mode 100644 index 0000000000000000000000000000000000000000..c48d9d94caaecf8d8f40b3e8667fb4100a78441e --- /dev/null +++ b/medical/md/PMC5105473.md @@ -0,0 +1,209 @@ + +## Course For Supervisors Of Occupation Centres & Institution Staffs + +The C.A.M.W.'s annual three weeks' Course for Supervisors and Institution Staffs, originally announced for July, has been postponed till the autumn. + +It will now be held [in London] from October 23rd to November 11th, and it will be non-residential. The fact that during this period the theoretical and specialised section of the Year's Course for Occupation Centre workers and Home Teachers will be in process, will make it possible for the students to take advantage of some of its lectures and classes in addition to those arranged for them separately. + +Staffordshire Study Week The Staffordshire Association for Mental Welfare are again arranging a Study Week for those engaged in the teaching of mental defectives in Institutions, Occupation Centres, Special Schools and Classes for backward children. + +The Course will be held from Monday, 2nd October, to Friday, 6th October, 1939. + +It will include lectures on the medical aspect of mental deficiency, delinquency and treatment of difficult children, and on the functions of the social worker, and there will be demonstrations and practical work in physical education. Practical classes in handwork and needlework will be held, and opportunities will be given for discussion in study groups. + +Full information can be obtained from: The Organiser of Centres, Crabbery Chambers, Crabbery Street, Stafford. + +The Burden Neurological Institute On May 12th, Sir Thomas Inskip opened the new Neurological Institute in the grounds of Stoke Park Colony, endowed by Mrs. R. G. Burden. + +The purpose of the Institute is " Research into the bodily substratum of mental diseases by the long and intensive study of a small number of cases." +Epilepsy will be one of the first problems to be attacked, and one of the two observation wards will be reserved for epileptics. + +The management of the Institute is in the hands of an autonomous com-mittee under the chairmanship of Sir Laurence Brock, and its work will be directed by Professor F. L. Golla. + +The equipment includes a theatre for brain surgery and a special radio-graphic unit devised by Lysholm, of Sweden. + +Board of Control The Board of Control, with the approval of the Minister of Health, have appointed Mr. T. R. Forsythe, M.B., Ch.B., D.P.M., Deputy Medical Superin-tendent of the Kent County Mental Hospital, Maidstone, to be a Commissioner to fill the vacancy caused by the appointment of Mr. A. E. Evans, M.B., D.P.H., +as a Chancery Visitor. + +Lay Psychotherapists and the British Medical Association Last year, it may be remembered, the British Medical Association appointed a special committee to "inquire and report upon the present medical equipment for dealing with mental health in this country, with particular reference to the treatment and prophylaxis of the psychoneurotic and allied disorders." The Committee's deliberations still continue, but their recommen-dations on the subject of the employment of lay psychotherapists have been embodied in the Association's Annual Report, permission to quote from which has been kindly given us by the Secretary. + +Prolonged consideration has been given to this matter, particularly to that part of it which is concerned with the functions of the clergy in regard to psychological treatment. Evidence has been received from individual clergymen, Protestant and Catholic, and it has been submitted that it is desirable that they should be trained in medical psychology so that they may be able +" not only to recognize cases which require psycho-therapeutic treatment rather than moral or spiritual direction, but also to resolve the mental conflicts of the normal individual which otherwise might develop into psychoneurotic disorder." +It has also been suggested that the clergy should co-operate with the medical profession" in undertaking the treatment of mental disorders which have their roots in religious problems," in so far as no cure can be complete until the patient has achieved a new spiritual adjustment. + +The Council " see no reason why the medical profession should not co-operate in the instruction of the clergy in the principles of medical psychology," +but do not accept the view that " the clergy should undertake sole respon-sibility for the treatment of cases which have a psychiatric symptomatology but which raise ethical or spiritual issues," although they agree that there may be some cases which could usefully be referred by medical practitioners for spiritual direction. + +With regard to the practice of psychotherapy by lay practioners, the Council have agreed that, in view of the present shortage of practitioners who have received medical training, such practice should be recognised under certain conditions. + +The Council's recommendations are as follows :? + +A. That the Representative Body approve the following principles: + +I. No lay psychotherapist should undertake treatment unless : +(i) The patient has been recommended to him by a registered medical practitioner, or has had his case investigated by such a practitioner at the request of the lay psychotherapist. + +(ii) The lay psychotherapist has undergone a prescribed and approved course of training. + +II. No medical practitioner should recommend a patient to a lay psychotherapist unless: +(i) The medical practitioner is himself a specialist in psychological medicine, or has referred the case to such a specialist. + +(ii) The medical practitioner has satisfied himself that the lay psychotherapist has undergone a prescribed and approved course of training. +III. Where treatment is undertaken by a medical practitioner and a lay psychotherapist in co-operation, the responsibility for the case should lie with the medical practitioner and there should be consultation from time to time between the medical practitioner and the lay psychotherapist. + +B. That the Representative Body expresses the opinion that lay psycho-therapists should undergo a special training of at least 2 years' duration on the basis of a curriculum to be agreed upon by the responsible bodies: and that the Association take the initiative in putting machinery in motion for the establishment of approved courses of training. + +It is not yet known whether these recommendations on so highly contro-versial a subject will be adopted as the policy of the Association, but they are to be considered by the Representative Body at a meeting to be held during July. + +A Physician's Philosophy In view of the issues raised by the British Medical Association's Committee on Mental Health referred to above, the following reflections of the Physician-Superintendent of the Crichton Royal Institution, Dumfries, in his Annual Report for 1938, would seem to be particularly opportune:? + +" In the practice of psychiatry, and particularly in dealing with the per-manently afflicted, it appears to me that a philosophy of medicine, guiding principles to the understanding and treatment of disease, is not enough; equally important is a philo.sophy of life, an appreciation of the ultimate values of the universe. Our choice is between realism and idealism. In this materialistic age in which applied science has played such a prominent part, it is probably not surprising that alike in philosophy, literature and art, realism holds .sway; . . . + +The world as we know it, . . . finite things in time and space, life and death on this earth, we are told that these are the only realities. They exist in their own right, with nothing behind them, nothing supporting them. Such a philosophy, with its neglect of aesthetic and moral values, religious experiences and intuitions, makes life seem particularly purpo.seless, if one lives amongst those whose world of sight and hearing is extremely individualistic and different from one's own. . . . The psychoses become particularly tragic if one must believe that the false beliefs of our patients deal with the ultimate realities of life." +After suggesting that, once a creed of idealism is accepted, a fundamental change of attitude to the psychotic patient follows, Dr. McCowan continues: +". . . . while it may not be the duty of the physician to discuss religion or philo.sophy with his patients, there are many, particularly of our neurotic patients, in whom discussion, and if need be, guidance on these subjects, is an essential part of treatment, if real integration of their personality is to be achieved. Again, his philosophy may well affect the attitude of the psychiatrist towards the incurable p.sychotic. To the realist, the incurable psychotic in an individual who may well be looked after as a matter of routine or even of interest, but scarcely as a worth-while human being. To the idealist, on the other hand, the psychotic does not differ from the physically ill patient: he is a human being who is doubtless fulfilling the purpose of the divine reality behind the scene as well a>s is the physician himself. Surely such a difference in attitude must tend to a difference in treatment." +Psycho-Analysis in Norway We learn from the Lancet, 20th May, 1939, that in Norway a Government order has recently been made restricting the employment of psycho-analytical methods of examination and treatment, to doctors who are specially authorised by the Department for Social Affairs, and prohibiting the use of such methods by foreign medical men without special permission from the Crown. + +In this measure, +" psycho-analysis " is defined as : +" any examination or treatment which is continued regularly for a considerable time, and whose object is to clarify, interpret, or otherwise influence unconscious mental processes." + +## Tavistock Clinic (Institute Of Medical Psychology)* + +It is disappointing to read in the Report of the Tavistock Clinic for 1938, that the site acquired for the erection of an In-Patients' Hospital has had to be sold, owing to lack of funds. A more modest plan for the provision of beds and facilities for treatment, research and teaching, has been substituted, but about the materialisation of even this plan the Report speaks guardedly and there is no indication of its certain fulfilment. + +The urgent necessity for expansion is shown by the fact that at the end of the year under review, there was a long waiting list of 480 children and adults accepted for treatment. The total number of new cases treated was 1,071 in comparison with 1,133 in 1937. + +The Director of Studies reports that in addition to the training of doctors for specialisation in psychotherapy, provision is made for the instruction of general practitioners and the number of medical officers from mental hospitals who take courses of training has increased. Amongst the lectures given to lay students was a series on problems of delinquency specially for magistrates, 34 of whom attended. + +The work of the Research Department during the year included a +" Follow- +Up +" of 104 cases of adults treated for delinquency or potential delinquency, and the completion of the investigation of the present position with regard to 500 cases of children treated between 1921 and 1924. Of the first group +(adult delinquents) 55.8% were recorded as +" Relieved" after a period of 3 years or longer; of the second group (children) improvement in the condition for which treatment was originally given was, after a similar period, still present in 77% of cases. + +The endowed research of the Clinic has continued to be concentrated on +" disorders in which it has long been suspected that a source of the pathological structural changes concerned, might be found in the prolonged disturbances of bodily function which accompany the chronic emotional states of certain psychological types." +In the spring of 1938, Sir Henry Brackenbury retired from the Chairman-ship of the Council, after 8 years' service, and his place was taken by Lord Alness. + +Institutions and the use of " Licence " +In the 80th Annual Report of the Royal Eastern Counties Institution, Colchester, one of the many interesting sections is that devoted to the subject of " Licence." +Dr. Turner reminds us that, so far as he knows, his Institution was the first to use this method of dealing with defectives and it is mentioned in a Report as long ago as 1866. Then it fell into abeyance and was not revived until 1919. On January 1st, 1939, the number on licence amounted to 17% +of the total number of patients on the books, and it increases every year. + +In reviewing the situation from the standpoint of his long experience, Dr. Turner writes that he isconvinced of two things +" :? + +" One is that Institution care is neither required, nor de.sirable, for all defectives even if it could be provided, but I would lay even greater stress on the importance of sending many more defectives than come now to institutions for training and stabilisation, so that they may successfully return to the world again." +" The second is, that of all forms of extra institutional care, licence, because of its elasticity, is far and away the most advantageous both to the patient and the community. The institution is and should be, for a far larger number of patients, a treatment centre for arresting and ultimately overcoming the difficulties reflected in the failure of the subnormal person to get along satisfactorily in the environment to which he belongs. The Institution will therefore always be lo.sing its best patients and having to keep the most dependent and least adjusted. All the older Institutions were founded with the ideal that they could educate and train the defective, so that he should return to the world. + +They were intended to be run as schools and not as custodial institutions." +". . . later, chiefly from the United States, came a wave of fear that the defective would so increase in numbers that the burden would be insupport-able. Thi.s brought in its train the idea of permanent care to prevent breeding. + +Fortunately now, that wave of fear has been banished amongst thinking people. + +We all now accept licence as a normal proceeding." +Dr. Turner recommends that girls sent on licence to domestic service should be placed near the Institution, as he thinks that it is essential to maintain close contact and that the friendly visits paid to the Matron and the opportunity of help and advice given by them, make all the difference between success and failure. Of the 31 girls in " living-in +" service at the time of writing the Report, all but 4 were within bus rides of one or other of the Institution Hostel Branches. + +It is interesting to read that out of all the girls sent out on licence since 1919 (the exact number is not given, but it is stated that it amounts to +" some hundred"), only 4 have returned to the institution on account of pregnancy. + +During the year under review, 96 out of the 191 cases on licence had to return to the Institution, but most of these returns were temporary and part of the normal working of the system, and were not due to failure. + +## A Danish Tribute To An English M.D. Colony + +Readers of " Mental Welfare " may remember that some time ago an article was published on the methods of training low-grade defectives used at Bridge Home, Witham?a branch of the Royal Eastern Counties Institution. + +In a Danish paper, Dr. Gunnar Wad, Medical Superintendent of the Ostifternes Aandsvageanstalt Institution, Ebberodgaard, draws attention to this article from which he quotes at length, and in describing a personal visit to Bridge Home he pays the following tribute to the work and to the man +(Dr. FitzGerald) to whom its initiation and success is largely due:? + +"The impression of this institution on me," Dr. Gunnar Wad writes, "was overwhelming and unforgettable. I had seen a number of other institutions in England where medium and high-grade defectives were treated by methods which only differed very slightly from what we do here at home. This wa.s, on the other hand, something quite new for me, something which I would not have thought possible if I had not seen it." +" For the first time in my life I gained an understanding of the just claims of low-grade defectives, not alone to nursing, but also to occupation and education, and in a manner which made them a part of human society. In this institution, thanks to all the work carried out, a greater fund of human happiness is found than I have seen in a corresponding place elsewhere. . . ." +"A few things are still for me inexplicable. I cannot understand how one can keep all patients employed, even the most excited, nor can I understand how one can in every case avoid using muffs, etc. I can only answer my own and the questions of others, by saying ' I have seen it with my own eyes.' +" +In attempting to +" +explain the inexplicable," Dr. Wad suggests that there is only one way of doing it, viz., by attributing what has been achieved to a medical superintendent who is " in possession of a special gift to inspire his staff, combined with enterprise, inventiveness and a feeling for and under-standing of the defectives far above the ordinary." + +## Mental Health Emergency Committee + +The work of the Mental Health Emergency Committee continues along the lines indicated in our April number. + +Its latest activity has been to approach all the Directors of Education and School Medical Officers, County and County Borough Medical Officers of Health, the Visiting Committees of Mental Hospitals, as well as Mental Deficiency Committees throughout England and Wales, with offers of assistance towards securing the help of educational psychologists and social workers skilled in dealing with cases of emotional disturbance both in adults and children if and when an emergency arises. A number of Local Authorities are already giving sympathetic consideration to the problems involved, and it is felt that the Committee's rapidly growing Register of Salaried Full-Time Social Workers in the Mental Health field should prove of great practical help towards their solution. + +Leicester Mental Hospital A copy of The Patients' Magazine, which is edited, printed, bound, published and contributed to, entirely by patients of this Hospital, has reached us, and we are glad to draw attention to a venture which should be more widely known. + +The magazine is issued quarterly, and is now in its 16th volume. It is attractively produced and its spirited accounts of Hospital activities give the impression of the existence of a real " esprit de corps." Illustrations of various Hospital +" Functions" add to the interest of this gallant little periodical. + +## Child Guidance Council + +In its Report for 1938, the Child Guidance Council draws attention to the widening conception of the province of " Child Guidance " which is beginning to be extended to the maternity and child welfare centre and to be accepted as a department of public health administration. In the Islington and Marylebone Centres, e.g., the work done by Dr. Mildred Creak and Dr. Grace Calver has resulted in the recognition and treatment of personality problems at a very early age, and has opened out avenues of enquiry offering great promise. + +There are now 54 Child Guidance Clinics in Great Britain recognised by the Council (a list of these is given in the Report) of which 16 are wholly maintained by Local Authorities. 10 Clinics are partially so maintained, and the remaining 28 are independent voluntary clinics, or clinics functioning as out-patient departments of voluntary hospitals or education departments of Universities. + +The Council announce the following forthcoming activities :? + +Year's Course of Training for Staffs of Children's Homes and Residential Schools: +This Course, arranged at the Battersea Polytechnic, in co-operation with the Child Guidance Council, begins on September 18th, 1939. The Course is a full-time non-residential one, open to men and women. + +It will be adapted to the special needs of the individual student and will include instructions and practical demonstrations in some or all of the following: Physiology, hygiene, normal psychology, and care of difficult and nervous children, home nursing and/or first aid handicrafts and household repairs, household management including cookery and dietetics, physical train-ing, mothercraft and/or catering and simple book-keeping. It will include observation visits to Maternity and Child Welfare Centres, Nursery Schools, Play Centres, Child Guidance Clinics, and a wide variety of Homes and of Day and Residential Schools. In addition, a certain number of sessions will be devoted to actual work with children of different ages. + +Further particulars can be obtained from the Principal, Battersea Poly-technic, London, S.W.ll. + +Course of Lectures at Newcastle A Course of Lectures on the " Understanding and Management of Children " for staffs of Children's Homes and Residential Schools, will be held at Newcastle-on-Tyne, from October 9th to 15th. Fee, ?1 Is. 0dv payable in advance. + +For preliminary notice and syllabus apply to the Secretary, Child Guidance Council, Woburn House, Upper Woburn Place, London, W.C.I. + +Institute for the Scientific Treatment of Delinquency The Institute's Annual Report for 1938 records a year of great expansion and development made possible by generous financial assistance received and by the facilities provided, as a result of the move to new premises, at 8 Portman Street, W.l. + +During the year, 166 new patients were seen, of whom 42 were treated. + +The total number of patients treated (old and new cases) was 106, and at the end of the year 26 were on the waiting list for treatment. In 48 other cases, advice was given without the patient being seen. An increase in the number of cases referred by Courts is noted, viz., 106 as against 92 the previous year. + +In 1938 (to give some examples of " Discharges and Disposals " as analysed in the Report) 7 cases were discharged as +" cured," i.e., as those in which +" some fundamental cause of the delinquency has been discovered and treated satisfactorily "; 26 were discharged as +" improved +" +; 23 were referred to other institutions; 24 were not suitable for psychological treatment, and 33 refused it. The largest diagnostic groups amongst the 166 new cases referred, were the "character cases" (56) and those classified as "psychoneurotic" (53). + +A table giving the types of offences for which patients were referred, gives the largest group as "Theft" (76), with "Sex Cases" (46) coming next in the list. + +The Institute's educational activities are increasing and a Week-end Lecture Course for Doctors was an innovation during the year. Another development in this branch of its work was the recognition of the Institute by the University of London as a centre for Extension Lectures, and in the autumn 57 students enrolled for a course of 24 lectures on +" Social Psychology," +forming part of the four-vear course for the University Diploma in Social Studies designed primarily for social workers and probation officers. + +## Special M.D. Schools In 1938 + +| given in regard to Special M.D. Schools :? | Residential | | | | | +|-----------------------------------------------|---------------|-----|----|-----|----| +| Day Schools | Schools | | | | | +| Maintained by Local Education Authorities | ... | 134 | _ | ... | 11 | +| Maintained by Non-Local Education Authorities | ... | 10 | | | | +| Total | 134 | ... | 21 | | | + +The total accommodation in these Schools, on March 31st, 1938, was 16,375 +(of which 4,246 was in schools maintained by the London County Council). This shows an actual decrease of 32 on the figures of the preceding year. + +## Report On The Voluntary Mental Health Services + +The final meeting of the Committee presided over by the Earl of Feversham, which for the past three years has been considering the position of the Voluntary Mental Health Services, was held on June 9th. + +It is hoped that the Committee's Report will be available within the next few weeks, and in our October issue, first place will be given to it. + +The price of the Report will, we are informed, be 2/6. Copies will be obtainable from the office of the Feversham Committee, 47 Whitehall, and from the C.A.M.W., 24 Buckingham Palace Road, S.W.I, or they can be ordered through any bookseller. + +## Scottish Training Week + +Under the Mental Deficiency Section of the Scottish Association for Mental Hygiene, a training week will be held in Glasgow from 9th to 13th October, 1939, for those engaged in nursing-care, teaching or visiting of ineducable children and adults, other than helpless ones. Lectures and practical work will cover most kinds of activity suitable for Occupation Centres, and visits will be paid to Centres in session, day and resident. The number of students will be limited, but anyone interested can receive particulars from Dr. Constance Hunter, Secretary, S.A.M.H., 23 Eglinton Crescent, Edinburgh. + +## South African Council For Mental Hygiene + +In our last issue, we reported the opening of two Occupation Centres in Johannesburg, under the auspices of the South African Council for Mental Hygiene. + +As we go to press, further news reaches us from the Secretary, Mrs. Netta Levine, who reports that the Government have now recognised the Centre, and a grant not exceeding ?250 per annum is to be made towards its maintenance by the Social Welfare Department. A second class is therefore to be started shortly, and plans are being considered for beginning the work also in Cape Town. + +The Council is hoping shortly to open a Hostel for stabilised feeble-minded male patients from institutions in whose case home conditions make discharge on leave impossible. In this venture also, the Department of Mental Hygiene is giving its support by a subsidy of a per capita grant of ?3 10s. per annum. It is proposed to limit the number of patients received to 16, in order that con-ditions may approximate as far as possible to a normal home. The underlying idea of the scheme is to re-establish these patients in community life, and when they are fully re-orientated and have proved themselves capable of earning their own living, to obtain their discharge from the provisions of the Act. \ No newline at end of file diff --git a/medical/md/PMC5297090.md b/medical/md/PMC5297090.md new file mode 100644 index 0000000000000000000000000000000000000000..03444ce9b4e49dec585ebc0048c59c2c951bcbcd --- /dev/null +++ b/medical/md/PMC5297090.md @@ -0,0 +1,25 @@ +# Periscope + +LEECH-THERAPY. + +No one can peruse the literature of a generation ag' , Marvelling at the prominent 'sional part leeches occupied m therapeusis of those ^ fam bygone days. In Scotland at ^ny Jv -t' looks and the Leeches keeps the memory ever green. Ana . as if leeches were coming into their own again. ouzon +^ the application of leeches acted partly by the quantity o re by leech extracted, which was not more than 10 to 15 cm., +the fact that from the leech-bite, long after the engorge fallen off, blood oozed drop by drop to the extent o 100 Less rapid than the old venesection the Wrhes application o constituted a small "bleeding." +The leech mnnhilia produces by its bite a condition of local has p the blood refuses to clot efficiently and blood from +^ +sue ?? + +gathered on a unurs glass slide or in a test-tube will remain many often many days, as fluid as when it was received. + +In 1884, a Haycraft showed that the head of the leech contained a substance inimical to coagulation, a substance which cou extracted by maceration, soluble in water but insou Intravenous injection of this solution in a rabbit or e^i 0o s n(j the coagulability of the blood, and even from such a sm an(^ as the puncture of the needle, gradual haemorrhage might even prove fatal. Weil and Boye showed that in continu^ t e r whole gamut of g symptoms of haemophilia might be pro uce , e and haematomata, dental haemorrhages, hsemarthrosis, haematu To this material man is more uniformly suscepti e an ^ and it has been observed that on the application of two to ?u , alone, wounds in the process of healing have recommence and severe epistaxis has occasionally followed. T^us ^ 0 the effect produced is not ^rranism only local but may affect t e w o e +.g and Weil and Boye have shown a coincident anaemia an a with excess of mononuclear cells. + +If, therefore, a condition ^lirWI in like haemophilia may e pr normal blood, could the substance which some produces it no^ a therapeutic use where thrombosis is the source of anger post- years surgeons have employed, perhaps empirically, eec es operative and in puerperal phlebitis. Leech-pro uce <- +seventy- yields a means of diminishing hypercoagulability. ^ ore three cases have been collected dolens. of its applicability to p To f?1*1 the lateral aspect of the thigh of the threatene +^ +ini leeches are even applied and this may be repeated two ay s a rapidly ?n a third occasion. The relief is very deci e , +219 diminishes, the temperature falls, cedema does not develop and the venous cords disappear. Instead of weeks of recumbency the patient is able to be out of bed within a fortnight. The two days' intermission is advisable, as the effect of the leeches appears about four to eight hours after application, reaches its maximum on the following day and persists for forty-eight hours. + +A prophylactic dose may be recommended immediately after parturition 'where thrombosis in varicose veins has given trouble in previous confinements. The post-auricular application of a leech in certain deep-seated ocular affections, hitherto empirical, now receives a reasonable explanation. Is it not possible that the more serious arterial thromboses might not yield to this treatment as the less serious venous affection does? Mouzon suggests its trial in intermittent c au ication.^ He considers that in other cases it might be effectively com ined with antispasmodic treatment. He warns that in cerebral cases the symptoms must be due to thrombosis and not to haemorrhage w ic the treatment would promote and he calls for scientific experimenta-tion of leech-therapy in its action on artificially produced vascular thrombi. + +J. Mouzon, "Le traitement des phlebitis thrombosantes par les sangsues" (La Presse medicate, Paris, 1927, xxxv., 677). \ No newline at end of file diff --git a/medical/md/PMC5402105.md b/medical/md/PMC5402105.md new file mode 100644 index 0000000000000000000000000000000000000000..6986a48cb446dec2aaf5a14005c75b38e00e6fc6 --- /dev/null +++ b/medical/md/PMC5402105.md @@ -0,0 +1,38 @@ +$\stackrel{a}{\mathrm{M}}\stackrel{C a s\theta}{\mathrm{B}}$. +Article IV.- -On a Case of Trance. By W. Allan Jamieson, M.B., Berwick-on-Tweed. + +Tiie exact nosological position of the following case is doubtful. + +It differs from true catalepsy inasmuch as there was an absence of plasticity in the limbs : thus the arms, though usually folded across the chest with the flexor muscles in a state of slight tonic contrac-tion, on being removed from that situation and raised up, dropped when no longer supported unresistingly by her side ; while the complexion was more highly tinted than in the waking state. It much more closely resembled somnambulism, the sleep being more profound than natural, though the imagination did not prompt to the performance of co-ordinate motor acts. That it was not feigned I am convinced, though some might call it a mere phase of hysteria, hysteric coma. From its indefiniteness I prefer the word trance, here used as expressing a morbid mental state lying on the borderland between somnambulism and catalepsy, in a person of hysteri-cal constitution. + +Catherine K., ?et. 16, domestic servant, a well-grown pretty girl, with dark hair, full lips, and brown eyes, shaded by drooping lids, of active lively disposition, though wayward temper. Her appetite had been capricious of late, but otherwise she was in good health up to the evening of the 25th February 1871. + +While in the passage leading up to her master's house shortly after six, she was struck a violent blow on the right cheek by a lad whom she had spoken sharply to for some rude conduct. Though the blow was painful and stunned her, it produced no other immediate effect, for she went down the street, delivered a message, and on her return did some household work. Shortly before seven she was found lying unconscious on the stair. She was removed to a sofa, cold water was dashed freely over her face, burnt feathers were applied to her nostrils, and sinapisms to her legs. These measures failing to rouse her, I was called in. + +I found her lying as if asleep, breathing quietly, with a pulse of 90, regular, and of good strength. The right cheek, over the malar bone, and round the eye, was a little swollen; and on touching this she shrank as if it painful. There was slight but constant quivering of both eyelids, which closed; on opening them the conjunctiva was insensible to touch. The pupil was moderately dilated and reacted to light. By-and-by there were slight symmet-rical clonic spasms of the legs and arms, which ceased when the mustard poultices were removed. No signs of consciousness being elicited by rough shaking or shouting, a pinch of snuff was gently blown up each nostril, but without causing any reflex action. I then threatened to bleed her, and after much preparation bared her arm, and drew a cold piece of iron across it, but there was not a trace of resistance. In deference to the wish of her relatives, a couple of leeches were put on the bruised part. The catamenia were said to be regular, though recurring every three weeks ; and it was nearly that time since their last appearance. She had a similar attack after a fright two years ago, lasting three hours and a half, from which she awoke crying. + +26th.?She awoke about 2 a.m., and again between 6 and 7, sat up for a short time while she arranged her dress and ate some biscuits, appearing quite rational, though complaining of pain in her limbs and heaviness in her head. The urinary secretion is nor-mal, only containing a slight leucorrhceal deposit; it is passed dur-ing the waking intervals. + +At half-past nine in the morning she was again in the state of trance, the pulse 90, temp. 98'6?. There was a peculiarity about her respiration: though always quiet, it presented a rhythmical arrangement; the inspirations gradually became fainter and fainter, till the cycle was closed about every five minutes by a deep sigh. + +During my visit she once called out, +" There he comes again, but never mind," in probable allusion to the lad who struck her. She did not again become conscious till 5 p.m., and then only for a short time. As the bowels had not acted since her seizure, a drop of cro-ton-oil was this evening placed upon her tongue. + +11th.?The medicine operated freely thrice, but the soporose con-dition continued without much change. During one of her rare waking intervals, she was led home, a distance of a hundred yards, and fell asleep on the way. In the evening, having propped her up in bed, I examined the fundus of the eye with the ophthalmo-scope. Owing to an irregular distribution of the retinal vessels in the right eye, and to her wincing when the eyelid was touched, the observation was made on the left. The colour of the fundus be-came paler as the region of the optic disc was approached, though whether this merely arose from deficiency of the superficial layer of choroidal epithelium, or from actual aneemia of the vessels of the choroid in that part,11 was unable to determine. Certainly the choroidal vessels very plainly visible even to their tolerably fine ramifications. There was, however, a marked contrast in size be-tween the retinal veins and arteries. Compared with their appear-1 Dr Hughlings-Jackson on the Retina in Sleep. See Carpenter's Physiology, 6th p. 590. + +ance after the attack had quite passed away, the veins larger and the arteries smaller, and at the time this was especially distinct, the veins standing boldly out against the white background afforded by the disc and orange-coloured choroid, while the arteries were tine and hairlike. There was no pulsation in either set of vessels. + +I watched for long before the disc came fully into view, but at last the eye remained sufficiently steady, with the optic nerve in the centre of the pupil, to allow me to assure myself of the correctness of the observation. + +28th.?At 10 a.m. was still in the state of trance, and had been so without waking since 5 p.m. yesterday. The skin of the right eyelid was to-day seen to be marked with reddish lines, and round the eye there was yellow subcutaneous staining. She awoke at eleven, soon again to relapse into unconsciousness; but at my evening visit she was sitting up in bed at length thoroughly roused. She declared she had never been asleep, said she remembered nothing since sitting down on the stair in her master's house on the evening of the 25th. + +Though several times asleep after this, the slumber was never again so profound, and she was almost quite well on the 3d March, on which day the catamenia appeared, though scantily. For a day or two after she was otherwise well, the vision of the right eye was rather indistinct. + +There are two points in this case (which in its duration reminds one of the classic legend of the nuptials of Alcmena and Jupiter) +deserving a moment's consideration. + +First, as to causation. Had the blow anything to do with the seizure ? The case appears to me to be an example of one of the rarer forms of the remote consequences of nerve-lesion. Hufeland1 relates a case of running and of catalepsy caused by a blow on the head. Attacks of hysteria often date their first appearance from a fall, or even from some injury so trivial apparently as to leave no visible trace. Again, there is a stage of incubation in hysteria and allied affections as truly pre-existent, though perhaps less evident, as in smallpox or measles. In many instances, this stage is marked by a degree of capriciousness as to diet, and this producing more or less detective nutrition favours the nervous instability, and uncommon reflex effects result in such persons from causes which in a healthy nervous system would be trifling. + +Secondly, as to the nature of the attack. The blow was followed by reflex vaso-motor changes; the circulation through the brain was impeded by the contraction of the arterial and dilatation of the venous system; renewal of tissue-change in the central nervous system was at its minimum, and a deep stupor was the result. By degrees this wore off, but the imperfection of vision, and the feeling of weight in the head, remaining after the return of consciousness, would show that the vascular system was slow in recovering its natural tension. + +1 Holmes's System of Surgery, 2d vol. iv. p. 191. \ No newline at end of file diff --git a/medical/md/PMC5402676.md b/medical/md/PMC5402676.md new file mode 100644 index 0000000000000000000000000000000000000000..76f4cd07229f83f8dfe1fce57820d5934f31858c --- /dev/null +++ b/medical/md/PMC5402676.md @@ -0,0 +1,388 @@ +Michela Balconi 1,2 and Maria Elide Vanutelli 1,2 1 Research Unit in Affective and Social Neuroscience, Catholic University of the Sacred Heart, Milan, Italy 2 Department of Psychology, Catholic University of the Sacred Heart, Milan, Italy + +## Abstract + +- .org 105 + +# Empathy In Negative + +and Positive Interpersonal Interactions. What is the Relationship Between Central (EEG, fNIRS) and Peripheral (Autonomic) Neurophysiological Responses? + +Emotional empathy is crucial to understand how we respond to interpersonal positive or negative situations. In the present research, we aim at identifying the neural networks and the autonomic responsiveness underlying the human ability to perceive and empathize with others' emotions when positive (cooperative) or negative (uncooperative) interactions are observed. A multimethodological approach was adopted to elucidate the reciprocal interplay of autonomic (peripheral) +and central (cortical) activities in empathic behavior. Electroencephalography (EEG, frequency band analysis) and hemodynamic (functional Near-Infrared Spectroscopy, fNIRS) activity were all recorded simultaneously with systemic skin conductance response (SCR) and heart rate (HR) measurements as potential biological markers of emotional empathy. Subjects were required to empathize in interpersonal interactions. As shown by fNIRS/EEG measures, negative situations elicited increased brain responses within the right prefrontal cortex (PFC), whereas positive situations elicited greater responses within the left PFC. Therefore, a relevant lateralization effect was induced by the specific valence (mainly for negative conditions) of the emotional interactions. Also, SCR was modulated by positive/negative conditions. Finally, EEG activity (mainly low-frequency theta and delta bands) intrinsically correlated with the cortical hemodynamic responsiveness, and they both predicted autonomic activity. The integrated central and autonomic measures better elucidated the significance of empathic behavior in interpersonal interactions. + +## Keywords + +interpersonal empathy, positive/negative interactions, fNIRS, brain oscillations; autonomic activity + +## Introduction + +The abilities to monitor and regulate emotional processes are parts of a functional model of empathic behavior (Chauhan, Mathias, & Critchley, 2008) which includes processes of *emotional resonance*. These are constituted by an affective response to another person, which often entails knowing what another person is feeling; sharing that person's emotional state; and, in some cases, having the intention to respond compassionately to another person's distress (Decety & Jackson, 2006; Hooker, Verosky, Germine, Knight, & D'Esposito, 2008; Ickes, 1997; Preston & de Waal, 2002). Specifically, the emotional behavior, in addition to the cognitive ability to share representations, constitute the basic components of empathy (Decety & Svetlova, 2012). + +However, limited previous studies explored empathy by using stimuli consisting of real interpersonal situations. In fact, previous research Corresponding author: Michela Balconi, Department of Psychology, Catholic University of the Sacred Heart, Largo Gemelli, 1, 20123, Milan, Italy. Phone: +39-2-72342586; fax: +30-2-72342280. E-mail: michela.balconi@unicatt.it E-mail: mariaelide.vanutelli@unicatt.it This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +mainly focalized on the emotional response to generic emotional cues and it did not include a specific empathic task (Balconi & Bortolotti, 2014; Balconi, Grippa, & Vanutelli, 2015a; Herrmann et al., 2008), or it explored facial expressions of emotions (Balconi & Canavesio, 2013; Herrmann et al., 2008) or empathy in specific domains (such as empathic responses to pain conditions, Avenanti, Sirigu, & Aglioti, 2010; Rêgo et al., 2015; Wang, Wang, Hu, & Li, 2014). In addition, exiguous research monitored analytically the effect induced by different types of empathic situations—that is, the positive versus negative valence of the situations in which the subjects were required to empathize (Balconi & Bortolotti, 2014; Herrmann et al., 2008; Silani & Singer, 2015). In one case, the valence effect was explored in an empathic context, although no specific effect was found in relation to both valence of the situation (positive or negative) and lateralization of brain activity (left or right) considered together. In other cases (see, e.g., Tullett, Harmon-Jones, & Inzlicht, 2012), significant right lateralized prefrontal patterns have been found in the case of empathy in negative circumstances. Here, the authors suggested the mediation of feelings of sadness in the development of the empathic mechanisms towards the suffering of other, together with the elicitation of prefrontal asymmetry. Nonetheless, results have often been proven to be inconsistent (see, e.g., Morelli & Lieberman, 2013). + +In the current study, we explored in an empathic context, both the valence of the situation (positive or negative) and the lateralization of brain activity (left or right). From a neurophysiological point of view, it has been established that empathic responses influence both cortical activity (Brüne et al., 2012; Decety & Jackson, 2006; Rameson & +Lieberman, 2009; Thirioux, Mercier, Blanke, & Berthoz, 2014) and autonomic physiological responsiveness (Balconi & Bortolotti, 2012b; Eisenberg et al., 1989; Prguda & Neumann, 2014). Indeed, as suggested by empathy models, the indubitable vantage of acquiring both autonomic and central activities is the possibility to better elucidate the reciprocal interplay of these two domains (Decety & Svetlova, 2012; Preston & de Waal, 2002). The multidimensionality of the construct of empathy makes it less compatible with single measures. However, so far central and peripheral measures were scarcely related to each other in empathy research (Balconi & Bortolotti, 2012b). The current study addresses this research gap. + +Concerning cortical activity correlates of empathy, previous neuroimaging studies on the emotional behavior in relation to empathy have revealed a range of areas activated in response to empathic interactions, specifically, during general emotional processing, the medial prefrontal cortex (MPFC, Seitz, Nickel, & Azari, 2006; ShamayTsoory & Aharon-Peretz, 2007) and the dorsolateral prefrontal cortex +(DLPFC, Balconi & Bortolotti, 2012a; Balconi, Bortolotti, & Gonzaga, 2011; Brüne et al., 2012; Damasio, Everitt, & Bishop, 1996; Davidson, 2002; Ochsner & Gross, 2005; Rameson, Morelli, & Lieberman, 2012). Moreover, electroencephalography (EEG) and lesion studies indicated that the prefrontal cortex (PFC) plays a prominent role in mediating empathy-related behaviors. Specifically, many studies reported a significant prefrontal involvement for the disruption of empathic behavior in the case of psychopathy (for a review see Pera-Guardiola et al., 2016). For example, as has been found by Howard and McCullagh (2007) in conditions involving both a categorization and a vigilance task with affective stimuli, psychopaths showed significantly smaller positive Slow Wave (pSW) amplitudes than healthy controls during the categorization task, where they were required to discriminate between living and nonliving stimuli, thus reflecting insensitivity to an affective mismatch between neutral backgrounds and positive pictures. Also, psychopaths showed a larger prefrontal negative event-related potential (ERP, N350), the amplitude of which positively correlated with the behavioral markers of psychopathy. Similarly, Kiehl, Hare, McDonald, and Brink (1999), by conducting a task comparing semantic and affective verbal information, reported greater centrofrontal negative-going wave amplitudes in psychopaths than controls. + +However, neither classical functional magnetic resonance imaging (fMRI) nor EEG seem to have completely uncovered in depth the physiological correlate of the emotional empathic experience, as both of these methods have their shortcomings: a low temporal resolution of fMRI and a low spatial resolution of activity below the cortical surface plus an insensitivity to the hemodynamic response of the EEG. + +Therefore, we applied optical imaging (i.e., near-infrared spectroscopy, NIRS) as a complementary method in the study of emotions and empathy. NIRS is particularly well-suited for evaluating PFC activity, which is among the regions involved in emotional processing (i.e., the frontopolar cortex and the DLPFC, Doi, Nishitani, & Shinohara, 2013). Due to its high temporal resolution, a spatial resolution exceeding that of the EEG, and its sensitivity for hemodynamic changes, NIRS seems well suited to study the temporally evolving representation and integration among complex, extended neural networks, of the empathic response. The temporal resolution of NIRS is high enough for measuring event-related hemodynamic responses (Elwell et al., 1993), and combined EEG/NIRS measurements allow for the complementary examination of neural as well as hemodynamic aspects of brain activation (Balconi et al., 2015a; Biallas, Trajkovic, Haensse, Marcar, & Wolf, 2012). + +Specifically, recent studies with functional NIRS (fNIRS) have identified the PFC as a key region in the experience and regulation of emotional responses (Brink et al., 2011; Nomura, Ogawa, & Nomura, 2010; Ogawa & Nomura, 2012). Based on this research, also a significant lateralization effect was found, related to the positive versus negative valence of the activating emotional context. Specifically, left PFC areas were more activated in response to positive or *approach* emotions, whereas right PFC areas were more activated in response to negative or *withdrawal* emotions (Balconi et al., 2015a; Balconi, Grippa, & Vanutelli, 2015b; Tullett et al., 2012). + +Concerning EEG, frequency band analysis contributed to elucidating the role of specific cortical areas, mainly with respect to the lateralization effect in emotional empathy processing, too. In fact, brain oscillations may furnish clear brain correlates of specific empathic contexts in terms of their valence (positive or negative) and in relation to cortical lateralization. However, the specific role of brain oscillations in affective and empathic behavior is partially unknown (Balconi & Lucchiari, 2006, 2008; Başar, 1999; Vanutelli & Balconi, 2015). Only few studies used brain oscillations to study empathy (Gutsell & Inzlicht, 2012; Moore, Gorodnitsky, & Pineda, 2012; Mu, Fan, Mao, & Han, 2008; Tullett et al., 2012). What is known from related investigations outside empathy research proper is that, regarding the alpha frequency band, lower-1 alpha desynchronizes in response to a warning stimulus (Klimesch, Doppelmayr, Russegger, Pachinger, & Schwaiger, 1998). Overall, changes in alpha power and lateralization effects related to these changes suggested that a right frontal unbalance is associated with negative emotions while relatively stronger left frontal activation is associated with positive emotions (Bekkedal, Rossi, & Panksepp, 2011). + +An anterior asymmetry was found in alpha activity that was explained as a correlate of changes in the affective state (Balconi, Brambilla, & Falbo, 2009a, 2009b; Davidson, 1998; Dimberg & Petterson, 2000). In addition, some studies showed that theta band power responds to prolonged visual emotional stimulation (Knyazev, 2007; Krause, Enticott, Zangen, & Fitzgerald, 2012). Therefore, the modulation of this frequency band may significantly contribute to the explanation of arousal effects on emotional cue comprehension (Bekkedal et al., 2011). In contrast, exiguous data concern the modulation of delta and beta band when considering the emotional significance of a stimulus (Karakaş, Erzengin, & Başar, 2000). In some cases, it was shown that delta could be a marker of novelty of the emotional cues and that it can respond to the exigency of stimulus updating in memory (Fernández et al., 1998). + +Finally, as markers of spontaneous and automatic empathic behavior, autonomic measures are very important for understanding the relationship between empathy and autonomic measures. It has been observed that different degrees of empathic experience may affect autonomic psychophysiological responses (Balconi, Falbo, & Conte, 2012; Prguda & Neumann, 2014). In those cases, participants imagined (a) a personal experience of fear or anger from their own past, (b) an equivalent experience from another person as if it were happening to them, or (c) a non-emotional experience from their own past (Ruby & +Decety, 2004). Autonomic differences were found between these conditions. Nevertheless, in this approach, only imagined (and not real) +empathic situations were proposed and this fact may have introduced important variations in the subjective responses. + +Systemic blood pressure (BP), heart rate (HR), and skin conductance response (SCR) were considered as potential biological markers of emotions in empathic behavior, and recorded simultaneously with EEG and NIRS (Tupak et al., 2014). Among the other dependent variables, SCR provides a useful measure of limbic function (Furmark, Fischer, Wik, Larsson, & Fredrikson, 1997; Lang, Davis, & Öhman, 2000). It is also a significant measure of arousal modulations, as has been demonstrated previously (Balconi et al., 2009a; Balconi & Pozzoli, 2008; Bradley & Lang, 2000). + +Also several NIRS studies underlined the association between PFC +activation and autonomic responses to emotional stimulation (see, e.g., Tanida, Katsuyama, & Sakatani, 2007). Likewise, during viewing of trauma-related video clips, increased hemodynamic activity +(oxy-hemoglobin, O2Hb) has been found to be positively correlated with heart rate change (Matsuo et al., 2003). Furthermore, Moghimi, Kushki, Guerguerian, and Chau (2012) have linked the steepness of the O2Hb peak to subjectively reported arousal levels, which is a widely accepted indicator of autonomic system activation (Matsuo et al., 2003; Roos, Robertson, Lochner, Vythilingum, & Stein, 2011). Moreover, a significant correlation between ventromedial prefrontal cortex (vmPFC) activation and SCR was found, based on stimulus content (its threatening value, Tupak et al., 2014). + +These previous studies supported the view that the prefrontal areas regulate autonomic reactions or somatic markers associated with emotional conditions. However, such research lacked a detailed and integrated analysis of all three levels (hemodynamic, electrophysiological, and autonomic) involved in emotional processing during empathic interactions. Only one previous study directly compared hemodynamic, EEG, and autonomic measures, but, firstly, it focused on generic emotional cues and, secondly, it was not on empathy (Balconi et al., 2015a). In contrast, the present research first of all clearly focused on empathic behavior by asking participants "to put themselves in the shoes of another person and try to feel what this person is feeling" (as reported in the procedural instructions). Secondly, the participants were required to observe situations where subjects performed specific interactions and not simply a generic emotional display (such as emotional pictures or faces). Therefore, in comparison with previous studies, a highly empathic task was included. + +In conclusion, in light of current knowledge on empathy, we propose an integration of cortical (EEG and fNIRS) measures with autonomic psychophysiological measures, as they have been shown to indicate the presence of emotional tuning between subjects. In the present study, EEG (frequency band analysis), systemic SCR and HR +were all recorded simultaneously with fNIRS measurements as potential biological markers of emotional responses to empathic situations during a natural and interpersonal situation in which positive versus negative contexts were represented. + +A consistent prefrontal activation was expected, as indicated by a hemodynamic modulation and brain oscillations. Both fNIRS and brain oscillations were supposed to elicit a significant PFC response to emotional interpersonal conditions. Specifically, as a correlate of an empathic response, we firstly expected a higher synchronous brain activity in low-frequency bands (delta and theta) and, in contrast, a desynchronization of the alpha band. Moreover, based on valence and lateralization effects of emotions (Balconi & Mazza, 2010; Russell, 2003), a significant and consistent higher prefrontal left activation was expected for positive emotional interactions, whereas a consistent higher prefrontal right activation was expected in response to negative interactions. Secondly, we expected that electrodermal activity (SCR) +and HR could be significant measures of implicit reactivity to emotional cues; they should consistently vary with emotional valence, with larger responses (increased SCR and HR) elicited in either negative or positive or both emotional conditions compared to neutral situations (Balconi et al., 2009a; Bradley & Lang, 2000; Lang, Greenwald, Bradley, & Hamm, 1993). + +Thirdly, we expected a high coherence between the three measures +(fNIRS, EEG, and autonomic variations). Significant correlations were hypothesized based on situational and interpersonal significance +(valence) of the empathic context. Indeed, we expected a relevant modulation within the left and right PFC for hemodynamic activity in concomitance with electrophysiological and autonomic response. + +## Materials And Methods Subjects + +Twenty-two subjects, 12 females and 10 males (Mage = 24.5 years; SD += 3.53; age range from 20 to 33 years) participated in the experiment. All subjects were right-handed (Edinburgh Handedness Inventory, Oldfield, 1971), with normal or corrected-to-normal visual acuity. Exclusion criteria were neurological or psychiatric pathologies of the subjects or their close family members. Specifically, they did not show deficits related to depression (Beck Depression Inventory II, BDI, Beck, Steer, & Brown, 1996) and to anxiety (State-Trait Anxiety Inventory, STAI, Spielberger, Gorsuch, Lushene, Vagg, & Jacobs, 1970): Exclusion criterion of the BDI Inventory was 19 points or lower (M = 8.95; SD = +0.46; score range from 2 to 12 points); for the STAI 39 points or lower (M = 28.45; SD = 1.03; score range from 27 to 45 points). No payment was provided for participation. Participants gave informed written consent and the research was approved by the Ethical Committee institution where the work was carried out. The experiment was conducted in accordance with the Declaration of Helsinki, and all the procedures were carried out with adequate understanding by the subjects. The Research Consent Form was submitted before participation in the study. + +## Stimuli + +Subjects were required to view affective images depicting real interpersonal situations which represented two people who interacted in a common and familiar situation (e.g., at home, in a workplace, or on a journey). Colored images (16 cm in width and 10 cm in height) representing positive, negative, and neutral interactions were selected. Twenty-four pictures were used for each type of interaction. Positive interactions represented positive and emotionally comfortable situations (such as a handshake between two people); negative interactions represented negative and emotionally uncomfortable situations (such as a quarrel between two people); neutral pictures represented interactions without a specific emotional valence (such as two people sitting on a couch, see Figure 1). All images were similar in their perceptual features (i.e., their luminance, complexity, i.e., number of details in the scene, and characters' genders: half of the actors were male and half were female). In order to validate the image dataset, a pre-experimental procedure was adopted. Each depicted scene was evaluated by four judges on valence and arousal dimensions, using the Self-Assessment Manikin Scale (SAM) with a five-point Likert scale (Bradley & Lang, 1994, 2007). Separately for each condition (positive, negative, and neutral), +ratings were averaged across all images presented. As shown by statistical analysis (two distinct repeated-measures analyses of variance +[ANOVAs] applied to valence and arousal), images firstly differed in terms of valence (positive: M = 4.56, SD = 0.34; negative: M = 1.33, SD = 0.26; neutral: M = 2.75, SD = 0.37)—positive interactions were more positively rated than the other two categories, negative interactions were more negatively rated than the other two categories, neutral images were rated to be of intermediate valence between the other two categories (for all significant contrast comparisons, p ≤ .01). Secondly, with respect to arousal, the positive and negative interactions (positive: M = 4.23, SD = 0.24; negative: M = 4.72, SD = 0.25; neutral: M = 1.77, SD = 0.31) were rated as more arousing than the neutral interactions (for all significant contrast comparisons, p ≤ .01). In contrast, no significant differences were revealed between positive and negative interactions (p = .32). + +## Procedure + +Subjects were seated in a dimly lit room, facing an LCD computer monitor that was placed at about 50 cm from the subject. The stimuli were presented using E-Prime 2.0 software (Psychology Software Tools, Inc) running on a laptop PC with a 15 in. screen (Acer TravelMate 250P). Images were presented in a random order in the center of the screen for 6 s, with an inter-stimulus interval of 8 s (see Figure 2). + +Participants were required to view each stimulus during fNIRS/ +EEG measures recording, and they were asked to attend to the interpersonal situations during the entire time of exposition, focusing on the emotional conditions which characterized the represented human actors. Moreover, they were required to empathize with the two persons interacting with each other ("Try to put yourself into the shoes of the persons and to experience their feelings in this situation"). In order to facilitate empathizing with the depicted actors, the two actors were of about the same age as the experimental subjects. + +Before scene presentations, a 2 min resting period was registered at the beginning of the experiment. Next, a familiarization phase followed, ![3_image_0.png](3_image_0.png) + +Figure 1. + +![4_image_0.png](4_image_0.png) + +Experimental setting with fNIRS, EEG, and autonomic measures. + +tional category), different from the images used in the experimental phase. After the experimental phase, subjects were required to rate the pictures with the SAM on valence and arousal dimensions. As shown by statistical analysis (two repeated-measures ANOVAs for the valence and arousal measures), images differed in terms of valence (with more positive evaluations of positive than negative and neutral interactions, with more negative evaluations of negative than positive and neutral interactions, and with intermediate evaluations of neutral compared to positive and neutral interactions) and arousal (with significant differences between positive and neutral interactions, and between negative and neutral interactions, showing a higher arousal rating for positive and negative interactions). For all paired comparisons significance was assumed for an alpha level of .01 or lower. + +A specific questionnaire was used in order to assess the subjects' self-rating on key aspects of the subjective evaluation of the empathic task. The questionnaire was used in a de-briefing post-experimental section (a five-point Likert scale for each item, from low to *high*). The aspects examined included the degree of experienced empathy ("How much did you put yourself into the shoes of the actors and felt what they felt in the depicted situation?"), personal emotional involvement ("How much did you feel emotionally involved in the situation?"), semantic attribution of the situation (positive, negative, and neutral, "How did you classify the interpersonal situation?"), and emotional significance (high or low, "Did you perceive an emotional significance of the situation?"). All subjects experienced a high sense of empathy (M = 4.11, SD = 0.26), were emotionally engaged in the task (M = 4.23, SD = 0.34), and were able to attribute a coherent emotional value to the pictures (for coherent semantic attribution of valence: M = 4.09, SD = +0.32; for emotional significance: M = 4.88, SD = 0.45). + +## Eeg: Frequency Band Analysis + +A 16-channel portable EEG-System (V-AMP, Brain Products) was used for data acquisition. An NIRS-EEG compatible ElectroCap with Ag/AgCl electrodes was used to record EEG from active scalp sites referred to earlobe (10/5 system of electrode placement). EEG activity was recorded from the following positions: AFF3, AFF4, Fz, AFp1, AFp2, C3, C4, Cz, P3, P4, Pz, T7, T8, O1, and O2 (for examples, see Figure 3). The cap was fixed with a chin strap to prevent shifting during the task. Additionally, one EOG electrode was placed on the lower side of the left eye. + +Data preprocessing has been conducted with BrainVision Analyzer 2 (Brainproducts). The data were recorded using a sampling rate of 500 Hz, with a notch filter of 50 Hz. The impedance of recording electrodes was monitored for each subject prior to data collection, and it was always kept below 5 kΩ (rejected epochs 4%). Blinks were also visually monitored. Ocular artefacts (eye movements and blinks) were corrected using an eye-movement correction algorithm that employs a regression analysis in combination with artefact averaging. After EOG correction and visual inspection, only artefact-free trials (not less than 22) were considered. To obtain a signal proportional to the power of the EEG frequency band, the filtered signal samples were squared and successively log-transformed (Pfurtscheller, 1992). Successively, the data were epoched, using a time window of 1 s and an average absolute power value was calculated for each electrode and condition. Artefact-free data have been used to compute power spectra for relevant EEG frequency bands by the Fast Fourier transform method (with a Hamming window of a length of 10%) that was used to obtain estimates of spectral power (μV2) in 1 Hz wide frequency bins for each electrode site. Spectral power values were averaged across all epochs and were then transformed to power density values for different frequency bands. An average of the pre-experimental absolute power (2 min) was used to determine the individual power without stimulation. From this reference power value, individual power changes during stimulus viewing were determined as the relative stimulus-related decreases or increases. Digital EEG data (from all 15 active channels) + +## Figure 3. + +![4_Image_1.Png](4_Image_1.Png) + +Locations of the prefrontal measurement channels of EEG and fNIRS. For fNIRS, emitter-detector distance was 30 mm for contiguous optodes and near-infrared light of two wavelengths (760 and 850 nm) were used. NIRS optodes were attached to the subject's head using a NIRS-EEG compatible cup, with respect to the international 10/5 system. + +were band-pass filtered in the following frequency bands: delta (0-3), theta (4-7), alpha (8-12), and beta (13-20). During data reduction, a bandpass filter was applied in the 0.01-50 Hz frequency band. + +## Fnirs + +- .org 110 software (Biopac Systems Inc) according to the manufacturer guidelines. ECG was converted to HR in number of beats per minute. The signal was low-pass filtered at 35 Hz and highpass filtered at 0.05 Hz for motor and ocular artefacts. For SCR, before attaching the electrodes, the skin was cleaned with alcohol and slightly abraded. The electrodes for SCR were attached to the distal phalanges of the first and second finger of the left hand. SCL was recorded using two Ag/AgCl electrodes and an isotonic gel. The signal was low-pass filtered at 10 Hz for motor, ocular, and biological artefacts. Ocular artefacts were then checked with a visual inspection to eventually eliminate specific elements. Trials with artefacts (2%) were excluded from the analysis. SCR elicited by each stimulus was registered continuously with a constant voltage. It was defined as the largest increase in conductance during emotional image presentation, with a cut-off of at least 0.3 μS in amplitude with respect to baseline (pre-stimulus) mean values. Baseline values were scored during the 2 min prior to task onset. + +fNIRS measurements were conducted with the NIRScout System (NIRx Medical Technologies, LLC) using a six-channel array of optodes +(four light sources/emitters and four detectors) covering the prefrontal area. Emitters were placed at AF3-AF4 and F5-F6 while detectors were placed at AFF1-AFF2 and F3-F4 (see Figure 3). Emitter-detector distance was 30 mm for contiguous optodes and a near-infrared light of two wavelengths (760 and 850 nm) was used. NIRS optodes were attached to the subject's head using a NIRS-EEG compatible cup, with respect to the international 10/5 system. + +With NIRStar Acquisition Software (NIRx Medical Technologies LLC), changes in the concentration of O2Hb and deoxygenated hemoglobin (HHb) were recorded from a 2 min starting baseline. Signals obtained from the six NIRS channels were measured with a sampling rate of 6.25 Hz and analyzed and transformed according to their wavelength and location, resulting in values for the changes in the concentration of O2Hb and HHb for each channel. Haemoglobin quantity is scaled in mM*mm, implying that all concentration changes depended on the path length of the NIR light in the brain. + +With Nirslab Software (v2014.05; NIRx Medical Technologies LLC) the raw data of O2Hb and HHb from individual channels were digitally band-pass filtered at 0.01–0.3 Hz. Successively, the mean concentration of each channel within a subject was calculated by averaging data across the trials for 6 s from trial onset. Based on the mean concentrations in the time series, we calculated the effect size in every condition for each channel within a subject. The effect sizes (Cohen's d) were calculated as the differences of the means of the baseline and trial divided by the SD of the baseline, d = (M1 + − M2 +)/SD1 +. Accordingly, M1 and M2 are the mean concentration values during the baseline and trial, and SD1 the SD of the baseline. The mean concentration value of the 2 s immediately before the trial was used as a baseline. Then, the effect sizes obtained from the six channels were averaged in order to increase the signal-to-noise ratio. Although the raw data of NIRS were originally relative values and could not be averaged directly across subjects or channels, normalized data, such as the effect sizes, could be averaged regardless of the units of measurement (Matsuda & Hiraki, 2006; Schroeter, Zysset, Kruggel, & Von Cramon, 2003; Shimada & +Hiraki, 2006). In fact, the effect size is not affected by differential pathlength factor (DPF, Schroeter et al., 2003). Instead of a block design, a continuous trial design was used in the present research. + +## Autonomic Measures + +Biopac MP 150 system (Biopac Systems Inc) was used to record the autonomic activity. Electrocardiography (ECG) was recorded continuously in lead 1 from two electrodes attached to the lower wrist, with the positive pole on the left arm and the negative pole on the right arm. One more reference electrode was placed over the left ankle. The ECG signal was sampled at 1,000 Hz with the Biopac Acknowledge 3.7.1 + +## Results + +The following set of analyses was performed on the data with SPSS software for Windows (version 18): A first set of repeated-measures ANOVAs was applied to each frequency band, a second set of analyses was applied to hemodynamic d values, and a third set of ANOVAs was applied to autonomic (HR, SCR) measures. Finally, stepwise multiple regression and correlational analyses (Pearson correlations) were applied to compare the three levels (band oscillations, d values, and autonomic measures). Bonferroni correction was inserted for multiple comparisons. + +## Eeg Frequency Band Analysis + +Frequency band data were entered into three-ways repeated-measures ANOVAs, with independent variables of Lateralization (two sides: left channels and right channels), Valence (3), and Localization (three sites: frontal, AFF3/AFF4 and AFp1/AFp2; temporo-central, C3/C4 and T7/ +T8; and parietal, P3/P4). Type I errors associated with inhomogeneity of variances were controlled by decreasing the degrees of freedom using Greenhouse-Geiser epsilon. Post hoc comparisons were successively applied to the data (contrast analyses for repeated-measures ANOVA). + +As shown by ANOVA, delta was modulated by valence, F(2, 42) = +6.16, p = .001, η2 = .27, and Lateralization × Valence, F(2, 42) = 7.23, p = .001, η2 = .29. No other main effect or interaction was statistically significant. Delta increased for negative and positive relative to neutral stimuli. Moreover, it increased for negative more than for positive interactions (for all comparisons, p ≤ .001). In addition, concerning the simple effects for the two-way interaction, significant differences were observed between positive and negative interactions, with increased delta within the right hemisphere for negative, F(2, 42) = 5.79, p = .001, η2 = .24, and within the left hemisphere for positive, F(2, 42) = 6.54, p = .001, η2 = .26, interactions (see Figure 4). + +For theta, the ANOVA revealed a significant main effect of valence, F(1, 13) = 6.56, p = .001, η2 = .33, and a significant Lateralization × + +![6_image_0.png](6_image_0.png) + +Figure 4. + +Valence interaction, F(2, 42) = 7.76, p = .001, η2 = .29. No other effect or interaction was statistically significant. Theta increased in response to negative relative to positive stimuli. Concerning the two-way interaction, significant differences were observed between positive and negative interactions, with increased delta within the right hemisphere for negative, F(2, 42) = 6.09, p = .001, η2 = .26, and within the left hemisphere for positive, F(2, 42) = 6.43, p = .001, η2 = .26, interactions (see Figure 4). + +Concerning the alpha band, the valence effect was statistically significant, F(2, 42) = 7.15, p = .001, η2 = .30. A generally decreased alpha (increased brain activity) was observed for positive and negative interactions. Finally, concerning beta, no significant effects were found (see Figure 4). + +## Fnirs + +The statistical analysis was applied to d—the dependent measure for O2Hb and HHb-concentrations. The analysis of HHb did not reveal any significant effects, and for this reason we report results for O2Hb values only. The lack of any significant effect for HHb may be due to the increase in O2Hb that is larger than the decrease in HHb (Wolf et al., 2002). D was subjected to a repeated-measures ANOVA, with Lateralization (2) and Valence (3) as independent variables. The data were averaged over left (Channel 1: AF3–F3; Channel 2: AF3–AFF1; Channel 3: F5–F3) and right (Channel 4: AF4–F4; Channel 5: AF4– AFF2; Channel 6: F6–F4) channels. + +As shown by the ANOVA, the effect of valence, F(2, 42) = 9.13, p +< .001, η2 = .41, and a Lateralization × Valence interaction, F(2, 42) = 8.13, p < .001, η2 = .32, were significant. No other effect or interaction was statistically significant. As shown by paired comparisons, negative and positive stimuli revealed increased d values in comparison to neutral interactions, F(1, 21) = 6.70, p < .001, η2 = .31, and F(1, 21) = +6.62, p < .001, η2 = .31, respectively. Moreover, negative interactions showed higher d values for negative than positive interactions, F(1, 21) = 7.50, p < .001, η2 = .32. Regarding the interaction effect, positive stimuli showed an increased brain activity within the left compared to the right hemisphere, F(1, 21) = 8.03, p < .001, η2 = .34, whereas negative stimuli showed an increased activity within the right compared to the left hemisphere, F(1, 21) = 8.88, p < .001, η2 = .35 (see Figure 5). In contrast, no significant differences were found for neutral interactions between left and right side, F(1, 21) = 1.16, p = .32, η2 = .16. + +## Autonomic Measures + +HR and SCR measures were analyzed with two separate repeatedmeasures ANOVAs, both with Valence (3) as an independent factor. + +![6_image_1.png](6_image_1.png) + +For SCR, the valence main effect was significant, F(2, 41) = 8.88, p < +.001, η2 = .32: Negative stimuli induced an increased SCR relative to positive and neutral conditions, F(1, 22) = 8.11, p < .001, η2 = .31, and F(1, 22) = 6.90, p < 0.001, η2 = .28, respectively. Moreover, the positive condition showed increased SCR values compared to neutral, F(1, 22) += 7.13, p < .001, η2 = .30. For HR, no effect or interaction was sig- + +## Figure 5. + +Hemodynamic states (O2Hb relative concentrations) as a function of size and valence (obtained with Nirslab Software, Data viewer section, Map tool). In response to negative stimuli, the concentration of O2Hb was higher for the right than the left side. Moreover, the concentration of O2Hb was higher in response to negative more than positive stimuli within the right side. + +Figure 6. +Mean values for SCR (up) and HR (down), with a significant effect shown for SCR based on positive versus negative valence. (M and SD reported. Asterisks mark statistical significance, with p ≤ .05.) +nificant (see Figure 6). No other effect or interaction was statistically significant. + +## Correlational Analyses + +Pearson's correlation analyses (across-subject correlations) were carried out on each frequency band power and d values. Correlations were calculated separately for each valence (positive/negative/neutral interactions) within the left and right prefrontal area. Extensive analyses were also applied to all the EEG and prefrontal fNIRS channels. However, since no significant effect was found in the posterior EEG channels, for the final analysis, we opted to compare the EEG and fNIRS data only for the prefrontal area. + +There was a significant positive correlation between d and theta (r += .491, p < .02, Variance Inflation factor [VIF] = .460) and between d and delta (r = .513, p < .01, VIF = .458) bands in response to negative stimuli within the right hemisphere. Moreover, significant positive correlations between d and theta (r = .561, p < .01, VIF = .511) and between d and delta (r = .544, p < .01, VIF = .493) bands in response to positive stimuli were observed within the left hemisphere. Finally, the alpha band showed a negative correlation with d (r = -.511, p < .01, VIF = .469) within the right hemisphere in response to negative stimuli. That is, cortical activation (alpha decreasing) was revealed within the right hemisphere for negative interactions (see Figure 7). No other correlations were statistically significant. + +## Regression Analysis + +Two stepwise multiple regression analyses were performed for positive and negative interactions. Predictor variables were Hemodynamic (d + +![7_image_0.png](7_image_0.png) + +![7_image_1.png](7_image_1.png) + +Scatter plots of correlational analyses between hemodynamic and EEG measures as a function of valence and lateralization. Each diamond corresponds to a single participant. + +## Table 1. + +Stepwise Multiple Regressions + +| a) | positive | negative | | | | | | | | | +|-----------|------------|------------|-------|-------|------|----------|-------|-------|-------|------| +| Predictor | d values | delta | theta | alpha | beta | d values | delta | theta | alpha | beta | +| Model | 1 | 2 | 3 | 4 | 5 | 1 | 2 | 3 | 4 | 5 | +| SCR R | 0.37 | 0.58 | 0.77 | 0.91 | 0.94 | 0.38 | 0.52 | 0.74 | 0.96 | 0.99 | +| R2 | 0.13 | 0.33 | 0.59 | 0.82 | 0.85 | 0.14 | 0.26 | 0.48 | 0.85 | 0.89 | +| β | 0.34 | 0.27 | 0.27 | −0.31 | 0.25 | 0.20 | 0.23 | 0.32 | −0.30 | 0.20 | +| std error | 0.18 | 0.20 | 0.22 | 0.18 | 0.23 | 0.15 | 0.20 | 0.11 | 0.21 | 0.26 | +| t | 2.12* | 1.84* | 1.63* | 1.75* | 0.98 | 2.35* | 1.87* | 1.99* | 2.01* | 0.75 | +| b) | positive | negative | | | | | | | | | +| Predictor | d values | delta | theta | alpha | beta | d values | delta | theta | alpha | beta | +| Model | 1 | 2 | 3 | 4 | 5 | 1 | 2 | 3 | 4 | 5 | +| HR R | 0.39 | 0.58 | 0.72 | 0.95 | 0.98 | 0.27 | 0.50 | 0.74 | 0.96 | 0.99 | +| R2 | 0.15 | 0.26 | 0.50 | 0.86 | 0.89 | 0.09 | 0.25 | 0.49 | 0.86 | 0.88 | +| β | 0.29 | 0.22 | 0.31 | −0.22 | 0.18 | 0.23 | 0.21 | 0.16 | −0.28 | 0.27 | +| std error | 0.25 | 0.20 | 0.26 | 0.20 | 0.21 | 0.20 | 0.25 | 0.24 | 0.20 | 0.17 | +| t | 1.98* | 1.83* | 1.78* | 1.98* | 0.77 | 2.01* | 1.94* | 2.04* | 2.08* | 0.83 | + +Note. (a) d, delta, theta, alpha and beta as predictor variables, SCR as predicted variable, and (b) d, delta, theta, alpha and beta as predictor variables, HR as predicted variable values) and EEG measurements, while the predicted variable was the Autonomic Modulation (separately for SCR and HR). In Table 1, we report the cumulative multiple correlations between predictors and predicted variables (R), cumulative proportion of explained variance (R2), +and the regression weights (β) for the regression equation at each step of the multivariate analysis. As shown in Table 1, delta, theta, and alpha frequency bands and d values predicted the SCR variations. Increased delta and theta and decreased alpha, as well as increased d were related to increased SCR in case of positive and negative conditions. Similarly, increased delta and theta and decreased alpha band powers, as well increased d were related to increased HR in response to positive and negative interactions. + +## Discussion + +The present research elucidated some main points to better comprehend the empathic response to interpersonal interactions. Our multilevel analysis, which included three measures (hemodynamic, electrophysiological, and autonomic), allowed us to investigate and support the significant key role of some specific brain areas—that is, the PFC, and some autonomic responses in empathic emotional behavior. Firstly, we found that the PFC was mainly recruited when the subjects empathized with actors in positive or negative interactions. + +Secondly, a lateralization effect was also revealed, as shown by both hemodynamic and brain oscillation modulations. Thirdly, the present data supported a significant valence effect, with increased PFC responses in the case of positive and negative interactions. Autonomic activity (SCR) was similarly responsive to the valence of the interactions, even if indistinctively for positive and negative pictures. Finally, a systematic combined modulation was detected for fNIRS and EEG measures, where both have a significant predictive role for autonomic (SCR and HR) activity, since, in the regression, both fNIRS and EEG were predictors of SCR and HR. + +The first effect we observed was related to the PFC, which was shown to be responsive to empathic situations where an emotional behavior is involved. O2Hb increased within the PFC. These results were in line with other results. For example, some recent studies revealed a significant contribution of the right DLPFC in response to positive and negative emotional faces. Along similar lines, right prefrontal stimulation +(high frequency repetitive transcranial magnetic stimulation, rTMS) +resulted in impaired disengagement from angry faces, with a significant DLPFC effect on attentional processing of emotional information +(De Raedt et al., 2010). It was also found that, when activated, the left DLPFC improved processing related to positive emotions and reduced negative emotional processing (Baeken et al., 2010). Neuroimaging studies have provided support for a functionally interactive network of cortico-limbic pathways that play a central role in the top-down regulation of emotions. Indeed, a large number of studies suggested that the PFC activates emotion regulation by inhibiting the amygdala (Siegle, Thompson, Carter, Steinhauer, & Thase, 2007). + +Results from previous fMRI studies indicated that the PFC is not only involved in emotion induction but also in emotion regulation. Moreover, by investigating the neural correlates of emotion regulation processes, NIRS studies underlined the role of the PFC. For example, the instruction to decrease the effect of negative stimuli by reinterpreting the displayed situation led to an increased PFC activation and a reduced activation of the amygdala (Banks, Eddy, Angstadt, Nathan, & Luan Phan, 2007; Eippert et al., 2007; Kalisch et al., 2005; Lévesque et al., 2003; Ochsner, Bunge, Gross, & Gabrieli, 2002; Ochsner et al., 2004; Phan et al., 2005). Herrmann et al. (2002) used NIRS to compare general emotional cue processing with processing of more specific facial patterns. They found increased medial PFC activity during an emotion induction paradigm which generated emotions by instructing participants to try to feel like a person whose facial expression was displayed. In accordance with these results, the instruction to remember emotional events leads to an increase of activation in the prefrontal brain areas (Ohtani, Matsuo, Kasai, Kato, & Kato, 2005). Furthermore, patients with post-traumatic stress disorder show increased PFC activation to disorder-related stimuli (Matsuo et al., 2003). In some cases, the social effect of emotional face processing was considered (Nomura et al., 2010). The study of Nomura et al. (2010) employed face stimuli and perspective-taking, and NIRS was used to show the individual differences in empathy that underlie the perspective taking function and the role of the right ventrolateral PFC. + +Although all these studies indicated an involvement of the PFC, for the first time in the present research, the empathic emotional response to interactional affective contexts was monitored. In addition, negative versus positive situations were systematically evaluated. Indeed, we revealed that emotional valence affected both hemodynamic activity and brain oscillations, with a more relevant impact for the negative interactions. In addition, this cortical activity was shown to be lateralized within the right hemisphere in response to negative situations and within the left hemisphere in response to positive stimuli. This result clearly supports the view of a lateralization effect in empathic responses to contexts of different valences when an empathic task was administered. + +Some specific brain oscillations (mainly delta and theta modulation) confirmed this lateralized activation effect of stimulus valence: +Low-frequency oscillations were mainly synchronized within the right and left side in response to negative and positive emotional interactions, respectively. The increased values of delta and theta that we found in response to positive and mainly negative interactions may support the hypothesis that delta plays a main role in regulating the attentional behavior in the case of salient stimuli. In line with this hypothesis, in previous studies, delta band was related to the relevance of the material being processed and to the degree of attention involved in visual stimuli processing (Balconi & Pozzoli, 2005; Keil et al., 2001). Therefore, in our case, brain responses to negative interactions could suggest that subjects could have perceived them as the most relevant emotional context, since they have a potentially threatening value. + +It should be noted that in the present research we did not find a significant and specific effect for higher frequency bands (beta). This is in contrast with previous research (Balconi & Pozzoli, 2009). These different results may be due to the adoption of different methodological approaches (e.g., task differences) and to different range limits used for the computation of the oscillations. + +A similar profile was observed for O2Hb measure, and the present results thus confirmed the homogeneity of the emotional empathic behavior in response to interpersonal situations by considering the hemodynamic level of analysis. These results were also supported by a consistent cortical lateralization effect for O2Hb, in combination with a specific prefrontal effect. A general left/right positive/negative distinction was observed in the subjects. That is, the subjects showed a distinct cortical lateralized response based on the emotional valence of the interactions: more left localized for positive situations; more right localized for negative situations. Indeed, increased brain activity was found to be based on stimulus valence. It has to be noted though that, compared with some previous research on neuroimaging and NIRS (Herrmann et al., 2008; Hoshi, 2009), we found that valence was relevant for hemispheric lateralization during processing of emotional cues. However, some differences were found based on valence (with increased activation for negative situations), as previously shown by EEG analysis. To account for the differences, we may assume that the most salient contexts to be processed are related to negative interpersonal interactions. Due to this higher degree of salience, higher cortical activation could have been evoked by more negative interactions. In general, it might be concluded that the fNIRS/EEG measures showed a broad sensitivity to the motivational significance of social interactions, varying as a function of the degree of negativity/positivity attributed to the emotional situations. A general right/negative association was observed in the subjects, and it was mainly supported by the right hemisphere—that is, negative, aversive interactions showed a more consistent lateralized brain activation when compared to other emotional situations (i.e., positive situations). Previous research underlined that human emotions are organized by two cortically lateralized systems: the appetitive and defensive motivation systems, presumably evolved from primitive approach and withdrawal tendencies +(Balconi & Bortolotti, 2014; Davidson, 1995; Davidson, Ekman, Saron, Senulis, & Friesen, 1990; Dickinson & Dearing, 1978; Lang, Bradley, & Cuthbert, 1990, 1997, 1998). + +In line with this theory, emotional activation fundamentally varies in centrally organized appetitive and aversive motivational systems that have evolved to mediate a wide range of adaptive behaviors that are necessary for an organism to survive (Bradley & Lang, 2007; Davidson et al., 1990; Lang et al., 1990). Most pleasant affects are held to be associated with the appetitive motivation system; unpleasant affects with defensive motivation (Cacioppo & Berntson, 1994). Specifically, aversive conditions were considered highly relevant for the survival since they include a threatening value (Fanselow, 1994; Russell, 1980). Also the autonomic behavior was related to empathic behavior, with an increased psychophysiological activity (higher SCR) for both positive and negative interactions. This response was attributed to general emotional involvement and to the ability to respond physiologically to the emotions displayed by other people in an interpersonal positive or negative situation. Indeed, as suggested by recent models of empathic behavior, a complex network of central and peripheral circuits supports the phylogenetic developments of a specific empathy-related response to conspecifics' emotional signs. The multiple elements of the empathic response are continuously modified during the social interactions and are contextually embedded (Decety & Svetlova, 2012). The relation between more central processes (mediated by PFC) and more peripheral processes (mediated by the autonomic system) confirmed the close relation existing between high order mechanisms (evolutionarily recent) +and the autonomic responsiveness (evolutionarily ancient). It was also underlined that behaviors specifically supported by arousal evolved earlier than the mechanisms supported by more complex cognitive processes (Decety & Svetlova, 2012). Moreover, it should be noted that relevant models of empathic behavior have pointed out that the emotional states related to empathy and the underlying neural mechanisms are similar in all mammals (Panksepp, 1998). + +The present results are also consistent with previously reported negative, empathy-related responses to unpleasant situations (Brown, Bradley, & Lang, 2006). Conflictual (negative) and cooperative (positive) situations were shown to be more powerful in eliciting empathic responses, presumably emotionally involving and significant, compared with neutral interpersonal conditions. In particular, the non-cooperative condition was negatively connoted, highly empathy-inducing, and able to produce a clear "negative" consonant autonomic reactivity. + +Moreover, positive and negative situations showed a relation between empathic emotional, psychophysiological, and central (both hemodynamic and EEG) measures. Indeed, it should be emphasized that important connections were found in the correlational analysis between hemodynamic and cortical EEG and the regression analyses between these two measures and the responses at the autonomic level. + +Firstly, the joined EEG-NIRS analysis revealed significant linear associations between the hemodynamic values and brain oscillations. + +The significant positive relation between NIRS and EEG measures may suggest, on the one hand, a general direct relation between these two measures and PFC activation since they synchronously varied within the prefrontal areas based on a situation's valence. On the other hand, the positive relation may support the connection between these two brain measures in response to empathic situations. More generally, the simultaneous registration of EEG and NIRS was found to be useful for studies on empathic behavior. A general link between electrophysiological effects and the regional hemodynamic changes was suggested based on present and past evidences (Balconi et al., 2015a; Herrmann et al., 2008; Schneider et al., 2014). + +To summarize, the significant correlations between EEG and NIRS +measures within PFC may suggest that a specific cortical prefrontal area supports empathic responsiveness. Indeed, whereas in band oscillations only a lateralization effect was found, the intrinsic relation between PFC activity observed in the EEG (mainly the low-frequency band) and the hemodynamic modulation may suggest the existence of a coherent prefrontal network for empathy. However, future research should explore the prefrontal localization of the EEG in more depth, also investigating potential cortical generator (e.g., with a LORETA approach) to define the reciprocal contribution by oscillations and hemodynamic measures. + +Secondly, regression analyses revealed that brain oscillations and hemodynamic variations might have affected autonomic responses by the subjects. That is, the PFC activity as marked by O2Hb increases and synchronous cortical activity (mainly for low-frequency bands) were significant factors, able to explain autonomic response modulation since subjects modified their autonomic parameters as a function of EEG/O2Hb changes in an empathic behavioral task. Specifically, increased SCR/HR was predicted by frequency band and hemodynamic activity in response to negative and positive interactions. + +To summarize, the direct relation between EEG and O2Hb, shown by correlational values, and the regression analysis, confirmed the interconnections between the three levels of processing (hemodynamic, electrophysiological, and autonomic). Indeed, the two analyses allowed respectively evidencing the direct relationship between the two independent measures (correlation analysis) and their consistent influence on autonomic responses (regression analysis). + +In the end, some limitations of this study and future suggestions for improved research should be considered. Firstly, future research should take into account the different roles that emotional and cognitive empathy might have in interpersonal interactions. 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Different time evolution of oxyhemoglobin and deoxyhemoglobin concentration changes in the visual and motor cortices during functional stimulation: A near-infrared spectroscopy study. *Neuroimage, 16*, 704–712. + +Received 09.09.2016 | Accepted 08.02.2017 \ No newline at end of file diff --git a/medical/md/PMC5417665.md b/medical/md/PMC5417665.md new file mode 100644 index 0000000000000000000000000000000000000000..cb76b53c2443e4ae14be946e81d8d6cf930ca948 --- /dev/null +++ b/medical/md/PMC5417665.md @@ -0,0 +1,155 @@ +Open Access Full Text Article + +# Case Report Open Access To Scientific And Medical Research Epidural Anesthesia For Labor And Delivery In A Patient With May–Hegglin Anomaly: A Case Report + +| Annas Muhammad Muzannara Mohammed Al Harbi Raza Rathore Nasser Tawfeeq Freddie Wambi Nasir Mahmood Sonia Albrechtova Vassilios Dimitriou | +|---------------------------------------------------------------------------------------------------------------------------------------------| + +Correspondence: Vassilios Dimitriou Department of Anesthesia, King Abdulaziz Medical City, National Guard Health Affairs, Riyadh 14611, Kingdom of Saudi Arabia Email vaskdimi58@gmail.com Abstract: We report a case of May–Hegglin anomaly (MHA) in a woman who had a successful labor and delivery under epidural anesthesia. MHA is an inherited thrombocytopenia easily misdiagnosed as idiopathic (immune) thrombocytopenic purpura (ITP). Early and appropriate diagnosis of MHA during pregnancy is essential for optimal maternal and neonatal delivery outcome. Additionally, it can avoid unnecessary diagnostic studies, such as bone marrow aspiration and biopsy, and even harmful therapies with corticosteroids, immunosuppressive agents, and splenectomy. Consequently, the most serious impacts of this disease are iatrogenic managements due to misdiagnosis. It seems that in patients with MHA, adequate clinical coagulation is far more dependent on adequate platelet function than any particular platelet count. The diagnosis of MHA may pose a challenge for clinicians managing pregnant women with thrombocytopenia. + +Keywords: May–Hegglin anomaly, thrombocytopenia, pregnancy + +## Introduction + +May–Hegglin anomaly (MHA) is a rare hematological disorder, inherited as an autosomal dominant trait, within the family of myosin heavy chain (MHC) disorders, including Fechtner syndrome, Sebastian syndrome, Epstein syndrome, and Alport-like syndromes.1–4 This group is also known as MYH9-related disorders (MYH9RDs), because all have largely overlapping phenotypes and result from mutations in the MYH9 gene on chromosome 22, which encodes the nonmuscle myosin heavy chainIIA (NMMHC-IIA) protein.3–5 NMMHC-IIA is a cytoplasmic protein with expression in many tissues, including platelets.1–4 MYH9RDs are characterized by varying degrees of thrombocytopenia, giant platelets, and large (2–5 μm), well-defined, basophilic, cytoplasmic inclusion bodies (resembling Döhle bodies) in the granulocytes.1–5 These inclusion bodies result from deposition of MHC within white blood cells. Interestingly, they are not seen in platelets, and their presence in leukocytes can differentiate MHA from idiopathic (immune) thrombocytopenic purpura (ITP).1–5 + +## Top Of Form + +MYH9RDs are considered very rare. The Italian Registry for MYH9RD indicates that the prevalence of the disorder in Italy is at least 3:1,000,000.6 Because mild forms are discovered incidentally and severe forms are often misdiagnosed or underreported as other disorders, the actual prevalence is expected to be higher. MYH9RD has been diagnosed worldwide, and there is no evidence of variation in prevalence across ethnic + +submit your manuscript | www.dovepress.com + +Dovepress + +53 + +© 2017 Muzannara et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work + +you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). + +Local and Regional Anesthesia 2017:10 53–58 + +populations.7 In general, the worldwide incidence of MHA +is unknown. + +Thrombocytopenia occurs in approximately 50% of the patients with MHA, and the clinical manifestations vary from mild bleeding not requiring specific treatment up to severe bleeding episodes following trauma or surgery that require blood products.1–3,8 However, despite severe thrombocytopenia in most patients, platelet function is frequently normal. + +Therefore, they are asymptomatic, discovered incidentally.1–3,8 Thrombocytopenia is common during pregnancy; however, MHA is very rare, with 40 cases reported in the literature.9 Diagnosis has been established prior to pregnancy in some women, but in the majority, the problem is first identified incidentally during pregnancy as thrombocytopenia on routine booking blood tests. Most of such cases, without careful inspection of blood smears and a thorough family and bleeding history, are initially misdiagnosed as refractoryto-treatment ITP.9 Consequently, the most serious impacts of this disease are iatrogenic managements due to misdiagnosis.3 The diagnosis of MHA may pose a challenge for clinicians managing pregnant women with thrombocytopenia. Herein, we report a case of MHA in a woman who had a successful labor and delivery under epidural anesthesia. The patient provided written informed consent for her data to be included in this case report. + +## Case Description + +The history of the patient starts 10 years ago on April 2006, when she presented as primigravida at 36 weeks gestation, booked for antenatal investigations. Patient was not known to have any medical illness or history of previous surgical procedures. Her platelet count was found to be 22×109/L, +and she was admitted as a case of possible ITP. Coagulation profile was within normal limits, and there was no history of bleeding tendency. Family history positive for bleeding disorders was not reported. She was started on steroid therapy (initially with prednisolone and later with dexamethasone); +however, the platelet count decreased to 19×109/L. After transfusion of 6 units of platelets, her platelet count was 20×109/L. Then, she received four cycles of rituximab (monoclonal antibody) and three doses of immunoglobulin, without response. Bone marrow biopsy revealed thalassemia trait and iron deficiency. Megakaryocytes and erythroblasts exhibited significant dysplasia. Differential diagnosis was between ITP and myelodysplastic syndrome. She had an uncomplicated cesarean section under general anesthesia, due to failure to labor progress. She received 18 units of platelets and 6 units of cryoprecipitate since the cesarean section. No bleeding tendency was observed perioperatively. Postoperatively, her platelet count was 65×109/L, with normal coagulation profile. + +She was discharged without any complication. + +After 3 months, a second bone marrow biopsy did not reveal additional pathology and confirmed the initial diagnosis (ITP). Her platelet count was 18×109/L, so the patient was scheduled for splenectomy due to refractory ITP. In August 2006, she was transfused with 8 units of platelets and underwent an uncomplicated laparoscopic splenectomy under general anesthesia, with minimal blood loss. The spleen pathology report was consistent with the clinical diagnosis of ITP. After splenectomy, she had a platelet count 8×109/L. Two months later, she showed improvement to 50×109/L and, at 6 months, to 100×109/L. However, then her platelet count decreased again and ranged between 7 and 20×109/L. + +In December 2007, the patient was then evaluated while she was on her second pregnancy at 2 months' gestation. Her platelet count was 7×109/L, and she was advised admission and the need for treatment. However, she signed against medical advice. She was transfused with 6 units of platelets and received intravenous (IV) immunoglobulin 55 g (1 g/kg) +and then was discharged with platelet count 45×109/L and on oral treatment with prednisolone. In June 2008, she had an uncomplicated labor and delivery that did not need any anesthetic management. During regular follow-up evaluations, the patient presented with hypothyroidism and started treatment with oral thyroxine. + +About 2 years later, in January 2011, during regular follow-up evaluations, microscopic assessment of a peripheral blood smear showed giant platelets and cytoplasmic inclusion bodies in the granulocytes. Therefore, the patient was diagnosed as suffering from MHA. Hemoglobin electrophoresis revealed findings consistent with alpha thalassemia trait and sickle cell trait with iron deficiency anemia. The patient had another bone marrow examination, which revealed that she did not have ITP and that her thrombocytopenia was due to *MYH9* gene-related hereditary macrothrombocytopenia. During her further course, she had two more pregnancies in 2011 and 2012, with spontaneous abortions. Her platelet count ranged between 8 and 20×109/L. + +In 2014, the patient presented for labor and delivery after her fifth pregnancy, with spontaneous rupture of membrane at 39 weeks' gestation. Her platelet count was 13×109/L, +and she was transfused with 6 units of platelets, which increased the platelet count to 24×109/L. The hematology consult was that the patient should be treated as a patient with a normal coagulation status and did not require any other specific treatment. Additionally, a neuraxial blockade + +![2_image_0.png](2_image_0.png) + +could be performed if needed. Once requested, an epidural catheter was easily inserted at L1–L2. The patient had an uncomplicated delivery and an uneventful hospital stay. She was discharged after 6 days. + +Figure 1 shows a graphic over a period of 9 years (2006– +2014), presenting the range of changes in platelet count, prothrombin time, and hematocrit of the patient. Platelet count was transiently increased only after massive platelet transfusions, while most of the time, it ranged between 10 and 20×109/L. At the same time, no significant changes were noted in prothrombin time and hematocrit, except during labor and deliveries. + +## Discussion + +We have presented a case of MHA in a woman who underwent a successful labor and delivery under epidural anesthesia. Review of literature revealed that neuraxial blockade has been rarely reported in the past in MHA cases. Spinal anesthesia for cesarean section has been applied in four cases and epidural analgesia applied for labor and delivery in five cases.10–13 Interestingly, there is still no consensus regarding the absolute lower limit of platelet count that confers safety for neuraxial anesthesia. Even the very recent (2016) guidelines for obstetrical anesthesia from the American Society of Anesthesiologists (ASA)14 avoid suggesting a clear guidance and just state: "A specific platelet count predictive of neuraxial anesthetic complications has not been determined." In general, a platelet count of ≥50×109/L is recommended for safe delivery.4 Therefore, in most cases, general anesthesia is preferred, due to severe thrombocytopenia and considering the risk of possible spinal or epidural hematoma.14–16 MYH9RDs result from mutations in the *MYH9* gene. + +Depending on the position of the causative mutation within the gene, the risk increases for syndromic clinical manifestations.3,17 Bleeding diathesis, high-tone hearing loss, glomerular nephropathy, and presenile cataract are the clinical features of MYH9RDs in descending order of frequency.5 Mutations in the neck region of the NMMHC-IIA protein are more likely associated with these comorbidities than mutations in the N- or C-terminal part of the gene.3,17 To date, at least 49 mutations of the *MYH9* gene have been identified.6 More specifically, MHA is caused by abnormalities in the *MYH9* gene located on chromosome 22q12–13 and encoding NMMHC–IIA.1–4 The diagnosis of MHA has been conventionally based on morphological criteria alone, by microscopic assessment of a peripheral blood smear after conventional staining (such as May-Grünwald-Giemsa staining), demonstrating a triad of giant platelets, thrombocytopenia, and inclusions in the cytoplasm of leukocytes.1–4 It is important to note that electronic cell counters work mainly based on the size and therefore they often classify very large platelets as erythrocytes. As a consequence, in MYH9RD, +these instruments greatly underestimate mean platelet volume, as well as platelet count.18,19 The pathogenesis of MHA is poorly understood. Reported platelet counts in MHA have ranged from less than 10×109/L +to normal levels, indicating variable expressivity; however, the platelet structure, function, and life span are usually normal.20 Additionally, no correlation was found between bleeding tendency and platelet count.2 A qualitative defect of platelets may be responsible for mild bleeding diathesis even in the absence of thrombocytopenia, while severe bleeding results from both qualitative and quantitative platelet defects. Bone marrow examination shows normal megakaryocytes. The cause of the thrombocytopenia in MHA is thought to be defective megakaryocyte maturation and fragmentation, which may account for production of large platelets.21 Thrombocytopenia is common during pregnancy. The most common causes include gestational thrombocytopenia, preeclamptic disorders of pregnancy and ITP. However, MHA is rare during pregnancy.9 Whenever a family history of thrombocytopenia is absent or unclear, evaluation of peripheral blood slides is a simple and effective tool to distinguish patients with MHA from those with ITP, as platelets are significantly larger in patients with MHA than in those with ITP. In particular, a mean platelet diameter >3.74 µm distinguishes MHA from ITP with 86% sensitivity and 87% specificity.22 The clinical spectrum of congenital thrombocytopenias in women ranges from severe bleeding tendency, recognized within the first few weeks of life, to mild conditions that may remain undetected even in adulthood. Positive bleeding history may include heavy menstrual bleeding, easy bruising, nose bleedings, bleeding after tooth extraction, and gum bleeding. Clinically, most women with MHA are asymptomatic, discovered incidentally, and require no specific treatment. In a recent systematic review of MHA during pregnancy, it was reported that 33% of women had no history of any bleeding symptoms.9 As the MHA is an autosomal dominant characteristic, the fetus has 50% chance of inheriting the pathogenic variant and presenting thrombocytopenia and MHA.1–5,23 Therefore, vaginal deliveries in women with severe thrombocytopenia should be considered at increased risk for neonatal intracranial bleeding.1,4 Approximately 65% of individuals diagnosed with MYH9RD have an affected parent.3–5 Prenatal diagnosis for pregnancies at increased risk is possible if the pathogenic variant in the family is known.4 The definitive diagnosis of MHA is limited to those with inclusion bodies in granulocytes, due to aggregates of abnormal NMMHCIIA, and no other organ dysfunction.8 Genetic analysis can be used if the evidences are nondiagnostic, but access to this test is generally only available through specialist molecular testing facilities. + +Differentiating between inherited and acquired thrombocytopenias may be difficult, and significant numbers of patients with MHA have been misdiagnosed with refractory ITP.3,24,25 In most cases, the first presentation is identified during pregnancy as thrombocytopenia during routine antenatal blood tests, with incidental finding of low platelet count. The biggest risk for these patients is that they are subjected to unnecessary, ineffective, and potentially harmful treatments based on the misdiagnosis of ITP.3 Consequently, the most serious impacts of this disease are iatrogenic managements due to misdiagnosis, and many of such patients suffer a lot before the final MHA diagnosis is established. Like in our case, a common scenario includes ineffective immunosuppressive (steroids) and intravenous immunoglobulin-G +treatment, unnecessary repeated platelet and cryoprecipitate transfusions, multiple painful bone marrow examinations, and eventually ineffective, unnecessary, and potentially harmful splenectomy for steroid-resistant ITP.26,27 Routine transfusions of platelet concentrates are used in patients with MHA, to transiently increase platelet count. However, this is unnecessary and carries associated risks of possible alloimmunization, producing antibodies against human leukocyte antigen (HLA) and subsequent refractoriness to platelet infusions.3–5 Therefore, platelets and blood should be ready available for use; however, they should be administered only if abnormal active bleeding occurs, which cannot be otherwise managed.4,28 When available, transfusing platelets from HLA-matched donors should be preferred, as this reduces the risk of alloimmunization.4,28 There is no known prevention or treatment for the nonhematopoietic consequences of MHA. Previous therapy with steroids and/or high-dose immunoglobulins had no effect on platelet count or bleeding diathesis.3–5,9 Hemostatic prophylaxis for labor and delivery may include desmopressin (DDAVP®), tranexamic acid, and cryoprecipitate transfusion.4,29 Preoperative use of desmopressin can be considered, because it has been proven to reduce the bleeding tendency in many acquired and congenital disorders of platelet function, including MHA.4,8,29 An assessment of the bleeding risk should be performed during the third trimester of pregnancy. Appropriate hemostatic cover can then be arranged to minimize the risk of primary postpartum hemorrhage. Women with no bleeding history and platelet count of at least 80–109×109/L are at low risk for bleeding and do not require platelet transfusion for labor and delivery.4 Desmopressin and/or tranexamic acid are generally adequate as hemostatic cover.4,29 However, women with a positive bleeding history or with platelet count less than 50×109/L may additionally require prophylactic platelet transfusion. Eventually, splenectomy, which is a treatment for refractory ITP, has not shown any improvement in the platelet count or any decrease in the bleeding tendency.26,27 Consequently, splenectomy is contraindicated in all hereditary macrothrombocytopenias, including MHA.26,27 + +## Conclusion + +MHA is an inherited thrombocytopenia that is easily misdiagnosed as ITP, if careful inspection of blood smear and family history are overlooked. Early and appropriate diagnosis of MHA during pregnancy is essential for optimal maternal and neonatal delivery outcome. Additionally, this can avoid unnecessary diagnostic studies, such as bone marrow aspiration and biopsy, and even harmful therapies with corticosteroids, immunosuppressive agents, and splenectomy. It seems that in patients with MHA, adequate clinical coagulation is far more dependent on adequate platelet function than any particular platelet count. A qualitative defect of platelets may be responsible for mild bleeding diathesis even in the absence of thrombocytopenia, while severe bleeding results from both qualitative and quantitative platelet defects. MHA should be suspected whenever a patient has a low platelet count or a bleeding diathesis of unknown origin. Early and appropriate diagnosis of MHA during pregnancy is essential for optimal maternal and neonatal delivery outcome. + +## Disclosure + +The authors report no conflict of interest in this work. + +## References + +1. Saito H, Kunishima S. May-Hegglin anomaly. *Am J Hematol*. 2008;83: +304–306. + +2. Noris P, Spedini P, Belletti S, Magrini U, Balduini CL. Thrombocytopenia, giant platelets, and leukocyte inclusion bodies (May-Hegglin anomaly): clinical and laboratory findings. *Am J Med*. 1998;104(4):355–360. + +3. Althaus K, Greinacher A. MYH9-related platelet disorders. Semin Thromb Hemost. 2009;35(2):189–203. + +4. Savoia A, Pecci A. MYH9-related disorders. *GeneReviews®* [Internet] +[updated July 16, 2015]. Available from https://www.ncbi.nlm.nih.gov/ books/NBK2689/. Accessed January 12, 2017. + +5. Balduini CL, Pecci A, Savoia A. Recent advances in the understanding and management of MYH9-related inherited thrombocytopenias. Br J Haematol. 2011;154(2):161–174. + +6. Saposnik B, Binard S, Fenneteau O, et al; French MYH9 networka. + +Mutation spectrum and genotype-phenotype correlations in a large French cohort of MYH9-related disorders. *Mol Genet Genomic Med*. 2014;2(4):297–312. + +7. Registro Italiano della Malattia MYH9-correlata [homepage on the Internet]. [updated June 2014]. Available from: http://www.registromyh9.org/. Accessed January 12, 2017. + +8. Sehbai AS, Abraham J, Brown VK. Perioperative management of a patient with May-Hegglin anomaly requiring craniotomy. *Am J Hematol*. 2005;79(4):303–308. + +9. Hussein BA, Gomez K, Kadir RA. May-Hegglin anomaly and pregnancy: +a systematic review. *Blood Coagul Fibrinolysis*. 2013;24(5):554–561. + +10. Fishman EB, Connors JM, Camann WR. Anesthetic management of seven deliveries in three sisters with the May-Hegglin anomaly. *Anesth* Analg. 2009;108(5):1603–1605. + +11. Duff P, Jackson MT. Pregnancy complicated by rhesus sensitization and the May-Hegglin anomaly. *Obstet Gynecol*. 1985;65(3 suppl):7S–10S. + +12. Kotelko DM. Anaesthesia for caesarean delivery in a patient with MayHegglin anomaly. *Can J Anaesth*. 1989;36(3 pt 1):328–330. 13. Nelson LH, Dewan DM, Mandell GL. Obstetric and anesthetic considerations in the May-Hegglin anomaly. A case report. *J Reprod Med*. + +1993;38(4):311–313. + +14. Practice guidelines for obstetric anesthesia: an updated report by the American Society of Anesthesiologists Task Force on Obstetric Anesthesia and the Society for Obstetric Anesthesia and Perinatology. Anesthesiology. 2016;124:270–300. + +15. Takabayashi R, Nishikido O, Nagano K, et al. Anesthetic management for cesarean delivery in a patient with May Hegglin anomaly. *Masui*. 2007;56:1198–1199. + +16. Garcia Vallejo G, Cabellos M, Kabiri M, Fraile JR, Cuesta J. Anaesthetic implications in a pregnant patient with an extreme thrombocytopenia due to a May-Hegglin anomaly: general or regional anaesthesia? *Rev* Esp Anestesiol Reanim. 2014;61:460–465. + +17. Pecci A, Panza E, Pujol-Moix N, et al. Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease. *Hum Mutat*. 2008;29(3):409–417. + +18. Althaus K, Greinacher A. MYH9-related platelet disorders. Semin Thromb Hemost. 2009;35(2):189–203. + +19. Urato AC, Repke JT. May-Hegglin anomaly: a case of vaginal delivery when both mother and fetus are affected. *Am J Obstet Gynecol*. 1998; 179(1):260–261. + +20. Hamilton RW, Shaikh BS, Ottie JN, et al. Platelet function, ultrastructure and survival in the May-Hegglin anomaly. *Am J Clin Pathol*. 1980; 74:663–668. + +21. Goodwin HA, Ginsburg AD. May–Hegglin anomaly: a defect in megakaryocyte fragmentation? *Br J Haematol*. 1974;26:117–127. 22. Noris P, Biino G, Pecci A, et al. Platelet diameters in inherited thrombocytopenias: analysis of 376 patients with all known disorders. *Blood*. + +2014;124(6):e4–e10. + +23. Seri M, Pecci A, Di Bari F, et al. MYH9-related disease: May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but represent a variable expression of a single illness. *Medicine*. 2003;82:203–215. + +24. Balduini CL, Noris P. Inherited thrombocytopenias. *Hematology*. + +2015;20:374–375. + +25. Balduini CL, Pecci A, Noris P. Diagnosis and management of inherited thrombocytopenias. *Semin Thromb Hemost*. 2013;39(2):161–171. + +26. Drachman JG. Inherited thrombocytopenia: when a low platelet count does not mean ITP. *Blood*. 2004;103(2):390–398. + +27. Balduini CL, Drachman JG. Role of splenectomy in inherited thrombocytopenias. *Blood*. 2004;104:1227. + +28. Chabane H, Gallais Y, Pathier D, Tchernia G, Gaussem P. Delivery management in a woman with thrombocytopenia of the May–Hegglin anomaly type. *Eur J Obstet Gynecol Reprod Biol*. 2001;99: +124–125. + +29. Rosen AC, Hafner E, Sternsite W, Kier P, Kyrle A. Das May-HegglinSyndrom in der Schwangerschaft. [Das May-Hegglin-Syndrom in der Schwangerschaft]. *Geburtshu Frauenheilk*. 1997;57:301–312. German. + + + +57 Local and Regional Anesthesia Publish your work in this journal Local and Regional Anesthesia is an international, peer-reviewed, open access journal publishing on the development, pharmacology, delivery and targeting and clinical use of local and regional anesthetics and analgesics. The journal is included in PubMed, and welcomes submitted papers covering original research, basic science, clinical studies, Submit your manuscript here: https://www.dovepress.com/local-and-regional-anesthesia-journal reviews and evaluations, guidelines, expert opinion and commentary, case reports and extended reports. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. + +Dovepress \ No newline at end of file diff --git a/medical/md/PMC5695379.md b/medical/md/PMC5695379.md new file mode 100644 index 0000000000000000000000000000000000000000..41160da29e5c311bc0cd66bd6dd9c21a47c699fd --- /dev/null +++ b/medical/md/PMC5695379.md @@ -0,0 +1,233 @@ +# Validation Of An Automated Delirium Prediction Model (Delirium Model (Demo)): An Observational **Study** + +Carlota Mestres Gonzalvo,1,2 Hugo A J M de Wit,1,3 Brigit P C van Oijen,1 Debbie S Deben,1 Kim P G M Hurkens,4 Wubbo J Mulder,5 Rob Janknegt,1 Jos M G A Schols,6 Frans R Verhey,7 Bjorn Winkens,8 Paul-Hugo M van der Kuy1,9 To cite: Mestres Gonzalvo C, +de Wit HAJM, van Oijen BPC, +et al. Validation of an automated delirium prediction model +(DElirium MOdel (DEMO)): an observational study. *BMJ Open* 2017;7:e016654. doi:10.1136/ bmjopen-2017-016654 +► Prepublication history and additional material for this paper are available online. To view these files, please visit the journal online (http://dx.doi. org/10.1136/bmjopen-2017016654). Received 10 March 2017 Revised 6 October 2017 Accepted 9 October 2017 + +## Abstract + +Objectives Delirium is an underdiagnosed, severe and costly disorder, and 30%–40% of cases can be prevented. + +A fully automated model to predict delirium (DEMO) in older people has been developed, and the objective of this study is to validate the model in a hospital setting. + +Setting Secondary care, one hospital with two locations. Design Observational study. Participants The study included 450 randomly selected patients over 60 years of age admitted to Zuyderland Medical Centre. Patients who presented with delirium on admission were excluded. + +Primary outcome measures Development of delirium through chart review. Results A total of 383 patients were included in this study. The analysis was performed for delirium within 1, 3 and 5 days after a DEMO score was obtained. Sensitivity was 87.1% (95% CI 0.756 to 0.939), 84.2% (95% CI 0.732 to 0.915) and 82.7% (95% CI 0.734 to 0.893) for 1, 3 and 5 days, respectively, after obtaining the DEMO score. Specificity was 77.9% (95% CI 0.729 to 0.882), 81.5% (95% CI 0.766 to 0.856) and 84.5% (95% CI 0.797 to 0.884) for 1, 3 and 5 days, respectively, after obtaining the DEMO score. Conclusion DEMO is a satisfactory prediction model but needs further prospective validation with in-person delirium confirmation. In the future, DEMO will be applied in clinical practice so that physicians will be aware of when a patient is at an increased risk of developing delirium, which will facilitate earlier recognition and diagnosis, and thus will allow the implementation of prevention measures. + +For numbered affiliations see + +![0_image_0.png](0_image_0.png) end of article. Correspondence to Carlota Mestres Gonzalvo; c.mestresgonzalvo@zuyderland. nl + +## Introduction + +A delirium or acute confused state is a transient attention and cognition disorder that develops over a short period of time and occurs mainly in hospitalised patients and people aged 60 years and over. Delirium is an underdiagnosed, severe (increased mortality), costly and often preventable disorder.1–3 Its severity and symptoms can vary considerably, but the main features are impaired cognitive and sensory functions, reduced consciousness and diminished + +## Strengths And Limitations Of This Study + +► A high risk of delirium can be predicted electronically by using DEMO (DElirium MOdel) with reasonably good sensitivity and specificity. + +► DEMO can be applied in clinical practice to facilitate earlier recognition and diagnosis of delirium. + +► Important factors that could predict delirium +(previous delirium, cognitive impairment, severity of disease, visual impairment, etc) are not included in this model because these data are not yet electronically available. + +attention. In addition, it is often accompanied by problems with psychomotor activity, the circadian rhythm and emotions. + +The prevalence and incidence of delirium in the general population differ widely depending on the setting. The overall prevalence in the community is estimated to be 1%–2%. In a hospital setting, this prevalence increases to 10%–31% at the time of hospital admission and 3%–29% during hospitalisation. The incidence increases up to 87% when more specialised populations, such as the elderly and people in postoperative, intensive care and/or palliative care, are considered.4–11 In 30%–40% of cases, delirium is preventable, which, along with its associated high costs (ranging from US$164billion to US$182billion per year), makes it a perfect target for interventions by healthcare professionals.1 4 12–15 As a result, a great number of screening tools have been developed and are widely used to detect the early onset of delirium, which can in turn allow treatment measures to be introduced in a timely manner.16–21 These tools help healthcare professionals to establish and quantify symptoms associated with delirium.19–23 Once the diagnosis has been established, the underlying medical condition can be targeted, and delirium can be managed appropriately. + +## Box **Delirium Model (Demo) And Cut-Off Point** + +DEMO score=1/(1+e−(linear predictor)) +DEMO score ≥14.1%→increased risk at delirium DEMO score <14.1% → no increased risk at delirium Linear predictor = -8.823+(0.081×V1)+(0.031×V2)+(0.248×V3)+(1.1 23×V4)+(0.286×V5)+(1.963×V6)+(0.359×V7)+(1.199×V8)+(0.413×V + +![1_image_0.png](1_image_0.png) + +There is no effective treatment for delirium.24 25 Preventing delirium is by far a more effective strategy to improve patient outcomes.1 4 26–29 Risk models have been used to identify patients at higher risk for delirium development because these patients would most likely benefit from delirium prevention. These models are based on manual evaluation of individual risk factors and may be difficult to implement, so automated models are preferable and more feasible.30–34 + +## Screening Instrument + +A fully automated model to predict delirium in older people (aged over 60 years) was developed at Zuyderland Medical Centre. This DElirium MOdel (DEMO) uses only electronically available data to predict the occurrence of delirium. The predictive variables include age, polypharmacy and the use of antidementia drugs, antidepressants, anti-Parkinson's agents, antidiabetic drugs, analgesia and/ or sleeping tablets (see box). This model can be applied hospital-wide and has an area under receiver operating characteristic (measure for model prediction quality) value of 0.770 (95% CI 0.736 to 0.804) with a sensitivity of 78.2% and a specificity of 63.7%, when 14.1% is used as a cut-off value for the predicted probability of developing delirium. DEMO was developed retrospectively but has not yet been validated.4 Therefore, the objective of this study is to validate DEMO in a hospital setting. To do so, the system's accuracy (main study parameter), that is, sensitivity (proportion of delirium patients who test positive) and specificity +(proportion of non-delirium patients who test negative), will be calculated. In addition to these parameters, the positive predictive value (PPV), negative predictive value (NPV), positive and negative likelihood ratios (LR+, LR−) with their 95%CI will be computed. + +Methods This is an observational study of the ability of DEMO to predict delirium in an elderly hospital population. It was conducted in Zuyderland Medical Centre (locations Sittard and Heerlen) in the period from January 2016 to October 2016. + +DEMO involves a daily analysis of all hospitalised patients aged ≥60 years at the different wards and predicts whether a patient is at risk of developing delirium in a 24-hour postanalysis period. The electronic patient record (EPR) was accessed at a later date to check for delirium diagnosis. In this study, DEMO was calculated prospectively, but the outcome was ascertained by chart review retrospectively. + +Although delirium diagnosis was determined by chart review, delirium documentation in our hospital is robust. At admission, patients are routinely screened for delirium, both in the emergency department and in the ward. The first screening is performed by a checklist (Inspectie voor de Gezondheidszorg=Dutch Healthcare Inspectorate, Veiligheids management systeem=Safety Management System and Dutch guideline for delirium).35 36 This checklist consists of three questions: does the patient need help with self-care?, has the patients previously suffered a delirium?, does the patients suffer from memory disorders? When one of the questions is positively answered, the patient is at risk of developing delirium; in this case, the Delirium Observation Screening Scale (DOSS) method20 is used to evaluate whether a patient has delirium and it is subsequently noted in the chart. + +Patients aged over 60 years who were admitted to Zuyderland were eligible for enrolment. From all patients admitted between 31 December 2015 and 31 October 2016, 450 patients were randomly selected (using https://www.randomlists.com/team-generator) and their charts extracted for review. Patients who, based on chart review, presented with delirium on admission were then excluded (see online supplementary figure 1). + +A search in the EPR was performed according to patient and date by using the following search terms: 'delirium', 'delirious', 'agitation', 'agitated', 'confused', 'confusion', 'restlessness', 'disturbed', 'disorientation', 'disoriented', 'apathy', 'hallucination', 'mistrust', 'haloperidol' and 'delirium prevention measures'. These search terms were discussed with an internist geriatrician, a professor of geriatric medicine and a professor of geriatric psychiatry. + +The search was performed by first identifying where the different words appeared in the EPR, and then, if any of these words appeared, the whole EPR during the admission period was read and interpreted by two authors (KH +(internist geriatrician) and CMG (hospital pharmacist)) to determine whether it was truly a delirium diagnosis. All notes were reviewed, including notes by physicians, nurses, physiotherapists and speech therapists. During the study, treating healthcare professionals (physicians, nurses, etc) were blinded to DEMO scores in order to avoid bias. If a diagnosis of delirium could not be established for a patient as a result of insufficient information + +| Table 1 | Test results of the prediction model (DEMO positive or negative) and diagnosis (delirium during admission or | | | | | | +|------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------|------------------------------------|----|-----|----|-----| +| no delirium during admission) within 1, 3 and 5 days after DEMO analysis Delirium within 1day after DEMO | No delirium within 5days after DEMO | | | | | | +| DEMO positive | 54 | 71 | 69 | 56 | 81 | 44 | +| DEMO negative | 8 | 250 | 11 | 247 | 17 | 241 | +| No delirium | Delirium | | | | | | +| within | within 3days | | | | | | +| 1day after DEMO after DEMO | No delirium within 3days after DEMO | Delirium within 5days after DEMO | | | | | +| DEMO, DElirium MOdel. | | | | | | | + +in the chart, this patient was excluded from the analysis. The date of delirium onset was determined by chart review. + +Delirium diagnosis based on chart review was then compared with the risk score from DEMO. The DEMO was dichotomised into two groups: high risk ≥14.1%,4 and low risk <14.1% for this analysis. A two-by-two table was then constructed to calculate true positive (TP), true negative (TN), false positive (FP) and false negative (FN) rates. + +The predictive value of DEMO was determined for delirium developing within 1, 3 and 5 days after the DEMO score was calculated. It had been developed to predict delirium within the next 24hours, but here we wished to also investigate whether its predictive value could be extended to 3 or 5days. + +In the study wherein the DEMO was developed, an incidence rate of 17.4% was used.4 Given the assumption of the same sensitivity of 0.75 (75%), we calculated that 33 delirium patients were needed based on the requirement that the lower limit of 95%CI would be at least 60% (width of 95%CI ≤0.30 (30%)). With regard to the specificity, the number of non-delirium patients would be much larger than the number of delirium patients, and hence, the width of the 95% CI for specificity would be <0.30. + +It was assumed that at least 332 patients would be needed to identify 33 delirium patients. Taking into account the exclusion criteria and the possibility of a smaller percentage of patients who would develop delirium, a sufficient number of patients were screened to obtain 33 delirium patients (ie, 450 patients). + +The sensitivity, specificity, PPV, NPV, LR+, LR− with corresponding 95% CIs were calculated with the use of an online calculator (http://vassarstats.net/clin1.html). + +The differences in PPV and NPV over time were tested using McNemar's test. The differences in age and gender between delirium and non-delirium groups were tested by using the independent-samples t-test and χ2 test, respectively. IBM SPSS statistics for Windows (V.23.0) was used to perform these tests. A two-sided p≤0.05 was considered statistically significant. + +## Results + +The study lasted 8months, for 450 patients chart review was undergone. Finally, a total of 383 patients were included, as 21 patients presented with delirium at admission, and for 46 patients there was insufficient information to determine delirium status (see online supplementary figure 1). The results of the diagnostic test (TP/FP/FN/ TN) for 1, 3 and 5 days after DEMO analysis are shown in table 1. The analysis, including prevalence estimates, sensitivity, specificity, PPV, NPV and LRs, is presented in table 2. Although sensitivity decreased and specificity increased if the period increased from 1day to 3 or 5 days after DEMO score was obtained, all values were rather high (sensitivity ≥0.827, specificity ≥0.779). PPV was statistically different p<0.001 for all three comparisons (1 vs + +| Table 2 | Estimates of the prevalence, sensitivity, specificity, PPV, NPV and LRs with corresponding 95% CIs 1, 3 and 5 days | | | | | | | | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|---------------------------|-------|-------------|-------|-------|--------|-------|-------| +| after DEMO analysis Day 1 after DEMO analysis | Day 3 after DEMO analysis | Day 5 after DEMO analysis | | | | | | | | +| 95% CI | 95% CI | 95% CI | | | | | | | | +| Lower | Upper | Lower | Upper | Upper | | | | | | +| limit | limit | limit | limit | Lower limit | limit | | | | | +| Prevalence | 16.2% | 0.127 | 0.204 | 18.8% | 0.150 | 0.221 | 25.6% | 0.213 | 0.303 | +| Sensitivity | 87.1% | 0.756 | 0.939 | 84.2% | 0.732 | 0.915 | 82.7% | 0.734 | 0.893 | +| Specificity | 77.9% | 0.729 | 0.822 | 81.5% | 0.766 | 0.856 | 84.5% | 0.797 | 0.884 | +| PPV | 43.20%* | 0.345 | 0.524 | 51.3%* | 0.419 | 0.607 | 64.8%* | 0.557 | 0.730 | +| NPV | 96.90% | 0.938 | 0.986 | 95.7% | 0.922 | 0.977 | 93.4% | 0.895 | 0.960 | +| LR+ | 3.938 | 3.140 | 4.939 | 4.560 | 3.526 | 5.898 | 5.354 | 4.020 | 7.129 | +| LR− | 0.166 | 0.087 | 0.317 | 0.193 | 0.112 | 0.332 | 0.205 | 0.133 | 0.316 | +| Estimated value | Estimated value | Estimated value | | | | | | | | +| *PPV: statistically different p<0.001 for all three comparisons (1 vs 3 days, 1 vs 5 days, 3 vs 5 days). DEMO, DElirium MOdel; LR, likelihood ratio; NPV, negative predictive value; PPV, positive predictive value. | | | | | | | | | | + +3 days, 1 vs 5 days, 3 vs 5 days), NPV was not statistically different p=0.25, 0.004, 0.031 for 1 vs 3 days, 1 vs 5 days and 3 vs 5 days, respectively. + +Patients who developed delirium within 5 days were significantly older (mean age 83.9 (SD 7.8)) compared with those who did not develop a delirium within 5 days (mean age 73.9 (SD 9.1); p<0.001). There was no significant difference in the percentage of males within the delirium and non-delirium groups (50.0% vs 50.1%, p=0.911). + +## Discussion And Conclusion + +In the current study, a previously developed model for predicting delirium has been validated. DEMO was calculated prospectively, and the outcome was ascertained by chart review retrospectively. Based on the current data and the high sensitivity and specificity, it can be concluded that DEMO is a satisfactory prediction model. + +Another strength of DEMO is that it predicts delirium within 5 days postanalysis on a daily basis. This is a novel concept, as most delirium prediction rules apply at admission but not daily. Even though it is not clear whether there is a definite advantage to predicting delirium on a daily basis, as this could lead to information overload, it could eventually be something that is tracked along with vital signs and intake/output. + +We found sensitivity and specificity rates that were higher than reported in the study by de Wit *et al*, which may be because his study only checked the patients' medical history for delirium and not the entire EPR. Moreover, de Wit *et al* had performed the search merely on the diagnosis of delirium. In the current study, the full EPR during the admission period was taken into account, and a wider set of terms was considered for delirium diagnosis. Furthermore, in the current study, in those cases in which delirium was not clear, these patients were excluded, whereas such patients had been included in the development of the delirium model.4 The present study does present some limitations. + +First, the validation of the DEMO depends on how and when a healthcare professional reports that a patient has developed delirium. It is well known that documentation of delirium is poor since the majority of delirium remains unrecognised by clinical teams.37 We therefore performed a wider search considering other words that might suggest delirium as delirium diagnosis and read through the whole EPR during the admission period. The number of delirium patients is noticeably higher than originally found, which can be explained by the search we performed. The DEMO is merely an aid to detect delirium, not a diagnostic tool by itself. Furthermore, for 46 patients there was insufficient information in the chart to determine delirium status, which could influence the generalisability of the present study. + +In addition, as mentioned in the study by Inouye *et al*, +38 using a chart review method has some limitations as it has a 30% false-positive rate and thus it is possible that patients with delirium at admission may have been included in the non-delirious cohort due to poor documentation in the chart. + +Furthermore, the checklist used to screen the patients is a non-validated tool. Nevertheless, after that first check, the DOSS is used. The DOSS method is a validated method used by nurses to screen for delirium. Its sensitivity ranges from 89% to 100%and its specificity ranges from 88% to 96.6%.20 39 40 The DOSS scores and its conclusion (delirium/non-delirium) are recorded in the chart. + +In that way, and taking into account that the chart is a complete document in which different healthcare professionals note their findings, makes the outcome more reliable and strengthens the validity of the present study. + +Another limitation of the present study is that this is a single-centre study (two hospital locations) located in the Netherlands and may not be generalisable in other settings. + +The DEMO uses only electronically available data. + +Other important factors that could predict a delirium (previous delirium, cognitive impairment, severity of disease, visual impairment, etc) are not included in this model because they were not electronically available. If these data were also made electronically available, the predictive quality of DEMO could be improved.22 23 27 30 Taking into account that the registration of such factors is becoming increasingly important and mandatory, it is only a matter of time until these important factors can be used in the DEMO.2 3 In addition, DEMO already uses an alternative way of identifying cognitive impairment by including medications used for dementia. + +The DEMO is a fully automated satisfactory prediction model that predicts delirium up to 5 days after analysis. The next step is to validate the DEMO in a cohort in which the outcome of delirium would be prospectively assessed in person and to use DEMO for retrospective measurements. In the future, DEMO will be applied to clinical practice so that physicians are alerted when a patient is at increased risk of developing delirium. This will facilitate earlier recognition and diagnosis and, thus, the implementation of prevention measures. + +## Author Affiliations + +1Department of Clinical Pharmacy, Pharmacology and Toxicology, Zuyderland Medical Centre, Sittard-Geleen, The Netherlands 2Department of Clinical Pharmacy, Elkerliek Hospital, Helmond, The Netherlands 3Department of Clinical Pharmacy, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands 4Section of Geriatric Medicine, Department of Internal Medicine, Zuyderland Medical Centre, Sittard-Geleen, The Netherlands 5Department of Internal Medicine, Maastricht University Medical Centre, Maastricht, The Netherlands 6Department of Family Medicine and Department of Health Services Research, CAPHRI-School for Public Health and Primary Care, Maastricht University, Maastricht, The Netherlands 7Department of Psychiatry and Neuropsychology, Alzheimer Centrum Limburg/ +School for Mental Health and Neurosciences, Maastricht University, Maastricht, The Netherlands 8Department of Methodology and Statistics, CAPHRI-School for Public Health and Primary Care, Maastricht University, Maastricht, The Netherlands 9Department of Hospital Pharmacy, Erasmus Medical Center, Rotterdam, The Netherlands Contributors All authors have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. They all have been involved in drafting the manuscript and revising it critically for important intellectual content. They all have given final approval of the version to be published; and they all agree to be 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. Conception or design of the work: CMG, HAJMdW, BPCvO, DSD, KPGMH, WJM, RJ, JMGAS, FRV, BW, PHMvdK. Data collection: CMG, DSD, KPGMH. Data analysis and interpretation: CMG, KPGMH, FRV, BW, PHMvdK. Drafting the article: CMG. Critical revision of the article: HAJMdW, BPCvO, JMGAS, FRV, BW, PHMvdK. Final approval of the version to be published: CMG, HAJMdW, BPCvO, DSD, KPGMH, WJM, RJ, JMGAS, FRV, BW, PHMvdK. Funding This work was supported by ZonMw (the Netherlands Organisation for Health Research and Development) grant number (113101001). Competing interests None declared. Ethics approval The medical ethics committee METC Z (Medisch Ethische Toetsings Commissie van Zuyderland en Zuyd Hogeschool, Zuyderland Medical Centre, Heerlen) approved this study. Provenance and peer review Not commissioned; externally peer reviewed. Data sharing statement All data are anonymised and will be confidentially handled. Only the investigators have access to the data. All patient data will be kept for as long as the project is being conducted. 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) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted. + +## References + +1. Inouye SK, Westendorp RG, Saczynski JS. Delirium in elderly people. + +Lancet 2014;383:911–22. + +2. National Institute for Health and Care Excellence. Delirium: diagnosis, prevention and management: NICE guidelines [CG103], 2010. + +3. Delirium for Adults Dutch Guideline. Richtlijn Delier Volwassenen. + +Nederlandse Vereniging voor Klinische Geriatrie, 2013. + +4. de Wit HA, Winkens B, Mestres Gonzalvo C, *et al*. The development of an automated ward independent delirium risk prediction model. Int J Clin Pharm 2016;38:915–23. + +5. Siddiqi N, House AO, Holmes JD. Occurrence and outcome of delirium in medical in-patients: a systematic literature review. Age Ageing 2006;35:350–64. + +6. Ryan DJ, O'Regan NA, Caoimh RÓ, *et al*. Delirium in an adult acute hospital population: predictors, prevalence and detection. *BMJ Open* 2013;3:e001772. + +7. Inouye SK. Delirium in hospitalized older patients. *Clin Geriatr Med* 1998;14:745–64. + +8. Bruce AJ, Ritchie CW, Blizard R, *et al*. The incidence of delirium associated with orthopedic surgery: a meta-analytic review. Int Psychogeriatr 2007;19:197–214. + +9. Girard TD, Ely EW. Delirium in the critically ill patient. Handb Clin Neurol 2008;90:39–56. + +10. Inouye SK. Delirium in older persons. *N Engl J Med* 2006;354:1157–65. + +11. Pisani MA, McNicoll L, Inouye SK. Cognitive impairment in the intensive care unit. *Clin Chest Med* 2003;24:727–37. + +12. Leslie DL, Marcantonio ER, Zhang Y, *et al*. One-year health care costs associated with delirium in the elderly population. Arch Intern Med 2008;168:27–32. + +13. Salluh JI, Wang H, Schneider EB, *et al*. Outcome of delirium in critically ill patients: systematic review and meta-analysis. BMJ 2015;350:h2538. + +14. WHO Regional Office for Europe. European hospital morbidity database. Copenhagen: World Health Organization, 2012. + +15. Organisation for Economic Co-operation and Development. OECD +health data 2012. Paris: Organisation for Economic Co-operation and Development, 2012. + +16. Adamis D, Sharma N, Whelan PJ, *et al*. Delirium scales: a review of current evidence. *Aging Ment Health* 2010;14:543–55. + +17. Wong CL, Holroyd-Leduc J, Simel DL, *et al*. Does this patient have delirium?: value of bedside instruments. *JAMA* 2010;304:779–86. + +18. Inouye SK, van Dyck CH, Alessi CA, *et al*. Clarifying confusion: +the confusion assessment method. A new method for detection of delirium. *Ann Intern Med* 1990;113:941–8. + +19. Han JH, Vasilevskis EE. Ultrabrief delirium assessments-are they ready for primetime? *J Hosp Med* 2015;10:694–5. + +20. Schuurmans MJ, Shortridge-Baggett LM, Duursma SA. The Delirium observation screening scale: a screening instrument for delirium. Res Theory Nurs Pract 2003;17:31–50. + +21. van Velthuijsen EL, Zwakhalen SM, Warnier RM, *et al*. Psychometric properties and feasibility of instruments for the detection of delirium in older hospitalized patients: a systematic review. *Int J Geriatr* Psychiatry 2016;31:974–89. + +22. Laurila JV, Laakkonen ML, Laurila JV, *et al*. Predisposing and precipitating factors for delirium in a frail geriatric population. J Psychosom Res 2008;65:249–54. + +23. Inouye SK. Predisposing and precipitating factors for delirium in hospitalized older patients. *Dement Geriatr Cogn Disord* 1999:10:393–400. + +24. AGS/NIA Delirium Conference Writing Group, Planning Committee and Faculty. The American Geriatrics Society/National Institute on aging bedside-to-bench conference: research agenda on Delirium in older adults. *J Am Geriatr Soc* 2015;63:843–52. + +25. Neufeld KJ, Yue J, Robinson TN, *et al*. Antipsychotic Medication for Prevention and Treatment of Delirium in Hospitalized Adults: A Systematic Review and Meta-Analysis. *J Am Geriatr Soc* 2016;64:705–14. + +26. Hipp DM, Ely EW. Pharmacological and nonpharmacological management of delirium in critically ill patients. *Neurotherapeutics* 2012;9:158–75. + +27. Young J, Leentjens AF, George J, *et al*. Systematic approaches to the prevention and management of patients with delirium. *J Psychosom* Res 2008;65:267–72. + +28. Teslyar P, Stock VM, Wilk CM, *et al*. Prophylaxis with antipsychotic medication reduces the risk of post-operative delirium in elderly patients: a meta-analysis. *Psychosomatics* 2013:54:124–31. + +29. Kalisvaart KJ, de Jonghe JF, Bogaards MJ, *et al*. Haloperidol prophylaxis for elderly hip-surgery patients at risk for delirium: a randomized placebo-controlled study. *J Am Geriatr Soc* 2005;53:1658–66. + +30. Kishi T. Antipsychotic medications for the treatment of delirium: +a systematic review and meta-analysis of randomised controlled trials. *J Neurol Neurosurg Psychiatry* 2016;87:767–74. + +31. van den Boogaard M, Pickkers P, Slooter AJ, *et al*. Development and validation of PRE-DELIRIC (PREdiction of DELIRium in ICu patients) delirium prediction model for intensive care patients: observational multicentre study. BMJ 2012;344:e420. + +32. Inouye SK, Viscoli CM, Horwitz RI, *et al*. A predictive model for delirium in hospitalized elderly medical patients based on admission characteristics. *Ann Intern Med* 1993;119:474–81. + +33. Carrasco MP, Villarroel L, Andrade M, *et al*. Development and validation of a delirium predictive score in older people. *Age Ageing* 2014;43:346–51. + +34. Douglas VC, Hessler CS, Dhaliwal G, *et al*. The AWOL tool: +derivation and validation of a delirium prediction rule. *J Hosp Med* 2013;8:493–9. + +35. Veiligheids Agenda. Platform voor patiëntveiligheid. http://www. + +vmszorg.nl/ (accessed 20 Jun 2017). + +36. Richtlijn delier Volwassen en ouderen. Guideline delirium adults and elderly (Richtlijn delier volwassen en ouderen). 2013 http://www. vmszorg.nl/_library/24018/Richtlijn%20Delier%20Volwassenen% 20en%20ouderen%202014.pdf 37. Collins N, Blanchard MR, Tookman A, *et al*. Detection of delirium in the acute hospital. *Age Ageing* 2010;39:131–5. + +38. Inouye SK, Leo-Summers L, Zhang Y, *et al*. A chart-based method for identification of delirium: validation compared with interviewer ratings using the confusion assessment method. *J Am Geriatr Soc* 2005;53:312–8. + +39. Gemert van LA, Schuurmans MJ. The Neecham confusion scale and the Delirium observation screening scale: capacity to discriminate and ease of use in clinical practice. *BMC Nurs* 2007;6:3. + +40. Koster S, Hensens AG, Oosterveld FG, *et al*. The delirium observation screening scale recognizes delirium early after cardiac surgery. *Eur J Cardiovasc Nurs* 2009;8:309–14. \ No newline at end of file diff --git a/medical/md/PMC5808842.md b/medical/md/PMC5808842.md new file mode 100644 index 0000000000000000000000000000000000000000..b91bcd896e28066152f04464b7ab33ced3d3d3a1 --- /dev/null +++ b/medical/md/PMC5808842.md @@ -0,0 +1,320 @@ +# Atezolizumab: Feasible Second-Line Therapy For Patients With Non-Small Cell Lung Cancer? A Review Of Efficacy, Safety And Place In Therapy + +Ther Adv Med Oncol 2017, Vol. 9(12) 769–779 DOI: 10.1177/ +1758834017741074 https://doi.org/10.1177/1758834017741074 https://doi.org/10.1177/1758834017741074 + +© The Author(s), 2017. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav Fanny Jean, Pascale Tomasini and Fabrice Barlesi Abstract: Advanced non-small cell lung cancer (NSCLC) prognosis is still poor and has recently been reformed by the development of immune checkpoint inhibitors and the approval of anti-PD-1 (programmed cell-death 1) treatments such as nivolumab and pembrolizumab in second line. More recently, atezolizumab (MDPL 3280A), a programmed cell-death-ligand 1 (PD-L1) inhibitor, was also studied in this setting. Here, we report a review of the literature assessing the efficacy, safety, and place of atezolizumab in the second-line treatment of advanced NSCLC. We performed a literature search of PubMed, American Society of Clinical Oncology, European Society of Medical Oncology and World Conference on Lung Cancer meetings. Atezolizumab showed a good tolerance profile and efficacy in comparison with docetaxel for second-line treatment of advanced NSCLC. Potential predictive biomarkers also have to be assessed. + +Keywords: atezolizumab, immunotherapy, lung cancer, PD-L1, second-line Received: 6 July 2017; revised manuscript accepted: 28 September 2017 + +## Introduction + +Lung cancer is the leading cause of cancer death worldwide.1 Indeed, non-small cell lung Cancer +(NSCLC) prognosis is poor with an overall survival rate at 5 years of 15% and until recently, treatment options in the second- or third-line setting for advanced diseases were limited.2,3 However, in the last few years, comprehension of cell biology has improved, and mechanisms allowing cancer cells to avoid immune destruction,4 relying both on innate and adaptive immunity, are better understood and addressed. Regarding adaptive immunity, there are two phases: during the first phase, dendritic cells interact with T cells which will then induce cancer cell destruction in the second phase. Inhibitory signals occur in both phases, involving membrane molecules of which the most important representative is the couple PD-1 (programmed cell-death 1) expressed by T cells with PD-L1 (programmed cell-death-ligand 1) on cancer cells. The last few years have seen the development and approval of anti-PD-1 therapies such as nivolumab in pretreated squamous5 and nonsquamous6 NSCLC, or pembrolizumab in previously treated, PD-L1-positive, advanced NSCLC.7 Therefore, recent guidelines established new recommendations including anti-PD-1 immunotherapy for second line treatment of NSCLC.8,9 However, PD-L1 blockade is distinct, since it allows PD-1 to bind its other ligand, PD-L2 which may be important in possibly preventing severe immune adverse effect events such as pneumonitis.10–12 Atezolizumab (MDPL 3280A) is a humanised engineered IgG1 monoclonal antibody targeting PD-L1 usually used with its specific companion immunohistochemistry (IHC) diagnostic assay, SP142 (Ventana Medical Systems, Inc. Arizona, USA), assessing PD-L1 expression both on tumour cells (TCs) and on tumour-infiltrating immune cells (ICs). + +The percentage of PD-L1-positive cells is expressed by a score including PD-L1 expression levels on both TCs and ICs. On TCs, TC3, TC2, TC1 and TC0 are respectively correlated with PD-L1 expression levels of ⩾50%; ⩾5% and <50%; ⩾1% +Correspondence to: Fabrice BARLESI Service d'Oncologie Multidisciplinaire & Innovations Thérapeutiques, Pôle Cardio-Vasculaire et Thoracique, Aix Marseille University, Assistance Publique - Hôpitaux de Marseille Hôpital Nord - Chemin des Bourrely, 13915 Marseille Cedex 20, France fabrice.barlesi@ap-hm.fr Fanny Jean Multidisciplinary Oncology and Therapeutic Innovations Department, Aix Marseille University, Marseille, France Pascale Tomasini Multidisciplinary Oncology and Therapeutic Innovations Department, Aix Marseille University, Marseille, France Aix Marseille University, Inserm U911 CRO2, Marseille, France and <5%; and <1%. On tumour-infiltrating ICs IC3, IC2, IC1 and IC0 are respectively correlated with PD-L1 expression levels of ⩾10%; ⩾5% and +<10%; ⩾1% and <5%; and <1%.13–16 In this article, we are proposing a review of the efficacy and safety of atezolizumab in locally advanced or metastatic NSCLC, focusing on its place as second-line treatment of NSCLC. + +## Methods + +A literature search of PubMed, ASCO (American Society of Clinical Oncology), ESMO (European Society of Medical Oncology) and WCLC (World Conference on Lung Cancer) meeting abstracts, as well as a review of ClinicalTrials.gov was conducted using the following terms: 'Atezolizumab', +'MDPL3280A', 'lung cancer' or 'Non-Small Cell Lung Cancer'. We chose to include clinical trials, meta-analyses and communications from international congresses between 2015 and 2017, since no clinical data were available on atezolizumab before 2013. + +## Results + +In PubMed, among 53 articles found, 4 were clinical trials. One phase I trial was excluded due to the lack of data compared with the others. Moreover, four articles were meta-analyses, including one with insufficient atezolizumab data, not reported here. Among congress reports, between 2015 and 2017, we found 19 abstracts. Two of them, being preliminary data further published in a larger trial or not reporting enough data about atezolizumab, were excluded. Figure 1 reports the flow chart of articles and reports included in this review. + +## Efficacy Phase I + +Herbst and colleagues conducted a phase I study17 aiming to evaluate the single-agent safety and tolerability of MDPL3280A/atezolizumab administered by intravenous infusion every 3 weeks (q3w) +to patients with locally advanced or metastatic solid tumours or haematological malignancies. A +total of 277 patients were included, mostly suffering from NSCLC (31%), renal cell carcinoma +(25%) and melanoma (16%). In the NSCLC subgroup, patients had a median age of 60 years and were mostly males (56%) performance status 0–1. A total of 76% had nonsquamous NSCLC, only 5% had central nervous system (CNS) +metastasis, 80% had a tobacco history and 55% had received at least three prior systemic regimens. In the dose-escalation phase of the study, patients received atezolizumab q3w from 0.01 mg/kg to 20 mg/kg and the expansion phase enrolled patients at 10 mg/kg, 15 mg/kg or 20 mg/kg q3w. Mean terminal serum half-life of 3 weeks was reached with a minimum dose of 1 mg/kg. A dose of 15 mg/kg q3w was sufficient to maintain target drug levels and the equivalent fixed dose of 1200 mg q3w was moved forward in clinical development of single-agent atezolizumab. In this phase I study, 21% of patients with NSCLC showed a confirmed response according to Response Evaluation Criteria in Solid Tumours, version 1.1 (RECIST 1.1). The median progression-free survival (PFS) of all patients was 18 weeks. The association between response to atezolizumab and tumour-infiltrating ICs PD-L1 expression reached statistical significance in NSCLC (p = 0.015). These data were confirmed in a Japanese phase I study in 2016.14 + +## Phase Ii - Poplar Trial + +POPLAR15 was a phase II clinical trial testing atezolizumab 1200 mg q3w *versus* docetaxel 75 mg/m² q3w in the treatment of stage IIIb–IV +NSCLC patients previously treated with platinum-based first-line chemotherapy. Overall survival (OS) favoured atezolizumab *versus* docetaxel with a hazard ratio (HR) of 0.73 [95% confidence interval (CI) 0.53–0.99], p = 0.040 in the intentto-treat (ITT) population. Increasing improvement in OS was correlated with increased PD-L1 expression. However, PFS was not significantly improved in the atezolizumab arm: HR = 0.94 (95% CI 0.72–1.23), p = 0.645 (ITT +population). An objective response rate (ORR) of 38% was noticed in the TC3 or IC3 subgroup. Objective responses with atezolizumab were durable, with a median duration of 14.3 months (11.6–nonestimable) compared with 7.2 months (5.6–12.5 months) for docetaxel. This gap between atezolizumab and docetaxel was even wider in updated data presented at ASCO congress in 2016.18 An ongoing phase II trial, BIRCH, is currently conducted in first or more lines of treatment in preselected patients with IC2/3 or TC2/3 PD-L1 + +![2_image_0.png](2_image_0.png) + +expression profile [ClinicalTrials.gov identifier: +NCT02031458].19,20 In the first-line subgroup, ORR was 19%; 6-month PFS was 46%; 6-month OS was 82%; whereas in the second line subgroup, ORR was 17%; 6-month PFS was 29% +and 6-month OS was 76%.19 + +## Phase Iii - Oak Trial + +The following phase III trial, OAK,16,21 highlighted the efficacy of atezolizumab in second-line treatment of NSCLC, with a median OS of 13.8 months in the atezolizumab arm (95% CI 11.8– 15.7) *versus* 9.6 months in the docetaxel arm +[(8.6–11.2); HR 0.73 (95% CI 0.62–0.87), p = +0.0003]. PFS was similar between treatment groups in the ITT population [HR 0.95 (95% CI 0.82–1.10)]. There was no difference regarding objective response between the two groups with an ORR of 14% with atezolizumab and 13% with docetaxel in the ITT population. Characteristics of TC3 or IC3 population were: median age of 64 years, mostly males (64.2%), White (77.4%), previous (65%) or current (19.7%) smokers, *EGFR* wild type (73.7%) and with nonsquamous NSCLC (70.1%). + +Treatment beyond progression (TBP) is authorized if the investigator deemed the patient to be receiving clinical benefit and if patients consented to continuation. Clinical benefit is defined by an absence of unacceptable toxicity, a symptomatic deterioration attributed to disease progression after an integrated assessment of radiographic data, biopsy results (if available) and clinical status. New data from the OAK trial22 suggest that TBP with atezolizumab is efficient, as presented in ASCO 2017, where a pool of patients continue to receive the anti-PD-L1 agent after disease progression if a clinical benefit was still present. + +Among 332 patients with PD while treated by atezolizumab, 51% (168) continued anti-PD-L1 therapy. A total of 7% achieved subsequent response from new baseline (at PD), 49% had stable target lesions and median of OS (mOS) was 12.7 months (95% CI 9.3–14.9) while those who received other anticancer therapy (chemotherapy or new line of immunotherapy) had an mOS of 8.8 months (95% CI 6.0–12.1). Safety profile seemed to be tolerable. Consequently, there would be an interest of using atezolizumab in postprogression prolongation of survival. + +## Subgroup Analyses Pd-L1 Expression + +In the POPLAR study,15 OS was correlated with PD-L1 expression level since OS in the TC1/2/3 or IC1/2/3 subgroups was higher in the atezolizumab [HR of 0.59 (95% CI 0.33–0.89), p = +0.014], whereas OS was not improved by atezolizumab in the TC0 and IC0 groups [HR 1.04 +(0.62–1.75), p = 0.871]. Unlike in POPLAR, the OAK study16,21,23 showed a survival advantage for atezolizumab *versus* docetaxel even in the TC0 or IC0 subgroups (45% of the patients) with an HR of 0.75 (95% CI 0.59– +0.96), p = 0.0215. It was consistent with the PD-L1 gene expression results: OS was improved by atezolizumab regardless of PD-L1 gene level expression. The difference in the two trials may be due to a statistically larger female population in the docetaxel group in POPLAR, overestimating the OS. + +These data were consistent with a meta-analysis of three clinical trials with anti-PD-1 or PD-L1 antibodies such as nivolumab or atezolizumab24 and showing a significant improvement in OS, +but not in PFS, except in the case of elevated levels of PD-L1 expression. + +The main results of the OAK and POPLAR trials are showed in Table 2. Clinical characteristics The survival advantage of atezolizumab over docetaxel was statistically the same regardless of pathology subtype (squamous or nonsquamous),16,21 the presence of CNS metastases at baseline, or tobacco smoking history.16,21 However, a trend emerged, revealing that some subgroups benefited even more from atezolizumab than the global population, such as the elderly population (⩾65 years)16,21 [median OS +was 14.1 months *versus* 9.2 months respectively, HR = 0.66 (95% CI 0.52–0.83) *versus* 0.80 (95% +CI 0.64–1.00)] or the CNS metastases population19 [HR of 0.54 (95% CI 0.31–0.94) *versus* 0.75 (0.63–0.89)]. On the contrary, there was a disadvantage of atezolizumab *versus* docetaxel in the *EGFR* (epidermal growth factor receptor) mutant population [OS HR 1.24 (95% CI 0.71–2.18)],21 as previously reported with other immune checkpoint inhibitors such as nivolumab or pembrolizumab [OS HR = 1.05 (95% CI 0.70–1.55), p +< 0.81] in a meta-analysis recently published.25 Other biomarkers In the phase II trial, POPLAR, OS in the atezolizumab population seemed to be correlated to higher gene level expression of PD-L1, PD-1, PD-L2, B7.1 and T-effector interferon gamma,15 but since it was not confirmed for PD-L1 in OAK, further analyses should be performed based on tumour mutation burden researches as shown in Rizvi and colleagues' study: high nonsynonymous mutation burden was associated with clinical benefit of pembrolizumab.26 + +## Safety + +In the phase I study by Herbst and colleagues,17 the maximum tolerated dose was not reached and no dose-limiting toxicities were observed. The rates of adverse effect events in clinical trials of atezolizumab are reported in Table 1. The most common side effects of any grade were fatigue (26.8%), decreased appetite (23.5%), cough (23.2%), asthenia (19%), dyspnoea (19%), nausea (17.7%), pyrexia (17.7%), constipation (17.6%), +diarrhoea (15.4%) and arthralgia (12%).16 Pooled safety analyses were conducted on patients suffering from asymptomatic untreated CNS metastases or stable previously treated brain metastases at baseline27 in several studies as POPLAR. A total of 27 patients were analysed: 44% had any neurological adverse event +(AE) *versus* 28% in the population with no CNS metastasis, but there were no more AEs of any type and no discontinued treatment due to AE + +| Table 1. Adverse event rates in the POPLAR and OAK trials in intention-to-treat populations. Trial name Arms Any grade adverse events (%) Grade 3–4 adverse events (%) Adverse events leading to dose modification, delay or interruption (%) | Immunerelated adverse events (%) | | | | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------|------|----|----|--------| +| Hervst et al.13 | Atezo | 94.9 | 39 | - | - | +| POPLAR | Atezo | 96 | 40 | 24 | 3.2* | +| Doc | 96 | 53 | 33 | - | | +| OAK | Atezo | 94 | 37 | 25 | 10.5** | +| Doc | 96 | 54 | 36 | - | | +| *Pneumonitis: 6; colitis: 2; hepatitis: 2. **Pneumonitis: 4; increased aspartate aminotransferase: 6; increased alanine aminotransferase: 6; colitis: 4; hepatitis: 1. Atezo, atezolizumab; Doc, docetaxel. | | | | | | + +## In The Subgroup Of Patients With Cns Metastases. + +especially in comparison with standard chemotherapy with docetaxel. + +PD-1/PD-L1 AEs compared with docetaxel in a meta-analysis had an odds ratio (OR) of 0.36 (95% +CI 0.28–0.46); p < 0.001, all grades included and of 0.18 (95% CI 0.14–0.22), p < 0.001 for grade 3–5 AEs, showing a better tolerability profile of immune checkpoint inhibitors in comparison with chemotherapy by docetaxel.28 Another meta-analysis compared anti-PD-1 and anti-PD-L1 tolerance profile and did not show any difference on AE rates (including immune AE and pneumonitis) on a population of more than 4400 patients.29 In conclusion, phase I–III trials showed a good tolerance profile and efficacy of atezolizumab for the treatment of pretreated NSCLC patients, + +## Discussion + +Based on clinical trials and meta-analyses, we report that atezolizumab is efficient and safe for second-line treatment of advanced NSCLC, irrespective of PD-L1 expression. But its place compared with other immune checkpoint inhibitors, including anti-PD-1 treatments already approved in this setting is still to be proven. Otherwise, relevance of PD-L1 expression according to SP142 IHC should be reassessed, since atezolizumab is effective in any PD-L1 expression group, and other predictive biomarkers should be studied. + +Furthermore, there are remaining questions regarding potential combination of atezolizumab with other therapeutics, such as systemic treatment + +| Table 2. Efficacy data of POPLAR and OAK trials on atezolizumab in intention-to-treat and TC3 and IC3 populations. Phase Population Group n OS HR p PFS HR p | ORR | | | | | | | | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------|-------------|----------|------|----------|------|--------|--------|------|-------|------| +| (months) | (95% CI ) | (months) | (95% CI) | (%) | | | | | | | | +| Fehrenbacher et al.15 (POPLAR) | II | ITT | Atezo | 144 | 12.6 | 0.73 | 0.04 | 2.7 | 0.94 | 0.645 | 14.6 | +| (0.53–0.99) | (0.72–1.23) | | | | | | | | | | | +| Docetaxel | 143 | 9.7 | 3 | 14.7 | | | | | | | | +| TC3 or IC3 | Atezo | 24 | 15.5 | 0.49 | - | 7.8 | 0.60 | 0.127 | 37.5 | | | +| (0.27–1.07) | (0.31–1.16) | | | | | | | | | | | +| Docetaxel | 23 | 11.1 | 3.9 | 13 | | | | | | | | +| Rittmeyer | III | ITT | Atezo | 425 | 13.8 | 0.73 | 0.0003 | 2.8 | 0.95 | 0.49 | 14 | +| et al.16 (OAK) | (0.62–0.87) | (0.82–1.10) | | | | | | | | | | +| Docetaxel | 425 | 9.6 | 4.0 | 13 | | | | | | | | +| TC3 or IC3 | Atezo | 72 | 20.5 | 0.41 | < 0.0001 | 4.2 | 0.63 | 0.0123 | 30.6 | | | +| (0.27–0.64) | (0.43–0.91) | | | | | | | | | | | +| Docetaxel | 65 | 8.9 | 3.3 | 10.8 | | | | | | | | +| Atezo, atezolizumab; CI, confidence interval; HR, hazard ratio; IC3, immune cell 3; ITT, intention to treat; OS, overall survival; ORR, objective response rate; PFS, progression-free survival; TC3, tumour cell 3. | | | | | | | | | | | | + +(chemotherapy, other immunotherapy, and targeted therapy), radiotherapy or surgery; the existence of a targeted population of interest and biomarkers to guide atezolizumab use; the place of atezolizumab in the treatment strategy of advanced NSCLC; the potential extension of atezolizumab use to other histology like small cell lung cancer (SCLC) or in other settings such as adjuvant or neoadjuvant treatment of early-stage NSCLC. All upcoming trials referring to atezolizumab are presented in Table 3. Atezolizumab is being tested alone in the neoadjuvant or adjuvant setting, in first-line treatment of NSCLC, in small SCLC or in association with other therapies. Atezolizumab combined with other treatments Atezolizumab is currently tested in association with chemotherapy, anti-VEGF, EGFR tyrosine kinase inhibitor, vaccine therapy, anti-CD38, antiCyclin Dependent Kinase (CDK)4/6 and radiation therapy (Table 2). Combinations of atezolizumab with chemotherapy shouldn't convey more toxicities than chemotherapy alone as shown as in a phase Ib study examining safety of the anti-PD-L1 associated with either paclitaxel, pemetrexed or weekly nab-paclitaxel.30 Other trials should provide more information about the efficacy and safety of the combination with chemotherapy.31,32 Targeted population and potential biomarkers Updated data from the BIRCH trial were presented at WCLC 2016,20 where the ORR in the investigated group was 32% for TC3 or IC3 and 24% for TC2/3 or IC2/3. In this last subgroup, median duration of response was 13.1 months, median OS was 20.1 months. ORR was surprisingly better in the *EGFR* mutant subgroup +(31%) than in the *EGFR* wild-type subgroup (20%) and ORR was also better in the *Kirsten* rat sarcoma virus oncogene (KRAS) mutant subgroup (27% *versus* 21% for *KRAS* wild-type NSCLC). The FIR [ClinicalTrials.gov identifier: NCT01846416] study is currently assessing the efficacy of atezolizumab on preselected patients with CNS metastasis at baseline. + +Companion diagnostic assays for anti-PD-1/PD-L1 therapies are discussed because of many differences and heterogeneity of measures and practical use. For instance, the nivolumab companion assay is Dako 28-8, targeting membrane tumour cells, with a threshold of positivity ⩾5%, pembrolizumab uses Dako 22C3 (Agilent, CA, United States), staining PD-L1 present on membrane tumour cells with a distinction for 'weak' expression (1–49%) and strong (⩾50%), SP142 attendant atezolizumab detect PD-L1 on tumour and tumour-infiltrating immune cells with a the positivity score TC1/2/3 or IC1/2/3.33 However, SP142, used with atezolizumab, is distinguishing itself due to its ability to detect PD-L1 expression on two different types of cells: TCs and tumour-infiltrating ICs. Furthermore, there is a strong correlation between PDL1 IHC status and PD-L1 messenger RNA +(mRNA) expression also with T-effector mRNA +expression.34 One interesting fact in PD-L1 expression is that its intrapatient heterogeneity is low in metachronous tissues, indicating distinct types of tumour samples, including fresh or archival, can be reliably used to assess PD-L1 expression.35 The phase I study of atezolizumab17 for the treatment of NSCLC, melanoma, renal cell carcinoma, other solid tumours and haematological malignancies showed that atezolizumab was more effective in patients with pre-existing immunity suppressed by PD-L1, thus immunotherapy helps to reinvigorate immune cells. Therefore, high levels of PD-L1 and cytotoxic T-lymphocyteassociated protein 4 (CTLA-4) expression at baseline, such as low levels of CX3CL-1 (fractalin) expression were correlated with a positive response, in all tumour types. In addition, in melanoma, great expression of interferon gamma and indoleamine 2,3-dioxygénase (IDO-1) or Chemokine (C-X-C motif) ligand 9 (CXCL-9) +was correlated with a good response to anti-PDL1. The authors of this phase I study proposed three models of nonresponders: (1) 'immunological ignorance' (little or no tumour-infiltrating immune cell infiltration); (2) 'nonfunctional immune response' (presence of an immune infiltrate with minimal to no expression of PD-L1); +and (3) 'excluded infiltrate' (presence of an immune infiltrate that resided solely around the outer edge of the tumour cell mass). + +| F Jean, P Tomasini et al. | | | | | | +|-------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------|----------------------------------------|---------------------------------------|-----------------|-------------| +| Table 3. Expected atezolizumab (MDPL3280A) trials. Histology Stage Histology Therapeutic place | Phase | Experimental arm | Control arm | ClinicalTrials. | | +| subgroup | gov identifier | | | | | +| NSCLC | Stage I | I | MDPL3280A + stereotaxic radiotherapy | NCT02599454 | | +| Stage | After adjuvant | | | | | +| Ib-IIIa | cisplatin-based chemotherapy | III | MDPL3280A | Best supportive | NCT02486718 | +| care | | | | | | +| Neoadjuvant +/– | II | MDPL3280A | NCT02927301 | | | +| adjuvant Neoadjuvant | II | MDPL3280A + | NCT02716038 | | | +| carboplatin + nabpaclitaxel | | | | | | +| Stage | Nonsquamous | First line | III | MDPL3280A + | | +| IIIb–IV | carboplatin + nab-paclitaxel | Carboplatin + | NCT02367781 | | | +| nab-paclitaxel | | | | | | +| III | MDPL3280A + carboplatin/cisplatin + pemetrexed Carboplatin/ cisplatin + pemetrexed | NCT02657434 | | | | +| III | MDPL3280A + carboplatin + paclitaxel +/– bevacizumab | Carboplatin + paclitaxel + bevacizumab | NCT02366143 | | | +| First line or higher | Ib–II | MDPL3280A + | NCT02630186 | | | +| in EGFR mutant | rociletinib | | | | | +| Squamous | First line | III | MDPL3280A + carboplatin + paclitaxel or nabpaclitaxel | Carboplatin + | NCT02367794 | +| nab-paclitaxel | | | | | | +| III | MDPL3280A + carboplatin/ cisplatin + gemcitabine | Carboplatin/ cisplatin + gemcitabine | NCT02409355 | | | +| Both | After previous | II | MDPL3280A | NCT03014648 | | +| treatment with PD1-directed therapy First line or higher | Ib | MDPL3280A + | NCT02013219 | | | +| alectinib or erlotinib | | | | | | +| First line or higher | II | MDPL3280A | NCT02031458 | | | +| in preselected PDL1 (+) | or NCT01846416 | | | | | +| First line or higher | II | CDX-1401 + MDPL3280A | NCT02495636 | | | +| in NY-ESO-1 (+) Second line or | IB-II | MDPL3280A + | MDPL3280A | NCT03023423 | | +| higher | daratumumab (antiCD38) | (Continued) | | | | + +| Table 3. (Continued) Histology Stage | Histology | Therapeutic place | Phase | Experimental arm | Control arm | ClinicalTrials. | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------|-------------------------------------|---------------------------------------|--------------------|---------------|-------------------| +| subgroup | gov identifier | | | | | | +| First line | I-II | MDPL3280A + | | | | | +| SCLC | Stage IIIb–IV | carboplatin + etoposide | Carboplatin– | NCT02748889 | | | +| etoposide | | | | | | | +| II | MDPL3280A + carboplatin + etoposide + trilaciclib | MDPL3280A + carboplatin + etoposide | NCT03041311 | | | | +| I–III | MDPL3280A + carboplatin + etoposide | Carboplatin + | NCT027633579 | | | | +| etoposide | | | | | | | +| Second line | II | MDPL3280A | Carboplatin + etoposide or topotecan | NCT03059667 | | | +| EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; NY-ESO-1, cancer-testis antigen; PD-1, programmed cell-death 1; PD-L1, programmed cell-death-ligand 1; SCLC, small cell lung cancer. | | | | | | | + +However, atezolizumab has shown efficacy in phase II and III studies regardless of SP142 PD-L1 expression results, and other biomarkers should be investigated further. For example, tumour mutation load, which was quantified by Kowanetz and colleagues using the Fundation Medicine 1 (FM1) panel of 315 cancer-related genes on tumour specimens of patients from the POPLAR, BIRCH and FIR studies. OS, PFS and ORR were improved in patients with increased mutation load treated with atezolizumab in both unselected and PD-L1-positive patients.36 PD-L2 expression also seems to be correlated with atezolizumab efficacy in NSCLC, melanoma, renal cell carcinoma and urothelial carcinoma but further evidence is required.37 Moreover, PD-L1 staining is usually performed on tumour samples and sometimes leads to rebiopsy, but PD-1/PD-L1/PD-L2 expression on peripheral blood cells could be an easiest way to characterize PD-L1 expression and predict response to atezolizumab.38 The place of atezolizumab in therapy and potential extension of use So far, atezolizumab was studied for secondline treatment of advanced NSCLC, such as previously approved other immune checkpoint inhibitors. + +A phase II study of atezolizumab as neoadjuvant and adjuvant therapy in patients with resectable NSCLC is currently ongoing39 aiming to enrol 180 patients with stage I, II and IIIa NSCLC prior to curative intent, who will receive two injections of atezolizumab 1200 mg before surgery. A second phase of this study will assess atezolizumab as an adjuvant therapy for patients who benefited from the neoadjuvant phase. A multicentre phase III double-blinded, placebocontrolled study, IMpower133 [ClinicalTrials. + +gov identifier: NCT02763579],40 will try to extend the indication of atezolizumab to SCLC treatment in first line and in association with standard chemotherapy: carboplatin–etoposide. + +## Conclusions + +Atezolizumab (MDPL3280A) clearly is an added value in the treatment of advanced stage pretreated NSCLC. Its interest in contrast with other immune checkpoint inhibitors relies on its efficacy, even in low or no PD-L1 expression subgroups. Considering the efficacy of anti-PD-1 such as pembrolizumab or nivolumab is overall higher in PD-L1-positive patients, atezolizumab might be preferable in PD-L1-negative patients. It will be necessary to consider other variant methods of PD-L1 testing used for each therapy to further explore this hypothesis. + +Nonetheless, toxicity profile does not seem to differ between anti-PD-1 and anti-PD-L1 treatments and, in order, there is no argument for choosing atezolizumab over pembrolizumab or nivolumab, based on toxicity profile. We are still far from fully understanding cancer immunity and the mechanisms leading to the success or not of immunotherapy agents. For this reason, we should investigate to a greater level the factors of failure, such as *EGFR*-mutant tumours. + +## Funding + +This research received no specific grant from any funding agency in the public, commercial, or notfor-profit sectors. + +## Conflict Of Interest Statement + +Consultations for Astra-Zeneca, Bristol-Myers Squibb, Boehringer–Ingelheim, Clovis Oncology, Eli Lilly Oncology, F. Hoffmann–La Roche Ltd., Novartis, Merck, MSD, Pierre Fabre and Pfizer. + +## References + +1. 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Paper presented at 2016 World Conference on Lung Cancer, December 2016; Vienna, Austria. Abstract P2.03b-014, Poster 5214. + +28. Wang C, Yu X and Wang W. A meta-analysis of efficacy and safety of antibodies targeting PD-1/PD-L1 in treatment of advanced NSCLC. Medicine 2016; 95: e5539. + +29. Pillai RN, Behera M, Owonikoko TK*, et al*. + +Evaluation of toxicity profile of PD-1 versus PD-L1 inhibitors in NSCLC. Paper presented at 2016 American Society of Clinical Oncology, June 2016; Chicago, USA. Abstract 9035. + +30. Camidge R, Liu SV, Powderly J*, et al*. + +Atezolizumab (MDPL3280A) combined with platinum-based chemotherapy in non-small cell lung cancer (NSCLC): a phase IB safety and efficacy update. Paper presented at 2015 World Conference on Lung Cancer, September 2015; Denver, CO, USA. Oral session, S173– +S260. + +31. Reck M, Papadimitrakopoulou VA, Capuzzo F, et al. Phase III clinical trials in chemotherapynaive patients with advanced NSCLC assessing the combination of atezolizumab with chemotherapy. + +Paper presented at 2016 European Society of Medical Oncology, October 2016; Copenhagen, Denmark. vi416–vi454. + +32. Papadimitrakopoulou VA, Cappuzzo F, Jotte RM, +et al. Phase III clinical trials of atezolizumab combined with chemotherapy in chemotherapy-naive patients with advanced NSCLC. Paper presented at 2016 American Society of Clinical Oncology, June 2016; Chicago, IL, USA. Abstract TPS9103. + +33. Texido C, Karachaliou N, Gonzalez-Cao M, +et al. Assays for predicting and monitoring responses to lung cancer immunotherapy. *Cancer* Biol Med 2015; 12: 87–95. + +34. Williams J, Kowanetz M, Koeppen H*, et al*. + +The SP142 PD-L1 IHC assay for atezolizumab reflects pre-existing immune status in NSCLC and correlates with PD-L1 mRNA. Paper presented at 2016 European Society of Medical Oncology, October 2016; Copenhagen, Denmark. Abstract 1171P. + +35. Chaft JE, Chao B, Akerley WL, et al. *Evaluation* of PD-L1 expression in metachronous tumour samples and FDG-PET as a predictive biomarker in Ph2 study (FIR) of atezolizumab (MDPL3280A). Paper presented at 2015 World Conference on Lung Cancer, September 2015; Denver, CO, USA. 1556–0864. + +36. Kowanetz M, Zou W, Shames DS, et al. *Tumour* mutation load assessed by Foundation One (FM1) is associated with improved efficacy of atezolizumab in patients with advanced NSCLC. Paper presented at 2016 European Society of Medical Oncology, October 2016; Copenhagen, Denmark. Abstract 77P. + +37. Schmid P, Hegde PS, Zou W*, et al*. + +Association of PD-L2 expression in human tumours with atezolizumab activity. Paper presented at 2016 American Society of Clinical Oncology, June 2016; Chicago, USA. Abstract 11506. + +38. Arrieta O and Montes-Servin E. Evaluation of PD1/PDL1 expression on peripheral blood cells subpopulations in patients with NSCLC. Paper presented at 2016 World Conference on Lung Cancer, December 2016; Vienna, Austria. Abstract MA 14.02. + +39. Owen D, Bunn PJ, Johnson B, et al. *A phase II* +study of atezolizumab as neoadjuvant and adjuvant therapy in patients with resectable non-small-cell lung cancer (NSCLC). Paper presented at 2016 World Conference on Lung Cancer, December 2016; Vienna, Austria. S1082. + +40. Horn L, Reck M, Mok TSK, et al. *A phase III* +study of atezolizumab with carboplatin plus etoposide in patients with extensive-stage small-cell lung cancer (IMpower 133). Paper presented at 2016 European Society of Medical Oncology, October 2016; Copenhagen, Denmark. vi493–vi496. + +Visit SAGE journals online journals.sagepub.com/ home/tam SAGE journals \ No newline at end of file diff --git a/medical/md/PMC5905384.md b/medical/md/PMC5905384.md new file mode 100644 index 0000000000000000000000000000000000000000..3dcf1b8c1357657072043ad50d00c507d9ffc65e --- /dev/null +++ b/medical/md/PMC5905384.md @@ -0,0 +1,297 @@ +# The Occurrence Of Apparent Bilateral Aldosterone Suppression In Adrenal Vein Sampling For Primary Aldosteronism + +Yui Shibayama,1 Norio Wada,1 Mitsuhide Naruse,2 Isao Kurihara,3 Hiroshi Ito,3 Takashi Yoneda,4 Yoshiyu Takeda,4 Hironobu Umakoshi,2 Mika Tsuiki,2 Takamasa Ichijo,5 Hisashi Fukuda,6 Takuyuki Katabami,6 Takanobu Yoshimoto,7 Yoshihiro Ogawa,7,8 Junji Kawashima,9 Yuichi Ohno,10 Masakatsu Sone,10 Megumi Fujita,11 Katsutoshi Takahashi,11,12 Hirotaka Shibata,13 Kohei Kamemura,14 Yuichi Fujii,15 Koichi Yamamoto,16 and Tomoko Suzuki17 1Department of Diabetes and Endocrinology, Sapporo City General Hospital, 060-8604 Sapporo, Japan; 2Department of Endocrinology, Metabolism, and Hypertension, Clinical Research Institute, National Hospital Organization Kyoto Medical Center, 612-8555 Kyoto, Japan; 3Division of Endocrinology, Metabolism, and Nephrology, Keio University, 160-8582 Tokyo, Japan; 4Division of Endocrinology and Hypertension, Department of Internal Medicine, Kanazawa University Graduate School of Medicine, 920-1192 Kanazawa, Japan; 5Department of Diabetes and Endocrinology, Saiseikai Yokohama Tobu Hospital, 230-8765 Yokohama, Japan; 6Division of Metabolism and Endocrinology, Department of Internal Medicine, St. Marianna University School of Medicine Yokohama City Seibu Hospital, 241-0811 Yokohama, Japan; 7Department of Molecular Endocrinology and Metabolism, Graduate School of Medicine, Tokyo Medical Dental University, 113-8582 Tokyo, Japan; 8Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, 812-8582 Fukuoka, Japan; 9Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, 860-8556 Kumamoto, Japan; 10Department of Diabetes, Endocrinology, and Nutrition, Kyoto University Graduate School of Medicine, 606-8501 Kyoto, Japan; 11Division of Nephrology and Endocrinology, The University of Tokyo, 113-0033 Tokyo, Japan; 12Department of Metabolism, Showa General Hospital, 187-8510 Kodaira, Japan; 13Department of Endocrinology, Metabolism, Rheumatology, and Nephrology, Faculty of Medicine, Oita University, 879-5593 Yuhu, Japan; 14Department of Cardiology, Akashi Medical Center, 674-0063 Akashi, Japan; 15Department of Cardiology, JR Hiroshima Hospital, 732-0057 Hiroshima, Japan; 16Department of Geriatric and General Medicine, Osaka University Graduate School of Medicine, 565-0871 Suita, Japan; and 17Department of Public Health, School of Medicine, International University of Health and Welfare, 286-8686 Narita, Japan Context: In adrenal venous sampling (AVS) for patients with primary aldosteronism (PA), apparent bilateral aldosterone suppression (ABAS), defined as lower aldosterone/cortisol ratios in the bilateral adrenal veins than that in the inferior vena cava, is occasionally experienced. ABAS is uninterpretable with respect to lateralization of excess aldosterone production. We previously reported that ABAS was not a rare phenomenon and was significantly reduced after adrenocorticotropic hormone (ACTH) administration. + +Objective: To validate the effects of ACTH administration and adding sampling positions in the left adrenal vein on the prevalence of ABAS in the larger Japan Primary Aldosteronism Study. Patients: The data from 1689 patients with PA who underwent AVS between January 2006 and October 2016 were studied. All patients in the previous study, the West Japan Adrenal Vein Sampling study, were excluded. + +Outcome Measurements: The prevalence of ABAS was investigated at two sampling positions in the left adrenal vein, the central vein and the common trunk, without and with ACTH administration. + +Results: The prevalence of ABAS with ACTH administration was significantly lower than that without ACTH administration [without ACTH vs with ACTH: 79/440 (18.0%) vs 45/591 (7.6%); P , 0.001]. With ACTH administration, the prevalence of ABAS was not different between the sampling position, at the central vein and at the common trunk [33/591 (5.6%) vs 32/591 (5.4%); P = 1.00]. Conclusions: The effectiveness of ACTH administration for the reduction of ABAS in AVS regardless of the sampling position in the left adrenal vein was confirmed in the larger cohort. + +Copyright © 2018 Endocrine Society This article has been published under the terms of the Creative Commons Attribution NonCommercial, No-Derivatives License (CC BY-NC-ND; https://creativecommons.org/licenses/by-ncnd/4.0/). + +Freeform/Key Words: adrenal vein sampling, aldosterone suppression, apparent bilateral, hypertension, primary aldosteronism Primary aldosteronism (PA) is the most common form of secondary endocrine hypertension, accounting for 5% to 10% of all hypertensive patients [1, 2]. In patients with PA, adrenal venous sampling (AVS) is recognized as the most reliable method for subtype diagnosis, and it is important for patients to have this procedure to determine the need for an adrenalectomy. To decide whether the disease is either unilateral or bilateral, two criteria are commonly used, the lateralization index (LI) and the contralateral ratio [3–6]. The LI is the ratio of aldosterone/cortisol (A/C) in the dominant adrenal vein relative to that in the nondominant adrenal vein. The contralateral ratio is the ratio of A/C between the nondominant adrenal vein and either the inferior vena cava (IVC) or the peripheral vein. If the A/C ratio in one adrenal vein is lower than that in either the IVC or the peripheral vein, the diagnosis is unilateral disease of the other side, because of the presence of a contralateral suppression. + +In some patients, the results of AVS were inconclusive because of the bilaterally lower A/C +ratios in the adrenal veins than that in IVC despite successful cannulation [7]. Wolley et al. [8] +reported that 2.6% of the patients who underwent AVS without adrenocorticotropic hormone +(ACTH) administration showed the same phenomenon. Recently, we demonstrated using the data from the West Japan Adrenal Vein Sampling (WAVES-J) study that this phenomenon was observed in 9.5% of the patients who underwent AVS without ACTH administration, and that in 92% of these patients, it was resolved by ACTH administration [9]. In this study, we term this phenomenon as apparent bilateral aldosterone suppression (ABAS). ABAS was thought to be developed by the blood sampling in quiescent phase of aldosterone from aldosterone-producing adenoma (APA) accompanying the stress-induced ACTH secretion and to be eliminated by ACTH administration. + +Although ACTH was administered, ABAS was still observed in a small number of patients. + +Some patients who did not show ABAS without ACTH administration demonstrated ABAS with ACTH administration. In the WAVES-J study, in most patients, the left adrenal vein samples were collected from the central vein during AVS. An additional sampling at the common trunk was performed in a subset of these patients. We reported a patient with ABAS without ACTH administration, where ABAS was resolved with an additional sampling at the common trunk. + +In this study, we analyzed the data of the Japan Primary Aldosteronism Study (JPAS), a multicenter collaborative study for patients with PA in Japan, from which the WAVES-J +study cohort had been derived. We excluded the data from the patients who had been included in the WAVES-J study to evaluate ABAS in the remaining, larger JPAS cohort. The JPAS also had many more patients sampled at both the central vein and the common trunk in AVS than that in the WAVES-J study. + +In this study, we examined the effects of ACTH administration and the addition of a second blood sampling position in the left adrenal vein on resolving ABAS. + +## 1. Patients And Methods A. Patients + +Twenty-seven centers participated in the JPAS. The patients with confirmed PA who underwent AVS from January 2006 to October 2016 were enrolled in the study. The baseline clinical findings, the results of the AVS, and the posttreatment outcomes were electronically collected using the WEB registry system, and data security and maintenance of registered data were outsourced to EPS Corporation (Tokyo, Japan). The study protocol was approved by the ethics committees of each center. + +The diagnostic procedure for PA was based on the guidelines of both the Japan Endocrine Society [10] and the Japan Society of Hypertension [11]. The screening for PA was performed based on the ratio of the plasma aldosterone concentration (PAC; pg/mL) to the plasma renin activity (PRA; ng/mL/h) (aldosterone renin ratio .200), after changing from potentially interfering antihypertensive drugs to calcium channel blockers and/or to a-blockers where applicable. The diagnosis of PA was established by at least one positive result of confirmatory testing, including the captopril challenge test, the upright-furosemide loading test, and the saline loading test. + +## B. Avs + +Blood samples obtained via AVS were generally collected before and at 30 minutes after ACTH administration from both adrenal veins and from the IVC at a point distal to the renal vein. The tip of the catheter was placed into the left adrenal vein at a point distal to the division of the left inferior phrenic vein, termed the central vein, and at the junction of the inferior phrenic vein and the left adrenal vein as a common trunk [12]. The protocol for ACTH +administration varied in each center. Eighteen centers used a bolus injection of 250 mg of cosyntropin. Eight centers used a bolus injection of 200 mg (or 250 mg) followed by continuous infusion of 50 mg (or 100 or 250 mg) of cosyntropin. One center used a continuous infusion of cosyntropin. In four centers, sampling of the left and right adrenal veins was performed simultaneously, whereas in the other centers, the sampling was done sequentially. Catheterization was judged to be successful if the selectivity index (the ratio of cortisol concentration between the adrenal vein and the IVC) was either more than 2 without ACTH administration or more than 5 with ACTH administration. + +Unilateral disease was defined if the LI was more than 4 both without and with ACTH +administration. ABAS was defined if the A/C ratios in adrenal veins were bilaterally lower than that in the IVC. + +## C. Analysis + +The data from the JPAS were studied retrospectively. The prevalence of ABAS in AVS with and without ACTH administration was determined. ABAS was investigated in two different vein sampling combinations (the right adrenal vein and the central vein of the left adrenal vein or the right adrenal vein and the common trunk of the left adrenal vein). The baseline characteristics, the results of the AVS, and the percentages of patients who underwent a unilateral adrenalectomy with ACTH administration were compared. + +We estimated the postoperative clinical and biochemical outcomes referring to the Primary Aldosteronism Surgery Outcome study over either a 6- or 12-month follow-up [13]. + +## D. Assay Methods + +PAC and PRA were measured by commercially available kits. PAC was determined by radioimmunoassay (SPAC-S Aldosterone Kit; Fuji Rebio, Co., LTD, Tokyo, Japan) in all centers. + +The reference range of PAC in the supine position was 3.0 to 15.9 ng/dL. PRA was measured by radioimmunoassay or enzyme immunoassay. The reference range of PRA in the supine position was 0.3 to 2.9 ng/mL/h (PRA radioimmunoassay kits; Fuji Rebio, Co., LTD) in 15 centers, 0.2 to 2.3 ng/mL/h (PRA enzyme immunoassay kits; Yamasa, Co., LTD, Choshi, Japan) in seven centers, and 0.2 to 2.7 ng/mL/h (PRA radioimmunoassay kits; Yamasa, Co., +LTD) in four centers. Plasma active renin concentration (ARC) was measured by immunoradiometric assay (Renin immunoradiometric assay kits, Fuji Rebio, Co., LTD) in one center. The reference range of ARC in the supine position was 2.5 to 21.4 pg/mL. ARC value was used for analysis after converting to PRA by dividing by 5 according to the Japan Endocrine Society guideline [10]. + +## E. Statistics + +The data were analyzed and compared using BellCurve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan). Continuous variables were expressed as either mean 6 standard deviation or median and interquartile range. Continuous variables were analyzed by either the one-way analysis of variance or the Kruskal-Wallis test, as appropriate. Comparison of the frequency among two groups was estimated by either the x 2 test or the Fisher's exact test, as appropriate. Statistical significance was achieved when the P value was ,0.05. + +## 2. Results + +A flowchart of this study is summarized in Fig. 1. In the JPAS, 1689 patients were confirmed as having PA. Seven hundred and forty-two patients underwent attempted cannulation of the right adrenal vein and both the central vein and the common trunk with ACTH administration. Five hundred and ninety-one patients were successfully cannulated at the right adrenal vein and both positions in the left adrenal vein. The prevalence of ABAS at either the central vein or the common trunk with ACTH administration was 45/591 (7.6%). The characteristics of patients with ACTH administration are summarized in Table 1. The percentages of male were higher in the patients with ABAS at both the central vein and the common trunk than that in the patients without ABAS (75% vs 48%; P = 0.02). The percentages of males were significantly higher than that of females in the patients who were + +![3_image_0.png](3_image_0.png) + +| Table 1. | Baseline Characteristics of the Patients With and Without ABAS in AVS ABAS at Both the Central Vein and the Common | Without ABAS | | | +|--------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|------------------|--------------------|---------------------| +| Trunk (n = 20) | (n = 546) | | | | +| ABAS at Only | | | | | +| the Central Vein (n = 13) | ABAS at Only the Common Trunk (n = 12) | | | | +| Age (y) | 53.8 6 11.8 | 57.7 6 11.2 | 56.6 6 15.6 | 55.6 6 10.9 | +| Sex (male/female) | 8 (62%)/5 (38%) | 9 (75%)/3 (25%) | 15 (75%)a /5 (25%) | 261 (48%)/285 (52%) | +| BMI | 28.2 [21.6–31.0] | 24.3 [22.3–28.1] | 24.7 [22.7–25.8] | 24.1 [21.8–27.2] | +| Duration of hypertension (y) | 19.6 6 9.0a | 14.8 6 11.6 | 10.0 6 9.2 | 8.4 6 9.0 | +| Systolic BP (mm Hg) | 153.2 6 23.7 | 152.8 6 28.9 | 159.0 6 31.0 | 154.9 6 24.3 | +| Diastolic BP (mm Hg) | 88.3 6 13.8 | 98.4 6 25.5 | 89.1 6 18.6 | 92.8 6 15.7 | +| PRA (ng/mL/h) | 0.4 [0.3–0.5] | 0.2 [0.1–0.5] | 0.2 [0.1–0.4] | 0.3 [0.2–0.6] | +| PAC (pg/mL) | 231 [184–399] | 279 [194–425] | 250 [132–298] | 184 [126–282] | +| ARR | 752 [467–1065] | 1765 [603–2860] | 960.9 [554–1639] | 521 [306–1125] | +| Serum potassium levels (mEq/L) | 3.5 [3.3–3.6]b | 3.0 [3.0–3.6]b | 3.7 [3.2–4.0] | 3.8 [3.5–4.1] | +| Potassium supplementation | 6 (46%)a | 7 (58%)a | 10 (50%)a | 103 (19%) | +| Blood urea nitrogen levels (mg/dL) | 14.3 6 4.3 | 14.8 6 4.0 | 14.3 6 3.7 | 13.4 6 3.9 | +| Serum creatinine levels (mg/dL) | 0.86 [0.7–0.9] | 0.76 [0.7–0.9] | 0.80 [0.7–0.9]a | 0.71 [0.6–0.9] | +| Adrenal nodule in CT | 10 (77%) | 11 (92%)a | 13 (65%) | 306 (56%) | +| Adrenalectomy | 8 (62%)a | 6 (50%) | 6 (30%) | 158 (29%) | +| Data are expressed as the mean 6 standard deviation, median [interquartile range], or number (percentage). P value | | | | | + +Data are expressed as the mean 6 standard deviation, median [interquartile range], or number (percentage). P value represents the patients with ABAS vs the patients without ABAS. + +Abbreviations: ARR, aldosterone renin ratio; BMI, body mass index; BP, blood pressure; CT, computed tomography. + +aSignificantly higher than the patients without ABAS, P , 0.05. bSignificantly lower than the patients without ABAS, P , 0.05. + +diagnosed as the unilateral subtype in AVS and did not have ABAS with ACTH administration (data not shown). Duration of hypertension was significantly longer in the patients with ABAS at the central vein than that in the patients without ABAS (19.6 6 9.0 vs 8.4 6 9.0 years; P , 0.001). The percentages of patients taking potassium supplementation was significantly higher in the patients with ABAS at either the central vein or the common trunk or with ABAS at both the central vein and the common trunk than that in the patients without ABAS (46%, 58%, 50% vs 19%; P = 0.03, P , 0.01, P , 0.01, respectively). The percentages of patients having an adrenalectomy was significantly higher in the patients with ABAS at only the central vein than that in the patients without ABAS (62% vs 29%; P = 0.03). The protocols of ACTH administration in AVS were not different between patients with and without ABAS +(data not shown). + +The frequency of ABAS is shown in Table 2. The prevalence of ABAS at either the central vein or the common trunk with ACTH administration was significantly lower than that without ACTH administration [without ACTH administration vs with ACTH administration; 79/440 (18.0%) vs 45/591 (7.6%), P , 0.001]. In contrast, there were no differences in the prevalence of ABAS at the central vein and at only the common trunk between without and with ACTH administration [without ACTH administration vs with ACTH administration; + +| Without ACTH | With ACTH | | | +|-----------------------------------------------------|----------------|---------------|--------| +| Administration | P Value | | | +| Administration | | | | +| ABAS at only the central vein | 14/440 (3.2%) | 13/591 (2.2%) | 0.4356 | +| ABAS at only the common trunk | 11/440 (2.5%) | 12/591 (2.0%) | 0.7705 | +| ABAS at both the central vein and the common trunk | 54/440 (12.3%) | 20/591 (3.4%) | ,0.001 | +| ABAS at either the central vein or the common trunk | 79/440 (18.0%) | 45/591 (7.6%) | ,0.001 | + +Data are expressed as numbers and percentages of patients with ABAS only at the central vein, only at the common trunk, both at the central vein and the common trunk, and either the central vein or the common trunk. + +ABAS at only the central vein, 14/440 (3.2%) vs 13/591 (2.2%); ABAS at only the common trunk, 11/440 (2.5%) vs 12/591 (2.0%); P = 0.33, P = 0.67, respectively]. With ACTH administration, the prevalence of ABAS between only the central vein and only the common trunk was not different [13/591 (2.2%) vs 12/591 (2.0%); P = 1.00]. When the patients with ABAS at both the central vein and the common trunk were added, the prevalence of ABAS +between the central vein and the common trunk was not significantly different [33/591 (5.6%) +vs 32/591 (5.4%); P = 1.00]. + +The details of AVS of patients with ABAS with ACTH administration are shown in Table 3. + +With ACTH administration, 11 of the 13 (85%) patients with ABAS at only the central vein showed an LI more than 4 at the common trunk. Similarly, 11 of the 12 (92%) patients with ABAS at only the common trunk showed an LI more than 4 at the central vein. Unilateral adrenalectomy was performed on eight (62%) of the patients with ABAS at only the central vein and on seven (58%) of the patients with ABAS at only the common trunk. All the patients had a left-side adrenalectomy. + +The AVS data of the patients with ABAS at both the central vein and the common trunk are shown in Table 4. In 16 of the 20 patients with ABAS at both the central vein and the common + +| ACTH Administration | ABAS at Both the Central Vein and the Common Trunk (n = 20) | | | +|--------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------|----------------------------------|-----------------------| +| ABAS at Only the | ABAS at Only the | | | +| Central Vein (n = 13) | Common Trunk (n = 12) | | | +| Without ACTH administration At the central vein LI . 4 | 3 (L3, R0)/5 (60%) | 4 (L4, R0)/7 (57%) | 5 (L1, R4)/11 (45%) | +| LI . 4 and concordant | 3 (L3, R0)/5 (60%) | 3 (L3, R0)/7 (43%) | 4 (L1, R3)/11 (36%) | +| with CT | | | | +| At the common trunk LI . 4 | 4 (L4, R0)/5 (80%) | 2 (L2, R0)/7 (29%) | 5 (L2, R3)/11 (45%) | +| LI . 4 and concordant | 3 (L3, R0)/5 (60%) | 1 (L1, R0)/7 (14%) | 3 (L1, R2)/11 (27%) | +| with CT | | | | +| With ACTH administration At the central vein LI . 4 | 2 (L2, R0)/13 (15%) | 11 (L11, R0)/12 (92%) | 1 (L0, R1)/20 (5%) | +| LI . 4 and concordant | 2 (L2, R0)/13 (15%) | 9 (L10, R0)/12 (75%) | 1 (L0, R1)/20 (5%) | +| with CT | | | | +| At the common trunk LI . 4 | 11 (L11, R0)/13 (85%) | 2 (L2, R0)/12 (17%) | 3 (L2, R1)/20 (9%) | +| LI . 4 and concordant | 9 (L9, R0)/13 (69%) | 1 (L1, R0)/12 (8%) | 3 (L1, R1)/20 (9%) | +| with CT | | | | +| CT findings | Left nodule (9)/bilateral | Left nodule (8)/right nodule | Left nodule (6)/right | +| nodules (1) | (2)/bilateral nodules (1) | nodule (5)/bilateral nodules (2) | | +| The side of adrenalectomy | Left (8) | Left (6) | Left (4)/right (2) | +| In each group (ABAS at only the central vein, ABAS at only the common trunk, and ABAS at both the central vein and | | | | + +| Without ACTH Administration | With ACTH Administration | | | | | | | | +|-------------------------------------------------------------------------------------------------------------------|----------------------------|--------------------|-------------------|---------------|---------------------|--------------------|--------------------|-----------------| +| Patient | RAV | Central Vein | Common Trunk | IVC | RAV | Central Vein | Common Trunk | IVC | +| 1 | 15,800/165 (95.6)a | 848/177 (4.8) | 4860/915 (5.3) | 1840/44 (41) | 76,700/2102 (36.5)a | 6300/1017 (6.2) | 8850/2032 (4.4) | 3400/60 (56.9) | +| 2 | 2040/441 (4.6) | 18,200/390 (46.7)a | 2600/120 (21.7)a | 347/11 (30.7) | 11,300/1989 (5.7) | 49,900/2264 (22.0) | 18,100/1452 (12.5) | 784/30 (26.3) | +| 3 | 251/43 (5.8) | 1090/85.3 (12.8) | 1430/95 (15.0) | 141/9 (15.3) | 2660/591 (4.5) | 3000/295 (10.2) | 2220/298 (7.4) | 378/30 (12.8) | +| 4 | 626/52 (12.0) | 29,400/37 (801.3) | 13,300/33 (409.1) | 514/22 (23.0) | 3830/1523 (2.5) | 3560/1768 (2) | 16,600/1949 (8.5) | 759/37 (21) | +| 5 | 2212/608 (3.6) | 3482/31 (113.1) | 2409/28 (87.5) | 701/14 (50.8) | 4194/940 (4.5) | 4756/823 (5.8) | 26,102/944 (27.6)a | 1556/26 (60) | +| 6 | n.d. | n.d. | n.d. | n.d. | 6020/559 (10.8) | 1530/337 (4.5) | 1450/325 (4.5) | 306/18 (17.4) | +| 7 | 1983/109 (18.2) | 5189/85 (61.2) | 6853/68 (100.8)a | 124/10 (13.1) | 10,451/1710 (6.1) | 14,321/1990 (7.2) | 7690/620 (12.4) | 175/14 (12.8) | +| 8 | 3530/58 (61.2)a | 1450/148 (9.8) | 2910/165 (17.6) | 409/20 (20.2) | 13,300/1100 (12.0) | 4450/799 (5.6) | 4530/1052 (4.3) | 569/32 (18.0) | +| 9 | 1850/42 (43.7)a | 2540/882 (2.9) | 2680/604 (4.4) | 215/11 (19.0) | 17,400/1611 (10.8) | 6440/1292 (5.0) | 5250/1149 (4.6) | 288/21 (13.6) | +| 10 | 4960/30 (167.6)a | 1210/426 (2.8) | 625/161 (3.9) | 201/9 (21.6) | 16,400/1835 (8.9) | 4120/1383 (3.0) | 896/177 (5.1) | 269/18 (15.1) | +| 11 | 1510/26 (58.2) | 1110/23 (48.2) | 936/18 (50.7) | 86/7 (12.8) | 20,700/1395 (14.8) | 17,600/1127 (15.6) | 11,100/819 (13.6) | 149/8 (18.3) | +| 12 | 5300/1100 (4.8) | 8076/1160 (7.0) | 6420/1120 (5.7) | 82/13.3 (6.2) | 13,500/1906 (7.1) | 7430/1050 (7.1) | 4190/830 (5.0) | 186/25 (7.4) | +| 13 | 350/67 (5.2) | 1060/168 (6.3) | 711/111 (6.4) | 72/8 (9.1) | 4180/680 (6.1) | 3680/617 (6.0) | 3810/656 (5.8) | 198/20 (10.2) | +| 14 | 2299/132 (17.4) | 573/50.2 (11.4) | 576/45 (12.9) | 414/10 (40.6) | 9150/572 (16.0) | 10,128/641 (15.8) | 6732/514 (13.1) | 541/17.3 (31.3) | +| 15 | 328/17 (19.8) | 512/145 (3.5) | 502/112 (4.5) | 211/10 (20.9) | 2670/566 (4.7) | 2060/353 (5.8) | 1950/323 (6.0) | 331/20 (16.6) | +| 16 | n.d. | n.d. | n.d. | n.d. | 2400/4068 (0.6) | 2010/1172 (1.7) | 7800/495 (15.8)a | 718/39 (18.3) | +| 17 | 136/18 (7.6) | 932/40 (23.3) | 1030/64 (16.1) | 238/19 (12.5) | 4780/948 (5.0) | 8300/911 (9.1) | 7350/748 (9.8) | 356/23 (15.8) | +| 18 | 110/26 (4.2) | 185/42 (4.4) | 344/57 (6.0) | 114/23 (5.0) | 1930/882 (2.2) | 864/420 (2.1) | 723/186 (3.9) | 118/21 (5.5) | +| 19 | n.d. | n.d. | n.d. | n.d. | 25,300/1734 (14.6) | 9510/846 (11.2) | 10,900/981 (11.1) | 449/27 (16.6) | +| 20 | n.d. | n.d. | n.d. | n.d. | 16,700/857 (19.5) | 14,300/815 (17.5) | 15,900/862 (18.4) | 389/20 (19.9) | +| Data are shown as A/C ratio (pg/mL, mg/dL, pg/mL per mg/dL, respectively). The number 1 to 3 and 7 to 14 patients | | | | | | | | | + +1 15,800/165 (95.6)a 848/177 (4.8) 4860/915 (5.3) 1840/44 (41) 76,700/2102 (36.5)a 6300/1017 (6.2) 8850/2032 (4.4) 3400/60 (56.9) + +2 2040/441 (4.6) 18,200/390 (46.7)a 2600/120 (21.7)a 347/11 (30.7) 11,300/1989 (5.7) 49,900/2264 (22.0) 18,100/1452 (12.5) 784/30 (26.3) + +3 251/43 (5.8) 1090/85.3 (12.8) 1430/95 (15.0) 141/9 (15.3) 2660/591 (4.5) 3000/295 (10.2) 2220/298 (7.4) 378/30 (12.8) 4 626/52 (12.0) 29,400/37 (801.3) 13,300/33 (409.1) 514/22 (23.0) 3830/1523 (2.5) 3560/1768 (2) 16,600/1949 (8.5) 759/37 (21) + +5 2212/608 (3.6) 3482/31 (113.1) 2409/28 (87.5) 701/14 (50.8) 4194/940 (4.5) 4756/823 (5.8) 26,102/944 (27.6)a 1556/26 (60) + +6 n.d. n.d. n.d. n.d. 6020/559 (10.8) 1530/337 (4.5) 1450/325 (4.5) 306/18 (17.4) + +7 1983/109 (18.2) 5189/85 (61.2) 6853/68 (100.8)a 124/10 (13.1) 10,451/1710 (6.1) 14,321/1990 (7.2) 7690/620 (12.4) 175/14 (12.8) 8 3530/58 (61.2)a 1450/148 (9.8) 2910/165 (17.6) 409/20 (20.2) 13,300/1100 (12.0) 4450/799 (5.6) 4530/1052 (4.3) 569/32 (18.0) 9 1850/42 (43.7)a 2540/882 (2.9) 2680/604 (4.4) 215/11 (19.0) 17,400/1611 (10.8) 6440/1292 (5.0) 5250/1149 (4.6) 288/21 (13.6) + +10 4960/30 (167.6)a 1210/426 (2.8) 625/161 (3.9) 201/9 (21.6) 16,400/1835 (8.9) 4120/1383 (3.0) 896/177 (5.1) 269/18 (15.1) + +11 1510/26 (58.2) 1110/23 (48.2) 936/18 (50.7) 86/7 (12.8) 20,700/1395 (14.8) 17,600/1127 (15.6) 11,100/819 (13.6) 149/8 (18.3) 12 5300/1100 (4.8) 8076/1160 (7.0) 6420/1120 (5.7) 82/13.3 (6.2) 13,500/1906 (7.1) 7430/1050 (7.1) 4190/830 (5.0) 186/25 (7.4) 13 350/67 (5.2) 1060/168 (6.3) 711/111 (6.4) 72/8 (9.1) 4180/680 (6.1) 3680/617 (6.0) 3810/656 (5.8) 198/20 (10.2) 14 2299/132 (17.4) 573/50.2 (11.4) 576/45 (12.9) 414/10 (40.6) 9150/572 (16.0) 10,128/641 (15.8) 6732/514 (13.1) 541/17.3 (31.3) + +15 328/17 (19.8) 512/145 (3.5) 502/112 (4.5) 211/10 (20.9) 2670/566 (4.7) 2060/353 (5.8) 1950/323 (6.0) 331/20 (16.6) + +16 n.d. n.d. n.d. n.d. 2400/4068 (0.6) 2010/1172 (1.7) 7800/495 (15.8)a 718/39 (18.3) + +17 136/18 (7.6) 932/40 (23.3) 1030/64 (16.1) 238/19 (12.5) 4780/948 (5.0) 8300/911 (9.1) 7350/748 (9.8) 356/23 (15.8) 18 110/26 (4.2) 185/42 (4.4) 344/57 (6.0) 114/23 (5.0) 1930/882 (2.2) 864/420 (2.1) 723/186 (3.9) 118/21 (5.5) + +19 n.d. n.d. n.d. n.d. 25,300/1734 (14.6) 9510/846 (11.2) 10,900/981 (11.1) 449/27 (16.6) 20 n.d. n.d. n.d. n.d. 16,700/857 (19.5) 14,300/815 (17.5) 15,900/862 (18.4) 389/20 (19.9) + +Data are shown as A/C ratio (pg/mL, mg/dL, pg/mL per mg/dL, respectively). The number 1 to 3 and 7 to 14 patients + +showed the selectivity index . 2 without ACTH administration at the right adrenal vein, the central vein, and the + +common trunk. The AVS data in the adrenal vein with a higher A/C ratio than that in the IVC are shown in bold characters. The number 1 to 6 patients underwent a unilateral adrenalectomy. The number 1, 2, and 7 to 10 patients were determined as having unilateral disease from the AVS data without ACTH administration. The number 3, 13, and 14 patients showed ABAS at both the central vein and the common trunk without ACTH administration. The number 5 and 16 patients with ABAS with ACTH administration were determined as having unilateral disease from + +the AVS data at the common trunk in the left adrenal vein without ACTH administration. Patient no. 1 was determined as having unilateral disease at the right adrenal vein with ACTH administration. + +Abbreviations: n.d., no data; RAV, right adrenal vein. + +aThe AVS data are determined as unilateral disease because of an LI more than 4. + +Table 4. The Details of AVS in the 20 Patients With ABAS at Both the Central Vein and the Common Trunk With ACTH Administration trunk, blood sampling was also performed without ACTH administration. Eleven of the 16 patients were successfully cannulated at both the two positions in the left adrenal vein, and six patients were determined as having unilateral disease (right dominant: four patients; left dominant: two patients). Two of the six patients underwent unilateral adrenalectomies. Three patients showed ABAS at both the central vein and the common trunk even without ACTH administration. + +In 2 of the 20 patients who had ABAS at both the central vein and the common trunk, a repeat AVS was performed. In one patient (patient no. 3; Table 4), an alternative drainage vein from the left adrenal tumor to the left renal vein through the accessary left adrenal vein with higher A/C ratio than that in the IVC was detected. In the other patients (patient no. 6; Table 4), an alternative drainage vein to the right renal vein with higher A/C ratio than that in the IVC was detected. + +The postoperative outcomes of the 20 patients with ABAS who underwent unilateral adrenalectomy were evaluated. Of the 15 patients with available outcome data after surgery, complete clinical success, partial clinical success, and absent clinical success was 33.3% (five patients, including patient no. 6; Table 4), 53.3% (eight patients, including patient no. 3; Table 4), and 13.3% (two patients), respectively. Of the 13 patients with available biochemical data, complete biochemical success and absent biochemical success was 92.3% (12 patients, including patients no. 3 and 6; Table 4) and 7.7% (one patient), respectively. + +## 3. Discussion + +In this study, we used a sample size that was much larger than that of the WAVES-J study. + +Because we investigated the left adrenal vein data at the two sampling positions, the frequency of ABAS was higher than that of the studies by Wolley et al. [8] and the WAVES-J +study. ABAS was more prevalent in male patients with PA. The exact reason for the sex difference remains unknown. However, it could simply be attributed to APA being more common in the patients with ABAS and male sex being more common in the patients with APA in the current study. + +The current study demonstrated that ACTH administration reduces the frequency of ABAS, as suggested in the WAVES-J study. From the results of this study, it is recommended to perform ACTH administration to reduce the inconclusive results of AVS by ABAS. + +The prevalence of ABAS was not different between the procedures of the sampling position in the left adrenal vein, at the central vein and at the common trunk. Umakoshi et al. [12] +reported that the concordance rate of subtype diagnosis in the central vein and the common trunk was more than 95%, using the data of the WAVES-J study. They concluded that in a view of its better diagnostic accuracy, technical ease, lower cost, and lower risk of vein rupture, sampling from the common trunk of the left adrenal vein might be preferable as the standard method of AVS [12]. Performing an additional sampling at the central vein after encountering ABAS in AVS sampled at only the common trunk could be a worthwhile method. + +In the WAVES-J study, we reported that a patient who showed ABAS both without and with ACTH administration had an alternative drainage vein from APA that was discovered by a repeat AVS. A higher aldosterone concentration and a higher A/C ratio in the alternative drainage vein was found in the patient [9]. There are also some reports of an alternative drainage vein from an adrenal tumor [14, 15]. In this study, 2 of the 20 patients with residual unresolved ABAS with ACTH administration received a repeat AVS. In one patient, an alternative drainage vein that flowed into the left renal vein was found, and in the other patient, an alternative drainage vein that flowed into the right renal vein was found. + +In this study, the six patients who had ABAS at both the central vein and the common trunk with ACTH administration did not show ABAS and had conclusive results of AVS without ACTH administration. The current study demonstrated that the phenomenon occurs on both sides of the adrenal vein. It is suggested that the phenomenon could be attributed to a different catheter position without and with ACTH administration. Carr et al. [16] reported that the catheter shifted during the sampling without and with ACTH administration due to the respiratory movements. Because a sequential procedure requires recannulation into the adrenal vein, the position of the catheter may shift more frequently during the procedure. In this study, a sequential AVS was performed in 17 of the 20 patients with ABAS at both the central vein and the common trunk. It is however not clear whether the results of AVS may change by a simultaneous procedure. + +Some patients showed ABAS only at the central vein or at the common trunk. ABAS only at the central vein could be attributed to an incidentally too-selective cannulation into the tributary that is not drained by APA [17–20]. ABAS only at the common trunk could be attributed to a dilution into the common trunk turn by a blood flow with low aldosterone concentration from the left inferior phrenic vein. + +The strength of this study is that we could show the frequency of ABAS with a much larger cohort than the WAVES-J study with ACTH administration. In addition, we could show the frequency of ABAS at the central vein and at the common trunk in the left adrenal vein. The limitation of this study is that the variation in the protocols of AVS among participating centers might have effects on the occurrence of ABAS. The effect on ABAS between a sequential and a simultaneous AVS could not be clarified. + +In conclusion, the effectiveness of ACTH administration for the reduction of ABAS in the AVS procedures, regardless of the sampling positions in the left adrenal vein, was proven in a large cohort. + +## Acknowledgments + +We thank the JPAS investigators Masanobu Yamada, Nobuya Inagaki, Hiromi Rakugi, Koichi Yamamoto, Shigeatu Hashimoto, Masayoshi Soma, Hiroki Kobayashi, Toshihiko Yanase, Michio Otsuki, Yuichi Matsuda, Atsushi Ogo, Ryuichi Sakamoto, Tatsuya Kai, Tomikazu Fukuoka, Shintaro Okamura, and Shozo Miyauchi. + +Financial Support: This work was supported in part by grants-in-aid for the Japan Primary Aldosteronism Study, including a Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development under Grant JP17ek0109122 and National Center for Global Health and Medicine Grant 27-1402. + +Correspondence: Norio Wada, MD, PhD, Department of Diabetes and Endocrinology, Sapporo City General Hospital, Kita 11, Nishi 13, Chuo-Ku, Sapporo 060-8604, Japan. E-mail: norio.wada@doc. + +city.sapporo.jp. + +Disclosure Summary: The authors have nothing to disclose. + +## References And Notes + +1. Young WF. Primary aldosteronism: renaissance of a syndrome. Clin Endocrinol (Oxf). 2007;66(5): +607–618. + +2. Hannemann A, Wallaschofski H. Prevalence of primary aldosteronism in patient's cohorts and in population-based studies: a review of the current literature. Horm Metab Res. 2012;44(3):157–162. + +3. Rossi GP, Auchus RJ, Brown M, Lenders JW, Naruse M, Plouin PF, Satoh F, Young WF Jr. An expert consensus statement on use of adrenal vein sampling for the subtyping of primary aldosteronism. + +Hypertension. 2014;63(1):151–160. + +4. Monticone S, Viola A, Rossato D, Veglio F, Reincke M, Gomez-Sanchez C, Mulatero P. Adrenal vein sampling in primary aldosteronism: towards a standardised protocol. Lancet Diabetes Endocrinol. 2015; 3(4):296–303. + +5. Doppman JL, Gill JR Jr. Hyperaldosteronism: sampling the adrenal veins. Radiology. 1996;198(2): +309–312. + +6. Espiner EA, Ross DG, Yandle TG, Richards AM, Hunt PJ. Predicting surgically remedial primary aldosteronism: role of adrenal scanning, posture testing, and adrenal vein sampling. J Clin Endocrinol Metab. 2003;88(8):3637–3644. + +7. Zelinka T, Masˇek M, Vlkov ´a J, Kasalick ´y M, Michalsk ´y D, Holaj R, Petr ´ak O, Sˇtrauch B, Rosa J, +Dvorˇakov ´ ´ a J, Widimsk ´y J Jr. Discrepant results of adrenal venous sampling in seven patients with primary aldosteronism. Kidney Blood Press Res. 2012;35(4):205–210. + +8. Wolley M, Gordon RD, Pimenta E, Daunt N, Slater GJ, Ahmed AH, Stowasser M. Repeating adrenal vein sampling when neither aldosterone/cortisol ratio exceeds peripheral yields a high incidence of aldosterone-producing adenoma. J Hypertens. 2013;31(10):2005–2009. + +9. Shibayama Y, Wada N, Umakoshi H, Ichijo T, Fujii Y, Kamemura K, Kai T, Sakamoto R, Ogo A, +Matsuda Y, Fukuoka T, Tsuiki M, Suzuki T, Naruse M. Bilateral aldosterone suppression and its resolution in adrenal vein sampling of patients with primary aldosteronism: analysis of data from the WAVES-J study. Clin Endocrinol (Oxf). 2016;85(5):696–702. + +10. Nishikawa T, Omura M, Satoh F, Shibata H, Takahashi K, Tamura N, Tanabe A; Task Force Committee on Primary Aldosteronism, The Japan Endocrine Society. Guidelines for the diagnosis and treatment of primary aldosteronism: the Japan Endocrine Society 2009. Endocr J. 2011;58(9):711–721. + +11. 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(4):253–390. + +12. Umakoshi H, Wada N, Ichijo T, Kamemura K, Matsuda Y, Fuji Y, Kai T, Fukuoka T, Sakamoto R, Ogo A, +Suzuki T, Tsuiki M, Naruse M; WAVES-J Study Group. Optimum position of left adrenal vein sampling for subtype diagnosis in primary aldosteronism. Clin Endocrinol (Oxf). 2015;83(6):768–773. + +13. Williams TA, Lenders JWM, Mulatero P, Burrello J, Rottenkolber M, Adolf C, Satoh F, Amar L, +Quinkler M, Deinum J, Beuschlein F, Kitamoto KK, Pham U, Morimoto R, Umakoshi H, Prejbisz A, +Kocjan T, Naruse M, Stowasser M, Nishikawa T, Young WF Jr, Gomez-Sanchez CE, Funder JW, +Reincke M; Primary Aldosteronism Surgery Outcome (PASO) Investigators. Outcomes after adrenalectomy for unilateral primary aldosteronism: an international consensus on outcome measures and analysis of remission rates in an international cohort. Lancet Diabetes Endocrinol. 2017;5(9):689–699. + +14. Parnaby CN, Galbraith N, O'Dwyer PJ. Experience in identifying the venous drainage of the adrenal gland during laparoscopic adrenalectomy. Clin Anat. 2008;21(7):660–665. + +15. Scholten A, Cisco RM, Vriens MR, Shen WT, Duh QY. Variant adrenal venous anatomy in 546 laparoscopic adrenalectomies. JAMA Surg. 2013;148(4):378–383. + +16. Carr CE, Cope C, Cohen DL, Fraker DL, Trerotola SO. Comparison of sequential versus simultaneous methods of adrenal venous sampling. J Vasc Interv Radiol. 2004;15(11):1245–1250. + +17. Satani N, Ota H, Seiji K, Morimoto R, Kudo M, Iwakura Y, Ono Y, Nezu M, Omata K, Ito S, Satoh F, +Takase K. Intra-adrenal aldosterone secretion: segmental adrenal venous sampling for localization. Radiology. 2016;278(1):265–274. + +18. Kitamoto T, Suematsu S, Yamazaki Y, Nakamura Y, Sasano H, Matsuzawa Y, Saito J, Omura M, +Nishikawa T. Clinical and steroidogenic characteristics of aldosterone-producing adenomas with ATPase or CACNA1D gene mutations. J Clin Endocrinol Metab. 2016;101(2):494–503. + +19. Satoh F, Morimoto R, Seiji K, Satani N, Ota H, Iwakura Y, Ono Y, Kudo M, Nezu M, Omata K, Tezuka Y, +Kawasaki Y, Ishidoya S, Arai Y, Takase K, Nakamura Y, McNamara K, Sasano H, Ito S. Is there a role for segmental adrenal venous sampling and adrenal sparing surgery in patients with primary aldosteronism? Eur J Endocrinol. 2015;173(4):465–477. + +20. Morimoto R, Satani N, Iwakura Y, Ono Y, Kudo M, Nezu M, Omata K, Tezuka Y, Seiji K, Ota H, +Kawasaki Y, Ishidoya S, Nakamura Y, Arai Y, Takase K, Sasano H, Ito S, Satoh F. A case of bilateral aldosterone-producing adenomas differentiated by segmental adrenal venous sampling for bilateral adrenal sparing surgery. J Hum Hypertens. 2016;30(6):379–385. \ No newline at end of file diff --git a/medical/md/PMC6138786.md b/medical/md/PMC6138786.md new file mode 100644 index 0000000000000000000000000000000000000000..6cf4e88857bca342351a3984119322cdd35b7f75 --- /dev/null +++ b/medical/md/PMC6138786.md @@ -0,0 +1,526 @@ +Available online at www.sciencedirect.com + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +![0_image_4.png](0_image_4.png) + +journal homepage: www.elsevier.com/locate/bj + +![0_image_3.png](0_image_3.png) + +Review Article: Special Edition + +![0_image_1.png](0_image_1.png) + +Danger signals, inflammasomes, and the intricate intracellular lives of chlamydiae Matthew A. Pettengill a,1, Ali Abdul-Sater b,1, Robson Coutinho-Silva c, David M. Ojcius c,d,* +a University of Rochester Medical Center, Rochester, USA +b School of Kinesiology and Health Science, York University, Toronto, Ontario, Canada c Laboratory of Immunophysiology, Carlos Chagas Filho Biophysics Institute of the Federal University of Rio de Janeiro, Brazil d Department of Biomedical Sciences, University of the Pacific, Arthur Dugoni School of Dentistry, San Francisco, USA +article info + +## Abstract + +| Article history: Received 4 April 2016 Accepted 13 July 2016 Available online 27 October 2016 Keywords: Innate immunity Chlamydia Immunology Inflammation Infection | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Chlamydiae are obligate intracellular bacterial pathogens, and as such are sensitive to alterations in the cellular physiology of their hosts. Chlamydial infections often cause pathologic consequences due to prolonged localized inflammation. Considerable advances have been made in the last few years regarding our understanding of how two key inflammation-associated signaling pathways influence the biology of Chlamydia infections: inflammation regulating purinergic signaling pathways significantly impact intracellular chlamydial development, and inflammasome activation modulates both chlamydial growth and infection mediated pro-inflammatory cytokine production. We review here elements of both pathways, presenting the latest developments contributing to our understanding of how chlamydial infections are influenced by inflammasomes and purinergic signaling. + +The bacterial family Chlamydiaceae includes several species which promiscuously or sporadically infect humans. Chlamydia trachomatis is the most common nationally notifiable infection in the USA [1], the most common bacterial cause of sexually transmitted infection worldwide [2], and also a prominent cause of preventable blindness following repeated conjunctival infections in the developing world [3]. C. trachomatis can also cause pneumonia in infants following exposure during birth [4]. Chlamydophila pneumoniae is one of the leading causes of pneumonia in the developed world [5] and may increase the risk of developing atherosclerotic lesions in coronary artery disease [6]. Chlamydophila psittaci, while primarily an avian pathogen, sporadically causes human pneumonia [7,8]. Chlamydophila abortus and Chlamydophila caviae zoonotic infections have been reported in humans but are rare [9,10]. Untreated C. trachomatis genital tract infection in women may ascend the endometrial endothelium to reach the fallopian tubes, with the associated chronic inflammation leading to pelvic inflammatory disease (PID), which may also cause miscarriage [11], ectopic pregnancy [12], or tubal scarring and infertility [13]. Repeated and chronic infection of the conjunctiva with C. trachomatis leads to recruitment of lymphocytes and the formation of follicles and inflammation mediated conjunctival thickening, which subsequently causes the deformation of the eyelids and corneal damage via scraping of the cornea by in-turned eyelashes [14]. Pneumonia caused by C. pneumoniae develops slowly and leads to inflammation of the lungs but with limited production of purulent sputum. C. pneumoniae also causes infections of the upper respiratory tract including pharyngitis, sinusitis, and bronchitis. Inflammation due to repeated infections with C. + +pneumoniae, or unrecognized and untreated infections, may contribute to chronic obstructive pulmonary disorder (COPD) +[15,16]. Human disease following infection with Chlamydiaceae species bears a consistent hallmark: chronic, localized inflammation. + +Two major pathways relevant to the induction and regulation of localized inflammation have recently received considerable research emphasis and have been demonstrated to be particularly relevant during Chlamydia infection: purinergic signaling, and the formation of macro-molecular inflammasomes. Here we review the basic concepts of purinergic signaling in the context of immune function and inflammation regulation, and inflammasome mediated inflammatory cytokine production, followed by an examination of the recent literature evaluating the impact of host purinergic signaling and inflammasome activation on chlamydial infection. + +## Extracellular Purines And Purinergic Receptors + +A wide range of extracellular purine concentrations are physiologically relevant [17], and they are met by a similarly broad spectrum of sensory affinity in the purinergic receptor families [18]. ATP is released from cells under normal physiologic conditions reaching nanomolar to low micromolar concentrations in the immediately adjacent extracellular space [19e21]. Higher concentrations of ATP or ADP result from various forms of cell stress [22], platelet degranulation +[23], or are present in tumor microenvironments [24]. The concentration of ATP in cells ranges from 3 to 10 mM, and thus in the context of cell damage or necrosis the neighboring cells are exposed to low millimolar levels of extracellular ATP and purine metabolites. ATP may be released via degranulation in cell types which produce ADP or ATP rich granules, via pannexin channels [25,26], or following cell damage. At colonized mucosal epithelial surfaces, ATP may also be directly released by bacteria [27]. ATP is also released from cervical epithelial cells in vitro during C. trachomatis infection, particularly during the late stages of inclusion development when there is likely more cellular stress [28]. + +Receptor mediated purine signaling is an evolutionarily conserved cellular function, and is involved in a wide variety of physiological processes in mammals including neurologic signaling, vascular function, and immune cell regulation. + +Receptors which recognize purine nucleotides and nucleosides are termed purinergic receptors. Purinergic receptors are grouped into families based on functional similarity: P1 receptors are engaged by the purine nucleoside adenosine, while P2 receptors are activated by nucleotides and are further subdivided into gated ion channels (P2X) or G-protein coupled seven transmembrane receptors (P2Y). + +Adenosine receptors couple via G-proteins to adenylyl cyclase to modulate cAMP generation in cells. A1 and A3 receptors associate with Gi proteins to inhibit adenylyl cyclase and prevent cAMP upregulation, whereas A2a and A2b receptors interact with Gs proteins to activate adenylyl cyclase, leading to elevated intracellular cAMP. Adenosine receptors are expressed in a wide variety of cell types, with particularly well described roles for A1 and A3 receptors in cardiac function, and for A2a receptors in immune cell function and A2b on epithelial and endothelial cells. A2b also mediates intracellular signaling via Gq and phospholipase C [29]. Recent excellent reviews have organized the great depth of studies related to adenosine receptor mediated regulation of immune cells [30,31]. + +P2X purinergic receptors are ligand gated ion channels which are activated by ATP, and possibly ADP in the case of P2X4, and are increasingly recognized to play a role in inflammation and immune cell function [32]. There are seven described P2X receptors, the best characterized being P2X7. P2X7 plays a critical role in the production of IL-1b and other inflammatory cytokines both in the context of infection and sterile inflammation [33,34]. Initial stimulation of P2X receptors leads to the opening of small-cation permeable pores (approximately 0.85 nm for P2X7), while prolonged ligation then leads to increased permeability to larger molecules (greater than 1 nm for P2X7) [35], either by P2X7 pore dilation or by P2X7 coupling to pannexin1 and the pannexin pore. + +P2Y receptors respond to purine and pyrimidine nucleotides and mediate intracellular signaling via regulation of cAMP or activation of PLC. Eight P2Y receptors have been characterized (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14), and while their activity relevant to inflammation and immune cell function is less well characterized than for P2X +receptors, some P2Y receptors are expressed on immune cells +[18,36,37]. + +While there is nuance to the roles that purine signaling plays in local and systemic immune regulation, in general, ATP mediates pro-inflammatory responses, and adenosine receptor stimulation is anti-inflammatory [38]. Thus soluble or cell surface associated extracellular enzymes play a critical role in determining the response to extracellular purines. + +Adenine nucleotides released from cells are dephosphorylated by members of several families of purine enzymes to generate adenosine. Enzymes such as CD39 (ENTPD1) and alkaline phosphatase dephosphorylate ATP and ADP to generate AMP, and CD73 (ecto-50-nucleotidase) or alkaline phosphatase dephosphorylates AMP to generate adenosine. Adenosine can be captured by adjacent cells via nucleoside transporters for purine salvage, or metabolized by adenosine deaminase to produce inosine. Enzymes regulating extracellular purine metabolism have been more completely described in recent reviews [39,40]. + +## Purinergic Receptor Stimulation During Chlamydial Infection + +In the last 10 years, there have been many investigations into the impact of purinergic receptor stimulation on chlamydial development in vitro. + +After work by other groups showed that purinergic receptor stimulation altered mycobacterial infection in host cells +[41,42], we demonstrated that P2X7 ligation (5 mM ATPe) +reduced growth of C. psittaci in J774 macrophages, while at the same time the chlamydial infection partially inhibited ATPe mediated apoptosis of host cells [43]. We then demonstrated that ATP stimulation of P2X7 on murine macrophages led to killing of intracellular Chlamydia muridarum [44], the murine equivalent of C. trachomatis. Ligation of P2X7 receptor with 0.5e5.0 mM ATPe caused fusion of chlamydial inclusions with host-cell lysosomes, leading to chlamydial death, an effect which was dependent on activation of phospholipase D (PLD). + +Additionally, P2X7 ligation suppresses C. muridarum infection in the preferred target cell of genitotropic Chlamydia species e cervical epithelial cells [45]. As was the case in murine macrophages, ATPe/P2X7 mediated chlamydial inhibition in murine cervical epithelial cells was at least partially dependent on PLD activation. In animal experiments with P2X7 wild-type and P2X7/- mice, P2X7 expression reduced the intensity, but not the duration, of vaginal infection, and pathology scores based on histological evaluation of endocervix, oviduct, and mesosalpinx tissue indicated increased inflammation in the P2X7/- mice following chlamydial infection [45]. + +While millimolar concentrations of ATPe are required for P2X7 mediated effects on Chlamydia infection, micromolar concentrations of ADPe and ATPe reversibly inhibit chlamydial development in human cervical epithelial cells via P2X4 ligation [28]. One hundred mM ADPe or ATPe applied to C. + +trachomatis infected epithelial cells at 1 h post-infection (hpi) +significantly inhibited chlamydial inclusion development through 24 h of infection, although by 48 hpi inclusion development was proceeding normally. If, however, repeated applications of ADPe or ATPe were made, inclusion development did not occur by 48 hpi unless the media was changed at 24 hpi. Thus, duration of exposure to micromolar concentrations of ADP or ATP determined the extent of impairment of chlamydial development. In the context of inflammation, ATPe (pro-inflammatory) and ADOe (anti-inflammatory) often have paradoxical effects; however, both inhibit development of C. trachomatis in human cervical epithelial cells at micromolar concentrations. ADOe suppression of chlamydial development is reversible, and is mediated by A2b receptor stimulation [46]. ADOe and ATPe also inhibit the development of C. trachomatis serovar E and Chlamydia pecorum in human epithelial cells [47]. It should be noted that two independent investigations have also demonstrated that millimolar concentrations of extracellular cyclic adenosine monophosphate (cAMP) inhibit chlamydial development, leading to the production of aberrant chlamydial bodies [47,48]. As no receptor for extracellular cAMP has been described, and cAMP is not cell permeable, this effect may possibly be mediated by purinergic receptor stimulation following degradation of extracellular cAMP. The mechanism by which purinergic stimulation of host cells alters chlamydial development has not been elucidated, and it is also not clear whether the alterations described in vitro would be deleterious or advantageous for chlamydial propagation in vivo. As an obligate intracellular bacterium with a substantially reduced genome, chlamydiae are particularly sensitive to changes in the metabolic environment of the host cell. Indeed, in the best described in vitro model of chlamydial persistence, it has been demonstrated that IFN-g stimulation of human epithelial host cells leads to restriction of intracellular tryptophan for which chlamydiae are auxotrophic [49,50]. Future studies are needed to evaluate the molecular basis for the impairment in chlamydial development following host cell exposure to elevated extracellular purines. + +## Inflammasomes And Inflammation + +The innate immune system mounts a rapid response to unique molecular signatures from the invading pathogen termed pathogen associated molecular patterns (PAMPs), +where it utilizes a set of germ-line encoded pattern recognition receptors (PRRs) to initiate a measured and appropriate inflammatory response in order to slow down the spread of infection and thereby allowing and contributing to the initiation of adaptive immunity. Furthermore, PRRs can detect danger signals termed danger associated molecular patterns +(DAMPs) released by damaged host cells (for a review, see Ref. + +[51]). PRRs can be membrane-bound like the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), which sample the extracellular milieu from the plasma membrane and within endosomes, or cytosolic like the nucleotidebinding and oligomerization domain (NOD)-like receptors +(NLRs), retinoid acid-inducible gene I (RIG-I)-like receptors +(RLRs) and AIM2-like receptors (ALRs), which survey intracellular PAMPs and DAMPs (reviewed in Refs. [52e54]). + +Typically, most PRRs (e.g. TLRs), following recognition of their PAMP, initiate distinct intracellular signaling cascades, which lead to induction of various programs of gene transcription including, but not limited to, co-stimulatory molecules which activate components of the adaptive immune response, production of reactive oxygen and nitrogen species, and expression of pro-inflammatory cytokines and chemokines [53,54]. On the other hand, NLRs e a family of 22 genes in humans and 34 genes in mice ecan detect not only intracellular PAMPs (e.g. NOD1, NOD2) [55], but also can sense endogenous DAMPs [52,56,57]. These latter NLRs, which include NLRP and NLRC proteins, are components of a macromolecular protein complex e the inflammasome e required for the activation of caspase-1 and the subsequent secretion of potent pro-inflammatory cytokines, including Interleukin (IL)-1b (IL-1b) and IL-18 (reviewed in Ref. [58]). + +Due to their high pro-inflammatory potency and their significant role in innate defense to invading pathogens, secretion of both IL-1b and IL-18 is a highly regulated process in order to avoid diseases associated with their excessive and chronic production, like autoimmune disorders and septic shock [59]. Both cytokines are produced as inactive and leaderless forms, pro-IL-1b and pro-IL-18, and require cleavage by caspase-1 to promote the unconventional secretion of their mature and biologically active forms [60e62]. Caspase-1, along with caspase-4 and caspase-5 in humans and caspase-11 in mice, constitute a family of inflammatory caspases that exist as inactive zymogens (pro-caspases). Activation of these caspases requires the activation of inflammasomes, which are a multimeric assembly of pro-caspase-1 and an NLR family member, either directly through CARDeCARD interaction or indirectly via the adapter protein ASC [63,64]. These studies led the way for the identification of additional means to nucleate the inflammasome by members of the RLR and ALR +families, and the assembly of non-canonical inflammasomes that lead to the activation of caspase-11 in mice and caspase4/5 in humans. The NLRP3 inflammasome (also known as NALP3, cryopyrin) is the best-characterized inflammasome to date and can be activated by wide variety of stimuli including PAMPs, DAMPs, pore-forming toxins, whole pathogens and environmental stressors (reviewed in Refs. [65e67]). Due to the large number of triggers and in order to prevent inadvertent activation, the NLRP3 inflammasome requires two signals for its optimal induction. PRR agonists (e.g. LPS) provide the first signal and cause NF-kB mediated upregulation of pro-IL-1b and components of the inflammasome, including NLRP3 [68]. The second signal can be provided by a large repertoire of +'danger signals' that include host derived DAMPs (e.g. ATP, +adenosine, hyaluronan, HMGB1, gout-associated uric acid crystals and amyloid-b fibrils [69e74]), environmental stressors (e.g. asbestos, silica crystals [75e77]), and PAMPs that gain access to the cytosol. These PAMPs can be derived from bacteria (e.g. toxins, RNA:DNA hybrids, type III secretion system effectors, cyclic-dinucleotides [58,67,70,78e85]), viruses (e.g. dsRNA, viral proteins [86e90]), fungi (e.g. hyphae, zymosan, mannan [91e94]), or Plasmodium, a malaria causing parasite (e.g. hemozoin [95]). Importantly, some intracellular pathogens can activate the NLRP3 inflammasome by providing both signals (e.g. C. trachomatis [58]). + +Despite this wide array of NLRP3 activators, researchers in the field generally agree that NLRP3 does not directly sense any of these stimuli, but instead detects specific host-derived molecular event(s), which then cause the assembly of the NLRP3 inflammasome. These upstream molecular events can be summarized into three main categories. (1) Events that perturb the homeostasis of intracellular cations, and include an increase in intracellular Ca2þ [79,96,97] or Kþ efflux, which usually occurs through the activated ATP-gated ion channel P2X7 or toxin-induced pores in plasma membrane [70,98e100]. (2) +Events that cause reactive oxygen species (ROS)-mediated oxidative stress and mitochondrial perturbation. Although the exact mechanism of ROS-mediated NLRP3 inflammasome activation remains controversial, there is a general consensus that mitochondrial ROS (mROS) production is the upstream trigger for NLRP3 activation following stimulation with several inducers (e.g. ATP, uric acid crystals, alum, nigericin, certain pathogens [65,101]). Elevated and prolonged release of mROS by damaged mitochondria [101] leads to oxidation of mitochondrial DNA, which when leaked to the cytosol binds directly to NLRP3, thereby promoting its activation [102,103]. (3) Release of Cathepsins from destabilized lysosomes following 'frustrated phagocytosis' of relatively large 'sterile' molecules (e.g. silica, alum and uric acid crystals, asbestos, amyloid-b-fibrils, malarial hemozoin [69,77]) also activate the NLRP3 inflammasome. Again, the exact mechanism of how this leads to NLRP3 inflammasome activation remains poorly understood. + +## Chlamydiae And Inflammasomes + +In response to Chlamydia infection, epithelial cells produce proinflammatory cytokines and chemokines in order to activate and recruit innate immune cells into the site of infection [104]. + +These immune cells, which include dendritic cells, macrophages, neutrophils and natural killer cells, in turn secrete cytokines including TNF-a and IL-1b in response to the infection [105e110]. Consistently, IL-1b deficient mice infected with C. muridarum displayed delayed clearance of the infection [111]. + +However, chronic and excessive production of these proinflammatory cytokines has been touted as the dominant culprit causing the pathology associated with chlamydial infections [112e115]. Consistently, caspase-1 deficient mice displayed reduced inflammatory damage in the urogenital tract, suggesting that it may contribute to the pathology of infection by Chlamydia [116]. Therefore, revealing the mechanism of IL1b production has important implications for understanding pathology associated with Chlamydia infection. + +Previous studies have shown that caspase-1 could be activated during chlamydial infection [107,109,111,116,117]. + +Indeed, C. trachomatis infection of cervical epithelial cells caused caspase-1 activation in a manner dependent on NLRP3 inflammasome activation [58]. Subsequent studies showed that human monocytic cell lines infected with C. trachomatis and C. muridarum secreted IL-1b following caspase-1 activation in an NLRP3-dependent fashion [78], while C. pneumoniae was the most potent inducer of IL-1b secretion in bone marrow derived murine macrophages [118]. These studies showed that Chlamydia infection alone was sufficient for inflammasome-dependent caspase-1 activation likely because an active Chlamydia infection can provide both signal one via the TLR2/MyD88 axis [119e121] in addition to signal two, activating caspase-1. The mechanism whereby chlamydial infections provide the second signal for NLRP3 inflammasome activation was thoroughly investigated. C. trachomatis induced inflammasome activation is dependent on Kþ efflux [58] although the role of the P2X7 receptor, which is required for Kþ efflux mediated inflammasome activation +[122] and plays an important role in Chlamydia infection [45], +remains to be investigated. Chlamydia-induced loss of intracellular potassium seems to trigger production of ROS, which appears to be essential for proper activation of the NLRP3 inflammasome [67]. Importantly, the mitochondrial associated protein, NLRX1, plays an important role in mediating ROS production following infection with C. trachomatis [78]. Moreover, C. pneumoniae mediated capase-1 activation required lysosomal acidification and cathepsin B release [118]. The ability of chlamydiae to activate the inflammasome is dependent on the chlamydial type III secretion system (T3SS) +[58]. Intriguingly, a chlamydial plasmid encoded protein, pORF5 (a.k.a. pgp3), which is secreted into the host cytosol +[123], might play a role in inflammasome activation [124]. + +Finally, the Rho family GTPase, Rac1, was shown to play a role in C. pneumoniae induced inflammasome activation [125]. + +![4_image_0.png](4_image_0.png) + +The role of the inflammasome in the control of chlamydial infection is still controversial. In a mouse model of C. pneumoniae lung infection, results showed that caspase-1 deficient mice displayed delayed pulmonary bacterial clearance of infection, which was associated with increased mortality compared to wild type mice, thereby indicating that inflammasome activation has an important role in the host response to the infection [83]. On the other hand, lung fibroblasts from ASC/- and caspase/- mice displayed resistance to infection by C. trachomatis, a result which was recapitulated by the caspase-1 inhibitor, YVAD [126]. In agreement, a recent report showed that IL-10 mediated increase in tubal damage and infertility in Chlamydia-infected mice is mediated by its ability to enhance NLRP3 inflammasome activation in infected DCs +[127]. Furthermore, in vivo challenge with C. muridarum in IL1R deficient mice showed that, while IL-1b plays an important role for clearance of genital chlamydial infection, infection of NLRP3/- and ASC/- mice showed that the inflammasome plays a limited role in this model [128]. + +Chlamydia is described as the 'perfect pathogen' due to its ability to evade host-cell defenses and utilize host resources to promote its own growth [129e131]. Consistently, Chlamydia subverts host lipids from the Golgi by inducing caspasemediated fragmentation of the Golgi apparatus [132]. Intriguingly, inflammasome-mediated caspase-1 activation following C. trachomatis infection is required for optimal intracellular growth of this pathogen [58]. Similarly, bonemarrow derived macrophages from NLRP3, ASC or caspase-1 deficient mice showed severely impaired intracellular growth of C. pneumoniae [133]. Intriguingly, both the caspase-1 inhibitor, YVAD, and caspase-4/5 (murine caspase-11 ortholog) inhibitor, WEHD, inhibited chlamydial growth, suggesting possible roles for both caspase-1 and non-canonical caspases (caspase-4/5 or caspase-11) in this process [58]. In fact, a recent study suggested that guanylate binding protein (GBP), which promotes caspase-11-dependent pyroptosis, might play a partial role in regulating the kinetics of inflammasome activation and affecting the ratio of IL-1b vs IL-18 production following C. muridarum infection [134]. Furthermore, treatment with probenecid, a pannexin-1 channel blocker that inhibits P2X7 receptor mediated inflammasome activation following Kþ efflux [135], directly inhibits chlamydial development in a dose-dependent and reversible manner, although a direct role for pannexin-1 is not clear [136]. + +In conclusion, although results from different laboratories show consistently that an NLRP3 inflammasome is activated by infection with different species of Chlamydia and that the inflammasome may be involved in the immune response to infection, much remains to be known of the effects that both canonical and non-canonical inflammasomes may have on metabolism of infected cells [Fig. 1]. 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Assessing a potential role of host Pannexin 1 during Chlamydia trachomatis infection. + +PLoS One 2013;8:e63732. \ No newline at end of file diff --git a/medical/md/PMC6514177.md b/medical/md/PMC6514177.md new file mode 100644 index 0000000000000000000000000000000000000000..f04a440ecff5295eabf99db44a1524c730dc1db4 --- /dev/null +++ b/medical/md/PMC6514177.md @@ -0,0 +1,408 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +![0_image_1.png](0_image_1.png) + +# Real-World Data To Identify Hypercholesterolemia Patients On Suboptimal Statin Therapy + +Kouji Kajinami1, Asuka Ozaki2, Yuki Tajima2, Shizuya Yamashita3, 4, Hidenori Arai5 and Tamio Teramoto6 1Department of Cardiology, Kanazawa Medical University, Ishikawa, Japan 2Sanofi, Tokyo, Japan 3Rinku General Medical Center, Osaka, Japan 4 Department of Community Medicine & Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan 5National Center for Geriatrics and Gerontology, Aichi, Japan 6Teikyo Academic Research Center, Tokyo, Japan Aim: **Statins are generally well-tolerated but some patients develop adverse events and down-titrate or discontinue statins. It is important to understand the frequency of dyslipidemia patients with the inability to continue** +statins. The aim of the present study was to identify the frequency of high-risk dyslipidemia patients who are unable to take or not taking statins for any reason using Japanese hospital claims database. + +Methods: **2,527,405 dyslipidemia patients with atherosclerotic cardiovascular disease were investigated between** +April 2008 and September 2017. Definition 1 included statin discontinuation or down-titration with non-statin lipid modifying therapy (LMT) prescription, rhabdomyolysis or muscle-related symptoms with statin downtitration or discontinuation, or prescription for ≥3 statin types. Definition 2 included all components of Definition 1 in addition to statin down-titration or discontinuation for any reason. Patients never given statins but who started non-statin LMT were considered as Definition 3. The achievement rate of the target LDL-C level was investigated. + +Results: **Among 54,296 patients with statin prescription, 2.32% and 48.38% patients were identified as Definition 1 and 2, respectively. Of eligible patients, 13.16% patients were identified as Definition 3. The achievement** +rate of target LDL-C level was lower in patients meeting each definition than not satisfying each definition. + +Conclusions: **There is a proportion of high-risk dyslipidemia patients unable to take or not taking statins for** +any reason, and it is associated with lower achievement rates of target LDL-C levels. Suboptimal management of LDL-C is directly associated with residual cardiovascular risk and implementation of alternative therapeutic options in addition to existing LMT is warranted. + +## See Editorial Vol. 26: 403-405 + +Key words: **Hypercholesterolemia, Claim data, Statin, Non-HDL-cholesterol, LDL-cholesterol** + +## Introduction + +Lowering low-density lipoprotein cholesterol (LDLC) level is one of the most widely available approaches to prevent atherosclerotic cardiovascular disease (ASCVD), +including coronary artery disease (CAD), which is a major cause of mortality in Japan and worldwide 1, 2). + +The Japan Atherosclerosis Society (JAS) guidelines recommend lowering LDL-C to <100 mg/dL in patients with a history of CAD and to <70 mg/dL (or by 50% +or more) in high-risk patients with heterozygous familial hypercholesterolemia or acute coronary syndrome for secondary prevention of CAD3). + +The JAS guidelines as well as those of other major international associations including the American College of Cardiology/American Heart Association (ACC/ +AHA), the European Society of Cardiology/European Atherosclerosis Society, the National Lipid Association, and the International Atherosclerosis Society recommend statins as the first-line treatment for dyslipidemia patients with elevated LDL-C levels 4-7). Asian patients often show an increased response to drug therapy compared to Western populations, leading to lowered doses of therapeutic drugs such as statins. The approved maximum dose of statin is lower (e.g., atorvastatin 40 mg/ +day and rosuvastatin 20 mg/day) as well as clinically used dose8, 9). Statins are generally well tolerated, and serious adverse events are infrequent10, 11); however, some patients experience statin-associated muscle symptoms +(SAMS) such as myositis and rhabdomyolysis and hepatic disorder that result in dose reductions or discontinuation of statin treatment 4-7, 12). The inability to continue an effective dosage or the discontinuation of a statin treatment may limit clinically important effects, such as CAD risk reductions13, 14). + +Statin intolerance is a term that is widely used to describe clinical phenomenon such as the inability to continue an effective dosage or the discontinuation of statin treatment due to adverse events, but there are no globally accepted criteria for statin intolerance. Previous studies have attempted to estimate the proportion of dyslipidemia patients unable to continue an effective dosage or who need to discontinue statin treatment using various criteria; results range from 1% to 30%15, 16). With the complexity of a dose-dependent relationship of adverse effects and more sensitive response in the Asian population to medications, the precise number of the inability to continue an effective dosage or the discontinuation of statin treatment due to adverse events is unknown. + +In an era of new alternative lipid-lowering therapies supported by emerging evidence, it is important to understand the frequency of dyslipidemia patients with an inability to continue an effective dosage of statins by quantifying patients with high cardiovascular risk who may be candidates for non-statin lipidmodifying therapy (LMT) such as PCSK9 inhibitors, cholesterol absorption inhibitors, and fibrates in Japan. + +## Aim + +The aim of the present study was to identify the frequency of dyslipidemia patients with a history of ASCVD such as CAD with an inability to continue an effective dosage or who need to discontinue statin treatment for any reason in Japanese clinical practice using a Japanese electrical medical record (EMR) database. Because lipid management in these patients might be poor, we also investigated the frequency of patients who achieved target values such as LDL-C recommended by the JAS guidelines3). + +## Methods Database + +This retrospective observational study was conducted using the EBM Provider, which contains hospital claims database stored in electronic hospital information systems constructed by Medical Data Vision Co., Ltd. (MDV; Tokyo, Japan). The EBM Provider covers approximately 20.8 million patients from 323 hospitals across Japan (as of December 2017) with bed numbers ranging from 20 to over 1000. This database also includes approximately 19% of all acute phase hospitals in Japan, including university hospitals, which use the Diagnostic Procedure Combination (DPC) payment system/Per-Diem Payment System. + +The EBM Provider includes anonymized patientlevel information on sex, age, previous medical history +(diagnosed diseases), medical procedure, and prescribed drug. Laboratory test values were also provided by 35 hospitals. Information about the diagnosis and drugs is extractable based on the International Classification of Disease (ICD)-10 codes and the Anatomical Therapeutic Chemical (ATC) code, respectively. + +## Patient Selection + +All patients diagnosed with ASCVD (ICD-10 code: I20, I21, I22, I23, I24, I25, I63, I65, I66, I67, I69, I70, I71, I72, I73, I74) between April 2008 and September 2017 (defined as the study period) were identified from the EBM Provider database. Patients who met all of the following criteria were included in the present study: 1) diagnosis of dyslipidemia (standardized Japanese disease codes: 2724036; high LDL cholesterolemia, 2720004; hypercholesterolemia, 8840108; essential hypercholesterolemia, 8833881; mixed hyperlipidemia, 2724007; hyperlipidemia, 8833722; hyperlipoproteinemia, 8844446; dyslipidemia, 2724012; essential hyperlipidemia, 2729002; disorder of lipid metabolism, 8845052; type1 diabetic hypercholesterolemia, 8845081; type2 diabetic hypercholesterolemia, 8838067; diabetic hypercholesterolemia) during the study period; 2) initiation of any statin (ATC code: C10A1, C11A1) +agents during the study period and initiation of nonstatin LMT (ATC code: C10, C11) if statin agents were not prescribed during the study period (as an index date); 3) at least 365 days of medical data available before and after the index date; 4) age at the index date over 20 years; 5) no prescription of LMT during the look-back period of 365 days before the index date; and 6) diagnosis of ASCVD before the index date. + +Patients with age at the index date younger than 20 years old and index date before April 2009 or after September 2016 were excluded from the study. + +Definition of dyslipidemia patients who were unable to take or were not on statin treatment for any reason was modified to fit a Japanese setting with reference to the definitions used in previous studies15, 16). + +In the present study, three definitions of dyslipidemia patients who were not able to take or not taking statin treatment for any reason were considered. + +Patients included in the present study who met any of following criteria between 365 days from index date were considered to meet the criteria for Definition 1. + +- Statin discontinuation (not prescribed within 90 days after the addition statin prescription days on last visit to the last visit date) with prescription of non-statin LMT +- Statin down-titration with prescription of nonstatin LMT. + +- Rhabdomyolysis with statin down-titration or discontinuation. + +- Muscle-related symptoms with statin down-titration or discontinuation. + +- Prescription for ≥ 3 types of statins. + +A dyslipidemia patient who was unable to take or was not taking statin treatment for any reason was considered to be down-titrated or discontinued without prescribing non-statin LMT. Definition 2 included the following criteria in addition to all components of Definition 1. + +- Down-titration of statin for any reason - Discontinuation of statin for any reason Patients who had never been prescribed statin but had started non-statin LMT during the study period were considered to satisfy the criteria for Definition 3. + +Details of each definition component are described in Table 1. + +## Study Measures + +The number and proportion of patients who met the criteria for each definition of dyslipidemia patients unable to take or not taking statin treatment due to any reason were identified. Patient characteristics (sex, age, previous disease history, medication use, and Charlson Comorbidity Index 17)) for Definitions 1, 2, and 3 during the 365 days before the index date were described and compared with those of patients with statin prescription who did not meet the criteria for Definitions 1 and 2 (defined as comparator of each definition). + +The number and proportion of patients who achieved the target laboratory test values were identified to confirm lipid controls for each definition and each comparator. The earliest LDL-C and non-highdensity lipoprotein (HDL-C) level recorded 365 days after the index date were used. The target value of each laboratory test parameter was referred the value of a patient with a history of CAD recommended by the JAS guidelines (LDL-C: <100 mg/dL, <70 mg/dL, +non-HDL-C: <130 mg/dL, <100 mg/dL). In addition, subgroup analysis of patients with a history of CAD and diabetes mellitus (DM) was also conducted. + +## Statistical Analysis + +For patient characteristics, categorical variables were presented as number of patients and percentages and continuous variables were presented as mean and standard deviation (SD). The chi-squared test was used for categorical variables and the Student t-test was used for continuous variables to evaluate group differences. + +The achievement of a target laboratory test value was presented as the number of patients and percentages. Since not all patients had laboratory data stored in the EBM Provider, the denominator of the achievement proportion was the number of patients with clinical laboratory values during the follow-up period. A +chi-square test was used to test differences between the groups. + +All analyses were conducted using SAS version 9.4 software (SAS Institute, Cary, North Carolina). When the p-value on the 2-sided test was <0.05, the difference was regarded as statistically significant. + +## Results + +A total of 2,527,405 patients diagnosed with ASCVD during the study period were identified from the EBM Provider. Among the 62,522 eligible patients who matched all inclusion criteria, 54,296 patients were started on statins and were included in the analysis based on Definitions 1 and 2. Among patients with statin prescription, 1260 patients (2.32%) were identified according to Definition 1; 26,271 patients (48.38%) +were identified as satisfying Definition 2 criteria. Among all eligible patients, 8226 patients (13.16%) were treated only with non-statin LMT (Definition 3). "Statin discontinuation with prescription of non-statin LMT" +(48.65%) and "Muscle-related symptoms with statin down-titration or discontinuation" (38.73%) were the most common reasons for satisfying Definition 1 criteria. "Discontinuation of statin for any reason" (92.91%) +was the most common reason for satisfying Definition 2 criteria (**Fig.1**). + +Patient characteristics for each definition and comparators are shown in **Tables 2** to 4. + +For the patients satisfying Definition 1 criteria and the comparators of Definition 1, the number was 1260 + +## Table 1. Components And Details Of Each Definition Group + +Definition 1 + +Component Details + +Statin discontinuation with prescription of + +non-statin LMT. + +Statin was discontinued and non-statin LMT was prescribed within 90 days after the + +expected next visit (the last visit day+prescription days on last visit) from the last visit. + +Statin down-titration with prescription of + +non-statin LMT. + +All of the following four criteria: - Prescribed non-statin LMT between 365 days from index date (defined as assessment + +period). + +- Had no statin prescription for the same or higher intensity (Supplementary Table 1) + +than the initial statin prescription on the same day or after the first LMT prescription + +and during the remainder of the assessment period. + +- Had a statin prescription at a lower intensity than the initial statin prescription any time + +before or within 7 days after the first non-statin LMT prescription. + +- Had ≥1 statin prescription at a lower intensity than the initial statin prescription including the statin prescription closest to and before the first non-statin LMT prescription + +and any subsequent statin prescription through the end of the assessment period. + +Rhabdomyolysis with statin down-titration or discontinuation.Had ≥1 inpatient or outpatient claim for rhabdomyolysis (standardized Japanese disease + +code: 8846452) in any position, followed by statin down-titration or discontinuation. + +Muscle-related symptoms with statin down-titration or discontinuation. + +Had ≥1 inpatient or outpatient claim or diagnosis for muscle related symptoms (Supplementary Table 2) in any position, followed by statin down-titration or discontinuation. + +Prescription for ≥3 types of statins. Switching between ≥3 types of statins. + +Definition 2 + +Down-titration of statin for any reason. Had a statin prescription at a lower intensity than the initial statin prescription during the + +assessment period with no subsequent statin prescription for the same or higher intensity + +than the initial statin prescription. + +Discontinuation of statin for any reason. The statin is not prescribed within 90 days after the expected next visit (the last visit day + ++prescription days on last visit). + +Definition 3 + +Not prescribed statin. The statin is not prescribed during study period. + +LMT: lipid-modifying therapy + +and 53,036, with a mean age of 69.9 and 71.2 years (p<0.001), and the proportion of males was 55.71% and 59.62% (p=0.005), respectively. The proportion of patients with CAD in the comparators of Definition 1 was higher than that of those satisfying Definition 1 criteria. + +For the patients satisfying Definition 2 criteria and the comparators of Definition 2, the number was 26,271 and 28,025, the mean ages were 72.8 and 69.5 years (p<0.001), and the proportion of males was 57.71% and 61.23% (p<0.001), respectively. The individual proportions of patients with diabetes, stroke, and chronic kidney disease satisfying Definition 2 criteria were higher than those in the comparators of Definition 2. + +For the patients satisfying Definition 3 criteria and the comparators of Definition 3, the number was 8226 and 54,296, the mean age was 69.9 and 71.1 years (p< 0.001), and the proportion of males was 56.54% and 59.53% (p<0.001), respectively. The proportion of patients with diabetes, CAD, stroke, and heart failure satisfying Definition 3 criteria was lower than that of the comparators of Definition 3. + +The frequency of patients satisfying each definition criteria and the comparators who achieved the target value recommended by the JAS guidelines for LDL-C +and non-HDL-C are shown in **Figs.2** to 4. + +For Definition 1, the achievement rates of LDL-C +<100 mg/dL and LDL-C <70 mg/dL were significantly lower than in the comparators (LDL-C <100 mg/dL: 36.03% vs. 62.99%; p<0.001, LDL-C <70 mg/dL: 10.29% vs. 19.14%; p=0.009). The achievement rates of non-HDL-C <130 mg/dL and nonHDL-C <100 mg/dL were also significantly lower than in the comparators (non-HDL-C <130 mg/dL: 45.59% +vs. 72.27%; p<0.001, non-HDL-C <100 mg/dL: +15.44% vs. 35.08%; p<0.001). + +For Definition 2, the achievement rates of LDL-C + +![4_image_0.png](4_image_0.png) + +Among the 62,522 eligible patients who met all the inclusion criteria, 54,296 patients were started on statins and were included in the analysis for Definition 1 and 2. Among patients with statin prescription, 1260 patients (2.32%) met Definition 1 criteria, 53,036 patients +(54,296–1260) were identified as the comparators of Definition 1, while 26,271 patients (48.38%) met Definition 2 criteria and 28,025 patients (54,296–26,271) comprised the comparators of Definition 2. Among all cligible patients, 8226 patients (13.16%) were identified to ment Definition 3 criteria and 54,296 patients (62,522–8,226) were identified as the comparators of Definition 3 (the same as for patients starting any statin agents). ASCVD: atherosclerotic cardiovascular disease. LMT: lipid-modifying therapy. + +| Table 2. Patient characteristics: Definition 1 Total patients | Definition 1 | Comparator of definition 1 | p-Value | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|----| +| N, % Sex Male (n, %) Female (n, %) Age Mean (SD) Previous disease history Diabetes (n, %) Coronary artery disease (n, %) Stroke (n, %) Heart failure (n, %) Peripheral artery disease (n, %) Hypothyroidism (n, %) Chronic kidney disease (n, %) Mild liver disease (n, %) Moderate/severe liver disease (n, %) Antihypertensive medication use (n, %) Cyclosporine use (n, %) Charlson comorbidity index Mean score (SD) | 54,296 (100.00%) | 1,260 (2.32%) | 53,036 (97.68%) | - | +| 32,321 (59.53%) 21,975 (40.47%) | 702 (55.71%) 558 (44.29%) | 31,619 (59.62%) 21,417 (40.38%) | 0.005 | | +| 71.1 (10.668) | 69.9 (11.154) | 71.2 (10.655) | <0.001 | | +| 11,927 (21.97%) 28,028 (51.62%) 4,204 (7.74%) 17,114 (31.52%) 12, 280 (22.62%) 2,119 (3.90%) 4,394 (8.09%) 6,910 (12.73%) 211 (0.39%) 33,067 (60.90%) 111 (0.20%) | 270 (21.43%) 602 (47.78%) 97 (7.70%) 382 (30.32%) 310 (24.60%) 56 (4.44%) 111 (8.81%) 190 (15.08%) 5 (0.40%) 768 (60.95%) 4 (0.32%) | 11,657 (21.98%) 27,426 (51.71%) 4,107 (7.74%) 16,732 (31.55%) 11,970 (22.57%) 2,063 (3.89%) 4,283 (8.08%) 6,720 (12.67%) 206 (0.39%) 32,299 (60.90%) 107 (0.20%) | 0.641 0.006 0.952 0.353 0.088 0.315 0.345 0.011 0.962 0.970 0.369 | | +| 2.8 (2.137) | 2.9 (2.130) | 2.8 (2.137) | 0.100 | | +| SD: standard deviation | | | | | + +| Table 3. Patient characteristics: Definition 2 Total patients | Definition 2 | Comparator of definition 2 | p-Value | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------|----| +| N, % Sex Male (n, %) Female (n, %) Age Mean (SD) Previous disease history Diabetes (n, %) Coronary artery disease (n, %) Stroke (n, %) Heart failure (n, %) Peripheral artery disease (n, %) Hypothyroidism (n, %) Chronic kidney disease (n, %) Mild liver disease (n, %) Moderate/severe liver disease (n, %) Antihypertensive medication use (n, %) Cyclosporine use (n, %) Charlson comorbidity index Mean score (SD) | 54,296 (100.00%) | 26,271 (48.38%) | 28,025 (51.62%) | - | +| 32,321 (59.53%) 21,975 (40.47%) | 15,161 (57.71%) 11,110 (42.29%) | 17,160 (61.23%) 10,865 (38.77%) | <0.001 | | +| 71.1 (10.668) | 72.8 (10.361) | 69.5 (10.709) | <0.001 | | +| 11,927 (21.97%) 28,028 (51.62%) 4,204 (7.74%) 17,114 (31.52%) 12,280 (22.62%) 2,119 (3.90%) 4,394 (8.09%) 6,910 (12.73%) 211 (0.39%) 33,067 (60.90%) 111 (0.20%) | 6,497 (24.73%) 13,665 (52.02%) 2,372 (9.03%) 7,958 (30.29%) 5,726 (21.80%) 960 (3.65%) 2,467 (9.39%) 2,763 (10.52%) 112 (0.43%) 14,227 (54.15%) 36 (0.14%) | 5,430 (19.38%) 14,363 (51.25%) 1,832 (6.54%) 9,156 (32.67%) 6,554 (23.39%) 1,159 (4.14%) 1,927 (6.88%) 4,147 (14.80%) 99 (0.35%) 18,840 (67.23%) 75 (0.27%) | <0.001 0.075 <0.001 <0.001 <0.001 0.004 <0.001 <0.001 0.171 <0.001 <0.001 | | +| 2.8 (2.137) | 2.8 (2.171) | 2.8 (2.105) | 0.470 | | +| SD: standard deviation | | | | | + +| Table 4. Patient characteristics: Definition 3 Total patients | Definition 3 | Comparator of definition 3* | p-Value | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------|----| +| N, % Sex Male (n, %) Female (n, %) Age Mean (SD) Previous disease history Diabetes (n, %) Coronary artery disease (n, %) Stroke (n, %) Heart failure (n, %) Peripheral artery disease (n, %) Hypothyroidism (n, %) Chronic kidney disease (n, %) Mild liver disease (n, %) Moderate/severe liver disease (n, %) Antihypertensive medication use (n, %) Cyclosporine use (n, %) Charlson comorbidity index Mean score (SD) | 62,522 (100.00%) | 8,226 (13.16%) | 54,296 (86.84%) | - | +| 36,972 (59.13%) 25,550 (40.87%) | 4,651 (56.54%) 3,575 (43.46%) | 32,321 (59.53%) 21,975 (40.47%) | <0.001 | | +| 71.0 (10.882) | 69.9 (12.153) | 71.1 (10.668) | <0.001 | | +| 13,318 (21.30%) 30,973 (49.54%) 4,694 (7.51%) 19,228 (30.75%) 15,033 (24.04%) 2,495 (3.99%) 5,022 (8.03%) 8,521 (13.63%) 276 (0.44%) 37,786 (60.44%) 140 (0.22%) | 1,391 (16.91%) 2,945 (35.80%) 490 (5.96%) 2,114 (25.70%) 2,753 (33.47%) 376 (4.57%) 628 (7.63%) 1,611 (19.58%) 65 (0.79%) 4,719 (57.37%) 29 (0.35%) | 11,927 (21.97%) 28,028 (51.62%) 4,204 (7.74%) 17,114 (31.52%) 12,280 (22.62%) 2,119 (3.90%) 4,394 (8.09%) 6,910 (12.73%) 211 (0.39%) 33,067 (60.90%) 111 (0.20%) | <0.001 <0.001 <0.001 <0.001 <0.001 0.004 0.154 <0.001 <0.001 <0.001 0.008 | | +| 2.8 (2.144) | 2.9 (2.184) | 2.8 (2.137) | 0.024 | | +| SD: standard deviation | | | | | + +<100 mg/dL were significantly lower than in the comparators (53.78% vs. 66.53%; p<0.001). The achievement rates of LDL-C <70 mg/dL were not significantly different (17.57% vs. 19.58%; p=0.079). The achievement rates of non-HDL-C <130 mg/dL and non-HDL-C <100 mg/dL were significantly lower than in the comparators (non-HDL-C <130 mg/dL: 63.72% +vs. 75.50%; p<0.001, non-HDL-C <100 mg/dL: 30.56% vs. 36.58%; p<0.001). + +With regard to the Definition 3 group, the achievement rates of LDL-C <100 mg/dL and LDL-C <70 mg/dL were significantly lower than in the comparators (LDL-C <100 mg/dL: 36.59% vs. 62.29%; p< +0.001, LDL-C <70 mg/dL: 6.56% vs. 18.91%; p< +0.001). The achievement rates of non-HDL-C <130 mg/dL and non-HDL-C <100 mg/dL were also significantly lower than in the comparators (non-HDL-C +<130 mg/dL: 45.06% vs. 71.57%; p<0.001, nonHDL-C <100 mg/dL: 13.54% vs. 34.57%; p<0.001). + +The frequencies of patients with a history of CAD +and DM who achieved the target value recommended by the JAS guidelines for LDL-C and non-HDL-C are shown in **Supplementary Figs.1** to 10. + +The achievement rates of patients with a history of disease were significantly higher than that of patients with no history of the diseases evaluated. + +## Discussion + +This retrospective observational study was conducted using the EBM Provider database, which contains hospital claims data stored in electronic hospital information systems. This provider was used to identify the frequency of dyslipidemia patients who had a history of ASCVD and who were unable to take or were not taking statin treatment for any reason. + +The number of patients who met the criteria for Definitions 1, 2, and 3 was 1260 (2.32%), 26,271 +(48.38%), and 8226 (13.16%), respectively. For Definition 1, 48.65% of patients had "statin discontinuation with prescription of non-statin LMT" significantly more frequently than "statin down-titration with prescription of non-statin LMT" (5.56%). Asian patients often show an increased response to drug therapy compared with Western populations, leading to lower approved and clinically used doses of therapeutic drugs such as statins, and with a lower approved maximum dose of statin (e.g., atorvastatin 40 mg/day and rosuvastatin 20 mg/day) as the clinically used dose8, 9). Therefore, it appears conceivable that there was a smaller number of patients who met the "statin down-titration with prescription of non-statin LMT" criteria in the study. + +"Discontinuation of statin for any reason" was very common and occurred in 92.91% of the patients + +![7_image_0.png](7_image_0.png) + +meeting Definition 2 criteria, and was observed in 24,408/54,296 (44.95%) of the patients who were started on any statins during the study period. As the EBM Provider collects information from each hospital separately, if patients who became stable of lipid control in the hospital move to other institutes such as clinic, the record of these patients will be terminated. + +This is a database limitation that patients moved to other institutes could not be follow-up. This would likely increase the discontinuation rate and thus lead to overestimation of the rate of medication discontinuation, especially for Definition 2. Similar to our analysis, Vinogradova *et al*. reported a statin discontinuation rate of 41% in a study of patients on statins for secondary prevention18). In a previous study in Japan using another claims database called Japan Medical Data Center (JMDC) database, Nagar *et al*., investigated treatment pattern and possible statin intolerance in 32.9% of ASCVD patients discontinuing statins 19). + +Different from the MDV database used in the current analysis, the JMDC database comprises retrospective claims data from the Japanese union-managed health insurance system (Health Insurance Association) and follows patients who move to other institutes, which may explain the reason for the lower discontinuation rate observed. However, even the JMDC database showed that over 30% of patients discontinued statins and supported the observation that "discontinuation of statins for any reason" was common. + +Definition 1 is a more stringent definition, which takes into consideration one of the most common adverse events, SAMS, as well as other signs of inability to take statins such as initiation of non-statin LMT +following statin discontinuation/down-titration. In the present study, in the population satisfying Definition 1 criteria, SAMS was identified in 38.73% of patients as a likely cause of statin discontinuation or downtitration. Although it was not possible to include elevation of CK in an algorithm, there was no case of statin discontinuation directly due to rhabdomyolysis in this database analysis. Chang *et al*. searched the EBM provider for cases of muscle toxicity, defined as occurrence of rhabdomyolysis or myositis, and estimated the incidence rate of muscle toxicity leading to statin discontinuation to be 0.17/1000 person-year 11). + +The results indicate that statin discontinuation due to + +![8_image_0.png](8_image_0.png) + +rhabdomyolysis is rare, which is consistent with the current analysis. + +In the present study, the definition of dyslipidemia patients who are unable to take or not taking statin treatment due to any reason was modified to adapt to the Japanese setting with regard to the definitions used in previous studies 15, 16). A previous study that compared the background of patients with high statin adherence and those with statin discontinuation or down-titration for any reason using Medicare claims data by Colantonio *et al*., indicated that 1.0% of patients (1320/134,863) corresponded to Definition 1, and 33.6% of patients (45,266/134,863) corresponded to Definition 2 in this analysis. In addition, a previous study comparing the incidence of CAD and recurrence of myocardial infarction (MI) in patients with a prior history of MI with high adherence to statin and statin discontinuation or down-titration for any reason using Medicare claim data by Serban *et al*., indicated that 1.65% of patients (1741/105,329) corresponded to Definition 1 in this analysis. + +The proportion of patients with Definition 1 in the present study was slightly higher than in previous studies. One of the reasons for this higher prevalence was considered to be a difference in the definition, that is, "Muscle-related symptoms with statin downtitration or discontinuation" was used as a component of Definition 1 in the present study, while in a previous study "antihyperlipidemic adverse event" was used. + +Since there is no code corresponding to "antihyperlipidemic adverse event" among the ICD-10 codes in Japan, "muscle-related symptoms" was used in the present study. In previous studies by Colantonio *et al*. and Serban *et al*., 11.7% and 11.4% patients met the definition of "Rhabdomyolysis with statin down-titration or discontinuation," respectively, while 7% and 1.2% patients met the definition of "antihyperlipidemic adverse event with statin down-titration or discontinuation," respectively. Had the definition "antihyperlipidemic adverse event" been used instead of "musclerelated symptoms" as in the present study, it is likely that the proportion of patients meeting the definition would also increase in previous studies. + +In the study by Serban *et al*., the proportion of patients satisfying the condition "prescription for ≥3 types of statins" was higher than in the present study + +![9_image_0.png](9_image_0.png) + +(59.3% vs. 10.3%) and that for "statin discontinuation with prescription of non-statin LMT" was lower than in the present study (17.0% vs. 48.7%). This may suggest a difference in clinical management involving switching to other statins more frequently in the US, +with switching to non-statin LMT being more frequent in Japan. The latter may reflect the cultural characteristics in Japan with a seemingly higher concern for the adverse effects and a lower threshold for discontinuing statins, which derives from clinical experience. + +The achievement rate of each LDL-C target was significantly lower in patients meeting each definition criteria compared to that in the comparators. This suggests that patients with a history of ASCVD who were unable to take or were not taking statin treatment for any reason were achieving poorer lipid control, leaving them at higher risk of recurrent cardiovascular events or progression of ASCVD. The achievement rate of the low level target value (LDL-C <100 mg/dL, nonHDL-C <130 mg/dL) of Definition 1 and Definition 3 were not markedly different. Definition 3 criteria were likely met by patients who were not prescribed statin because they had mild dyslipidemia and low LDL-C levels. + +When comparing patients with or without a history of CAD and DM, the achievement rates of LDL-C and non-HDL-C target levels in patients with a history of each disease were higher than that of patients with no history of either disease. A likely explanation can be the lower target LDL-C levels recommended by the JAS guidelines3), and the positive life-style changes made as a consequence of the diagnosis of CAD and DM. However, the trend of LDL-C and non-HCL-C +target level achievement remained consistent with higher rates of achievement in patients who did not satisfy the definition criteria. + +Although non-statin LMT (i.e., a cholesterol absorption inhibitor and fibrates) could be prescribed for patients who are unable to take or are not taking statin treatment for any reasons, the present study suggested that fewer patients on non-statin LMT achieved the target lipid levels recommended by the JAS guidelines compared to patients on statins 3). Novel PCSK9 inhibitors, which show greater efficacy for lowering LDLC levels, may represent a potential option for those patients unable to take statins for any reason3, 20). + +The present study also attempted to analyze the rate of MI and incidence rate of CAD as in the study by Serban *et al*. However, these results have not been reported herein to avoid misinterpretation of the clinical characteristics due to the limitations of the database. + +## Limitations + +There were several limitations in the present study. + +First, as mentioned above, the EBM Provider collects information from each hospital separately. If patients are transferred to other institutions for any reason, their records will be terminated in the database. This can lead to increase and overestimation of the rate of medication discontinuation. + +Second, the clinical laboratory test values included in the EBM Provider were specified by only 35 of the 323 medical institutions for which data were provided. Therefore, it could not be considered to reflect the data of the entire patient cohort included in the EBM Provider. This limitation influences both the generalizability and internal validity of the present study. + +Third, since the definition of dyslipidemia patients who are unable to take or not taking statin treatment for any reason was not clearly defined in the established guidelines, the definitions used in the present study were based on hypothesis. Therefore, patients who had any other reason to discontinue treatment, which was not directly considered in the definitions, were not included. Previous studies have used various methods to define patients with dyslipidemia who were unable to take or were not on statin treatment. A previous study using the US Administrative Database by Schluman *et al*. categorized patients on the basis of changes in the statin prescription patterns (e.g., number of statin prescription, discontinuation), AE, and clinical laboratory test value (creatine kinase) 21). In a study using the IMS LifeLink PharMetrics Plus commercial claims, Quek *et al*. defined statin treatment intolerance and/or ineffectiveness in high CVD risk patients with type 2 diabetes on the basis of only changes in statin prescriptions22). In the present study, the definitions used by Serban *et al*. and Colantonio *et al*. + +were adopted to integrate the information on potential adverse effects and rhabdomyolysis in addition to the prescription data. However, there was no code corresponding to "antihyperlipidemic adverse event" among the ICD-10 codes in Japan, and "muscle-related symptoms" was used for Definitions 1 and 2 in the present study. The ICD codes for "muscle-related symptoms" were carefully selected and were analyzed in relationship to statin prescription; however, this process does not exclude all the unrelated diagnoses. + +Although there were several limitations, as described above, the EBM Provider managed by the MDV +comprised approximately 19% of all acute phase hospitals using the DPC system and was not biased towards a specific area in Japan. Thus, generalizability of the results for in-hospital treatment may be considered to be high. + +## Conclusion + +The present study showed the risk of being unable to continue lipid-lowering therapy with statins exists to a certain degree in dyslipidemia patients and is associated with lower rates of target LDL-C and non-HDL-C level achievement, leaving patients untreated or undertreated. Suboptimal management of LDL-C and non-HDL-C is directly associated with residual cardiovascular event risk and implementation of alternative therapeutic options in addition to cholesterol absorption inhibitor, fibrates, and PCSK9 inhibitors is warranted. + +## Acknowledgments And Notice Of Grant Support + +The present study was funded by Sanofi. + +## Conflict Of Interest Statement + +K. Kajinami received a research grant from Bristol-Myers Squibb, scholarship grants from Takeda pharmaceutical Company Limited, Daiichi Sankyo Company, Limited, Sumitomo DainipponPharma Co., Ltd., +Boehringer Ingelheim, Fujifilm, Otsuka Pharmaceutical and St. Jude Medical. + +A. Ozaki and Y. Tajima are employees of Sanofi. S. Yamashita received honoraria and scholarship grants from Sanofi, Kowa Company Limited, Otsuka Pharmaceutical, Shionogi, Bayer Yakuhin Limited, MSD +K.K., Takeda pharmaceutical Company Limited, Sanwa Kagaku Kenkyusho, Ono Pharmaceutical, Astellas Pharma Inc, Daiichi-Sankyo Company, Astra-Zeneca KK, Medical Review, Skylight Biotech, Kaken Pharmaceutical, Pfizer, Bristol-Myers Squibb, Amgen Astellas Biopharma K.K., Aegerion Pharmaceuticals, Toa Eiyo, Nippon Boehringer Ingelheim, National lnstitute of Biomedical lnnovation, Kyowa Medex, Mochida Pharmaceutical, Hayashibara, Teijin Pharma and Kissei Pharmaceutical. + +H. Arai received honoraria from Daiichi Sankyo Company, Limited, MSD K.K., Kowa Company Limited, Astellas Pharma Inc, Amgen Astellas Biopharma K.K. and Chugai Pharmaceutical Co., Ltd. + +T. Teramoto received a research grant from Amgen Astellas Biopharma K.K., scholarship grants from Daiichi Sankyo Company, Limited, Takeda pharmaceutical Company Limited, Astellas Pharma Inc, Kissei KK +and Shionogi & Co. Limited, honoraria for lecture fees from Bayer Yakuhin Limited, Pfizer Japan Limited, Takeda pharmaceutical Company Limited, Astellas Pharma Inc, Kowa company Limited, Sanofi K.K., MSD +K.K., Amgen Astellas Biopharma K.K. and Astra-Zeneca KK, fees for promotion materials from Daiichi Sankyo Company Limited, courses endowed from Kowa company Limited, MSD K.K., Bayer Yakuhin Limited and Mochida Yakuhin KK. + +## References + +1) Estimates for 2000-2015: cause-specific mortality. Available at: http://www.who.int/healthinfo/global_burden_disease/ +estimates/en/index1.html. Accessed August 01, 2018 2) Population Survey Report 2016. Available at: http://www. + +mhlw.go.jp/toukei/saikin/hw/jinkou/kakutei16/dl/10_h6. + +pdf. 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BMJ Open, 2013; 3: e002040 12) Foody JM, Joyce AT, Rudolph AE, Liu LZ, and Benner JS: +Persistence of atorvastatin and simvastatin among patients with and without prior cardiovascular diseases: a US managed care study. Curr Med Res Opin, 2008; 24: 1987-2000 13) Cholesterol Treatment Trialists (CTT) Collaboration, Baigent C, Blackwell L, Emberson J, Holland LE, Reith C, Bhala N, Peto R, Barnes EH, Keech A, Simes J, and Collins R: +Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials. Lancet, 2010; 376: 1670-1681 14) Shalev V, Chodick G, Silber H, Kokia E, Jan J, and Heymann AD: Continuation of statin treatment and all-cause mortality: a population-based cohort study. Arch Intern Med, 2009; 169: 260-268 15) Serban MC, Colantonio LD, Manthripragada AD, Monda KL, Bittner VA, Banach M, Chen L, Huang L, Dent R, +Kent ST, Muntner P, and Rosenson RS: Statin Intolerance and Risk of Coronary Heart Events and All-Cause Mortality Following Myocardial Infarction. J Am Coll Cardiol, 2017; 69: 1386-1395 16) Colantonio LD, Kent ST, Huang L, Chen L, Monda KL, +Serban MC, Manthripragada A, Kilgore ML, Rosenson RS, and Muntner P: Algorithms to Identify Statin Intolerance in Medicare Administrative Claim Data. Cardiovasc Drugs Ther, 2016; 30: 525-533 17) Quan H, Sundararajan V, Halfon P, Fong A, Burnand B, +Luthi JC, Saunders LD, Beck CA, Feasby TE, and Ghali WA, Coding algorithms for defining comorbidities in ICD9-CM and ICD-10 administrative data. Med Care, 2005; 43: 1130-1139 18) Vinogradova Y, Coupland C, Brindle P, and Hippisley-Cox J: Discontinuation and restarting in patients on statin treatment: prospective open cohort study using a primary care database. BMJ, 2016; 353: i3305 19) Nagar SP, Rane PP, Fox KM, Meyers J, Davis K, Beaubrun A, Inomata H, Qian Y, and Kajinami K: Treatment Patterns, Statin Intolerance, and Subsequent Cardiovascular Events Among Japanese Patients With High Cardiovascular Risk Initiating Statin Therapy. Circ J, 2018; 82: +1008-1016 20) Teramoto T, Kondo A, Kiyosue A, Harada-Shiba M, Ishigaki Y, Tobita K, Kawabata Y, Ozaki A, Baccara-Dinet MT, and Sata M: Efficacy and safety of alirocumab in patients with hypercholesterolemia not adequately controlled with non-statin lipid-lowering therapy or the lowest strength of statin: ODYSSEY NIPPON study design and rationale. Lipids Health Dis, 2017; 16: 121 21) Schulman KL, Lamerato LE, Dalal MR, Sung J, Jhaveri M, Koren A, Mallya UG, and Foody JM: Development and Validation of Algorithms to Identify Statin Intolerance in a US Administrative Database. Value Health, 2016; 19: +852-860 22) Quek RG, Fox KM, Wang L, Li L, Gandra SR, and Wong ND: Lipid-lowering treatment patterns among patients with type 2 diabetes mellitus with high cardiovascular disease risk. BMJ Open Diabetes Res Care. 2015; 23; 3: e000132 + +| Supplementary Table 1. Categorization of high-, moderate-, and low-intensity statins* Statin type High-intensity Moderate-intensity Low-intensity (mg/day) (mg/day) (mg/day) 5 2.5 5-10 10-20 20-40 1 *The intensity for each statin type in accordance with the American College of Cardiology/American Heart Atorvastatin Rosuvastatin Simvastatin Pravastatin Fluvastatin Pitavastatin 30-40 15-20 10-20 5-10 20 - 60 2-4 - - - | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +| Supplementary Table 2. The list of muscle related symptom standardized | standardized | | | | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| ICD-10 | Disease name | ICD-10 | | | | +| Japanese | Japanese | Disease name | | | | +| code | code | | | | | +| disease code | disease code | | | | | +| M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M609 M626 M628 M628 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 | 7280003 7280005 7291060 7291058 7289022 8837265 7291040 7291095 7291059 7289021 7291078 7291088 7291009 7104008 7291010 7291028 7291064 8842239 7291080 7298036 8831347 8841635 7291015 7291003 8833247 7291068 8840783 7291092 7231018 7289005 8835426 7291039 7291021 7291055 8837285 7291032 8839366 7248003 7291062 7291047 7291085 | Myositis Femoral region myositis Psoitis Thigh myositis Biceps brachii myositis Greater pectoral myositis Brachial myositis Cervical myositis Rectus femoris myositis Triceps brachii myositis Rectus abdominis myositis Gluteal myositis Acute myositis Localized myositis Sternocleidomastoid Nuchal myositis Spine myositis Masseter myositis Abdominal myositis Muscle fatigue Shoulder discomfort Rhabdomyolysis Muscle pain Muscle pain of the lower extremity Shoulder muscle pain Muscular backache Psoas myalgia Neck-shoulder muscle pain Cervical muscle pain Chest muscle pain Upper limb muscle pain Upper arm muscle pain Scapular region muscle pain Multiple muscle pain Thigh muscle pain Lumbar muscle pain syndrome Gastrocnemius pain Thoracodorsal muscle pain Gluteal region muscle pain Forearm muscle pain Intercostal muscle pain | M791 M791 M791 M791 M791 M791 M791 M791 M791 M791 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 M796 | 7231008 7291049 8831297 7291012 8839751 7231006 7291011 8835524 7291063 8835552 7295003 8837336 8831316 8836736 8835547 7295022 7295020 8835434 8836617 8831096 8840187 8834411 7295058 7295073 7295033 7295032 7295015 7295045 7295074 7295077 7295007 7295024 7295016 7295021 7295061 7295075 7299023 7295041 7295056 7295017 | Nuchal muscle pain Trapezius muscle pain Triceps surae muscle pain Thoracoabdominal muscle pain Abdominal wall muscle pain Nucha muscle pain Sternocleidomastoid muscle pain Triceps brachii muscle pain Cephalic muscle pain Biceps brachii muscle pain Melosalgia Meralgia Lower leg pain Podalgia Brachialgia Hand pain Finger pain Arm pain Forearm pain Heel pain Hallucis pain Toe pain Thumb pain Forefoot pain Acrotarsium pain Plantar pain Thigh pain Digitus medius pain Metatarsal pain Indicis pain Annularis pain Minimus Pain Melalgia Opisthenar pain Sural pain Metapodium pain Poditis Inner thigh pain Thenar region pain Distal extremity pain | + +![14_image_0.png](14_image_0.png) + +For patients with and without history of CAD (patients with statin prescription), 2895 (2895/28,028: 10.33%) and 2404 (2,404/26,268: 9.15%) had available LDL-C data, while 2938 (2938/28,028: 10.48%) and 2243 patients +(2243/26,268: 8.54%) had non-HDL-C data available, respectively. The achievement rates of LDL-C < 100 mg/dL and LDL-C < 70 mg/dL in patients with history of CAD were significantly higher than those in patients with no history (LDL-C < 100 mg/dL: 67.15% vs. 56.45%; p < 0.001, LDL-C < 70 mg/dL: 22.94% vs. 14.06%; p < 0.001). + +The achievement rates of non-HDL-C < 130 mg/dL and non-HDL-C < 100 mg/dL in patients with a history of CAD were also significantly higher than those in patients with no history (non-HDL-C < 130 mg/dL: 75.90% vs. + +65.89%; p<0.001, non-HDL-C < 100 mg/dL: 38.46% vs. 29.47%; p<0.001). CAD: coronary artery disease, LDL- C: low-density lipoprotein, non-HDL-C: non-high-density lipoprotein. + +![15_image_0.png](15_image_0.png) ![16_image_0.png](16_image_0.png) + +For patients who satisfied the criteria for Definition 2 with and without history of CAD, and the comparators of Definition 2 with and without history of CAD, 1016 (1016/13,665: 7.44%), 743 (743/12,606: 5.89%), 1879 (1879/14,363: 10.30%), and 1661 (1661/13,662: + +![17_image_0.png](17_image_0.png) + +For patients with and without a history of CAD (all eligible patients), 3259 (3259/30,973: 10.52%) and 2909 patients (2909/31,549: 9.22%) had available LDL-C data, while 3301 (3301/30,973: 10.66%) and 2670 +(2670/31,549: 8.46%) had non-HDL-C data available, respectively. The achievement rates of LDL-C < 100 mg/dL and LDL-C <70 mg/dL in patients with a history of CAD were significantly higher than those in patients with no history (LDL-C < 100 mg/dL- 64.16% vs. 52.53%; p < 0.001, LDL-C < 70 mg/dL- 21.20% vs. 12.65%; p < 0.001). + +The achievement rates of non-HDLC < 130 mg/dL and non-HDL-C < 100 mg/dL in patients with a history of CAD were also significantly higher than those in patients with no history (non-HDL-C < 130 mg/dL: 73.13% vs. + +61.80%; p<0.001, non-HDL-C < 100 mg/dL: 35.90% vs. 26.70%; p<0.001). CAD: coronary artery disease, LDL- +C: low-density lipoprotein, non-HDL-C: non-high-density lipoprotein. + +![18_image_0.png](18_image_0.png) + +For patients who met the criteria for Definition 3 with and without a history of CAD, and the comparators of Definition 3 with and without a history of CAD, 364 (364/2945: 12.36%), 505 (505/5281: 9.56%), 2895 (2895/28,028: 10.33%) and 2404 (2404/26,268: 9.15%) had available LDL-C data, while 363 (363/2945: 12.33%), 427 (427/5281 8.09%), 2938 (2938/28,028: 10.48%), and 2243 (2243/26,268: 8.54%) had available non-HDL-C data, respectively. For patients satisfying the Definition 3 criteria, the achievement rate of LDL-C < 100 mg/dL in those with a history of CAD was significantly higher than those with no history (40.38% vs. 33.86%; p = 0.049) and that of LDL-C +< 70 mg/dL was also higher than in those with no history (7.42% vs. 5.94%; p =0.386). The achievement rate of non-HDL-C < 130 mg/dL +was significantly higher than that in those with no history (50.69% vs. 40.28%; p =0.003) and that of non-HDL-C < 100 mg/dL was higher than in those with no history (15.15% vs. 12.18%; p =0.224). For the comparators of Definition 3, the achievement rates of LDL-C < 100 mg/dL and LDL-C < 70 mg/dL in those with a history of CAD were significantly higher than in those with no history (LDL-C < 100 mg/ +dL: 67.15% vs. 56.45%; p < 0.001, LDL-C < 70 mg/dL: 22.94% vs. 14.06%; p < 0.001). The achievement rates of non-HDL-C < 130 mg/ +dL and non-HDL-C < 100 mg/dL were also significantly higher than in those with no history (non-HDL-C < 130 mg/dL. 75.90% vs. 65.89%; p < 0.001, non-HDL-C < 100 mg/dL: 38.46% vs. 29.47%; p < 0.001). CAD: coronary artery disease, LDL-C: low-density lipoprotein, non-HDL-C: non-high-density lipoprotein. + +![19_image_0.png](19_image_0.png) + +![20_image_0.png](20_image_0.png) + +For patients who satisfied the criteria of Definition 1 with and without a history of DM, and the comparators of Definition 2 with and without a history of DM, 23 (23/270: 8.52%), 113 (113/990: 11.41%), 1149 (1149/11,657: 9.86%), and 4014 (4014/41,379: 9.70%) had available LDL-C data, while 38 (38/270:14.07%), 98 (98/990: 9.90%), 1445 (1445/11,657: 12.40%), and 3600 (3600/41,379: 8.70%) had vs. 70.00%; p <0.001, non-HDL-C <100 mg/dL: 40.28% vs. 33.00%; p <0.001). DM: diabetes mellitus, LDL-C: low-density lipoprotein, non-HDL-C: non-high-density lipoprotein. + +![21_image_0.png](21_image_0.png) + +For patients satisfying the criteria for Definition 2 with and without a history of DM, and the comparators of Definition 2 with and without a history of DM, 494 (494/6497: 7.60%), 1265 (1265/19,774: 6.40%), 678 (678/5430: 12.49%), and 2862 (2862/22,595: 12.67%) had + +![22_image_0.png](22_image_0.png) + +For patients with and without a history of DM (all eligible patients), 1288 (1288/13,318: 9.67%) and 4880 +(4880/49,204: 9.92%) had available LDL-C data, while 1639 (1639/13,318: 12.31%) and 4332 (4332/49,204: +80%) had non-HDL-C data available, respectively. The achievement rates of LDL-C < 100 mg/dL and LDL-C +< 70 mg/dL in patients with a history of DM were significantly higher than in those with no history (LDL-C < 100 mg/dL: 69.25% vs. 55.88%; p<0.001, LDL-C <70 mg/dL: 25.93% vs. 14.86%; p<0.001). The achievement rates of non-HDL-C < 130 mg/dL and non-HDL-C < 100 mg/dL in those with a history of DM were also significantly higher than in those with no history (non-HDL-C < 130 mg/dL. 75.29% vs. 65.33%; p< 0.001, non-HDL-C < 100 mg/dL: 37.83% vs. 29.50%; p<0.001). DM: diabetes mellitus, LDL-C: low-density lipoprotein, non-HDL-C: nonhigh-density lipoprotein. + +![23_image_0.png](23_image_0.png) + diff --git a/medical/md/PMC6657939.md b/medical/md/PMC6657939.md new file mode 100644 index 0000000000000000000000000000000000000000..8bd2baa873a14c0a4e5c782f9dc478387a53b9f5 --- /dev/null +++ b/medical/md/PMC6657939.md @@ -0,0 +1,224 @@ +# Original Article Fgf-17 From Hypoxic Human Wharton'S Jelly-Derived Mesenchymal Stem Cells Is Responsible For Maintenance Of Cell Proliferation At Late Passages + +Kyu-Hyun Han1, Min-Hee Kim1, Gun-Jae Jeong1, Ae-Kyeong Kim1, Jong Wook Chang2, Dong-ik Kim1 1Division of Vascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 2*Stem Cell & Regenerative Medicine Institute, Research Institute for Future Medicine, Samsung Medical Center, Seoul, Korea* Background and Objectives: Although it is well known that hypoxic culture conditions enhance proliferation of human mesenchymal stem cells, the exact mechanism is not fully understood. In this study, we investigated the effect of fibroblast growth factor (FGF)-17 from hypoxic human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) on cell proliferation at late passages. + +Methods and Results: hWJ-MSCs were cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS) +in normoxic (21% O2) and hypoxic (1% O2) conditions. Protein antibody array was performed to analyze secretory proteins in conditioned medium from normoxic and hypoxic hWJ-MSCs at passage 10. Cell proliferation of hypoxic hWJ-MSCs was increased compared with normoxic hWJ-MSCs from passage 7 to 10, and expression of secretory FGF-17 was highly increased in conditioned medium from hypoxic hWJ-MSCs at passage 10. Knockdown of FGF-17 in hypoxic and normoxic hWJ-MSCs decreased cell proliferation, whereas treatment of hypoxic and normoxic hWJ-MSCs with recombinant protein FGF-17 increased their proliferation. Signal transduction of FGF-17 in hypoxic and normoxic hWJ-MSCs involved the ERK1/2 pathway. Cell phenotypes were not changed under either condition. Differentiation-related genes adiponectin, *Runx2*, and *chondroadherin* were downregulated in normoxic hWJ-MSCs treated with rFGF-17, and upregulated by siFGF-17. Expression of alkaline phosphatase (ALP), *Runx2*, and *chondroadherin* was upregulated in hypoxic hWJ-MSCs, and this effect was rescued by transfection with siFGF-17. Only *chondroadherin* was upregulated in hypoxic hWJ-MSCs with rFGF-17. Conclusions: In hypoxic culture conditions, FGF-17 from hypoxic hWJ-MSCs contributes to the maintenance of high proliferation at late passages through the ERK1/2 pathway. Keywords: FGF-17, Hypoxic culture, Mesenchymal stem cells, Proliferation Received: May 12, 2018, Revised: February 27, 2019, Accepted: March 4, 2019, Published online: April 30, 2019 Correspondence to Dong-ik Kim Division of Vascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea Tel: +82-2-3410-3467, Fax: +82-2-3410-0040 E-mail: dikim@skku.edu 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. + +Copyright ⓒ 2019 by the Korean Society for Stem Cell Research + +## Introduction + + Hypoxic culture conditions enhance the proliferation or survival of several cell types such as tumor cells (1, 2), endothelial cells (3), fibroblasts (4), neural stem cells (5), +and mesenchymal stem cells (6). Moreover, cellular proliferation is maintained at a high level in long-term hypoxic culture (7). + + Although a signal pathway dependent on hypoxia-inducible factor (HIF)-1α or HIF-2α has been reported as the major mechanism for high proliferation in hypoxic conditions (8, 9), identification of other involved signal pathways or molecules is required to understand the response and function of cells under hypoxic condition. The secretome from mesenchymal stem cells in hypoxic culture condition shows beneficial effects on the cells themselves or neighboring cells through autocrine or paracrine signaling (10-12). + + Previous studies have reported that fibroblast growth factor (FGF)-17 is expressed in the embryonic brain (13). Moreover, FGF17 increased the proliferation of carcinoma cells (14) and leukemic cells (15), and inhibited the differentiation of oligodendrocyte progenitor cells (16). However, the role of FGF-17 in human mesenchymal stem cells cultured in hypoxic conditions has not yet been investigated. + + In this study, we aimed to investigate the role of FGF-17 secreted by human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) cultured in hypoxic conditions at late passages based on protein profiling of conditioned medium (CM) of hypoxic hWJ-MSCs. + +## Materials And Methods Cell Cultures + + This study was approved by the Institutional Review Board of Samsung Medical Center and informed consent was obtained from pregnant mothers (IRB. No.201607-102). hWJ-MSCs were isolated according to the procedure specified in a previous report (17) and cultured in Alpha Minimum Essential Medium (α-MEM, Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco) +and 0.5% antibiotic/antimycotic (Gibco) at 37oC in normoxic (21% O2) or hypoxic (1% O2) culture conditions with 5% CO2. Media changes and passaging for both conditions were performed in a normoxic environment. When the culture reached 90% confluency, the cells were washed twice with phosphate buffered saline (PBS) and dissociated with 0.25% trypsin-EDTA (Gibco) for 2 minutes at 37oC. Dissociated cells were treated with medium to block trypsin-EDTA activity. After centrifugation, cell pellets were resuspended with medium and seeded at 3,000 cells/cm2. Cell images were acquired with a model CKX41 inverted microscope (Olympus Corporation, Tokyo, Japan). For the supplementary experiments on the role of FGF-17 in hWJ-MSCs, 2 of hWJ-MSCs were purchased from PromoCell (Heidelberg, Germany) and named as hWJMSCs-1 and hWJ-MSCs-2. + +## Protein Antibody Array Of Cm From Hwj-Mscs + + Normoxic or hypoxic hWJ-MSCs at passage 10 were seeded in 100-mm diameter culture plates containing complete medium at a density of 3,000 cells/cm2. When the cells reached 90% confluency the medium was replaced with 12 ml of serum-free medium. After incubation for 18 h, CM was concentrated by centrifugation at 5,000×g for 1 h using an Amicon Ultra-15 centrifugal filter (Millipore, Billerica, MA, USA) with a size cut-off of 3,000 Dalton. + +Expression of proteins in CM from hWJ-MSCs was measured with a RayBio® Label-based (L-Series) Human Antibody Array (Raybiotech, Inc., Norcross, GA, USA) from E-biogen (Kyung Hee Business Center, Kyung Hee University, Seoul, Korea). The result was acquired from a single sample analysis. Upregulated and downregulated proteins are listed in Fig. 1B, 1C and Table 1. + +## Elisa + + Expression of FGF-17 in CM from normoxic hWJ-MSCs or hypoxic hWJ-MSCs at passage 10 was measured using Human FGF-17 ELISA Kit, Cusabio Technology, LLC, College Park, MD, USA) by manufacture's manual. Briefly, 100 ul of 1:3 diluted CM from normoxic or hypoxic hWJ-MSCs at passage 10 were added to each well of 96 well plate coated with anti-FGF-17 antibody, and incubated for 2 h at 37oC. In the next step, biotin-antibodies were added to each well for 1 h at 37oC. After washing wells with washing buffer, HRP-avidins were added to each well for 1 h at 37oC. TMB substrates were added to each well for 15 min and stop solutions were added. The optical density of each well was determined using a microplate reader xMark microplate spectrophotometer (BioRad, Hercules, CA, USA) set to 450 nm. + +## Treatment With Sifgf-17 And Rfgf-17 + + For transfection with siRNA, 20 pmol of siFGF-17 (sense, CGACUGAAGGCCUUGCAGA; antisense, UCUG +CAAGGCCUUCAGUCG; Bioneer, Daejeon, Korea) was mixed with 1 ul of Lipofectamin 2000 (Invitrogen, Carlsbad, CA, USA) for 20 minutes at room temperature, and then added to normoxic hWJ-MSCs at passage 7 and hypoxic hWJ-MSCs at passage 10. After 6 h, the medium was replaced with fresh complete medium, and the cells were cultured for 48h. Negative control siRNA was used for the control group (SN-1013, Bioneer, Daejeon, Korea). For rFGF-17 treatment, normoxic hWJ-MSCs at passage 7 and hypoxic hWJ-MSCs at passage 10 were treated with 250, 500, 1,000 ng/ml of rFGF-17 (PeproTech Inc., Rocky Hill, NJ, USA) for 48 h. + + For both treatments, cell counting, BrdU proliferation assay, quantitative real-time polymerase chain reaction (QRT-PCR), western blot, and flow cytometry analysis were performed after incubation for 48 h, as described + +![2_image_0.png](2_image_0.png) + +## Cell Proliferation Assay + + Cells from passages 6 to 10 were seeded at 3,000 cells/cm2 in normoxic or hypoxic culture conditions. Cells were removed by trypsinization at 90% confluency for each passage and cell numbers were counted using Trypan blue 0.5% solution (Biowest, Riverside, MO, USA) and a hemocytometer. A BrdU cell proliferation assay kit (Millipore) was used to analyze cell proliferation according to the manufacturer's manuals. Briefly, normoxic or hypoxic hWJ-MSCs were seeded in 96-well plates at 3,000 cells/cm2, incubated overnight, and treated with 1×BrdU solution for 24 h. The medium was removed and the cells were treated with Fixing/Denaturing Solution (Millipore) for 30 min. Cells were incubated with 1×detection antibody and 1×HRP-conjugated secondary antibody (Millipore) for 1.5 h and washed with 1×Wash Buffer (Millipore) After incubation with TMB Substrate (Millipore) for 30 min, STOP solution (Millipore) was added and the absorbance of BrdU was measured at 450 nm using xMark Microplate Spectrophotometer (Bio-Rad, Hercules, CA, USA). + +Fig. 1. Enhanced proliferation of hypoxic hWJ-MSCs and levels of secretory proteins at late passages. (A) +Cell numbers of normoxic and hypoxic hWJ-MSCs from passage 6 to 10. Results are expressed as the mean±SEM from three independent experiments (n=3, *p<0.05, t-test). + +(B) Scatterplot of protein expression levels between normoxic and hypoxic hWJ-MSCs at passage 10. The red line above represents upregulated proteins (>2 fold), and the green line below represents downregulated proteins (>2 fold) in conditioned medium from hypoxic hWJMSCs compared with normoxic hWJMSCs. (C) Clustering of upregulated proteins and downregulated proteins in conditioned medium from hypoxic hWJ-MSCs compared with normoxic hWJ-MSCs at passage 10 (fold change +>2.0). Results were from a single sample analysis. (D) Absolute amount of FGF-17 in CM from normoxic hWJ-MSCs and hypoxic hWJ-MSCs +(1:3 diluted CM) (n=3, **p<0.01, t-test). (E) Expression of FGFR-1, -2, -3 and -4 in normoxic and hypoxic hWJ-MSCs at passage 10. Nor-MSC, +normoxic mesenchymal stem cells; Hypo-MSC, hypoxic mesenchymal stem cells. + +| Table 1. Secretory proteins that were upregulated or downregulated (>2-fold) in conditioned medium from hypoxic hWJMSCs compared with normoxic hWJ-MSCs | Table 2. Primer sequences used for QRT-PCR Primer name Sequence (5' → 3') PCNA Forward TCCTGTGCAAAAGACGGAGT PCNA Reverse CATCCTCGATCTTGGGAGCC Ki67 Forward CAGATGATGGAGCCCGGAA Ki67 Reverse TGCACACCTCTTGACACTTCC Adiponectin Forward AACATGCCCATTCGCTTTACC Adiponectin Reverse TAGGCAAAGTAGTACAGCCCA ALP Forward TACAAGGTGGTGGGCGGTGAACGA ALP Reverse TGGCGCAGGGGCACAGCAGAC Runx2 Forward TCTTCACAAATCCTCCCC Runx2 Reverse TGGATTAAAAGGACTTGG Chondroadherin Forward GGACCACAACAAGGTCACTGA Chondroadherin Reverse GTGGAATTTGGCGAGGTTCTC SOX9 Forward GAACGCACATCAAGACGGAG SOX9 Reverse TCTCGTTGATTTCGCTGCTC β-actin Forward AGTCCTGTGGCATCCACGAA β-actin Reverse GATCCACACGGAGTACTTGC | | | +|-----------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|----| +| Name | Fold change | SwissProt ID | | +| FGF-17 | +5.008 | O60258 | | +| VEGF/VEGFA | +4.117 | P15692 | | +| IGFBP-3 | +3.340 | P17936 | | +| IL-1 R3/IL-1 R AcP | +2.382 | Q9NPH3 | | +| Chordin-like 1 | +2.220 | Q9BU40 | | +| EN-RAGE | +2.051 | P80511 | | +| CTGF/CCN2 | −2.039 | P29279 | | +| MMP-7 | −2.082 | P09237 | | +| ErbB2 | −2.093 | P04626 | | +| Ubiquitin+1 | −2.098 | P0CG47 | | +| TSLP R, TSLP Receptor | −2.107 | Q9HC73 | | +| TRANCE | −2.151 | O14788 | | +| sFRP-1 | −2.272 | Q8N474 | | +| Follistatin-like 1 | −2.289 | Q12841 | | +| TGF-beta 3 | −2.437 | P10600 | | +| ANG-1 | −2.538 | Q15389 | | +| TCCR/WSX-1/IL27Ra | −2.634 | Q6UWB1 | | +| Tie-1 | −2.662 | P35590 | | +| LRP-6 | −2.760 | O75581 | | +| XEDAR/TNFRSF27 | −3.084 | Q9HAV5 | | +| OPG/TNFRSF11B | −3.142 | O00300 | | +| Smad 1 | −3.200 | Q15797 | | +| Grb2 | −3.286 | P62993 | | +| IL-15 R alpha | −3.676 | Q13261 | | +| MMP-1 | −4.022 | P03956 | | +| Activin A | −4.197 | P08476 | | +| FGF-20 | −4.402 | Q9NP95 | | +| EDA-A2 | −8.219 | Q92838 | | +| IL-17B | −9.443 | Q9UHF5 | | +| IL-23 | −10.144 | Q9NPF7 | Quantitative real-time polymerase chain reaction (QRT-PCR) Total RNA was extracted from cells using a RNeasy Plus Mini Kit (Qiagen GmbH, Hilden, Germany) and 200 ng of total RNA was used for reverse transcription with a PrimeScript RT reagent kit (Takara, Bio, Shiga, Japan) according to the manufacturer's manuals. Real- time PCR was conducted using SYBR Green Realtime PCR Master Mix (Toyobo Co., LTD, Osaka, Japan) with 100 ng template DNA in a 7900 HT Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) using reaction conditions of denaturation at 95o C for 15 s and extension at 60o C for 34 s for 40 cycles. The primer sequences are listed in Table 2. | + +## Cell Viability Assay + + 3,000 cells/cm2 of normoxic hWJ-MSCs at passage 7 or hypoxic hWJ-MSCs at passage 10 were seeded into the well of 96-well white plates. After 48 h, culture medium was removed and serum free medium was added to the wells. After 1 day incubation, 500 ng/ml of rFGF-17 was treated to the cells for 48 h. For the inhibition of secretory FGF-17 from hypoxic hWJ-MSCs at passage 10, 6,000 cells/cm2 of hypoxic hWJ-MSCs were incubated in 96-well white plates for 48 h. Then, 30 ug/ml of blocking antibody of FGF-17 (R&D Systems, Minneapolis, MN, USA) in serum free medium was treated to the cells for 48 h. In the next step, 100 ul of CellTiter-Glo assay 2.0 reagents (Promega, Madison, WI, USA) was treated to the cells. After 10 min incubation, luminescence from cells was measured with GLOMAX Multi Detection System (Promega, Sunnyvale, CA, USA). + +## Western Blotting + + For the analysis of FGF-17 receptors on normoxic hWJMSCs and hypoxic hWJ-MSCs at passage 10, cell lysates were harvested from both kinds of cells. For the analysis of intracellular signaling related with FGF-17, cell lysates were harvested from normoxic hWJ-MSCs treated with rFGF-17 and transfected with siRNA against FGF-17 at passage 7 or hypoxic hWJ-MSCs treated with rFGF-17 and transfected with siRNA against FGF-17 at passage 10 using lysis buffer (20 mM HEPES pH 7.6, 20% Glycerol, 250 mM NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 2 mM PMSF, 1mM DTT, 1 mM NaF and 1 mM Na3VO4) with protease inhibitor cocktail (Roche Diagnostics GmbH, +Mannheim, Germany). Quantification of proteins in lysates was performed with Quick Start Bradford 1×Dye Reagent (Bio-Rad), and absorbance was measured at 450 nm using xMark Microplate Spectrophotometer. Protein samples were boiled at 95oC for 15 min and 20 ug of protein from each sample was subjected to SDS–PAGE. + +Separated proteins in the gel were transferred to a nitrocellulose membrane, which was incubated for 1 h with 5% +bovine serum albumin (Abcam, Cambridge, UK) in 1×TBS solution (Intron Biotechnology, Seoul, Korea) with 0.1% +Tween 20 (Sigma-Aldrich, St Louis, MO, USA). The membrane was washed with 1×TBST and incubated overnight at 4oC with the following primary antibodies: anti-FGFR-1, FGFR-2, FGFR-3 and FGFR-4 (1:1,000; Cusabio Technology, LLC, College Park, MD, USA), anti-phospho AKT +(S473) (1:2,000; Cell Signaling Technology, MA, USA), +anti-phospho ERK1/2 (Thr202/Tyr204) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti- ERK1/2 +(1:1000; Abcam, Cambridge, UK), anti-phospho STAT3 +(Y705) (1:1,000; Cell Signaling Technology), anti-GAPDH (1:10,000; Abcam, Cambridge, UK), anti-P21 (1:1,000; Cell Signaling Technology), anti-P27 rabbit antibody (1:1,000; Cell Signaling Technology), anti-P53 (1:500; Santa Cruz Biotechnology), and anti-FGF-17 mouse (1:500; Santa Cruz Biotechnology) antibody. After incubation with goat anti-mouse or -rabbit HRP-conjugated antibody (1:10,000; Bethyl, Montgomery, TX, USA) for 1 h at room temperature, the expression of proteins was visualized using WESTSAVE UP (Abfrontier, Seoul, Korea) and developed with Automatic X-RAY Film Processor (JPI Healthcare Co, Ltd., Seoul, Korea). + +## Flow Cytometry + + Normoxic hWJ-MSCs treated with rFGF-17 and transfected with siFGF-17 at passage 7 or hypoxic hWJ-MSCs treated with rFGF-17 and transfected with siFGF-17 at passage 10 were harvested and washed with 1×PBS (Intron Biotechnology, Seoul, Korea). Normoxic hWJ-MSCs not treated with rFGF-17 and transfected with negative control siRNA or hypoxic hWJ-MSCs not treated with rFGF-17 and transfected with negative control siRNA +were used as respective control groups. Cells were fixed with BD Cytofix Fixation Buffer (BD Biosciences, Piscataway, NJ, USA) and stained with V450 mouse anti-human CD31 (1:20), fluorescein isothiocyanate +(FITC) mouse anti-human CD34 (1:20), phycoerythrin +(PE)-CyTM7 mouse anti-human CD44 (1:20), V500 mouse anti-human CD45 (1:20), PerCP-CyTM5.5 mouse anti-human CD73 (1:20), PE mouse anti-human CD90 (1:20), +APC mouse anti-human CD105 (1 : 20), and V450 mouse anti-human CD144 (1:20) (BD Biosciences) antibodies for 30 min at room temperature. Cells were washed twice with Stain Buffer (BD Biosciences) and analyzed with a FACSVerseTM flow cytometer (BD Biosciences) and Flowjo software (Treestar, San Carlos, CA, USA). + +## Statistical Analysis + + Data analysis and statistics (t-tests) were conducted with GraphPad Prism version 6.01 (San Diego, CA, USA) and a p-value<0.05 was considered statistically significant. + +## Results Hwj-Mscs Cultured In Hypoxic Condition Showed High Proliferation And Secretion Of Fgf-17 At Late Passages + + In this study, we defined passage 6 as early passage, passages 7 and 8 as middle passages, and passages 9 and 10 as late passages for normoxic and hypoxic hWJ-MSCs. When normoxic and hypoxic hWJ-MSCs were cultured from passage 6 to 10, cell numbers of hypoxic hWJ-MSCs were highly maintained from passage 7 to 10 compared with normoxic hWJ-MSCs (Fig. 1A). To investigate the factors related to maintenance of high proliferation of hypoxic hWJ-MSCs, we compared proteins in CM from hypoxic hWJ-MSCs and normoxic hWJ-MSCs at passage 10 using protein antibody array. The signal intensities of proteins that were upregulated or downregulated by at least 2-fold were analyzed in a scatter plot (Fig. 1B). FGF-17 +(+5.008 fold), VEGF/VEGFA (+4.117 fold), IGFBP3 (+ +3.340 fold), IL-1 R3/IL-1 R AcP (+2.382 fold), ChordinLike 1 (+2.220 fold), and EN-RAGE (+2.051 fold) were highly expressed in CM of hypoxic hWJ-MSCs (Fig. 1C, +Table 1). We focused on the highest expression of FGF-17 in CM from hypoxic hWJ-MSCs, and confirmed that the absolute amount of FGF-17 in CM from hypoxic hWJMSCs was significantly higher than in CM from normoxic hWJ-MSCs (Fig. 1D). Furthermore, expression of FGF-17 in hypoxic hWJ-MSCs-1 and -2 was higher than normoxic hWJ-MSCs-1 and -2 (Supplemental Fig. S1A, S2A). Expression of the receptors of FGF-17 such as FGFR-1, -2, +-3 and -4 was confirmed without different expression level in both kinds of cells (Fig. 1E). + +## Fgf-17 Of Hypoxic Hwj-Mscs Had A Role In The Increase Of Cell Proliferation + + Based on results showing the highest expression of FGF-17 in CM from hypoxic hWJ-MSCs and maintenance of high proliferation of hypoxic hWJ-MSCs at passage 10, we investigated the relationship between FGF-17 and proliferation of hypoxic hWJ-MSCs using siRNA knockdown. Expression of FGF-17 was efficiently decreased when FGF-17 was knocked down in hypoxic hWJ-MSCs by siRNA treatment (Fig. 2A). Furthermore, + +![5_image_0.png](5_image_0.png) + +cell numbers (Fig. 2B, 2C), BrdU incorporation (Fig. 2D), and expression of proliferation-related gene *Ki67* (Fig. 2E) +were significantly reduced in hypoxic hWJ-MSCs transfected with siRNA of FGF-17. When hypoxic hWJ-MSCs were treated with rFGF-17 for 48 h, cell proliferation (Fig. 2F, 2G) and cell viability (Fig. 2H) were increased in a dose-dependent manner up to 500 ng/ml. Furthermore, there were consistent results on the role of FGF-17 in hypoxic hWJ-MSCs-1 and -2 (Supplementary Fig. S1B∼G, +S2B∼G). When secretory FGF-17 from hypoxic hWJ-MSCs was inhibited by blocking antibody of FGF-17, cell viability was significantly decreased (Fig. 2I). + +## Fgf-17 Of Normoxic Hwj-Mscs Had A Role In The Increase Of Cell Proliferation + + Based on the high levels of FGF-17 in conditioned medium from hypoxic hWJ-MSCs, we investigated the effects of FGF-17 on normoxic hWJ-MSCs at passage 7. Expression of FGF-17 was efficiently decreased when FGF-17 was knocked down in normoxic hWJ-MSCs (Fig. 3A). Cell numbers (Fig. 3B, 3C) and BrdU incorporation (Fig. 3D) of normoxic hWJ-MSCs with transfected with siRNA of FGF-17 were significantly reduced. Treatment of normoxic hWJ-MSCs with rFGF-17 for 48 h increased cell proliferation in a dose-dependent manner up to 500 ng/ml (Fig. 3E, 3F). At 1,000 ng/ml rFGF-17, the proliferation of normoxic hWJ-MSCs was decreased, but without sig- + +![6_image_0.png](6_image_0.png) + +nificance (Fig. 3E, 3F). Cell viability and expression of PCNA and *Ki67* increased after treatment with 500 ng/ml rFGF-17 (Fig. 3G, 3H). Furthermore, there were consistent results on the role of FGF-17 in normoxic hWJ-MSCs-1 and -2 (Supplementary Fig. S1H∼M, S2H ∼M). + +## Fgf-17 Affects The Erk Signaling Pathway In Hypoxic And Normoxic Hwj-Mscs + + To investigate signal transduction pathways of FGF-17 in normoxic or hypoxic hWJ-MSCs, we analyzed the expression of the following proteins related to cell proliferation or survival: phospho-AKT (S473), phospho-ERK1/2 +(Thr202/Tyr204), and phospho-STAT3 (Y705). Knockdown of FGF-17 in hypoxic (Fig. 4A, 4B) or nomoxic hWJ-MSCs +(Fig. 4E, 4F) decreased expression of phospho-ERK1/2 +(Thr202/Tyr204), but had no effect on levels of phospho-AKT (S473) and phospho-STAT3 (Y705). + + Treatment of hypoxic (Fig. 4C, 4D) or normoxic hWJMSCs (Fig. 4G, 4H) with 500 ng/ml rFGF-17 for 48 h resulted in increased expression of phospho-ERK1/2 (Thr202/ Tyr204), whereas phospho-AKT (S473) and phosphoSTAT3 (Y705) were not changed. Based on the relationship between high proliferation and expression of FGF-17 from hypoxic hWJ-MSCs at late passages, we investigated whether FGF-17 was associated with senescence of normoxic and hypoxic hWJ-MSCs. However, expression of senescence-related proteins p21, p27, and p53 was not + +![7_image_0.png](7_image_0.png) + +changed in hypoxic hWJ-MSCs transfected with siFGF-17 +(Fig. 4A) and treated with rFGF-17 (Fig. 4C) or in normoxic hWJ-MSCs treated with siFGF-17 (Fig. 4E) and transfected with rFGF-17 (Fig. 4G). + +## Effects Of Fgf-17 On Characterization Of Hwj-Mscs + + To investigate the effect of FGF-17 on phenotypic characterization of hWJ-MSCs, we analyzed phenotype markers of not only hypoxic hWJ-MSCs transfected with siFGF-17 and treated with rFGF-17 (Fig. 5A, 5B) but also normoxic hWJ-MSCs transfected with siFGF-17 and treated with rFGF-17 (Fig. 5C, 5D). Under both conditions, phenotypes were not changed by FGF-17. The increased expression of osteogenesis-related gene ALP, *Runx2*, and chondrogenesis-related genes *Chondroadherin* in hypoxic hWJ-MSCs was significantly decreased by knockdown of FGF-17 (Fig. 5E), and expression of *Adiponectin* and *SOX9* was decreased but without significance (Fig. 5E). When rFGF-17 was treated to hypoxic hWJ-MSCs, *Chondroadherin* was increased (Fig. 5F). When FGF-17 was knocked down in normoxic hWJ-MSCs, expression of adipogenesis-related gene *Adiponectin*, osteogenesis-related gene *Runx2*, and chondrogenesis-related genes *Chondroadherin* were increased, whereas chondrogenesis-related genes *SOX9* was decreased (Fig. 5G). Expression of the adipogenesis-related gene *adiponectin*, osteogenesis-related gene *Runx2*, and chondrogenesis-related genes *chondroadherin* and *SOX9* was significantly decreased in normoxic hWJ-MSCs treated with rFGF-17 and expression of the osteogenesis-related gene ALP was decreased without significance (Fig. 5H). + +![8_image_0.png](8_image_0.png) + +## Discussion + + In this study we show that increased expression of FGF-17 in CM from hypoxic hWJ-MSCs plays a role in maintenance of high proliferation of human mesenchymal stem cells at late passages. + + In general, it is well known that the proliferation and survival of cells decreases with increased passage numbers under normal cell culture conditions (18). The mechanisms related to decreased cell proliferation have been reported to be senescence (18), apoptosis (18), DNA mutation (19), +and cell transformation (20). In contrast, it has been reported that hypoxic cell culture conditions not only enhance proliferation and survival of cells, but also influence the secretome of mesenchymal stem cells (10-12). According to our protein antibody array, secretory FGF-17 from hypoxic hWJ-MSCs was highly expressed at passage 10 (late passage), and knockdown of FGF-17 decreased cell proliferation of hypoxic hWJ-MSCs. These findings indicate that FGF-17 from hypoxic hWJ-MSCs plays a role in maintaining high proliferation at late passages. In addition, treatment of normoxic hWJ-MSCs at passage 7 (middle passage) with 250 ng/ml or 500 ng/ml rFGF-17 increased cell proliferation, whereas 1,000 ng/ml rFGF-17 did not further increase proliferation. This may suggest that there is a threshold in the working concentration of FGF-17 for effects on normoxic hWJ-MSCs at the cellular level. According to protein antibody array, VEGF, IGFBP-3, IL-1 R3, Chordin-Like 1, and EN-RAGE were upregulated at least 2-fold in CM from hypoxic hWJ-MSCs at passage 10 compared with normoxic hWJ-MSCs. It has been reported that VEGF and Chordin-Like 1 are related to increased cell proliferation (21, 22) and these may be additional candidate molecules for the maintenance of high proliferation of hypoxic hWJ-MSCs at late passages. + +Interestingly, previous studies showed that IGFBP-3 and EN-RAGE play a role in inhibiting cell proliferation (23, 24). It is possible that the positive effect of FGF-17, VEGF, and Chordin-Like 1 overcome the negative effect of IGFBP-3 and EN-RAGE for maintenance of high proliferation in late passages of hypoxic hWJ-MSCs. We will further investigate the roles of these molecules. In a previous study, FGF-4 and associated activation of the ERK1/2 pathway triggered a switch from self-renewal to lineage commitment in embryonic stem cells (25). In contrast to FGF-4, even though treatment with FGF-17 increased the expression of phospho-ERK1/2 in normoxic hWJ-MSCs, the expression of differentiation genes was decreased. This suggests that FGF-4 and FGF-17 may have different roles in stem cell function even though they are both members of the FGF family. It has been reported that expression of FGF-2, FGF-11 and FGF-23 included in fibroblast growth factor family were regulated by hypoxia-inducible factor (HIF-1α) in hypoxic condition (26-28). + +For further study, we will investigate the relation between FGF-17 and HIF-1α. + + We presumed that the co-relationship between high expression of FGF-17 and maintenance of cell proliferation at late passages might result from inhibition of cell senescence by FGF-17. However, expression of senescence-related proteins (p21, p27, and p53) was not changed in normoxic hWJ-MSCs treated with siFGF-17 and rFGF-17 at passage 7 or hypoxic hWJ-MSCs treated with siFGF-17 and rFGF-17 at passage 10. + + Furthermore, under the same conditions the phenotypes of normoxic hWJ-MSCs were not altered and expression levels of differentiation-related genes *adiponectin* +(adipogenesis), *Runx2* (osteogenesis), and *chondroadherin* and *SOX9* (chondrogenesis) were decreased by treatment of rFGF-17. These results provide supportive evidence for the use of rFGF-17 as a supplement in culture medium of human mesenchymal stem cells in order to enhance the expansion of cells with stemness. + + Interestingly, in hypoxic hWJ-MSCs the expression of differentiation genes was increased (significantly for ALP, +Runx2 and *chondroadherin*; non-significantly for *adiponectin* and *SOX9*) compared with normoxic hWJ-MSCs, whereas knockdown of FGF-17 in hypoxic hWJ-MSCs decreased the expression of these molecules (significantly for ALP, +Runx2, and *chondroadherin*; non-significantly for adiponectin and SOX9). This suggests that the effects of FGF-17 on differentiation-related genes are different between normoxic and hypoxic hWJ-MSCs and further studies will be necessary to investigate the mechanisms underlying these differential activities. + + In conclusion, FGF-17 from hypoxic hWJ-MSCs plays a role in maintenance of their high proliferation at late passages through the ERK1/2 pathway. And it will be useful as a supplement for the maintenance of proliferation and expansion of hWJ-MSCs under long-term culture conditions. + +## Acknowledgments + + This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), Ministry of Health and Welfare, Republic of Korea (HI17C1728), and by a grant from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea +(HI14C3484). + +## Potential Conflicts Of Interest + + The authors have no conflicting financial interest. + +## Supplementary Materials + + Supplementary data including two figures can be found with this article online at http://pdf.medrang.co.kr/paper/ +pdf/IJSC/IJSC-12-s18042.pdf. + +## References + +1. Liu Y, Yan W, Tohme S, Chen M, Fu Y, Tian D, Lotze M, Tang D, Tsung A. 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Bone marrow-derived mesenchymal stem cells modified with IGFBP-3 inhibit the proliferation of pulmonary artery smooth muscle cells. Int J Mol Med 2017;39:223-230 24. Hofmann Bowman M, Wilk J, Heydemann A, Kim G, +Rehman J, Lodato JA, Raman J, McNally EM. S100A12 mediates aortic wall remodeling and aortic aneurysm. Circ Res 2010;106:145-154 25. Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, +Meloche S, Smith A. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 2007;134:2895-2902 26. Conte C, Riant E, Toutain C, Pujol F, Arnal JF, Lenfant F, Prats AC. FGF2 translationally induced by hypoxia is involved in negative and positive feedback loops with HIF1alpha. PLoS One 2008;3:e3078 27. Yang J, Kim WJ, Jun HO, Lee EJ, Lee KW, Jeong JY, Lee SW. Hypoxia-induced fibroblast growth factor 11 stimulates capillary-like endothelial tube formation. Oncol Rep 2015;34:2745-2751 28. Zhang Q, Doucet M, Tomlinson RE, Han X, Quarles LD, +Collins MT, Clemens TL. The hypoxia-inducible factor-1α activates ectopic production of fibroblast growth factor 23 in tumor-induced osteomalacia. Bone Res 2016;4:16011 \ No newline at end of file diff --git a/medical/md/PMC6667870.md b/medical/md/PMC6667870.md new file mode 100644 index 0000000000000000000000000000000000000000..a5ca545502cf26b1e097770b04b54a2b83d2aa5d --- /dev/null +++ b/medical/md/PMC6667870.md @@ -0,0 +1,108 @@ +Are Baseball Statistics an Appropriate Tool for Assessing Return to Play in Injured Players: An Analysis of Statistically Variability in Healthy Players Using a Machine Learning Approach Ayoosh Pareek, MD, Chad W. Parkes, MD, Alexey A. Leontovich, Ph.D., Christopher D. Bernard, BS, Aaron John Krych, MD, Diane L. Dahm, MD, Christopher L. Camp, MD Mayo Clinic, Rochester, MN, USA +Objectives: Traditional pitching statistics (ERA, WHIP, etc) have been used as surrogates for pitcher performance without being validated. Even amongst healthy pitchers, the normal variability of these parameters has not yet been established. The purpose of this study was to determine the normal variability of basic and advanced pitching statistics in non-injured Major League Baseball (MLB) pitchers. It is our hope that this work will serve as the foundation for the identification and implementation of validated, pitcher dependent statistical measures that can be used to assess return to play performance following injury. + +Methods: Publicly available data from MLB Statcast and Pitch/Fx databases was used to analyze all non-injured MLB pitchers during 2015 and 2016 seasons who pitcher greater than 100 innings each season without injury. Traditional and advanced baseball pitching statistics were analyzed. The variability of each parameter was assessed by computing coefficient of variation (CV) between individual pitchers and across all pitchers. A CV below 10 is typically indicative of a relatively constant parameter, and parameters with a CV > 10 are generally considered inconsistent and unreliable. + +Results: A total of 118 pitchers met all inclusion criteria. For each of these healthy pitchers, 38 basic/traditional parameters and 17 advanced parameters were analyzed. Of the traditional pitcher statistics, only 1 (3%) demonstrated a CV value < 10 (average fastball velocity [FBv]; CV 1.5) (Figure 1). In advanced statistics, 9 of 17 (53%) variables demonstrated acceptable consistency as evidenced by a CV value < 10 (Figure 2). Release position from plate (release_pos_y) along with velocity from the plate (vy0) where the two most constant advanced parameters. When separated by pitch type, these two parameters were the most constant (lowest CV) in every pitch type. Conclusion: The validity and variability of baseball statistics as surrogate markers for performance after injury/surgery have not yet been evaluated. It is critical that baseball statistics undergo proper vetting prior to being used to assess recovery. This study reveals average fastball velocity and release position from the plate to be the least variable basic and advanced baseball statistics in MLB pitchers. In total, only 10 of the 55 statistics analyzed demonstrated acceptable consistency and reliability. This study can be further used to determine the minimum time that each of these variables needs to be followed to ensure an appropriate sample size is obtained to detect significant differences in pre- and post- injury performance. + +Figure 1. Variability for each traditional MLB pitcher statistic sorted in ascending order. Lower values indicate + +| less variation. | | | | +|--------------------------------------------------------|-------------------------------------|------------------------------------|-------| +| Variability of Traditional Pitching Game Statistics in | | | | +| Non-Injured Pitchers | | | | +| Rank | Variability | | | +| est to | Parameter | Parameter Definition | | +| most +sriable) | Coeffici | | | +| FBv | Average Fast Ball Velocty | 1.5 | | +| 1 | | | | +| 2 | FB% | Fastball % | 23.1 | +| 3 | LOB% | Left on Base % | 32.0 | +| 4 | TBF | Total Batters Faced | 54.5 | +| 5 | Pitches | Number of Pitches | 56.4 | +| 6 | Strices | Number of Strikes | 56.5 | +| 7 | Bals | Number of Balls | 67.4 | +| 8 | K9 | Strikeouts Over 9 Innings | 7.9 | +| Expected Fielding Adjusted Pitching | | | | +| XFIP. | 81.3 | | | +| 9 | Adjusted for Park Effects | | | +| 10 | Expected Fielding Adjusted Pitching | 81.4 | | +| xFIP | | | | +| 11 | SIERA | Skill Adjusted Earned Run Aversge | 83.4 | +| v | 83.5 | | | +| K% | Striceout % | | | +| 13 | K/BB | Strikeouts per Walk | 888 | +| 14 | BAA | Batting Average Against | 90.3 | +| 15 | K | Number of Strikeouts | 94.2 | +| 16 | BABIP | Batting Average in Balls in Play | 95.2 | +| 17 | H | Number of Hits allowed | 106.6 | +| 18 | WHIP | Walks plus Hits per Inning Pitched | 109.0 | +| Fielding Adjusted Pitching Adjusted for | | | | +| 19 | FIP. | 133.2 | | +| Park/League Effects | | | | +| 20 | Fielding Adjusted Pitching | 133.3 | | +| FIP | | | | +| 21 | B% | Walk % | 150.2 | +| 2 | GS | Games Started | 151.0 | +| 23 | K-88% | Strikeout-Wak% | 153.8 | +| 24 | BB | Number of Walks | 154.3 | +| 25 | B/9 | Walks per 9 Innings | 172.3 | +| 26 | R | Runs alowed | 177.6 | +| 27 | ER | Earned Runs allowed | 183.9 | +| Eamed Run Average adjusted for | | | | +| 28 | ERA- | 20.0 | | +| Park/League Effects | | | | +| Earned Run Average (Runs over 9 | | | | +| 29 | ERA | 21.9 | | +| inngs) | | | | +| 30 | HR | Home Runs allowed | 279.0 | +| 31 | HLD | Number of holds | 285.5 | +| 32 | HR9 | Home Runs alowed over 9 Innings | 306.8 | +| 33 | W | Wins | 353.9 | +| 34 | 452.1 | | | +| L | LOSSES | | | +| 35 | HBP | Ht by Pitch | 476.7 | +| 36 | BS | Blown Saves | 528.0 | +| 37 | C | Complete Games | 553.4 | +| 38 | SV | Saves | 567.2 | + +| Figure 2. Variability for each advanced MLB pitcher statistic. Lower values indicate less variation | | | | +|-------------------------------------------------------------------------------------------------------|----------------------|--------------------------------|-------| +| Variability of Advanced Pitch Statistics (cumulative) | | | | +| Rank | | | | +| (east to | Variability | | | +| Parameter | Parameter Definition | | | +| most | Coefficient | | | +| variable) | release_pos_y | Release Position from Plate | 0.8 | +| 1 | | | | +| 2 | vy0 | Velocity from Pitcher to Plate | 3.5 | +| 3 | release_speed | Release Speed | 3.6 | +| 4 | effective_speed | Effective Speed | 3.8 | +| 5 | release_extension | Release Extension | 6.9 | +| 6 | release_spin_rate | Spin Rate | 7.0 | +| Force experienced in Plan from | | | | +| 7 | ay | 7.1 | | +| Pitcher to Plate | | | | +| 8 | plate_z | Plate Position Vertical | 7.5 | +| 9 | release_pos_z | Release Position Vertical | 7.7 | +| Force experienced in the Vertical | | | | +| 10 | az | 16.3 | | +| Plane | | | | +| Vertical Distance traveled from | | | | +| 11 | pfx_z | 32.3 | | +| release to front of plate | | | | +| 12 | vz0 | Velocity in Vertical Plane | 34.8 | +| 13 | release_pos_x | Release Position Horizontal | 269.1 | +| Vertical Distance traveled from | | | | +| 14 | pfx_x | 293.4 | | +| release to front of plate | | | | +| 15 | vx0 | Velocity in Horizontal Plane | 319.1 | +| Force/Drag in the Horizontal | | | | +| 16 | ax | 657.1 | | +| Plane | | | | +| 17 | plate_x | Plate Position Horizontal | 790.6 | + +The Orthopaedic Journal of Sports Medicine, 7(7)(suppl 5) +DOI: 10.1177/2325967119S00397 +©The Author(s) 2019 \ No newline at end of file diff --git a/medical/md/PMC6686438.md b/medical/md/PMC6686438.md new file mode 100644 index 0000000000000000000000000000000000000000..f853f243dcf62520c09cfbd6ee0a95401ca540c4 --- /dev/null +++ b/medical/md/PMC6686438.md @@ -0,0 +1,405 @@ +# Administration Of Rctrp9 Attenuates Neuronal Apoptosis Through Adipor1/ Pi3K/Akt Signaling Pathway After Ich In Mice Lianhua Zhao1,2, John H. Zhang2, Prativa Sherchan2, Paul R. Krafft3, Wei Zhao1, Sa Wang4, Shengpan Chen2,5, Zaiyu Guo1, And Jiping Tang2 + +1 Department of Neurology, Tianjin TEDA Hospital, Tianjin, China 2 Department of Physiology and Pharmacology, Loma Linda University, Loma Linda, CA, USA +3 Department of Neurological Surgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA +4 Department of Neurology, Affilicated Wenling Hospital of Wenzhou Medical University, Wenling, Zhejiang, China 5 Department of Neurosurgery, Affiliated Haikou Hospital, Xiangya School of Medicine, Central South University, Haikou, China Submitted: October 15, 2018. Revised: December 3, 2018. Accepted: December 11, 2018. + +Corresponding Authors: +Jiping Tang, MD, Department of Physiology and Pharmacology, Loma Linda University, 11041 Campus St, Loma Linda, CA 92354, USA; Zaiyu Guo, MD, +PhD, Department of Neurosurgery, Tianjin TEDA Hospital, No.65 The 3th Avenue, Tianjin TEDA, China. Emails: jtang@llu.edu; guozai_yu@163.com Cell Transplantation 2019, Vol. 28(6) 756–766 ª The Author(s) 2019 Article reuse guidelines: +sagepub.com/journals-permissions DOI: 10.1177/0963689718822809 journals.sagepub.com/home/cll + +## Abstract + +Targeting neuronal apoptosis after intracerebral hemorrhage (ICH) may be an important therapeutic strategy for ICH +patients. Emerging evidence indicates that C1q/TNF-Related Protein 9 (CTRP9), a newly discovered adiponectin receptor agonist, exerts neuroprotection in cerebrovascular disease. The aim of this study was to investigate the anti-apoptotic role of CTRP9 after experimental ICH and to explore the underlying molecular mechanisms. ICH was induced in mice via intrastriatal injection of bacterial collagenase. Recombinant CTRP9 (rCTRP9) was administrated intranasally at 1 h after ICH. To elucidate the underlying mechanisms, adiponectin receptor1 small interfering ribonucleic acid (AdipoR1 siRNA) and selective PI3 K +inhibitor LY294002 were administered prior to rCTRP9 treatment. Western blots, neurofunctional assessments, immunofluorescence staining, and Fluoro-Jade C (FJC) staining experiments were performed. Administration of rCTRP9 significantly improved both short- and long-term neurofunctional behavior after ICH. RCTRP9 treatment significantly increased the expression of AdipoR1, PI3 K, p-Akt, and Bcl-2, while at the same time was found to decrease the expression of Bax in the brain, which was reversed by inhibition of AdipoR1 and PI3 K. The neuroprotective effect of rCTRP9 after ICH was mediated by attenuation of neuronal apoptosis via the AdipoR1/PI3K/Akt signaling pathway; therefore, rCTRP9 should be further evaluated as a potential therapeutic agent for ICH patients. + +## Keywords + +intracerebral hemorrhage, adiponectin receptor 1, C1q/TNF-related protein 9, neuronal apoptosis, PI3K/Akt signaling + +## Introduction + +Intracerebral hemorrhage (ICH) is a stroke subtype with a high mortality and morbidity rate that accounts for 10–15% +of all strokes worldwide1–3. Despite its devastating socioeconomic burden on individuals and society, ICH remains an unresolved medical problem with little improvement in patient outcomes over the past 20 years4. Regarding the pathophysiology of ICH, it is generally accepted that the intraparenchymal hematoma compressing the surrounding brain tissue is considered to elicit the primary brain injury; then red blood cell debris and other clot components lead to secondary brain injury (SBI), which involves a series of molecular processes including inflammatory responses, activation of apoptosis cascades, ischemia, blood–brain barrier disruption, and brain edema formation. Apoptosis is the main form of cell death within the brain region surrounding the intracerebral hematoma5,6. Furthermore, it has been Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.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 pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). + +shown that the extent of neuronal apoptosis is closely related to impaired neurological functions7,8. Therefore, inhibiting neuronal apoptosis after ICH may alleviate neurological deficits and improve overall patient outcomes. + +Adiponectin (APN) is an adipose-derived secretory serum protein that has been shown to alter multiple functions in the peripheral and central nervous systems9–11. However, APN +knock-out mice exhibit highly variable phenotypes12. Because APN has a C-terminal globular domain with sequence homology to the immune complement protein C1q, it belongs to the larger family of C1q proteins13. C1q/TNF-related proteins +(CTRPs) have similar structural and biochemical properties as APN14–16. Of all identified CTRPs, CTRP9 shares the highest degree of sequence identity (54%) with APN at the presumed functional globular domain15. + +APN exerts its physiological effects by binding to and thereby activating its specific receptors. Currently two APN +receptors, AdipoR1 and AdipoR2, have been identified. AdipoR1 is widely expressed in the central nervous system +(CNS), especially on neurons17. It has been reported that AdipoR1 agonists participate in neuroprotective actions against ischemic stroke, particularly by reducing neuronal apoptosis18. Two recent studies suggest that the anti-apoptotic effects of AdipoR1 agonists are mediated via the phosphatidylinositol 3-kinase (PI3 K)/ protein kinase B (Akt) signaling pathway19,20. It has been shown that CTRP9 exerts a high affinity to AdipoR121, and emerging evidence suggests that CTRP9 has the potential to carry out neuroprotective functions on diverse CNS disease22. However, the anti-apoptotic effects of CTRP9 following ICH have not yet been illustrated. + +The present study aimed to assess the effects of CTRP9 on neurofunctional outcomes and neuronal apoptosis in mice subjected to experimental ICH. The mechanisms of the observed effects were then explored using AdipoR1 siRNA and PI3 K inhibitor, LY294002. + +## Materials And Methods Animals + +A total of 144 male CD1 mice (8 weeks old, weight 30–40 g; Charles River, Wilmington, MA, USA) were housed in a temperature and humidity-controlled room with a 12-hour light/dark cycle and free access to food and water. All animals were acclimated to the environment for a minimum of 3 days before being subjected to ICH induction or sham surgery. The experimental protocols and procedures for this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Loma Linda University +(IACUC No. 8160049) and were in compliance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. + +## Ich Model And Study Design + +Experimental ICH was induced in mice by intrastriatal injection of bacterial collagenase, as previously reported23. + +Briefly, mice were anesthetized with a mixture of ketamine +(100 mg/kg) and xylazine (10 mg/kg) (2:1, intraperitoneal injection) and positioned prone in a stereotaxic head frame +(Kopf Instruments, Tujunga, CA, USA). A 1-mm cranial burr hole was drilled, and a 26-gauge needle on a 10-ml Hamilton syringe was inserted stereotactically into the right striatum (coordinates 0.2 mm posterior, 2.2 mm lateral to the bregma, and 3.5 mm below the dura). Bacterial collagenase type VII-S (0.075 units) (Sigma-Aldrich, St. Louis, MO, +USA) dissolved in 0.5 ml sterile phosphate-buffered saline (PBS) was infused into the brain at a rate of 0.167 ml/min with an infusion pump (Stoelting, Harvard Apparatus, Holliston, MA, USA). The needle was left in place for an additional 5 min after the injection to prevent possible leakage of the collagenase solution before being withdrawn slowly at a rate of 1 mm/min. The cranial burr hole was sealed with bone wax, the scalp was sutured, and 0.4 ml of normal saline was injected subcutaneously to avoid postsurgical dehydration. Mice were allowed to recover fully under close observation. The sham operation was performed following the same protocol but involved needle insertion only. + +All mice were randomly assigned to the following four separate experiments as shown in the timeline of the experimental design (Additional file 1: Fig. S1). Experimental groups, animal numbers, and mortality rates of this current study are summarized in the supplemental material (Additional file 2: Table S1). + +Experiment 1. To determine the time course of endogenous AdipoR1 and CTRP9 expression in the brain of mice subjected to ICH, n¼36 mice were divided into six groups +(n¼6/group) including sham as well as ICH after 3, 6, 12, 24, and 72 h. Two additional mice were used for double immunohistochemistry staining of AdipoR1 combined with specific markers for neurons, astrocytes, and microglia at 24 h after ICH. + +Experiment 2. To evaluate the short-term neurofunctional deficits and the extent of neuronal apoptosis, n¼27 mice were randomly divided into three groups (n¼9/group): sham, ICHþvehicle (PBS), ICHþrCTRP9 (0.1 mg/g). Recombinant CTRP9 (rCTRP9) was administered intranasally at 1 h post-ICH. Neurofunctional tests and Fluoro-Jade C (FJC) +staining were conducted at 24 h post-ICH. + +Experiment 3. To assess long-term neurofunctional deficits, n¼24 mice were divided into three groups (n¼8/group): +sham, ICHþvehicle (PBS), ICHþrCTRP9 (0.1 mg/g). The foot-fault test and rotarod test were performed on days 7, 14, and 21 post-ICH, and the Morris water maze test was conducted on days 21–25 post-ICH. + +Experiment 4. To verify the anti-apoptotic mechanism of rCTRP9, n¼42 mice were divided into seven groups (n¼6/ +group): sham, ICHþvehicle (PBS), ICHþrCTRP9 (0.1 mg/g), +ICHþrCTRP9þAdipoR1 siRNA, ICHþrCTRP9þScramble siRNA (Scr siRNA), ICHþrCTRP9þLY294002 (specific PI3 K inhibitor), ICHþrCTRP9þDMSO (5 ml of 5% dimethyl sulfoxide in PBS). LY294002 and DMSO were delivered intracerebroventricularly 30 min before ICH24 and scrambled siRNA and AdipoR1 siRNA were injected intracerebroventricularly 48 h before ICH25. Neurofunctional testing and western blot were performed at 24 h post-ICH. + +## Western Blot Analysis + +Western blot was performed as previously described26. + +Briefly, mice were transcardially perfused with ice-cold PBS +under deep anesthesia, and brains were removed and separated into two hemispheres at 24 h after ICH induction or sham surgeries. The ipsilateral/right brain hemispheres were homogenized in RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and centrifuged at 4C for 30 min at 14,000 g. The supernatant was collected, and the protein concentration was determined using a detergent compatible assay (DC protein assay, Bio-Rad Laboratories, CA, USA). Equal amounts of protein were loaded on an SDS-PAGE gel and run using electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked and incubated at 4C overnight with the following primary antibodies targeting: CTRP9 (1:500, NBP2-46834, Novus, Centennial, CO, USA), AdipoR1 (1:1000, ab126611, Abcam, Cambridge MA, USA), PI3 K (1:1000, ab182651, Abcam), p-Akt (1:1000, \#9271, Cell Signaling Technology, Boston (CST), MA, USA), Akt (1:2000, \#9271, CST), Bcl-2 +(1:1000, \#2764, CST), Bax (1:500, NBP1-28566, Novus Biologicals) and actin (1:4000, Santa Cruz Biotechnology, Dallas, TX, USA). Appropriate secondary antibodies (1:3000, Santa Cruz Biotechnology; 1:5000, Abcam) were selected to incubate with the membrane for 2 h at room temperature. The bands were probed with an ECL Plus chemiluminescence regent kit (Amersham Biosciences, Arlington Heights, PA, +USA) and visualized with the image system (Bio-Rad, Versa Doc, model 4000). Relative density of the protein immunoblot images were analyzed by ImageJ software (ImageJ 1.4, National Institutes of Health (NIH), Bethesda, MD, USA). + +## Immunofluorescence Staining + +The brain samples used for immunohistochemistry staining were prepared according to a previously described protocol27. Briefly, animals were deeply anesthetized at 24 h after ICH and transcardially perfused with 100 ml ice-cold PBS followed by 60 ml of 10% paraformaldehyde. The whole brain was collected and fixed in 10% paraformaldehyde for 24 h and dehydrated in a 30% sucrose solution for 3 days. + +After being kept frozen at –80C, brains were cut into 8 mmthick coronal sections on a cryostat (LM3050 S; Leica Microsystems, Bannockburn, Germany). Immunofluorescence staining was conducted as previously described28. + +Briefly, brain samples were incubated overnight at 4-C with the primary antibodies including anti-Iba-1 (1:200, ab178847, Abcam), anti-NeuN (1:200, ab177487, Abcam) +and anti-GFAP (1:200, ab16997, Abcam), and anti-AdipoR1 (1:200, ab126611, Abcam). The corresponding secondary antibodies (1:500, Jackson Immunoresearch, West Grove, PA, USA) were added to the brain sections and allowed to incubate at room temperature for 2 h. The sections were visualized and photographed by using a fluorescence microscope (Leica Microsystems). + +## Short-Term Neurofunctional Assessments + +Short-term neurofunctional behavior was assessed with the modified Garcia score, forelimb placement test, and corner turn test involving an independent researcher blinded to the experimental groups. All tests were performed at 24 h after ICH induction or sham surgery, as previously described29,30. + +The Garcia score includes seven individual tests that evaluate spontaneous activity, vibrissae proprioception, axial sensation, symmetry of limb movement, lateral turning, forelimb walking, and climbing. Each subtest was given a score ranging from 0 to 3, with a composite maximum score of 21 (no neurological deficits). The forelimb placement test was used to assess the animals' responsiveness to vibrissae stimulation, and results were expressed as a percentage of the number of successful left forepaw placements out of 10 stimulations, normalized to the mean of sham performance. + +For the corner turn test, animals were allowed to advance into a 30- corner and exit by turning either to the left or right. + +Choice of turning was recorded for a total of 10 trials, and a score was calculated as number of left turns/all trials 100. + +## Fluoro-Jade C Staining + +Degenerating neurons were evaluated by FJC staining as previously described utilizing a modified FJC ready-todilute staining kit (Biosensis, USA) according to the manufacturer's instructions31,32. Briefly, FJC-positive neurons were counted in six sections per brain at 200 magnification by an independent observer. Quantified analysis was performed with Image J software (Image J 1.4, NIH, Bethesda, MD, USA). The data were presented as the average number of FJC-positive neurons in the fields (cells/mm2). + +## Drug Administration + +Recombinant CTRP9 (rCTRP9) (cat\# H00338872-P01, Novus Biologicals) dissolved in PBS was administered intranasally at 1 h post-ICH as previously reported27. A total volume of 20 ml of rCTRP9 solution was administered intranasally. Mice under anesthesia were placed in a supine position and rCTRP9 solution was administered in drops (5 ml/ +drop) every 2 min, alternating between the left and right nares, over a period of approximately 20 min. The ICHþvehicle group was given an equal volume of PBS +intranasally. The method for drug administration was the same for short- and long-term experiments. + +## Long-Term Neurofunctional Assessments + +To evaluate the long-term neurofunctional outcomes after ICH, the foot-fault and rotarod tests were performed within the first, second, and third week post-ICH as previously described33. These tests specifically evaluate the rodents' sensorimotor function, coordination, and balance. Water maze tests were performed on days 21 to 25 post-ICH to evaluate memory and spatial learning as previously described34. + +## Intracerebroventricular Injection + +Intracerebroventricular administration was performed as previously described35. AdipoR1 siRNA (cat\# 4390771) and Scramble siRNA (scr siRNA) (cat\# 4390843, Life Technologies, Carlsbad, CA, USA) were prepared at 500 pmol in RNAsefree suspension buffer and were infused (5 ml of the siRNAs) +48 h before ICH modeling in ICHþrCTRP9þAdipoR1 siRNA +and ICHþrCTRP9þScr siRNA mice, respectively. LY294002 (Selleck Chemicals, Houston, TX, USA) was prepared at 50 mmol/l in PBS (contains 25% DMSO), and 5 ml of the DMSO and LY294002 were infused 30 min before ICH induction in ICHþrCTRP9þLY294002 and ICHþrCTRP9þDMSO mice, respectively. The 26-gauge needle of a 10-ml Hamilton syringe was inserted into the left lateral ventricle through a cranial burr hole at the following coordinates relative to bregma: 0.3 mm posterior, 1.0 mm lateral, and 2.3 mm below the dura. A microinfusion pump was used for the intracerebroventricular administrations at a rate of 0.667 ml/min. The needle was left in place for an additional 5 min after the end of infusion before being slowly withdrawn over 3 min. The burr hole was sealed with bone wax. + +## Statistical Analysis + +All data were expressed as the mean and standard deviation +(mean + SD). Statistical analysis was performed with Graph Pad Prism (Graph Pad Software Inc., San Diego, CA, USA). + +Data were analyzed by one-way analysis of variance +(ANOVA) followed by multiple comparisons between groups using Tukey's post-hoc test. A P-value of less than 0.05 was considered statistically significant. + +## Results Animal Mortality And Exclusion + +None of the sham animals died prior to euthanasia. The total animal mortality rate in this study was 11.3% (13/115). The mortality rate was not significantly different between the experimental groups (Additional file 2: Table S1). + +## The Endogenous Expressions Time Course Of Adipor1 And Ctrp9 After Ich Western Blot Was Performed To Assess The Protein Expression Of Adipor1 And Ctrp9 In The Ipsilateral/Right Cerebral + +hemispheres of sham animals or at 3, 6, 12, 24, and 72 h after ICH induction. The endogenous expressions of AdipoR1 and CTRP9 continuously increased after surgery, reached a peak at 24 h, and then decreased again at 72 h after ICH induction (p<0.05 compared with sham, Fig. 1A, +B). Double immunofluorescence staining was performed to detect the localization of AdipoR1 on neurons (NeuN), +microglia/macrophages (Iba-1), and astrocytes (GFAP) at 24 h after ICH. AdipoR1 was mainly expressed on neurons and astrocytes after ICH (Fig. 1C). + +## Rctrp9 Ameliorated Short-Term Neurofunctional Deficits After Ich + +ICH induction significantly reduced the composite modified Garcia score, as well as performances of the forelimb placement and corner turn test at 24 h after surgery when compared with sham animals. The neurofunctional impairments were significantly ameliorated by administration of rCTRP9 +(0.1 mg/g) as seen in the ICHþrCTRP9 group, (p<0.05 compared with the vehicle group, Fig. 2A–C). + +## Rctrp9 Attenuated Neuronal Apoptosis After Ich + +FJC was used to evaluate the severity of neuronal degeneration and apoptosis in the ipsilateral hemisphere at 24 h after ICH (Fig. 3B). ICH induction resulted in a significantly increased number of FJC-positive cells (ICHþvehicle) when compared with sham animals (p<0.05); however, rCTRP9 treatment (0.1 mg/g) significantly reduced the number of apoptotic cells as demonstrated in the ICHþrCTRP9 group (p<0.05 compared with ICHþvehicle, Fig. 3A and C). + +## Rctrp9 Improved Long-Term Neurofunctional Outcomes After Ich + +At the first, second, and third week after surgery, mice randomized to the ICHþvehicle group demonstrated significantly more foot faults of the left forelimb as well as shorter falling latencies than those animals randomized to the sham group (p<0.05, Fig. 4A, B). However, animals in the ICHþrCTRP9 group demonstrated significantly fewer missteps with the left forelimb as well as longer walking periods on the rotarod before falling when compared with the vehicle group (p<0.05, Fig. 4A, B). + +During Morris water maze testing, the swim distance and escape latency measured for vehicle animals to find a platform within a pool of water were found significantly increased when compared with the sham group (p<0.05, Fig. + +4C, D). However, a significantly shorter swim distance and escape latency was observed in the ICHþrCTRP9 group on days 3 to 5 of testing (p<0.05 compared with ICHþvehicle, Fig. 4C, D). + +760 Cell Transplantation 28(6) + +![4_image_0.png](4_image_0.png) + +## Adipor1 Sirna And Pi3 K Inhibitor Reversed The Neurofunctional Improvements Seen With Rctrp9 Therapy + +Administration of rCTRP9 resulted in improved neurofunctional performances after ICH, which was assessed via neurofunctional tests including the modified Garcia test, forelimb placement test, and corner turn test at 24 h after ICH. The neurofunctional improvements seen with rCTRP9 therapy were reversed by administration of AdipoR1 siRNA +and specific PI3 K inhibitor LY294002 (p<0.05 compared with ICHþrCTRP9, Fig. 5A-C). + +## Rctrp9 Attenuated Neuronal Apoptosis Through Adipor1/Pi3K/Akt Signaling After Ich + +Administration of rCTRP9 resulted in significantly greater protein expressions of AdipoR1 and PI3 K within the affected brain hemispheres of treated ICH animals when compared with the sham and ICHþvehicle groups at 24 h + +![5_image_0.png](5_image_0.png) + +![5_image_1.png](5_image_1.png) + +after surgery (p<0.05, Fig. 6A, B). Moreover, the expressions of phosphorylated Akt, Bcl-2 were significantly increased, while the expression of Bax was significantly decreased in the ICHþrCTRP9 group when compared with the ICHþvehicle group at 24 h after surgery (p<0.05, Fig. + +6A, B). However, inhibition of AdipoR1 with AdipoR1 siRNA significantly decreased the expression of AdipoR1, PI3 K, phosphorylated Akt and Bcl-2, and increased the expression of Bax when compared with the ICHþrCTRP9þ scr siRNA group at 24 h after ICH (p<0.05, Fig. 6B). Similarly, pretreatment with LY294002 significantly decreased the expression of PI3 K and the expression of the downstream molecules p-Akt, as well as Bcl-2 and increased the expression of Bax in the ICHþrCTRP9þLY294002 group when compared with the ICHþrCTRP9þDMSO group at 24 h after ICH (p<0.05, Fig. 6B). + +## Discussion + +In this present study, we first investigated the potential antiapoptosis effect of rCTRP9 and explored the possible underlying mechanism of rCTRP9 after ICH. We discovered that the expression of AdipoR1 and rCTRP9 increased after ICH induction. AdipoR1 was found to be expressed in neurons, astrocytes, and microglia cells. Administration of rCTRP9 attenuated neuronal apoptosis and preserved neurological + +![6_image_0.png](6_image_0.png) + +![6_image_1.png](6_image_1.png) + +function after ICH. Administration of rCTRP9 was associated with upregulation of AdipoR1, PI3 K, p-Akt, and Bcl-2, and downregulation of Bax after ICH. However, blockage of AdipoR1 and PI3 K reversed the beneficial effects of rCTRP9 on neurofunctional deficits and neuronal apoptosis. Conclusively, our findings suggest that administration of rCTRP9 might attenuate neuronal apoptosis after ICH, +which was, at least in part, mediated by the AdipoR1/ +PI3K/Akt signaling pathway. + +The APN receptor is an emerging potential target in diverse CNS disease36–38. Activation of APN receptors results in adequate regulation of cellular metabolism, and + +![7_image_0.png](7_image_0.png) + +exerts anti-atherosclerosis and anti-inflammatory effects39–41. + +Although APN receptors have been shown to exist abundantly in the brain, their exact role in brain diseases remains unclear. Two APN receptors have been identified: AdipoR1 and AdipoR2. Recent studies reported that AdipoR1 and AdipoR2 are both expressed within cortical neurons of mice, with AdipoR1 being more pronounced than AdipoR242. + +Furthermore, AdipoR1 binds to globular APN with higher affinity than AdipoR2. AdipoR2 has an intermediate binding affinity for both globular APN and full-length APN17. + +Previous studies revealed that AdipoR1 was widely expressed in neurons, astrocytes, and microglial cells43. + +Furthermore, our results indicate that the expression of AdipoR1 increases within 24 h following ICH. Based on our immunohistochemistry data, the expression of AdipoR1 was observed in neurons, astrocytes, and microglia, which is consistent with the results of the aforementioned studies. The increase in AdipoR1 expression within the ipsilateral brain hemisphere after ICH inductions requires further explanation; however, we suggest that the observed change in AdipoR1 expression might be a response to the stress reaction induced by ICH. Moreover, the expression of AdipoR1 was further elevated after administration of rCTRP9. The effects of rCTRP9 on AdipoR1 expression were consistent with the results of a previous study, which showed that APN +increased the expression of AdipoR1 and protected the brain tissue against the detrimental effects of ischemic stroke44. + +APN is an adipokine that is predominantly secreted by adipocytes. It has numerous functions, including regulation of metabolism, anti-inflammation, protection against endothelial dysfunction, and protection against ischemic injury40,41. The CTRPs are a newly discovered, highly conserved family of APN paralogs, containing 15 members +(CTRP1 to CTRP15). This specific protein family exhibits similar structure as APN, consisting of four distinct domains including a N-terminal signal peptide, a short variable domain, a collagen-like domain, and a C-terminal C1q-like globular domain45,46. Of all CTRP paralogs, CTRP9 shows the highest degree of amino acid identity to APN in its globular C1q domain. In the present study, we demonstrated for the first time that CTRP9 has anti-apoptotic effects protecting neurons from ICH-induced SBI. Furthermore, we evaluated the temporal expression of endogenous CTRP9 within 72 h after experimental ICH. The results indicated that CTRP9 expression increased after ICH and reached the highest level at 24 h from ICH induction. + +It is generally believed that neuronal apoptosis is a crucial process leading to secondary neurological damage after ICH47,48. The anti-apoptotic effects of AdipoR1 have been reported in an obese model44. It has been reported that APN +receptor activation attenuates human hepatocytes, HepG2 cells apoptosis via the PI3K/Akt pathway49. Two recent studies suggest that the anti-apoptotic effects of AdipoR1 agonists are mediated via PI3K/Akt signaling pathway50. + +Although anti-apoptotic effects of APN have been previously reported in many disease models51–53, the underlying mechanism remains unclear. PI3 K is a protein tyrosine kinase activated by a cytokine receptor. Activated PI3 K +activates cytoplasmic Akt via phosphorylation, thereby regulating expression of genes involved in cell survival, proliferation, cell-cycle progression, and angiogenesis in cerebral development and disorders54,55. It has been reported that activated PI3K/Akt signaling improves neurological function and attenuates neuronal apoptosis in murine models of subarachnoid hemorrhage (SAH)34. The present study showed that CTRP9 treatment attenuated neuronal apoptosis following ICH. Specifically, CTRP9 treatment upregulated the expression of the targeted proteins on the pathway (AdipoR1, PI3 K, and p-Akt) as well as the anti-apoptotic molecular marker (Bcl-2) and downregulated the expression of the pro-apoptotic molecular marker (Bax). When AdipoR1 was silenced by AdipoR1 siRNA pretreatment, expression of AdipoR1, PI3 K, p-Akt, Bcl-2 was suppressed and expression of Bax was enhanced, suggesting that anti-apoptotic effects of CTRP9 were abolished. Similarly, pretreatment with a PI3 K inhibitor blocked the activation of PI3 K and p-Akt by rCTRP9, leading to decreased expression of antiapoptotic marker (Bcl-2) and increased expression of pro-apoptotic marker (Bax). These findings suggest that activation of PI3K/Akt signaling pathway underlies the antiapoptotic effects of CTRP9. + +There were some limitations in our study. First, the present study did not rule out the possible neuroprotective effects of CTRP9 through preservation of the blood–brain barrier or alleviation of neuroinflammation. Therefore, further studies must be conducted to explore the aforementioned possibilities. Second, the pathophysiology of neuronal apoptosis after ICH is a complex network. Other downstream factors such as mitogen-activated protein kinase (MAPK), adenosine monophosphate-activated protein kinase (AMPK) may be modulated by rCTRP9 administration after ICH, which was not explored in this study. Another limitation is that we have evaluated the total expression of PI3 K rather than its activated phosphorylated form. + +In the current study, we focused on AdipoR1/PI3K/Akt signaling in regards to anti-apoptotic molecular mechanisms. Further studies need to be conducted to explore other potential signaling pathways contributed by rCTRP9 and AdipoR1 activation. Third, in our experiments, we used adult CD1 mice to mimic the progression of ICH; however, ICH tends to occur in older patients with hypertension, vascular disorders, and cerebral amyloid angiopathy56. We did not evaluate the effects of rCTRP9 in different age groups or in animals with systemic co-morbidities. + +## Conclusions + +In summary, we showed for the first time that rCTRP9 administration improved neurological function and reduced neuronal apoptosis through AdipoR1/PI3K/Akt pathway in an experimental ICH model. Therefore, administration of rCTRP9 may be a promising therapeutic strategy in ICH +management. Further studies need to evaluate further mechanisms and applications of rCTRP9 in the CNS. + +## Authors' Contributions + +JHZ, LHZ, and JPT conceived and designed the study. LHZ, SPC, JPT and PS performed the experiments and collected the data. LHZ, PS, WZ, PK, and JHZ contributed to analyzing the data and drafting this article. All authors read and approved the final manuscript. + +## Ethical Approval + +All animal experiments in this study were performed with approval of the Institutional Animal Care and Use Committee (IACUC) at Loma Linda University (IACUC No. 8160049). + +## Statement Of Human And Animal Rights + +All procedures in this study followed the Health's Guide for the Care and Use of Laboratory Animals (National Research Council) and complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for reporting in vivo experiments. + +## Statement Of Informed Consent + +There are no human subjects in this article and informed consent is not applicable. + +## Declaration Of Conflicting Interests + +The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. + +## Funding + +The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study is supported partially by grants from National Institutes of Health (NS091042 and NS082184) to Dr. J. 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Transl Stroke Res. 2019;10(2):231–239. \ No newline at end of file diff --git a/medical/md/PMC6735024.md b/medical/md/PMC6735024.md new file mode 100644 index 0000000000000000000000000000000000000000..b71560360a06fc3fe78d31df90412f3d14ff1dc9 --- /dev/null +++ b/medical/md/PMC6735024.md @@ -0,0 +1,109 @@ +62 + +# The Contribution Of Faith-Based Health Organisations To Public Health Cristiane Schumann, André Stroppa And Alexander Moreira-Almeida + +Research Center in Spirituality and Health (NUPES), School of Medicine, Federal University of Juiz de Fora (UFJF), Brazil, email alex.ma@ufjf.edu.br Religiousness and spirituality are recognised as important factors to consider in both health services research and clinical practice. A large and growing number of studies have examined the relationship between, for example, religiousness and spirituality and physical and mental health, and many of these point to a positive relationship between them. Increased psychological wellbeing, lower prevalence of depression, substance misuse and suicidal ideation, as well as better physical health, are reported in those who are religious when compared with control groups (Koenig *et al*, 2001). + +In spite of the current recognition of the importance of this relationship, its clinical applications have not been well explored. In particular, few studies have analysed the nature of the work carried out by faith-based health organisations, or investigated their public health impact. The purpose of this brief paper therefore is to consider some of the work familiar to the authors in this field. + +## Health Services Provided By Faithbased Organisations + +Since early times, medicine and religion have been closely associated. In ancient Mesopotamia, Egypt, India, China, Greece and the Americas, priests were commonly responsible for healing practices. There was great respect for medicine among the Jews, who were forbidden to live in towns where there was no doctor. During the Middle Ages, medicine was taught in schools maintained by the Church, or in monasteries; and in Europe many hospitals were founded, most of them connected with religious orders (often Catholic or Islamic). They were regarded as charitable guest-houses and instruments of pious benevolence; medicine was seen as evidence of God's love in the face of human suffering. This tradition of caring for sick people through religious hospitals has continued until the present time, in the numerous religious-based charitable hospitals that currently exist across the world (Risse, 1999; Koenig *et al*, 2001). According to the Catholic News Agency (2010), the Catholic Church manages 26% of healthcare provision in the world, through a total of 117000 facilities such as hospitals, clinics and orphanages. + +The European Study of the Epidemiology of Mental Disorders compared European and US data and found that, among those consulting with mental health problems during the previous year, 6.9% of Europeans had seen religious advisers (ranging from 12.2% in Germany to 2.1% in Spain), compared with 18.8% in the USA (Kovess-Masfety *et al*, 2010). + +A study carried out by the World Health Organization (WHO) estimated that between 30 and 70% of the health infrastructure in Africa is currently owned by faithbased organisations. Yet there is often little cooperation between these organisations and mainstream public health programmes. Christian hospitals and health centres, for example, provide about 40% of HIV care and treatment services in Lesotho, and almost a third of the HIV/AIDS treatment facilities in Zambia (Karpf, 2007). + +A report on integrating mental health into primary care by the WHO and the World Organization of Family Doctors (Wonca) (2008) specifically commented on the importance of mental health services provided by faith-based nongovernmental organisations in Australia and in Uganda, where 59% of respondents sought help from religious leaders for depression, compared with 0.6% who consulted a traditional healer and 2.3% who visited a public health facility. + +A study that analysed trends in the healthcare literature on spiritual, pastoral and chaplain care on MEDLINE between 1980 and 2006 found that there was an increase in the rate of published articles in this field, especially on spiritual care, and that this was particularly noticeable in nursing, mental health and general healthcare journals. However, in the last decade there was a decrease in the number of papers published within the field of pastoral care (Harding *et al*, 2008). + +In Brazil, where healthcare facilities provided by religious organisations are common, a census carried out by the Brazilian government in 2002 found that there were 1718 'philanthropic hospitals' in Brazil, mostly run by religious organisations, with 155503 beds, which represented 32% of all Brazilian hospitals. Most of these provided a service for low-income patients and were in small towns (over a half of these were the only hospital in the town) (Crisóstomo, 2002). Their funding was mostly from the Unified Health System (SUS), although some services were financed by private health insurance. In Brazil there is a universal health system (the SUS) funded by the government, which provides basic and complex healthcare. It is commonly a partnership between public and private sectors in terms of service provision. + +Psychiatric hospitals related to spiritism (a French branch of spiritualism common in Brazil that accepts mediumship, believes in reincarnation and 'Jesus ethics', and has a strong emphasis on the practice of charity) are common in Brazil; about 50 were built throughout the country between 1930 and 1970. The majority of philanthropic mental health institutions located in the state of São Paulo (the most populous state in Brazil) are linked to spiritism (Moreira-Almeida & Lotufo Neto, 2005). + +In most of the Catholic hospitals there is a chapel for the celebration of mass; visits are made by a priest for the distribution of the Holy Communion, and there are duty chaplains who respect the beliefs of non-Christian patients, their families and other visitors. Spiritist hospitals also offer counselling to patients, 'passes' (laying on of hands), talks based on ethics and spiritual issues, prayers, and other forms of spiritual healing. + +## Impact On Health + +A systematic literature review of articles published between 1990 and 2000 identified 53 studies evaluating the effectiveness of faith-based health programmes, all of them from the USA. It showed that most were focused on primary prevention (50.9%), general health maintenance (25.5%), cardiovascular health (20.7%) or cancer (18.9%). Results showed reductions in cholesterol and blood pressure, weight and disease symptoms, and increases in the use of mammography and breast self-examination - suggesting that this kind of intervention can improve the health of the population (DeHaven *et al*, 2004). A very clear example of the role of religious organisations in health promotion and prevention is the work of the Children's Pastoral Care (CPC), an organ of the National Conference of Brazilian Bishops (CNBB). The CNBB has been identified as one of the most important organisations around the world working in health, nutrition and education of children under 6 years of age; its work involves families and communities, and the CNBB tracks monthly over 1.5 million children in about 3300 towns all over Brazil. The data from an information system that collects and analyses the indicators of coverage and the impact of the CPC have shown a positive effect of these interventions, when compared with areas not covered by the CPC. In 2006, approximately 260000 volunteers supported the development of 1.8 million children and the care of 95000 pregnant women in more than 42000 communities in Brazil. + +In a study conducted in the city of Criciúma (Brazil) with 2208 children below 3 years of age, it was found that pastoral visits were significantly associated with increased maternal knowledge about appropriate feeding methods during a child's episode of diarrhoea, the contraindications to formula milk, the interpretation of the growth curve and increased knowledge of immunisation schedules. There was also an increase in the duration of breast-feeding and the later introduction of bottle-feeding. In addition, the researchers found an increased number of infant weighings in the quarter that preceded the survey, and in the possession of measuring spoons for oral rehydration (Neumann *et al*, 2003). + +Another study also found that mothers followed by CPC, +when compared with a control group, had better knowledge about the management of diarrhoea and attended more prenatal consultations (at least six); their children were born with higher birthweight and they received iron supplementation more often during pregnancy (Neumann *et al*, 2002, 2003). + +The mortality rate among children less than 1 year old in communities where there is CPC provision is up to 50% lower than in those where the CPC is not present. It has also been observed that there is a reduction in violence and criminality in the areas served by the CPC. In order to disseminate the CPC programme more widely, the International Pastoral Care was established. Other Latin American countries, the Caribbean and Haiti already have branches of this organisation contributing to improvements in healthcare and education. Children's Pastoral Care was nominated for the Nobel Peace Prize 2011 in recognition of the work done by its founder, the paediatrician Dr Zilda Arns Neumann, and the achievements of the CPC (Coordenação Nacional da Pastoral da Criança, 2011). + +## Conclusion + +Given these findings, and despite the overall shortage of studies on this subject, it is clear that faith-based health organisations are relevant to any consideration of public health worldwide, especially in low-income countries. There is limited but consistent evidence that points to their positive effect on health-related outcome measures. However, there is a need to know more about the nature and motivations of these organisations and to assess in greater detail their impact on healthcare provision and on public health (DeHaven *et al*, 2004). + +Although there are some instances of mistrust between religious communities and health services, the data presented above would suggest that it would be very fruitful to foster this partnership for health promotion. Further studies in this vital field should therefore be undertaken, as faithbased healthcare is an important source of health provision worldwide. + +## References + +International Psychiatry **Volume 8** +63 Catholic News Agency (2010) Catholic hospitals comprise one quarter of world's healthcare, council reports. See http://www. catholicnewsagency.com/news/catholic_hospitals_represent_26_ percent_of_worlds_health_facilities_reports_pontifical_council/ (accessed June 2011). + +Coordenação Nacional da Pastoral da Criança (2011) Pastoral da Criança - CNBB. Rede Brasileira de Informação e Documentação sobre Infância e Adolescência. REIBIDIA. See http://www.rebidia.org.br/ index.php?option=com_content&view=article&id=211&Itemid=122 (accessed June 2011). + +Crisóstomo, M. (2002) *Hospitais Filantrópicos no Brasil*. [*Philanthropic* Hospitals in Brazil.] BNDES. See http://www.bndes.gov.br/SiteBNDES/ export/sites/default/bndes_pt/Galerias/Arquivos/conhecimento/bndes_ social/bndes_social5_3.pdf (accessed June 2011). + +DeHaven, M. J., Hunter, I. B., Wilder, L., *et al* (2004) Health programs in faith-based organizations: are they effective? American Journal of Public Health, 94, 1030–1036. + +Harding, S. R., Flannelly, K. J., Galek, K., *et al* (2008) Spiritual care, pastoral care, and chaplains: trends in the health care literature. Journal of Health Care Chaplaincy, 14, 99–117. + +Karpf, T. (2007) Faith-based organizations play a major role in HIV/AIDS +care and treatment in sub-Saharan Africa. WHO Media Centre. See http://www.who.int/mediacentre/news/notes/2007/np05/en (accessed June 2011). + +Koenig, H. G., McCullough, M. & Larson, D. B. (2001) *Handbook of* Religion and Health: A Century of Research Reviewed. Oxford University Press. + +Kovess-Masfety, V., Dezetter, A., de Graaf, R., *et al* (2010) ESEMeD/ +MHEDEA 2000 Investigators. Religious advisors' role in mental health care in the European Study of the Epidemiology of Mental Disorders survey. *Social Psychiatry and Psychiatric Epidemiology*, 45, 989–998. + +Moreira-Almeida, A. & Lotufo Neto, F. (2005) Spiritist views of mental disorders in Brazil. *Transcultural Psychiatry*, 42, 570–579. + +64 Neumann, N. A., Victora, C. G., Valle, N. J., *et al* (2002) Impacto da Pastoral da Criança sobre a nutrição de menores de cinco anos no Maranhão: uma análise multinível. [Impact of the Children's Pastoral Care on the nutrition of five-year-olds in Maranhão: a multilevel analysis.] *Revista Brasileira de Epidemiologia*, 5, 30–40. + +Neumann, N. A., Tanaka, O. U., Victora, C. G., *et al* (2003) Qualidade e eqüidade da atenção ao pré-natal e ao parto em Criciúma, Santa Catarina, Sul do Brasil. [Quality and equity of prenatal and childbirth services in Criciúma, Santa Catarina, South Brazil.] *Revista Brasileira de* Epidemiologia, 6, 307–318. + +Risse, G. B. (1999) *Mending Bodies, Saving Souls: A History of Hospitals*. + +Oxford University Press. + +WHO & Wonca (2008) *Integrating Mental Health into Primary* Care: A Global Perspective. See http://whqlibdoc.who.int/ publications/2008/9789241563680_eng.pdf (accessed June 2011). + +Country profile + +## Mental Health In Argentina Daniel Moldavsky1 Md Pgdippsych (Tel Aviv), Ceri Savage2 **Ma Phd Dclinpsy,** Enrique Stein3 Md Dipph And Andy Blake4 **Md Mph** + +1Specialist in Psychiatry (Argentina), Consultant Psychiatrist, Millbrook Mental Health Unit, Kings Mill Hospital, Sutton-in-Ashfield, UK, +email Daniel.moldavsky@nottshc.nhs.uk; 2Clinical Psychologist (UK) and Visiting Research Fellow, Institute of Cognitive Neurology, Buenos Aires, Argentina; 3Specialist in Psychiatry, Professor of Public Health, Comahue National University, Neuquen, Argentina; 4Specialist in Psychiatry (Argentina), Consultant for the National Parliamentary Commission for Mental Health, Argentina Argentina, the second largest country in South America, is a federation of 23 provinces and its capital, the autonomous city of Buenos Aires. Its population is a little over 40 million, 50% of whom reside in its five largest metropolitan areas. The rural areas are extensively underpopulated. The city of Buenos Aires and its suburbs contain 15.5 million inhabitants, making it one of the largest urban areas in the world. + +Although Argentina belongs to the high- to middleincome countries according to the World Bank, its socioeconomic inequalities are extensive. Sharp contrasts exist between the urban and rural areas. Between 29 and 33% of its population live below the poverty line. + +During the 20th century, the country saw several military coups and administrations in between periods of precarious democratic government. Military regimes, repression of the opposition, hyperinflation and several collapses of the economy took a heavy toll on the mental health field. Importantly, more than one generation of professionals and academics were effectively exiled abroad, damaging the country's scientific system and research capacities. Argentina is currently experiencing its longest unbroken period of democracy in its history, and the most recent signs indicate a trend for academics and professionals to return, in response to improving conditions and government incentives, but the consequences of the earlier turbulent history cannot be ignored. + +Argentina as yet lacks a national system for data collection for mental health. Each province has its own system. Data pertain mainly to the public system, and exclude the substantial private sector, making the information incomplete. + +## Policy And Legislation + +The National Mental Health Authority provides advice to government on policy and mental health legislation. However, it is not involved in planning, monitoring or evaluating the quality of services. These domains are under the independent responsibility of each province. + +In 2010, the National Parliament approved the Law for Mental Health Services, which covered service planning and policies, mental health in primary care, as well as monitoring of compulsory admissions to hospitals. It is not yet clear how this change will affect existing services. + +A survey by the World Health Organization (WHO) on the state of mental health services in Argentina gathered much useful information (some reported below), although it covered only ten provinces. Nine of these have explicit mental health policies drafted. A Federal Plan for Mental Health was drafted in January 2008, but is still vague and not widely supported by all the provincial authorities. + +The budget for mental health is below WHO recommended levels. Allocations vary among the provinces, from 0.5 to 5.0%. Nationally, 68% of the mental health budget goes to psychiatric hospitals, leaving community services underresourced. + +Six provinces have an official list of essential medications. + +However, the lack of clinical guidelines leaves the availability of medical treatment potentially subject to market mechanisms, with pharmaceutical companies exerting pressure for the approval of medication that is not necessarily supported by clinical evidence. + +## Service Delivery + +Health services are delivered by several systems that coexist in a structure that is subject to little formal regulation, being essentially a free market health economy. The federal state bears no responsibility for health cover for the population, which is instead devolved to provincial control. Each province has a public health sector that gives free cover. Its resources are limited. + +The national social security system is financed by contributions from employers and workers. However, people who are unemployed are not covered by this. Private insurance plans, taken out independently by individuals, cover more than 12% of the population, mainly in urban areas. Pensioners and people with a disability receive cover provided by the + \ No newline at end of file diff --git a/medical/md/PMC6754583.md b/medical/md/PMC6754583.md new file mode 100644 index 0000000000000000000000000000000000000000..83e81b16a613a0473bb35287ddeea4987b75509b --- /dev/null +++ b/medical/md/PMC6754583.md @@ -0,0 +1,433 @@ +# Multiplexed Protein Force Spectroscopy Reveals Equilibrium Protein Folding Dynamics And The Low-Force Response Of Von Willebrand Factor + +Achim Löfa,b, Philipp U. Walkera,b, Steffen M. Sedlaka,b, Sophia Grubera,b, Tobias Obserc, Maria A. Brehmc, Martin Benoita,b,1, and Jan Lipferta,b,1 aDepartment of Physics, LMU Munich (Ludwig Maximilian University of Munich), 80799 Munich, Germany; bCenter for NanoScience, LMU Munich, 80799 Munich, Germany; and cDepartment of Pediatric Hematology and Oncology, University Medical Center Hamburg–Eppendorf, 20246 Hamburg, Germany Edited by Taekjip Ha, Johns Hopkins University, Baltimore, MD, and approved August 1, 2019 (received for review February 1, 2019) +Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force regulation, and function. So far, the field has relied primarily on atomic force microscope and optical tweezers assays that, while powerful, are limited in force resolution, throughput, and require feedback for constant force measurements. Here, we present a modular approach based on magnetic tweezers (MT) for highly multiplexed protein force spectroscopy. Our approach uses elastin-like polypeptide linkers for the specific attachment of proteins, requiring only short peptide tags on the protein of interest. The assay extends protein force spectroscopy into the low force (<1 pN) regime and enables parallel and ultra-stable measurements at constant forces. We present unfolding and refolding data for the small, single-domain protein ddFLN4, commonly used as a molecular fingerprint in force spectroscopy, and for the large, multidomain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. For both proteins, our measurements reveal exponential force dependencies of unfolding and refolding rates. We directly resolve the stabilization of the VWF +A2 domain by Ca2+ and discover transitions in the VWF C domain stem at low forces that likely constitute the first steps of VWF's mechano-activation. Probing the force-dependent lifetime of biotin–streptavidin bonds, we find that monovalent streptavidin constructs with specific attachment geometry are significantly more force stable than commercial, multivalent streptavidin. We expect our modular approach to enable multiplexed force-spectroscopy measurements for a wide range of proteins, in particular in the physiologically relevant low-force regime. + +protein folding | force spectroscopy | magnetic tweezers | von Willebrand factor Mechanical forces acting on proteins or ligand–receptor pairs are an integral part of many biological processes. + +Often the physiological functions of proteins are critically regulated by force: Examples include the mechano-activation of enzymes, force-regulated exposure of cryptic binding sites, and forcedependent unfolding and refolding of protein domains as "strain absorbers" to dissipate mechanical stress (1, 2). A well-studied protein "strain absorber" is the 4th domain of the F-actin cross-linking filamin rod of Dictyostelium discoideum (ddFLN4), which exhibits extraordinarily fast refolding, facilitated by an intermediate state along the refolding pathway (3, 4). Another remarkable example of physiological force regulation occurs in the large, multimeric glycoprotein von Willebrand factor (VWF) in the vasculature. VWF's hemostatic function is regulated by increased hydrodynamic forces occurring upon blood vessel injury. Activation of VWF relies on a complex interplay of force-induced conformational changes, both of single domains and of the large-scale protein conformation (5–7), +while down-regulation of VWF is based on mechano-enzymatic cleavage at a cryptic binding site only accessible upon unfolding of VWF's A2 domain (8). While many of the individual transitions in VWF have been probed in detail, their interplay, and thus our understanding of how full-length VWF reacts to external forces in the bloodstream, remains incomplete. Since + +![0_image_0.png](0_image_0.png) + +hydrodynamic peak forces grow as the square of the contour length (5, 8), transitions that release contour length at low forces are expected to be particularly relevant for VWF's physiological function, as they will initiate a cascade of increasing forces that trigger additional transitions with further contour length release. Recent work using atomic force microscopy +(AFM) imaging has suggested large-scale transitions in the VWF C-domain stem that, however, could not be detected in AFM-based force spectroscopy, due to its limited force resolution (9, 10). + +Most insights into the mechanical properties and regulation of proteins and their complexes at the single-molecule level have been obtained from force-spectroscopy experiments using atomic force microscopes or optical tweezers (OT). While AFM and OT force-spectroscopy measurements have provided unprecedented insights, they also have important shortcomings (11). AFM measurements cannot resolve forces below ∼10 pN; OT provide excellent spatiotemporal resolution even for forces down to ∼1 pN, +but are not capable of measuring many molecules in parallel (11). + +In addition, both AFM and OT intrinsically control position and not force, such that constant-force measurements require active Significance The physiological function of proteins is often critically regulated by mechanical forces acting on them. Single-molecule manipulation techniques such as atomic force microscopy or optical tweezers have enabled unprecedented insights into the molecular mechanisms underlying such force regulation. Current limitations include the resolution at low forces and low throughput. + +We here introduce a versatile, modular approach for force measurements on proteins in magnetic tweezers that allows for probing dozens of single molecules in parallel, in a wide force range including very low forces <1 pN. We demonstrate the utility of this assay by elucidating regulatory low-force transitions within von Willebrand factor, a vascular protein that is activated for its critical role in hemostasis by hydrodynamic forces in the bloodstream. + +Author contributions: A.L., P.U.W., M.A.B., M.B., and J.L. designed research; A.L., P.U.W., +S.M.S., S.G., and T.O. performed research; S.M.S. and T.O. contributed new reagents/ analytic tools; A.L., P.U.W., S.M.S., S.G., and J.L. analyzed data; and A.L., P.U.W., S.G., M.A.B., M.B., and J.L. wrote the paper. The authors declare no conflict of interest. + +This article is a PNAS Direct Submission. + +This open access article is distributed under Creative Commons Attribution-NonCommercialNoDerivatives License 4.0 (CC BY-NC-ND). + +1To whom correspondence may be addressed. Email: Martin.Benoit@physik.unimuenchen.de or Jan.Lipfert@lmu.de. + +This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + +1073/pnas.1901794116/-/DCSupplemental. + +Published online August 28, 2019. + +feedback (12). A passive force-clamp mode of operation for OT +has been demonstrated, but is limited to a narrow range of molecular extensions (13). + +Magnetic tweezers (MT) are a single-molecule force-spectroscopy technique that can overcome these shortcomings. In MT, molecules of interest are tethered between a surface and superparamagnetic beads (11, 14, 15) (SI Appendix, Fig. S1). External magnetic fields exert precisely controlled forces (16) in the range of ∼0.01–100 pN, and previous work has demonstrated camerabased tracking for tens to hundreds of nucleic acid-tethered beads simultaneously (17–19) in (x, y, z) with ∼1-nm spatial resolution and, recently, also up to ≤1-ms temporal resolution (20–22). MT +naturally operate in constant force mode, i.e., the applied force is constant during the measurement (to within 0.01%; SI Appendix, Fig. S2), as long as the external magnetic field is not actively changed, with excellent sensitivity in particular at low forces. In addition, MT enable long-term, stable, and robust measurements, as they do not suffer from heating or photo-damage and as instrument drift can be compensated by using nonmagnetic beads immobilized to the surface as references (11, 21, 23, 24). + +Despite these advantages, MT so far have mainly been employed to investigate nucleic acid tethers. A key challenge in applying MT to protein force spectroscopy remains to tether approximately nanometer-sized proteins between much larger, +∼1-μm-sized beads and the surface, while avoiding unspecific surface interactions and ideally with a large number of usable tethers in each field of view. Previous MT studies on proteins, therefore, mostly employed large protein constructs, often engineered as polyproteins with repeats of, e.g., titin Ig or protein L domains (23, 25–29). Both covalent and noncovalent strategies have been utilized for attaching proteins to the surface in MT. Noncovalent approaches are either based on antibodies +(30–34) or His-tag Cu2+-NTA chemistry (35, 36), but have the disadvantage of limited force stability compared to covalent attachment. High force stability is especially desirable for measurements over extended periods of time—for instance, in order to study protein folding and unfolding dynamics at equilibrium. + +The most common covalent immobilization strategy, based on linking Halo-tagged proteins directly to a surface coated with Halo-tag amine ligands (23, 25, 26, 28, 29, 37), has the problem that unfolding of the tag can contribute undesired signals in addition to specific signals of the protein of interest (25). Recent studies have circumvented this by replacing the Halo-tag system with the Spy-tag–SpyCatcher system (27, 38). Still, as the protein is immobilized onto the surface without a linker, this approach may be prone to unspecific interactions or bias (re)folding (39). Recently, fusion proteins—including, e.g., titin I27 domains flanking the protein of interest or a short DNA tether attached to the protein of interest—have been used as linkers to increase the distance between magnetic bead and surface (27). The use of large fusion proteins not only requires major protein modification, but also again has the disadvantage that the spacer domains potentially complicate the analysis due to their unfolding and refolding transitions. Similarly, double-stranded DNA undergoes an overstretching transition around 65 pN (40), giving a large extension signal in this force range. In summary, currently employed approaches suffer from a low number of usable tethers, as so far there are no reports of multiplexed protein unfolding and refolding measurements in MT. + +Here, we present a versatile, modular protein attachment strategy for single-molecule MT force spectroscopy. Our tethering protocol uses an elastin-like polypeptide (ELP) linker (41) that ensures efficient attachment to the surface while minimizing unspecific interactions, both critical prerequisites for high-throughput parallel measurements. In our approach, the protein of interest requires only short [1 and 11 amino acids (aa)] peptide tags for coupling to the linker and bead, respectively, avoiding the need for large fusion proteins and providing a general attachment protocol that is independent of protein size. We demonstrate the versatility of our attachment strategy by applying it to a small, single-domain protein, ddFLN4 (100 aa), and a very large, multidomain protein, dimeric full-length VWF (∼4,000 aa). For both proteins, we achieve a high yield of specific tethers—that is, a large number of singlemolecule tethers that exhibit characteristic unfolding and refolding signatures and can be measured in parallel in a single field of view. + +Our highly parallel, ultra-stable measurements of repeated unfolding and refolding resolve outstanding questions about the respective folding pathways and stabilities and reveal how forces in the range of 1 pN are sufficient to open up the VWF C-domain stem. In addition, we leverage the ability of our assay to apply constant forces over extended periods of time to many molecules in parallel to probe the stability of the biotin–streptavidin receptor–ligand system. We anticipate our tethering strategy to be applicable to a wide range of proteins and, furthermore, expect it to be of immediate use for other parallel force-spectroscopy techniques, such as single-molecule centrifugation (42, 43) or acoustic force spectroscopy (44), extending their capabilities toward protein force spectroscopy. + +## Results Site-Specific And Efficient Tethering Of Proteins With Elp Linkers. Our + +attachment strategy uses an unstructured ELP linker (41, 45) with a contour length of ∼120 nm (∼300 aa) and functional groups at its termini that we utilize as spacers for immobilizing the protein of interest to the bottom glass slide of the flow cell and to reduce unspecific protein–surface (41) and bead–surface interactions (Fig. 1A). + +We chose the ELP peptide linkers over polyethylene glycol +(PEG) linkers, which are widely used in AFM-based force spectroscopy (9, 46–48), and over DNA linkers, as typically used for OT measurements (49–51) and in a recent MT study (27), for several reasons. During systematic tests with PEG-coated surfaces, unspecific binding of beads remained a major problem, even in the absence of the protein of interest (SI Appendix, Fig. + +S3), severely limiting the number of useable tethers per field of view. In contrast, we found the ELP linkers to provide excellent passivation (SI Appendix, Fig. S3), in line with AFM-based measurements comparing PEG- and ELP-based linkers (41). Disadvantages of DNA linkers include 1) the fact that covalent DNA– +protein coupling schemes typically provide only low yields (52, 53); +2) the DNA overstretching transition around 65 pN that severely complicates measurements in this force range, in contrast to ELP +linkers (compare SI Appendix, Figs. S2B and S4); and 3) the observation that DNA interacts with many proteins; in particular, VWF has been reported to directly interact with DNA via its A1 domain (54). Compared to peptide tethers, DNA linkers—or even engineered DNA bundles (55)—provide a lower level of intrinsic Brownian fluctuations at high forces due to their stiffness. + +However, at forces <1 pN, peptide linkers have a significantly lower level of intrinsic extension fluctuations (SI Appendix, Fig. S5). + +In our attachment scheme, the ELP linker is attached to a glass slide functionalized with thiol-reactive maleimide groups via an Nterminal cysteine (see Materials and Methods for details of the coupling protocol). The ELP linker carries a C-terminal LPETGG +motif that allows for site-specific and covalent ligation to the protein of interest via an N-terminal glycine residue in a reaction catalyzed (56) by the enzyme sortase A. For coupling to the bead, the protein of interest is further engineered to carry an 11-aa ybbRtag (57) at its C terminus that is covalently attached to CoA–biotin in the sfp phosphopantetheinyl transferase reaction. Finally, the biotin label forms a high-affinity noncovalent bond to streptavidinfunctionalized beads. Our approach requires only short peptide tags on the protein of interest that can readily be introduced by standard molecular cloning methods and have been shown to be compatible with expression and folding of a large range of proteins +(46, 56–59). The coupling can also be inverted, by coupling a protein with an N-terminal ybbR-tag and a C-terminal LPETGG +COMPUTATIONAL BIOLOGY +BIOPHYSICS AND + +![2_image_0.png](2_image_0.png) + +sequence to an ELP linker with an N-terminal glycine and a Cterminal cysteine (SI Appendix, Fig. S6). + +Here, we apply our tethering protocol to 2 very different protein systems: the small ddFLN4 domain and large, full-length dimeric VWF. We obtained comparable and efficient tethering of beads with a large number of specific, single-molecule tethers in both cases. Typically, in a single field of view (∼440 × 330 μm2) +of our MT setup (Fig. 1B; see Materials and Methods and SI +Appendix, Figs. S1 and S2 for details on the setup), 50–100 tethered beads are tracked in parallel, of which 30–50 tethers exhibit characteristic unfolding and refolding signatures (Fig. + +1C). The beads that do not show characteristic signatures are likely anchored to the surface by multiple protein tethers, since in control measurements without the protein of interest added, there is essentially no unspecific binding of beads to the surface (0–1 beads per field of view; SI Appendix, Fig. S3). The fraction of specific tethers attached via a single protein can be increased by decreasing the density of proteins immobilized on the surface. However, decreasing the protein concentration will also result in a decrease of the number of single-tethered beads. Optimizing our conditions, we achieved fractions of up to ∼60% specific, singleprotein tethers, while still obtaining a large number of tethered beads at the same time. The most efficient flow cell exhibited 50 specific out of 85 beads within the single field of view measured. Three-State Folding and Unfolding of ddFLN4. We first applied our tethering protocol to the Ig-fold ddFLN4 domain (Fig. 2A), +which exhibits a characteristic 3-state unfolding pattern that has been extensively studied in AFM experiments (3, 4) and is routinely employed as a molecular fingerprint in AFM forcespectroscopy experiments (47, 60, 61). To characterize unfolding and (re)folding in our MT assay, we recorded time traces of tether extension under different, constant forces. In a typical measurement (Fig. 2B), the force was increased from an initial low value (0.5 pN) that allows for (re)folding, to a high value +(25 pN in Fig. 2B) that promotes unfolding, and subsequently decreased to a moderate value (6.5 and 7.5 pN in Fig. 2B) to directly monitor refolding. Subsequently, this cycle was repeated multiple times with variable force levels to collect statistics. + +Unfolding and refolding of ddFLN4 were observed as clear double steps in the traces—i.e., as an increase or decrease of the tether extension in 2 separate steps that we interpret as transition between the native (N) and intermediate (I) and between the intermediate and unfolded (U) states, respectively (Fig. 2 B, +Insets). We analyzed the changes in extension for the transitions N ↔ I and I ↔ U as well as for the full transition N ↔ U for many different clamped forces (Fig. 2C). The resulting force– +extension profiles are well described by fits of the worm-like chain (WLC) model with a fixed persistence length of 0.5 nm, yielding contour length values (mean ± SD) of 15.0 ± 0.1, 18.3 ± 0.1, and 31.9 ± 0.2 nm, in excellent agreement with values reported from AFM (3, 4). + +Our data are fully consistent with previous work that found unfolding of the ddFLN4 domain to proceed via a mandatory, short-lived intermediate state: In a first unfolding step, strands A +and B (42 aa; blue in Fig. 2A) detach and unfold, with strands C–G +(58 aa; red in Fig. 2A) forming a less stable intermediate state +(3), which quickly unfolds in the second unfolding step. Folding of ddFLN4 was also suggested to proceed via an intermediate state that is most likely structurally identical or very similar to the intermediate populated during unfolding (4). In our dataset, unfolding (data points > 8 pN) and refolding (data points ≤ +8 pN) are well described by a single WLC curve, confirming that the intermediate states populated during unfolding and folding are structurally very similar or identical. Importantly, no other features except the double steps originating from ddFLN4 were observed in the force range probed (SI Appendix, Fig. S7), +showing that the other components of our tethering strategy do not interfere with the measurements. + +Our force-clamp measurements allowed us to directly determine the rates of all transitions (Materials and Methods and SI Appendix, Fig. S8). For unfolding (Fig. 2D), we observed the rate for the first transition, N → I, to increase with increasing force from ∼0.001 s−1 at 9 pN to ∼0.2 s−1 at 31 pN. We fitted the rates to a single-barrier kinetic model, in which the rate is given by k(F) = k0 exp(F·Δx/kBT), +where F is the applied force, k0 the rate at zero force, Δx the distance to the transition state, kB the Boltzmann constant, and T +the absolute temperature (62). We find k0,N → I = 1.5 ×10−4 s +−1 +(1.3–1.8 × 10−4 s +−1) and ΔxN → I = 0.99 ± 0.03 nm (mean ± SD for all rates and Δx values). The measured rates for full unfolding N → U +are essentially identical to those for the transition N → I (Fig. 2D), +owing to the fact that the rates for the second transition, I → U +(k0,I → U = 0.25 s−1 [0.19–0.32 s−1]; ΔxI → U = 0.32 ± 0.04 nm), are at least 1 order of magnitude faster (Fig. 2D), implying that the transition N → I is the rate-limiting step for unfolding. + +For refolding in the force range from 2 to 8 pN (Fig. 2E), the rates for the first substep U → I (k0,U −> I = 0.52 s−1 [0.34–0.79 s−1]; +ΔxU −> I = 3.6 ± 0.3 nm) are again essentially identical to the rates for full refolding U → N (Fig. 2E), and rates for the second transition I → N (1,500 s−1 [950–2,500 s−1]; ΔxI −> N of 3.5 ± +0.3 nm) are at least 3 orders of magnitude higher compared to the first transition, implying that again the first transition, U → I, +is rate-limiting (Fig. 2F). For forces below 6 pN, the intermediate + +![3_image_0.png](3_image_0.png) + +state was too short-lived to be reliably detected, so that rates were determined separately only for forces ≥6 pN. + +We compared our force clamp results to the rates at zero force reported from fits of a single barrier kinetic model to constant pulling speed AFM measurements (4). For unfolding, the rates for the second step k0,I −> U are in excellent agreement (0.25 and 0.33 s−1 in MT and AFM, respectively), yet the zero force rates for the first step k0,N −> I appear, in contrast, to differ significantly +(1.5 × 10−4 vs. 0.28 s−1). However, in AFM measurements with low pulling speeds of 1 nm/s, a markedly higher mechanical strength of ddFLN4 has been observed and explained by switching to a second unfolding pathway (63). The reported zero-force rate +(for full unfolding) from AFM of 1.4 × 10−4 s +−1 agrees with our results. Thus, our data support the existence of a second unfolding pathway and suggest that differences between the 2 pathways can be largely attributed to the first unfolding step N → I. + +For refolding, a direct comparison is less straightforward, as refolding in AFM experiments has been measured at zero force and not under load (4). The rates obtained from MT and AFM +differ significantly (k0,U −> I: 0.52 vs. 55 s−1; k0,I −> N: 1,500 vs. + +179 s−1), which might indicate different folding pathways in the presence and absence of force. Intriguingly, however, in both cases, the same intermediate state appears to be populated during folding. Whereas the first step of folding—and thereby also full folding—is markedly slowed down by force, the second step of folding is almost 10-fold sped up, suggesting a prealignment of the structured portion of the intermediate state by force that allows for faster folding of strands A and B in the second folding step. Since ddFLN4 in vivo is positioned within actin-cross-linking + +![4_image_0.png](4_image_0.png) + +filamin and under tensile load, it appears plausible that a forceinduced prealignment of the intermediate state might play a physiological role. + +Ultra-Stable Equilibrium Measurements of ddFLN4 Unfolding and Refolding. By determining the force for which the fitted rates for full unfolding and refolding (Fig. 2 D and E; black lines) +intersect, we predicted the equilibrium force at which the probabilities of ddFLN4 being in the unfolded and folded states are equal to ∼7.3 pN (Fig. 3A). We tested this prediction by measuring at constant forces close to the predicted equilibrium force. Since the predicted rates at equilibrium are only ∼3 h−1 +(Fig. 3A), we performed very long measurements (55 h in Fig. 3B +and 144 h in SI Appendix, Fig. S9), harnessing the excellent force and drift stability of MT. We observed repeated transitions between the unfolded and folded states, with the system spending approximately half of the time in each of the 2 states (Fig. 3B), as expected for a measurement close to equilibrium. Examining the traces close to equilibrium in detail, we observed repeated transitions not only N ↔ U via the I state (Fig. 3C, left and center traces), but also from the U and N states into the I state that return to the initial state (Fig. 3C, right trace, and SI Appendix, Fig. S9, Inset), again strongly suggesting that the same intermediate state is populated during unfolding and folding. Finally, we note that even for the very long measurements reported here, no significant change of ddFLN4's force response over time was observed, indicating reliable, correct refolding of the domain without any hysteresis effects, both for the long equilibrium measurement at constant force and for repeated cycles of unfolding and refolding under varying load (SI Appendix, Fig. S10). The long-term stability combined with its very characteristic 3-state unfolding signature make ddFLN4 an ideal fingerprint for the identification of single-molecule tethers. + +Lifetime of Biotin–Streptavidin Interactions for Multivalent and Monovalent Streptavidin. Having established ddFLN4 as an ideal fingerprint and having demonstrated the ability to apply constant forces over extended periods of time to multiple tethers in parallel, we utilized our assay to investigate not only protein folding and refolding, but also ligand–protein receptor interactions. As a proof-of-concept measurement, and to validate our tethering approach, we directly probed the stability of the high-affinity, noncovalent biotin–streptavidin interaction under constant force. + +Since all other linkages in our tethering protocol consisted of mechanically stable covalent bonds, we used ddFLN4-tethered beads to apply different high forces (45–65 pN) to the biotin– +streptavidin bond and monitored the time until bead rupture. In the analysis, we only took into account beads that showed the specific ddFLN4 unfolding signature in 2 short force plateaus of 25 pN at the beginning of the measurement. Importantly, the number of beads that ruptured already during these initial short plateaus was small (<3.5%). For commercially available streptavidincoated beads (Dynabeads M-270 Streptavidin; Invitrogen), we found the survival fraction to decay with time in a complex, multiexponential fashion (Fig. 4A) for all forces probed, suggesting the existence of several populations of the biotin–streptavidin interaction with different lifetimes. To quantify the lifetimes involved, we fit the fastest and slowest decaying populations by linear regression to the logarithm of the first and last 20% of data points, respectively (lines in Fig. 4A). Over the studied force range, the lifetime of the fastest decaying population ranged from ∼100 s at 65 pN to ∼2,100 s at 45 pN, whereas the lifetime of the slowest decaying population was +∼50-fold higher, increasing from ∼5,000 s at 65 pN to ∼68,000 s at 45 pN (Fig. 4C). For both populations, the lifetime was found to increase exponentially with decreasing force (Fig. 4C). Already for a force of 20 pN, extrapolated lifetimes were well above a day, and offrates at zero force were in the range of 10−7 to 10−8 s +−1, consistent with the fact that beads remained stably bound for hours or days in our force-spectroscopy measurements at forces ≤20 pN (Figs. 2, 3, and 5). + +We hypothesized that the different populations and multiexponential lifetimes for commercially available streptavidinfunctionalized beads originate from the biotin–streptavidin complex being loaded with force in different geometries that result from the tetravalency of streptavidin (61). Indeed, for measurements with custom-made beads functionalized with a monovalent version of streptavidin (61) in a well-defined geometry using a C-terminal tag (48), the survival fraction was well described by a single-exponential decay (Fig. 4B). We chose immobilization of the monovalent streptavidin construct via the C terminus of its functional subunit, as it has been recently demonstrated in AFM force-spectroscopy measurements that + +![5_image_0.png](5_image_0.png) + +the monovalent streptavidin–biotin complex can withstand markedly higher forces when loaded with force from the C terminus as compared to pulling from the N terminus (48). Indeed, we found the lifetime of the custom-made monovalent streptavidin beads to be 24,000 s (∼6.7 h) at 65 pN (Fig. 4B), similar to and even exceeding that of the 20% longest-lived commercially available beads (Fig. 4C). + +Force-Clamp Measurements on Full-Length VWF Dimers. Having demonstrated our attachment approach on a small, wellcharacterized protein, we next applied it to large (∼500 kDa) +dimeric constructs of full-length VWF. Dimers, the smallest + +![5_image_1.png](5_image_1.png) repeating subunits of VWF multimers, consist of 2 multidomain monomers that are C-terminally linked via disulfide bonds and have a contour length of ∼130 nm between their N termini +(5, 64) (Fig. 5A). Since different peptide tags at the 2 N termini are required for attaching dimers in the desired pulling geometry +(Fig. 5A), we genetically engineered heterodimers consisting of 2 different monomers that are N-terminally modified with a ybbR-tag or a sortase motif GG, respectively (Materials and Methods). After tethering in the MT, we recorded time traces of + +Fig. 5. Folding and unfolding of A2 domains within VWF dimers. (A) Schematic structure of a VWF dimer, consisting of 2 ∼65-nm, multidomain monomers Cterminally connected by disulfide bonds. The 2 A2 domains, which can unfold under force (A, Inset), are shown in blue. Arrows indicate the direction of force acting on the 2 N termini during MT experiments. (B) Segments from a ∼30-h-long extension vs. + +time trace of a VWF dimer tether subjected to alternating intervals of high force (here 12 pN), +allowing for A2 unfolding; of intermediate force +(here 3, 4, or 5 pN), allowing for direct observation of A2 refolding; and of low force (0.5 pN) to ensure refolding. Unfolding and refolding of the 2 A2 domains are observed as 2 independent positive or negative steps in the trace, respectively. Dashed lines in B, Insets indicate extension levels with none, one, or both of the A2 domains unfolded. (C) Force–extension curves of A2 (un)folding, in near-physiologic buffer containing Ca2+ (black) and in buffer without Ca2+ and with 10 mM EDTA (red). Data points are obtained by Gaussian fits to step extension histograms (C, Lower Right Inset) at each constant force. Data points above 5 pN are from unfolding (based on 62–632 and 40–747 events for the near-physiologic and EDTA case, respectively, obtained from 13–53 independent tethers), and data points up to 5 pN are from refolding (41–120 and 49– +158 events for the near-physiologic and EDTA case, respectively, obtained from 10–19 independent tethers). Error bars correspond to the FWHM of Gaussian fits, divided by the square root of counts. Lines are fits of the WLC model. C, Upper Left Inset shows a global WLC fit to all data points. (D) Rates of unfolding (circles) and refolding (triangles) at different constant forces for nearphysiologic (black) and EDTA (red) buffer. Error bars correspond to 95% CIs of exponential fits to the fraction of observed events as a function of time (Materials and Methods). Lines are fits of a single-barrier kinetic model, yielding rates at zero force k0 and distances to the transition state Δx for unfolding and refolding as indicated. +BIOPHYSICS AND +VWF dimers with alternating plateaus of high force (Fig. 5B, 6– +20 pN) and moderate force (Fig. 5B, 2–5 pN). In most cases, we observed 2 unfolding and 2 refolding steps in the high and moderate force traces, respectively, with extension values matching the expected values for unfolding of the A2 domains (∼180 aa each) +that have been previously probed in isolation in OT (8, 52). Observation of domain (un)folding only for the 2 A2 domains is consistent with the prediction that all domains of VWF except A2 are protected against unfolding by long-range disulfide bonds +(64, 65) and with the results of recent AFM studies (9, 10). + +In addition to the steps attributed to A2 unfolding and refolding, we less frequently observed larger steps (SI Appendix, Fig. + +S11; 70–80 nm at ∼11 pN), which we attributed to the dissociation of a strong intermonomer interaction mediated by the D4 domains that has recently been identified in AFM force measurements in approximately one-half of all VWF dimers under near-physiologic conditions (9, 10). Consistent with their assignment to the D4mediated intermonomer interaction, the large unfolding steps occurred much less frequently in the absence of divalent ions, which have been shown to be critical for the intermonomer interaction (9, 10), and were absent for mutant constructs lacking the D4 domain (delD4; SI Appendix, Figs. S11 and S12). The dissociation of the intermonomer interaction was in some casesafter intermittent relaxation to a low force—observed repeatedly for the same molecule, implying reversibility of the interaction (SI +Appendix, Fig. S11). Whereas in the constant pulling speed AFM +measurements dissociation of this interaction had always occurred at much higher forces than—and therefore after—A2 unfolding, in our constant force measurements, we observed dissociation of the D4-mediated intermonomer interaction in the same force range as A2 unfolding, suggesting a steep force-loading rate dependence for the intermonomer interaction. In fact, in the constant-force measurements, we repeatedly observed dissociation of this interaction, even before unfolding of one or both of the A2 domains (SI Appendix, Fig. S11). Opening of the D4-mediated intermonomer interaction in the same force range as A2 unfolding suggests that it might play an important role for regulation of VWF's hemostatic activity at physiologically relevant forces in the bloodstream. + +Calcium Binding Stabilizes the VWF A2 Domain. We next used our assay to elucidate the controversial impact of calcium on A2 stability (52, 66, 67). We performed measurements both in buffer mimicking the physiological pH and salt concentrations of the vasculature ("near-physiologic"; pH 7.4, 150 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2) and in buffer lacking divalent ions and supplemented with 10 mM EDTA. First, we analyzed the change in extension upon A2 unfolding and refolding for different constant forces. For both buffer conditions, the resulting force–extension profiles (Fig. 5C), combining data from unfolding (data points > 6.5 pN) and from refolding (data points < 5 pN), are well described by a single WLC curve. The WLC fits yielded values for contour length and persistence length of 75.0 and 0.42 nm (95% CI: 70.8–79.2 and 0.37– +0.46 nm) for near-physiologic buffer, and of 68.5 and 0.50 nm +(62.7–74.3 and 0.41–0.58 nm) for the EDTA buffer, and thus showed no significant difference, indicating that calcium has no effect on the extension of the unfolded state. A WLC fit to the combined data from both buffer conditions (Fig. 5 C, Inset) +yielded contour and persistence length values of 71.9 and 0.45 nm (68.3–75.4 and 0.41–0.50 nm). The contour length increments determined from the MT measurements on full-length dimeric VWF are in excellent agreement with OT unfolding studies on isolated A2 domains (8, 52, 66), suggesting that complete A2 unfolding is not obstructed by the presence of other domains. Control measurements using the same attachment protocol and ddFLN4 under the same buffer conditions found no difference in the force response for the different buffer conditions (SI Appendix, Fig. S13). + +Next, we studied the kinetics of A2 unfolding and refolding. In the case of unfolding, rates were approximately 2- to 4-fold higher for the EDTA buffer in the force range probed, 6.5–17 pN +(Fig. 5D, circles). For both buffer conditions, rates increased exponentially with increasing force, with a slightly stronger dependence on force for the EDTA condition. Fitting a singlebarrier kinetic model yielded values for the unfolding rate at zero force kunf,0 = 3.6 × 10−5 s +−1 (1.8–7.1 × 10−5 s +−1) and 7.8 ×10−5 s +−1 (5.1–12 ×10−5 s +−1) and distances to the transition state Δxunf = 2.45 ± 0.22 and 2.60 ± 0.15 nm in the presence and absence of Ca2+, respectively. The rates measured in our constant force assay were 2 orders of magnitude slower than the rates determined in near-physiologic buffer in OT measurements on isolated A2 domains. While in principle, this difference might indicate stabilization of A2 by neighboring domains, we deem it likely that it at least partially results from the transformation of rupture force distributions measured in the OT by using the Dudko–Hummer–Szabo method (68), which is sensitive to the elastic response of employed flexible linkers. In our traces, we did not observe any features that corresponded to dissociation of potential interactions of A2 with neighboring domains. For refolding against external forces of 2–5 pN, rates were approximately 2- to 6-fold higher in the presence of Ca2+ (Fig. 5D, triangles) and decreased exponentially with force, with a more pronounced force dependence in the presence of Ca2+, which was reflected by the higher value of Δx of 6.80 ± 0.56 nm compared to 4.73 ± 0.26 nm in the absence of Ca2+. The refolding rate at zero force in the presence of Ca2+ kref,0 = 5.1 s−1 +(2.9–8.7 s−1) was 20-fold higher than in the absence of Ca2+, +kref,0 = 0.23 s−1 (0.18–0.28 s−1), indicating that calcium substantially speeds up folding of A2. + +Taken together, our results demonstrate that A2 is stabilized by the presence of Ca2+ by increasing the refolding rate and stabilizing against unfolding compared to the conditions without Ca2+. The observed increases in the refolding rates in our experiments are in quantitative agreement with a previous report using OT on isolated A2 domains (52). Importantly, we directly observed refolding under mechanical load, even in the absence of Ca2+ (SI Appendix, Fig. S14), in contrast to a previous study (66). The role of Ca2+ in the stabilization against unfolding is controversial: We observed a modest reduction in the unfolding rate by Ca2+, which is consistent with the low-force data found in a previous OT study (52), which, however, reported no statistically significant change in the unfolding rate with and without Ca2+ overall, possibly as their assay might have lacked the sensitivity to resolve small differences. In contrast, we found no evidence for a long-lived intermediate in the unfolding pathway in the presence of Ca2+ that was reported by another study using OT (66). Finally, we occasionally observed tethers that only showed the unfolding and refolding signal of one A2 domain (SI Appendix, Fig. S15). In such tethers, refolding of one A2 domain may be inhibited due to cis–trans isomerization of a cis-proline, as reported in a previous OT study (8). + +Transitions in the VWF Stem at Low Forces. AFM imaging (9, 10) +and electron microscopy (69) suggest that the VWF stem consisting of 6 C domains can open and close in a zipper-like fashion +(Fig. 6 A and B). However, transitions of the VWF stem have not been observed directly. To probe for interactions in the VWF stem, we subjected dimeric VWF tethers that had shown the characteristic A2 unfolding pattern to different levels of low forces. At forces of ∼1 pN, we observed repeated, reversible transitions with a maximum contour length increase of ∼50 nm that were consistent with fully unzipping and rezipping the VWF +stem (Fig. 6C and SI Appendix, Fig. S16). Increasing force in the range 0.5–1.5 pN systematically shifted the population toward higher tether extensions, which we interpreted as less compact +"unzipped" conformations of the VWF stem. Control measurements with ddFLN4-tethered beads using the same linkers, attachment protocols, and buffer conditions never exhibited such transitions (SI Appendix, Fig. S7). In addition, the large change in extension at low forces makes it highly unlikely that the observed transitions originated from domain unfolding events (for comparison, a tether extension of <5 nm would be expected for unfolding of an A2 domain at a force of ∼1 pN, due to the WLC +stretching behavior of the unfolded protein chain). + +To characterize the overall force response of the VWF stem, we carried out a simple 2-state analysis and determined the fraction of data points above and below the midpoint between the 2 extreme values of extension (Fig. 6C, dashed lines). The fraction in the high-extension "up" state increased systematically with increasing force, and the data are well described by a simple 2-state model (Fig. 6D) with a midpoint force of F1/2 = 1.00 ± +0.24 pN (mean ± SD from 21 molecules; Fig. 6E). The fitted F1/2 is very close to the value for the onset of VWF multimer elongation predicted by Brownian hydrodynamics simulations (70). + +While the 2-state analysis enables direct determination of the characteristic forces required to open the stem, a close inspection of the extension time traces (Fig. 6C) suggests that the stem does not behave as a strict 2-state system, but that, instead, the extension takes a range of values with fast transitions between different levels of extension. This multistate behavior with different levels of stem zipping is fully consistent with observations from AFM imaging that show that the stem frequently populates partially open configurations with different numbers of C-domains interacting (Fig. 6B and refs. 9 and 10). In order to quantify the rapid transitions in the VWF stem, we analyzed extension time traces close to F1/2 with a step-finding algorithm developed for detecting transitions in single-molecule time traces (71) (Fig. 7A). + +The analysis revealed a broad distribution of step sizes with a maximum around 15 nm (Fig. 7B and SI Appendix, Fig. S17 A and B). + +Interestingly, steps >40 nm were rare (2.6% of all steps); similarly, small steps were also relatively rare (e.g., 5.8% of steps are <7 nm). Analysis of AFM images has shown that the unzipped VWF stem shows WLC behavior with a contour length of 70 nm and a bending persistence length of 6.4 nm (9), which implies a total extension change upon unzipping of ∼42 nm at 1 pN, in good agreement with the overall extension changes observed in the time traces (Fig. 6C). The fact that steps >40 nm were only rarely observed suggests that direct transitions between the fully open and completely closed conformations are rare and that most transitions pass through partially zipped intermediate configurations. We note that the most common step size of ∼14 nm was + +![7_image_0.png](7_image_0.png) + +Schematic of closing and opening of the stem (domains C1–C6, yellow) of VWF dimers in a zipper-like fashion due to interactions between the C domains. (B) AFM images of single VWF dimers adsorbed onto a surface under near-physiologic buffer conditions. Arrowheads indicate C-terminal ends of dimers, where the 2 constituting monomers are linked. In thermal equilibrium and in the absence of force, dimer stems exhibit conformations ranging from fully open to fully closed. It should be noted that in approximately onehalf of all dimers, the stem region is firmly closed by the D4-mediated intermonomer interaction (9, 10) (Scale bars: 20 nm; height range of color scale: 2.4 nm.) (C) Extension vs. time traces of VWF dimer constructs subjected to low forces in MT. Fast reversible transitions between a maximum and minimum value of extension, ∼50 nm apart, are observed that we attribute to closing and opening of the C-domain stem. Dashed lines indicate the midpoint between the 2 extreme values of extension. Traces are raw data recorded at 58 Hz. (D) Fraction of data points in the up state, i.e., above the midpoint lines indicated in C as a function of applied force. The black line is a fit of the expression {1 + exp[−Δx·(F − F1/2)/(kBT)]}−1 with F1/2 the midpoint force and Δx a length-scale parameter characterizing the cooperativity of the transition. (E) Histogram of F1/2 values determined from fits as shown in D. The red line is a Gaussian fit with mean ± SD = 1.00 ± 0.24 pN. The fitted values for Δx gave 28.6 ± 15.9 nm (mean ± SD from 21 molecules). + +![7_image_1.png](7_image_1.png) + +consistent with the expected length increase for pairs of C domains on each monomer zipping and unzipping. + +The dwell times between steps were exponentially distributed, with a characteristic time of τ = 0.28 s ± 0.006 s (mean ± 95% +CI; Fig. 7C). The fact that the dwell times were well described by a single exponential suggests that the transitions occur between substates of similar individual lifetimes. To investigate the transitions in the VWF stem at high resolution, we recorded extension time traces of the VWF dimer constructs using highspeed, camera-based tracking at 1 kHz (Fig. 7D). The high-speed data revealed overall extension changes, step sizes, and dwell times that were in good agreement, within experimental error, with the results from tracking at 58 Hz (SI Appendix, Fig. S17 C–F). Importantly, molecular interactions that break and release contour length at low forces are expected to be particularly relevant for VWF's physiological function, as these are likely the first interactions to open under shear flow and to set off a cascade of increased contour length and increased force, since hydrodynamic peak forces grow as the square of the contour length (5, 8). + +## Discussion + +We have introduced an approach for single-molecule forcespectroscopy measurements on proteins using MT. Our protocol enables multiplexed measurements through high-yield, ultra-stable tethering of proteins requiring only short peptide tags. As a proofof-concept measurement, we probed the 3-state unfolding and refolding of ddFLN4. Our measurements at constant force are overall in excellent agreement with constant pulling-speed AFM +experiments and confirm the existence of a low-loading rate pathway for unfolding (4, 63). While for ddFLN4, we observed exponential force dependencies of unfolding and refolding rates, we note that the precise determination of rates at low, constant forces afforded by our assay should allow detection of deviations from such exponential Bell model-type behavior (72) expected for other systems and testing of more complex models, e.g., involving transitions from catch-bond to slip-bond behavior (73, 74), +switching between different protein unfolding or receptor–ligand dissociation pathways (75–77), or taking into account structural– +elastic properties of the investigated molecules (73, 78). + +Applying our method to the large, force-regulated protein VWF +confirmed several structural transitions previously observed in AFM and OT force-spectroscopy measurements, including D4 and A2 unfolding. In addition, our data show how Ca2+ stabilizes the A2 domain by reducing the unfolding and increasing the refolding rate. Finally, our measurements reveal transitions between multiple extension levels in the VWF stem at forces of ∼1 pN, which likely constitute critical first steps in the stretch response of VWF under physiological shear flow. Future measurements under a range of solution conditions, in particular varying pH, and using VWF +mutants should help to further identify the distinct contributions and roles of the different C domains. + +Using the ability of our assay to apply constant forces over long periods of time to multiple tethers in parallel, we probed the stability of streptavidin–biotin bonds, a widely used ligand–receptor system. We found that commercially available, multivalent streptavidin (far) exceeds the requirements of typical constant force force-spectroscopy measurements (with lifetimes ≥1 d at forces ≤20 pN), but has a multiexponential lifetime distribution. + +Monovalent, site-specifically attached streptavidin, in contrast, exhibits a single-exponential lifetime distribution with extremely high force stability, making it an attractive approach for force spectroscopy on systems that require high forces over extended periods of time. Ultimately, one could also replace the biotin– +streptavidin bond with a covalent linkage to even further enhance the force and also chemical stability of the attachment protocol. + +In conclusion, we have demonstrated the versatility and power of single-molecule protein force-spectroscopy measurements using MT. Our method provides a high yield of ultra-stable specific single-molecule tethers that can be probed in parallel at constant forces over extended periods of time. Given ongoing improvements in camera technology, we expect that the number of protein tethers that can be measured in parallel will further increase by at least an order of magnitude in the near future. In addition, we anticipate that our tethering strategy will enable multiplexed protein force spectroscopy using other singlemolecule methods such as acoustic and centrifugal force spectroscopy as well. Since the approach is modular and only requires minimal modifications to the protein of interest, we anticipate it to be applicable to a wide range of proteins. We expect MT force spectroscopy to, in particular, give access to the physiologically relevant low force (<1 pN) regime and to provide a wealth of insights into the mechanics and force regulation of proteins. + +## Materials And Methods + +Experimental Procedures, Data Analysis, and Engineering of Recombinant Proteins. Please refer to the SI Appendix for details of how tagged ddFLN4, hetero-bifunctional VWF dimer constructs, regular and inverted ELP linkers, and monovalent streptavidin were prepared, purified, and coupled. Measurements used a custom-built MT setup described in ref. 79; forces in the MT +were calibrated by analysis of transverse fluctuations using long DNA tethers +(80). The largest source of force uncertainty was the bead-to-bead variation, which we found to be on the order of ≤10% for the beads used in this study (SI +Appendix, Fig. S2), in line with several reports (16, 81, 82). Details of the MT assay, AFM imaging, and data analysis are given in SI Appendix. Buffers. Measurements on ddFLN4 and measurements on VWF dimers under +"near-physiologic" conditions were performed in buffer containing 20 mM +Hepes, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 0.1% Tween-20, at pH +7.4. 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Graph. + +14, 33–38 (1996). \ No newline at end of file diff --git a/medical/md/PMC7393756.md b/medical/md/PMC7393756.md new file mode 100644 index 0000000000000000000000000000000000000000..c28ee1ea830ba658185cf88950e7ee937e87de69 --- /dev/null +++ b/medical/md/PMC7393756.md @@ -0,0 +1,123 @@ + + +![0_image_0.png](0_image_0.png) + +Obstet Gynecol Sci 2020;63(4):543-547 + +![0_image_1.png](0_image_1.png) + +![0_image_2.png](0_image_2.png) https://doi.org/10.5468/ogs.19189 pISSN 2287-8572 · eISSN 2287-8580 + +# Granulomatous Peritonitis Caused By Iatrogenic Spillage Of Ovarian Dermoid Cystectomy: A Case Report And Literature Review + +## Hyo-Eun Kim, Md1, Minji Seo, Md2, Jae Young Kwack, Md2, Yong-Soon Kwon, Md, Phd2 + +Department of Obstetrics and Gynecology, 1Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, 2Nowon Eulji Medical Center, Eulji University, Seoul, Korea A 39-year-old nulliparous woman experienced continuous mild fever and abdominal pain since undergoing laparoscopic ovarian dermoid cystectomy 3 months previously in a local hospital. Abdominal computed tomography revealed diffuse heterogeneous fat infiltrations with numerous micronodules in the greater and lesser omentum, combined with ascites with thickening of the parietal peritoneum. The patient underwent exploratory laparoscopy, which included partial pelvic peritonectomy, excision of granulomas, and adhesiolysis with massive irrigation. The patient was treated successfully with laparoscopic surgery and all reproductive structures were spared without operative complications. To avoid peritonitis, complete removal of cyst contents and massive irrigation should be performed during ovarian dermoid cystectomy. Conservative surgical treatment may be a good choice for treating granulomatous peritonitis induced by iatrogenic rupture. + +Keywords: Iatrogenic disease; Teratoma, ovarian; Peritonitis + +## Introduction + +Ovarian dermoid cyst (ODC) is one of the most common benign ovarian neoplasms [1]. Ovarian cystectomy or oophorectomy is usually performed to treat associated pain symptoms to exclude malignancy and avoid potential complications such as torsion and spontaneous rupture. Laparoscopic ovarian cystectomy has become a routine procedure and has replaced traditional laparotomic approaches [2]. However, despite the advantages of laparoscopic treatment, there is an increased risk for intraperitoneal spillage of cyst contents that can potentially result in chemical peritonitis. Prolonged chemical peritonitis rarely results in the formation of granuloma in the peritoneum, otherwise known as granulomatous peritonitis (GP). To our knowledge, there has been only one reported case of prolonged GP, which was treated with repeated laparotomic surgeries [3]. The case described herein would be the second of iatrogenic GP due to spillage of ODC contents. However, it is the first to describe successful treatment of iatrogenic GP using conservative laparoscopic surgery. + +## Case Report + +A 39-year-old nulliparous woman visited the department of infection with symptoms of daily night fever and abdominal pain. She had a history of laparoscopic ovarian cystectomy, performed 3 months previously in a local hospital, due to an ODC. She also complained of other gastrointestinal symptoms including nausea, dyspepsia, intermittent constipation, and abdominal discomfort; however, she did not experience pelvic pain. Physical examination revealed mild tenderness Received: 2019.10.05. Revised: 2019.11.12. Accepted: 2019.12.18. Corresponding author: Jae Young Kwack, MD Department of Obstetrics and Gynecology, Nowon Eulji Medical Center, Eulji University, 68 Hangeulbiseok-ro, Nowon-gu, Seoul 01830, Korea E-mail: podogazi@naver.com https://orcid.org/0000-0001-6685-5894 Articles published in Obstet Gynecol Sci are open-access, distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. + +Copyright © 2020 Korean Society of Obstetrics and Gynecology noted throughout the abdomen. Laboratory investigations revealed increased levels of inflammatory markers, including the tumor marker cancer antigen 125 (CA-125), which was elevated to 189.8 U/mL. + +Abdominal computed tomography (CT) revealed diffuse heterogeneous, smudged, fat infiltrations with numerous micronodules in the greater and lesser omentum, along with ascites with thickening of the parietal peritoneum, and there was tubular enhancing granulation tissue in the right lower quadrant abdominal wall (Fig. 1A). The differential diagnosis included tuberculous peritonitis, malignant peritonitis, and GP, which in all cases involve elevated levels of CA-125. Consequently, the patient was referred to the department of general surgery and gynecology for simultaneous evaluation of the peritoneal and pelvic cavities, including biopsies of the nodules revealed on abdominal CT. + +The first laparoscopic surgery was performed in the department of general surgery. The operation revealed the presence of multiple nodular lesions, 1–3 mm in size, in the peritoneum and omentum. Laparoscopic findings revealed pelvic adhesion and approximately 500 mL of serous ascites (Fig. + +1B). In addition to the peritoneum and omentum excision biopsies, ascites fluid was sent for cytological examination. + +The patient experienced persistent fever, even after the first laparoscopic surgery, and was transferred to the department of gynecology for exploration of the pelvic cavity and subsequent gynecological surgical management. The second laparoscopic surgery was performed 2 weeks after the first operation. Operative findings included severe adhesions in the omentum, bowel to the peritoneum, uterus, and tube to ovary, with multiple granulomatous nodules in the lesser and greater omentum (Fig. 1C). Peritoneal and pelvic adhesiolysis was performed, in which prominent granulomas were removed, and the peritoneal and pelvic cavities were copi- + +![1_image_0.png](1_image_0.png) + +ously irrigated. Frozen biopsy of the omental tissue revealed granulomatous inflammatory processes consistent with foreign-body reaction. However, no spillage of ODC materials trapped within the operative field was suspected. + +Laboratory investigations revealed no evidence of malignancy or signs of tuberculosis in the ascites. The surgical specimens exhibited multifocal fibrotic changes with inflammatory cell infiltration (Fig. 2A), foreign-body type granulomas encompassing hair-like foreign materials (Fig. 2B and C), +multinucleated giant cells within the granulomas (Fig. 2C), +and non-caseating granulomas with chronic inflammatory cells (Fig. 2D). Biopsies of prominent nodules collected from the right pelvis and right para-colic gutter demonstrated a microscopic appearance similar to the surgical specimens. Interestingly, any suspected spillage of ODC contents was not observed either in laparoscopic exploration or in the irrigated ascites fluids. However, microscopic examination revealed hair-like materials in the resected granulomas. + +The patient was started on a soft diet on postoperative day 3 and experienced significantly decreased gastrointestinal symptoms. The level of C-reactive protein increased to 7.77 mg/dL on postoperative day 1 but returned to the normal range (0.98 mg/dL) 1 week later. The patient was discharged on postoperative day 4 of the second laparoscopic surgery. + +She remained free of symptoms at the 3-month follow-up, with no additional postoperative management. + +## Discussion + +ODC is one of the most common benign ovarian neoplasms present during childbearing years [1]. Such cysts are usually benign teratomas that often contain diverse types of tissue including hair, teeth, thyroid parenchyma, and cystic components. Ovarian cystectomy is used to treat pain and avoid complications such as torsion and spontaneous rupture. + +Spontaneous rupture and rupture during cystectomy can lead to chemical peritonitis, as observed in the present case. + +Laparoscopy is regarded to be a safe and efficient method for the diagnosis and treatment of ODCs by proficient laparoscopic surgeons. This procedure offers many advantages including less postoperative pain, lower risk for wound complication, earlier ambulation, and more rapid convalescence. The risk for adhesion formation is also reduced using this procedure. However, in the case of an ODC, the laparoscopic approach could result in chemical peritonitis caused by spilled contents of a ruptured dermoid cyst. Gynecological laparoscopy is generally performed with the patient in the Trendelenburg position, and there can be difficulty in suctioning contents after rupture, which can be hidden in the upper abdominal space [4]. If spillage of ODC contents is suspected, irrigation and changing patient position to the reverse Trendelenburg will help spilled contents flow downward from the upper abdomen. + +Leakage of sebaceous material and hair follicles from the intraperitoneal rupture of mature cystic teratoma causes an aseptic inflammatory peritoneal reaction, specifically, chemical peritonitis. Although we could not find materials that induced GP on gross inspection in the second operation, microscopic examination revealed hair-like materials, from which we inferred the possibility of a small amount of spillage of ODC contents during laparoscopic ovarian cystectomy. + +![2_image_0.png](2_image_0.png) + +Prolonged chemical peritonitis leads to granuloma formation in the peritoneum [4]. Chronic GP, however, is one of the catastrophic complications caused by intraoperative leaking of dermoid cyst contents. Moreover, the condition often appears along with severe adhesions, and ascites that resemble tuberculosis peritonitis or malignancy [5]. + +The occurrence of GP is very rare. Shawki et al. [6] reviewed 496 laparoscopic dermoid cystectomies in which spillage occurred in 324 (65.3%) cases. Despite the high incidence of spillage, however, chronic GP developed in only 1 (0.2%) +patient. There are a few reports describing chronic GP due to spontaneous rupture of ODC [7,8]. To manage chronic GP due to spontaneous dermoid cyst rupture, hysterectomy with salpingo-oophorectomy or omentectomy using a laparotomy approach have been performed due to a clinical pattern that mimics advanced ovarian malignancy [7,8]. + +To the best of our knowledge, the first case of iatrogenic GP caused by the spillage of ODC contents during laparoscopic surgery was reported by Huss et al. [3]. They treated the patient with 2 laparotomies: one trans-rectal and the other via xipho-pubic access. + +The signs and symptoms of GP may be subtle and marginal in the early period of chemical peritonitis; however, the patient would complain of progressive abdominal distention, lower abdominal pain, and gastrointestinal disturbances such as anorexia, nausea, vomiting, and diarrhea [7]. + +Chronic GP usually exhibits unusual radiological findings such as ascites and omental infiltration and thickening of parietal peritoneum on CT scan. These findings may be confused with the peritoneal spread of advanced malignancy and tuberculosis [5]. Thus, ascites fluid analysis includes acidfast bacillus culture and staining, and/or cytology to rule out the presence of other malignancies; in addition, bacterial or fungal culture tests should also be evaluated. Significant elevation in serum CA-125 levels occurs in patients with tuberculous peritonitis and aseptic GP [9]. + +In the present case, conservative laparoscopic surgerysparing the uterus and both ovaries by only removing the prominent inflammatory tissues and copious lavage—proved to be sufficient treatment for prolonged chemical peritonitis induced by dermoid cyst spillage. Granulomas encompassing foreign materials are relatively large and the surface mucosa is contagious and should be removed. If a patient experiences persistent gastrointestinal or pelvic symptoms and has suspicious findings of aggravated peritonitis on ultrasonography, repeated laparoscopic surgeries should be performed. Ruptured teratomas with peritonitis may be difficult to discriminate from malignancies [10]. In case of malignancy, thorough rinsing can reduce the possibility of direct seeding of tumor cells. As such, massive irrigation is essential when malignant tumor cannot be absolutely excluded [11]. In addition, copious saline lavage has been shown to decrease the risk for adhesion and inflammation [12]. Another technical point to prevent GP is careful dissection of the cyst and use of an endoscopic retrieval bag to remove cysts via a port site to prevent spillage [13]. + +In conclusion, surgeons should perform the technique in a less traumatic manner and be cautious about cystic rupture to prevent spillage. If, however, spillage occurs, complete removal of cyst contents and massive irrigation should be considered key measures to avoid peritonitis. It is apparent that an early conservative surgical approach with laparoscopy, involving removal of peritoneal granuloma and copious lavage, can result in successful treatment of advanced chemical peritonitis. + +## Conflict Of Interest + +No potential conflict of interest relevant to this article was reported. + +## Ethical Approval + +This study was approved by Institutional Review Board of Ulsan University Hospital (approval number 2018-03-035). + +## Patient Consent + +Written informed consent was obtained from each participant. + +## References + + 1. Comerci JT Jr, Licciardi F, Bergh PA, Gregori C, Breen JL. + +Mature cystic teratoma: a clinicopathologic evaluation of 517 cases and review of the literature. Obstet Gynecol 1994;84:22-8. + + 2. Shamshirsaz AA, Shamshirsaz AA, Vibhakar JL, +Broadwell C, Van Voorhis BJ. Laparoscopic management of chemical peritonitis caused by dermoid cyst spillage. + +JSLS 2011;15:403-5. + + 3. 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BJOG 2010;117:1027-30. \ No newline at end of file diff --git a/medical/md/PMC7559647.md b/medical/md/PMC7559647.md new file mode 100644 index 0000000000000000000000000000000000000000..d9fd781ee70e0078261d3e6b2de5435a99c4d4a8 --- /dev/null +++ b/medical/md/PMC7559647.md @@ -0,0 +1,186 @@ +World Journal of W J C C *Clinical Cases* + +![0_image_0.png](0_image_0.png) + +Submit a Manuscript: https://www.f6publishing.com *World J Clin Cases* 2020 October 6; 8(19): 4303-4310 DOI: 10.12998/wjcc.v8.i19.4303 ISSN 2307-8960 (online) + +| MINIREVIEWS | +|---------------| + +Liver injury in COVID-19: A minireview + +## Jian-Nan Zhao, Ying Fan, Shuo-Dong Wu + +ORCID number: Jian-Nan Zhao 00000003-0217-2757; Ying Fan 00000002-2925-507X; Shuo-Dong Wu 0000-0002-1984-4057. + +Jian-Nan Zhao, Ying Fan, Shuo-Dong Wu, Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China Corresponding author: Ying Fan, MD, PhD, Doctor, Professor, Department of General Surgery, Shengjing Hospital of China Medical University, No. 36 Sanhao Street, Heping District, Shenyang 110004, Liaoning Province, China. coolingpine78@163.com + +## Abstract + +| Author contributions: Zhao JN drafted and revised the manuscript; Fan Y and Wu SD reviewed and revised the manuscript; All authors read and approved the final version of the manuscript. Conflict-of-interest statement: Authors declare no conflict of interests for this article. | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +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/licenses /by-nc/4.0/ + +| Manuscript source: Invited manuscript Received: April 30, 2020 Peer-review started: April 30, 2020 First decision: May 21, 2020 Revised: June 1, 2020 | +|---------------------------------------------------------------------------------------------------------------------------------------------------------| + +In December 2019, an outbreak of unexplained pneumonia was reported in Wuhan, China. The World Health Organization officially named this disease as novel coronavirus disease 2019 (COVID-19). Liver injury was observed in patients with COVID-19, and its severity varied depending on disease severity, geographical area, and patient age. Systemic inflammatory response, immune damage, ischemia-reperfusion injury, viral direct damage, drug induce, mechanical ventilation, and underlying diseases may contribute to liver injury. Although, in most cases, mild liver dysfunction is observed, which is usually temporary and does not require special treatment, the importance of monitoring liver injury should be emphasized for doctors. The risk of COVID-19 infection of liver transplantation recipients caused more and more concerns. In this article, we aimed to review the available literature on liver injury in COVID-19 to highlight the importance of monitoring and treating liver injury in COVID-19. + +Key Words: Liver injury; COVID-19; Novel coronavirus; SARS-CoV-2; Hepatoprotective drugs; Pandemic ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved. + +Core Tip: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2. Liver injury was observed in COVID-19 patients especially in severe and critical types. Liver injury presented differentially based on disease severity, geographical areas, and patient age. It may be related to systemic inflammatory response, immune damage, ischemia-reperfusion injury, viral direct damage, drug induce, mechanical ventilation, and underlying diseases. Special treatment is unnecessary in most mild liver injury cases and only one or two types of hepatoprotective drugs should be used to treat significant liver injury. Monitoring liver function is important parameter during the treatment of COVID-19 as it affects survival and recovery of patients, and its significance should be emphasized for doctors treating the disease. + +| Accepted: September 8, 2020 Article in press: September 8, 2020 Published online: October 6, 2020 P-Reviewer: Milovanovic T, Rossi RE S-Editor: Zhang L L-Editor: Filipodia P-Editor: Wang LL | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Citation: Zhao JN, Fan Y, Wu SD. Liver injury in COVID-19: A minireview. *World J Clin* Cases 2020; 8(19): 4303-4310 URL: https://www.wjgnet.com/2307-8960/full/v8/i19/4303.htm DOI: https://dx.doi.org/10.12998/wjcc.v8.i19.4303 + +## Introduction + +An outbreak of unexplained pneumonia occurred in Wuhan, China beginning in December 2019. The causative pathogen, a novel coronavirus, was subsequently identified by high-throughput sequencing technology[1,2]. The novel coronavirus was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)[3], and the disease was named 2019 novel coronavirus disease (COVID-19) by the World Health Organization. COVID-19 is transmitted by droplets and aerosols via mouth mucus membranes[4]. Because of its worldwide spread, the World Health Organization declared COVID-19 a pandemic, and it has since spread to at least 210 countries and infected more than 2700000 people globally as of the end of April 2020. Fever and dry cough were the main clinical characteristics of COVID-19. Diarrhea was also reported in some patients. Liver injury was observed in several cases with gastrointestinal (GI) symptoms. It may be related to liver injury in COVID-19 as different degrees of liver dysfunction were observed in a proportion of patients with COVID-19[5-7]. This article aimed to review the available literature on liver injury in COVID-19. + +## Clinical Manifestations + +Nausea, vomiting, and diarrhea are the main GI clinical manifestations of COVID-19. The incidence of liver injury in patients with COVID-19 with GI symptoms were significantly higher than that in those without GI symptoms (17.57% vs 8.84%, P = +0.035)[8]. Similarly, the rate of chronic liver disease in patients with COVID-19 with GI +symptoms was also significantly higher than that of those without GI symptoms +(10.81% vs 2.95%, P = 0.004)[8]. It might indicate that chronic liver disease was closely related to GI symptoms. Meanwhile, chronic liver disease increased the level of alanine transaminase and aspartate transaminase to aggravate the liver injury in the patients with COVID-19. The incidence of liver injury in COVID-19, which was mainly indicated by abnormal alanine transaminase/aspartate transaminase levels, ranged from 14.8% to 53%[9]. The severity of liver injury was most obvious in the 2 nd wk of the course of disease, and it may be related to the viral infection of the regenerated liver cells from the bile duct with high angiotensin-converting enzyme 2 (ACE2) +expression[10]. + +The degree of liver injury varied with different types of COVID-19. The incidence of liver injury in patients with severe COVID-19 was significantly higher than that in patients showing mild symptoms[4,5,11]. Additionally, incidence of liver injury was reported in 58.06%[12] and 78%[13] of the total COVID-19 related deaths. Wan *et al*[14] +found that liver injury was more significant in severe patients as compared to those with mild symptoms. Likewise, Ma *et al*[15] also reported a significant difference between the liver function of patients in the intensive care unit and those out of the intensive care unit. Using multiple regression analysis, Yao *et al*[10]found that the liver injury was only associated with critical type patients, and they concluded that a higher proportion of liver injury was detected in severe patients, and critical type was an independent risk factor for liver injury. Moreover, Jin *et al*[8]reported that the incidence of the severe and critical types was also significantly increased in patients with COVID-19 with GI symptoms than in those without GI symptoms (22.97% vs 8.14%, P < 0.001), indicating increased incidence of liver injury in severe and critical types. + +Liver injury in COVID-19 also varies based on geographical area and age of patients. A study revealed that liver dysfunction was detected in only three patients +(3.75%) in Jiangsu province, which was lower than that observed in Wuhan[16]. As the place where the outbreak of COVID-19 occurred, the number of patients in Wuhan surged, which exceeded the existing capacity of treatment in the early stage. So a proportion of patients managed in Wuhan were severe and critical types whose liver injury was significant. In other places such as Jiangsu province, the number of patients was fewer, and early detection and treatment made the liver injury mild when compared with patients from Wuhan. Zhu *et al*[17] observed thrombocytopenia along with liver dysfunction in two out of ten neonates. Additionally, in a single center observational study, Sun *et al*[18]found liver dysfunction in four out of eight pediatric patients with severe COVID-19. Thus, the management of severe and critical pediatric patients should be valued. + +## Pathology + +Liver injury in COVID-19 can cause characteristic pathological changes that can be revealed during patient autopsy. Xu *et al*[19]reported that moderate microvesicular steatosis and mild lobular and portal activity were detected in the postmortem liver biopsies of a patient with COVID-19; this indicated that either SARS-CoV-2 infection or drug induction caused the liver injury. Minimally invasive autopsies of several organs or tissues from three patients who died due to COVID-19 were performed[20]. + +Hepatocyte degeneration, focal necrosis, and cholestasis of cholangioles were observed along with bile suppository in the small bile duct, which might have been the secondary injury caused by viral infection. However, in both these studies, viral inclusions were not observed, and SARS-CoV-2 was not detected in the liver tissue. + +## Mechanism Of Liver Injury + +Liver injury may be related to systemic inflammatory response and immune damage. + +Xu *et al*[19]found decreased peripheral CD4 and CD8 T cell counts with hyperactivated status, which may be indicative of severe immune injury in the patient with COVID19. Other studies have also observed that liver injury was not dangerous during the early stage of the disease; however, liver deterioration suddenly occurred during the late stage, which was considered to be related to systemic inflammatory response syndrome (SIRS) and activated immune system due to viral infection[21]. SIRS and immune damage can cause multiple organ damage including the liver. Yao *et al*[10] +found that patients with lower absolute value of lymphocyte were more likely to suffer liver dysfunction, which suggested that immune damage might cause liver injury (Figure 1). + +Ischemia-reperfusion injury can also contribute to liver injury. Hypoxia and shock caused by the complications of COVID-19, such as acute respiratory distress syndrome, SIRS, and multiple organ dysfunction syndrome, can lead to ischemiareperfusion injury of the liver[21]. Another study found that hepatocyte necrosis and inflammatory cell infiltration caused by ischemia and hypoxia can be observed not only in the liver transplantation specimens but also in *vivo* and in *vitro* models of liver ischemia and hypoxia[22]. Under conditions of shock and hypoxia, there is a continuous increase in reactive oxygen species. Reactive oxygen species and its peroxidation products act as second messengers to activate transcription factors sensitive to redox reactions, and further initiate the release of various proinflammatory factors, leading to liver damage[23]. + +The novel coronavirus may damage the liver function directly. As the viral RNA can be detected in the blood specimens of the patients with COVID-19[24], and the liver is an organ with abundant blood supply, thus providing the virus with an opportunity to cause direct liver injury. It is now known that ACE2 is the receptor for cell entry of SARS-CoV-2 +[25]. ACE2 was found to be expressed in both liver and bile duct cells[26]. + +Up-regulated expression of ACE2 in hepatic cells due to compensatory hyperplasia of bile duct epithelial cells that was observed in patients with COVID-19 might be related to viral infection of neoplastic hepatocytes[27]. However, the ACE2 expression was found to be higher in bile duct cells than in liver cells[9]indicating that damage and dysfunction of bile duct cells induced hepatocyte dysfunction and subsequent liver injury. Thus, liver injury is caused by bile duct damage and dysfunction rather than hepatocyte damage. However, as there is no significant increase of markers, such as alkaline phosphatase and gamma-glutamyl transpeptidase[15], associated with bile duct cell injury reported in patients with COVID-19, direct virus damage to hepatocytes and biliary duct cells may be limited. + +Liver injury may be induced by medication, which is a likely reason for the varied extent of liver dysfunction observed in patients with COVID-19 based on different types and geographical areas[28]. Until now, no "wonder drug" has been developed to treat COVID-19, and supporting therapy is the mainstay of its management. Fever is the main symptom of COVID-19, and acetaminophen, a common constituent of antipyretic drugs, is recognized as a common liver damage-causing drug and may be + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +one of the important causes of liver dysfunction[21]. In addition, antivirals, antibiotics, steroids, herbal medications, as well as hormones are widely used for the treatment of COVID-19[29,30]. Antivirals such as oseltamivir, abidor, lopinavir, and ritonavir were found to have adverse reactions such as liver injury[21]. Moreover, similar findings were observed in a recent study where lopinavir/ritonavir caused liver injury in patients with COVID-19[31]. In detail, the odds of liver injury using lopinavir/ritonavir was increased by four fold[30]. Another study reported that liver injury was more likely to occur in patients treated with multiple kinds of drugs as well as large amounts of hormones (P = 0.002, P = 0.031, respectively)[10]. This may explain that liver injury was more commonly observed in severe and critical types because less drugs are usually administered to patients with mild disease. As drug metabolism occurs in the liver and kidneys, liver injury may impair their metabolism, excretion, dosing, and effective concentrations, leading to increased risk of toxicity due to the use of antiviral drugs[32]. + +Therefore, we should attach importance to the monitoring of liver function in COVID19. + +In the cases of patients receiving mechanical ventilation, high levels of positive and expiratory pressure can increase the right atrial pressure and impede venous return and subsequently cause hepatic congestion, which may participate in liver injury. However, clinical data show that many patients with COVID-19 who do not require mechanical ventilation also have liver dysfunction[33]. This observation may support the higher incidence of liver injury in severe and critical patients than in other mild cases. + +Furthermore, underlying diseases may contribute to liver injury. Individuals suffering from underlying diseases, such as chronic liver disease, are more likely to be infected by the SARS-CoV-2 as they have lower immune function. Similarly, a guideline proposed by the Asian-Pacific Association for the Study of the Liver suggested that patients with advanced cirrhosis, hepatic cell cancer, autoimmune liver diseases, and post liver transplantation were more susceptible to COVID-19 infection because of being immuno-compromised[34]. A study on COVID-19, found that out of 22 patients with liver injury, the proportion of injury was significantly higher (P = 0.011) in 16 patients (72.7%) with underlying diseases than that in patients without underlying diseases[10]. Half of the patients with abnormal liver test results also tended to have chronic liver diseases, including nonalcoholic fatty liver disease, alcoholic liver disease, and chronic hepatitis B (P = 0.001)[30]. Another study about GI symptoms in COVID-19 reported that the incidence of chronic liver disease (10.81%) in patients with GI symptoms was significantly higher than that (2.95%) in patients without GI +symptoms (P = 0.004)[8], which indicates a potential relationship between chronic liver disease and liver injury. However, few studies reported the occurrence of liver failure in the chronic liver disease patients with COVID-19[9]. + +## Management + +The mainstay for management of liver injury in COVID-19 is to inhibit the inflammatory response and correct the hypoxemia in order to prevent the SIRS and multiple organ dysfunction syndrome[21]. Most cases with COVID-19 show mild liver damage, which is usually temporary and returns to normal without any special treatment[28](Table 1). Targeted hepatoprotective therapy is necessary in patients with severe liver injury[21]. Clinically used liver protective drugs include polyene phosphatidylcholine, glycyrrhizin preparation, ursodeoxycholic acid, and s-adenosine methionine. One or two of these drugs were used according to the liver function of the patient. As we have previously discussed, the use of several drug types is not recommended. As prevention is preferable to management, the monitoring of liver function to avoid liver damage is a key role in the treatment of COVID-19. + +## Liver Transplantation + +With the outbreak of COVID-19, the risk of COVID-19 infection and management of liver transplantation (LT) recipients have recently begun to attract increased attention. The impacts of COVID-19 on LT recipients may different. The infection of COVID-19 may be more severe in the perioperative or early post-transplant period because of high immunosuppressive levels. As for long-term post-transplant patients, diabetes, hypertension, and other comorbidities may be associated with severe COVID-19[12,35]. A +study suggested that immunosuppressed patients did not have an increased risk of severe complications of COVID-19 when compared with the general population[36]. Just as mentioned above, a reactive innate immune response might cause severe clinical manifestations, such as liver injury, SIRS, and multiple organ damage, and immunosuppression might have a protective effect[37]. Compared with non-transplant patients, it was observed that COVID-19 infection was no worse in LT patients, and they had lower risks of hyperinflammation and cytokine storm[38], which might indicate that the host response to infection may be modified by immunosuppression[34]. Based on current studies, some recommendations were given below[39]. + +For LT recipients without COVID-19, there is no need to adjust the immunosuppressive dosage. Keep current immunosuppressant dosage and monitor the condition of patients with mild to moderate COVID-19. Reducing the dosage of calcineurin inhibitor and stopping anti-metabolic drugs can be considered in the patients with severe or rapidly progressing COVID-19. + +## Conclusion + +The novel infectious disease, COVID-19, is known to mainly present with fever, dry cough, and in some cases GI symptoms. Liver injury is observed in all types of COVID-19, especially in severe and critical types, which may be related to systemic inflammatory response, immune damage, ischemia-reperfusion injury, viral direct damage, drug induce, mechanical ventilation, and underlying diseases. Therefore, more attention should be paid to the monitoring of liver function in the management of COVID-19. However, until now, the mechanism of liver injury in COVID-19 has not been elucidated. Therefore, further studies need to be carried out. Diagnostic criteria, pathogenesis, clinical characteristics, treatment, and prognosis of liver injury in COVID-19 should be clarified in further clinical trials[40]. Finally, while facing this new + +| Table 1 Related data of liver injury Ref. Object | Group 1 | Group 2 | P value | | +|----------------------------------------------------|----------------------------|----------------------------|--------------------------------|-------| +| Jin et al[8] | GI symptoms | Without GI symptoms | | | +| Incidence of liver injury | 17.57% | 8.84% | 0.035 | | +| Rate of chronic liver disease | 10.81% | 2.95% | 0.004 | | +| Incidence of the severe and critical types | 22.97% | 8.14% | < 0.001 | | +| Yao et al[10] | Critical type patients (%) | Liver injury group (77.3%) | Non-liver injury group (27.8%) | 0.002 | +| Wan et al[14] | Mild cases | Severe cases | | | +| Aspartate aminotransferase, U/L (≤ 40) | 84% | 62.5% | 0.0005 | | +| Lactate dehydrogenase, U/L (> 250) | 29% | 75% | 0.0055 | | + +GI: Gastrointestinal. + +unknown infectious disease, the importance of prevention by wearing masks, washing hands, and staying at home needs to be emphasized for the public. + +## References + +1 Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N, Bi Y, Ma X, Zhan F, Wang L, Hu T, Zhou H, Hu Z, Zhou W, Zhao L, Chen J, Meng Y, Wang J, Lin Y, Yuan J, Xie Z, Ma J, Liu WJ, Wang D, Xu W, Holmes EC, Gao GF, Wu G, Chen W, Shi W, Tan W. 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Coronaviruses and Immunosuppressed Patients: The Facts during the Third Epidemic. *Liver* Transpl 2020; 26: 832-834 [PMID: 32196933 DOI: 10.1002/lt.25756] +36 Bhoori S, Rossi RE, Citterio D, Mazzaferro V. COVID-19 in long-term liver transplant patients: preliminary experience from an Italian transplant centre in Lombardy. *Lancet Gastroenterol Hepatol* 2020; 5: 532-533 [PMID: 32278366 DOI: 10.1016/S2468-1253(20)30116-3] +37 Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, Tan W. Detection of SARS-CoV-2 in Different Types of Clinical Specimens. *JAMA* 2020 [PMID: 32159775 DOI: 10.1001/jama.2020.3786] +38 Liu H, He X, Wang Y, Zhou S, Zhang D, Zhu J, He Q, Zhu Z, Li G, Sun L, Wang J, Cheng G, Liu Z, Lau G. Management of COVID-19 in patients after liver transplantation: Beijing working party for liver transplantation. *Hepatol Int* 2020; 14: 432-436 [PMID: 32277387 DOI: 10.1007/s12072-020-10043-z] +39 Chinese Digestion Association; Chinese Medical Doctor Association; Chinese Society of Hepatology; Chinese Medical Association. [The protocol for prevention, diagnosis and treatment of liver injury in coronavirus disease 2019]. *Zhonghua Gan Zang Bing Za Zhi* 2020; 28: 217-221 [PMID: 32306655 DOI: +10.3760/cma.j.cn501113-20200309-00095] +40 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/PMC7648165.md b/medical/md/PMC7648165.md new file mode 100644 index 0000000000000000000000000000000000000000..4fb36e241648ddbb87782d418b95c0b7192314eb --- /dev/null +++ b/medical/md/PMC7648165.md @@ -0,0 +1,194 @@ +Contents lists available at ScienceDirect + +![0_image_3.png](0_image_3.png) + +![0_image_2.png](0_image_2.png) + +journal homepage: www.elsevier.com/locate/jctube +Variation in tuberculosis treatment outcomes and treatment supervision + +![0_image_1.png](0_image_1.png) + +practices in Uganda Christopher A. Bergera,1, Alex Kityamuwesib,1, Rebecca Crowdera, Maureen Lamunub, Lynn Kunihira Tinkab, Joseph Ggitab, Agnes Sanyu Nakateb, Catherine Namaleb, Denis Oyukub, Katherine Chenc, Stavia Turyahabwed, Adithya Cattamanchia,b, Achilles Katambab,e,⁎ +a Division of Pulmonary and Critical Care Medicine and Center for Tuberculosis, San Francisco General Hospital, University of California San Francisco, San Francisco, USA +b *Uganda Tuberculosis Implementation Research Consortium, Kampala, Uganda* c *University of California Berkeley, Berkeley, USA* d *Uganda National Tuberculosis and Leprosy Programme, Kampala, Uganda* e *Clinical Epidemiology & Biostatistics Unit, Department of Medicine, Makerere University College of Health Sciences, Kampala, Uganda* + +| A R T I C L E I N F O Keywords: Tuberculosis Quality of care Practice variation | +|---------------------------------------------------------------------------------------| + +| ABSTRACT | +|------------| + +Background: Variation in healthcare delivery is increasingly recognized as an important metric of healthcare quality. Directly observed therapy (DOT) has been the standard of care for tuberculosis (TB) treatment supervision for decades based on World Health Organization (WHO) guidelines. However, variation in implementation of DOT and associated TB treatment supervision practices remains poorly defined. + +Methods: We collected individual patient data from TB treatment registers at 18 TB treatment units in Uganda including District Health Centers, District Hospitals, and Regional Referral Hospitals. We also administered a survey and did observations of TB treatment supervision practices by health workers at each site. We describe variation in TB treatment outcomes and TB treatment supervision practices. Results: Of 2767 patients treated for TB across the 18 clinical sites between January 1 and December 31, 2017, 1740 (62.9%) were men, most were of working age (median 35 years, interquartile range [IQR] 27 - 46), 2546 (92.0%) had a new TB diagnosis, and nearly half (45.9%, n = 1283) were HIV positive. The pooled treatment success proportion was 69.4% (95% confidence interval [CI] 67.8 - 71.1) but there was substantial variation across sites (range 42.6 - 87.6%, I-squared 92.7%, p < 0.001). The survey and observation of TB treatment practices revealed that the majority of sites practice community-based DOT (66.7%, n = 12) and request a family member, who receives no additional training or supervision, to serve as a treatment supporter (77.8%, +n = 14). At TB medication refill visits, all sites screen for side effects and most assess adherence via self-report (83.3%, n = 15). Only 7 (38.9%) sites followed-up patients who missed appointments using either phone calls +(22.2%, n = 4/7) or community health workers (16.7%, n = 3/7). All 18 sites counseled patients at treatment initiation, but none provided additional counseling at refill visits other than addressing poor adherence or missed appointments. + +Conclusion: There was substantial variation in implementation of DOT, including observation and documentation of daily dosing, training and supervision of treatment supporters, and follow-up for missed clinic visits. + +Identifying best practices and reducing uncontrolled variation in the delivery of TB treatment is critical to improving treatment outcomes. + +1. Introduction tuberculosis (TB) treatment services, continues to be below the 90% +target established by the World Health Organization (WHO) in most high burden countries [1]. Improving the quality of healthcare services Treatment success rate, a key metric for assessing the quality of +⁎ Corresponding author at: Clinical Epidemiology & Biostatistics Unit, Department of Medicine, Makerere University College of Health Sciences, P.O. BOX 7062, Kampala Uganda. + +E-mail address: axk95@case.edu (A. Katamba). 1 Authors contributed equally. + +![0_image_0.png](0_image_0.png) + +2405-5794/ 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/). + +requires understanding and reducing variation in the workflows and processes of care [2,3]. Renowned physicist and statistician W. Edwards Deming has noted that "uncontrolled variation is the enemy of quality." +Although directly observed therapy (DOT) has been a core component of the WHO strategy for improving TB treatment outcomes since 1994 +[4], there are limited data on facility-level variation in implementation of DOT and associated TB treatment supervision practices within routine care settings in high-burden countries. + +Similar to most high burden countries, TB Diagnostic and Treatment Units (DTUs) affiliated with the Uganda National TB and Leprosy Program (NTLP) use a mix of facility- and community-based Directly Observed Therapy Short-course (DOTS) models for TB treatment management. Once registered, most TB patients are asked to name a treatment supporter and are provided with a 2-week supply of medicines in the intensive phase and a one-month supply of medicines in the continuation phase. Patients are expected to take medicines with observation by the treatment supporter and return to clinic bi-weekly +(intensive phase) or monthly (continuation phase) to check on side effects and to obtain refills. DTU staff are expected to assess adherence via patient self-report at refill visits, and to call or visit patients who do not return for refills [4–6]. + +To better understand the quality of TB treatment services and inform the integration and scale-up of newer strategies such as digital adherence technologies, we collected patient- and facility-level data from health centers in rural and *peri*-urban Uganda to understand the extent of variation in TB treatment outcomes and how TB treatment is delivered. + +## 2. Methods 2.1. Study Setting And Population + +We conducted a cross-sectional study at 18 Uganda NTLP-affiliated TB treatment units. Study sites were chosen in consultation with the Uganda NTLP in preparation for a planned stepped-wedge cluster randomized trial of 99DOTS, a low-cost digital adherence technology [7]. + +The study sites were eligible for the cluster RCT if they 1) diagnosed > 10 pulmonary TB cases per month in 2016–2017, 2) were not located within Kampala District, 3) were located within 225 km of Kampala, and 4) had a treatment success rate of < 80%. A total of 23 sites met these criteria, of which 18 were chosen in consultation with the Uganda NTLP. The 18 study sites were located in 15 districts (10 in Central, 7 in Eastern and 1 in Western Uganda), with 10 situated in General hospitals, 5 in Regional Referral hospitals, and 3 in District Health Centers. + +We included data on all adults (≥18 years of age) initiating TB +treatment between January 1 and December 31, 2017 at the 18 treatment units. We excluded patients being treated for extrapulmonary TB +or drug-resistant TB, and patients transferred to or from another TB treatment unit. In addition, we administered a survey to the "TB Focal Person" or another senior staff member within the TB treatment unit at each participating site. + +The study was reviewed and approved by Institutional Review Boards (IRBs) at the University of California San Francisco (San Francisco, USA) and Makerere University School of Public Health (Kampala, Uganda), and by the Uganda National Council for Science and Technology. All survey participants were consented prior to survey administration and the IRBs granted a waiver of consent for extraction of patient-level data from TB treatment registers. + +## 2.2. Study Procedures + +Trained research staff visited all study sites to photograph TB +treatment registers and upload photos to a secure REDCap server. + +Project staff then performed single extraction of patient-level data including demographic, clinical, and TB treatment outcome data from the photos for all eligible patients initiating TB treatment using a standardized data extraction form (Supplementary Fig. 1). All data were subsequently reviewed by a data analyst for quality control and any errors or inconsistencies fixed utilizing the treatment registers or by calling health center staff for missing data. + +During the site visits, project staff also conducted repeated observations of TB treatment visits and administered a survey to the TB +focal person and/or a TB treatment unit staff member after obtaining informed consent. The survey was developed in consultation with local and international experts to assess the organization of clinical services including practices around facility- or community-based DOT, and support systems in place for TB patients and their families (Supplementary Fig. 2). For clinical service organization, we collected information about the use of community- vs. facility-based DOT, and any patient reminder systems. For both facility- and community-based DOT, we asked how often patients had scheduled visits during the intensive and continuation phases of treatment, work-flow, patient wait times, how doses are observed and recorded, how adherence and side effects are assessed and followed-up, and whether treatment supporters are utilized. For support systems, we inquired what type of counseling patients receive, and if any social or monetary support systems are in place. Observations of routine clinical care were done by trained medical officers with experience in TB treatment. + +## 2.3. Definitions + +Treatment outcomes were defined according to standard WHO definitions [8]. We considered treatment success to include patients who were recorded as having been cured or completed treatment. + +## 2.4. Data Analysis + +The data were cleaned and analyzed using Stata version 15.0 (Stata Corporation, USA). Summary statistics such as median and interquartile range (IQR) or proportion and 95% confidence interval (CI) were used to describe characteristics of the study population. The pooled treatment success proportion and 95% CI across sites was calculated using a random effects model. Heterogeneity between health facilities was assessed using the Chi-squared test and the I-squared statistic. + +## 3. Results 3.1. Demographic And Clinical Characteristics + +Of 2767 eligible patients initiated on treatment for pulmonary TB +during the study period, median age was 35 years (IQR 27 - 46 years), +the majority (62.9%, n = 1740) were male, 1283 (45.9%) had HIV coinfection and 1587 (57.4%) were bacteriologically-confirmed. The proportion male (57.7% vs 64.3% vs 62.8%), proportion HIV-positive +(42.6% vs 49.5% vs 43.8%), proportion with bacteriologically-confirmed TB (59.7% vs 57.4% vs 56.6%) and median age (33 vs 35 vs 36 years) were similar when examined across Health Centers, General Hospitals, and Regional Referral Hospitals, respectively. + +## 3.2. Tb Treatment Outcomes + +There was significant variation in TB treatment outcomes across health facilities, with treatment success rates ranging from 42.6% to 87.6% (I-squared 92.7%, p < 0.001). Most of the variation occurred at District Hospitals (I-squared 92.9%, p < 0.001) and Regional Referral Hospitals (I-squared 91.9%, p < 0.001). The pooled proportion of TB +patients treated successfully was 69.4% (95% CI 67.8 - 71.1), and was lower at Regional Referral Hospitals (61.2%, 95% CI 58.4 - 64.0) compared to District General Hospitals (72.7%, 95% CI 70.4 - 75.1) and District Health Centers (78.3%, 95% CI 73.8 - 82.7) (Fig. 1). + +Fig. 1. Forest Plot of Treatment Success by Health Center. Forest plot showing percentage of successful tuberculosis treatment outcomes from January 1st to December 31st, 2017 and corresponding 95% confidence intervals (CI) for 18 health facilities in Uganda. The health facilities are grouped into Level IV Health Centers, District General Hospitals (Level V), and Regional Referral Hospitals. The total overall treatment success rate for the 2767 patients in the study was 69.4% +(95% CI 67.8 - 71.1%). The dotted vertical line at 90 is the curative threshold recommended by the World Health Organization for achieving effective control of tuberculosis. There was statistically significant heterogeneity in treatment success across the three levels of health facility (I-squared 92.7%, p < 0.001). Compared to Level IV Health Centers, General and Regional Referral Hospitals had lower treatment success rates (78.3%, 95% CI 73.8 - 82.7 vs 72.7%, 95%CI 70.4 - 75.1 and 61.2%, 95% CI 58.4 - 64.0, respectively). Unlike Level IV Health Centers (I-squared 1.8%, p = 0.381), there were statistically significant heterogeneity in treatment success rate within General (I-squared 92.9%, p < 0.001) and Regional Referral Hospitals (I-squared 91.9%, p < 0.001). Abbreviations: TSR, Treatment Success Rate; CI, Confidence Interval. + +## 3.3. Tb Treatment Supervision + +Our assessment of TB treatment supervision practices also revealed substantial variation across health facilities (Table 1). The majority of health facilities practiced community-based DOTS (CB-DOTS) only (66.7%, n = 12). The remaining facilities reported admitting very sick patients (N = 6) or all new TB patients (N = 2) to the inpatient facility for several weeks prior to transitioning to CB-DOTS following hospital discharge. Treatment supporters in CB-DOTS were typically a family member (77.8%, n = 14) who received no additional training beyond what was discussed at the initial visit with the patient and no direct supervision by health workers. While the majority of health facilities asked treatment supporters to observe daily swallowing of medication (88.9%, n = 16), only 5 facilities had government-issued TB treatment cards for tracking ingestion, and none confirmed that treatment supporters were observing doses after the initial visit. Community health workers (CHWs) served as treatment supporters at two (11.1%) health facilities and received incentives to do so at both facilities. Most health facilities assessed adherence at refill visits via self-report (83.3%, +n = 15), but none evaluated adherence via pill count or another objective measure. For patients who do not return for refill visits only four facilities (22.2%) regularly called patients and three (16.7%) traced patients with the help of a CHW. In order to support patients, all health facilities (100%, n = 18) reported providing TB counseling at the initial visit, but any subsequent counseling was largely limited to adherence assessment and reminders to not miss refill appointments. Posted TB educational content was present at 10 facilities (55.6%). No facility provided social or monetary support to TB patients. + +## 4. Discussion + +We used survey and observational data to examine sub-optimal and varied TB treatment outcomes at health facilities in Uganda with substantial variation in the processes of TB treatment. With increasing recognition that unplanned or random variation in processes of care are a key contributor to poor quality care and outcomes, this study demonstrates there is an urgent need to identify and monitor the implementation of best practices for TB treatment supervision. + +Furthermore, the suboptimal implementation of DOTS highlights the need for other methods of adherence monitoring and support, such as digital adherence technologies. + +A *meta*-analysis examining seven categories of adherence interventions identified that TB treatment outcomes improved with the use of CB-DOTS and trained health workers, as well as the use of patient education, incentives and enablers, psychological interventions, reminders and tracers and digital technologies [9]. With the exception of CB-DOTS, the remaining categories of adherence interventions were either not used or used inconsistently in our study. Moreover, the implementation of CB-DOTS and practices related to adherence assessment and patient counseling and follow-up at refill visits were highly + +| Table 1 Tuberculosis Treatment Supervision Practices. | Overall N = 18 (%) | +|----------------------------------------------------------------------------------------------------------------|-----------------------| +| Organization of Clinical Services and DOT Practices Form of DOT during intensive phase? Community-based only | 12 (66.7) | +| Community-based +/- inpatient admission | 6 (33.3) | +| Who observes daily dosing in the community? Family Member | 14 (77.8) | +| Community Health Worker | 2 (11.1) | +| Either | 2 (11.1) | +| Treatment supporters: Receive specialized treatment supporter training? | 0 (0) | +| Routine supervision by clinic staff? | 0 (0) | +| Receive any incentive? | 2 (11.1) * | +| Asked to observe patients swallow meds? | 16 (88.9) | +| Asked to record daily doses on the treatment card? | 6 (33.3) | +| If a patient misses a refill visit, how are they contacted? Phone call | 4 (22.2) | +| SMS | 0 (0) | +| Community Health Worker | 3 (16.7) | +| Use of daily adherence reminders? Yes | 0 (0) | +| Refill Visit practices: Screen for side effects at each refill visit? | 18 (100) | +| Assess adherence via self-report? | 15 (83.3) | +| Assess adherence via pill count? | 0 (0) | +| Support Systems Frequency of TB counseling: At treatment initiation only | 18 (100) | +| At all clinic visits | 0 (0) | +| What tools are used for TB Counseling? Trained TB Counselor | 18 (100) | +| Educational Handout for Patient | 0 (0) | +| Posted Educational Content | 10 (55.6) | +| Does your facility provide any social or monetary support? Yes | 0 (0) | +| *Only those treatment supporters who were community health workers, and | | + +*Only those treatment supporters who were community health workers, and not family members, received any incentive for their work at two facilities. + +Abbreviations: DOT, directly observed therapy; TB, tuberculosis; CB-DOT, +community-based directly observed therapy; FB-DOT, facility-based directly observed therapy; SMS, short messaging service. + +varied. Increasing uptake of effective treatment practices, and measuring and reducing variation in their delivery, are critical to improving the quality of TB treatment services and TB treatment outcomes. In the context of COVID-19, extended time between clinic visits for medication refills further reduces the feasibility and potential utility of DOT +and highlights the need for alternative strategies for adherence support and monitoring. + +Our study has several important limitations. Due to the small number of health facilities included, we were unable to analyze associations between specific TB treatment supervision practices and improved TB treatment outcomes. Further research is needed on how best to reduce variation in the processes of TB treatment. The small sample size also potentially limits the generalizability of our findings. + +With recent evidence suggesting that adherence is the strongest predictor of treatment outcomes [10], it is increasingly important to define and scale-up effective TB treatment supervision practices in high-burden countries. While guidelines have traditionally shied away from dictating "best-practices" for DOTS in order to permit flexibility, this has allowed for incredible variation in the delivery of TB treatment. + +Increased efforts are needed to define best practices and to measure and reduce unplanned variation in TB treatment services in order to improve the quality of care and ultimately achieve the goal of improved TB treatment outcomes. + +## Funding + +This project is supported by the Stop TB Partnership's TB REACH +initiative and is funded by the Government of Canada, the Bill & Melinda Gates Foundation, and the United States Agency for International Development. Additional funding was provided by the UCSF Nina Ireland Program for Lung Health. + +## 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. + +## Acknowledgements None. Author Contributions + +ST, AC, and AKa conceived of the study. AKi, ML, LKT, JG, ASN, CN, +DO, and KC collected and entered data. CB, AKi, RC, and AC analyzed data. CB, AKi RC, AC, and AKa drafted the manuscript. All authors critically reviewed and revised the manuscript. + +## Appendix A. Supplementary Data + +Supplementary data to this article can be found online at https:// +doi.org/10.1016/j.jctube.2020.100184. + +## References + +[1] World Health Organization. International Standards for Tuberculosis Care (ISTC) and the Patients' Charter for Tuberculosis Care. In. https://www.who.int/tb/ publications/2006/istc/en/. Geneva, Switzerland: WHO Press; 2006. + +[2] Ferguson J. Reducing Unwanted Variation in Healthcare Clears the Way for Outcomes Improvement. Health Catalyst Products and Services 2017. Published February 2, 2017. Accessed May 29, 2020. + +[3] Keech S. Decrease and Smooth Variation to Improve Flow. [4] Bhandari R. International standards for tuberculosis care (ISTC) and patients' charter: New Advances in Tuberculosis Care. Health Prospect. 2011;10:43–5. + +[5] Ministy of Health. National TB and Leprosy Program Guidelines. In: MOH, ed. http:// +library.health.go.ug/publications/tuberculosis/uganda-national-guidelinestuberculosis-infection-control-health-care. Kampala, Uganda: MOH; 2017. + +[6] World Health Organization. Guidelines for treatment of tuberculosis - 4th edition. In. + +https://www.who.int/tb/publications/2010/9789241547833/en/. Geneva: WHO Press; 2010. + +[7] Everwell Health Solutions. 99DOTS, low-cost monitoring and improving medication adherence. https://www.99dots.org Web site. Accessed2019. + +[8] World Health Organization. Definitions and reporting framework for tuberculosis 2013 revision (updated December 2014). http://apps.who.int/iris/bitstream/ 10665/79199/1/9789241505345_eng.pdf. Accessed April 2020. + +[9] Alipanah N, Jarlsberg L, Miller C, et al. Adherence interventions and outcomes of tuberculosis treatment: A systematic review and meta-analysis of trials and observational studies. PLoS Med. 2018;15(7):e1002595. + +[10] Imperial MZ, Nahid P, Phillips PPJ, et al. A patient-level pooled analysis of treatment-shortening regimens for drug-susceptible pulmonary tuberculosis. Nat. Med. + +2018;24(11):1708–15. + +4 \ No newline at end of file diff --git a/medical/md/PMC7910716.md b/medical/md/PMC7910716.md new file mode 100644 index 0000000000000000000000000000000000000000..31ba1b8f15cfb0292c525ca5ffd84a1205cda3b5 --- /dev/null +++ b/medical/md/PMC7910716.md @@ -0,0 +1,233 @@ +Contents lists available at ScienceDirect + +![0_image_1.png](0_image_1.png) + +![0_image_2.png](0_image_2.png) + +![0_image_3.png](0_image_3.png) + +journal homepage: www.jsesinternational.org +Validation and reliability of a Japanese version of the Simple Shoulder + +![0_image_0.png](0_image_0.png) Test: a cross-sectional study Takuya Sekiguchi, MD, PhD a,b, Yoshihiro Hagiwara, MD, PhD b,*, Akira Ando, MD, PhD c, Kenji Kanazawa, MD, PhD d, Kazuaki Suzuki, MD b, Masashi Koide, MD, PhD c, Yutaka Yabe, MD, PhD b, Satsuki Onoda, MD, PhD e, Eiji Itoi, MD, PhD b a Department of Orthopedic Surgery, JR Sendai Hospital, Sendai, Japan b Department of Orthopedic Surgery, Tohoku University School of Medicine, Sendai, Japan c Department of Orthopedic Surgery, Matsuda Hospital, Sendai, Japan d Department of Orthopedic Surgery, Miyagi South Medical Center, Sendai, Japan e Department of Orthopedic Surgery, Iwate Prefectural Central Hospital, Morioka, Japan article info Keywords: +Shoulder Simple Shoulder Test cross-cultural comparison patient-reported outcome measures validity Japan Level of Evidence: Basic Science Study; Validation of Outcome Instruments Background: The Simple Shoulder Test (SST) is a widely used patient-reported outcome assessment. The purpose of this study was to develop and validate a Japanese version of the SST (SST-Jp). + +Methods: A two-stage observational study was conducted to validate the cross-cultural adaptation of the SST. A total of 100 patients with shoulder disorders completed the SST-Jp; the Disability of Arm, Shoulder, and Hand assessment; and the Medical Outcomes Short-Form 36 (SF-36) at an initial visit. + +Thirty-four of the patients repeated the SST-Jp one week after the first examination. The test-retest reliability was quantified using the interclass correlation coefficient, and Cronbach's alpha (a) was calculated to assess internal consistency. Construct validity was assessed using Spearman's rank correlation coefficient. + +Results: The internal consistency of the SST-Jp was very high (a ¼ 0.826). The interclass correlation coefficient of the SST-Jp was also high (0.859). There was a strong, positive correlation between the Disability of Arm, Shoulder, and Hand and the SST-Jp (r ¼ 0.717, P < .001). The SST-Jp was significantly correlated with most of the SF-36 subscales. The correlations of the SST-Jp with physical subscales of the SF-36 were stronger than those with the other subscales. Conclusions: The SST-Jp was found to be a valid and reliable measurement for shoulder joint pain and function assessment among the Japanese population. + +© 2020 The Author(s). Published by Elsevier Inc. on behalf of American Shoulder and Elbow Surgeons. + +This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). + +Shoulder disorders, such as rotator cuff tear and frozen shoulder, are the second most common musculoskeletal disorders.3 The prevalence of shoulder pain ranges from 6.9% to 26% in the general population,17 and more specifically, the prevalence of neck and shoulder pain ranges from 10% to 48% in Japan.10,25,26 Although shoulder disorders are not life-threatening, they can limit the activities of daily living (ADLs) and negatively impact quality of life.15 Furthermore, shoulder disability may disrupt psychological and social well-being.22 This study was approved by the institutional review board of the authors' affiliated institutions (Cyubyou 1352). + +* Corresponding author: Yoshihiro Hagiwara, MD, PhD, Department of Orthopaedic Surgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. + +E-mail address: hagi@med.tohoku.ac.jp (Y. Hagiwara). + +Patient-reported outcomes (PROs) have been widely used in clinical practice and research to evaluate treatments to reduce the discrepancy between clinical evaluation and patient perception. There are many different PROs to evaluate shoulder conditions, such as the Disabilities of the Arm, Shoulder, and Hand Questionnaire (DASH);12 Western Ontario Shoulder Instability Index;14 Shoulder Pain and Disability Index;23 and Simple Shoulder Test +(SST).16 The SST is one of the most commonly used instruments, in part, because it is a simple and short PRO tool. It has been tested in various clinical settings6,24 and can be completed within 3 minutes, and patients can complete it before the clinical visit, making it very practical.16 For proper use of the SST in noneEnglish-speaking populations, it is necessary to use a translated version of the SST that is culturally adapted and properly evaluated. The need for validated translations has become more essential with the growing number of multicenter and multinational studies, which provide more statistical power for randomized controlled trials.2,9 The SST + +## + +has been translated into several languages.5,18,20,21,28 Although a Japanese version of the SST (SST-Jp) has not been developed and validated, studies using the SST in Japanese populations without cultural adaptation and validation have been published. Cultural adaptation and validation of the SST would benefit Japanese clinicians, surgeons, and patients by improving the rigor of and evidence for a standardized SST-Jp. + +Therefore, the purpose of this study was to translate and culturally adapt the SST for use in Japan and evaluate its validity and reliability in Japanese patients with shoulder pain and dysfunction. + +## Materials And Methods + +This study was conducted in two phases. In phase 1, crosscultural adaptation was conducted according to guidelines of the American Association of Orthopaedic Surgeons.2 In phase 2, we assessed the measurement properties in a Japanese population. + +## Cross-Cultural Adaptation + +The SST-Jp was developed according to the American Association of Orthopaedic Surgeons guidelines for cross-cultural adaptation.2 The process consisted of five steps, each of which was documented with a written report. First, the English version of the SST was translated into Japanese by two bilingual translators (forward translation). One of the translators was an experienced shoulder surgeon and aware of the concept of the translation, while the other was a professional translator without a medical background who was not aware or informed of the medical concepts. + +For step 2, the two translations were synthesized into one after discussion with the study team. In step 3, the synthesized Japanese version was translated into English by two professional translators +(back translation). In step 4, any inconsistencies between the original and the back-translated English version were resolved in the second draft of the questionnaire. Finally, all versions were evaluated and a pilot test was conducted. For the pilot study, the SST-Jp was administered to 30 patients with shoulder dysfunction. After analysis of the pilot test, results were found to be acceptable, and the final version of the SST-Jp was confirmed (Supplemental Appendix). + +## Patients + +We recruited patients with shoulder disorders who were admitted to one of four hospitals between February and October 2019. Patients who were 18 years or older, native Japanese speakers, and able to complete the questionnaire by themselves were included in the study. The exclusion criteria were a previous major trauma (e.g., fracture and dislocation) on the affected shoulder or girdle; impairment in the cervical spine, elbow, and/or hand affecting the shoulder function; a history of inflammatory arthritis, physical and/or mental dysfunction as an underlying disease; or a regional tumor and metastasis. Patients completed the following questionnaires: the SST-Jp, the official Japanese version of the 36-item short-form health survey (SF-36 v2),7,8 and the Japanese Society for Surgery of the Hand version of the DASH.13 Basic characteristics such as sex, age, affected side, and diagnosis were also recorded. A series of 100 patients with shoulder disorders were included in the study. Among the patients, 36 were readministered the SST-Jp after one week. These were surgical patients who returned for a preoperative appointment and did not have additional treatments in the previous week. This study was approved by the institutional review board of the authors' affiliated institutions. All patients provided written, informed consent before participating in this study. + +## Assessments + +The SST is a shoulder function scale consisting of 12 questions with 'yes' or 'no' answers to assess the ability to tolerate or perform ADLs. The SST scores range from 0 (worst) to 100 (best) reported as the percentage of answered items to which the patient responds in the affirmative.1 The SF-36 is a widely used instrument that measures 8 subscales of health including physical functioning (PF), role physical (RP), bodily pain (BP), general health (GH), vitality, social functioning (SF), role emotional, and mental health. Each subscale score was transformed into a 0 to 100 scale where a lower score indicated worse quality of life and more severe disability.7,8 The DASH score is a general upper extremity score used for patients with any condition related to the joints of the upper extremities. It consists of 30 items with a total score ranging from 0 (no disability) +to 100 (worst).13 + +## Statistical Analyses + +Continuous variables are summarized as means ±standard deviation. Categorical variables are summarized as numbers (%). Floor and ceiling effects were evaluated when 15% or more of the subjects achieved the lowest (0) or highest (100) possible points on the SST-Jp.19 Internal consistency was assessed by Cronbach's alpha (a), which was calculated at an anticipated value ranging between 0.65 and 0.95.4 Intraclass correlation coefficients (ICCs) were calculated to assess the test-retest reliability where values > 0.7 were assumed to sufficiently support test-retest reliability.27 Measurement error is expressed as the standard error of measurement (SEM) or the minimal detectable change (MDC). The SEM was calculated using the square root of the variance between the measurements and the error variance of the ICC. The MDC was calculated as the SEM*1.96√2. + +The DASH and SF-36 scores were used to assess the construct validity of the SST-Jp. Construct validity was evaluated using Spearman's rank correlation coefficients because they do not rely on normal distributions (using the Kolmogorov-Smirnov test). + +Correlation coefficients were categorized as very weak or negligible +(below 0.20), weak or low (between 0.20 and 0.40), moderate (between 0.40 and 0.70), strong or high (between 0.70 and 0.90), +and very strong or very high (above 0.90).11 All tests were 2-tailed, and P < .05 was considered statistically significant. All statistical analyses were performed using SPSS, version 24.0 (SPSS Japan Inc., Tokyo, Japan). + +## Results Demographic Data + +The mean age of the participants was 64.3 years (range: 56 to 72 years), and 46% (n ¼ 46) were women. In 53 cases, pain and/or dysfunction was in the right shoulder. The disorders were classified as rotator cuff tear in 59 cases, frozen shoulder in 25 cases, shoulder periarthritis in 9 cases, osteoarthritis in 2 cases, and "other" in 5 cases (Table I). The SST-Jp showed no missing data. In total, 3% of the participants reported the worst possible score (0 points) and 5.0% reported the best possible score (100 points). + +## Reliability + +Table II summarizes the reliability of the SST-Jp. The internal consistency for the 12 questions in the SST-Jp was high (a ¼ 0.826). + +The ICC of the SST-Jp was 0.859 (95% confidence interval: +0.715e0.930; MDC ¼ 3.39%). + + JSES International 5 (2021) 334e337 + +| Table III Correlation of the SST-Jp with the DASH and SF-36 (validity) Instrument | r (P value)* SST-Jp | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------| +| DASH | 0.717 (P < .001) | +| SF-36 subscales Physical function | 0.493 (P < .001) | +| Role physical | 0.545 (P < .001) | +| Bodily pain | 0.545 (P < .001) | +| General health | 0.200 (P ¼ .046) | +| Vitality | 0.223 (P ¼ .025) | +| Social function | 0.345 (P ¼ .005) | +| Role emotion | 0.350 (P < .001) | +| Mental health | 0.307 (P < .001) | +| SST-Jp, Japanese version of the Simple Shoulder Test, DASH, Disability of Arm, Shoulder and Hand, SF-36, Short-Form 36. * Calculated using Spearman's rank correlation coefficient of the SST-Jp with DASH and SF-36. | | +| Table I Baseline characteristics of the participants Characteristics (n ¼ 100) | Total | +| Sex, % Male | 54.0 | +| Female | 46.0 | +| Age (years), mean (SD) | 64.3 (10.5) | +| Affected side, % Right | 53.0 | +| Left | 47.0 | +| Diagnosis, % Rotator cuff tear | 59.0 | +| Frozen shoulder | 25.0 | +| Periarthritis | 9.0 | +| Osteoarthritis | 2.0 | +| Others | 5.0 | +| SD, standard deviation. | | + +## Validity + +The validity results of the SST-Jp are shown in Table III. There was a strong correlation between the DASH and the SST-Jp (r ¼ +0.717). The SST-Jp was shown to have a significant correlation with all SF-36 subscales, as follows: a moderate correlation with the PF +(r ¼ 0.493), RP (r ¼ 0.545), and BP (r ¼ 0.545) subscales and a weak correlation with the GH (r ¼ 0.200), vitality (r ¼ 0.223), SF (r ¼ +0.279), SF (r ¼ 0.345), role emotional (r ¼ 0.350), and GH (r ¼ 0.307) +subscales. + +## Discussion + +This study aimed to culturally adapt and validate the SST for use in Japan. The results show that the SST-Jp presented in this study has high internal consistency and reliability for the subscores and the total score and, therefore, would be a reliable and valid measure of shoulder pain and function that can be implemented in Japanese clinical practice. In addition, the SST-Jp showed a strong correlation with the DASH and moderate correlation with the SF-36 physical subscales, including the PF, RP, and BP. + +The values for internal consistency in the present study were similar to those reported from other studies to adapt the SST for different cultural contexts.5,18,20,21,28 The ICC indicated good reproducibility in the present study, and the value was similar or slightly higher than the values obtained in the other versions of the SST.5,18,20,21,28 In addition, the construct validity of the SST-Jp was good, as evidenced by a strong correlation between the SST-Jp and DASH, which was similar or higher than that observed in the Dutch, Spanish, and Persian versions of the SST (r ¼ 0.60e0.74).5,20,28 Although the SST-Jp showed moderate correlation with the physical subscales, such as PF, RP, and BP, they were lower than the correlations observed between the SST-Jp and the DASH. Furthermore, other subscales of the SF-36 showed a weak correlation with the SST-Jp. The SST-Jp was assumed to reflect the pain and function of the shoulder better than the general health. These results are consistent with the results obtained with the Persian version of the SST.5 + +| Cronbach's alpha and ICC of the SST-Jp (reliability) Instrument Cronbach's alpha ICC | 95% CI | | | +|-------------------------------------------------------------------------------------------------------------------------|----------|-------|-------------| +| SST-Jp | 0.826 | 0.859 | 0.715e0.930 | +| SST-Jp, Japanese version of the Simple Shoulder Test, ICC, intraclass correlation coefficient, CI, confidence interval. | | | | + +One strength of this study was the standardized method for cross-cultural adaptation. Furthermore, the participants were recruited from 4 medical centers, which reduced potential selection bias. However, this study has several limitations to consider. First, owing to the cross-sectional design, the sensitivity to change +(responsiveness) and the minimal clinical difference in responsiveness (perceptibility) were not analyzed. Second, the mean age of this study's participants was slightly high. Third, a sample size calculation was not performed before study recruitment to determine the optimal sample size for robust statistical analysis. + +Although 100 patients can be considered an excellent sample size, 36 patients for the test-retest reliability assessment is only moderately good.27 It would be necessary to increase the number of patients, especially to strengthen the test-retest reliability outcome. + +## Conclusion + +The proposed SST-Jp has satisfactory internal consistency, testretest reliability, and construct validity. Therefore, we recommend the SST-Jp for evaluating patients with shoulder dysfunction in the Japanese population. + +## Supplementary Data + +Supplementary data to this article can be found online at https://doi.org/10.1016/j.jseint.2020.10.018. + +## Acknowledgments + +The authors would like to thank the study participants and Editage (www.editage.com) for English language editing. + +## Disclaimer + +The authors, their immediate families, and any research foundations with which they are affiliated have not received any financial payments or other benefits from any commercial entity related to the subject of this article. + +## References + +1. Angst F, Schwyzer HK, Aeschlimann A, Simmen BR, Goldhahn J. Measures of adult shoulder function: Disabilities of the arm, shoulder, and hand questionnaire (DASH) and its short version (QuickDASH), shoulder pain and disability index (SPADI), American Shoulder and Elbow Surgeons (ASES) Society standardized shoulder assessment form, constant (Murley) score (CS), simple shoulder test (SST), Oxford shoulder score (OSS), shoulder disability questionnaire (SDQ), and Western Ontario shoulder instability index (WOSI). Arthritis Care Res (Hoboken) 2011;63:S174. https://doi.org/10.1002/acr.20630. + +2. Beaton DE, Bombardier C, Guillemin F, Ferraz MB. Guidelines for the process of cross-cultural adaptation of self-report measures. Spine (Phila Pa 1976) +2000;25:3186-91. + +3. Burbank KM, Stevenson JH, Czarnecki GR, Dorfman J. Chronic shouler pain part I: Evaluation and diagnosis. Am Fam Physician 2008;77:453-60. + +4. Cronbach LJ. Coefficient alpha and the internal structure of tests. Psychometrika 1951;16:297-334. + +5. Ebrahimzadeh MH, Vahedi E, Baradaran A, Birjandinejad A, SeyyedHoseinian SH, Bagheri F, et al. Psychometric properties of the Persian version of the Simple Shoulder Test (SST) Questionnaire. Arch Bone Jt Surg 2016;4:387-92. + +6. Ekeberg OM, Bautz-Holter E, Tveita EK, Keller A, Juel NG, Brox JI. Agreement, reliability and validity in 3 shoulder questionnaires in patients with rotator cuff disease. BMC Musculoskelet Disord 2008;9:68. https://doi.org/10.1186/14712474-9-68. + +7. Fukuhara S, Bito S, Green J, Hsiao A, Kurokawa K. Translation, adaptation, and validation of the SF-36 Health Survey for use in Japan. J Clin Epidemiol 1998;51:1037-44. + +8. Fukuhara S, Ware JE Jr, Kosinski M, Wada S, Gandek B. Psychometric and clinical tests of validity of the Japanese SF-36 Health Survey. J Clin Epidemiol 1998;51:1045-53. + +9. Guillemin F, Bombardier C, Beaton D. Cross-cultural adaptation of healthrelated quality of life measures: literature review and proposed guidelines. + +J Clin Epidemiol 1993;46:1417-32. + +10. Hagiwara Y, Sekiguchi T, Yabe Y, Sugawara Y, Watanabe T, Kanazawa K, et al. + +Living status, economic hardship and sleep disturbance were associated with subjective shoulder pain in survivors of the Great East Japan Earthquake: A +cross sectional study. J Orthop Sci 2017;22:442-6. https://doi.org/10.1016/ j.jos.2016.12.027. + +11. Haldorsen B, Svege I, Roe Y, Bergland A. Reliability and validity of the Norwegian version of the Disabilities of the Arm, Shoulder and Hand questionnaire in patients with shoulder impingement syndrome. BMC Musculoskelet Disord 2014;15:78. https://doi.org/10.1186/1471-2474-15-78. + +12. Hudak PL, Amadio PC, Bombardier C. Development of an upper extremity outcome measure: the DASH (disabilities of the arm, shoulder and hand) [corrected]. The Upper Extremity Collaborative Group (UECG). Am J Ind Med 1996;29:602-8. + +13. Imaeda T, Toh S, Nakao Y, Nishida J, Hirata H, Ijichi M, et al. Validation of the Japanese Society for Surgery of the Hand version of the Disability of the Arm, Shoulder, and Hand questionnaire. J Orthop Sci 2005;10:353-9. https://doi.org/ 10.1007/s00776-005-0917-5. + +14. Kirkley A, Alvarez C, Griffin S. The development and evaluation of a diseasespecific quality-of-life questionnaire for disorders of the rotator cuff: The Western Ontario Rotator Cuff Index. Clin J Sport Med 2003;13:84-92. https:// +doi.org/10.1097/00042752-200303000-00004. + +15. Largacha M, Parsons IMt, Campbell B, Titelman RM, Smith KL, Matsen F 3rd. + +Deficits in shoulder function and general health associated with sixteen common shoulder diagnoses: a study of 2674 patients. J Shoulder Elbow Surg 2006;15:30-9. https://doi.org/10.1016/j.jse.2005.04.006. + + +16. Lippitt SB, Harryman DT, Matsen FA, Lippitt S, Lippit S. A practical tool for evaluating function: The simple shoulder test. The shoulder: a balance of mobility and stability. Rosemont (IL): American Academy of Orthopaedic Surgeons; 1993. + +17. Luime J, Koes B, Hendriksen I, Burdorf A, Verhagen A, Miedema H, et al. + +Prevalence and incidence of shoulder pain in the general population; a systematic review. Scand J Rheumatol 2004;33:73-81. https://doi.org/10.1080/ +03009740310004667. + +18. Marchese C, Cristalli G, Pichi B, Manciocco V, Mercante G, Pellini R, et al. Italian cross-cultural adaptation and validation of three different scales for the evaluation of shoulder pain and dysfunction after neck dissection: University of California-Los Angeles (UCLA) Shoulder Scale, Shoulder Pain and Disability Index (SPADI) and Simple Shoulder Test (SST). Acta Otorhinolaryngol Ital 2012;32:12. + +19. McHorney CA, Tarlov AR. Individual-patient monitoring in clinical practice: are available health status surveys adequate? Qual Life Res 1995;4: +293-307. + +20. Membrilla-Mesa M, Tejero-Fernandez V, Cuesta-Vargas A, Arroyo-Morales M. + +Validation and reliability of a Spanish version of Simple Shoulder Test (SST-Sp). + +Qual Life Res 2015;24:411-6. https://doi.org/10.1007/s11136-014-0760-2. + +21. Neto JOB, Gesser RL, Steglich V, Ferreira APB, Gandhi M, Vissoci JR, et al. + +Validation of the Simple Shoulder Test in a Portuguese-Brazilian population. Is the latent variable structure and validation of the Simple Shoulder Test Stable across cultures? PLoS One 2013;8:e62890. https://doi.org/10.1371/ +journal.pone.0062890. + +22. Paananen M, Taimela S, Auvinen J, Tammelin T, Zitting P, Karppinen J. Impact of self-reported musculoskeletal pain on health-related quality of life among young adults. Pain Med 2011;12:9-17. https://doi.org/10.1111/j.15264637.2010.01029.x. + +23. Roach KE, Budiman-Mak E, Songsiridej N, Lertratanakul Y. Development of a shoulder pain and disability index. Arthritis Rheum 1991;4:143-9. + +24. Schmidt S, Ferrer M, Gonzalez M, Gonz alez N, Valderas JM, Alonso J, et al. + +Evaluation of shoulder-specific patient-reported outcome measures: a systematic and standardized comparison of available evidence. J Shoulder Elbow Surg 2014;23:434-44. https://doi.org/10.1016/j.jse.2013.09.029. + +25. Sekiguchi T, Hagiwara Y, Sugawara Y, Tomata Y, Tanji F, Watanabe T, et al. + +Influence of subjective economic hardship on new onset of neck pain (socalled: katakori) in the chronic phase of the Great East Japan Earthquake: A +prospective cohort study. J Orthop Sci 2018;23:758-64. https://doi.org/ +10.1016/j.jos.2018.04.011. + +26. Takasawa E, Yamamoto A, Kobayashi T, Tajika T, Shitara H, Ichinose T, et al. + +Characteristics of neck and shoulder pain in the Japanese general population. J Orthop Sci 2015;20:403-9. https://doi.org/10.1007/s00776-014-0676-2. + +27. Terwee CB, Mokkink LB, Knol DL, Ostelo RW, Bouter LM, de Vet HC. Rating the methodological quality in systematic reviews of studies on measurement properties: a scoring system for the COSMIN checklist. Qual Life Res 2012;21: 651-7. https://doi.org/10.1007/s11136-011-9960-1. + +28. van Kampen DA, van Beers LW, Scholtes VA, Terwee CB, Willems WJ. Validation of the Dutch version of the Simple Shoulder Test. J Shoulder Elbow Surg 2012;21:808-14. https://doi.org/10.1016/j.jse.2011.09.026. \ No newline at end of file diff --git a/medical/md/PMC7954265.md b/medical/md/PMC7954265.md new file mode 100644 index 0000000000000000000000000000000000000000..f7f6ce127862662bc73d61dcc98f8e3906dd0504 --- /dev/null +++ b/medical/md/PMC7954265.md @@ -0,0 +1,183 @@ + + +![0_image_1.png](0_image_1.png) + +Ilio-femoral venous thrombosis with hereditary + +![0_image_0.png](0_image_0.png) + +antithrombin deficiency: a case report of rare thrombotic disease and successful treatment with catheter directed thrombolysis Hirokazu Miyashita 1, Kazuki Tobita1*,Eriko Morishita2, and Shigeru Saito 1 1Department of Cardiology, Shonan Kamakura General Hospital, 1370-1 Okamoto, Kamakura 247-8533, Japan; and 2Department of Clinical Laboratory Science, Kanazawa University School of Medical Science, Kanazawa, Japan Received 26 June 2020; first decision 24 August 2020; accepted 21 November 2020 + +| Background | Hereditary antithrombin (AT) deficiency is an uncommon autosomal dominant thrombogenic disorder, which can cause venous thromboembolism (VTE). Although conservative treatment options for hereditary AT deficiencyassociated VTE such as anticoagulation (warfarin, direct oral anticoagulant, or heparin), intravenous thrombolysis, and recombinant AT are well known, interventional treatment options have not been reported so far. | +|--------------|---| +| Case summary A 19-year-old man with a family history of thrombogenic diseases, referred to our hospital with left leg pain, was diagnosed with AT deficiency-associated VTE. In the absence of symptomatic relief with intravenous thrombolysis and anticoagulation, he received venous intervention and catheter directed thrombolysis (CDT) for 4 days for left iliac venous thrombosis. Following a second venous intervention, venous thrombus disappeared almost entirely on cross-sectional imaging, and his symptoms improved. He was discharged on apixaban and has been recurrence-free for one and a half years Discussion This case presents CDT and maintenance therapy with apixaban as possible treatment options for VTE in patients with hereditary AT deficiency, especially following failure of conservative therapy. Individual risks and benefits should be considered when CDT is performed for acute VTE in patients with AT deficiency. Keywords Case report - Hereditary antithrombin deficiency - Venous thromboembolism - Deep vein thrombosis - Catheter directed thrombolysis | | + +## Learning Points + +- When a young patient developed venous thromboembolism (VTE), antithrombin deficiency should be considered as differential diagnosis. - Catheter directed thrombolysis and venous intervention should be considered for acute VTE irrespective of the underlying diseases when conservative treatments are not effective. + +. + +. + +. . . + +. + +. . . . + +. + +. . + +. . + +. + +. + +. + +. + +. + +. . + +. . . . . . + +. . . . + +. + +. . + +. Introduction Hereditary antithrombin (AT) deficiency is an uncommon autosomal dominant thrombogenic disorder, with an estimated prevalence in the general population of 0.02–0.2%.1 Individuals with hereditary AT +deficiency are at an increased risk of venous thromboembolism +(VTE) (odds ratio 16.3; 95% confidence interval 9.9–26.7).2 Owing to its low prevalence, developing a treatment strategy for VTE in patients with AT deficiency is challenging. Herein, we report a successful venous interventional treatment for a patient with AT deficiency and acute VTE. + +## Timeline + +Laboratory investigations were as follows: D-dimer level, 23.1 lg/ +mL (normal < 1.0 lg/mL); AT activity, 49.3% (normal, 75–125%); protein C/S activity and lupus anticoagulant, normal range; and antibodies related to antiphospholipid antibody syndrome, negative. Echocar diogram showed no signs of right heart overload. Duplex ultrasound of his left leg showed thrombotic occlusion from the left femoral vein to left iliac vein. + +Based on his family history and AT levels, a diagnosis of hereditary AT deficiency was made, and following consent, anticoagulant therapy was initiated. We prescribed apixaban 10 mg twice a day as loading dose for 7 days along with 275 000 units/kg of monteplase intravenously. Monteplase was administrated only once. Owing to + +| Timeline Timeline Description Five days prior to admission The patient, who had a family history of thrombogenic disease, developed acute left leg pain. Day 0 Vital signs were stable, but he complained of swelling, continuous pain, and tenderness of his left thigh to left groin. Pulmonary embolism and thrombotic occlusion in left iliac to femoral vein were confirmed by contrast enhanced computerized tomography (CT) and duplex ultrasound. Day 1 Inferior vena cava (IVC) filter deployment and intravenous monteplase administration was performed, and apixaban 10 mg 2 per day was prescribed. Day 3 Symptoms hadn't changed, and follow-up CT showed large amount of thrombus in left iliac and left femoral vein. Day 4 First venous intervention was performed and FOUNTAIN INFUSION catheterTM (Merit Medical, USA) was deployed from left femoral to iliac vein. Days 5–7 Catheter directed thrombolysis (CDT) was performed for 3 days. The CDT infusion contents were continuous heparin and argatroban, with intermittent urokinase four times a day. Day 8 Second venous intervention, which included thrombo-aspiration in left femoral and iliac vein and balloon dilatation, was performed. Day 11 Follow-up CT showed that left iliac vein thrombus disappeared. Day 12 IVC filter was removed. Day 17 He discharged on apixaban 5 mg twice a day. Two months after discharge Genetic test for hereditary antithrombin deficiency proved to be positive. | +|---| + +## Case Presentation + +A 19-year-old man without any medical history was referred to our hospital, in August 2018, with acute left leg pain that developed over 5 days. His father and paternal relatives had thrombotic disease; however, genetic diagnosis hadn't been performed. The patient did not report any other transient or reversible risk factors of venous thrombosis and did not take any medication. Contrast enhanced computed tomography (CT) performed at the referring hospital identified evidence of pulmonary embolus (Figure 1), and presence of venous thrombus extending from the left popliteal vein to the inferior vena cava (IVC). + +His vital signs were as follows: blood pressure, 133/68 mmHg; heart rate, 79/min, regular; oxygen saturation at room air, 99%; and respiratory rate, 22/min. Cardiac and pulmonary auscultation were normal. There was no elevated jugular venous pulse, and no peripheral oedema. The patient had swelling involving the left thigh and groin, which was tender to touch. + +the large size of the venous thrombus, a Gu¨nther Tulip IVC filter (Cook Medical, USA) was deployed on the first day of admission (Day 1). His symptoms failed to improve despite 2 days of treatment. Follow-up contrast enhanced CT on Day 3 showed no change in the size of the thrombus (Figure 2). Hence, we decided to perform venous intervention on Day 4 to reduce the risk of development of postthrombotic syndrome (PTS). + +We employed a trans-popliteal vein approach with 6 Fr introducer sheath (Terumo, Japan). Preliminary angiogram identified total occlusion of the left femoral vein with thrombus (Figure 3A). Despite repeated thrombo-aspiration with 6 Fr Mach1 (Boston Scientific, USA), the deep femoral vein remained occluded with thrombus (Figure 3B). Subsequently, we deployed a Fountain infusion catheter (Merit Medical, USA) through his femoral vein into the iliac vein and injected 120 000 units of urokinase. This was followed by continuous injections of heparin (14 000 units per day) and argatroban (20 mg per day), and intermittent injection of urokinase (60 000 units four times a day) as catheter directed thrombolysis (CDT). On Day 8, + +![2_image_0.png](2_image_0.png) + +after removing the Fountain infusion catheter, we performed thrombo-aspiration again and dilated the vein with 7.0/200 mm and 8.0/80 mm semi-compliant balloon, which resulted in improved venous flow (Video 1). We performed an intravascular ultrasound as the venous angiogram showed proximal iliac vein stenosis, which revealed that iliac vein compression between the lumbar spine and iliac artery (Figure 4). + +Follow-up CT on Day 11 confirmed clearance of almost all venous thrombus (Figure 5). We removed the IVC filter on Day 12. The patient was discharged on Day 17 with apixaban 5 mg twice a day, which was considered lifelong prescription. Two months after his hospitalization, genetic testing confirmed hereditary AT deficiency (Exon 6, c.1171C>T, p.Arg359Ter). The patient has completed 18 months of follow-up without any recurrence of VTE or other treatment related complications. + +![2_image_1.png](2_image_1.png) + +## Discussion + +This case demonstrates successful CDT for ilio-femoral venous thrombosis in a patient with AT deficiency. + +Management of acute VTE in patients with AT deficiency is not different from that of VTE in general.3 Thrombolytics, heparin or fondaparinux, and vitamin K antagonists are the conventional treatment modalities, although there is a theoretical risk of heparin resistance in patients with AT deficiency.4 Currently, direct oral anticoagulants (DOACs) are also prescribed for the treatment of VTE.5 A current consensus document for deep vein thrombosis (DVT) from the European Society of Cardiology (ESC) recommends anticoagulation for all patients with proximal DVT, and adjuvant CDT for selected patients.6 However, it does not provide any details regarding the management of patients with hereditary AT deficiency. Recent case reports described the successful use of rivaroxaban in the treatment of VTE in a patient with hereditary AT deficiency.7,8 DOACs are effective for VTE treatment regardless of anti-thrombin deficiency status, as they directly affect factor Xa. One randomized trial demonstrated decreased incidence of subsequent PTS but increased bleeding episodes with the use of CDT in addition to anticoagulation for patients with proximal DVT.9 In contrast, another randomized trial did not demonstrate any substantial benefit of pharmacochemical therapy, which was defined as catheter or device-mediated intrathrombus delivery of recombinant tissue plasminogen activator and thrombus aspiration or maceration, and CDT over anticoagulation.10 However, neither of these studies included patients with AT deficiency. Therefore, the strategy for acute VTE and DVT in patients with AT deficiency, especially with the advent of novel treatment options, can only be empirical owing to the small number of cases. In the current case, we performed CDT following the failure of + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +![3_image_2.png](3_image_2.png) + +. . . + +. + +. + +. + +. + +. + +. . . + +. + +. + +. + +. . . + +. . . + +. anticoagulation therapy. Furthermore, the patient also satisfied the ESC criteria for CDT,6 and was at low risk of bleeding owing to his age and physical status. + +According to the current guidelines, the indications for IVC filter placement are: anticoagulation is absolutely contraindicated in patients with proximal DVT, recurrent pulmonary embolism despite adequate anticoagulation, and as primary prophylaxis in patients with a high risk of VTE.6,11 However, the effect of IVC filter placement on CDT has never been discussed. We considered that CDT has a risk of destabilization of thrombus, which may develop pulmonary embolus. Our use of IVC filter was not based on existing guidelines. + +Although our patient did not develop any complications related to IVC filters, their use should be carefully considered. There is a need for further studies focusing on IVC filter placement and CDT. + +![4_image_0.png](4_image_0.png) + +Figure 5 Follow-up computed tomography on Day 11. + +Thrombus in left iliac vein had disappeared (red dotted line). + +The duration of anticoagulation is a potential area of difficulty for this patient. Since hereditary AT deficiency is neither transient nor reversible, the patient has a high estimated risk for long-term recurrence of VTE.11 This patient also had iliac venous compression syndrome (IVCS), which may have been an additional contributor to VTE. Left iliac vein stenting may appear to be optimal treatment for IVCS.12 However, we were reluctant to deploy a stent owing to his age and underlying thrombogenic condition. Our opinion was that this patient required lifelong anticoagulant therapy owing to the irreversible risk factor of AT deficiency, even in the absence of IVCS. + +In conclusion, this case presents CDT as an effective option in treating acute DVT in patients with AT deficiency. As for any DVT, CDT should be considered in the event of failure of anticoagulant treatment alone. This strategy is important for prevent PTS. There is a need for further studies to standardize the treatment of DVT in patients with AT deficiency. + +## Lead Author Biography + +![4_Image_1.Png](4_Image_1.Png) + +Hirokazu Miyashita graduated from Kanazawa University in 2014. He was trained as a clinical resident in Shonan Kamakura General Hospital for 2 years, and joined department of cardiology at the same hospital in 2016. He has learned interventional cardiology, peripheral artery diseases, and structure heart diseases. + +Since 2020, he is studying abroad as a research fellow in Helsinki University Hospital. + +## Supplementary Material + +Supplementary material is available at European Heart Journal - Case Reports online. Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Supplementary data. Consent: The authors confirm that written consent for submission and publication of this case report including images and associated text has been obtained from the patient in line with COPE guidance. Conflict of interest: S.S. reports lecture fees from TERUMO Corporation, Japan. All other authors declare that there is no conflict of interest. + +Funding: none declared. + +## References + +1. Tait RC, Walker ID, Perry DJ, Islam SI, Daly ME, McCall F et al. Prevalence of antithrombin deficiency in the healthy population. Br J Haematol 1994;87: 106–112. + +2. Di Minno MN, Ambrosino P, Ageno W, Rosendaal F, Di Minno G, Dentali F. + +Natural anticoagulants deficiency and the risk of venous thromboembolism: a meta-analysis of observational studies. Thromb Res 2015;135:923–932. + +3. Patnaik MM, Moll S. Inherited antithrombin deficiency: a review. Haemophilia 2008;14:1229–1239. + +4. Schulman S, Tengborn L. Treatment of venous thromboembolism in patients with congenital deficiency of antithrombin III. Thromb Haemost 1992;68:634–636. + +5. Becattini C, Agnelli G. Treatment of venous thromboembolism with new anticoagulant agents. J Am Coll Cardiol 2016;67:1941–1955. + +6. Mazzolai L, Aboyans V, Ageno W, Agnelli G, Alatri A, Bauersachs R et al. + +Diagnosis and management of acute deep vein thrombosis: a joint consensus document from the European Society of Cardiology working groups of aorta and peripheral vascular diseases and pulmonary circulation and right ventricular function. Eur Heart J 2018;39:4208–4218. + +7. Minami K, Kumagai K, Sugai Y, Nakamura K, Naito S, Oshima S. Efficacy of oral factor Xa inhibitor for venous thromboembolism in a patient with antithrombin deficiency. Intern Med 2018;57:2025–2028. + +8. Yamaguchi J, Hara N, Yamaguchi T, Nagata Y, Nozato T, Miyamoto T. Successful treatment of a massive pulmonary embolism using rivaroxaban in a patient with antithrombin III deficiency. J Cardiol Cases 2017;16:144–147. + +9. Haig Y, Enden T, Grøtta O, Kløw NE, Slagsvold CE, Ghanima W et al. Postthrombotic syndrome after catheter-directed thrombolysis for deep vein thrombosis (CaVenT): 5-year follow-up results of an open-label, randomised controlled trial. Lancet Haematol 2016;3:e64–e71. + +10. Vedantham S, Goldhaber SZ, Julian JA, Kahn SR, Jaff MR, Cohen DJ et al. + +Pharmacomechanical catheter-directed thrombolysis for deep-vein thrombosis. N Engl J Med 2017;377:2240–2252. + +11. Konstantinides SV, Meyer G, Becattini C, Bueno H, Geersing GJ, Harjola VP et al.; ESC Scientific Document Group. 2019 ESC Guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the European Respiratory Society (ERS). Eur Heart J 2020;41:543–603. + +12. Radaideh Q, Patel NM, Shammas NW. Iliac vein compression: epidemiology, diagnosis and treatment. Vasc Health Risk Manag 2019;15:115–122. \ No newline at end of file diff --git a/medical/md/PMC9054581.md b/medical/md/PMC9054581.md new file mode 100644 index 0000000000000000000000000000000000000000..95b5f58bec34b72e9a61e054677b4cf64ba250c5 --- /dev/null +++ b/medical/md/PMC9054581.md @@ -0,0 +1,271 @@ +Cite this: RSC Adv., 2020, 10, 22440 + +# Towards 3D-Lithium Ion Microbatteries Based On + +![0_Image_0.Png](0_Image_0.Png) Silicon/Graphite Blend Anodes Using A Dispenser Printing Technique† + +Mathias Drews, a Sebastian Tepner,a Peter Haberzettl,a Harald Gentischer,a Witali Beichel,a Matthias Breitwieser,bc Severin Vierrath b and Daniel Biro*a In this work we present for the first time high capacity silicon/carbon–graphite blend slurries designed for application in 3D-printed lithium ion microbatteries (3D-MLIBs). The correlation between electrochemical and rheological properties of the corresponding slurries was systematically investigated with the prospect of production by an automated dispensing process. A variation of the binder content (carboxymethyl cellulose/styrene–butadiene rubber, CMC/SBR) between 6 wt%, 12 wt%, 18 wt% and 24 wt% in the anode slurry proved to be crucial for the printing process. Regarding the rheological properties increasing binder content leads to increased viscosity and yield stress values promising printed structures with high aspect ratios. Consequently, interdigital 3D-printed micro anode structures with increasing aspect ratios were printed with increasing binder content. For printed 6-layer structures aspect ratios of 6.5 were achieved with anode slurries containing 24 wt% binder. Electrochemical results from planar coin cell measurements showed that anodes containing 12 wt% CMC/SBR binder content exhibited stable cycling at the highest charge capacities of 484 mA h g1 at a current rate of C/4. Furthermore, at 4C the cells showed high capacity retention of 89% compared to cycling at C/4. Based on this study and the given material formulation we recommend 18 wt% CMC/SBR as the best trade-off between electrochemical and rheological properties for future work with fully 3D-printed MLIBs. + +Received 8th April 2020 Accepted 27th May 2020 DOI: 10.1039/d0ra03161e rsc.li/rsc-advances + +## Introduction + +The increasing market of small electronic devices in the eld of the internet of things (IOT) such as wearables, smart electronics and medical implants requires suitable energy storage devices. + +Lithium ion batteries (LIB) are currently the batteries of choice for microelectronics, due to their high gravimetric (W h kg1) +and volumetric energies (W h l1).1 However, conventionally designed LIBs (i.e. pouch, prismatic, cylindrical) are difficult to miniaturize. On the other hand supercapacitors and thin lm batteries can be miniaturized and are already used in microelectronic devices, but at the expense of energy density.2–5 For thin lm batteries energy and power density can be increased by achieving 3D-structured cathodes.6,7 Additive manufactured microbatteries are a promising technology that can handle the integration problem into electronic devices and have the aFraunhofer Institute for Solar Energy Systems ISE, Heidenhofstraße 2, 79110 Freiburg im Breisgau, Germany. E-mail: daniel.biro@ise.fraunhofer.de; Tel: +49-761-4588-5246 bElectrochemical Energy Systems, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg im Breisgau, Germany cHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg im Breisgau, Germany +† Electronic supplementary information (ESI) available. See DOI: +10.1039/d0ra03161e potential to reach high power at high energy density levels simultaneously. To achieve this, the principle idea of 3Dmanufactured batteries is to decouple ionic and electronic transport processes. In the additive manufacturing process active material is deposited in the z-direction of the current collector which leads to tall and preferable small 3D-battery structures. Such high aspect ratio (AR) electrodes are considered to have the advantage of increased energy per footprint area whereas the electrode thickness and therefore the ionic pathway is kept constant. Thus, energy density can be increased by a steady value of power density where the major rate limiting factor is the electric resistance of the electrodes.8 In conventional planar electrode batteries either power or energy density can be optimized by adjusting the electrode thickness.9 The basic idea of 3D-printed microbatteries is illustrated in Fig. 1. + +There are several different ways to manufacture 3D-battery structures e.g. by using electrodeposition,10,11 inkjet12,13 and dispenser printing techniques.8,14–16 Additive manufacturing based on dispenser printing represents a simple and low cost microbattery production process.17 Furthermore, slurry formulation can be adopted from industrial battery cell production by adjusting the amount or type of specic components. Fu et al. + +presented an interdigitated lithium ion microbattery (IMLIB) +based on lithium iron phosphate/graphene oxide (LFP/GO) cathodes and lithium-titanate/graphene oxide (LTO/GO) + +![1_image_0.png](1_image_0.png) + +anodes with 10 wt% of PVDF binder. The printed full cell with a size of 7 3 mm2 showed limited cycling behavior <20 cycles with a capacity of 100 mA h g1 at a current rate of 50 mA g1. + +15 So far, the smallest printed 3D-battery structures using the dispenser printing approach were published by Sun et al. in 2013.8 The cell consisting of an LTO anode and LFP cathode and had dimensions of approximately 1 1 mm2 (5 anode and cathode ngers) at a height of approx. 190 mm. Wei et al. + +manufactured biphasic electrodes for customized planar lithium ion batteries with electrode thicknesses up to 1 mm. + +Areal energy density could be increased by more than one order of magnitude at constant areal power densities compared to electrodes with 0.05 mm thickness.14 With respect to slurry development for 3D-printed batteries Li et al. showed that the detailed investigation of electrochemistry and slurry rheology is crucial for a successful production of additive manufactured microbatteries.18 However, in the eld of suspension rheology and its impact on printability by the dispensing method Pospischil et al. demonstrated that there are more rheological parameters (e.g. yield stress), which need to be investigated, especially, when high aspect ratio dispensing is desired.19 Furthermore, in another work the same authors showed that aspect ratios of dispensed structures are strongly inuenced by the underlying surface properties and process parameters for a variety of different photovoltaics applications.20 Based on experiences from photovoltaic applications, we investigate in this study customized Si/C–graphite blend slurries as anode for 3D-MLIBs unprecedented in scientic reports. + +As it was already stated earlier in this manuscript energy density of 3D-printed batteries can be increased by depositing active material in the z-direction whereas power density of the cell remains constant. In addition to this fundamental property of additive manufactured batteries we have chosen high capacity Si/C–graphite blend anodes to increase the energy density of the cell not only from a geometrical but also a material perspective. The investigation of the corresponding slurries includes a systematic investigation of the correlation between slurry rheology and electrochemical battery characteristics. + +Rheological investigations are key to adjust dispenser printing properties regarding the quality of printed structures. The amount of binder was considered a crucial factor inuencing the electrochemical and rheological properties. Consequently, Si/C–graphite blend anode slurries were prepared with varying amounts of binder and investigated regarding the electrochemical rate performance, rheological behavior and the quality of the printed interdigitated structures. + +## Experimental Materials And Slurry Preparation + +For the preparation of Si/C (made from crystalline silicon nanoparticles APS z 100 nm, 99%, plasma synthesized, Alfa Aesar)–graphite blend anode slurries carboxymethyl cellulose +(CMC, Merck group) was suspended in deionized water by magnetic stirring for 6 h. When the dispersion was fully homogenized the styrene–butadiene rubber (SBR) dispersion +(85 wt% water, Targray group) was added. The ratio between CMC and SBR was 50/50 (wt%/wt%) and slurries with a binder content of 6, 12, 18 and 24 wt% were investigated. A binder content of 6 wt% was chosen since this value is close to planar industry-orientated Si/C–graphite blend anodes. However, binder contents of lower than 6 wt% were found to have relatively low viscosities and were thus suggested to be scarcely relevant for 3D-printing processes. In pretests, slurries with binder a content of 24 wt% were found to be difficult to process by laboratory equipment such as magnetic stirrers whereby a homogenization of the slurry via magnetic stirring could not necessarily be ensured. Aer another 30 min of stirring the Si/C, +graphite (APS 7 - 11 mm Alfa Aesar, 99%) and carbon black (Alfa Aesar, 100% compressed, 99.9%) were added. The amount of silicon in the blended material was aimed to be 12 wt%. For a detailed overview of the composition of each slurry see Table 1. Here solid fractions are listed for dispenser printing experiment. For rheological measurements values are given only for slurries with the highest solid content (aimed to be 40 wt%). In the ensuing sections the term "40 wt%" acts as an abbreviation for these numbers. Corresponding values for slurries with lower solid content can be found in the section regarding rheological characterization. For electrochemical measurements solid contents of 40 wt% were desired but not specically measured. All components were mixed (DAC 150/ +250, Speedmixer, Germany) at 3500 rpm for 2 min (2 times with 10 min rest). A three roll mill (80E, Exakt Advanced Technologies, Germany) with a large roll distance of 10 mm and a small roll distance of 5 mm was used to adjust the particle size of the components and to homogenize the slurry. The process time in the three roll mill was minimized (60–90 s) to have as less water evaporation as possible. For the determination of the solid fraction three samples were taken from the three roll mill and weighed before and aer drying. For rheology and dispenser printing the slurries were used immediately. For anode preparation the slurries were coated on copper foil via doctor blade technique with a wet lm thickness of 50 mm and dried at 313.15 K overnight. Aer predrying 14 mm coins were punched out and dried under vacuum at 393.15 K overnight. + +| Solid fraction [wt%] | | | | | | | +|------------------------|------------|----------------|--------------------|-----------|-----------|---------------------| +| Binder content | Si/C [wt%] | Graphite [wt%] | Carbon black [wt%] | CMC [wt%] | SBR [wt%] | Rheology/dispensing | +| 6 wt% | 18.8 | 69.5 | 5.7 | 3.0 | 3.0 | 42.5/41.7 | +| 12 wt% | 17.6 | 65.0 | 5.4 | 6.0 | 6.0 | 42.6/42.3 | +| 18 wt% | 16.4 | 60.6 | 5.0 | 9.0 | 9.0 | 44.1/43.4 | +| 24 wt% | 15.2 | 56.2 | 4.6 | 12.0 | 12.0 | 43.1/43.8 | + +## Physiochemical Characterization + +BET surface measurements of the Si/C material were performed on a QuadraSorb Station 3 (Quantachrome Instruments) +instrument. Raman measurements were conducted on an Alpha500 (WITec, Germany) confocal Raman microscope with a laser wavelength of 532 nm. X-ray diffraction patterns were collected using a Miniex 600 (Rigaku, Japan) with a copper source (l ¼ 1.54 A), a stepwidth of 0.02 ˚ and speed of 0.1 min1 operating at 40 kV and 15 mA. For particle size distribution measurements an LS 13 320 Laser Diffraction Particle Size Analyzer from Beckman Coulter was used.21,22 The samples were mixed thoroughly as aqueous colloid suspensions containing tensides to prevent the formation of aggregates. The colloid suspensions were dispersed by an ultrasonic lance before measurement (5 times for 60 s each). + +## Rheological Characterization + +For rheological measurements a commercial rotational rheometer (Anton Paar, Germany) was used. Two types of measurements were conducted to determine the shear rate dependent viscosity and the yield stress for each slurry. A measurement protocol according to Tepner et al. was used in this study.23,24 For both measurements, a plate–plate geometry was chosen. The upper plate had a surface roughness of 2–4 mm and was 25 mm in diameter at a measurement gap of 500 mm. + +All slurries were investigated regarding their shear viscosity. For this a successive shear controlled measurement protocol from 0.1 s1 to 200 s1 was chosen with 40 points per decade. Prior to the measurement, the edge of the rotating plate was cleared of excess slurry as described in literature.23 To investigate the yield stress of all slurries the procedure was similar with the exception that the measurement was shear stress controlled in the range from 1 to 10 000 Pa with 40 points per decade. Finally the critical yield stress was determined using the tangent intersection method.25 The mean values of three measurements are presented. All measurements were done at 25 C. To avoid preshearing each measurement was done with a new batch of slurry. + +## 3D-Dispenser Printing And Subsequent Analysis Of The Structure + +The dispensing experiments for the 3D-microbatteries were conducted on a commercially available table robot (VR3303, Vieweg, Germany). The robot allows for a precise relative movement between the dispensing nozzle and the substrate in 3 dimensions with an accuracy of 8 mm in the x,y-direction and 1 mm in the z-direction. Each structure was printed on a copper substrate. Before processing the copper was cleaned in an 18% +HCl solution. The offset between substrate and nozzle was set to 240 mm and the step size in the z-direction (rise of the nozzle aer each printed layer) was set to 140 mm. Depending on the rheological parameters of each slurry, it was necessary to evaluate the correct pressure in order to ensure a sufficient volume ow rate for a given speed (vx,y) of the table robot.26,27 Here the pressure was set to 1.2, 1.6, 2.0 and 3.2 bar for the sample with 6 wt%, 12 wt%, 18 wt% and 24 wt% binder, respectively. The diameter of the micro nozzle (Vieweg, Germany) was 110 mm. + +Fig. 2 shows a simple sketch of a section of an interdigitated battery structure including dimensioning (dening anode and cathode does not play an important role in this case). As it was already discussed high aspect ratios increase the energy density per footprint area. Thus, high ARs were desired by dispensing 8 layers on top of each other and the development of the AR was investigated at 2-, 4-, 6- and 8-layered structures. + +3D-printed anode structures were dried for 24 h at ambient conditions. A scanning electron microscope (Auriga 60, Zeiss, Germany) was used at an acceleration voltage of 7.0 kV to image the microstructure of the samples. A confocal laser microscope +(Lext Series, Olympus) was employed to measure the mean average height and the mean average width of the dispensed structures. The capacity per footprint area was calculated using the mass of the anode considering the active fraction and the base area of the structure. Regarding the latter the nger width was taken from microscopy analysis whereas nger lengths + +![2_image_0.png](2_image_0.png) + +were not directly measured but rather taken from the pathlength the dispensing robot travelled. + +## Electrochemical Characterization + +Half cells were assembled in an argon lled glovebox (E-Line, GS Glovebox System, Germany, O2 < 3 ppm, H2O < 0.5 ppm) +in CR2032 coin cells using Si/C–graphite blend electrodes, lithium metal as counter electrode, 1 mm glass bre separator +(EL-Cell, Germany) soaked with 150 ml LP30 (1 M LiPF6 in ethylene carbonate (EC)/dimethyl carbonate (DMC) 50/50 (v%/ +v%)) + 10 wt% (uoroethylene carbonate) FEC (Merck group) as electrolyte. Rate capability tests were performed on a BCS-805 +(BioLogic, France) battery tester in the voltage range between 1.5 V and 0.01 V in CC setup with a CV step at the lower cut-off voltage and 10 min rest aer each lithiation step. Formation cycles included two cycles at C/10 and two cycles at C/5. Cells were cycled at C/4, C/2, 1C, 2C and 4C each for 10 cycles. + +## Results And Discussion Material Composition Of Si/C Slurries + +The Si/C–graphite blend materials were characterized by Raman spectroscopy, X-ray diffraction and nitrogen physisorption experiments (BET surface). While bulk silicon shows a Raman mode at 521 cm1, the corresponding mode of the Si/C sample used in this study has a signal at 515 cm1. This shi is commonly known and due to the silicon particle size. Silicon nanoparticles show a shi to lower wavenumbers compared to the signal of bulk silicon.28,29 The carbon related D- and G-band is shown at 1350 cm1 and 1585 cm1, respectively30,31 (see Fig. S1†). The ratio between the area of the D- and G-band (A(D)/ +A(G)) is calculated as 2.5 0.6 which suggests a relatively high amount of disordered carbon in the Si/C anode material. In the XRD spectrum all silicon- and carbon-related peaks could be assigned to the corresponding reection planes (see Fig. S2†). + +The BET surface area of the Si/C particles was determined to be 14.1 m2 g1, which is in good accordance with literature values.32,33 + +## Particle Size Distribution + +Particle size has a major impact on both, the electrochemical properties and the printing processes. Si/C particles were ball milled in order to adjust particle size with respect to electrochemical and dispenser printing optimization. It is commonly known that smaller particle sizes promote electrochemical performance.34,35 Regarding dispensing processes particles need to reach sufficiently small sizes to prevent clogging of the nozzle.19 The latter is extremely important for industrial processes, where the breakdown of machines and signicant downtime is unacceptable in terms of production cost. However, Si/C particles should not be milled too small to maintain a homogeneous composite material and prevent fresh silicon surface formation. The latter would be detrimental for battery cell performance. Particle size distributions of the Si/C +composite material aer ball milling and exemplary of a Si/C– +graphite slurry with 18 wt% binder (only values of slurry with 18 wt% binder are shown since this slurry will be recommended for fully printed 3D-MLIBs) are given in the ESI.† D10 and D90 values of the Si/C composite are evaluated to 2.1 0.2 mm and 43.5 0.3 mm, respectively. Corresponding values for the slurry are 1.9 0.2 mm and 17.4 0.3 mm. The D90 values of the slurry are signicantly smaller compared to the pure composite which refers to the impact of the three roll mill on the slurry production. Since the Si/C composite are suggested to represent the biggest particles in the slurry, changes in characteristics of the particle size distribution are mostly attributed to the crushing of the Si/C particles. It needs to be mentioned that the three roll mill process is absolute necessary, since only aer this additional treatment the corresponding slurries become extractable through a 110 mm nozzle. + +## Rheological Properties Of Si/C Slurries + +Rheological investigation is a powerful instrument and essential to characterize slurries with respect to dispenser printing processes. Viscosity and yield stress values are signicant quantities for prediction of high AR structures and can be measured to estimate slurry behavior in micro nozzle openings. + +In Fig. 3a, the shear rate dependent viscosity for the different types of slurries is presented, revealing a strong non Newtonian ow behavior. Especially at low shear rates (101 to 100 s 1), the viscosity increases with the amount of binder in the sample. + +Regarding dispenser printing processes it was shown that slurries should have signicantly increased viscosities compared to slurries from standard coating processes and are in the range of 103 to 104 Pa at 1 s1. + +8,14,15 Thus the binder content should be increased to guarantee a high viscosity aer nozzle exit when thixotropic effects are neglected.19 During the dispensing process the shear rate in the micro nozzle correlates linearly with the process speed. For industrial dispenser process speeds the shear rate is surpassing 5000 s1, which cannot be measured with commercial rotational rheometers due to signicant sample spillage at high rotation speeds.23,36 Here all paste samples show similar viscosities at high shear rates (see Fig. 3a) but differ signicantly during dispensing experiments in terms of their printed line geometry. Thus the shear rate dependent viscosity is not the dominant factor when it comes to high speed dispensing through nozzle openings in the micrometer range. Pospischil et al. showed that the shape of the printed structure strongly correlates with the yield stress of the slurry, which describes the necessary shear stress where the slurry transitions from elastic- to plastic-deformation.19 Therefore, yield stress measurements are crucial for dispenser printing processes and presented in Fig. 3b. At a given shear stress, the rate of deformation changes from linear towards an exponential rate of increase. The intersection point denes the yield stress and is calculated by the tangent intersection method.25 In Fig. 3c, measurements of the yield stress for all corresponding combinations of the solid and the binder content are shown. The yield stress increases with increasing solid and binder content. A combination of both is quite promising to generate tall AR structures. However, increasing the solid content in the paste is limited because a sufficient + +![4_image_0.png](4_image_0.png) + +printability requires a certain content of the binder/solvent mixture which dictates the shear thinning nature of the paste and therefore the paste ow within the micro nozzle. To reach preferably high yield stresses for the dispenser printing experiments, only slurries with 40 wt% solid content were used. For lower solid contents of the sample with 6 wt% binder the measurement procedure of the yield stress does not create sufficient results because the yield stress value is too low. Thus, it is hardly possible to measure the elastic behavior of the sample, which fullls Hooke's Law in a reproducible manner. + +Therefore, yield stress values for the 6 wt% binder sample with a solid content below 40 wt% are not presented. + +## Dispenser Printed 3D-Structures + +High aspect ratio structures are promising for application in microbatteries with high energy and power density per footprint area. To investigate 3D-printed Si/C–graphite anodes with respect to their ARs, interdigitated structures were dispensed on copper substrates. Fig. 4 shows electron microscope images of printed 8-layer structures with slurries containing 12 wt%, 18 wt% and 24 wt% CMC/SBR binder. A structure with 6 wt% +binder is not illustrated since the single ngers overlapped and could not be resolved (for 2 and 4 layers it was possible to print apparent interdigitated structures). This can be attributed to low values of the corresponding yield stress (see Fig. 3b and c). + +Obviously the AR value (apparent as "sharpness" in Fig. 4a–c) of the structure increases with increasing binder content. + +Apart from that, all printed structures show strong adhesion to the copper substrate which is attributed to the adhesion properties of the CMC/SBR binder.37 Fig. 5a shows the height and the width values of printed structures in dependence of the binder content and the number of layers. All values were determined at two positions (see Fig. 4c) using a confocal laser microscope. Regarding the height of the structures it can be seen, that the values increase with increasing binder content, which is consistent with the increased viscosity (in the low shear rate regime) and yield stress. As expected, height values of all samples containing $12 wt% binder increase linearly with similar slopes. The sample with 6 wt% binder suggests a steeper increase of height. However, two data points might be insufficient to make a reliable comparison. The linear increase of height values is in good accordance with literature values.8,15,16 Additionally, Fu et al. presented values of ca. 1000 mm (height) at 6 layers which is very close to the data published here.15 In contrary, the width increases with decreasing binder content. It was suggested, that wider structures are due to a stronger melting tendency of the slurry. With respect to the rheological results it can be stated that higher rates of melting + +![5_image_0.png](5_image_0.png) + +![5_image_1.png](5_image_1.png) + +are related to decreased yield stress values and vice versa. + +19,27 Interestingly samples with 18 wt% and 24 wt% binder show constant values at all number of layers. In contrast to that, samples with 6 wt% and 12 wt% binder show slightly increasing values which suggests melting due to lower yield stress. Similar behavior of high yield stress slurries is reported in literature.8 Finally the resulting ARs are shown in Fig. 5b. In accordance with Fig. 5a ARs increase with increasing binder content and number of layers. It can be seen that the unsteady development of the AR values of the sample with 24 wt% binder is mainly due to minor uctuations of the corresponding width. A printed 6layer structure with 24 wt% binder shows the highest AR value of 6.5. The above presented dispensing results, i.e. height and width value evolution of the printed structures, are in accordance with rheological investigations, in particular with yield stress values. Here, it was conrmed that higher binder contents are necessary to get taller AR structures. Regarding the electrochemical characterization of the given anode structure with 18 wt% binder content the capacity per footprint area was calculated as 0.14 mA h mm2. For anode structures with binder content of 6, 12, 24 wt% the capacity per footprint area was not calculated since the corresponding anodes are not recommended for application in 3D-printed batteries and were thus not characterized in more detail. + +## Electrochemical Performance + +To investigate the electrochemical performance of the Si/C– +graphite blend anodes for application in 3D-printed MLIBs cycling tests were performed using a standard coin cell setup. + +High capacity values at high current rates are crucial for MLIBs, thus rate capability tests can deliver the corresponding information. Fig. 6a shows capacity values depending on the binder content and C-rates. All blended anodes show increased charge capacity over the standard graphite anode whereas the anode with 12 wt% binder has the highest capacity increase by about 30% at C/2. Anodes with 12 wt% binder show stable cycling at C/ +4 with 484 mA h g1 and capacity retention of 99.4% aer 65 cycles. This result is in good agreement with recently published and industry-orientated silicon based anode materials.38–40 With increasing C-rate the capacity of the cells decreases which is a well-known behavior and can be explained by kinetic limitations and pronounced ohmic drop behavior.41 Interestingly, capacity values decrease with increasing binder content >12 wt%. Possible reasons for that are increased electrical resistance caused by the relatively huge amount of electrically insulating binder.42 For a more detailed discussion regarding internal resistances of the anodes see ESI.† +Anodes with 6 wt% and 12 wt% binder at a current rate of 4C +show high capacity retentions of 90% and 89% compared to cycling at C/4 which suggests lower internal resistance compared to anodes with higher binder content. Cell results are summarized for better comparison in Fig. 6b. The overall capacity retention aer 65 cycles of all samples is close to 100%, +which indicates minor irreversible capacity losses from fast charging. The sample with 6 wt% binder has the lowest capacity at C/4. Since the capacity drop with increasing C-rates is + +![6_image_0.png](6_image_0.png) + +comparable to the sample with 12 wt% binder the low capacity does likely not correspond to high internal resistance. A counteractive process might occur which leads to the low capacity values. A hypothesis could be low percolation of the anode and weak connectivity of active particles to the current collector.18 It can be summarized that a Si/C–graphite blend slurry containing 12 wt% of CMC/SBR binder has the best performance regarding capacity and rate capability behavior. Based on this study a contrary development of electrochemical and rheological/ printing properties could be found which indicates clearly that neither slurries with the lowest binder content (6 wt%) nor the highest (24 wt%) can be successfully applied in 3D-printed batteries. Thus, based on the given material formulation a slurry with 18 wt% binder content is recommended for 3Dprinted IMLIBs since it combines high yield stress values +(around 1500 Pa) and aspect ratios of close to 6 within an 8-layer structure with high capacity values (466 mA h g1 at C/4) and considerable rate performance (around 427 mA h/g at 2C). To make lower binder content and higher capacity anodes suitable for 3D-printed IMLIBs, future study will focus on enhancing the printability of slurries by use of viscosity-increasing additives. + +## Conclusion + +In this work we presented for the rst time tailor-made Si/C +composite blended graphite anode slurries for 3D-printed lithium ion microbatteries using a scalable dispensing process. Solvent and binder content were adapted to adjust the rheological properties regarding dispensing performance. Corresponding lithium metal cells with 12 wt% of binder were characterized by the best capacity and rate performance behavior using a coin cell setup. Although slurries had a relatively wide particle size distribution they could be successfully and reproducibly extruded through 110 mm commercially available micro nozzles without clogging. Rheological investigations, in particular the yield stress measurements, revealed that higher binder content for the dispensing process is favorable. In this regard 3D-printed interdigitated anode structures with 24 wt% binder showed high aspect ratios up to 6.5, which suggests them to be highly promising in fully printed 3D- +IMLIBs. A fundamental trade-off between electrochemical and dispensing characteristics was found, whereby electrochemically optimized slurries lead to poor rheological and dispensing properties and vice versa. Thus, based on this study and the given material composition Si/C–graphite blend slurries with 18 wt% binder are recommended for application in 3D-IMLIB +because it combines high capacity values (466 mA h g1 at C/ 4) and considerable rate performance (around 427 mA h g1 at 2C) with AR values close to 6 within an 8-layered structure. However, future work will focus on enabling the printability of lower binder content anodes with enhanced electrochemical performance by using viscosity-increasing additives. In summary, CMC/SBR is not only a suitable system for silicon based anodes from an electrochemical point of view but also because of the promoting ad- and cohesion properties in the dispenser printing process. + +## Conflicts Of Interest + +The authors declare no competing nancial interests. + +## Acknowledgements + +The Baden-Wuerttemberg Stiung is gratefully acknowledged for nancing part of this work within the research project "ADD +SPICE (IAF-04)". + +## References + +1 Y. Wang, B. Liu, Q. Li, et al., Lithium and lithium ion batteries for applications in microelectronic devices: A +review, J. Power Sources, 2015, 286, 330–345. + +2 M. Roberts, P. Johns, J. Owen, et al., 3D lithium ion batteries—from fundamentals to fabrication, J. Mater. + +Chem., 2011, 21, 9876. + +3 J. Bates, N. Dudney, B. Neudecker, et al., Thin-lm lithium and lithium-ion batteries, Solid State Ionics, 2000, 135, 33–45. + +4 P. Simon, Y. Gogotsi and B. 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Power Sources, 2002, 112, 61–66. \ No newline at end of file diff --git a/medical/md/PMC9078861.md b/medical/md/PMC9078861.md new file mode 100644 index 0000000000000000000000000000000000000000..ebf06e0c572d767034cbc9011ec0874c8e680846 --- /dev/null +++ b/medical/md/PMC9078861.md @@ -0,0 +1,101 @@ +Open Access Full Text Article + +# Review The Anti-Inflammatory Effects Of Cannabidiol (Cbd) On Acne + +Nadia Peyravian 1, Sapna Deo1, Sylvia Daunert 1, Joaquin J Jimenez1,2 1Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA; 2Phillip Frost Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA Correspondence: Joaquin J Jimenez, Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA, Tel +1 305 243 6586, Email j.jimenez@med.miami.edu Abstract: Acne is the most common skin condition in the United States and affects approximately 85% of people ages 12–24. As a multifactorial disease, the pathogenesis of acne involves overproduction of sebum, irregular shedding of the cutaneous cells, accretion of *Cutibacterium acnes* at the pilosebaceous unit, and inflammation. To date, conventional therapies for acne include topical retinoids, over-the-counter bactericidal agents, and systematic treatments, such as oral antibiotics and isotretinoin. However, the potential for significant side effects and risk of antibiotic resistance remain limitations in these therapies, in turn reducing patient compliance and adherence to acne treatment regimens. Therefore, the use of natural plant-derived treatments or phytotherapeutics as an alternative or adjuvant to conventional treatments is attractive to patients due to their safety and minimal risk for side effects. Cannabidiol (CBD) is a non-psychoactive phytocannabinoid of the *Cannabis sativa* (hemp) plant. The therapeutic use of CBD has been implicated in many diseases with an inflammatory aspect, including cancers, neurodegeneration, immunological disorders, and dermatological diseases. However, the use of CBD for acne treatment remains a novel window of opportunity. Herein, we summarize the available and relevant data, highlighting the potential use of CBD in acne for its anti-inflammatory properties. To that extent, CBD and other *cannabis* constituents such as cannabis seeds were found to reduce inflammation and expression of inflammatory cytokines including TNF-α and IL-1β when evaluated in acne-like conditions. Treatment with these *cannabis* extracts was also found to be safe and well tolerated, further strengthening the prospect of CBD as an anti-inflammatory therapeutic for acne. + +Keywords: cannabidiol, CBD, acne, inflammation, therapeutic + +## Introduction + +As the 8th most prevalent disease, Acne vulgaris (acne) affects an estimated 9.4% of the world's population.1 In the United States alone, acne is estimated to affect at least 40 million individuals and is the most common skin disorder.2,3 Although most common in adolescents, acne may begin in younger children and persist through adulthood as well.2 Unfortunately, this widespread disease is associated with discomfort, scarring of the skin, as well as emotional and psychological distresses.1 Furthermore, by affecting 85% of adolescents, acne is often associated with significant psychosocial effects including, negative self-perception and self-esteem in addition to decreased social interactions and overall quality of life.4 Despite this, treatment options and treatment compliance remain barriers.5,6 + +## Acne + +Acne is a multifactorial disease; therefore, the onset and progression of the disease can be triggered by an interplay of many factors including stress, and hormonal and nutritional alterations.7,8 Pathologically, acne is an inflammatory disease affecting the pilosebaceous units. The pilosebaceous unit consists of the hair follicle and shaft, and sebaceous gland; these sebaceous pores are found on the face, chest, and back.9,10 Excess sebum production (hyperseborrhea), abnormal shedding of the cutaneous cells (hyperkeratinization), accumulation of Cutibacterium acnes *(C. acnes*) at the pilosebaceous unit, and inflammation are elemental to the disruption of sebaceous gland activity and pathogenesis of acne +(Figure 1).7,10,11 Androgen hormones, particularly testosterone, stimulate sebum production and are linked to the + + + +![1_image_0.png](1_image_0.png) + +Figure 1 Acne pathogenesis. Interplay of increased sebum production, accumulation of *C. acnes*, hyperkeratinization, and inflammation resulting in the formation of comedones. Created with BioRender.com. + +formation of comedones.12 Comedones are formed when the pilosebaceous units become plugged with sebum and can be classified as open comedones (blackheads) or closed comedones (whiteheads). Follicular hyperkeratinization occurs when cutaneous cells that line the sebaceous follicle, primarily keratinocytes, do not become cohesive with one another, resulting in rapid shedding of these cells, and subsequent microcomedone formation.13 Colonization of *C. acnes* at the sebaceous follicle may also lead to comedogenesis and subsequent presentation of acne. *C. acnes*, formerly termed Propionibacterium acnes (*P. acnes*), is a gram-positive anaerobic commensal bacteria found as part of the skin's microbiota in all individuals to maintain skin homeostasis.7,14 However, colonization of *C. acnes* at the pilosebaceous units, particularly in areas densely packed with sebaceous follicles that harbor a lipid-rich environment, is a pathological hallmark of acne.12 While *C. acnes* is present cutaneously in all individuals, the onset of acne is dependent on individual immunological responses to the bacteria, triggering the formation of comedones and the release of inflammatory mediators.12 Detection of *C. acnes* initiates an inflammatory response by the immune system, triggering the release of inflammatory cells, such as lymphocytes and neutrophils, and their subsequent release of inflammatory chemicals, such as reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-α).15 Consequently, these inflammatory mediators contribute to the inflammation and the subsequent chemical insult to the follicle epithelium and proximate dermis.12,16 + +## Current Treatments For Acne + +The management of acne involves topical and systematic treatments (Table 1). Topical retinoids such as tretinoin and erythromycin are commonly prescribed as comedolytic agents and for their anti-inflammatory properties.10 Topical overthe-counter formulations containing salicylic acid and benzoyl peroxide are also used as bactericidal agents to treat mildto-moderate acne.3 Systematic treatment of acne primarily includes tetracycline family antibiotics, such as minocycline + +| Mild Acne | Moderate Acne | Severe Acne | | +|-------------|--------------------------------------------------------------------------|-------------------------------------------------------------------|---------------------------------------| +| Topical | Bactericidal agents (Benzoyl Peroxide, Salicylic Acid) Topical retinoids | Topical retinoids (Tetracycline, Tretinoin, Erythromycin) | Bactericidal agents Topical retinoids | +| Systematic | - | Oral Tetracyclines (Minocycline, Doxycycline) Oral contraceptives | Isotretinoin Oral Contraceptives | + +and doxycycline.17 These oral antibiotics are commonly prescribed for moderate-to-severe acne. In patients with very severe acne in which antibiotics are no longer effective, oral Isotretinoin is often prescribed to control the presence of C. acnes, and to reduce sebum production and inflammation.10,18 Additionally, oral contraceptives, such as those with low-dose estrogen, may also be prescribed for persistent acne for anti-androgenic effects.10 Nonetheless, there are significant limitations to these conventional treatments, leading to low patient adherence and compliance.5,19 For example, topical retinoids may irritate the skin, with side effects including burning, drying, itching, and redness.5,19 These effects may lead to early termination of the treatment by the patient. Furthermore, long-term use of topical and oral antibiotics may lead to bacterial and antimicrobial antibiotic resistance.5,6 Although generally regarded as safe, Isotretinoin is also known to exhibit embryotoxic and teratogenic properties.5,19 These potential adverse side effects may deter patients from commencing or continuing their acne treatment, therefore highlighting the need for new, natural, and safe therapies. As many plant derivatives have already been widely investigated for their innate anti-inflammatory properties for the treatment of acne, herein, we draw attention to Cannabidiol as a potential anti-inflammatory phytotherapeutic for acne. + +## Cannabidiol + +Cannabidiol (CBD) is a non-psychoactive biochemical constituent of the *Cannabis sativa* (hemp) plant. In recent years, the investigation and use of CBD for its anti-inflammatory and immunomodulatory effects has become extensive amongst the scientific community.20 The therapeutic potential of CBD has been extensively supported in various immunological, neurodegenerative, cancer, cardiovascular, and dermatological diseases, as conditions with an inflammatory component.21 To date, Epidiolex remains the first and sole FDA-approved prescription CBD drug used to treat seizures. Mechanistically, CBD decreases inflammation and subsequent cytokine production through modulation of the immune system and suppression of immune responses.22 We have previously reviewed the antiinflammatory and immune suppressive effects of CBD through modulation of critical signaling pathways and receptors.23 As CBD does not have a high affinity for CB1 and CB2 receptors, the pharmacological activity of CBD is suggested to be independent of these receptors. Instead, CBD has been studied to modulate pro-inflammatory transient receptor potential vanilloid 1 channel (TRPV1), such that treatment with TRPV1 antagonists reversed the anti-inflammatory and immune suppressive effects of CBD (Figure 2).23–25 CBD also modulates the Janus kinase/ +signal transducers and activators of transcription (JAK/STAT) signaling pathway, a fundamental regulator of immune responses, by inhibiting cytokines that facilitate the activation of the cascade (Figure 2).23,26,27 Since constitutive activation of JAK/STAT has been implicated in the pathogenesis of immunological diseases and cancers, CBDinduced reduction of pro-inflammatory signaling is desirable.27,28 To that extent, CBD has been shown to suppress inflammatory cytokine initiators of JAK/STAT, such as TNF-α, Interferon-γ (IFN- γ), Interleukin-1 (IL-1), IL-2, and IL-6 in vitro and in vivo.23,26,28 CBD has been shown to prevent nucleotide-binding oligomerization domain-like receptors (NLR) inflammasome complex activation, suppressing the production of pro-inflammatory cytokines, such as IL-1β and IL-18 that are critical in regulating NF-κB, MAPK, and IFN pro-inflammatory pathways +(Figure 2).23,29,30 Furthermore, CBD has also been studied to modulate adenosine signaling by inhibiting rapid + +![3_image_0.png](3_image_0.png) + +cellular uptake of adenosine by the equilibrative nucleoside transporter (Figure 2) (ENT).31–33 Adenosine is a purine nucleoside that exhibits protective effects and attenuates inflammation by activating G protein-coupled adenosine receptor, A2A. + +23,33,34 By inhibiting intracellular adenosine uptake, CBD promotes endogenous adenosine signaling as a protective mechanism during inflammatory and immune response.23,35 Taken together, a mechanistic understanding of the pharmacological reduction of inflammation by CBD further supports therapeutic exploration of CBD as a novel and unique anti-inflammatory option. Next, we specifically highlight the potential use of CBD for acne treatment.23 + +## Cbd And Acne + +In a study evaluating the effects of CBD on human sebaceous glands, CBD was evaluated in the context of immortalized human sebocytes under "acne-like" conditions.20 In this study, CBD was shown to dose-dependently inhibit excessive lipid synthesis (lipogenesis) in sebocyte cultures that were pre-treated with "pro-acne" inducing inflammatory compounds such as arachidonic acid, linoleic acid, and testosterone.20 The anti-inflammatory effects of CBD were also observed in the reduction of inflammatory cytokine TNF-α. Lipopolysaccharides36 were used to pre-treat sebocytes to induce "pro-acne" inflammatory conditions. Subsequently, TNF-α concentrations in sebocytes were measured after 24 h with or without CBD (10 μM).20 Treatment with CBD was found to decrease TNF-α mRNA expression and the expression of other inflammatory cytokines including, IL-1β and IL-6 in the sebocytes.20 Results from this study strongly demonstrate the clinical potential of CBD in the treatment of acne.20 In a separate study, the potential anti-acne effects of another cannabis plant derivative, hemp seed hexane extracts (HSHE), were evaluated in an in vitro model of acne.37 Like CBD, hemp seed extracts also do not exhibit psychotropic properties and are suggested to have potential antiinflammatory effects. In a human keratinocyte cell line, 0.6% was HSHE reduced *C. acnes*-induced inflammation in human keratinocytes through down-regulation of pro-inflammatory cytokines, IL-1β and IL-8.37 Additionally, 0.6% +HSHE was found to significantly inhibit overproduction of inflammatory radical nitric oxide in the same cells, further supporting the conventional development of hemp-derived constituents, such as HSHE as natural, safe, and effective antiinflammatory acne treatments.37 In a human study evaluating the safety and efficacy of cannabis seed extracts for acne, 3% cannabis seed extracts were found to reduce inflammation-induced redness of the skin, known as erythema.38 Changes in skin erythema were measured every 2 weeks for a total of 12 weeks. Treatment with the 3% cannabis seeds extract cream was found to significantly reduce skin erythema after each 2-week observation, and a notable gradual reduction in erythema was observed across the 12-week study.38 Furthermore, the application of 3% cannabis seeds extract cream on the cheeks, twice a day, for 12 weeks was found to be safe, well tolerated, non-allergenic, and nonirritating in the patient volunteers.38 + +## Discussion + +Acne is the most prevalent condition in the US, affecting nearly 50 million people.2,3 As a widespread disease that affects most people at some point in their lifetime, the effects of acne include significant irritation and scarring of the skin, in addition to emotional and psychosocial distress. Furthermore, treatment options and compliance remain challenges due to the potential adverse side effects of conventional treatments and the increasing emergence of antibiotic resistance, suggesting the need for new, natural, and safe acne therapies. As CBD possesses antiinflammatory activities, CBD has been widely studied in various inflammatory and immunological disorders, including Alzheimer's diseases, diabetes, multiple sclerosis, and rheumatoid arthritis, setting a precedent for investigation of CBD in other inflammatory conditions, such as acne. Understanding of the pharmacodynamics of CBD reveals modulation of critical inflammatory and immunological pathways and receptors leading to a decrease in inflammation and subsequent cytokine production, further supporting the anti-inflammatory effects of CBD. Evaluation of CBD in acne-like conditions revealed suppressed expression of pro-inflammatory cytokines, TNF-α, IL-1β, and IL- 6. CBD also reduced excessive lipid synthesis. + +Like CBD, hemp seed extracts are also derived from *cannabis*, and while they are suggested to have antiinflammatory properties, they do not exhibit psychoactive effects. Nevertheless, hemp seed extracts have also been shown to reduce *C. acnes*-induced inflammation and inflammatory cytokine production of IL-1β and IL-8. Additionally, these hemp seed extracts also significantly protect cells from overproduction of inflammatory radicals and nitric oxides, further supporting the use of hemp-derived constituents as potential natural, safe, and effective anti-inflammatory therapies. Moreover, in a small patient study, treatment with another *cannabis* derivative, cannabis seed extract cream, was found to be efficacious in reducing skin inflammation, as well as safe and non-irritating. Accordingly, the use of CBD for reducing inflammation in acne is supported and should be further explored. Since the pathogenesis of acne is complex and multifactorial, limitations include the lack of reliable animal models to mimic acne conditions and test therapeutics.39 Additionally, there is an evident need for large-scale human studies to understand the impact of CBD on acne at the clinical level.40 Future studies may also optimize the concentration and delivery of CBD for the treatment of acne. In conclusion, CBD may be beneficial as an adjuvant anti-inflammatory treatment for acne. + +## Acknowledgments + +SD thanks the Miller School of Medicine of the University of Miami for the Lucille P. Markey Chair in Biochemistry and Molecular Biology. + +## Funding + +This research received no external funding. + +## Disclosure + +The authors declare no conflicts of interest in this work. + +## References + +6. Castillo DE, Nanda S, Keri JE. 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Cannabinoids Delta(9)-tetrahydrocannabinol and cannabidiol differentially inhibit the lipopolysaccharide-activated NF-kappaB and interferon-beta/STAT proinflammatory pathways in BV-2 microglial cells. *J Biol Chem*. 2010;285 (3):1616–1626. doi:10.1074/jbc.M109.069294 28. Weiss L, Zeira M, Reich S, et al. Cannabidiol lowers incidence of diabetes in non-obese diabetic mice. *Autoimmunity*. 2006;39(2):143–151. + +doi:10.1080/08916930500356674 29. Liu C, Ma H, Slitt AL, Seeram NP. Inhibitory effect of cannabidiol on the activation of NLRP3 Inflammasome is associated with its modulation of the P2X7 receptor in human monocytes. *J Nat Prod*. 2020;83(6):2025–2029. doi:10.1021/acs.jnatprod.0c00138 30. Libro R, Scionti D, Diomede F, et al. Cannabidiol modulates the immunophenotype and inhibits the activation of the inflammasome in human gingival mesenchymal stem cells. *Front Physiol*. 2016;7:559. doi:10.3389/fphys.2016.00559 31. de Almeida DL, Devi LA. Diversity of molecular targets and signaling pathways for CBD. *Pharmacol Res Perspect*. 2020;8(6):e00682. + +doi:10.1002/prp2.682 32. Carrier EJ, Auchampach JA, Hillard CJ. Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. *Proc Natl Acad Sci U S A*. 2006;103(20):7895–7900. doi:10.1073/pnas.0511232103 33. Liou GI, Auchampach JA, Hillard CJ, et al. Mediation of cannabidiol anti-inflammation in the retina by equilibrative nucleoside transporter and A2A adenosine receptor. *Invest Ophthalmol Vis Sci*. 2008;49(12):5526–5531. doi:10.1167/iovs.08-2196 34. Hasko G, Cronstein BN. Adenosine: an endogenous regulator of innate immunity. *Trends Immunol*. 2004;25(1):33–39. doi:10.1016/j.it.2003.11.003 35. Mecha M, Feliú A, Iñigo PM, et al. Cannabidiol provides long-lasting protection against the deleterious effects of inflammation in a viral model of multiple sclerosis: a role for A2A receptors. *Neurobiol Dis*. 2013;59:141–150. doi:10.1016/j.nbd.2013.06.016 36. Gottlieb JF, Benedetti F, Geoffroy PA, et al. The chronotherapeutic treatment of bipolar disorders: a systematic review and practice recommendations from the ISBD task force on chronotherapy and chronobiology. *Bipolar Disord*. 2019;21(8):741–773. doi:10.1111/bdi.12847 37. Jin S, Lee MY, Chen W. The ameliorative effect of hemp seed hexane extracts on the Propionibacterium acnes-induced inflammation and lipogenesis in sebocytes. *PLoS One*. 2018;13(8):e0202933. doi:10.1371/journal.pone.0202933 38. Ali A, Akhtar N. The safety and efficacy of 3% Cannabis seeds extract cream for reduction of human cheek skin sebum and erythema content. Pak J Pharm Sci. 2015;28(4):1389–1395. + +39. Olah A, Markovics A, Szabó-Papp J, et al. Differential effectiveness of selected non-psychotropic phytocannabinoids on human sebocyte functions implicates their introduction in dry/seborrhoeic skin and acne treatment. *Exp Dermatol*. 2016;25(9):701–707. doi:10.1111/exd.13042 40. Baswan SM, Klosner AE, Glynn K, et al. Therapeutic Potential of Cannabidiol (CBD) for skin health and disorders. Clin Cosmet Investig Dermatol. 2020;13:927–942. doi:10.2147/CCID.S286411 Journal of Inflammation Research **Dovepress** Publish your work in this journal The Journal of Inflammation Research is an international, peer-reviewed open-access journal that welcomes laboratory and clinical findings on the molecular basis, cell biology and pharmacology of inflammation including original research, reviews, symposium reports, hypothesis formation and commentaries on: acute/chronic inflammation; mediators of inflammation; cellular processes; molecular mechanisms; pharmacology and novel anti-inflammatory drugs; clinical conditions involving inflammation. 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/PMC9219330.md b/medical/md/PMC9219330.md new file mode 100644 index 0000000000000000000000000000000000000000..fa75564f277fd5fb64ecaac016363876ba20d73e --- /dev/null +++ b/medical/md/PMC9219330.md @@ -0,0 +1,359 @@ +Contents lists available at ScienceDirect + +![0_image_0.png](0_image_0.png) + +# Sports Medicine And Health Science + +![0_Image_2.Png](0_Image_2.Png) + +journal homepage: www.keaipublishing.com/en/journals/sports-medicine-and-health-science/ +Original Article + +![0_image_1.png](0_image_1.png) + +Lifestyle behaviours of immigrant and Australian children: Evidence from a nationally representative sample Shahnawaz Ahmed a,*, Riaz Uddin a,b, Jenny Ziviani a, Sjaan Gomersall a, Asaduzzaman Khan a,** +a School of Health and Rehabilitation Sciences, The University of Queensland, Brisbane, Australia b Institute for Physical Activity and Nutrition (IPAN), Deakin University, Geelong, Australia ARTICLE INFO + +| Keywords: Physical activity Screen time Sleep Fruit Vegetable HIC LMIC | +|--------------------------------------------------------------------------| + +This study aimed to examine the prevalence of physical activity (PA), screen time (ST), sleep, and fruit and vegetable intake of children with low-and-middle-income countries (LMIC) parents, high-income countries (HIC), and Australian children, and whether these behaviours are associated with their immigrant status. Data for this study were from wave 7 of the Birth cohort (B cohort) of the Longitudinal Study of Australian Children. We used generalized estimating equations (GEE) to examine associations between health behaviours and immigrant status. The models were adjusted for a number of covariates. Children with LMIC parents had lower odds of meeting PA +and sleep recommendations and higher odds ratio (OR) of meeting fruit and vegetable intake, and ST recommendations than Australian or HIC children. Children with LMIC parents had one-third the odds of meeting the PA recommendations (OR 0.39 [95%CI 0.22–0.70]) than Australian children. No significant differences were observed in lifestyle behaviours among children with HIC parents and Australian children. Lifestyle behaviours of children with LMIC parents differed from those of HIC and Australian children. However, children with HIC +parents and Australian children had comparable lifestyle behaviours. Identified disparities in lifestyle behaviours among immigrant children can inform strategies to bring equity in Australian children's lifestyles. Our study underscores the importance of culturally appropriate targeted interventions to promote PA and sleep of children with LMIC parents. + +| ABSTRACT | +|------------| + +## Introduction + +Unhealthy lifestyle behaviours such as insufficient physical activity +(PA), unhealthy diet, high screen time (ST), and inadequate sleep have been linked with a higher risk of childhood-onset of obesity, poor psychosocial health, poor academic performance, cardiovascular risk factors, metabolic syndrome, and type 2 diabetes mellitus.1–5 Although behavioural risk factors such as insufficient PA, high ST, inadequate sleep, and unhealthy diet are established in childhood, there is also evidence that they will continue to present into adulthood.6–8 For example, childhood metabolic risk factors have been linked to long-term adverse health effects such as cardiovascular diseases in early adulthood and continue over adult years.7,8 Childhood, therefore, establishes the foundation for future health, growth, and wellbeing.9 Despite the evidence to support the importance of health behaviours in childhood, many children fail to attain the recommended health behaviours.10–13 For example, globally, in 2016, 81% of school-going adolescents aged 11–17 years did not accumulate the recommended 60 min of daily moderate-to vigorous-intensity PA (MVPA).14 Children and adolescents aged 5–18 years from developed countries also exceed the recommendations of 2 h or less of ST daily.15 Furthermore, a review from 20 countries among children aged 5-18-year-old reported a decline of more than 1 h of sleep duration over the study period (1905–2008).13 Available evidence from Australia demonstrates that children's adherence rates are 7% for recommended daily vegetable intake (5-serves/- +day) and 79% for fruit intake (2-serves/day).16 Thus, efforts to promote healthy behaviours during childhood could be the most cost-effective and feasible approach for achieving long-term health and wellbeing.17 Most studies of health behaviours in children have been conducted, and only a few have included multiple health behaviours. The International Study of Childhood Obesity, Lifestyle, and the Environment project examined PA, ST, and sleep duration in relation to adiposity, dietary patterns, and quality of life of children aged 11–13 years from 12 countries including Australia.18,19 However, the most recent Australian Report Card on Physical Activity for Children and Youth shows that +* Corresponding author. School of Health and Rehabilitation Sciences, The University of Queensland, Brisbane, QLD 4072, Australia. + +** Corresponding author. School of Health and Rehabilitation Sciences, The University of Queensland, Brisbane, QLD, 4072, Australia. + +E-mail addresses: shahnawaz.ahmed@uqconnect.edu.au (S. Ahmed), a.khan2@uq.edu.au (A. Khan). + +https://doi.org/10.1016/j.smhs.2022.02.002 Received 14 October 2021; Received in revised form 14 February 2022; Accepted 15 February 2022 Available online 23 February 2022 2666-3376/© 2022 Chengdu Sport University. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. + +| Abbreviations B cohort Birth cohort AIFS Australian Institute of Family Studies ABS Australian Bureau of Statistics K cohort Kindergarten cohort BMI body mass index DSS Department of Social Services HIC High-income countries LMIC Low- and middle-income countries LSAC Longitudinal Study of Australian Children PA Physical activity ST Screen time | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +15%–41% and 32% are meeting the MVPA and ST recommendations, respectively.20 A recent study found that 79% met their recommended daily intake of fruit; 56% followed the sleep guidelines, 20% met their PA guidelines, 18% adhered to the guidelines for ST, and only 7% of Australian children were meeting their recommended daily intake of vegetables.16 Thus, it is important to understand lifestyle behaviours, which can inform policies pointing at optimising the health and wellbeing of children. + +Globally, international migration has grown rapidly; however, little is known about the immigrant child's lifestyle behaviours specifically. + +Recent systematic review suggests that immigrant children are underrepresented in health behaviour research globally, and prior studies typically have not specified the country of origin of immigrants.21,22 Australia has one of the most culturally diverse populations in the world,23 and immigrant children represent one-third of the total child population in Australia.24 The impact of these multifaceted issues on immigrants' health behaviours in the Australian context is poorly understood. While limited, research from Australia indicates that immigrant children are less active, more involved in ST, and have low fruit and vegetable consumption.25 However, this study did not explore all the behaviours together, did not compare PA, ST and sleep behaviours with the Australian Guidelines, and there are no data about the sleep behaviour of immigrant children in Australia. + +The pattern of immigration from different countries has changed over time in Australia: most notably, Asian immigrants of mostly lowand–middle-income backgrounds have begun to arrive at an accelerated rate in recent years.23,25,26 Available evidence suggests a lower prevalence of PA among immigrants from developing countries such as those in Asia and Africa than immigrants from developed countries such as the United Kingdom (UK), Ireland, and New Zealand.27 However, these studies are limited to PA only and have not looked at high-income countries (HIC) and low-and middle-income countries (LMIC) immigrants separately. An earlier systematic review on immigrant children in the United States of America (USA) reported that immigrant children from Asian backgrounds consumed more fruit and vegetables but did not separate their analysis according to HIC and LMIC.28 In the present study, we aimed to estimate the prevalence of PA, ST, sleep, and fruit and vegetable intake of immigrant and Australian children. Further, we sought to examine if these behaviours vary among children of immigrant (HIC and LMIC) backgrounds and Australian children. + +## Material And Methods Study Participants And Recruitment + +This study used data from the Longitudinal Study of Australian Children (LSAC), which tracks a cohort of infants from birth (B cohort) and a cohort of children from 4 to 5 years of age (K cohort) with biennial data collection from 2004.29 The present study was based on data from + +| MVPA | Moderate-to vigorous-intensity PA | +|--------|----------------------------------------| +| CI | Confidence intervals | +| GEE | Generalized estimating equations | +| SD | Standard Deviation | +| SES | Socio-economic status | +| GNI | Gross national income | +| SEP | Socio-economic position | +| SA2 | Statistical Area 2 | +| SPSS | Statistical Package for Social Science | +| USA | United States of America | +| UK | United Kingdom | + +Wave 7 of the B-cohort of the LSAC, collected in 2016. Wave 7 was the first wave that captured whether participating children achieved compliance with the recommended 60 min of MVPA per day along with information on ST, sleep, and diet and therefore offered the first opportunity to examine the PA, ST, sleep, and dietary habits simultaneously. A +detailed description of the LSAC methodology has been published elsewhere.30 The study sample size consisted of 3381 children aged 12–13 years. + +## Outcome Measures + +Physical activity. A single item was used to capture participating children's PA; specifically, children were asked: "About how many days each week do you do at least 60 min of moderate or vigorous physical activity?" According to the current Australian guidelines, sufficient PA is defined as accumulating 60 min MVPA per day for children aged 5–17 years.16 The study children completed this item by responding with the number of days per week they did 60 min of MVPA. PA was dichotomized as either meeting (60 min/day for 7 days) or not meeting (< 60 min/day for 7 days) the PA guidelines. Screen time. Study children were asked to report recreational ST using two items: (a) "Total minutes of TV watched on an average week at home?" (b) "Total minutes per week of electronic gaming on an average at home?". According to the current Australian guidelines, recreational ST was defined as no more than 2 h per day for children aged 5–17 years16 and subsequently classified as meeting or not meeting guidelines. + +Sleep. Study children were asked to indicate their typical time of sleep onset and typical wake-up time during school days and non-school days. + +Children answered the following questions (a) "About what time do you fall asleep on a usual school night?", (b) "About what time do you wake up in the morning on a usual school day?", (c) "About what time do you fall asleep on the nights when you do not have school the next day?", and +(d) "About what time do you wake up in the morning on the days when you do not have school?". According to the current Australian guidelines: an uninterrupted 9–11 h of sleep per night is recommended for children aged 5–13 years and 10 h for those aged 14–17 years16 and subsequently classified as meeting or not meeting guidelines. + +Fruit and vegetable intake. Study children were asked to indicate their fruit and vegetable consumption as follows: "Think about yesterday, how often did you have fresh fruit?" "Think about yesterday, how often did you have cooked vegetables?" "Think about yesterday, how often did you have raw vegetables?" Response options included "not at all", "once", +"twice", and "more than twice". Then classified vegetables and fruit as intake 2 times/day or not intake ( 2 times/day). + +| Table 1 Characteristics of immigrant children from low-and-middle-income countries (LMIC), high-income countries (HIC) and Australian children. Characteristics Australian (n ¼ 2260) HIC (n ¼ 619) | LMIC (n ¼ 343) | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|--------------|--------------| +| Mean age in years (SD) | 12.48 (0.51) | 12.49 (0.51) | 12.43 (0.50) | +| Female, n (%) | 1116 (49.4) | 306 (49.4) | 157 (45.8) | +| Region of residence, n (%) Metropolitan area | 1177 (52.1) | 440 (71.3) | 289 (85.0) | +| Non-metropolitan area | 1082 (47.9) | 177 (28.7) | 51 (15.0) | +| Child's mother speaks English language at home, n (%) | 2149 (97.5) | 540 (91.2) | 125 (43.4) | +| Family rules about quantity of TV, n (%) | 1331 (60.4) | 371 (61.2) | 214 (64.3) | +| High SEP, n (%) | 554 (29.9) | 180 (34.4) | 100 (34.8) | +| Middle SEP, n (%) | 961 (51.9) | 275 (52.6) | 121 (42.2) | +| Low SEP, n (%) | 336 (18.2) | 68 (13.0) | 66 (23.0) | +| Mother's weight status, n (%) Normal weight | 780 (36.1) | 236 (40.1) | 144 (45.0) | +| Overweight | 638 (29.5) | 166 (28.2) | 61 (19.1) | +| Obese | 106 (4.9) | 24 (4.1) | 17 (5.3) | +| Maternal employment status, n (%) Employed | 1784 (78.9) | 477 (77.1) | 269 (78.4) | +| Child BMI, mean (SD) | 20.3 (3.7) | 20.4 (3.8) | 20.3 (3.7) | +| Child participated in team sport, n (%) | 1107 (49.3) | 312 (50.7) | 133 (39.9) | +| Numbers may not add to total sample size (n ¼ 3381) due to missing values. Excluded from the analysis (n ¼ 159), comprising mixed immigrant background (n ¼ 36), | | | | + +Immigrant status. Children were classified as Australians if they were born in Australia with both Australian-born parents. Children were classified as immigrants if they were born in Australia and one or more of their parents were born overseas.22,31 Immigrant children were then further classified according to the economic development of their parent's (mother or father or both) birth country. Parents can play an important role in increasing a child's lifestyle behaviours, and mothers generally take on more childcare responsibilities.32 Parental socio-economic status (SES) influences children's and adolescents' healthy lifestyles; for example, higher screen time is more likely among children with low parental SES than children with higher SES backgrounds.33 To our knowledge, only one study with representative samples has looked at immigrant children by the socio-economic development of mothers' country of origin,25 where the outcome was obesity-related risk factors. The classification was based on gross national income (GNI), using the World Bank Classification, 2018.34 Children were classified as immigrants of a low-and-middle-income country +(LMIC) if one or more of their parents were born in LMIC (hereafter LMIC +children). Children were classified as immigrants of high-income countries (HIC) if one or more of their parents was born in a HIC (hereafter HIC children). A child was classified as mixed background when one parent comes from an LMIC and another from a HIC. + +## Covariates + +Based on previous research,25 potential covariates were identified for the analyses. Child's age, sex, body mass index (BMI), area of residence, number of siblings, child's mother's language spoken at home, participation in team sports, family rules about quantity of television (TV), and socio-economic position (SEP) were considered as covariates. Family SEP +summarized the social and economic capital available to families and included combined annual family income, both parent's employment status and education.26,35 + +## Statistical Analysis + +Proportions of study children with sufficient PA, ST, and sleep and consumption of fruit, raw vegetable, and cooked vegetable were compared between immigrant status using chi-square tests. A series of generalized estimating equations (GEE) with an exchangeable correlation were used to examine the associations of PA, ST, sleep, and fruit and vegetable intake with different population groups (children of LMIC, HIC +immigrant and Australian parents). GEE was used to take into account the nested structure of the data, as children were nested within Statistical Area 2 (SA2) identifiers to model spatial clustering. A robust standard error estimator was used to reduce the influence of outliers. A set of covariates initially considered for adjustments included: age, sex, BMI, language spoken at home, area of residence, family rules about quantity of TV, participation in team sports, SEP, PA, and ST. Covariates were examined to identify any collinearity issues. There were no collinearity issues associated with the above covariates. The results for the current GEE models are presented as odds ratios (ORs) and their 95% confidence intervals (CI). All statistical analyses were performed using Statistical Package for Social Science (IBM SPSS, version 25; Chicago, IL, USA). + +## Results + +Of the final analytical sample (n ¼ 3222), 2260 (70.1%) were children of Australian parents (reference group), 619 (19.2%) were HIC +children, and 343 (10.7%) were LMIC children. Thirty-six children (1%) +were from a mixed background. The ratio of mother vs. father as an immigrant was 17.7% vs. 17.9%, respectively. Table 1 presents the summary characteristics of the study participants. The mean age of the study children was 12.4 years (SD: 0.514), and 48.2% were female. The study participants who lived in metropolitan areas were 85.0%, 71.3%, and 52.1% for LMIC, HIC and Australian children, respectively. English was spoken at home by 43.4% of mothers from LMIC compared to 91.2% from HIC mothers and 97.5% for Australian mothers. + +The proportion of children meeting lifestyle behaviour guidelines is presented in Fig. 1. About 6%, 11%, and 11% of children meeting PA +recommendations were from LMIC, HIC, Australian children, respectively. A third (37%) of children from LMIC met the recommendations for ST, followed by children of HIC (29%), and Australian (27%) parents. + +Seventy-six percent of HIC children met sleep recommendations of 9–11 h of sleep per day, followed by 75% and 71%, Australian and LMIC children, respectively. Sixty-three percent of children of LMIC, 59%, and 55% of children from HIC and Australia, respectively, consumed fruits 2 times/day. About 34%, 32% and 28% of children who consumed cooked vegetables 2 times/day were from LMIC, HIC, and Australia, respectively. Twenty-four percent of LMIC children, 23% and 20% of children from HIC and Australian parents, respectively, consumed raw vegetables 2 times/day. + +GEE modelling (presented in Table 2) revealed that compared to Australian children, LMIC children had lower odds of meeting the PA +recommendations (OR 0.39 [95%CI 0.22–0.70], p ¼ 0.002). LMIC children had lower odds of meeting sleep recommendations (0.70 [95%CI +0.52–0.95], p ¼ 0.021) than Australian children. LMIC children had higher odds of meeting ST recommendations (1.86 [95%CI 1.38–2.51], + + + +![3_image_0.png](3_image_0.png) + +p ¼ 0.001) than Australian children. LMIC children also had higher odds of frequent fruit ( 2 times/day) (1.54 [95%CI 1.16–2.05], p ¼ 0.002) +and cooked vegetable ( 2 times/day) (1.51 [95%CI 1.15–1.98], p ¼ +0.003) consumption than Australian children. No statistically significant findings were observed in raw vegetable intake of immigrant children from LMIC and Australian children. Among the adjusted covariates, sex, ST, sleep, PA, BMI, and SEP were significant in the analyses (Table 2). + +Additional analyses on examining the role of duration of immigration on lifestyle behaviours across HIC and LMIC immigrants found that duration of immigration was not significant in any of the behaviours examined +(p ¼ 0.536–0.966). + +Our study found that compared to HIC children, LMIC children had lower odds of meeting PA (0.32 [95%CI 0.15–0.66], p ¼ 0.002) and sleep recommendations (0.62 [95%CI 0.44–0.86], p ¼ 0.004) and higher odds of meeting ST (1.52 [95%CI 1.08–2.15], p ¼ 0.015), fruit intake (1.33 +[95%CI 0.97–1.82], p ¼ 0.072), and cooked vegetable intake (1.54 [95% +CI 1.13–2.09], p ¼ 0.006) (presented in Supplementary Table 1) recommendations. No statistically significant findings were observed in raw vegetable intake of children of immigrant parents from LMIC and HIC +(Supplementary Table 1). Our study also identified no statistically significant differences between HIC children and Australian children in relation to the odds of meeting recommendations on PA, ST, sleep, and fruit and vegetable intake. + +## Discussion + +The present study has examined the lifestyle behaviours of Australian and immigrant children aged 12–13 years. Overall, the compliance with guidelines on PA, ST, and vegetable consumption is low across all groups. There were lower odds of meeting PA and sleep guidelines among LMIC children compared to HIC and Australian children. Meeting ST, fruit and cooked vegetable consumption guidelines were more common in LMIC children than HIC and Australian children. + +LMIC children had lower odds of meeting PA recommendations than Australian and HIC children in the present study. Our findings are consistent with research conducted from developed countries that demonstrate immigrant children are less likely to engage in PA compared to native children.36–38 However, the studies did not classify immigrant children according to parental socio-economic background. The significant differences identified in the prevalence of PA between LMIC and Australian children could be partly due to predisposing factors such as culturally bound values and beliefs, attitudes, environmental factors +(access/availability), and lack of motivation to engage in PA, or perceived importance of PA among LMIC immigrants.27,39 Our study also identified significant differences in PA between the immigrant groups. + +This could be partly due to immigrant parents from LMIC are lacking knowledge on PA's health benefits or perhaps the high cost of attending gym or lack of culturally sensitive physical activity facilities. Another potential explanation might be HIC children (such as the United Kingdom, Ireland, and New Zealand) experiencing the advantages of better socio-economic backgrounds and similar physical activity infrastructure from their country of origin. Immigrant parents who do not use English as their preferred language may not be able to access PA promotional materials available in English or utilise the services and opportunities that support and promote PA for their children. Our study reported a lack of difference in PA between immigrant children from HIC and Australian children. Our study, therefore, underscores future research to understand factors responsible for the lower level of PA in immigrant children from LMIC, specifically. + +Our study found that immigrant children from LMIC had higher odds of meeting ST recommendations compared to HIC and Australian children. This could be partly due to immigrant parents being more concerned about their children's educational success and encouraging them to spend more time in education40; screening devices may be allowed to be used for educational purposes, not for recreation. In addition, immigrant families may experience financial difficulties, and not able to make the screen devices accessible to their children. This finding contradicts previous studies from the USA and UK, where immigrant children and adolescents were more engaged in screen-based activities than native-born children36,41; however, these studies did not differentiate between HIC and LMIC groups. Using a nationally representative sample of USA adolescents, a recent study reported no significant association + +| Table 2 Examination of lifestyle behaviours between immigrant children from low-and-middle-income countries (LMIC), high-income countries (HIC) and Australian children. Characteristics Model 1 Model 2 Model 3 Model 4 Model 5 Model 6 Meeting PA guidelines (60 min/day for 7 days MVPA): Yes/No Raw vegetable intake ( 2 times/day): Yes/ No Adjusted OR pvalue Adjusted OR pvalue Adjusted OR pvalue Adjusted OR pvalue Adjusted OR pvalue Adjusted OR pvalue (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) Immigrant status Australian 1.00 (Ref.) 1.00 (Ref.) 1.00 (Ref.) 1.00 (Ref.) 1.00 (Ref.) 1.00 (Ref.) HIC 0.97 (0.70–1.36) 0.890 1.14 (0.91–1.44) 0.241 0.94 (0.74–1.19) 0.622 1.15 (0.93–1.41) 0.183 1.25 (1.01–1.56) 0.038 1.16 (0.91–1.48) 0.215 LMIC 0.39 (0.22–0.70) 0.002 1.86 (1.38–2.51) 0.001 0.70 (0.52–0.95) 0.021 1.54 (1.16–2.05) 0.002 1.51 (1.15–1.98) 0.003 1.26 (0.92–1.73) 0.138 Age 1.09 (083–1.43) 0.532 1.10 (083–0.91) 0.287 0.72 (0.60–0.87) 0.001 0.92 (0.77–1.08) 0.338 0.98 (0.82–1.17) 0.856 1.09 (0.89–1.33) 0.385 Sex (Female) 0.46 (0.35–0.62) 0.001 2.46 (2.39–3.50) 0.001 0.96 (0.79–1.16) 0.686 1.37 (1.16–1.02) 0.001 0.96 (0.80–1.15) 0.702 1.06 (0.86–1.30) 0.537 BMI 0.96 (0.92–1.00) 0.088 0.95 (0.93–0.98) 0.002 0.96 (0.94–0.98) 0.003 1.00 (0.98–1.02) 0.592 1.01 (0.99–1.04) 0.197 1.00 (0.97–1.03) 0.881 SEP 0.91 (0.80–1.04) 0.202 1.50 (1.35–1.67) 0.001 1.11 (1.00–1.22) 0.035 1.14 (1.05–1.24) 0.002 1.12 (1.01–1.23) 0.019 1.19 (1.05–1.34) 0.004 1.00 0.001 - – 1.00 0.249 1.00 0.001 1.00 0.258 1.00 0.002 ST: 2 h/day (Ref.) Meeting ST guidelines Meeting sleep guidelines (9–11 h/ ( 2 h/day): Yes/No day): Yes/No Fruit intake ( 2 Cooked vegetable times/day): Yes/No intake ( 2 times/day): Yes/No > 2 h/day 0.54 (0.40–0.74) 0.87 (0.70–1.09) 0.59 (0.49–0.72) 0.89 (0.73–1.08) 0.70 (0.56–0.87) Sleep: 9–11 h/day 1.00 0.054 1.00 0.249 - – 1.00 0.475 1.00 0.597 1.00 0.869 (Ref.) < 9 h and >11 h 1.34 (0.99–1.82) 0.87 (0.70–1.09) 0.93 (0.76–1.13) 1.05 (0.86–1.29) 0.98 (0.77–1.24) - – 1.00 0.001 1.00 0.249 1.00 0.001 1.00 0.243 1.00 0.246 PA: 60 min/day for 7 days MVPA (Ref.) 60 min/day for 0.55 (0.40–0.74) 0.87 (0.70–1.09) 0.61 (0.45–0.80) 0.84 (0.63–1.12) 0.82 < 7 days MVPA (0.60–1.13) (0.64–0.95) 0.014 - –– –– - 0.95 (0.71–1.26) 0.737 0.60 (0.49–0.73) 0.001 0.78 Family rules about quantity of TV: Yes Model 1: adjusted for age, sex, ST, sleep, duration of immigrant status, BMI, Family rules about quantity of TV and SEP. | +|---| + +Model 1: adjusted for age, sex, ST, sleep, duration of immigrant status, BMI, Family rules about quantity of TV and SEP. + +Model 2: adjusted for age, sex, sleep, duration of immigrant status, BMI, PA, Family rules about quantity of TV and SEP. Model 3: adjusted for age, sex, PA, ST, duration of immigrant status, BMI, Family rules about quantity of TV and SEP. Model 4, 5, and 6: adjusted for age, sex, PA, ST, sleep, duration of immigrant status, BMI, and SEP. + +HIC¼ High-income countries, LMIC ¼ Low- and middle-income countries, PA ¼ physical activity, ST ¼ screen-time, BMI ¼ Body Mass Index, SEP ¼ Socio-economic Position, Ref. ¼ Reference. + +between ST use and immigration status.42 Our study only examined ST, +determined by watching TV and playing electronic games. With the expanding opportunities for media engagement, future research would benefit from examining a wider range of ST, such as smartphone use or the usage of social media platforms that are popular among teens, to explore this question further. + +A recent global systematic review found that only one study had been conducted on sleep behaviour, with immigrant children in the Netherlands reporting less sleep per night than their native-born equivalents.43 Cultural practices such as parenting style and room sharing +(multiple persons sleeping in one room) have been shown to influence sleep behaviours, such as sleep onset and sleep duration.43,44 In our study, immigrant children from LMIC had lower odds of meeting sleep guidelines than HIC and Australian children. Irregular bedtimes and no set family routine may contribute to disparities in sleep quality and duration. Lack of parental knowledge about healthy sleep habits and the significance of bedtime routines may also be associated with harmful sleep practices.45 A recent qualitative study among Brazilian mothers living in the USA also reported inconsistencies in mothers' awareness of the importance of sleep and their parenting practices.46 It is important for the health and wellbeing of LMIC children that we better understand the facilitators and barriers to them having optimal sleep behaviours. + +Immigrant children are influenced by the food culture of the host country and social contexts such as schools or peers, which potentially lead to changes in their dietary habits.47 Our study found low consumption of vegetables in all groups, while immigrant children from LMIC had higher odds of consuming more fruit and cooked vegetable than those from HIC children and Australian children. This finding is consistent with previous research in Australia, which reported that vegetable consumption was highest among immigrant children, while also contradicts with our study that fruit consumption did not differ between immigrant and Australian children.25 Immigrants carry over their dietary preferences and practices but often begin to include westernized foods into their diet.28 A recent systematic review reported Asian children who lived in the USA to have high intakes of fruits, vegetables, white rice, high fat and high sugar foods.28 One probable reason for dietary changes over time among immigrant children is acculturation, the process by which individuals from one cultural context adapt to another cultural context.48 Alternatively, parents of HIC children are likely to have come from a country where the diet has already become more westernized, and so the acculturation process may have started even before arrival to Australia. It should be noted that the measures for fruit and vegetable consumption used in this study were determined by the frequency of consumption, not by serving size. Therefore, the study child may have eaten more or less than one serving of fruit and vegetables on each occasion. Thus, more comprehensive research, including consideration of serving sizes, is needed to inform prevention and intervention efforts. + +The findings point towards the evidence about the disparities in lifestyle behaviours among children with an immigrant background and can inform strategies to bring equity in the lifestyle of Australian children irrespective of their immigrant background. However, each group +(Australian, HIC, and LMIC) has areas to improve on, and special considerations such as language and cultural differences need to consider among immigrant children from LMIC. + +This study's strength is analysing an extensive, nationally representative sample of Australian children. The clustering of data was considered using GEE modelling. However, the study has some limitations that should be acknowledged. Our study assessed only children aged 12–13 years, which constrains the generalization of the study findings beyond this age range. The cross-sectional design of the current study does not allow for any cause-effect interpretation. All lifestyle behaviour data were self-reported in our study. ST was measured using time watching TV and playing electronic games and did not include time spent on social media, video-chatting or browsing the internet, which is likely to have underestimated the overall ST. Sleep duration was measured using sleep onset and wake-up time, which may not indicate uninterrupted sleep. + +## Conclusions + +Our findings highlight that HIC and Australian children have comparable lifestyle behaviours while differences exist in LMIC children compared to HIC and Australian children. LMIC children had lower odds of meeting PA and sleep guidelines, but higher odds of meeting ST, cooked vegetable, and fruit consumption guidelines compared to HIC and Australian children. Our study underscores the importance of targeted, culturally appropriate intervention strategies for promoting and supporting adherence to the health behaviour guidelines among Australian children, with particular emphasis on LMIC children. + +## Submission Statement + +The work has neither been published nor submitted elsewhere and will not submit to any other journal under review in this journal (except in the form of an abstract or as part of a published lecture or academic thesis), its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere including electronically in the same form, in English or any other language, without the written consent of the copyright-holder. + +## Authors' Contributions + +SA and AK conceptualized and designed the study and carried out the analysis. SA, AK, JZ, RU, and SG interpreted the data. SA led the manuscript writing. AK, JZ, RU, and SG reviewed, revised, and approved the final manuscript as submitted. + +## Ethical Approval Statement + +The LSAC received ethics approval from the Australian Institute of Family Studies Ethics Committee (AIFS 15-01), which stated that for each participating child, their parent/guardian provided written informed consent. This secondary analysis was approved by The University of Queensland Human Ethics Committee (2021/HE000253). + +## Conflict Of 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. + +## Acknowledgments + +This paper uses data from Growing Up in Australia, the Longitudinal Study of Australian Children, which was conducted in partnership with + +## + +the Department of Social Services (DSS), the Australian Institute of Family Studies (AIFS) and the Australian Bureau of Statistics (ABS). The authors thank the LSAC study participants, staff, and others relevant personalities for their contributions. SA is supported by the Australian Government Research Training Scholarship and Research Training Tuition Fee Offset Scholarship. RU is supported by an Alfred Deakin Postdoctoral Research Fellowship. + +## Appendix A. Supplementary Data + +Supplementary data to this article can be found online at https://do i.org/10.1016/j.smhs.2022.02.002. + +## References + +1. Leech RM, McNaughton SA, Timperio A. The clustering of diet, physical activity and sedentary behavior in children and adolescents: a review. Int J Behav Nutr Phys Activ. + +2014;11:4. https://doi.org/10.1186/1479-5868-11-4. + +2. Kell KP, Cardel MI, Bohan Brown MM, et al. Added sugars in the diet are positively associated with diastolic blood pressure and triglycerides in children. Am J Clin Nutr. + +2014;100(1):46–52. https://doi.org/10.3945/ajcn.113.076505. + +3. Carson V, Stone M, Faulkner G. 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Appl Psychol. 1997;46(1): +5–34. https://doi.org/10.1111/j.1464-0597.1997.tb01087.x. \ No newline at end of file diff --git a/medical/md/PMC9251660.md b/medical/md/PMC9251660.md new file mode 100644 index 0000000000000000000000000000000000000000..e9be293e5eb38f620c021dd0dcf88ad5e9338ae0 --- /dev/null +++ b/medical/md/PMC9251660.md @@ -0,0 +1,245 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +# Tuberculosis Among Patients With Systemic Lupus + +## Erythematosus In Indonesia: A Cohort Study + +Laniyati Hamijoyo,1,2, Edhyana Sahiratmadja,3,4, Nadia G. Ghassani,2, Guntur Darmawan,5, Evan Susandi,6, Reinout van Crevel,7, Philip C. Hill,8, + +## And Bachti Alisjahbana4,9, + +1Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine, Universitas Padjadjaran/Dr. Hasan Sadikin General Hospital, Bandung, Indonesia, 2Study Center of Immunology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia, 3Department of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia, 4Research Center for Care and Control of Infectious Diseases, Universitas Padjadjaran, Bandung, Indonesia, 5Department of Internal Medicine, Faculty of Medicine, Krida Wacana Christian University, Jakarta, Indonesia, 6Department of Internal Medicine, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia, 7Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands, 8Centre for International Health, University of Otago, Dunedin, New Zealand, and 9Division of Tropical Diseases, Department of Internal Medicine, Faculty of Medicine, Universitas Padjadjaran/Dr. Hasan Sadikin General Hospital, Bandung, Indonesia Background. In previous studies, researchers have identified systemic lupus erythematosus (SLE) as a risk factor for tuberculosis (TB), but data from TB-endemic countries are still relatively scarce. We examined TB in a large cohort of SLE patients in Indonesia. + +Methods. All patients registered in a lupus registry of the top referral hospital for West Java between 2008 and 2020 were included. Data on SLE characteristics and treatment were retrieved from the registry, and data on TB diagnosis, localization, and outcome were extracted from medical records. Cox-proportional hazard model was used to examine risk factors for development of TB. + +Results. Among 1278 SLE patients observed over a total of 4804 patient-years, 131 patients experienced 138 episodes of TB, +a median of 2 years (interquartile range, 0.6–5.4) after diagnosis of SLE. A total of 113 patients (81.9%) had pulmonary involvement and 61 (44.2%) had extrapulmonary involvement, with disseminated disease in 26 of 138 episodes (18.8%), and 13 of 131 patients (9.9%) died from TB. The estimated TB incidence was 2873 cases per 100 000 person years. In multivariate cox regression analysis, development of TB was associated with household TB contact (hazard ratio [HR], 7.20; 95% confidence interval [CI], 4.05–12.80), pulse methylprednisolone therapy (HR, 1.64; 95% CI, 1.01–2.67), and age ≤25 years old at SLE +diagnosis (HR, 1.54; 95% CI, 1.00–2.35). + +Conclusions. There is a high burden of TB in SLE patients in this TB-endemic setting, underlining the need for evaluation or implementation of TB preventive strategies. + +Keywords. Indonesia; risk factors; systemic lupus erythematosus; tuberculosis. + +Infections are a leading cause of morbidity and mortality among patients treated with immunosuppressive agents. An infectious complication of particular interest is tuberculosis (TB), which is reported to be more frequent in patients with rheumatic diseases including systemic lupus erythematosus (SLE), +especially for those living in TB-endemic settings [1,2]. + +Development of TB may either be a result of Mycobacterium tuberculosis infection earlier in life, with reactivation of latent TB infection under immunosuppressive therapy, or newly acquired infection with primary progression to active TB [3]. Moreover, due to immunosuppressive therapy, extrapulmonary or disseminated TB may be more common, diagnosis of TB may be more challenging, and treatment outcomes might be worse in patients with SLE [4,5]. + +Indonesia has the second highest number of TB cases globally [6], but little is known about the TB burden among SLE patients in Indonesia. Therefore, in this study, we evaluated incidence, clinical characteristics, and risk factors for TB in SLE patients in an urban setting in Indonesia. + +## Methods Study Design And Settings + +This was a retrospective cohort study involving all SLE patients who were registered from 2008 to 2020 in the Lupus Registry at Dr. Hasan Sadikin General Hospital, Bandung, Indonesia, the largest referral hospital for West Java, a province with 41 million inhabitant. In brief, the Lupus Registry is a database relating to SLE patients from the time they were first diagnosed onwards. Included are data on clinical manifestations, laboratory test results, treatment, and clinical outcome [7]. + +Comorbidities are recorded both at onset (entry in the registry) +and during follow-up. However, details of TB (such as specific TB manifestations, microbiological testing etc) were retrieved from patients' medical records). + +## Data Collection + +Patients were included if they fulfilled American College of Rheumatology 1997 and/or Systemic Lupus International Collaborating Clinics 2012 Classification Criteria for SLE. + +They were excluded if there was no date of SLE diagnosis or date of birth. History of Bacille Calmette-Guerin (BCG) vaccination, smoking, and close contact to an infectious TB patient were retrieved by interview when patients entered the SLE registry. The SLE starting date was recorded as the first time an individual was diagnosed with SLE. Most of our patients visit the outpatient clinic monthly initially, then every 3 months when SLE is in remission. Recorded visits of each patient were collected from the registry until their last visit in December 2020. Patients were considered lost to follow-up if they did not have any visit from December 2018 to the end of the study date and were not known to have died. Medications used for SLE were extracted from the registry and included corticosteroids as well as complications and their treatment, such as vasculitis, nephritis, and hemodialysis. Recorded information on other comorbidities, such as diabetes, was limited and not included in the analysis. Histopathology, sputum smear microscopy for acid-fast bacilli, and sputum molecular TB diagnostic testing by Xpert MTB/Rif (which was introduced in approximately 2012) were used to define a diagnosis of definite TB; sputum M tuberculosis culture is not routinely performed in Indonesian hospitals. + +Clinical TB (without microbiological confirmation) was defined as TB diagnosed or anti-TB treatment started by the attending physician. + +## Patient Consent Statement + +Written consent was obtained from all of patients involved in this study. The registry had received ethical clearance from the Ethic Committee of University of Padjadjaran (number 036/UN6.KEP/EC/2021). + +## Statistical Analysis + +Comparisons between presenting clinical features and the prevalence of various clinical manifestations were made using the χ 2 or Fisher exact tests. The unpaired Students' t test was used to compare parametric continuous data between 2 groups. When normal distribution or equal variance could not be assumed, the Mann-Whitney U test was used instead. Correlation between various continuous data was evaluated by Spearman's rank correlation test. Estimates of the survival of the patients were studied by life table analysis using the Kaplan-Meier method. Survival curves with the presence and absence of a TB event were compared using the non-parametric log-rank test. Univariate and multivariate analyses of the predictive factors for TB were conducted using a Cox proportional hazard regression model. Statistical significance was defined as a P value of less than .05, 2-tailed, without correction for multiple testing. + +## Results + +Among a total of 1293 registered SLE patients, 1278 could be included in the study, most of whom were young females +(Table 1). Nephritis was the most commonly reported complication (45.3%). Almost all patients used corticosteroids, and many had previously received pulse methylprednisolone (MP) or other immunosuppressive therapy (Table 1). + +Regarding possible other risk factors for TB, human immunodeficiency virus (HIV) and diabetes were not systematically screened for, but few reported smoking or a history of TB in the household. There were 130 (10.2%) patients with a history of TB when they were diagnosed with SLE, and 18 of them developed another episode of TB after diagnosed SLE. + +## Table 1. Baseline Characteristic And Treatment Of Systemic Lupus Erythematosus Patientsa + +| Total SLE Patients | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------| +| Characteristics | n = 1278 | +| Female gender | 1123 (95.7) | +| Median disease duration, years (IQR) | 2.3 (0.5–5.8) | +| Median age diagnosed, years (IQR) | 27 (22–35) | +| Age of SLE diagnosis ≤25 years old | 552 (43.2) | +| .25 years old | 726 (56.8) | +| BCG scar, n/N (%) | 230/388 (59.3) | +| Household TB contact, n/N (%) | 29/711 (4.1) | +| Smoking (current/ever), n/N (%) | 69/756 (9.1) | +| History of TB before SLE diagnosis | 130 (10.2) | +| Complication Nephritis | 579 (45.3) | +| Vasculitis | 85 (6.7) | +| Hemodialysis | 18 (1.4) | +| Medication Corticosteroids | 1247 (97.6) | +| Chloroquine/hydroxychloroquine | 675 (52.8) | +| Azathioprine | 557 (43.6) | +| Cyclophosphamide | 293 (22.9) | +| Mycophenolate mofetil/mycophenolate acid | 287 (22.5) | +| Ever methylprednisolone pulse therapy | 239 (18.7) | +| Methotrexate | 82 (6.4) | +| Cyclosporine | 84 (6.6) | +| Rituximab | 9 (0.7) | +| Abbreviations: BCG, Bacille Calmette-Guerin; IQR, interquartile range; SLE, systemic lupus erythematosus; TB, tuberculosis. a Data are presented as percentage unless stated otherwise. Data for BCG scar, household TB contacts, and smoking status were only available for 388, 711, and 756 individuals, | | + +| Proportion With | | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------|----------------------| +| Disease Localization | Number (%) | Definite TB (n/N, %) | +| Pulmonary and extrapulmonary | 36 (26.1) | | +| Pulmonary only | 77 (55.8) | | +| Extrapulmonary only | 25 (18.1) | | +| Pulmonary involvement | 113 (81.9) | 61/85 (71.8) | +| Extrapulmonary involvement | 61 (44.2) | | +| Meningeal | 11 (8.0) | 2/3 (66.7) | +| Pleural | 9 (6.5) | | +| Pericardial | 1 (0.7) | | +| Lymphadenitis | 21 (15.2) | 11/11(100) | +| Miliary | 15 (10.9) | | +| Abdominal | 6 (4.3) | 3/3 (100) | +| Genitourinary | 1 (0.7) | 1/1(100) | +| Mucocutaneous | 2 (1.4) | | +| Musculoskeletal | 5 (3.6) | | +| Abbreviations: SLE, systemic lupus erythematosus; TB, tuberculosis. a Definite TB: positive to at least 1 of histopathology, sputum microscopy, or Xpert MTB/Rif molecular testing. The value is the positive results of the tests that have been done. | | | + +Patients were followed for a median of 2.3 years (interquartile range [IQR], 0.5–5.8). During follow-up, 138 episodes of TB + +![2_image_0.png](2_image_0.png) + +were recorded in 131 patients; 7 patients experienced 2 episodes of TB during follow-up. Most patients presented with (1) +pulmonary TB or (2) a combination of pulmonary and extrapulmonary disease (Table 2). Microbiological examination could be verified in 85 of 113 (65%) patients with pulmonary disease, 61 of 85 (72%) of whom had confirmed TB by positive sputum smear microscopy (47 or 64 tested) and/or sputum Xpert TB/ +RIF testing (47 or 64 tested). Three patients who developed pulmonary TB were diagnosed with rifampicin-resistant TB, based on sputum Xpert TB/Rif molecular testing. Among 62 patients with extrapulmonary involvement, most suffered from lymphadenitis (n = 22), miliary disease (n = 15), or meningitis (n = +11); bacteriological or histopathological confirmation of TB +was found in 17 of these patients. Thirteen of the 131 diagnosed TB patients (9.9%) died during their episode of active TB. + +With 138 episodes of TB disease during 4804 person years of follow-up, the incidence was 2873 (95% confidence interval +[CI], 2400–3345) cases per 100 000 person years. The median duration until the diagnosis of TB after SLE diagnosis was 2 years (IQR, 0.6–5.4). There were 6 patients diagnosed with TB within 2 weeks after diagnosis of SLE. The incidence rate of TB was 48.5/1000 patients in the first year after SLE diagnosis, diminishing to 30/1000 patients by year 5, and remaining relatively constant after that (data in the Supplement). The estimated cumulative TB incidence at 15 years was 29.0% +(Figure 1). + +| n (%) n (%) Crude HR 95% CI for Crude HR P Value Adjusted HR 95% CI for Adjusted HR P Value Adjusted HR 95% CI for Adjusted HR P Value | Gender Male 55 7 (12.7) 48 (87.3) 1.47 .69–3.15 .323 1.27 .53–3.02 .595 Female 1223 124 (10.1) 1099 (89.9) 1 (ref) 1 (ref) Age of SLE Diagnosis ≤25 years old 552 69 (12.5) 483 (87.5) 1.54 1.09–2.17 .014* 1.57 1.02–2.42 .040* 1.54 1.00–2.35 .049* .25 years old 726 62 (8.5) 664 (91.5) 1 (ref) 1 (ref) BCG (n = 388) Yes 230 44 (19.1) 185 (80.9) 0.63 .42–.95 .026* No 158 50 (31.6) 108 (68.4) 1 (ref) History of TB Yes 130 18 (13.8) 112 (86.2) 1.24 .75–2.04 .400 1.14 .59–2.18 .702 No 1148 113 (9.8) 1035 (90.2) 1 (ref) 1 (ref) Household TB Contact (n = 711) Yes 29 15 (51.7) 14 (48.3) 7.17 4.08–12.60 ,.001* 6.83 3.64–12.79 ,.001* 7.20 4.05–12.80 ,.001* No 682 74 (10.9) 608 (89.1) 1 (ref) 1 (ref) 1 (ref) Hemodialysis Yes 18 3 (16.7) 15 (83.3) 1.64 .52–5.17 .395 2.92 .88–9.67 .080 No 1260 128 (10.2) 1132 (89.8) 1 (ref) 1 (ref) Nephritis Yes 579 53 (9.2) 526 (90.8) 0.91 .64–1.29 .595 1.26 .77–2.05 .356 No 699 78 (11.2) 621 (88.8) 1 (ref) 1 (ref) Vasculitis Yes 85 11 (12.9) 74 (87.1) 1.24 .67–2.30 .497 1.21 .59–2.45 .603 No 1193 120 (10.1) 1073 (89.9) 1 (ref) Ever MP Pulse Yes 239 28 (11.7) 211 (88.3) 1.62 1.06–2.46 .026* 1.63 .98–2.70 .060 1.64 1.01–2.67 .047* No 1039 103 (9.9) 936 (90.1) 1 (ref) 1 (ref) 1 (ref) Corticosteroid Yes 1247 131 (10.5) 1116 (89.5) No 31 0 (0) 31 (100) Azathioprine Yes 557 61 (11) 496 (89) 0.73 .52–1.04 .078 0.88 .56–1.38 .582 No 721 70 (9.7) 651 (90.3) 1 (ref) 1 (ref) Methotrexate Yes 82 11 (13.4) 71 (86.6) 0.94 .51–1.76 .856 0.86 .39–1.89 .699 No 1196 120 (10) 1076 (90) 1 (ref) 1 (ref) Cyclosporin | +|------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Initial Model Final Model | | +| Multivariate Analysis | Univariate Analysis | +| Table 3. Factors Associated With Occurrence of Tuberculosis During Follow-up | Variable Total | +| TB (n = 131) Non-TB (n = 1147) | | + +![4_image_0.png](4_image_0.png) + +In this cohort of approximately 1300 SLE patients in West Java, Indonesia, TB was a common complication. Although most patients presented with pulmonary disease, almost one fifth of cases presented with disseminated miliary TB or TB meningitis. + +Younger age (≤25 years) at the time of SLE diagnosis, highdose pulse corticosteroid therapy, and history of a TB contact were identified as risk factors for development of TB. These data clearly show the burden of TB in SLE patients living in TB-endemic countries and the need for awareness and mitigating measures. + +Our study is the largest reported so far from a TB-endemic country, but the incidence of TB in SLE patients in this study was comparable to studies from India (309 SLE patients; TB incidence 2450/100 000 person-years), Malaysia (102 SLE patients; TB incidence 1067/100 000 person-years), and Korea (283 SLE +patients; TB incidence 790/100 000 person-years) [1]. The incidence was much higher than the current estimated TB incidence in Indonesia of 312/100 000, or the estimated incidence of 349/ +100 000 in 2008, when thefirst patients entered this cohort [6]. + +The younger age at SLE diagnosis along with the disease itself and additional exposure to immunosuppressive therapy result in an increased susceptibility to TB. Significantfindings regarding younger age were noted to increase the risk of TB in this study. Bacille Calmette-Guerin vaccine, which used to be given Table 3. ContinuedVariable Totalin newborns, has proved to protect against TB in young +Significant differences were found between patients who developed TB and those who did not (Table 3). In multivariate cox regression analysis, household TB contact (hazard ratio +[HR], 7.20; 95% CI, 4.05–12.80), a history of methylprednisolone pulse therapy (HR, 1.64; 95% CI, 1.01–2.67), and age ≤25 years old at SLE diagnosis (HR, 1.54; 95% CI, 1.00–2.35) +were independently and significantly associated with development of TB, whereas a history of BCG vaccination (HR, 0.63; 95% CI, .42–.95) was associated with a lower risk (HR, 0.67; 95% CI, .43–1.04) (Table 3). The cumulative TB incidence was significantly higher in patients without a BCG scar and those who were under age 25 years at time of diagnosis (Figure 2A and B, increasing steadily over time). Those who had a known TB contact and those who had received MP pulse therapy also showed significant differences in cumulative incidence, which plateaued after the first few years of follow-up (Figure 2C and D), in keeping with MP pulse therapy mostly being used early in SLE treatment, and TB contact history being taken at the time of recruitment. Of the 1278 lupus patients in this cohort, 131 died during the study period. A total of 567 patients did not visit the rheumatology outpatient clinic in the final 2 years of the study period (2018–2020); they most likely continued their care at a lower-level health facility and were considered lost to follow-up. + +## Discussion + +![5_image_0.png](5_image_0.png) + +children, but its efficacy varies against pulmonary TB in adults +[8,9]. In a review of several observational studies, researchers reported that BCG induced trained immunity against TB, +which might involve epigenetic and metabolic changes of innate immune cells [10]. Our data demonstrated a significant protective effect of BCG (HR, 0.63; P = .026) on TB development among SLE patients, but multivariable analysis could not be performed due to missing data. + +Autoimmune diseases including SLE, especially where immunosuppressive medications are needed based on disease activity, may amplify the risk of TB. As shown in previous studies, the use of steroid and or immunosuppressive drugs implies serious potential risk factors for infections [2,11,12]. Active TB was also reported to be more frequent in patients with other rheumatic diseases, including rheumatoid arthritis, Sjogren's syndrome, and systemic vasculitis, especially for those living in TB-endemic settings [2]. Corticosteroid and immunosuppressive agents are the mainstay treatment in SLE. A retrospective casecontrol study in Colombia showed that 12 months of cumulative steroid dose ≥1830 mg almost tripled the risk of TB [13]. A +13-year cohort study of SLE patients in Taiwan has shown that corticosteroid treatment was associated with a more than 10-fold increased risk of TB [14]. In our study, almost all patients used corticosteroids, but those patients who also received pulse methylprednisolone treatment had an almost 2-fold increased risk of TB, in line with a longitudinal study from China [15]. + +Evidence of risk of TB associated with the use of other immunosuppressive drugs in patients with SLE is mixed [16,17]. + +Cyclophosphamide was associated with an almost 3-fold risk of TB in a cohort study in Taiwan [14]. In contrast, consistent with our study, there was no increase in risk in a case-control study in Hong Kong, where azathioprine, cyclophosphamide, and cyclosporin were the immunosuppressive agents used +[18]. In univariate analysis, chloroquine or hydroxychloroquine use was associated with a lower risk of developing TB +among our SLE patients, but this finding may also be a result of confounding by indication, because these drugs are mostly used for milder disease. We did not specifically measure disease activity as a risk factor, but nephritis, as a marker of higher disease activity, was not significantly associated with the development of TB. Yang et al [14], showed a similar finding in SLE +patients with renal complications. In contrast, a study from Tam et al [18] showed nephritis was associated with over twice the risk of TB. + +## Conclusions + +Our study has several limitations. First, some relevant clinical data were not recorded in our database, such as average daily and cumulative dose of corticosteroids [18,19]. Second, data on other risk factors for TB such as diabetes and HIV infection were incomplete, noting that diabetes prevalence is likely to be low in this young population (as shown in the low proportion with glycosuria), and that the HIV prevalence in the general population in Indonesia is estimated to be only 0.3%. We did not actively perform TB-case finding for all patients. In addition, bacteriological assessments were incomplete, noting that routine TB diagnosis in Indonesia does not include sputum culture, and that Xpert TB, which was introduced in 2012, is still not widely implemented [20]. Finally, subjects were patients at tertiary referral hospital, limiting the generalizability of the findings of the study and loss to follow-up cases from the registry. However, our study can still conclude that there is a very high burden of TB among SLE patients in this TB-endemic setting. This finding reinforces the need for studies evaluating the benefits or implementation of latent tuberculosis infection screening and TB-preventive therapy in TB-endemic countries. + +## Supplementary Data + +Supplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. + +## Acknowledgments + +We are grateful to all members of the Lupus Study Group in the Immunology Study Center of Faculty of Medicine Universitas Padjadjaran, who helped with collecting data and entering information into the Hasan Sadikin Lupus Registry. We thank Dr. Alfons Den Broeder, rheumatologist and clinical epidemiologist from Sint Marteenkliniek Nijmegen, for valuable input to this manuscript. + +Authors contributions. L. H. contributed to conception, design, data collection, and interpretation and drafted and revised the manuscript. E. Sa., +R. v. C., P. C. H., and B. A. contributed to conception, design, and data interpretation and drafted and revised the manuscript. G. D. drafted and revised manuscript. N. G. G. contributed to data collections and interpretation and revised the manuscript. E. Su. contributed to data analysis and revised the manuscript. + +Financial support. This work was funded by the Internal Research Fund of University of Padjadjaran Bandung Indonesia. + +Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. + +## References + +1. Erdozain JG, Ruiz-Irastorza G, Egurbide M, Martinez-Berriotxoa A, Aguirre C. + +High risk of tuberculosis in systemic lupus erythematosus. Lupus 2006; 15:232–5. + +2. Lu MC, Lai CL, Tsai CC, Koo M, Lai NS. Increased risk of pulmonary and extra-pulmonary tuberculosis in patients with rheumatic diseases. Int J Tuberc Lung Dis 2015; 19:1500–6. + +3. Delogu G, Goletti D. The spectrum of tuberculosis infection: new perspectives in the era of biologics. J Rheumatol 2014; 91:11–6. + +4. Hou CL, Tsai YC, Chen LC, Huang JL. Tuberculosis infection in patients with systemic lupus erythematosus: pulmonary and extra-pulmonary infection compared. + +Clin Rheumatol 2008; 27:557–63. + +5. Prabu V, Agrawal S. Systemic lupus erythematosus and tuberculosis: a review of complex interactions of complicated diseases. J Postgrad Med 2010; 56:244–50. + +6. World Health Organization. Global Tuberculosis Report 2020. Available at: +https://www.who.int/publications/i/item/9789240013131/. Accessed 9 August 2021. + +7. Hamijoyo L, Candrianita S, Rahmadi A, et al. The clinical characteristics of systemic lupus erythematosus patients in Indonesia: a cohort registry from an Indonesia-based tertiary referral hospital. Lupus 2019; 28:1604–9. + +8. Davenne T, Mcshane H. Why don't we have an effective tuberculosis vaccine yet? + +Expert Rev Vaccines 2016; 15:1009–13. + +9. Shimeles E, Enquselassie F, Aseffa A, et al. Risk factors for tuberculosis: a casecontrol study in Addis Ababa, Ethiopia. PLoS One 2019; 14:e0214235. + +10. Foster M, Hill P, Setiabudiawan T, Koeken VACM, Alisjahbana B, van Crevel R. + +BCG-induced protection against Mycobacterium tuberculosis infection: evidence, mechanisms, and implications for next-generation vaccines. Immunol Rev 2021; 301:122–44. + +11. Danza A, Ruiz-Irastorza G. Infection risk in systemic lupus erythematosus patients: susceptibility factors and preventive strategies. Lupus 2013; 22: +1286–94. + +12. Jung JY, Yoon D, Choi Y, Kim HA, Suh CH. Associated clinical factors for serious infections in patients with systemic lupus erythematosus. Sci Rep 2019; 9: +9704. + +13. González-Naranjo LA, Coral-Enríquez JA, Restrepo-Escobar M, et al. Factors associated with active tuberculosis in Colombian patients with systemic lupus erythematosus: a case-control study. Clin Rheumatol 2021; 40:181–91. + +14. Yang S, Lai Y, Huang M, Tsai CS, Wang JL. Corticosteroid dose and the risk of opportunistic infection in a national systemic lupus erythematosus cohort. + +Lupus 2018; 27:1819–27. + +15. Xiao X, Da G, Xie X, et al. Tuberculosis in patients with systemic lupus erythematosus–a 37-year longitudinal survey-based study. J Intern Med 2021; 290: +101–15. + +16. Rúa-Figueroa Í, López-Longo J, Galindo-Izquierdo M, et al. Incidence, associated factors and clinical impact of severe infections in a large, multicentric cohort of patients with systemic lupus erythematosus. Semin Arthritis Rheum 2017; 47: +38–45. + +17. Feldman CH, Hiraki LT, Winkelmayer WC, et al. Serious infections among adult Medicaid beneficiaries with systemic lupus erythematosus and lupus nephritis. + +Arthritis Rheumatol 2015; 67:1577–85. + +18. Tam LS, Li EK, Wong SM, Szeto CC. Risk factors and clinical features for tuberculosis among patients with systemic lupus erythematosus in Hong Kong. Scand J +Rheum 2002; 31:296–300. + +19. Sayarlioglu M, Inanc M, Kamali S, et al. Tuberculosis in Turkish patients with systemic lupus erythematosus: increased frequency of extrapulmonary localization. + +Lupus 2004; 13:274–8. + +20. van Kampen SC, Susanto NH, Simon S, et al. Effects of introducing Xpert MTB/ +RIF on diagnosis and treatment of drug-resistant tuberculosis patients in Indonesia: a pre-post intervention study. PLoS One 2015; 10:e0123536. \ No newline at end of file diff --git a/medical/md/PMC9433691.md b/medical/md/PMC9433691.md new file mode 100644 index 0000000000000000000000000000000000000000..e9c10bff77036d15283515880c0fc7814a09589b --- /dev/null +++ b/medical/md/PMC9433691.md @@ -0,0 +1,211 @@ + + +![0_image_1.png](0_image_1.png) + +![0_image_2.png](0_image_2.png) + +![0_image_3.png](0_image_3.png) + +| ARTICLE INFO Keywords: Orthodontic tooth movement Orthodontic compressive force Bone resorption Osteoclast differentiation GSK-3β/β-catenin signaling pathway | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------| + +| ABSTRACT by regulating the GSK-3β/β-catenin pathway. | +|--------------------------------------------------------| + +Research article Compressive force regulates GSK-3β in osteoclasts contributing to alveolar bone resorption during orthodontic tooth movement in vivo Yan Liu a,b, Ke Wu a, Xing Cui a, Yelin Mao a,* +a The Affiliated Stomatology Hospital of Suzhou Vocational Health College, Suzhou, 215000, China b The First People's Hospital of Changzhou, Changzhou, 213003, China https://doi.org/10.1016/j.heliyon.2022.e10379 + +![0_image_0.png](0_image_0.png) + +Received 19 January 2022; Received in revised form 11 April 2022; Accepted 15 August 2022 2405-8440/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). + +Background: Orthodontic tooth movement mainly depends on biological and mechanical reactions in the periodontium, such as the indispensable reconstruction process of the periodontal ligament and alveolar bone. To explore whether orthodontic compressive force can induce bone resorption during orthodontic tooth movement by regulating the GSK-3β/β-catenin pathway. + +Methods: We established orthodontic tooth movement models in Sprague-Dawley rats. In addition, compressive force-induced bone resorption that occurred during orthodontic tooth movement was analyzed by HE staining and micro-CT. The number and distribution of osteoclasts were observed by TRAP staining. Furthermore, pressureinduced bone resorption mediated by the GSK-3β/β-catenin signaling pathway was analyzed by immunohistochemistry. + +Results: As shown by the micro-CT results, bone parameters, such as bone mineral density (BMD), the bone volume fraction (BV/TV), and trabecular thickness (Tb. Th), were significantly decreased under orthodontic compressive force stimulation, in contrast with the dramatically increased trabecular spacing (Tb. Sp). During the process of tooth movement, the compressive force can induce bone resorption on the side with the force, which increases the expression of phosphorylated Ser-GSK-3β and activation of the β-catenin signaling pathway. Additionally, downregulation of the GSK-3β activity further caused the downregulation of bone parameters, leading to bone loss. The TRAP staining and immunohistochemistry staining results indicated that orthodontic compressive force influenced osteoclast formation and the secretion of osteoclast-related cytokines, matrix metallopeptidase 9 +(MMP-9) and receptor activator of nuclear factor-κB ligands (RANKLs), which is also related to the duration of orthodontic force. + +Conclusions: These results indicated that the GSK-3β inhibitor can promote osteoclast formation on the side with orthodontic compressive force. In addition, the activation of the GSK-3β/β-catenin signaling pathway contributes to bone reconstruction caused by orthodontic compressive force. Therefore, the GSK-3β/β-catenin signaling pathway can be a potential target for further clinical applications. + +tension side forms a vacancy, and the new bone can fill this vacancy [4]. + +During orthodontic teeth movement, on the compression side of the periodontal ligament, osteoblasts proliferate in large numbers to generate and deposit new bone tissue along the alveolar bone; on the tension side where the periodontal ligament is compressed, a large number of osteoclasts accumulate, causing the alveolar bone to resorb. + +Bone resorption on the compression side provides a movable space for orthodontics. Hence, osteoclasts play an important role in the process of transforming mechanical force into biological signals. A study showed that osteoclasts and the RANKL of osteoclast precursor cells can sequentially bind receptors on the osteoclast membrane [5]. This 1. Introduction In orthodontic clinical treatment, the major purpose of intraoral or extraoral orthodontic devices is to move teeth to achieve ideal orthodontic effects [1]. Orthodontic tooth movement mainly depends on biological and mechanical reactions in the periodontium, such as the indispensable reconstruction process of the periodontal ligament and alveolar bone [2]. Similar to that of other bone tissues, the process of moving teeth mainly involves osteoclasts and osteoblasts [3]. These detailed steps, including the activation of osteoclasts on the pressure side, lead to bone resorption. When a tooth moves, the alveolar bone on the +* Corresponding author. + +E-mail address: yelin_mao@126.com (Y. Mao). + +Contents lists available at ScienceDirect journal homepage: www.cell.com/heliyon combination promotes the activation of osteoclasts and forms a unique signal cascade [6]. Furthermore, RANKL upregulation can promote the secretion of matrix metalloproteinases-9 (MMP-9) and induce bone resorption [7]. In recent years, research has found that mechanical stimulation has a certain regulatory effect on periodontal ligament cells, thereby affecting the reconstruction of periodontal tissue on the side with tension [8]. The situation is opposite on the side with compressive force; the gaps among the periodontal ligament on the compressive force side are relatively small, the fibers are relatively concentrated, the blood vessels are compressed, and osteoclasts are easily differentiated +[9]. Therefore, osteoclasts on the compressive force side of the orthodontic process are essential, but their underlying mechanism is still unclear. + +In our previous study, we found that the tension force can induce bone formation via regulated GSK3β, and the binding between Wnt protein and its transmembrane receptor can lead to the inhibition of GSK-3β and the stabilization of β-catenin. In addition, β-catenin accumulation in the nucleus activates downstream Wnt target genes, leading to osteogenic differentiation [10]. However, the role of GSK3β on the side with compressive force is less known. A better understanding of the mechanisms by which GSK-3β regulates osteoclast formation on the compressive side is very important. In this study, we found that a GSK-3β inhibitor could accelerate osteoclast formation on the compressive side. Furthermore, the promoted nuclear translocation of β-catenin results in decreased RANKL expression by inhibiting GSK-3β. Finally, the activation of the GSK-3β/β-catenin signaling pathway is beneficial to orthodontic compressive force-induced bone reconstruction (Scheme 1). + +## 2. Materials And Methods 2.1. Experimental Animals + +![1_Image_0.Png](1_Image_0.Png) + +Thirty Sprague-Dawley (SD) male rats that were 8 weeks old and had body weights of 200–250 g were purchased from the Experimental Animal Center of Soochow University (Suzhou, China). The animal handling and surgical procedures were conducted in accordance with protocols approved by the Ethics Committee at the First Affiliated Hospital of Soochow University (201709A472). + +2.2. Establishing orthodontic tooth movement models and drug administration procedures The SD rats were divided into the following groups: SD rats treated with orthodontic tooth movement (OTM) (denoted as OTM, n ¼ 15), LiCl (denoted as OTM þ LiCl, n ¼ 5), ICG001 (denoted as OTM þ ICG001, n ¼ +5), and LiCl and ICG001 (denoted as OTM þ LiCl þ ICG001, n ¼ 5). The rats were anesthetized by intraperitoneal administration of 1.5% pentobarbital sodium. Then, the OTM model was established by applying a closed nitinol spring (50-g force) between the maxillary incisor and left first molar to cause the first molar to move to the middle. LiCl (200 mg/ +kg/day) was intragastrically administered to the rats. Additionally, ICG001 (10 μg/day) was locally injected into the mucoperiosteal of the left first molars. Rats in the OTM group were sacrificed at days 0, 7 and 14 (n ¼ 5 at each time point, denoted as the control, 7 d and 14 d). At each point, rats were euthanized, and the whole maxilla, including the left first, secondly, and third molars, was collected. Micro CT was used to analyze the histology. The distance between the nearest points of dental crown of the first and second molars was measured on the digital radiographs. + +## 2.3. Micro Ct Scanning And Quantified Analysis + +High-resolution micro-CT (SkyScan 1176, Belgium) scanned the maxilla with fixed parameters (resolution: 18 μm, voltage: 80 kV, current: +100 μA). After scanning, three-dimensional image reconstructions were performed. For the quantified analysis, the region of interest (ROI) was the alveolar bone area located in the middle of the mesial cheek root. The top of the cube (500 μm - 500 μm - 500 μm) was located at the interface between 1/3rd of the tooth root and 1/3rd of the root tip. After scanning, three-dimensional (3D) digital image reconstruction was carried out using the supporting analysis software (Mimics). Then the resident quantitative analysis software (CTAn, Skyscan) was used to obtain the following parameters: bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb. Th) and trabecular spacing (Tb. Sp). + +2.4. Hematoxylin-eosin staining and tartrate-resistant acid phosphatase staining After decalcification, the tissues were embedded in paraffin and sliced. Then, these specimens were dewaxed with xylene and stained with hematoxylin for 5 min. The residual hematoxylin was cleaned with deionized (DI) water, and then the specimen was immersed in a 75% +hydrochloric acid ethanol solution for 30 s. After being cleaned with DI +water for 2 min, these specimens were immersed in ammonia for 30 s. Then, the specimens were washed with DI water and stained with eosin dye for 2 min. Finally, these specimens were successively immersed in 95% ethanol and xylene for two cycles (1 min for each solution) and sealed with neutral resin. + +The specimens were fixed with fixative for 30 s and then washed with DI water for 1 min. Then, the specimens were immersed in the prepared TRAP staining solution and incubated for 1 h at 37 C under light protection. After that, the residual TRAP staining solution was cleaned with DI water. Finally, the specimens were stained with hematoxylin and sealed with neutral resin. + +## 2.5. Immunohistochemistry + +After Micro-CT scanning, the maxillae were dissected and trimmed into smaller blocks containing the first molars. All blocks were decalcified in 10% (w/v) ethylene diamine tetraacetic acid for 3 weeks, and then embedded in paraffin. The blocks were cross-sectioned to 5 μm, mounted on protein-coated glass slides, dewaxed. For immunohistological staining, the specimens were incubated with GSK-3β antibody +(1:200, ab32391), phosphorylation-Ser9-GSK-3β antibody (1:250, ab75814), β-catenin (1:500, ab32572), RANKL antibody (1:500, sc71955), MMP-9 antibody (1:500, ab10375), and sclerostin antibody +(1:100, ab63097) overnight at 4 C. + +Then, the specimens were washed and incubated with the secondary antibody (coupled with biotin) for 30 min. After that, the specimens were further incubated with avidin-biotinase for another 30 min and processed with a 3,3N-diaminobenzidine tertrahydrochloride (DAB) color development kit. Cells showing brown particles in the cytoplasm or intracell were marked as positive cells, and 10 random views were selected to count the number of positive cells. The results were calculated by two different observers separately. Briefly, the histomorphometric analysis was performed on the most central section. A round-shaped region of interest was defined as previously recommended. Using 20- magnification, the bone tissue was circled by the operator, and the software automatically recorded and calculated the determined area with positive cells. + +## 2.6. Statistical Analysis + +Non-parametric T test was used to measure significant differences among the treatment groups (*p values <0.05, and **p values <0.01). + +Data were taken as the mean standard error. + +## 3. Results + +3.1. Compressive force-induced bone resorption during the process of orthodontic tooth movement As the H&E staining and micro-CT 3D reconstruction results indicated when orthodontic compressive force was applied, there was an + +![2_image_0.png](2_image_0.png) + +increased gap between the first molar that experienced mesial movement and the second molar, as shown in Figure 1A. Under orthodontic compressive force, the periodontal ligament fibers of rats became arranged irregularly with an incomplete morphology. After an orthodontic compressive force of 7 d, the periodontal space was very narrow; the alveolar bone was destroyed, and the bony edge was obviously absorbed with resorption lacuna. In contrast, rats in the control group had regular arrangements of periodontal ligament fibers and uniform periodontal spaces. The fibroblasts were evenly distributed in the periodontal ligament fibers with a clear cytoplasmic nucleus structure. After an orthodontic compressive force for 14 d, the rats had an obviously larger ~7.32 fold (P < 0.01) periodontal space between the first molar and the second molar than that of the rats without an orthodontic compressive force (control group), as shown in Figure 1A. Additionally, the alveolar bone treated with orthodontic compressive force was analyzed by micro-CT. Specifically, BMD (~0.68 fold, P < 0.01), BV/TV (~0.65 fold, P < 0.01) and Tb. Th (~0.73 fold, P < 0.01) significantly decreased with the stimulation of orthodontic compressive force, but the Tb. Sp (~1.55 fold, P < 0.01) and orthodontic tooth movement (~7.32 fold, P < 0.01) was dramatically increased in 14 d (Figure 1B to 1F), which indicates that orthodontic compressive forces can induce bone resorption on the pressured side, leading to bone loss with time pass. + +The TRAP and HE staining results further indicated that the orthodontic compressive force affected the formation of osteoclasts on the pressured side. Specifically, as shown in Figures 2A and 2B, compared with that of the control group, more TRAP-positive cells (~3.34 fold, P < 0.01) on the pressured side were observed in the rats treated with orthodontic compressive force in 7 d with obvious alveolar bone resorption. Importantly, most of the osteoclasts are distributed at the pressured side of the alveolar bone edge and few osteoclasts were also observed at the pressured side of the remote end of the alveolar bone, which indicates that subtle bone resorption occurred in the remote end. + +However, after treatment for 14 d, the markers of TRAP, MMP-9 and RANKL decreased significantly (P < 0.01) (Figure 2C to 2F). These results revealed that the orthodontic compressive force at the pressured side affected the formation of osteoclasts and osteoclast-related cytokines. + +![3_image_0.png](3_image_0.png) + +3.2. The relationship between the GSK-3β/β-catenin signaling pathway and compressive force side-bone resorption during orthodontic tooth movement As shown in Figure 2G, GSK-3β staining results were more significant for the pressure side than for the control group at different time points. + +The semiquantitative results indicated that the expression level of GSK3β decreased after treatment for 7 d (~0.35 fold) but increased after treatment for 14 d (~0.45 fold) compared with control group (P < 0.01) +(Figure 2H). However, the expression of phosphorylated Ser9-GSK-3β (pGSK) was obviously increased in the pressure-exposed alveolar bone after treatment for 7 d (~6.71 fold), and then the expression level decreased after treatment for 14 d (~3.13 fold) with control group (P < 0.01) +(Figures 2I and 2J). Since GSK-3β plays an important role in β-catenin expression, we further explored the expression of β-catenin. As shown in Figures 2K and 2L, the expression of β-catenin increased on the pressure side after 7 d (~2.07 fold, P < 0.01) compared with control group. These results revealed that the relationship between the GSK-3β/β-catenin signaling pathway and pressure side bone resorption was time-dependent during the process of orthodontic tooth movement. + +3.3. Pressure-induced bone resorption mediated by the GSK-3β/β-catenin signaling pathway To explore whether compressive force can induce bone resorption by mediating the GSK-3β/β-catenin signaling pathway during the process of orthodontic tooth movement, the orthodontic compressive force-treated rats were be treated by the GSK-3β inhibitor (LiCl) for 7 d. The distance of tooth movement is affected by many factors, due to differences in the crown structure, the measurement results sometimes may not reflect the accurate movement of the teeth (Figure 3B). Therefore, the Micro-CT +results at the alveolar bone and the roots could show the changes more accurately. As shown in Figure 3C to 3F, LiCl-treated rats exhibited significantly decreased, BV/TV and Tb. Th (~0.78 fold and ~0.79 fold, P +< 0.01) indicating bone loss, which is consistent with the decrease in new bone deposition seen in the HE staining results (Figure 3A). Additionally, for the TRAP staining results, osteoclasts were observed in the pressure side periodontal tissue in all the experimental rats (Figure 3G). However, the percentage of positive cells was different among these groups, and the rats treated with OTM þ LiCl showed the highest percentage of positive cells. + +The expression of MMP-9 and RANKL was evaluated by immunochemistry staining to explore whether the enhanced osteoclasts could contribute to the decreased bone density. As shown in Figure 4A to 4D, +daily administration of LiCl obviously enhanced the percentage of MMP9- and RANKL-positive cells (~1.37 fold and ~1.40 fold, P < 0.01) in the pressure side alveolar bone. Moreover, after treatment of LiCl for 7 d, the population of β-catenin- and P-GSK positive cells increased (~1.37 fold and ~1.87 fold, P < 0.01) with the decreased number of GSK-3β-positive cells (~0.45 fold, P < 0.01) compared with OTM group, and the GSK-3β/ +β-catenin signaling pathway was activated. After local administration of ICG001 for 7 d, the GSK-3β/β-catenin signaling pathway was inhibited, leading to decreased numbers of β-catenin- and P-GSK positive cells +(~0.68 fold and ~0.80 fold, P < 0.01) and increased numbers of GSK-3βpositive cells (~3.85 fold, P < 0.01) compared with OTM þ LiCl group +(Figures 4E-4J). Moreover, the MMP-9- and RANKL-positive populations were reduced and had improved bone mass. All these results indicated that the GSK-3β/β-catenin signaling pathway mediated bone resorption on the pressure side during the process of orthodontic tooth movement. + +## 4. Discussion + +Orthodontic tooth movement is a type of biological response that + +![4_image_0.png](4_image_0.png) + +allows the reconstruction of alveolar bone and periodontal tissue under mechanical stimulation [11]. The orthodontic compressive force causes bone formation and bone resorption during the process of orthodontic tooth movement. When the process reaches a certain balance, the normal morphology and function of alveolar bone can be maintained. Additionally, one of the most important factors in orthodontic tooth movement is bone resorption caused by osteoclasts +[12]. Recently, many studies have focused on how osteoclast differentiation influences orthodontic tooth movement and the mechanism behind this process [13]. According to our results, the orthodontic compressive force interacts with osteoclast-induced bone resorption. + +Specifically, mechanical stimulation contributes to osteoclast activation and bone resorption, but long-term mechanical stimulation weakens bone resorption. + +In addition, with an orthodontic compressive force, the periodontal ligament can process mechanical stimulation to biological signals, which further leads to a series of biological responses, such as changes in periodontal tissue metabolism and the reconstruction of alveolar bone +[14]. This is a very complicated process involving various signal transduction molecules and pathways, diverse matrix proteins and cytokines +[15, 16]. With the knowledge that RANKL/RANK signaling is essential for osteoclast formation, major efforts have been made to identify the signaling pathways that are activated downstream and to determine the full extent of the involvement of RANKL in osteoclast biology and common bone diseases. During the differentiation and maturation of osteoclasts, RANKLs produced by bone cells also participate in orthodontic tooth movement [17]. In a previous study, as an osteoclast differentiation factor, the production of RANKL was positively correlated with the duration of mechanical stimulation [18]. On the one hand, MMPs can degrade the ECM, and on the other hand, MMPs can induce bone resorption and regulate the activity of osteoclasts [19]. In particular, MMPs can specifically degrade nonmineralized cartilage and release vascular endothelial growth factor, which leads to osteoclast activation and delayed osteoclast recruitment [20]. Our immunohistochemistry results indicate a positive correlation between the positive cell population of MMP-9 and RANKL and the duration of mechanical stimulation. + +After an orthodontic compressive force of 7 d, alveolar bone reconstruction reached the most active status, which is also consistent with previous research. + + + +![5_image_0.png](5_image_0.png) + +The periodontal ligament can process mechanical stimulation to biological signals. The Wnt signaling pathway plays an important role in this process; specifically, mechanical stimulation can activate this pathway, leading to stimulation duration and stimulation forcedependent bone reconstruction [21]. In mouse bone cells, only short-term mechanical stimulation can induce the activation of the Wnt/β-catenin signaling pathway, and long-term stimulation significantly downregulates the expression level of Wnt/β-catenin [22]. + +Moreover, on the pressure side, a time-dependent relationship was observed between mechanical stimulation and the GSK-3β/β-catenin signaling pathway. GSK-3β is a multifunctional protein kinase that plays an important role in signal transmission and cell differentiation, mediating multiple signaling pathways. GSK-3β can activate osteoclasts in the process of orthodontic tooth movement, and it can also accelerate the root resorption. Some research has also indicated that LiCl can prevent bone loss. ICG001 competes with β-catenin in combination with CBP and inhibits TCF/β-catenin-mediated transcription, which can be used to specifically evaluate the interaction between CBP/β-catenin and the Wnt signaling pathway [23]. In addition, we further investigated GSK-3β-mediated osteoclast bone resorption on the pressure side during orthodontic tooth movement. As the TRAP staining results revealed, after 7 d of mechanical stimulation, the OTM þ LiCl treatment groups had the most osteoclast populations; in contrast, the OTM þ ICG001 treatment groups had the least number of osteoclast populations. After antagonism of LiCl, bone resorption was enhanced; however, ICG001 inhibited bone resorption. Since ICG001 is an inhibitor of β-catenin, it can further inhibit the expression of β-catenin. Therefore, we considered the positive correlation between the activation of the GSK-3β/β-catenin signaling pathway and bone resorption at the tooth pressure side. β-catenin is a key factor in mediating the Wnt signaling pathway; in addition, the Wnt signaling pathway has a close relationship to osteoclasts [24]. The activity of β-catenin is mediated by the phosphorylation of GSK-3β. When phosphorylation occurs at the Ser9 position of GSK-3β, GSK-3β loses its ability to activate β-catenin. The resulting GSK-3β/β-catenin can directly or indirectly influences osteoclast formation. However, the mechanism is still unclear, and some researchers have found that increasing osteoclasts with β-catenin gene silencing will induce accelerated bone resorption and decelerated bone formation [25]. Compared to our previous study, the activity of GSK-3β/β-catenin signaling pathway was positively correlated with bone formation on tension sites and bone resorption on the compressive force side during orthodontic tooth movement. GSK-3β inhibitors can accelerate alveolar bone remodeling and can be used as targets for clinical orthodontic treatment. In addition, we can use cell stretching model to explore the effect of GSK-3β/β-catenin on osteoblasts in vitro. However, the specific role and molecular mechanism still need to be further explored. + +In conclusion, our results indicate that the GSK-3β inhibitor can promote osteoclast formation on the side with orthodontic compressive force. In addition, the activation of the GSK-3β/β-catenin signaling pathway contributes to bone reconstruction caused by orthodontic compressive force. Therefore, the GSK-3β/β-catenin signaling pathway can be a potential target for further clinical applications. + +## Declarations + +Author contribution statement Yan Liu; Ke Wu: Analyzed and interpreted the data; Performed the experiments; Wrote the paper. + +Xing Cui: Analyzed and interpreted the data; Wrote the paper. + +Yelin Mao: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper. + +Funding statement This work was supported by the 2022 Suzhou Medical and Health Technology Innovation Project, The First Batch of Key Medical Supported Projects in Suzhou (SZFCXK202127) and the 2017 Suzhou Clinical Medical Expert Team Introduced Project. Data availability statement Data will be made available on request. + +Declaration of interest's statement The authors declare no conflict of interest. + +Additional information No additional information is available for this paper. + +Acknowledgements The Authors would like to say thank to The Affiliated Stomatology Hospital of Suzhou Vocational Health College and Funding support. + +## References + +[1] J. An, Y. Li, Z. Liu, R. Wang, B. Zhang, A micro-CT study of microstructure change of alveolar bone during orthodontic tooth movement under different force magnitudes in rats, Exp. Ther. Med. 13 (2017) 1793–1798. + +[2] M. Di Domenico, F. D'Apuzzo, A. Feola, L. Cito, A. Monsurro, G.M. Pierantoni, L. Berrino, A. De Rosa, A. Polimeni, L. Perillo, Cytokines and VEGF induction in orthodontic movement in animal models, J. Biomed. Biotechnol. (2012), 201689. + +[3] Y. Zhao, H.L. Wang, T.T. Li, F. Yang, C.M. 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Sci. 21 (2020). \ No newline at end of file diff --git a/medical/md/PMC9546561.md b/medical/md/PMC9546561.md new file mode 100644 index 0000000000000000000000000000000000000000..b4db348bf0b1bf5aef14d40db13f9b66881bc695 --- /dev/null +++ b/medical/md/PMC9546561.md @@ -0,0 +1,364 @@ +# Omnimodal Topological Polarization Of Bilayer Networks: Analysis + +![0_Image_0.Png](0_Image_0.Png) In The Maxwell Limit And Experiments On A 3D-Printed Prototype + +Mohammad Chararaa,1 , James McInerneyb,1,2 , Kai Sunb , Xiaoming Maob,2, and Stefano Gonellaa,2 Edited by Yonggang Huang, Northwestern University, Glencoe, IL; received May 9, 2022; accepted August 24, 2022 Periodic networks on the verge of mechanical instability, called Maxwell lattices, are known to exhibit zero-frequency modes localized to their boundaries. Topologically polarized Maxwell lattices, in particular, focus these zero modes to one of their boundaries in a manner that is protected against disorder by the reciprocal-space topology of the lattice's band structure. Here, we introduce a class of mechanical bilayers as a model system for designing topologically protected edge modes that couple in-plane dilational and shearing modes to out-of-plane flexural modes, a paradigm that we refer to as +"omnimodal polarization." While these structures exhibit a high-dimensional design space that makes it difficult to predict the topological polarization of generic geometries, we are able to identify a family of mirror-symmetric bilayers that inherit the in-plane modal localization of their constitutive monolayers, whose topological polarization can be determined analytically. Importantly, the coupling between the layers results in the emergence of omnimodal polarization, whereby in-plane and out-of-plane edge modes localize on the same edge. We demonstrate these theoretical results by fabricating a mirror-symmetric, topologically polarized kagome bilayer consisting of a network of elastic beams via additive manufacturing and confirm this finite-frequency polarization via finite element analysis and laser-vibrometry experiments. + +topological mechanics | mechanical metamaterials | flexural modes Topological phases of matter have properties that are characterized by a topological invariant, in contrast to conventional phases of matter that are characterized by symmetry breaking. Importantly, these topological phases are defined by the reciprocal-space band structure of the lattice and are generically immune to disorder. For example, certain metallic compounds have bulk band structures that classify them as topological insulators possessing conducting states localized to their boundary (1, 2). Similarly, a variety of mechanical systems can exhibit topologically protected, one-way elastic waves (3–15), +which can be exploited for vibration isolation or waveguiding in mechanical metamaterials (16, 17). + +A special class of topological mechanical metamaterials, called "Maxwell lattices," +have equal numbers of constraints and degrees of freedom, which places them near the onset of the rigidity transition (18–20). Interestingly, such Maxwell lattices can exhibit zero-frequency modes that localize to their boundaries (21–29)—e.g., edges in two dimensions or surfaces in three dimensions. The relative number of such zero modes on opposite sides of the lattice specifies the "topological polarization" of the structure and distinguishes between distinct topological phases. The main consequence of this topological polarization is the ability of the lattice to display an excess of softness on a particular edge without requiring generalized softness of the entire system, thereby preserving load-bearing capabilities. The fact that the polarization is topological implies that the phenomenon is not a property specific to the edge and, therefore, subjected to the variability of the edge properties; rather, it is a property of the bulk that manifests at the edges (i.e., the "bulk-edge correspondence"), and, therefore, it is guaranteed to persist as long as the topological properties of the bulk remain unchanged. Moreover, these topologically polarized Maxwell lattices possess a number of additional notable properties, including transformability (30, 31), mechanical solitons (32, 33), mechanical Weyl modes (26, 28, 29, 34), stress focusing (35), and robustness against fracture (36). + +The experimental realization of these topologically polarized Maxwell lattices remains a challenge. The existence of nontrivial zero modes requires finely tuned joints that eliminate friction and bending stiffness, which can be realized by using pin-jointed truss structures for planar configurations (25, 32). In contrast, structural realizations, such as those fabricated via additive manufacturing, feature structural elements whose kinematics exhibit additional interactions that overconstrain Maxwell geometries. While this implies that such structural Maxwell lattices cannot possess zero modes, the boundary modes predicted for ideal Maxwell lattices migrate to finite frequencies. For example, ref. 37 + +## Significance + +![0_Image_1.Png](0_Image_1.Png) + +While the mechanical properties of materials are canonically dictated by their composition, in the metamaterial paradigm, structural materials are endowed with unconventional properties via the spatial arrangement and connectivity of their geometric constituents. In this vein, we introduce a family of bilayer metamaterials that display a dichotomous response to vibrations imparted on opposite edges. We prove that this edge-selectivity has topological origins, a property that ultimately grants them robustness against imperfections and disorder. We confirm our predictions on a laser-sintered prototype, whose fabrication characteristics minimize the lossiness typically associated with additive manufacturing processes. This exercise provides a proof-of-concept for the implementation of a broader class of plate-like cellular structures featuring edge-selective energy localization across a variety of modes of operation. + +Author contributions: M.C., J.M., K.S., X.M., and S.G. designed research; M.C. and J.M. performed research; M.C. + +analyzed data; and M.C., J.M., X.M., and S.G. wrote the paper. + +The authors declare no competing interest. This article is a PNAS Direct Submission. Copyright © 2022 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 +(CC BY-NC-ND). + +1M.C. and J.M. contributed equally to this work. + +2To whom correspondence may be addressed. Email: +jmcinern@umich.edu, maox@umich.edu, or sgonella@ umn.edu. + +This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas. 2208051119/-/DCSupplemental. + +Published September 26, 2022. + +investigates a topologically polarized, structural kagome lattice realized via waterjet cutting of a polymer sheet and experimentally demonstrates a combined edge- and frequency-selectivity of the mechanical response. Importantly, heuristic theoretical models show that this polarization can vanish when the joints become too stiff, thereby leading to bulk wave propagation at all frequencies +(38). + +Moreover, although the in-plane (IP), topologically polarized modes of two-dimensional (2D) Maxwell lattices are well understood, fundamental questions remain about the mechanics of 2D Maxwell lattices embedded in three-dimensional (3D) space. + +This is the scenario of thin plate-like structures, in which flexural modes dominate the low-energy mechanical response, due to the relative scaling of their bending and stretching moduli (39). This feature can be manipulated for the deployment and transformation of mechanical structures by programming particular flexural modes into the sheet, such as the rigid folding modes of origami metamaterials (40–42). However, triangulated origami, which lies at the Maxwell point, possesses a hidden symmetry that pairs edge modes on opposite sides of the sheet, thereby preventing its topological polarization (43). While this hidden symmetry can be broken by introducing an equal number of quadrilateral faces and holes (44), thereby yielding topologically polarized Maxwell kirigami (45), these quadrilateral faces exhibit additional lowenergy modes, due to bending of the faces (46, 47), which are not necessarily localized. Furthermore, such topologically polarized Maxwell kirigami sheets are challenging to realize experimentally, and their large design space makes it difficult to characterize polarized geometries analytically. Thus, the state of the art suggests that the simultaneous topological polarization of IP and out-ofplane (OOP) edge modes remains an important question, both theoretically and experimentally. + +The mechanical bilayers introduced in ref. 48 present one possible solution. Such bilayers are designed heuristically to have one topologically polarized layer and one unpolarized layer. This polarization mismatch presents an incompatibility between the IP effective stiffness of the two layers, so that the IP actuation of the edge modes couples to OOP flexural deformations that localize at the same edge. However, this approach presents a few major limitations. First, the polarization of flexural waves requires activation of the IP strains through a force applied directly in the plane of the topological layer, while OOP actuation leads strictly to bulk wave propagation, thus limiting the functional versatility of this configuration as a wave-control device. Second, because this bilayer is constructed by attaching the two layers as continua, the system is not close to the Maxwell connectivity, and, thus, the modes of this bilayer are not characterized by a topological invariant, implying that the observed polarization is generally weak and does not feature any topological protection. + +The present manuscript addresses the topological polarization of Maxwell bilayer geometries, both in theory and in experiment. + +We design a bilayer lattice that exactly satisfies the Maxwell criterion when modeled as point masses connected by harmonic springs, permitting the rigorous definition of a topological index. + +Furthermore, we choose a subset of Maxwell bilayer geometries that are characterized by a mirror symmetry between the top and bottom layers, along with a particular choice of interlayer connections. We show that these two features endow the bilayer with simultaneous IP and OOP edge modes, whose localization is protected by a topological index and is inherited from the planar geometry of the individual layers, a condition we refer to as +"omnimodal polarization." We then consider the polarization of a lattice with the same geometry, but composed of elastic beams, where boundary conditions at the joints become incompatible with the zero modes without bending the beams, and show via finite element analysis that the polarized edge modes persist at finite frequencies. Finally, we corroborate these predictions through laser-vibrometry experiments on a 3D-printed prototype. + +## Results And Discussion + +Topological Polarization of Maxwell Bilayers. This section formally introduces the geometry of mirror-symmetric kagome bilayers and explores their topological polarization in the idealized spring–mass model. Importantly, we distinguish between "coplanar" bilayers, where the vertices of a single layer lie in a plane, and "noncoplanar" bilayers, where the vertices of each single layer lie at different heights. We show that the localized OOP +response of coplanar bilayers can be characterized analytically by the antisymmetric activation of the topological edge modes of their constituent monolayers; however, these coplanar bilayers also possess bulk zero modes that compromise the mode selection and topological protection of their edge modes. In contrast, noncoplanar bilayers possess strongly localized edge modes in the short-wavelength regime, and their bulk zero modes are restricted to zero-dimensional points, so that the edge modes are topologically protected. These results indicate that the imposed mirror symmetry effectively reduces the design space of kagome bilayers to achieve omnimodal topological polarization by leveraging the well-characterized topological polarization of planar kagome monolayers (*SI Appendix*). + +Figs. 1A and 2A show examples of coplanar and noncoplanar, mirror-symmetric kagome bilayers, respectively. Both are composed of unit cells that contain six vertices, specified by the IP positions r1 = (0, 0, 0), r2 = (1, 0.3, 0), and r3 = (0.5, √3/2 + +0.6, 0) and the layer separation h = 1.5 as r±i = ri ± (h/2 + ηi)ˆz , where +(−)specifies a vertex in the upper (lower) layer. The vertex-dependent height modulation, ηi, distinguishes the coplanar geometry, for which ηi = 0, from the noncoplanar geometry, for which we choose η1 = 0.1 and η2 = η3 = 0. Since each vertex has three spatial degrees of freedom, six interlayer bonds must be introduced for the bilayers to achieve Maxwell coordination in the bulk and eliminate trivial zero modes. We utilize the particular choice, which creates three pairs of adjoined vertices out of the six vertices in each unit cell [r−2 connects to r+1 in cells (0, 0) +and (1, 0), r−3 connects to r+2 in cells (0, 0) and (−1, 1), and r−1 connects to r+3 in cells (0, 0) and (0, −1)], which enables greater control over the topological polarization, in contrast to alternative choices. Each cell is related to its neighbors by the lattice vectors, +-1 = (2, 0, 0) and -2 = (1, √3, 0), so that the position of vertex i in cell n = (n1, n2) is r±i (n) = r±i + n1-1 + n2-2. Note that the analysis in this section is scale-independent, so that the lengths are written in dimensionless units. + +In the spring–mass model, the mechanical response of such bilayers is characterized by their normal modes. This model considers each vertex to have a mass m and each edge to be a spring with stiffness k. The infinitesimal displacements, Ui = (ui, vi, wi) and Uj = (uj , vj , wj), of vertices i and j, respectively, extend the spring adjoining these vertices to first order by an amount ij = ˆrij · (Uj − Ui), thereby leading to a restoring force directed along their shared edge, fij = −kij rˆij , which causes the masses to oscillate about their equilibrium position with frequency ω = -k/m. Thus, these dynamical quantities are nondimensionalized by introducing the timescale -m/k, and, hence, we take k = m = 1. The linear operator, B, called the "compatibility matrix," maps the vector of all displacements, + +![2_image_0.png](2_image_0.png) +Fig. 1. Coplanar, mirror-symmetric kagome bilayer. (A) An illustration of the lattice geometry with planar vertex positions, ri; lattice vectors, -1,2; and layer separation, h. (B) Eigenfrequency, ω, for the lowest-energy band over the 2D Brillouin zone with wavenumbers, ξ = (ξ1, ξ2), directed along the corresponding lattice vectors, revealing lines of bulk modes along wavevectors that exhibit the vertex-pair modes in Eq. 5. (C) The decay rate of zero modes, κ2, along the -2-direction as a function of the wavenumber, ξ1, in the -1-direction. The dashed lines indicate the decay rates of the constitutive planar kagome lattices, at which the edge modes are doubly degenerate. (D) Norm of vertex displacements, |U(n2)|, in each cell for the six zero modes with periodic boundary conditions ξ1 = π. (E) Illustration of the six zero modes in a supercell with periodic boundary conditions ξ1 = π in the -1-direction, where the size of the arrows denotes the relative displacements of the vertices. + +U = (U1, U2, ...), to the vector of all extensions, = (ij , ik , ...), so that such oscillations correspond to the eigenmodes, KU = ω2U, of the stiffness matrix, K = BT B +(see *SI Appendix* for remarks on notation). Since the bilayers are periodic, their normal modes are Bloch-periodic in the bulk. Thus, the compatibility matrix can be diagonalized into blocks in reciprocal space B(ξ) for each wavevector ξ = (ξ1, ξ2), where the wavenumbers ξ1,2 are defined along the corresponding lattice vector -1,2 (*Materials and Methods*). The corresponding displacements vary between cells as U(n) = Ueiξ·n, where U is an eigenvector of the Bloch-periodic stiffness matrix K(ξ) = B(ξ)†B(ξ), with † denoting Hermitian conjugation. + +The Maxwell coordination implies that one zero mode arises for each bond cut from the boundary in a finite-sized realization. + +Generically, these zero modes localize to an edge of the bilayer and are characterized by complex-valued wavenumbers, where the imaginary part, κ = Imξ, specifies the decay rate. Such edge modes can be realized in a "supercell" with periodic boundary conditions in the -1-direction and a finite number of cells with open boundary conditions in the -2-direction. The vertex displacements then grow (decay) across the supercell as e−κ2n2 for κ2 < 0 (κ2 > 0). The topological aspect of these modes arises from a bulk–boundary correspondence, whereby the topology of the Brillouin zone dictates the localization of edge modes. In the absence of bulk zero modes, this is quantified by an integer-valued topological invariant, called the "winding number" + +$$\nu_{2}(\xi_{1})=\frac{1}{2\pi}\int_{-\pi}^{+\pi}d\xi_{2}\frac{\partial}{\partial\xi_{2}}\mathrm{Im}\log\operatorname*{det}\mathbf{B}(\xi_{1},\xi_{2}),\qquad\mathbf{[1]}$$ + +which determines the relative number of zero modes localized to each edge (*Materials and Methods*). This type of winding number was introduced in ref. 21 for IP topological floppy modes. + +The compatibility matrix for generic bilayers maps IP () and OOP (⊥) degrees of freedom in the upper (+) and lower (−) layers to constraints on the upper (+), lower (−), and interlayer +(±) bonds: + +$$\begin{pmatrix}\epsilon^{-}\\ \epsilon^{+}\\ \epsilon^{\pm}\end{pmatrix}=\begin{pmatrix}\mathbf{B}_{\parallel}^{-}&\mathbf{0}&\mathbf{B}_{\perp}^{-}&\mathbf{0}\\ \mathbf{0}&\mathbf{B}_{\parallel}^{+}&\mathbf{0}&\mathbf{B}_{\perp}^{+}\\ \mathbf{B}_{\parallel}^{\pm}&\mathbf{B}_{\perp}^{\pm}&\end{pmatrix}\begin{pmatrix}\mathbf{U}_{\parallel}^{-}\\ \mathbf{U}_{\parallel}^{+}\\ \mathbf{U}_{\perp}^{-}\\ \mathbf{U}_{\perp}^{+}\end{pmatrix}.\qquad\mathbf{[2]}$$ +$$[{\mathfrak{H}}]$$ + +Importantly, the mirror symmetry implies that the intralayer, IP constraints are equal for each layer, B−- = B+- , so that the normal modes of the constitutive planar monolayers are normal modes of the bilayer with strictly IP displacements. This means that the topologically polarized zero modes of the planar monolayers are inherited by both the coplanar and noncoplanar mirrorsymmetric bilayers as symmetric modes that displace vertices in the top and bottom layers identically: + +$$\mathbf{U}_{\mathrm{sym}}=(+\mathbf{U}_{\parallel},+\mathbf{U}_{\parallel},\mathbf{0},\mathbf{0}).$$ + +Usym = (+U-, +U-, 0, 0). [3] +The character of these symmetric modes for the coplanar (noncoplanar) bilayer is shown by the decay rates in Fig. 1C (Fig. 2C), +which overlap with the black dashed lines corresponding to the decay rates of the planar monolayer, and the two strictly IP mode shapes in Fig. 1E (Fig. 2E), where the size of the arrows indicate the relative displacements. Since the mirror-symmetric kagome bilayer has six total intercellular bonds along the -2-direction (two from each layer and another two adjoining the layers together), it can exhibit up to six zero modes localized to a single edge for any value of the transverse wavenumber ξ1. The character of the four remaining zero modes depends on the magnitude of any height modulations and the assignment of interlayer connections. + +![3_image_0.png](3_image_0.png) +For coplanar bilayers, the intralayer, OOP constraints are trivially satisfied, B−⊥U−⊥ = −B+⊥U+⊥ = 0, so that the OOP displacements are only constrained by the interlayer connections. + +This simplification allows the four remaining modes to be categorized as two types: 1) antisymmetric modes that couple IP +and OOP deformations of the bilayer and 2) bulk modes that displace isolated sets of vertices (which depend on the choice of interlayer connections) OOP. The antisymmetric modes are a straightforward generalization of the symmetric modes, where the zero modes of the planar monolayers are activated antisymmetrically between the two layers, which generates incompatibility that can only be resolved by simultaneously displacing the vertices OOP, as illustrated by the two antisymmetric mode shapes in Fig. 1E: +Uasym = (+U-, −U-, U−⊥, U+⊥), [4] +where U+⊥ = U−⊥ due to asymmetry of the interlayer connections. + +The flexural nature of these antisymmetric modes can be understood in analogy to the bending of an elastic plate, where the extension of the top of the plate is accompanied by contraction of the bottom of the plate. The key aspect in the mirror-symmetric bilayer is that this deformation localizes according to the topological polarization of the planar monolayers; thus, the corresponding decay rates are doubly degenerate, as shown in Fig. 1C. Since the interlayer connections only couple isolated pairs of vertices in the unit cell, which connect to one another along rows across the whole lattice, the coplanar bilayer possesses zero modes that displace these pairs of vertices strictly OOP + +$$\mathbf{U}_{\mathrm{pair}}=(\mathbf{0},\mathbf{0},\delta_{i}^{-},\delta_{j}^{+}),$$ +$\downarrow$ -. +Upair = (0, 0, δ−i , δ+j ), [5] +where δ−i = δ+j are the isolated OOP displacements of vertices i and j in the lower and upper layers, respectively, which are connected via interlayer bonds, as illustrated by the two strictly OOP mode shapes in Fig. 1E. These vertex-pair modes exist at wavevectors perpendicular to the lines formed by these vertex pairs, so that the whole line displaces OOP by the same amount, stretching no bonds: δi(n) = δieξ·n-= δi. The vertexpair modes in Eq. 5 are given by δ−3 = δ+1 for ξ2 = 0, δ−2 = δ+3 for ξ2 = ξ1, and δ−1 = δ+2 at ξ1 = 0, as shown by the lines of bulk modes apparent in the vanishing eigenfrequencies of the lowest band shown in Fig. 1B and the doubly degenerate lines of κ2 = 0 at ξ1 = 0 shown in Fig. 1C. Note that alternative choices of interlayer connections can change the character of these trivial modes, such as the connections that exhibit a flat band discussed in *SI Appendix*. Importantly, the vertex-pair modes are bulk zero modes that inhibit the calculation of the winding number, so that the four edge modes (the symmetric and antisymmetric modes) +cannot be explicitly shown to be topologically robust and make it difficult to selectively actuate the edge modes. + +For the noncoplanar bilayer, the four zero modes do not admit the simple analytical descriptions that they do for the coplanar bilayer. This is because the height modulation, ηi, breaks the geometric singularity that gives rise to these bulk modes and makes the intralayer edges constrain OOP displacements, B+⊥ = +−B−⊥ = 0. This height modulation lifts the lines of bulk zero modes corresponding to the vertex-pair modes in Eq. 5 and introduces Weyl points, labeled by the blue markers in Fig. 2B, at the origin of the Brillouin zone that move to larger wavenumbers as the magnitude of the modulation increases. Consequently, the antisymmetric modes in Eq. 4 remain strongly localized, and the vertex-pair modes in Eq. 5 become weakly localized in the shortwavelength regime, as indicated by their respective decay rates in Fig. 2C, even though the character of their mode shapes changes, as shown in Fig. 2E: The strongly localized modes retain their OOP character, but no longer exhibit an antisymmetry between the top and bottom layers, and the weakly localized modes acquire an IP component. The significance is that now, all six of the zero modes of the noncoplanar bilayer localize to the top edge at short wavelength, and four of these zero modes exhibit both IP and OOP character. As a result, the winding number is well-defined, as shown in Fig. 2D, away from the Weyl points, where it undergoes discontinuities labeled by the blue markers that correspond to a mode moving to the opposite edge, thus yielding the desired omnimodal topological polarization. The exact location of these Weyl points is highly nontrivial to predict analytically from the geometry, which can lead to undesirable zero modes on the edge opposite of the polarization direction. Importantly, the present choice of interlayer connections limits such undesirable effects, whereas alternative choices can lead to more dramatic changes, including the presence of zero modes strongly localized to the opposite edge (*SI Appendix*). + +The analysis of the topologically polarized, mirror-symmetric kagome bilayer presented here can be extended to bilayers composed of topological square lattices (25, 34), bilayers without mirror symmetry that do not exhibit strictly IP modes, and bilayers with interlayer connections that yield flat bands in the coplanar limit (*SI Appendix*).Thus, investigations of less restrictive geometries could provide greater control over the presence of bulk modes and exhibit more strongly polarized flexural modes. + +However, the mirror-symmetric case provides an intuitive explanation for the existence of omnimodal edge modes in the coplanar limit and dramatically reduces the design space for topologically polarized, flexural mechanical metamaterials. + +Edge Modes in Structural Bilayers: Supercell Analysis Simulations. The spring–mass model presented in the previous section provides a tool for analytical treatment of the bilayer and assessing its topological roots. In terms of realistic physical systems, this framework also captures the behavior of truss-based networks of rods connected by ideal joints, which are extremely difficult to fabricate. Here, we consider a realistic structural bilayer obtained via additive manufacturing—and therefore amenable to physical testing—more appropriately modeled as a network of elastic beams. Importantly, these beams meet at internal clamps, which locally prevent their free relative rotation, in contrast to the perfect hinges in the spring–mass model for the ideal Maxwell bilayer. As a result, the system becomes overconstrained, despite nominally maintaining the critical coordination number, and we cannot formally calculate a topological invariant. + +However, we elucidate the bilayer's ability to polarize IP and OOP modes, and, through a systematic parametric study, we can provide evidence that its behavior possesses "topological origin," rooted in the topological polarization of its ideal Maxwell counterpart. + +To this end, we construct the 10 × 13-cell kagome bilayer prototype shown in Fig. 3, featuring the geometry and connectivity of the noncoplanar bilayer in Fig. 2, via selective laser sintering (SLS) with glass-fiber-reinforced nylon (*Materials and Methods*). In the structural lattice, the springs connecting the vertices are replaced with beams of radius R = 1.5 mm, whose mass is distributed along the length of the beam, rather than lumped at the vertices. + +This lattice features an interlayer height h = 3 cm and a height modulation η1 = 2 mm. Note that solid flanges are fabricated on the left and right edges of the lattice to act as clamping sites for the experiments presented in the following section. + +We model the connections of the structural lattice as 3D Timoshenko beams (49, 50), subject to clamped boundary conditions, which preserve the relative angles between beams meeting at a joint and invoke bending of the beams (*Materials and Methods*). More generally, Timoshenko beams are capable of undergoing + +![4_image_0.png](4_image_0.png) + +axial, flexural, shear, and torsional deformations. Note that the choice of the Timoshenko model is inspired by a criterion of generality, and we obtain nearly identical results using the Euler– +Bernoulli model, which omits shear (an effect that becomes more relevant for short and stubby beams). We discretize each bond via the finite element method framework (*Materials and Methods*) into multiple beam elements, whose nodes each feature three positional degrees of freedom and three cross-sectional rotations (*SI Appendix*). We then construct a supercell by aggregating 10 cells in the -2 direction and applying free boundary conditions at the ends and Bloch-periodic boundary conditions with respect to wavenumber ξ1 in the -1 direction (*Materials and Methods*) to calculate the bilayer's band diagram. The resulting supercell band diagram is shown in Fig. 4A. We seek the edge modes among the low-frequency modes that fall below the bulk band near the edge of the Brillouin zone, in analogy with the scenario described in ref. + +48 (the relevant portions of the branches are plotted in black). The mode shapes of the lowest four modes at ξ1 = π are shown where the IP deformation is shown with color intensity proportional to displacement along yˆ (the Cartesian direction normal to the open boundaries of the supercell and lattice), and the OOP deformation is shown with color proportional to displacement along zˆ. + +Both the IP and the OOP deformation exhibit strong localization at the top end of the supercell, with a comparable decay rate. This localization is quantified by the norm of the total nodal displacements in each cell, which is shown to increase exponentially toward the top edge of the supercell (n2 = 10) in Fig. 4D. The most striking observation is that all four modes below the bulk band are localized. This is a considerable qualitative improvement over the results presented in ref. 48, in which only two polarized edge modes were found and, more importantly, + +![5_image_0.png](5_image_0.png) + +where the overall signature of polarization in the flexural response was significantly milder. Moreover, the localization of these modes highlights that the top and bottom edges of this bilayer serve as the floppy and nonfloppy edges, respectively, consistent with the predictions from the ideal Maxwell lattice. + +Although four low-frequency modes localize in the structural lattice, the ideal Maxwell case predicts six total edge modes. Since these zero modes involve unimpeded rotations about the perfect hinges with no elongation of the springs, which is disallowed in the structural model due to the clamped boundary conditions at the beam ends, the beam's behavior should approach (albeit never reach) the behavior of the ideal Maxwell case when the cost of beam bending becomes negligible (to the cost of beam extension). This limit can be achieved by tuning the beams' aspect ratio (AR = R/L, where L is the length of the shortest bond in the unit cell) because the axial stiffness scales with R2, while the bending stiffness scales with R4. Thus, as the AR diminishes, the bending stiffness becomes minute, and a greater amount of flexural deformation is allowed in the vicinity of the structural hinges. Indeed, in Fig. 4C, we compare the frequency of the topologically polarized modes as a function of AR, monitoring the frequencies of the first six modes (until they enter the bulk band and can no longer be identified), finding that as AR decreases, the frequencies approach zero. The band diagram of the low-AR case (Fig. 4B) shows that as we decrease AR, two additional modes drop from the bulk band and become localized, as confirmed by plotting the norm of the total displacements in each cell, shown in Fig. 4 B, *Inset*. Note that a 100-unit-cell supercell was used here due to the low decay rate exhibited by some of the modes, which require a large number of cells to appreciate. To confirm the contrast with the results applicable to our prototype, decay rates for a 100-unit-cell supercell with the geometric characteristics +(i.e., AR) of the prototype are shown in *SI Appendix*, Fig. S3; Wave Propagation: Experiments and Full-Scale Simulations. + +We now experimentally demonstrate the activation of the polarized modes in the bilayer prototype shown in Fig. 3. To this end, we excite the bilayer with, first, IP and, later, OOP +tone bursts, in the frequency range of the topological edge modes found via supercell analysis, and we perform 3D laser Doppler vibrometer measurements on the joints of the bilayer surface. We verify the proper activation of the desired bulk or edge modes through a morphological inspection of the wavefield data and a spectral analysis of the transformed wave response in the ξ1 − ω plane. Furthermore, we ensure that the edge-selective polarization is frequency-selective, thus providing additional evidence that the activated modes are topological in nature. All results are corroborated by full-scale wave propagation simulations of a bilayer geometry, discretized by 3D Timoshenko beams. + +The prototype is excited with a five-cycle burst applied at the tip of the centermost cell of the top and bottom edge, respectively. We excite the structure at a carrier frequency of 900 Hz, which lies in the frequency range 760 to 1,050 Hz, where the topological modes fold and display the highest density of states, to maximize their signatures in the resulting wavefield. The relevant region of the band diagram is shown by the black portions of branches 1 to 4 in Fig. 4A. + +Results for IP excitation with a carrier frequency at 900 Hz prescribed at the bottom edge are shown in Fig. 5 A and B, and corresponding results for excitation at the top edge are shown in Fig. 5 E and F. Snapshots of the resulting OOP wavefields show strong asymmetry between the top- and bottom-edge excitations: +The top-edge excitation produces highly localized deformation at that edge, while excitation at the bottom edge produces a bulk-like + +![6_image_0.png](6_image_0.png) + +flexural wave. This corroborates the results from the ideal Maxwell case and the supercell, where the top edge exhibits floppy edge behavior, while the bottom edge is nonfloppy. + +We verify activation of the topological modes via spectral analysis of the response. We collect the time histories of the OOP +displacement at evenly spaced points along the edge where the excitation is applied, perform a 2D discrete Fourier transform +(DFT) on this spatiotemporal data matrix, and superimpose the contours of the resulting spectral amplitude surfaces onto the supercell band diagram to infer which modes are predominantly activated. The DFT analysis corroborates the conclusions made from the inspection of the wavefields, supporting the notion of topological polarization. Excitation at the floppy edge produces a spectral signature that is spread across the branches associated with the edge modes. In contrast, excitation of the nonfloppy edge results in activation of longer wavelength modes (at lower values of ξ1) that belong to the flexural bulk band. + +These results are confirmed by simulations performed on a full-scale model of the bilayer, discretized with 3D Timoshenko beam elements (with the same element characteristics used in the supercell analysis). The results for the same excitation conditions used in the experiments (excitation from the nonfloppy edge in Fig. 5 C and D and from the floppy edge in Fig. 5 G and H) match the experiments qualitatively, with only a relatively small deviation in frequency that can be attributed to some inevitable discrepancies in material properties between the model and prototype (due to material-property variability resulting from the additive manufacturing process) and to other nonidealities in the geometry of the specimen. + +Asymmetric behavior for flexural waves triggered by IP excitation is observed in the bilayer presented in ref. 48, albeit with a significantly milder polarization signature. However, in that case, such asymmetry requires a direct IP strain activation of the polarized layer and is completely lost when the excitation is prescribed OOP. We repeat the experiments with the current bilayer using an OOP tone burst to test the robustness of the asymmetry achievable against changes in the orientation of the excitation force (i.e., IP vs. OOP). We show the resulting wavefields and DFTs for excitations from the nonfloppy (Fig. 5 I and J) and floppy (Fig. 5 M and N) edges. The results are consistent with their IP excitation counterparts: The wavefield snapshots reveal a highly polarized response with displacement localized to the edge when we excite at the floppy edge and propagation deep into the bulk for excitation at the nonfloppy edge. Again, the DFT plots confirm this dichotomy of the mode activation. The magnitude of this result can be truly appreciated by recalling that the topological character of the bilayer stems from the geometry of its two kagome layers. The dichotomous behavior between the edges documented in Fig. 5 confirms that the coupling provided by the interlayer connections is very effective in transferring the topological character to the flexural modes, yielding omnimodal polarization encompassing both IP and OOP behavior.The results are again confirmed by full-scale numerical simulations (Fig. 5 K, +L, O, and P), with the DFTs showing, even more clearly in this case, topological polarization of the edge modes. + +For completeness, we calculate the kinetic energy transmissibility of the lattice over a wide range of frequencies to assess the level of frequency selectivity of the omnimodal polarization achievable in a spectral region containing the polarized modes +(Fig. 6). We perform full-scale wave propagation simulations on the Timoshenko beam-discretized lattice model using IP (solid lines) and OOP excitations (dashed lines) applied at the floppy (blue lines) and nonfloppy (orange lines) edges. We sweep the frequency of excitation, and, for each frequency, we normalize the kinetic energy reaching the output edge by that sampled along the + +![7_image_0.png](7_image_0.png) + +input edge over a time range in which the wave can be considered transient. The polarization, which manifests as edge-selectivity of localization, is conveniently identifiable as the gap between each pair of lines (i.e., solid and dashed). The largest difference in transmissibility happens in the frequency range where the polarized modes are flattest on the band diagram (∼900 Hz) +for both types of excitation. The persistence of edge-selectivity at frequencies lower and higher than the precise spectral regions of the edge modes is explainable by invoking the following considerations. At lower frequencies, the polarized modes are still present, albeit much longer in wavelength and, thus, competing with nearby bulk-like modes in the same spectral regions of the band diagram, which makes them harder to isolate from the modally dense response. Moreover, it is worth noting that the tone bursts used in the simulations and experiments are Hanning-windowed, resulting in the inevitable activation of a finite frequency band about the carrier. At higher frequencies, such widening of the excitation spectrum is especially pronounced, spreading the wave's energy over a band of frequencies higher and lower than the intended carrier frequency, which results in partial activation of the polarized modes. The pairs of lines eventually coalesce, signifying that symmetric wave behavior is re-established when the frequency of excitation enters far enough into the bulk band, and all the excited phonons are bulk-like in nature. Finally, the consistent drop in transmissibility from all lines as the frequency increases can be explained by the decrease in wave speed with the increase of the activated frequencies, which reduces the kinetic energy effectively reaching the output side within the calculated time frame. + +## Concluding Remarks + +We have introduced a broad family of topological mechanical metamaterials, referred to herein as mirror-symmetric Maxwell bilayers, and explored their mechanical response via analytical calculations, numerical simulations, and experimental testing. We have provided an analytical theory of how omnimodal topological polarization arises for coupled IP and OOP deformations of ideal Maxwell bilayers in the spring–mass model. We have also shown that these modes remain localized at finite frequencies in structural Maxwell bilayers. Finally, we have experimentally verified the existence of this omnimodal polarization via laser vibrometry experiments. + +An important aspect of the geometry studied in the present manuscript is the mirror symmetry and the particular choice of interlayer connections that leverages the known topological polarization of the IP mechanics of Maxwell lattices and provides a design principle for mechanical bilayers with topological polarization in both the IP and OOP domains; however, more generic geometries may yield stronger polarizations and could be determined by applying tools such as machine learning to navigate the high-dimensional design space. Furthermore, the connection between the topologically protected zero modes in the spring–mass model and the edge-localized modes at finite frequencies in the beam model warrants further study. It is a special manifestation of a more general class of dilution mechanisms observed in topological mechanics problems in going from ideal to structural lattices; e.g., ref. 37. The present manuscript utilizes the aspect ratio of the beams as an effective parameter to distinguish between regions where the low-frequency, shortwavelengths modes localize to a particular edge, but does not explicitly prove that the floppiness is topologically protected. The primary difficulties toward achieving this goal are the fact that the beam model is overconstrained, so that the winding number cannot be computed. The continuum approximation developed in ref. 51 could potentially be extended to address this issue to show the topological protection of long-wavelength edge modes in the structural lattice. Simplified models that treat these bonds as filaments with bending and stretching rigidity (52, 53) may also provide insight toward this detail. + +The topologically polarized bilayers explored in this manuscript offer functionalities in ways not achievable by previous mechanical metamaterials. The particular utility of these structures is their topological polarization, which endows the materials with insulating characteristics. Importantly, the topological protection against defects, damage, and wear and tear of the material represents an attribute of significant practical utility for the design of structural lattices intended to bear load and be subjected to dynamic excitations, such as impact and vibrations. In addition, this property can yield stress localization at topological domain walls. Such stress focusing has been implemented theoretically in 2D planar lattices (27) and experimentally in 3D cellular lattices +(54), and the results presented herein could be utilized to focus stress from flexural deformations of the lattice in a similar manner. + +This stress focusing could be used to redistribute mass toward the high-stress regions during the design process, thereby enhancing the strength against failure of the structure and the ability to resist shape change. Furthermore, the individual layers possess nonlinear Guest–Hutchinson modes in the spring–mass limit (25, 30), whose interplay with flexural modes may be used to control the curvature of the bilayer. + +## Materials And Methods + +Zero Modes in the Spring–Mass Model. The zero modes of a spring–mass networks span the null space of the compatibility matrix,which can be considered under periodic boundary conditions. For bulk modes,this is achieved by performing the Fourier transformation: + +$$\mathrm{i}e^{-i\xi\cdot\mathbf{n}},$$ + +## B(Ξ) = N B(N)E −Iξ·N, [6] + +where the Bloch factor −iξ·n is nontrivial only for bonds that connect to adjacent cells. Thus, the assignment of such intercellular connections determines which particular Bloch factors appear in the compatibility matrix, and the appropriate choice is the "symmetric gauge," where there are an equal number of factors e iξ1,2 and e +−iξ1,2 , which eliminates superfluous phase factors in the description of the edge modes (21, 24, 51). For edge modes, the Fourier transform in Eq. 6 is extended to the Laplace transform to allow for complex-valued wavevectors. + +The Maxwell criterion for the lattice implies that each block of the compatibility matrix is square; therefore, its null space is nonempty at wavevectors for which its determinant vanishes: det B(ξ) = 0. In general, this determinant is a Laurent polynomial in the Bloch factors,cm1,m2 e i(m1ξ1+m2ξ2), where the sum is taken from m1,2 = −3 to m1,2 = 3 in the symmetric gauge, and cm1,m2 are real-valued coefficients that depend on the lattice geometry. Thus, for each value of ξ1 in the Brillouin zone, there are up to six complex values of ξ2 for which this determinant vanishes and there exists an edge mode. Consequently, the phase of the determinant of the compatibility matrix changes over any loop enclosing such a zero (55). In particular, the winding number in Eq. 1 considers a contour over the bulk modes κ2 = 0, which bound the modes localized to the top edge, κ2 < 0, and those localized to the bottom edge, κ2 > 0. The gauge symmetry implies that a winding number of ν = 0 corresponds to three modes on each edge, whereas a winding number of ν = −3 corresponds to six modes on the top edge. Finally, this contour cannot be computed when it crosses a zero, as is the case in the presence of bulk zero modes. + +Lattice Details. The lattice is 3D-printed by using SLS (56) (DTM Sinterstation) +by Stratasys Direct. The lattice is first modeled by using the Solidworks software and exported as an ".STL" file at a resolution finer than that of the SLS process used (0.76 mm in x − y and 0.1 mm in z). SLS consists of depositing a fine layer of powder material(Stratasys Nylon 12-glass-fiber-reinforced nylon; Young's modulus = 2.896 GPa and Poisson's ratio = 0.35), which is then melted by a laser into a solid layer following the cross-section of a given design, bonding it to all previously deposited layers. This printing process produces a monolithic structure, while the more commonly commercially available fused deposition modeling often results in imperfect bonding between deposited layers and adjacent deposited lines that can detrimentally affect wave-transport properties. + +These considerations were key factors in the selection of SLS as the fabrication method for the current task. + +The slenderness ratio and cell size are decided concurrently, making sure that the thickness of the beams is within the resolution of the SLS 3D-printing process and that the beams are sufficiently slender to prevent their flexural behavior from completely overwhelming the lattice response. The lattice dimensions are selected to be large enough to appreciate the topologically induced decay phenomenon, within the dimensions of the 3D-printer's fabrication print bed +(67.31 × 34.29 cm). + +Computational Beam Model. The bilayer connections are modeled as 3D +Timoshenko beams (50). Recall that that each lattice bond is discretized into multiple beam elements, featuring three positional degrees of freedom and three cross-sectional rotations. The stiffness matrix of a single beam element is given by Kb = L BTbkBb dL, where L is the length of the beam element; Bb is a matrix of derivatives of the shape functions, which approximate the compatibility relations within one element; and k is a diagonal constitutive matrix of elastic constants, featuring rigidity contributions for axial deformation (scaling with R2), lateral deflection (scaling with R4) (encompassing the effects of both shear and bending deformability encoded in the beam model), and torsional deformation +(scaling with R4). The shape functions used in the calculation of the elemental stiffness matrix Kb are linear in axial and torsional deformations, cubic in flexural deformation, and quadratic in rotations. Stiffness matrices of each individual beam are assembled into a global stiffness matrix for the entire system, which is used for computational analysis. Note that, for each discretized beam element, Bb plays a role analogous to a single row of the compatibility matrix in the spring– +mass counterpart, which quantifies the constraints for a single spring. Further details of the framework used in this work can be found in *SI Appendix*, and a full, pedagogical account can be found in ref. 50. + +We discretized each beam into 9 to 11 elements, depending on the bond length (sufficient discretization to allow for appropriate mode visualization), +resulting in a model with 3,240 and 29,376 nodes for the supercell and fullscale lattice models, respectively. For supercell calculations of 100 unit cells, the beams were discretized into only 5 elements due to computational constraints; however, comparing the results of the higher and lower discretization reveals a change of less than 2% for all the relevant modes (lowest 6), meaning that this alternative discretization is accurate in capturing the spectral characteristics of the modes, despite offering a lower spatial resolution of the mode shapes. Supercell Construction and Band Diagram Calculation. To calculate the band diagram of the supercell, we consider a supercell consisting of 10 cells, discretized by using 3D Timoshenko beam elements and connected in the +-2 direction with free boundary conditions at the ends, and Bloch-periodic boundary conditions with respect to wavenumber ξ1 in the -1 direction. We extract the Bloch-reduced arrays of generalized nodal displacements U˜ and cross-sectional rotations Θ˜ in the supercell as the modes of K˜b(ξ1) +U˜ Θ˜T= +ω2M˜ b(ξ1) +U˜ Θ˜T, where K˜b(ξ1)[M˜ b(ξ1)] is the stiffness (mass) matrix of the supercell under Bloch conditions. + +The 3D Laser Doppler Vibrometer Experiments. The experimental setup is shown in Fig. 7. A 3D laser Doppler vibrometer (Polytec PSV-400-3D) is used to measure the velocity of the joints of the beams (the selected scan sites) +on the face of the lattice closest to the lasers, where reflective tape is applied to increase the material reflectivity, in order to reduce noise in the data. An electromechanical shaker (Bruel & Kjær Type 4810), internally triggered by ¨ the vibrometry setup through an amplifier (Bruel & Kjær Type 2718), probes ¨ the lattice through a stinger at the desired excitation location, and a five-cycle + +![9_image_0.png](9_image_0.png) + +testing of the 3D-printed bilayer lattice. +Hann-windowed burst, with a carrier frequency of 300, 900, or 1,300 Hz is fired into the structure, while velocity is measured by the laser heads at a scan point. The process is repeated for each scan point, automatically moving the lasers 1. M. Z. Hasan, C. L. Kane, Colloquium: Topological insulators. *Rev. Mod. Phys.* 82, 3045 (2010). 2. X. L. Qi, S. C. Zhang, Topological insulators and superconductors. *Rev. Mod. Phys.* 83, 1057 (2011). 3. E. Prodan, C. Prodan, Topological phonon modes and their role in dynamic instability of microtubules. + +Phys. Rev. Lett. 103, 248101 (2009). + +4. R. Susstrunk, S. D. Huber, PHYSICS. 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Lett.* +120, 068003 (2018). + +to subsequent scan locations in a prescribed sequence and providing enough relaxation time between scans to ensure that bursts have fully dissipated by damping before the next measurement is taken. + +The velocity data collected are decomposed into ˆx,ˆy, and ˆz components by using the Euler angles internally calculated by the vibrometry software. This data are further processed in MATLAB to recreate the wave fields and DFT plots. The temporal data of the spaces between the scan points is interpolated by MATLAB postprocessing using a triangulation-based linear-interpolation algorithm. + +It should be noted that this interpolation favors the creation of bulk-appearing wave fronts, which implies that the wave fields for experiments with excitation at the floppy side could potentially be experiencing higher localization than what is shown in the result figures. + +Data, Materials, and Software Availability. All study data are included in the article and/or supporting information. + +ACKNOWLEDGMENTS. This work was supported by NSF Grant EFRI-1741618 +(to M.C. and S.G.) and the Office of Naval Research Multidisciplinary University Research Initiative N00014-20-1-2479 (to J.M., K.S., and X.M.) and leveraged the High Performance Computing systems at the Minnesota Supercomputing Institute. + +Author affiliations: aDepartment of Civil, Environmental, and Geo-Engineering, University of Minnesota, Minneapolis, MN 55455; and bDepartment of Physics, University of Michigan, Ann Arbor, MI 48109 28. J. E. Socolar, T. C. Lubensky, C. L. Kane, Mechanical graphene. *New J. Phys.* 19, 025003 (2017). 29. G. Baardink, A. Souslov, J. Paulose, V. Vitelli, Localizing softness and stress along loops in 3D +topological metamaterials. *Proc. Natl. Acad. Sci. U.S.A.* 115, 489–494 (2018). + +30. S. Guest, J. Hutchinson, On the determinacy of repetitive structures.*J. Mech. Phys. Solids* 51, 383–391 (2003). + +31. 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Kumar, Selective laser sintering: A qualitative and objective approach.JOM 55, 43–47 (2003). \ No newline at end of file diff --git a/medical/md/PMC9746884.md b/medical/md/PMC9746884.md new file mode 100644 index 0000000000000000000000000000000000000000..82d3b166f43d5576496066325167ec4583703f07 --- /dev/null +++ b/medical/md/PMC9746884.md @@ -0,0 +1,304 @@ +DOI: 10.1002/trc2.12335 RESEARCH ARTICLE + +# Models Of Depressive Pseudoamnestic Disorder + +Jongwoo Choi1 Seonjoo Lee1,2,3 Jeffrey N. Motter4 **Hyun Kim4** Howard Andrews2,3 P. Murali Doraiswamy5 D. P. Devanand4 **Terry E. Goldberg3,4,6** +1Division of Mental Health Data Science, New York State Psychiatric Institute, New York, New York, USA +2Department of Biostatistics, Mailman School of Public Health, Columbia University, New York, New York, USA +3Department of Psychiatry, Columbia University Medical Center, New York, New York, USA +4Division of Geriatric Psychiatry, New York State Psychiatric Institute, New York, New York, USA +5Department of Psychiatry and Behavioral Sciences, Duke University School of Medicine, Durham, North Carolina, USA +6Department of Anesthesiology, Columbia University Medical Center, New York, New York, USA +Correspondence Terry Goldberg, Medical Psychology in Psychiatry and Anesthesiology, Division of Geriatric Psychiatry, 1051 Riverside Drive, Rm. + +2409, New York, NY 10032, USA. Email: teg2117@cumc.columbia.edu All study procedures have been conducted in New York State Psychiatric Institute +(NYSPI)/Columbia University Medical Center and Duke University Medical Center. Analyses were performed at NYSPI. + +## Abstract + +Objective: Little effort has been made in the past to validate depressive pseudodementia based on hypothesis-driven approaches. We extended this concept to individuals with amnestic Mild Cognitive Impairment and Major Depression, that is, pseudodepressive amnestic disorder. We tested two hypotheses consistent with the presentations and mechanisms associated with this potential syndrome: improvements in cognition would be significantly correlated with improvements in depression after treatment (Hypothesis 1), and if not confirmed, the presence of such an association could be identified once moderator variables were taken into account (Hypothesis 2). + +Methods: Within a clinical trial, 61 individuals received open label serotonin reuptake inhibitor (citalopram or venlafaxine) treatment over a 16-week period. Selective Reminding Test and Hamilton Depression scale were conducted serially to measure change in memory and depression, respectively. Magnetic resonance imaging, other cognitive measures (Alzheimer's Disease Assessment Scale–Cognitive and speed of processing tests), and additional depression measure (Beck Depression Inventory +[BDI]) were also administered. + +Results: No significant associations between improvement in depression and improvement in cognition were observed. Sensitivity analyses with other cognitive measures, the BDI, and exclusion of possible "placebo" responders were negative as well. There were no significant moderation effects for baseline Hamilton Rating Scale for Depression as a measure of symptom severity or age. *APOE* ε4 genotype and white matter hyperintensity burden yielded counter-intuitive, albeit marginally significant results. Conclusions: Negative findings cast doubt on the frequency of depressive pseudoamnestic disorder in older populations with documented depression and memory impairments. + +KEYWORDS +Clinical trial, depression, memory, mild cognitive impairment, pseudodementia 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. *Alzheimer's & Dementia: Translational Research & Clinical Interventions* published by Wiley Periodicals LLC on behalf of Alzheimer's Association. + +## 1 Introduction + +The concept of depressive pseudodementia has a known history. It was initially proposed by Kiloh in 1961 and reached prominence in the 1970s when it was applied to a variety of diagnoses.1 Depressive pseudodementia in particular refers to memory impairments and functional status compromises, mainly in depressed older individuals, that improve with treatment of depression. Thus, they are reversible. This view is complicated but not negated by the fact that older individuals have increased risk for both depression and dementia and that about 51% of Alzheimer's disease (AD) cases may initially present with apathy, memory problems, and mood changes that can be viewed as "depressive."2–5 There are also cognitive concomitants of depression, including executive function, speed of processing, and memory, that may not completely resolve after an episode. Additionally, latelife depression may differ from depression in younger individuals due to findings implicating small vessel disease in the brain that may contribute to late-life cognitive impairments.6,7 While some older studies validated the concept of pseudodementia, most were conducted in the 1960s and 1970s when tricyclic antidepressants were a first-line antidepressant. These are known to have anticholinergic effects that might reduce cognitive gain. Furthermore, patients were generally younger (<70 years), carried various diagnoses including psychosis, and received limited cognitive assessments. + +Recent studies of depressive pseudodementia have reached contrasting or equivocal conclusions. Butters et al.8 found that older depressed patients did not show greater improvement on multiple cognitive tests than a non-depressed control group despite large improvements in depression. However, their second study demonstrated an improvement on the Dementia Rating Scale's initiation/perseveration and conceptualization in the depressed group, though performance was not normalized.9 Nevertheless, Butters et al. went on to suggest that pseudodementia was a rare occurrence. In contrast, Barch et al.10 found evidence for state-related changes in cognition in older depressed individuals treated with sertraline. The largest improvements were in measures of memory and executive function. However, given the nature of the tests used (word lists and stories; card sorting) practice effects were likely unavoidable. + +Other developments have made pseudodementia conceptually problematic. First, there is an increasing realization that mild cognitive impairment (MCI) may represent a transitional phase of AD and so cognitive impairments found in the context of depression in late life may be related to neurodegeneration. Second, depression itself is a risk factor for AD and may be present 15 to 20 years prior to a dementia diagnosis.11 Additionally, depression and its concomitant symptom apathy are frequent comorbidities of AD; depression has been also related to amyloid pathology.12 Here, we extend this line of work to what we term pseudodepressive amnestic disorder. The current study was therefore implemented in a group of patients with Major Depressive Disorder and psychometrically and clinically diagnosed amnestic MCI who were assessed longitudinally. Such patients do not have the more generalized and severe impairments in cognition and function that AD patients demon- + +## Research In Context + +1. **Systematic Review**: The authors reviewed the literature using traditional (e.g., PubMed) sources, references in recent papers, and their own knowledge. While there is an older literature on depressive pseudodementia, there were no studies on what we term depressive pseudoamnestic disorder, that is, major depression in individuals with amnestic mild cognitive impairment (MCI). + +Relevant citations to the broader field, including critical reviews, are made. + +2. **Interpretation**: Our study was hypothesis-driven as we examined evidence for a systematic relationship between changes in depression level and cognition in this MCI +group. We did not find such a relationship, casting doubt on the utility of the concept. + +3. **Future Directions**: Our study proposes a framework to study depressive pseudodementia and pseudoamnestic disorder. We recommend use of a placebo control group and better control of practice effects to further refine work in this area. + +strate. This may increase the likelihood that improvements in mood and cognition might be both observed and correlated. In this study we take a hypothesis-driven approach to validate what may be implicit assumptions that are contained in previous literature. Thus, we evaluated whether changes (improvements) in depression covary with changes +(improvements) in cognition among the MCI participants with depression during serotonin reuptake inhibitor (SRI) or selective SRI (SSRI) +treatment. We further examined multiple potential moderators of this relationship. Last, we examined whether cognitive improvements were present, general, and not consistent in their profile with a practice effect. + +## 2 Methods 2.1 Participants + +The trial was conducted at New York State Psychiatric Institute +(NYSPI)/Columbia University Medical Center (N = 41) and Duke University Medical Center (N = 40). Among the 81 patients aged 55 to 95 years with depression and cognitive impairment, two patients did not complete baseline procedures, leaving a sample of 79 patients. + +The trial was registered on clinicaltrials.gov (identifier: NCT01658228) +and the study was conducted from October 2012 to August 2016. + +Inclusion criteria for participants were the following: (1) met diagnosis for the Diagnostic and Statistical Manual of Mental Disorders +(DSM)-IV major depression or dysthymic disorder by SCID-P evaluation, with a minimum score of 14 on the 24-item Hamilton Rating Scale for Depression (HAM-D); (2) presented with subjective memory or other cognitive complaints; (3) had a score of ≥21 on the Folstein Mini- +Mental State Exam (MMSE), with memory score meeting amnestic MCI +criteria (≤11 on the Logical Memory-II subscale from the Wechsler Memory Scale-Revised; or≥1.5 standard deviations [SDs] below norms on the Selective Reminding Test [SRT, immediate or delayed recall]); (4) received 0.5 from the Clinical Dementia Rating; and (5) were willing and capable of giving informed consent. + +Patients were excluded if they had other serious major pre-existing mental or neurological disorders, including dementia, schizophrenia, schizoaffective disorder, psychotic depression, other psychosis, bipolar I disorder, alcohol or substance abuse or dependence in the past 6 months, active suicidal ideation or suicide attempt, use of medications known to cause cognitive impairment, any acute medical illness, uncontrolled hypertension, current use of effective antidepressants, current use of cholinesterase inhibitors or memantine, and electrocardiogram QTc interval greater than 460 msec. The overall trial design is described in a previous paper.13 version of the Fazekas scale that included deep white matter intensities (WMH), subcortical gray matter hyperintensities in the basal ganglia, and periventricular hyperintensities using MRIcro software.14 We dichotomized the total hyperintensity volume into high and low burden using a median split of the data. + +## 2.3 Treatment + +In 16 weeks of open-label antidepressant therapy (a pre-phase for a randomized clinical trial), citalopram was started at 10 mg and raised to 20 mg daily after 2 weeks in 36 of the participants. If there was lack of antidepressant response by 8 weeks, citalopram was switched to venlafaxine 37.5 mg daily, with the dose raised weekly over 4 weeks to reach 225 mg daily or the maximum tolerated dose in 25 of the participants. Twenty participants began the trial on venlafaxine and remained on it. Other antidepressants were prescribed for patients with nonresponse or intolerability to citalopram and venlafaxine. We found 18 patients who switched antidepressant drugs from baseline to week 16: thirteen from citalopram to venlafaxine, three from citalopram to another drug, and two from venlafaxine to another drug. Other drugs included fluoxetine, escitalopram, duloxetine, and bupropion. + +A second phase of the trial involving the cholinesterase inhibitor donepezil was not analyzed here as it was not relevant to the focus of this study and has been reported elsewhere.15 + +## 2.4 Statistical Analyses 2.2 Measures + +The assessments for depression, HAM-D and Beck Depression Inventory-II (BDI), were collected at each study visit for a total of 10 ratings from baseline to the week 16 endpoint. Response to treatment was defined as ≥50% decrease in HAM-D scores in the 16 weeks. + +The three primary outcome measures were the Free and Cued SRT (a test of verbal list learning 12-item 6-trial version) total immediate recall score, modified Alzheimer's Disease Assessment Scale– Cognitive (ADAS-Cog; 13-item version that assays multiple cognitive domains including verbal memory [free recall and word recognition], visual motor abilities, verbal comprehension, praxis, and naming) total score, and MMSE total score. These three measures were administered at baseline and the week 16 endpoint. The following are secondary measures used in exploratory analyses: Letter and Animal verbal fluency (VF) test, which measures word generation in a 60 second time period; Wechsler Adult Intelligence Scale—Third Edition, Digit Symbol subtest, used to evaluate processing speed (involving using a key to code numbers and symbols); Trail Making Test A (Trails A), which served as an estimate of attention and speed of processing (in which numbers on a page had to be connected in order); and Trail Making Test B (Trails B), a measure of speed and executive function (in which numbers and letters must be connected in alternating sequence). + +The apolipoprotein E (*APOE*) genotype was determined by Prevention Genetics using blood samples processed by the Columbia University Human Genetics Resource Core. + +Magnetic resonance imaging (MRI) scans were acquired on a Signa 3 Tesla whole body scanner (GE Medical Systems, Milwaukee, WI) using an identical model at the two sites (Columbia, Duke). As described elsewhere in detail,12 sequences involved a T1 spoiled gradient recalled acquisition in steady state aligned to the long axis of the hippocampus, and T2 fluid-attenuated inversion recovery (FLAIR). Hyperintensity volumes on axial FLAIR images were measured on a semiautomated The study will test the following two hypotheses. First, as Hypothesis (Hyp) 1, improvements in cognition would be significantly correlated with improvements in depression. Multiple sensitivity analyses could examine whether cognitive domains might be differentially sensitive to antidepressant efficacy and that changes in cognition and depression are differentially imparted by drug type (SSRIs vs. norepinephrine/serotonin SRIs), depression severity, or exclusion of participants who demonstrated an early and large expectancy response (hereafter called "placebo" responders). Second (as Hyp 2), other factors (e.g., *APOE*, WMH, age, depression severity at baseline) might moderate such systematic changes in both depression and cognition. WMH may be associated with or cause late life depression and might modulate the relationship between depression symptom change and cognition change. Age is also a crucial variable in pseudodementia given that the earlier literature showed that cognitive improvements were greater in younger individuals.16 This study has 80% power to detect r = .35 (moderate) correlation at the 5% significance level. We reported regression coefficients with 95% confidence intervals (CIs) since a 95% CI contains all the null hypotheses that would not be rejected at the 5% level. Descriptive statistics for demographic and clinical variables at baseline across the whole sample were reported. The chi-square and the two-sample t-test were used to compare the group differences between 61 participants who completed the 16-week open-label antidepressant treatment and + +| TABLE 1 | Demographic characteristics and cognition for included and excluded participants at baseline (n = 79) Excluded (n = 18) Included (n = 61) | Overall at Week 0 (n = 79) | | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|-----------------------|-------------| +| Mean (SD) or Freq (%) | Mean (SD) or Freq (%) | P | Mean (SD) or Freq (%) | | +| Sex Female | 7 (38.9%) | 31 (50.8%) | .53 | 38 (48.1%) | +| Male | 11 (61.1%) | 30 (49.2%) | 41 (51.9%) | | +| Age (years) | 65.8 (9.53) | 69.8 (8.66) | .12 | 68.9 (8.97) | +| Race Black/African-American | 4 (22.2%) | 8 (13.1%) | .48 | 12 (15.2%) | +| Caucasian | 13 (72.2%) | 45 (73.8%) | 58 (73.4%) | | +| Hispanic | 1 (5.6%) | 8 (13.1%) | 9 (11.4%) | | +| Education (years) | 15.1 (2.41) | 15.4 (2.88) | .64 | 15.3 (2.77) | +| APOE ε4 allelea No | 12 (85.7%) | 39 (65.0%) | .24 | 51 (68.9%) | +| Yes | 2 (14.3%) | 21 (35.0%) | 23 (31.1%) | | +| HAM-D | 23.6 (4.02) | 22.9 (5.40) | .57 | 23.0 (5.10) | +| BDI Total Score | 26.9 (10.5) | 20.3 (8.14) | .02 | 21.8 (9.10) | +| Folstein MMSE Total Score | 28.1 (1.97) | 27.8 (1.96) | .58 | 27.9 (1.95) | +| Modified ADAS-Cog Score | 11.9 (3.35) | 13.0 (5.34) | .33 | 12.7 (4.96) | +| SRT Total Recall | 45.2 (8.02) | 41.8 (10.7) | .15 | 42.6 (10.2) | +| WAIS-R Digit Symbol Test | 43.9 (8.95) | 41.4 (13.0) | .35 | 42.0 (12.2) | +| Trails A Time | 45.4 (17.2) | 47.0 (21.0) | .75 | 46.6 (20.1) | +| Trails B Time | 113 (34.3) | 135 (70.5) | .083 | 130 (64.5) | +| Fluency Animal Total | 20.1 (4.32) | 16.7 (4.50) | .01 | 17.5 (4.65) | +| Abbreviations: APOE, apolipoprotein E; ADAS-Cog, Alzheimer's Disease Assessment Scale–Cognitive; BDI, Beck Depression Inventory; HAM-D, Hamilton Rating Scale for Depression; MMSE, Mini-Mental State Exam; SRT, Selective Reminding Test; WAIS-R, Wechsler Adult Intelligence Scale, revised. | | | | | + +Abbreviations: *APOE*, apolipoprotein E; ADAS-Cog, Alzheimer's Disease Assessment Scale–Cognitive; BDI, Beck Depression Inventory; HAM-D, Hamilton Rating Scale for Depression; MMSE, Mini-Mental State Exam; SRT, Selective Reminding Test; WAIS-R, Wechsler Adult Intelligence Scale, revised. + +aFive participants (4 were excluded and 1 was followed) had missing values. + +18 subjects who did not complete it. Similar analyses were performed to examine the differences between the 12 placebo responders (at least 50% decrease in HAM-D scores in the first 2 weeks) and 49 non-placebo responders, and to examine the differences between APOE ε4 carriers versus non-carriers. Additionally, pre-post changes in the depression and cognitive measures were tested using paired Wilcoxon's signed rank test to minimize outlier effects. + +For Hyp 1, separate linear regressions were used to estimate the association between each change in depression measures (primary: ΔHAM-D; secondary: ΔBDI) and changes in cognition measures +(primary: ΔMMSE, ΔADAS-cog, ΔSRT total recall; secondary: ΔDigit Symbol, ΔTrails A, ΔTrails B, ΔVF). The predictor was ΔHAM-D and the dependent measures were ΔSRT Total recall, ΔADAS-Cog, and ΔMMSE. We also examined whether antidepressant response (binary variable indicating ≥ 50% reduction in HAM-D from baseline) over 16 weeks was associated with the change in cognition measures. The same linear regression models were performed on the sample without placebo responders (N = 12), that is, those individuals who evinced a large very rapid antidepressant response, as inclusion might influence the opportunity to observe antidepressant biological effects as a sensitivity analysis. Last, we examined ΔBDI as a secondary analysis. All models were adjusted for each baseline cognition measure, as well as for age, gender, race, education, and antidepressant medication at baseline. For effect size comparison, standardized coefficients were reported. + +For Hyp 2, we then examined a series of variables that could act as moderators of the relationship. For these, we examined the moderator as a main effect and, critically, as an interaction term with ΔHAM-D. + +Moderators were separately examined with *APOE* ε4, WMH, baseline HAM-D, and continuous and dichotomized age (> 69 vs. < = 69). For the significant moderators, we performed a post hoc contrast analysis to quantify the effect of ΔHAM-D by moderator groups. + +## 3 Results 3.1 Demographic Characteristics + +Seventy-nine participants were enrolled in the study, and 61 participants completed the 16-week open-label antidepressant treatment and were included for the analysis. We focused on these individuals with complete data in order to minimize modeling or imputation and to allow for correlational analyses. Additionally, these patients went on to participate in the randomized trial of donepezil. The 61 participants had lower BDI (Table 1, P = .02) and had lower animal fluency total (Table 1, P = .01) at baseline compared to the 18 participants who did not complete the trial. The two groups did not otherwise differ in age, sex, race, education, *APOE* ε4 status, and other cognition measures. Among the participants included in the study (n = 61), there were no differences between *APOE* ε4 positive and negative groups in either demographics or cognition (Table S1 in the Supporting Information). Change in ADAS-Cog was associated with years of education +(Table S2, r = –0.35, P < .01). In contrast, changes in other cognition and depression measures were not associated with other demographic variables. + +## 3.2 General Trends + +First, the general trends in ΔHAM-D from baseline and every 4 weeks thereafter until 16 weeks are shown as a spaghetti plot in Figure 1. BDI +showed a similar improving pattern (data not shown).We also show the ΔSRT from baseline to 16 weeks, as well as other cognitive measures, in Table 2. + +Cognitive improvement across measures was variable, but none worsened. MMSE, ADAS-Cog, SRT Total Recall, Digit Symbol, and Trails B improved significantly (Table 2). Improvements in other cognitive measures involving speed of processing and/or fluency (Trails A and fluency measures) were non-significant. + +Examination of effect sizes demonstrated a stepwise profile in which SRT Total Recall gains were largest (Cohen's d = 0.34) and speed of processing gains (in timed tests including digit symbol, verbal fluency, and Trails A and B) were smallest (Cohen's d = 0.18). ADAS-Cog improvement was intermediate (Cohen's d = .20). + +## 3.3 Hyp 1: Association Between Change In Cognition And Change In Depression For Primary Outcomes + +Small and non-significant correlations were found between ΔHAM-D +or antidepressant responder status and changes in primary cognitive + +![4_image_0.png](4_image_0.png) + +![4_image_1.png](4_image_1.png) + +TABLE 2 Depression and cognitive measures at baseline and + +week 16 + +| week 16 | Baseline | Week 16 | | | | +|----------------------|-------------|-------------|---------|------------|--------| +| (n = 61) | (n = 61) | | | | | +| Mean (SD) | Mean (SD) | P-valuea | | | | +| HAM-D | 22.9 (5.40) | 9.69 (5.21) | <.001 | | | +| BDI | 20.3 (8.14) | 10.3 (6.55) | <.001 | | | +| Folstein MMSE Total | 27.8 (1.96) | 28.4 (1.93) | .018 | | | +| Score | | | | | | +| Modified ADAS-Cog | 13.0 (5.34) | 11.8 (6.06) | .045 | | | +| Score | | | | | | +| SRT Total Recall | 41.8 (10.7) | 45.6 (11.2) | <.001 | | | +| WAIS-R Digit Symbol | 41.4 (13.0) | 42.6 (12.9) | .035 | | | +| Testb | | | | | | +| Trails A Time | 47.0 (21.0) | 43.7 (19.3) | .17 | | | +| Trails B Time | 135 (70.5) | 120 (62.2) | .009 | | | +| Verbal Fluency Total | 16.7 (4.50) | 17.5 (5.37) | .18 | | | +| Abbreviations: | ADAS-Cog, | Alzheimer's | Disease | Assessment | Scale– | + +Abbreviations: ADAS-Cog, Alzheimer's Disease Assessment Scale– Cognitive; BDI, Beck Depression Inventory; HAM-D, Hamilton Rating Scale for Depression; MMSE, Mini-Mental State Exam; SRT, Selective Reminding Test; WAIS-R, Wechsler Adult Intelligence Scale, revised. + +aWilcoxon's rank sum test was conducted on the change score. + +bOne outlier was identified at week 0, although the assessment was legitimate. If we exclude that subject, the means (SD) of weeks 0 and 16 changed to 40.567 (11.197) and 42.683 (13.018) with P = 0.01. + +measures (ΔMMSE, ΔADAS-Cog, and ΔSRT Total Recall, Figure 2A–C, +all |r| < 0.07). No associations were found when we adjusted for baseline cognitive score, age, gender, race, education years, and antidepressant medication at baseline in multiple regression models (Table 3). Beyond our main cognition measures, we measured multiple tests assaying domains such as speed of processing, executive function, and semantic fluency. Consistently, no associations were found between change in these secondary measures and either change in depression measures or antidepressant efficacy (Table S3). + +## 3.4 Sensitivity Analyses + +We next conducted an analysis after excluding possible placebo responders (n = 12), that is, those who had a 50% improvement in HAM-D scores in the first 2 weeks. These participants had worse baseline depressive symptoms compared to the non-placebo responders (n += 49) (Table S3, all P < .001), but there were no differences in depression or cognition change scores. After excluding them, 29 (59%) of the 49 subjects remaining at week 16 had shown a response to open-label antidepressant treatment. We found no relationships between either change in depression measures or antidepressant responder status and change in cognitive measures (Table S3) in this refined group. + +We examined the influence of outliers on the primary outcomes. All five primary outcomes normally distributed with few outliers. Using the 3 × interquartile range (IQR) beyond first and third quartiles as the thresholds of the extreme outliers, there were no extreme outliers. + +![5_image_0.png](5_image_0.png) + +Using the 1.5 × IQR beyond first and third quartiles as the thresholds of outliers, two HAM-D change scores were identified as outliers. We repeated all analyses after excluding two outliers in HAM-D, and the results were highly similar to the original analysis. + +## 3.5 Hyp 2: Moderator Analyses + +There were no moderation effects for the following variables: WMH or baseline HAM-D as a measure of symptom severity for any of the cognitive measures (Table 3, Moderator Analysis). For *APOE* ε4 we observed a significant interaction for ADAS-Cog only (β = 0.61, 95% CI = 0.07 to 1.15, P = .028). The post hoc contrast analysis showed that HAM- +D improvements in the *APOE* e4 carriers were related to cognitive improvements (β = 0.57, 95% CI = 0.12 to 1.02, P = .01), while no such relationship was found in the non-carriers (β = –0.04, 95% CI = –0.33 to 0.26, P = .80). No significant association was found for other cognitive measures. We also found a significant interaction with WMH for ADAS-Cog (β = 0.72, 95% CI = 0.06 to 1.38, P = .038), such that HAM-D +improvements in the patients with high WMH were related to cognitive improvements (β = 0.71, 95% CI = 0.09 to 1.33, P = .03), while no such relationship was found in the low WMH group (β = –0.01, 95% CI += –0.37 to 0.35, P = 0.95). Associations with other cognitive measures were non-significant. We further examined antidepressant response in the two WMH groups and found them to be nearly identical. Last, we did not find a significant effect for age (treated categorically after median split) as a moderator. These results are shown in Table 3. + +individuals grossly. Here we premised two testable hypotheses. In our primary model of depressive pseudoamnestic disorder (Hyp 1) we posited that treatment-induced changes in depression should covary with changes in cognition. In Hyp 2 we examined whether other factors might amplify or obscure such a response. + +We found little support for Hyp 1, in that we did not find a significant relationship between change in depression and change in cognition. This held true irrespective of whether we examined depression change as continuous or as categorical (responders vs. non-responders) or whether we examined clinician- or patient-rated depression symptomatology. It also held true irrespective of cognitive domain (memory, global, speed) and in more sophisticated regression models with multiple adjustments for baseline demographics. + +For Hyp 2, we examined potential moderators of the relationship between change in depressive symptoms and change in cognition and found non-significant results for age and depression severity. For*APOE*, +results were counterintuitive in that depression and cognitive changes scores were significantly associated only in ε4 carriers, the group most likely to have features of prodromal AD and progressive neurodegenerative processes. Moreover, findings were negative for the MMSE and SRT. Age associated WMHs, including signal in deep white matter and periventricular white matter, have been linked to treatment of refractory late-life depression by a number of researchers,17,18 though this finding has not been uniform.7,18 In our study, individuals with a greater WMH demonstrated a significant relationship between depressive symptom change and change in ADAS-Cog, but not change in memory, MMSE, or speed measures. Both of these findings require more study to determine their validity. + +We found improvement in memory (SRT), ADAS-Cog (a compos- + +## 4 Discussion + +Many prior studies of pseudodementia have not fully articulated any hypotheses. They tended to examine cognitive changes in depressed ite of multiple tests of memory, naming, praxis, and drawing), but not speed of processing. These are in our estimation more likely to be due to practice effects because (1) memory is the domain most susceptible to practice effects and is present even in MCI populations; (2) the dura- + +![6_image_0.png](6_image_0.png) + +![6_image_1.png](6_image_1.png) + +tion between tests was relatively short; (3) alternate forms were not used (these have attenuated practice effects); and (4) the speed domain showed the smallest gain, and this is also the least likely to demonstrate practice effects.19–21 Our study adds substantively to the literature on depressionassociated cognitive impairments in old age. We took a hypothesisbased approach in that we sought to determine if a principled relationship existed between gains in cognition and improvement in depressive symptoms. We also examined multiple potential moderator effects. + +Last, our sample was large and included only subjects who met psychometric criteria for amnestic MCI and Major Depressive Disorder. As noted, we believed this group was ideally suited to test our hypotheses about pseudodementia. We found little evidence in its favor and so view our findings as largely negative; we also suggest that this study is broadly consistent with other studies that have demonstrated that depression increases the risk for dementia and may perhaps be a very early symptom of neurodegeneration.22,23 In a systematic review, Brodaty and Connors2 found collectively that 38% of depressive pseudodementia patients progressed to what they termed frank dementia. In the study with the largest N and longest follow-up, Kral and Emery observed that after an initially good response to antidepressants, 89% of the large sample (N = 44) eventually progressed to dementia of the Alzheimer's type during an extended follow-up period. Many of the studies included in the Brodaty and Connors review were small and had short follow up periods compared to Kral and Emery. + +This study has several limitations. First, we did not utilize biomarkers (e.g., cerebrospinal fluid, amyloid-β, or tau) to diagnose MCI. + +However, *APOE* ε4 may serve as a useful proxy for these.24 Second, we did not have a placebo control group that could be directly compared to the antidepressant group on the magnitude of cognitive gains. We also did not have a depressive pseudodementia group that could be contrasted with the literature more directly. + +In conclusion, we conducted this study in a group of older individuals, who by virtue of their documented memory impairment, responsivity to antidepressant treatment, and serial broad-based cognitive assessment over 16 weeks, were well-positioned to test the hypotheses about depressive pseudoamnestic disorder. Our negative findings cast doubt on the frequency of this syndrome in older populations with documented depression and memory impairments. + +## Acknowledgments + +Terry E. Goldberg: NIA R01AG051346. Davangere P. Devanand: NIA R01AG040093. P. Murali Doraiswamy: NIA R01AG04009301. Jeffrey N. Motter: NIMH 2T32MH020004-21. Hyun Kim: +5T32MH020004-22. + +## Conflicts Of Interest + +P. Murali Doraiswamy has received research grants (through Duke University) and advisory/board/speaking fees from several companies; P. Murali Doraiswamy owns shares in several companies and is a coinventor on patents relating to dementia which are not discussed here. + +Davangere P. Devanand is Scientific Adviser to Acadia, Eisai, Genentech, and Sunovion, and is on the Data and Safety Monitoring Board for Green Valley. All other authors declare no conflicts or disclosures. Author disclosures are available in the supporting information. + +## References + +1. Snowdon J, Kiloh L. Pseudodementia, a term for its time: the impact of Leslie Kiloh's 1961 paper. *Australas Psychiatry.* 2011;19:391-397. + +2. Brodaty H, Connors MH. Pseudodementia, pseudo-pseudodementia, and pseudodepression. *Alzheimers Dement (Amst)*. 2020;12:e12027. + +3. Lyketsos CG, Olin J. Depression in Alzheimer's disease: overview and treatment. *Biol Psychiatry*. 2002;52:243-252. + +4. Rutherford BR, Taylor WD, Brown PJ, et al. Biological aging and the future of geriatric psychiatry. *J Gerontol A Biol Sci Med Sci*. + +2017;72:343-352. + +5. Starkstein SE, Ingram L, Garau ML, et al. On the overlap between apathy and depression in dementia. *J Neurol Neurosurg Psychiatry*. + +2005;76:1070-1074. + +6. Alexopoulos GS. Mechanisms and treatment of late-life depression. + +Transl Psychiatry. 2019;9:188. + +7. Taylor WD, Schultz SK, Panaite V, et al. Perspectives on the management of vascular depression. *Am J Psychiatry*. 2018;175:1169-1175. + +8. Butters MA, Becker JT, Nebes RD, et al. Changes in cognitive functioning following treatment of late-life depression. *Am J Psychiatry*. + +2000;157:1949-1954. + +9. Koenig AM, DeLozier IJ, Zmuda MD, et al. Neuropsychological functioning in the acute and remitted States of late-life depression. J +Alzheimers Dis. 2015;45:175-185. + +10. Barch DM, D'Angelo G, Pieper C, et al. Cognitive improvement following treatment in late-life depression: relationship to vascular risk and age of onset. *Am J Geriatr Psychiatry*. 2012;20:682-690. + +11. Caselli RJ, Langlais BT, Dueck AC, et al. Neuropsychological decline up to 20 years before incident mild cognitive impairment. *Alzheimers* Dement. 2020;16:512-523. + +12. Wilson RS, Boyle PA, Capuano AW, et al. Late-life depression is not associated with dementia-related pathology. *Neuropsychology*. + +2016;30:135-142. + +13. Devanand DP, Pelton GH, D'Antonio K, et al. Donepezil treatment in patients with depression and cognitive impairment on stable antidepressant treatment: a randomized controlled trial. Am J Geriatr Psychiatry. 2018;26:1050-1060. + +14. Motter JN, Pelton GH, D'Antonio K, et al. Clinical and radiological characteristics of early versus late mild cognitive impairment in patients with comorbid depressive disorder. *Int J Geriatr Psychiatry*. + +2018;33:1604-1612 15. Ahern E, Semkovska M. Cognitive functioning in the first-episode of major depressive disorder: a systematic review and meta-analysis. + +Neuropsychology. 2017;31:52-72 16. Duff K, Lyketsos CG, Beglinger LJ, et al. Practice effects predict cognitive outcome in amnestic mild cognitive impairment. *Am J Geriatr* Psychiatry. 2011;19:932-939. + +17. Sneed JR, Culang-Reinliebb ME, Brickman AM, et al. Roose MRI signal hyperintensities and failure to remit following antidepressant treatment. *J Affect Disord*. 2011;125:315-320 18. Salloway S, Boyle PA, Correia S, et al. The relationship of MRI subcortical hyperintensities to treatment response in a trial of sertraline in geriatric depressed outpatients. *Am J Geriatr Psychiatry*. 2002;10:107111. + +19. Calamia M, Markon K, Tranel D. Scoring higher the second time around: meta-analyses of practice effects in neuropsychological assessment. *Clin Neuropsychol*. 2012;26:543-570 20. Goldberg TE, Harvey PD, Wesnes KA, et al. Practice effects due to serial cognitive assessment: implications for preclinical Alzheimer's disease randomized controlled trials. *Alzheimers Dement (Amst)*. 2015;1:103-111. + +21. Mathews M, Abner E, Kryscio R, et al. Diagnostic accuracy and practice effects in the National Alzheimer's Coordinating Center Uniform Data Set neuropsychological battery. *Alzheimers Dement*. 2014;10:675-683. + +22. Pomara N, Bruno D, Osorio RS, et al. State-dependent alterations in cerebrospinal fluid Aβ42 levels in cognitively intact elderly with latelife major depression. *Neuroreport*. 2016;27:1068-1071. + +23. Sheline YI, Disabato BM, Hranilovich J, et al. Treatment course with antidepressant therapy in late-life depression. *Am J Psychiatry*. 2012;169:1185-1193. + +24. Jack CR, Wiste HJ, Therneau TM, et al. Associations of Amyloid, Tau, and neurodegeneration biomarker profiles with rates of memory decline among individuals without Dementia. *JAMA*. 2019;321:23162325. + +25. Gunning-Dixon FM, Walton M, Cheng J, et al. MRI signal hyperintensities and treatment remission of geriatric depression. *J Affect Disord*. + +2010;126:395-401. + +26. Prado CE, Watt S, Crowe SF. A meta-analysis of the effects of antidepressants on cognitive functioning in depressed and non-depressed samples. *Neuropsychol Rev*. 2018;28:32-72. + +27. Rosenblat JD, Kakar R, McIntyre RS. The cognitive effects of antidepressants in major depressive disorder: a systematic review and meta-analysis of randomized clinical trials. *Int J Neuropsychopharmacol*. + +2015;19:pyv082. + +28. Rock PL, Rosier JP, Riedel WJ, et al. Cognitive impairment in depression: a systematic review and meta-analysis. *Psychol Med*. 2014;44:2029-2040 + +## Supporting Information + +Additional supporting information can be found online in the Supporting Information section at the end of this article. + +How to cite this article: Choi J, Lee S, Motter JN, et al. Models of depressive pseudoamnestic disorder. *Alzheimer's Dement*. + +2022;8:e12335. https://doi.org/10.1002/trc2.12335 \ No newline at end of file diff --git a/medical/md/PMC9760240.md b/medical/md/PMC9760240.md new file mode 100644 index 0000000000000000000000000000000000000000..b2dd4fc07ac8b814cb3d00b1be06c7a147c5c2a0 --- /dev/null +++ b/medical/md/PMC9760240.md @@ -0,0 +1,141 @@ + +## Review + +doi: 10.5455/medarh.2022.76.373-376 MED ARCH. 2022 OCT; 76(5): 373-376 RECEIVED: SEP 24, 2022 ACCEPTED: OCT 20, 2022 Faculty of Law, Jadara University, Jadara, Jordan Corresponding author: Hisham Jadallah Mansour Shakhatreh. Faculty of Law, Jadara University, Jordan. E-mail: dr.hishamshakhatreh@yahoo.com. ORCID ID: http//www.orcid.org/0000-0001-86935744. + +## © 2022 Hisham Jadallah Mansour Shakhatreh, Ali Jabbar Salih, Khaled Khalaf Abed Rabbo Aldrou, Farouq Ahmad Faleh Alazzam, Mohammad Saleh Bani Issa + +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. + +## Medico-Legal Aspects Of Abortion: Updates Of The Literature + +Hisham Jadallah Mansour Shakhatreh, Ali Jabbar Salih, Khaled Khalaf Abed Rabbo Aldrou, Farouq Ahmad Faleh Alazzam, Mohammad Saleh Bani Issa + +## Abstract + +Background: Abortion is an increasingly important issue since it is discussed from multiple points of view including medical, social, religious, and legal aspects. Abortion is an old topic that is continuously discussed from various perspectives including medicine, laws, and society. Abortion is an illegal in general terms, but it can be carried out under certain medical conditions for the interest of pregnant woman, if the pregnancy is threatening her life.. **Objective:** The aim of this article is updates of the literature about medico-legal aspects of the abortion. **Methods:** This is descriptive study about medico-legal aspects of the abortion based on searching this topic in the scientific literature deposited in on-line databases. **Results and Discussion:** . At global level, more and more women are opting to terminate their pregnancies for societal reasons. It is estimated that 50 million unborn babies are terminated every year across the globe, causing the lives of 200,000 pregnant women and the agony of millions more people. It is a difficult decision to conduct abortion. In this study, a comprehensive review of the literature was made to discuss abortion from medical, legal, and cultural points of view. In general, abortion was legally discussed because legal aspects involve the various perspectives. **Conclusion:** From religious points of view, abortion implies ending the life of the fetus. The right of life is to be kept. The international laws do not permit abortion and criminalize the parties involved in its existence unless it is recognized it is crucial to save the life of pregnant woman.. Keywords: abortion, legal, medical, religion, culture. + +## 1. **Background** + +Induced Globally, more and more women are opting to terminate their pregnancies for societal reasons. It is estimated that 50 million unborn babies are terminated every year across the globe, causing the lives of 200,000 pregnant women and the agony of millions more people. Abortion in violation of the law can result in very serious problems. In many parts of the world, abortion is still practiced as a method of population control. The decision to terminate a pregnancy due to a medical condition is hardly common and accounts for just a small percentage of total abortions. In this study, the various perspectives on abortion, as well as its history, its development over the course of time, and the current legal situations, are discussed. The situation in Islamic countries as well as the impact of Islamic fatwas on abortion are the focal points of this discussion (1). + +## 2. **Definition Of Abortion** + +There is a startling variety of definitions used in the legal systems of many countries, as well as in medical practice. Expulsion of the conceptus prior to the 28th week of pregnancy is what Sir Stanley Clayton and John Newton designate as "abortion" in their pamphlet titled "A Pocket Obstetrics," which was published in 1972. This viewpoint is still upheld by British legal precedent (2). Abortion is defined by Ralph Benson in the "Handbook of Obstetrics and Gynecology" (3) as "the termination of pregnancy before the fetus is viable." When the fetus weighs slightly more than 600 grams, technically speaking, viability is achieved between the ages of 23 and 24 weeks. In the United States, the laws differ from state to state, but in general, an abortion can be performed on the patient at any time during the first trimester of pregnancy. During the second trimester, however, there are more limits placed on specific medically justified circumstances. Abortion is defined by the World Health Organization (WHO) as "the expulsion or extraction from its mother of a fetus or embryo weighing less than 500 grams." (4). + +Abortion, according to contemporary consensus in the medical community, is the process of delivering a conceptus prior to the fetus's viability, which is defined as 20 weeks of pregnancy or a fetus weighing 500 grams or more. In several states, the weight requirement for viability has recently been reduced to 300 grams (4). + +The general population typically refers to an abortion as a miscarriage when they mean an abortion that occurred naturally, but the term abortion refers to an abortion that was induced, regardless of whether or not it was legal (1). + +Abortion was defined as the termination of a pregnancy before viability in the 1982 edition of the Encyclopedia Britannica. Viability was defined as a fetus weighing at least 1000 grams or having been pregnant for more than 20 weeks (1). + +## 3. **Legal Framework** + +International law has always viewed abortion rights within a medico-legal paradigm, the notion that legal and medical control guarantee safe abortion. Self-managed abortion (SMA), supported by feminist action, challenges this paradigm. SMA action has revealed that more than medical service delivery matters for safe abortion and questioned legal abortion control beyond criminal restrictions. This study discusses how abortion rights have begun to depart from the medico-legal paradigm and support SMA action in a field shift. SMA activism reimagines abortion rights in human rights objectives relating to structural violence and injustice, collective organizing and international solidarity, and political engagement (5). + +International law has always described abortion rights as 'safe and legal abortion.' The end of abortion decriminalization is legalization or a regulatory structure for legal abortion (6). Legalization refers to legal laws governing who, where, and how an abortion can be provided and accessed. A regulated system is believed to ensure safe abortions. Abortion rights have focused on legal abortion grounds and access to services under these grounds (7). Human rights require states to include safe, legal abortion into health care (Assis and Erdman, 2021). The medico-legal paradigm represents a global public health and human rights truth that unlawful abortions are hazardous (8). In 1990, the WHO defined "unsafe abortion" as being performed by "individuals lacking the essential expertise or in an environment that does not adhere to minimal medical standards, or both" (World Health Organization, 1993). Illegal abortion didn't meet these standards. This scheme succeeded for decades since abortions outside these conditions were invasive and risky. WHO has revised its safe and unsafe abortion scheme to incorporate a risk gradient (9). While preserving the safe/unsafe binary, it acknowledged that informal use of abortion medicines (mifepristone and/ or misoprostol) has made illegal abortion safer (10). + +Decriminalization and legalization are coterminous within the medico-legal paradigm. In this perspective, human rights standards argue for decriminalizing and legalizing abortion (11, 12). These standards are supported and rationalized by a focus on maternal mortality and morbidity, health-related harms of criminalization, which designate medical systems as the remedial state institution for violations (13, 14). + +In a classic formulation of the paradigm, the Special Rapporteur on the Right to Health warned that "safe abortion will not immediately be available upon decriminalization unless States create conditions under which they may be provided," namely establishing clinics, training physicians and health-care workers, enacting licensing requirements, and ensuring the availability of medicines and equipment (15). + +## 4. **The Provision Of Abortion As A** Necessary Health Service + +After the COVID-19 pandemic, when sexual and reproductive health services were disrupted, disempowering individuals and exposing them to preventable health risks, the World Health Organization (WHO) +added comprehensive abortion care to the list of essential health services in recent technical publications and guidance issued in the aftermath of the pandemic. The new recommendation emphasizes that reaching the Sustainable Development Goal goal 3.8 of universal health coverage requires not only access to essential health services of high quality but also access to critical medicines at reasonable prices. It acknowledges that expanding access to abortion care is an essential component of creating a favorable environment for universal health coverage, the goal of which is "to accelerate efforts to ensure that all people and communities receive the full spectrum of essential, quality health services" without experiencing undue monetary strain. In addition, WHO recognizes, in terms of the regulation of abortion, that +"unlike other essential health services, abortion is commonly regulated to varying degrees through the criminal law in addition to regulation under health-care law." +This is something that is different from other essential health services. The elimination of all forms of criminal regulation pertaining to abortion is a recommendation made within the scope of this guideline (16). + +## 5. **Accountability** + +The World Health Organization (WHO) acknowledges that responsibility is one of the most important factors in ensuring that sexual and reproductive health and rights are safeguarded, respected, and realized. The recommendation makes notice of the fact that accountability can take on a variety of forms in the process of ensuring that women have access to safe abortions. One of these forms is ensuring that abortion laws and policies comply with human rights standards. It explains that this should be accomplished by recording and monitoring health outcomes related to abortion laws and policies and reporting them to national human rights institutions. Additionally, it should be accomplished by ensuring that all individuals have access to justice, which includes an accessible mechanism "to challenge denial of abortion in a timely manner" and a meaningful and effective remedy in cases where the individual's rights have been violated. Restitution, compensation, rehabilitation, satisfaction, and guarantees of non-repetition can all fall under the category of remedies. Remedies can also involve changes to laws and policies. In the guideline, it is emphasized that an environment that is enabling should feature the regular review and reform of laws and policies on abortion, including the decriminalization of services and "forms of behavior that can be performed only by women, such as abortion." This is because abortion is a form of behaviour that can only be performed by women (16). + +## 6. **Religion And Abortion** + +Both a person's individual bioethical decision about whether or not to have an abortion and a nation's legal framework for abortion are heavily influenced by religious beliefs and practices. Despite this, there hasn't been enough research done to develop a comprehensive knowledge of the Islamic stance. This research began with an in-depth and methodical examination of Islam's stance on abortion. This was accomplished by looking at the two most authoritative biblical sources (i.e. the Quran and the Sunnah) in addition to other elements that provided relevant information (i.e. contemporary fatwas, Islamic mysticism and broader Islamic principles, interest groups, and transnational Islamic organizations). There is no clear biblical prohibition on abortion, despite the fact that Islamic jurisprudence does not support the practice. There is a wide range of opinion on the topic of abortion, and a significant number of religious academics advocate for the legalization of the procedure under certain conditions and at certain ages of gestation. However, there is remarkable heterogeneity in regards to other circumstances (such as preserving physical or mental health, foetal impairment, rape, or social or economic reasons), and later gestational development of the foetus. It is generally agreed that the least blameworthy abortion is when the life of the pregnant woman is threatened and when 120 days have not lapsed. In the second part of this research project, a comparative evaluation of abortion laws across nations with a Muslim-majority population was carried out. It was discovered that the majority of nations take a conservative view on the issue, since 18 of the 47 countries studied do not permit abortions under any circumstances other than when the life of the pregnant woman is in danger. However, there was a significant amount of variation between countries, and ten of those countries permitted abortion "upon request." There is a discussion on the discursive aspects that could allow for the creation of policy in nations with a predominantly Muslim population, as well as on future research that could improve the study of abortion rights. Particularly, more lenient abortion laws may be achieved by disabusing individuals of the misconception that the most authoritative texts unambiguously oppose abortion, highlighting more lenient interpretations that exist in certain Islamic legal schools, emphasizing significant actors that support abortion, and being mindful of policy frames that will not be well received in Muslim-majority countries (17). + +## 7. **Abortion Perspectives In Some** Arabic And Islamic Countries + +7.1. Jordanian perspectives of abortion According to the report by the United Nations Development Programme (2022), Articles 321–325 of the Penal Code make abortion illegal for anybody to undergo an abortion, including a woman who has been sexually assaulted. Any individual who engages in the practice of abortion is liable to a possible sentence of one to three years in prison. If the offender is a member of the medical community, the punishment will be enhanced by one-third. A woman who induces her own miscarriage or who allows another woman to induce her miscarriage faces a possible prison sentence of between six months and three years. In order to protect the lady's reputation, the penalties for getting an abortion are less severe if the procedure is carried out by the woman herself, a descendant, or a relative up to the third degree (Article 324). + +According to Article 322(2), a person who performs an abortion and is responsible for the death of the woman who had the abortion faces a minimum of five years in prison and a sentence of forced labor at a temporary location. Article 323(1) of the law stipulates that anybody who knowingly terminates the pregnancy of a woman without her agreement is subject to a sentence of hard labor for a period of time not to exceed ten years. According to Article 323(2), the minimum sentence for an abortion that results in the woman's death must not be less than ten years in prison. Article 12 of Public Health Law No. 47 of 2008 makes it illegal for doctors to counsel patients on abortion or to perform abortions, with the exception of situations in which it is required to protect the life or health of the pregnant woman. The law does not provide a precise definition of a woman's health but rather refers to health in general, including mental and physical well. Since 1993, numerous fatwas addressing various aspects of abortion have been published (18). + +According to freedom house organization, in Jordan, having an abortion is against the law, with a few exceptions, such as when it is necessary to protect the life or health of the mother. Women who perform abortions on themselves face penalties ranging from six months to three years in prison under the provisions of Article 321 of the penal code. Those who perform abortions on others, including doctors, face penalties ranging from one year to three years in prison under the provisions of Article 322. The person who is responsible for an abortion that results in the death of the mother gets a mandatory minimum term of five years in jail. According to Article 323, individuals who carry out an abortion on a woman against her will face a possible penalty of up to ten years in jail, with the potential for a minimum sentence of the same length if the woman passes away. If a woman performs her own abortion in order to protect her family's honor, or if the individual carrying out the abortion does so in order to protect the honor of a female relative, the woman or the individual may be eligible for reduced sentences under Article 324, which range from three months to two years (19). + +7.2. Egyptian perspectives of abortion Articles 260–264 of Egypt's Penal Code from 1937 make it illegal to have an abortion in the country (20). + +Despite this, Article 61 of the Penal Code allows for exceptions to be made in circumstances of need. This provision has traditionally been interpreted to allow for abortions that are required to preserve the life of the pregnant woman. This exception has, in certain circumstances, been broadened to cover pregnancies that pose risks to the health of the pregnant woman as well as pregnancies that result in the impairment of the fetus (21). In these kinds of situations, a doctor is only allowed to execute an abortion if they have received permission from two different specialists, or the woman's life is in immediate danger (1, 22- 23). Anyone who assists in the termination of a pregnancy is subject to criminal prosecution, including medical professionals who participate in such activities. Because the prosecution has the burden of proving both that the lady was pregnant and that the pregnancy was terminated in some manner, convictions for this crime are extremely rare (24). + +7.3. Tunisian perspective of abortion In 1973, Tunisia was the first Muslim, African, and Arab country to liberalize its legal system, coming in ahead of France, Germany, and the United States of America in the process. In point of fact, Tunisia legalizes abortion "upon request" up to 12 weeks into a pregnancy, and the procedure is offered free of charge inside the country's public healthcare system (25). This stands in stark contrast to the majority of other Muslim-dominated countries, the majority of which only permit abortions in the event that the pregnant woman's life is in danger. Notably, Asman (26) examined the legal frameworks of three Muslim nations (i.e. Egypt, Kuwait, and Tunisia) and found that the abortion laws in these nations had gotten less restrictive over time. + +## 8. **Conclusion** + +Abortion is an old topic that is continuously discussed from various perspectives including medicine, laws, and society. Abortion is an illegal in general terms, but it can be carried out under certain medical conditions for the interest of pregnant woman, if the pregnancy is threatening her life. From religious points of view, abortion implies ending the life of the fetus. The right of life is to be kept. The international laws do not permit abortion and criminalize the parties involved in its existence unless it is recognized it is crucial to save the life of pregnant woman. + +- **Author's contribution**: All authors were invoved in the preparation of this article. Final proofreading was made by the T. + +C. and I. M. + +- **Conflict of interest**: None declared. - **Financial support and sponsorship**: Nil + +## References + +1. Albar MA. Induced abortion from an islamic perspective: is it criminal or just elective?. Journal of Family and Community Medicine. + +2001; 8(3): 25–35. + +2. Encyclopedia Britannica. 1982; 2: 1069–1072. Available at: https:// +hr.wikipedia.org/wiki/Encyclop%C3%A6dia_Britannica. Accessed: +September 10th 20223. Benson R. Handbook of Obstetrics and Gynecology. 6th edition. Middle East Edition, Lebanon. Lange Medical Publication. 1977: 260 pp. + +4. Bennett M. Abortion. In: Hacker N, Moore JG, editors. Essentials of Obstetrics and Gynecology. 3rd ed. Philadelphia: Saunders Co.; 1998: 477–486. + +5. Mariana Prandini Assis, Joanna N. Erdman. Abortion rights beyond the medico-legal paradigm. Global Public Health. 2021. https://doi. org/10.1080/17441692.2021.1971278. + +6. Berer M. Abortion Law and Policy Around the World: In Search of Decriminalization. Health and human rights. 2017; 19(1): 13–27. + +7. Erdman JN, Cook RJ. Decriminalization of abortion - A human rights imperative. Best practice and research. Clinical Obstetrics and Gynaecology. 2020; 62: 11–24. Available at: https://doi.org/10.1016/j.bpobgyn.2019.05.004. Accesse: September 10th 2022.ز 8. Berer M. National laws and unsafe abortion: The parameters of change. + +Reproductive Health Matters. 2004; 12(24, Suppl..): 1–8. Available at: https://doi.org/10.1016/S0968-8080(04)24024-1. Accessed: September 10th 2022. + +9. Ganatra B, Tunçalp Ö, Johnston HB, Johnson BR, Gülmezoglu AM, +Temmerman M. From concept to measurement: Operationalizing WHO's definition of unsafe abortion. Bulletin of the World Health Organization. 2014; 92(3): 155. Available at: https://doi.org/10.2471/ BLT.14.136333. Accessed at: September 10th 2022. + +10. Singh S. Hospital admissions resulting from unsafe abortion: Estimates from 13 developing countries. Lancet. 2006; 368(9550): 1887– +1892. Available at: https://doi.org/10.1016/S0140-6736(06)69778-X. Accessed: September 10th 2022.ز 11. UN Special Rapporteur on the right of everyone to the enjoyment of the highest attainable standard of physical and mental health. 2011: Report (A/66/254). https://undocs.org/A/66/254. + +12. UN Working Group on the issue of discrimination against women in law and in practice. 2016: Report (A/HRC/32/44). https://undocs. + +org/A/HRC/32/44. + +13. UN Committee on Economic Social and Cultural Rights. 2016; General Comment No. 22 (E/C.12/GC/22). + +14. UN Working Group on the issue of discrimination against women in law and in practice. 2016: Report (A/HRC/32/44). https://undocs. + +org/A/HRC/32/44. + +15. Erdman, J. N. Access to information on safe abortion: A harm reduction and human rights approach. Harvard Journal of Law and Gender, 2011: 34(2), 413–462. + +16. https://reproductiverights.org/wp-content/uploads/2022. + +17. Shapiro G. K. Abortion law in Muslim-majority countries: an overview of the Islamic discourse with policy implications. Health policy and planning, 2014; 29(4), 483–494. https://doi.org/10.1093/ +heapol/czt040. + +18. https://www.undp.org/sites/g/files/zskgke326/files/migration/arabstates/Jordan-Country-Assessment—-English.pdf, retrieved in 22/9/2022. + +19. https://www.freedomhouse.org, retrieved in 22/9/2022. + +20. https://www.unescwa.org/sites/default/files/inline-files/egypt, retrieved in 24/9/2022. + +21. https://www.un.org/esa/population/publications/abortion/doc/ +egypts1.doc, retrieved in 24/9/2022. + +22. Hathout H. Topics in Islamic Medicine. Kuwait: Islamic Medicine Organization; 1984; 93–135. + +23. Ahmed WD. Oath of Muslim Physician. JIMA. 1998; 20:11–4. 24. Potts M, Diggory P. Abortion. 2nd edition. Cambridge Univer-sity Press; Textbook of Contraceptive Practice. 1983: 274–367. + +25. Hessini L. Islam and abortion: the diversity of discourses and practices, IDS Bulletin, 2008, 39: 18-27. + +26. Asman O.. Abortion in Islamic countries—legal and religious aspects. + +Medicine and law. 2004, 23(1): 73–89. \ No newline at end of file diff --git a/medical/md_images/PMC10035009/10_image_0.png b/medical/md_images/PMC10035009/10_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..1512e569c2b71a31ed43d69a05f39eb6d8ccb9d8 --- /dev/null +++ b/medical/md_images/PMC10035009/10_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:60b2624a24e43865882fcf9eab3c32b966a4369cd5d4db2533eb0e3a6ae66924 +size 73034 diff --git a/medical/md_images/PMC10035009/5_image_1.png b/medical/md_images/PMC10035009/5_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..8c53c8f454f837a7a1108823687f50b74b2de043 --- /dev/null +++ b/medical/md_images/PMC10035009/5_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:30d015a4656a3f417dc3dd38a69f928fdbed30727817c27cd341222fc5163832 +size 17575 diff --git a/medical/md_images/PMC10077408/PMC10077408.md b/medical/md_images/PMC10077408/PMC10077408.md new file mode 100644 index 0000000000000000000000000000000000000000..5d49df4e8877d481700a68c358e6f97454995ec3 --- /dev/null +++ b/medical/md_images/PMC10077408/PMC10077408.md @@ -0,0 +1,407 @@ +DOI: 10.1002/nop2.1579 RESEARCH ARTICLE + +# Value Of Social Activities And Prerequisites For Continued Participation Of Rural Older Adults: A Qualitative Study + +Ryuta Onishi1 | Risa Takashima2 | Kazuko Saeki1 | Michiyo **Hirano**3 1Faculty of Nursing, Toyama Prefectural University, Toyama-ken, Japan 2Department of Rehabilitation Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan 3Department of Comprehensive Development Nursing, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan Correspondence Michiyo Hirano, Department of Comprehensive Development Nursing, Faculty of Health Sciences, Hokkaido University, Kita 12, Nishi 5, Kita-ku, Sapporo-shi, Hokkaido 060-0812, Japan. Email: mihirano@med.hokudai.ac.jp Funding information Japan Society for the Promotion of Science, Grant/Award Number: JP18H03103; Nihon Unisys + +## Abstract + +Aims: To explore the value of social activities and the prerequisites for continuous participation among rural older adults based on their experiences. Design: Qualitative, descriptive design. Methods: Individual semi-structured interviews were conducted with 14 older adults from voluntary community salons in a rural area in Japan. Participants were selected through purposeful sampling. Data were analyzed using qualitative content analysis. The consolidated criteria for reporting qualitative research (COREQ) checklist were used. Results: Four values of social activities were identified: "Mutual benefit connectedness," "Preventing and coping with aging," "Making life brilliant 'now'," and "Building a safety net." Three prerequisites for activities were identified: "Do not overreach," "Do not infringe on personal territory," and "Do not go against community norms." Social activities provide rural older adults with reciprocity ties, coping with aging, enhanced daily lives, and a social safety net. Continued participation in rural social activities requires adherence to cultural norms and moderation of relationship distances. Community and public health nurses are expected to promote social activities that incorporate these values and adhere to these prerequisites. + +KEYWORDS +older adults, rural community, social activity, social engagement, social participation, social values + +## 1  | **Introduction** + +With the rapid increase in longevity worldwide (United Nations, 2022), improving older adults' health and well-being in rural areas is a global concern and the critical goal of nursing practice. As Japan rapidly approaches a super-ageing society, it faces numerous related challenges, and its contingency efforts, including improving quality of life and treatment, have attracted global attention (Arai et al., 2012, 2015). Thus, a paramount concern is how older adults can continue to live happily despite experiencing an age-related decline (Arai et al., 2012). Focusing on older adults in rural areas in Japan and understanding what factors can improve their well-being can help resolve challenges in gerontological health care, which the world will likely face in the future. + +## 1.1  |  **Background** + +Older adults' well-being can be improved by promoting their participation in social activities. According to Levasseur et al. (2010), social This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2022 The Authors. *Nursing Open* published by John Wiley & Sons Ltd. + +What does this paper contribute to the wider global clinical community? + +- Emphasizes the importance of nursing practice, focusing on social participation, for improving rural older adults' well-being in a super-ageing society. + +- Elucidates the direction of nursing practices needed to promote social participation among rural older adults. +previous studies—is beneficial. "Value" is defined as an attribute or performance of a specific object that attracts the subject of action (Mita, 1996). Takashima et al. (2020) reported that the values of social activities for older men in urban Japan were "health as a resource and reward for social activities," "feeling I am still useful," "feeling that something is my responsibility," "feeling of time well spent," and "finding interest through interactions." Extending these findings to rural older adults can understand the value of social activity for them. A strategy for promoting social participation unique to rural areas can thus be considered. + +## 2.1  |  **Aims And Objective** + +This study aimed to explore the value of social activities and the prerequisites for continuous participation among rural older adults based on their experiences. Furthermore, this study focused on the experiences of older adults participating in Japanese "salons." In Japan, older adults are familiar with activities in communitybased centres (i.e., "salons"), where they gather to create arts and crafts, play games, and engage in health-and enjoyment-promoting activities (Hikichi et al., 2017). "Voluntary community salon activities" in Japan are resident-centred, wherein participants play active roles and thus experience an expansion of social relations (Oda et al., 2020). Older adults who participate in voluntary community salon activities in a rural area are expected to have more experience participating in social activities and are thus more likely to perceive the value of social activities. Understanding the value that these activities hold for participants can help elucidate their significance in the health and lives of rural older adults. Further, exploring the conditions that encourage continued participation in these activities can inform nursing practice strategies to support and promote social participation. + +## 3  | **Methods** 3.1  |  **Design** + +This study used a descriptive qualitative design (Sandelowski, 2000) that comprehensively summarizes events in everyday, straightforward terms. We adopted this qualitative approach because we activities such as paid or unpaid jobs, volunteering, and participating in social groups provide interaction with others in the community. This occurs across six levels of taxonomy: (1) performing an activity in preparation for connecting with others; (2) being with others (alone, but with people around); (3) interacting with others (social contact) without performing a specific activity together; (4) performing an activity with others (collaborating to accomplish the same goal); (5) helping others; and (6) contributing to society. Participation in social activity is associated with vigorous physical activity (Kikuchi et al., 2017), prevention of physical frailty (Abe et al., 2020), reduced risk of declining ability to perform activities of daily living (Fujihara et al., 2022; Kanamori et al., 2014), good physical health (Tomioka et al., 2017), prevention of cognitive decline (Bourassa et al., 2017; Cai, 2022; Saito et al., 2018; Sun & Lyu, 2020), and decreased loneliness (Niedzwiedz et al., 2016; Zhao & Wu, 2022) and depression (Amagasa et al., 2017; Lee & Kim, 2014). The cumulative effect of positive psychological and social health outcomes improves older adults' overall well-being (Hoshino et al., 2020; Steinbach, 1992) and enhances their quality of life (He et al., 2017; Lu et al., 2022). Notably, participation in social activities reduces mortality (Arnau et al., 2016; Fain et al., 2022; Kim et al., 2019) and increases survival (Holtzman et al., 2004) among older adults. + +Environmental, social, and individual factors promote or inhibit participation in social activities (Aroogh & Shahboulaghi, 2020). Environmental factors include access to community resources, such as public transportation, and services to meet daily needs, such as household goods and medical care (Levasseur et al., 2015). Social factors comprise social support (Levasseur et al., 2015), social cohesion (Latham & Clarke, 2018), and relationship with the neighbourhood (Nemoto et al., 2018). Individual factors consist of age, gender, disease or disability, mental health, economic status, employment status, and education attainment (Nemoto et al., 2018; Pinto & Neri, 2017). + +Moreover, compared with urban areas, rural areas have specific barriers related to environmental and social factors for social participation. Specifically, rural areas have limited social and medical resources, constraining access to them (Farmer et al., 2012; Strasser, 2003). Reduced social resources and population in rural areas tend to isolate older adults (Slama, 2004). Additionally, people in these areas lack privacy and anonymity and are prone to peer pressure due to the fear of gossip (Slama, 2004). This results from the relationship proximity inherent in rural areas and may become a barrier to people's participation in social activities. Thus, adequate transportation, information about activities and services, and social support networks are salient factors for enhancing social participation among older adults in rural areas (Levasseur et al., 2020). + +## 2  |  **The Study** + +To promote the participation of rural older adults in social activities, understanding the value of social activities as perceived by the parties involved—in addition to the objective factors indicated by aimed to highlight the value of social activity among rural older adults in Japan. + +## 3.2  |  **Theoretical Framework** + +Symbolic interactionism was adopted as the theoretical paradigm in this study. Symbolic interactionism focuses on meanings that mediate the interpretation of interactions within dynamic social activities (Blumer, 1969). This qualitative study assumed that rural older adults in Japan act according to the meaning attached to their interpretations of interactions within their social activity, the community, and society. The consolidated criteria for reporting qualitative research (COREQ) checklist (Tong et al., 2007) was used to ensure the quality and transparency of this study. + +## 3.3  |  **Sampling And Recruitment** + +To target older adults who fully realize the value of social activities, the participants were recruited by purposive sampling with the cooperation of the salon directors. First, a public health nurse introduced the authors to the salon. Second, the authors met the four directors and requested an introduction to the potential participants in writing and verbally. The directors explained the outline of the research to all members and introduced 14 participants (including themselves) who had provided informed consent for participation. The researchers explained the study's outline verbally and in writing to participants. + +## 3.4  |  **Population And Sample** + +Participants included 14 older adults who were residents of Town A and engaged in the local salon. Town A is a rural area in Japan with a population of approximately 6000 and an ageing population of nearly 27%. The community salon is less than 10 years old; it comprises 19 members and holds monthly activities. Four men serve as directors and plan activities. Three members are responsible for cooking lunch at each gathering, and everyone eats at the table together. After lunch, members engage in recreational activities, such as exercise, games, and casual conversation. Kitchen duty is rotated, with all participants undertaking cooking duties every few months. We started interacting with the salon members in August 2018 to gain their trust. + +## 3.5  |  **Data Abstraction** + +Between October and December 2018, the authors conducted individual 40- to 90-minute semi-structured interviews with participants at their community salon so that they could express themselves comfortably. Interviewers were the first (man, Ph.D.), second +(woman, Ph.D.), and fourth (woman, Ph.D.) authors. The interviewers are university researchers with extensive interviewing experience in qualitative research and practical experience as healthcare professionals. + +Participants initially reported their name, age, family structure, and life history, including occupational history, on the demographic questionnaire. Interviews were conducted in Japanese based on an interview guide (Table 1), a flexible tool to encourage free-flowing conversations. The study authors originally developed the interview guide by considering the participants' characteristics to achieve the study objectives. + +## 3.6  |  **Inclusion And Exclusion Criteria** + +Criteria for participation in the study were age 65 or older and not being treated for dementia. Nineteen older adults were active in the salon, but four members declined to participate in interview survey for their personal reasons. In addition, one director participated in a separate interview survey and was therefore excluded from recruitment. + +## 3.7  |  **Data Sources/Collection** + +Participants were asked about their interactions with family members, non-family members, and participation in various social activities in the community daily, weekly, or monthly. They were asked to reflect on their life in the past year, their interactions and + +## Table 1 Interview Guide + +Questions (translated to English from the original Japanese interview guide) 1. First, could you think about your daily life for a day, a week, or a month? + +1. Please tell me about your interactions with your family members in detail. + +2. Do you interact with people other than your family members? + +Please tell me about it in detail. + +3. Do you ever join a social gathering or organization in the community? Please tell me about it in detail. + +2. Second, could you think about your life for a year? + +1. Please tell me about your interactions with your family members in detail. + +2. Do you interact with people other than your family members? + +Please tell me about it in detail. + +3. Do you ever join a social gathering or organization in the community? Please tell me about it in detail. + +3. What do you think about connecting with society? 4. What do you think about connecting with people other than your family members? + +5. Is there anything you are working on for your well-being? + +## 3.8  |  **Data Analysis** 3.10  |  **Rigour** 4  |  **Findings** 4.1  |  **Participants** 4.2  |  **Value Of Social Activities** 3.9  |  **Ethical Considerations** + +| TABLE 2 Examples of analysis (coding tree) Categories Subcategories Final codes Building a safety net Social ties with this community to protect my livelihood Maintaining connections with community members to avoid isolation Creating relationships with people who can help me in times of need Interaction with people in daily life with reasonable boundaries Interactions with community members to bring emotional stability An opportunity to make people in the community care about me Opportunities to gain approval from the community members Creating interactions with people who are friendly and helpful | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +engagement with other people, and their perceptions of being connected with society and others outside their families. Additionally, the interview guide was refined after obtaining prior confirmation from the salon director and by conducting pilot interviews with two older adults. With the participants' permission, interviewers recorded the interviews using a digital voice recorder. This study used an inductive approach for qualitative content analysis. Qualitative content analysis focuses on the subject and context of the text and offers opportunities to analyse manifest and descriptive content besides latent and interpretative content (Graneheim & Lundman, 2004). With an inductive approach, the researcher seeks similarities and differences within the data and then organizes data into categories and/or themes with varying levels of abstraction and interpretation (Graneheim et al., 2017). + +The first author conducted data analysis as follows. First, recorded data were transcribed verbatim and repeatedly read to grasp its context. Second, open coding was performed with a focus on the descriptions of value and prerequisites, and memos were made by continually asking the questions: "What is this?" and "What does this mean?" Codes were extracted based on the memos and focusing on the context and meaning of the data. Codes were compiled into sub-categories by revisiting the data and theoretical notes to examine their similarities and differences. Furthermore, subcategories were integrated with categories that involved more comprehensive concepts. During this process, the data sensitivity was refined by continuing to make memos. An example of analysis from coding to category extraction is shown in Table 2. + +Specifically, the first author initially extracted seven participants' 2018, no. 18–39). The participants were assured of anonymity, data confidentiality, voluntary participation, and the publication of the results—verbally and in writing. All the participants provided written informed consent for participation and were free to withdraw at any time. To increase the trustworthiness and credibility of the data analysis (Guba & Lincoln, 1989; Lincoln & Guba, 1985), the authors participated multiple times in the salon activities attended by the participants to build relationships. We undertook a continuous comparative analysis and reviewed the results together until a consensus was reached. We also used member checking, which involves sharing the analysis results with some participants for review. + +codes and subcategories from the interview data, which included directors and other salon members. Next, based on the subcategories derived from the initial seven participants' data, we analysed the data of the remaining seven participants. We revised the label names of the original subcategories and confirmed the appearance of new subcategories. No new subcategories emerged in the data from the latter seven participants. Finally, subcategories were merged into categories. Codes, subcategories, and categories were assessed and agreed upon by the three co-authors. When deepening the analysis, we found that participants perceived the value of voluntary community salon activity and other social activities to be equivalent; therefore, both codes were integrated to create subcategories and categories. Data analysis was conducted in Japanese, and the results were translated into English. + +A summary of the study participants is presented in Table 3. The content analysis identified four values of social activity and three prerequisites of continuous social activity for older adults who participate in salons in a rural community (Table 4). The four values of social activity were as follows: "Mutual benefit connectedness," "Preventing and coping with ageing," "Making life brilliant 'now'" and "Building a safety net." +This study was approved by the Ethics Review Committee of the faculty of Health Sciences, Hokkaido University (approval date: July 31, + +## 3278 |    Et Al. + +| TABLE 3 Characteristics of the study participants Variables Item | n (%) mean (SD) | | | +|-----------------------------------------------------------------------------|----------------------------|------------------------|----| +| Age (years) | 81.1 (4.80) | | | +| Sex | Men | 6 (42.9) | | +| Women | 8 (57.1) | | | +| Family composition | Single | 9 (64.3) | | +| Only a couple | 2 (14.3) | | | +| Two or three generations | 3 (21.4) | | | +| Birthplace | Town A | 7 (50.0) | | +| Outside town A | 6 (42.9) | | | +| Unknown | 1 (7.1) | | | +| Social activities that participants described (Other than salon activity) | In groups or organizations | Senior citizens' clubs | 7 | +| Neighbourhood associations | 4 | | | +| Senior colleges | 3 | | | +| Activity as a welfare commissioner | 2 | | | +| Activities at shrines | 2 | | | +| Volunteer activities | 1 | | | +| Religious activities | 1 | | | +| Day-care | 1 | | | +| Widow support group | 1 | | | +| Activities of the Cultural Property Protection | 1 | | | +| Committee | | | | +| Activities of the Gymnastics Federation | 1 | | | +| Activities at the agricultural cooperative | 1 | | | +| Women's association | 1 | | | +| A group enjoying a hot spring | 1 | | | +| Group of gate ball players | 1 | | | +| In informal interactions | Interaction with friends | 4 | | +| Interaction at the kitchen garden | 4 | | | +| Taking a walk | 2 | | | +| Picking edible wild plants | 1 | | | +| Park golf | 1 | | | +| Interaction with helpers Interaction with neighbours | 1 | | | +| Interaction at the Post Office | 1 | | | +| Hobby-related activities | 1 | | | +| Commuting to hospital | 1 | | | + +## 4.2.1  |  Mutual Benefit Connectedness + +The participants experienced happiness by sharing time with their peers and ascribed value to sharing small pleasures, such as meals and games. Community activities, such as neighbourhood associations, were valuable opportunities for community residents to share happy occasions. Spending time with people in the community reinforced the study participants' sense of belongingness. + +The fun part of a neighborhood association is getting together to eat delicious food at New Year's or eating Jingisukan together during the cherry blossom viewing season. Everyone is still having fun. + +[ID 6] +The participants experienced joy when they gathered casually with community members, as their time together was congenial and harmonious. They highly valued casual conversations with + +| TABLE 4 Results of qualitative content analysis Categories | Subcategories | | +|------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------| +| Value | Mutual benefit connectedness | Happy time spent with community mate Chance to embody a spirit of contribution | +| Preventing and coping with ageing | Demonstration of self-worth fostered through life Source for active ageing | | +| Making life brilliant "now" | Having fun daily, arising from interesting experiences Part of my everyday life in this community | | +| Building a safety net | Social ties with this community to protect my livelihood Interactions with community members for engendering emotional stability | | +| Prerequisites | Do not overreach | Activities to participate in while relaxing Activities to maintain own pace without strain | +| Do not infringe on personal territory | Maintenance of a neutral relationship with community members Mutual thoughtfulness in interaction with community members | | +| Do not violate community norms | Restriction of participation in activities by local norms Compliance with potential rules for activities in this community | | +| members while eating from the communal pot in the salon. Sharing | young, and I have always been impressed with them. | | +| a place where everyone could engage in casual conversation was a | They are very lively (when they are on kitchen duty). | | +| source of enjoyment for all salon members. | Their eyes glisten when using a kitchen knife (a skill learned in one's former days). | | +| Whenever I show up here (at the voluntary commu | [ID 1] | | +| nity salon), I enjoy doing something or the other and | | | + +having lunch together. There is an old friend here who I can talk to! It has been a pleasure to spend time talking with him about a lot of things. + +[ID 2] +Furthermore, participants wished to contribute to the community and its members, especially those in need. Social activities were perceived as opportunities to embody these wishes. + +I am going to participate in any gatherings in the community. People are never happy on their own. I +believe that everyone should be happy, whether it is family, loved ones, friends, or neighbors. So, if my friends are in trouble, I want to do something to help. + +[ID 9] + +## 4.2.2  |  Preventing And Coping With Ageing + +When participating in social activities, participants valued using the wealth of their life experiences. They utilized their skills acquired in the past to remain active in the salon. For example, some salon participants used to work as cooks and used their refined cooking skills when they were assigned cooking duty at the salon. + +Cooking skills of the ladies who cook here (in the salon) are the result of their training when they were Social activities were also crucial in preventing physical and mental frailty and remaining as youthful as possible. For the participants, who were accustomed to listening to sermons at the temple, social activities were meant to involve lifelong learning. Some community activities included health lectures, where the participants gained knowledge on how to improve their health. The participants also realized that active interaction with community members through social activities was linked to maintaining their cognitive function. + +I am an older adult, so I think my cognitive function will gradually decline. That is why I think it's good to participate, to be able to talk with people in the community, like at the voluntary community salon activities. + +[ID 5] + +## 4.2.3  |  Making Life Brilliant "Now" + +Social activities were linked to enriching the participants' present times. The annual trip with community members was an opportunity to experience the extraordinary, and the participants looked forward to it. Additionally, kitchen duty at the salon required trial and error, such as considering the preferences of community members, and the participants saw this as an interesting and unusual experience. The novel experiences through social activities added fun to the participants' lives. + +## 4.3.1  |  Do Not Overreach + +The participants hoped to engage in social activities as much as possible, based on their health and physical strength. As participants aged and realized that their bodies were deteriorating, they were less inclined to participate in their usual activities that were beyond their health and physical capacity. However, they continued to participate in activities that they could manage and proactively chose whether to continue. + +I have not gone on trips organized by the senior citizens' club as I have a back problem. Now, if it is a day trip, I can go, but if it is an overnight trip, I have to decline. I can walk if I rest adequately, but it is hard to keep up with the pace of others. + +[ID 6] + +## 4.3.2  |  Do Not Infringe On Personal Territory + +The participants believed that boundaries were necessary to maintain relationships with community members. Having interpersonal boundaries in rural communities ensured participants maintained an appropriate distance. In the community, rumours spread quickly; thus, participants tried to avoid gossiping about others. Some participants were concerned that rumours about others would spread because of them; hence, they would only participate in activities with close friends. Since theirs is a small community, the participants believed it is essential to build relationships where community members care about each other without getting too involved. + +It is not a good idea to get into a deep relationship with people in the community. I hear both good and bad rumors, so I try not to get too deep into a relationship. This is a harmonious relationship. + +[ID 13] + +## 4.3.3  |  Do Not Violate Community Norms + +Participants must strictly follow the rules of social activity to continue their activities. The participants said many people had quit the voluntary community salons because they did not want to be on kitchen duty. The community members had self-selected social activities if they could not strictly follow the implicit norms. Further, participants were influenced by ingrained community norms when choosing social activities, such as joining a senior citizens club after retiring and getting older. The latent normative consciousness of the community was one factor that dictated participants' social participation. + +I know the members of the senior citizen's club would share the food they made at home with everyone during their spa trips. It is the season for tasty pickles, so they bring me tasty pickles. And we enjoy asking each other, 'How did you pick it?'. + +[ID 7] +Social activities were also a part of the participants' daily lives in their communities. The participants took the salon and other community activities for granted, stating that those activities were not special. Social activities were an important component of the participants' daily lives. + +(Because of actively participating in various activities such as senior citizen clubs and gateball gatherings regularly), I'm pretty busy, so my friends say, 'You're not home at all'. + +[ID 8] + +## 4.2.4  |  Building A Safety Net + +Participants' interactions with community members through their social activities helped form social networks with a safety net function to enable continued residence in their communities. The participants engaged in social activities to connect with community members due to a fear of isolation. + +Whenever there is a gathering, I try to attend. I think I cannot endure an isolated life without community members because I feel lonely and uneasy. + +[ID 9] +In particular, the participants perceived that their daily interaction with neighbours, as an extension of their neighbourhood association activities, helped them in times of emergency. + +I am always indebted to the people in the neighborhood. I am far away from my relatives. I know the neighbors rush to me if I encounter trouble. Just the other day, when the electricity went out in my house due to the earthquake, my neighbors came to check on me, asking if I was okay. + +[ID 8] + +## 4.3  |  **Prerequisites For Continuous Participation In** Social Activities + +Three prerequisites of continuous participation in social activities were as follows: "Do not overreach," "Do not infringe on personal territory" and "Do not violate community norms." + +## 5  | **Discussion** + +I thought that eventually, I would have to join a senior citizens' club, too. Since I was old (enough to be a community member), I joined a senior citizen club. + +well-being of older adults (Ingersoll-Dayton et al., 2001), and social activities present opportunities for them to obtain happiness by enriching their daily enjoyment. + +Fourth, the participants saw "building a safety net" as another value of participating in social activities. Participation prevented them from being socially isolated, which they are especially at risk of in rural areas (Slama, 2004) because of the inconvenience of transportation and lack of opportunities for informal interactions (Harvey, 2007). Social activities increase older adults' social networks and security. For instance, participants received support during emergencies from the community members that they had cultivated a relationship with through social activities. Thus, social activities can ensure that rural older adults continue to live in their communities without the risk of isolation. + +The participants had three prerequisites for continued partici- +This study contributes findings on the value of social activities as captured by the lived experiences of older adults, compared to previous research that largely focused on outcomes from social participation and superficial facilitating and inhibiting factors. The value of social activities and the preconditions for continued participation may provide suggestions for nursing practice to stimulate social participation among rural older adults. + +From the narratives of rural older adults who participated in voluntary community salon activities, we derived four values that they placed on social activities. First, "mutual benefit connectedness" was an important factor. Specifically, the opportunity to eat lunch together at the salon or neighbourhood association symbolized reciprocity. This shared mealtime was special in that it symbolized their emotional and social connection with others (Shiraz et al., 2020). For older adults with a sense of belonging to the region and its community members, this is an essential factor in improving their well-being (Harvey, 2007). Social activities may be valuable in forming close relationships among rural older adults and improving their well-being. + +Reciprocity refers to the relationship of mutual support among community members. The participants desired to contribute to their community and its people, and social activities were a valuable opportunity to actualize that desire. For older adults, altruistic contributions are a strategy for maintaining mental health (Shiraz et al., 2020). Moreover, it generates a sense of being needed, which empowers their positive attitudes and pride (Lewis, 2014). + +Contributing through social activities may be vital in maintaining older adults' psychological well-being. + +Second, the participants saw "preventing and coping with ageing" as another value of social activities, through opportunities to prevent physical and mental decline and learn new things. Previous studies have reported that "keep moving" and "keep learning" are strategies older adults use to adapt to declining physical health (Shiraz et al., 2020). Social activities may assist in acclimating to changing health and increasing frailty. For participants, social activities demonstrated the inherent strength they had developed in their lives. The feeling of self-efficacy from a long life is a factor that older adults perceive as successful ageing (Zanjari et al., 2016). Exercising their abilities and enhancing their self-efficacy through social activities may be a valuable way to facilitate their successful ageing experiences. + +Third, the participants also saw "making life brilliant 'now'" as a pation in social activities. First, since the participants were aware of their declining physical function and ill health, they required activities to be within their capabilities, that is, "Do not overreach." Older adults who experience declining physical functions adapt by avoiding overdoing activities, taking breaks, and maintaining their own pace in their daily lives (Nicholson et al., 2013). Hence, they need to exert control over their level of participation in social activities for continued participation. + +Other conditions were "Do not infringe on personal territory" and "Do not violate community norms," which ensured that the participants maintained harmony in the unique human relationships that arise from rural culture. Harmonious relationships with people who form a social network, such as friends, are important for older people to maintain psychological health (Shiraz et al., 2020). However, in rural communities, there are specific difficulties in harmonizing relationships. This includes the lack of privacy and exclusion of those who do not conform to the rural identity (Harvey, 2007). Thus, participants likely would adopt strategies to counter the negative aspects of relationships and culture that are unique to rural areas while continuing to live in their rural communities. Therefore, activities must be implemented without interfering with these strategies to achieve continued participation in social activities. + +This study's prerequisites for continuous participation in social activities were internal factors for the participants. Combining the results of this study with those of previous studies (e.g., Levasseur et al., 2015) on external environmental factors of social participation is expected to strengthen strategies for promoting social participation among rural older adults. + +This study emphasizes the importance of nursing practice, with the promotion of social participation as a focal point, for improving the well-being of rural older adults in a super-ageing society. Furthermore, this study provides insights into the direction of nursing practices needed to promote social participation among rural older adults. + +value of social activities, as they added novel experiences to their daily lives. These activities became necessities for living, and the novel experiences, including excursions, enhanced the participants' regular lives. Enjoyment in life is an essential factor in defining the To promote rural older adults' participation in social activities, it is vital to produce resources for social activities that provide the four values identified in this study. Community nurses and public +[ID 6] + +## 3282 |    Et Al. + +health nurses should promote social activities that contribute to building reciprocity, which would serve as a foundation for rural older adults to have richer ageing experiences and daily lives. This role may become even more important in an ageing society. In addition, the psychological well-being of older adults can be improved by creating a place in the community for social activities that embody the spirit of reciprocity. This would also lead to a strategy to promote health promotion for older adults living in rural communities. + +Furthermore, it is crucial to consider these social activities and their values and prerequisites in the context of the COVID-19 pandemic. Since 2020, social distancing and other preventive measures have forced older adults to reduce their face-to-face social activities and thus experience isolation (Chen, 2020; Tyrrell & Williams, 2020). Nurses must understand that these circumstances reduce older adults' opportunities to find value in social activities. A potential solution would be to use information and communication technology (ICT) to create non-face-to-face connections, which have gained prominence during the pandemic (Banskota et al., 2020; Smith et al., 2020). In the community development context, community and public health nurses must create opportunities for social activities using ICT that provide the value of social activities indicated by this study. + +## 5.1  |  **Strengths And Limitations Of The Work** + +Although this study provided new insights, it exhibited some limitations. First, the results are likely influenced by socio-cultural influences in rural Japan. The transferability of this study will be limited to older adults in countries and regions with collectivist cultures like Japan. Second, the analysis results are limited to saturation among participants within a single rural community salon. Although the continuing comparative analysis confirmed that no new concepts were derived from the data obtained from the study participants, this was limited to the single salon that participated in this study. Third, because three interviewers conducted the data collection for this paper, we cannot eliminate the possibility of differences in the quality and content of the data across interviews. Although the interview guide may have helped to ensure the homogeneity of the interviews, differences in the interviewers' skills and relationships with the study participants may have affected the data. + +activities can be achieved by investigating the experiences of older adults who are reluctant to participate—and non-participants—in social activities. + +## 6  |  **Conclusion** + +This study explored the value of social activities for older adults who participate in voluntary community salon activities in rural communities. Social activities provide rural older adults with reciprocity ties, coping with ageing, enhanced daily lives, and a social safety net. Continued participation in rural social activities requires adherence to cultural norms and moderation of relationship distances. Community and public health nurses are expected to promote social activities that incorporate these values and adhere to these prerequisites. This ensures a healthy, secure, and rich ageing experience for older adults. This strengthens the promotion of psychological wellbeing and health promotion among rural older adults. + +## Author Contributions + +All authors listed meet the authorship criteria according to the latest guidelines of the International Committee of Medical Journal Editors and agree with the manuscript. **Conceptualization**: Ryuta Onishi, Risa Takashima, Kazuko Saeki, Michiyo Hirano; Data curation: Ryuta Onishi; **Formal analysis**: Ryuta Onishi; Funding acquisition: Michiyo Hirano; **Investigation**: Risa Takashima, Ryuta Onishi, Michiyo Hirano; **Methodology**: Risa Takashima, Ryuta Onishi, Michiyo Hirano; **Project administration**: Michiyo Hirano; **Resources**: Ryuta Onishi; **Supervision**: Kazuko Saeki, Michiyo Hirano, Risa Takashima; **Validation**: Ryuta Onishi, Risa Takashima, Kazuko Saeki, Michiyo Hirano; **Writing - original draft**: Ryuta Onishi; Writing - review & editing: Ryuta Onishi, Risa Takashima, Kazuko Saeki, Michiyo Hirano. + +## Acknowledgements + +We offer special thanks to the participants who cooperated with this study. This work was supported by JSPS KAKENHI (grant number JP18H03103). In addition, we received support from Nihon Unisys, Ltd. for the research expenses. + +## Conflict Of Interest + +This study received unrestricted research grants from Nihon Unisys, Ltd. A researcher from Nihon Unisys, Ltd. played a role in data collection. + +## 5.2  |  **Recommendations And For Further Research** Data Availability Statement Research Data Are Not Shared. + +An important area for future research is to explore the value of social activities based on the experiences of a broader range of older adults. Although rich narratives were obtained from older adults who regularly participate in senior citizen salons, the value of social activities identified by this study was solely from an insider's perspective. A more comprehensive clarification of the value of social + +## Statement + +There has long been a wealth of previous research on social participation of the **older adults**, the topic of this study. 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Rural culture is a diversity issue. *Minnesota Psychologist*, +53, 9–13. + +Smith, M. L., Steinman, L. E., & Casey, E. A. (2020). Combatting social isolation among older adults in a time of physical distancing: The COVID-19 social connectivity paradox. *Frontiers in Public Health*, 8, 403. https://doi.org/10.3389/fpubh.2020.00403 Steinbach, U. (1992). Social networks, institutionalization, and mortality among elderly people in the United States. *Journal of Gerontology*, 47, S183–S190. https://doi.org/10.1093/geronj/47.4.s183 Strasser, R. (2003). Rural health around the world: Challenges and solutions. *Family Practice*, 20, 457–463. https://doi.org/10.1093/fampr a/cmg422 Sun, J., & Lyu, S. (2020). Social participation and urban-rural disparity in mental health among older adults in China. *Journal of* Affective Disorders, 274, 399–404. https://doi.org/10.1016/j. jad.2020.05.091 Takashima, R., Onishi, R., Saeki, K., & Hirano, M. (2020). The values and meanings of social activities for older urban men after retirement. *PLoS One*, 15(11), e0242859. https://doi.org/10.1371/journ al.pone.0242859 Tomioka, K., Kurumatani, N., & Hosoi, H. (2017). Positive and negative influences of social participation on physical and mental health among community-dwelling elderly aged 65–70 years: A cross-sectional study in Japan. *BMC Geriatrics*, 17, 111. https://doi. + +org/10.1186/s12877-017-0502-8 Tong, A., Sainsbury, P., & Craig, J. (2007). Consolidated criteria for reporting qualitative research (COREQ): A 32-item checklist for interviews and focus groups. *International Journal for Quality in Health* Care, 19(6), 349–357. https://doi.org/10.1093/intqhc/mzm042 Tyrrell, C. J., & Williams, K. N. (2020). The paradox of social distanc-ing: Implications for older adults in the context of COVID-19. + +Psychological Trauma: Theory, Research, Practice and Policy, 12, S214–S216. https://doi.org/10.1037/tra0000845 United Nations. (2022). World population prospects 2022 summary of results. https://www.un.org/development/desa/pd/sites/www. un.org.development.desa.pd/files/wpp2022_summary_of_resul ts.pdf Zanjari, N., Sharifian Sani, M., Hosseini Chavoshi, M., Rafiey, H., & +Mohammadi Shahboulaghi, F. (2016). Perceptions of successful ageing among Iranian elders: Insights from a qualitative study. *The* International Journal of Aging & Human Development, 83, 381–401. https://doi.org/10.1177/0091415016657559 Zhao, L., & Wu, L. (2022). The association between social participation and loneliness of the Chinese older adults over time—The mediating effect of social support. International Journal of Environmental Research and Public Health, 19(2), 815. https://doi.org/10.3390/ ijerph19020815 + +## Supporting Information + +Additional supporting information can be found online in the Supporting Information section at the end of this article. + +How to cite this article: Onishi, R., Takashima, R., Saeki, K., & Hirano, M. (2023). 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10, + "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/PMC10243522/PMC10243522.md b/medical/md_images/PMC10243522/PMC10243522.md new file mode 100644 index 0000000000000000000000000000000000000000..0262f712faa82de1b198c0fa79013abdc8853713 --- /dev/null +++ b/medical/md_images/PMC10243522/PMC10243522.md @@ -0,0 +1,123 @@ +# Case Report + +1 Universidade de São Paulo, Faculdade de Medicina, Departamento de Clínica Médica, São Paulo, São Paulo, Brazil 2 Universidade de São Paulo, Faculdade de Medicina, Departamento de Patologia, São Paulo, São Paulo, Brazil 3 Universidade de São Paulo, Faculdade de Medicina, Divisão de Moléstias Infecciosas e Transmissíveis, São Paulo, São Paulo, Brazil Correspondence to: Amaro Nunes DuarteNeto Universidade de São Paulo, Faculdade de Medicina, Departamento de Patologia, Av. Dr. Arnaldo 455, Sala 1161, Cerqueira César, CEP 01246903, São Paulo, SP, Brazil Tel: +55 11 30617322 E-mail: amaro.ndneto@hc.fm.usp.br Received: 16 December 2022 Accepted: 20 April 2023 What else in times of COVID-19? The role of minimally invasive autopsy for the differential diagnosis of acute respiratory failure in a case of kala-azar João Carlos Geber-Júnior 1**, Renata Aparecida de Almeida Monteiro** 2, João Wilson Pedro da Rocha 1, Edson Luiz Társia Duarte 3, Elizabete Nicodemo3, Olavo Munhoz3, Edison Ferreira de Paiva1, Thais Mauad2**, Luiz** Fernando Ferraz da Silva 2, Paulo Hilario Nascimento Saldiva 2**, Marisa** Dolhnikoff 2**, Amaro Nunes Duarte-Neto** 2 + +## Abstract + +Visceral leishmaniasis (VL) is a chronic vector-borne zoonotic disease caused by trypanosomatids, considered endemic in 98 countries, mainly associated with poverty. About 50,000–90,000 cases of VL occur annually worldwide, and Brazil has the second largest number of cases in the world. The clinical picture of VL is fever, hepatosplenomegaly, and pancytopenia, progressing to death in 90% of cases due to secondary infections and multiorgan failure, if left untreated. We describe the case of a 25-year-old female who lived in the metropolitan area of Sao Paulo, who had recently taken touristic trips to several rural areas in Southeastern Brazil and was diagnosed post-mortem. During the hospitalization in a hospital reference for the treatment of COVID-19, the patient developed acute respiratory failure, with chest radiographic changes, and died due to refractory shock. The ultrasoundguided minimally invasive autopsy diagnosed VL (macrophages containing amastigote forms of Leishmania in the spleen, liver and bone marrow), as well as pneumonia and bloodstream infection by gram-negative bacilli. KEYWORDS: Visceral leishmaniasis. Kala-azar. Pneumonia. Autopsy. Minimally invasive autopsy. + +## Introduction + +Visceral leishmaniasis (VL), also known as kala-azar, is a chronic vector-borne zoonotic disease caused by protozoa from the genus *Leishmania* and is considered a neglected tropical disease (NTD), endemic in 98 countries in Africa, America, Asia, and Europe1. It is estimated that about 50,000–90,000 cases of VL occur annually worldwide. In 2018, 95% of the cases occurred in only ten countries: Brazil, China, Ethiopia, India, Iraq, Kenya, Nepal, Somalia, Sudan and South Sudan2. VL has already been identified in 12 Latin American countries3. + +The etiologic agent of leishmaniasis is *L. donovani* in Asia and Africa, and L. infantum in the Mediterranean Basin, the Middle East, Central Asia, South America and Central America1. The disease mainly affects populations in poverty and can be fatal (90%) if there is a delay in diagnosis and initiation of treatment. + +Since the first description of VL in South America in 1913, it has been considered one of the most important endemic diseases in Brazil4, where it is also considered a reemerging disease, accounting for 97% of the cases reported in the Americas2. In 2014, a total of 3,453 cases of VL were confirmed in the country, with a lethality rate of 6.7% and related medical costs of almost US$ 2 million for the Brazilian public health system - 40% and 22% of this amount allocated for hospitalizations and treatment, respectively5. The clinical hallmark of VL is the classic triad of fever, hepatosplenomegaly and pancytopenia6. However, the clinical course can be variable and even challenging for some experienced physicians, as it depends not only on the parasite burden, but also on the hostpathogen interaction. We report a case of a young woman who traveled around rural areas of Southeastern Brazil and acquired visceral leishmaniasis, which took a long time to be diagnosed. She was hospitalized in the Hospital das Clinicas of the Faculdade de Medicina da Universidade de Sao Paulo (HCFMUSP) during the COVID-19 pandemic presenting febrile hepatosplenomegaly and died due to acute respiratory insufficiency. The ultrasound guidedminimally invasive autopsy (MIA-US) diagnosed kala-azar and suppurative bacterial pneumonia. + +## Case Report + +A 25-year-old female who was referred to a + +![1_image_0.png](1_image_0.png) + +rheumatologic outpatient clinic, complaining of fever for 8 months, initially daily but changing to an irregular pattern along the course, predominantly in the afternoon, and associated with arthralgia in small and large joints, malaise, pallor, increased abdominal volume, and involuntary weight loss (20 kilograms during the period). The patient lived in the metropolitan area of Sao Paulo. Laboratory tests showed pancytopenia associated with increased erythrocyte sedimentation rate and C-reactive protein, polyclonal gammopathy, and a weakly positive rheumatoid factor. The abdominal tomography showed great splenomegaly (reaching > 26 cm in greater diameter) and hepatomegaly. A bone marrow aspirate was performed and did not show any hematological diseases or etiological agents. The initial clinical diagnosis was Still's disease, and she was treated with a 2-month course of prednisone 1 mg/kg/day (tapered to 0.6 mg/kg/day) and methotrexate (MTX) 15 mg/week. + +The patient progressed with significant worsening of symptoms and required hospitalization due to poor general condition, presenting cachexia, persistent fever, abdominal distension, and pancytopenia. She was admitted to HCFMUSP, in a ward reserved for cases without clinical suspicion of COVID-19 ('non-COVID-19 area'). Upon admission, we retrieved a detailed clinical history, showing that she toured to many places, from the north and coastal areas of Sao Paulo State to the south region of Minas Gerais State, all considered endemic areas for leishmaniasis in Southeastern Brazil (Figure 1), from September 2018 to September 2019. A new laboratory work-up showed no helminthic diseases in stool samples, and serologies for systemic mycoses were non-reactive. The initial blood sample taken for Polymerase Chain Reaction of KDNA for *Leishmania* was negative on the third day of hospitalization (DH). On the eighth DH, the patient progressed with dyspnea associated with ground-glass opacity and consolidations on the chest tomography. In this context, SARS-CoV-2, influenza A and influenza B infections were ruled out by molecular tests, and piperacillin-tazobactam was started. Blood cultures were negative, and the patient presented an improvement in respiratory status after the antibiotic treatment. On the thirteenth DH, serologies to detect anti-*Leishmania* antibodies were collected (rapid immunochromatographic test for qualitative detection of anti-recombinant antigen K39 and indirect immunofluorescence), showing positive results. At this time, the patient was taking 10 mg/day of prednisone, without MTX for 45 days. Treatment with liposomal amphotericin 300 mg/day was started, but on the second day of treatment, the patient developed hypoglycemia, torpor, dyspnea, and shock, evolving within a few hours to cardiac arrest, unresponsive to cardiopulmonary resuscitation maneuvers. An autopsy was requested to clarify the cause of the acute respiratory failure, with pulmonary thromboembolism, nosocomial infection or COVID-19 as the main clinical hypotheses. + +The MIA-US was performed after written consent from the next-of-kin, using a portable SonoSite M-Turbo R (Fujifilm, Bothell, WA, USA) ultrasound, with C60x (5–2 MHz Convex) multifrequency broadband transducers and DICOM standard images, to guide the tissue sampling from the liver, spleen, lungs, heart and kidneys, with Tru-Cut semi-automatic coaxial needles (14G; 20 cm in length). Additional sampling of skeletal muscle (*quadriceps femoris*) and skin (left thigh) was performed with a 5-mm punch needle; the brain was sampled through transsphenoidal puncture, and the bone marrow through sternal puncture. This autopsy protocol was approved by the HCFMUSP Ethical Committee (protocol Nº 3951.904) and was previously described in detail7. + +The US showed hepatomegaly, splenomegaly and lungs with irregular and thickened pleura, B lines, and subpleural hyperechogenicity, indicative of pneumonia (Figure 2A). Microscopic analysis showed: hypocellular bone marrow aspirate with hemophagocytosis and several macrophages containing amastigote forms of Leishmania spp. (Figure 2B); spleen with necrotic red pulp and hyperplastic sinusoidal cells containing amastigotes (Figure 2C); liver with hyperplastic Kupffer cells containing amastigotes, hepatic steatosis, and gram-negative bacilli in vessels. The immunohistochemistry labeled amastigotes forms, using as the primary antibody a polyclonal anti- +Leishmania in the bone marrow, liver and spleen samples (Figure 2D). The lungs exhibited pulmonary edema and foci of suppurative pneumonia (Figure 2E), with numerous gram-negative bacilli in the alveoli and vessels (Figure 2F). Other findings included: acute tubular necrosis, hyperplasia of mesangial cells, and reactive microglia. + +## Discussion + +This case shows two important aspects: first, that the delayed diagnosis of VL affected the patient's outcome, and second, that MIA-US is useful for clarifying the immediate and underlying cause of death in patients with febrile splenomegaly and pancytopenia, when the conventional autopsy is not available. + +Despite the old paradigm that leishmaniasis is strongly associated with rural areas and poverty, the transmission of the disease has become increasingly frequent at the interfaces between urban and rural areas in Brazil, throughout the 20th century. The case exemplifies a real problem in the practice of travel and tropical medicine in Brazil: the delay in the diagnosis of certain endemic and neglected diseases in individuals living in large urban centers, not considering them as susceptible to those diseases, even if the past history points to visiting transmission areas. The false premise that VL affects only those living in rural and poor areas can underestimate the value of displacement data in the clinical history, leading to a low clinical suspicion and diagnostic and therapeutic delay, resulting in unfavorable outcomes8. This issue was recently addressed, showing that a prolonged time between the onset of symptoms and the definitive diagnosis of VL occurs in rural and peri-urban areas, among adult and elderly patients, associated with several visits to primary care services, and jeopardizes the definitive diagnosis, mainly in referral hospitals9. This diagnostic delay explains why the VL mortality rate has not declined in recent years, although modern diagnostic and treatment techniques are available on the Brazilian public system. In this specific case, additional confounding factors delayed the final definitive diagnosis, which certainly influenced the negative outcome of our case: the negativity of the first laboratory tests for leishmaniasis, the initial misdiagnosis of Still's disease and the immunosuppressive therapy. + +The clinical presentation of VL results from factors such as virulence, genetic background, and immune status of the host. The triad fever–hepatosplenomegaly– pancytopenia is the classic presentation of VL, however it is only present in the minority of cases6. For instance, the asymptomatic infection can be common in endemic areas, reaching up to 63% for *L. donovani*10 and 34% for L. infantum11. In individuals with clinical suspicion and + +![3_image_0.png](3_image_0.png) + +epidemiological criteria, tissue collection by aspiration or biopsy is recommended for histopathological study, cultures, and molecular tests to demonstrate the presence of the parasite12. Although splenic aspirate is the most sensitive method, lymph node, liver and marrow aspiration samples can be performed accordingly, although there is wide variability in sensitivity13,14. The first choice is usually the marrow or splenic aspirate12. The decision will depend on the patient's clinical conditions as potential complications can be lethal15,16. In our case, two marrow aspirates were negative. Accordingly, the bone marrow aspirate sensitivity for diagnosing VL is correlated with the duration of the microscopic exam by the pathologist, the sensitivity being 40.2%, 65.5%, 89.7%, 92%, and 95.4% at 1, 5, 20, 30, and 60 min, respectively16. + +As VL causes a breakdown in the reticuloendothelial system, the disease may mimic autoimmune diseases, leading to a missed diagnosis. Moreover, it has been shown that in immunosuppressive status, especially related to HIV infection, the accuracy of the ELISA and blood sample PCR for the diagnosis of VL can be decreased17. As seen in the case described here (sepsis by gram-negative bacilli), infections (mainly pneumonia) are the main causes of death among patients with VL (41%), followed by bleeding (38%) and respiratory failure (5%)6. Pancytopenia, hemophagocytosis and malnutrition are involved in the predisposition to infections in these cases. + +Regarding the autopsy procedure, during the current epidemic of SARS-CoV-2 infection in Brazil, a conventional autopsy (CA) was not recommended due to the lack of autopsy rooms with biosafety level III in the country. To overcome this problem, we employed a safe alternative for professionals - the MIA-US approach, in order to diagnose the *causa mortis* in suspected fatal cases to minimize excesses of mortality falsely attributed to COVID-19 and to investigate the pathology of this novel disease, in various organs12. Our group had previously used this autopsy methodology during the sylvatic yellow fever (YF) epidemic that affected the peri-urban region of Sao Paulo city in 2018. We obtained 100% of agreement between MIA-US and the gold-standard CA for determining the underlying cause of death (YF and other diseases) and the immediate cause of death in 20 cases18. During the COVID-19 pandemic, an acute respiratory infectious disease with systemic repercussion, we used the MIA-US method to diagnose the *causa mortis* and to study the pathogenesis of this novel disease7. Given those successful results, it was possible to infer that MIA-US would have good accuracy for *post-mortem* diagnosis in future epidemics by other respiratory agents (such as measles, influenza, hantavirus, and others) or hemorrhagic fever. + +VL is one of the main etiologies of another relevant clinical-pathological syndrome in tropical regions: febrile splenomegaly, associated or not with hepatomegaly and anemia, which also includes schistosomiasis, malaria, brucellosis, typhoid, and leukosis as differential diagnoses. In the case of our study, MIA-US was able to identify both the underlying (VL) and the immediate cause of death (gram-negative bacilli pneumonia and sepsis) of the patient. + +MIA can replace CA in low-resource settings, obtaining satisfactory diagnostic samples for determining the cause of death. Nevertheless, in a comparative study between MIA and CA, both methods had an overall agreement of 75.9%, with higher concordance for neoplasia (81.3%) and infectious diseases (78.8%), and lesser for other diseases (56.2%)19. Finally, MIA presents a better performance when it is associated with ultrasound to guide the tissue sampling, rather than collection using only anatomical reference points20. + +## Conclusion + +To conclude, the delay in the diagnosis and treatment of VL is a serious problem in Brazil. A detailed medical history, including a history of travel to areas of VL transmission, can help strengthen clinical suspicion and reduce the mortality rate from this disease. The MIA-US can allow the *postmortem* diagnosis of diseases that progress with febrile splenomegaly and respiratory failure, such as VL, in scenarios with limited resources to perform a conventional autopsy. + +## Acknowledgments + +The authors acknowledge all healthcare providers involved in the care of the patients with coronavirus disease 2019 (COVID-19); the hospitals and the Sao Paulo Autopsy Service staff involved in the Coronavirus Crisis Task Force during the epidemic season; and the Nucleo de Vigilancia Epidemiologica for providing the hospital data during the epidemic period. The authors also acknowledge and are deeply thankful to all relatives and legal representatives who consented to the post-mortem examinations of their beloved relatives who died during the COVID-19 pandemic. + +The authors are thankful to Prof. Francisco ChiaravallotiNeto for assistance in the geopositioning chart of Figure 1. + +## Authors' Contributions + +JCGJ: provided medical care, conceptualization, methodology, formal analysis, and writing (review and editing); RAMA: minimally invasive autopsy analysis, data curation, formal analysis, investigation, figures, and writing (review and editing); JWPR: provided medical care and writing (review and editing); ELTD: provided medical care, review and editing; EN: provided medical care, review and editing; OM: provided medical care, review and editing; EFP: provided medical care, review and editing; TM: visualization, data curation and writing (review and editing); LFFS: supervision, project administration, and writing (review and editing); PHNS: supervision, project administration, autopsy analysis, and writing (review and editing); MD: supervision, project administration, autopsy analysis, and writing (review and editing); ANDN: conceptualization, methodology, autopsy analysis, figures, and writing (review and editing). + +## Conflict Of Interests + +The authors declare that they have no competing financial interests or personal relationships that influence the work reported in this article. + +## Funding + +Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, grant Nº 2020/10281-0, recipient PHNS; Bill and Melinda Gates Foundation, grant Nº INV002396, recipient PHNS; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, grant Nº 401825/2020-5, recipient PHNS; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, grant Nº 316485/2021-7, recipient MD. + +## References + +1. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet. + +2018;392:951-70. + +2. World Health Organization. Leishmaniasis. [cited 2023 Apr 24]. + +Available from: https://www.who.int/en/news-room/factsheets/detail/leishmaniasis 3. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. + +Departamento de Vigilância Epidemiológica. Manual de vigilância e controle da leishmaniose visceral. Brasília: Ministério da Saúde; 2014. [cited 2023 Apr 24]. Available from: https://bvsms.saude.gov.br/bvs/publicacoes/manual_ vigilancia_controle_leishmaniose_visceral_1edicao.pdf 4. Penna HA. Leishmaniose visceral no Brasil. Bras Med. + +1934;48:949-50. + +5. Carvalho IP, Peixoto HM, Romero GA, Oliveira MR. Cost of visceral leishmaniasis care in Brazil. Trop Med Int Health. 2017;22:1579-89. + +6. Costa CH, Werneck GL, Costa DL, Holanda TA, Aguiar GB, +Carvalho AS, et al. Is severe visceral leishmaniasis a systemic inflammatory response syndrome? A case control study. Rev Soc Bras Med Trop. 2010;43:386-92. + +7. Monteiro RA, Duarte-Neto AN, Silva LF, Oliveira EP, Theodoro Filho J, Santos GA, et al. Ultrasound-guided minimally invasive autopsies: a protocol for the study of pulmonary and systemic involvement of COVID-19. Clinics. 2020;75:e1972. + +8. Conti RV, Lane VF, Montebello L, Pinto Junior VL. Visceral leishmaniasis epidemiologic evolution in timeframes, based on demographic changes and scientific achievements in Brazil. J Vector Borne Dis. 2016;53:99-104. + +9. Luz JG, Carvalho AG, Naves DB, Dias JV, Fontes CJ. Where, when, and how the diagnosis of human visceral leishmaniasis is defined: answers from the Brazilian control program. Mem Inst Oswaldo Cruz. 2019;28;e190253. + +10. Srivastava P, Gidwani K, Picado A, Van der Auwera G, Tiwary P, Ostyn B, et al. Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India. Trop Med Int Health. 2013;18:548-54. + +11. Marques LH, Rocha IC, Reis IA, Cunha GM, Oliveira E, +Pfeilsticker TR, et al. Leishmania infantum : illness, transmission profile and risk factors for asymptomatic infection in an endemic metropolis in Brazil. Parasitology. 2017;144:546-56. + +12. Aronson N, Herwaldt BL, Libman M, Pearson R, Lopez-Velez R, Weina P, et al. Diagnosis and treatment of leishmaniasis: clinical practice guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH). Am J Trop Med Hyg. 2017;96:24-45. + +13. Ho EA, Soong TH, Li Y. Comparative merits of sternum, spleen and liver punctures in the study of human visceral leishmaniasis. Trans R Soc Trop Med Hyg. 1948;41:629-36. + +14. Zijlstra EE, Ali MS, El-Hassan AM, El-Toum IA, Satti M, Ghalib HW, et al. Kala-azar: a comparative study of parasitological methods and the direct agglutination test in diagnosis. Trans R Soc Trop Med Hyg. 1992;86:505-7. + +15. Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. + +Clin Diagn Lab Immunol. 2002;9:951-8. + +16. Silva MR, Stewart JM, Costa CH. Sensitivity of bone marrow aspirates in the diagnosis of visceral leishmaniasis. Am J Trop Med Hyg. 2005;72:811-4. + +17. van Griensven J, Carrillo E, López-Vélez R, Lynen L, Moreno J. Leishmaniasis in immunosuppressed individuals. Clin Microbiol Infect. 2014;20:286-99. + +18. Duarte-Neto AN, Monteiro RA, Johnsson J, Cunha MP, Pour SZ, Saraiva AC, et al. Ultrasound-guided minimally invasive autopsy as a tool for rapid post-mortem diagnosis in the 2018 Sao Paulo yellow fever epidemic: correlation with conventional autopsy. PLoS Negl Trop Dis. 2019;13:e0007625. + +19. Castillo P, Martínez MJ, Ussene E, Jordao D, Lovane L, Ismail MR, et al. Validity of a minimally invasive autopsy for cause of death determination in adults in Mozambique: an observational study. PLoS Med. 2016;13:e1002171. + +20. Cox JA, Lukande RL, Kalungi S, Van de Vijver K, Van Marck E, +Nelson AM, et al. Practice of percutaneous needle autopsy; a descriptive study reporting experiences from Uganda. 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b/medical/md_images/PMC4387352/PMC4387352.md new file mode 100644 index 0000000000000000000000000000000000000000..0261c26f638767f011abca754731b9cb5a8b0955 --- /dev/null +++ b/medical/md_images/PMC4387352/PMC4387352.md @@ -0,0 +1,108 @@ + +## Giant Cells Glioblastoma: Case Report And Pathological Analysis From This Uncommon Subtype Of Glioma Telmo A.B. Belsuzarri,1 + +João F.M. Araujo,1 Aguinaldo P. Catanoce,1 Maick W.F. Neves,1 Rodrigo A.S. Sola,1 Juliano N. Navarro,1 Leandro G. Brito,1 Nilton R. Silva Junior,1 Luis Otavio C. Pontelli,1 Luiz Gustavo A. Mattos,1 Tiago F. Gonçales,1 **Wolnei M. Zeviani,1** Renata M.B. Marques2 1**Department of Neurosurgery and** 2**Department of Pathology, Maternity** Hospital of Celso Pierro, Pontifical Catholic University of Campinas, São Paulo, Brazil + +## Abstract + +Glioblastoma multiforme (GBM) is the most common glial tumor of the brain system; nevertheless, the giant cell (GC) subtype is uncommon. Recent reviews report for an incidence of 1% in adults and 3% in children. The GCs usually have a better prognosis than GBM and also an increasing long-term survival rate. It is known that the diagnosis of this tumor is due to its histological findings and patterns, such as the unusual increased number of giant cells. Unfortunately, due to its rarity, the immunohistochemical and cytogenetical analysis of this tumor is not well known. Some authors also suggest that there are few subtypes of GCs and their patterns of aggressiveness could be due to cytogenetical markers. It is recognized that maximum safe resection treatment and adjuvant radiotherapy can improve survival rate (5-13 months) similar to GBM patients. + +## Introduction + +Glioblastoma multiforme (GBM) is a highgrade glioma of the central nervous system associated with high morbity and/or mortality. According to the WHO classification of brain tumors, there are two subtypes of GBM: the giant cells and the gliosarcomas. Giant cell glioblastoma (GC) is an uncommon subtype of GBMs accounting for 1% of them. Due to the rarity of the cases, epidemiology, natural history and follow up, it is not well defined. It has been hypothesized that GC has a better outcome and higher survival rates than GBM, due to several reasons, such as the age of diagnosis, histopathological and genetic differences etc.. The standard treatment for GBM is the Stupp protocol with maximum safe resection, adjuvant radiotherapy and temozolamide. Nevertheless, due to its rarity, the clinical and prognostic significance of the treatment with Stupp protocol and the extent of resection (EOR) is not well known. However, there is evidence to suggest that EOR and adjuvant therapy serve as prognosticators for long-term survival. + +A literature review was made by searching in several databases (Pubmed, Bireme and Cochrane) the following key words: glioblastoma multiforme, giant cell glioblastoma, genetic, immunohistochemical, long-term survivals The objectives of this study are i) to report a case of giant cell glioblastoma with clinical, radiological and histology analysis, ii) review the immunohistochemical and genetic findings in GC to be compared to GBM, iii) to discuss the possible reasons of different outcomes. + +## Case Report + +A 58-year-old white male, with a history of complete right hemiparesis associated with Broca's aphasia, was referred to our hospital, without family suspicion of stroke. At initial examination, patient had a Glasgow:12, with motor deficit and aphasia. Computed tomography (CT) scan was performed: it showed a 3cm-diameter mass lesion with edema, enhancement with contrast and midline deviation of 5 mm. A magnetic resonance imaging +(MRI) was also performed, showing an extended lesion with infiltration of the pre/pos central gyri, the posterior limb of the internal capsule with a heterogenic surface, small cysts and apparent necrosis inside (Figure 1A-C). After examination patient was diagnosed with GBM and surgery was performed. + +After surgery, the patient had a worsening of the motor deficit, remaining with the motor aphasia and therefore underwent another CT scan (Figure 1D). The pathology conclusion was a GCG (Figure 2). He was then referred for adjuvant radio/chemotherapy, with 10 months follow up. + +## Discussion + +Glioblastoma multiforme is the most common primary brain tumor, but the GC subtype is uncommon and also has a different prognosis. In a recent review was found that only 1% +Correspondence: Telmo Augusto Barba Belsuzarri, Av. John Boyd Dunlop s/n. Campinas, São Paulo 13060-803, Brazil. Fax: +55.193.343.8600. E-mail: telmobelsuzarri@hotmail.com Key words: glioblastoma multiforme, giant cell glioblastoma, genetic, immunohistochemical, long-term survival. Contributions: TABB, design of the work, data acquisition and analysis; JFMA and APC, critical revision; MWF, RASS, JNN, LGB, NRSJ, LOCP, LGAM, TFG, WMZ, RMB, data collection. + +Conflict of interest: the authors declare no potential conflict of interest. + +Received for publication: 12 September 2014. Revision received: 17 December 2014. + +Accepted for publication: 18 December 2014. + +This work is licensed under a Creative Commons Attribution NonCommercial 3.0 License (CC BYNC 3.0). + +©Copyright T.A.B. Belsuzarri et al., 2015 Licensee PAGEPress, Italy Rare Tumors 2015; 7:5634 doi:10.4081/rt.2015.5634 of 16,430 cases were GCs,1 which is also proved by other authors.2,3 Some other studies account for 5% of GBM,4,5 In the literature there are less than 100 cases reported.6 The mean age is 51 years, 11 years less than GBM, and about 27.5% of GCs occur in patients with less than 40 years. The most common location are the frontal and temporal lobe, and rarely the multifocal.1 At pediatric age group GBM is about 5-10% of all intracranial neoplasms and GC's are 3% of GBM's at this age group.7 Even though both tumors have poor outcomes and overall survival rate, an increased median survival is observed in GC, with 11 months compared to 8 months in GBM. Also, long term survival (5 years) is more frequent in GC (12,3%) than GBM (3,4%).1,4 Taemin and Rutkowski reported a recent series of 20 patients,8 with a median age of 47.9 years and a median survival of 15.4 months, congruent to other reports.1 As firstly described by Meyer,9 in histological analysis GBM has a predominance of multinucleated giant cells with abundant cytoplasm, which can reach up to 500 nm with abundant reticulin fibers, and it was used to be called monstrocellular brain tumor.7 Its pathology diagnosis is made by morphology and histological architecture. However, recent studies have also shown that among GC, there are immunohistochemical differences that could lead to GCs subtypes, and that could maybe explain the increased survival rate in some cases.10,11 At immunohistochemical analysis, both giant cells/non-giant cells are positive for glial fibrillary acidic protein (GFAP), S100 protein and vimentin; in giant cells nuclei are positive to proliferation of cell nuclear antigen (PCNA) and Ki-67;12 so there is no specific immunohistochemical for this kind of tumor. At cytogenetic and molecular genetic analyses, microsatellite instability is more frequent in GC than GBM (30% vs 7.8%), +TP53 mutation is observed in 83.3%, but epidermal growth factor receptor is uncommon +(8.3%). Chromosome 10 deletion is found in all non-giant cells of the GC; at GBM it is found in 45%. GC has more p53 mutations than GBM, +but p16 deletion, MD2M and CD4K amplifications are rare.10,11,13,14 Long-term survival (LTS), considered as more than 3 years survival, has been reported in about 3-5% of all GBM. Kraus *et al*. found that about 25% of all LTS were actually mistaken with anaplastic oligodendroglioma. The LTS +is associated with the disease free period, suggesting that are different grown factors and courses.15-17 The giant cell variety is associated with the presence of giant cell subtype in LTS patients,2 in almost 30% of the cases.3,14,18 Some authors suggest that extremely long-term survival case series could have been misclassified with the pleomorphic xanthoastrocytoma, which is most common in younger patients and has better outcomes; but in a recent review, it was proved that excluding patients with less that 40 years, the survival median rate was unchanged.1,4 There are several hypothesis for the increased survival rate and number of LTS patients. Some authors suggest that the increased survival rate is due to younger presenting age in patients with GC. Others suggest that the margins and circumscription of the GC lesion are more visible and this could lead to better resection.1 However, Taemin and Rutkowski found a overall mortality of 75% and ![1_image_0.png](1_image_0.png) + +On the other hand, it is believed that the immunohistochemical and genetic differences, as discussed before, could lead to a better prognosis. + +The surgical management is still uncertain due to the rarity of this cases, but is known that maximum safe resection treatment and adjuvant radiotherapy can improve survival rate from 5 to 13 months, similarly to GBM patients.1 + +## Conclusions + +Giant cell glioblastoma is a rare subtype of classic GBM with different pathology, genetic and immunohistochemical markers and a better prognosis, but it is still under study. Due to its rarity, case series and reports are necessary to understand the tumor behavior. Recent retrospective reviews have shown an increased survival rate of GCG and maximum safe surgical treatment associated with adjuvant radiotherapy seems to be the best choice. + +## References + +1. Kozak KR, Moody JS. Giant cell glioblastoma: a glioblastoma subtype with distinct epidemiology and superior prognosis. Neuro Oncol 2009;11:833-41. + +2. Burger PC, Vollmer RT. Histologic factors of prognostic significance in the glioblastoma multiforme. Cancer 1980;46:1179-86. + +3. Shinojima N, Kochi M, Hamada JI, et al. + +The influence of sex and the presence of giant cells on postoperative long-term survival in adult patients with supratentorial glioblastoma multiforme. J Neurosurg 2004;101:219-26. + +4. Folgueral JMV, Mascarenhas L, Costa JA, +et al. Giant cell glioblastoma: review of the literature and illustrated case. Neurocirugía (Astur).2008 19:343-9. + +5. Palma L, Celli P, Maleci A, et al. Malignant monstrocellular brain tumors: a study of 42 surgically treated cases. Acta Neurochir (Wien) 1989;97:17-25. + +6. Ohgaki H, Peraud A, Nakazato Y, et al, ed. + +World Health Organization classification of tumours: pathology and genetics of tumours of the nervous system. Lyon: IARC Press; 2000. pp. 40-41. + +7. Borkar SA, Lakshmiprasad G, Subbarao KC, et al. Giant cell glioblastoma in pediatric age group: report of two cases. J +Pediatr Neurosci 2013;8:38-40. + +8. Oh T, Rutkowski MJ, Safaee M, et al. + +Survival outcomes of giant cell glioblastoma: institutional experience in the management of 20 patients. J Clin Neurosci 2014;21:2129-34. + +9. Meyer O. Ein besonder typus von riesenzellengliom. Frankf Z Pathol 1913;14:185203. + +10. Temme A, Geiger KD, Wiedemuth R, et al. + +Giant cell glioblastoma is associated with altered aurora b expression and concomitant p53 mutation. J Neuropathol Exp + +## Neurol 2010;69:632-42. + +11. Martinez R, Roggendorf W, Baretton G, et al. Cytogenetic and molecular genetic analyses of giant cell glioblastoma multiforme reveal distinct profiles in giant cell and non-giant cell subpopulations. Cancer Genet Cytogenet 2007;175:26-34. + +12. Katoh M, Aida T, Sugimoto S, et al. + +Immunohistochemical analysis of giant cell glioblastoma. Pathol Int 1995;45:27582. + +13. Kraus JA, Wenghoefer M, Schmidt MC, et al. Long-term survival of glioblastoma multiforme: importance of histopathological reevaluation. J Neurol 2000;247:455-60. + +14. Naydenov E, Bussarsky V, Nachev S, et al. + +Long-term survival of a patient with giant cell glioblastoma: case report and review of the literature. Case Rep Oncol 2009;2;10310. + +15. Filippini G, Falcone C, Boiardi A, et al. + +Prognostic factors for survival in 676 consecutive patients with newly diagnosed primary glioblastoma. Neuro Oncol 2008;10:79-87. + +16. Mineo JF, Quintin-Roue I, Lucas B, et al. + +[Glioblastomas: clinical study and search for prognostic factors]. Neurochirurgie 2002;48:500-9. [Article in French]. + +17. Chandler KL, Prados MD, Malec M, Wilson CB. Long-term survival in patients with glioblastoma multiforme. Neurosurgery 1993;32:716-20. + +18. Deb P, Sharma, MC, Mahapatra, AK, et al. + +Glioblastoma multiforme with long term survival. Neurol India 2005 53:329-32. \ No newline at end of file diff --git a/medical/md_images/PMC4389433/1_image_1.png b/medical/md_images/PMC4389433/1_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..7688fbf9fae30b4a0c4602cdfecd09980d962e88 --- /dev/null +++ b/medical/md_images/PMC4389433/1_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e3c45b028c64eaae570a0a40f3e15b3c93c64b732a58db880dc329aaaf1d42c6 +size 34107 diff --git a/medical/md_images/PMC5145293/0_image_1.png b/medical/md_images/PMC5145293/0_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..676982a1af1636e18dd04f3b6902c8b04d3107cd --- /dev/null +++ b/medical/md_images/PMC5145293/0_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:eb58ff6c8867256a45cc98eba17d4a3cfc738ec587f9f53b87d66f59b3e98302 +size 598 diff --git a/medical/md_images/PMC5156477/0_image_0.png b/medical/md_images/PMC5156477/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..885b7c66654af6222d0ce1842cdbd4e3e230fd54 --- /dev/null +++ b/medical/md_images/PMC5156477/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:62212619ebcca9e733b8fde0bbdd6bc5a02daf9a20807520b502d61ef7b0054a +size 8747 diff --git a/medical/md_images/PMC5156477/0_image_2.png b/medical/md_images/PMC5156477/0_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..191c613883eb16866f02077025596a0855090aa5 --- /dev/null +++ b/medical/md_images/PMC5156477/0_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:ed9155719289848e732199c0b53a0f4acba64397c08f22bae9aa7f1c3c98cbad +size 3047 diff --git a/medical/md_images/PMC5275604/PMC5275604.md b/medical/md_images/PMC5275604/PMC5275604.md new file mode 100644 index 0000000000000000000000000000000000000000..f998f85810c22336e50a467968679fb8ed3eef31 --- /dev/null +++ b/medical/md_images/PMC5275604/PMC5275604.md @@ -0,0 +1,18 @@ +Royai, Colleges of Physicians and Surgeons, Edinburgh.- 11 +-The following gentle- . + +men passed their final examination for the double qualification in Medicine and I +Surgery at the sittings held in April and May 1886, and were admitted L.R.C.P. and i S. Ed.:?David Denholm Black, Cockburnspatli ; Frederick Buckham, Hereford ; +John Connell Knock, Rochestown, Co. Cork ; Alfred Owen Davies, Barmouth; Henry Caudwell, Wallingford, Berks ; Sidney Edward Percy Cade, Cork ; Thomas Valentine Devey, Wolsingliam, Darlington ; Jonathan Clarke, Cork ; William Henry Carter, Aberfell ; Devereux Gwynne Huglies, Llandilo; Henry Benson Goulding, Dublin ; James Angus Haggart, Edinburgh ; David Hume, Edinburgh ; +Thomas Joseph Jones, Rotherham; William John Morton, Edinburgh ; Edwin Priestly, Saintfield, Co. Down ; James Williamson Patrick, Derrylane, Belturbet, Co. + +Cavan ; Alexander William Mackenzie, Musselburgh ; James M'Gaw, Castlewellan, Co. Down; Charles William Reilly, Roscrea; Alexander Smith, Blackrock, Co. Cork; Alexander Rajagopal, Edinburgh; Henry Powell, Dublin; John Joseph Tuohey, St Just, Penzance ; William Shaw, Islandmagee, Co. Antrim; Henry Hugh Williams, Holyhead ; William James Van der Vyver, Edinburgh ; Charles Wicks, Newcastleon-Tyne; Robert John Willis, Belfast. + +Royal College of Physicians, Edinburgh.?The following gentlemen passed their final examination at the sittings held on 8th May 1886, and were duly admitted L. R C.P.Ed. :?Arthur Somers Bradford, Chippenham, Wilts ; Henry Wentworth Windsor-Aubrey, Hull; Williatn Duncan, Frome; Robert Henry Currey, Liverpool. + +Royal Colleges of Physicians and Surgeons, Edinburgh, and Faculty of Physicians and Surgeons, Glasgow.?The following gentlemen passed their fin?' +examination for the triple qualification in Medicine and Surgery at the sittings held in April and May 1886, and were admitted L.R.C.P. andS. Ed. and L.F.P. and S. Glas-gow:?Horace Kelly Young, Canterbury; Arnold Edward Thorpe, Northampton; John Wingfield, Edinburgh; William Edward Bennett, Leeds ; George Robert Nelson, Glasgow ; William Colquhoun, Bowling; Edward Armitage, Ben Rhydding* Yorkshire; Geo. Alex. Armstrong, Victoria, Australia; Alfred Duckworth, Accring" ton; Charles Edward Dew, Weston-Super-Mare; James Gordon, Annalong, ^0* Down; Adelbert Hanna, M.D., Edinburgh; Kobert Nelson Horton, London; James Gardner Holmes, Leeds; James Lychlander, Edinburgh; John Lyon, Glasgow > +William Murray Mackay, Eshe, Durham ; Sydney Rumboll, Edinburgh ; Percy J?nn Wilkinson, Manchester ; Alfred Henry Whittell, Leeds. + +Royal College of Surgeons, Edinburgh.?The following gentlemen passed the'* +final examination at the sittings held in April and May 1886, and were admits L.R.C.S.Ed.:?James M'Nish, Walsall; Wm. Henry Clarke, Macclesfield; Jai"g Sinclair Tait, Newfoundland. 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2Centro de Investigacio´ n Biome´dica en Red Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain; and 3Institut d'Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain Received 17 May 2019; first decision 13 June 2019; accepted 1 October 2019; online publish-ahead-of-print 22 October 2019 + +. + +. . . + +. + +. . . . + +. + +. . . + +. + +. . . + +. . . + +. + +. . . + +. + +. . . . . + +. + +. . + +. + +. + +. + +| Background | In recent years, subcutaneous implantable cardioverter-defibrillator (S-ICD) implants have progressively increased and have been shown to be safe and highly successful, affording low reintervention rates regardless of the technique used. | +|--------------|---| +| Case summary We present a case of S-ICD implantation in a patient diagnosed with idiopathic ventricular fibrillation. In the first follow-up consultation the patient showed appropriate detection parameters in the three configurations. However, chest X-ray revealed lead displacement with a tip migration from the manubrium area of the sternum to the xiphoid process Discussion This case highlights the importance of performing at least one chest X-ray during the first weeks after S-ICD implantation, allowing the detection of a problem such as lead displacement, which can lead to undersensing of ventricular arrhythmias or S-ICD oversensing. Keywords Cardiac devices - S-ICD - X-ray - Case report | | + +## Learning Points + +- Lead dislodgement after subcutaneous implantable cardioverter-defibrillator (S-ICD) implantation is a rare complication. + +- Routine device checks may be not sufficient to ensure adequate S-ICD performance. + +- A chest X-ray within the first weeks after implantation is needed to assess correct lead position and complement device revision. + +## Introduction + +Subcutaneous implantable cardioverter-defibrillators (S-ICDs) are alternatives to transvenous implantable cardioverter-defibrillators (ICDs) for preventing sudden cardiac death in patients who do not require bradycardia or antitachycardia pacing.1 These devices were developed to eliminate the primary risks of transvenous ICDs, such as lead dysfunction or device infection, both associated with increased morbi-mortality.2,3 + +## Timeline + +| Day | Hour | Event | +|-------|--------|---------| +| 0 Subcutaneous implantable cardioverter-defibrillator (S-ICD) implant after aborted sudden death and normal cardiovascular studies. 13 10:30 First follow-up consultation and detection of electrode displacement. 16 08:30 Reintervention for S-ICD implant with the three incisions technique. 17 10:00 Device and X-ray control, patient discharge. 45 10:00 Follow-up consultation demonstrating normal functioning of the S-ICD. | | | + +## Case Presentation + +. . + +. + +. + +. . + +. + +. + +. . . + +. + +. + +. + +. + +. + +. + +. . + +. . . + +. + +. + +. . + +. + +. . . . . + +. + +. + +. . + +. . . . . . + +. + +. + +. . + +. . . + +. + +. + +. . + +. + +. + +. . . . + +. + +. + +. . + +. + +. + +. . . + +. + +. + +. + +. + +. + +. + +. + +. + +. + +. . + +. + +. + +. . + +. + +. + +. . . + +. + +. + +. + +. + +. + +. . . + +. . + +. . + +. + +. + +. + +. + +. + +. + +. + +. . + +. + +. . . . + +. . . . . + +. + +. . . + +. + +. + +. The S-ICD implant procedure has been shown to be safe and the implant success rate high, accompanied by low reintervention rates regardless the technique used.4,5 In addition, the design of high-pass filters (SMARTpass filtering) has drastically reduced T-wave oversensing.6 A 29-year-old man with no previous history of cardiovascular disease (weight 76 kg; height 177 cm; body mass index 24) presented with out-of-hospital cardiac arrest. The rhythm initially recorded was ventricular fibrillation. He was admitted to another centre where a complete cardiac study (echocardiogram, cardiac magnetic resonance, ajmaline test, and coronary angiography) was performed. The specific cause of cardiac arrest was not identified. According to European Society of Cardiology (ESC) guidelines, implantable cardioverterdefibrillator (ICD) implantation is recommended for survivors of idiopathic ventricular fibrillation (Class of recommendation I; Level of evidence B)1; therefore, an S-ICD (EMBLEM; Boston Scientific) was implanted using the two-incision technique. The secondary vector (distal-tip-to-can) was the chosen configuration, and the SMARTPass sensing filter was also activated. The patient was discharged and referred to our centre 13 days later for device and clinical follow-up. + +At this 13-day follow-up, no clinical complications were revealed. + +Assessment of the S-ICD electrograms revealed unremarkable results (Figure 1). Neither arrhythmias nor oversensing episodes were identified, and the secondary vector continued as the configuration of choice to obtain adequate signal amplitude. The SMARTPass filter remained activated (Figure 2). + +According to our institutional protocol, a chest X-ray was performed, which surprisingly revealed an important inferior lead displacement (by about 9 cm). This lead dislodgement had not been previously detected during device interrogation, because the distal electrode moved to the proximal electrode position, so the detection of the device remained unchanged (Figure 3). + +The heart did not interpose between the coil and the ICD generator; therefore, the lead was replaced. During reintervention, an SICD lead was found to be stitched without sufficient firmness at the xiphoid level, allowing displacement of the distal electrode 2 cm above the xiphoid appendage. To reduce the risk of displacement of the new lead, the three-incision technique was performed, fixing the lead tip to the underlying fascia and muscle at the manubriosternal junction, 1 cm left of the midline. Further dislodgement was not subsequently observed (Figure 4). + +## Discussion + +This case shows that S-ICD lead displacement can occur without detection by remote device monitoring (Latitude) or routine device revision. This should be kept in mind because important lead displacement can have important repercussions in the treatment of ventricular arrhythmias. Proper functioning of the S-ICD is directly related to the position of its components (device-electrode) in the thorax and their relationships with the heart in the presence of malignant arrhythmias. + +The displacement of an S-ICD lead is a rare complication (0.8– +1.7%), and its primary aetiology is inadequate fixation of the suture olive in the muscular plane. This migration usually manifests itself by producing inappropriate discharges as a result of extracardiac detection signals. Three circumstances necessitate intraoperative S-ICD +lead repositioning: (i) when the defibrillation threshold is not met +(failure of a defibrillation test); (ii) when sensing fails in the three vectors; and (iii) when the myocardium does not interpose between the coil and the generator.7–10 + +![1_image_0.png](1_image_0.png) + +## Informe Sumario Boston, Ocientific + +| ocientific | Versión de Software de Dispositivo: 3.1.539 | +|------------------------------------------------|-----------------------------------------------| +| Modelo del Dispositivo: A219 EMBLEM™ MRI S-ICD | | +| Nombre del paciente: | N° de Serie del Dispositivo: 233063 | +| Fecha del Ultimo Segumento: | 70/02/2018 | +| Fecha de Seguimiento: 10/03/2018 | N° de Modelo del Electrodo: 3501 | +| Fecha del Implante: 09/20/2018 | N° de Serie del Electrodo: 122973 | +| Parámetros de dispositivo programable | | +| Configuración actual del dispositivo | Configuración inicial del dispositivo | +| Tratamiento: ON | Tratamiento: ON | +| Zona de descarga: 240 bpm | Zona de descarga: 250 bpm | +| Zona de Descarga Condicional: 210 bpm | Zona de Descarga Condicional: 200 bpm | +| Estimulación Posterior a la Descarga: ON | Estimulación Posterior a la Descarga: ON | +| SMART Pass: ON | SMART Pass: ON | + +Ajuste de ganancia: 1X Configuración de detección: Secundaria Se imprimió informe: 10/03/2018 1:52 PM +Versión de Software de Programador: 4.04 Ajuste de ganancia: 1X +Configuracion de deteccion: Secundaria Polaridad de la descarga: STD +Desde el último seguimiento Episodios no tratados: 0 Episodios tratados: 0 Cant. de descargas administradas: 1 Resumen del episodio Desde el implante Episodios no tratados: 0 Episodios tratados: 0 Cant. de descargas administradas: 2 Estado de la impedancia del electrodo Estado de la pila Vida útil restante de la pila para ERI: 100% + +![2_image_0.png](2_image_0.png) + +Figure 2 Thirteen days post-implant. Device interrogation does not show alerts; the SMARTPass is activated. + +Figure 3 Posteroanterior and lateral chest X-ray showing lead displacement from its original position (red arrow) to the lower parasternal region +(black arrows). + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +Table 1 Comparison between S-ICD implant + +techniques + +| techniques Advantages | Disadvantages | +|-------------------------|-----------------| +| Two - Shorter procedure incisions time. - Greater comfort; aesthetically superior. - Poor distal lead support. - Distal lead support. - Longer procedure Three incisions time. - The high sternal wound is associated with superficial infections and discomfort. | | + +In this case, the secondary configuration (tip-electrode-to-can) +likely acted as a primary configuration (proximal-electrode-to-can), +given that the distal electrode of the lead was placed where the proximal electrode had previously been. The excellent signal obtained in both positions did not produce inappropriate shocks, and the SMARTPass filter remained activated. However, in ventricular arrhythmia, the shock could have been ineffective, given that the coil was in a low position, thus excluding the cardiac structure between it and the can. + +Since its initial description, the two-incision technique has been shown to provide technical and aesthetic advantages over the threeincision technique (Table 1) +11; however, it has not been shown to reduce the frequency of electrode displacement.2 In our case, the three-incision technique was chosen at reintervention to provide greater distal support to the electrode. + +In the S-ICD System IDE Clinical Investigation, device interrogations and chest X-rays were performed at hospital discharge and at 30, 90, and 180 days after implantation.13 However, in other studies performed in a real-world setting, no information about performing chest X-rays during follow-up was provided.2,4,12,13 Our protocol specifies two X-rays during follow-up (at 4 weeks and again between the 6th and 12th months). + +## Conclusions + +This case highlights the importance of performing at least one chest X-ray during the first weeks after implantation of an S-ICD, allowing detection of a problem such as lead displacement, which can lead to undersensing ventricular arrhythmias or S-ICD oversensing. Realworld, multicentre registries are needed to better explore the causes and best management of S-ICD complications. + +## Lead Author Biography + +Jose Apolo is a cardiologist and EP fellow of the arrhythmia section of Hospital ClinicBarcelona, he has worked in training and research programmes at the A´lvaro Cunqueiro Hospital-Vigo and Clinico San Carlos HospitalMadrid, currently, he is focused on the cardiac devices and atrial fibrillation research. + +## Supplementary Material + +Supplementary material is available at European Heart Journal - Case Reports online. Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Supplementary data. + +## Conflict Of Interest: None Declared. + +. + +. + +. . + +. . . + +. + +. + +. + +. + +. + +. . + +. + +. . + +. + +. + +. . + +. . . + +. + +. . + +. + +. + +. . + +. . . + +. + +. + +. + +. + +. + +. . . . + +. + +. . + +. + +. + +. . . + +. . . + +. + +. + +. + +. + +. + +. . . + +. . . + +. . . . . + +. + +. . + +. + +. + +. + +. + +. Consent: The author/s confirm that written consent for submission and publication of this case report including image(s) and associated text has been obtained from the patient in line with COPE guidance. + +References 1. Priori SG, Blomstrom-Lundqvist C, Mazzanti A, Mazzanti A, Blom N, Borggrefe M, Camm J, Elliott PM, Fitzsimons D, Hatala R, Hindricks G, Kirchhof P, Kjeldsen K, Kuck KH, Hernandez-Madrid A, Nikolaou N, Norekva˚l TM, Spaulding C, Van Veldhuisen DJ; ESC Scientific Document Group. 2015 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death: the task force for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death of the European Society of Cardiology (ESC). Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC). Eur Heart J 2015;36:2793–2867. + +2. Lambiase PD, Barr C, Theuns DA, Knops R, Neuzil P, Johansen JB, Hood M, +Pedersen S, Ka¨a¨b S, Murgatroyd F, Reeve HL, Carter N, Boersma L; EFFORTLESS Investigators. Worldwide experience with a totally subcutaneous implantable defibrillator: early results from the EFFORTLESS S-ICD Registry. Eur Heart J 2014;35:1657–1665. + +3. San Antonio R, Guasch E, Chipa-Ccasani F, Apolo J, Pujol-Lopez M, Fernandez H, Trotta O, Niebla M, Borra`s R, Trucco E, Arbelo E, Roca-Luque I, Brugada J, +Mont L, Tolosana JM. Failure-free survival of the Riata implantable cardioverterdefibrillator lead after a very long-term follow-up. Indian Pacing Electrophysiol J +2019;19:140–144. + +4. Burke MC, Gold MR, Knight BP, Barr CS, Theuns DAMJ, Boersma LVA, Knops RE, Weiss R, Leon AR, Herre JM, Husby M, Stein KM, Lambiase PD. Safety and efficacy of the Totally Subcutaneous Implantable Defibrillator: 2-year results from a pooled analysis of the IDE Study and EFFORTLESS Registry. J Am Coll Cardiol 2015;65:1605–1615. + +5. Brouwer TF, Miller MA, Quast A-FBE, Palaniswamy C, Dukkipati SR, Reddy V, +Wilde AA, Willner JM, Knops RE. Implantation of the subcutaneous implantable cardioverter-defibrillator: an evaluation of 4 implantation techniques. Circ Arrhythm Electrophysiol 2017;10:e004663. + +6. Tachibana M, Nishii N, Banba K, Fujita S, Ikeda E, Okawa K, Morita H, Ito H. + +SMART pass will prevent inappropriate operation of S-ICD. J Arrhythmia 2019; 35:86–91. + +7. Dabiri Abkenari L, Theuns DA, Valk SD, Van Belle Y, de Groot NM, Haitsma D, +Muskens-Heemskerk A, Szili-Torok T, Jordaens L. Clinical experience with a novel subcutaneous implantable defibrillator system in a single center. Clin Res Cardiol 2011;100:737–744. + +8. Aydin A, Hartel F, Schlu¨ter M, Butter C, Ko¨ be J, Seifert M, Gosau N, Hoffmann B, Hoffmann M, Vettorazzi E, Wilke I, Wegscheider K, Reichenspurner H, +Eckardt L, Steven D, Willems S. Shock efficacy of the subcutaneous ICD for prevention of sudden cardiac death: initial multicenter experience. Circ Arrhythm Electrophysiol 2012;5:913–919. + +9. Kobe J, Reinke F, Meyer C, Shin DI, Martens E, Kaab S, et al. Implantation and follow-up of totally subcutaneous versus conventional implantable cardioverter-defibrillators: a multicenter case-control study. Heart Rhythm 2013; 10:29–36. + +10. Sugumar H, Tung MK, Lee AJ, Bashir J, Lane C. Subcutaneous ICD lead position affects defibrillation threshold. HeartRhythm Case Rep 2018;4:29–30. + +11. Knops RE, Olde Nordkamp LR, de Groot JR, Wilde AA. Two-incision technique for implantation of the subcutaneous implantable cardioverter-defibrillator. Heart Rhythm 2013;10:1240–1243. + +12. Weiss R, Knight BP, Gold MR, Leon AR, Herre JM, Hood M, Rashtian M, +Kremers M, Crozier I, Lee KL, Smith W, Burke MC. Safety and efficacy of a totally subcutaneous implantable-cardioverter defibrillator. Circulation 2013;128: +944–953. + +13. Bettin M, Rath B, Ellermann C, Leitz P, Reinke F, Ko¨ be J, Eckardt L, Frommeyer G. Follow-up of the first patients with a totally subcutaneous ICD in Germany from implantation till battery depletion. Clin Res Cardiol 2019;108: 16–21. \ No newline at end of file diff --git a/medical/md_images/PMC7056617/PMC7056617.md b/medical/md_images/PMC7056617/PMC7056617.md new file mode 100644 index 0000000000000000000000000000000000000000..dc32e13dd5ef24166e441c3a81ddc387c6bccbeb --- /dev/null +++ b/medical/md_images/PMC7056617/PMC7056617.md @@ -0,0 +1,292 @@ + + +![0_image_0.png](0_image_0.png) + +See corresponding commentary on page 425. + +High Compared with Moderate Protein Intake Reduces Adaptive Thermogenesis and Induces a Negative Energy Balance during Long-term Weight-Loss Maintenance in Participants with Prediabetes in the Postobese State: A PREVIEW Study Mathijs Drummen,1 Lea Tischmann,1 Blandine Gatta-Cherifi,2 Mikael Fogelholm,3 Anne Raben,4 Tanja C Adam,1 and Margriet S Westerterp-Plantenga1 1Faculty of Health, Medicine and Life Sciences, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht UMC+, Maastricht University, Maastricht, Netherlands; 2Department of Endocrinology, Diabetology and Nutrition, Universite de Bordeaux, Bordeaux, France; 3Department of Food and Nutrition Sciences, University of Helsinki, Helsinki, Finland; and 4Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark + +## Abstract + +Background: Weight loss has been associated with adaptations in energy expenditure. Identifying factors that counteract these adaptations are important for long-term weight loss and weight maintenance. Objective: The aim of this study was to investigate whether increased protein/carbohydrate ratio would reduce adaptive thermogenesis (AT) and the expected positive energy balance (EB) during weight maintenance after weight loss in participants with prediabetes in the postobese state. + +Methods: In 38 participants, the effects of 2 diets differing in protein/carbohydrate ratio on energy expenditure and respiratory quotient (RQ) were assessed during 48-h respiration chamber measurements ∼34 mo after weight loss. + +Participants consumed a high-protein (HP) diet (n = 20; 13 women/7 men; age: 64.0 ± 6.2 y; BMI: 28.9 ± 4.0 kg/m 2) +with 25:45:30% or a moderate-protein (MP) diet (n = 18; 9 women/9 men; age: 65.1 ± 5.8 y; BMI: 29.0 ± 3.8 kg/m 2) +with 15:55:30% of energy from protein:carbohydrate:fat. Predicted resting energy expenditure (REEp) was calculated based on fat-free mass and fat mass. AT was assessed by subtracting measured resting energy expenditure (REE) +from REEp. The main outcomes included differences in components of energy expenditure, substrate oxidation, and AT between groups. + +Results: EB (MP = 0.2 ± 0.9 MJ/d; HP = −0.5 ± 0.9 MJ/d) and RQ (MP = 0.84 ± 0.02; HP = 0.82 ± 0.02) were reduced and REE (MP: 7.3 ± 0.2 MJ/d compared with HP: 7.8 ± 0.2 MJ/d) was increased in the HP group compared with the MP group (P < 0.05). REE was not different from REEp in the HP group, whereas REE was lower than REEp in the MP group (P < 0.05). Furthermore, EB was positively related to AT (rs = 0.74; P < 0.001) and RQ (rs = 0.47; P < 0.01) +in the whole group of participants. + +Conclusions: In conclusion, an HP diet compared with an MP diet led to a negative EB and counteracted AT ∼34 mo after weight loss, in participants with prediabetes in the postobese state. These results indicate the relevance of compliance to an increased protein/carbohydrate ratio for long-term weight maintenance after weight loss. The trial was registered at clinicaltrials.gov as NCT01777893. *J Nutr* 2020;150:458–463. + +Keywords: protein, energy expenditure, adaptive thermogenesis, energy balance, weight loss, weight maintenance, obesity + +## Introduction + +The increasing prevalence of obesity is a major problem in our modern world and has been associated with numerous comorbidities such as type 2 diabetes and cardiovascular diseases. The most straightforward remedy for obesity is weight loss, but the success of long-term weight-loss maintenance is poor (1). An important factor that prevents long-term weightloss maintenance may be metabolic adaptation, also called adaptive thermogenesis (AT). AT leads to a reduction in energy expenditure greater than that predicted based on reductions in fat-free mass (FFM) and/or fat mass (FM) (2). Adaptations in energy expenditure have been reported lasting for multiple years after a weight-loss period, but may even persist permanently (3– +6). + +Therefore, reducing AT in the postobese condition after weight loss may be pivotal for long-term weight-loss maintenance and several factors have been shown to reduce AT. These factors include cold-induced brown adipose tissue activation +(7), physical activity (8), capsaicinoids (9–14) and tea catechins (15–17). Another factor that may reduce AT after weight loss is high protein (HP) intake (18–21). An HP diet has been shown to increase FFM, diet-induced energy expenditure (DEE), resting energy expenditure (REE), and total energy expenditure (TEE) +(22, 23). After energy restriction, an HP intake supported by a concomitant increase in physical activity was able to preserve REE despite a reduction in FM and FFM (24). Furthermore, a 12-wk HP diet (30% of energy from protein) was able to maintain sleeping metabolic rate (SMR), DEE, and TEE +compared to a low-protein diet (5% of energy from protein) in weight-stable individuals (25). However, it remains unclear whether HP intake after weight loss is able to counteract AT. + +In the present study, we investigated the effects of a controlled HP diet compared with a moderate protein (MP) +diet on components of energy expenditure, energy balance (EB), and AT at the end of the PREVIEW study intervention in a fully controlled respiratory chamber assessment. The PREVIEW study, the PREVention of diabetes through lifestyle Intervention and population studies in Europe and around the World (FP7- +KBBE-2012, no. 312,057) consisted of a 3-y weight loss and weight maintenance intervention in 4 intervention groups, differing in dietary [high-protein, low-glycemic index (HP) compared with moderate-protein, moderate-glycemic index +(MP)] and physical activity guidelines (26). The primary endpoint of the PREVIEW study was the incidence of type 2 diabetes over 3 y analyzed by diet and subsequently according to physical activity and the combination of diet and physical activity. Secondary outcomes included changes in body weight, body composition, and insulin resistance. The main outcomes of the current study included differences in components of energy expenditure, substrate oxidation, and AT between the 2 diet groups. We hypothesized that a controlled HP diet would counteract AT and increase energy expenditure. + +## Methods + +The Medical Ethical Committee of Maastricht University approved the study (clinicaltrials.gov identifier NCT01777893), and all subjects gave written informed consent. + +## Participants + +Based on voluntary willingness to participate, 40 individuals (20 from the HP group and 20 from MP the group) were recruited from the PREVIEW population at Maastricht University in the Netherlands, of whom 2 dropped out because of lack of time (**Supplemental Figure 1**). + +For the general PREVIEW study, participants underwent screening that included anthropometric measurements as described in Fogelholm et al (26). The PREVIEW intervention study is a multicenter randomized controlled trial aimed at finding an effective lifestyle intervention to prevent the development of type 2 diabetes mellitus in individuals with prediabetes as defined by the American Diabetes Association criteria +(27): fasting plasma glucose 5.6–6.9 mmol/L and/or 7.8–11.0 mmol/L at 2 h after an oral-glucose-tolerance test of 75 g glucose, with a fasting plasma glucose concentration <7.0 mmol/L. + +## Study Design + +The PREVIEW intervention consisted of 2 phases: an 8-wk weightloss period using a low energy diet, followed by a 34-mo weight maintenance period with instructions to follow the guidelines of 1 of the 4 intervention groups: an MP, moderate-glycemic index (GI) diet or an HP, low-GI diet, combined with either moderate-intensity or highintensity physical activity. Close to the last clinical investigation day of the weight maintenance period, participants stayed in the respiration chamber for 48 h. The 38 participants that underwent the respiration chamber measurements lost a mean of 11.1 ± 3.6 kg (11.9 ± 2.5%) +during the weight-loss period and the minimum weight-loss percentage was 8.1%. The mean body weight at the time of the respiration chamber measurements was 5.5 ± 6.2 kg lower compared to baseline, corresponding with a BMI of 28.9 ± 3.9 kg/m2. There were no differences between groups regarding the changes in body weight during the PREVIEW intervention. + +## Respiration Chamber + +Subjects arrived at the Metabolic Research Unit Maastricht research facilities in the morning having fasted from 22:00 the night before. + +The respiration chamber session started at 09:30 and stopped 2 d later at 09:30. The respiration chamber is an airtight chamber of 14 m3 furnished with a bed, chair, desk with computer, TV, telephone, intercom, sink, and toilet. The climate inside the chamber is controlled. O2 consumption and CO2 production were continuously measured by open-circuit ventilated indirect calorimetry (28). The room was ventilated with fresh air at a rate of 70– +80 L/min. Flow was measured using electronically modified dry gasmeters (G6, gasmeterfabriek Schlumberger). The concentrations of O2 and CO2 were measured with dual pairs of infrared CO2 analyzers (ABB/Hartman&Braun Uras) and paramagnetic O2 analyzers +(Servomex 4100, and ABB/Hartman&Braun Magnos) (28). During each 15-min period, 6 samples of outgoing air, 1 sample of fresh air, zero gas and calibration gas were measured. The gas samples to be measured were selected by a computer that also stored and processed the data (28). Physical activity was continuously measured by use of an ActiSleep+ (ActiGraph LLC) accelerometer worn on the hip and expressed as counts-per-minute (CPM). Subjects had fixed bedtimes between 23:30 and 07:30. In the daytime, they were not allowed to sleep or to perform exercise. Meals were offered at stated times (breakfast: +09:00, lunch: 13:00, dinner: 17:45), and subjects were instructed to finish these within 30 min. + +## Dietary Intervention + +During the weight maintenance period, dietary intervention groups comprised an MP group with 15/55/30% of energy from protein/carbohydrate/fat and a moderate dietary GI (≥56), and an HP +group with 25/45/30% of energy from protein/carbohydrate/fat, with a low dietary GI (≤50). Both diets were consumed ad libitum with respect to energy, but with the instruction to maintain the achieved body weight. + +Additional weight loss was allowed. More detailed information on the weight-loss period, dietary guidelines, and of the intervention groups has been reported before (26, 29). + +In the respiration chamber, participants received an MP or HP +diet, corresponding with their dietary intervention instructions during the PREVIEW study. The basis of the meals was the same between groups, combined with either carbohydrate-rich or protein-rich food items to keep menus comparable. Originally in the PREVIEW study, subjects were randomly allocated to the HP or MP diet groups by stratification on sex, age, and BMI. Protein was completely exchanged with carbohydrate, resulting in the relative fat content being similar in the 2 groups. The diets consisted of commercially available food items and were provided individually in EB. Individual daily energy requirements were calculated as the basal metabolic rate using the FFM and FM (30) multiplied by a physical activity level of 1.35 (25). Daily energy intake was divided over 3 meals, with breakfast containing 20%, and lunch and dinner 40% each. During all measurements in the respiration chamber, the meals within each condition had the same macronutrient composition.Water consumption was allowed ad libitum between the meals; no other foods or beverages were available. + +## Energy Expenditure And Respiratory Quotient + +TEE and the separate components of energy expenditure, SMR, DEE, +and activity-induced energy expenditure (AEE) were calculated during the 48-h stay in the respiration chamber. O2 consumption and CO2 production were used to calculate TEE according to the formula of Weir (31). SMR was expressed as the lowest mean TEE during 3 consecutive hours between 00:00 and 07:00. The mean accelerometer CPM corresponding with the SMR was assumed to represent the inactive state. Simple linear regression analysis based on the relation between TEE and the corresponding accelerometer CPM was used to calculate REE (32). DEE was determined by subtracting SMR from REE. AEE was calculated by subtracting REE from TEE (33). Energy expended for thermoregulation was neglected, because subjects were staying in the respiration chamber in the thermoneutral condition. + +EB was determined as the difference between energy intake and TEE. + +Predicted resting energy expenditure (REEp) was calculated as basal metabolic rate based on the FFM and FM (30) and diet-induced thermogenesis calculated as 9.7% of the energy intake for the MP +group and 11.2% for the HP group, based on median values for the thermogenic effect of separate nutrients (carbohydrate 10% EI, fat 1.5% EI, protein 25% EI) (34). AT was calculated by subtracting REE +from REEp. Respiratory quotient (RQ) was calculated by dividing CO2 production by O2 consumption as a measure of substrate oxidation. The amounts of carbohydrate and fat oxidized were calculated from CO2 production, O2 consumption, and protein oxidation with the formulas of Carpenter (35). Carbohydrate and fat balances were determined as the difference between intake and oxidation. + +## Biomarker Of Protein Intake + +Nitrogen excretion, measured from 24-h urine collections during the respiration chamber measurements, was used as an estimate of protein intake. Urine was collected in 2-L urine bottles containing 10 mL of diluted hydrochloric acid (4 mmol/L) to prevent nitrogen loss through evaporation. The total volume of the 24-h urine was recorded. Urine was gently mixed, and samples were taken and frozen at −20◦C until analysis. Nitrogen concentrations were measured with an elemental analyzer (CHN-O-Rapid, Heraeus, in the Netherlands, and Integra COBAS 400 plus, Roche Diagnostics GmbH, in the USA). Total nitrogen output was calculated as 24-h urinary nitrogen plus 10% to account for normal losses via feces and other miscellaneous losses. Nitrogen excretion was multiplied by 6.25 to determine protein oxidation. + +Protein balance was determined as the difference between intake and oxidation. + +## Body Weight And Body Composition + +Body weight and composition were determined with subjects in the fasted state before entering the respiration chamber. Body weight was measured using a calibrated scale (Life Measurement Inc.). Body composition was determined based on body density measured via airdisplacement plethysmography with the BodPod system (BOD POD, +Life Measurement Inc.), with use of Siri's equation for body density (36). Height was measured using a wall-mounted stadiometer to the nearest 0.1 cm (Seca, model 222). + +## Statistical Analysis + +Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, IBM Corp.) and GraphPad Prism (GraphPad Prism version 7.00 for Windows, GraphPad Software). Data are presented as mean (SD). Normality of the parameters was assessed using the Shapiro-Wilk test and outliers were detected with the use of box plots in SPSS. Sample size calculation showed that at least 16 participants per group were needed to show an interaction effect of dietary intervention and time on 24-h DEE in this parallel study. + +For the calculations we used an α of 0.05, a β of 0.10, and effect size of 0.4, based on a respiration chamber study by Westerterp et al. (33). In the whole group, protein oxidation, BMI, and AEE were not normally distributed. In the HP group, protein oxidation, fat oxidation, BMI, FFM, body-fat percentage, TEE, and AEE were not normally distributed. For these reasons, nonparametric tests were used for the analyses. Differences in subject characteristics, nitrogen excretion, body weight and body composition, energy expenditure and its components, and substrate oxidation between the diet groups were assessed using the Mann-Whitney U test. Differences in slopes and y-intercepts of the FFM and REE/REEp regression lines between the groups were assessed with linear regression analysis using GraphPad Prism. For the purpose of physiological comparativeness, intercepts of these regression lines were reported for the mean FFM. To assess the y-intercepts, the mean FFM was subtracted from the individual FFM values. Associations between EB and other parameters were investigated using Spearman correlation analysis. + +## Results Macronutrient Intake, Oxidation, And Balance In The 2 Groups + +The different macronutrient composition of the diets resulted in differences in protein and carbohydrate balance between the groups (**Figure 1**). Protein intake and oxidation were higher in the HP group and the protein balance was more positive compared to the MP group (P < 0.01). Carbohydrate intake and oxidation were lower and the carbohydrate balance was less positive in the HP group compared to the MP group (P < 0.05). There was a trend for a lower fat balance in the HP group compared to the MP group (P = 0.077). + +## Anthropometric Variables, Energy Expenditure, And Rq + +Anthropometric variables, energy expenditure, and RQ were assessed for the MP and HP groups (Table 1, **Figure 2**). There were no significant differences in BMI, FM, FFM, body-fat percentage, energy intake, or energy expenditure between the groups. EB was significantly lower in the HP group compared to the MP group (P = 0.015). The negative EB in the HP group was also significantly different from zero (P = 0.020). + +To determine AT, REE of the 2 groups and the predicted REE as functions of FFM were compared (**Figure 3**). For both groups, the slopes of the measured REE regression were not significantly different from the predicted regression (REEp). The intercept of + +![3_image_0.png](3_image_0.png) + +the MP regression line, however, was significantly lower than the intercept of the predicted regression line (REE: 7.3 ± 0.2 MJ/d compared with REEp: 7.8 ± 0.1 MJ/d; P = 0.006). The intercept of the HP regression line was not significantly different from the intercept of the REEp regression line (REE: 7.8 ± 0.2 MJ/d compared with REEp: 8.0 ± 0.1 MJ/d; P = 0.332). The intercepts between the MP and HP regression lines were significantly different (MP: 7.3 ± 0.2 MJ/d compared with HP: 7.8 ± 0.2 MJ/d; P = 0.030). The mean RQ was lower in the HP group compared to the MP group (P = 0.004). In the whole group of participants, EB was not related to any of the anthropometric variables, but was inversely associated with DEE (rs = −0.40; P = 0.014) and positively associated with AT (rs = 0.74; P < 0.001) (**Figure 4**) and RQ (rs = 0.47; P = 0.003) (**Figure 5**). + +## Discussion + +Measurements of energy expenditure showed that a 48-h HP diet compared with an MP diet induced a negative EB and increased REE. The HP diet was able to counteract AT during weight maintenance after weight loss, whereas the MP diet TABLE 1 Anthropometric variables, energy expenditure, and respiratory quotient in the MP and HP groups1 + +| MP (n = 18) | HP (n = 20) | P value | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|--------------|-------| +| Sex, f/m | 9/9 | 13/7 | | +| Age, y | 65.1 ± 5.8 | 64.0 ± 6.2 | 0.553 | +| BMI, kg/m2 | 29.0 ± 3.8 | 28.9 ± 4.0 | 0.942 | +| Fat-free mass, kg | 52.5 ± 10.9 | 50.8 ± 11.3 | 0.553 | +| Fat mass, kg | 33.9 ± 7.7 | 34.8 ± 8.8 | 0.740 | +| Body fat, % | 39.3 ± 7.4 | 40.7 ± 7.7 | 0.593 | +| Calculated EI, MJ/d | 9.4 ± 1.7 | 9.3 ± 1.6 | 0.573 | +| EB, MJ/d | 0.2 ± 0.9 | − 0.5 ± 0.9 | 0.015 | +| RQ | 0.84 ± 0.02 | 0.82 ± 0.02 | 0.004 | +| Activity, CPM | 133.3 ± 34.1 | 155.0 ± 36.6 | 0.087 | +| 1Values are means ± SDs. Differences between groups were assessed by means of Mann-Whitney U tests. CPM, counts-per-minute; EB, energy balance; EI, energy | | | | + +was not. In addition, AT was positively associated with EB, + +![3_image_1.png](3_image_1.png) indicating its potential to reduce weight regain after weight loss and prevent weight cycling. This study shows that these effects are robust enough to hold up with a parallel study design, in overweight prediabetic participants, in the postobese state. + +Although HP diets have been previously shown to acutely increase fat oxidation (37), the reduced RQ in the HP group is likely the result of the negative EB, because a negative balance Protein intake counteracts adaptive thermogenesis 461 + +![4_image_0.png](4_image_0.png) + +results in increased fat oxidation (38, 39). Long-term negative EB leads to weight loss. This underlines the impact of increasing the protein/carbohydrate ratio to prevent AT and combat overweight/obesity by supporting body weight maintenance. HP +diets are able to affect EB via increased satiety and increased thermogenesis and have favorable effects on body composition +(22). The lower EB in the HP group compared to the MP group can be explained by reduced AT in the HP group. + +The observed AT was 0.5 MJ/d in the MP group. These numbers are in line with results that have been described before +(4, 40, 41), indicating that AT remains present even up to 34 mo after an 8-wk weight loss period during which participants lost 11.9% of their body weight on average. The HP diet was able to increase REE relative to the MP diet and the REE in the HP group was equal to the REEp. These results suggest that the HP +diet was able to completely abolish the effect of the AT. + +In long-term interventions it has been shown that up to 90% of weight-reduced individuals return to their previous body weight (1, 42) and weight cycling has been associated with increased mortality (43). AT has been suggested to play + +![4_image_1.png](4_image_1.png) + +462 Drummen et al. + +an important role in this process (44). Previous research has identified factors that are able to counteract AT including coldinduced brown adipose tissue activation (7), physical activity +(8), capsaicinoids (9–14), and tea catechins (15–17). However, many of these factors affect AT by directly increasing energy expenditure. The counteracting effects of HP intake on AT may partly be explained by the higher thermogenic cost of protein. + +Because we incorporated a higher diet-induced thermogenesis for the HP group [HP: 11.2% compared with MP: 9.7% (34)] +in the calculations to predict energy expenditure, the increases in REE of the HP diet compared with the MP diet exceeded the increased thermic effect of protein. The effects of protein intake on REE as a function of FFM may be mediated via upregulation of uncoupling proteins (UCP) (45), which in turn would support increased energy expenditure (46). Corroborating this cascade and in accordance with our results, a short-term study in rats showed that protein intake led to higher night-time energy expenditure, lower RQ, and higher mRNA expression values for UCP2 and UCP3 (47). Moreover, night-time energy expenditure was positively correlated with UCP mRNA expression. + +In the current study, the effects of differences in protein intake on EB and AT were assessed once at the end of the PREVIEW intervention period. + +In conclusion, an HP diet compared with an MP diet led to a negative EB and counteracted AT ∼34 mo after weight loss, in participants with prediabetes being in the postobese state. These results indicate the relevance of compliance to an increased protein/carbohydrate ratio for long-term weight maintenance after weight loss. + +## Acknowledgments + +The authors' responsibilities were as follows—AR, MF, TCA, +and MSW-P: designed research; MD, LT, and BG-C: conducted research; MD and LT: analyzed data; MD, TCA, and MSWP: wrote the paper; AR and MSW-P: primary responsibility for final content; and all authors: read and approved the final manuscript. + +## References + +1. Wing RR, Phelan S. Long-term weight loss maintenance. 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The acute effects of a lunch containing capsaicin on energy and substrate utilisation, hormones, and satiety. Eur J Nutr 2009;48:229–34. + +13. Smeets AJ, Janssens PL, Westerterp-Plantenga MS. Addition of capsaicin and exchange of carbohydrate with protein counteract energy intake restriction effects on fullness and energy expenditure. J Nutr 2013;143:442–7. + +14. Janssens PL, Hursel R, Martens EA, Westerterp-Plantenga MS. Acute effects of capsaicin on energy expenditure and fat oxidation in negative energy balance. PLoS One 2013;8:e67786. + +15. Westerterp-Plantenga MS, Lejeune MP, Kovacs EM. Body weight loss and weight maintenance in relation to habitual caffeine intake and green tea supplementation. Obes Res 2005;13:1195–204. + +16. Hursel R, Westerterp-Plantenga MS. Thermogenic ingredients and body weight regulation. Int J Obes (Lond) 2010;34:659–69. + +17. Janssens PL, Hursel R, Westerterp-Plantenga MS. Nutraceuticals for body-weight management: the role of green tea catechins. Physiol Behav 2016;162:83–7. + +18. Clamp LD, Hume DJ, Lambert EV, Kroff J. Successful and unsuccessful weight-loss maintainers: strategies to counteract metabolic compensation following weight loss. J Nutr Sci 2018;7:e20. + +19. Westerterp-Plantenga MS, Rolland V, Wilson SA, Westerterp KR. + +Satiety related to 24 h diet-induced thermogenesis during high protein/carbohydrate vs high fat diets measured in a respiration chamber. Eur J Clin Nutr 1999;53:495–502. + +20. Westerterp-Plantenga MS, Lejeune MP, Nijs I, van Ooijen M, Kovacs EM. High protein intake sustains weight maintenance after body weight loss in humans. Int J Obes Relat Metab Disord 2004;28:57–64. + +21. Soenen S, Martens EA, Hochstenbach-Waelen A, Lemmens SG, +Westerterp-Plantenga MS. Normal protein intake is required for body weight loss and weight maintenance, and elevated protein intake for additional preservation of resting energy expenditure and fat free mass. J Nutr 2013;143:591–6. + +22. Drummen M, Tischmann L, Gatta-Cherifi B, Adam T, WesterterpPlantenga M. Dietary protein and energy balance in relation to obesity and co-morbidities. Front Endocrinol (Lausanne) 2018;9:443. + +23. Mikkelsen PB, Toubro S, Astrup A. Effect of fat-reduced diets on 24h energy expenditure: comparisons between animal protein, vegetable protein, and carbohydrate. Am J Clin Nutr 2000;72:1135–41. + +24. Pasiakos SM, Mettel JB, West K, Lofgren IE, Fernandez ML, Koo SI, +Rodriguez NR. Maintenance of resting energy expenditure after weight loss in premenopausal women: potential benefits of a high-protein, reduced-calorie diet. Metabolism 2008;57:458–64. + +25. Martens EA, Gonnissen HK, Gatta-Cherifi B, Janssens PL, WesterterpPlantenga MS. Maintenance of energy expenditure on high-protein vs. high-carbohydrate diets at a constant body weight may prevent a positive energy balance. Clin Nutr 2015;34:968–75. + +26. Fogelholm M, Larsen TM, Westerterp-Plantenga M, Macdonald I, +Martinez JA, Boyadjieva N, Poppitt S, Schlicht W, Stratton G, +Sundvall J, et al. PREVIEW: Prevention of Diabetes through Lifestyle Intervention and Population Studies in Europe and around the World. Design, methods, and baseline participant description of an adult cohort enrolled into a three-year randomised clinical trial. Nutrients 2017;9:E632. + +27. American Diabetes Association. 2. Classification and diagnosis of diabetes. Diabetes Care 2018;41:S13–27. + +28. Schoffelen PF,Westerterp KR, Saris WH, Ten Hoor F. A dual-respiration chamber system with automated calibration. J Appl Physiol (1985) +1997;83:2064–72. + +29. Christensen P, Meinert Larsen T, Westerterp-Plantenga M, Macdonald I, Martinez JA, Handjiev S, Poppitt S, Hansen S, Ritz C, Astrup A, et al. Men and women respond differently to rapid weight loss: metabolic outcomes of a multi-centre intervention study after a low-energy diet in 2500 overweight, individuals with pre-diabetes (PREVIEW). Diabetes Obes Metab 2018;20:2840–51. + +30. Westerterp KR, Donkers JH, Fredrix EW, Boekhoudt P. Energy intake, physical activity and body weight: a simulation model. Br J Nutr 1995;73:337–47. + +31. Weir JB. New methods for calculating metabolic rate with special reference to protein metabolism. Nutrition 1990;6:213–21. + +32. Tataranni PA, Larson DE, Snitker S, Ravussin E. Thermic effect of food in humans: methods and results from use of a respiratory chamber. Am J Clin Nutr 1995;61:1013–9. + +33. Westerterp KR, Wilson SA, Rolland V. Diet induced thermogenesis measured over 24 h in a respiration chamber: effect of diet composition. + +Int J Obes Relat Metab Disord 1999;23:287–92. + +34. Tappy L. Thermic effect of food and sympathetic nervous system activity in humans. Reprod Nutr Dev 1996;36:391–7. + +35. Brouwer E. On simple formulae for calculating the heat expenditure and the quantities of carbohydrate and fat oxidized in metabolism of men and animals, from gaseous exchange (oxygen intake and carbonic acid output) and urine-N. Acta Physiol Pharmacol Neerl 1957;6: +795–802. + +36. Siri WE. Body composition from fluid spaces and density: analysis of methods. 1961. Nutrition 1993;9:480–91; discussion, 92. + +37. Stepien M, Gaudichon C, Fromentin G, Even P, Tome D, AzzoutMarniche D. Increasing protein at the expense of carbohydrate in the diet down-regulates glucose utilization as glucose sparing effect in rats. + +PLoS One 2011;6:e14664. + +38. Westerterp KR. Food quotient, respiratory quotient, and energy balance. + +Am J Clin Nutr 1993;57:759S–64S; discussion 64S-65S. + +39. Rumpler WV, Seale JL, Miles CW, Bodwell CE. Energy-intake restriction and diet-composition effects on energy expenditure in men. + +Am J Clin Nutr 1991;53:430–6. + +40. Leibel RL, Rosenbaum M, Hirsch J. Changes in energy expenditure resulting from altered body weight. N Engl J Med 1995;332: +621–8. + +41. Weyer C, Walford RL, Harper IT, Milner M, MacCallum T, +Tataranni PA, Ravussin E. Energy metabolism after 2 y of energy restriction: the biosphere 2 experiment. Am J Clin Nutr 2000;72: +946–53. + +42. McGuire MT, Wing RR, Hill JO. The prevalence of weight loss maintenance among American adults. Int J Obes Relat Metab Disord 1999;23:1314–9. + +43. Moon JH, Lim S, Choi SH, Oh TJ, Jang HC, Park KS, Cho NH. + +Body-weight fluctuation and incident diabetes mellitus, cardiovascular disease, and mortality: a 16-year prospective cohort study. J Clin Endocrinol Metab 2019;104:639–46. + +44. Major GC, Doucet E, Trayhurn P, Astrup A, Tremblay A. Clinical significance of adaptive thermogenesis. Int J Obes (Lond) 2007;31: +204–12. + +45. Petzke KJ, Friedrich M, Metges CC, Klaus S. 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J Nutr Biochem 2007;18:400–7. \ No newline at end of file diff --git a/medical/md_images/PMC7056617/PMC7056617_meta.json b/medical/md_images/PMC7056617/PMC7056617_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..fb5574712699429ca0723c00a9825d036a7d84b1 --- /dev/null +++ b/medical/md_images/PMC7056617/PMC7056617_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 6, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 0, + "code": 0, + "table": 1, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC7225764/PMC7225764.md b/medical/md_images/PMC7225764/PMC7225764.md new file mode 100644 index 0000000000000000000000000000000000000000..aa28f4ecf071a3bfc0271f91d0468963ae98cffa --- /dev/null +++ b/medical/md_images/PMC7225764/PMC7225764.md @@ -0,0 +1,101 @@ + +## Case Report + +# Peritoneal Dialysis Using A Bicarbonate-Buffered Dialysate In A Child With An Inborn Error Of Metabolism Presenting With Severe Acidosis + +Anjali Kalbhande1, Uday Gajare2, Preeti Shanbag3 + +## Ab S T Rac T + +Metabolic acidosis is observed in the pediatric intensive care unit (PICU) in several conditions including sepsis, intoxications, and severe catabolic states. It is occasionally seen due to acute decompensation in an inborn error of metabolism (IEM). Persistent acidosis results in a decrease in myocardial contractility, cardiac output, and catecholamine responsiveness. The mainstay of treatment of metabolic acidosis has been intravenous sodium bicarbonate infusion. However, the large amounts of sodium bicarbonate sometimes required can be hazardous resulting in hypernatremia, hypervolemia, and hyperosmolality. We report a 3-year child who presented with persistent lactic acidosis due to an IEM whom we treated with peritoneal dialysis (PD) using a bicarbonate-buffered dialysate. The child recovered uneventfully within 72 hours of dialysis. Peritoneal dialysis using a bicarbonate-buffered dialysate is a safe and simple method of treating persistent severe acidosis in the PICU. + +Keywords: Acute peritoneal dialysis, Bicarbonate-buffered dialysate, Inborn error of metabolism, Severe acidosis. + +Indian Journal of Critical Care Medicine (2020): 10.5005/jp-journals-10071-23372 + +## Bac Kg R O U N D + +Metabolic acidosis is observed in a large number of conditions in the pediatric intensive care unit (PICU). These include infections, severe catabolic states, tissue anoxia, severe dehydration, and intoxication. It may occasionally be observed during decompensation in an unrecognized inborn error of metabolism (IEM). A decrease in arterial blood pH below 7.1 has been demonstrated to be associated with marked decrease in myocardial contractility, cardiac output, and catecholamine responsiveness. The resultant cardiovascular depression leads to tissue hypoperfusion and lactic acid production.1–3 Control of acidosis is, therefore, essential in the management of these patients. The routine use of sodium bicarbonate in the treatment of most causes of severe acidemia, diabetic ketoacidosis, lactic acidosis, and cardiac arrest has been controversial due to conflicting data on the beneficial and detrimental effects in experimental animals as well as patients.4 The large amounts of sodium bicarbonate that are often required could cause serious problems which include sodium overload and cardiac failure, hypernatremia and hyperosmolality, impaired oxygen delivery to the tissues and aggravation of cerebrospinal fluid acidosis, and development of metabolic alkalosis following the correction of lactic acidosis.4 Thus, the need for administration of large amounts of sodium bicarbonate, on the one hand, and the potential hazards of this treatment, on the other hand, present a difficult therapeutic dilemma. + +We present our experience of using peritoneal dialysis (PD) with a bicarbonate-buffered dialysate for treatment of severe acidosis in a 3-year-old girl who presented with severe lactic acidosis with ketosis. + +## Cas E **De S C R I P T I O N** + +A 3-year-old Muslim girl from Solapur was admitted with fever, diarrhea, and vomiting for 1 day. Abdominal pain and breathlessness 1–3Department of Pediatrics, ESI-Postgraduate Institute of Medical Science and Research, Model Hospital, Mumbai, Maharashtra, India Corresponding Author: Preeti Shanbag, Department of Pediatrics, ESI-Postgraduate Institute of Medical Science and Research, Model Hospital, Mumbai, Maharashtra, India, Phone: +91 9869128012, e-mail: pshanbag@gmail.com How to cite this article: Kalbhande A, Gajare U, Shanbag P. Peritoneal Dialysis Using a Bicarbonate-buffered Dialysate in a Child with an Inborn Error of Metabolism Presenting with Severe Acidosis. Indian J Crit Care Med 2020;24(3):200–202. Source of support: Nil + +## Conflict Of Interest: None + +were present for 4 hours prior to admission. There was no history of polyuria, polydipsia, loss of consciousness, or convulsions in the past. The child had been hospitalized at 13 months of age with a similar history. Diagnosis at that time had been sepsis with hypoglycemia and metabolic acidosis. She was the third child of a consanguineous marriage (the parents were first cousins). Two older siblings, a 14-year-old boy and a 9-year-old girl were alive and well. There was no family history of a similar problem nor a history of deaths in infancy or early childhood. The child was born normally at term. The neonatal period was uncomplicated. The child was developmentally normal. Immunization was appropriate for age. + +On examination, the child was drowsy. Heart rate was 160/minute. Peripheral pulses were feeble. Respiratory rate was 68/minute, acidotic. Blood pressure was 94/60 mm Hg in the right upper limb. The extremities were cool with a capillary refill time of more than 2 seconds. The oral mucosa was dry, but skin turgor was normal. Weight and height were 13.5 kg and 92 cm, respectively, and both above the 50th percentile for age. + +There was no icterus nor any other signs of liver cell failure. There were no dysmorphic features. On examination of the abdomen, the +© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons. org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and non-commercial reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. + +liver was palpable 2 cm below the right costal margin in the midclavicular line. There was no splenomegaly. Other systems were essentially normal. There was no peculiar odor to the urine. + +Investigations at admission showed hemoglobin of 11.4 mg/dL; +total white blood cell count of 18,220/mm3 with a differential count of neutrophils 68%, lymphocytes 29%, and monocytes 3%; and a platelet count of 540,000/mm3. Peripheral smear was negative for malarial parasites. Blood glucose was 315 mg/dL. Serial arterial blood gases and electrolytes are shown in Table 1. Anion gap at admission was 25.4 mEq/L (normal 8–16 mEq/L). Serum creatinine was 0.8 mg/dL which decreased to 0.4 mg/dL at 24 hours. Serum calcium was 9.1 mg/dL and serum uric acid 8.5 mg/dL. Serum ammonia was 57 μmol/L (normal in children: 11–35 μmol/L), and plasma lactate was 14.4 mmol/L. Serum-free fatty acids and 3-OH butyric acid were elevated. Blood culture was sterile. Liver function tests were normal. + +A diagnosis of diabetic ketoacidosis was initially made, and the child given fluid boluses. However, blood sugars dropped to 123 mg/dL at 1 hour and were un-recordable at 2 hours; hence, insulin was not started. A diagnosis of an IEM was considered especially with the past history of hypoglycemia and acidosis at 13 months of age. Blood and urine were collected for metabolic workup. Despite three bicarbonate corrections given over a period of 16 hours, there was persistent severe acidosis; hence, a decision was taken to start PD with a bicarbonate-buffered dialysate. Since bicarbonate-buffered dialysate cannot be stored, it was prepared just before use by adding 260 mL of 5% dextrose and 40 mL of 8.4% soda bicarbonate to 700 mL of normal saline (dialysate containing sodium 147 mEq/L and bicarbonate 40 mEq/L). + +A rigid PD catheter was inserted percutaneously into the peritoneal cavity through a small midline incision 4 cm below the umbilicus under local anesthesia, using sterile precautions. Peritoneal dialysis was initiated with a fill volume of 30 mL/kg and dwell times of 45 minutes. The child was started on intravenous fluids and an empiric antibiotic—ceftriaxone, pending the blood culture report. Vital signs, mental status, and urine output were monitored prior to, during, and after dialysis as were the arterial blood gases, serum electrolytes, and blood sugars. The child regained consciousness within 6 hours of initiation of dialysis. Metabolic acidosis was under control within 48 hours, and hence, PD was discontinued. + +Blood tandem mass spectrometry of blood was normal. Gas chromatography/mass spectrometry of urine showed significantly elevated lactate, 2-OH butyrate, 3-OH butyrate, acetoacetate, and glycerol. + +A defec t in gluconeogenesis, probably a fruc tose 1,6 biphosphatase deficiency was considered in view of hypoglycemia, high anion gap acidosis, hyperlactatemia, ketosis, and presence of glycerol in the urine. Enzyme studies were not available. Genetic studies will be sent as soon as financially feasible. The child was discharged on a fructose-restricted diet with advice to the parents to avoid prolonged fasting. At last follow-up at 10 months, the child is doing well, with no further episodes of decompensation. + +## Di S C U S S I O N + +We report here a case of a child with an IEM who presented with severe metabolic acidosis and was managed with PD with a bicarbonate-buffered dialysate. While PD with bicarbonate added to the PD fluid is a well-known procedure, this is an often forgotten modality of treatment and is useful in small setups where facilities for hemodialysis (HD)/continuous renal replacement therapy (CRRT) are not available. + +Our patient presented with hyperglycemia in this admission. + +Paksu et al.5 described a patient with fructose 1,6-biphosphatase who presented with hyperglycemia. They attributed this to stress hyperglycemia associated with sepsis. In our patient, however, the blood culture was sterile. Hyperglycemia was probably due to a dextrose bolus the child had received prior to admission to our PICU. Despite correction of subsequent hypoglycemia and giving three corrections with intravenous sodium bicarbonate over a 16-hour period, our patient continued to have altered sensorium and persistent acidosis. We, therefore, decided to do PD with a bicarbonate-buffered dialysate. + +Commercial dialysates available for acute PD contain acetate as the buffer. However, acetate metabolism may be abnormal in patients with severe lactic acidosis. Bicarbonate is a physiologic buffer requiring no metabolic conversion and, hence, is the logical choice for the treatment of metabolic acidosis. With PD, the net sodium and water transfers are negligible due to the lack of significant concentration and osmolar gradients for sodium. However, significant diffusion of bicarbonate and lactate takes place as a result of their marked concentration gradients. Thus, effective removal of lactate and delivery of bicarbonate can be obtained without undesirable changes in the osmolality or the volume of body fluids. Since solute transfer is dependent on the gradient, the rate of bicarbonate delivery during dialysis is self-limited, i.e., the delivery rate decreases with decrease in acidosis and ceases when the metabolic acidosis is corrected. The serum bicarbonate concentration cannot, therefore, rise above that of the dialysate. Undesirable rapid changes of plasma bicarbonate and blood pH, which are commonly encountered with intravenous bicarbonate administration, can be avoided. + +| Table 1: Serial arterial blood gases and electrolytes of the patient Hours 0 2 8 12 | 16 | 36 | 48 | 60 | 72 | | | | | +|---------------------------------------------------------------------------------------|-------|-------|-------|------|------|-------|-------|------|-------| +| pH | 7.05 | 7.23 | 7.12 | 7.15 | 7.13 | 7.28 | 7.30 | 7.33 | 7.36 | +| pCO2 (mm Hg) | 8 | 13 | 16 | 14 | 8 | 13 | 14 | 28 | 33 | +| pO2 (mm Hg) | 86 | 88 | 92 | 98 | 102 | 108 | 104 | 106 | 102 | +| Serum HCO3 (mEq/L) | <3 | 5.3 | 6.7 | 6.2 | <3 | 6.1 | 11.5 | 16.7 | 19.7 | +| Base excess | −26 | −20.4 | −22.4 | −24 | −25 | −18.8 | −19.5 | −10 | −0.62 | +| Serum Na+ (mEq/L) | 133 | 134.9 | 137 | 140 | 148 | 143 | 140 | 138 | 137 | +| Serum K+ (mEq/L) | 4.2 | 4.1 | 3.7 | 3.1 | 2.6 | 3.2 | 3.5 | 3.7 | 4.0 | +| Serum Cl− (mEq/L) | 100.5 | 101.4 | 102 | 104 | 105 | 102 | 101 | 102 | 104 | + +Our patient presented with severe lactic acidosis. Peritoneal dialysis with a bicarbonate-buffered dialysate, along with the appropriate supportive measures, resulted in marked improvement of the acid–base parameters. We did not observe hypervolemia or hypernatremia in our patient. Vaziri et al. have also used PD using a bicarbonate-buffered dialysate for the treatment of lactic acidosis in adults, with no adverse effects.6 A diagnosis of an IEM in a child may be delayed as the result of nonspecific symptoms. Definitive diagnosis is based on the detection of abnormal levels of amino acids or organic acids, or their metabolites, in urine and/or serum specimens. This may require an additional 24–72 hours, further delaying the initiation of specific medical therapy until the exact metabolic defect is identified. Previous studies have shown that neurologic and developmental morbidities of children with acute metabolic disturbances caused by inborn errors of metabolism increase with prolonged duration of the metabolic derangement.7,8 Thus, children with a suspected or confirmed IEM often require therapy to achieve rapid correction of their metabolic disturbance. Exchange transfusions, PD, HD, and CRRT have been used for this purpose; HD and CRRT are preferred since they allow for the efficient removal of toxic metabolites to safer levels, while diagnostic studies are being performed.9,10 Most studies have focussed on reduction of hyperammonemia where CRRT is more effective than PD for improving the prognosis.9,10 + +## Co N C Lu S I O N + +We conclude that PD with a bicarbonate-buffered dialysate is a useful modality for the treatment of persistent severe acidosis in the PICU. + +## Re F E R E N C E S + +1. Mitchell JH, Wildenthal K, Johnson RL Jr. The effects of acid-base disturbances on cardiovascular and pulmonary function. Kidney Int 1972;1(5):375–389. DOI: 10.1038/ki.1972.48. + +2. Orchard CH, Kentish JC. Effects of changes of pH on the contractile function of cardiac muscle. Am J Physiol 1990;258(6 Pt 1):C967–C981. + +DOI: 10.1152/ajpcell.1990.258.6.C967. + +3. Campbell GS, Houle DB, Crisp NWJr, Weil MH, Brown EBJr. Depressed response to intravenous sympathicomimetic agents in humans during acidosis. Dis Chest 1958;33(1):18–22. DOI: 10.1378/chest.33.1.18. + +4. Kraut JA, Kurtz I. Use of base in the treatment of severe acidemic states. + +Am J Kidney Dis 2001;38(4):703–727. DOI: 10.1053/ajkd.2001.27688. + +5. Paksu MS, Kalkan G, Asilioglu N, Paksu S, Dinler G. Gluconeogenesis defect Presenting with resistant hyperglycemis and acidosis mimicking diabetic ketoacidosis. Pediatr Emerg Care 2011;27(12): 1180–1181. DOI: 10.1097/PEC.0b013e31823b412d. + +6. Vaziri ND, Ness R, Wellikson L, Barton C, Greep N. Bicarbonatebuffered peritoneal dialysis an effective adjunct in the treatment of lactic acidosis. Am J Med 1979;67(3):392–396. DOI: 10.1016/00029343(79)90784-8. + +7. Surtees RA, Matthews EE, Leonard JV. Neurologic outcome of propionic acidemia. Pediatr Neurol 1992;8(5):333–337. DOI: 10.1016/0887-8994(92)90085-d. + +8. Msall M, Batshaw ML, Suss R, Brusilow SW, Mellits ED. Neurologic outcome in children with inborn errors of urea synthesis: outcome of ureacycle enzymopathies. N Engl J Med 1984;310(23):1500–1505. DOI: 10.1056/NEJM198406073102304. + +9. Picca S, Dionisi-Vici C, Abeni D, Pastore A, Rizzo C, Orzalesi M, et al. + +Extracorporeal dialysis in neonatal hyperammonemia: modalities and prognostic indicators. Pediatr Nephrol 2001;16(11):862–867. DOI: 10.1007/s004670100702. + +10. Schaefer F, Straube E, Oh J, Mehls O, Mayatepeck E. Dialysis in neonates with inborn errors of metabolism. Nephrol Dial Transplant 1999;14(4):910–918. DOI: 10.1093/ndt/14.4.910. \ No newline at end of file diff --git a/medical/md_images/PMC7225764/PMC7225764_meta.json b/medical/md_images/PMC7225764/PMC7225764_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..10b6da964fe197766570c7d823f388b1c925d853 --- /dev/null +++ b/medical/md_images/PMC7225764/PMC7225764_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 3, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 4, + "code": 0, + "table": 1, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC7392567/PMC7392567.md b/medical/md_images/PMC7392567/PMC7392567.md new file mode 100644 index 0000000000000000000000000000000000000000..b242d6f714564b112d05e4cf08bd2cd58a969707 --- /dev/null +++ b/medical/md_images/PMC7392567/PMC7392567.md @@ -0,0 +1,63 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +![0_image_3.png](0_image_3.png) + +![0_image_1.png](0_image_1.png) + +![0_image_4.png](0_image_4.png) + +# Is Whole-Mount Section In Rectal Cancer Effective For Measuring Lateral Margin? + +Jun Seong Chung, Han Deok Kwak, Jae Kyun Ju Department of Surgery, Chonnam National University Hospital, Gwangju, Korea + +## See Articles On Page 172-177 + +Total mesorectal excision (TME) was first described by Heald et al. [1] in 1982, and since then, TME has been the standard technique of rectal cancer surgery [2]. Since the introduction of this technique, it has contributed to reducing local recurrence and improving survival after rectal cancer surgery [3-5]. However, local recurrence remains a serious problem after rectal cancer surgery and affects the patient's quality of life [6]. Several studies reported that circumferential resection margin (CRM) involvement is a powerful predictor of local recurrence [6, 7]. Therefore, in order to evaluate the exact radical resection of rectal cancer, the accuracy of CRM involvement and quality of completeness of TME +are the most important [8, 9]. According to the 8th edition of the American Joint Committee on Cancer staging manual published in 2018, a new nodal stage called "N1c" has been added. N1c indicates that the local lymph nodes are not positive, but there are tumor deposits in the subserosa, mesentery, or perirectal/mesorectal tissues. This means that tumor invasion into mesorectal tissue affects local recurrence and prognosis of rectal cancer. Several studies [10, 11] suggested that whole-mount sections provide a more accurate and effective assessment of rectal cancer, especially in the spread of the mesorectal region of the discontinuous microscopic tumor nodules compared to a conventional section. In addition, all whole-mount sections showed the overall morphological characteristics of the surgical specimen including the primary tumor, the bowel wall and the mesorectum. In a recent study, whole-mount sections were used to analyze lateral margins Correspondence to: Jae Kyun Ju, M.D. Department of Surgery, Chonnam National University Hospital, 42 Jaebong-ro, Dong-gu, Gwangju 61469, Korea Tel: +82-62-220-6456, Fax: +82-62-227-1635 E-mail: jkju@jnu.ac.kr ORCID: https://orcid.org/0000-0003-1605-3310 +© 2020 The Korean Society of Coloproctology This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 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. + +and micrometastasis more accurately for radical resection of rectal cancer [12]. They categorized the patients with perirectal tissue invasion by the 3 groups according to the length of the lateral margin (LM: 1 mm, 1.5 mm, 2 mm). They analyzed recurrence, overall survival, disease specific survival and clinicopathologic features including lymphovascular invasion, perineural invasion and micrometastasis according to each group. In all groups, lymphovascular invasion was significantly frequent and the N state was higher when LM was short. There were more micrometastasis in group LM ≤1 mm (53.3% ≤1 mm vs. 26.6% >1 mm, P= +0.039). And overall survival was worse when LM was short in each analysis based on 1 mm and 1.5 mm (P <0.001). In all groups, disease specific survival was worse when LM was short. + +In summary, the results of improvement in local recurrence and survival through TME technique in the treatment of rectal cancer are obvious, but inadequate resection margin or incomplete CRM +causes poor prognosis of rectal cancer. Therefore, a precise pathologic examination method for accurate resection margin and incomplete CRM in rectal cancer is needed. According to recent study results, specimen analysis through the whole-mount section may be a more effective alternative to conventional section analysis for lateral margin measurement. Also, based on continuous additional studies, it may have a good effect on the prognosis of rectal cancer patients. + +## Conflict Of Interest + +No potential conflicts of interest relevant to this article were reported. + +## References + +1. Heald RJ, Husband EM, Ryall RD. The mesorectum in rectal cancer surgery-the clue to pelvic recurrence? Br J Surg 1982;69:613-6. 2. Reynolds JV, Joyce WP, Dolan J, Sheahan K, Hyland JM. Pathological evidence in support of total mesorectal excision in the management of rectal cancer. Br J Surg 1996;83:1112-5. + +3. Arbman G, Nilsson E, Hallböök O, Sjödahl R. Local recurrence following total mesorectal excision for rectal cancer. Br J Surg + +![1_image_0.png](1_image_0.png) + +1996;83:375-9. + +4. Heald RJ, Ryall RDH. Recurrence and survival after total mesorectal excision for rectal cancer. Lancet 1986;1:1479-82. + +5. Peeters KC, Marijnen CA, Nagtegaal ID, Kranenbarg EK, Putter H, Wiggers T, et al. The TME trial after a median follow‐up of 6years: increased local control but no survival benefit in irradiated patients with resectable rectal carcinoma. Ann Surg 2007;246: +693-701. + +6. Nagtegaal ID, Marijnen CA, Kranenbarg EK, van de Velde CJ, +van Krieken JH; Pathology Review Committee, et al. Circumferential margin involvement is still an important predictor of local recurrence in rectal carcinoma: not one millimeter but two millimeters is the limit. Am J Surg Pathol 2002;26:350-7. + +7. Agger EA, Jörgren FH, Lydrup MLA, Buchwald PL. Risk of local recurrence of rectal cancer and circumferential resection margin: population‐based cohort study. Br J Surg 2020;107:580-5. + +8. Quirke P, Steele R, Monson J, Grieve R, Khanna S, Couture J, et al. Effect of the plane of surgery achieved on local recurrence in patients with operable rectal cancer: a prospective study using data from the MRC CR07 and NCIC-CTG CO16 randomised clinical trial. Lancet 2009;373:821-8. + +9. Nagtegaal ID, Quirke P. What is the role for circumferential margin in the modern treatment of rectal cancer. Br J Surg 2009;96: +982-9. + +10. Shimada Y, Takii Y. Clinical impact of mesorectal extranodal cancer tissue in rectal cancer: detailed pathological assessment using whole-mount sections. Dis Colon Rectum 2010;53:771-8. + +11. Wang Z, Zhou Z, Wang C, Zhao G, Chen Y, Gao H, et al. Microscopic spread of low rectal cancer in regions of the mesorectum: +detailed pathological assessment with whole-mount sections. Int J Colorectal Dis 2005;20:231-7. + +12. Nam S, Hong Y, Choi YJ, Kang JG. Clinicopathologic analysis of lateral margin measured by whole-mount section in T3 rectal cancer. Ann Coloproctol 2020;39:172-7. \ No newline at end of file diff --git a/medical/md_images/PMC7392567/PMC7392567_meta.json b/medical/md_images/PMC7392567/PMC7392567_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..6dd43e7d719b53517c1649b1f67f77f2d7690c4e --- /dev/null +++ b/medical/md_images/PMC7392567/PMC7392567_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 2, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 0, + "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/PMC7692953/4_image_0.png b/medical/md_images/PMC7692953/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..84262ab95f168f53e08fdf8b190904519ebba5a3 --- /dev/null +++ b/medical/md_images/PMC7692953/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8b7a1246ebd1d3426f66c3e6c36c122415b61a28f1d4745baa213e21c71da981 +size 88228 diff --git a/medical/md_images/PMC7692953/PMC7692953.md b/medical/md_images/PMC7692953/PMC7692953.md new file mode 100644 index 0000000000000000000000000000000000000000..908129ec32d3b491c6071157fbead5db5a4a6a8d --- /dev/null +++ b/medical/md_images/PMC7692953/PMC7692953.md @@ -0,0 +1,281 @@ +# Heterogeneous Catalysis Hot Paper + +How to cite: Angew. Chem. Int. Ed. 2020, 59, 20973– 20979 International Edition: doi.org/10.1002/anie.202005616 German Edition: doi.org/10.1002/ange.202005616 + +# Scaling Platinum-Catalyzed Hydrogen Dissociation On Corrugated Surfaces Sabine V. Auras, Richard Van Lent, Dima Bashlakov +, Jessika M. Piçeiros Bastidas+, Tycho Roorda+, Rick Spierenburg+, And Ludo B. F. Juurlink* + +Abstract: We determine absolute reactivities for dissociation at low coordinated Pt sites. Two curved Pt(111) single-crystal surfaces allow us to probe either straight or highly kinked step edges with molecules impinging at a low impact energy. A model extracts the average reactivity of inner and outer kink atoms, which is compared to the reactivity of straight A- and Btype steps. Local surface coordination numbers do not adequately capture reactivity trends for H2 dissociation. We utilize the increase of reactivity with step density to determine the area over which a step causes increased dissociation. This step-type specific reactive area extends beyond the step edge onto the (111) terrace. It defines the reaction cross-section for H2 dissociation at the step, bypassing assumptions about contributions of individual types of surface atoms. Our results stress the non-local nature of H2interaction with a surface and provide insight into reactivity differences for nearly identical step sites. + +## Introduction + +For gas-phase reactions involving the smallest of molecules, H2, accurate theoretical descriptions are nowadays available and match experimental results. + +[1] However, the same remains challenging for gas-surface reactions. + +[2] The involvement of many atoms in the surface complicates the description of energy dissipation mechanisms, amongst others. The chemically accurate prediction of reactivity for the highly activated H2 dissociation on Cu(111), using quantum dynamics calculations and density functional theory (DFT)- +based potentials, is exceptional.[3] In other cases, simplifica- +[*] S. V. Auras, Dr. R. van Lent, J. M. PiÇeiros Bastidas,[+] T. Roorda,[+] +R. Spierenburg,[+] Assoc. Prof. L. B. F. Juurlink Leiden Institute of Chemistry, Leiden University P.O. Box 9502, 2300 RA Leiden (The Netherlands) E-mail: l.juurlink@chem.leidenuniv.nl Dr. D. Bashlakov[+] +ILTPE, National Academy of Sciences of Ukraine 47 Nauky Ave., Kharkiv 61103 (Ukraine) +[ ++] These authors contributed equally to this work. + +Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/anie.202005616. T 2020 The Authors. Published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial 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. + +tions in the description of the system are needed to lower the computational effort, and show continuing advancements. A +relevant example is the successful application of quasiclassical trajectory calculations for the more reactive H2 dissociation on Pt(111).[4] +Reactions on high Miller index surfaces, such as regularly stepped surfaces, have often been claimed to be more relevant to real-world chemistry, for example, heterogeneous catalysis. Such vicinal surfaces contain lower coordinated surface atoms, for example, forming steps in terraces and kinks in steps, which resemble edges and corners on actual catalyst particles. Accurate calculations based on periodic potentials then require larger unit cells, hence much higher computational effort. The chemically accurate description of H2 dissociation on Cu(211) is the state of the art.[5] The dissociation of a larger molecule, methane, can be described with chemical accuracy by ab initio molecular dynamics on select Pt surfaces, but the same approach does not describe reaction on a kinked surface to the same accuracy. + +[6–8] +The computational effort required to accurately describe the simplest dissociative event on well-defined surface structures highlights the challenges to constructing an accurate kinetic description for heterogeneous catalysis. Different methods are necessary to consider all surface sites available on a catalyst particle simultaneously. A recent addition to various approaches (see Ref. [9] for a thorough discussion) is the idea that one may generalize the reactivity dependence of surface sites on their coordination number.[10–12] Coordination numbers are commonly used in organic and inorganic chemistry. Consequently, it can seem intuitively logical to apply it to gas-surface interactions. To capture different surface geometries of nearly identical surface sites,ageneralized coordination number, CN, was introduced. It includes the coordination shell of all atoms neighboring the surface atom involved in the dissociation event. This concept was successfully applied in a study of bond energies of H- and Ocontaining adsorbates on Pt particles. The authors suggested it to be a predictive descriptor linking geometric arrangement of a surface to adsorption properties. As CN can simply be calculated by hand, it does not require electronic structure calculations. + +Surface coordination approaches shift the focus in our chemical thinking about bond breaking from the impinging molecule probing a surface potential, towards the local properties of the site of reaction. While for many systems, this may be acceptable and appropriate, we wonder whether it applies to the dissociative adsorption of H2. First, the two types of straight step edges occurring on (111) facets of fcc Angew. Chem. Int. Ed. 2020, 59, 20973–20979 T 2020 The by GmbH 20973 metals, for example, Pt or Ni, provide the same coordination numbers at the upper step edge, but are found to be chemically unique in desorption of H2 (see, for example, Refs. [13,14]). Second, the quantum dynamical nature of the molecule may allow it to probe the surrounding potential energy surface (PES) bypassing the highly localized nature of the collision as imposed by a coordination number approach. + +Dynamical calculations for H2interaction with Pt(211) by Baerends and co-workers suggested this: At low collisional energy, dissociation predominantly occurs via a dynamical chemisorbed molecular state that dissociates barrier-free at the upper edge of a step even though its center of mass impinged at the inner cusp. + +[15, 16] +Recent advances in the use of curved surfaces of single crystal samples allow us to critically evaluate the relation between dissociative reaction dynamics and the structure of the surface. + +[17,18] Here, we use two Pt curved single crystals exposing surface atoms with various coordination numbers and local surface geometries to determine whether the dissociation of molecular hydrogen on platinum follows trends predicted by coordination number approaches. We experimentally determine the dissociation probability for molecular hydrogen along the curvatures and relate the absolute reactivity to the local structure of steps and kinks. + +## Results And Discussion + +We impinge D2from a molecular beam onto the curved + +![1_image_0.png](1_image_0.png) + +surfaces of two Pt crystals to determine the initial sticking probability (S0). As D2 chemisorbs dissociatively at the applied surface temperature of 155 K, sticking probabilities correspond to dissociation probabilities. The schematics of the curved samples and the relative size of the beam impinging on the surface with a rectangular profile are shown in Figure 1 a. Both Pt crystals contain the (111) surface at their apex and curve 15.58 to either side. The macroscopic curvature of the Pt crystals results from the introduction of a single step type with smoothly increasing density when moving toward the crystalQs edges. The supersonic beam always impinges normal to the (111) terraces. + +The two curved surfaces contain three different step structures. On the crystal with a [11¯0] rotational axis, (001)- +oriented (or A-type) steps and (110)-oriented (or B-type) steps occur separately on the two sides of the apex. The second crystal has its rotational axis oriented along [112¯], +exhibiting facets of the (201) and (021) planes, forming kinked steps. As their direction lies halfway in between A- and Btype steps, the kinked steps consist of short, alternating sections of A- and B-steps connected by inner and outer kinks. The kinked steps give rise to chiral stepped surfaces with opposite handedness on the two sides of the crystal. The chirality is not expected to impact reactivity for the achiral H2. + +We therefore refer to the kinked steps on either side of the crystal as {210}-oriented steps (we use Miller indices {hkl} to indicate the set of equivalent surfaces). A- and B-type and kinked step structures in their ideal form are shown in Figure 1 b. Note that we show the fully kinked step facets for the {210} case. + +Figure 1 c plots S0 of D2 as a function of step density for an average kinetic energy of 9.3 meV. The hatched area indicates where the crystal surface becomes more narrow than the beam due to the shape of our samples. The data are averages of multiple measurements on the same location of a curved surface. Error bars reflect one standard deviation of the average value. For the crystal with kinked steps, data from both sides of the crystal are included, but do not vary appreciably. At the (111) apices of the two crystals we measure an S +ð111Þ +0 of 0.011 : 0.018 and 0.032 : 0.013 respectively, in good agreement with previous studies on flat Pt(111) +surfaces for similar collision energies. + +[19–21] To the sides of the crystals, S0increases linearly with step density for all step types over the range where the molecular beam fits onto the crystal surface. Scalability of the reactivity of stepped surfaces was previously suggested for A-type steps. + +[15] Here S0 can be described for all stepped surfaces as [Eq. (1)]: + +## S0Ðsdþ ¼ S Ð111Þ 0 Þ S0? Sd Ð1Þ + +where SD is the step density and S0the rate of increasing S0 with step density (that is, the slope). S0 quantifies the reactivity of the step type at the incident energy used here (see the Supporting Information for a detailed discussion on the relation of S0 and the sticking probabilities at steps S +step 0). + +We apply linear fits to the data with appropriate weighting to compare S0for these step types. Here, we exclude data from the hatched area and fix S +ð111Þ +0 at the average of the two values determined for (111) on the two separate single crystals, that is, 0.025. For the crystal with kinked steps, we use data of both sides in a single fit. The resulting linear fits are displayed with colors matching the data. The extracted values for S0 are listed in Table 1. While S0for B-type and {210} steps are almost equal, that is, about 0.6 nm, the A-type steps show a significantly smaller increase in reactivity with step density, that is, about 0.4 nm. + +While it is sometimes assumed that kinks would be more reactive than straight step edges, which in turn would be more reactive than flat terraces, our data at first glance does not represent this trend. However, the S0 value we obtain for the +{210} steps here does not necessarily reflect dissociation at ideal fully-kinked step. The kinked steps present on the surface may differ from the representation provided in Figure 1 a. The surface can maintain its overall crystallographic orientation while breaking the fully kinked step into larger segments (or microfacets) of A- and B-type steps, as observed for kinked Ag steps. + +[22] Figure 2 a illustrates two slightly longer segments. The number of kink sites is reduced from the fully kinked edge (section a) by introducing either 1 atom (section b) or 2 atoms (section c) in between kinks. Increasing the segment lengths decreases the kink density. To better understand the observed reactivity trend, we hereinafter determine the structural composition of the {210} steps using STM and apply a model that deconvolutes the contribution of inner and outer kinks. + +Typical STM images with step arrays ranging from a step density of 0.086 nm@1to 0.909 nm@1are shown in Figure 2 b–e. + +| Table 1: Step types and their coordination (CN) and generalized coordination numbers (CN) at the upper step edge.[a] Step type Coordination (upper S0 [nm] a [nm] s0 [nm2 ] edge) CN CN A/ (001) 7 5.50 0.387 0.277 0.107 B/ (110) 7 5.50 0.597 0.277 0.165 real kinked/ {210} 0.593 0.48 0.285 fully kinked (max) 8*/ 6 + 6.17*/ 4.83+ 0.726 0.48 0.348 [a] For the kinked step coordination numbers for the inner (*) and outer | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +A/ (001) 7 5.50 0.387 0.277 0.107 B/ (110) 7 5.50 0.597 0.277 0.165 real kinked/ {210} 0.593 0.48 0.285 fully kinked (max) 8*/ 6 ++6.17*/ 4.83+0.726 0.48 0.348 +[a] For the kinked step coordination numbers for the inner (*) and outer +(+) kinks are listed. Rates of increasing sticking with step density (S0) +are determined by linear fits of the S0 data in Figure 1 for the first three rows. The value of S0in the bottom row is derived from Equation (4). Multiplying S0 with unit cells widths of the stepped surfaces (a) gives the resulting reaction cross sections (s0) of the three types of steps. Note that the unit cell width is considerably larger for the kinked step. + +We determine the outline of the step (shown as colored lines) +by taking line profiles and determining the local height gradient. We then determined the length of all segments separated by inner and outer kinks along the step edge. This process is repeated for multiple steps in the image. Histograms on the right side of Figure 2 are normalized to represent the average percentage of segments with a length of one, two, three atoms, and so on. They are shown separately for both A- (blue) and B-type (red) microfacets. Each consecutive bin in the histograms represents a microfacet of one additional atom length. The lettering of the bins corresponds to the sections indicated in Figure 2 a. + +The histograms in Figure 2 show that approximately half of the segments forming {210} steps on Pt are ideal, that is, they are fully-kinked. Roughly 40 to 60% of the sections are in bin a. The remaining sections are longer, but very rarely do we observe segments of more than 5 atoms. All histograms show a similar smooth decrease in observed frequency with microfacet length from bin b onward. The distributions are independent of the average terrace width over the probed range of 43.0 atom rows in Figure 2 b to 4.1 atom rows in Figure 2 e. This confirms that the linear increase in S0 observed in Figure 1 does not result from changes in the structure of {210} steps, but is exclusively caused by increasing step density. Furthermore, the distributions of A- and B-type facets are similar and suggest no preference for longer or shorter segments of one microfacet type despite varying line free energies of A- and B-type steps. + +[23] +From the distributions in Figure 2 and the atomic compositions for microfacets with varying lengths (Supporting Information, Table S2), we calculate the average number of kink (NK), A-type (NA), and B-type (NB) atoms in an edge consisting of 100 segments in {001} orientation and 100 segments in {110} orientation. The total number of atoms in these 200 segments is given in Equation (2): +N ¼ NA þ NB þ NK ð2Þ +The fractional occurrence of kinks along a step is given by NK/N. It consistently lies between 0.4 and 0.5 in all STM +images (see the Supporting Information). Assuming that additional atoms in microfacets exhibit the reactivity of the A- (blue) or B-type steps (red), we now define the reactivity of {210} steps (S +f210g 0) as a weighted average of reactivities of kink atoms, A-type atoms (S +A +0 +), and B-type atoms (S +B +0 +) in all segments and set it equal to the experimentally determined value from Table 1 [Eq. (3)]: + +## S F210G 0 ¼ Nks K 0 Þ Nas A 0 Þ Nbs B 0 N¼ 0:593Nm Ð3Þ + +We solve for S +K +0using the data from individual STM +images and the already known values for S +A 0 and S +B 0 +. Table S3 in the Supporting Information lists the results. The average value of S +K +0is 0.726 nm. As our model enforces lower reactivities for all A- and B-type atoms in longer segments of kinked steps, it is the upper limit for the average reactivity of inner and outer kinks [Eq. (4)]: + +``` +S + K + 0 ¼ ðS + Kin + + 0 þ S + Kout + + 0Þ=2 ¼ 0:726nm ð4Þ + +``` + +Angew. Chem. Int. Ed. 2020, 59, 20973–20979 T 2020 The by GmbH 20975 + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +Figure 2. a) Kinked step edge with different widths of its {001}- and {110}-oriented segments. In a, segments are only one atom wide. Each atom +is either at an inner or an outer kink site (purple). In b, two atom wide segments are formed. Half of the atoms are still kink sites, but in each This maximum reactivity for the ideal or fully kinked step edge is shown in Table 1 and used to define the upper limit of average kink reactivity in Figure 3 (light purple line). This graph also shows a lower limit. It is derived from assuming that the facets along the kinked step edge act as type A and B steps with no additional reactivity of kinks. This situation is identical to a {210} step edge that is fully reconstructed, containing large stretches of both the A-type and B-type steps (with a kink density comparable to that typically occurring in A- or B-type steps). Its slope is given by [Eq. (5)]: + +## 1 + +2 + +## ? Ðs A 0 Þ S B 0 + +Þ ? 1:16 ¼ 0:570nm ð5Þ +The factor 1.16 accounts for the increased length of the step compared to a straight line connecting the first and last atom of a {210} step. The range of S +K +0 as defined by these two extremes is indicated as the purple shaded area (in between the lines representing no kinks and only kinks) in Figure 3 b. + +The reactivity of the kink sites may hence indeed exceed that of straight step edges, as expected by the reduced number of nearest numbers at the outer kinks. The lower reactivity of the A-type step, however, remains unexpected from a coordination number viewpoint. As listed in Table 1, the number of nearest neighbors (coordination number CN) is identical for atoms forming the A- and B-type steps, that is, CN = 7. On fully kinked steps, CN varies for the inner and outer kinks, being 8 and 6 respectively. Depending on their relative contribution to reactivity, their weighted average may coincide or deviate from straight steps. Based on CN alone, however, it cannot coincide with the B-type and deviate from the A-type. In fact, the difference in reactivity for the A and B-types already disagrees with CN. The inconsistency is also not resolved when considering coordination of atoms in the lower plane of the step, as discussed in the Supporting Information. + +As CN fails to predict the reactivity trend, we verify whether the generalized coordination number, CN, captures it. CN averages over all CN values belonging to the nearest neighbors of an atom, hence capturing more of the surroundings of the surface atom involved in the chemical event.[10] +Table 1 presents values for CN. Again, reactivity is not represented properly. On the basis of upper edge atoms alone, the three edge types would be identical in reactivity if the average of inner and outer kinks is used. If one considers only the outer kinks, then kinked edges should have been considerably more reactive than both A- and B-type edges and the latter should have been identical in reactivity. Although our analysis suggests that fully kinked steps are likely more reactive than B-type steps, approaches considering reactivity of each atom site individually continue to fail representing the clear variations in reactivity of all three step types. + +Abandoning atomistic approaches, where we consider the local reactivity of each surface atom, we swing our viewpoint back to a non-local nature of the interaction of D2 with the stepped surface as suggested by earlier dynamical calculations. Earlier experimental and theoretical dynamics studies using vicinal Pt surfaces suggest that three dissociation mechanisms occur in parallel.[15, 24–26] First, dissociation at the upper edge is direct and non-activated. Second, molecules impinging into the bottom cusp of the step may be dynamically trapped and react by following shallow potential wells leading toward the upper edge. Third, molecules impinging on (111) terrace sites encounter modest activation barriers. While the value of S +ð111Þ +0 was shown to be small at low collision energies, a linear combination of three contributions explains the observed dependence on step density shown in Figures 1 and 3. The slope (S0) then gives the average length +(measured normal to the edge) over which the step contributes to reactivity. It represents the cross section for the dissociation mechanisms related to steps, but reduced to a single spatial dimension. + +Consequently, multiplying this length by the unit cell width (a) yields the initial reaction cross section (s0) +[Eq. (6)]: +s0 ¼ S0 > a ð6Þ + +![4_image_0.png](4_image_0.png) + +The values we obtain for s0 per step type are included in Table 1. They are also illustrated as white areas at the different edge types in Figure 3 a. They may be compared to the area of molecular wells and the holes leading to nonactivated dissociation in a PES. For the A-type, we have shown previously that it corresponds well to the reactive area of the PES calculated for H2/Pt(211).[17] Our data suggests sizes of these areas for the B-type step and the corrugated {210}-type step. Both extend further onto the lower terrace than for the A-type step. Note that the larger s0 value for the +{210} steps in Table 1 is partially caused by a larger unit cell width a. + +The step-type dependence of s0for D2 colliding at less than than 10 meV in kinetic energy indicates that the distance over which steps may induce dynamical trapping and/or barrier-free dissociation critically depends on the local atomic arrangement in the step. For the A-type step, dissociation on the upper edge was found to be entirely barrier-free. + +[15] It is likely that the upper edges for the B-type and kinked edges also have these holes in the potential. The difference we find for the various edges must then result from the capability to trap molecular hydrogen in the cusp of the step. For A-type steps, the molecular adsorption wells that cause trapping with subsequent dissociation are then the smallest. B-type steps trap and dissociate molecular hydrogen over a longer distance. This may be caused by the relative shifts of the upper and second layers of Pt atoms at these steps. For the B-type step, the lateral shift is 2/3 of the distance between atom rows parallel to the edge, whereas it is only 1/3 for the A-type. Kinks appear to cause an even larger disturbance to the local potential with deeper and/or wider wells. + +Finally, in Figure 3 b, we extrapolate the fits with slope S0 to predict reactivity beyond the range offered by our curved crystals to the highest possible step density for each step type, as indicated by the dashed lines. Our predictions for A-type steps agree reasonably well with the only published data obtained for surfaces with very short terraces, added as blue squares in Figure 3 b. + +[24] +Surfaces featuring short terraces of only a few atom rows, such as those that we approach with the extrapolation in Figure 3 b are particularly interesting to heterogeneous catalysis, as they are featured on the surface of real nanoparticles. + +[27] Only the equilibrium shape of very small metal particles of < 1 nm size one would expect to find sharp edges that do not feature a cusp to the adjacent terrace. There, the reactivities may vary from the ones derived here, due to the absence of dynamical trapping at the cusp. An impinging H2 wavepacket would thus experience a smaller cross-section with such an edge than the ones derived here. For larger Pt nanoparticles, as well as regularly stepped Pt surfaces, we expect the chemical step size S0to be a reliable descriptor of reactivity towards D2 dissociation. + +## Conclusion + +Our examination of Pt-catalyzed hydrogen dissociation on surfaces with close-packed and highly-kinked steps yields reactivities for different surface structural features. STM +analysis of kinked steps allows us to extract an average reactivity for inner and outer kinks. Results show that kinks likely aid dissociation more than atoms in straight steps. However, the measured reactivity difference for the two close-packed step types is not adequately captured by models that rely on counting (nearest) neighbors of various types of surface atoms. We believe that the origin of the discrepancy is the dynamical nature of the interaction. For a light-weight molecule interacting at low collision energy with a surface that is characterized by (at most) modest barriers to dissociation, molecular wells close to the dissociation site increase the dissociation probability. We therefore consider the interaction in terms of a cross section, s0. It reflects the physical area near the step where impingement leads to trapping and dissociation as found in earlier theoretical dynamics studies. The average length of this area, S0, measured normal to the step, is derived directly from the experimentally determined dependence of reactivity on step density. We believe that the reactivities determined here, including the rule of thumb resulting from extrapolating experimental data to the smallest unit cells, provide a useful tool for modeling dissociation on vicinal surfaces with potentially large unit cells. Furthermore, they allow careful prediction of reactivities on industrially relevant catalyst sites by bridging the whole range of stepped Pt surfaces. + +## Acknowledgements + +This work was financially supported by the Leiden Institute of Chemistry and the Netherlands Organisation for Scientific Research. The authors thank G.J. Kroes and H.F. Busnengo for helpful discussions. + +## Conflict Of Interest + +The authors declare no conflict of interest. Keywords: corrugated surfaces · heterogeneous catalysis · reaction cross-sections · reaction mechanisms · surface chemistry + +[1] H. C. Schewe, Q. Ma, N. Vanhaecke, X. Wang, J. K. los, M. H. Alexander, S. Y. Van De Meerakker, G. Meijer, A. + +Van Der Avoird, P. J. Dagdigian, J. Chem. Phys. 2015, 142, 204310. + +[2] G. J. Kroes, C. D&az, Chem. Soc. Rev. 2016, 45, 3658 - 3700. + +[3] C. D&az, E. Pijper, R. A. Olsen, H. F. Busnengo, D. J. Auerbach, G. J. Kroes, Science 2009, 326, 832 - 834. + +[4] E. Nour Ghassemi, M. Wijzenbroek, M. F. 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+|----------------------------------------------------------------------------------------|------------| +| Keywords: Urofacial syndrome Ochoa syndrome Voiding dysfunction Neurogenic bladder | Dr. Ochoa proposed a condition known as Urofacial syndrome (UFS) which is characterized by the abnormal facial expression while smiling along with dysfunctional lower urinary tract in 1987. This study investigated 7 years old boy who presented with recurrent urine retention, dysuria, and inversion facial expression. Radiological and genetic work-up diagnosed him with UFS. | + +## Introduction + +In 1987, Ochoa and Gorlin described a syndrome in a group of children with congenital obstructive uropathy. It is a rare autosomal recessive disease. It includes complications like urinary tract infection +(UTI), urgency, enuresis, constipation, bladder trabeculation, and vesicoureteral reflux (VUR). Additionally, inversion of facial expression while smiling or laughing is also characteristic of the syndrome.1,2 Although its rarity, this condition has to be well thought-out in the differential diagnosis of kids with enuresis and recurrent UTI. + +We are reporting this case due to its rarity, increase the awareness about the special facial characteristic to facilitate the diagnosis and to share our experience with the management and outcome in such cases. + +## Case Presentation + +A 7-year-old boy was referred to the outpatient department of our hospital as a case of recurrent urine retention, incontinence, dysuria, and constipation. On physical examination, a characteristic of inversion facial expression was noticed (Fig. 1), palpable bladder, and abdomenpelvic ultrasound showed severe bilateral hydronephrosis with thinning of the parenchyma and distended bladder with irregular outline (Fig. 2). Upon admission, the creatinine level was 1.65 mg/dL and for that Foleys catheter was inserted. Later, voiding cystourethrogram (VCUG) showed thickened trabeculated bladder with irregular outline (pine cone bladder), good capacity with increased sphincter tone and left grade 4 VUR (Fig. 3). Mercaptoacetyltriglycine (MAG3) scan was conducted with Foley catheter showed severe renal impairment with bilateral renal poor function. After two weeks of management with the nephrology team, creatinine came down to 1.23 mg/dL and hydronephrosis slightly improved. Cystoscopy was done and showed normal anterior urethra, wide posterior urethra, normal external sphincter, no posterior urethral valve, severely trabeculated bladder with multiple saccules, and diverticula. Genetic testing showed, a homozygous pathogenic variant that was identified in the HPSE2 gene, this confirmed the diagnosis of USF. The patient has a positive family history with one similarly affected sibling, consanguineous parents (second cousins). The parents were counseled about clean intermittent catheterization and discharged home. During follow up with a regular bedside US, the hydronephrosis is slightly improved also the creatinine level back to a normal level. The patient planned for urodynamic study and appendicovesicostomy in the future. + +## Discussion + +UFS is characterized by detrusor - sphincter discoordination, severe bladder dysfunction with a distorted facial expression while smiling. Although UFS is rare, it is crucial to distinguish this problem in patients with repetitive UTI, constipation, and incontinence.3 The relationship between distorted facial expression and voiding dysfunction remains unclear. It denotes a discrete article. The Center of weeping and snickering are situated at the upper pone midbrain Available online 14 April 2021 2214-4420/© 2021 The Author(s). 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/). + +![1_image_1.png](1_image_1.png) + +proximally to the ordinary micturition center. So, it hypothesized that neurologic lesion in this area would probably affect both regions, which is resulted in this rare syndrome.4 It is worthy of note, that an abnormal facial expression is related to a defect in the coordination between the facial muscle. Interestingly, brain magnetic resonance images (MRI) of UFS patients were unremarkable without lesions in the brain or normal spine revealed non-neurological pathogenesis of disease course.5 Genetically, UFS is autosomal recessive disorder, which is a heterogeneous condition resulting from both alleles of a gene pathogenic variants in either *HPSE2 (*inactive heparanase-2) or *LRIG2 (*Leucine-rich repeats and immunoglobulin-like domains protein 2).1 Mapping of the causative gene located in the HPSE2 gene at chromosome 10q23-q24, and the LRGI2 gene located at chromosome 1p13.2.4 The molecular pathogenesis currently is unknown. Previously, it had been thought that UFS feature could result from abnormality in the central nervous system.2 However, recently it is considered more likely that UFS syndrome related to an abnormality in the peripheral neurodevelopment or function because both genes (HPSE2 and LRIG2) are localize in the peripheral nervous system.5 + +![1_image_0.png](1_image_0.png) + +![1_image_2.png](1_image_2.png) + +2 Todate, no conventional diagnostic criteria has been published yet, but the diagnosis can be determined by the presence of the clinical feature involving the urinary bladder and facial feature in addition to identification of the pathogenic variants in either HPSE2 or *LRIG2*. + +1 The severity of the disease is a wide spectrum range from mild +(represent facial finding with mild or no voiding dysfunction symptoms) +to severe obstructive feature, including urine retention, high post-void residual, hydroureteronephrosis, VUR, and renal insufficiency.3 Which is demonstrated in our patient. + +Early recognition of UFS is crucial to prevent deterioration of the urinary tract through simple physical examination of the facial features and to consider this syndrome in the differential diagnosis. + +The mainstay of treatment is to prevent urinary tract deterioration by control bladder emptying. As we did in our patient by using clean intermittent catheterization. The administration of drugs can be added based on urodynamic study findings. Surgical intervention such as bladder augmentation or urinary diversion is an alternative to prevent further damage to the kidneys.2,3 Which is a long-term treatment planned for our patient. + +## Conclusion + +USF is autosomal recessive disease, non-neurogenic bladder dysfunction, can be suspected simply from distorted facial expressions. Early recognition of this syndrome and treatment will prevent further deterioration in renal function. + +## References + +1. Newman WG, Woolf AS. Urofacial syndrome. In: Adam MP, Ardinger HH, Pagon RA, +et al., eds. GeneReviews® *[Internet]*. Seattle (WA): University of Washington, Seattle; 2013:1993–2021. PMID: 23967498. + +2. Ochoa B. Can a congenital dysfunctional bladder be diagnosed from a smile? The Ochoa syndrome updated. *Pediatr Nephrol*. 2004;19(1):6–12. https://doi.org/ +10.1007/s00467-003-1291-1. + +3. Stamatiou K, Karakos C. Urofacial syndrome: a subset of neurogenic bladder dysfunction syndromes? *Indian J Urol*. 2010;26(4):582. https://doi.org/10.4103/ 0970-1591.74469. + +4. Al-Qahtani FN. Ochoa syndrome: new features. *Saudi J. Kidney Dis.Transpl.* 2003;14 +(1):61. + +5. Tu Y, Yang P, Yang J, et al. Clinical and genetic characteristics for the urofacial syndrome (UFS). *Int J Clin Exp Pathol*. 2014;7:1842–1848. \ No newline at end of file diff --git a/medical/md_images/PMC8082552/PMC8082552_meta.json b/medical/md_images/PMC8082552/PMC8082552_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..de3193505af4e8da8135300870bf565a599ce46d --- /dev/null +++ b/medical/md_images/PMC8082552/PMC8082552_meta.json @@ -0,0 +1,106 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Urofacial syndrome: Uncommon and unforeseen cause of lower urinary tract dysfunction in children", + "level": 0, + "is_closed": false, + "n_kids": 5, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 629.5350341796875, + 0.0 + ] + }, + { + "title": "Introduction", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 431.0329895019531, + 0.0 + ] + }, + { + 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+1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:7d1378e0acf98fea52d242e14fa7b05c8bd176b543029b10abeeeb0a3a27bb88 +size 43503 diff --git a/medical/md_images/PMC8810022/PMC8810022.md b/medical/md_images/PMC8810022/PMC8810022.md new file mode 100644 index 0000000000000000000000000000000000000000..26e162c0a5a66b65c9a2aafe06aadd4ca69d1284 --- /dev/null +++ b/medical/md_images/PMC8810022/PMC8810022.md @@ -0,0 +1,246 @@ +# Human Umbilical Vein Endothelial Cells Derived-Exosomes Promote Osteosarcoma Cell Stemness By Activating Notch Signaling Pathway + +Jian Yanga,\#, Yong Hua,\#, Lu Wangb, Xiangran Suna, Ling Yua, and Weichun Guoa aDepartments of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei, China; bDepartments of Gynecology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China ABSTRACT +Osteosarcoma is one of the most common primary malignant tumors of bone in adolescents. + +Human umbilical vein endothelial cells (HUVECs) derived exosomes are associated with osteosarcoma cell stemness. Little is known about the function of HUVECs-exosomes in osteosarcoma cell stemness. This work aimed to investigate the mechanism of action of HUVECs-exosomes in regulating stem cell-like phenotypes of osteosarcoma cells. HUVECs were treated with GW4869 (exosome inhibitor). Human osteosarcoma cells (U2OS and 143B) were treated with HUVECs supernatant, HUVECs-exosomes with or without RO4929097 (γ secretase inhibitor, used to block Notch signaling pathway). We found that HUVECs supernatant and HUVECs-exosomes enhanced the proportions of STRO-1+CD117+ cells and the expression of stem cell-related proteins Oct4 and Sox2. Both HUVECs supernatant and HUVECs-exosomes promoted the sarcosphere formation efficiency of U2OS and 143B cells. These stem-like phenotypes of U2OS and 143B cells conferred by HUVECs-exosomes were repressed by GW4869. Moreover, HUVECs-exosomes promoted the expression of Notch1, Hes1 and Hey1 in the U2OS and 143B cells. RO4929097 treatment reversed the impact of HUVECs-exosomes on Notch1, Hes1, and Hey1 expression by inhibiting Notch1 signaling pathway. In conclusion, this work demonstrated that HUVECs-exosomes promoted cell stemness in osteosarcoma through activating Notch signaling pathway. Thus, our data reveal the mechanism of HUVECs-exosomes in regulating cell stemness of osteosarcoma, and provide a theoretical basis for osteosarcoma treatment by exosomes. + +ARTICLE HISTORY + +![0_image_0.png](0_image_0.png) + +Received 23 August 2021 Revised 5 November 2021 Accepted 5 November 2021 KEYWORDS +Human umbilical vein endothelial cells; exosomes; cell stemness; Notch signaling pathway; osteosarcoma + +## Introduction + +Osteosarcoma is a kind of mesenchymal tissue tumor, and one of the common primary malignant tumors of bone in adolescents [1]. Osteosarcoma has obvious heterogeneity, high invasive ability, and the tendency for early metastasis [2]. Cancer stem cells (CSCs) are cells that have the potential to regenerate tumors. Osteosarcoma cells with stem cell-like characteristics are closely associated with the malignant phenotype of osteosarcoma [3]. Clarifying the regulation mechanism of stem cell-like phenotype in osteosarcoma cells may be of great significance for seeking new therapeutic strategies for osteosarcoma. + +CSCs rely on the microenvironment to maintain their biological characteristics [4]. It also continuously affects and modifies the microenvironment. + +The vascular microenvironment of CSCs includes some cells located next to vein endothelial cells, + +![0_image_1.png](0_image_1.png) such as CSCs, vein endothelial cells, immune cells, and fibroblasts. Among them, endothelial cells have a crucial role in the tumor microenvironment, and have the ability to promote tumor metastasis [5]. Osteosarcoma is a malignant vascular tumor, and osteosarcoma cells can undergo hematogenous metastasis through the bloodstream. Thus, it is very important to study the interaction between vein endothelial cells and stem cell-like phenotypes in osteosarcoma cells. Previous studies have reported that the vein endothelial cell supernatant induces tumor cells to acquire a CSC-like characteristics [6,7]. Moreover, Lu et al. have found that endothelial cells activate neurogenic locus notch homolog protein 1 (Notch1) signaling pathway by secreting CONTACT Weichun Guo guo_wc@126.com Departments of Orthopedics, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan, Hubei 430060, China +\#These authors considered to be co-first authors. + +Supplemental data for this article can be accessed here. + +soluble form of Jagged-1, which contributes to promote the stem cell-like characteristics of colorectal cancer cells [8]. In glioma, the vein endothelial cells in the tumor microenvironment promote stemness of glioma cells and accelerate the progression of glioma by activating the Hedgehog signaling pathway [9]. Vein endothelial cells also induce glioma cells to differentiate into stem celllike glioma cells through secreting bFGF [10]. In liver cancer, lymphatic endothelial cells promote the self-renewal and immune escape of liver cancer stem cells through secreting IL-17A [11]. Whether endothelial cells can affect osteosarcoma cells is still unknown. + +Additionally, endothelial cells also affect the function of target cells in a paracrine or endocrine manner by producing exosomes [12,13]. Exosomes are one of the main components of the tumor microenvironment, which carries biomolecules such as proteins, RNA, lipids. Exosomes interact with target cells, participate in cell communication, and guide various physiological and pathological processes. Previously, we have found that the activated Notch signaling pathway enhances the expression of cancer stem cell surface markers (cluster of differentiation 9, CD117; stromal cell antigen 1, Stro-1) and stem cell-related genes (octamer-binding transcription factor 4, Oct4; SRY-box transcription factor 2, Sox2), and promotes stem cell-like characteristics in osteosarcoma cells +[14]. Whether exosomes can affect stem cell-like characteristics of osteosarcoma cells by regulating Notch signaling pathway has not been reported. + +We hypothesized that human umbilical vein endothelial cell (HUVEC)-derived exosomes may regulate cell stemness of osteosarcoma by regulating Notch signaling pathway. Thus, the aim of this work was to investigate the functional role of HUVECs-exosomes in regulating cell stemness of osteosarcoma. This work confirmed that HUVECs can activate the Notch signaling pathway through exosome manner, thereby regulating the stem celllike characteristics in osteosarcoma. + +## Materials And Methods Cell Culture + +HUVECs and human osteosarcoma cell lines (U2OS and 143B) were purchased from China Centre for Type Culture Collection (CCTCC; Wuhan, China). These cells were cultured in Dulbecco's Modified Eagle's medium (DMEM; Gibco, Camarillo, CA, USA) containing 10% +fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Solarbio, Beijing, China). Cells were cultured in a constant temperature incubator at 37°C and 5% CO2. + +HUVECs were incubated with 10 μM +GW4869 (exosome inhibitor; Sigma-Aldrich, St. Louis, MO, USA) or 0.1% DMSO for 24 h. + +GW4869 is a cell-permeable, noncompetitive NSmase (neutral sphingomyelinase) inhibitor. GW4869 blocks the ceramide-mediated sprouting of multivesicular bodies, thereby inhibiting the release of exosomes from multivesicular bodies. Thus, GW4869 is a commonly used as an exosome inhibitor [15]. For co-culture, U2OS and 143B cells were incubated with the HUVEC supernatant, HUVEC-derived exosomes (VECs-Exo) or PBS. U2OS and 143B +cells were treated with 1 mM RO4929097 (γ secretase inhibitor; Sigma-Aldrich) for 24 h. + +The Notch receptor is cleaved by furin convertase, ADAM and γ-secretase to produce Notch intracellular domain. The Notch intracellular domain is released to the nucleus and then activates the transcription of related genes +[16]. Thus, RO4929097 blocks the Notch signaling pathway by inhibiting the activity of γ secretase. + +## Isolation And Identification Of Exosomes + +Exosomes were isolated from HUVEC supernatant by ultracentrifugation as previous reported +[17]. HUVECs were seed into six-welll plates and cultured in exosome-free DMEM at 37°C +and 5% CO2 for 24 h. HUVEC supernatant was collected by centrifugation, and then filtrated through a 0.22 µm filter to remove the debris. To collect the pellets, the HUVEC supernatant were centrifuged at 200,000 g, 4°C for 2 h. The collected pellets were washed with PBS for several times, and then dissolved in PBS. The ultrastructure and particle size of BMSCs-exosomes was analyzed using transmission electron microscopy (Thermo Fisher Scientific, Waltham, MA, +USA) or NanoSight nanoparticle tracking analysis (NanoSight, Salisbury, UK). The expression of exosome positive proteins, cluster of differentiation 9 (CD9) and apoptosis-linked gene-2 interacting protein X (ALIX), and exosome negative protein, cis-Golgi matrix protein +(GM130), was assessed to identify exosomes by performing Western blot (WB). Exosomes are positive for CD9 and ALIX, and negative for GM130. + +## Flow Cytometry + +Flow cytometry was performed to examine the proportions of STRO-1+CD117+ cells in U2OS +and 143B cells as previous described [18]. U2OS and 143B cells were cultured in DMEM +medium, and the cell suspension (106 cells/ +100 μL) was incubated with 10 μL STRO-1-PE or CD117-APC (Abcam) at 4°C for 30 min in darkness. After that, the proportions of STRO1+CD117+ cells in U2OS and 143B cells were detected using a BD FACScaliber flow cytometer (BD Biosciences, San Jose, CA, USA). + +The fluorescent intensity was analyzed using Cell Quest Software (BD Biosciences). + +## Quantitative Real-Time Pcr (Qrt-Pcr) + +The qRT-PCR was performed to assess gene expression following the description of previous study [19]. Total RNA was extracted from cells using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). ND-1000 spectrophotometer (Thermo Fisher Scientific) was used to examine the concentration of RNA. RNA integrity was examined by 1.5% agarose gel electrophoresis. Complementary DNA was generated applying PrimeScript™ RT reagent Kit (Takara, Tokyo, Japan) as the protocol described. The relative expression of genes was estimated by qRT-PCR using TB Green® Premix Ex Taq II (Tli RNaseH +Plus) (Takara). The PCR system (50 μL) contained 4 μL cDNA, 25 μL Ex Taq II, 2 μL of 10 μM forward and reverse primers, 1 μL ROX Reference Dye and 16 μL sterile water. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 5 min; followed by 40 cycles of 15 s at °C, 1 min at 60°C and 30 s at 72°C; and a final extension for 10 min at 72°C. The primer sequences were shown in Table 1. The qRTPCR was carried out on a 7900 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). The results were analyzed using 2−∆∆CT method for quantification. + +## Wb + +WB was performed to assess protein expression according to the description of previous study +[19]. Total protein was extracted from exosomes or cells using Exosomal RNA and Protein Extraction Kit (Nwbiotec, Beijing, China) or Total Protein Extraction Kit (Solarbio). The concentration of proteins was examined using BCA Protein Assay Kit +(Solarbio). Protein samples (25 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred onto the polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk at room temperature for 1 h. Then, the membranes were incubated with the primary antibodies, CD9 (1:1000; Proteintech, Wuhan, China), ALIX (1:1000; Proteintech), GM130 (1:2000; Proteintech), Oct4 (1:1000; Proteintech), Sox2 (1:1000; Proteintech), Notch1 (1:1000; Proteintech), Hes family bHLH transcription factor 1 (Hes1; 1:1000; Abcam, Cambridge, MA, USA) or Hes-related family BHLH transcription factor with YRPW motif 1 (Hey1; 1:1000; Abcam) at 4°C for 12 h. After washed with Tris Buffered saline Tween, horseradish peroxidase-conjugated second antibody (1:5000; Proteintech) was incubated with the membranes. β-actin antibody (1:5000; Proteintech) was used as a reference protein for normalization. The WB bands were visualized by electrochemiluminescence detection + +| Gene | Forward sequence (5ʹ-3ʹ) | Reverse sequence (5ʹ-3ʹ) | +|---------|----------------------------|----------------------------| +| Sox2 | AGAACCCCAAGATGCACAAC | GGGCAGCGTGTACTTATCCT | +| Oct4 | AGCGATCAAGCAGCGACTAT | AGAGTGGTGACGGAGACAGG | +| β-actin | TGACGTGGACATCCGCAAAG | CTGGAAGGTGGACAGCGAGG | + +reagent (Beyotime, Shanghai, China). The data were analyzed by Image J software. + +## Tumor Spheroid Assay + +Tumor spheroid assay was carried out to detect the sarcosphere formation efficiency of U2OS and 143B cells as previous described [20]. U2OS and 143B cells were seeded into an ultra-low attachment plate with 5000 cells each well. Each well contained DMEM, B27 supplement (1: 50; Invitrogen, Carlsbad, CA, USA), +20 ng/mL human basic fibroblast growth factor (bFGF; Sigma-Aldrich) and 20 ng/mL human epidermal growth factor (EGF; Invitrogen). bFGF is widely distributed in various tissues and is an important cell proliferation and differentiation regulator, which can stimulate proliferation of various cells. Similarly, EGF is an active substance in the human organism that promotes cell proliferation and differentiation. Thus, the role of bFGF and EGF is to promote proliferation of U2OS and 143B cells. U2OS and 143B cells were cultured at 37°C and 5% +CO2 for 2 weeks, and 100 µL DMEM was added into each well every 2–3 days. After culture, colonies with a diameter ≥ 50 μm were regarded as sarcospheres. The spheres were quantified and photographed under an inverted-phase contrast microscopy (Leica, Wetzlar, Germany). + +## Statistical Analysis + +Each assay was performed for three times. All data reported as mean ± standard deviation. SPSS 22.0 statistical software (IBM, Armonk, NY, USA) was used for statistical analysis. Two-tailed Student's t test and one-way ANOVA was used to analyze the statistical difference. P < 0.05 was considered as a significant difference. + +## Results + +Osteosarcoma cells with stem cell-like characteristics are closely associated with the malignant phenotype of osteosarcoma. This work aimed to investigate whether HUVECs-exosomes can affect cell stemness of osteosarcoma and osteosarcoma progression. We hypothesized that HUVECs-exosomes may regulate cell stemness of osteosarcoma through Notch signaling pathway. We treated osteosarcoma cells with HUVECs-exosomes, and examined the impact of HUVECs-exosomes on the stem cell-like characteristics of osteosarcoma cells. The data revealed that HUVECs-exosomes activated Notch signaling pathway, which contributed to promote cell stemness in osteosarcoma. + +## Huvecs Promoted Osteosarcoma Cell Stemness Through The Secretion Of Exosomes + +To explore whether HUVEC-exosomes can regulate osteosarcoma cell stemness, GW4869 was used to inhibit the secretion of exosomes from HUVECs. Then, flow cytometry was performed to examine the proportions of STRO-1+CD117+ cells in the U2OS +and 143B cells. Compared with normal U2OS and 143B cells, coculture-DMSO and coculture-GW4869 significantly enhanced the proportions of STRO-1- +CD117+ cells in U2OS and 143B cells. GW4869 treatment caused a decrease of STRO-1+CD117+ cell proportions in U2OS and 143B cells (Figure 1a-b). Moreover, the expression of stem cell-related proteins Oct4 and Sox2 in the U2OS and 143B cells was examined by qRT-PCR. The mRNA expression of Oct4 and Sox2 was notably enhanced in both coculture-DMSO +and coculture-GW4869 groups. Coculture-DMSOmediated up-regulation of Oct4 and Sox2 was reversed by coculture-GW4869 treatment (Figure 1cd). Consistently, coculture-DMSO and cocultureGW4869 groups displayed an up-regulation of Oct4 and Sox2 proteins. The expression of Oct4 and Sox2 proteins in the U2OS and 143B cells was decreased by coculture-GW4869 (Figure 1e-g). Subsequently, we assessed the sarcosphere formation efficiency of U2OS and 143B cells by tumor spheroid assay. Figure 1h-i reveals that both coculture-DMSO and coculture-GW4869 promoted the sarcosphere formation efficiency of U2OS and 143B cells. However, coculture-GW4869 impaired the impact of coculture-DMSO on sarcosphere formation efficiency of U2OS and 143B cells (Figure 1h-i). Taken together, HUVECs promoted osteosarcoma cell stemness by secreting exosomes. + +![4_image_0.png](4_image_0.png) + +## Identification Of Exosomes Derived From Huvecs + +To further explore the biological role of HUVECsexosomes in osteosarcoma cell stemness, we separated exosomes from HUVECs, and identified the ultrastructure and particle size of exosomes. Figures 2a-b showed that exosomes exhibited a typical cup-shaped morphological feature formed by a lipid bilayer membrane, and the diameter of exosomes was approximately 102 nm. Then, WB was performed to examine the expression of exosome markers. The expression of exosome-positive proteins CD9 and ALIX were increased in exosomes. The exosome-negative protein GM130 was highly expressed in HUVEC lysate, while GM130 was not expressed in exosomes (Figure 2b). + +These data showed that exosomes were successfully separated from HUVECs and can be used for further analysis. + +## Huvecs-Exosomes Promoted Osteosarcoma Cell Stemness By Activating Notch Signaling Pathway + +U2OS and 143B cells were treated with HUVECderived exosomes to explore the molecular mechanism of HUVEC-exosomes in regulating osteosarcoma cell stemness. The results obtained from flow cytomerty revealed that the proportions of STRO-1+CD117+ cells were notably enhanced in the U2OS and 143B cells in the presence of + +![5_image_0.png](5_image_0.png) + +VECs-Exo (Figure 3a-b). Then, we performed qRT-PCR and WB to estimate the gene and protein expression of Oct4 and Sox2 in the U2OS and 143B cells. VECs-Exo treatment led to an increase in the mRNA and protein expression of Oct4 and Sox2 in the U2OS and 143B cells (Figure 3c-g). The results of tumor spheroid assay showed that VECs-Exo treatment significantly enhanced the sarcosphere formation efficiency of U2OS and 143B cells (Figure 3h). In addition, we determined the expression of Notch signaling pathway-related proteins by WB analysis. VECs-Exo-treated U2OS and 143B cells displayed an up-regulation of Notch1, Hes1, and Hey1 (Figure 3i-k). + +Next, U2OS and 143B cells were treated with RO4929097 to inhibit Notch signaling pathway in the presence of VECs-Exo. The results of flow cytomerty showed that RO4929097 treatment significantly reduced the proportions of STRO-1- +CD117+ cells in the U2OS and 143B cells +(Figure 4a-b). Compared with VECs-Exo treatment, RO4929097 treatment caused a decrease in the mRNA expression of Oct4 and Sox2 in the VECs-Exo-treated U2OS and 143B cells (Figures 4c-d). Oct4 and Sox2 proteins were also decreased in VECs-Exo-treated U2OS and 143B cells following RO4929097 treatment (Figure 4e-g). In addition, compared with VECs-Exo treatment, the sarcosphere formation efficiency of U2OS and 143B cells was notably repressed by RO4929097 treatment (Figure 4h). + +These data taken together revealed that HUVECs-exosomes promoted osteosarcoma cell stemness by activating Notch signaling pathway. + +![6_image_0.png](6_image_0.png) + +## Discussion + +Osteosarcoma cells with stem cell-like properties have been reported to play a vital role in the occurrence, development, recurrence, and drug resistance of osteosarcoma [21]. Accumulating researches have confirmed the biological role of exosomes in cancer stem cells. Cancer stem cellderived exosomes function as a carcinogenic factor and promote the stem-like phenotype in papillary + +![7_image_0.png](7_image_0.png) + +thyroid carcinoma through delivering long noncoding RNA DOCK9-AS2 [22]. MiR-500a-3p is highly expressed in the plasma exosomes of gastric cancer patients. Exosomal miR-500a-3p promotes stem-like phenotype and cisplatin resistance of gastric cancer, which attributes to repress FBXW7 expression [23]. For example, Li et al. have demonstrated that exosomal PSGR enhances the expression of Sox2 and Oct4a, and promotes epithelial-mesenchymal transition in low aggressive prostate cancer cells [24]. Consistently, we found that HUVECs-exosomes enhanced the proportions of STRO-1+CD117+ cells and the expression of Oct4 and Sox2 in the U2OS and 143B cells. + +HUVECs-exosomes also enhanced the sarcosphere formation efficiency of U2OS and 143B cells. STRO-1 and CD117 are important markers for identification of stem cell-like osteosarcoma cells [25]. Oct4 and Sox2 are two main transcription factors that associated with self-renewal and differentiation of stem cells. Oct4 is expressed in the undifferentiated embryonic stem cells, embryonic cells and embryonic germ cells [26]. As a common stem gene, Sox2 regulates the self-renewal of different types of stem cells and tissues [27]. Thus, our data suggested that HUVECs supernatant promoted stem-like phenotype of U2OS and 143B +cells. However, GW4869 treatment impaired the impact of HUVECs-exosomes on the stem-like phenotype of U2OS and 143B cells. GW4869 inhibits the release of exosomes from multivesicular bodies by blocking the sprouting of multivesicular bodies [15]. All these findings demonstrated that HUVECs promoted stem-like phenotype of U2OS and 143B cells by secreting exosomes. + +Notch signaling pathway is highly conserved in evolution, and participates in regulating cell proliferation, survival, apoptosis, and differentiation +[28]. The dysfunction of Notch signaling pathway usually hinders cell differentiation and leads to malignant transformation [29,30]. A previous report has found that Notch signaling pathway is abnormally expressed in osteosarcoma [31]. Inhibition of Notch signaling pathway accelerates the tumor growth of osteosarcoma by regulating the balance between Th1 and Th2 cells and promoting M2 polarization of tumor-associated macrophages [32]. Jin et al. have found that activation of Notch signaling pathway is contribute to cell stemness of osteosarcoma, and CSC-induced recurrence in osteosarcoma by activating Notch signaling pathway [33]. Consistently, we also confirmed the involvement of Notch signaling pathway in cell stemness of osteosarcoma. HUVECsexosomes activated Notch signaling pathway and enhanced the stem-like phenotype of U2OS and 143B cells. However, RO4929097 treatment reversed the impact of HUVECs-exosomes on stem-like phenotypes by inhibiting Notch signaling pathway. Therefore, these results supported that HUVECs-exosomes promoted stem-like phenotype of osteosarcoma cells through activating Notch signaling pathway. + +Stem cell-derived extracellular vesicles have a crucial role in maintaining stemness, differentiation and repairing tissue injuries [34,35]. Additionally, previous study has found that glioblastoma stem cellderived exosomes promote stemness and tumorigenicity of non-glioblastoma stem cell-like glioma cells by activating Notch1 signaling pathway [36]. Our data revealed that HUVECs-exosomes promoted osteosarcoma cell stemness by activating Notch signaling pathway. Mu et al. have demonstrated that murine osteosarcoma cell-derived exosomes are rich in Notch-activating factors, and activate Notch signaling pathway in muscle-derived stem cells [37]. Thus, we speculated that exosomes derived from osteosarcoma cells may promote stemness of itself through activating Notch signaling pathway. We will further explore whether osteosarcoma cell-exosomes can affect cell stemness of itself in our further work. + +## Conclusions + +In conclusion, this work demonstrated that HUVECs-exosomes promoted cell stemness in osteosarcoma through activating Notch signaling pathway. Thus, our data reveal the molecular mechanism of HUVECs-exosomes in regulating cell stemness of osteosarcoma, and provides a theoretical basis for exosomes as a treatment for osteosarcoma. + +## Data Availability + +The data used to support the findings of this study are available from the corresponding author on reasonable request. + +## Authors' Contributions + +JY designed the study; JY, YH, LW, XS, LY, WG performed the experiments; JY, YH analyzed the data; JY drafted the paper. All authors read and approved the paper. + +## Disclosure Statement + +No potential conflict of interest was reported by the author(s). + +## Funding + +This study was supported in part by Grants from the Natural Science Foundation of Hubei Province (grant No. + +2019CFB175) and Innovative Seed Funds for Medical School of Wuhan University (grant No. TFZZ2018030). + +## References + +[1] Xu N, Kang Y, Wang W, et al. The prognostic role of CD133 expression in patients with osteosarcoma. Clin Exp Med. 2020;20(2):261–267. + +[2] Sadykova LR, Ntekim AI, Muyangwa-Semenova M, et al. Epidemiology and risk factors of osteosarcoma. Cancer Invest. 2020;38(5):259–269. + +[3] Brown HK, Tellez-Gabriel M, Heymann D. Cancer stem cells in osteosarcoma. Cancer Lett. 2017;386: 189–195. + +[4] Das M, Law S. Role of tumor microenvironment in cancer stem cell chemoresistance and recurrence. Int J Biochem Cell Biol. 2018;103:115–124. +[5] Chouaib S, Kieda C, Benlalam H, et al. 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+|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Shoena Lucas a, Patrice H. Lalive a,b, Agustina M. Lascano a,b,* +a Faculty of Medicine, University of Geneva, Geneva, Switzerland b *Division of Neurology, Department of Clinical Neuroscience, Geneva University Hospitals, Geneva, Switzerland* Background: Recently, an association between painful tonic spasms (PTS) and Neuromyelitis Optica Spectrum Disorder (NMOSD) was established. + +Objective: To describe the clinical characteristics of PTS in NMOSD based on a video recording and to provide a literature review on the topic. + +Methods: We report a case of a 38 years-old woman with a diagnosis of NMOSD and positive aquaporin-4 IgG antibody status who developed PTS five weeks after an episode of longitudinal extensive transverse myelitis +(LETM). + +Results: Repetitive, brief, and painful episodes of muscle contraction were observed on the patient's left hand, spreading to the left arm, and then extending to the four limbs. While pregabalin and topiramate had no influence on these episodes, the patient responded to carbamazepine (CBZ), without symptom recurrence after one year. Conclusions: PTS in association with LETM can be considered typical for NMOSD. Although the exact mechanism is unknown, ephaptic transmission after spinal cord damage and excitatory soluble factors released during acute inflammation responses are sought to be involved. Symptomatic treatment with CBZ achieved remission of spams in our case. + +the author, who examined the patient. This report is conducted in compliance to Swiss Federal Act on Research involving Human Beings that wave ethic approval for case report of less than five patients. Patient's consent to publication was obtained. + +Neuromyelitis Optica Spectrum Disorder (NMOSD) is a rare relapsing inflammatory disorder affecting the central nervous system (CNS). + +Suggestive NMOSD-related symptoms include acute onset bilateral and severe visual loss, complete forms of acute myelitis, area postrema syndrome with uncontrollable hiccups or nausea and vomiting [1]. Interestingly, a set of brief and stereotyped neurological symptoms can appear at short a temporal distance of the first relapse. Paroxysmal symptoms were previously described in CNS autoimmune disorders mimicking epileptic fits or transient ischemic events [2,3]. However, limited literature exists on the association of paroxysmal phenomena and NMOSD. We discuss the association of PTS in NMOSD, provide a video recording of its characteristics and, finally, tackle treatment approach by means of an illustrative case. + +A 38 years-old female patient was admitted to the division of Neurology at the University Hospitals of Geneva due to acute onset of tetraparesis with left hemibody predominance and walking impairment. Additional symptoms included saddle hypoesthesia associated with fecal and urinary incontinence and dysesthesia/allodynia of the left hemiface and left upper limb. Three months earlier, she developed intractable hiccups, nausea, and vomiting. An initial diagnosis of *H. pylori* gastritis was suspected and treated with antibiotics and proton pump inhibitor. + +At admission, neurologic exam showed tetraparesis scored according to the Medical Research Council's strength assessment as follows: left elbow flexion 3/5, left fingers abduction 1/5, and left fingers flexion 2/ +5, others muscle groups of the upper and lower limbs were scored at 4/5. + +Clinical and ancillary tests description were personally retrieved by +* Corresponding author at: Division of Neurology, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1205 Geneva, Switzerland. + +E-mail address: Agustina.Lascano@hcuge.ch (A.M. Lascano). + +https://doi.org/10.1016/j.ensci.2023.100443 Received 27 November 2022; Received in revised form 23 December 2022; Accepted 8 January 2023 Available online 13 January 2023 2405-6502/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). + +# Paroxysmal Painful Tonic Spasms In Neuromyelitis Optica Spectrum Disorder + +journal homepage: www.elsevier.com/locate/ensci + +![0_image_5.png](0_image_5.png) + +Case report + +![0_image_1.png](0_image_1.png) + +![0_image_2.png](0_image_2.png) + +ARTICLE INFO +She presented with left upper limb hypoesthesia, four limb ataxia and + +![1_image_0.png](1_image_0.png) increased deep tendon reflexes. Plantar reflexes were in extension. The rest of the neurologic examination was normal. + +Brain magnetic resonance imaging (MRI) revealed increased signal on T2-weighted sequences of the left antero-lateral bulb and the area postrema (Fig. 1 A-B) with gadolinium enhancement on T1-weighted sequences (Fig. 1 C). Spinal cord MRI showed gadolinium enhancement at C1-T1 level (Fig. 1 D-E). Lumbar puncture displayed 21 M/L +leucocytes with lympho-monocytic predominance, 0.68 g/L of proteins, and no oligoclonal bands. Intravenous methylprednisolone was initiated at 1 g q.d. for five days, followed by 1 mg/kg/day of prednisone which was tapered down for 2.5 months associated with seven plasmapheresis sessions on alternate days for 14 days. + +Serology was negative for active/recent infection. Blood tests for autoimmune diseases showed positive anti-AQP4 IgG, anti-SSA/Ro, anti-SSB/La, rheumatoid factors IgM and IgA, and antinuclear (ANA) +autoantibodies. Immunological workup was completed with an MRI +sialography showing signs of chronic sialadenitis and Sjogren ¨ 's syndrome was diagnosed due to xerosis, polyarthralgia and positive antibody testing. + +Given typical clinical characteristic (i.e., area postrema syndrome and longitudinal extensive transverse myelitis or LETM), AQP-4 IgG positive status and neuroimagery findings, the diagnosis of NMOSD was made and rituximab treatment was started at 1 g i.v. and repeated two weeks later and then, every six months. + +Five weeks after LETM onset, the patient presented with painful tonic spasms that started in left hand, rapidly spread to the left arm, and finally involving the four limbs, lasting less than a minute, at a frequency of 9–14 times a day (see Video 1). Pain intensity was assessed at 9–10/10 on the Visual Analog Scale and Quality of Life Scale of American Chronic Pain Association was rated at 5/10. Intense emotions and rapid and sudden movements triggered the spasms. Antispastic medication was initiated with baclofen 20 mg t.i.d. and pregabalin 150 mg b.i.d, followed by tolperisone 150 mg b.i.d. Due to limited symptom control, carbamazepine (CBZ) 100 mg q.d. was initiated, the dose of baclofene was decreased and tolperisone was discontinued, resulting in prompt and complete remission. CBZ was discontinued four months after onset due to lympho-neutropenia and was switched to topiramate 75 mg b.i.d. + +showing reappearance of PTS. For that reason, CBZ was reintroduced at 100 mg q.d. and topiramate was slowly discontinued showing complete remission. Treatment was pursued for a ten-month period and stopped by the patient. Three years after CBZ discontinuation, there is no recurrence of PTS and both clinical and radiological workup is stable. + +## 4. **Discussion** + +Paroxysmal movement disorders in autoimmune neurological disorders were first described in the late 50's by Matthews and were previously referred to as "tonic seizures". It was described as a transient neurological symptoms of rapid onset, brief duration and increased frequency [2]. Other terms are used to describe the same movement disorder, including paroxysmal dystonia [4], tonic spasm [5] or paroxysmal painful tonic spasm (PTS) [6]. + +Unravelling the mechanism of PTS remains a challenge. Ekbom et al. + +reported two multiple sclerosis (MS) patients with spinal cord lesions presenting with sensory-motor seizures [7]. During the attacks, patients 2 showed unilateral tonic spasms, accompanied with a tonic posture of the limbs, and preceded/followed by contralateral sensory disturbances. + +The combination of ipsilateral motor and contralateral sensitive complaints can be explained by an impairment of the lateral funiculus at the spinal cord level, which gathers fibres of the lateral corticospinal and spinothalamic tracts. Given the spatio-temporal correlation, a transversally spread activation of the damaged axons known as ephaptic transmission was proposed as the physiopathological mechanism underlying PTS in demyelinating disorders [3]. + +From a biological standpoint, it was hypothesized that the release of excitatory soluble factors during acute inflammation provoke "irritation" of demyelinated axons in MS active plaques, therefore, facilitating the spreading of the anomalous transmission [5,8]. Furthermore, a correlation between PTS and MRI lesions was described by Maimone et al., who reported a patient developing tonic spasms on the left hemibody during an MS relapse associated with a new MRI hyperintense T2-weighted lesion located on the posterior limb of the right internal capsule [8]. + +The occurrence of PTS in NMOSD varies from 14% to 95% [5,10]. + +This can be explained by the difference in the study design, due to their retrospective nature. While comparing the prevalence of PTS in NMOSD +and MS groups, a recent cross-sectional study reported a higher incidence in NMOSD, up to 22 times more [4]. Kim and collaborators proposed that the association of PTS and acute myelitis allows to distinguish NMOSD from MS with a specificity of 98.7% [6]. We hypothesize that PTS is more frequent in NMOSD due to the different pathological mechanisms of demyelination and disease behaviour - i.e., astrocytopathy with secondary and severe demyelination versus demyelination as a result of oligodendrocyte destruction in MS and the presence of extensive spinal cord lesions, rarely observed in MS [11]. + +Potential risk factors of PTS in NMOSD show that only the presence of acute myelitis is related with a higher probability of developing PTS +[6]. In our patient, PTS occurred five weeks after LETM and lasted for several months, which corresponds to the recovery phase. This suggests that partial remyelination is more likely to be the origin of spasms rather than the demyelination. No association was established with age at disease onset, sex, annualized relapse rate, presence of optic nerve damage at disease onset, expanded disability status scale score, spinal cord lesion extension and presence of brain MRI lesions, in a cohort of ten NMOSD patients [6]. + +Concerning PTS presentation, the onset is abrupt, lasting few seconds, and can be precipitated by a stimulus such as sudden movement, particularly after a long resting period, or triggered by emotional factors +[5]. In a recent review, it was reported that PTS can involve one side of the body (3/13 patients, 23%), the four limbs (8/13 patients, 61.5%) and, more rarely, the neck and upper limbs exclusively [12]. No major differences were observed between NMOSD and MS patients in terms of clinical presentation and movement disorder's characteristic. These paroxysmal events are usually painful (9/13, 69%), constituting a major source of disability [12] as it occurred in our patient. Table S1 (supplementary material) summarises the clinical features and therapy used in neuromyelitis optica patients with PTS. + +Treatment response of PTS in NMOSD is remarkable while administering phenytoin, topiramate, benzodiazepine, gabapentine and, specially, CBZ [6,10]. However, several studies show higher efficacy of topiramate and CBZ versus gabapentin or pregabalin [6,13]. The response observed with CBZ, usually occurs within the first week, achieving cessation of PTS in most cases [6,9,10]. Similarly, our patient fully responded to low doses of CBZ remission immediately after treatment onset, but not to topiramate and/or pregabaline. CBZ and topiramate are anti-epileptic drugs blocking voltage-gated sodium channels, limiting inward flux of sodium, and stabilizing the membrane [14]. Through these mechanisms the abovementioned drugs can achieve inhibition of the ephaptic transmission occurring at the level of demyelinated lesions. + +We reported and recorded a case of PTS during recovery phase after an NMOSD relapse. Our goal is to increase awareness on this not so infrequent symptom appearing in association with NMOSD and responding to CBZ administration. + +Supplementary data to this article can be found online at https://doi. + +org/10.1016/j.ensci.2023.100443. + +## Funding Sources + +No targeted funding reported. + +## Declaration Of Competing Interest + +SL has nothing to disclose. AML received consulting fees for ADC +therapeutics SA not related to this study. PHL received honoraria for speaking for Biogen-Idec, CSL Behring, Merck Serono, Novartis, SanofiAventis, TEVA, Roche; consulting fees from Biogen-Idec, Geneuro, Genzyme, Merck Serono, Novartis, Sanofi-Aventis, TEVA; research grants from Biogen-Idec, Merck Serono, Novartis nonrelated to this study. + +## Acknowledgments + +We would like to express our gratitude to Dr. Marjolaine Uginet for her assistance during the video recording. We thank the participant who volunteered for this case presentation. + +## References + +[1] D.M. Wingerchuk, B. Banwell, J.L. Bennett, et al., International consensus diagnostic criteria for neuromyelitis optica spectrum disorders, Neurology 85 +(2015) 177–189. + +[2] W.B. Matthews, Tonic seizures in disseminated sclerosis, Brain 81 (1958) 193–206. + +[3] P.O. Ostermann, C.E. Westerberg, Paroxysmal attacks in multiple sclerosis, Brain 98 (1975) 189–202. + +[4] C. Candeias da Silva, D.B. Bichuetti, S.M.C. Azevedo Silva, et al., Movement disorders in multiple sclerosis and neuromyelitis optica: a clinical marker of neurological disability, Parkinsonism Relat. Disord. 51 (2018) 73–78. + +[5] L. Abaroa, S.A. Rodriguez-Quiroga, L. Melamud, et al., Tonic spasms are a common clinical manifestation in patients with neuromyelitis optica, Arq. Neuropsiquiatr. + +71 (2013) 280–283. + +[6] S.M. Kim, M.J. Go, J.J. Sung, et al., Painful tonic spasm in neuromyelitis optica: +incidence, diagnostic utility, and clinical characteristics, Arch. Neurol. 69 (2012) +1026–1031. + +[7] K.A. Ekbom, C.E. Westerberg, P.O. Osterman, Focal sensory-motor seizures of spinal origin, Lancet 1 (1968) 67. + +[8] D. Maimone, A.T. Reder, F. Finocchiaro, E. Recupero, Internal capsule plaque and tonic spasms in multiple sclerosis, Arch. Neurol. 48 (1991) 427–429. + +[9] E. Carnero Contentti, F. Leguizamon, J.P. Hryb, et al., Neuromyelitis optica: +association with paroxysmal painful tonic spasms, Neurologia 31 (2016) 511–515. + +[10] N. Usmani, G. Bedi, B.L. Lam, W.A. Sheremata, Association between paroxysmal tonic spasms and neuromyelitis optica, Arch. Neurol. 69 (2012) 121–124. + +[11] C.F. Lucchinetti, Y. Guo, B.F. Popescu, et al., The pathology of an autoimmune astrocytopathy: lessons learned from neuromyelitis optica, Brain Pathol. 24 (2014) +83–97. + +[12] H. Abboud, H.H. Fernandez, M.A. Mealy, M. Levy, Spinal movement disorders in neuromyelitis optica: an under-recognized phenomenon, Mov. Disord. Clin. Pract. + +3 (2016) 596–602. + +[13] J. Liu, Q. Zhang, Z. Lian, et al., Painful tonic spasm in neuromyelitis optica spectrum disorders: prevalence, clinical implications and treatment options, Mult. + +Scler. Relat. Disord. 17 (2017) 99–102. + +[14] S. Iida, M. Nakamura, R. Wate, et al., Successful treatment of paroxysmal tonic spasms with topiramate in a patient with neuromyelitis optica, Mult. Scler. 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