diff --git a/medical/md/PMC10275788.md b/medical/md/PMC10275788.md new file mode 100644 index 0000000000000000000000000000000000000000..0ab083f4e7bd4cfaf5ae89d9efd6f81c51aad93a --- /dev/null +++ b/medical/md/PMC10275788.md @@ -0,0 +1,458 @@ + + +![0_image_3.png](0_image_3.png) + +![0_image_4.png](0_image_4.png) + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +![0_image_5.png](0_image_5.png) + +![0_image_6.png](0_image_6.png) + +## 1. **Introduction** + +| ABSTRACT | +|------------| + +| Keywords: Digital technology Joint graduation design Collaborative ability Evaluation system Structure | +|---------------------------------------------------------------------------------------------------------------| + +Research on the construction of a collaborative ability evaluation + +![0_image_1.png](0_image_1.png) + +system for the joint graduation design of new engineering specialty groups based on digital technology Jiandong Tian *, Guifang He Suzhou Vocational Institute of Industrial Technology, Jiangsu, 215104, China Conducting research on the construction of a collaborative ability evaluation system for the joint graduation design of new engineering specialty groups based on digital technology holds great practical relevance. In this paper, which is based on a comprehensive analysis and research of the current situation pertaining to the joint graduation design of college graduates in China and elsewhere and on the construction of a collaborative ability evaluation system, combined with the talent training program of the joint graduation design, the Delphi method and the analytic hierarchy process (AHP) are adopted to establish a hierarchical structure model of the collaborative ability evaluation system for joint graduation design. In this system, collaborative abilities in the areas of cognition, behavior and emergency management are used as the criteria level evaluation indices. Additionally, collaborative ability in regard to targets, to knowledge, to relationships, to software, to the workflow, to organization, to culture, to learning and to conflict are used as evaluation indices. The comparison judgment matrix of the evaluation indices is constructed at the collaborative ability criterion level and at the index level. By calculating the maximum eigenvalue and corresponding eigenvector of the judgment matrix, the weight assignment of the evaluation indices is obtained, and the evaluation indices are sorted. Finally, the related research content is evaluated. The research results show that the key evaluation indicators for the collaborative ability evaluation system of joint graduation design that need to be considered are easy to determine, and these indicators provide a theoretical reference for the reform of graduation design teaching of new engineering specialty groups. + +The rapid development of artificial intelligence and the digital economy has driven a new round of technological revolution and industrial transformation. College level engineering education needs to be adapted to the development of such new technologies, new industries, new business forms and new models, both in China and throughout the world. Therefore, new engineering has emerged as the times require. Compared with traditional engineering, new engineering pays greater attention to cultivating high-quality composite talent with strong practical ability, strong innovation ability and strong international competitiveness. New engineering specialty groups are composed around several related specialties with the same engineering objects, similar technical fields or similar https://doi.org/10.1016/j.heliyon.2023.e16855 Received 26 December 2022; Received in revised form 24 May 2023; Accepted 31 May 2023 Available online 1 June 2023 2405-8440/© 2023 The Authors. 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/). + +ARTICLE INFO +* Corresponding author. + +E-mail addresses: tjd168@163.com (J. Tian), 3582927230@qq.com (G. He). + +professional disciplinary bases, using key specialties as the core. The joint graduation design of new engineering specialty groups is a practical teaching tool, the quality of practical teaching directly affects the cultivation of comprehensive skills for college students participating in the joint graduation design of the new engineering specialty groups. Although the joint graduation design maintains consistent engineering objects and similar professional foundations, there are still differences regarding professional knowledge among different majors, as well as timing differences in the graduation design. Strengthening communication between different majors, improving the comprehensive literacy of graduates, cultivating students' cognitive, behavioral, and strain collaborative abilities, and forming a closed loop of students' development capabilities are the main objectives behind the cultivation of high-quality composite application-oriented professionals. Therefore, it is of critical practical importance to conduct research on the construction of a collaborative evaluation system for graduation design based on digital technology and new engineering specialty groups. + +In recent years, there have been many studies conducted in China and elsewhere on the construction of an evaluation system for the joint graduation design of colleges. Engineering education in colleges needs to be adapted to the development of new technologies, new industries, new business forms and new models, both in China and elsewhere [1,2]. Ao et al. used Cronbach's alpha coefficient and principal component analysis to evaluate 2777 undergraduate graduation design results based on building information modelling +(BIM) technology in architecture and obtained common factors affecting the undergraduate graduation design of architecture-related disciplines, such as teacher ability, college atmosphere, team cooperation, personal ability, mastery of BIM technology, social environment incentive and achievement demand. Finally, the results of the ordered logistic regression showed that the comprehensive ability of instructors, level of team cooperation, personal abilities and the achievement needs of the participants significantly affected the evaluation of the graduation design [3]. Zhao & Teng believe that there are disadvantages in the teaching mode used in the traditional graduation design. In addition to their practical experience with the new mode emerging in the joint graduation design of college programs, Zhao & Teng provided new ideas and methods for joint graduation design; explored innovation in and the characteristics of design topics, cross professional communication, etc.; discussed the new problems faced by multidisciplinary joint graduation design; and proposed improvement measures based on various aspects to comprehensively improve the quality of the graduation design of related majors [4]. Taking the graduation design of electrical engineering-related majors as an example, Li et al. + +systematically studied the overall structure of the multidisciplinary cross graduation design mode, introduced diversified forms of evaluation and examination pertaining to graduation, established an evaluation index for joint graduation design, and adopted the method of combining learning process evaluation and comprehensive quality evaluation to carry out scientific and reasonable evaluations based on various aspects of the entire graduation design process, including student achievements, behavioral performance and final abilities mastered. The whole evaluation process for graduation design sets higher requirements for students' innovation ability, independent learning capacity and level of engagement in team cooperation [5]. Wang et al. proposed a collaborative graduation design mode for different majors in civil engineering based on a production–education integration platform. On the basis of the production–education integration platform that is jointly established by universities and enterprises, the advantageous resources of all parties were optimized, the division of labor and cooperation content of all parties was clarified, the management and control mechanisms for professional coordination was strengthened, and the management and control of node links, comprehensive evaluation mechanism, and safeguard measures were considered. This mode was closely combined with the teaching characteristics in civil engineering and served to greatly improve students' independent innovation and team cooperation abilities [6]. Wang et al. believed that BIM technology has been increasingly widely used in the design, construction, operation and maintenance of construction projects. Joint graduation design is an important means of cultivating student skills in analysis and practical problem solving by using the BIM technology knowledge learned through the program and serves as an important measure for promoting improvements in students' practical innovation ability and comprehension quality. The research contents, methods and results of a joint graduation design based on BIM technology are proposed. Practice shows that the development of a joint graduation design benefits students, enterprises and schools, leading to fruitful results and good social effects [7]. + +Most of the literature has studied the ideas and methods behind joint graduation designs and the results that they produce, but there has been little research in which the construction of a collaborative ability evaluation system for the joint graduation design of new engineering specialty groups based on digital technology has been systematically studied [8–10]. In this paper, the Delphi method and the AHP are used to evaluate the collaborative ability of joint graduation design students based on the aspects of collaborative ability in regard to cognition, collaborative ability in regard to behavior and collaborative ability in regard emergency management. The corresponding evaluation system is constructed and combined with a questionnaire survey, thus providing a theoretical reference for graduation design teaching reform against the background of the construction of new engineering specialty groups in colleges. + +## 2. **Theoretical Framework** 2.1. Digital Technology + +Digital technology refers to any technology that uses "0′′ and "1′′ digital codes to express, transmit and process information through computers, optical cables, communication satellites and other equipment. Digital technology generally includes digital coding, digital compression, digital transmission, digital modulation and demodulation. It converts things in the objective world into binary 0s and 1s, the only machine language that can be recognized by a computer, and realizes a series of subsequent processing operations through technical means to realize information digitization. With the rapid development and popularization of digital technology, various industries worldwide have created increasing economic value and social benefits through the use of this technology. Nelson et al. conducted a cross-sectional study on 52 COVID-19 mobile applications from virtual stores for Android and iOS operating system smart phones by using Fisher's exact test and the Cram´er V test. In the 23 countries sampled, there are 11 languages to choose from and 9 topics related to COVID-19, most of which involve "case monitoring" and "symptoms, prevention and care." There is a link between the target audience and the symptoms, prevention and care subtheme, as well as a link between patient monitoring and professional training. COVID-19 mobile applications are available free of charge in the major communication languages in the countries of the five selected continents; however, for people with disabilities, accessibility is limited [11]. With the application of digital technology in various processes involving dental implants, a new type of dental implant treatment mode with high efficiency, comfort and accuracy has been formed. In this mode, the digital technology and equipment used for tooth defect treatment can obtain more accurate preoperative information, assist in conducting implant operations and impression repair, and complete personalized abutment design and production, thus generating significant value [12–14]. + +## 2.2. New Engineering Specialty Groups + +Facing the needs inherent to future industrial development, maintain focus on the internet and artificial intelligence at the core of this development and relying on a discipline that has a strong foundation in colleges, new engineering specialty groups are clusters that are formed by numerous related specialties with the same engineering object, a similar technical nature, closely related content and a strong internal logic. They are professional clusters within the same service field and the same professional foundation that share related professional positions. Each specialty of a new engineering specialty group has the same disciplinary foundation, which is reflected in teaching, so each specialty completes student training tasks within the same system. There is a large amount of common equipment in experimental training facilities, as well as a considerable number of common experimental training projects [15]. Based on digital technology, facing regional or industrial key industries and reliant on the advantages and characteristics of colleges, the dynamic adjustment and self-improvement level of the professional group of the docking industry have been improved, which can further promote the professional resource integration and structural optimization of colleges. The agglomeration effect and service function of the professional group are brought into play to realize the functions and advantages of the comprehensive integration of structural elements on the talent supply side and the industrial demand side [16]. Shang et al. proposed exploring the training mode of new engineering professionals under the background of specialty groups, making full use of the construction foundation of interdisciplinary resource sharing and actively carrying out the construction of the curricular system, thereby teaching teams and practice platforms along with industry enterprises to provide an accumulation of experience for improving the quality of talent training and developing the mode and practice of industry–education integration and the collaborative education among new engineering specialty groups [17]. + +## 2.3. Collaborative Ability Of Joint Graduation Design + +The collaborative ability within joint graduation design means that the joint graduation design of multiple specialties cultivates the professional quality of multidisciplinary teams in such a way that focuses on cultivating students' collaborative ability in regard to synergistically analyzing complex problems by using the basic theories and skills learned, as well as by applying collaborative ability to synergistically solve practical problems through the use of modern technology and tools, and by applying collaborative ability in regard emergency management to synergistically cope with unexpected problems by using practical experience that is accumulated through daily and focused thinking [18]. Collaborative ability in regard to cognition refers to the ability of students of various specialties to comprehensively use their basic knowledge learned through studying in their discipline to analyze complex problems that correspond to different task stages by establishing a task-oriented framework of joint graduation design and forming a collaborative-oriented group cognition of students of various specialties on the basis of students' individual thought cognition. + +Collaborative ability in regard to cognition mainly pertains to targets, knowledge and relationships. Collaborative ability in regard to targets is generally investigated from two perspectives: target realization and multiobjective coordination. Collaborative ability in regard to knowledge mainly refers to the basic knowledge of the objectives to be achieved and the collaborative understanding ability regarding the specialty and related specialties. Collaborative ability in regard to relationships refers to the clear degree of relationship cognition and role positioning among different specialties and students [19]. Collaborative ability in regard to behavior refers to the ability to effectively carry out cooperative behaviors such as value acquisition and creation on the basis of ideological coordination and with the help of digital technologies such as the internet and related software through coordination among the behavioral elements of a joint graduation design specialty group to achieve an effect that transcends the professional activities of a single discipline and to integrate the resources and information related to the task. Collaborative ability in regard to behavior mainly regards software, the workflow and overall organization. Collaborative ability in regard to software refers to the ability to understand the basic knowledge of the relevant software and to solve obstacles to collaboration presented by the software. The interaction between a user and software is a basic indicator for collaboration ability. Collaborative ability in regard to the workflow refers to workflow problems, work handover standards, work completion efficiency, and resource utilization. Collaborative ability in regard to organization refers to the level of cooperation between disciplines, the level of specialty collaboration within each specialty group, and the degree of organizational standardization [20]. Collaborative ability in regard emergency management refers to the ability of students of various disciplines to use their accumulated specialty knowledge and practical experience to solve the design content conflicts between different specialties and the challenges of changes in the external environment that often occur in the process of completing the joint graduation design task of a specialty group. Collaborative ability in regard emergency management mainly regards culture, learning and conflict. Collaborative ability in regard to culture refers to the degree of specialty knowledge integration within the specialty group, including the trust between different specialties, the value orientation, and the decision-making of students from various specialties in the specialty group. Collaborative ability in regard to learning refers to the ability of students from various specialties in the specialty group to learn new knowledge through collaborative communication and to solve new problems using new software. Collaborative ability in regard to conflict refers to the ability to balance, manage and coordinate the trade-offs between various possible conflict problems and solutions in the process of completing the joint graduation design task [21]. + +## 2.4. The Delphi Method And Theahp + +The Delphi method was invented by Norman Dalky and Olaf Helmer of RAND Corporation in 1964 and applied to prediction analysis. It is an expert evaluation method named after the ancient Greek city of Delphi [22]. The basic process of applying this method to collect expert evaluation opinions is as follows: (1) first, the evaluation object must be defined, and the experts in the corresponding field should be invited to form an expert group to assess the evaluation object. The experts should have high levels of authority, experience and theoretical expertise, and the number of experts should generally not exceed 20. (2) The questionnaire should provide the expert group with the relevant rules, relevant information, and research questions. (3) The members of the expert group should score the evaluation objects and anonymously provide evaluation opinions according to the evaluation rules and their own understanding. (4) The organizer should summarize the evaluation opinions of the experts and feed them back to the expert group. Each member then conducts a second evaluation round according to the summary results of the first round. The organizer collects, summarizes and feeds back the evaluation opinions one by one, generally through3 to 4 rounds, until the evaluation opinions of the members of the expert group are generally consistent. + +The AHP was formally proposed by T.L. Saaty, an American operations research scientist, in the mid-1970s. This method has a wide range of practicability for the decision-making analysis of various complex problems, and it is the most commonly used method for determining index systems in China and elsewhere. It decomposes the decision-making problem into different hierarchical structures according to the order of the general objective, the subobjectives of each level, the evaluation criteria and the specific alternative schemes. Then, the eigenvector of the judgment matrix is solved to obtain the priority weight of each element of each level over an element of the previous level. Finally, the weighted sum method is used to merge each alternative scheme stepwise to obtain the final weight of the general objective. The final scheme with the largest weight is the best scheme [23]. The steps of the AHP are as follows: + +(1) The hierarchical structure model of the evaluation system is constructed, and the evaluation indices are hierarchized. + +(2) A comparison judgment matrix is constructed. After the hierarchical structure is constructed, the lower indicators have a clear subordinate relationship with the upper indicators. The comparison matrix is obtained by analyzing the ratio of the relative importance of each indicator based on the indicators of the previous level. A 1–9 scale is used for comparing the relative importance of *tij* and *tji.* See Table 1 for the specific meanings used. +After the values of *tij* and *tji* are determined, the judgment matrix is obtained as follows: + +T = +⎡ +⎢⎢⎢⎢⎢⎣ +t11 t12 ⋯ t1n + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +tn1 tn2 ⋯ tnn +⎤ +⎥⎥⎥⎥⎥⎦(1) +The relative weights of the indices in the criterion layer and the index layer are determined through using the judgment matrix. + +(3) The maximum eigenvalue of the judgment matrix and the corresponding eigenvector are calculated. + +Each column of judgment matrix T is normalized: + +$$\mathbf{(1)}$$ + +| Table 1 The meaning of the AHP scale. The scale of tij and tji | The definition (comparing Factors i and j) | +|--------------------------------------------------------------------|---------------------------------------------------------------------------------------| +| 1 | Factor i is as important as Factor j. | +| 3 | Factor i is slightly more important than Factor j. | +| 5 | Factor i is much more important than Factor j. | +| 7 | Factor i is extremely more important than Factor j. | +| 9 | Factor i is absolutely more important than Factor j. | +| 2, 4, 6, 8 | The intermediate value of two adjacent judgments | +| The reciprocal | If Factor i is compared with Factor j, the judgment value is tji = 1/tij, and tii = 1 | + + $$\text{(2)}$$ . +(3) $$\mathbf{a}$$ + $$T'=\left[\begin{array}{ccccc}{t}_{11}&&\\ {t}_{11}&&{t}_{12}^{'}&\cdots&{t}_{1n}^{'}\\ {t}_{21}&&{t}_{22}^{'}&\cdots&{t}_{2n}^{'}\\ ⋮&⋮&\ddots&⋮\\ {t}_{n1}&&{t}_{n2}^{'}&\cdots&{t}_{nn}^{'}\end{array}\right]=\left[\begin{array}{c}{t}_{11}\\ \sum\limits_{i=1}^{n}{t}_{i1}\\ \sum\limits_{i=1}^{n}{t}_{i1}\\ ⋮\\ {t}_{n1}^{'}&\cdots&{t}_{nn}^{'}\end{array}\right]$$ $$\left[\begin{array}{c}{t}_{n1}\\ \sum\limits_{i=1}^{n}{t}_{i1}\\ \sum\limits_{i=1}^{n}{t}_{i1}\end{array}\right]$$ + +$\begin{array}{ccc}\dfrac{t_{12}}{\sum\limits_{i=1}^n t_{i2}}&\hdots\\ \\ \dfrac{t_{22}}{\sum\limits_{i=1}^n t_{i2}}&\hdots\\ \\ \vdots&&\\ \dfrac{t_{n2}}{\sum\limits_{i=1}^n t_{i2}}&\hdots\\ \end{array}$ . +⋯ t1n +∑n i=1 tin ∑n i=1 tin ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ t2n ∑n i=1 tin ⋮ +⋯ tnn +(i = 1, 2, ⋯, n) (2) +The sum of all values is calculated by line. + +$$T^{\prime\prime}=\left[\begin{array}{c}t_{1}\\ t_{2}\\ \vdots\\ t_{n}\end{array}\right]=\left[\begin{array}{c}\dot{t}_{11}+\dot{t}_{12}+\cdots+\dot{t}_{1n}\\ \dot{t}_{21}+\dot{t}_{22}+\cdots+\dot{t}_{2n}\\ \vdots\\ \dot{t}_{n1}+\dot{t}_{n2}+\cdots+\dot{t}_{nn}\end{array}\right]=\left[\begin{array}{c}\sum_{j=1}^{n}\dot{t}_{ij}\\ \sum_{j=1}^{n}\dot{t}_{ij}\\ \vdots\\ \sum_{j=1}^{n}\dot{t}_{ij}\end{array}\right](j=1,2,\cdots,n)$$ + +(j = 1, 2, ⋯, n) (3) + +$$\mathbf{\Sigma}(4)$$ + +Matrix T′′ is normalized to obtain the eigenvector ω corresponding to the maximum eigenvalue λmax. + +ω = ⎡ ⎢⎢⎢⎢⎣ ω1 ω2 ⋮ ωn ⎤ ⎥⎥⎥⎥⎦ = ⎡ ⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣ t1 ∑n i=1 ti tn ∑n i=1 ti ⎤ ⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ t2 ∑n i=1 ti ⋮ M = T × ω = ⎡ ⎢⎢⎢⎢⎢⎣ t11 t12 ⋯ t1n t21 ⋮ t22 ⋮ ⋯ ⋱ t2n ⋮ tn1 tn2 ⋯ tnn ⎤ ⎥⎥⎥⎥⎥⎦ ⎡ ⎢⎢⎢⎢⎣ ω1 ω2 ⋮ ωn ⎤ ⎥⎥⎥⎥⎦ = ⎡ ⎢⎢⎢⎢⎢⎣ M1 M2 ⋮ Mn ⎤ The maximum eigenvalue λmax of judgment matrix T is calculated. +(i = 1, 2, ⋯, n) (4) +⎥⎥⎥⎥⎥⎦(5) +λmax = 1n +∑n i=1 Mi ωi +(i = 1, 2, ⋯, n) (6) +λmax—T represents the maximum eigenvalue of the judgment matrix. + +ω—The is the eigenvector corresponding to the maximum eigenvalue. + +(4) Consistency test of the judgment matrix. + +Table 2 Value of the average random consistency index RI. + +(5) The hierarchical model of the evaluation system is used to draw the index weight table of the evaluation system and to evaluate the related research content. + +n 1 2 3 4 5 6 7 8 9 10 11 RI 0 0 0.58 0.9 1.12 1.24 1.32 1.41 1.45 1.49 1.51 + +$$(5)$$ + +$$(6)$$ + +5 + +$$Cl=\frac{\lambda_{\max}-n}{n-1}$$ +n − 1 (7) +6 +CR = CI +RI (8) +CI—C is the consistency index. + +RI—A is the average random consistency index. See Table 2 for details. + +CR— stands for random consistency. + +When CR < 0.10, the judgment matrix has a satisfactory consistency; otherwise, it needs to be adjusted to eliminate excessive inconsistency until it is satisfactory. + +Li & Zhou used an improved AHP to determine the relative weight of each factor by establishing a multifactor evaluation system to evaluate the abilities and comprehensive scores of graduate students with a professional degree in clinical medicine. The research results show that the improved AHP evaluation model can be used to reasonably evaluate the comprehensive abilities of graduate students with a professional degree in clinical medicine, providing a basis for objectively analyzing the various ability levels of such graduate students [24]. After obtaining the results of three rounds of expert consultation through the Delphi method, Shen et al. + +constructed an evaluation index system for the target achievement degree of industry–education integration by using the AHP. The organizational guarantee, industrial enterprises' investment in vocational education, diversified school operational system, school–enterprise collaborative education, effect of industry–education integration, and contribution to regional economic development and industrial upgrading were all used as first-level indicators. Governance organization, the school operational form, school–enterprise collaboration, financial input, personnel input, technological innovation, talent training and talent quality were all used as second-level indicators, and there were a total of 27 third-level indicators [25]. + +## 3. **Construction Of A Collaborative Ability Evaluation System For The Joint Graduation Design Of New Engineering Specialty** Groups Based On Digital Technology + +3.1. *Connotation of the collaborative ability of the joint graduation design of new engineering specialty groups based on digital technology* The collaborative ability of the joint graduation design of new engineering specialty groups based on digital technology refers to the collaborative ability to train students in cognition, behavior and emergency management by implementing a joint graduation project in a new engineering specialty group according to the joint talent training scheme of new engineering specialty groups and the professional training objectives of all disciplines. It combines the common characteristics of engineering objects and similar professional bases among specialty groups, uses modern technologies and tools such as the internet and artificial intelligence, and forms a closed loop of student development ability to achieve the training target of high-quality and characteristic composite applicationoriented professionals. The evaluation indicators of collaborative ability in regard to cognition mainly regard targets, knowledge and relationships. The evaluation indices of collaborative ability in regard to behavior mainly regard software, workflow and organization. The evaluation indicators of collaborative ability in regard to emergency management mainly regard culture learning and conflict [26]. + +3.2. Construction of the collaborative ability evaluation system for the joint graduation design of new engineering specialty groups based on digital technology In recent years, there have been many studies in China and elsewhere on the construction of an evaluation system for the joint graduation design of colleges [27–29]. Based on an analysis of the relevant research in China and elsewhere regarding the collaborative ability evaluation system of joint graduation design in general, as well as that of new engineering specialty groups in China's application-oriented universities, in combination with the actual problems encountered in this design, the Delphi method is applied to conduct an expert questionnaire survey to quantify the unanimous expert opinions collected, and an AHP is used to build the hierarchical model of the evaluation system, as well as to sort and assign weights to the evaluation indices. Finally, the index weight table of the evaluation system is developed to evaluate the relevant research content. + +| Table 3 Judgment matrix A of the collaboration ability evaluation system. A A1 A2 | A3 | | | +|---------------------------------------------------------------------------------------|------|-----|-----| +| A1 | a11 | a12 | a13 | +| A2 | a21 | a22 | a23 | +| A3 | a31 | a32 | a33 | +| In the table. a12 indicates the importance of Factor A1 compared with Factor A2. | | | | + +(1) The hierarchical structure model of the evaluation system is established, as shown in Fig. 1. + +Table 4 + +Judgment matrix B of collaborative ability with regard to cognition. + +B B1 B2 B3 + +B1 b11 b12 b13 + +B2 b21 b22 b23 + +B3 b31 b32 b33 + +In the table. + +b12 indicates the importance of Factor B1 compared with Factor B2. b21 indicates the importance of Factor B2 compared with Factor B1. + +Fig. 1. Hierarchical structure model of the evaluation system. + +(2) According to the meaning of the AHP scale listed in Table 1 of Section 2, a questionnaire on the evaluation indicators of the collaborative ability of joint graduation projects is formulated. The members of the expert group are required to anonymously complete the questionnaire according to the evaluation rules and their own understanding. The questionnaire is distributed, collected, summarized and fed back many times until the evaluation opinions of the members of the expert group tend to be consistent. The evaluation system of collaboration ability and the judgment matrices of collaborative ability pertaining to cognition, behavior and emergency management are constructed, and they are displayed as judgment matrices A, B, C and D, respectively. See Table 3, Table 4, Table 5 and Table 6 for details. + +The meanings of the other symbols used in the table are the same as those used above. The meanings of the other symbols used in the table are the same as those used above. The meanings of the other symbols used in the table are the same as those used above. The meanings of the other symbols used in the table are the same as those used above. + +| Judgment matrix C of collaborative ability in regard to behavior. C C1 C2 | C3 | | | +|------------------------------------------------------------------------------|------|-----|-----| +| C1 | c11 | c12 | c13 | +| C2 | c21 | c22 | c23 | +| C3 | c31 | c32 | c33 | +| In the table. | | | | + +| Judgment matrix D of collaborative ability in regard emergency management. D D1 D2 | D3 | | | +|---------------------------------------------------------------------------------------|------|-----|-----| +| D1 | d11 | d12 | d13 | +| D2 | d21 | d22 | d23 | +| D3 | d31 | d32 | d33 | +| In the table. d12 indicates the importance of Factor D1 compared with Factor D2. | | | | + +(3) The maximum eigenvalue of the judgment matrix and the corresponding eigenvector are calculated. + +The eigenvector a^ = ( aft, a 2 , a 2 , a 2 ) 1 corresponding to the maximum eigenvalue of judgment matrix A , the eigenvector ω = ( ω f , ω f , ω f ) to the maximum eigenvalue of judgment matrix C and the eigenvector ω P = ( ωγ, ωγ, ωγ, ωγ, ωγ, corresponding to the maximum eigenvalue of judgment matrix D are calculated based on Formulas (2), (3), (4), (5) and (6) in Section 2 , respectively. The maximum eigenvalue +, of judgment matrix A , the maximum eigenvalue λ ,,, of judgment matrix B , the maximum eigenvalue λ ,,,, of judgment matrix C +and the maximum eigenvalue λ pax of judgment matrix D are calculated based on the eigenvector. + +(4) Consistency test of the judgment matrix. + +Based on Formulas (7) and (8) in Section 2 , the consistency of judgment matrices A , B , C and D is verified, and the consistency indicators of judgment matrices A, B, C and D are calculated to be CR,CR,CR,CR, and CR,, respectively. + +$$CR_{A}=\frac{\lambda_{\max}^{A}-3}{1.16},CR_{B}=\frac{\lambda_{\max}^{B}-3}{1.16},CR_{C}=\frac{\lambda_{\max}^{C}-3}{1.16},CR_{D}=\frac{\lambda_{\max}^{D}-3}{1.16}$$ + +(9) +We then judge whether the values of CRA,CRB,CRC and CRD are less than 0.1. When they are less than 0.1, the judgment matrices have satisfactory consistency. Otherwise, they need to be adjusted to eliminate excessive inconsistency until they are satisfactory. + +(5) Using the hierarchical structure model of the evaluation system, the index weight table of the evaluation system is developed, and the related research content is evaluated. + +After the judgment matrix passes the consistency test, based on the hierarchical structure model of the evaluation system detailed in step (1) of Section 3.2 and in combination with the eigenvectors ω A , ω P , ω C , and ω P as calculated in step (3) of Section 3.2 , the index weight table of the evaluation system is developed. + +![7_image_0.png](7_image_0.png) + +Schedule + +| July, August, September | | +|---------------------------|-----------------------| +| and October of each year | | +| July, August, September | | +| and October of each year | | +| November and December | | +| of each year, January and | | +| February of the next year | | +| November and December | | +| of each year, January and | | +| February of the next year | | +| November and December | | +| of each year, January and | | +| February of the next year | | +| November and December | | +| of each year, January and | | +| February of the next year | | +| March, April, may and | | +| June of the next year | | +| Project | March, April, may and | +| valuation | June of the next year | + +Fig. 2. The collaborative process of an office building project used in the joint graduation design of the architectural engineering specialty group. + +## 4. **Experimental Design** 4.1. Basic Information Of The Experimental Background + +An application-oriented university in Jiangsu, Chinacomprises11 secondary colleges: the College of Mechanical and Electrical Engineering, College of Architectural Planning, College of Precision Manufacturing, College of Logistics Engineering, College of Artificial Intelligence, College of Software Engineering, College of Information Engineering, College of Civil Engineering, College of Economics and Trade, College of Materials Engineering and College of Business Administration. To accelerate the upgrading and transformation of traditional disciplines, high-quality engineering and technical talent needs to be cultivated, and new-type engineering talent needs to be provided for the development of the new economy. The university has established a new engineering specialty group for mechanical manufacturing from some specialties in the college of mechanical and electrical engineering, the college of precision manufacturing, and the college of materials engineering by leveraging successful experience with interdisciplinarity, infiltration and the integration of disciplines in China and elsewhere. The college of artificial intelligence, college of software engineering and college of information engineering will form a new engineering specialty group for computer science, and the college of mechanical and electrical engineering, college of building planning and college of civil engineering will form a new engineering specialty group for building engineering. According to the requirements of the joint talent training program of new engineering specialty groups, modern IT methods are applied to joint graduation projects in eight specialties: heating and ventilation engineering, water supply and drainage engineering, and electrical engineering in the College of Mechanical and Electrical Engineering; architecture in the College of Architectural Planning; and structural engineering, engineering management, engineering cost, and surveying engineering in the College of Civil Engineering. Additionally, the projects involve surveying and mapping as well as the design, construction and information management of an office building project. The specific collaboration process, organizational structure, task division and schedule are shown in Fig. 2. To build an evaluation system for the collaborative ability of joint graduation design, the collaborative ability of the joint graduation design of new engineering specialty groups was evaluated in combination with the responses to the evaluation questionnaire of the collaborative ability of students in the joint graduation design of the 2020, 2021 and 2022 sessions. The evaluation grade division table of the collaborative ability of students participating in joint graduation design is shown in Table 8. Before we conducted this study, we reported it to the Ethics Approval Committee of Suzhou Vocational Institute of Industrial Technology and received permission from the committee to conduct the research,document number: SIIT20220021. In this study, all participants were volunteers who provided written informed consent. Furthermore, they knew that they had the right to withdraw from the study at any time during the experiment. We used numbers to refer to the participants instead of their names. Their data were only used for the purpose of research.. 4.2. *Construction of the evaluation system for the collaborative ability of a specialty group in the joint graduation design of an office* building project +(1) The first step was establishing a hierarchical structure model of the evaluation system for the collaborative ability of the joint graduation design of an office building project This hierarchical structure model is displayed in Fig. 1 of Section 3.2 and is not repeated here. + +| Table 7 Index weights of the evaluation system. Target layer Criterion layer | Weight | Index layer | Weight | Comprehensive weight | | +|-----------------------------------------------------------------------------------------------|------------------------------------------|---------------------------------|---------------------------------|-------------------------|-------------| +| Evaluation system of | Collaborative ability in cognition | ωA 1 | Collaborative ability regarding | ωB 1 | ωA 1 × ωB 1 | +| collaborative ability | targets Collaborative ability regarding | ωB 2 | ωA 1 × ωB 2 | | | +| knowledge Collaborative ability regarding | ωB 3 | ωA 1 × ωB 3 | | | | +| relationships | | | | | | +| Collaborative ability in behavior | ωA 2 | Collaborative ability regarding | ωC 1 | ωA 2 × ωC 1 | | +| software Collaborative ability regarding | ωC 2 | ωA 2 × ωC 2 | | | | +| workflow Collaborative ability regarding | ωC 3 | ωA 2 × ωC 3 | | | | +| organization | | | | | | +| Collaborative ability in emergency | ωA 3 | Collaborative ability regarding | ωD 1 | ωA 3 × ωD 1 | | +| management | culture Collaborative ability regarding | ωD 2 | ωA 3 × ωD 2 | | | +| learning Collaborative ability regarding | ωD 3 | ωA 3 × ωD 3 | | | | +| conflict | | | | | | +| Based on the data listed in Table 7, the values of the evaluation index weights are analyzed. | | | | | | + +| Evaluation grade division table of the collaborative ability of students in the joint graduation design. The evaluation grade The score range The grade description A 0–3 Very low collaborative ability B 3–5 Low collaborative ability C 5–7 General collaborative ability D 7–9 High collaborative ability E 9–10 Very high collaborative ability Note: The score range includes the lower limit but does not include the upper limit. | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +(2) Ten experts were asked to complete an anonymous questionnaire on the evaluation indicators of the collaborative ability of the joint graduation design of an office building project. The questionnaire went through five circulation processes including distribution, collection and summary, and finally, the unanimous opinions of the experts were synthesized in the final evaluation. Two pairs of comparison judgment matrices for the collaborative ability evaluation system of joint graduation design, collaborative ability in cognition, collaborative ability in behavior and collaborative ability in emergency management are constructed, i.e., judgment matrices A, B, C and D. +⎡ +⎢⎣ +1 2 2 +$$\begin{array}{r r r}{1}&{2}&{2}\\ {1/2}&{1}&{1}\\ {1/2}&{1}&{1}\end{array}\quad B=\left[\begin{array}{r r}{1}\\ {1/2}\\ {1/2}\end{array}\right]$$ +$$A=$$ +1/2 1 1/2 +1/2 2 1 +⎤ ⎥⎦C = ⎡ ⎢⎣ 1 1/2 2 2 1 2 1/2 1/2 1 ⎤ ⎥⎦D = ⎡ ⎢⎣ 1 1/2 1/2 2 1 2 2 1/2 1 ⎤ ⎥⎦ +(3) The maximum characteristic roots λAmax, λBmax, λCmax and λDmax of judgment matrices*A, B*, C and D and the corresponding eigenvectors ωA, ωB, ωC and ωD are calculated. + +Each column of judgment matrix A is normalized: + +A ′ = ⎡ ⎢⎣ 0.500 0.500 0.500 0.250 0.250 0.250 0.250 0.250 0.250 ⎤ ⎥⎦̅̅̅̅̅̅̅̅̅̅̅̅→ Calculating the sum by row ⎡⎢⎣ 1.50 0.75 0.75 ⎤ ⎥⎦̅̅̅̅̅̅̅̅̅→ Normalization processing ⎡⎢⎣ 0.50 0.25 0.25 ⎤ ⎥⎦ = ωA MA = A × ωA = ⎡ ⎢⎣ 1 2 2 1/2 1 1 1/2 1 1 ⎤ ⎥⎦ ⎡ ⎢⎣ 0.50 0.25 0.25 ⎤ ⎥⎦ = ⎡ ⎢⎣ 1.50 0.75 0.75 ⎤ ⎥⎦ +The maximum eigenvalue λAmax of judgment matrix A is calculated. + +$$\lambda_{\mathrm{max}}^{A}={\frac{1}{3}}\sum_{i=1}^{3}{\frac{M_{i}}{\omega_{i}}}={\frac{1}{3}}\left({\frac{1.5}{0.5}}\right)$$ +0.25 + +$$\cdot+{\frac{0.75}{0.25}}$$ +$$-{\frac{0.75}{0.25}}\biggr)=3$$ + +Similarly, the following values can be calculated: +ωB = (0.49, 0.198, 0.312) +T; ωC = (0.312, 0.49, 0.198) +T; ωD = (0.198, 0.49, 0.312) +T + +$$\lambda_{\mathrm{{rms}}}^{B}=3$$ +$\mathbf{ma}$ +λBmax = 3.054; λCmax = 3.054; λDmax=3.054 +(4) Consistency test of judgment matrices *A, B*, C and D. + +$\begin{array}{c}\mbox{$\rm C_{A}$}=\frac{\mbox{$\rm C_{A}$}}{0.58}=\frac{0}{0.58}=0\\ \mbox{$\rm C_{B}$}=\mbox{$\rm C_{C}$}=\mbox{$\rm C_{D}$}=\frac{3.054-3}{2}=0.027\\ \mbox{$\rm C_{B}$}=\mbox{$\rm C_{C}$}=\mbox{$\rm C_{D}$}=\frac{0.027}{0.58}=0.047\\ \end{array}$ +Because *CRA* <0.10 and CRB = CRC = CRD < 0.1, judgment matrices *A, B, C* and D have satisfactory consistency. + +(5) Judgment matrices *A, B, C* and D all pass the consistency test. Combined with the evaluation system hierarchy model in Fig. 1 in Subsection 3.2, the index weight table of the collaborative ability evaluation system for the joint graduation design of an office building project is developed, and the relevant research content is evaluated. + +Based on the weight values of the evaluation indicators listed in Table 9, it is concluded that collaborative ability regarding cognition has the largest weight among all evaluation indicators at the criterion layer and that collaborative ability regarding targets has the largest weight among all evaluation indicators at the indicator layer. + +(6) Twenty questionnaires to evaluate collaborative ability were administered to those students who participated in the joint graduation project in 2020, 2021 and 2022, and the questionnaires were collected and summarized. According to the statistical analysis of the questionnaires, among those students who participated in the joint graduation design in 2020, 6 students were evaluated as showing general collaboration ability, and 14 were evaluated as showing slightly high collaboration ability. The highest collaborative ability score was 8.0, the lowest score was 6.1, and the average score was 7.2. In 2021, 5 students were evaluated as showing general collaborative ability, and 15 were evaluated as showing slightly high collaborative ability. The highest collaborative ability score was 8.5, the lowest score was 6.6, and the average score was 7.7. In 2022, 3 students were evaluated as showing general collaborative ability, and 17 were evaluated as showing slightly high collaborative ability. The highest collaborative ability score was 8.9, the lowest score was 7.0, and the average score was 8.1. The distribution of evaluation indicators of the collaborative ability of students participating in joint graduation design in 2022 is shown in Table 10. The comparison chart of the collaborative ability scores of students participating in joint graduation design over the three years is shown in Fig. 3, and the distribution chart of collaborative ability evaluation grades is shown in Fig. 4. + +## 5. **Discussion** + +Fig. 2shows that specialty groups have collaborative needs not only in regard to different fields of professional knowledge but also regarding scheduling in following the joint graduation design. The students with an engineering surveying specialty should first carry out field mapping of the construction project, draw the topographic map of the construction project site and the BIM model of the site, and provide them to the students of the architectural planning and design specialty. The students of the architectural planning and design specialty should then draw the construction and BIM model of the construction project according to the functional requirements of the construction unit and provide them to the students of the architectural structure, heating and ventilation engineering, water supply and drainage engineering and electrical engineering specialties. The students of the engineering surveying specialty and architectural planning and design specialty must complete the design task in July, August, September and October of that year. The students of the building structure, heating and ventilation engineering, water supply and drainage engineering and electrical engineering specialties design building structure construction drawings, heating and ventilation engineering construction drawings, water supply and drainage engineering construction drawings, electrical engineering construction drawings and corresponding BIM models through the relevant calculations and must complete the design tasks in November and December of that year and January and February of the following year. After the BIM models designed by the students of the above specialties are verified following collaboration, all construction drawings and corresponding BIM models are provided to the students of the engineering management and engineering cost specialties for the detailed design of the construction BIM models and engineering pricing, and these students must complete the design tasks in March, April, May and June of that year. + +The judgment matrices A, B, C, and D corresponding to the collaborative ability evaluation system of the joint graduation design of an office building project all passed the consistency test, and the weight values of each evaluation index of the collaborative ability of + +| Table 9 The index weight table of the evaluation system. The target layer The criterion layer | The | The indicator layer | The | +|--------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------|-----------------------| +| weight | weight | | | +| The evaluation system for the collaborative ability of the joint graduation design of an office building project | Collaborative ability | 0.5 | Collaborative ability | +| regarding cognition | regarding targets Collaborative ability regarding knowledge Collaborative ability regarding relationships | | | +| Collaborative ability | 0.25 | Collaborative ability | | +| regarding behavior | regarding software Collaborative ability regarding workflow Collaborative ability regarding organization | | | +| Collaborative ability regarding emergency management | 0.25 | Collaborative ability regarding culture Collaborative ability regarding learning Collaborative ability regarding conflict | | + +| Table 10 The score distribution of the collaborative ability evaluation indicators of students who participated in joint graduation design in 2022. The criterion layer The average The indicator layer The highest The lowest The average score score score score Collaborative ability regarding cognition 4.05 Collaborative ability regarding 2.1805 1.7150 1.9845 target Collaborative ability regarding 0.8811 0.6930 0.8019 knowledge Collaborative ability regarding 1.3884 1.0920 1.2636 relationships Collaborative ability regarding behavior 2.025 Collaborative ability regarding 0.6942 0.5460 0.6318 software Collaborative ability regarding the 1.0947 0.8610 0.9923 workflow Collaborative ability regarding 0.4361 0.3430 0.4010 organization Collaborative ability regarding emergency 2.025 Collaborative ability regarding 0.4361 0.3430 0.4010 management culture Collaborative ability regarding 1.0947 0.8610 0.9923 learning Collaborative ability regarding 0.6942 0.5460 0.6318 conflict | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +![11_image_0.png](11_image_0.png) + +Fig. 3. Comparison chart of the collaboration ability scores of students who participated in joint graduation design over three years. + +![11_image_1.png](11_image_1.png) + +Fig. 4. The distribution of the collaborative ability evaluation grades of students who participated in joint graduation design over three years. + +joint graduation design were obtained. Table 9 shows that collaborative ability regarding cognition had the largest weight among the three evaluation indicators in the criterion layer, with a weight value of 0.5. Among the nine evaluation indicators in the indicator layer, the weights of collaborative ability regarding targets, workflow and learning were relatively large, with weight values of0.49. + +However, after combining the weights of the evaluation indicators in the criterion layer, the comprehensive weight of collaborative ability regarding targets among the nine evaluation indicators in the indicator layer was the largest, and its comprehensive weight value was 0.245.The comprehensive weights of the two evaluation indices of collaborative ability regarding workflow and that regarding learning were each 0.123. Table 10 presents the distribution of the scores of the evaluation indicators for the collaborative ability of students participating in joint graduation design in 2022. Additionally, Table 10 shows that collaborative ability regarding cognition scored the highest among the three evaluation indicators at the criterion layer, with a score of 4.05. Among the nine evaluation indicators in the indicator layer, collaborative ability regarding targets scored the highest, with the highest, lowest and average scores of 2.1805, 1.715 and 1.9845, respectively. Therefore, in the process of cultivating the collaborative ability of the joint graduation design of specialty groups, not only should the cultivation of the individual ideological cognitive collaborative ability of students participating in joint graduation design be considered but also the cultivation of the macro cognitive collaborative ability regarding the project goals based on the realization of their own targets should receive more attention. + +Figs. 3 and 4 were designed based on the statistical results of the 20 collaborative ability evaluation questionnaires distributed to the students participating in the joint graduation design over the three years of 2020, 2021 and 2022. Fig. 3shows that the highest, lowest and average scores of the students participating in joint graduation design over these three years slowly increased each year. This result indicates that the teaching practice of the joint graduation design of new engineering specialty groups based on digital technology is gradually being accepted and recognized by students and that the talent training scheme of joint graduation design has been undergoing gradual improvement. Fig. 4shows that the evaluation grades of the collaborative ability of the students participating in joint graduation design over the past three years were all concentrated at two levels, i.e., general and high collaborative ability, and there were no lower, low or higher collaborative ability levels. These results indicate that the students participating in the joint graduation design were curious about the new graduation design method from the initial implementation of the joint graduation design teaching practice of the engineering specialty groups based on digital technology and that they gradually accepted this teaching method. The collaborative ability evaluation level of the students participating in joint graduation design has not reached the level of higher collaborative ability. These results indicate that there is still room for further improvement in the training of collaborative ability regarding cognition, behavior and emergency management in the joint graduation design teaching method of new engineering specialty groups based on digital technology. Fig. 4 shows that higher collaborative ability underwent a slow upward trend year by year, while general collaborative ability showed a slow downward trend year by year. + +## 6. **Conclusion** + +In this paper, joint graduation design based on digital technology is carried out in new engineering specialty groups. Based on the weight and order of the evaluation indicators of the hierarchy model of the collaborative ability evaluation system for joint graduation design obtained through use of the Delphi method and AHP, the key evaluation indicators that need to be considered in the criterion and indicator layers of the joint graduation design collaborative ability evaluation system structure model, the aspects needing improvement and optimization, and the specific contents needing supplementation are analyzed. This study provides reliable basic data for the revision of the joint graduation design talent training program of new engineering specialty groups based on digital technology. However, both of these statistical analysis methods share a common problem. Both the distribution of the questionnaires and the expert scores are affected by subjective factors. In the future, graduation project scores should be improved by incorporating objective factors. + +Research on the construction of a collaborative ability evaluation system for the joint graduation design of new engineering specialty groups based on digital technology is carried out in the context of university specialty groups. Much of the collaborative work is performed through face-to-face coordination. In particular, the problems found in the collision inspection of the BIM models of different specialties and the problems encountered when the BIM model of the building, BIM model of the building structure and BIM installation model are converted into the graphic calculation model, reinforcement calculation model and installation calculation model required by the project cost discipline still need to be solved offline. Under the current situation of the COVID-19 pandemic, building a collaborative ability evaluation system for joint graduation design online is a topic for future research. + +## Ethical Statement + +Not applicable. + +Consent statement Not applicable. + +## Author Contribution Statement + +Tian Jiandong: Conceived and designed the experiments; Wrote the paper. He Guifang: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data. + +## Data Availability Statement + +Data included in article/supp. material/referenced in article. + +## Declaration Of Competing Interest + +We declare that we have no any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work, there is no potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. + +## Acknowledgments + +This research was financially supported by the Jiangsu Provincial College Graduates' Employment and the Entrepreneurship Research Project of the Jiangsu Provincial Department of Education, project number JCKT-C-20210505. + +## Appendix A. **Supplementary Data** + +Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e16855. + +## References + +[1] H. Liu, S. Fang, Z. Zhang, D. Li, K. Lin, J. Wang, Mfdnet: collaborative poses perception and matrix Fisher distribution for head pose estimation, IEEE Trans. + +Multimed. 24 (2021) 2449–2460. + +[2] H. Liu, T. Liu, Z. Zhang, A. Kumar, ARHPE: asymmetric relation-aware representation learning for head pose estimation in industrial human-machine interaction, IEEE Trans. Ind. Inf. 18 (10) (2022) 7107–7117. + +[3] Y. Ao, Y. Liu, L. Tan, L. Tan, M. Zhang, Q. Feng, J. Zhong, Y. Wang, L. Zhao, I. Martek, Factors driving BIM learning performance: research on Chinese sixth national BIM graduation design innovation competition of colleges and universities, Buildings 11 (12) (2021) 616. + +[4] X. Zhao, L. Teng, Research and practice on the teaching mode of multi school joint graduation design—taking the architecture speciality of Liaoning Province as an Example, J. Shenyang Univ. (Soc. Sci. Ed.) 23 (3) (2021) 337–342. + +[5] M. Li, J. Wang, Z. Gan, K. Zeng, X. Han, Discussion on evaluation method of joint graduation design, J. North China Univ. Technol. (Soc. Sci. Ed.) 20 (5) (2020) +108–133. + +[6] S. Wang, Y. Zhou, X. Zheng, G. Yang, C. Lin, Research on the collaborative graduation design mode of civil engineering based on the integration of industry and education, J. Qinzhou Univ. 34 (3) (2019) 67–71. + +[7] W. Wang, H. Li, Y. Liu, Application and practice of BIM technology in school enterprise joint graduation design, High. Arch. Educ. 27 (6) (2018) 161–166. + +[8] H. Liu, H. Nie, Z. Zhang, Y.F. Li, Anisotropic angle distribution learning for head pose estimation and attention understanding in human-computer interaction, Neurocomputing 433 (2021) 310–322. + +[9] H. Liu, C. Zheng, D. Li, X. Shen, K. Lin, J. Wang, Z. Zhang, Z. Zhang, N. Xiong, EDMF: efficient deep matrix factorization with review feature learning for industrial recommender system, IEEE Trans. Ind. Inf. 18 (7) (2022) 4361–4371. + +[10] T. Liu, B. Yang, H. Liu, J. Ju, J. Tang, S. Subramanian, Z.L. Zhang, GMDL: toward precise head pose estimation via Gaussian mixed distribution learning for students' attention understanding, Infrared Phys. Technol. 122 (2022), 104099. + +[11] M.G.N. Nelson, G.M.S. Guilherme, U.B. Luciana, C.N.P. Juliana, H.B.H. Amanda, M.B. Lívia, COVID-19 and digital technology: mobile applications available for download in smart phones, Texto & Contexto Enfermagem 29 (4) (2020) 1–11. + +[12] B. Chang, Application of digital technology in oral implantation, Med. Equip. 35 (2) (2022) 192–193. + +[13] H. Liu, C. Zheng, D. Li, Z. Zhang, K. Lin, X. Shen, N. Xiong, J. Wang, Multi-perspective social recommendation method with graph representation learning, Neurocomputing 468 (2022) 469–481. + +[14] H. Liu, T. Liu, Y. Chen, Z. Zhang, Y.F. Li, EHPE: skeleton cues-based Gaussian coordinate encoding for efficient human pose estimation, IEEE Trans. Multimed. + +(2023) 1–12. + +[15] X. Xu, Y. Shen, S. Zhong, Y. Jiang, C. Zhang, Exploration and practice of new engineering mode and innovative talent training —new engineering Π type scheme of Harbin Institute of Technology, Res. High. Educ. Eng. (2) (2020) 18–24. + +[16] J. Ding, Y. Chen, The thinking and operating mechanism of "building colleges by groups" in higher vocational colleges, Res. High. Educ. Eng. 3 (2020) 122–125. + +:. + +[17] X. Shang, H. Ji, H. Wu, K. Li, X. Jin, Q. Xia, Exploration of the mode of production education integration and collaborative education of new engineering disciplines in the context of professional clusters - taking the robotics major of Beijing Union University as an example, Sci. Technol. Innov. 11 (2022) 49–51. + +[18] M. Zhou, M. Dun, Z. Lu, H. Zhang, Practice and thinking of the multi subject cooperative education mode in the undergraduate joint graduation design of urban and rural planning, High. Arch. Educ. 30 (5) (2021) 146–154. + +[19] B. Wang, Y. Song, G. Zhu, Theoretical Research and Model Construction of Collaborative Cognition in Collaborative Work, the 11th Academic Conference of Network and Data Communication of China Computer Society, 2001, pp. 132–137, 2001. + +[20] Z. Xue, An Empirical Study on the Relationship between Multi Team Trust, Collaborative Behavior and Collaborative Innovation Performance of Internet Enterprises, Zhejiang Business University, 2017. + +[21] F. Xue, L. Geng, F. Yan, W. Liu, Research on the cultivation of college students' innovation and entrepreneurship ability and teaching reform - based on the analysis of the 2017 "Challenge Cup" college students' entrepreneurship competition, J. Hebei Agric. Univ. (Agric. For. Educ. Ed.) 20 (2) (2018) 5–8. + +[22] H. Guo, S. Huang, C. Wang, A decision method of ship overall plan group based on improved Delphi method, J. Shanghai Jiaot. Univ. 48 (4) (2014) 515–519; +[a] R. Hu, X. Chen, S. Xue, Strategies for universities to promote interdisciplinary integration - taking MIT as an example, J. Huazhong Agric. Univ. (Soc. Sci. Ed.) 3 (2012) 105–110. + +[23] Y. Liu, H. Zhang, T. Zhang, N. Li, Q. Wang, W. Wang, Y. Xu, R. Zhang, K. Wang, Evaluation of the comprehensive carrying capacity of resources and environment in the economic circle of Shandong provincial capital city agglomeration based on the analytic hierarchy process (AHP), 2019 Annual Conference of Science and Technology of Chinese Academy of Environmental Sciences —Environmental Engineering Technology Innovation and Application Sub Forum 8 (2019). + +[24] Q. Li, X. Zhou, Improved analytic hierarchy process for evaluating the comprehensive ability of clinical medical postgraduates, Modern Med. Health 35 (10) +(2019) 1583–1585. + +## + +[25] Q. Shen, R. Ouyang, Y. Ouyang, Construction of evaluation index system for achieving the goal of integration of industry and education based on Delphi method and analytic hierarchy process, Higher Educ. Explor. (12) (2021) 104–108. + +[26] Q. Zhou, Diversification and leap forward —preliminary exploration of the teaching mode of the joint graduation design of architecture, Educ. Teach. Forum +(50) (2018) 71–73. + +[27] H. Liu, C. Zhang, Y. Deng, B. Xie, T. Liu, Z. Zhang, Y.F. Li, TransIFC: invariant cues-aware feature concentration learning for efficient fine-grained bird image classification, IEEE Trans. Multimed. (2023) 1–14. + +[28] Z. Li, H. Liu, Z. Zhang, T. Liu, N. Xiong, Learning knowledge graph embedding with heterogeneous relation attention networks, IEEE Transact. Neural Networks Learn. Syst. 33 (8) (2022) 3961–3973. + +[29] T. Liu, J. Wang, B. Yang, X. Wang, Facial expression recognition method with multi-label distribution learning for non-verbal behavior understanding in the classroom, Infrared Phys. Technol. 112 (2021), 103594. \ No newline at end of file diff --git a/medical/md/PMC10320094.md b/medical/md/PMC10320094.md new file mode 100644 index 0000000000000000000000000000000000000000..7b19a60ad1a73627cb7ab4f45b656b0cd9652139 --- /dev/null +++ b/medical/md/PMC10320094.md @@ -0,0 +1,520 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +![0_image_3.png](0_image_3.png) + +![0_image_5.png](0_image_5.png) + +![0_image_6.png](0_image_6.png) + +## Abstract 1. **Introduction** + +| Keywords: Confidence International education Education decision-making International students | +|-----------------------------------------------------------------------------------------------------| + +Why confidence leads to swifter tertiary education choices: A + +![0_image_2.png](0_image_2.png) + +qualitative study of the international tertiary education industry of Australia Udo Rainer Gottlieb *, Amanda Beatson Queensland University of Technology, Business School, 2 George Street, Brisbane 4000, Australia This paper investigates international students' confidence as a base for imminent tertiary education decisions. International students are a highly sought-after commodity, particularly during and after a global pandemic when the income streams of tertiary education providers are limited. + +In-depth interviews were conducted with students who sought guidance towards an international study experience to explore the guiding research questions, (1) how does confidence impact tertiary education decisions of international students and (2) what is the relationship between confidence and time to make a tertiary education decision? Set within the context of the international tertiary education industry of Australia, the original contribution stems from identifying that guidance towards an international study experience is affected by confidence in; guidance counselors, the brand name of the university, and the tertiary education decision itself. The identified confidence characteristics in this study have an inverse relationship with the length of time taken for the students' decision-making process. This leads to swifter finalizations of tertiary education decisions by students increasing returns on education providers' admission activities. + +In high uncertainty environments, such as the (post)-COVID-19 world, service mega-disruptions have led to unexpected disruptions of entire service eco-systems [1]. Education service providers are facing immense financial pressures due to lockdowns, rapidly changing delivery modes, and a significant drop in international students [2]. Within the international education industry COVID-19 was the biggest crisis to have ever hit and has caused the most significant disruption to international student flows in history [3] +including student recruitment [4], content delivery methods [5,6], and student retention [7]. Given the recent removal of international border restrictions around the world, there is a heightened sense of urgency now to regain a lot of the international tertiary student market lost through the COVID-19 pandemic [8]. This is due not only because of the boost to tertiary education providers and their bottom line, but also because of the flow on effect of international students for tourism, job creation, skilled migration, and general economic benefits [9]. + +Under externally threating situations, such as anxiety about disease (e.g., COVID-19), individuals are known to increase variety seeking behaviors [10], therefore, it is likely that this extends to the international tertiary education industry as well, with students likely to exhibit an increased desire for variety seeking. Consequently, they would search for additional information on potential https://doi.org/10.1016/j.heliyon.2023.e17484 Received 30 October 2022; Received in revised form 14 June 2023; Accepted 20 June 2023 Available online 21 June 2023 2405-8440/© 2023 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/). + +Contents lists available at ScienceDirect + +![0_image_4.png](0_image_4.png) + +ARTICLE INFO +* Corresponding author. + +E-mail addresses: udo.gottlieb@qut.edu.au (U. Rainer Gottlieb), a.beatson@qut.edu.au (A. Beatson). + +international study experiences and education service providers, prior to making decisions. This variety seeking inclination results in increased exposure to different choices and time lags, adding further challenges to an already competitive and disrupted marketplace +[11,12]. Within this context, this research examines international students who seek guidance on international study experiences for tertiary education. Specifically, it explores the role of confidence in international students' tertiary education decisions and its temporal relationship to decision-making time. + +Confidence has been widely researched [13] and so have individuals' decision-making processes [14]. Specifically, confidence contributes to success, commitment, and goal achievement with regards to career development and is widely studied [15–17]. The global COVID-19 pandemic is still having unprecedented impact on governments, businesses, institutions of learning, and the general population alike. It caught everyone in the tertiary education industry, management, academics, and students unaware [18]. In such uncertain times when rules and regulations change frequently and individuals perceive the world to be in constant flux, a high level of confidence can contribute to a reduction of perceived risk [19]. Confidence is the expectation to not get disappointed [13] and provides certainty for future actions [20]. This is important, since individuals' decisions and behaviors are influenced by the levels of risk they perceive [19]. + +Within the tertiary education sector, the role of confidence has been studied by considering various stakeholders such as health professionals [21], students [22,23], and teachers [24]. These studies focus on future positive outcomes at an individual level. Within this space, organizational benefits to education service providers remain scant. Further, current research does not present a temporal assessment of confidence and decision-making time which may enhance organizational benefits. Considering these gaps, this paper conceptualizes the impact of confidence and its relationship with time by contextualizing it to students entering the tertiary education system. This specific context allows for a direct decision outcome (i.e., tertiary education decision) that can be determined by investigating the temporal implications of the decision. Therefore, by drawing on concepts such as the purchase decision-making process, time, and confidence this study explores the research questions, (1) how does confidence impact tertiary education decisions of international students and (2) what is the relationship between confidence and time to make a tertiary education decision? + +Underpinned by the Theory of Planned Behavior [25], theoretically, this paper proposes a relationship between confidence and decision-making time, putting into perspective the role confidence plays in a wider institutional context. Specifically, it proposes that the time taken to make a tertiary education decision is shortened if confidence persists during the process. The Theory of Planned Behavior (TPB) proposes that the likelihood of an individual acting on a behavior, in this instance enrolling at a particular university, is influenced by their attitude toward the behavior, subjective norms and perception of behavioral control, via their behavioral intentions [25]. The likelihood of action is stronger when their attitude is more favorable, in other words they have more confidence in their decision [26]. Therefore, this study is looking at how a decision can be strengthened through confidence in that decision, which is the strength of the attitude toward the behavior. Practically, this paper makes a case for confidence to be considered a facilitator of international students' tertiary education decision. As a further contribution, this current research will show that providing guidance in the form of interactions with education and guidance counsellors at international tertiary education shows encourages confidence in students across three identified internal and external factors. Guidance provided at these education shows includes information about career outcomes after graduation, what to expect when undertaking the course of study, opportunities that arise during the course including internships, placements and other work integrated learning opportunities, expectations regarding salary and employability, and other such information as required by the student. + +In the proceeding sections, first, the relevant literature will be discussed with particular focus on the concept of confidence and time. This is followed by the project's methodology, findings, and the discussion with implications and future research opportunities. + +## 2. **Literature Review** 2.1. Confidence + +Confidence is well established within decision-making processes [27] with the majority of research focusing on the relationship between subjective confidence and an objective outcome measure i.e., purchase decision [28]. Despite this, literature is yet to agree on a definition for the construct of confidence [29]. Whilst Blomqvist [30] defines confidence as a "state of mind" where one expects an outcome to occur with certainty, confidence is also considered as the belief that "everything is under control" and hence as the expectation that one does not get disappointed [19]. Since this research links confidence with future tertiary education decisions, it follows the definition of confidence as "a future directed call to action with a linked expectation of future actions by third party/ies and one's own future directed abilities" [31] (p. 284) underpinned by the Theory of Planned Behavior [32]. + +Confidence as a construct therefore refers to the magnitude of one's belief without any stipulation of the source of the belief [33]. + +The construct of confidence is broad since it can refer to sources internal and/or external to one's own and captures the amassed evidence for a decision [34]. Confidence is likely to vary based on perceived quantity and quality of information collected to make the decision [35]. When uncertainty is reduced, individuals are likely to incur greater confidence in decision-making [36]. Examples from the tertiary education industry demonstrate that confidence with regard to engaging in specific learning experiences such as e-learning +[37], clinical training [38–40], and peer teaching [41] impacts student performance. In terms of tertiary education decisions, self-confidence is a contributor for choosing science, technology, engineering, and mathematics (STEM) related study courses [42]. + +Knowledge, beliefs, attitudes, judgments, and expectations are primary factors of confidence, with confidence seen as having three key characteristics; constancy, certainty propensities, and anticipatory reflection [27,43]. Constancy refers to patterns related to an issue or domain specific area where a confidence assessment is ascribed. Certainty propensities express a set of beliefs based on consistency in past patterns supported by degrees of certainty. Anticipatory reflection signifies looking forward in time and conveys the 2 transferability of patterns into a future time [27]. Therefore, confidence is not independent of an individual's experiences, but is conceptualized as reflective conditioning imbedded in foresight which guides behavior. + +It is important to distinguish confidence from self-efficacy, one of the widely researched constructs in educational research and frequently misstated as equivalent to confidence. While these two constructs may be related, they are in fact two distinct constructs +[44,45]. Within education settings self-efficacy has been defined as "beliefs in one's capabilities to organize and execute the courses of action required to produce given attainments" [page 3, 46]. It evidently strengthens academic performance as well [47] and can work as a coping mechanism within learning environments [48]. Hence, self-efficacy only refers to sources of beliefs internal to one's own, whereas confidence incorporates both internal sources and external sources of certainty about future actions [19,20]. Due to this multifaceted nature, confidence is a valuable construct that can aid in predicting and comprehending future behavior [44]. + +## 2.2. Time And Confidence + +The concept of time is a fundamental part of the notion of confidence, as confidence reflects the amount of certainty one has about the future [20], which is highly relevant in the increasingly uncertain and unpredictable context of careers [49] and relevant career decisions. All future action is based on the feeling of assuredness or confidence about the decision to be made and therefore provides a temporal framing for the decision. The concept of time is also linked to any decision-making process due to the underlying confidence in making a decision e.g., a tertiary education decision. A decision in itself takes time to make, there is a lead-up time, and a time allocated to the post-decision evaluation. Researchers have investigated the concept of time and how it influences individuals' decisions [50]. For example, time was researched as a scarce resource and how limited time impacts individuals' decision-making [51]. + +Time is considered both an antecedent and a consequence of a decision [52]. For example, research can investigate whether factors such as complexity impact the amount of time spent on a decision, or whether time spent deciding impacts evaluation of that decision. Relevant research mostly focused either on decisions about time and/or on the decision-making process and thus decision time [50]. Particularly within the tertiary education industry, students are active information seekers [53]. They have a plethora of choices available to them 24/7 [54] and can make instant career development decisions. Therefore, time emerges as a key consideration in the tertiary education decision process. + +## 2.3. Service Interactions And Confidence + +One area touted for understanding individuals' needs and identifying required information from these individuals is interactions with staff [55]. These interactions, such as among actors within the tertiary education industry as in this current research, are at the center of any high involvement service. Career advisors and guidance counselors can influence these interactions to promote emotions in the form of confidence resulting in positive service experiences [56]. Positive emotions can include trust in the knowledge provided by the career advisors and guidance counselors, gratitude in the support provided for completing course and scholarship applications and hope regarding what the future entails after completing the course [57]. The decision-making literature focuses on psychological +(emotions, beliefs, intentions, etc.) and non-psychological (time, marketing mix, etc.) factors that influence decision-making processes. + +Yet, literature still lacks knowledge on the impact of confidence on tertiary education decisions. + +Research has shown that in the tertiary education industry, prospective students have as few as three alternatives in their evoked set of choices they eventually consider choosing from Ref. [58]. Therefore, this research argues that helping prospective students increase their confidence while making the right tertiary education decisions will enable them to reduce the decision-making time needed and to be more satisfied with their decision for longer. In turn, it will reduce prospective students' exposure and response to stimuli from competing tertiary education providers and increase the likelihood of a decision in favor of the guidance offering tertiary education provider. + +## 3. **Methodology** + +The objectives of this research were to identify how confidence impacts international students' perception of their tertiary education decisions, the choice process, as well as its impact on the student recruitment processes of tertiary education providers. This research project extends the literature by evaluating the impact of confidence as a future directed emotion with a direct impact on tertiary education decisions creating temporal and financial benefits for education service providers. + +## 3.1. Research Design + +Since confidence and its impact on tertiary education decisions are underrepresented in the literature with little attention to students entering or moving within the tertiary education system, an exploratory approach to the research design was justified. As the research focused on participants' experiences and their rich and detailed descriptions of these, semi-structured in-depth interviews were considered the most appropriate [59]. Interview questions were designed to solicit information from the participants about the decision-making process they underwent when deciding on an international study experience, in particular what elements were most influential in the process. + +## 3.2. Sample + +The data was obtained from students who had previously visited international tertiary education shows where multiple education providers from around the world interacted with prospective students. These international tertiary education shows formed part of their tertiary education choice process for an international study experience at a university in Australia. Fourteen interviews were conducted over a seven-month timeframe in 2018 once the students had made their choice and had arrived in Australia to take up their study. This breadth of time helped manage any potential selection bias by ensuring a variety of participants. However, through reflexivity it is acknowledged that there will always be a level of bias in such qualitative work despite the experience of the interviewers [60]. + +The Australian international tertiary education industry was chosen since Australia together with other Anglophone countries have the longest-lasting and well-established international student markets [61]. Since all participants interviewed were enrolled at a university in Australia and accessible on campus, purposive sampling was utilized using convenience data collection [62]. This style of homogenous, purposive sample is typically adopted in qualitative research to maximize the insights gained from the sample of interest +[63]. The sample is selected to highlight the conditions under which the phenomena of interest operate, not the generalizability of the findings to other settings [64]. Whilst purposive sampling was favored over population-based probability sampling due to cost reasons this may have exposed the research project to increased selection bias and/or sampling error but given that the goal of the research was to investigate the phenomenon of interest in detail, these potential limitations were deemed not to be substantial [64]. + +## 3.3. Participant Recruitment + +Teaching staff across several undergraduate and postgraduate degrees promoted the research project before the start of their lectures. The students were advised that the researchers were looking for students who participated in a tertiary education show before committing to enrol in the degree they were currently studying. Amongst others, the students were further informed of the purpose of the study, that ethics approval was obtained, sample questions, approximate time involvement, and that they would receive a AU$20 shopping voucher for their time. Interested students were then directed to email the research team to be considered. + +This resulted in a sample population comprising 14 interviewees. Eight participants self-identified as female and six as male. They were all between the ages of 22 and 38 years of age. The interviewees' countries of origin span across four continents (i.e., Egypt, France, Germany, Singapore, Vietnam, China, Indonesia, India, and USA). The interviewees were enrolled in undergraduate (eight) +and postgraduate (six) degrees. One interviewee identified as studying in a research degree (PhD) and the remaining 13 interviewees were studying in coursework degrees (Bachelor and Master). + +## 3.4. Data Collection + +After two initial pilot interviews, followed by minor adjustments to the standardized interview guide, 12 additional interviews were + +![3_image_0.png](3_image_0.png) + +conducted independently by two researchers. These interviews lasted between 30 and 40 min. In total 14 audiotaped face-to-face interviews took place. Thematic saturation [65] was used as an indicator for when to conclude the interviewing process. These interviews were subsequently professionally transcribed. With a homogeneous sample and good quality data, 12 interviews are normally sufficient to reach data saturation [66]. Exploratory studies in social sciences are best completed with small samples, allowing for improved validity of the in-depth and detailed analysis within the social setting [67]. + +Both interviewers are highly experienced in designing and conducting in-depth, semi-structured interviews. The interviewers were academic staff from a Business School in a major Australian university. In line with Corbin and Strauss [68], the interview guide evolved throughout the data collection process to ensure it captured any new concepts that emerged. Sample questions included, "what elements were most influential to your decision-making process", "tell me about your experience when making a decision" and "what were the outcomes of your decision-making experience". At this stage the participants were also probed for the length of time it took them to make the decision. The appendix outlines the interview guide. + +## 3.5. Data Analysis + +The data analysis was "conducted in a precise, consistent and exhaustive manner" [69]. The research process of thematic analysis followed Lincoln and Guba [70] utilizing the six phases of Braun and Clarke [71] and their 15-point checklist, including the organization and description of the data. See Fig. 1 for these six phases. Categories and themes were identified through rigorous analysis conducted by both researchers. The data was first sorted and coded using NVivo 11, a qualitative data analysis software program from QSR International and a codebook developed by the researchers. This thematic content analysis resulted in the identification of emerging categories, themes, and findings. Thematic analysis is a widely used analytic method in qualitative research that enables a thorough explanation of the data by classifying, analyzing, and recording of patterns [71]. An additional count of the themes was conducted following Miles and Huberman [72] to provide further information. + +Rigor was established through dependability, credibility, and confirmability as outlined below [70]. + +- Dependability: an audit trail was established outlining the decisions made, such as the justification for the sample as well as the method used, including the questions in the interview guide [73]. Furthermore, consultation between the researchers occurred regularly throughout the data collection and analysis phases. Although the researchers conducted the analysis individually, they compared their findings and any discrepancies to validate findings and to minimize potential bias [74]. + +- Credibility: to ensure credibility through the analysis phase, ideas were garnered through literature searches and discussions with other researchers, stretching the authors' ideas such as the identification of theoretical frameworks and concepts (see phases 2–5 in Fig. 1). Regular peer debriefings about the outcomes were also a part of the process [73]. + +- Confirmability: was achieved by utilizing a reflexive approach, where all decisions undertaken were recorded in such a manner enabling confirmation from others (outlined in phases 2–5 in Fig. 1). + +Ethical approval was obtained from the researchers' home university (University Human Research Ethics Committee), and the research was conducted in line with standard ethical guidelines and the National Statement on Ethical Conduct in Human Research. + +## 4. **Findings** + +This research project explored actual tertiary education decisions with 14 participants who had embarked on an international study experience. To analyze the influence of confidence on tertiary education decisions, prospective students who attended international tertiary education shows in search of guidance were interviewed. Through data analysis, three components of confidence were identified as having an impact on the tertiary education decisions of prospective students. Thematic content analysis revealed that confidence was mentioned across all participants 48 times. Further scrutiny of the identified mentionings of confidence, revealed three separate components of confidence; "confidence in the decision to be made", "confidence in the education provider's brand", and + +| Interview# | Newly identified emotion construct - Confidence Confidence main construct Confidence Sub constructs Confidence in the tertiary education decision | Education provider's brand | Guidance counselors | | +|--------------|-------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|-----------------------|----| +| 1 | 10 | 6 | 3 | 1 | +| 2 | 7 | 3 | 2 | 2 | +| 3 | 6 | 3 | 2 | 1 | +| 4 | 5 | 1 | 1 | 3 | +| 5 | 4 | 2 | 2 | 0 | +| 6 | 3 | 1 | 0 | 2 | +| 7 | 3 | 1 | 0 | 2 | +| 8 | 3 | 0 | 1 | 2 | +| 9 | 2 | 1 | 1 | 0 | +| 10 | 2 | 0 | 2 | 0 | +| 11 | 1 | 1 | 0 | 0 | +| 12 | 1 | 0 | 0 | 1 | +| 13 | 1 | 0 | 1 | 0 | +| 14 | 0 | 0 | 0 | 0 | +| ∑ | 48 | 19 | 15 | 14 | + +"confidence in the education provider's guidance counselors". These three components will be discussed in turn. Each of these components of confidence were independent of each other with no discussion of them interlinking. The component with the highest mentionings was "confidence in the decision to be made". See Table 1 for a breakdown of numbers for a participant level. + +These three components map to the three characteristics related to patterns and propensities identified by Simintiras et al. [27]; +constancy, certainty propensities and anticipatory reflection. These components represented both controllable aspects of confidence (external factors to the participant) and non-controllable aspects of confidence (internal factors to the participant). As such, the current work consolidates past findings and extends this work by identifying the findings within the tertiary education decision process and exploring the relationship between the components of confidence, time to a decision and resulting benefits for the education service provider. + +## 4.1. Confidence - Clarification On The Decision To Be Made + +The most frequent confidence component to be discussed in the interviews was the notion of being confident in the tertiary education decision participants were making. The participants suggested they came to the education shows to seek final guidance around their decision and to reduce the options or to confirm an underlying prior idea, reflecting the search for constancy in patterns [27] +evidenced through prior research by the participants. Choice is important; however, choice overload may result in lower confidence in the eventual decision made [75]. Although they might not come with a final evoked set [58] to choose from, they have identified criteria to evaluate the tertiary education options available to them. Given that perceived quality of education emerged as an increasing consideration by students [76], directed efforts such as education shows can be used to disseminate such information strengthening students' decision-making confidence. + +Confidence in the tertiary education decision to be made was mentioned the most with 19 out of 48 total mentionings. Hence, the data suggests that prospective students within international tertiary education choose to attend international tertiary education shows towards the end of their choice process when the initial information search stage has already been concluded and a narrowing down of options towards an evoked set is in the process. + +Everything was clear to me, so I decided. Actually, I didn't have time to browsing all the booths on that day because I got what I want within the timeframe, so I think I told my husband, I say, "Okay, we know everything, so I think we don't have much time, we should go home, prepare application and everything," so we didn't browse much, just got what we want and go home. + +(Interview 2) +… because I knew that I wanted to go deeper into this career and to do that I had to bridge my various knowledge gaps and that was through further studies, so I knew that I wanted to go there. So the trade show helped me to better understand the expectations and living standards in Australia, and yeah it really compelled my decision ultimately. (Interview 9) +I felt more assured that I had really shortlisted a few universities rather than - because I came in with more in mind, with more choices in mind, but I left with a shortlist of one, so that reduced my choice. (Interview 1) + +## 4.2. Confidence - Education Provider'S Brand Recognition + +The second most frequently identified component of confidence was the feeling of confidence the participants had in the brand of the university. University brand image is a key consideration of students across cultures [76]. Aligning with this, the participants put confidence in well-known brand names suggesting alignment with certainty propensities and pattern reoccurrence [27]. + +Students visiting international tertiary education shows appear to be also somewhat influenced by their peers. These influences span across areas of what they feel about the recognizable brand names, and which university they put confidence into delivering the course they are interested in. Interestingly, word of mouth appears to be more useful than other marketing efforts in a tertiary education context [77]. The transcripts reveal that potential international students trust their peers as part of their in-group and that they are inclined to put confidence in their peer's decisions [78]. + +I went to this [university], that's one. Because from my seniors I heard that - about [university] and all that they conduct these things, they will apply for visas or they'll help you in each and every stage, so that's why I went to that place because … they will be conducting the [language] examination, it means they are good authorities (Interview 13) +I think I would not be that interested in a university that I never heard before. … I found the name that I'm interested in before I +attended, yeah, because there are lists of the universities that will attend the show. Even when I ask my friends about it, they say is a large amount of universities is coming through. How many of them? So each one make up the whole first impression of whether people want to go or are not going. (Interview 6) + +## 4.3. Confidence - Guidance Counselors + +The final component of confidence which emerged through the interviews was the importance placed on the interactions they had with the education provider's guidance counselors rather than through interactions with other entities such as educational agents or through university websites. The participants referred to the confidence gained in their decision based on the style of interactivity [79] they had with the counselors. + +Students perceive an information overload when trying to identify and choose the right education provider to pursue their career goal. The participants indicated that third parties, such as educational agents, are only broadly supportive regarding the tertiary education choice process. They frequently lack knowledge and skills when it comes to guidance and career development options. + +Students initially try to compensate for the lack of rich information with individual information searches online. In doing so the exhibiting education providers' websites are used, as well as electronic word of mouth seeking behavior via social media [80]. + +The findings indicate that interactions with guidance counselors at international tertiary education shows play a part in establishing the students' confidence and perception of the degree/course they are searching information for and therefore, initiate the relationship building even before students sign up with a particular tertiary education provider. This early relationship building captures the anticipatory reflection evident in the characteristics of confidence [27]. + +I think it's a matter of the person in charge. I'm still thinking if that staff wasn't the person I talked to during the trade show, I +may not even choose [name of university] because he was really convincing. And in all the trade shows, I met him. So I came to meet him three times. So he knows all the information he has told me and now what's built up from there. (Interview 4) +I think, it was the boost, you can say, that I got the confidence that yes I can get the scholarship. The confidence from that that yes I can get the scholarship, they saw my profile and everything so they said that, you will definitely get a scholarship. + +(Interview 12) +… you can actually go talk to a person who's representing the university, which was quite good. And I also wanted to know my chances, because like what if I'm not accepted, so like, "This is my score, so where do I stand?" (Interview 6) +Actually, I want to talk directly to the institutions' representatives. I want them directly address my concerns. Because I kept emailing back and forwards with some of the staff … and I didn't get really good answer from those staff, I know they need authority from their manager, or something like that, so I come directly, on that day I really want a good answer so I can make application straight away. (Interview 1) Actually, the staff really knowledgeable. When I switch from education to social work, he can tell me straight away, he open the booklet and he show me the requirements of social work degree, English requirements, application due date for international student. He make it really clear about all the requirements, so it's really good for me. (Interview 1) + +## 4.4. Temporal Relationship Between Exposure To Guidance, Confidence, And Tertiary Education Decision + +Due to decision processes that regularly vary in length and complexity, the performance of international trade show participation has been questioned previously [81]. To address this previous questioning, the current research investigated the time it took the interviewed students from the exposure of the guidance provided at an international tertiary education show to the finalization of their tertiary education choice process and signing up to an international study experience. + +The research data shows students displayed a significant spread of the temporal relationships between receiving guidance and the time the tertiary education choice process was finalized. Time and related pressures have been previously identified as decision stressors that impact decision quality [82]. Findings reveal that confidence built through international trade shows contributes to reducing decision-making time influencing students' decision quality through the improvement of the decision-making process. Since all participants were active students at an Australian university, the focus of this research was how swiftly they made their actual finalized tertiary education decisions as an outcome of the guidance provided to them, therefore actual behavior around this decision was considered. The temporal range included finalization of the decision through an on-the-spot commitment during the international tertiary education show, to a decision that took place five years after participation as seen in Table 2. Almost 80% (11) of participants indicated that they had committed to an international study experience within a month of receiving guidance, with almost half of these + +| Table 2 Impact of confidence on the temporal link between career guidance received & career development decision. Interview # Confidence TOTAL Time between career guidance received and finalization of career development decision 1 10 At the TE show 2 7 1 day 3 6 1 week 4 5 1 week 5 4 1 month 6 3 1 week 7 3 1 month 8 3 1 month 9 2 1 month 10 2 1 month 11 1 1 month 12 1 2 months 13 1 4 months 14 0 5 years | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +7 participants (five) having made their decision within a week of receiving guidance at the international tertiary education show. + +## 4.5. Impact Of "Confidence" On Students' Tertiary Education Decisions + +One of the aims of this research was to investigate the role confidence played as a base for international students' tertiary education decision outcomes. This was accomplished by comparing the participants' references to the various components of confidence experienced during their visit to an international tertiary education show and their exposure to relevant guidance, with the time it took the participants to finalize their tertiary education choice process. The findings show a relationship between the confidence gained from the international tertiary education show, and the duration of the final tertiary education decision. Table 2 depicts confidence as a proposed base for guidance outcomes. Accordingly, the more mentionings the participants made regarding confidence, the faster they finalized their tertiary education choice process. The highest number of confidence mentionings resulted in an on-the-spot decision for one participant. The participant who did not express any form of confidence when recalling the guidance s/he received took five years to reach a tertiary education decision based on the guidance received at the international tertiary education show. + +Between the previously mentioned two extreme cases, all remaining participants mentioned various amounts of confidence, whereby, the lower the amount of confidence exhibited the longer the time between receiving guidance and the finalization of the tertiary education choice process. The only exception is Participant 6 who, with a lower number of mentionings of confidence depicted a faster decision than Participant 5 who ranked higher in confidence yet took longer to finalize her/his decision. Thus, the amount of confidence appears to suggest a temporal connection between receiving guidance, the confidence gained through that process and the final decision. + +The following sections delineate the discussion and implications as to the role of confidence on the tertiary education choice process. The revelation that the temporal relationship between guidance and a tertiary education decision is influenced by the amount of confidence perceived by students is novel and emerges as a significant contribution of this paper. + +## 5. **Discussion** + +This research answers the questions, (1) how does confidence impact tertiary education decisions of international students and (2) +what is the relationship between confidence and time to make a tertiary education decision? Previous research has shown that confidence influences individuals' decision-making behavior [31]. This research builds on the literature identifying confidence as strengthening the tertiary education decision process whereby the greater the amount of confidence experienced the shorter the tertiary education decision timeframe. This is supported by TPB, demonstrating that when students have a stronger positive attitude toward their decision, they are more likely to act on the decision, hence shortening the time to making the education decision-making process. + +In response to question 1, how does confidence impact tertiary education decisions of international students, the results revealed that although international students have access to a lot of information about different universities and study options, it appears that participants rely a great deal on guidance counselling obtained at international tertiary education shows to build confidence in their final decision. This research identified three specific confidence factors; confidence about the tertiary education decision to be made, confidence in the interactions with the university's guidance counselors, and confidence in the university's brand recognition, as influencing the confidence that persists through to the decision-making point. These three components reflect the three theoretical characteristics of confidence previously identified by Simintiras et al. [27]. The challenge for tertiary education providers is that only two of these elements (i.e., guidance counselors and brand) are to some degree within their control. The other factor, confidence in the tertiary education decision to be made, is a subjective perception of one's future actions and is internal to the student. However, as the two factors of guidance counselors and brand name inevitably influence students' final decision, tertiary education providers may use the resulting halo effect to their advantage. + +## 5.1. Confidence In The Tertiary Education Decision + +Confidence appears to be a key contributor in students' choice of tertiary education and their prospective career [42]. Still, research on how confidence influences their decision process remains limited. To this end, through its findings this research suggests that confidence aids international students' tertiary education decision process by providing affirmations to their previous research and perceptions. Evidently, this confidence is influenced by organizational level factors that include guidance counselors and the institution's brand recognition encountered at international tertiary education shows. + +## 5.2. Confidence In Guidance Counselors + +Guidance counselors emerged as contributing to strengthening international students' confidence in a context of tertiary education decision-making. The interactions at international tertiary education shows between students and guidance counselors from exhibiting education providers generated responses such as trust [31] and gratitude [83] which were linked to the feeling of confidence the participants displayed. This supports research in the education sector that links interactions of students and staff with a reduction in anxiety and an increase in confidence [84] and previous research highlighting the importance of interactions with employees in establishing positive responses towards an organization [55]. This same finding emerged in the current research, with participants indicating that confidence in the information provided by guidance counselors resulted in a favorable disposition toward their tertiary education decision. Within tertiary education, a lack of specific degree and course knowledge is compensated for by the education providers' facilitation of engaging guidance counselors. Research has shown that students have a perceived need for positive relationships with staff of tertiary education providers such as guidance counselors [85,86]. This element suggests that education providers should ensure appropriate training of staff representing them at international tertiary education shows, as it is knowledge acquired through these interactions that translates to confidence in the student's final decision. This study confirms students' expectation of guidance counselors to provide them with accurate and immediate information about courses, progression, and further opportunities like scholarships. It appears also that the manner that this information is delivered is also important, not just the information itself. This includes the convincing style and tone of speech used at tertiary education shows just as it is required in other frontline informational exchanges [87]. This assured style of service delivery is echoed in other service research and is linked with the degree of training and appropriate recruitment of staff [see for example, 88]. + +## 5.3. Confidence In The Education Provider'S Brand Recognition + +Education provider brand and the confidence attached to it emerged as strengthening tertiary education decisions as well. This finding provides further justification for investments in brand name and university ranking efforts within the tertiary education industry. Rankings have become a powerful influencer within tertiary education [89]. The global push of tertiary education providers to be well presented across a number of international rankings seems to support this development [90]. When an education provider is highly ranked, the likelihood of the education providers' brand to get recognized at education shows is therefore of considerable benefit to the attempts to get students (and their families [91]) to walk onto the booth and to engage with the exhibiting education provider. Universities have focused more recently on rankings and accreditation of their programs to differentiate themselves [92] for student recruitment, attract a higher caliber of staff and as an indicator of quality [93]. This more objective measure of university quality [94] was seen by international students as a way of establishing confidence in their decision. If the brand was recognized and that connection was made during marketing communication and service interactions such as international tertiary education shows, confidence in the tertiary education decision can be enhanced. Education providers need to ensure continued efforts in improving their rankings and accreditation levels while translating these efforts into meaningful information for prospective international students +[76]. Rankings and accreditations have a favorable response from potential employers [93] and perhaps through student case studies, career progression stories and the like, guidance counselors can use this information to further confidence in the brand name. + +## 5.4. The Temporal Relationship Between Guidance, Confidence, And Time + +Addressing research question 2, what is the relationship between confidence and time to make a tertiary education decision, the findings revealed the connection between confidence and the time difference between students receiving guidance at an international tertiary education show and the finalization of their tertiary education decision. Findings show that the higher the confidence level of participants during the international tertiary education show attendance, the shorter the time between that event and the finalization of decisions. Within an education context, research has shown that if education providers help increase their students' confidence levels in their academic performance, then students are more likely to engage with the subject matter [95] and the higher their confidence levels the stronger their decision-making behavior [96]. A similar link is likely for students seeking guidance on their tertiary education choices. When students engage with guidance counselors, they are more likely to be confident in the tertiary education decision they are making. A key component for an organization's competitive advantage is the effective delivery of services +[97]. Therefore, this research envisions the confidence construct to provide an alternative method for academic and industry led research into the assessment of guidance outcomes. + +## 5.5. Theoretical Implications + +This research examined international students within tertiary education who sought guidance towards an international study experience. Prospective students attend international tertiary education shows to gain further information about tertiary education decisions. By exploring the role of confidence in this interaction, the inverse relationship between exposure to information and time taken to make a tertiary education decision is highlighted. This finding is supported by TPB in that the stronger the attitude toward a behavior, the more likely the behavior will be enacted [98]. Thus, a possible temporal relationship between guidance, confidence and decision is proposed. This helps further comprehension of international students' decision-making process directing academic attention to confidence within this process. Furthermore, the three components of confidence; guidance counselors, the brand name of the university, and the tertiary education decision itself, reflect the theoretical characteristics of constancy, certainty propensities, and anticipatory reflection proposed by Simintiras et al. [27] indicating parallels between consumer behavior research and education marketing research. + +The findings provide a first indication for the elements that academics can use as antecedents when trying to ascertain how to understand the confidence levels perceived by students as part of their tertiary education choice process. Specifically, these implications are of particular significance within high uncertainty environments such as the international tertiary education environment but also during events that may cause general uncertainty such as a global pandemic. Furthermore, the findings have direct relevance to academics working on guidance outcomes for students (i.e., linearity/changes, choice) as well as for career guidance outcomes for tertiary education institutions that focus on student recruitment and measure this success in student admission numbers. The findings further support the development of a generally applicable guidance outcomes scale based on the components of confidence as revealed in this study. + +The theoretical understanding into confidence is enhanced by examining the construct of confidence in a contemporary service setting, such as international tertiary education shows. As confidence reflects belief not only in one's own assuredness, but also that of the organization, it is important to investigate confidence within multiple contexts. This paper contributes to a growing body of literature of confidence research in a variety of service contexts, such as tourism and hospitality, the public service sector, and banking +[99–101]. + +## 5.6. Managerial Implications + +This research shows that confidence can be used as a base for the likelihood of an imminent tertiary education decision. This is of significance as the career development choice process can be long and complex with many individual events and milestones along its way, as well as a significant number of alternatives to consider [102]. Similarly, within the tertiary education industry as the context of this study, students typically have an evoked set of at least three universities to choose from Ref. [58]. This evoked set, and desire for variety seeking, is also likely to be increased by students due to such perceived threats as COVID-19, posing additional challenges and time delays for education providers trying to attract prospective students. Therefore, the more an education provider can influence the confidence levels of its prospective students, the closer the temporal gap between receiving guidance and making a tertiary education decision. This temporal proximity limits the number of alternatives within a student's evoked set of tertiary education providers to choose from and maintains a higher level of confidence in the decision by reducing the prospective students' exposure and response to stimuli from competing tertiary education providers. From a tertiary education provider's point of view this provides for higher student admission numbers and a better conversion ratio. + +Another strategy that can support closing the temporal link while maintaining confidence in the decision-making process that tertiary education providers with international branch campuses undertook through COVID-19 was offering students to begin a degree at a branch campus, and then transfer at a later stage to the home campus. This is a strategy that can be leveraged more with additional improvements to online learning and support resources and reduce the uncertainty in the decision process and increase confidence levels for prospective students. + +The data suggests that the two confidence components that managers can influence (i.e., education provider's brand and guidance counselors) might have the highest likelihood for tertiary education providers to pro-actively influence the confidence perceptions of their current and prospective students. Specifically, guidance counselors and other front-line staff can focus their efforts on their communications strategies to ensure that the tertiary education provider's messages are consistent and in support of a coherent brand reputation whilst also providing adequate guidance to the individual student. Within the context of international tertiary education shows and more generally within the context of the education industry a new focus on the construct of confidence might offer practitioners a new avenue to evaluate the performance of their guidance counselors and other front-line staff. + +Additionally, the research shows that the education provider's guidance counselors need to ensure a consistent and prompt service experience. This could be achieved by rigorous and prompt follow-up processes after the initial contact with a student. The participants indicated that the expectation for instant responses and instant answers when communicating with an exhibiting tertiary education provider is a differentiating factor and has direct impact on their tertiary education choice process. As a conclusion, this research presents tertiary education decision, guidance counselors and brand name as strengthening confidence of prospective students who seek guidance through international tertiary education shows. This confidence translates to swifter decision-making within this context as well. Thus, a temporal relationship between guidance, confidence and tertiary education decision is suggested. + +## 5.7. Limitations And Future Research + +Although the research presented is comprehensive, some limitations exist in research design given the qualitative nature of the study. The interviews were with students who all chose the same university as their final international study option and the students were being asked to reflect on an earlier decision, which may have resulted in them not as accurately remembering their thought processes of the time. Although every effort was made to avoid selection bias, to use a broad cross-section of participants, and ask them to accurately reflect on their decision, this does not necessarily constitute a representative sample of the education industry globally although this emphasis on the conditions under which the concepts operate, not with generalizability, is a key focus of qualitative insights [64]. Students choosing international study experiences in countries other than Australia could, however, be considered for future research, as well as students whose aim is a domestic study experience. Interviewing students directly at the tradeshow could also limit the impact of memory on decision making. + +While this research was conducted prior to the COVID-19 pandemic, the results still highlight important insights into the role of confidence in making tertiary education decisions and how best to enhance this for tertiary students. With the general consensus that Australia will continue its focus on international student recruitment despite increased competition from other countries, insights into optimizing student confidence in their decision-making process is valuable. To extend the generalizability of the study however, future research may like to collect more data post the COVID-19 pandemic. While Australia did not experience the extensive challenges other countries did, it would still be beneficial to extend the findings and potentially make comparisons pre- and post-pandemic. Furthermore, it would be advantageous to investigate how health fears impact students' confidence levels. While Australia has not experienced the numbers of deaths or COVID-19 related cases as other countries have, this may impact aspects of confidence and the time to decide. Data collected now after individuals have come to a level of acceptance of the COVID-19 pandemic would also help extend the findings. + +Extending the research to include a quantitative investigation would also provide generalizability beyond the limitations of a qualitative study. It could be of benefit to investigate students who did not acquire any guidance prior to making their study decision to see if the same findings hold. Specifically, an experimental study could investigate both a control group of students who did not receive any guidance, and a group who did receive guidance. The components of confidence could be manipulated with these two groups to see if the results hold. Furthermore, a broader quantitative investigation could also overcome potential bias in the results from the small sample. While the focus of this research was on confidence, other factors beyond confidence might also act as indicators for tertiary education decisions as well as self-beliefs such as self-efficacy and self-esteem. Future research should investigate this and assess alternative indicators. The research into confidence as a construct for a perceived value concept as a base for a tertiary education decision within the tertiary education choice process was exploratory in nature and should be replicated. This research has highlighted several under-researched areas and will hopefully be able to help academics as well as practitioners extend research in this area. + +## Name Of The Approving Ethics Committee + +University Human Research Ethics Committee (UHREC). + +Ethics approval number 1500000301. + +Production notes Author contribution statement Udo Gottlieb, Amanda Beatson: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper. + +Data availability statement Data will be made available on request. + +Additional information No additional information is available for this paper. + +## 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. + +## Appendix. **Interview Guide** + +1. What tertiary education trade show did you attend & when? + +2. Why did you attend this particular tertiary education trade show? + +a. What motivated you to attend? + +b. What were your objectives for attending the trade show? + +c. When did you make the decision to attend [university]? + +3. Please tell me about your experience when you attended this trade show. + +a. What did you like about this particular trade show? + +b. What did you dislike about this particular trade show? + +c. Tell me about your experience when making a decision. + +4. What were the outcomes of your decision-making experience? + +a. What elements of the trade show were most influential to your experience/decision-making processes? + +b. What were the outcomes of attending this trade show besides from signing up with [university]? + +5. Did you achieve your initial objectives? + +## References + +[1] S. Kabadayi, E. O'Connor Genevieve, S. Tuzovic, Viewpoint: The Impact Of Coronavirus On Service Ecosystems As Service Mega-Disruptions, 34(6), 2020, pp. 809–817. + +[2] S. Krishnamurthy, The future of business education: a commentary in the shadow of the Covid-19 pandemic, J. Bus. Res. 117 (2020) 1–5. + +[3] C. Ziguras, L. Tran, The Coronavirus Outbreak Is the Biggest Crisis Ever to Hit International Education, The Conversation, 2020, 10th Sept. 2021]; Available from: https://theconversation.com/the-coronavirus-outbreak-is-the-biggest-crisis-ever-to-hit-international-education-131138. + +[4] M.R.C. Haas, et al., Reimagining residency selection: Part 1—a practical guide to recruitment in the post-COVID-19 era, J. Grad. Med. Edu. 12 (5) (2020) +539–544. + +[5] S. Brammer, T. Clark, COVID-19 and management education: reflections on challenges, opportunities, and potential futures, Br. J. Manag. 31 (3) (2020) 453. + +[6] R.H. Merola, et al., Through the looking glass: how the COVID-19 pandemic changed international branch campuses' academic experience and home campus relationship, J. Stud. Int. Educ. (2022), 10283153211070112. + +[7] K.L. Willis, B. Holmes, N. Burwell, Improving graduate student recruitment, retention, and professional development during COVID-19, Am. J. Educ. Res. 10 +(2) (2022) 81–84. + +[8] P. Hurley, International Students Are Coming Back And It's Not Just Universities Sighing With Relief. The Conversation [cited 2022 27th Feb. 2022]; Available from:, 2022 https://theconversation.com/international-students-are-coming-back-and-its-not-just-universities-sighing-with-relief-176530. + +[9] M.B. Chowdhury, Internationalisation of education and its effect on economic growth and development, World Econ. 45 (1) (2022) 200–219. + +[10] J. Kim, Impact of the perceived threat of COVID-19 on variety-seeking, Australas. Market J. 28 (3) (2020) 108–116. + +[11] H. de Wit, P.G. Altbach, Internationalization in higher education: global trends and recommendations for its future, Pol. Rev. High. Education 5 (1) (2021) +28–46. + +[12] K.H.D. Tang, Impacts of COVID-19 on primary, secondary and tertiary education: a comprehensive review and recommendations for educational practices, Educ. Res. Pol. Pract. 22 (1) (2023) 23–61. + +[13] J.M. Barbalet, Social emotions: confidence, trust and loyalty, Int. J. Sociol. Soc. Pol. 16 (9/10) (1996) 75–96. [14] E. Sirakaya, A.G. Woodside, Building and testing theories of decision making by travellers, Tourism Manag. 26 (6) (2005) 815–832. [15] H. Zacher, Individual difference predictors of change in career adaptability over time, J. Vocat. Behav. 84 (2) (2014) 188–198. + +[16] T. da Cunha Moreira, R.A.M. Ambiel, Antecedents and consequents of professional choice self-efficacy: a study with Brazilian students, Int. J. Educ. Vocat. + +Guid. 18 (2) (2018) 149–163. + +[17] M.C. Ginevra, et al., Stimulating resources to cope with challenging times and new realities: effectiveness of a career intervention, Int. J. Educ. Vocat. Guid. 17 +(1) (2017) 77–96. + +[18] B. Giridharan, Engaging with students and faculties online in the era of the corona virus pandemic: a higher education perspective, J. Human. Soci. Sci. Res. 2 +(2020) 103–110. + +[19] M. Siegrist, H. Gutscher, T.C. Earle, Perception of risk: the influence of general trust, and general confidence, J. Risk Res. 8 (2) (2005) 145–156. [20] J.M. Barbalet, Confidence: time and emotion in the sociology of action, J. Theor. Soc. Behav. 23 (3) (1993) 229–247. + +[21] M. Gottlieb, et al., Confidence-competence alignment and the role of self-confidence in medical education: a conceptual review, Med. Educ. 56 (1) (2022) +37–47. + +[22] C. Blackmore, et al., Effects of postgraduate medical education "boot camps" on clinical skills, knowledge, and confidence: a meta-analysis, J. Grad. Med. Educ. + +6 (4) (2014) 643–652. + +[23] B. Jobst, et al., The Faith-Factor in Design Thinking: Creative Confidence through Education at the Design Thinking Schools Potsdam and Stanford? Design Thinking Research: Measuring Performance in Context, 2012, pp. 35–46. + +[24] I. Sadler, The role of self-confidence in learning to teach in higher education, Innovat. Educ. Teach. Int. 50 (2) (2013) 157–166. [25] I. Ajzen, The theory of planned behavior, Organ. Behav. Hum. Decis. Process. 50 (2) (1991) 179–211. + +[26] L.E. Davis, et al., The decision of African American students to complete high school: an application of the theory of planned behavior, J. Educ. Psychol. 94 +(2002) 810–819. + +[27] A.C. Simintiras, et al., Redefining confidence for consumer behavior research, Psychol. Market. 31 (6) (2014) 426–439. [28] J.P. Schuldt, et al., Confidence in dyadic decision making: the role of individual differences, J. Behav. Decis. Making 30 (2) (2017) 168–180. + +[29] E. Oney, G. Oksuzoglu-Guven, Confidence: a critical review of the literature and an alternative perspective for general and specific self-confidence, Psychol. + +Rep. 116 (1) (2015) 149–163. + +[30] K. Blomqvist, The many faces of trust, Scand. J. Manag. 13 (3) (1997) 271–286. + +[31] U. Gottlieb, A. Beatson, High on emotion! Perceived value: influencing decision-making processes at international student recruitment trade shows, J. Market. + +High Educ. 28 (2) (2018) 282–297. + +[32] I. Ajzen, From intentions to actions: a theory of planned behavior, in: J. Kuhl, J. Beckmann (Eds.), Action Control: from Cognition to Behavior, Springer Berlin Heidelberg, Berlin, Heidelberg, 1985, pp. 11–39. + +[33] A. Bandura, Self-efficacy: the exercise of control, Educ. Adm. Q. 34 (1) (1998) 153–158. [34] K. Desender, A. Boldt, N. Yeung, Subjective confidence predicts information seeking in decision making, Psychol. Sci. 29 (5) (2018) 761–778. [35] I.E. Berger, The nature of attitude accessibility and attitude confidence: a triangulated experiment, J. Consum. Psychol. 1 (2) (1992) 103–123. + +[36] K.-Y. Goh, C.-S. Heng, Z. Lin, Social media brand community and consumer behavior: quantifying the relative impact of user-and marketer-generated content, Inf. Syst. Res. 24 (1) (2013) 88–107. + +[37] W. Wagiran, et al., Determining the e-learning readiness of higher education students: a study during the COVID-19 pandemic, Heliyon 8 (10) (2022), e11160. + +[38] K.A.N. Calica, R.E. Paterson, The listening project: a qualitative study on the experiences of pre-registered nurses during the Covid-19 pandemic in Scotland, Heliyon (2023), e12664. + +[39] S.A. Kim, et al., Effect of Korean nursing students' experience of incivility in clinical settings on critical thinking, Heliyon 6 (7) (2020), e04367. + +[40] I. Thepwongsa, et al., Uptake and effectiveness of online diabetes continuing education, Persp. Thai Gener. Pract. Train. (2021). Available at: SSRN 3907924. [41] K. Martins, A. Wagg, E. Afonso, Gaining supervision skills in pre-registration nursing through peer teaching: an evaluative survey, Heliyon 8 (11) (2022), +e11398. + +[42] S. Verdugo-Castro, A. García-Holgado, M.C. Sanchez-G ´ omez, ´ The Gender Gap in Higher STEM Studies: A Systematic Literature Review, Heliyon, 2022, e10300. + +[43] C. Civera, et al., Customers' judgments and misjudgments of corporate responsibility communication: a cross-country investigation of the effects on confidence and trust within the banking sector, Psychol. Market. 35 (2) (2018) 138–149. + +[44] L. Stankov, et al., Confidence: a better predictor of academic achievement than self-efficacy, self-concept and anxiety? Learn. Indiv Differ 22 (6) (2012) +747–758. + +[45] J. Van Dyk, S.L. Siedlecki, J.J. Fitzpatrick, Frontline nurse managers' confidence and self-efficacy, J. Nurs. Manag. 24 (4) (2016) 533–539. + +[46] A. Bandura, Self-Efficacy : the Exercise of Control, W.H. Freeman, New York, 1997. + +[47] M.H.A. Al-Abyadh, H.A.H. Abdel Azeem, Academic achievement: influences of university students' self-management and perceived self-efficacy, J. Intell. 10 +(3) (2022) 55. + +[48] J.W. Peltier, P.R. Chennamaneni, K.N. Barber, Student anxiety, preparation, and learning framework for responding to external crises: the moderating role of self-efficacy as a coping mechanism, J. Market. Educ. 44 (2) (2022) 149–165. + +[49] I. Grosemans, N. De Cuyper, Career competencies in the transition from higher education to the labor market: examining developmental trajectories, J. Vocat. + +Behav. 128 (2021), 103602. + +[50] P.P. Van de Calseyde, G. Keren, M. Zeelenberg, Decision time as information in judgment and choice, Organ. Behav. Hum. Decis. Process. 125 (2) (2014) +113–122. + +[51] G.A. Haynes, Testing the boundaries of the choice overload phenomenon: the effect of number of options and time pressure on decision difficulty and satisfaction, Psychol. Market. 26 (3) (2009) 204–212. + +[52] J. Jacoby, G.J. Szybillo, C.K. Berning, Time and consumer behavior: an interdisciplinary overview, J. Consum. Res. 2 (4) (1976) 320–339. [53] A. Fagerstrøm, G. Ghinea, Co-creation of value in higher education: using social network marketing in the recruitment of students, J. High Educ. Pol. Manag. + +35 (1) (2013) 45–53. + +[54] E.A. Eff, C.C. Klein, R. Kyle, Identifying the best buys in US higher education, Res. High. Educ. 53 (8) (2012) 860–887. + +[55] M.D. Hartline, O.C. Ferrell, The management of customer-contact service employees: an empirical investigation, J. Market. 60 (4) (1996) 52. + +[56] V. Bindroo, R.G. Pillai, M.J. Arnold, Valence- and arousal-focused emotional clusters among highly-satisfied customers, J. Manag. Issues 27 (1–4) (2015) 43. + +[57] R.S. Lazarus, Emotion and Adaptation, Oxford University Press, New York, 1991. + +[58] A.L. Stephenson, A. Heckert, D.B. Yerger, College choice and the university brand: exploring the consumer decision framework, High Educ. 71 (4) (2016) +489–503. + +[59] I. Seidman, Interviewing as Qualitative Research : a Guide for Researchers in Education and the Social Sciences, fourth ed., Teachers College Press, New York, 2013. + +[60] J.W. Creswell, C.N. Poth, Qualitative Inquiry and Research Design: Choosing Among Five Approaches, Sage publications, 2016. + +[61] S. Wilkins, Transnational higher education in the 21st century, J. Stud. Int. Educ. 20 (1) (2016) 3–7. + +[62] W.G. Zikmund, Business Research Methods, 5th., The Dryden Press, Orlando, Florida, 1997. + +[63] O.C. Robinson, Sampling in interview-based qualitative research: a theoretical and practical guide, Qual. Res. Psychol. 11 (1) (2014) 25–41. + +[64] M.B. Miles, A.M. Huberman, Qualitative Data Analysis: an Expanded Sourcebook, sage, 1994. + +[65] M. O'Reilly, N. Parker, 'Unsatisfactory Saturation': a critical exploration of the notion of saturated sample sizes in qualitative research, Qual. Res. 13 (2) (2013) +190–197. + +[66] G. Guest, A. Bunce, L. Johnson, How many interviews are enough?:an experiment with data saturation and variability, Field Methods 18 (1) (2006) 59–82. [67] M. Crouch, H. McKenzie, The logic of small samples in interview-based qualitative research, Soc. Sci. Inf. 45 (4) (2006) 483–499. [68] J.M. Corbin, A. Strauss, Grounded theory research: procedures, canons, and evaluative criteria, Qual. Sociol. 13 (1) (1990) 3–21. + +[69] L.S. Nowell, et al., Thematic analysis: striving to meet the trustworthiness criteria, Int. J. Qual. Methods 16 (1) (2017). + +[70] Y.S. Lincoln, E.G. Guba, Naturalistic Inquiry, Sage Publications, Beverly Hills, Calif, 1985. + +[71] V. Braun, V. Clarke, Using thematic analysis in psychology, Qual. Res. Psychol. 3 (2) (2006) 77–101. + +[72] M.B. Miles, A.M. Huberman, J. Saldana, Qualitative Data Analysis : a Methods Sourcebook, third ed., SAGE Publications, Inc, Thousand Oaks, Califorinia, 2014. + +[73] C. Seale, The Quality of Qualitative Research, The Quality of Qualitative Research, 1999, pp. 1–224. + +[74] S. Spiggle, Analysis and interpretation of qualitative data in consumer research, J. Consum. Res. 21 (3) (1994) 491–503. + +[75] B. Denizci Guillet, A. Mattila, L. Gao, The effects of choice set size and information filtering mechanisms on online hotel booking, Int. J. Hospit. Manag. 87 +(2020), 102379. + +[76] M. Mourad, H. Meshreki, S. Sarofim, Brand equity in higher education: comparative analysis, Stud. High Educ. 45 (1) (2020) 209–231. + +[77] D. Amani, I have to choose this university: understanding perceived usefulness of word of mouth (WOM) in choosing universities among students of higher education, Serv. Market. Q. 43 (1) (2022) 1–16. + +[78] Z. Keshf, S. Khanum, It is a very difficult process": career service providers' perspective on career counseling in Pakistani universities, Heliyon 8 (1) (2022), +e08766. + +[79] F.Z. Xu, Y. Wang, Enhancing employee innovation through customer engagement: the role of customer interactivity, employee affect, and motivations, J. Hospit. Tourism Res. 44 (2) (2020) 351–376. + +[80] T.D. Le, A.R. Dobele, L.J. Robinson, Information sought by prospective students from social media electronic word-of-mouth during the university choice process, J. High Educ. Pol. Manag. 41 (1) (2019) 18–34. + +[81] T.M. Smith, K. Hama, P.M. Smith, The effect of successful trade show attendance on future show interest: exploring Japanese attendee perspectives of domestic and offshore international events, J. Bus. Ind. Market. 18 (4/5) (2003) 403–418. + +[82] G. Phillips-Wren, M. Adya, Decision making under stress: the role of information overload, time pressure, complexity, and uncertainty, J. Decis. Syst. 29 (sup1) +(2020) 213–225. + +[83] S.B. Algoe, Find, remind, and bind: the functions of gratitude in everyday relationships, Soci. Pers. Psych. Compass 6 (6) (2012) 455–469. + +[84] B. Mills, et al., A pilot evaluation of simulation-based interprofessional education for occupational therapy, speech pathology and dietetic students: +improvements in attitudes and confidence, J. Interprof. Care 34 (4) (2020) 472–480. + +[85] I. Snijders, et al., Relationship quality time: the validation of a relationship quality scale in higher education, High Educ. Res. Dev. 37 (2) (2018) 404–417. + +[86] V. Tsiligiris, C. Hill, A prospective model for aligning educational quality and student experience in international higher education, Stud. High Educ. 46 (2) +(2021) 228–244. + +[87] J.L. Burton, J.R. Mosteller, K.E. Hale, Using linguistics to inform influencer marketing in services, J. Serv. Market. 35 (2) (2021) 222–236. + +[88] S.-H. Tsaur, Y.-C. Lin, Promoting service quality in tourist hotels: the role of HRM practices and service behavior, 1982, Tourism Manag. 25 (4) (2004) +471–481. + +[89] J. Kim, The functions and dysfunctions of college rankings: an analysis of institutional expenditure, Res. High. Educ. 59 (1) (2018) 54–87. + +[90] T. Luque-Martínez, N. Faraoni, Meta-ranking to position world universities, Stud. High Educ. (2019) 1–15. + +[91] S.S. Batool, S. Ghayas, Process of career identity formation among adolescents: components and factors, Heliyon 6 (9) (2020), e04905. + +[92] S. Hashim, N. Mohd Yasin, S.A. Ya'kob, What constitutes student–university brand relationship? Malaysian students' perspective, J. Market. High Educ. 30 (2) +(2020) 180–202. + +[93] J.E. Trapnell, AACSB International accreditation: the value proposition and a look to the future, J. Manag. Dev. 26 (1) (2007) 67–72. + +[94] J. Vaughn, Accreditation, Commercial Rankings, and New Approaches to Assessing the Quality of University Research and Education Programmes in the United States. Higher education in Europe. Enseignement sup´erieur en Europe, Vysshee obrazovanie v Evrope 27 (4) (2002) 433–441. + +[95] M. Atherton, Measuring confidence levels of male and female students in open access enabling courses, Issues Educ. Res. 25 (2) (2015) 81–98. [96] O. Akbari, J. Sahibzada, Students' self-confidence and its impacts on their learning process, Am. Int. J. Soci. Sci. Res. 5 (1) (2020) 1–15. [97] F.F. Reichheld, W.E. Sasser Jr., Zero defection: quality comes to services, Harv. Bus. Rev. (1990) 105–112. Sept.-Oct. [98] J. Wang, B.W. Ritchie, Understanding accommodation managers' crisis planning intention: an application of the theory of planned behaviour, Tourism Manag. + +33 (5) (2012) 1057–1067. + +[99] P. Flanagan, R. Johnston, D. Talbot, Customer confidence: the development of a "pre-experience" concept, Int. J. Serv. Ind. Manag. 16 (4) (2005) 373–384. + +[100] C. Valeria, Can tourism confidence index improve tourism demand forecasts? J. Tourism Futur. 2 (1) (2016) 6–21. + +[101] B. Howcroft, R. Hamilton, P. Hewer, Customer involvement and interaction in retail banking: an examination of risk and confidence in the purchase of financial products, J. Serv. Market. 21 (7) (2007) 481–491. + +[102] A. Vinhas, D. Bowman, Online/offline information search patterns and outcomes for services, J. Serv. Market. 33 (7) (2019) 753–770. \ No newline at end of file diff --git a/medical/md/PMC10839798.md b/medical/md/PMC10839798.md new file mode 100644 index 0000000000000000000000000000000000000000..ac29c8804405d3c26720a6856d6536049063f6cf --- /dev/null +++ b/medical/md/PMC10839798.md @@ -0,0 +1,333 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_2.png](0_image_2.png) + +journal homepage: www.cell.com/heliyon + +![0_image_3.png](0_image_3.png) + +## Abstract 1. **Introduction** + +| Keywords: Cutaneous squamous cell carcinoma microRNA-124 Snail family transcriptional repressor 2 Invasion Immunotherapy determinant | +|---------------------------------------------------------------------------------------------------------------------------------------------| + +Research article MicroRNA-124 plays an inhibitory role in cutaneous squamous cell carcinoma cells via targeting SNAI2, an immunotherapy determinant Hao Feng a,1, Xing Hu a,1, Renli Yan b, Xiaomin Jia c, Hao Feng d, Nan Zhang e, Xiao Chen e,* +a *Department of Dermatology, Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha, Hunan* Province, 410000, China b Surgery Center of Women and Children's Hospital, Qingdao University, Qingdao, Shandong, China c Department of Pathology, Lhasa People's Hospital, Lhasa, 850, Tibet, 850000, China d State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China e *Department of Medical Cosmetology, The First People's Hospital of Changde City, Changde, Hunan Province, 415000, China* +* Corresponding author. + +![0_image_1.png](0_image_1.png) + +E-mail addresses: doctorfenghao@126.com (H. Feng), huxing@hunnu.edu.cn (X. Hu), yanliren151@163.com (R. Yan), 445399103@qq.com +(X. Jia), fenghao@hunnu.edu.cn (H. Feng), 251010572@qq.com (N. Zhang), chenxiao1980118@163.com (X. Chen). 1 Contributed equally. + +Contents lists available at ScienceDirect Purpose: MicroRNAs (miRs) play multiple roles during cutaneous squamous cell carcinoma (CSCC) progression. Previous studies suggest miR-124 could inhibit cancer development in CSCC. + +Methods: Obtained 63 pairs of CSCC and adjacent tissues for analysis. Cultured HaCaT and two CSCC cell lines (A431 and SCL-1) in DMEM (10 % FBS). Transfected cells using Lipofectamine 2000 with various miR-124 mimics, inhibitors, or Snail family transcriptional repressor 2 (SNAI2) +expression plasmid. Performed a series of assays, including real-time quantitative PCR, Western blot, CCK8, wound healing, transwell, and luciferase reporter gene assay, to examine the effects of miR-124 on CSCC cells. Results: An evident downregulation of miR-124 in CSCC tissues, which was related to advanced disease stage and nodal metastasis. Overexpressing miR-124 could reduce the proliferation, migration, and invasion abilities of CSCC cells. It was verified that miR124 targets the SNAI2 in CSCC cells. Moreover, ectopic expression of SNAI2 rescued the suppressive effects on CSCC cells induced by miR-124 overexpression. Furthermore, miR-124 increased cell sensitivity to cisplatin. Besides, SNAI2 is a critical factor in the immune-related aspects of CSCC and its modulation may influence the response to immunotherapy. Conclusion: We demonstrate that miR-124 inhibits CSCC progression through downregulating SNAI2, and thus it may be a molecular candidate for treating CSCC in the clinic. + +The second most frequent type of dermal cancer is called cutaneous squamous cell carcinoma (CSCC) [1]. CSCC is more aggressive compared to other skin cancers. Although great efforts have been made in CSCC treatment, the overall survival of CSCC patients at a https://doi.org/10.1016/j.heliyon.2024.e24671 Received 10 November 2023; Received in revised form 21 December 2023; Accepted 11 January 2024 Available online 15 January 2024 2405-8440/© 2024 The Authors. 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/). + +ARTICLE INFO + +## H. Feng Et Al. + +later stage has not been improved [2–4]. Several signaling pathways regulate the progression of CSCC cells. However, proper targets in CSCC treatment are still to be identified [5,6]. Thus, exploring the molecular targets associated with CSCC and developing novel therapeutic strategies are urgently needed [7–9]. + +MicroRNAs (miRs) could bind to the 3′ or 5' untranslated region (UTR) of corresponding target mRNAs, resulting in translation repression [10,11]. They participate in cell proliferation, migration, and invasion [11–15]. Recently, many miRs have been dysregulated in CSCC, either promoting or suppressing cancer development [16,17]. For instance, Zhou et al. reported that miR-365 promoted CSCC progression by inhibiting the expression of nuclear factor I/B [18]. MiR-142 also functions as a tumor promoter in CSCC by inhibiting PTEN [19]. Others like MiR-203 and Mir-204 act as an inhibiting role in CSCC targeting c-myc and SHP2 respectively [20,21]. + +MiR-124 is a microRNA that has been extensively studied in the context of cancer. It is known to act as a tumor suppressor in several types of cancer, including breast cancer, glioblastoma, and colorectal cancer. MiR-124 regulates various cellular processes involved in cancer progression, such as cell proliferation, migration, invasion, and apoptosis. MiR-124 is also thought to be a candidate in regulating CSCC development. It is significantly downregulated in CSCC previously [22]. Moreover, Yamane et al. reported that miR-124 downregulation in CSCC increased tumor cell proliferation by activating the ERK signaling pathway [23]. According to these earlier research, miR-124 may function as a tumor suppressor in CSCC. However, the detailed mechanism of miR-124 underlying the progression of CSCC cells remains unknown. Therefore, we wish to reveal other molecular targets of miR-124 in CSCC. + +## 2. **Materials And Methods** 2.1. Clinical Sample + +The study received authorization from the Ethics Committee of Changde First People's Hospital in China, and participants provided their informed consent. Between 2014 and 2016, we gathered 63 sets of cutaneous squamous cell carcinoma (CSCC) samples and their corresponding adjacent tissues. These specimens were preserved at − 80 ◦C following surgical removal for later analysis and experimentation. In this study, the inclusion criteria were patients who underwent surgery for CSCC without prior chemotherapy or radiotherapy. The diagnosis of CSCC was validated by histopathological studies, along with the capacity to give informed consent and sufficient organ function. Exclusion criteria included previous treatment with targeted or immunotherapy agents, presence of other malignancies, active infections, or any medical condition that could interfere with study participation or interpretation of results. + +## 2.2. Cell Culture In Vitro + +HaCaT is a human skin cell line. A431 and SCL-1 are CSCC cell lines (Cell Bank of the Chinese Academy of Sciences). To foster cellular development and multiplication, these cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM) (supplied by ThermoFisher, Waltham, MA, USA) enriched with 10 % fetal bovine serum (FBS) (also from ThermoFisher). The incubation process took place at 37 ◦C with 5 % carbon dioxide. + +## 2.3. Cell Transfection + +Please see the supplementary materials for the detailed methods. + +2.4. *Real-time quantitative PCR (RT-qPCR)* +Please see the supplementary materials for the detailed methods [24]. + +2.5. *Western blot* Please see the supplementary materials for the detailed methods. + +2.5.1. *CCK8 assays* CSCC cells were transfected and seeded into a 96-well plate for varying time intervals as specified. Afterward, the cells were treated with cck8 assay solution obtained from Sigma and further incubated for 3 h at 37 ◦C. Finally, the wells' optical density (OD) was determined with a microplate reader. + +## 2.6. Wound Healing Assays + +The cells were scraped with a 100 μl pipette tip and rinsed with DMEM once they reached 95 % confluency. Cells were cultured in FBS-free DMEM for 24 h, after which the scratch width was evaluated. + +## 2.7. Transwell Assays + +Please see the supplementary materials for the detailed methods. + +![2_image_0.png](2_image_0.png) + +| Table 1 Association between the MiR-124 expression and clinicopathologic characteristics in cutaneous squamous cell carcinoma. Variables Number (n = 63) Low expression (n = 35) High expression (n = 28) | P value | | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|----|----| +| Age (years) | 0.452 | | | +| ≤ 55 | 37 | 19 | 18 | +| > 55 | 26 | 16 | 10 | +| Tumor size (cm) | 0.134 | | | +| ≤ 5 | 29 | 13 | 16 | +| > 5 | 34 | 22 | 12 | +| Differentiation | 0.199 | | | +| Well to moderately | 51 | 26 | 25 | +| Poor | 12 | 9 | 3 | +| Lymph node metastasis | 0.029* | | | +| Present | 18 | 14 | 4 | +| Absent | 45 | 21 | 24 | +| TNM stage | 0.031* | | | +| I-II | 42 | 19 | 23 | +| III | 21 | 16 | 5 | + +2.8. *Luciferase reporter assay* Please see the supplementary materials for the detailed methods. + +2.9. *Tumor immuno-related analysis on SNAI2* The dataset GSE53462 was retrieved from the GEO database. KEGG pathways were subjected to GSEA focusing on SNAI2 [25]. GO +terms were analyzed using GSVA, emphasizing SNAI2. Using the ESTIMATE algorithm, ESTIMATE, Immune, and Stromal scores were determined [26]. The 28 immune cell types were analyzed using the ssGSEA algorithm [27,28]. + +## 2.10. Statistical Analysis + +Data was shown as mean ± SD and analyzed by SPSS 21 software. Tumor immuno-related analysis on SNAI2 was conducted using R +software. Data between the two groups was compared using a student t-test. Correlations were assessed using the Pearson correlation test. Data among multiple groups was compared using a one-way ANOVA. The chi-square test was used to assess correlations. P<0.05 was considered statistically different. + +## 3. **Results** 3.1. Downregulation Of Mir-124 In Cscc + +To elucidate MiR-124's involvement in cutaneous squamous cell carcinoma (CSCC), we assessed its expression in both CSCC lesions + +![3_image_0.png](3_image_0.png) + +and surrounding healthy tissue. Our analysis revealed a markedly reduced presence of MiR-124 in the CSCC samples compared to the n-cancerous counterparts, a disparity supported by statistical evidence ( Fig. 1A ). This pattern of under-expression was further confirmed in a comparative study of two CSCC cell lines against normal skin cells, with the former showing a substantial decrease in MIR-124 levels ( Fig. 1B ). Data from 63 patient samples, encompassing both cancerous and adjacent healthy tissues, were scrutinized. + +The comparative analysis of these samples demonstrated a significant correlation between MiR-124 expression and key clinical parameters, including lymph node metastasis and TNM classification ( Table 1 ). These observations point to a potential role for MiR-124 in the development and progression of CSCC. + +## 3.2. Mir-124 Inhibits The Malignant Phenotypes Of Cscc Cells + +To investigate MiR-124's function in CSCC, we initially altered its expression in vitro within cultured CSCC cells by introducing MiR-124 mimics. Post-treatment, a significant elevation in MiR-124 levels was observed ( Fig. 2A ). Subsequent proliferation assays +(CCK-8) revealed a notable decrease in cell growth in the MiR-124 augmented group when contrasted with the miR-NC (negative + +![4_image_0.png](4_image_0.png) + +control) group (Fig. 2B and C). Moreover, wound healing assays indicated a substantial reduction in cellular migration for the MiR-124 enhanced group versus the miR-NC group (Fig. 2D). Additionally, the invasion capabilities of the cells with MiR-124 upregulation were markedly diminished compared to the miR-NC group (Fig. 2E). These findings imply that MiR-124 plays a regulatory role in the proliferation, migration, and invasion of CSCC cells. + +## 3.3. Mir-124 Directly Targets Snai2 In Cscc Cells + +In our quest to decode MiR-124's mechanism within CSCC, we consulted the TargetScan database, which indicated SNAI2 as a potential target gene of MiR-124. The predicted interaction sites are depicted in Fig. 3A, highlighting the possible areas of MiR-124 attachment on SNAI2. Given that miRNAs typically function by binding to the 3′ untranslated regions (UTRs) of their target genes, we engineered mutations at these predicted sites to create both wild-type (WT) and mutant (MT) plasmids (Fig. 3B). Utilizing a dualluciferase reporter assay for empirical validation, we observed a pronounced reduction in luciferase activity in the cohort coexpressing MiR-124 and WT SNAI2, as opposed to the mutant or control groups (Fig. 3C and D). This reduction indicates a specific binding between MiR-124 and the SNAI2 gene. + +## 3.4. Mir-124 Negatively Affects Snai2 Expression In Cscc Cells + +Building on the predictive data suggesting MiR-124's targeting of SNAI2, we evaluated SNAI2 expression in CSCC cells under the influence of MiR-124 mimics and inhibitors to ascertain MiR-124's regulatory impact. The application of MiR-124 mimics resulted in a notable downregulation of SNAI2 at both mRNA and protein levels (Fig. 4A and B). Conversely, the introduction of MiR-124 inhibitors caused a significant suppression of MiR-124 expression relative to the control inhibitor group (Fig. 4C), which was accompanied by a marked increase in SNAI2 mRNA and protein expression (Fig. 4D and E). These outcomes reinforce the hypothesis that MiR-124 directly modulates SNAI2 expression in CSCC cells. + +## 3.5. Snai2 Rescued The Mir-124'S Effects On Cscc Cells + +To deepen our understanding of how MiR-124's interaction with SNAI2 influences CSCC cellular behavior, we co-transfected CSCC +cells with SNAI2 overexpression plasmids alongside MiR-124 mimics. This co-transfection led to a substantial increase in SNAI2's mRNA and protein levels in the MiR-124 + SNAI2 group when compared with the MiR-124 mimic control (NC) group (Fig. 5A and B). + +![5_image_0.png](5_image_0.png) + +Further analysis using the CCK-8 assay showed a significant enhancement in cell proliferation in the MiR-124 + SNAI2 group relative to the MiR-124 NC group (Fig. 5C and D). Moreover, the wound healing assay indicated a considerable increase in migration capability in the MiR-124 + SNAI2 group versus the MiR-124 NC group (Fig. 5E). In line with these findings, the invasion assay also demonstrated a significant elevation in invasive potential in the MiR-124 + SNAI2 group compared to the MiR-124 NC group (Fig. 5F). These results imply that SNAI2 overexpression can counteract the suppressive effects of MiR-124 on the proliferation, migration, and invasion of CSCC cells. + +## 3.6. Mir-124 Increases The Sensitivity Of Cscc Cells To Cisplatin + +The conducted studies have established that elevating MiR-124 levels markedly curtails the proliferation, migration, and invasion capabilities of CSCC cells. To determine if MiR-124 also heightens the responsiveness of CSCC cells to cisplatin, we administered this chemotherapeutic agent alongside transfection with MiR-124 mimics. The CCK-8 assay outcomes indicated a significant suppression of cell proliferation in the groups treated with cisplatin alone and in combination with miR-NC, compared to the control. Notably, the cisplatin + MiR-124 group exhibited the most pronounced reduction in cell proliferation among all groups tested (Fig. 6A and B). This evidence points to MiR-124's potential role in bolstering the susceptibility of CSCC cells to the cytotoxic effects of cisplatin. + +## 3.7. Function Annotation Of Snai2 + +To comprehensively define the pathogenic roles of SNAI2, we paid special attention to the tumor immuno-related functions of SNAI2. GSEA of KEGG pathways on SNAI2 revealed that T cell signaling, B cell signaling, and immunotherapy response were significantly associated with SNAI2 (Fig. 7A). GSVA of GO pathways on SNAI2 revealed that T cell activation, B cell activation, and immune response were significantly associated with SNAI2 (Fig. 7B). + +## 3.8. Tumor Immuno-Related Characteristics Of Snai2 + +The analysis revealed a substantial inverse relationship between SNAI2 expression and the ESTIMATE scores, which include Immune and Stromal scores, as depicted in Fig. 8A. Additionally, a significant negative correlation was also discerned between SNAI2 levels and the presence of immune cells within the same study (Fig. 8B). These correlations suggest that higher SNAI2 expression may be associated with a lower immune and stromal cell infiltration in the tumor microenvironment. + +![6_image_0.png](6_image_0.png) + +Fig. 5. Demonstration of how SNAI2 can counteract the effects of MiR-124 on CSCC cells. (A–B) These sections detail the mRNA and protein + +![7_image_0.png](7_image_0.png) + +expression levels of SNAI2. The uncropped blots of Fig. 5B are provided in Supplementary Figs. S5 and S6. (C, D) Measurements of cell proliferation are reported. (E) The assay results for cell migration are provided. (F) The data for cell invasion are depicted. With a sample size of n = 4, the results consistently show that the overexpression of SNAI2 can reverse the inhibitory effects of MiR-124 on the proliferation, migration, and invasion of CSCC cells, as evidenced by the significant changes in these cellular behaviors (**P<0.01). This suggests that SNAI2 plays a crucial role in modulating the tumor-suppressive functions of MiR-124 in the context of CSCC. + +## 4. **Discussion** + +In our study, we delved into the molecular dynamics of miR-124 and its association with the progression of CSCC. We established that miR-124 is significantly underexpressed in CSCC tissues, a phenomenon linked with advanced lymph node metastasis and higher clinical stages. The reintroduction of miR-124 curtailed the proliferation, invasion, and migration of CSCC cells. Furthermore, we identified SNAI2 as a gene directly downregulated by miR-124. Counteracting the inhibitory influence of miR-124, the overexpression of SNAI2 mitigated its suppressive effects on CSCC cell behavior. Additionally, enhancing miR-124 levels augmented the sensitivity of these cells to the chemotherapeutic agent cisplatin. + +Previous research has indicated that miR-124 is frequently suppressed in various prevalent human cancers, where it typically acts as a tumor suppressor. For example, in cervical carcinoma, miR-124 exhibits downregulation and is known to impede cancer cell proliferation, invasion, migration, and the epithelial-mesenchymal transition (EMT) process by specifically targeting AEG-1 [29,30]. It also shows negative regulatory effects on bladder cancer cell proliferation and motility, as well as angiogenesis [31]. Our investigation revealed that miR-124 levels were markedly reduced in CSCC tissues relative to adjacent non-tumorous tissues, as well as in CSCC cell lines. This observation aligns with previous reports indicating a downregulation of miR-124 in CSCC A431 cells when compared to normal human epidermal keratinocytes [22]. Besides, hypermethylation of other promoter regions rather than the miR-124 promoter may be the main cause of miR-124 downregulation in CSCC [23]. We then for the first time observed that miR-124 downregulation was closely related to CSCC progression, which further suggests that miR-124 may play a key role in CSCC. + +After our findings on miR-124 downregulation, we noted that its ectopic expression led to a decrease in CSCC cell proliferation, invasion, and migration. This corroborates the findings of Yamane et al. who also observed that the introduction of miR-124 into CSCC +cells resulted in the inhibition of cell proliferation [23]. Considering these results alongside our own, it is suggested that miR-124 exerts a suppressive influence on the progression of CSCC. We then proceeded to identify potential targets of miR-124. Through bioinformatics analysis and luciferase reporter assays, we verified that SNAI2 is a direct target of miR-124 in A431 and SCL-1 cells. SNAI2 is known to act as a transcriptional repressor in various cellular contexts [32]. Previous studies have shown that SNAI2 could repress E-cadherin transcription, and thus promote EMT and tumor metastasis [32,33]. Moreover, the oncogenic role of SNAI2 has been reported in head and neck squamous cell carcinoma [33], tongue squamous cell carcinoma [34], and esophageal squamous cell carcinoma [35]. Invasive CSCC cells were reported to display high migratory and invasive abilities with high SNAI2 expression levels +[36]. But till now, the detailed role of SNAI2 in CSCC remains unknown. The inverse relationship between miR-124 and SNAI2 protein levels in CSCC cells leads us to propose that SNAI2 may operate as a downstream mediator of miR-124's effects in these cells. Indeed, the upregulation of SNAI2 reversed the suppressive impact of miR-124 on CSCC cell proliferation, migration, and invasion, substantiating our hypothesis. In addition, the miR-124/SNAI2 signaling has also been found in NSCLC [37] and breast cancer [38]. + +Immunotherapy is a critical advancement in cancer treatment, offering targeted and potentially less toxic options and reliable markers are essential for predicting patient response and personalizing therapy [39–41]. In terms of cancer immunity, SNAI2 has been found to suppress the expression of major histocompatibility complex class I (MHC-I) molecules on cancer cells [42,43]. MHC-I +molecules are responsible for presenting antigens to cytotoxic T cells, which play a crucial role in immune surveillance and elimination of cancer cells [44]. By downregulating MHC-I expression, SNAI2 helps cancer cells evade immune recognition and destruction. + +Furthermore, SNAI2 has been implicated in the regulation of immune checkpoint molecules, such as programmed death-ligand 1 + +![8_image_0.png](8_image_0.png) + +(PD-L1). PD-L1 is often upregulated in cancer cells and interacts with programmed cell death protein 1 (PD-1) on T cells, leading to T cell exhaustion and immune evasion. SNAI2 has been shown to directly bind to the PD-L1 promoter and enhance its expression, thereby promoting immune evasion. In terms of immunotherapy response [ 45 , 46 ], SNAI2 is associated with resistance to immune checkpoint blockade therapies, such as anti-PD-1/PD-L1 antibodies. High expression of SNAI2 in tumor cells has been correlated with poor response to these therapies. This suggests that targeting SNAI2 or its downstream signaling pathways could potentially enhance the efficacy of immunotherapy. Overall, the role of SNAI2 in modulating immune responses in the context of immunotherapy against CSCC +is complex and context-dependent. Depending on the specific conditions and signaling pathways involved, it can have both pro-tumorigenic and anti-tumorigenic effects. Further research is needed to fully understand the mechanisms underlying SNAI2's role in immune modulation and to explore its potential as a therapeutic target in CSCC immunotherapy. + +To sum up, the downregulation of miR-124 in CSCC suggests that it may play a significant role in the malignant progression of this cancer type. This finding highlights the potential importance of miR-124 as a therapeutic target in CSCC. Furthermore, the study suggests that miR-124 can inhibit CSCC in vitro by targeting SNAI2. This indicates that miR-124 may have a tumor-suppressive effect by regulating the expression of SNAI2. Additionally, the increased cell sensitivity to cisplatin, a commonly used chemotherapy drug, further supports the potential therapeutic value of miR-124 in CSCC treatment. Considering these findings, miR-124 holds promise as a + +![9_image_0.png](9_image_0.png) + +![9_image_1.png](9_image_1.png) + +![9_image_2.png](9_image_2.png) + +therapeutic candidate for CSCC. Further research and clinical trials are needed to explore its efficacy and safety in human patients. If proven effective, miR-124 could potentially be used as a targeted therapy for CSCC, either alone or in combination with existing treatment modalities. Moreover, the study also suggests that SNAI2 may serve as an immunotherapy determinant. This implies that targeting SNAI2 in immunotherapy strategies against CSCC could potentially enhance the effectiveness of these treatments. Further investigations are required to fully understand the underlying mechanisms and potential clinical implications of SNAI2 in immunotherapy for CSCC. In conclusion, the findings of this study provide valuable insights into the role of miR-124 and SNAI2 in CSCC. The downregulation of miR-124 and the potential therapeutic effects of targeting SNAI2 highlight the importance of further research in this area. These results pave the way for the development of novel therapeutic approaches, including the potential use of miR-124 as a therapeutic candidate and the exploration of SNAI2 as an immunotherapy determinant in CSCC. + +There are also some limitations of our study. Firstly, in vivo validation is further needed to confirm the pathogenic roles of SNAI2 and miR-124. Secondly, the specific mechanisms of how SNAI2 is connected to the immune activity and immunotherapy response in CSCC are expected to be revealed by more in vitro and in vivo experiments. Thirdly, more datasets could be included for performing the external validation of SNAI2. + +## Ethical Statement + +The study was conducted by the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Changde First People's Hospital in China (Approval No. YX-2020-081-01). Written informed consent was taken from all the patients. + +## Data Sharing Statement + +The original data could be obtained from the corresponding author. + +## Data Availability Statement + +Not applicable. + +## Credit Authorship Contribution Statement + +Hao Feng: Writing - review & editing, Writing - original draft, Formal analysis, Data curation, Conceptualization. **Xing Hu:** +Writing - review & editing, Writing - original draft, Formal analysis, Data curation, Conceptualization. **Renli Yan:** Writing - review & +editing, Validation, Software. **Xiaomin Jia:** Writing - review & editing, Validation, Software, Methodology. **Hao Feng:** Writing - +review & editing, Validation, Software, Methodology, Investigation. **Nan Zhang:** Writing - review & editing, Validation, Software, Methodology. **Xiao Chen:** Writing - review & editing, Writing - original draft, Supervision, Methodology, Investigation, Funding acquisition. + +## 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 + +Supported by Hunan Provincial Natural Science Foundation 2020JJ8001, 2021JJ30399, 2023JJ30344, Hunan Provincial Health Commission Foundation 202104122479, Lhasa Health Technology Program Foundation LSKJ202104. + +## Appendix A. **Supplementary Data** + +Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e24671. + +## References + +[1] Y. Li, Z. Tai, J. Ma, F. Miao, R. Xin, C. Shen, M. Shen, Q. Zhu, Z. Chen, Lycorine transfersomes modified with cell-penetrating peptides for topical treatment of cutaneous squamous cell carcinoma, J. Nanobiotechnol. 21 (1) (2023) 139, https://doi.org/10.1186/s12951-023-01877-4. + +[2] J. Cheng, S. Yan, Prognostic variables in high-risk cutaneous squamous cell carcinoma: a review, J. Cutan. Pathol. 43 (11) (2016) 994–1004, https://doi.org/ +10.1111/cup.12766. + +[3] R.L. Siegel, K.D. Miller, H.E. Fuchs, A. Jemal, Cancer statistics, 2021, CA Cancer J Clin 71 (1) (2021) 7–33, https://doi.org/10.3322/caac.21654. + +[4] H. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J Clin 71 (3) (2021) 209–249, https://doi.org/10.3322/caac.21660. + +[5] E. Kumah, K. Bibee, Modelling cutaneous squamous cell carcinoma for laboratory research, Exp. Dermatol. 32 (2) (2023) 117–125, https://doi.org/10.1111/ +exd.14706. + +[6] J. Galambus, K.Y. Tsai, Molecular and immune targets in cutaneous squamous cell carcinoma, Mol. Carcinog. 62 (1) (2023) 38–51, https://doi.org/10.1002/ +mc.23451. + +[7] M.C.G. Winge, L.N. Kellman, K. Guo, J.Y. Tang, S.M. Swetter, S.Z. Aasi, K.Y. Sarin, A.L.S. Chang, P.A. Khavari, Advances in cutaneous squamous cell carcinoma, Nat. Rev. Cancer 23 (7) (2023) 430–449, https://doi.org/10.1038/s41568-023-00583-5. + +[8] C.Y. Chong, M.S. Goh, S.V. Porceddu, D. Rischin, A.M. Lim, The current treatment landscape of cutaneous squamous cell carcinoma, Am. J. Clin. Dermatol. 24 +(1) (2023) 25–40, https://doi.org/10.1007/s40257-022-00742-8. + +[9] E.C. Cozma, L.M. Banciu, C. Soare, S.M. Cretoiu, Update on the molecular pathology of cutaneous squamous cell carcinoma, Int. J. Mol. Sci. 24 (7) (2023), +https://doi.org/10.3390/ijms24076646. + +[10] Y. Zhou, C. Yang, K. Wang, X. Liu, Q. Liu, MicroRNA-33b inhibits the proliferation and migration of osteosarcoma cells via targeting hypoxia-inducible factor1alpha, Oncol. Res. 25 (3) (2017) 397–405, https://doi.org/10.3727/096504016X14743337535446. + +[11] V. Ambros, The functions of animal microRNAs, Nature 431 (7006) (2004) 350–355, https://doi.org/10.1038/nature02871. + +[12] Y. Zhang, Q. Dai, F. Zeng, H. Liu, MALAT1 promotes the proliferation and metastasis of osteosarcoma cells by activating the Rac1/JNK pathway via targeting MiR-509, Oncol. Res. (2018), https://doi.org/10.3727/096504017X14957939026111. + +[13] M. Liu, D. Wang, N. Li, MicroRNA-20b downregulates HIF-1alpha and inhibits the proliferation and invasion of osteosarcoma cells, Oncol. Res. 23 (5) (2016) +257–266, https://doi.org/10.3727/096504016X14562725373752. + +[14] X. Liu, J. Li, Z. Yu, J. Li, R. Sun, Q. Kan, miR-935 promotes liver cancer cell proliferation and migration by targeting SOX7, Oncol. Res. 25 (3) (2017) 427–435, https://doi.org/10.3727/096504016X14747300207374. + +H. Feng et al. + +[15] C. Luo, J. Qiu, miR-181a inhibits cervical cancer development via downregulating GRP78, Oncol. Res. 25 (8) (2017) 1341–1348, https://doi.org/10.3727/ +096504017X14867268787969. + +[16] L. Zhou, R. Gao, Y. Wang, M. Zhou, Z. Ding, Loss of BAX by miR-365 promotes cutaneous squamous cell carcinoma progression by suppressing apoptosis, Int. J. + +Mol. Sci. 18 (6) (2017), https://doi.org/10.3390/ijms18061157. + +[17] N. Lin, Y. Zhou, X. Lian, Y. Tu, MicroRNA-31 functions as an oncogenic microRNA in cutaneous squamous cell carcinoma cells by targeting RhoTBT1, Oncol. + +Lett. 13 (3) (2017) 1078–1082, https://doi.org/10.3892/ol.2017.5554. + +[18] M. Zhou, L. Zhou, L. Zheng, L. Guo, Y. Wang, H. Liu, C. Ou, Z. Ding, miR-365 promotes cutaneous squamous cell carcinoma (CSCC) through targeting nuclear factor I/B (NFIB), PLoS One 9 (6) (2014) e100620, https://doi.org/10.1371/journal.pone.0100620. + +[19] X. Bai, Y. Zhou, P. Chen, M. Yang, J. Xu, MicroRNA-142-5p induces cancer stem cell-like properties of cutaneous squamous cell carcinoma via inhibiting PTEN, +J. Cell. Biochem. 119 (2) (2018) 2179–2188, https://doi.org/10.1002/jcb.26379. + +[20] W. Lohcharoenkal, M. Harada, J. Loven, F. Meisgen, N.X. Landen, L. Zhang, J. Lapins, K.D. Mahapatra, H. Shi, L. Nissinen, V.M. Kahari, M. Stahle, E. Sonkoly, D. Grander, M. Arsenian-Henriksson, A. Pivarcsi, MicroRNA-203 inversely correlates with differentiation grade, targets c-MYC, and functions as a tumor suppressor in cSCC, J. Invest. Dermatol. 136 (12) (2016) 2485–2494, https://doi.org/10.1016/j.jid.2016.06.630. + +[21] A. Toll, R. Salgado, B. Espinet, A. Diaz-Lagares, E. Hernandez-Ruiz, E. Andrades, J. Sandoval, M. Esteller, R.M. Pujol, I. Hernandez-Munoz, MiR-204 silencing in intraepithelial to invasive cutaneous squamous cell carcinoma progression, Mol. Cancer 15 (1) (2016) 53, https://doi.org/10.1186/s12943-016-0537-z. + +[22] M. Harada, M. Jinnin, Z. Wang, A. Hirano, Y. Tomizawa, T. Kira, T. Igata, S. Masuguchi, S. Fukushima, H. Ihn, The expression of miR-124 increases in aged skin to cause cell senescence and it decreases in squamous cell carcinoma, Biosci Trends 10 (6) (2017) 454–459, https://doi.org/10.5582/bst.2016.01102. + +[23] K. Yamane, M. Jinnin, T. Etoh, Y. Kobayashi, N. Shimozono, S. Fukushima, S. Masuguchi, K. Maruo, Y. Inoue, T. Ishihara, J. Aoi, Y. Oike, H. Ihn, Downregulation of miR-124/-214 in cutaneous squamous cell carcinoma mediates abnormal cell proliferation via the induction of ERK, J. Mol. Med. (Berl.) 91 (1) +(2013) 69–81, https://doi.org/10.1007/s00109-012-0935-7. + +[24] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (4) +(2001) 402–408, https://doi.org/10.1006/meth.2001.1262. + +[25] X.N. Wu, D. Su, Y.D. Mei, M.Q. Xu, H. Zhang, Z.Y. Wang, L.L. Li, L. Peng, J.Y. Jiang, J.Y. Yang, D.J. Li, H. Cao, Z.W. Xia, W.J. Zeng, Q. Cheng, N. Zhang, Identified lung adenocarcinoma metabolic phenotypes and their association with tumor immune microenvironment, Cancer Immunol. Immunother. 70 (10) +(2021) 2835–2850, https://doi.org/10.1007/s00262-021-02896-6. + +[26] K. Yoshihara, M. Shahmoradgoli, E. Martinez, R. Vegesna, H. Kim, W. Torres-Garcia, V. Trevino, H. Shen, P.W. Laird, D.A. Levine, S.L. Carter, G. Getz, K. Stemke-Hale, G.B. Mills, R.G. Verhaak, Inferring tumour purity and stromal and immune cell admixture from expression data, Nat. Commun. 4 (2013) 2612, https://doi.org/10.1038/ncomms3612. + +[27] S. Li, N. Zhang, S. Liu, H. Zhang, J. Liu, Y. Qi, Q. Zhang, X. Li, ITGA5 is a novel oncogenic biomarker and correlates with tumor immune microenvironment in gliomas, Front. Oncol. 12 (2022) 844144, https://doi.org/10.3389/fonc.2022.844144. + +[28] P. Charoentong, F. Finotello, M. Angelova, C. Mayer, M. Efremova, D. Rieder, H. Hackl, Z. Trajanoski, Pan-cancer immunogenomic Analyses reveal genotypeimmunophenotype relationships and predictors of response to checkpoint blockade, Cell Rep. 18 (1) (2017) 248–262, https://doi.org/10.1016/j. + +celrep.2016.12.019. + +[29] X. Zhang, D. Cai, L. Meng, B. Wang, MicroRNA-124 inhibits proliferation, invasion, migration and epithelial-mesenchymal transition of cervical carcinoma cells by targeting astrocyte-elevated gene-1, Oncol. Rep. 36 (4) (2016) 2321–2328, https://doi.org/10.3892/or.2016.5025. + +[30] N. Zhang, H. Zhang, Z. Liu, Z. Dai, W. Wu, R. Zhou, S. Li, Z. Wang, X. Liang, J. Wen, X. Zhang, B. Zhang, S. Ouyang, J. Zhang, P. Luo, X. Li, Q. Cheng, An artificial intelligence network-guided signature for predicting outcome and immunotherapy response in lung adenocarcinoma patients based on 26 machine learning algorithms, Cell Prolif. 56 (4) (2023) e13409, https://doi.org/10.1111/cpr.13409. + +[31] X. Wang, Q. Wu, B. Xu, P. Wang, W. Fan, Y. Cai, X. Gu, F. Meng, MiR-124 exerts tumor suppressive functions on the cell proliferation, motility and angiogenesis of bladder cancer by fine-tuning UHRF1, FEBS J. 282 (22) (2015) 4376–4388, https://doi.org/10.1111/febs.13502. + +[32] J. Li, S. Li, R. Chen, X. Lu, Increased risk of poor survival in ovarian cancer patients with high expression of SNAI2 and lymphovascular space invasion, Oncotarget 8 (6) (2017) 9672–9685, https://doi.org/10.18632/oncotarget.14192. + +[33] L. Wang, Y. Jia, Z. Jiang, W. Gao, B. Wang, FSCN1 is upregulated by SNAI2 and promotes epithelial to mesenchymal transition in head and neck squamous cell carcinoma, Cell Biol. Int. 41 (8) (2017) 833–841, https://doi.org/10.1002/cbin.10786. + +[34] C. Wang, X. Liu, H. Huang, H. Ma, W. Cai, J. Hou, L. Huang, Y. Dai, T. Yu, X. Zhou, Deregulation of Snai2 is associated with metastasis and poor prognosis in tongue squamous cell carcinoma, Int. J. Cancer 130 (10) (2012) 2249–2258, https://doi.org/10.1002/ijc.26226. + +[35] C. Kestens, P.D. Siersema, G.J. Offerhaus, J.W. van Baal, BMP4 signaling is able to induce an epithelial-mesenchymal transition-like phenotype in barrett's esophagus and esophageal adenocarcinoma through induction of SNAIL2, PLoS One 11 (5) (2016) e0155754, https://doi.org/10.1371/journal.pone.0155754. + +[36] K. Barrette, S. Van Kelst, J. Wouters, V. Marasigan, S. Fieuws, P. Agostinis, J. van den Oord, M. Garmyn, Epithelial-mesenchymal transition during invasion of cutaneous squamous cell carcinoma is paralleled by AKT activation, Br. J. Dermatol. 171 (5) (2014) 1014–1021, https://doi.org/10.1111/bjd.12967. + +[37] F.Y. Hu, X.N. Cao, Q.Z. Xu, Y. Deng, S.Y. Lai, J. Ma, J.B. Hu, miR-124 modulates gefitinib resistance through SNAI2 and STAT3 in non-small cell lung cancer, J Huazhong Univ Sci Technolog Med Sci 36 (6) (2016) 839–845, https://doi.org/10.1007/s11596-016-1672-x. + +[38] S. Du, H. Li, X. Sun, D. Li, Y. Yang, Z. Tao, Q. Li, K. Liu, MicroRNA-124 inhibits cell proliferation and migration by regulating SNAI2 in breast cancer, Oncol. Rep. + +36 (6) (2016) 3259–3266, https://doi.org/10.3892/or.2016.5163. + +[39] B. Yan, S. Wang, C. Liu, N. Wen, H. Li, Y. Zhang, H. Wang, Z. Xi, Y. Lv, H. Fan, X. Liu, Engineering magnetic nano-manipulators for boosting cancer immunotherapy, J. Nanobiotechnol. 20 (1) (2022) 547, https://doi.org/10.1186/s12951-022-01760-8. + +[40] S.R. Li, F.Y. Huo, H.Q. Wang, J. Wang, C. Xu, B. Liu, L.L. Bu, Recent advances in porous nanomaterials-based drug delivery systems for cancer immunotherapy, J. Nanobiotechnol. 20 (1) (2022) 277, https://doi.org/10.1186/s12951-022-01489-4. + +[41] Z. Wang, X. Wang, N. Zhang, H. Zhang, Z. Dai, M. Zhang, S. Feng, Q. Cheng, Pentraxin 3 promotes glioblastoma progression by negative regulating cells autophagy, Front. Cell Dev. Biol. 8 (2020), https://doi.org/10.3389/fcell.2020.00795. + +[42] D. Zhang, Z. Jiang, J. Hu, X. Sun, Y. Zheng, Y. Shen, Comprehensively prognostic and immunological analysis of snail family transcriptional repressor 2 in pancancer and identification in pancreatic carcinoma, Front. Immunol. 14 (2023) 1117585, https://doi.org/10.3389/fimmu.2023.1117585. + +[43] P.D. Pioli, S.K. Whiteside, J.J. Weis, J.H. Weis, Snai2 and Snai3 transcriptionally regulate cellular fitness and functionality of T cell lineages through distinct gene programs, Immunobiology 221 (5) (2016) 618–633, https://doi.org/10.1016/j.imbio.2016.01.007. + +[44] J. Zhang, Z. Wang, X. Zhang, Z. Dai, W. Zhi-Peng, J. Yu, Y. Peng, W. Wu, N. Zhang, P. Luo, J. Zhang, Z. Liu, S. Feng, H. Zhang, Q. Cheng, Large-scale single-cell and bulk sequencing Analyses reveal the prognostic value and immune aspects of CD147 in pan-cancer, Front. Immunol. 13 (2022) 810471, https://doi.org/ 10.3389/fimmu.2022.810471. + +[45] Z. Luo, K.M. Eichinger, A. Zhang, S. Li, Targeting cancer metabolic pathways for improving chemotherapy and immunotherapy, Cancer Lett. 575 (2023) +216396, https://doi.org/10.1016/j.canlet.2023.216396. + +[46] J. Shen, H. Yang, X. Qiao, Y. Chen, L. Zheng, J. Lin, J. Lang, Q. Yu, Z. Wang, The E3 ubiquitin ligase TRIM17 promotes gastric cancer survival and progression via controlling BAX stability and antagonizing apoptosis, Cell Death Differ. 30 (10) (2023) 2322–2335, https://doi.org/10.1038/s41418-023-01221-1. \ No newline at end of file diff --git a/medical/md/PMC10952263.md b/medical/md/PMC10952263.md new file mode 100644 index 0000000000000000000000000000000000000000..97c7fd85acbec34443c06da4665491061ed4884e --- /dev/null +++ b/medical/md/PMC10952263.md @@ -0,0 +1,192 @@ +# Elective Spinal Surgery In New South Wales Adults, 2001–20, By Procedure Funding Type: A Cross-Sectional Study + +Duong Thuy Tran1 , Adriane M Lewin2 , Louisa Jorm1, Ian A Harris1,3 The known: The benefits of spinal decompression and fusion for people with degenerative conditions are dubious, but procedure rates in high income countries have risen substantially in recent decades. The new: Rates of privately funded spinal fusion and decompression procedures have increased much more rapidly in New South Wales than those of procedures that are publicly funded or covered by workers' compensation. The implications: Differences in procedure rates suggest that too many privately funded or too few publicly funded spinal procedures are undertaken. The influence of financial considerations, access to specialist care, and patient and surgeon preferences should be investigated. + +Degenerative spinal disorders and the associated chronic back and leg pain are common health problems.1,2 Their prevalence increases with age,2 and people may choose surgery if non-surgical management has not provided relief.2-4 Although the benefits of spinal decompression and fusion for people with degenerative conditions are debated,3,4 use of these procedures in high income countries has risen substantially in recent decades, but there are significant variations in their use within and between countries.3,5-9 Factors such as population ageing, overuse of diagnostic imaging, and greater expectations about the outcomes achievable with advanced technology may explain the increase in surgery volumes.3,4 However, consensus about the indications for spinal surgery is limited, as is high quality evidence regarding its clinical effectiveness.3,10 Private health insurance does not cover all non-surgical treatments for degenerative spine conditions, but can facilitate timely access to surgery as a private patient.3 In Australia, the rate of privately funded spinal fusions increased by 167% during 1997–2006 (from 7.7 to 20.5 per 100000 population), while the publicly funded rate increased by only 2% (from 5.1 to 5.2 per 100 000 population).9 During 2012–18, 83% of spine procedures were performed in the private sector (as were about two-thirds of hip and knee replacement procedures),3 suggesting that financial considerations are important and that access to surgical interventions may consequently be inequitable.3,4,9 In Australia, workers' compensation provides health insurance for people who sustain workplace-related injuries.11 A study of workers' compensation claims in New South Wales during 2010–18 reported spinal surgery rates of 15–29 per 100 000 covered workers per year; they declined during 2011–15 before rising again.1 The study did not compare procedure rates for insured workers with those for publicly and privately funded patients. + +## Abstract + +Objective: To investigate elective rates of spinal fusion, decompression, and disc replacement procedures for people with degenerative conditions, by funding type (public, private, workers' compensation). Design, setting: Cross-sectional study; analysis of hospitals admissions data extracted from the New South Wales Admitted Patient Data Collection. Participants: All adults who underwent elective spinal surgery (spinal fusion, decompression, disc replacement) in NSW, 1 July 2001 - 30 June 2020. Main outcome measures: Crude and age- and sex-adjusted procedure rates, by procedure, funding type, and year; annual change in rates, 2001–20, expressed as incidence rate ratios (IRRs). Results: During 2001–20, 155 088 procedures in 129 525 adults were eligible for our analysis: 53 606 fusion, 100 225 decompression, and 1257 disc replacement procedures. The privately funded fusion procedure rate increased from 26.6 to 109.5 per 100 000 insured adults (per year: IRR, 1.06; 95% confidence interval [CI], 1.05–1.07); the workers' compensation procedure rate increased from 6.1 to 15.8 per 100 000 covered adults (IRR, 1.04; 95% CI, 1.01–1.06); the publicly funded procedure rate increased from 5.6 to 12.4 per 100 000 adults (IRR, 1.03; 95% CI, 1.01–1.06), and from 10.5 to 22.1 per 100 000 adults without hospital cover private health insurance (IRR, 1.03; 95% CI, 1.01–1.05). The privately funded decompression procedure rate increased from 93.4 to 153.6 per 100 000 people (IRR, 1.02; 95% CI, 1.01–1.03); the workers' compensation procedure rate declined from 19.7 to 16.7 per 100 000 people (IRR, 0.98; 95% CI, 0.96–0.99), and the publicly funded procedure rate did not change significantly. The privately funded disc replacement procedure rate increased from 6.2 per million in 2010–11 to 38.4 per million people in 2019–20, but did not significantly change for the other two funding groups. The age- and sex-adjusted rates for privately and publicly funded fusion and decompression procedures were similar to the crude rates. Conclusions: Privately funded spinal surgery rates continue to be larger than for publicly funded procedures, and they have also increased more rapidly. These differences may indicate that some privately funded procedures are unnecessary, or that the number of publicly funded procedures does not reflect clinical need. + +The Medical Services Advisory Committee recommended in 2011 that artificial disc replacement be publicly subsidised in Australia.12 Little is known about the uptake of this procedure since this time. Given the doubtful evidence for the benefit of many spinal procedures, financial incentives may be important for decisions regarding spinal surgery. We therefore investigated the rates of spinal fusion, decompression, and disc replacement procedures for degenerative spinal conditions in Australia during 2001–20 by funding type (public, private, workers' compensation). + +- 2023 + +## Methods + +We undertook a retrospective observational (cross-sectional) study, analysing NSW hospitals admissions data for 1 July 2001 - 30 June 2020, extracted from the NSW Admitted Patient Data Collection (APDC), for all adults who underwent elective procedures for degenerative spinal conditions (Supporting Information, table 1). The APDC includes a record for each separation from a public or private hospital, including information on the demographic characteristics of the patient, diagnoses, procedures, external causes of injuries, urgency of admission, payment at discharge, referral, and discharge status. Diagnoses were coded according to the International Classification of Diseases, tenth revision, Australian modification (ICD-10-AM);13 procedures were coded according to Australian Classification of Health Interventions.14 We report our study in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology guidelines.15 + +## Inclusion And Exclusion Criteria + +We classified procedures using a hierarchical definition: any procedure involving fusion (fusion with or without disc replacement, with or without decompression) was classified as a fusion procedure; a disc replacement procedure with or without decompression was classified as a disc replacement; and decompression alone (decompression without fusion or disc replacement) was classified as a decompression procedure. To include only elective procedures for adults with degenerative spine disorders, we excluded procedures for people under 18 years of age, emergency admissions, procedures for spinal fracture, trauma, tumour, lesion, or congenital deformities, revisions of scoliosis or kyphosis surgery, and revision or removal of spinal fixation or intervertebral disc prostheses (Supporting Information, table 2). + +Under the National Health Reform Agreement,16 Australian citizens and eligible residents can select public or private care on admission to hospital. A public patient in a public hospital receives cost-free treatment (public funding). When available, a patient can receive private care in a public or private hospital, with costs covered by private health insurance, the patient, and the Medicare rebate (private funding). Hospital treatment for work-related conditions is covered by workers' compensation insurance. We classified procedure funding as public funding, private funding, or workers' compensation according to funding status at hospital discharge as recorded in the APDC, without regard to hospital type. Funding for the small number of procedures paid by the Department of Veterans' Affairs were classified as private funding. We excluded procedures covered by insurance schemes other than workers' compensation (eg, vehicle accident insurance). + +## Statistical Analyses + +All analyses were undertaken in SAS 9.4. We summarise patient characteristics as numbers and proportions or as means with standard deviations (SDs). We calculated procedure rates with 95% confidence intervals (CIs) with respect to the appropriate NSW adult population, by funding scheme and financial year. The number of people covered by workers' compensation was supplied by the State Insurance Regulatory Agency. + +The numbers with private health insurance (hospital treatment coverage) were derived from membership data published by the Australian Prudential Regulation Authority.17 As some public patients have private health insurance, we calculated both lower and upper bound rates for publicly funded procedures. For lower bound rates, we included all NSW adults18 (ie, all people eligible for public sector surgery); for upper bound rates, we included only those without private hospital health insurance cover (ie, we subtracted the number with private health insurance from the total adult population number). To compare rates by funding scheme, age- and sex-adjusted procedure rates were directly standardised to the NSW +population using 2016 national census data18 (Supporting Information, table 3). When comparing workers' compensation rates with those of the other two groups, we restricted the analysis to procedures for people of working age (18–64 years). We estimated annual incidence rate ratios (IRRs) with 95% CIs in generalised linear models (PROC GENMOD, Poisson distribution, log-link function, log of the denominator as an offset term, α = 0.05). Standardised rates and IRR estimates were not calculated for disc replacement procedures because of their small numbers. + +## Ethics Approval + +The human research ethics committees of NSW Population and Health Services Research (HREC/18/CIPHS/56), the Aboriginal Health and Medical Research Council of NSW (1503/19), and the Australian Institute of Health and Welfare (EO2018/2/431) approved the study. + +## Results + +During 1 July 2001 - 30 June 2020, 200962 spinal procedures were performed in 161 566 people in NSW, of which 155 088 procedures in 129 525 adults were eligible for our analysis: 53606 fusion (including 40 380 with decompression [75.3%]), 100225 decompression, and 1257 disc replacement procedures. In total, 65.9% of procedures were privately funded, 22.8% were publicly funded, and 11.1% were funded by workers' compensation (Box 1, Box 2). + +The mean age of people who underwent fusion procedures was 59.1 years (SD, 14.8 years), for decompression procedures 57.4 years (SD, 16.3 years), and for disc replacements 43.3 years (SD, 9.7 years); 48.1% of fusion procedures, 56.9% of decompressions, and 55.6% of disc replacements were undertaken in men (Box 2). The mean age of people who underwent procedures funded by workers' compensation was 46.2 years (SD, 11.1 years), + +![1_image_0.png](1_image_0.png) + +| 2 Demographic and clinical characteristics of adults who underwent elective procedures for degenerative spinal conditions in New South Wales, 2001–20, by procedure type* Characteristic Fusion Decompression Disc replacement All procedures All procedures 53 606 100 225 1257 155 088 Sex (men) 25 792 (48.1%) 57 062 (56.9%) 699 (55.6%) 83 553 (53.9%) Age (years), mean (SD) 59.1 (14.8) 57.4 (16.3) 43.3 (9.7) 57.9 (15.9) 18–29 years 1593 (3.0%) 4942 (4.9%) 112 (8.9%) 6647 (4.3%) 30–34 years 1634 (3.0%) 5372 (5.4%) 140 (11.1%) 7146 (4.6%) 35–39 years 2796 (5.2%) 7028 (7.0%) 208 (16.5%) 10 032 (6.5%) 40–44 years 4116 (7.7%) 8271 (8.3%) 267 (21.2%) 12 654 (8.2%) 45–49 years 5065 (9.4%) 8849 (8.8%) 251 (20.0%) 14 165 (9.1%) 50–54 years 5348 (10.0%) 9061 (9.0%) 139 (11.1%) 14 548 (9.4%) 55–59 years 5667 (10.6%) 9579 (9.6%) 83 (6.6%) 15 329 (9.9%) 60–64 years 6342 (11.8%) 10 234 (10.2%) 36 (2.9%) 16 612 (10.7%) 65 years and over 21 045 (39.3%) 36 889 (36.8%) 21 (1.7%) 57 955 (37.4%) Living with a partner 35 632 (66.5%) 67 692 (67.5%) 840 (66.8%) 104 164 (67.2%) Funding scheme Private 35 355 (66.0%) 65 991 (65.8%) 820 (65.2%) 102 166 (65.9%) Public 11 558 (21.6%) 23 535 (23.5%) 194 (15.4%) 35 287 (22.8%) Workers' compensation 6693 (12.5%) 10 669 (10.7%) 243 (19.3%) 17 635 (11.4%) Remoteness of residence† Major cities 40 068 (74.7%) 75 928 (75.8%) 991 (78.8%) 116 987 (75.4%) Inner regional 9827 (18.3%) 18 083 (18.0%) 197 (15.7%) 28 107 (18.1%) Outer regional/remote/very remote 2231 (4.2%) 4596 (4.6%) 30 (2.4%) 6857 (4.4%) Missing data 1480 (2.8%) 1618 (1.6%) 39 (3.1%) 3137 (2.0%) Principal diagnosis Intervertebral disc disorders 17 323 (32.3%) 43 709 (43.6%) 846 (67.3%) 61 878 (39.9%) Spondylopathies 16 005 (29.9%) 33 854 (33.8%) 134 (10.7%) 49 993 (32.2%) Backpain 6970 (13.0%) 13 531 (13.5%) 197 (15.7%) 20 698 (13.3%) Deforming dorsopathies 9130 (17.0%) 732 (0.7%) 6 (0.5%) 9868 (6.4%) Disorders of central nervous system, nerve root 1976 (3.7%) 5275 (5.3%) 50 (4.0%) 7301 (4.7%) Symptoms involved nervous and musculoskeletal 545 (1.0%) 568 (0.6%) 7 (0.6%) 1120 (0.7%) systems Others 1657 (3.1%) 2556 (2.6%) 17 (1.4%) 4230 (2.7%) Financial year 2001–02 1003 (1.9%) 3444 (3.4%) NA 4447 (2.9%) 2002–03 1162 (2.2%) 3983 (4.0%) NA 5145 (3.3%) 2003–04 1392 (2.6%) 3798 (3.8%) NA 5190 (3.3%) 2004–05 1427 (2.7%) 3764 (3.8%) NA 5191 (3.3%) 2005–06 1670 (3.1%) 3986 (4.0%) NA 5656 (3.6%) 2006–07 1879 (3.5%) 4315 (4.3%) NA 6194 (4.0%) 2007–08 2088 (3.9%) 4365 (4.4%) NA 6453 (4.2%) 2008–09 2304 (4.3%) 4772 (4.8%) NA 7076 (4.6%) 2009–10 2737 (5.1%) 5311 (5.3%) NA 8048 (5.2%) 2010–11 3081 (5.7%) 5742 (5.7%) 32 (2.5%) 8855 (5.7%) 2011–12 3307 (6.2%) 5676 (5.7%) 38 (3.0%) 9021 (5.8%) 2012–13 3399 (6.3%) 5981 (6.0%) 68 (5.4%) 9448 (6.1%) Continues | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +305 + +| 2 Continued Characteristic | Fusion | Decompression | Disc replacement | All procedures | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------|-----------------|--------------------|------------------| +| 2013–14 | 3710 (6.9%) | 6353 (6.3%) | 162 (12.9%) | 10 225 (6.6%) | +| 2014–15 | 3942 (7.4%) | 6613 (6.6%) | 134 (10.7%) | 10 689 (6.9%) | +| 2015–16 | 3802 (7.1%) | 6390 (6.4%) | 132 (10.5%) | 10 324 (6.7%) | +| 2016–17 | 3853 (7.2%) | 6397 (6.4%) | 155 (12.3%) | 10 405 (6.7%) | +| 2017–18 | 4266 (8.0%) | 6599 (6.6%) | 193 (15.4%) | 11 058 (7.1%) | +| 2018–19 | 4340 (8.1%) | 6671 (6.7%) | 189 (15.0%) | 11 200 (7.2%) | +| 2019–20 | 4244 (7.9%) | 6065 (6.1%) | 154 (12.3%) | 10 463 (6.7%) | +| NA = not applicable (no public funding for artificial disc replacement until 2010–11). * Characteristics by funding type are provided in the Supporting Information, table 4. † Based on the Australian Statistical Geography Standard 2016 classification;19 remoteness categories were mapped to the Statistical Area Level 2 of residential postcodes. ◆ | | | | | + +for privately funded procedures 59.8 years (SD, 15.8 years), and for publicly funded procedures 58.2 years (SD, 15.6 years). + +## Procedure Rates (Crude) + +The privately funded fusion procedure rate increased from 26.6 per 100 000 in 2001–02 to 109.5 per 100 000 insured adults in 2019–20 (per year: IRR, 1.06; 95% CI, 1.05–1.07), and the workers' compensation procedure rate from 6.1 to 15.8 per 100 000 covered adults (IRR, 1.04; 95% CI, 1.01–1.06). The lower bound for the publicly funded procedure rate increased from 5.6 to 12.4 per 100 000 people (IRR, 1.03; 95% CI, 1.01–1.06), the upper bound rate from 10.5 to 22.1 per 100 000 people (IRR, 1.03; 95% CI, 1.01–1.05) (Box 3). The privately funded fusion procedure rate was highest for people aged 65 years or more (176 [95% CI, 173.8–179.2] per 100 000 insured persons; Supporting Information, table 6). + +The privately funded decompression procedure rate increased from 93.4 per 100000 people in 2001–02 to 175.9 per 100000 insured adults in 2014–15, then declined to 153.6 per 100000 people in 2019–20 (per year: IRR, 1.02; 95% CI, 1.01–1.03). The workers' compensation rate declined from 19.7 to 16.7 per 100000 covered adults (IRR, 0.98; 95% CI, 0.96–0.99). There was no significant change in the lower (from 19.0 to 22.4 per 100 000 people; IRR, 1.00; 95% CI, 0.98–1.01) or upper bound rates (35.4 to 39.8 per 100000 people; IRR, 1.00; 95% CI, 0.98–1.01) for publicly funded procedures (Box 3). The privately funded decompression procedure rate was highest for people aged 65 years or more (288.9 [95% CI, 285.5–292.4] per 100000 insured persons; Supporting Information, table 6). + +3 Adults who underwent elective procedures for degenerative spinal conditions in New South Wales, 2001–20, by procedure type and funding type: crude rates, with 95% ![3_image_0.png](3_image_0.png) + + * Denominators for publicly funded, lower bound procedure rates: NSW people aged 18 years or more;18 publicly funded, upper bound rates: NSW people aged 18 years or more who did not have hospital cover private health insurance;17,18 privately funded procedure rates: NSW people aged 18 years or more who had hospital cover private health insurance;17 workers' compensation procedure: NSW people aged 18 or more with workers' compensation cover. Prior to April 2011, few disc replacement procedures were undertaken (any funding type), and most were associated with claims for a fusion or for an indication other than degenerative spine disorders.20 The data for these graphs are included in the Supporting Information, table 5. ◆ +The privately funded disc replacement procedure rate increased from 6.2 per million in 2010–11 to 38.4 per million insured adults in 2019–20, but did not markedly change for the other two funding groups during this period (Box 3). + +## Discussion Procedure Rates (Age- And Sex-Adjusted) + +The age- and sex-adjusted rates for privately and publicly funded fusion and decompression procedures were similar to the crude rates (Supporting Information, figure 1, table 8). + +The adjusted fusion and decompression procedure rates for people of working age (18–64 years) funded by workers' compensation lay between the lower and upper bounds for the adjusted publicly funded procedure rate. The adjusted privately funded fusion procedure rates were higher than for procedures funded by workers' compensation in both 2001–02 (20.1 v 5.8 per 100 000 people) and 2019–20 (64.3 v 16.3 per 100 000 people), +as were the adjusted decompression rates (2001–02: 68.0 v 19.1 per 100 000 people; 2019–20: 104.8 v 16.7 per 100 000 people) +(Box 4). + +Research 4 Working age adults (18–64 years) who underwent elective fusion or decompression procedures in New South Wales, 2001–20, by funding type: age- and sex-adjusted ![4_image_0.png](4_image_0.png) + +- 2023 + * Denominators for publicly funded, lower bound procedure rates: NSW people aged 18–64 years;18 publicly funded, upper bound rates: NSW people aged 18–64 years who did not have hospital cover private health insurance;17,18 privately funded procedure rates: NSW people aged 18–64 years who had hospital cover private health insurance;17 workers' compensation procedure: NSW people aged 18–64 years with workers' compensation cover. Standardisation for age and sex based on 2016 national census data for NSW.17 The data for these graphs are included in the Supporting Information, table 9. ◆ +During 2001–20, rates of privately funded spinal fusion, decompression, and (from 2010–11) disc replacement procedures increased substantially in NSW, whereas changes for procedures that were publicly funded or covered by workers' compensation were smaller. The greatest rise was in privately funded fusion procedures, the rate of which increased throughout the study period; the decompression procedure rate peaked in 2014–15. Unlike many similar studies, we included surgery for all spinal regions, not just the lumbar spine; however, about 70% of fusions and 88% of decompressions undertaken in Australia during 2010–2018 were lumbar procedures.1 Our findings are consistent with reports that spinal surgery rates have increased over the past two decades, particularly lumbar fusions and procedures for people over 65 years of age.5-9,21 In the United States, the lumbar fusion rate rose during 2004–2015 from 60.4 to 79.8 per 100000 population (privately insured, uninsured, and governmentsubsidised patients),6 and from 30 to 110 per 100 000 among Medicare beneficiaries (people aged 65 years or more without health insurance) +during 1992–2003.21 Reported rates were lower and increases smaller in countries with universal health care systems than in the United States. In Finland, the lumbar fusion rate increased from 9 to 30 per 100 000 population during 1997– +2018;7 in Norway, it rose from 13.6 to 21 per 100 000 population during 1997– 2013, and the lumbar decompression rate increased from 64.3 to 88.9 per 100 000;5 in the United Kingdom, the lumbar surgery rate increased from 24.5 to 48.8 per 100000 population during 1999–2013.8 The privately funded decompression rate declined while the privately funded fusion rate increased during 2015–20. Studies of lumbar spinal stenosis surgery have found similar patterns. United States studies found declining decompression rates and increased rates of complex spinal fusion during 2002–2009.22,23 An Australian study found a small increase in the use of decompression and a fourfold increase in the use of complex fusion during 2003–2013.24 Our finding of a decline in the rate of decompressions covered by workers' compensation is consistent with an earlier study.1 The decline in the spinal fusion rate for this group might be partially explained by the 2018 reform of the Medicare spinal surgery billing schedule (ie, sixty new MBS items with new rules to ensure claiming consistency and reduce billing ambiguity).25 Spinal surgery rates are rising despite the lack of evidence that spinal fusion is an effective treatment for degenerative conditions.26,27 The costs of the procedures are high, as are the risks of complications. The increases in privately funded fusion (312%) and decompression rates (64%) in NSW during 2001–20 are unlikely to be explained by the increased uptake of private health insurance during this period (25% increase).17 While the rising fusion rate may be related to changes in the conditions treated and the availability of less invasive methods, these factors would not explain differences in change by funding type. The differences in publicly and privately funded procedure rates in NSW suggest that practice is determined by the preferences of the patient and the surgeon rather than by evidence of effectiveness. The adjusted rates of fusion and decompression procedures covered by workers' compensation were similar to those for publicly funded procedures, but the people in the two groups may have different health profiles; further, people covered by workers' compensation can also be treated in the public and private hospital systems. + +## Limitations + +Although diagnoses were recorded in the data sources, we could not identify specific indications for procedures or the spinal regions in which surgery was undertaken (cervical, thoracic, lumbar). Information about patient pain management, procedural techniques, and devices was not available to us. + +## Conclusion + +The privately funded spinal surgery rate continues to be larger than the publicly funded procedure rate, and it has also increased more rapidly. These differences may indicate that some privately funded procedures are unnecessary, or that the number of publicly funded procedures does not reflect clinical need. The factors that influence whether spinal surgery is undertaken should be further investigated, including financial considerations, access to specialist care and surgery, and the preferences of patients and physicians with respect to surgical and non-surgical care. Our findings could assist the evaluation of spinal surgery policies and the planning of services,3 and also contribute to reducing low value care.10 Acknowledgements: We thank the NSW Ministry of Health for providing access to the data we analysed, the NSW Centre for Health Record Linkage for conducting record linkage, the State Insurance Regulatory Agency, and the Australian Orthopaedic Association Research Foundation, the Australia and New Zealand Low Back Pain Research Network, and the National Health and Medical Research Council (APP1162833) for financial support. Duong Thuy Tran is supported by the Australia and New Zealand Low Back Pain Research Network. The funding sources were not involved in study design, data collection, analysis, or interpretation, reporting, or publication. + +Open access: Open access publishing facilitated by University of New South Wales, as part of the Wiley - University of New South Wales agreement via the Council of Australian University Librarians. + +Competing interests: No relevant disclosures. ■ +Received 2 March 2023, accepted 25 May 2023 +© 2023 The Authors. Medical Journal of Australia published by John Wiley & Sons Australia, Ltd on behalf of AMPCo Pty Ltd. + +This is an open access article under the terms of the Creative Commons AttributionNonCommercial-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. + +- 2023 1 Lewin AM, Fearnside M, Kuru R, et al. Rates, costs, return to work and reoperation following spinal surgery in a workers' compensation cohort in New South Wales, 2010–2018: a cohort study using administrative data. BMC Health Serv Res 2021; 21: 955. + +2 Wong AYL, Karppinen J, Samartzis D. Low back pain in older adults: risk factors, management options and future directions. Scoliosis Spinal Disord 2017; 12: 14. + +3 Australian Commission on Safety and Quality in Health Care and Australian Institute of Health and Welfare. The fourth Australian atlas of healthcare variation. Sydney: ACSQHC, 2021. + +https://www.safetyandquality.gov.au/ourwork/healthcare-variation/fourth-atlas-2021 +(viewed June 2023). + +4 Evans L, O'Donohoe T, Morokoff A, +Drummond K. The role of spinal surgery in the treatment of low back pain. Med J Aust 2023; 218: 40-45. https://www.mja.com.au/ journal/2023/218/1/role-spinal-surgery-treat ment-low-back-pain 5 Grotle M, Småstuen M, Fjeld O, et al. Lumbar spine surgery across 15 years: trends, complications and reoperations in a longitudinal observational study from Norway. BMJ Open 2019; 9: e028743. + +6 Martin BI, Mirza SK, Spina N, et al. Trends in lumbar fusion procedure rates and associated hospital costs for degenerative spinal diseases in the United States, 2004 to 2015. Spine (Phila Pa 1976) 2019; 44: 369-376. + +7 Ponkilainen VT, Huttunen TT, Neva MH, et al. + +National trends in lumbar spine decompression +and fusion surgery in Finland, 1997–2018. Acta Orthop 2021; 92: 199-203. + +8 Sivasubramaniam V, Patel HC, Ozdemir BA, +Papadopoulos MC. Trends in hospital admissions and surgical procedures for degenerative lumbar spine disease in England: a 15-year time-series study. BMJ Open 2015; 5: e009011. + +9 Harris IA, Dao ATT. Trends of spinal fusion surgery in Australia: 1997 to 2006. ANZ J Surg 2009; 79: 783-788. + +10 Buchbinder R, Underwood M, Hartvigsen J, et al. + +The Lancet series call to action to reduce low value care for low back pain: an update. Pain 2020; 161 (Suppl 1): S57-S64. + +11 State Insurance Regulatory Agency (NSW). + +Workers compensation insurance. https:// www.sira.nsw.gov.au/insurance-coverage/ workers-compensation-insurance (viewed Aug 2022). + +12 Medical Services Advisory Committee. + +Application 1145: artificial intervertebral disc replacement in patients with cervical degenerative disc disease [public summary document]. 29 Nov 2011. http://www.msac.gov. au/internet/msac/publishing.nsf/Content/FD416 0B6D7E29A41CA25801000123B84/$File/1145_ PSD.pdf (viewed Sept 2022). + +13 Australian Consortium for Classification Development. The international statistical classification of diseases and related health problems, tenth revision, Australian modification. Tenth edition. Adelaide: IHPA; Lane Publishing, 2016. + +14 Australian Consortium for Classification Development. Australian classification of health interventions, tenth edition. Adelaide: IHPA; Lane Publishing, 2017. + +15 von Elm E, Altman DG, Egger M, et al; STROBE +Initiative. Strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. BMJ 2007; 335: 806-808. + +16 Australian Department of Health and Aged Care. + +2020–25 National Health Reform Agreement (NHRA): Australian Government; 2023. https:// www.health.gov.au/our-work/2020-25-natio nal-health-reform-agreement-nhra (viewed Jan 2023). + +17 Australian Prudential Regulation Authority. + +Private health insurance membership trends. Mar 2023. https://www.apra.gov.au/sites/defau lt/files/2023-05/Quarterly%20Private%20 Health%20Insurance%20Membership%20Tre nds%20March%202023.xlsx (viewed July 2023). + +18 Australian Bureau of Statistics. Estimated resident population by state, age and sex, 2001 onwards. 2022. https://explore.data.abs.gov.au/ ?fs[0]=People%2C0%7CPopulation%23POPUL ATION%23&pg=0&fc=People (viewed Oct 2022). + +19 Australian Bureau of Statistics. 1270.0.55.005. + +Australian Statistical Geography Standard (ASGS), volume 5: remoteness structure, July 2016. 16 Mar 2018. https://www.abs.gov.au/ ausstats/abs@.nsf/mf/1270.0.55.005 (viewed Oct 2022). + +20 Medical Services Advisory Committee. Review of interim funded service. Artificial intervertebral disc replacement: lumbar. MSAC application 1090.1: assessment report. Apr 2011. http:// www.msac.gov.au/internet/msac/publishing. +308 nsf/Content/5DE3C448FF252171CA2580100 0123B66/$File/1090.1-Assessment-Report.pdf +(viewed July 2023). + +21 Weinstein JN, Lurie JD, Olson PR, et al. United States' trends and regional variations in lumbar spine surgery: 1992–2003. Spine (Phila Pa 1976) +2006; 31: 2707-2714. + +22 Bae HW, Rajaee SS, Kanim LE. Nationwide trends in the surgical management of lumbar spinal stenosis. Spine (Phila Pa 1976) 2013; 38: +916-926. + +23 Deyo RA, Mirza SK, Martin BI, et al. Trends, major medical complications, and charges associated with surgery for lumbar spinal stenosis in older adults. JAMA 2010; 303: 1259-1265. + +24 Machado GC, Maher CG, Ferreira PH, et al. + +Trends, complications, and costs for hospital admission and surgery for lumbar spinal stenosis. + +Spine (Phila Pa 1976) 2017; 42: 1737-1743. + +25 Australian Department of Health and Aged Care. Changes to MBS items for spinal surgery services. Updated 12 Oct 2018. http://www. + +mbsonline.gov.au/internet/mbsonline/publi shing.nsf/Content/773E298D6CD05602CA25 831A00000E58/$File/Spine%20Surgery%20 Quick%20reference%20guide.pdf (viewed Sept 2022). + +26 Harris IA, Traeger A, Stanford R, et al. Lumbar spine fusion: what is the evidence? Intern Med J 2018; 48: 1430-1434. + +27 Harris IA, Sidhu V, Mittal R, Adie S. Surgery for chronic musculoskeletal pain: the question of evidence. Pain 2020; 161 (Suppl 1): S95-S103. ■ + +## Supporting Information + +Additional Supporting Information is included with the online version of this article. + +- 2023 \ No newline at end of file diff --git a/medical/md/PMC2626663.md b/medical/md/PMC2626663.md new file mode 100644 index 0000000000000000000000000000000000000000..7ada1a8bb72fcbe964d6cbbc7108cf4da8ba5f9e --- /dev/null +++ b/medical/md/PMC2626663.md @@ -0,0 +1,255 @@ +The Journal of Experimental Medicine CORRESPONDENCE N.D. Huntington: nhunting@pasteur.fr + +# Il-15 Trans-Presentation Promotes Human Nk Cell Development And Diff Erentiation In Vivo + +Nicholas D. Huntington , +1,2 Nicolas Legrand , +4 Nuno L. Alves , +1,2 Barbara Jaron , +3 Kees Weijer , +4 Ariane Plet , +5 Erwan Corcuff , +1,2 Erwan Mortier , +5 Yannick Jacques , +5 Hergen Spits , +4 and James P. Di Santo 1,2 1 Cytokine and Lymphoid Development Unit, Immunology Department, 2 Institut National de la Sant é et de la Recherche M é dicale (INSERM) U668, 3 INSERM U883, Unit é de R é gulation Immunitaire et Vaccinologie, Institut Pasteur, Paris 75724, France 4 Academic Medical Center, University of Amsterdam, 1100 DD, Amsterdam, Netherlands 5 INSERM U892, Groupe de Recherche Cytokines et R é cepteurs, Institut de Biologie, Nantes F-44093, France The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self - major histocompatability complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefi ned. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2 -/ -c -/ - **mice** +transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor (IL-15R ) signifi cantly augmented human NK cells. IL-15 - IL-15R complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16 +KIR + NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56 hiCD16 -KIR - to CD56 loCD16 +KIR -**, and** +fi nally to CD56 loCD16 +KIR +. These data provide the fi rst evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15 – +responsive cells during immunotherapy strategies. + +NK cells participate in host protection by eliminating cells with altered expression of self - MHC class I (MHC-I), which can result from viral infection or transformation ( 1 ). Although we are beginning to appreciate a role for viral and stress-induced ligands in NK cell activation, the best described regulatory mechanism of NK cell activity is the expression of inhibitory receptors for self - MHC-I ligands by mature NK cells with high cytotoxic potential. In man, killer Ig-like receptors (KIRs) +recognizing the classical MHC-I molecules HLA-A, -B, or -C are expressed on the predominate peripheral NK cell (CD56 loCD16 +) +subset, which possesses abundant intracellular perforin and granzymes and displays spontaneous cytotoxicity ( 1 ). In contrast, CD56 hiCD16 - NK cells rarely express KIRs, and because they are more prevalent in blood early after bone marrow transplant ( 2, 3 ), give rise to CD56 loCD16 + NK cells when transferred into NOD/SCID mice ( 4 ), and have longer telomeres than CD56 loCD16 + NK cells ( 5 ), it is likely that CD56 hiCD16 - NK cells are (or contain within this population) precursors of CD56 loCD16 + NK cells; however, the tools to defi nitively prove this hypothesis are lacking. + +H. Spits ' s present address is Genentech, Inc., South San Francisco, CA 94080. + +Although inhibitory KIRs control reactivity of mature NK cells, their expression also infl uences the functional maturation of developing NK cells, as NK cells expressing at least one KIR-recognizing self - MHC-I have a lower threshold of activation and appear more functional than NK cells expressing no KIRs or those only expressing KIRs recognizing nonself - MHC-I ligands ( 6 ). In addition, patients lacking the transporter associated with antigen processing have dramatically reduced MHC-I surface expression and, consequently, hyporesponsive NK cells to MHC-I - defi cient cells ( 7 ). KIR + +NK cells are present in these patients, indicating that normal MHC-I expression itself is not required for KIR expression ( 7 ). This phenomenon termed " licensing "or " disarming "has been better characterized in mice, and suggests a role for KIR - self - MHC-I interactions during human NK cell development. Given the importance of KIR expression in regulating NK cell function, knowledge of elements infl uencing KIR acquisition would improve our understanding and clinical approaches to diseases where KIR and HLA haplotypes infl uence susceptibility, progression, or outcome of autoimmune/infl ammatory disease, cancer, hematopoietic grafts, and infections ( 8 ). + +Because KIRs are expressed on mature CD56 loCD16 + +NK cells, factors that infl uence NK cell homeostasis could potentially infl uence KIR acquisition in vivo. An elegant series of mouse studies using gene targeting and bone marrow chimeras have revealed that NK cell development requires IL-15R - expressing cells to chaperone IL-15 to the surface, where it is bioactive and signifi cantly more potent in inducing activation and proliferation of IL-15R - expressing cells via trans-presentation ( 9, 10 ). When not bound to IL-15R , +IL-15 appears to have a minimal eff ect on NK cell homeostasis in vivo ( 11, 12 ). In humans, mutations in IL-15R have not been reported; however, NK cells are dramatically reduced in patients carrying mutations in the common chain +( c) cytokine receptor (used in IL-15, -7, -4, -9, -2, and -21 signal transduction), Jak3, or the shared IL-2/-15R , whereas they are present in IL-7R - defi cient patients, suggesting that IL-15 may regulate human NK cell development ( 13, 14 ). + +In vivo studies of NK cells have been largely restricted to mice, and although this line of experimentation is valuable, some of this knowledge is not transferable to human NK cell biology. A clear example of this is NK cell development, where the kinetics, frequency, and phenotype are clearly different between species ( 1 ). An intermediate between mouse and human in vivo studies exists in the form of human immune system (HIS) mice. A recently developed HIS mouse model is the engraftment of newborn BALB/c Rag2 -/ -c -/ - +mice with human hematopoietic stem cells (HSCs) from fetal liver or cord blood ( 15, 16 ). BALB/c Rag2 -/ -c -/ - HIS +mice represent a practical HIS model with high human chimerism, most lymphocyte lineages generated, and adaptive immune responses occasionally evoked ( 15, 16 ). + +In this study, using HIS BALB/c Rag2 -/ -c -/ - mice engrafted with fetal liver HSCs, we study the in vivo role of IL-15 trans-presentation in human NK cell homeostasis. + +## Results And Discussion Human Hematopoietic Chimerism And Nk Cell Development In His Mice + +Human HSCs engrafted into newborn BALB/c Rag2 -/ -c -/ - +mice develop into mature myeloid and lymphoid cells ( 15, 16 ). + +We used this approach to investigate human NK cell development in vivo . 8 - 12 wk after HSC engraftment, HIS mice displayed human hematopoietic chimerism in all organs analyzed, with human thymopoiesis, B lymphopoiesis, and myelopoiesis evident ( Fig. 1, A and B,and Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20082013/DC1). + +Using an NK cell - specifi c antibody (anti-NKp46), we identifi ed human NK cells in all lymphoid organs of HIS mice, although at low frequencies, typically between 0.3 and 1.5% of human lymphocytes ( Fig. 1 A ). Both CD56 hiCD16 - and CD56 loCD16 + NK cells subsets are present, with the majority of NK cells having the latter phenotype ( Fig. 1 C ). Interestingly, some CD16 + cells expressed CD56 at levels indistinguishable from non - NK cells. These CD56 lo cells are NK +cells, as they express NKp46, NKG2D, CD94, and, like their counterparts in man, KIRs, whereas CD56 hiCD16 - cells are rarely KIRs + ( Fig. 1 C ). NKp46 + cells present a phenotype consistent with peripheral NK cells in man including expression of CD122 (IL-2R ), NKG2A, CD161, but not CD3 +( Fig. 1 D ). In vivo - generated human NK cells express high levels of intracellular granzyme B and IFN- when stimulated with IL-12 and -18 ex vivo, and they degranulate when co-cultured with K562 human leukemia cells as determined by expression of CD107a ( Fig. 1 E ). + +IL-15 is a pleiotropic cytokine essential for mouse NK +cell development ( 10 ). Given the sparse number of human NK cells, we hypothesized that IL-15 availability in this HIS model might be suboptimal. HIS mice represent a hybrid human - mouse system where cytokine receptor compatibility between species may not exist. Specifi cally, although human IL-15 (hIL-15) induces survival and proliferation of mouse NK cells ( 17, 18 ), it is not clear whether the reverse is true. This is a critical question, as we expect most of the IL-15 in HIS mice to be mouse-derived given the low human myeloid ( 15 ) or absent epithelial/stromal cell chimerism (cells likely to be sources of IL-15) ( 10, 19 ). We found that human NK cells cultured in vitro with hIL-15 proliferated extensively (predominately the CD56 hiCD16 - subset); this was in contrast to those cultured in mouse IL-15 (mIL-15), which itself suffi ciently induced proliferation of mouse NK cells ( Fig. 1 Fand Fig. S2, available at http://www.jem.org/cgi/ content/full/jem.20082013/DC1). Given that mIL-15 is trans-presented in vivo, we investigated the eff ect of culturing human NK cells with low concentrations of IL-15 alone or IL-15 trans-presented by IL-15R -Fc of the same or alternate species. Irrespective of which species of IL-15R was used, mIL-15 failed to induce human NK cell proliferation, although some cells survived when mIL-15 was combined with hIL-15R -Fc ( Fig. 1 G ). In contrast, hIL-15R was clearly superior in inducing NK cell proliferation compared with mIL-15R when combined with hIL-15 ( Fig. 1 G ). + +26 IL-15 TRANS-PRESENTATION DRIVES HUMAN NK CELL DIFFERENTIATION | Huntington et al. + +![2_image_0.png](2_image_0.png) + +![3_image_0.png](3_image_0.png) + +Similarly, activated human, but not mouse, myeloid cells were able to induce human NK cell proliferation in vitro (Fig. S2). Last, human NK cells are observed in athymic HIS mice +(Rag2 -/ -c -/ -FOXN1 -/ -), ruling out a major role for T cell - derived IL-2 driving NK cell development ( Fig. 1 H ). + +Collectively, these data indicate that the few resident NK cells in HIS mice are dependent on the available hIL-15. Indeed, the number of human NK cells in the spleen of HIS +mice could be further reduced after treatment with neutralizing antibody against human, but not mouse, IL-15 (Fig. S2). + +Furthermore, hIL-15R + cells known to express IL-15 and support NK cell development, such as members of the myeloid lineage, are found in the bone marrow of HIS mice +(unpublished data) ( 10, 15 ). Given this last point, it is possible that enhancing the chimerism of hIL-15/-15R - expressing cells will improve human NK cell development in HIS mice. + +Given the likely dependence of human NK cells on hIL-15, we next treated HIS mice with the same concentration of hIL-15 alone or hIL-15 preincubated with hIL-15R -Fc to mimic IL-15 trans-presentation in vivo. Consistent with mouse studies ( 11, 12 ), we observed a signifi cant increase in NK cell frequency when hIL-15 was complexed to IL-15R -Fc, but not when administered alone ( Fig. 2 A ).Increased NK lymphopoiesis by hIL-15+IL-15R -Fc compared with IL-15 alone was associated with increased Bcl-xL expression and cell proliferation, as demonstrated by BrdU uptake and Ki-67 expression (Fig. 2 B ). These data indicate that IL-15R - mediated human NK cells responses are more readily evoked in vivo when IL-15 is complexed to IL-15R -Fc. + +We next compared two diff erent IL-15R agonists, both mimicking IL-15 trans-presentation, hIL-15+IL-15R -Fc, and RLI (hIL-15 covalently linked to an hIL-15R extended sushi domain, but lacking any Fc fragment) ( 11, 12, 20 ). Four injections of 2.5 μ g hIL-15R agonists (one per week) resulted in a signifi cant increase in the number of NKp46 + cells in all organs ( Fig. 2 C ). Both these agonists markedly enhanced NK cell in vivo uptake of BrdU; expression of Bcl-xL, Ki-67, and CD69; and resulted in delayed apoptosis of NK cells when withdrawn from cytokines ( Fig. 2, D and E , and not depicted), suggesting both proliferation and survival result from IL-15+IL-15R binding to NK cells in vivo. Human CD8 T cells were also signifi cantly augmented in HIS mice and had an obvious increase in BrdU uptake after treatment with RLI ( Fig. 2 F ). Interestingly, RLI that activates both the IL-15R / and IL-15R / / was consistently more eff ective in vivo than noncovalent association of hIL-15+hIL-15R -Fc that activates IL-15R / alone. This observation is in agreement with in vitro studies showing RLI functions more effi - ciently than the noncovalent associations of IL-15 + sushi domain or other soluble forms of IL-15R ( 20, 21 ). The evident eff ect of RLI compared with IL-15+IL-15R -Fc also indicates that the Fc protein does not contribute to the augmented NK lymphopoiesis and rules out any activation of human NK cells through CD16. + +Although IL-15 is known to heighten NK cell cytotoxicity and induce proliferation, it also protects cells from apoptosis (the latter possible at lower concentrations) by suppressing proapoptotic Bim and elevating Bcl-2 family members such as Bcl-xL and Mcl-1 ( 18, 22, 23 ). A clear in vivo eff ect of RLI and IL-15+IL-15R -Fc was the up-regulation of Bcl-xL +in NK cells ( Fig. 2, B and E ). We next asked if enhanced survival could improve NK cell reconstitution in the limiting hIL-15 environment of HIS mice. To address this, HSCs were infected with a bicistronic retrovirus encoding the prosurvival protein Bcl-xL and GFP (to detect infected cells) in vitro before engrafting newborn BALB/c Rag2 -/ -c -/ - +mice. Ectopic expression of Bcl-xL in human HSCs resulted in a signifi cant increase in thymocyte and splenocyte cellularity 8 wk after engraftment, with Bcl-xL - transduced cells +(GFP +) representing a greater proportion of hCD45 + cells compared with control transduced cells in all organs ( Fig. 3, A - C ).NKp46 + cells were signifi cantly increased in thymus and spleen of Bcl-xL HIS mice compared with control HIS +mice; however, this appeared to be primarily a result of increased cellularity in these organs as the percentage of NK +cells largely unchanged ( Fig. 3, D and E ). In addition, although a greater proportion of NK cells were GFP + in BclxL - infected mice compared with controls, no accumulation of Bcl-xL - expressing NK cells was observed among the most mature subset (CD56 loCD16 +). Lastly, the number of NK +cells in HIS mice engrafted with Bcl-xL - infected HSCs, in terms of absolute numbers and fold diff erence compared with control, were substantially lower than mice treated with transpresented IL-15. These fi ndings indicate that IL-15/IL-15R +eff ects extend beyond providing survival signals in promoting human NK cell development in vivo. + +A consistent observation in all lymphoid organs after treatment with trans-presented IL-15 was the skewing of NK cell maturation toward the more diff erentiated CD56 loCD16 + +phenotype. In particular, we observed signifi cant and specifi c increases in the frequency of CD16 + NK cells and the ratio of CD16 +/CD16 - NK cells 7 d after the fi nal treatment ( Fig. 4, A and B ).This in vivo accumulation is consistent with a model where CD56 loCD16 + NK cells represents the terminal stage of NK cell development and suggests that trans-presented IL-15 + +![5_image_0.png](5_image_0.png) promotes this diff erentiation. CD16 expression is associated with human NK cell diff erentiation and increased cyto toxicity by means of increased intracellular eff ector granules and the ability to perform antibody-dependent cell cytotoxicity. IL-15 itself is known to enhance NK cell cytotoxicity via up-regulating eff ector molecules, such as IFN- , perforin, and granzymes +( 24 ). Strikingly, among the enhanced CD16 + population after IL-15 trans-presentation treatment, the percentage of NK cells expressing KIRs was also signifi cantly elevated, resulting in a large increase in the KIR + NK cell pool, an eff ect that is also more prominent with RLI ( Fig. 4 A ). The fraction of KIR + +NK cells after exogenous IL-15 trans-presentation was typically highest in the thymus and elevated compared with normal frequencies in human blood (typically 50 - 60%). This may result from greater IL-15 concentrations in our model compared with humans; however, the frequency of KIR + NK cells in human thymus has not been reported using all existing commercial antibodies to KIRs. In contrast to IL-15 trans-presentation, no enhanced NK cell diff erentiation and induction of KIR expression was observed in HIS mice treated with IL-15 alone or in those engrafted with Bcl-xL - expressing HSCs ( Fig. 4 C ). Furthermore, the increase in KIR + NK cells did not represent an expansion of one CD56 loCD16 +KIR + NK clone, as CD56 loCD16 + NK cells expressing a combination of 1 - 5 diff erent KIR members were present in this population ( Fig. 4 D ). + +It is unlikely the accumulation of CD56 loCD16 + NK cells results from a preferential expansion of this subset in response to hIL-15 trans-presentation as both CD56 loCD16 + and CD56 hiCD16 - NK cell subsets had a similar level of incorporated BrdU during treatment ( Fig. 5 A ).Although one cannot distinguish between CD16 + NK cells that have incorporated BrdU and BrdU + cells that have up-regulated CD16, it is most likely that the latter accounts for most of the BrdU +CD16 + +NK cells as CD56 loCD16 + NK cells are refractory to IL-15 stimulation in vitro ( 25, 26 ), especially when compared with CD56 hiCD16 - NK cells ( Fig. 1 E ). To test this hypothesis, we sorted and transferred either highly purifi ed CD56 hi CD16 -KIRs - or CD56 loCD16 +KIRs - NK cells from fetal spleen into BALB/c Rag2 -/ -c -/ - in the presence or absence of RLI treatment. Although we were unable to recover the donor cells in mice receiving PBS alone, clear populations of donor cells were recovered from RLI-treated mice 7 d after transfer ( Fig. 5 B ). Consistent with our hypothesis, trans-presented IL-15 induced the diff erentiation of CD56 hiCD16 - NK cells, with between 34 and 40% of recovered NK cells becoming CD16 + ( Fig. 5 B ). In addition a fraction of the CD16 + NK cells also now expressed KIRs. Similarly, whereas CD56 loCD16 + NK cells retained this phenotype in the presence of RLI after transfer, expression of KIRs were acquired by a fraction of these CD56 loCD16 + NK cells that lacked KIR expression before transfer ( Fig. 5 B ). The absence of NK cells in non-RLI treated recipients clearly shows that the available mouse IL-15 is inadequate to support human NK cell survival and highlights the importance of a source of hIL-15 in HIS mice in promoting NK cell diff erentiation. + +Binding of IL-15 to NK cells ultimately activates signaling pathways stemming from STAT5 phosphorylation, such as activation of NF- B, induction of cyclin D, down-regulation of proapoptotic proteins, and up-regulation of Bcl-2 family members and cytolytic granules ( 18, 24, 27 ). Although it is clear how these signaling events promote cell division, survival, and eff ector functions, it is not known how these pathways regulate acquisition of KIRs. Coordination between granzyme/perforin induction and KIR expression would be one means to limit NK cell activity during diff erentiation, and whereas most NK cells express other inhibitory receptors for MHC-I (CD94/NKG2A and LILR) throughout development, it appears acquisition of KIRs is a late event and likely unique 30 IL-15 TRANS-PRESENTATION DRIVES HUMAN NK CELL DIFFERENTIATION | Huntington et al. + +compared with other forms of MHC-I - mediated inhibition. + +Collectively, these data provide the fi rst in vivo demonstration that trans-presented IL-15 is essential for NK cell survival and subsequent diff erentiation and that KIR expression on mature NK cells is dynamically regulated by IL-15 signaling. + +## Concluding Remarks + +![6_Image_0.Png](6_Image_0.Png) + +Mouse studies show that IL-15 functions as a membranebound cytokine that can only be present at the cell surface and support NK cell development when bound to IL-15R on the same cell ( 9, 10 ). The clear eff ect of hIL-15R to enhance hIL-15 activity in vivo suggests this is also likely in man. Our fi ndings using a novel HIS mouse approach demonstrates that hIL-15 trans-presentation is necessary to promote human NK +cell development and diff erentiation in vivo. The failure to observe accumulation of CD16 +KIR + NK cells in HIS mice with HSCs ectopically expressing Bcl-xL suggests that NK cell survival alone is not suffi cient to promote diff erentiation and a source of hIL-15 is essential for this process. + +Factors regulating KIR expression on developing NK cells in vivo are undefi ned. Our study provides the fi rst evidence that trans-presented IL-15 induces KIR expression on CD56 loCD16 + NK cells in vivo and suggests that KIRs are expressed after CD16 and that acquisition of KIRs represents a further step in NK cell diff erentiation. Interestingly, expression of human self - MHC-I on a fraction of the hematopoietic cells in HIS mice appears suffi cient in generating phenotypically mature KIR + NK cells. Our fi ndings suggest use of hIL-15R +agonists in vivo may augment human NK cell diff erentiation, and subsequently functionality, and could benefi t patients with Figure 5. IL-15-dependent development of CD16 +KIR + NK cells + +![7_image_0.png](7_image_0.png) + +from CD16 -KIR - **precursors in vivo.** (A) NK cell subsets in the bone marrow of RLI-treated HIS mice from Fig. 2 D were analyzed for BrdU +uptake using fl ow cytometry. FACS plots are representative of two individual experiments of three to fi ve mice per treatment group engrafted with two different CD34 + HSC sources. (B) CD56 hi CD16 - KIRs - or CD56 lo CD16 + KIRs - NK cells were FACS sorted from fetal spleen (CD45 + CD3 - ) and transferred intrahepatically into 1-wk-old sublethally irradiated Rag2 - / - c - / - recipients for 7 d. Mice were treated i.p. with 2.5 μ g RLI or PBS on day 0 and 4 after transfer. Sorted KIRs - NK cells lacked KIR-2DL2/3/1/-2DS1/2/4/-3DL1/S1 expression. Data are representative of four mice per NK subset transferred and two mice per treatment group. + +underrepresented populations of mature cytotoxic KIR + NK +cells and improve NK cell –or CD8 T cell - based cancer immuno therapy strategies. The effi ciency of IL-15R agonists, particularly RLI, in augmenting human NK cell development in HIS mice will enable us to more readily dissect the role of IL-15-dependent lymphocytes (NK cell, memory CD8 T cell, NK T cell, and / T cell) responses to human pathogens and disease in vivo. Given obvious cross talk between innate and adaptive immune cells, having robust reconstitution of IL-15 - dependent cells in vivo improves the accuracy and application of HIS mice for studying human immune responses. Collectively, our results defi ne an essential role for IL-15 in human NK cell development in vivo and demonstrate the effi cacy of IL-15 - IL-15R complexes in promoting human NK cell homeostasis, suggesting hIL-15 is likely bound and trans-presented by IL-15R - expressing cells in man. + +## Materials And Methods + +Mice. C57BL/6 mice were purchased from Harlan. Rag2 -/ -c -/ - mice were backcrossed onto the BALB/c background and BALB/c Rag2 -/ -c -/ - +mice on a Nude background were bred and maintained in isolators with autoclaved food and water under a protocol approved by the AMC-UVA. + +Mice with a HIS were generated as previously described ( 15, 16 ). In brief, newborn (3 - 5-d-old) Rag2 -/ -c -/ - mice received sublethal (3.3 Gy) total body irradiation from a Cs source, and were injected intrahepatically with 10 5 sorted CD34 +CD38 - or 5 ×10 5 CD34 + human fetal liver cells. All manipulations of HIS mice were performed under laminar fl ow. Mouse experiments were approved by an institutional committee at the Institut Pasteur and validated by the French Ministry of Agriculture. + +In vivo treatments. HIS mice were injected i.p. with 100 μ l of hIL-15 +(2.5 μ g) and hIL-15R -Fc (7.5 μ g) + hIL-15 (2.5 μ g) both from R & D Systems, 2.5 μ g RLI ( 20 ), or PBS alone, commencing at a minimum of 6 wk after reconstitution. 100 μ l of 10 mg/ml of BrdU from BrdU fl ow kit (BD) was injected i.p. daily 2 d before sacrifi cing mice. For NK cell transfer assays, 3 ×10 4 CD56 hiCD16 -KIRs - or 2 ×10 5 CD56 loCD16 +KIRs - NK cells were sorted from human CD45 +CD3 - fetal splenocytes (15-wk gestation) and transferred intrheaptically into 1-wk-old sublethally irradiated BALB/c Rag2 -/ -c -/ - mice. A cocktail of KIR antibodies recognizing KIR2DL2/3/1/-2DS1/2/4/-3DL1/S1 was used. Recipients were then treated i.p. with PBS or 2.5 μ g of RLI on day 0 and 4 after transfer. IL-15 in vivo neutralization was performed by i.p. administration of 50 μ g of goat anti - hIL-15 or 25 μ g of goat anti - mouse IL-15 (both from R & D Systems) every second day for 7 d. Flow cytometry analysis for cell surface and intracellular markers. Cell suspensions were labeled with mAb against the following human cell surface markers: CD3 (SK7), CD4 (SK3), CD34 (581), CD8 (SK1), CD19 (HIB19), CD10 (HI10a), CD38 (HB7), NKG2D (1D11), NKp46 (9E2), CD16 (3G8), CD161 (DX12), CD56 (B159), HLA-DR (L243), HLA-A/ B/C (G46-2.6), CD117 (YB5.B8), IgD (IA6-2), IgM (G20-127), CD14 +(M5E2), CD122 (Mik- 3), TCR- / (T10B9.1A-31), CD127 (hIL-7RM21), CD11c (B-ly6), CD7 (M-T701), CD107a (1D4B), CD45 (2D1), +CD69 (L78), Bcl-2 (Bcl-2/100), IFN- (XMG1.2), BrdU (B44), and Ki67 +(B56) from BD; KIR2DL2/L3 (DX27), KIR3DL1 (DX9), CD94 (DX22), NKp44 (p44-8), CD27 (O323), CD62L (DREG-56), and NKp30 (P30-15) from BioLegend; KIR2DS4 (FES172), KIR2DL1/DS1 (EB6B), KIR2DL2/ L3/DS2 (GL183), KIR3DL1/DS1 (Z27.3.7), Bcl-xL (7B2.5), and CD159a +(NKG2A; Z199) from Beckman Coulter; granzyme B (GB11) from Invitrogen; and CD11b (ICRF44), CD25 (BC96), CD116 (4H1), CD83 (HB15e), anti - mouse NK1.1, CD11b, CD11c, F480, and DX5 from eBioscience. Intracellular staining was performed after fi xation and permeabilization of the cellular suspensions using Perm/Wash and Cytofi x/Cytoperm reagents from BD according to the manufacturer ' s instruction. For BrdU detection, cells were incubated for 1 h at 37 ° C with 30 μ g DNase from BrdU fl ow kits (BD). All washings and reagent dilutions were done with PBS containing 2% +FCS. All acquisitions were performed using LSRII, Canto 1, or Canto 2 cytometers, cell sorting was performed using FACSAria, and all machines were interfaced to the FACSDiva software (BD). + +Cell preparation. Human fetal liver was obtained from elective abortions, with gestational age ranging from 14 to 20 wk. Experiments using human fetal liver cells were approved by the Medical and Ethical Committees at the Institut Pasteur and AMC-UvA and performed in full compliance with French law. Single-cell suspensions of fetal material were achieved by mechanical disruption using a Stomacher Biomaster laboratory system (Seward). Magnetic enrichment of CD34 + cells ( > 98% pure) was performed by using the CD34 Progenitor Cell Isolation kit (Miltenyi Biotech, Auburn, CA), after preparation of single-cell suspension and isolation of mononuclear cells by density gradient centrifugation over FicollHypaque (Nycomed Pharma). Cell suspensions were prepared in RPMI medium with 2% FCS. Single-cell suspensions of mouse organs were prepared as previously described ( 17 ). + +In vitro assays. Human NK cells were purifi ed from donor blood buff y coat prepared by density gradient centrifugation over Ficoll-Hypaque (Nycomed Pharma) using anti-CD56 magnetic beads (Miltenyi Biotech). C57BL/6 splenic NK cells were purifi ed by anti-DX5 magnetic beads (Miltenyi Biotech). Purifi ed cells were loaded with 5 μ M CFSE (Invitrogen) and cultured at 2 ×10 5 cells/ml in RPMI with 10% FCS and 10 ng/ml rhIL-15 +(rhIL-15; R & D Systems) or 30 ng/ml rmIL-15 (Peprotech) for 3 or 5 d. Alternatively, 10 4 CD56 + NK cells purifi ed from human peripheral blood were labeled with CFSE and cultured for 72 h in combinations of human or mouse IL-15 that had been preincubated with human or mouse IL-15R - Fc for 1 h at 4 ° C. The fi nal concentration of IL-15 and IL-15R -Fc were 32 IL-15 TRANS-PRESENTATION DRIVES HUMAN NK CELL DIFFERENTIATION | Huntington et al. + +5 and 20 ng/ml, respectively. In vitro restimulation of HIS-derived NK cells was performed using CD56 + NK cells purifi ed by magnetic beads (Miltenyi Biotech) from a cell suspension of spleen and bone marrow from seven HIS +mice pooled together. NK cells were cultured at 2.5 ×10 5 cells/ml in RPMI +supplemented with 10% FCS, rhIL-15 (5 ng/ml), and either rhIL-12 (5 ng/ml) + +rhIL-18 (20 ng/ml; R &D Systems) or 2.5 ×10 6 K562 cells/ml (American Type Culture Collection) for 18 h, with brefeldin A (Sigma-Aldrich) added for the last 4 h of culture. Retroviral Bcl-xL expression. Human fetal liver cells were prepared and modifi ed by retroviral transduction as follows. The sorted CD34 +CD38 - +fetal liver cells were cultured overnight in IMDM (Invitrogen) supplemented with Yssel ' s medium, 5% normal human serum, 20 ng/ml human stem cell factor, 20 ng/ml human thrombopoietin, and 20 ng/ml human IL-7 (PeproTech). The next day, cells were incubated for 6 - 8 h with control LZRS +IRES-GFP or LZRS Bcl-xL-IRES-GFP virus supernatant in fi bronectincoated plates (30 μ g/ml; Takara Biomedicals). The cell bulk was then inoculated intrahepatically to the newborn recipients. The cDNA sequence encoding Bcl-xL, initially provided by the laboratory of J.G. Collard (The Netherlands Cancer Institute, Amsterdam, Netherlands) was inserted into the multiple cloning sites of LZRS vector upstream of an internal ribosomal entry site and enhanced GFP ( 28 ). Control vectors were empty LZRS IRES. Retroviral supernatants were produced as previously described ( 29 ) using the 293T-based Phoenix packaging cell line ( 30 ). Online supplemental material. Fig. S1 shows lymphocyte reconstitution in HIS mice. Fig. S2 shows in vitro NK cell cultures and in vivo IL-15 neutralization. Online supplemental material is available at http://www.jem +.org/cgi/content/full/jem.20082013/DC1. + + We acknowledge the Bloemenhove Clinic (Heemstede, The Netherlands) for providing fetal tissues. We would like to thank Allison Bordack, Dr. Jean-Jacques Mention, Dr. Ferenc Scheeren, Joran Volmer, and Jenny Meerding for reagents and technical assistance and Prof. Andreas Strasser for critiques on the manuscript. + + This work is supported by grants from Institut Pasteur, Institut National de la Sant é et de la Recherche M é dicale, Ligue National Contre le Cancer, National Health and Medical Research Council of Australia, The Menzies Foundation, and a Grand Challenges in Global Health grant from the Bill and Melinda Gates Foundation. Authors from the Institut Pasteur and Academic Medical Center are part of the Human Vaccine Consortium " Grand Challenges in Global Health: Devise Reliable Testing Systems for New Vaccines:" (http://www.hv-consortium.org). + + The authors have no confl icting fi nancial interest. + +## Submitted: 9 September 2008 Accepted: 24 November 2008 References + + 1 . Huntington , N.D. , C.A. Vosshenrich , and J.P. Di Santo . 2007 . + +Developmental pathways that generate natural-killer-cell diversity in mice and humans. *Nat. Rev. Immunol.* 7 : 703 - 714 . + + 2 . Jacobs , R. , M. Stoll , G. Stratmann , R. Leo , H. Link , and R.E. Schmidt . + +1992 . CD16- CD56+ natural killer cells after bone marrow transplantation. *Blood.* 79 : 3239 - 3244 . + + 3 . Gottschalk , L.R. , R.A. Bray , H. Kaizer , and H.M. Gebel . 1990 . Two populations of CD56 (Leu-19)+/CD16+ cells in bone marrow transplant recipients. *Bone Marrow Transplant.* 5 : 259 - 264 . + + 4 . Chan , A. , D.L. Hong , A. Atzberger , S. Kollnberger , A.D. Filer , C.D. + +Buckley , A. McMichael , T. Enver , and P. Bowness . 2007 . CD56bright human NK cells diff erentiate into CD56dim cells: role of contact with peripheral fi broblasts. *J. Immunol.* 179 : 89 - 94 . + + 5 . Romagnani , C. , K. Juelke , M. Falco , B. Morandi , A. D ' Agostino , +R. Costa , G. Ratto , G. Forte , P. Carrega , G. Lui , et al . 2007 . + +CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. + +J. Immunol. 178 : 4947 - 4955 . + + 6 . Anfossi , N. , P. Andre , S. Guia , C.S. Falk , S. Roetynck , C.A. Stewart , +V. Breso , C. Frassati , D. Reviron , D. Middleton , et al . 2006 . Human NK cell education by inhibitory receptors for MHC class I. *Immunity.* 25 : 331 - 342 . + + 7 . de la Salle , H. , D. Hanau , D. Fricker , A. Urlacher , A. Kelly , J. + +Salamero , S.H. Powis , L. Donato , H. Bausinger , M. Laforet , et al . 1994 . Homozygous human TAP peptide transporter mutation in HLA class I defi ciency. *Science.* 265 : 237 - 241 . + + 8 . Carrington , M. , and M.P. Martin . 2006 . The impact of variation at the KIR gene cluster on human disease. *Curr. Top. Microbiol. Immunol.* 298 : 225 - 257 . + + 9 . Mortier , E. , T. Woo , R. Advincula , S. Gozalo , and A. Ma . 2008 . IL15R chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation. *J. Exp. Med.* 205 : 1213 - 1225 . + +10 . Ma , A. , R. Koka , and P. Burkett . 2006 . Diverse functions of IL2, IL-15, and IL-7 in lymphoid homeostasis. *Annu. Rev. Immunol.* +24 : 657 - 679 . + +11 . Rubinstein , M.P. , M. Kovar , J.F. Purton , J.H. Cho , O. Boyman , C.D. + +Surh , and J. Sprent . 2006 . Converting IL-15 to a superagonist by binding to soluble IL-15R { alpha } . *Proc. Natl. Acad. Sci. USA.* 103 : 9166 - 9171 . + +12 . Stoklasek , T.A. , K.S. Schluns , and L. Lefrancois . 2006 . Combined IL15/IL-15Ralpha immunotherapy maximizes IL-15 activity in vivo. + +J. Immunol. 177 : 6072 - 6080 . + +13 . Buckley , R.H. 2004 . Molecular defects in human severe combined immunodefi ciency and approaches to immune reconstitution. *Annu. Rev.* +Immunol. 22 : 625 - 655 . + +14 . Gilmour , K.C. , H. Fujii , T. Cranston , E.G. Davies , C. Kinnon , and H.B. Gaspar . 2001 . Defective expression of the interleukin-2/interleukin-15 receptor beta subunit leads to a natural killer cell-defi cient form of severe combined immunodefi ciency. *Blood.* 98 : 877 - 879 . + +15 . Gimeno , R. , K. Weijer , A. Voordouw , C.H. Uittenbogaart , N. Legrand , +N.L. Alves , E. Wijnands , B. Blom , and H. Spits . 2004 . Monitoring the eff ect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2 -/ - gammac -/ - mice: functional inactivation of p53 in developing T cells. *Blood.* 104 : 3886 - 3893 . + +16 . Traggiai , E. , L. Chicha , L. Mazzucchelli , L. Bronz , J.C. Piff aretti , A. + +Lanzavecchia , and M.G. Manz . 2004 . Development of a human adaptive immune system in cord blood cell-transplanted mice. *Science.* 304 : +104 - 107 . + +17 . Huntington , N.D. , Y. Xu , S.L. Nutt , and D.M. Tarlinton . 2005 . A requirement for CD45 distinguishes Ly49D-mediated cytokine and chemokine production from killing in primary natural killer cells. *J. Exp.* +Med. 201 : 1421 - 1433 . + +18 . Huntington , N.D. , H. Puthalakath , P. Gunn , E. Naik , E.M. Michalak , +M.J. Smyth , H. Tabarias , M.A. Degli-Esposti , G. Dewson , S.N. Willis , +et al . 2007 . Interleukin 15-mediated survival of natural killer cells is determined by interactions among Bim, Noxa and Mcl-1. *Nat. Immunol.* +8 : 856 - 863 . + +19 . Fehniger , T.A. , and M.A. Caligiuri . 2001 . Interleukin 15: biology and relevance to human disease. *Blood.* 97 : 14 - 32 . + +20 . Mortier , E. , A. Quemener , P. Vusio , I. Lorenzen , Y. Boublik , J. + +Grotzinger , A. Plet , and Y. Jacques . 2006 . Soluble interleukin-15 receptor alpha (IL-15R alpha)-sushi as a selective and potent agonist of IL-15 action through IL-15R beta/gamma. Hyperagonist IL-15 x IL15R alpha fusion proteins. *J. Biol. Chem.* 281 : 1612 - 1619 . + +21 . Bouchaud , G. , L. Garrigue-Antar , V. Sole , A. Quemener , Y. Boublik , +E. Mortier , H. Perdreau , Y. Jacques , and A. Plet . 2008 . The exon-3-encoded domain of IL-15ralpha contributes to IL-15 high-affi nity binding and is crucial for the IL-15 antagonistic eff ect of soluble IL-15Ralpha. J. Mol. Biol. 382 : 1 - 12 . + +22 . Carson , W.E. , T.A. Fehniger , S. Haldar , K. Eckhert , M.J. Lindemann , +C.F. Lai , C.M. Croce , H. Baumann , and M.A. Caligiuri . 1997 . A potential role for interleukin-15 in the regulation of human natural killer cell survival. *J. Clin. Invest.* 99 : 937 - 943 . + +23 . Zheng , X. , Y. Wang , H. Wei , B. Ling , R. Sun , and Z. Tian . 2008 . BclxL is associated with the anti-apoptotic eff ect of IL-15 on the survival of CD56(dim) natural killer cells . *Mol. Immunol*. 45 : 2559 - 2569 . + +24 . Fehniger , T.A. , S.F. Cai , X. Cao , A.J. Bredemeyer , R.M. Presti , A.R. + +French , and T.J. Ley . 2007 . Acquisition of murine NK cell cytotoxicity requires the translation of a pre-existing pool of granzyme B and perforin mRNAs. *Immunity.* 26 : 798 - 811 . + +25 . Carson , W.E. , J.G. Giri , M.J. Lindemann , M.L. Linett , M. Ahdieh , R. + +Paxton , D. Anderson , J. Eisenmann , K. Grabstein , and M.A. Caligiuri . 1994 . Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. *J. Exp. Med.* 180 : 1395 - 1403 . + +26 . Cooley , S. , F. Xiao , M. Pitt , M. Gleason , V. McCullar , T.L. Bergemann , +K.L. McQueen , L.A. Guethlein , P. Parham , and J.S. Miller . 2007 . + +A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature. *Blood.* +110 : 578 - 586 . + +27 . Giron-Michel , J. , M. Giuliani , M. Fogli , D. Brouty-Boye , S. Ferrini , +F. Baychelier , P. Eid , C. Lebousse-Kerdiles , D. Durali , R. Biassoni , +et al . 2005 . Membrane-bound and soluble IL-15/IL-15Ralpha complexes display diff erential signaling and functions on human hematopoietic progenitors. *Blood.* 106 : 2302 - 2310 . + +28 . Jaleco , A.C. , A.P. Stegmann , M.H. Heemskerk , F. Couwenberg , A.Q. + +Bakker , K. Weijer , and H. Spits . 1999 . Genetic modifi cation of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3. *Blood.* 94 : 2637 - 2646 . + +29 . Heemskerk , M.H. , B. Blom , G. Nolan , A.P. Stegmann , A.Q. Bakker , +K. Weijer , P.C. Res , and H. Spits . 1997 . Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3. *J. Exp. Med.* 186 : 1597 - 1602 . + +30 . Kinsella , T.M. , and G.P. Nolan . 1996 . Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. *Hum. Gene Ther.* 7 : 1405 - 1413 . \ No newline at end of file diff --git a/medical/md/PMC2692018.md b/medical/md/PMC2692018.md new file mode 100644 index 0000000000000000000000000000000000000000..50f62a1b4fa4923b9f5676b97b171767bb77506f --- /dev/null +++ b/medical/md/PMC2692018.md @@ -0,0 +1,282 @@ +Journal of Experimental Botany, Vol. 60, No. 9, pp. 2763–2771, 2009 + +![0_image_0.png](0_image_0.png) + +doi:10.1093/jxb/erp132 Advance Access publication 18 May, 2009 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) +RESEARCH PAPER + +# The Expression Of Caffeic Acid 3-O-Methyltransferase In Two Wheat Genotypes Differing In Lodging Resistance + +## Qing-Hu Ma* + +Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, 20 Nanxin Cun, Xiangshan, Beijing 100093, China Received 7 January 2009; Revised 9 March 2009; Accepted 4 April 2009 + +## Abstract + +Stem lodging-resistance is an important phenotype in crop production. In the present study, the expression of the wheat COMT gene (TaCM) was determined in basal second internodes of lodging-resistant (H4564) and lodgingsusceptible (C6001) cultivars at stem elongation, heading, and milky endosperm corresponding to Zadoks stages Z37, Z60, and Z75, respectively. The TaCM protein levels were analysed by protein gel blot and COMT enzyme activity was determined during the same stem developmental stages. TaCM mRNA levels were higher in H4546 from elongation to the milky stages and in C6001 the TaCM mRNA levels decreased markedly at the heading and milky stages. The TaCM protein levels and COMT activity were also higher in H4564 than that in C6001 at the heading and milky stages. These results corresponded to a higher lignin content measured by the Klason method and stem strength and a lower lodging index in H4564 than in C6001 at the heading and milky stages. Therefore, the TaCM mRNA levels, protein levels, and enzyme activity in developing wheat stems were associated with stem strength and lodging index in these two wheat cultivars. Southern analysis in a different population suggested that a TaCM locus was located in the distal region of chromosome 3BL, which has less investigated by QTL for lodging-resistant phenotype. + +Key words: Caffeic acid 3-O-methyltransferase, lignin biosynthesis, lodging resistance, Triticum aestivum L. + +## Introduction + +Crop lodging is a complicated phenomenon that is influenced by many factors from physiology and genetics, to husbandry and environment. Lodging is a major problem in crop production which causes poor grain filling and yield loss, reduces grain quality and mechanical harvesting efficiency (see review by Berry et al., 2004, and references therein). The dwarfness of the crop using dwarfing genes and the application of plant growth regulators have been very effective tools for reducing the risk of lodging and maintaining a steady yield. However, they have not completely eradicated lodging risk. Furthermore, a reduction in plant height for lodging resistance may reduce the photosynthetic capacity of a canopy which would then limit the final yield (Wu et al., 2002). An analysis of the dwarfing genes on crop yield has shown that the optimum plant height for maximum photosynthetic capacity is between 70 cm and 100 cm in wheat (Flintham et al., 1997). Therefore, alternative strategies for further improvement in lodging resistance of crops must be developed. + +According to Sterling et al. (2003), two types of lodging occur in wheat. Stem lodging results from the bending or breaking of the lower culm internodes, while root lodging refers to intact and unbroken culms leaning from the crown due to failure of root anchorage on ground. Stem strength, the bending or breaking strength of the culm, is important for stem lodging resistance, particularly for the basal internodes of crops (Zuber et al., 1999). Increasing stem strength will be a very promising strategy to breed crops with high lodging resistance and yield. Except for stem weight and diameter which are closely related to physical strength, stem strength is also the product of its chemical and biochemical components. Stem tissues of crops mainly contain the storage and structural carbohydrates. Recent reports have shown that higher starch accumulation in the + +## 2764 | Ma + +stem tissues is responsible for the higher lodging resistance in rice during typhoons (Kashiwagi and Ishimaru, 2004; Kashiwagi et al., 2006; Ishimaru et al., 2008). In theory, the structural carbohydrates (cellulose and lignin) should also play the (even more) important roles for material strength in stems. + +Lignin is a phenolic cell wall polymer closely linked to cellulose and hemicelluloses, and is, second to cellulose, the most abundant biopolymer on earth. In plants, lignin mainly deposits in the walls of certain specialized cells such as tracheary elements, sclerenchyma, and phloem fibres. + +This leads to a dramatic change in cell wall properties, which impart strength and structural support to the wall and assists in the transport of water and nutrients within xylem tissue by decreasing the permeability of the cell wall +(Boerjan et al., 2003). It has long been proposed that lignin synthesis might be related to stem strength. This issue is especially important in monocotyledonous crop plants, where weak stem strength will lead to a lodging phenotype +(Hai et al., 2005). To date, however, there is little information at the molecular level as to how the regulation of lignin synthesis affects crop lodging. Reduced lignin levels have been observed in maize brown-midrib (bm) mutants and this has been associated with reduced stem strength. Recent work has confirmed that the bm3 mutant harboured a mutation in the gene for caffeic acid 3-O-methyltransferase (EC 2.1.1.68, COMT) (Vignols et al., 1995), while bm1 was shown to affect directly expression of the cinnamyl alcohol dehydrogenase (EC 1.1.1.195, CAD) (Halpin et al., 1998). + +Some brittleness mutants have been reported in barley +(Kokubo et al., 1991) and rice (Kinoshita, 1998). A +COBRA-like regulation protein has been reported to be involved in brittle stalk mutants in rice (Li et al., 2003) and maize (Sindhu et al., 2007). The actions of these mutants on gene levels, however, are largely unknown. + +A cDNA fragment for COMT from wheat has been isolated and RNA gel blot analysis showed that this gene was differentially expressed in lodging-sensitive and -resistant cultivars (Ma et al., 2002). The full-length cDNA for this COMT, TaCM, was obtained by screening wheat stem cDNA library and the functional expression and transgenic approach have demonstrated that TaCM is an authentic COMT involved in lignin biosynthesis (Ma and Xu, 2008). + +The analyses of TaCM gene expression, enzyme activity, and protein content in relation to the lodging-resistant phenotype in wheat is reported here. The TaCM locus was mapped in the chromosome. + +## Materials And Methods Plant Materials + +Wheat (Triticum aestivum L.) plants were grown in a naturally lit greenhouse with normal irrigation and fertilization. + +Two cultivars, C6001 and H4564, were chosen for our analyses. C6001 and H4564 developed from the same pedigree of Jimai from the Hebei province of China. Both cultivars have the same growth period (240 d) and vernalization type (winter), similar morphological and agricultural phenotypes (with plant height of 75–80 cm, diameter of the mature stem of 2.97–3.27 mm, kernel number per spike of 18–20, and grain number per kernel of 28–36 with mature seed weight 38–41 g per 1000 seeds), but differ in stem lodging phenotype, in which C6001 is lodgingsensitive while H4564 is lodging-resistant. The stem lodging level (as judged by the angle of the stem to the vertical position of more than 30o) just before harvest was 25–30% +for C6001 and 2–4% for H4564 in the flood irrigation field with 10–15% and 0%, respectively, in dry farmland. The majority of lodgings observed are due to stem breakage so they belong to stem lodging predominates. Basal second internodes were collected from the wheat plants with three internodes, and then collected at 3 week intervals at the stem elongation, heading, and milky endosperm stages, corresponding to Zadoks stages (Zadoks et al., 1974) Z37, Z60, and Z75, respectively. Previous investigations have shown that the second internodes play the more important role in wheat lodging resistance (Zuber et al., 1999; Dong et al., 2003). + +## Rna Isolation, Northern Blot, And Rt-Pcr Analysis + +Total RNA was isolated from wheat internode tissue by TRI reagent (Molecular Research Center, Inc, Cincinnati, USA) following the manufacturer's instructions. Ten lg of each total RNA sample was electrophoresed on formaldehyde 1.4% (w/v) agarose gels. RNA was blotted onto Hybond-N+ membrane (Amersham, GE Healthcare BioSciences Corp., Piscataway, NJ 08855-1327, USA) using the standard transfer procedure of Sambrook et al. (1989). The blots were hybridized at 42 oC with 63 SSC, 53 Denhardt, 0.5% SDS, 100 lg ml1 salmon sperm DNA with 50% +formamide, and washed with 0.13 SSC plus 0.1% SDS at 65 C. Probes were 32P-labelled using a Ready-to-Go DNA +Labeling Kit (Amersham). RNA hybridization signals were normalized by a soybean 18S ribosomal RNA. + +The first-strand cDNA was synthesized using the RT-PCR +kit (TakaRa, Japan) with total RNA. Reverse transcription reactions were carried out at 42 C for 60 min and terminated by heating to 95 C for 10 min. One microlitre of the reaction mixture was used as a template in the PCR +reaction. The parameters for 30 cycles were 95 C for 1 min, 55 C for 1 min, and 72 C for 1.5 min, with a final extension at 72 C for 10 min. The PCR products were loaded onto 1% agarose gel to visualize the amplified cDNAs. Primers for wheat TaCM were: sense primer 5\#-AAGGTCCTCATGGAGAGCTG-3\# and antisense primer 5\#-CGGCAGGATGCATCCACGGA-3\#. Primers for a-tubulin were: sense primer 5\#-CTCTACTGCCTCGAGCAT-3\# and antisense primer 5\#-GAGGAAAGCATGAAGTGG-3\#. + +## Enzyme Extraction And Assay + +Enzyme extraction followed Parvathi et al. (2001). In brief, wheat internode tissue was ground in liquid nitrogen and then the tissue powder was mixed with the extraction buffer +(100 mM TRIS-HCl pH 7.5, 0.2 mM MgCl2, 2 mM DTT, +10% glycerol) and incubated at 4 C for 1 h. The samples were spun at 12 000 g for 10 min at 4 C. The extracts were desalted on PD-10 columns (Pharmacia, Piscataway, NJ08855-1327, USA). The soluble protein fractions were used for the COMT enzyme assay. COMT enzyme activities were measured according to the method of Ni et al. (1996). + +Protein concentrations were determined by the Bradford assay (Bradford, 1976) with bovine serum albumin as the standard. + +## Protein Gel Blot Analysis + +Protein samples were resolved by 12% SDS-PAGE, then electro-transferred onto nitrocellulose membrane. The membrane was incubated in blocking buffer (PBS containing 0.05% Tween 20 and 5% skim milk) at room temperature overnight, and then incubated with rabbit anti-alfalfa COMT serum at 1:10 000 dilution (Kersey et al., 1999) in blocking buffer for 2 h. The membranes were washed with PBST (PBS with 0.05% Tween 20) and incubated with peroxidase-conjugated goat anti-rabbit polyclonal antibodies (Bio-Rad Laboratories Beijing Ltd., Beijing 100086, China) at 1:10 000 dilution in PBST for 1 h. The signals were detected with a chemiluminescent reaction reagent +(ECL, Amersham) according to the manufacturer's protocol. + +## Lignin Content Analysis + +Lignin content in air-dried basal second internodes was quantitatively measured by using the Klason method (Kirk and Obst, 1988) and expressed as a percentage of the cell wall residue (% CWR). + +## Mapping Analysis + +The mapping population consists of 114 recombinant inbred lines (RILs) and has been the subject of an extensive genome mapping effort by investigators of the International Triticeae Mapping Initiative (ITMI) (Marino et al., 1996). + +Genomic DNA isolation from Chinese Spring NT lines, W-7984, Opata 85, and the RILs, restriction enzyme digestion, gel electrophoresis, Southern blotting, and hybridization were performed as described in Faris et al. + +(2000). TaCM cDNA (GenBank accession no. EF413031) +was 32P-labelled using a Ready-to-Go DNA Labelling Kit +(Amersham) to be used as the probe. The genetic mapping and linkage relationships were analysed as reported previously (Ma, 2007). + +## Stem Strength And Lodging Index Determination + +Wheat basal second internodes were collected from the field. The stem strength and lodging index (LI) was determined according to the method of Wang and Li +(1996). In brief, stem strength was measured using a three point bending method where the freshly collected 5 cm stem segments (span more than 103 the diameter) were placed horizontally and mounted on two supports in the rack of the apparatus. The force exerted to break the stem was recorded with a universal force testing device (model DCKZ300, Sichuan, China) to determine the stem strength (S). + +The fresh weight (G) of the aerial part, and height (H) to the centre of gravity (measured by balancing the whole plant in the centre position) of each single wheat plant were determined. LI was calculated by following formula: +LI¼G3H/S. Fifteen stem samples were determined. + +Student's t test for independent samples (Geng and Hills, 1989) was applied to determine the difference between C6001 and H4564 cultivars and the probability value was estimated at the P0.05 and P0.01 levels. Data were shown as mean 6standard deviation. All statistical analyses were conducted using the SPSS 11.0 software package. + +## Results + +Stem strength and lodging index at different developmental stages in wheat stem As the basal internodes play a crucial role in stem lodging resistance in wheat, the basal second internodes were used to determine the stem strength and lodging index from three different developing stages (Table 1). The stem strength of H4564 was always higher than that of C6001, and this difference was particularly remarkable at the heading and Table 1. Comparison of stem bending strength (g), lodging index, Klason lignin content (as % CWR), and COMT activity (pkat mg-1 protein) of basal second internodes in wheat cultivars H4564 and C6001 at different developmental stages n.d. means not detected. Data represent means 6standard deviation (n¼15 for stem strength and lodging index, n¼4 for lignin content and COMT activity). Significant differences at P0.05 and P0.01 levels between the two cultivars in the same stage are marked by * and **, +respectively. + +| Developmental stages | Cultivars | Stem bending strength | Lodging index | Lignin content | COMT activity | +|------------------------|-------------|-------------------------|-----------------|------------------|-----------------| +| Elongation | C6001 | 167612 | n.d. | 2.8560.37 | 12.661.33 | +| H4564 | 183625* | n.d. | 2.9360.31 | 13.461.21 | | +| Heading | C6001 | 288621 | 0.9760.08 | 4.5461.24 | 26.861.05 | +| H4564 | 562641** | 0.5460.04** | 7.5562.08* | 35.562.10** | | +| Milky | C6001 | 523641 | 0.6360.05 | 10.2061.68 | 90.465.07 | +| H4564 | 933681** | 0.3760.03** | 14.4962.30* | 131.368.94** | | + +milky stages, which was more than 95.1% and 78.4%, respectively. Consistent with the high stem strength, the lodging index of H4564 was significant lower than that of C6001, consistent with its lodging-resistant phenotype. + +Lignin accumulations in relation to wheat stem development To determine whether lignin synthesis was associated with stem strength and lodging resistance, lignin content was analysed in the basal second internodes at different developmental stages in the two wheat cultivars (Table 1). + +From elongation to the milky stage, the content of Klason lignin increased accordingly, reflecting active lignin synthesis during wheat stem maturity. The Klason lignin content of H4564 was significantly higher than that of C6001 at the heading and milky stages, more than 66.3% and 42.1%, +respectively. By contrast, there was no remarkable difference for lignin content between the two cultivars at the elongation stage. + +## Expression Of The Tacm Gene At Different Developmental Stages In Wheat Stem + +RT-PCR analysis showed that the TaCM gene was constitutively expression in stem, leaf, and root tissues (Fig. 1), +indicating that TaCM is associated with the constitutive lignification process in various vegetative growing tissues. TaCM gene expression was analysed in the basal second internodes at different stem developmental stages by Northern blot hybridization (Fig. 2). At the elongation stage, the TaCM mRNA levels in C6001 and H4564 were similar high. At the heading and milky stages, the TaCM +mRNA levels in H4564 stem still remained very high, while these mRNA levels declined very markedly in C6001. + +Quantification of the TaCM1 mRNA levels with a Phosphor Image with 18S rRNA normalization confirmed these results. By contrast to the stem tissues, the TaCM mRNA levels in leaf tissues of the two wheat cultivars did not show much difference at different developmental stages (data not shown). + +COMT enzyme activity at different developmental stages in wheat stems COMT enzyme activity was analysed in the basal second internodes at different developmental stages of the wheat stem (Table 1). The results showed that the COMT activity increased continuously from the elongation to the milky stage, consistent with active lignin synthesis during stem development in wheat. COMT activity showed little difference between C6001 and H4564 at the elongation stage. + +However, COMT activity in H4564 was much higher than that in C6001 at the heading and milky stages, more than 32.5% and 45.2%, respectively. + +TaCM enzyme protein at different developmental stages in wheat stems TaCM protein levels in the basal second internodes of + +![3_image_0.png](3_image_0.png) + +wheat stems were determined by protein gel blot analysis by using alfalfa monospecific polyclonal antisera against COMT (Fig. 3). A single band was detected from wheat stem samples; indicating that only one COMT protein isozyme was present in the stem tissue of wheat. This is in accordance with the genetic mapping results (Fig. 4), +suggesting that TaCM exists as a single-copy gene in the wheat genome. The immunological reactivity of COMT protein from wheat stem extracts was similar to that of recombinant TaCM protein expressed in E. coli. This further supports a single COMT protein relating to the TaCM gene is present in stem tissues of wheat. + +The TaCM protein levels increased with the maturity of the wheat stem tissues, from the elongation to the milky + +![3_image_1.png](3_image_1.png) + +![4_image_0.png](4_image_0.png) + +stages. By comparison between H4564 and C6001, the TaCM protein levels were similar at the elongation stage. + +However, these levels were higher in H4564 than in C6001 at the heading and milky stages (Fig. 3). This is in good agreement with the results of COMT activity. + +## Chromosome Location Of Tacm + +Chromosome location of the TaCM gene was investigated by genetic mapping. Southern analysis with TaCM cDNA +inserts produced two bands with EcoRI digestion. Nullitetrasomic analyses localized TaCM to group-3 chromosmes. Linkage mapping using ITMI mapping population indicated that the TaCM locus was mapped to the proximal region of the distal region of chromosome 3B at a position between markers Xtam63b and Xmwg11a (Fig. 4). These results suggest that TaCM is present in a single locus in the wheat genome B, consistent with the data from RT-PCR, +Northern hybridization, and protein gel blot analyses. + +## Discussion + +Lodging resistance in crops represents a quantitative trait that is suggested to be involved in the actions of many genes and is difficult to assess on a phenotypic basis (Keller et al., 1999; Berry et al., 2004). Therefore, to link the actions of a single gene directly to the lodging resistant phenotype will not only be very useful in elucidating this complex agronomic trait but also helpful in developing lodgingresistant cultivars in crop-breeding programmes. + +In theory, lignin determines the physical strength of plants, as a lower lignin content causes stems to be soft and brittle. Some mutants called bm were obtained which were shown to be associated with low lignin quantity and low stem strength. The first bm mutant was described in maize as early as 1924. Based on morphological and genetic analyses, four mutants named bm1, bm2, bm3, and bm4 + +![4_image_1.png](4_image_1.png) + +were described in maize (Cherney et al., 1991; Barrie`re et al., 2004). These mutations were found to be located on chromosomes 1, 4, 5, and 9, respectively (Kuc et al., 1968). + +## 2768 | Ma + +However, until recently, the bm3 mutation was demonstrated to occur in the gene encoding COMT (Vignols et al., +1995). Since the COMT protein is encoded by a single gene in maize, the results indicated that the bm3 mutation had arisen from a retrotransposon event or a deletion of part of the COMT gene that had been cloned previously (Collazo et al., 1992). As the maize COMT gene is known to be strongly expressed in roots and to a lesser extent in the stems and leaves, the reason why this mutation leads to the visible phenotype in stem and leaf tissues is still questionable. Another mutant, bm1, was demonstrated to be closely associated with, if not identical to, the gene encoding CAD +(Halpin et al., 1998). CAD activity was reduced in bm1 mutant plants but the strong reduction was observed in root +(90–97% reduction) and leaf midrib (70% reduction) tissues, not in stem tissues. Furthermore, two bands were detected in Western and Northern blot hybridization although Southern hybridization suggested that there was only a single CAD gene in the maize genome. Clearly, the issue of lignin synthesis with plant tissue strength and lodging resistance, particularly in stems, still needs intensive investigation. + +The natural mutation for lignin synthesis in wheat is still lacking. For this reason, it was hoped to use another approach to explore the relationship between lignin synthesis and the wheat stem lodging-resistant phenotype. A +COMT cDNA was cloned from wheat, namely TaCM. Its biochemical identification as an authentic COMT enzyme involved in lignin biosynthesis was confirmed by an analysis of enzyme activity and a transgenic approach (Ma and Xu, 2008). Genetic mapping indicated that the TaCM gene was present as a single locus in the wheat genome (Fig. 4). In contrast to maize, the wheat TaCM gene was actively expressed in stem tissues, suggesting that it played a role in lignin biosynthesis and stem development (Fig. 1). The TaCM gene expression was kept fairly high from the elongation to the milky stages in the basal second internodes of the lodging-resistant cultivar (H4564), but it declined markedly at the heading and milky stages in the lodging-sensitive cultivar (C6001) (Fig. 2). In agreement with these results, TaCM protein levels (Fig. 3) and COMT activity (Table 1) exhibited the significant differences between H4564 and C6001 at the heading and milky stages. + +The TaCM gene exists as a single copy in the wheat genome. Furthermore, only a single band was detected in wheat stem tissues using an anti-alfalfa COMT antibody, and this band exhibited a similar immunological reaction with the recombinant TaCM protein that was expressed in E. coli. These results suggest that the COMT protein and activity detected in wheat basal second internodes is likely to corresponding to the TaCM gene. Therefore, high TaCM +gene expression will lead the more TaCM protein and enzyme activity, which results in more lignin accumulation in the basal second internodes of the H4564 cultivar and, subsequently, makes these internodes have strong strength and exhibit high lodging resistance. + +The comparison of the different parameters between C6001 to H4564 cultivars revealed that stem strength and lodging index showed good consistency. The difference in the lignin content between two cultivars was in proportion to that of stem strength and lodging index, and this difference was remarkable at the heading stage rather than at the milky stage. The total increase in lignin content from C6001 to H4564 was a little lower than that of the stem strength and lodging index, which may reflect that a small increase in lignin content will result in a high increase in stem strength and lodging resistance. TaCM enzyme activity, however, exhibited a slightly different trend with the other three parameters. In one aspect, TaCM activity showed more increase at the milky stage than at the heading stage. In another aspect, the total increase of TaCM activity from C6001 to H4564 was less than these other parameters, for example, a 32.5% increase in TaCM activity versus 95.65%, 78.6%, and 66.3% in stem strength, lodging index, and lignin content, respectively, at the heading stage. This reflects that other genes may also be involved in lignin biosynthesis and contribute to the stem strength and lodging resistance. In fact, of two CCR genes that have been analysed in wheat, CCR1 has also been shown to contribute to lignin biosynthesis and stem strength (Ma and Tian, 2005; Ma, 2007). What and how CCR1 and TaCM genes influence this process is an interesting issue and will warrant further investigation. It suggests that different genes will together regulate the whole lignin biosynthesis pathway, and then lead to lignin accumulation in certain tissues at the proper developmental time, subsequently making the stem have high strength and a lodging-resistant phenotype. TaCM will be a regulating point or marker for what takes place in the whole lignin biosynthesis pathway. + +In general, lignin or cellulose in the cell wall determines the physical strength of plant tissues, particular for lignin. + +Cellulose has good tensile strength, but it is soft and flexible in nature, while lignin is much like a polyether network, with a large molecular weight, random structure, and an enormous capacity to cross-link with cellulose together with the process of thickening plant secondary walls, which makes the plant tissue's mechanical intensity and supportable ability. In practice, however, the roles of lignin and cellulose in crop lodging resistance are not consistent. In maize, evidence from bm mutants have shown that lignin is involved in lodging resistance (Vignols et al., 1995; Halpin et al., 1998). In rice, the brittle culm1 (bc1) mutant, which altered the expression of a COBRA-like protein, causes a reduction in cell wall thickness and mechanical strength. + +The bc1 mutant, however, exhibits a reduction in cellulose content but an increase in lignin level, suggesting that cellulose instead of lignin plays an important role in the cell wall mechanical strength (Li et al., 2003). By contrast, the maize brittle stalk2 (bk2) mutant, which is involved in a COBRA-like protein similar to rice bc1, exhibits a similar cellulose content as the wild type in developing stems and a loss of staining lignin in the vascular bundles and sclereid layers (Sindhu et al., 2007). Furthermore, two loci have been identified that are responsible for the higher starch accumulation and, consequently, for the higher lodging resistance during typhoons in the lower and upper rice stems, respectively, which show that a high accumulation of starch in the stem instead of structural carbohydrates such as lignin and cellulose, plays the vital role for lodging resistance in rice (Kashiwagi and Ishimaru, 2004; Kashiwagi et al., 2006; Ishimaru et al., 2008). The different roles of lignin in lodging resistance between rice versus maize and wheat are unclear at present. It is possible that the aquatic versus the terrestrial environments that are used to cultivate these crops may affect the contribution of lignin and cellulose to mechanical strength in crop stems. + +Analysis of quantitative trait loci (QTLs) can reveal the genetic basis of relationships among traits, this is particularly important for comprehensive understanding of complex traits such as lodging (Cuthbert et al., 2008). + +Comparisons among QTLs for lodging traits have been made in several crop plants. In barley, one QTL has been associated with grain yield, plant height, and lodging severity (Spaner et al., 1999). Two loci on chromosome 5, namely prl5 and lrt5, have been identified for the improved lodging resistance of the lower and upper stems, respectively, in rice (Kashiwagi and Ishimaru, 2004; Ishimaru et al., 2008). In addition, multiple QTLs for stem diameter are required for the best pushing resistance but only a single QTL (namely sdm8) has been identified to contribute to thicker stems towards lodging resistance in rice (Kashiwagi et al., 2008). Nine lodging resistant QTLs have been identified, but five of them have been shown to be coincident with QTLs for plant height in a population between wheat and spelt (Keller et al., 1999). In another report using the doubled haploid population derived from two Canadian wheat cultivars, three QTLs have been identified for lodging resistance in chromosome 4D, 5A, +and 6D in wheat. One of three QTLs for lodging coincides with a plant height QTL which is located on chromosome 4DS. Only QTL QLdg.crc-5A detected in this report is in a similar position to a QTL for lodging on chromosome 5AL identified by Keller et al. (1999). This QTL is also associated with a yield QTL (Huang et al., 2006). Using a population of 132 F12 RILs derived by single-seed descent from a cross between the Chinese facultative wheat Ning7840 and the US soft red winter wheat Clark, three QTLs for lodging score were identified in linkage groups 1B, 4AL, and 5A (Marza et al., 2006). The QTL in 5A is in the similar location as QTL of lodging resistance reported by Keller et al. (1999) and Huang et al. (2006). Using a population of 96 doubled haploid lines from the cross 'Milan'3'Catbird' wheat, QTL for lodging and associated traits were found on chromosomes IB, ID, 2B, 2D, 4B, 4D, +6D, and 7D (Verma et al., 2005). A doubled-haploid population was generated from the cross RL44523'AC Domain' spring wheat, and three QTLs were identified to control plant lodging in chromosomes 4B, 4D, and 3D, +respectively (McCartney et al., 2005). A doubled-haploid population was derived from anther culture of the wheat cross CA9613 and H1488 and stem strength and related basal internode traits were measured at the milk stage in this population. A total of six QTLs for stem strength, culm wall thickness, pith diameter, and stem diameter were identified on chromosomes 1A, 2D, 3A, and 3B, respectively (Hai et al., 2005). These data indicate that multiple genes are involved in lodging resistance, consistent with physiological analysis. As there is low resolution power of QTL experiments, it is difficult from the above data to detect the real number of genes that regulate the lodging resistant trait. The TaCM gene identified is the first genetic locus with a clear biochemical function that links with lodging phenotype in wheat. It is interesting that the TaCM +locus is located on chromosome 3B, which is not overlapped by any other lodging QTLs that have been reported previously. Chromosome 3B is also mentioned as one of the locations of a stem solidness gene (Lanning et al., 2006) and two QTLs for stem strength and diameter (Hai et al., 2005), although they do not overlap with the TaCM locus. + +Therefore, TaCM plus stem strength, diameter, and solidness loci in chromosome 3B will be useful for the rapid development of lodging-resistant genotypes using a markerassisted selection approach. It would be an interesting further experiment to test the relationship among these loci and whether they co-segregates with a stem lodging phenotype. + +In conclusion, a COMT gene, namely TaCM, was characterized from wheat. Its mRNA levels were examined in lodging-resistant (H4564) and lodging-sensitive (C6001) +cultivars in the basal second internodes at different stem developmental stages. TaCM mRNA levels in H4564 remained constantly high from the elongation to the milky stages, while they declined markedly in C6001 at the heading and milky stages. TaCM protein levels, determined by protein gel blot, were higher in H4564 than that in C6001 at the heading and milky stages. COMT activity was also 33–45% higher in H4564 than that in C6001 at the heading and milky stages. These corresponded to the higher Klason lignin content (about 42–66%), higher stem strength +(about 78–95%), and lower lodging index (about 70–80%) in H4564 than that in C6001 during these developmental stages. This suggests that the regulation on TaCM gene expression is involved in lignin synthesis and subsequently contributes to stem strength and the lodging-resistant trait in wheat C6001 and H4564 cultivars. The TaCM locus was genetically mapped to chromosome 3B which would be useful for the rapid development of lodging-resistant genotypes using a marker-assisted selection approach. + +## Acknowledgements + +This work was supported by grants from the National High Technology and Research Development Program of PR +China ('863' programme, No. 2007AA10Z101), the Chinese National Special Foundation for Transgenic Plant Research and Commercialization (No. 2008ZX08002-003), the National Natural Science Foundation of China (No. 30671134, No. 30671043, No. 30570133), the Natural Science Foundation of Beijing (No. 6082018), and the Innovation Project of the Chinese Academy of Sciences. The author thanks Dr Richard Dixon of the Noble Foundation (USA) for providing the anti-alfalfa COMT serum. + +## References + +Barrie`re Y, Ralph J, Me´ china V, Guillaumie S, Grabber JH, +Argillier O, Chabbert B, Lapierre C. 2004. Genetic and molecular basis of grass cell-wall degradability. II. Lessons from brown-midrib mutants. Comptes Rendus Biologies 327, 847–860. + +Berry PM, Sterling M, Spink JH, Baker CJ, Sylvester-Bradley R, +Mooney SJ, Tams AR, Ennos AR. 2004. Understanding and reducing lodging in cereals. Advances in Agronomy 84, 217–271. + +Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein– +dye binding. Analytical Biochemistry 72, 248–254. + +Boerjan W, Ralph J, Baucher M. 2003. Lignin biosynthesis. Annual Review of Plant Biology 54, 519–546. + +Cherney JH, Cherney DJR, Akin DE, Axtell JD. 1991. Potential of brown-midrib, low-lignin mutants for improving forage quality. Advances in Agronomy 46, 157–198. + +Collazo P, Montoliu L, Puigdomenech P, Rigau J. 1992. Structure and expression of the lignin O-methyltransferase gene from Zea mays L. Plant Molecular Biology 20, 857–867. Cuthbert JL, Somers DJ, Brule-Babel AL, Brown PD, Crow GH. 2008. Molecular mapping of quantitative trait loci for yield and yield components in spring wheat (Triticum aestivum L.). Theoretical and Applied Genetics 117, 595–608. + +Dong Q, Wang AP, Liang SM. 2003. Study on the architectural characteristics of wheat stalks. Journal of Shanxi Agricultural University 2003, 188–191. Faris JD, Haen KM, Gill BS. 2000. Saturation mapping of a gene rich recombinant hot spot region in wheat. Genetics 154, 823–835. + +Flintham JE, Borner A, Worland AJ, Gale MD. 1997. Optimizing wheat grain yield: effects of Rht (gibberellin-insensitive) dwarfing genes. Journal of Agricultural Science, Cambridge 128, 11–25. Geng S, Hills FG. 1989. Biometrics in agriculture science. Dubuque, Iowa: Kendell Hunt Publisher. Hai L, Guo HJ, Xiao SH, Jiang GL, Zhang XY, Yan CS, Xin ZY, Jia JZ. 2005. Quantitative trait loci (QTL) of stem strength and related traits in a doubled-haploid population of wheat (Triticum aestivum L.). Euphytica 141, 1–9. + +Halpin C, Holt K, Chojecki J, Oliver D, Chabbert B, Monties B, +Edwards K, Barakats AA, Foxon GA. 1998. Brown-midrib maize (bm1): a mutation affecting the cinnamyl alcohol dehydrogenase gene. The Plant Journal 14, 545–553. Huang XQ, Cloutier S, Lycar L, Radovanovic N, Humphreys DG, Noll JS, Somers DJ, Brown PD. 2006. Molecular detection of QTLs for agronomic and quality traits in a doubled haploid population derived from two Canadian wheats (Triticum aestivum L.). Theoretical and Applied Genetics 113, 753–766. + +Ishimaru K, Togawa E, Ookawa T, Kashiwagi T, Madoka Y, +Hirotsu N. 2008. New target for rice lodging resistance and its effect in a typhoon. Planta 227, 601–609. + +Kashiwagi T, Ishimaru K. 2004. Identification and functional analysis of a locus for improvement of lodging resistance in rice. Plant Physiology 134, 676–683. Kashiwagi T, Madoka Y, Hirotsu N, Ishimaru K. 2006. Locus prl5 improves lodging resistance of rice by delaying senescence and increasing carbohydrate reaccumulation. Plant Physiology and Biochemistry 44, 152–157. + +Kashiwagi T, Togawa E, Hirotsu N, Ishimaru K. 2008. Improvement of lodging resistance with QTLs for stem diameter in rice (Oryza sativa L.). Theoretical and Applied Genetics 117, 749–757. Keller C, Karutz M, Schmid JE, Stamp P, Winzeler M, Keller B, Messmer MM. 1999. Quantitative trait loci for lodging resistance in a segregating wheat x spelt population. Theoretical and Applied Genetics 98, 1171–1182. + +Kersey R, Inoue K, Schubert KR, Dixon RA. 1999. Immunolocalization of two lignin O-methyltransferases in stems of alfalfa (Medicago sativa L.). Protoplasma 209, 46–57. + +Kinoshita T. 1998. Linkage mapping using mutant genes in rice. Rice Genetics Newsletter 15, 13–74. + +Kirk TK, Obst JR. 1988. Lignin determination. Methods in Enzymology 161, 87–101. + +Kokubo A, Sakuri N, Kuraishi S, Takeda K. 1991. Culm brittleness of barley (Hordeum vulgare L.) mutants is caused by smaller number of cellulose molecules in cell wall. Plant Physiology 97, 689–701. + +Kuc J, Nelson OE, Flanagan P. 1968. Degradation of abnormal lignins in the brown-midrib mutants and double mutants of maize. Phytochemistry 7, 1435–1436. Lanning SP, Fox P, Elser J, Martin JM, Blake NK, Talbert LE. + +2006. Microsatellite markers associated with a secondary stem solidness locus in wheat. Crop Science 46, 1701–1703. Li YH, Qian Q, Zhou YH, et al. 2003. BRITTLE CULM1, which encodes a COBRA-like protein, affects the mechanical properties of rice plants. The Plant Cell 15, 2020–2031. Ma QH. 2007. Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat. Journal of Experimental Botany 58, 2011–2021. + +Ma QH, Tian B. 2005. Biochemical characterization of a cinnamoylCoA reductase from wheat. Biological Chemistry 386, 553–560. Ma QH, Xu Y. 2008. Characterization of a caffeic acid 3-Omethyltransferase from wheat and its function in lignin biosynthesis. + +Biochimie 90, 515–524. + +Ma QH, Xu Y, Lin ZB, He P. 2002. Cloning of cDNA encoding COMT from wheat which is differentially expressed in lodging-sensitive and +-resistant cultivars. Journal of Experimental Botany 53, 2281–2282. + +Marino CL, Nelson JC, Lu YH, Sorrells ME, Leroy P, Tuleen NA, +Lopes CR, Hart GE. 1996. Molecular genetic maps of the group 6 chromosomes of hexaploid wheat (Triticum aestivum L. em Thell). + +Genome 39, 359–366. + +Marza F, Bai GH, Carver BF, Zhou WC. 2006. Quantitative trait loci for yield and related traits in the wheat population Ning78403Clark. Theoretical and Applied Genetics 112, 688–698. McCartney CA, Somers DJ, Humphreys DG, Lukow O, Ames N, Noll J, Cloutier S, McCallum BD. 2005. Mapping quantitative trait loci controlling agronomic traits in the spring wheat cross RL44523'AC Domain'. Genome 48, 870–883. Ni W, Sewalt VJH, Korth KL, Blount JW, Balance GM, Dixon RA. + +1996. Stress responses in alfalfa (Medicago sativa L.). XXI. Activation of caffeic acid 3-O-methyltransferase and caffeonyl CoA 3-O-methyltransferase genes does not contribute changes in metabolite accumulation in elicitor-treated cell suspension cultures. Plant Physiology 112, 717–729. Parvathi K, Chen F, Guo DJ, Blount JW, Dixon RA. 2001. Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols. The Plant Journal 25, 193–202. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Sindhu A, Langewisch T, Olek A, Multani DS, McCann MC, Vermerris W, Carpita NC, Johal G. 2007. Maize brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity. Plant Physiology 145, 1444–1459. + +Spaner D, Rossnagel BG, Legge WG, et al. 1999. Verification of a quantitative trait locus affecting agronomic traits in two-row barley. + +Crop Science 39, 248–252. + +Sterling M, Baker CJ, Berry PM, Wade A. 2003. An experimental investigation of the lodging of wheat. Agricultural and Forest Meteorology 119, 149–165. + +Verma V, Worland AJ, Sayers EJ, Fish L, Caligari PDS, Snape JW. 2005. Identification and characterization of quantitative trait loci related to lodging resistance and associated traits in bread wheat. Plant Breeding 124, 234–241. + +Vignols F, Rigau J, Torres MA, Capeliades M, Puigdomenech P. + +1995. The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase. The Plant Cell 7, 407–416. + +Wang Y, Li QQ. 1995. Evaluation method of stem lodging resistance in wheat. Acta Agriculturae Boreall Sinica 10, 84–88. + +Wu TY, Xie LQ, Yang XJ, Zhang CY, Chen RF. 2002. Study on the correlations between the components of the plant height and the yield and other traits of wheat. Journal of Agricultural University of Hebei 25, 10–18. + +Zadoks JC, Chang TT, Konzak CF. 1974. A decimal code for the growth stages of cereals. Weed Research 14, 415–421. + +Zuber U, Winzeler H, Messmer MM, Keller B, Schmid JE, +Stamp P. 1999. Morphological traits associated with lodging resistance of spring wheat (Triticum aestivum L.). Journal of Agronomy and Crop Science 182, 17–24. \ No newline at end of file diff --git a/medical/md/PMC2827795.md b/medical/md/PMC2827795.md new file mode 100644 index 0000000000000000000000000000000000000000..3b93e0e623f72ddfea41615f521737ff113689e2 --- /dev/null +++ b/medical/md/PMC2827795.md @@ -0,0 +1,205 @@ +# Impaired Ca2+-Handling In Hif-1Α+/− Mice As A Consequence Of Pressure Overload + +Monique Silter & Harald Kögler & Anke Zieseniss & Jörg Wilting & Katrin Schäfer & +Karl Toischer & Adam G. Rokita & Gerhard Breves & Lars S. Maier & +Dörthe M. Katschinski Received: 28 July 2009 /Revised: 7 October 2009 /Accepted: 13 October 2009 / Published online: 8 November 2009 +\# The Author(s) 2009. This article is published with open access at Springerlink.com Abstract The hypoxia-inducible factor (HIF)-1 is critically involved in the cellular adaptation to a decrease in oxygen availability. The influence of HIF-1α for the development of cardiac hypertrophy and cardiac function that occurs in response to sustained pressure overload has been mainly attributed to a challenged cardiac angiogenesis and cardiac hypertrophy up to now. Hif-1α+/+ and Hif-1α+/− mice were studied regarding left ventricular hypertrophy and cardiac function after being subjected to transverse aortic constriction +(TAC). After TAC, both Hif-1α+/+ and Hif-1α+/− mice developed left ventricular hypertrophy with increased poste- +Electronic supplementary material The online version of this article +(doi:10.1007/s00424-009-0748-x) contains supplementary material, which is available to authorized users. + +M. Silter : A. Zieseniss: D. M. Katschinski (*) +Department of Cardiovascular Physiology, Heart Center Georg-August University Göttingen, Humboldtallee 23, 37073 Göttingen, Germany e-mail: katschinski@physiol.med.uni-goettingen.de H. Kögler : K. Schäfer : K. Toischer : A. G. Rokita : L. S. Maier Department of Cardiology and Pneumology, Heart Center Georg-August University Göttingen, Robert-Koch Str. 40, 37075 Göttingen, Germany J. Wilting Department of Anatomy and Cell Biology, Georg-August University Göttingen, Kreuzbergring 36, 37075 Göttingen, Germany G. Breves Department of Physiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15/102, 30173 Hannover, Germany rior wall thickness, septum thickness and increased left ventricular weight to a similar extent. No significant difference in cardiac vessel density was observed between Hif-1α+/+ and Hif-1α+/− mice. However, only the Hif-1α+/− +mice developed severe heart failure as revealed by a significantly reduced fractional shortening mostly due to increased end-systolic left ventricular diameter. On the single cell level this correlated with reduced myocyte shortenings, decreased intracellular Ca2+-transients and SR-Ca2+ content in myocytes of Hif-1a+/− mice. Thus, HIF-1α can be critically involved in the preservation of cardiac function after chronic pressure overload without affecting cardiac hypertrophy. This effect is mediated via HIF-dependent modulation of cardiac calcium handling and contractility. + +Keywords Hypoxia . Transverse aortic constriction . + +Cardiac hypertrophy . Hypoxia-inducible factor. Heart failure + +## Introduction + +An intact oxygen homeostasis in the heart is important not only for cardiac development but also for adaptation of the adult myocardium to a decrease in oxygen supply [18, 21]. + +Hypoxia affects cardiac vessel density and cell fate of cardiomyocytes [10]. Like in other tissues, hypoxia initiates a hypoxia-inducible gene expression programme, including genes regulating angiogenesis, anaerobic glycolysis, etc., in the heart [14]. The hypoxia-inducible factor (HIF)-1 is the transcriptional master regulator for these hypoxia-inducible genes [36]. At the molecular level, hypoxia is sensed by three oxygen-, iron- and 2-oxoglutarate-dependent prolyl-4hydroxylase domain (PHD) enzymes. The basis for the hypoxia-induced stability and activity of HIF-1 is the oxygen-dependent, PHD-mediated hydroxylation of distinct prolyl residues of the HIF-1α subunit [11, 12, 25]. + +Hydroxylated HIF-1α is rapidly ubiquitinated by the von Hippel–Lindau E3 ligase complex and degraded in the proteasome [26]. + +The ventricular geometry of the heart is a major determinant for myocardial oxygen consumption. Accordingly, myocardial oxygen consumption is proportional to ventricular wall tension, which is increased as a consequence of pressure overload [17]. Sustained pressure overload, which occurs for example in chronic arterial hypertension or aortic valve stenosis, initially causes left ventricular hypertrophy and subsequently heart failure. + +Regarding cardiac pathophysiology, the role of HIF-1α for cardiac adaptation to ischemia has been predominantly analyzed so far [5, 16]. Less is known, however, about the functional involvement of HIF-1α for the adaptation to sustained pressure overload, although increased protein levels of HIF-1α have been observed in heart samples from various models of pathologic cardiac hypertrophy [19]. + +Homozygous inactivation of the Hif1α gene causes embryonic lethality because of failed cardiac and vascular development, whereas heterozygous Hif-1α+/− mice develop normally. Partial HIF-1α deficiency in Hif-1α+/− mice results in an impaired response to continuous or intermittent systemic hypoxia including pulmonary hypertension [33, 38]. However, under resting conditions Hif-1α+/− mice do not demonstrate an appreciable cardiovascular phenotype. + +This is in contrast to HIF-1α cardiac-specific knockout mice (MCL2v-Cre x Hif-1α loxP mice), which present a reduction in cardiac vascularity already under resting conditions [31]. Cardiac vessel density is critically involved in the development of cardiac hypertrophy in chronic pressure overload. To gain insight if HIF-1α affects cardiac hypertrophy and function in response to chronic pressure overload independent from cardiac vascularity, Hif-1α+/− +mice were used in a model of transverse aortic constriction +(TAC). Ca2+ handling is critically involved in mechanical load-dependent cardiac function. Therefore, Hif-1α+/+ and Hif-1α+/− mice were analyzed regarding indicators for cardiac hypertrophy in vivo (posterior wall thickness +(PWT), septum thickness (ST), left ventricular weight +(LVW) and cardiac angiogenesis), heart failure (left ventricular endsystolic (LVESD) and enddiastolic diameter +(LVEDD), fractional shortening (FS)) and myocyte function in vitro (cell shortening and Ca2+-transients) after TAC. + +## Materials And Methods + +Animals and surgical intervention All protocols regarding animal experimentation were approved by the Niedersächsische Landesamt für Verbraucherschutz und Lebensmittelsicherheit (33.42502-105/06). + +Surgical intervention was performed with littermate mice that were either WT or heterozygous for the Hif-1α mutant allele in which exon 2 has been replaced with a neoR gene. + +Hif-1α+/− mice were kindly provided by R. Johnson +(University of California, USA) and are described in [30]. + +HIF-1α protein levels in the heart of the Hif-1α+/− mice were reduced by 43%±18.7% as determined by immunoblots. Pressure overload was induced by TAC in 12–14week-old female and 8–10-week-old male mice. Mice were anaesthetized by intraperitoneal injection of a mixture of xylazine and ketamine. The aorta was constricted with polyviolene non-absorbable braided nylon strings (5-0 USP) using blunted 25-gauge (male mice) and 26-gauge +(female mice) needles as placeholders that were removed after ligation. After aortic constriction, the chest was closed and mice were allowed to recover from anaesthesia. Shamoperated animals were anaesthetized and handled like the TAC-operated animals with the exception of the aortic constriction. The following number of animals were treated with TAC or sham and underwent follow up analysis by echocardiography for 11 weeks: TAC: male Hif-1α+/+ n=7, male Hif-1α+/− n=3, female Hif-1α+/+ n=5, female Hif1α+/− n=10; sham: male Hif-1α+/+ n=5, male Hif-1α+/− n= +6, female Hif-1α+/+ n=5, female Hif-1α+/− n=6. + +## Echocardiography + +Two-dimensional images and M-mode tracings were recorded from the parasternal long axis view at midpapillary level (Vevo 660™, VS-0 M-VE660 version 3.1, Visual Sonics). Heart rate, PWT, ST, LVESD and LVEDD were determined. FS of the left ventricle was defined as the LVEDD minus LVESD divided by the LVEDD. FS was used as marker for cardiac contractile function. + +## Immunofluorescence Analysis + +Hearts were fixed in 4% paraformaldehyde for 10–20 min. + +They were rinsed in phosphate-buffered saline (PBS), transferred to 5% and 15% sucrose in PBS, and embedded in Tissue Freeze Medium (Sakura Finetek Europe, NL). + +Cryosections of 16 μm thickness were prepared. Nonspecific binding of antibodies was blocked by incubation with 1% bovine serum albumin (BSA) for 1 h before incubation with anti-vascular endothelial growth factor receptor (VEGFR)-2-antibodies (rat-anti-mouse; BD Pharmingen, San Diego, USA; 1:100), or monoclonal rat antimouse CD31 antibodies (SantaCruz Biotechnology, Santa Cruz, USA; 1:50). The sections were incubated with the primary antibodies for 1 h or at 4 C overnight, respectively. After rinsing, secondary antibodies (Alexa 488- or 555conjugated goat anti-rat; Molecular Probes, Leiden, The Netherlands) were applied at 1:200 dilution for VEGFR-2 or 1:400 dilution for CD31 for 1 h. Cell nuclei were counterstained with DAPI. Then, sections were mounted under coverslips with Fluoromount-G (Southern Biotechnology Associates, Birmingham, GB). They were studied with epifluorescent microscope (Axio Imager Z1, Zeiss, Goettingen, Germany). Capillaries were counted in at least five different fields per mouse and quantified as capillaries/mm2. + +## Western Blot + +Heart tissue was rapidly homogenized in a buffer containing 4 M Urea, 140 mM Tris (pH 6.8), 1% SDS, 2% NP-40 and protease inhibitors (Roche). Protein concentrations were quantified (Bio-Rad, DC Protein Assay). For immunoblot analysis protein samples were resolved by SDS/ +PAGE and transferred onto nitrocellulose membranes +(Amersham Biosciences) by semi-dry blotting (PeqLab). + +Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase +(SERCA), HIF-1α, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected using mouse antiSERCA (Dianova), rabbit anti-HIF-1α (Novus) and rabbit anti-GAPDH (Cell Signalling) antibodies followed by a goat horseradish peroxidase (HRP)-labelled anti-mouse or antirabbit antibody (Santa Cruz Biotechnology). Chemiluminescence detection of HRP was performed by incubation of the membranes with 100 mM Tris/HCl (pH 8.5), 2.65 mM +H2O2, 0.45 mM luminol and 0.625 mM coumaric acid for 1 min at room temperature and analysed by imaging with a chemiluminescence camera (LAS3000; Fujifilm). + +Isolation of cardiomyocytes, cardiomyocyte shortening and intracellular Ca2+-measurements Isolation of adult mouse ventricular myocytes was carried out as described previously [23]. Briefly, hearts were excised from mice that were anaesthetized in a gas chamber with isoflurane. Hearts were mounted on a Langendorffperfusion apparatus driven by gravity and perfused with nominally Ca2+-free Tyrode's solution containing (in millimolar) NaCl, 115; KCl, 4.7; KH2PO4, 0.6; Na2HPO4; MgSO4, 1.2; NaHCO3, 12; KHCO3, 10; HEPES, 10; taurine, 30; 2,3-butanedionemonoxime, 10; glucose, 5.5 +(pH 7.46) for 2–4 min at 37 C. Perfusion was then switched to the same solution containing Liberase blendzyme 1 (Roche) 0.25 mg/ml and Trypsin 0.14 mg/ml with perfusion continuing until the heart became flaccid (7–12 min). + +Ventricular tissue was removed, dispersed, filtered, and suspensions were rinsed several times. After Ca2+ reintroduction (stepwise increase to 0.8 mM), isolated myocytes were then plated onto superfusion chambers, with the glass bottoms treated with laminin to allow cell adhesion and used for immediate measurements. + +Shortening and [Ca]i measurements were performed simultaneously and performed as reported previously [23] +using a fluorescence detection system (IonOptix Corp., Milton, MA). Myocytes were loaded with fluo-3 by incubation with 10 μM of the acetoxymethyl ester (AM) +form of the dye (Molecular Probes, Eugene, OR) for 20 min at room temperature in darkness. The dye was excited with a wavelength at 480±15 nm using a 75 W xenon arc lamp +(Ushio, Japan) on the stage of a Nikon Eclipse TE200-U +inverted microscope. Emitted fluorescence was measured using a photomultiplier (at 535±20 nm; IonOptix Corp., +Milton, MA). From the raw fluorescence, ΔF/F0 was calculated by dividing through the baseline fluorescence +(F0), after subtraction of the background fluorescence. + +Myocytes were field-stimulated (voltage 25% above threshold) at 1, 2 and 4 Hz and 37 C until steady-state was achieved, and only those cells exhibiting stable steady-state contractions were included in the study. To measure myocyte shortening, cells were simultaneously transilluminated by red light (>650 nm, to avoid interference with fluorescence measurements), and shortening was measured using a sarcomere length detection system (IonOptix Corp., +Milton, MA). In case of determining SR Ca2+ content, cells were stimulated with 10 mM caffeine. + +## Statistical Analysis + +Data are shown as mean ± SEM. Multiple comparison was performed by two-way ANOVA analysis followed by the Bonferroni test for comparisons of means. In case of comparing the progress of cardiac hypertrophy (PWT and ST) and cardiac function (FS, LVEDD and LVESD) overtime paired t tests were performed. Values of p<0.05 were considered to be statistically significant. + +## Results + +No difference in angiogenesis between Hif-1α+/+ +and Hif-1α+/− mice following sustained pressure overload The vascular endothelial growth factor is a HIF-1 target gene and represents one of the major growth factors in reference to angiogenesis [24]. The spatio-temporal expression of its tyrosine kinase receptor VEGFR-2 in endothelial cells is indicative of an angiogenetic process. In cardiacspecific HIF-1α knockout mice, an influence of HIF-1α on cardiac vessel density after TAC has been described before +[31]. To analyze if a partial deletion of the HIF-1α expression affects cardiac angiogenesis following sustained pressure overload, TAC intervention was performed in Hif1α+/+ and Hif-1α+/− female and male mice. Subsequently, VEGRF-2 expression in Hif-1α+/+ and Hif-1α+/− mice was analyzed (Fig. 1a). The VEGFR-2 expression in Hif-1α+/+ +versus Hif-1α+/− mice did not show any apparent differences, indicating that angiogenesis was not impaired in the heterozygous mice. This result was substantiated by the fact that vessel density as measured by CD31 staining did not differ comparing Hif-1α+/+ and Hif-1α+/− mice (Fig. 1b and c). + +No difference in left ventricular hypertrophy in Hif-1α+/+ +and Hif-1α+/− mice in response to sustained pressure overload It has recently been hypothesized, that cardiac vessel density and cardiac hypertrophy are mutually related [13, 35]. Hif-1α+/+ and Hif-1α+/− mice developed cardiac hypertrophy to a similar extent after TAC. This finding is in line with the above-demonstrated data, in which we could not detect a difference in cardiac vessel density comparing TAC-treated Hif-1α+/+ with TAC-treated Hif1α+/− mice. Left ventricular hypertrophy was indicated by significantly increased PWT and ST in TAC-treated female and male mice (Fig. 2a and b, respectively). In shamoperated animals, no significant differences in PWT or ST +compared with pre-treatment values were observed (Suppl Fig. 1a and b). Additionally, post-mortem LVW and total heart weights (HW) were determined as indicators for cardiac hypertrophy (Fig. 3). In Hif-1α+/+ and Hif-1α+/− +mice increased LVW/HW-ratios after TAC were observed. In line with the data obtained in the echocardiography analyses, there were no significant differences comparing the LVW/HW-ratios of Hif-1α+/+ and Hif-1α+/− mice. + +Impaired cardiac function in Hif-1α+/− mice as a consequence of sustained pressure overload In contrast to cardiac hypertrophy, there was an impact of HIF-1α on heart function after TAC as indicated by the FS +of Hif-1α+/+ and Hif-1α+/− mice (Fig. 4). In Hif-1α+/+ mice, the FS did not significantly change over the course of eleven weeks after TAC compared with pre-treatment values. In Hif-1α+/− mice, however, FS was significantly reduced starting from 8 or 3 weeks after TAC in female +(Fig. 4a) and male (Fig. 4b) mice, respectively, while reduced FS was mainly due to a significantly increased LVESD. The TAC-induced heart failure in the Hif-1α +/− +mice was further substantiated by the finding that lung weights were significantly increased 15 weeks after TAC +intervention in the Hif-1α+/− compared with Hif-1α+/+ mice (Hif-1α+/+ mice: 0.15 g±0.008; Hif-1α+/− mice: 0.18 g± +0.011, p<0.05). Since cardiac hypertrophy was similar in Hif-1α+/+ and Hif-1α+/− mice, it has to be assumed that the development of heart failure in the Hif-1α+/− mice occurred independent of cardiac hypertrophy. In sham-treated female and male mice, no significant difference in + +![3_image_0.png](3_image_0.png) + +![4_image_0.png](4_image_0.png) + +LVEDD, LVESD and FS was observed (Suppl Fig. 2a and b). + +Impaired myocyte contractility and Ca2+ handling in cardiomyocytes of Hif-1α+/− mice Intact Ca2+ handling is critically important for heart function under resting conditions as well as during adaptation towards sustained pressure overload [20]. Therefore, we determined mechanical performance and cytosolic Ca2+ dynamics of isolated cardiomyocytes. The reduced contractility observed as a consequence of pressure overload in the Hif-1α+/− mice by echocardiography was also detectable at the cellular level. The contractile behaviour of Hif-1α+/− cardiomyocytes, isolated 3 weeks after TAC from male mice, displayed a reduction in myocyte shortening compared with cardiomyocytes isolated from Hif-1α+/+ mice (Fig. 5a). This correlated with abnormalities in Ca2+- +transients of the Hif-1α+/− cells. Hif-1α+/− cardiomyocytes isolated from TAC-treated mice demonstrated significantly reduced intracellular Ca2+-transients when stimulated with 1–4 Hz (Fig. 5b). Twitch relaxation and [Ca2+]i decline were significantly impaired and thus slower in cardiomyocytes isolated from TAC- versus sham-treated mice. + +However, there was no difference comparing cardiomyocytes isolated from Hif-1α+/+ or Hif-1α+/− mice (Fig. 5c and d). + +During excitation–contraction coupling, the Ca2+ entry via voltage dependent L-type Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (Ca2+-induced Ca2+ release), which then leads to activation of the myofilaments and contraction of the cell. + +Alterations in the amplitude of the Ca2+ transients and of the Fig. 3 HIF-1α does not affect + +![4_image_1.png](4_image_1.png) + +the left ventricular hypertrophy after transverse aortic constriction +(TAC). Male (a) and female (b) +Hif-1α+/+ and Hif-1α+/- mice underwent TAC or shamintervention. Fifteen weeks after the intervention, the hearts were excised and left ventricular weights (LVW) as well as total heart weights (HW) were determined + +![5_image_0.png](5_image_0.png) + +![5_image_1.png](5_image_1.png) + +subsequent contraction may result from changes in the SR +Ca2+ content. In line with the decreased Ca2+ transients SR +Ca2+ content was indeed significantly decreased in cardiomyocytes isolated of TAC-treated versus sham-treated HIF-1α+/− mice, whereas no significant difference was observed in cardiomyocytes isolated of TAC-treated versus sham-treated HIF-1α+/+ mice (Fig. 5e). The SERCA is the major pump, which is responsible for Ca2+ reuptake into the SR. SERCA protein levels, however, did not differ comparing HIF-1α+/+ and HIF-1α+/− mice (Fig. 5f). Taken collectively, HIF-1α+/− mice demonstrated significantly diminished Ca2+ +transients and fractional shortenings after TAC treatment most likely due to a decrease in SR Ca2+ content. + +## Discussion + +HIF-1 has been assigned an important role in coordinating gene expression with cardiac tissue oxygen tensions [32]. A +decrease in the oxygen availability is apparent during the course of ischemic heart disease. Under these hypoxic conditions HIF-1 plays an important role in the cellular adaptation of cardiomyocytes [22]. Examples of HIF-1mediated adaptations include increased expression of VEGF to promote angiogenesis, glucose transporter 1 to enhance glucose uptake and glycolytic enzymes to facilitate glucose metabolism. Patients with HIF1A point mutations +(HIF1A.2 or HIF1A.50, which result in both cases in HIF- +1α proteins with a diminished transactivation activity) +develop less coronary artery collaterals compared with patients with the wild type protein [8, 28]. A protective role of HIF-1α in the ischemic adult heart was further demonstrated in Hif-1α+/− mice. Hif-1α+/− animals have a reduction in the HIF-1α protein expression of ∼30–50%. + +Under resting conditions, they do not present an appreciable cardiovascular phenotype. However, in case of an acute ischemic stimulus they have a complete loss of ischemia pre-conditioning-induced cardioprotection [4]. Using the Hif-1α+/− mice in a TAC model, our data indicate that HIF1α likewise confers protection of cardiac tissue in case of sustained pressure overload. + +Hif-1α+/− mice developed a severe impairment of FS +after aortic constriction in sharp contrast to their respective wild type littermates. The PWT and ST after TAC, however, did not differ comparing Hif-1α+/− and Hif-1α+/+ mice. The impact of HIF-1α for cardiac hypertrophy is discussed controversially in the literature. In Tet-inducible cardiacspecific Hif-1α knockout mice mechanical load-induced cardiac hypertrophy is impaired, which correlates with a decreased cardiac vessel density [31]. Huang et al., however, demonstrated a mild hypertrophy with increased wall thickness and heart weight to body weight ratios in resting constitutive cardiac specific Hif-1α knockout mice despite of cardiac hypovascularization [10]. In the present study, we did not observe an impaired TAC-induced cardiac angiogenesis and hypertrophy comparing Hif-1α+/+ and + +![6_image_0.png](6_image_0.png) + +Hif-1α+/− mice. The discrepancies regarding the impact of HIF-1α on cardiac hypertrophy may rely on the onset and extent of the Hif-1α knockout in the different genetic mouse models used. + +Regarding the development of heart failure, female and male mice responded differently to the applied pressure overload in our study. Transition to heart failure developed 8–11 weeks after onset of pressure overload in female mice, whereas FS was decreased already 3 weeks after onset of pressure overload in male mice. A gender-dependent difference in the extent of heart failure as a consequence of mechanical load is in line with the literature [37]. Nevertheless, the data obtained in our study have to be analyzed with caution regarding gender effects, since female and male mice differed regarding the extent of the TAC applied and the age at which they were analyzed. + +Our results provide insights into the role of HIF-1α in the development of heart failure. Our findings link HIF-1α to an altered SR Ca2+ handling since we found a decreased SR Ca2+ content in the TAC-treated HIF-1α+/− mice. + +Abnormal SR Ca2+ uptake and release lead to depressed +[Ca2+]i, which eventually results in the contractile phenotype in heart failure. Altered Ca2+-handling in the Hif-1α+/− +mice thus could be contributing to the observed heart failure in chronic pressure overload. + +In our study, we found a decreased SR Ca2+ content in the Hif-1α+/− cardiomyocytes after TAC. In this regard, it is interesting to note that it has been previously reported that HIF-1α modulates Ca2+ signalling during T-cell receptor stimulation in thymocytes [27]. This has been attributed to an increased expression of SERCA in pVHL-depleted thymocytes, which could be reversed by inhibiting HIF- +1α stabilization. Up to now, the molecular link between HIF-1α and SERCA expression is not clear. However, it has been excluded that SERCA is a direct transcriptional target of HIF-1 [27]. In our loss of function mouse model, we could not observe a HIF-related difference in SERCA +protein levels, when comparing heart samples from Hif1α+/− and Hif-1α+/+ mice. SR-Ca2+ content is determined by Ca2+ release as well as SR Ca2+ reuptake. The reuptake is mainly due to SERCA function, which is highly regulated by for example post-translational modifications as well as its regulator protein phospholamban. Thus, the decrease in SR Ca2+ content after TAC in the Hif-1α+/- +mice may rely on an increased SR Ca2+ release or a diminished SERCA function. Taken together, it is tempting to speculate that HIF-1α plays a more general role in Ca2+ +handling, which affects adaptation of cardiomyocytes to various forms of mechanical stress. Nevertheless, further studies are needed, to define the molecular role of HIF-1α for cardiac Ca2+-handling and cardiomyocyte function in detail. + +HIF-1α can be stabilized by inhibiting the activity of the HIFα-regulating PHDs independent of the oxygen concentration [3, 6, 15]. Applying inhibitors of the PHDs has been demonstrated to induce tissue protection in case of ischemic diseases in the central nervous [1, 34], renal [2, 7], gastrointestinal [29] and cardiovascular system [9]. Recent reports describing the consequences of inhibiting the activity of PHDs in the heart have proven that HIF-1α is a central component for this protection [5]. These data imply that short-term elevation of HIF-1α levels in response to hypoxia or ischemia drives beneficial adaptive processes. + +The presented data indicate that HIF-1α has a likewise positive effect on the cardiac function in case of adaptation towards chronic pressure overload. Therefore, subsequent studies will aim to analyze, if stabilizing HIF-1α in the heart via inhibiting PHD activity will positively influence cardiac function in case of increased mechanical load. + +Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ka 1269/9-1 to DMK and HK and Klinische Forschergruppe 155 to LSM and KS). We acknowledge the excellent technical help from Sabine Krull. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. + +## References + +1. Baranova O, Miranda LF, Pichiule P, Dragatsis I, Johnson RS, +Chavez JC (2007) Neuron-specific inactivation of the hypoxia inducible factor-1α increases brain injury in a mouse model of transient focal cerebral ischemia. J Neurosci 27:6320–6332 2. Bernhardt WM et al (2006) Preconditional activation of hypoxiainducible factors ameliorates ischemic acute renal failure. J Am Soc Nephrol 17:1970–1978 3. Bernhardt WM, Warnecke C, Willam C, Tanaka T, Wiesener MS, +Eckardt KU (2007) Organ protection by hypoxia and hypoxiainducible factors. Methods Enzymol 435:221–245 4. Cai Z, Zhong H, Bosch-Marce M, Fox-Talbot K, Wang L, Wei C, +Trush MA, Semenza GL (2008) Complete loss of ischaemic preconditioning-induced cardioprotection in mice with partial deficiency of HIF-1α. Cardiovasc Res 77:463–470 5. Eckle T, Kohler D, Lehmann R, El Kasmi K, Eltzschig HK +(2008) Hypoxia-inducible factor-1 is central to cardioprotection: a new paradigm for ischemic preconditioning. Circulation 118:166–175 6. Fraisl P, Aragones J, Carmeliet P (2009) Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory disease. Nat Rev Drug Discov 8:139–152 7. Hill P, Shukla D, Tran MG, Aragones J, Cook HT, Carmeliet P, +Maxwell PH (2008) Inhibition of hypoxia inducible factor hydroxylases protects against renal ischemia-reperfusion injury. J +Am Soc Nephrol 19:39–46 8. Hlatky MA et al (2007) Polymorphisms in hypoxia inducible factor 1 and the initial clinical presentation of coronary disease. + +Am Heart J 154:1035–1042 9. Huang M et al (2008) Short hairpin RNA interference therapy for ischemic heart disease. Circulation 118:S226–S233 10. Huang Y, Hickey RP, Yeh JL, Liu D, Dadak A, Young LH, Johnson RS, Giordano FJ (2004) Cardiac myocyte-specific HIF-1α deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart. Faseb J 18:1138–1140 11. Ivan M et al (2001) HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292:464–468 12. Jaakkola P et al (2001) Targeting of HIF-α to the von Hippel– +Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472 13. Jiang Y et al (2007) Dietary copper supplementation reverses hypertrophic cardiomyopathy induced by chronic pressure overload in mice. J Exp Med 204:657–666 14. Kaelin WG Jr, Ratcliffe PJ (2008) Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 30:393–402 15. Katschinski DM (2009) In vivo functions of the prolyl-4hydroxylase domain oxygen sensors: direct route to the treatment of anaemia and the protection of ischaemic tissues. Acta Physiol +(Oxf) 195:407–414 16. Kido M, Du L, Sullivan CC, Li X, Deutsch R, Jamieson SW, +Thistlethwaite PA (2005) Hypoxia-inducible factor 1α reduces infarction and attenuates progression of cardiac dysfunction after myocardial infarction in the mouse. J Am Coll Cardiol 46:2116– +2124 17. Kissling G (1992) Mechanical determinants of myocardial oxygen consumption with special reference to external work and efficiency. Cardiovasc Res 26:886–892 18. Krishnan J, Ahuja P, Bodenmann S, Knapik D, Perriard E, Krek W, Perriard JC (2008) Essential role of developmentally activated hypoxia-inducible factor 1α for cardiac morphogenesis and function. Circ Res 103:1139–1146 19. Krishnan J et al (2009) Activation of a HIF1α-PPARγ axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy. Cell Metab 9:512–524 20. Lehnart SE, Maier LS, Hasenfuss G (2009) Abnormalities of calcium metabolism and myocardial contractility depression in the failing heart. Heart Fail Rev. doi:10.1007/s10741-009-9146-x 21. Liu H, Fisher SA (2008) Hypoxia-inducible transcription factor1α triggers an autocrine survival pathway during embryonic cardiac outflow tract remodeling. Circ Res 102:1331–1339 +22. Loor G, Schumacker PT (2008) Role of hypoxia-inducible factor in cell survival during myocardial ischemia-reperfusion. Cell Death Differ 15:686–690 23. Maier LS, Zhang T, Chen L, DeSantiago J, Brown JH, Bers DM +(2003) Transgenic CaMKIIdeltaC overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release. Circ Res 92:904–911 24. Marti HH (2005) Angiogenesis—a self-adapting principle in hypoxia. Exs 94:163–180 25. Masson N, Ratcliffe PJ (2003) HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O2 levels. + +J Cell Sci 116:3041–3049 26. Maxwell PH et al (1999) The tumour suppressor protein VHL +targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399:271–275 27. Neumann AK, Yang J, Biju MP, Joseph SK, Johnson RS, Haase VH, Freedman BD, Turka LA (2005) Hypoxia inducible factor 1 α regulates T cell receptor signal transduction. Proc Natl Acad Sci U S A 102:17071–17076 28. Resar JR et al (2005) Hypoxia-inducible factor 1α polymorphism and coronary collaterals in patients with ischemic heart disease. + +Chest 128:787–791 29. Robinson A, Keely S, Karhausen J, Gerich ME, Furuta GT, +Colgan SP (2008) Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition. Gastroenterology 134:145–155 30. Ryan HE, Lo J, Johnson RS (1998) HIF-1α is required for solid tumor formation and embryonic vascularization. EMBO J +17:3005–3015 31. Sano M et al (2007) p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature 446:444– +448 32. Semenza GL (2004) O2-regulated gene expression: transcriptional control of cardiorespiratory physiology by HIF-1. J Appl Physiol 96:1173–1177 33. Semenza GL (2006) Regulation of physiological responses to continuous and intermittent hypoxia by hypoxia-inducible factor 1. Exp Physiol 91:803–806 34. Siddiq A et al (2005) Hypoxia-inducible factor prolyl 4hydroxylase inhibition. A target for neuroprotection in the central nervous system. J Biol Chem 280:41732–41743 35. Tirziu D et al (2007) Myocardial hypertrophy in the absence of external stimuli is induced by angiogenesis in mice. J Clin Invest 117:3188–3197 36. Wenger RH, Stiehl DP, Camenisch G (2005) Integration of oxygen signaling at the consensus HRE. Sci STKE 2005:re12 37. Witt H et al (2008) Sex-specific pathways in early cardiac response to pressure overload in mice. J Mol Med 86:1013–1024 38. Yu AY et al (1999) Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1α. J Clin Invest 103:691–696 \ No newline at end of file diff --git a/medical/md/PMC3503469.md b/medical/md/PMC3503469.md new file mode 100644 index 0000000000000000000000000000000000000000..5f187901c5ab1bd7738b1860324b7050565ebe6a --- /dev/null +++ b/medical/md/PMC3503469.md @@ -0,0 +1,314 @@ +# Socioeconomic Inequalities In Childhood Exposure To Secondhand Smoke Before And After Smoke-Free Legislation In Three Uk Countries + +Graham F. Moore1, Dorothy Currie2, Gillian Gilmore3, Jo C. Holliday1, Laurence Moore1 1DECIPHer, School of Social Sciences, Cardiff University, 1-3 Museum Place, Cardiff, Wales CF10 3BD, UK +2Child and Adolescent Health Research Unit, School of Medicine, University of St Andrews, Medical and Biological Sciences Building, North Haugh, St Andrews, Fife KY16 9TF, UK 3Health Intelligence, Public Health Agency, 18 Ormeau Avenue, Belfast BT2 8HS, UK +Address correspondence to Graham F. Moore, E-mail: mooreg@cardiff.ac.uk + +## Abstract + +Background Secondhand smoke (SHS) exposure is higher among lower socioeconomic status (SES) children. Legislation restricting smoking in public places has been associated with reduced childhood SHS exposure and increased smoke-free homes. This paper examines socioeconomic patterning in these changes. Methods Repeated cross-sectional survey of 10 867 schoolchildren in 304 primary schools in Scotland, Wales and Northern Ireland. Children provided saliva for cotinine assay, completing questionnaires before and 12 months after legislation. Results SHS exposure was highest, and private smoking restrictions least frequently reported, among lower SES children. Proportions of saliva samples containing ,0.1 ng/ml (i.e. undetectable) cotinine increased from 31.0 to 41.0%. Although across the whole SES spectrum, there was no evidence of displacement of smoking into the home or increased SHS exposure, socioeconomic inequality in the likelihood of samples containing detectable levels of cotinine increased. Among children from the poorest families, 96.9% of post-legislation samples contained detectable cotinine, compared with 38.2% among the most affluent. Socioeconomic gradients at higher exposure levels remained unchanged. Among children from the poorest families, one in three samples contained .3 ng/ml cotinine. Smoking restrictions in homes and cars increased, although socioeconomic patterning remained. Conclusions Urgent action is needed to reduce inequalities in SHS exposure. Such action should include emphasis on reducing smoking in cars and homes. Keywords smoking, socioeconomic status, young people + +## Introduction + +Childhood secondhand smoke (SHS) exposure increases the risk of respiratory problems1,2 and arterial damage.3 Indeed, the World Health Organization (WHO) state that 'scientific evidence has unequivocally established that exposure to tobacco smoke causes death, disease and disability'.4 In all UK countries, childhood SHS exposure is highest amongst children from lower socioeconomic status (SES) families,5 - 8 representing a key mechanism in the intergenerational reproduction of health inequalities. + +In line with WHO calls for increased 'protection from exposure to tobacco smoke in indoor workplaces, public transport, indoor public places and, as appropriate, other public places',4 all UK countries have now implemented legislation prohibiting smoking in enclosed public places and workplaces (Scotland March 2006, Wales March 2007, Northern Ireland (NI) April 2007 and England July 2007). + +Graham F. Moore, Research Associate Dorothy Currie, Senior Statistician Gillian Gilmore, Health Intelligence Manager Jo C. Holliday, Research Fellow Laurence Moore, Professor of Public Health Improvement Legislation appears to be achieving its aim of protecting workers and the public from SHS.9,10 However, opponents hypothesized that it would displace smoking into the home, increasing childhood SHS exposure. Though displacement has been reported in Hong Kong11 and the USA,12 this hypothesis has found no support in UK studies.8,13 - 15 While substantial post-legislation declines in exposure have been limited to Scotland, in no country did exposure increase following legislation. + +However, although no longitudinal trend data are available elsewhere in the UK, Health Survey for England data indicate that childhood SHS exposure was declining prior to legislation, and doing so most rapidly amongst highly exposed groups (e.g. lower SES children), leading to a narrowing of inequalities.7 While there is no evidence that legislation has harmfully impacted children from lower SES families, evidence is accumulating that greater post-legislation declines in exposure have occurred amongst children at relatively low risk, potentially widening inequalities. In Wales and England, increased proportions of samples containing no detectable cotinine were accompanied by marginal overall reductions.14,15 Reductions in Scotland and Wales have been greatest amongst children of non-smokers;13,14 findings echoed internationally in a recent US study.16 In Scotland, while significant reductions were observed in all SES +groups, declines as a proportion of baseline exposure were lowest among children from low SES families.4 In Wales, proportions of samples with no detectable cotinine increased only amongst children from more affluent families.5,6 Recognition of the need to target children at risk from SHS has led to increased emphasis on private spaces, with the Department of Health17 aiming to encourage two-thirds of smoking parents to adopt smoke-free homes by 2020. + +Jarvis et al. + +15 suggest that rather than displacing smoking into the home, legislation has contributed to denormalizing smoking near children and growing support for reducing smoking in private spaces. Indeed, post-legislation increases in smoke-free homes have been reported among smoking and non-smoking parents in Scotland, and smoking parents in England.15,18 Although most research into private smoking restrictions focuses on homes, attention is increasingly turning to dangers of smoking in cars, as reflected in the British Medical Association's19 call to ban smoking in all vehicles. + +One study showed that adolescents regularly exposed to SHS in cars exhibited symptoms of nicotine dependence.20 Whilst not distinguishing between adult and child passengers, one study found that 29% of UK smokers smoked with non-smoking passengers present;21 lower than in Canada (34%) and the USA (44%), but identical to Australia. + +Given that smoking is higher among lower SES parents, with smoking restrictions in private spaces less common,18,22 encouraging uptake of smoke-free homes and cars may reduce socioeconomic inequalities. However, little attention has been paid to whether adoption of private smoking restrictions following legislation has been patterned by SES. Negotiation of such restrictions is perhaps easier for parents with fewer smokers in their social network.23 Given the higher prevalence of smoking amongst less affluent adults, denormalizing smoking near children may cause more rapid adoption of smoking restrictions amongst more affluent adults. Hence, patterning in uptake of private smoking restrictions represents a potential mechanism through which trends towards increased inequality following legislation may have occurred. + +This paper reports data from the Scottish, Welsh and Northern Irish studies of changes in Child Exposure to Environmental Tobacco Smoke (CHETS), repeated crosssectional studies examining associations of smoke-free legislation with children's SHS exposure.8,13,14 Through pooling data from three countries, it has enhanced power to examine socioeconomic patterning in SHS exposure and parental restrictions on smoking in private spaces before and after legislation. + +## Methods Sample + +Participants were 10 867 non-smokers (self-reported nonsmokers providing saliva samples containing ,15 ng/ml cotinine)24 in their final year at 304 primary schools in Scotland (n ¼ 111), Wales (n ¼ 71) and NI (n ¼ 122). + +## Measures + +Survey year The year of data collection ( pre- vs. post-legislation) was a proxy for smoke-free legislation. + +## Shs Exposure + +Salivary cotinine (a metabolite of nicotine) is a well-validated biomarker of SHS exposure in the previous 72 h.25 Anonymous samples were assayed using capillary gas chromatography with a detection limit of 0.1 ng/ml.26 + +## Socioeconomic Status + +The family affluence scale (FAS)27 asks children whether they have their own bedroom (no ¼ 0, yes ¼ 1), number of family computers and holidays in the past year (none ¼ 0, once ¼ 1, two ¼ 2, more than two ¼ 3) and how many cars their family own (none ¼ 0, one ¼ 1, two or more ¼ 2). + +The summed scale provides a measure of affluence (from 0, least affluent to 9, most affluent). + +## Smoking Restrictions In The Home + +Children were asked whether smoking was allowed inside their home. Response options were (i) no, smoking is not allowed at all, (ii) smoking is allowed in certain areas only, +(iii) smoking is allowed anywhere in our home, (iv) smoking is only allowed on special occasions in our home, (v) I don't know. Responses (ii) and (iv) represented 'partial smoking restriction'. Children who selected 'I don't know' (n ¼ 952) +were excluded from the analysis of smoking restrictions as a dependent variable, though retained as a separate category in cotinine analyses. + +## Smoking Restrictions In Cars + +Children were asked 'Are people allowed to smoke in your car, van or truck?'. Options were 'yes', 'no', 'I don't know' or 'don't have a car, van or truck'. Children whose family did not have a car (n ¼ 941) or did not know whether smoking was allowed (n ¼ 1251) were excluded from the analysis treating car-smoking restrictions as a dependent variable, though retained as a separate category in cotinine analyses. + +## Parental Smoking + +Children were asked to indicate how often each parent figure smoked ('most days', 'sometimes', 'doesn't', 'don't know' or 'don't have or see'). Parents were considered smokers if the child indicated that they smoked most days or sometimes. Children were classified as living with no smoking parent figures, smoking father figure only, mother figure only or two smoking parents. + +## Age + +Age was calculated by subtracting dates of birth from survey date. + +## Procedures + +Sampling and data collection are detailed elsewhere.8,13,14 CHETS were repeated cross-sectional studies of schoolchildren in their final year of primary school. As Scottish legislation was introduced 1 year earlier, follow-up coincided with pre-legislation collections in Wales and NI, +which replicated the Scottish study. Consent was sought via a letter to the headteacher of each school. Parents or guardians were sent a letter and information sheet and asked to inform the school if they did not wish their child to participate. Students completed questionnaires, with researchers collecting anonymous saliva samples during or after completion of questionnaires, using cottonwool swabs of a Salivettew. All samples were processed in the same laboratory. The studies received approval from the University of Edinburgh's School of Education Ethics Committee, Cardiff University School of Social Sciences Research Ethics Committee and NI Office of Research Ethics. The pooled data set retained items whose wording and response options were identical in each country, with variables recreated from raw data. + +## Statistical Analysis + +As almost 40% of children, and most from some subgroups, provided samples containing no detectable cotinine, adoption of linear regression would require imputed values to be used for an excessively large proportion of cases. Hence, data were divided into categories, with multinomial regressions, as adopted in Wales,6 favoured over the linear approach adopted in Scotland,13 where fewer concentrations lay ,0.1 ng/ml. Concentrations below the limit of detection +(0.1 ng/ml) were considered 'low', 0.1 –0.5 ng/ml 'medium' and above 0.5 ng/ml 'high' (to create approximately equal tertiles). The terms 'low', 'medium' and 'high' refer to the position of the child's cotinine value in the distribution, rather than associated risk. Medium was the base category, allowing examination of whether movement from the middle of the distribution down was matched by movement from the top to the middle. Proportions of children with each FAS score providing samples above a range of cotinine cut-points before and after legislation are presented graphically to illustrate the change in socioeconomic gradients, and an FAS - survey year interaction term entered into regression models. + +Further multinomial regression models examined the change in home-smoking restrictions, with full restriction the base category. Binary logistic regression models examined car-based smoking. Relative risk ratios (multinomial models) and odds ratios (binary models) are presented along with 95% confidence intervals. Models are presented for each country and the combined sample. All models adjust for age. Combined analyses include dummy variables to adjust for country. Percentages drawn from the combined sample are weighted to account for differential sampling fractions across the three countries. Svy settings of Stata 11 were used to account for clustering. + +## Results Response Rates + +Country-specific response rates are reported elsewhere.8,13,14 Of 586 schools approached, 320/304 (54/51%) participated at baseline/follow-up. Participating schools within each country were representative in terms of the percentage of children entitled to free school meals (a common marker of socioeconomic deprivation). Within these schools, 5946/ 7311 (81%) pupils at baseline and 5803/7044 (82%) at follow-up provided useable saliva samples and completed the survey. After excluding children in lower year groups +(n ¼ 100), with cotinine concentration .15ng/ml (n ¼ 80; range 15 –383.9 ng/ml), self-reported smokers (n ¼ 157), +children who did not complete the smoking question +(n ¼ 83), did not report living either with their father and mother, in a single-parent family or in a step-family +(n ¼ 215), or from schools who participated at baseline only +(n ¼ 245), the data set comprised 10 867 children (5347 baseline/5520 follow-up). + +## Sample Description + +Children averaged 11.2 (SD ¼ 0.40) years at both time points, with 50.0% (n ¼ 2672) boys at baseline and 50.3% (n ¼ 2775) at follow-up. Most reported living with their father and mother (73.8%; n ¼ 3942 baseline, 74.0%; +n ¼ 4065 at follow-up). The only characteristic varying significantly between survey years was SES, with affluence slightly higher at follow-up. + +## Cotinine Concentrations + +Percentages of children with undetectable concentrations increased from 31.0 (n ¼ 1715) to 41.0% (n ¼ 2251) following legislation overall, and from 20.1 to 34.2, 44.9 to 51.0 and from 38.6 to 42.9% in Scotland, Wales and NI, respectively. This increase at the bottom of the distribution was accompanied largely by decreases in samples containing 0.1 –0.5 ng/ml of cotinine, with smaller declines observed in the percentages of samples containing higher levels of cotinine (see Fig. 1). Regression analysis indicated that the relative risk of children's samples containing no detectable cotinine increased significantly following legislation +(Table 1). The relative risk of providing a sample containing a 'high' cotinine concentration also increased significantly, reflecting the failure of higher concentrations to decline to the same extent as more moderate exposure levels. Country-specific models indicate that this non-linear decline occurred in all countries. Increases in the likelihood of children providing samples with no detectable cotinine remain + +![3_image_0.png](3_image_0.png) + +significant after entry of terms for parental smoking and private smoking restrictions (Model 2; Table 1). + +## Patterning In Shs Exposure, And Post-Legislation Changes In Exposure, By Ses + +The relative risk of children's samples containing no detectable cotinine increased significantly as SES increased +(Table 1), whilst the relative risk of samples containing a +'high' cotinine concentration fell. These associations were almost identical in all countries, remaining significant after entry of terms for parental smoking and private smoking restrictions (Table 1; Model 2). + +Figure 2 shows the percentage of children with each FAS +score providing samples with cotinine levels above a range of thresholds, before and after legislation. Before legislation almost all children from the poorest families provided samples with a detectable level of cotinine (92.7%), whilst substantially fewer children from the most affluent families did (56.6%). However, this inequality appears to have widened following legislation, with percentages of samples above the limit of detection ranging from 96.9 to 38.2% for the least and most affluent children, respectively, after legislation. Gradients for higher exposure levels remained relatively unchanged. For example, at both time points, the percentage of children from the poorest families providing samples containing .3 ng/ml of cotinine, is 10 times higher than amongst the most affluent group (33.3 vs. 3.0% +post-legislation). + +Entry of an FAS - survey year interaction term into Model 1 (Table 1) indicates significantly increased inequality in the likelihood of a child providing samples with no + +| Scotland | Wales | NI | Alla | | | | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------|------------------------------------------------------------------------------------------------|-------------------|-------------------------------------|-------------------------------------|-------------------------------------|-------------------| +| Low vs. | High vs. | Low vs. | High vs. | Low vs. | High vs. | Low vs. | High vs. | +| medium | medium | medium | medium | medium | medium | medium | medium | +| cotinine | cotinine | cotinine | cotinine | cotinine | cotinine | cotinine | cotinine | +| Model 1 | n ¼ 4245 | n ¼ 2654 | n ¼ 3712 | n ¼ 10 611 | | | | +| Main effects (Step 1) Survey yearb | 2.28 (1.83–2.86) 1.22 (1.04 –1.43) | 1.36 (1.12 –1.65) | 1.15 (0.93 –1.41) | 1.36 (1.15 –1.60) 1.36 (1.14 –1.62) | 1.63 (1.45 –1.83) 1.25 (1.13 –1.39) | | | +| FASc | 1.14 (1.08–1.19) 0.78 (0.75 –0.81) | 1.16 (1.10 –1.22) | 0.82 (0.76 –0.88) | 1.14 (1.09 –1.20) 0.79 (0.74 –0.83) | 1.14 (1.11 –1.17) 0.79 (0.77 –0.81) | | | +| Interaction effects (Step 2) FAS by survey year 1.08 (1.00 –1.18) | 1.03 (0.95 –1.12) | 1.11 (0.99 –1.24) | 1.03 (0.91 –1.17) | 1.11 (1.01 –1.21) | 1.10 (0.99 –1.22) | 1.10 (1.05 –1.16) | 1.05 (0.99 –1.11) | +| Model 2d | n ¼ 4125 | n ¼ 2520 | n ¼ 3616 | n ¼ 10261 | | | | +| Main effects (Step 1) Survey year | 2.34 (1.87–2.93) | 0.95 (0.78 –1.16) | 1.38 (1.10 –1.73) | 1.18 (0.92 –1.52) | 1.42 (1.18 –1.70) 1.41 (1.17 –1.70) | 1.70 (1.50 –1.93) 1.16 (1.03 –1.31) | | +| FAS | 1.09 (1.03–1.15) 0.92 (0.87 –0.97) | 1.11 (1.04 –1.18) | 0.89 (0.82 –0.97) | 1.11 (1.05 –1.17) 0.85 (0.80 –0.91) | 1.10 (1.06 –1.13) 0.89 (0.86 –0.92) | | | +| Parentale smoking Father only | 0.51 (0.36–0.73) 2.53 (1.94 –3.30) | 0.34 (0.24 –0.48) | 2.13 (1.51 –3.02) | 0.35 (0.27 –0.44) 2.22 (1.68 –2.93) | 0.39 (0.33 –0.47) 2.39 (2.02 –2.82) | | | +| Mother only | 0.45 (0.29–0.71) 8.28 (6.46 –10.62) | 0.21 (0.13 –0.34) | 4.68 (3.28 –6.68) | 0.25 (0.18 –0.36) 6.02 (4.57 –7.93) | 0.28 (0.22 –0.36) 6.48 (5.51 –7.62) | | | +| Both | 0.24 (0.12–0.48) 11.81 (8.41 –16.59) 0.21 (0.09 –0.47) | 12.09 (8.08 –18.08) 0.16 (0.09 –0.28) 8.79 (6.52 –11.83) 0.19 (0.13 –0.27) 10.78 (8.90 –13.05) | | | | | | +| Interaction effects (Step 2) FAS by survey year 1.09 (1.00 –1.20) | 1.01 (0.91 –1.12) | 1.16 (1.02 –1.33) | 1.04 (0.90 –1.20) | 1.14 (1.04 –1.26) | 1.06 (0.94 –1.19) | 1.13 (1.06 –1.20) | 1.02 (0.96 –1.09) | +| Significant relative risk ratios (at the 95% level) are highlighted in bold. All models are adjusted for age. aModels from the combined sample are adjusted for country. | | | | | | | | + +Table 1 Relative risk ratios from multinomial logistic regression indicating the change in the risk of providing a sample with no detectable cotinine (low vs. medium cotinine) or .0.5 ng/ml of cotinine (high vs. medium cotinine) relative to the likelihood of a sample containing 0.1 –0.5 ng/ml aModels from the combined sample are adjusted for country. + +bPre-legislation ¼ 0, post-legislation ¼ 1. + +cHigher FAS score ¼ higher socioeconomic status. + +dModel 2 adjusts also for smoking restriction level in homes and cars (estimates not shown). + +eReference category is 'neither parent figure smokes'. + +SES AND CHILDHOOD SECONDHAND SMOKE EXPOSURE +603 + +![5_image_0.png](5_image_0.png) + +detectable cotinine for the combined sample. Trends toward widening inequality in the likelihood of a child's sample containing no detectable cotinine were observed for individual countries, though lay near the border of significance +(Scotland P ¼ 0.06, Wales P ¼ 0.07, NI P ¼ 0.03). After adjustment for parental smoking and private smoking restrictions, a significant interaction remains (Model 2). There was no significant change in inequality in the relative risk of providing samples with 'high' cotinine levels in any country. + +## Private Smoking Restrictions Smoking In The Home + +Before legislation, 51.9% (n ¼ 2499) of children reported living in homes where smoking was not allowed, while 31.3% (n ¼ 1509) reported partial restrictions and 16.8% +(n ¼ 805) that smoking was allowed throughout. Following legislation, the proportion of children reporting no home smoking restrictions fell to12.7% (n ¼ 612) accompanied by slight increases in full (55.1%; n ¼ 2705) and partial (32.2%; n ¼ 1642) restrictions. Percentages reporting no smoking restrictions fell from 18.5 to 14.2, from 14.2 to 11.4 and from 16.4 to 11.0% in Scotland, Wales and NI, respectively, whilst percentages reporting full smoking restrictions increased from 47.3 to 51.2, from 60.0 to 63.2 and from 51.7 to 52.4%. Among children of smokers, 18.3% +(n ¼ 370) reported that smoking was not allowed in their home before legislation, with 45.5% (n ¼ 892) reporting partial restrictions and 36.2% (n ¼ 687) that smoking was allowed throughout their home. After legislation, the percentage of these children reporting no restrictions fell to 27.4% (n ¼ 524), accompanied by an increase in partial restrictions to 53.3% (n ¼ 1087), and a small increase in the prevalence of full restrictions increasing (n ¼ 404; 19.3%). + +Regression analyses indicate that overall, the relative risk of reporting unrestricted smoking, rather than full smoking restrictions, decreased significantly following legislation, whilst the relative risk of partial restrictions did not change +(see Table 2). Decreased risk of unrestricted smoking remained significant after adjustment for parents' smoking +(Model 2), whilst only in Wales did decreased risk of unrestricted smoking fail to reach significance in either model. + +## Smoking In Cars + +Approximately 28.5% (n ¼ 1225) of children (29.1% in Scotland, 25.4% in Wales and 32.0% in NI) reported that smoking was allowed inside their car before legislation, declining to 25.7% (n ¼ 1148) after legislation (and to 26.1, 22.7 and 29.1% in Scotland, Wales and NI, respectively). Regression analyses indicate that whilst in no individual country did this reduction reach statistical significance, for the combined data set, this was significant (see Table 2). After adjustment for parental smoking, declines in Scotland, NI as well as the combined sample were significant (Model 2; Table 2). + +| full restrictions and the odds of smoking being allowed in the car Scotland (n ¼ 3894) | Wales (n ¼ 2358) | NI (n ¼ 3391) | Alla (n ¼ 9643) | | | | | | +|-------------------------------------------------------------------------------------------------------------------------------|---------------------|----------------------|---------------------|-----------------------|---------------------|---------------------|---------------------|---------------------| +| Partial vs. full | None vs. full | Partial vs. full | None vs. full | Partial vs. full | None vs. full | Partial vs. full | None vs. full | | +| Smoking in the home (Model 1) Main effects (Step 1) Surveyb year 0.96 (0.83 –1.10) | 0.76 (0.62–0.93) | 0.96 (0.79 –1.16) | 0.79 (0.60–1.04) | 1.17 (1.00 –1.38) | 0.71 (0.57 –0.89) | 1.04 (0.95–1.15) | 0.74 (0.65 –0.84) | | +| FASc | 0.79 (0.76 –0.83) | 0.70 (0.66–0.74) | 0.83 (0.78 –0.88) | 0.73 (0.67 –0.79) | 0.86 (0.82 –0.90) | 0.78 (0.73 –0.83) | 0.81 (0.79–0.84) | 0.73 (0.70 –0.76) | +| Interaction effects (Step 2) FAS year | 0.93 (0.87 –1.00) | 1.00 (0.90–1.10) | 0.96 (0.87 –1.06) | 0.99 (0.86–1.14) | 1.07 (0.98 –1.17) | 1.06 (0.95 –1.19) | 0.98 (0.94–1.03) | 1.02 (0.96 –1.09) | +| Scotland (n ¼ 3803) | Wales (n ¼ 2275) | NI (n ¼ 3320) | All (n ¼ 9398) | | | | | | +| Smoking in the home (Model 2, main effects only) Survey year | 0.84 (0.71 –0.99) | 0.59 (0.46–0.75) | 0.98 (0.80 –1.20) | 0.82 (0.59–1.14) | 1.12 (0.92 –1.36) | 0.64 (0.49 –0.84) | 0.97 (0.87–1.08) | 0.66 (0.55 –0.77) | +| FAS | 0.84 (0.80 –0.88) | 0.78 (0.72–0.83) | 0.89 (0.84 –0.95) | 0.82 (0.75 –0.90) | 0.93 (0.89 –0.98) | 0.90 (0.85 –0.97) | 0.88 (0.86–0.91) | 0.83 (0.79 –0.86) | +| Parentald smoking Father only | 5.90 (4.44 –7.86) | 20.47 (14.16–29.60) | 6.17 (4.50 –8.44) | 17.92 (9.87–32.54) | 4.91 (3.74 –6.44) | 8.76 (5.96 –12.86) | 5.50 (4.66–6.49) | 14.24 (11.23–18.05) | +| Mother only | 14.24 (10.79–18.79) | 60.90 (42.21–87.84) | 14.97 (10.58–21.19) | 67.68 (37.74–121.38) | 8,91 (6.79 –11.70) | 26.02 (17.88–37.88) | 11.82 (9.95 –14.05) | 43.66 (34.22–55.69) | +| Both | 16.53 (11.66–23.44) | 92.14 (61.36–138.36) | 22.78 (15.41–33.65) | 140.03 (72.52–270.39) | 12.63 (9.36 –17.04) | 31.29 (20.74–47.19) | 16.22 (13.30–19.78) | 68.48 (52.52–89.30) | +| Scotland (n ¼ 3345) | Wales (n ¼ 2066) | NI (n ¼ 3022) | All (n ¼ 8433) | | | | | | +| Smoking in cars (Model 1) Main effects (Step 1) Survey year | 0.88 (0.76–1.03) | 0.90 (0.72 –1.12) | 0.88 (0.74–1.04) | 0.89 (0.80–0.98) | | | | | +| FAS | 0.79 (0.75–0.83) | 0.80 (0.75 –0.85) | 0.85 (0.81–0.90) | 0.82 (0.79–0.84) | | | | | +| Interaction effects (Step 2) FAS year | 1.04 (0.93–1.15) | 0.93 (0.83 –1.05) | 1.04 (0.95–1.14) | 1.02 (0.96–1.08) | | | | | +| Scotland (n ¼ 3263) | Wales (n ¼ 1993) | NI (n ¼ 2960) | All (n ¼ 8216) | | | | | | +| Smoking in cars (Model 2, main effects only) Survey year | 0.81 (0.66–0.99) | 0.98 (0.74 –1.29) | 0.80 (0.65–0.99) | 0.84 (0.74–0.95) | | | | | +| FAS | 0.88 (0.83–0.94) | 0.89 (0.82 –0.96) | 0.92 (0.87–0.99) | 0.90 (0.87–0.93) | | | | | +| Parental smoking Father only | 16.30 (12.27–22.63) | 16.89 (11.72–24.36) | 13.62 (10.44–17.77) | 15.14 (12.80–17.91) | | | | | +| Mother only | 18.36 (13.79–24.43) | 23.40 (16.13–33.97) | 11.83 (9.11–15.36) | 16.00 (13.46–19.01) | | | | | +| Both | 32.51 (23.87–44.30) | 55.50 (36.24–85.01) | 20.36 (15.35–27.02) | 30.51 (25.30–36.79) | | | | | +| Significant relative risk ratios and odds ratios (at the 95% level) are highlighted in bold. All models are adjusted for age. | | | | | | | | | + +Table 2 Relative risk ratios and odds ratios (and 95% CIs) from multinomial and binary logistic regression models showing the likelihood of a child reporting partial or no smoking restrictions relative to the likelihood of reporting SES AND CHILDHOOD SECONDHAND SMOKE EXPOSURE +605 + +## Patterning In Private Smoking Restrictions, And Postlegislation Changes, By Ses + +In all countries, and the combined data set, as SES +increased, the likelihood of partial or no home smoking restrictions (rather than full smoking restrictions), decreased significantly, whilst the odds of smoking being allowed inside the family car also decreased significantly (Table 2). These trends remained after adjustment for parental smoking (Model 2). Entry of an interaction term for FAS - +survey year into models for home and car-based smoking restrictions indicated no change in inequality following legislation. Following legislation, 26.3% of children scoring 1 on FAS reported living in a fully smoke-free home, climbing to 72.0% for those scoring 9. Percentages reporting that smoking was not allowed in their car ranged from 51.7 (least affluent) to 83.0% (most affluent). + +## Discussion Main Findings Of This Study + +Following legislation, proportions of children providing samples containing no detectable cotinine increased in all countries, though little change occurred at higher ends of the distribution. Furthermore, socioeconomic inequality in the likelihood of a child's sample containing detectable traces of cotinine increased. Hence, declines in exposure occurred predominantly among children with low exposure before legislation, and from more affluent families. + +Substantial socioeconomic gradients in proportions of children with higher SHS exposure levels remained unchanged. + +Overall, declines in the percentage of children reporting that smoking was allowed throughout their home were observed following legislation. However, among children of smokers, full smoking restrictions increased by only 1%, +with reductions in the percentage of children reporting no home smoking restrictions matched largely by increases in partial restrictions. Small declines in reports that smoking was allowed in their family's car were also observed, reaching significance in the pooled sample. + +Nevertheless, after legislation little more than half of children, including only one in five children living with smoking parents, reported that smoking was not allowed in their home at all, while 1 in 4 children whose family owned a car reported that smoking was allowed in their car. While post-legislation changes in smoking restrictions in cars or homes were not patterned by socioeconomic status, with no suggestion of displacement of smoking into the home across the SES spectrum, smoking restrictions in private spaces remain substantially more common among children from higher SES families. + +## What Is Already Known On This Topic + +In all UK countries, childhood SHS exposure decreases as socioeconomic status increases.5 - 8 In no UK countries, and for no SES subgroups, did childhood SHS exposure increase following legislation.8,13 - 15 However, there is growing evidence that the greatest post-legislation declines in SHS +exposure occurred amongst children at least risk.13,14 There is some previous evidence that adoption of smoke-free homes increased following legislation.18 + +## What This Study Adds + +While no longitudinal trend data are available for UK countries in this study, the post-legislation increases in inequality in SHS exposure run counter to trends for greater declines amongst those most exposed reported in the years leading up to legislation in England.15 There was no significant change in inequality in relation to proportions of children with higher levels of SHS exposure. However, following legislation, 1 in 12 children provided saliva samples containing more cotinine than samples collected from Scottish bar workers prior to smoke-free legislation (2.94 ng/ml),28 including almost 1 in 3 children from the poorest families (10 times the proportion among children at the opposite end of the socio-economic spectrum). Hence, while the growing proportions of children providing samples with no detectable cotinine are encouraging, sustained emphasis on reducing SHS exposure amongst the sizeable minority continuing to have high levels of exposure remains a priority in reducing health inequalities. As argued by Frohlich and Potvin,29 where unequal responses to whole-population approaches lead to an exacerbation of inequality, there is a strong case for complementary intervention to address the needs of 'vulnerable' populations, such as children from lower SES families. + +Despite small overall increases in adoption of smoke-free homes, the home remains a key source of SHS exposure for many children, with persistent inequalities in the prevalence of restrictions on smoking in homes. Hence, efforts to encourage smoke-free homes are crucial in attempting to reduce health inequalities. Legislation against smoking in the home is unlikely to prove acceptable,30 and reducing inequalities in exposure may require intervention to support adoption of voluntary smoking restrictions in lower SES +households.31,32 However, extending legislation to cars carrying children, as introduced in Australia,33 and for which there appears to be significant public support in the UK,34,35 may contribute to reducing inequalities. + +## Funding Limitations Of This Study + +The study brings together three large data sets, harmonizing their analyses to allow comparability between three UK countries, benefitting from use of salivary cotinine as a primary outcome; a well-validated indicator of SHS exposure.25 However, our ability to attribute change to smoke-free legislation is weakened by the absence of a counterfactual. A longitudinal study may have facilitated examination of change over time, but would have made it impossible to distinguish between changes due to increased age, or due to legislation. Inclusion of a narrow age group, though enhancing internal validity, limits generalizability. Finally, reliance upon self-reports on some measures is perhaps liable to social desirability biases, although it is hoped that any error would have been equal at both time points. + +## Authors' Roles + +G.M. led the development of the paper plan, pooled the data sets, conducted statistical analysis and wrote the manuscript. D.C. and L.M. advised on statistical analysis. All authors contributed to the development of paper plans and provided comments on drafts of the manuscript. + +## Acknowledgements + +The CHETS Wales team would like to thank: Anna Hamilton and Hayley Collicott (study administrators), +Heather Rothwell, Claire Pimm, Nancy West, Ellie Byrne, Rachel Clark, Margaret Humphries, Keith Humphries, Rosie Salazar (Research Assistants); and Janine Hale and Chris Roberts (Welsh Government, Public Health Branch, Social Research Division). The CHETS Northern Ireland team would like to thank Malcolm Buchanan for school recruitment; and the study development/implementation group (Naomi McCay, Lindsay MacDonald and Diana Gossrau-Breen). The CHETS Scotland team would like to thank the study advisory group. We would also like to thank all schools and students who participated in the research in each of the three countries, ABS laboratories +(www.abslabs.com) who analysed salivary cotinine collected in all countries, and MVA Consultancy for managing the fieldwork in the Scotland and Northern Ireland arms of the study. + +The work was supported by the Centre for the Development and Evaluation of Complex Interventions for Public Health Improvement (DECIPHer), a UKCRC Public Health Research: Centre of Excellence. Funding from the British Heart Foundation, Cancer Research UK, Economic and Social Research Council (RES-590-28-0005), Medical Research Council, the Welsh Assembly Government and the Wellcome Trust (WT087640MA), under the auspices of the UK Clinical Research Collaboration, is gratefully acknowledged. For the original studies, including collection and processing of data samples, CHETS Wales was funded by the Welsh Government (grant 216/2005). CHETS Scotland was funded by NHS Health Scotland and the Scottish Government. CHETS NI was funded by the Department of Health, Social Services and Safety, Northern Ireland. + +## References + +1 Vork KL, Broadwin RL, Blaisdell RJ. Developing asthma in childhood from exposure to secondhand tobacco smoke: insights from a meta-regression. Environ Health Perspect 2007;115(10):1394 –400. + +2 Pattenden S, Antova T, Neuberger M et al. Parental smoking and children's respiratory health: independent effects of prenatal and postnatal exposure. Tob Control 2006;15(4):294 –301. + +3 Kallio K, Jokinen E, Saarinen M et al. Arterial intima-media thickness, endothelial function, and apolipoproteins in adolescents frequently exposed to tobacco smoke. Circ Cardiovasc Qual Outcomes 2010;3(2):196 –203. + +4 World Health Organisation. WHO Framework Convention on Tobacco Control. Geneva: WHO Document Production Services, 2005. + +5 Akhtar PC, Haw SJ, Levin KA et al. Socioeconomic differences in second-hand smoke exposure among children in Scotland after introduction of the smoke-free legislation. J Epidemiol Community Health 2010;64(4):341 –6. + +6 Moore GF, Holliday JC, Moore LAR. Socioeconomic patterning in changes in child exposure to secondhand smoke after implementation of smoke-free legislation in Wales. Nicotine Tob Res 2011;13(3):903–10. + +7 Sims M, Tomkins S, Judge K et al. Trends in and predictors of second-hand smoke exposure indexed by cotinine in children in England from 1996 to 2006. Addiction 2010;105(3):543–53. + +8 Health Promotion Agency. Childhood Exposure to Tobacco Smoke +(CHETS) in Northern Ireland. Belfast: Health Promotion Agency for Northern Ireland, 2009. + +9 Haw SJ, Gruer L. Changes in exposure of adult non-smokers to secondhand smoke after implementation of smoke-free legislation in Scotland: national cross sectional survey. BMJ +2007;335(7619):549–52. + +10 Allwright S, Paul G, Greiner B et al. Legislation for smoke-free workplaces and health of bar workers in Ireland: before and after study. BMJ 2005;331(7525):1117 –22. + +23 Norman GJ, Ribisl KM, Howard-Pitney B et al. Smoking bans in the home and car: do those who really need them have them? Prev Med 1999;29(6):581 –9. + +24 West R, Zatonski W, Przewozniak K et al. Can we trust national smoking prevalence figures? Discrepancies between biochemically assessed and self-reported smoking rates in three countries. Cancer Epidemiol Biomarker Prev 2007;16(4):820 –2. + +25 Dolcini MM, Adler NE, Lee P et al. An assessment of the validity of adolescent self-reported smoking using three biological indicators. Nicotine Tob Res 2003;5(4):473 –83. + +26 Feyerabend C, Russell M. A rapid gas-liquid chromatographic method for the determination of cotinine and nicotine in biological fluids. J Pharm Pharmacol 1990;42:450–2. + +27 Currie C, Molcho M, Boyce W et al. Researching health inequalities in adolescents: the development of the Health Behaviour in School-Aged Children (HBSC) Family Affluence Scale. Soc Sci Med 2008;66(6):1429–36. + +28 Semple S, Maccalman L, Naji AA et al. Bar workers' exposure to second-hand smoke: the effect of Scottish smoke-free legislation on occupational exposure. Ann Occup Hyg 2007;51:571–80. + +29 Frohlich KL, Potvin L. The inequality paradox: the population approach and vulnerable populations. Am J Public Health 2008;98(2):216–21. + +30 Chapman S. The future of smoke-free legislation. BMJ +2007;335(7619):521–2. + +31 Priest N, Roseby R, Waters E et al. Family and carer smoking control programmes for reducing children's exposure to environmental tobacco smoke. Cochrane Database Syst Rev 2008(4) +CD001746. + +32 Ritchie D, Amos A, Phillips R et al. Action to achieve smoke-free homes—an exploration of experts' views. BMC Public Health 2009;9(1):112. + +33 Freeman B, Chapman S, Storey P. Banning smoking in cars carrying children: an analytical history of a public health advocacy campaign. + +Aus NZ J Public Health 2008;32(1):60–5. + +34 Hitchman SC, Fong GT, Zanna MP et al. Support and correlates of support for banning smoking in cars with children: findings from the ITC Four Country Survey. Eur J Public Health 2011;21(3):360–5. + +35 Thomson G, Wilson N. Public attitudes to laws for smoke-free private vehicles: a brief review. Tob Control 2009;18(4):256–61. + +11 Ho SY, Wang MP, Lo WS et al. Comprehensive smoke-free legislation and displacement of smoking into the homes of young children in Hong Kong. Tob Control 2010;19(2):129–33. + +12 Adda J, Cornaglia FJ. The effect of taxes and bans on passive smoking. IZA Discussion Paper No. 2191. 2006. Available at SSRN: http://ssrn.com/abstract=919963. Last accessed December 2011. + +13 Akhtar PC, Currie DB, Currie CE et al. Changes in child exposure to environmental tobacco smoke (CHETS) study after implementation of smoke-free legislation in Scotland: national cross sectional survey. BMJ 2007;335(7619):545 –9. + +14 Holliday JC, Moore GF, Moore L. Changes in child exposure to secondhand smoke after implementation of smoke-free legislation in Wales: a repeated cross-sectional study. BMC Public Health 2009;9(1):430. + +15 Jarvis MJ, Sims M, Gilmore A et al. Impact of smoke-free legislation on children's exposure to secondhand smoke: cotinine data from the Health Survey for England. Tob Cont 2012;21:18–23. + +16 Dove MS, Dockery DW, Connolly GN. Smoke-free air laws and secondhand smoke exposure among nonsmoking youth. Pediatrics 2010;126(1):80–7. + +17 Department of Health. A Smokefree Future: A Comprehensive Tobacco Control Strategy for England. London, Department of Health, 2010. + +18 Akhtar PC, Haw SJ, Currie DB et al. Smoking restrictions in the home and secondhand smoke exposure among primary schoolchildren before and after introduction of the Scottish smoke-free legislation. Tob Control 2009;18(5):409 –15. + +19 British Medical Association Board of Science. Smoking in Vehicles: +A Briefing from the Board of Science. London, British Medical Association, 2011. + +20 Belanger M, O'Loughlin J, Okoli CTC et al. Nicotine dependence symptoms among young never-smokers exposed to secondhand tobacco smoke. Addict Behav 2008;33(12):1557–63. + +21 Hitchman SC, Fong GT, Borland R et al. Predictors of smoking in cars with nonsmokers: findings from the 2007 Wave of the International Tobacco Control Four Country Survey. Nicotine Tob Res 2010;12(4):374–80. + +22 Borland R, Yong HH, Cummings KM et al. Determinants and consequences of smoke-free homes: findings from the International Tobacco Control (ITC) Four Country Survey. Tob Control 2006;15:42–50. \ No newline at end of file diff --git a/medical/md/PMC3790206.md b/medical/md/PMC3790206.md new file mode 100644 index 0000000000000000000000000000000000000000..cef566fe7a3cff6b62a60b45226304cb91273ffa --- /dev/null +++ b/medical/md/PMC3790206.md @@ -0,0 +1,258 @@ +OPEN + +![0_image_1.png](0_image_1.png) + +SUBJECT AREAS: +METAMATERIALS +NANOPARTICLES +RAMAN SPECTROSCOPY +NANOPHOTONICS AND +PLASMONICS +Received 13 May 2013 Accepted 18 September 2013 Published 4 October 2013 Correspondence and requests for materials should be addressed to K.B.C. (kcrozier@seas. + +harvard.edu) + +# Wafer-Scale Metasurface For Total Power + +![0_Image_0.Png](0_Image_0.Png) + +![0_Image_2.Png](0_Image_2.Png) + +![0_Image_3.Png](0_Image_3.Png) Absorption, Local Field Enhancement And Single Molecule Raman Spectroscopy + +Dongxing Wang1, Wenqi Zhu1, Michael D. Best2, Jon P. Camden2 & Kenneth B. Crozier1 1School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA, 2Department of Chemistry, University of Tennessee, Knoxville, TN, 37996-1600, USA. + +The ability to detect molecules at low concentrations is highly desired for applications that range from basic science to healthcare. Considerable interest also exists for ultrathin materials with high optical absorption, e.g. for microbolometers and thermal emitters. Metal nanostructures present opportunities to achieve both purposes. Metal nanoparticles can generate gigantic field enhancements, sufficient for the Raman spectroscopy of single molecules. Thin layers containing metal nanostructures (''metasurfaces'') can achieve near-total power absorption at visible and near-infrared wavelengths. Thus far, however, both aims (i.e. single molecule Raman and total power absorption) have only been achieved using metal nanostructures produced by techniques (high resolution lithography or colloidal synthesis) that are complex and/or difficult to implement over large areas. Here, we demonstrate a metasurface that achieves the near-perfect absorption of visible-wavelength light and enables the Raman spectroscopy of single molecules. Our metasurface is fabricated using thin film depositions, and is of unprecedented (wafer-scale) extent. + +T +he realization of strong and tunable optical absorption is advantageous for applications that include photon detection1, solar energy conversion2, and photo-induced water splitting3. Previous studies4–9 have shown that metasurfaces enable efficient optical absorption at visible and infrared wavelengths. With the exception of Ref. 9, however, their fabrication involved electron beam lithography, a time-consuming and costly method that is only suitable over small areas (usually ,1 mm2). Ref. 9 aimed to address this by instead employing silver nanocubes, but their chemical synthesis is by no means straightforward. Here, we report the realization of metasurfaces with near-complete absorption and record areas (4 inch wafers, i.e. ,80 cm2) using standard sputtering and evaporation techniques. These consist of silver nanoparticle islands formed over a silver mirror, with an SiO2 spacer layer, and are termed ''SIOM metasurfaces''. We show that their reflectance can be tuned by adjusting parameters that include the spacer thickness and the evaporation rate, which in turn modifies the silver nanoparticle island morphology. We discuss the physical interpretation of the perfect absorption phenomenon in terms of equivalent electric and magnetic surface currents. These are enabled by the SIOM metasurface supporting both electric and magnetic resonances. The perfect absorption behavior is accompanied by huge local field enhancement that is advantageous for surface-enhanced Raman scattering (SERS)10–13. We perform SERS measurements of molecules adsorbed to the SIOM metasurfaces and determine the enhancement factors (EFs), i.e. the factors by which the Raman signals from molecules on the SIOM metasurface are increased compared to molecules in the pure liquid form (i.e. no metasurface). We find that the SIOM metasurfaces exhibit EFs that are more than one order of magnitude larger than those of silver nanoparticle islands on glass. We furthermore demonstrate, for the first time, single molecule SERS with a metasurface. The large extent of our SIOM metasurface enables the observation of an unprecedented number (.7000) of single molecule SERS events. + +## Results + +The SIOM metasurface we introduce is schematically illustrated as Figure 1a. The starting substrate is silicon, onto which silver and SiO2 layers are deposited by evaporation and sputtering, respectively. Silver nanoparticle islands are then formed by evaporating silver at a slow rate. Further details are provided in the Methods section. + +We note that our approach involves no complex or expensive fabrication procedures, and is inherently suitable for the producing substrates with large areas. Indeed, the SIOM metasurfaces are on 4 inch wafers (Figure 1b), only limited by the sizes of the holders in our evaporator and sputterer. We furthermore note that we choose to employ a Si wafer as the underlying substrate for convenience. The silver mirror, however, is of sufficient + +![1_image_0.png](1_image_0.png) + +![1_image_1.png](1_image_1.png) + +thickness so that there is almost no penetration of light into the Si substrate. The optical properties of the substrate are therefore unimportant, meaning that even more economical substrate materials (e.g. + +plastic or steel) could be used. + +An SEM image of a portion of the SIOM metasurface is shown in Figure 1c. The SEM image verifies that the silver nanoparticle islands are formed on the SiO2 layer. It is seen from Figure 1b that the fabricated SIOM metasurface appears red in color. To understand this phenomenon, we measure its reflectance (Figure 2a, Methods). + +The reflection spectrum (Figure 2a) shows a dip at l < 525 nm at which the reflectance is very small. As we discuss further below, the dip is related to the excitation of localized surface plasmons on the silver nanoparticle islands, but these are strongly modified by the presence of the silver mirror. As the reflectance is below 10% over a band extending from l < 450 nm to 600 nm, the SIOM appears red in color (Figure 2b). To understand the origin of the reflection spectrum dip, we analyze the sizes of the nanoparticle islands. We randomly pick a 5 mm 3 3.5 mm region from the SEM of the SIOM +substrate, import the image into MATLAB (The Mathworks Inc.), +and find the effective radius of each silver nanoparticle island within this region. The effective radius is the radius of a circle that would have the same area as the area of the nanoparticle island, as found from analysis of the SEM image. The effective radii found in this way are tabulated in a histogram (Figure 2b), and can be seen to follow a Gaussian distribution (red fitted curve) that peaks at an effective radius of 15.4 nm and has a standard deviation of 6 nm. We check other areas of the SIOM substrate and obtain similar results. It is likely that this size dispersion contributes to the broad width of the reflection dip. + +From Figure 2a, it can be seen that reflectance from the SIOM +substrate is very low, being below 1% at a wavelength of l 5 525 nm. + +Indeed, the SIOM metasurface achieves near total power absorption. The perfect absorption phenomenon can be interpreted as arising from the metasurface inducing equivalent magnetic and electric surface currents that produce far-fields that interfere destructively9. To explore this interpretation, we randomly choose a 1 mm 3 1 mm region of the SEM of Figure 1c, and simulate the fields under plane wave illumination using the finite difference time domain (FDTD) method. The equivalent magnetic and electric surface currents are found from the field distribution using the surface equivalence theorem14. These current distributions are shown as Figure S1 of the Supplementary Materials. It can be seen that the regions of enhanced electric currents are co-located with those of the enhanced magnetic currents. This originates from the SIOM metasurface simultaneously supporting electric and magnetic resonances; we discuss this point further below. These results show that, at the simulated resonance of l 5 530 nm, the total effective magnetic and electric currents, i.e. + +each integrated over the unit cell, are in a ratio (383 1 1.2i ohm) that is close to 377 ohm, i.e. the impedance of free space. As discussed further in the Supplementary Materials, the results therefore predict destructive cancellation of the far-fields produced by these currents, leading to the total absorption phenomenon. We furthermore note that the reflection spectrum predicted by the simulation is in very reasonable agreement with the experimentally-measured spectrum +(Figure S1b of Supplementary Materials). + +The simultaneous existence of magnetic and electric resonances has been previously been studied in relation to total absorption15. To further explore the near-total absorption achieved by the SIOM substrate, we study a simplified model consisting of a silver disk (radius 29 nm) formed on a silver mirror, with an SiO2 spacer layer (50 nm thick) between them. The disks are assumed to be on a square lattice (340 nm period). The results (Figure S2, Supplementary Materials) +reveal that a current loop is formed between silver islands and mirror when illuminated at l 5 525 nm, meaning that the structure has a magnetic resonance. It can also be seen that an electric resonance occurs at the same wavelength (Figure S2b, Supplementary Materials). These resonances generate equivalent electric and magnetic surface currents that (Figure S3, Supplementary Materials), +when integrated over a unit cell, are in a ratio (381 1 7i ohm) very close to 377 ohm, meaning that near-total absorption is also predicted for the simplified SIOM metasurface. This is discussed in further detail in the Supplementary Materials. + +The SIOM metasurface reflectance spectrum is readily tuned by modifying the fabrication process. Varying the evaporation rate presents a means for doing this, as it in turn modifies the sizes of the nanoparticle islands that are produced16,17. In Figure S4 (Supplementary Material), the reflectance spectra of SIOM metasurfaces produced with different silver evaporation rates are shown. It can be seen that increasing the evaporation rate red-shifts the reflectance dip position, which indicates the silver nanoparticle islands increase in size with evaporation rate. Annealing after deposition is another means for modifying the sizes of the silver nanoparticle islands18,19. + +One would also expect that modifying the thickness of the SiO2 layer would permit the reflectance spectrum to be tuned, due to the important role of the nanoparticle island-film coupling in this system. In Figure 3 we show reflectance spectra for SIOM substrates fabricated using methods that are identical, apart from the spacer layer thickness being varied from 30 to 65 nm. It can be seen that the reflectance dip blue-shifts with increasing spacer layer thickness. + +This is in agreement with the previous theoretical studies20,21 that show that the coupling between an optical antenna and mirror (i.e. + +silver mirror here) reduces with their separation, leading to a blueshift of the plasmon resonance. + +![2_image_0.png](2_image_0.png) + +Because our SIOM metasurface achieve near-total optical absorption in a very thin region at its surface, it is intuitive to expect that huge local field enhancement will occur around the silver island nanoparticles. This makes it an ideal candidate for SERS. SERS substrates patterned with high resolution lithography (e.g. e-beam) +achieve very high performance and reproducibility22–29, but have high cost and small active areas. For many applications, however, large area and low cost are critical. This has motivated the development of SERS substrates patterned by methods other than high resolution lithography, e.g. with nanomaterials such as nanospheres30,31 or nanoshells32. Our SIOM metasurfaces, however, present further advantages in this regard, however, as their fabrication does not involve lithography, nor the use of nanomaterials, but rather standard thin film deposition methods. The SIOM metasurface generates larger SERS enhancements than its simpler counterpart that consists of silver islands on an SiO2 substrate. This can be understood by considering the excitation and scattered fields in the SERS process. + +The SIOM metasurface achieves near-total absorption, meaning that the coupling between excitation fields and the plasmonic resonance of the silver islands is more efficient for the SIOM metasurface than for the simpler SERS substrates (silver islands on glass). For the SIOM metasurface, therefore, the field enhancement of the excitation is stronger. This is confirmed by simulations of the electric field intensities produced by the SIOM and by its counterpart consisting of silver islands on glass (Figure S5, Supplementary Materials). The simulation results reveal that SIOM generates peak electric field intensities about ,3.3 times larger than those produced by silver islands on glass. In addition to enhancement of the incident field, the silver mirror reshapes the SERS emission patterns so that the emission is mostly radiated into air26,33, rather than into the substrate. + +For comparison purposes, we characterize the SERS performance not only of our SIOM metasurfaces (Figure 1b), but also that of a substrate consisting of silver nanoparticle islands on indium tin oxide coated glass. The latter is fabricated by evaporating silver at the same rate as that used for the SIOM metasurface. This produces silver nanoparticle islands very similar to those of the SIOM metasurface, as confirmed by scanning electron microscopy (Figure S6, Supplementary Materials). Both types of samples are coated with benzenethiol, and SERS measurements are performed (further details in Methods section) under identical conditions. The results (Figure 4a) show that the SERS signals (and hence signal-to-noise ratios) of all Raman lines are much larger for the SIOM metasurface than for the silver island nanoparticles on glass. This indicates that the SIOM metasurface produces considerably stronger SERS +enhancements than do the silver nanoparticle islands on SiO2. To quantify the improvement, we analyze the 1072 cm21 and 1586 cm21 Raman lines. The SERS enhancement factor of these two Raman lines for the SIOM metasurface and the silver nanoparticle islands on glass are calculated by comparing the SERS measurements of Figure 4a with measurements made on pure benzenethiol liquid using the technique shown in a previous study24 and described in the Methods section. + +The experimentally-determined SERS enhancement factors of the 1072 cm21 and 1586 cm21 Raman lines of the SIOM metasurface are 4.3 3 107, and 4.4 3 107, respectively. By contrast, for the same Raman lines, the silver island nanoparticles on glass exhibit measured enhancement factors of 2.5 3 106 and 1.8 3 106. The enhancement factors of the 1072 cm21 and 1586 cm21 Raman lines of the SIOM +metasurface are therefore respectively 17 and 24 times larger than those of the silver island nanoparticles on glass. The more than one order of magnitude boost in enhancement factor results from improvement in the coupling between the incident light and plasmon resonance of the SIOM metasurface. The Raman scattering is furthermore efficiently collected due to the presence of the silver mirror. + +We next investigate the uniformity in SERS enhancement with position on the SIOM metasurface. A 300 mm 3 300 mm region of + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +Figure 4 | SERS measurement. (a). SERS spectra measured from SIOM +metasurface (blue line) and silver island nanoparticles on SiO2 (red line) +coated with benzenethiol. For display purposes, Raman spectrum from SIOM metasurface is shifted vertically by 7000 Raman counts and Raman spectrum from silver nanoparticle islands on glass is enlarged by 5 times. + +(b). Mapping of SERS signal of 1586 cm21 Raman line over SIOM +metasurface. Plotted quantity is intensity of 1586 cm21 Raman line with background subtracted. + +the SIOM metasurface is scanned with a step size of 30 mm, with a SERS spectrum obtained at each stage position. The measurement parameters are described in the Methods section. We plot the intensity of the 1586 cm21 Raman line with the background removed as a function of stage position (Figure 4b). It can be seen that the SERS enhancement is very uniform, with the standard deviation being only +,4% of the mean. The other Raman lines and the other regions of the SIOM metasurface also show similar results. We anticipate that the very high degree of uniformity of the SIOM metasurface would be advantageous for many applications. + +The initial reports of single molecule SERS (SMSERS)12,13 garnered considerable interest, but the demonstration of a large area substrate with SMSERS sensitivity has proven elusive. Furthermore, previous reports of SMSERS have been performed almost entirely using aggregates of Ag colloidal nanoparticles that are very heterogeneous, with generally fewer than 1% being SMSERS-active34. Here, we demonstrate that our SIOM metasurfaces have SMSERS sensitivity. + +This is performed using the isotopologue method35,36. We introduce two forms of Rhodamine 6G (R6G-d0 and R6G-d4) to the SIOM +substrates at low concentration (0.2 nM). As discussed further below, this allows us to prove that the SERS spectra we then measure are predominantly from single molecules. + +The isotopologue method we use for verifying that SMSERS is achieved35 employs two forms of Rhodamine 6G that differ only in the isotopic composition of their atoms, with the hydrogen atoms in R6G-d0 being replaced by deuterium atoms in R6G-d4. The isotopologues adsorb to the metasurface with identical affinities, yet can be distinguished by shifts in their vibrational features. The Raman line of R6G-d0 at ,620 cm21 shifts to ,610 cm21 in R6G-d4, for example. + +This presents a means for determining whether a measured SERS spectrum originates from one or from both types of molecules. If the SMSERS regime has been achieved, and the SERS spectra are from predominantly single molecules, then these spectra should indicate the presence of chiefly one isotopologue (R6G-d0 or R6G-d4), with both types of molecules being simultaneously present only infrequently. + +After the R6G isotopologues are introduced, the SIOM metasurface is scanned and SERS measurements are made (see Methods section). Measurements are made at 50625 stage positions, of which 7059 yield spectra that clearly contain Raman features. The criterion for a spectrum containing Raman features is that the height of the Raman peak (after background subtraction) is larger than the sum of the average value and one standard deviation. The average value and standard deviation are found from analysis of the spectra obtained at all 50625 stage positions. Representative spectra are included as Figure 5a. Each measured SERS spectrum is classified into one of three categories: it contains no R6G features, or it contains only R6Gd0 features, or it contains only R6G-d4 features, or it contains both R6G-d0 and d4 features. The results are shown as a histogram in Figure 5b. That the measured spectra contain features of both isotopologues simultaneously on an infrequent basis indicates that the SMSERS regime is achieved. This can be further quantified with a Poisson-binomial model35. The histogram of Figure 5b shows that the number of events with no molecules (null events), R6G-d0 only, both R6G-d0 and d4, and R6G-d4 events are in a ratio of 4356653407526753385, i.e. a normalized ratio of ,163512.85 1512.7. From the SEM of the SIOM metasurface (Figure 1c), it can be seen that many nanoparticles would be within the laser spot employed in the SERS measurements. It is entirely possible, therefore, that within the laser spot, there are general several locations of the SIOM metasurfaces at which the enhancement is sufficiently strong so that SMSERS is possible. One may think of the union of these regions as the hot spot region. A Poisson-Binomial model with on average 0.15 molecules within the hot spot region predicts that 86.1% will contain no molecules, 6.7% will contain only R6G-d0 molecules, 6.7% will contain only R6G-d4 molecules, and 0.52% will contain both R6G-d0 and R6G-d4. Such a model would therefore predict that, for the 50625 measurement points, the numbers with no molecules, R6G-d0 only, both R6G-d0 and d4, and R6G-d4 only would be 4357353394526453394, i.e. a normalized ratio of +,165512.8551512.85. This is in very reasonable agreement with the experimentally-measured ratio, confirming the validity of the model. The model further predicts that the 6.7% of events containing only one isotopologue comprises 6.45% having solely one molecule, with the remaining 0.25% having two or more molecules. One may therefore conclude that ,96.3% of SERS spectra containing features of only one isotopologue originate from a single molecule, with the remaining ,3.7% originating from two or more molecules. + +## Discussion + +In conclusion, we demonstrate a visible-wavelength metasurface that achieves near-total power absorption and exhibits single molecule sensitivity. Our SIOM metasurface consists of silver nanoparticle islands over a silver mirror, and is therefore readily fabricated on a wafer-scale using standard thin film depositions. The SIOM metasurface supports magnetic and electric resonances. These can be considered to generate effective electric and magnetic currents that + +![4_image_0.png](4_image_0.png) + +Figure 5 | Experimental results of SMSERS on SIOM metasurface. (a). Representative Raman spectra of R6G-d0 molecule and R6G-d4 molecule at single molecule level. R6G-d4 spectrum is shifted vertically by 2000 counts for display purposes. (b). Histogram of single molecule level events: null (purple), R6G-d0 molecule only (blue), both R6G-d0 and R6G-d4 molecules (green), and R6G-d4 molecule only (red). + +generate far-fields that cancel, leading to zero reflection and complete absorption. We show that the spectral properties of the SIOM metasurface can be tuned by modifying its geometry via changing the fabrication parameters. As one might intuitively expect, the total absorption property is accompanied by very strong local field enhancement. We demonstrate that this is highly advantageous for SERS. Measurements show that our SIOM metasurfaces exhibit SERS +enhancement factors that are more than one order of magnitude higher than those of silver nanoparticle islands on glass. The results also demonstrate a very high degree of uniformity over the SIOM substrate, with the standard deviation in SERS signal being only +,4% of the mean. We demonstrate that the high enhancement factor permits the detection of the Raman spectra of single molecules. Due to the uniformity of our SIOM substrate and its large area, we are able to present an unparalleled number (.7000) of single molecule events. + +We anticipate that, due to their high performance, large area, and low fabrication cost, SIOM metasurfaces could be highly appropriate for many applications in SERS, such as food safety monitoring37–39, explosive detection40,41, and pharmaceutical quality control42,43. + +## Methods + +Fabrication. The fabrication of SIOM metasurface begins with evaporation (1 A˚/sec) +of titanium to a thickness of 5 nm on a silicon substrate to form an adhesion layer. + +After this, silver is deposited to a thickness of 120 nm on the titanium layer by evaporation (1 A˚/sec). A 50-nm thick SiO2 layer is then deposited by sputtering (0.4 A˚/sec). Silver is then evaporated at a slow rate (0.2 A˚/sec), thereby producing silver nanoparticle islands. The duration of this evaporation step is such that the amount of silver deposited is equivalent to the amount that would be contained in 6.5 nm thick film, as measured by crystal oscillation monitor. These four steps complete the fabrication of the SIOM metasurface. Reflection spectra measurement. In the reflection spectra measurements, the SIOM metasurface is illuminated by light from a halogen lamp focused by a microscope objective (NA 5 0.15, magnification 53). The reflected light from the metasurface is collected by the same objective lens and analyzed by the spectrometer equipped with a thermoelectrically-cooled charge couple device (CCD). The reflected light from an area of the same sample containing only the SiO2 spacer and silver mirror (i.e. no silver nanoparticle islands) is measured as reference. The normalized reflection spectrum is then found by dividing the spectrum measured from the SIOM by the reference spectrum. This is then multiplied by the theoretical reflection spectrum of the sample containing only the SiO2 spacer and silver mirror found using the method of Ref. 14. This yields the SIOM reflection spectrum in absolute terms. SERS measurement. The SERS measurement with benzenethiol are performed as follows. The samples are immersed in a solution of benzenethiol in ethanol (3 3 1023 mol/L) for 1 hour. This results in a self-assembled monolayer of benzenethiol on the silver islands. After removal from the benzenethiol solution, the samples are rinsed in ethanol and blown dry with nitrogen. SERS measurements are made with a Raman microscope (Horiba Jobin Yvon, LabRam) equipped with a spectrometer and a thermoelectrically-cooled charge coupled device (CCD). The excitation laser wavelength is l 5 532 nm and the power impinging upon the substrate is 0.03 mW. + +These parameters are used for the measurements both on the SIOM metasurface and on the silver nanoparticle islands on glass. An objective lens (NA 5 0.15, magnification 53) is used to focus the excitation laser on the sample and collect the emitted Raman signals. The signal accumulation time for the CCD detector is 10 seconds. + +For the SMSERS measurement, the SIOM substrate is soaked in a solution containing the isotopologues R6G-d0 and R6G-d4 in methanol (0.2 nM each) for 12 hours, washed in methanol, then blown dry with nitrogen. The SIOM substrate is then scanned in the Raman microscope with a step size of 4 mm across an 896 mm 3 896 mm region of SIOM metasurface. In the microscope, a laser beam (l 5 532 nm) +is focused onto the substrate with a microscope objective (NA 5 0.9, magnification 1003). The laser power incident on the chip is 0.03 mW. The same objective lens is used to collect the Raman signal into a spectrometer equipped with CCD sensor. The integration time of acquiring the Raman spectrum for each scan position is 0.5 seconds. + +SERS enhancement factor. Measurements are made of Raman scattering from pure benzenethiol liquid sandwiched between a microscope slide and a coverslip. The benzenethiol thickness (500 mm) is defined using a spacer to separate the slide and coverslip. The SERS enhancement factor is then given by EF~ ISRES=NSERS +IREF =NREF +, where ISERS and IREF are the signal intensities measured in the SERS and Raman experiments, respectively. These intensities are the heights of the Raman lines in the spectra with the background subtracted.NSERS andNREF are the numbers of molecules in the SERS and Raman experiments, respectively. NSERS is given by the area of the exposed silver surfaces of the SIOM substrate or simple silver islands multiplied by the packing density of benzenethiol (6.8 3 1014 cm22) +44. To estimate the area of the exposed silver surfaces, we model each silver nanoparticle island as a cylindrical pillar whose radius is chosen to yield a cross sectional area matching that of the silver nanoparticle island, as determined from the SEM image. According to the measurements made using a crystal oscillation monitor, the amount of silver deposited is equivalent to that contained in a 6.5 nm thick film. The heights of the pillars are therefore estimated by equating their volume to that of a 6.5 nm thick film. For the SIOM metasurface shown in Figure 1c for example, this yields an estimated height of 14 nm. The estimated surface area of the exposed silver surfaces is taken as the areas of the tops and sides of the cylinders. NREF is given by the confocal collection volume of the microscope multiplied by the density of pure benzenethiol (9.8 mol/L). The collection volume is found by measuring the Raman signal from a silicon wafer as a function of its position along the optical axis24. + +1. Niesler, F. B. P., Gansel, J. K., Fischbach, S. & Wegener, M. Metamaterial metalbased bolometers. Appl. Phys. Lett. 100, 203508 (2012). + +2. Zhu, J.et al. Optical absorption enhancement in amorphous silicon nanowire and nanocone arrays. Nano Lett. 9, 279–82 (2009). + +3. Thomann, I. et al. Plasmon enhanced solar-to-fuel energy conversion. Nano Lett. + +11, 3440–6 (2011). + +4. Hao, J. et al. High performance optical absorber based on a plasmonic metamaterial. Appl. Phys. Lett. 96, 251104 (2010). + +5. Avitzour, Y., Urzhumov, Y. & Shvets, G. Wide-angle infrared absorber based on a negative-index plasmonic metamaterial. Phys. Rev. B 79, 045131 (2009). + +6. Liu, N., Mesch, M., Weiss, T., Hentschel, M. & Giessen, H. Infrared perfect absorber and its application as plasmonic sensor. Nano Lett. 10, 2342–8 (2010). + +7. Wu, C. et al. Large-area wide-angle spectrally selective plasmonic absorber. Phys. + +Rev. B 84, 1–7 (2011). + +8. Tittl, A. et al. Palladium-based plasmonic perfect absorber in the visible wavelength range and its application to hydrogen sensing. Nano Lett. 11, 4366–9 (2011). + +9. Moreau, A. et al. Controlled-reflectance surfaces with film-coupled colloidal nanoantennas. Nature 492, 86–89 (2012). + +10. Fleischmann, M., Hendra, P. J., McQuillan, A. J. Raman spectra of pyridine absorbed at a silver electrode. Chem. Phys. Lett. 26, 163–6 (1974). + +11. Jeanmaire, D. L. & Van Duyne, R. P. Surface Raman electrochemistry part I. + +heterocyclic, aromatic and aliphatic amines adsorbed on the anodized silver electrode. J. Electroanal. Chem. and Interfacial Electrochem. 84, 1–20 (1977). + +12. Nie, S. & Emory, S. R. Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 275, 1102–1106 (1997). + +13. Kneipp, K. et al. Single molecule detection using surface-enhanced Raman scattering (SERS). Phys. Rev. Lett. 78, 1667–1670 (1997). + +14. Kong, J. A. Electromagnetic wave theory. (Wiley, 1990). 15. Dayal, G. & Ramakrishna, S. Design of highly absorbing metamaterials for Infrared frequencies. Opt. Express 20, 17503–17508 (2012). + +16. Nishikawa, Y., Nagasawa, T., Fujiwara, K. & Osawa, M. Silver island films for surface-enhanced infrared absorption spectroscopy: effect of island morphology on the absorption enhancement. Vib. Spectrosc. 6, 43–53 (1993). + +17. Aroca, R., Ross, D. & Domingo, C. Surface-Enhanced Infrared Spectroscopy. Appl. + +Spectrosc. 58, 324A–338A (2004). + +18. Wang, Q. et al. Plasmonic-enhanced quantum efficiency of europium complex using annealed silver island films. J. Opt. Soc. Am. B 28, 220–4 (2011). + +19. Royer, P., Goudonnet, J. P., Warmack, R. J. & Ferrell, T. L. Substrate effects on surface-plasmon spectra in metal-island film. Phys. Rev. B 35, 3753–3759 (1987). + +20. Le´veˆque, G. & Martin, O. J. F. Tunable composite nanoparticle for plasmonics. + +Opt. Lett. 31, 2750–2 (2006). + +21. Le´veˆque, G. & Martin, O. J. F. Optical interactions in a plasmonic particle coupled to a metallic film. Opt. Express 14, 9971–81 (2006). + +22. Crozier, K. B., Sundaramurthy, A., Kino, G. S. & Quate, C. F. Optical antennas: +Resonators for local field enhancement. J. Appl. Phys. 94, 4632–4642 (2003). + +23. Banaee, M. G. & Crozier, K. B. Gold nanorings as substrates for surface-enhanced Raman scattering. Opt. Lett. 35, 760–2 (2010). + +24. Chu, Y., Banaee, M. G. & Crozier, K. B. Double-Resonance plasmon substrates for surface-enhanced Raman scattering stokes frequencies. ACS Nano 4, 2804–2810 +(2010). + +25. Zhu, W., Banaee, M. G., Wang, D., Chu, Y. & Crozier, K. B. Lithographically fabricated optical antennas with gaps well below 10 nm. Small 7, 1761–6 (2011). + +26. Wang, D., Zhu, W., Chu, Y. & Crozier, K. B. High directivity optical antenna substrates for surface enhanced Raman scattering. Adv. Mater. 24, 4376–80 (2012). + +27. Seok, T. J. et al. Radiation engineering of optical antennas for maximum field enhancement. Nano Lett. 11, 2606–10 (2011). + +28. Smythe, E., Dickey, M., Bao, J., Whitesides, G. M. & Capasso, F. Optical antenna arrays on a fiber facet for in situ surface-enhanced Raman scattering detection. Nano Lett. 9, 1132–1138 (2009). + +29. Zhang, W., Fischer, H., Schmid, T., Zenobi, R. & Martin, O. J. F. Mode-Selective Surface-Enhanced Raman Spectroscopy Using Nanofabricated Plasmonic Dipole Antennas. J. Phys. Chem. C 113, 14672–14675 (2009). + +30. Biggs, K. B., Camden, J. P., Anker, J. N. & Van Duyne, R. P. Surface-enhanced Raman spectroscopy of benzenethiol adsorbed from the gas phase onto silver film over nanosphere surfaces: determination of the sticking probability and detection limit time. J. Phys. Chem. A 113, 4581–6 (2009). + +31. Ormonde, A. D., Hicks, E. C. M., Castillo, J. & Van Duyne, R. P. Nanosphere lithography: fabrication of large-area Ag nanoparticle arrays by convective selfassembly and their characterization by scanning UV-visible extinction spectroscopy. Langmuir 20, 6927–31 (2004). + +32. Jackson, J. B., Westcott, S. L., Hirsch, L. R., West, J. L. & Halas, N. J. Controlling the surface enhanced Raman effect via the nanoshell geometry. Appl. Phys. Lett. 82, 257 (2003). + +33. Ahmed, A. & Gordon, R. Directivity enhanced Raman spectroscopy using nanoantennas. Nano Lett. 11, 1800–3 (2011). + +34. Camden, J. P. et al. Probing the structure of single-molecule surface-enhanced Raman scattering hot spots. J. Amer. Chem. Soc. 130, 12616–7 (2008). + +35. Dieringer, J. A., Lettan, R. B., Scheidt, K. A. & Van Duyne, R. P. A frequency domain existence proof of single-molecule surface-enhanced Raman spectroscopy. J. Amer. Chem. Soc. 129, 16249–56 (2007). + +36. Kleinman, S. L. et al. Single-molecule surface-enhanced Raman spectroscopy of crystal violet isotopologues: theory and experiment. J. Amer. Chem. Soc. 133, 4115–22 (2011). + +37. Liu, Y. et al. Potential of Raman spectroscopy and imaging methods for rapid and routine screening of the presence of melamine in animal feed and foods. Appl. + +Spectrosc. 63, 477–480 (2009). + +38. He, L. Application of surface enhanced Raman spectroscopy to food safety issues. + +148 (ProQuest, UMI Dissertation Publishing: 2012). + +39. Qin, J., Chao, K. & Kim, M. S. Raman chemical imaging system for food safety and quality inspection. T. ASABE 53, 1873–1882 (2010). + +40. Cheng, C., Li, Z., Liu, W. & Wu, J. Stand-Off explosives detection based on Raman spectroscopy. CORD Conf. Proc. 1–4 (2010). + +41. Izake, E. L. Forensic and homeland security applications of modern portable Raman spectroscopy. Forensic Sci. Int. 202, 1–8 (2010). + +42. Vankeirsbilck, T. et al. Applications of Raman spectroscopy in pharmaceutical analysis. TrAC Trends Anal. Chem. 21, 869–877 (2002). + +43. Wartewig, S. & Neubert, R. H. H. Pharmaceutical applications of Mid-IR and Raman spectroscopy. Adv. Drug Deliv. Rev. 57, 1144–70 (2005). + +44. McFarland, A. D., Young, M. A., Dieringer, J. A. & Van Duyne, R. P. Wavelengthscanned surface-enhanced Raman excitation spectroscopy. J. Phys. Chem. B 109, 11279–85 (2005). + +## Acknowledgments + +This work was supported by the National Science Foundation (NSF, grant numbers ECCS-0747560 and ECCS-1201687), the Harvard Quantum Optics Center, and by the Center for Excitonics, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science and Office of Basic Energy Sciences under Award Number DE-SC0001088. This work was also supported by the UT/ORNL Joint Institute for Advanced Materials, and the U.S. Department of Energy, Office of Basic Energy Sciences, under Award Number DESC0004792 (J.P.C.) and the National Science Foundation under Awards CHE-0954297 and DMR-0906752 (M.D.B.). Fabrication work was carried out in the Harvard Center for Nanoscale Systems, which is supported by the NSF. + +Author contributions D.W., W.Z., J.P.C. and K.B.C. developed the concept. D.W. performed fabrication and simulations. D.W. and W.Z. performed experiments and data analysis. J.P.C. and M.D.B. + +produced and characterized the isotopologues. D.W. and K.B.C. wrote the manuscript. + +## Additional Information Supplementary Information Accompanies This Paper At Http://Www.Nature.Com/ + +scientificreports Competing financial interests: The authors declare no competing financial interests. How to cite this article: Wang, D., Zhu, W., Best, M.D., Camden, J.P. & Crozier, K.B. + +Wafer-scale metasurface for total power absorption, local field enhancement and single molecule Raman spectroscopy. Sci. Rep. 3, 2867; DOI:10.1038/srep02867 (2013). + +This work is licensed under a Creative Commons AttributionNonCommercial-NoDerivs 3.0 Unported license. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0 \ No newline at end of file diff --git a/medical/md/PMC4027918.md b/medical/md/PMC4027918.md new file mode 100644 index 0000000000000000000000000000000000000000..bd1dead2dbc6e8ac48949b93499e307c88479b4b --- /dev/null +++ b/medical/md/PMC4027918.md @@ -0,0 +1,276 @@ +Open Access Full Text Article + +# O R Iginal Resea R C H Open Access To Scientific And Medical Research Blood And Urine Levels Of Heavy Metal Pollutants In Female And Male Patients With Coronary Artery Disease + +Michael Sponder1 Monika Fritzer-Szekeres2 Rodrig Marculescu2 Martina Mittlböck3 Maria Uhl4 Birgit Köhler-Vallant5 Jeanette Strametz-Juranek1 1Department of Cardiology, Medical University of Vienna, Vienna, Austria; 2Department of Medical-Chemical Laboratory Analysis, Medical University of Vienna, Vienna, Austria; 3Department of Medical Statistics, Informatics and Intelligent Systems, Section for Clinical Biometrics, Medical University of Vienna, Vienna, Austria; 4Department of Pollutants and Human, Environment Agency Austria, Vienna, Austria; 5Department of Inorganic Analysis, Environment Agency Austria, Vienna, Austria Correspondence: Michael Sponder Medical University of Vienna, Department of Cardiology, Währinger Gürtel 18-20, 1090 Vienna, Austria Tel +43 650 261 93 93 Fax +43 40 400 42 16 Email michael.sponder@meduniwien.ac.at Background: Heavy metal pollutants such as cadmium (Cd), lead (Pb), and mercury (Hg) are rarely the subjects of cardiovascular research although they have been suspected for decades to negatively impact the circulatory system. + +Methods: Apart from detailed anamnestic data, urinary levels of Cd and full blood levels of Pb and Hg were measured in 53 female (mean age: 68.04±7.03 years) and 111 male (mean age: 60.68±11.43 years) nonsmoking or never-smoking patients with angiographically verified and precisely quantified coronary artery disease (CAD). + +Results: Although Cd was quantifiable in 68.3% of subjects, only 34.1% of these patients exceeded the critical 1 µg/L Human Biomonitoring (HBM)-I level. Median Pb (20 µg/L) and Hg +(0.55 µg/L) levels were lower than the HBM-I, as well as reference levels of Pb. Wine consumption was the main source for Pb, fish and wine consumption for Hg, and previous nicotine abuse for Cd. There was no correlation between Cd, Pb, or Hg and severity of CAD although severity correlated positively with atherosclerosis parameters (uric acid, creatinine, triglycerides, blood urea nitrogen, C-reactive protein) and negatively with high density lipoprotein cholesterol. + +Conclusion: Cd levels detected in CAD patients were high compared to German and European reference levels but it could not be proven that urine levels of Cd and blood levels of Hg or Pb played a major role in the genesis of CAD, particularly when compared to well-known biomarkers such as blood pressure, glucose, and lipids. + +Keywords: cadmium, plumbum, mercury, coronary artery disease, pollutants + +## Introduction + +According to the European Cardiovascular Disease Statistics 20121 cardiovascular disease (CVD) is responsible for 47% of all deaths in Europe and 40% of deaths in the European Union. The most common coronary manifestations of atherosclerosis are acute coronary syndromes such as myocardial infarction or sudden cardiac death and stable angina pectoris. Of course the mortality rate for CVD strongly depends on several risk factors but in general, men are more likely to die of CVD at a rate of 2.5–4.5:1.2 Independent of sex-specific differences in risk factors, the absolute level of coronary artery disease (CAD) in men is higher than in women, although the dose response of most risk factors is the same in women and men, even after menopause, a fact that has been known for a long time.3,4 Traditional risk factors causing or promoting CAD are nicotine abuse, diabetes mellitus, arterial hypertension, dyslipidemia, physical inactivity, and overweight/obesity.5 However, heavy metal pollutants such as cadmium (Cd), lead (Pb), and mercury (Hg) may also contribute to the genesis of coronary atherosclerosis but they are far less commonly subjected to cardiovascular research http://dx.doi.org/10.2147/VHRM.S61510 although the first hints for this assumption were found nearly a century ago.6 Cd was associated with an increased risk for suffering hypertension, cardiac arrest, and stroke.7–9 Messner et al10 found a significant association between Cd levels in serum and intima media thickness in female individuals aged 18–22 years. Prozialeck11 suggested E-cadherin, a C++-binding protein localized at the junctions between vascular endothelial cells, as a target for Cd toxicity. Cd is also able to induce the expression of intercellular adhesion molecule 1, an adhesion molecule playing an important role in inflammation.12 Furthermore, Cd was shown to indirectly generate superoxide and hydroxyl radicals.13 Similar to Cd, increased Hg exposure was also suspected to lead to hypertension and cerebral artery occlusion.14–16 Wojciechowski and Kowalski,17 working on rabbits, showed that inhaling Hg vapor over 3 months caused electrocardiogram (ECG) abnormalities, thrombotic lesions in small and medium caliber blood vessels, and necrotic foci. Guallar et al18 performed a case-controlled study in eight European countries and Israel and found that amounts of Hg in toenails were 15% higher in a group of patients with myocardial infarction compared to controls. Again, similar to Cd, Salonen et al19 found a correlation between Hg amounts in hair and intima media thickness in men from eastern Finland. Like Cd and Hg, Pb measured in blood and bone (tibia and patella) was associated with hypertension, probably by affecting the renin–angiotensin system, increasing angiotensin converting enzyme levels20,21 or by influencing the beta-adrenergic system as suggested by Tsao et al.22 Another explanation may be an impairment of the balance between vasoconstrictive and vasodilatative factors to the benefit of vasoconstriction.23 Furthermore, a damaging effect of Pb on the endothelium may be due to its potential to increase reactive oxygen species. Reactive oxygen species may also be involved in blood pressure increase by increasing Ca++ concentration in endothelial cells or by the inactivation of nitrogen oxide.24 The results of the mentioned studies indicate that Cd, Hg, and Pb may increase cardiovascular risk by promoting atherosclerosis per se or by ameliorating the circumstances for atherosclerotic processes. + +The present study measured Cd levels in urine and Hg and Pb levels in blood, and correlated them with the severity of CAD with regard to sex and sex-specific aspects, and compared them with other well established cardiovascular risk factors, eg, blood pressure, blood glucose, and lipids. + +## Materials And Methods + +In total, 111 male and 53 female never-smoking or exsmoking (for at least 7 years) patients with angiographically verified CAD of differing severity were recruited at the Department of Cardiology, Medical University of Vienna. The study group consisted of 49.4% of patients with chronic CAD, defined as at least one coronary ischemic event in the past documented by coronary angiography, and 50.6% of patients who had never had an ischemic event. + +Detailed anamnestic data were collected by means of an interview taking into account exposure to heavy metal sources like housing, nutrition, past smoking behavior, amalgam fillings, and other factors. Alcohol intake was quantified in units +(1 unit = 0.02 L of spirits, 0.25 L of wine, or 0.3 L of beer). + +Meat and fish intake were quantified by times/week. Tap water consumption was measured in L/week. Blood pressure was measured at least three times during the in-patient stay. An ECG was taken, routine laboratory parameters were measured, and all patients had to undergo an angiography for diagnostic and/or therapeutic reasons on the grounds of their underlying disease. The coronary arteries were divided into 17 segments (left main, proximal/medial/distal left anterior descending artery, ramus circumflexus, first and second marginal branch, posterolateral branch, first and second diagonal branch, proximal/medial/distal left circumflex artery, proximal/medial/ +distal right coronary artery, ramus interventricularis posterior) and stenosis grade for each segment was measured. A simple 3-point grading system (coronary score) was developed considering both frequency and severity of CAD: 0 points for non-stenosed or only calcified segments, 1 point for each stenosis from 30%–,50%, 2 points for each stenosis from 50%–,70%, and 3 points for each stenosis .70%. Hg and Pb levels were measured in full blood, and Cd was measured in urine. The outcrop of full blood samples for the determination of Pb and Hg was done by 2 mL ultrapure water and 2 mL nitric acid (68% subboiled). The sample aliquot was 0.5 mL and the backfilling volume was 20 mL. The determination of Pb and Cd was performed by inductively coupled plasma mass spectrometry according to the ÖNORM EN ISO 17294-2. The determination of Hg was done by atomic fluorescence spectroscopy according to the ÖNORM EN 17852. The outcrop of urine samples for the determination of Cd was done by 2 mL ultrapure water and 2 mL nitric acid (68% subboiled). The sample aliquot was 5 mL and the backfilling volume was 20 mL. The detection/quantification limits were 0.4/2 µg/L for Pb, 0.067/0.13 µg/L for Hg, and 0.12/0.40 µg/L for Cd. In case the quantification limit was undercut, the following expected amounts were used: Cd, 0.3 µg/L; Pb, 1 µg/L; Hg, 0.1 µg/L. + +Cd and Hg Human Biomonitoring (HBM)-I and -II values and the Pb reference values from the German Environmental Agency25,26 were used for comparison. + +HBM values are derived on the basis of toxicological and epidemiological studies and are a tool to quickly assess whether exposure related health effects can be expected. However, they do not reflect the total body burden of heavy metal pollutants. At a concentration level higher than the HBM-I it is suggested that analytic results be verified to identify specific sources of exposure and to reduce the exposure in an adequate way. At levels higher than HBM-II values immediate action to reduce exposure is required. The reference values permit the assessment of exposure of individuals or population groups compared to ubiquitous background exposure. In the case of Pb, the German Commission has suspended the HBM values for Pb in blood due to the fact that no threshold can be derived for the persisting adverse effects on brain development and also due to the carcinogenicity of Pb compounds.27,28 Statistical analysis was done with SPSS 20.0 (IBM +Corporation, Armonk, NY, USA). Continuous and normally distributed data are described by mean ± standard deviation, group differences are tested by independent sample t-test, and correlation is calculated using Pearson's correlation coefficient. Continuous data with skew distribution or outliers are described by median, first and third quartile, and minimum and maximum values. Group differences are tested by Mann–Whitney U test and correlations were calculated using Spearman's correlation. For data with values below the quantification limit a value below quantification limit was imputed +(the same value for all observations) and the nonparametric Mann–Whitney U test was used. All tests were performed two-sided and P\#0.05 were considered significant. The protocol was approved by the Ethical Commission of the Medical University of Vienna and informed consent was obtained from patients. + +## Results + +Data concerning age, body mass index (BMI), heart rate, blood pressure, and ECG are shown in Table 1. Male patients were significantly younger compared to female patients +(P,0.01). There was no significant difference between BMI (P=0.22), systolic blood pressure (P=0.88), heart rate +(P=0.99), and ECG data. However, men showed a significantly higher diastolic blood pressure compared to women +(P,0.01). Atrial fibrillation was observed in 13 male and three female patients, PQ in patients without atrial fibrillation was 169.07±28.98 ms in male and 155.14±29.22 ms in female patients (P,0.01). The prevalence of traditional cardiovascular risk factors is shown in Table 2. The prevalence of hypertension and previous nicotine abuse was higher in + +| electrocardiogram data Female | Male | Total | P-value | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|--------------|--------------|-------| +| Age (years) | 68.04±7.03 | 60.68±11.43 | 63.05±10.96 | ,0.01 | +| BMI (kg/m2 ) | 27.28±5.35 | 28.40±5.50 | 28.04±5.46 | 0.22 | +| SBP (mmHg) | 131.26±16.08 | 130.86±15.36 | 130.99±15.55 | 0.88 | +| DBP (mmHg) | 72.15±8.28 | 78.41±11.35 | 76.39±10.84 | ,0.01 | +| Heart rate | 70.57±14.01 | 70.53±12.85 | 70.54±13.19 | 0.99 | +| (bpm) PQ (ms) | 155.14±29.22 | 169.07±28.96 | 164.36±29.70 | ,0.01 | +| QTc (ms) | 453.08±41.57 | 440.33±43.22 | 444.45±42.98 | 0.08 | +| QRS (ms) | 104.74±28.59 | 106.01±44.62 | 105.60±40.06 | 0.85 | +| Notes: Anthropometric data, systolic and diastolic blood pressure, heart rate, and electrocardiogram data of the coronary artery disease patients. Data is presented as | | | | | + +male patients whereas female patients had a higher prevalence of diabetes mellitus and physical inactivity, defined as less than 3 hours of walking per week. + +Parameters of special interest from routine laboratory analyses are shown in Table 3. Severity of CAD is described in detail in the Materials and methods. Female patients had a mean coronary score of 6.43±5.87 points with a maximum of 24 points. Male patients had a mean coronary score of 8.46±5.29 with a maximum of 29 points. + +The levels of Cd, Pb, and Hg among the CAD patients were compared to levels within the German population as well as with HBM-I/II and reference levels.25,26 Mean Cd, Pb, and Hg levels in the German population were 0.335 µg/L, 35.6 µg/L, and 0.86 µg/L, respectively. The HBM-I level for Cd is 1 µg/L and 5 µg/L for Hg. The HBM-II levels are 4 µg/L and 15 µg/L for Cd and Hg, respectively. There are no HBM levels for Pb. Reference levels are 0.8 µg/L for Cd, 2.0 µg/L for Hg, and 70 and 90 µg/L for Pb in women and men, respectively. The heavy metal levels in the CAD patients are + +| Table 2 Prevalence of traditional risk factors Female Male Total | P-value | | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|-----------|------------|-------| +| Hypertension | 94.3 (50) | 99.3 (98) | 90.2 (148) | 0.22 | +| Pos Fam history | 54.7 (29) | 55.0 (61) | 54.9 (90) | 0.98 | +| Diabetes mellitus | 37.8 (20) | 22.5 (25) | 45.0 (17) | 0.04 | +| Diabetes (no insulin) | 20.8 (11) | 12.6 (14) | 15.2 (25) | | +| Diabetes (insulin) | 17.0 (9) | 9.9 (11) | 12.2 (20) | | +| Hyperlipidemia | 88.7 (47) | 89.2 (99) | 89.0 (146) | 0.92 | +| Previous MI/stroke | 41.5 (22) | 40.5 (35) | 40.9 (67) | 0.97 | +| Ex-smoking | 39.6 (21) | 65.8 (73) | 57.3 (94) | ,0.01 | +| Physical inactivity | 30.2 (16) | 27.9 (31) | 28.7 (47) | 0.77 | +| Notes: Prevalence in percent (n) of traditional cardiovascular risk factors for female and male coronary artery disease patients. Physical inactivity was defined as ,3 hours of walking per week. P-values show the sex-specific statistical difference. | | | | | + +Sponder et al + +| Table 3 Important routine laboratory parameters Female | Male | Total | P-value | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|------------------|------------------|-------| +| Uric acid (mg/dL) | 5.85±1.89 | 6.64±2.03 | 6.39±2.01 | 0.02 | +| Triglycerides (mg/dL) | 131 (97–169) | 130 (94–183) | 130 (95–176) | 0.60 | +| Cholesterol (mg/dL) | 19.222±48.37 | 168.45±48.63 | 175.98±49.66 | ,0.01 | +| HDL (mg/dL) | 54.83±15.58 | 41.36±10.21 | 45.92±13.81 | ,0.01 | +| LDL (mg/dL) | 111.98±41.73 | 100.91±38.14 | 104.65±39.59 | 0.12 | +| LDL/HDL (mg/dL) | 2.14±0.88 | 2.56±1.09 | 2.42±1.04 | 0.02 | +| HbA1c (rel %) | 6.1 (5.7–7.0) | 5.9 (5.4–6.4) | 6.0 (5.5–6.9) | 0.10 | +| CRP (mg/dL) | 0.50 (0.17–1.60) | 0.62 (0.22–2.84) | 0.59 (0.20–2.42) | 0.36 | +| Notes: Extract of routine laboratory parameters of special interest. Data are presented as mean ± standard deviation or median (first to third quartile). P-values show the sex-specific statistical difference. Abbreviations: CRP, C-reactive protein; HDL, high density lipoprotein; HbA1c, glycated hemoglobin; LDL, low density lipoprotein. | | | | | + +shown in Table 4. Cd was quantifiable in 68.3% of patients, Pb in 100% of patients, and Hg in 83.5% of patients, and the remaining patients were below the quantification limit. Concerning Cd, female patients had higher levels compared to men but men showed higher levels of Pb and Hg for which the difference was significant (P=0.02). The P-values (and correlation coefficients) for the correlations of the coronary score with Cd, Pb, and Hg in female patients was 0.64 (0.07), +0.06 (−0.26), and 0.51 (0.09); for male patients it was 0.65 (−0.05), 0.79 (0.03), and 0.06 (−0.18), respectively. Analyzing the whole population, the P-values were 0.82 (−0.02), 0.50 (−0.05), and 0.60 (−0.04) for Cd, Pb, and Hg, respectively. + +After splitting the population into patients with chronic CAD (stents, bypasses) and patients who suffered their first event, again there was no significant correlation observable for Cd, + +| Table 4 Heavy metals in coronary artery disease patients Female Male P-value Total Cd n (%) 67.9 68.5 68.3 Median (first/ 0.72 0.65 0.84 0.67 third quartile) (,0.40/1.45) (,0.40/1.50) (0.30/1.50) Minimum/ ,0.40/16.00 ,0.40/27.00 ,0.40/27.00 maximum Pb n (%) 100 100 100 Median (first/ 19.0 21 0.26 20.0 third quartile) (12.0/28.5) (15.0/31.0) (14.0/30.8) Minimum/ 4.9/65.0 6.6/240.0 4.9/240.0 maximum Hg n (%) 81.1 84.7 83.5 Median (first/ 0.34 0.53 0.02 0.42 third quartile) (0.17/0.34) (0.22/0.92) (0.20/0.82) Minimum/ ,0.13/2.10 ,0.13/11.00 ,0.13/11.00 maximum Notes: Heavy metal analysis in coronary artery disease patients including number | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Notes: Heavy metal analysis in coronary artery disease patients including number of patients (%) in whom each metal could be quantified. Data is presented as median (first to third quartile), minimum and maximum values, and P-value of sex-specific differences. For cadmium the first quartile is below the detection limit of 0.40 μg/L. + +Abbreviations: Cd, cadmium; Hg, mercury; Pb, lead. + +Pb, or Hg. Performing multivariate analyses (with regard to the cardiovascular risk factors) did not show a significant correlation between the severity of CAD and the heavy metal levels. On the contrary, the coronary score correlated positively with laboratory markers that are associated with CAD, eg, uric acid (P=0.03), creatinine (P=0.01), blood urea nitrogen (BUN) (P=0.03), triglycerides (P=0.03), C-reative protein +(CRP) (P,0.01), pro brain natriuretic peptide (proBNP) (P,0.01), and fibrinogen Clauss (P,0.01), and negatively with high density lipoprotein (HDL) (P=0.02). Furthermore, it correlated positively with BMI (P=0.02). + +Compared to mean Cd levels that were found in the German population (0.335 µg/L urine), the CAD patients showed much higher median levels (0.67 µg/L) and 34.1% of the CAD patients exceeded the HBM-I level of 1 µg/L +and 8.5% of CAD patients exceeded the HBM-II level of 4 µg/L. Concerning mean Pb and Hg levels in the German population (35.6 and 0.86 µg/L full blood, respectively), +CAD patients showed much lower median levels (20 and 0.42 µg/L, respectively) and HBM-I/II levels were exceeded only by single cases. + +Concerning nutrition, significant positive correlations were found for Pb and the number of consumed units of red wine (P=001) and white wine (P,0.01) per week and for Hg with white wine (P,0.01) and fish consumption (P,0.01). + +Ex-smoking patients had significantly higher amounts of Cd in urine compared to patients who never smoked (0.84 versus 0.58 µg/L, respectively; P=0.03). Also Pb was higher in full blood in ex-smokers compared to never-smokers but without statistical significance (21.5 versus 19.5 µg/L, respectively; P=0.15). Patients with amalgam fillings showed significantly higher levels of Hg compared to patients without amalgam fillings (0.51 versus 0.27 µg/L, respectively; P,0.01). + +Patients living in a big city (.100,000 citizens) had median Cd levels of 0.62 µg/L, Pb levels of 20.0 µg/L, and Hg levels of 0.43 µg/L. Patients living in a small town had median Cd levels of 0.82 µg/L, Pb levels of 21.5 µg/L, and Hg levels of 0.47 µg/L, and patients who lived in rural areas had median Cd levels of 0.87 µg/L, Pb levels of 22.5 µg/L, and Hg levels of 0.33 µg/L. + +## Discussion + +Although heavy metal exposure by ubiquitous pollutants has frequently been associated with atherosclerosis, Cd, Hg, and Pb also play a tangential role in cardiovascular research and have been overshadowed by heavy weights such as hypertension, diabetes mellitus, and physical inactivity. This study is the first to correlate urine levels of Cd and blood levels of Hg and Pb with the extent of severity of CAD in 164 CAD patients. + +The main sources of contamination (dependent on the metal) are industry, traffic, tobacco consumption, and agriculture/nutrition. Heavy metals are long lasting substances which persist for decades, are transported within environmental media, and are partly accumulated in individuals after incorporation.29 Within CAD patients, Pb levels and the number of consumed units of red and white wine per week, and Hg levels and white wine and fish consumption correlated positively. Concerning red wine consumption and Pb, this correlation has been shown before,30–32 even within the female Austrian population.33 However, a connection between blood Hg levels and white wine consumption is hard to find in literature. On the contrary, much research has been done investigating the influence of fish consumption and Hg levels, especially methyl-mercury. The correlation between Hg amounts in blood and fish consumption has been described in large studies (eg, in the Canadian population34 and US children and women35). However, although Hg intake by fish consumption seems unequivocal the cardioprotective effects of a fish-rich diet seem to predominate. The Diet and Reinfarction Trial36 studied the influence of fish oil intake on total mortality and myocardial infarction (MI) in more than 2,000 men who had already suffered an MI with a follow-up period of 2 years. Patients who were asked to consume fish 2 times/week showed a 29% reduction in total mortality which was mostly driven by a 32% reduction in fatal MI. Other studies showed similar results18,37–39 and considering these results it seems that the positive effect of fish consumption on the cardiovascular system predominates but might be diminished by Hg content. + +A further source of pollution, this time for Cd, seems to be nicotine consumption and second hand smoking, as shown in several studies,40–42 and this was also observed in our CAD patients. Although we recruited only patients who never smoked or stopped smoking at least 7 years ago, ex-smokers had about 76% higher levels, probably due to bioaccumulation of Cd. Furthermore, we found significantly higher levels of Hg in patients with amalgam fillings compared to patients without amalgam fillings, although this topic cannot escape controversy. However, concerning the place of residence (urban, suburban, rural) we found no connection to Cd, Hg, or Pb. + +As mentioned before, the core of this study was the investigation of heavy metal levels in blood and urine in patients with CAD of different, but exactly quantified severity, by means of angiography. Firstly, it has to be said that the CAD patients showed much higher median urinary Cd levels compared to levels that were found in the German population.25 Furthermore, it was striking that although Cd was only quantifiable in 68.3% of patients, nearly 34.1% of all patients exceeded the HBM-I level of 1 µg/L in urine. Within the European HBM study Demo-COPHES (consortium to perform human biomonitoring on a European scale), mean levels in women +(age ,45 years) were 0.22 µg/L and the 90% percentile was 0.62.43 Concerning Pb and Hg, we found levels which were much lower in nearly all patients compared to both the German population25 and reference levels,26 respectively. Messner et al10 found a significant association of Cd levels in serum and intima media thickness in young healthy female individuals as a sign for early atherosclerosis.44 We analyzed urinary Cd and blood Hg and Pb amounts and quantified the severity of CAD but found no correlations. To this extent, this appears as rather surprising as the system of CAD quantification (described in the Materials and Methods section) seems to be appropriate because the score correlated positively with several (established) parameters of atherosclerosis (uric acid, creatinine, triglycerides, BUN, CRP, BMI, proBNP) and negatively with HDL cholesterol, even when these patients were under medication. One point of criticism may be that patients with long-term CAD and previous stent implants were also recruited. However, within the study group 50.6% had never had a cardiovascular event and there was again no observable correlation between heavy metals and severity of CAD. + +Unfortunately, although literature delivers interesting findings, the comparison of data is often difficult because heavy metal amounts are measured in various media (full blood, urine, hair, toenails, liver/kidney tissue). It is well recognized that once individuals are exposed to Cd, Pb, or Hg the metals can be detected in blood and urine but are quickly sequestered into tissue.45 It is therefore difficult to reveal the total burden of Cd, Hg, and Pb on the body. However, as the vascular system was in the foreground of this study the measurement of Hg and Pb in blood and Cd in urine was obvious despite the fact that several circumstances46 may affect the levels in these media. + +Furthermore, analysis is partly made in animals or humans which/who were confronted with enormous and (for "normal" populations) unrealistic heavy metal exposure, eg, rabbits which were fed with 8 mg Cd/kg/day over 8 months47 or Hg determination in workers of a chlorine producing factory.48 The present study is the first that assesses Cd, Pb, and Hg levels in CAD patients with precisely quantified CAD. Considering the results of this study, it seems very probable that nutrition +(above all, fish and alcohol consumption) and smoking are the main sources of heavy metal incorporation but it seems unlikely that Cd, Hg, or Pb, in quite low amounts, play a major role in the genesis of CAD, particularly when compared to the influence of blood pressure, blood glucose, and lipid profile in Austria. + +## Limitations + +Circulating heavy metal levels are influenced by numerous circumstances making it hard to control. Due to the dynamics of heavy metal pollutants, a single blood/urine sample does not reflect the total body burden but as the vasculature damage was in the foreground of this study it seemed the most appropriate. + +## Funding Sources + +This study was funded by means of the Medical University of Vienna and the Environment Agency Austria. + +## Acknowledgments + +The authors give special thanks to the laboratory team of the Medical University of Vienna. + +## Disclosure + +The authors have no disclosure to make. + +## References + +1. European Cardiovascular Disease Statistics 2012 [webpage on the Internet]. Brussels: European Heart Network; 2012. Available from: http://www.ehnheart.org/cvd-statistics.html. Accessed April 6, 2014. + +2. Kalin MF, Zumoff B. Sex hormones and coronary disease: a review of the clinical studies. *Steroids*. 1990;55(8):330–352. + +3. Isles CG, Hole DJ, Hawthorne VM, Lever AF. Relation between coronary risk and coronary mortality in women of the Renfrew and Paisley survey: +comparison with men. *Lancet*. 1992;339(8795):702–706. + +4. Wingard DL, Barrett-Connor EL, Scheidt-Nave C, McPhillips JB. + +Prevalence of cardiovascular and renal complications in older adults with normal or impaired glucose tolerance or NIDDM. A population-based study. *Diabetes Care*. 1993;16(7):1022–1025. + +5. Fifth Joint Task Force of the European Society of Cardiology; European Association of Echocardiography; et al. European Guidelines on cardiovascular disease prevention in clinical practice (version 2012): The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts). + +Eur J Prev Cardiol. 2012;19(4):585–667. + +6. Alsberg CL, Schwartze EW. Pharmacological action of Cd. + +Pharmacology. 1919;13:504–509. + +7. Bhatnagar A. Environmental cardiology: studying mechanistic links between pollution and heart disease. *Circ Res*. + +2006;99(7):692–705. + +8. Navas-Acien A, Silbergeld EK, Sharrett R, Calderon-Aranda E, Selvin E, +Guallar E. Metals in urine and peripheral arterial disease. Environ Health Perspect. 2005;113(2):164–169. + +9. Tellez-Plaza M, Navas-Acien A, Crainiceanu CM, Guallar E. Cadmium exposure and hypertension in the 1999–2004 National Health and Nutrition Examination Survey (NHANES). *Environ Health Perspect*. 2008;116(1):51–56. + +10. Messner B, Knoflach M, Seubert A, et al. Cadmium is a novel and independent risk factor for early atherosclerosis mechanisms and in vivo relevance. *Arterioscler Thromb Vasc Biol*. 2009;29(9):1392–1398. + +11. Prozialeck WC. Evidence that E-cadherin may be a target for cadmium toxicity in epithelial cells. *Toxicol Appl Pharmacol*. 2000;164(3): 231–249. + +12. Seok SM, Park DH, Kim YC, et al. COX-2 is associated with cadmiuminduced ICAM-1 expression in cerebrovascular endothelial cells. Toxicol Lett. 2006;165(3):212–220. + +13. Galan A, Garcia-Bermejo L, Troyano A, et al. The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-rays). Eur J Cell Biol. 2001;80(4):312–320. + +14. Choi AL, Weihe P, Budtz-Jorgensen E, et al. Methylmercury exposure and adverse cardiovascular effects in Faroese whaling men. *Environ* Health Perspect. 2009;117(3):367–372. + +15. Valera B, Dewailly E, Poirier P. Cardiac autonomic activity and blood pressure among Nunavik Inuit adults exposed to environmental mercury: a cross-sectional study. *Environ Health*. 2008;7:29. + +16. Fillion M, Mergler D, Sousa Passos CJ, Larribe F, Lemire M, +Guimaraes JR. A preliminary study of mercury exposure and blood pressure in the Brazilian Amazon. *Environ Health*. 2006;5:29. + +17. Wojciechowski J, Kowalski W. Cardiac and aortic lesions in chronic experimental poisoning with mercury vapors. *Pol Med Sci Hist Bull*. 1975;15(2):255–260. + +18. Guallar E, Sanz-Gallardo MI, van't Veer P, et al. Mercury, fish oils, and the risk of myocardial infarction. *N Engl J Med*. 2002;347(22): 1747–1754. + +19. Salonen JT, Seppanen K, Lakka TA, Salonen R, Kaplan GA. Mercury accumulation and accelerated progression of carotid atherosclerosis: a population-based prospective 4-year follow-up study in men in eastern Finland. *Atherosclerosis*. 2000;148(2):265–273. + +20. Alghasham AA, Meki AR, Ismail HA. Association of blood lead level with elevated blood pressure in hypertensive patients. Int J Health Sci (Qassim). 2011;5(1):17–27. + +21. Hu H, Aro A, Payton M, et al. The relationship of bone and blood lead to hypertension. The Normative Aging Study. *JAMA*. 1996;275(15): 1171–1176. + +22. Tsao DA, Yu HS, Cheng JT, Ho CK, Chang HR. The change of betaadrenergic system in lead-induced hypertension. *Toxicol Appl Pharmacol*. 2000;164(2):127–133. + +23. Khalil-Manesh F, Gonick HC, Weiler EW, Prins B, Weber MA, +Purdy RE. Lead-induced hypertension: possible role of endothelial factors. *Am J Hypertens*. 1993;6(9):723–729. + +24. Farmand F, Ehdaie A, Roberts CK, Sindhu RK. Lead-induced dysregulation of superoxide dismutases, catalase, glutathione peroxidase, and guanylate cyclase. *Environ Res*. + +2005;98(1):33–39. + +25. Umwelt-Survey. Umwelt-Survey/Bd.7: 1998. Arsen, Schwer-und Edelmetalle in Blut und Urin der Bevölkerung in Deutschland +[Arsenic, heavy and precious metals in blood and urine of the population in Germany]. Available from: http://www.umweltbundesamt. + +de/publikationen/umwelt-survey-1998-4. Accessed April 28, 2014. German. + +26. Bundesgesundheitsbl-Gesundheitsforsch-Gesundheitsschutz. + +Aktualisierung der Referenzwerte für Blei, Cadmium und Quecksilber im Blut und Urin von Erwachsenen - Stellungnahme der Kommission "Human-Biomonitoring" des Umweltbundesamtes [Update on the reference values *for lead, cadmium and mercury in the blood and* urine of adults - Commission Opinion on "Human Biomonitoring" of the Federal Environment Agency]. 2003. Available from: http:// +www.umweltbundesamt.at/fileadmin/site/umweltthemen/gesundheit/ +Humanbiomonitoring.pdf. Accessed April 28, 2014. German. + +27. Schulz C, Wilhelm M, Heudorf U, Kolossa-Gehring M; Human Biomonitoring Commission of the German Federal Environment Agency. Update of the reference and HBM values derived by the German Human Biomonitoring Commission. *Int J Hyg Environ Health*. 2011;215(1):26–35. + +28. Wilhelm M, Heinzow B, Angerer J, Schulz C. Reassessment of critical lead effects by the German Human Biomonitoring Commission results in suspension of the human biomonitoring values (HBM I and HBM II) for lead in blood of children and adults. *Int J Hyg Environ Health*. 2010;213(4):265–269. + +29. Bressler JP, Olivi L, Cheong JH, Kim Y, Bannona D. Divalent metal transporter 1 in lead and cadmium transport. *Ann N Y Acad Sci*. 2004;1012:142–152. + +30. Tahvonen R. Lead and cadmium in beverages consumed in Finland. + +Food Addit Contam. 1998;15(4):446–450. + +31. Cvetkovic J, Arpadjan S, Karadjova I, Stafilov T. Determination of cadmium in wine by electrothermal atomic absorption spectrometry. Acta Pharm. 2006;56(1):69–77. + +32. Tariba B. Metals in wine - impact on wine quality and health outcomes. + +Biol Trace Elem Res. 2011;144(1–3):143–156. + +33. Gundacker C, Komarnicki G, Zodl B, Forster C, Schuster E, +Wittmann K. Whole blood mercury and selenium concentrations in a selected Austrian population: does gender matter? *Sci Total Environ*. 2006;372(1):76–86. + +34. Lye E, Legrand M, Clarke J, Probert A. Blood total mercury concentrations in the Canadian population: Canadian Health Measures Survey cycle 1, 2007–2009. *Can J Public Health*. 2013;104(3):e246–e251. + +35. Schober SE, Sinks TH, Jones RL, et al. Blood mercury levels in US children and women of childbearing age, 1999–2000. *JAMA*. 2003;289(13):1667–1674. + +36. Burr ML, Fehily AM, Gilbert JF, et al. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). *Lancet*. 1989;2(8666):757–761. + +37. Rissanen T, Voutilainen S, Nyyssonen K, Lakka TA, Salonen JT. Fish oil-derived fatty acids, docosahexaenoic acid and docosapentaenoic acid, and the risk of acute coronary events: the Kuopio ischaemic heart disease risk factor study. *Circulation*. 2000;102(22):2677–2679. + +38. Chan HM, Egeland GM. Fish consumption, mercury exposure, and heart diseases. *Nutr Rev*. 2004;62(2):68–72. + +39. Stern AH. A review of the studies of the cardiovascular health effects of methylmercury with consideration of their suitability for risk assessment. *Environ Res*. 2005;98(1):133–142. + +40. Tellez-Plaza M, Navas-Acien A, Caldwell KL, Menke A, Muntner P, +Guallar E. Reduction in cadmium exposure in the United States population, 1988–2008: the contribution of declining smoking rates. Environ Health Perspect. 2012;120(2):204–209. + +41. Leung TF, Chan IH, Liu TC, Lam CW, Wong GW. Relationship between passive smoking exposure and urinary heavy metals and lung functions in preschool children. *Pediatr Pulmonol*. 2013;48(11):1089–1097. + +42. McElroy JA, Shafer MM, Trentham-Dietz A, Hampton JM, +Newcomb PA. Urinary cadmium levels and tobacco smoke exposure in women age 20–69 years in the United States. *J Toxicol Environ Health A*. 2007;70(20):1779–1782. + +43. COPHES: consortium to perform human biomonitoring on a European scale (COPHES). *Cent Eur J Public Health*. 2012;20(4):300. + +44. Magyar MT, Szikszai Z, Balla J, et al. Early-onset carotid atherosclerosis is associated with increased intima-media thickness and elevated serum levels of inflammatory markers. *Stroke*. 2003;34(1):58–63. + +45. Archibeque-Engle SL, Tessari JD, Winn DT, Keefe TJ, Nett TM, +Zheng T. Comparison of organochlorine pesticide and polychlorinated biphenyl residues in human breast adipose tissue and serum. *J Toxicol* Environ Health. 1997;52(4):285–293. + +46. Jandacek RJ, Anderson N, Liu M, Zheng S, Yang Q, Tso P. Effects of yo-yo diet, caloric restriction, and olestra on tissue distribution of hexachlorobenzene. *Am J Physiol Gastrointest Liver Physiol*. 2005;288(2):G292–G299. + +47. Subramanyam G, Bhaskar M, Govindappa S. The role of cadmium in induction of atherosclerosis in rabbits. *Indian Heart J*. 1992;44(3): 177–180. + +48. Skoczynska A, Jedrejko M, Martynowicz H, et al. [The cardiovascular risk in chemical factory workers exposed to mercury vapor]. *Med Pr*. 2010;61(4):381–391. Polish. + +## Vascular Health And Risk Management Dovepress + +Publish your work in this journal Vascular Health and Risk Management is an international, peerreviewed journal of therapeutics and risk management, focusing on concise rapid reporting of clinical studies on the processes involved in the maintenance of vascular health; the monitoring, prevention and treatment of vascular disease and its sequelae; and the involvement of Submit your manuscript here: http://www.dovepress.com/vascular-health-and-risk-management-journal metabolic disorders, particularly diabetes. This journal is indexed on PubMed Central and MedLine. 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. \ No newline at end of file diff --git a/medical/md/PMC4206733.md b/medical/md/PMC4206733.md new file mode 100644 index 0000000000000000000000000000000000000000..a7a4f01134201099726c1c8362361a114676a6e9 --- /dev/null +++ b/medical/md/PMC4206733.md @@ -0,0 +1,216 @@ +# —Original— Identification And Characterization Of Vancomycin-Resistant Enterococcus **Species** Frequently Isolated From Laboratory Mice + +Hitoki Yamanaka1), Toshikazu Takagi1), Makiko Ohsawa1), Naoto Yamamoto1), Noriaki Kubo1), Takahira Takemoto1), Shoko Sasano1), Ritsuko Masuyama2), and Kazutaka Ohsawa1) +1)Division of Comparative Medicine, Center for Frontier Life Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan 2)Department of Molecular Bone Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan +(Received 9 October 2013 / Accepted 28 January 2014) +Address corresponding: H. Yamanaka, Division of Comparative Medicine, Center for Frontier Life Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan Exp. Anim. 63(3), 297–304, 2014 Abstract: To determine the prevalence of drug resistant bacteria colonizing laboratory mice, we isolated and characterized vancomycin-resistant *Enterococcus* species (VRE) from commercially available mice. A total of 24 VRE isolates were obtained from 19 of 21 mouse strains supplied by 4 commercial breeding companies. Of these, 19 isolates of *E. gallinarum* and 5 isolates of *E. casseliflavus* possessing the *vanC1* and *vanC2/3* genes intrinsically, exhibited intermediate resistance to vancomycin respectively. In addition, these isolates also exhibited diverse resistant patterns to erythromycin, tetracycline, and ciprofloxacin, whereas the use of antibiotics had not been undertaken in mouse strains tested in this study. Although 6 virulence-associated genes (ace, asa, cylA, efaA, esp, and gelE) and secretion of gelatinase and hemolysin were not detected in all isolates, 23 of 24 isolates including the isolates of *E. casselifalvus* secreted ATP into culture supernatants. Since secretion of ATP by bacteria resident in the intestinal tract modulates the local immune responses, the prevalence of ATP-secreting VRE in mice therefore needs to be considered in animal experiments that alter the gut microflora by use of antibiotics. + +Key words: ATP, laboratory mice, prevalence, vancomycin-resistant *Enterococcus*, virulence factor + +## Introduction + +Enterococcus species are intestinal bacteria colonizing not only humans, mammals, and birds but also insects. They were regarded as harmless to humans and mammals for a long time. However, they have recently been recognized to be important opportunistic pathogens, causing nosocomial infections of the urinary tract, endocarditis, bacteremia, and central nervous system infections [4, 8]. + +In addition, *Enterococcus* species are intrinsically resistance to a number of antimicrobial agents such as β-lactams and aminoglycosides [13], and there are increasingly more acquired antibiotic resistant species to other antimicrobial agents by mutation or acquisition of foreign genetic material via transfer of plasmids and transposons, including the vancomycin-resistant gene +[1]. The emergence of vancomycin-resistant *Enterococcus* (VRE) has been linked to an increased use of glycopeptide antibiotics in human medicine and the use of the glycopeptide avoparcin as a growth promoter in livestock +[1]. Therefore, the outbreak of VRE in humans is possibly associated with the occurrence of VRE in livestock, although the relationship remains unclear. + +In laboratory animals, a few reports describing the detection of antibiotic resistant bacteria have been published that indicated a low incidence of drug resistance in laboratory animals, and no correlation between the emergence of the drug resistance in laboratory animals and those in humans or farm animals [11]. The detection of drug resistant *E. faecalis* and *E. faecium* in laboratory mice and rats has also been reported, and these enterococci showed resistance to β-lactams, aminogly-cosides, tetracycline, or erythromycin [10, 23, 27]. + +Therefore, the detailed investigations are needed to clarify the characteristics of VRE currently colonizing commercially produced mice used in biomedical research. + +A number of virulence factors of *Enterococcus* species have been identified in the isolates obtained from clinical specimens. These include collagen binding cell wall protein (ace), aggregation substance (asa), endocarditis-specific antigen (efaA) and extracellular surface protein +(esp), as well as gelatinase, and cytolysin (hemolysin) +that are associated with biofilm formation and cytotoxicity, respectively. In addition, several *Enterococcus* spe-cies of intestinal microflora have the ability to secrete a large amount of ATP [12, 16]. High levels of ATP in the gastrointestinal tract derived from intestinal microflora can modulate the balance of helper T cell (Th) immune responses of intestinal mucosa, leading to inflammatory bowel diseases [2, 22]. + +In the present study, VRE possessing a vancomycinresistant gene were isolated from laboratory mouse strains (including some immunodeficient strains) obtained from commercial breeding companies. To characterize these VRE isolates, their sensitivity to various antibiotics and several virulence-associated factors including secretion of ATP were investigated. The data provide information on the characteristics of VRE colonizing in intestinal tract of laboratory mice and raises concerns regarding the potential influence of VRE colonization on the results of some animal experiments. + +## Materials And Methods + +Isolation of VRE from laboratory mice Fresh feces were collected from mice immediately after delivery from four different commercial breeding companies between October 2011 and February 2012 (Table 1). The use of antibiotics had not been undertaken in the mouse strains tested in this study. Feces were homogenized with 1 ml of PBS by vortexing for 5 min and centrifugation at 6,000 ×g for 10 sec. Fifty µl of supernatants were spread on VR-EF plates containing 6 µg/ml−1 of vancomycin (Nissui, Tokyo, Japan) and in-cubated at 37°C for 2 days under aerobic conditions. + +Single colony isolation was performed using brain-heart infusion agar (Becton Dickinson, MD, USA) containing 6 µg/ml−1 of vancomycin (Wako, Osaka, Japan). Animal care and experimental procedures were performed in accordance with the Regulations for Animal Experimentation of Nagasaki University and with approval of the Institutional Animal Care and Use Committee. + +Detection of antibiotic resistance and virulenceassociated genes Vancomycin-resistant genes, vanA, vanB, *vanC1*, and vanC2/3 were detected by PCR using specific primers +[6]. All reactions were performed in a final reaction volume of 20 µl using bacterial cells as template, 10 µl of EmeraldAmpTM PCR Master Mix (TAKARA BIO INC., +Japan), and 1 µM of each primer. The PCR reaction was preheated at 95°C for 5 min and then subjected to 30 cycles consisting of denaturation at 95°C for 1 min, primer annealing at 54°C for 1 min, and extension at 72°C for 1 min, with final extension at 72°C for 7 min. + +The virulence-associated genes ace, efaA, asa, *cylA*, +esp, and *gelE* were also detected by PCR with specific primers as described [26]. The PCR reaction was preheated at 95°C for 5 min and then subjected to 30 cycles consisting of denaturation at 95°C for 30 sec, primer annealing at 58°C for 45 sec, and extension at 72°C for 1 min, with final extension at 72°C for 7 min. PCR prod-ucts were visualized on 1.5% agarose gels. + +Identification of Enterococcus species For identification of bacteria species, gram staining, catalase production and then rapid ID 32 STREP API (bioMérieux, March l'Etoile, France) were performed. If necessary, further identification was performed by detection and sequencing analysis of 16S rRNA genes using a universal primer pair (forward, 5′- AGAGTTTGATCCTGGCTCAG-3′; reverse, 5′- ACTACCAGGGTATCTAATC-3′). The PCR products were purified using LaboPassTM Gel kit (COSMO GENETECH CO., Ltd.., +Korea) according to the manufacturer's instructions. DNA sequences were determined with primers described above using ABI PRISM BigDyeTM Terminator v3.1 + +| from 4 commercial breeding companies | | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------|----------|-----|--------------------------------------------------|---------------------------------------------------| +| Source | Straina) | Sex | Age (week) | Enterococcus species | +| Identified (type of van gene) | | | | | +| Company 1 | A/J | F | 6 | E. gallinarum (VanC1) E. casseliflavus (VanC2/3) | +| C57BL/6N | M/F | 4/8 | Not detected | | +| ddY | M | 6 | E. gallinarum (VanC1) | | +| BALB/c | M | 8 | E. gallinarum (VanC1) E. casseliflavus (VanC2/3) | | +| ICR | F | 8 | E. gallinarum (VanC1) | | +| C3H/HeJ | F | 8 | E. gallinarum (VanC1) | | +| Company 2 | ddY | F | 6 | E. gallinarum (VanC1) E. casseliflavus (VanC2/3) | +| BALB/c | M/F | ≥48 | E. gallinarum (VanC1) E. casseliflavus (VanC2/3) | | +| Company 3 | ICR | M | 5 | E. gallinarum (VanC1) | +| C3H/HeJ | M/F | 8 | E. gallinarum (VanC1) | | +| ICR(MCH) | F | 4 | E. gallinarum (VanC1) | | +| 129+Ter/Sv | M | ≥48 | E. gallinarum (VanC1) | | +| BALB/c-nu/nub) | F | 6 | E. gallinarum (VanC1) | | +| Company 4 | ICR | F | 8 | E. gallinarum (VanC1) | +| BALB/c | F | 8 | E. gallinarum (VanC1) | | +| C57BL/6N | M | 8 | E. gallinarum (VanC1) E. casseliflavus (VanC2/3) | | +| C57BL/6J | M | 5 | E. gallinarum (VanC1) | | +| CBA/J | M | 6 | E. gallinarum (VanC1) | | +| SJL/J | M | 5 | E. gallinarum (VanC1) | | +| NOD/SCIDb) | M | 6 | Not detected | | +| BALB/c-nu/nub) | F | 5 | E. gallinarum (VanC1) | | +| a) The antibiotic treatment had not been undertaken in these mouse strains. b) These mouse strains are immunodeficiency. F, female; M, male. | | | | | + +Cycle Sequencing Kits (Applied Biosystems, USA). The identity of the 16S rRNA gene sequences was verified by comparison to *Enterococcus* sequences in the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST). Phenotype characterization of Enterococcus species Gelatinase activity was assessed by inoculation of brain heart infusion broth containing 3% gelatin (Difco Lab., MI, USA), which was then incubated in 37°C for 2 days in aerobic conditions [5]. Positive gelatinase activity was determined by degradation of the gelatin to liquid after 1–1.5 h cooling of the tubes at 4°C. Hemolysin activity was determined by streaking bacterial cultures on a blood agar plate with 5% sheep blood (bioMérieux, March l'Etoile, France) and incubation for 2 days under aerobic conditions. Hemolytic activity was classified as β-hemolysis (a clear zone of hydrolysis around the colonies), α-hemolysis (partial hydrolysis including greenish zones around the colonies), or γ-hemolysis (no reaction). When β-hemolysis was observed, the strain was consid- + +## Ered Positive For Hemolytic Activity. Antibiotic Susceptibility + +The minimal inhibitory concentration (MIC) for vancomycin and teicoplanin of each *Enterococcus* isolate was determined using the E-test (bioMérieux, March l'Etoile, France). Antibiotic susceptibilities to ampicillin (AM 10), erythromycin (E 15), chloramphenicol (C 30), tetracycline (TE 30), ciprofloxacin (CIP 5), linezolid (LZD 30), gentamycin (GM 120), and streptomycin +(STM 300) were performed using Sensi-DiscTM(Becton Dickinson, MD, USA). These tests were performed ac-cording to the Clinical and Laboratory Standards Institute (CLSI) documents M31-A2 (2002) and M100-S22 +(2012). Bacterial suspensions in phosphate-buffered saline were adjusted to a turbidity of 0.5 McFarland standard, spread onto Mueller-Hinton agar (Nissui, To-kyo, Japan), and incubated for 24–48 h at 37°C under aerobic conditions. After incubation, MICs were read from where the edge of the inhibition ellipse intersected the strip and the sensitivities graded as sensitive (S), +intermediate (I) or resistant (R) by measuring the diameter of the inhibition zone. *E. faecalis* ATCC 29212 (JCM +7783), *E. casseliflavus* ATCC 25788 (JCM 8723), and E. gallinarum ATCC 49573 (JCM 8728) provided by Japan Collection and Microorganisms, RIKEN BRC +which is participating in the National BioResource Project of the MEXT, Japan, were used as controls for tests. + +## Measurement Of Atp + +Isolates were cultured in serum-free RPMI medium +(Gibco, CA, USA) at 37°C for 18 h under aerobic conditions with shaking [2, 16]. After checking growth levels spectrophotometrically at 590 nm, the culture media was centrifuged at 13,000 ×g for 5 min and the supernatants collected. To remove the bacterial cells, supernatants were filtered with a 0.22-µm membrane (Millipore Japan, Tokyo, Japan). ATP concentrations in culture supernatants were measured using an ATP assay kit (Promega, WI, USA) according to the manufacturer's protocols. + +Table 2. MICs of 24 *Enterococcus* species isolates to glycopeptides + +| Isolates | Type of | MICs (μg/ml-1) | | +|--------------------------------------------------------------|-----------|----------------------------|------------| +| (no. of isolates) | van gene | Vancomycina) Teicoplaninb) | | +| E. gallinarum (19) | VanC1 | 6 (1) | ≤0.75 (19) | +| 8–12 (18) | | | | +| E. casseliflavus (5) | VanC2/3 | 6 (5) | ≤0.75 (5) | +| a) The intermediate breakpoint of MIC for vancomycin is 8–16 | | | | + +Results Detection of VRE from laboratory mice Fecal samples were collected from 21 inbred and outbred mouse strains purchased from 4 commercial breeding companies. The tested mouse strains included those commonly used in biomedical research as well as a number of immunodeficient strains. VRE were detected in 19 of 21 mouse strains, excepting C57BL/6N and NOD-SCID mice obtained from Companies 1 and 4, respectively (Table 1). The VRE isolates were identified as E. gallinarum or *E. casseliflavus* possessing the vancomycin-resistant gene, vanC1 or *vanC2/3*, respectively. *E. gallinarum* possessing *vanC1* was present in all 19 positive mouse strains while 5 mouse strains were also colonized with *E. casseliflavus* possessing *vanC2/3*. + +## Antimicrobial Susceptibility Of Vre + +The susceptibility of 24 isolates to vancomycin and teicoplanin were determined as MICs by the E-test method (Table 2). MICs to vancomycin for 18 of 19 E. gallinarum isolates possessing *vanC1* gene were 8–12 µg/ml−1 exhibiting intermediate resistance. The MICs to vancomycin for all 5 *E. casseliflavus* isolates possessing vanC2/3 gene were 6 µg/ml−1. The MICs of all 24 isolates were less than 0.75 µg/ml−1 indicating sensitivity to teicoplanin. + +The susceptibility of the 24 isolates to other antibiotics was evaluated by the disk diffusion method (Table 3). All isolates were susceptible to ampicillin, chloramphenicol, and linezolid, whereas *E. casseliflavus* isolate only exhibited an intermediate resistance to chloram-phenicol. No isolates exhibited high-level resistance to + +| Table 3. Antibiotic resistance of 24 Enterococcus species isolates from laboratory mice supplied from 4 breeding companies No. of resistant isolates (no. of intermediate isolates) to: Source Isolates (no. tested) AM 10 E 15 C 30 TE 30 CIP 5 LZD 30 GM 120a) STM 300a) Company 1 E. gallinarum (5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (5) 0 (0) 0 (0) 0 (0) E. casseliflavus (2) 0 (0) 0 (2) 0 (0) 0 (0) 0 (2) 0 (0) 0 (0) 0 (0) Company 2 E. gallinarum (2) 0 (0) 0 (0) 0 (0) 1 (0) 0 (2) 0 (0) 0 (0) 0 (0) E. casseliflavus (2) 0 (0) 1 (1) 0 (0) 0 (0) 2 (0) 0 (0) 0 (0) 0 (0) Company 3 E. gallinarum (5) 0 (0) 0 (2) 0 (0) 0 (0) 0 (5) 0 (0) 0 (0) 0 (0) Company 4 E. gallinarum (7) 0 (0) 0 (0) 0 (0) 1 (0) 1 (6) 0 (0) 0 (0) 0 (0) E. casseliflavus (1) 0 (0) 0 (1) 0 (1) 0 (0) 1 (0) 0 (0) 0 (0) 0 (0) E. gallinarum ATCC49573 S S S R S S S S E. casseliflavus ATCC25788 S I S S I S S S a) These tests are for high-level aminoglycoside resistant Enterococcus species. AM, ampicillin; E, erythromycin; C, chloramphenicol; | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +![4_image_0.png](4_image_0.png) + +the aminoglycosides gentamycin (GM 120) or strepto-mycin (STM 300). However, all *E. gallinarum* and E. casseliflavus isolates exhibited either resistantce or in-termediate resistance to ciprofloxacin, while the two reference strains were sensitive or intermediate resistant, respectively. For erythromycin, 2 out of 5 *E. gallinarum* isolates from mice from company 3 and all E. casseli-*flavus* isolates exhibited intermediate resistance except for one *E. casseliflavus* isolate from company 2 that was resistant. Although the reference strain of *E. gallinarum* exhibited resistance to tetracycline, only 2 of 19 isolates from companies 2 and 4 were resistant. No E. casseli-*flavus* isolates exhibited resistance to tetracycline, including the reference strain. + +Detection of virulence genes and secreting virulence factors No virulence-associated genes (ace, efaA, asa, *cylA*, +esp, and *gelE*) or gelatinase activity were detected in any of the 24 *Enterococcus* isolates. Since all 24 isolates exhibited α-hemolysis on 5% sheep blood agar, all isolates were negative for hemolytic activity (data not shown). + +ATP in the intestinal tract secreted by intestinal mi-croflora has recently shown to modulate the immune response of the intestinal lamina propria [2, 22]. Therefore, the ability of the *Enterococcus* isolates to secrete ATP was determined. As shown in Fig. 1, higher levels of ATP secretion were detected in each culture medium of all 5 *E. casseliflavus* isolates and 18 of 19 *E. gallinarum* isolates in comparison with that of *E. faecalis* ATCC 29212. Similarly, the two reference strains E. gallinarum ATCC 49573 and *E. casseliflavus* ATCC +25788 both secreted ATP. Furthermore, oxygen was dispensable for ATP secretion of these strains, since ATP in culture media under anaerobic condition were comparably detected with those of aerobic condition (data not shown). + +## Discussion + +In the present study, 19 *E. gallinarum* possessing vanC1, and 5 *E. casseliflavus* possessing *vanC2/3* were isolated from 19 of 21 mouse strains. These two *Enterococcus* species are generally found in the intestinal tract of mammals along with other *Enterococcus* species such as *E. faecalis* and *E. faecium*. The vancomycin-resistant genes *vanC1* and *vanC2/3* were detected in our isolates. + +Fig. 1. Detection of ATP in the culture supernatants of vancomycin resistant *Enterococcus* isolates. ATP concentrations in culture supernatants were determined using a luciferase reaction method and the culture density of isolates was determined spectrophotometrically. The 19 *E. gallinarum* isolates (open diamond) and 5 *E. casseliflavus* isolates +(open triangle) were tested; each symbol represents the value of an individual isolate. Reference strains of *E. faecalis* (filled circle), *E. gallinarum* (filled diamond), and E. + +casseliflavus (filled triangle) were also tested. +These genes impart low level resistance to vancomycin, and are located chromosomally and so are not readily transferable [8]. In contrast, *Enterococcus* species possessing either the vanA or *vanB* gene typically show high-level resistance to glycopeptides, and these genes are present not only on the chromosome but also be able to locate on transposons or plasmids, these are then read-ily transferred to other microorganisms [19, 31]. Thus, the emergence of VRE possessing the vanA or *vanB* gene is clinically important, especially *E. faecalis* and *E. faecium* species since these species are harboring various virulence factors. In this study, neither of these two VRE species nor other *Enterococcus* species carrying the *vanA* or *vanB* gene were detected in commercially bred mice. From the early 1990s, detection of VRE possessing the vanA or *vanB* gene has increased in both humans and livestock in Europe and the United States as well as in Japan [1, 9, 24, 25, 32]. However, these VRE have not been detected in livestock in Japan after the use of avoparcin was banned [20, 21]. The present results indicate that the emergences and outbreaks of VRE in humans and farm animals have not extended to commercial laboratory mouse breeding operations. + +Isolates of *Enterococcus* from laboratory mouse strains exhibited diverse antibiotic resistant characteristics when tested for resistance to erythromycin, tetracycline, and ciprofloxacin. In addition to intrinsic resistance to a number of antibiotics including β-lactams and aminoglycosides, the occurrence of acquired resistance among *Enterococcus* species to macrolides, tetracyclines, and quinolones by receipt of resistant gene, such as erythromycin (erm) or tetracycline (tet) resistance genes, and the accumulation of sporadic mutations in DNA gyrase (*gyrA*) or topoisomerase (*parC*) gene have been demonstrated [14, 17, 28]. Furthermore, these antibiotic resistance genes are found in the transferable regions of DNA which can be exchanged among different species [14]. Therefore, the VRE isolates in this study exhibiting resistance to antibiotics might possess transferable antibiotic resistant gene or chromosomal gene with mutations. However, since microflora in laboratory mice used in this study had not undergone a selective pressure by the use of antibiotics, the mechanism to achieve the antibiotic resistance is unclear. The previous study showed the detection of antibiotic resistant in laboratory mice which had not been administered with antibiotics [27]. Therefore, the resistant genes in those mice might be inherited stably in the laboratory mice to this day. In addition to the identifications of resistance genes in our isolates, further studies are required to clarify the source and the prevalence of these transferable resistant genes in laboratory mice. + +Although none of 6 virulence-associated genes we tested for were detected in any *Enterococcus* isolates, secretion of ATP at very high levels was detected in supernatants of *E. gallinarum* and *E. casseliflavus* cultures. + +ATP has been demonstrated to modulate immune cell functions via activation of the ATP receptors P2X and P2Y, leading to the development of excessive immunemediated and inflammatory disease [7, 15, 18, 30]. Furthermore, ATP in the intestinal tract induces activation of Th17 responses and mast cells in the intestinal mucosa, leading to inflammatory bowel diseases including ulcerative colitis and Crohn's disease [2, 22]. The source of ATP in the intestinal tract has been shown to be intestinal bacteria including several *Enterococcus* species +(including *E. gallinarum*) [12, 16]. Our present study showed *E. casseliflavus* may also be a plausible source of ATP in the intestinal tract. Since use of antibiotics can induce abnormal proliferation of VRE via low-induction of an innate immune response to eliminate the Gram-positive bacteria [3, 29], these two VRE species in the gastrointestinal tract may induce inflammatory diseases via accumulation of a large amount of ATP following use of antibiotics. + +In conclusion, the present study has shown that E. + +gallinarum and *E. casseliflavus* possessing *vanC* genes intrinsically were detected frequently in laboratory mice including immunocompromised mouse strain. These VRE isolates exhibited intermediate resistance to vancomycin, and several isolates also showed resistance to erythromycin, tetracycline, and/or ciprofloxacin, while the use of antibiotics had not been undertaken in all mouse strains. Additional studies are needed to clarify the mechanisms of drug-resistances in *Enterococcus* present in laboratory animals. Furthermore, since the present study has firstly elucidated that *E. casseliflavus* has the ability to secrete ATP, this species is consequently considered as a source of ATP in intestinal tracts like several *Enterococcus* species including *E. gallinarum*. Therefore, the potential role of VRE as pathogens and their ability to influence physiological/immunological responses should be considered by researchers using such colonized mice, especially when experiments involve the use antibiotics. + +## Acknowledgments + +The authors thank to Prof. R. Eberle (Center for Veterinary Health Sciences, Oklahoma State University) for helpful discussions and critical assessment of the manuscript, and Mr. Masashi Sakamoto and Ms. Yuko Matsuura (Nagasaki University) for technical assistance. + +This work was supported by Grant-in-aid for Scientific Research (C) (no. 25450444) from Japan Society for the Promotion of Science. + +## References + +[CrossRef] +5. Dahlén, G., Blomqvist, S., Almståhl, A., and Carlén, A. + +2012. Virulence factors and antibiotic susceptibility in enterococci isolated from oral mucosal and deep infections. J. + +Oral Microbiol. 4. + +6. Dutka-Malen, S., Evers, S., and Courvalin, P. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. *J. Clin. Microbiol.* 33: 24–27. [Medline] +7. Ferrari, D., Pizzirani, C., Adinolfi, E., Lemoli, R.M., Curti, A., Idzko, M., Panther, E., and Di Virgilio, F. 2006. The P2X7 receptor: a key player in IL-1 processing and release. + +J. Immunol. 176: 3877–3883. [Medline] [CrossRef] +8. Fisher, K. and Phillips, C. 2009. The ecology, epidemiology and virulence of Enterococcus. *Microbiology* 155: 1749– +1757. [Medline] [CrossRef] +9. Fujita, N., Yoshimura, M., Komori, T., Tanimoto, K., and Ike, Y. 1998. First report of the isolation of high-level vancomycin-resistant Enterococcus faecium from a patient in Japan. *Antimicrob. Agents Chemother.* 42: 2150. [Medline] +10. Goo, J.S., Jang, M.K., Shim, S.B., Jee, S.W., Lee, S.H., +Bae, C.J., Park, S., Kim, K.J., Kim, J.E., Hwang, I.S., Lee, H.R., Choi, S.I., Lee, Y.J., Lim, C.J., and Hwang, D.Y. 2012. Monitoring of antibiotic resistance in bacteria isolated from laboratory animals. *Lab. Anim. Res.* 28: 141–145. [Medline] [CrossRef] +11. Hansen, A.K. and Velschow, S. 2000. Antibiotic resistance in bacterial isolates from laboratory animal colonies naive to antibiotic treatment. *Lab. Anim.* 34: 413–422. [Medline] [CrossRef] +12. Hironaka, I., Iwase, T., Sugimoto, S., Okuda, K., Tajima, A., +Yanaga, K., and Mizunoe, Y. 2013. Glucose triggers ATP +secretion from bacteria in a growth-phase-dependent manner. *Appl. Environ. Microbiol.* 79: 2328–2335. [Medline] +[CrossRef] +13. Hollenbeck, B.L. and Rice, L.B. 2012. Intrinsic and acquired resistance mechanisms in enterococcus. *Virulence* 3: 421– 433. [Medline] [CrossRef] +14. Huys, G., D'Haene, K., Collard, J.M., and Swings, J. 2004. + +Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food. Appl. Environ. + +Microbiol. 70: 1555–1562. [Medline] [CrossRef] +15. Idzko, M., Hammad, H., van Nimwegen, M., Kool, M., Willart, M.A., Muskens, F., Hoogsteden, H.C., Luttmann, W., +Ferrari, D., Di Virgilio, F., Virchow, J.C. Jr., and Lambrecht, B.N. 2007. Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells. Nat. + +Med. 13: 913–919. [Medline] [CrossRef] +16. Iwase, T., Shinji, H., Tajima, A., Sato, F., Tamura, T., Iwamoto, T., Yoneda, M., and Mizunoe, Y. 2010. Isolation and iden-tification of ATP-secreting bacteria from mice and humans. + +J. Clin. Microbiol. 48: 1949–1951. [Medline] [CrossRef] +17. Kanematsu, E., Deguchi, T., Yasuda, M., Kawamura, T., +Nishino, Y., and Kawada, Y. 1998. Alterations in the GyrA +subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV associated with quinolone resistance in Enterococcus faecalis. *Antimicrob. Agents Chemother.* 42: 433– +435. [Medline] +18. Khakh, B.S. and North, R.A. 2006. P2X receptors as cellsurface ATP sensors in health and disease. *Nature* 442: 527– +532. [Medline] [CrossRef] +19. Klare, I., Konstabel, C., Badstübner, D., Werner, G., and Witte, W. 2003. Occurrence and spread of antibiotic resistances in Enterococcus faecium. *Int. J. Food Microbiol.* 88: +269–290. [Medline] [CrossRef] +20. Kojima, A., Asai, T., Ishihara, K., Morioka, A., Akimoto, K., +Sugimoto, Y., Sato, T., Tamura, Y., and Takahashi, T. 2009. + +National monitoring for antimicrobial resistance among indicator bacteria isolated from food-producing animals in Japan. *J. Vet. Med. Sci.* 71: 1301–1308. [Medline] [CrossRef] +21. Kojima, A., Morioka, A., Kijima, M., Ishihara, K., Asai, T., Fujisawa, T., Tamura, Y., and Takahashi, T. 2010. Classification and antimicrobial susceptibilities of enterococcus species isolated from apparently healthy food-producing animals in Japan. *Zoonoses Public Health* 57: 137–141. [Medline] [CrossRef] +22. Kurashima, Y., Amiya, T., Nochi, T., Fujisawa, K., Haraguchi, T., Iba, H., Tsutsui, H., Sato, S., Nakajima, S., Iijima, H., +Kubo, M., Kunisawa, J., and Kiyono, H. 2012. Extracellular ATP mediates mast cell-dependent intestinal inflammation through P2X7 purinoceptors. *Nat. Commun.* 3: 1034. [Medline] [CrossRef] +23. Maejima, K., Urano, T., Tamura, H., and Terakado, N. 1980. + +Drug resistance of organisms isolated from feces of laboratory mice and rats. *Jikken Dobutsu* 29: 71–75. [Medline] +24. Nilsson, O. 2012. Vancomycin resistant enterococci in farm animals − occurrence and importance. *Infect. Ecol. Epidemiol.* 2. + +25. Oana, K., Kawakami, Y., Ohnishi, M., Ishikawa, M., Hirota, M., Tozuka, M., Atarashi, K., Baba, K., Fujiki, K., Okazaki, M., Honda, T., and Hayashi, T. 2001. Molecular and epide-miological study of the first outbreak of vanB type vancomycin-resistant Enterococcus faecalis in Japan. *Jpn. J. Infect.* +Dis. 54: 17–22. [Medline] +26. Sedgley, C.M., Molander, A., Flannagan, S.E., Nagel, A.C., +Appelbe, O.K., Clewell, D.B., and Dahlén, G. 2005. Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp. *Oral Microbiol. Immunol.* 20: 10–19. + +[Medline] [CrossRef] +27. Shimoda, K., Maejima, K., and Urano, T. 1984. Drug resistance in Streptococcus faecalis, Streptococcus faecium and Staphylococcus epidermidis isolated from laboratory animals. *Jikken Dobutsu* 33: 351–355. [Medline] +28. Tankovic, J., Mahjoubi, F., Courvalin, P., Duval, J., and Leclerco, R. 1996. Development of fluoroquinolone resistance in Enterococcus faecalis and role of mutations in the DNA gyrase gyrA gene. *Antimicrob. Agents Chemother.* 40: +2558–2561. [Medline] +29. Ubeda, C., Taur, Y., Jenq, R.R., Equinda, M.J., Son, T., +Samstein, M., Viale, A., Socci, N.D., van den Brink, M.R., Kamboj, M., and Pamer, E.G. 2010. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans. *J. Clin. Invest.* 120: 4332–4341. [Medline] [CrossRef] +30. Wilhelm, K., Ganesan, J., Müller, T., Dürr, C., Grimm, M., +Beilhack, A., Krempl, C.D., Sorichter, S., Gerlach, U.V., Jüttner, E., Zerweck, A., Gärtner, F., Pellegatti, P., Di Virgilio, F., Ferrari, D., Kambham, N., Fisch, P., Finke, J., Idzko, M., and Zeiser, R. 2010. Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R. *Nat. Med.* 16: 1434– 1438. [Medline] [CrossRef] +31. Williams, J.J. and Hergenrother, P.J. 2008. Exposing plasmids as the Achilles' heel of drug-resistant bacteria. *Curr.* Opin. Chem. Biol. 12: 389–399. [Medline] [CrossRef] +32. Yoshimura, H., Ishimaru, M., Endoh, Y.S., Suginaka, M., +and Yamatani, S. 1998. Isolation of glycopeptide-resistant enterococci from chicken in Japan. *Antimicrob. Agents Chemother.* 42: 3333. [Medline] \ No newline at end of file diff --git a/medical/md/PMC4323959.md b/medical/md/PMC4323959.md new file mode 100644 index 0000000000000000000000000000000000000000..f6f5b5cfc3fe267800b9f1be99925b98b8e4663a --- /dev/null +++ b/medical/md/PMC4323959.md @@ -0,0 +1,118 @@ +# Original Article + +![0_Image_0.Png](0_Image_0.Png) Management And Functional Outcome Of Intramedullary Spinal Cord Tumors: A **Prospective** Clinical Study + +Raj Kumar, Sumit Banerjee1 Deoartment of Neurosurgery, 1Department of Orthopedics, AIIMS, Rishikesh, Uttarakhand, India + +## Abstract + +Aim: Intramedullary spinal cord tumors (IMSCT) are rare neoplasms of central nervous system but require proper evaluation and management to ensure a good outcome. This study was carried out to evaluate the functional outcome of IMSCT following surgery and to decipher the factors affecting optimal outcome of these cases. + +Materials and Methods: A prospective clinical study was carried out at a tertiary care center from 2003 to 2012. Forty three patients with intramedullary tumors diagnosed on magnetic resonance imaging were included. Their clinical details, neurological findings and demographic data were recorded. The patients were then subjected to surgery and adjuvant radiotherapy. The patients were followedup clinically and radiologically, and all parameters examined and recorded. + +Results: Sensory and motor impairment was present preoperatively in majority of patients (n = 39 and n = 38, 90.7% and 88.4%, respectively). Gross total excision was performed in 30 cases (69.76%). The most common histological diagnosis was ependymoma (n = 21, 48.8%). Postoperatively 32 patients (74.4%) were in McCormick functional Grade I or II improving from 13 cases (30.2%) in Grade I or II preoperatively. Fifteen of 17 patients in Medical Research Council (MRC) Grade III and 10 out of 12 patients in Grade MRC IV improved. No mortality was recorded during the entire period of follow-up (mean: 22, range: 3-96 months). Eight patients (18.6%) had recurrence till the last follow-up visit. + +Conclusions: Preoperative neurological grade was the most important predictor of functional outcome. Gross tumor excision was the best surgical modality to improve event free survival. High-grade tumors had higher rates of recurrence but no effect on functional outcome. + +Key words: Ependymoma, functional outcome, intramedullary tumor, radical excision + +## Introduction + +Intramedullary spinal cord tumors (IMSCT) are rare accounting for about 6% of all central nervous system tumors. They arise from the neural tissue of the spinal cord with propensity for the variable degree of neurological compromise. Intramedullary position poses complications in the surgical excision of these tumors because of their intricate origin. The clinical signs + +![0_image_1.png](0_image_1.png) + +www.asianjns.org + +| Access this article online Website: www.asianjns.org DOI: 10.4103/1793-5482.146591 | +|----------------------------------------------------------------------------------------| + +Address for correspondence: +Dr. Sumit Banerjee, 5/3, Type IV Flats, Residential Complex, AIIMS, Rishikesh ‑ 249 201, Uttarakhand, India. E‑mail: drsumitbanerjee@gmail.com and symptoms of these tumors are due to mass effect and neurological dysfunction.[1] The lack of any specific clinical characteristic often precludes early diagnosis of the lesion and results in most patients being diagnosed with advanced neurological impairments such as paralysis and bladder incontinence.[2] Radiological evaluation with magnetic resonance imaging (MRI) has however led to decreased lag time in the diagnosis of such lesions.[3] The management of IMSCTs is mainly surgical, however, the proximity and intimate involvement of neural tracts in the tumor makes surgery difficult and increases the surgical morbidity. The aggressive surgical procedures result in deterioration of the neurological status and hence a more conservative excision of the tumor with adjuvant radiotherapy may be an available alternative.[4,5] +Multiple studies have proven that optimum surgical excision of the tumor provides the best outcome.[6-8] In tumors which have definite plane there is a general consensus toward total excision but controversy still exists between radical tumor excision and near total excisions with adjuvant therapy particularly in tumors with ill-defined margins/planes. The role of adjuvant therapy is also variable in different histotypes and depends on the totality of excision of tumors. The role of chemotherapy is still questionable in spinal tumors. With these points in mind we studied the clinical presentation, histological diagnosis, management and functional outcome of patients presenting with IMSCTs. + +## Materials And Methods + +The study was a prospective clinical study carried out at a tertiary care center from 2003 to 2012. All patients presenting with neurological clinical features were evaluated thoroughly by academic residents of neurosurgery. They were subjected to systematic physical and neurological evaluation, and radio-diagnostic studies were performed and any doubtful features were checked by an academic faculty. The standard diagnostic tool was MRI (with or without contrast). Fifty-one patients with suspected intramedullary spinal mass were further evaluated on MRI of which 48 had spinal intramedullary tumors, three patients with recurrence were excluded. These three patients had been treated elsewhere, and the exact nature and details of their treatment were not available. The clinical details and demographic data of each case were recorded. Their motor and sensory functions were documented. The motor power grading was done using the Medical Research Council (MRC) grading. The sensory compromises were recorded quantitatively. Sphincter functions were assessed and recorded. The level of lesion was recorded. The patients were then subjected to surgery after obtaining anesthetic clearance following requisite investigations for anesthetic and other considerations. All patients were operated upon by a single senior neuro-surgeon. All efforts were made to remove the tumor intoto Gross total excision was defined as no visual evidence of tumor on the surgical field on operating microscope. Near total excision was defined as <10% residual tumor mass following surgery. Sub-total excision involved removal of 50-90% of tumor tissue by the end of surgical procedure. The surgeries were carried out under an operating microscope. The surgeon decided on his clinical experience and visual confirmation the extent of dissection and resection required. The excised tumor mass was then examined by two senior pathologists concurrently to arrive at a histological diagnosis. Postoperatively patients were subjected to adjuvant radiotherapy. The patients were then followed-up clinically and radiologically at 6 weeks, 3, 6, and 12 months and thereafter depending on neurological progression. Only those patients having a follow-up of more than 3 months were included in the present series. Neurological examination was carried out at each follow-up and recorded in terms of improvement, deterioration or stabilization. Any evidence of recurrence was duly noted and diligently followed-up with further diagnostic work-up. All complications were noted and treated as required. + +McCormick's functional classification was used to assess the functional outcome of the patients postsurgery.[9] The data generated was subjected to statistical analysis using IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp and two-tailed Fisher's t-test was used to calculate significance. + +## Observations + +Of the total 48 patients presenting with intramedullary tumors, intraoperatively and following the histo-pathological diagnosis one case with tuberculoma and two cases with benign growths (lipoma) were excluded. Two patients were lost to follow-up and were not included in the analysis. The remaining 43 patients were assessed for the study. Male patients constituted a majority of the study group (72%, n = 31). The patients ranged from pediatric age group (3 years) to geriatric population (63 years) with a mean age of 27.4 years. Being a tertiary care center and a referral hospital most patients presented to us with long duration of symptoms, (mean = 25.6 months, range - 1-45 months). The most common clinical presentation was back pain (upper and lower) which was documented in 82% patients (n = 34). On detailed neurological examination, nearly all patients had some degree of impairment [Table 1]. Sensory derangement was present in 39 patients(90.7%) but dissociated sensory loss was seen in only 19 cases (44.1%). Sphincteric functions were impaired in 22 cases (51.2%). While motor deterioration was seen in 38 patients (88.4%) with most common preoperative power grade being MRC Grade III (n = 17, 39.5%). Dorsal spine was most commonly affected region with 22 cases (51.6%) followed by cervical spine (n = 12, 27.9%) [Figure 1]. Gross total excision was performed in 30 cases (69.76%) the rest being near total excisions (n = 13, 30.2%). Fourteen patients (32.5%) were subjected to adjuvant radio-therapy in addition to surgery. On histopathological diagnosis the most common tumor type was ependymoma (n = 21, 48.8%) followed by astrocytoma (n = 12) cases [Figure 2]. The rest were hemangioblastomas (n = 5, 11%), scwannomas (n = 3) and gangliogliomas (n = 2). Of the astrocytomas, eight were anaplastic/high grade; similarly seven ependymomas were high grade on histopathological examination. Immediately after surgery 65% of cases (n = 28) showed a transient decline in the neurological status due to cord handling and edema, which stabilized over the recovery period. Neurological evaluation was then carried out at discharge and subsequently at every follow-up visit. Treatment led to an improvement in muscle power in 28 cases (65.1%) with only five cases showing deterioration while 10 cases remained stable, similarly 27 cases (62.8%) showed sensory improvement and four patients showed deterioration at average follow-up shown in Figure 3. Bladder incontinence persisted in five cases, pressure sore occurred in two cases and impotence was complained postoperatively by one case. The mean follow-up period was 22months(range: 3-96months). By the last follow-up only 8 patients (18.6%) had tumor recurrence proven in MRI. + +## Results Functional Outcome + +It was one of the most important facets of the study. Postoperatively patients improved by an average of one MRC grade. Fifteen of 17 patients in Grade III and 10 out of 12 patients in Grade IV improved and had near-normal functional muscle power [Table 2]. Only three patients out of nine (33%) with Grade I or Grade II showed improvement in functional muscle power. Bowel and bladder continence improved or was stabilized in 38 cases (88.4%). Similarly, protective sensation was restored in eight cases and maintained in 31 patients. The ability to walk with/without support and carryout activities of personal hygiene and daily living was improved in 19 out of 30 patients with impairment (63.3%). Preoperaive McCormick classification showed 13 cases (30.2%) in Grade I or II, which improved to 32 cases (74.4%) postoperatively [Figure 4]. + +![2_image_2.png](2_image_2.png) + +![2_image_3.png](2_image_3.png) + +Survival and recurrence No mortality was recorded during the entire period of study. More important was maintenance of the improvement in the + +Table **1: Preoperative neurological status** + +![2_image_0.png](2_image_0.png) + +Table 2: Pre and postoperative McCormick functional ![2_image_1.png](2_image_1.png) + +status of the patient during the follow-up. We measured the survival outcomes in terms of event free survival, that is, the absence of deterioration of functional results, absence of new sign and symptom or radiological evidence of tumor recurrence. Thirty-five patients (81.4%) had an event free survival till their last follow-up visit. Of the eight patients with recurrence three had clinical deterioration rest were diagnosed on radiology during follow-up visits. The average duration between surgery and detection of recurrence in this study was 34.4 months (range: 9-84 months). Of the eight cases with recurrence three had undergone gross total excision, the remaining five had undergone near total excision followed by radiotherapy. Thus, the percentage of patients who had a recurrence following gross total excision (GTE) was 10% as compared to 38.5% (5 out of 13) in cases with near total excision (NTE) plus radiotherapy. Thus, GTE was significantly better than NTE plus radiotherapy for management of IMSCTs (P = 0.0415). With respect to tumor type anaplastic astrocytoma had the most recurrences(n = 3) but was not statistically significant (P = 0.68) + +## Discussion + +Spinal tumors have always been a challenge for the treating surgeon. The rarity of IMSCTs notwithstanding,[2,6] they have always posed a question regarding their optimum management. Most authors have reported a delayed presentation and diagnosis of IMSCTs because they have no specific symptoms, and the patient is often treated symptomatically initially and the full radiological and clinical examination is frequently not performed.[2,7] the most common symptom in this study, as well as others in literature, is low back ache. Radiating low-back ache is not characteristic of intramedullary tumors and is more associated with extramedullary pathologies such as, disc disease, spondylitis, etc., However, it is seen in a number of cases of intramedullary tumors and hence the need for an exhaustive clinical examination and radiological work-up in cases of radicular back pain. MRI is the preferred method of diagnosis[1,3] and we found it quite useful not just for diagnosis, but also for preoperative planning and early detection of recurrent lesions. Any procedure or surgical management in neurosurgical oncology is judged by the improvement in functional status after surgery, the overall improvement in survival and the incidence of recurrence. With these parameters in mind, the results of the study were deciphered. Surgery on spinal cord tumors is fraught with the risk of postoperative morbidity, and the surgeon is always in a dilemma between aggressive radical resection and preserving functional neural mass. Conservative surgical excision coupled with radiotherapy was once promulgated as a viable alternative to radical surgery and consequent neurological morbidity.[10,11] Nowadays, the standard protocol is for gross total excision as it has been proven by many authors to be superior, in this study also, it was significantly better (P = 0.0415) in terms of rate of recurrence. The developments in neurosurgical techniques, particularly micro dissection and use of somatosensory evoked potential and motor evoked potential intraoperatively have vastly reduced the incidence of iatrogenic damage.[12,13] We routinely used the operating microscope. The experience of the surgeon is an important factor in reducing intraoperative damage and in experienced hands the surgical morbidity is minimal. The role of radiotherapy as adjuvant is also under debate. Most centers continue to advice adjuvant radiotherapy in cases of IMSCT, and there have been reports in the past suggesting its role in improving long-term outcome.[5] Latest studies however lay emphasis on gross tumor excision and relegate the role of adjuvant radiotherapy in inoperable tumors, high-grade/ +malignant tumors and inadequately resected tumors.[2,7,14] In this study too we found that radiotherapy didn't affect the rate of recurrence postresection, and the eventual outcome was inferior to those with GTE. However, we do support the use of adjuvant radiotherapy in high-grade astrocytomas and ependymomas. The recurrence rate in this study was 18.6% at the last follow-up, this is in agreement with the data published in the literature with recurrence rates ranging from 10% to 22%.[6,10,13] +The association of tumor type with recurrence is not that consistent. Most intramedullary tumors of spinal cord are either benign or low-grade and hence the rate of recurrence is more dependent upon the extent of surgical resection as micro-residual tumors (which are frequently left even after GTE) probably didn't recur frequently. On the other hand high grade astrocytomas and malignant tumors are associated with an increased risk of recurrence.[6,15] In this study too high-grade astrocytomas had the highest number of recurrence (3 out of 8, 37.5%) though not statistically significant and were associated with an inferior eventual outcome. There has been consistency in the literature regarding the importance of preoperative neurological status and its bearing on the eventual functional outcome.[6,7,13] In this study, too patients with Grade III and IV muscle power showed significant improvement and far exceeded the improvement in patients with lower MRC grading. This could be because a good preoperative status collaborates with less infiltration of tumor mass into neural tracts and recent tumor effect which can be reversed with surgery. Furthermore, the fact that even a single-grade improvement in muscle power for Grade III results in significant functional improvement is instrumental in the improved outcome in these patients. This again emphasizes the role of early diagnosis and timely, aggressive surgical management in IMSCTs. A pertinent point in this regard is that our being tertiary center patients presented to us late which led to a deterioration in their neurological status, thus the importance of an effective referral system. + +## Conclusions + +Intramedullary spinal cord tumors are rare neuroaxial neoplasms but require aggressive diagnostic and surgical management duly to ensure the best outcome. Even malignant lesions such as anaplastic astrocytomas and high-grade ependymomas occupy this location. The most important predictor of functional outcome is preoperative neurological status and hence referral system and other efforts must be made to ensure early diagnosis and prompt surgical management. Most IMSCTs are histologically of low-grade. Tumor type seemingly has no effect on functional outcome; however high-grade tumors are associated with a higher rate of recurrence. Gross total excision of the tumor offers the best chance for an improved event free survival and is definitely superior to sub-total excision and adjuvant radiotherapy. The role of radiotherapy is limited to high-grade tumors and in cases where radical excision is not achieved or in inoperable cases. Meticulous clinical and radiological follow-up is needed to ensure early detection and timely treatment of recurrence ensures prolonged survival and reduces morbidity in patients. + +## References + +1. Kane PJ, el‑Mahdy W, Singh A, Powell MP, Crockard HA. Spinal intradural tumours: Part II - Intramedullary. Br J Neurosurg 1999;13:558‑63. + +2. Malhotra N, BHowmick D, Whitfield P. Intramedullary spinal cord tumours: Diagnosis, treatment, and outcomes. Adv Clin Neurosci Rehabil 2010;10:21‑5. + +3. Jallo GI, Freed D, Epstein F. Intramedullary spinal cord tumors in children. Childs Nerv Syst 2003;19:641‑9. + +4. Chun HC, Schmidt‑Ullrich RK, Wolfson A, Tercilla OF, Sagerman RH, +King GA. External beam radiotherapy for primary spinal cord tumors. J Neurooncol 1990;9:211‑7. + +5. Linstadt DE, Wara WM, Leibel SA, Gutin PH, Wilson CB, Sheline GE. + +Postoperative radiotherapy of primary spinal cord tumors. Int J Radiat Oncol Biol Phys 1989;16:1397‑403. + +6. Constantini S, Miller DC, Allen JC, Rorke LB, Freed D, Epstein FJ. + +Radical excision of intramedullary spinal cord tumors: Surgical morbidity and long‑term follow‑up evaluation in 164 children and young adults. J Neurosurg 2000;93:183‑93. + +7. Sandalcioglu IE, Gasser T, Asgari S, Lazorisak A, Engelhorn T, +Egelhof T, et al. Functional outcome after surgical treatment of intramedullary spinal cord tumors: Experience with 78 patients. Spinal Cord 2005;43:34‑41. + +8. Hoshimaru M, Koyama T, Hashimoto N, Kikuchi H. Results of microsurgical treatment for intramedullary spinal cord ependymomas: Analysis of 36 cases. Neurosurgery 1999;44:264‑9. + +9. McCormick PC, Stein BM. Intramedullary tumors in adults. In: +Stein BM, McCormick PC, editors. Neurosurgery Clinics in North America: Intradural Spinal Surgery. Vol. 1. Philadelphia: WB Saunders Co.; 1990. p. 609‑30. + +10. Houten JK, Cooper PR. Spinal cord astrocytomas: Presentation, management and outcome. J Neurooncol 2000;47:219‑24. + +11. Wood EH, Berne AS, Taveras JM. The value of radiation therapy in the management of intrinsic tumors of the spinal cord. Radiology 1954;63:11‑24. + +12. Wiedemayer H, Fauser B, Sandalcioglu IE, Schäfer H, Stolke D. The impact of neurophysiological intraoperative monitoring on surgical decisions: A critical analysis of 423 cases. J Neurosurg 2002;96:255‑62. + +13. Garcés‑Ambrossi GL, McGirt MJ, Mehta VA, Sciubba DM, +Witham TF, Bydon A, et al. Factors associated with progression‑free survival and long‑term neurological outcome after resection of intramedullary spinal cord tumors: Analysis of 101 consecutive cases. J Neurosurg Spine 2009;11:591‑9. + +14. Jallo GI, Danish S, Velasquez L, Epstein F. Intramedullary low‑grade astrocytomas: Long‑term outcome following radical surgery. J Neurooncol 2001;53:61‑6. + +15. Cohen AR, Wisoff JH, Allen JC, Epstein F. Malignant astrocytomas of the spinal cord. J Neurosurg 1989;70:50‑4. + +How to cite this article: Kumar R, Banerjee S. Management and functional outcome of intramedullary spinal cord tumors: A prospective clinical study. Asian J Neurosurg 2014;9:177-81. + +Source of Support: Nil, **Conflict of Interest:** None declared. + +## "Quick Response Code" Link For Full Text Articles + +The journal issue has a unique new feature for reaching to the journal's website without typing a single letter. Each article on its first page has a "Quick Response Code". Using any mobile or other hand-held device with camera and GPRS/other internet source, one can reach to the full text of that particular article on the journal's website. Start a QR-code reading software (see list of free applications from http://tinyurl.com/yzlh2tc) and point the camera to the QR-code printed in the journal. It will automatically take you to the HTML full text of that article. One can also use a desktop or laptop with web camera for similar functionality. See http://tinyurl.com/2bw7fn3 or http://tinyurl.com/3ysr3me for the free applications. \ No newline at end of file diff --git a/medical/md/PMC5013291.md b/medical/md/PMC5013291.md new file mode 100644 index 0000000000000000000000000000000000000000..0f55bad9f2c4d4d82abd37229a86dfb3bf674696 --- /dev/null +++ b/medical/md/PMC5013291.md @@ -0,0 +1,137 @@ + of Case 26 +Contents lists available at ScienceDirect + +![0_image_0.png](0_image_0.png) + +![0_image_4.png](0_image_4.png) + +![0_image_5.png](0_image_5.png) + +# International Journal Of Surgery Case Reports + +![0_Image_2.Png](0_Image_2.Png) + +![0_Image_3.Png](0_Image_3.Png) + +journal homepage: www.casereports.com Congenital internal hernia through defect in the falciform ligament in + +![0_image_1.png](0_image_1.png) + +adult: A case report and review of the literature Simona Macina∗, Tommaso Testa, Caterina Losacco University of study of Genoa (Italy) IRCCS, Largo Benzi 10, 16132 Genova, Italy a r t i c l e i n f o Article history: +Received 22 March 2016 Accepted 1 May 2016 Available online 17 May 2016 Keywords: +Internal hernia Falciform ligament a b s t r a c t INTRODUCTION: The incidence of occlusion syndrome caused by internal hernia is very rare, in particular when the defect is congenital discovered in adults with no previous abdominal surgery. PRESENTATION OF CASE: We present a case of a 31 year-old female patient who presented with acute abdominal pain and mechanical obstruction. The patient had never undergone abdominal surgery. + +DISCUSSION: On diagnostic laparoscopy, it was found a herniation of a loop of small bowel through a hole in the falciform ligament. The obstruction was solved by the division of part of falciform ligament without intestinal resection. CONCLUSION: Internal hernia is a very uncommon pathology, most often discovered in pediatric age because of congenital abnormalities, it must be included in the differential diagnosis in adults. Preoperative diagnosis is difficult. The diagnostic laparoscopic approach has shown to be the best. + +© 2016 Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +## 1. **Introduction** + +An internal abdominal herniation is a protrusion of an abdominal organ, through a mesenteric or peritoneal hole [1], they have no sac and can include the colon, the omentum and most often the small bowel [2]. Internal hernia is rare, estimated to be 2% of all causes of internal obstruction [3], and among these through the falciform ligament is extremely uncommon. Internal abdominal hernia can be constitutional related to congenital defects or complete failure in the embryological development[4–6], acquired related to trauma and iatrogenic caused by surgical procedures. Except urgency, clinical presentation is often aspecific: chronic digestion disorders, pain after feeding, mild abdominal pain due to intermittent bowel obstruction [7,12]. The symptoms may mimic peptic ulcer, biliary pathologies and abdominal angina [8]. Therefore preoperative diagnosis is uncommon, Ct scan is routinally performed in Emergency Department. + +## 2. Case **Report** + +31-year-old woman was admitted to the Emergency department with complaints of: epigastric pain with nausea, suddenly arised and no associated with feeding and central abdominal distension. No other relevant history, except of previous episodes of abdominal pain, treated with antispastic drugs. Per abdomen, distension and pain in the epigastrium at the palpation were present, bowel sounds were present in all quadrant of the abdomen with hyperperistaltism in epigastrium. The systemic examination was normal. + +## 2.1. Investigations + +Complete blood count: leukocytosis (12,36 × 10E9/L)with neutrophilia (10,90 × 10E9/L); routine biochemistry showed increased values of LDH (221 U/L) and CPK (242 U/L); X-ray, chest: negative; abdomen: gas distension of some intestinal loops in epigastric and right-hypochondriac region with a single air-fluid level in epigastric region; computed tomography: interposition of right colon between anterior abdominal wall and liver. Gas distension of some ileal loops which are located between liver and anterior abdominal wall; in left paramedian area one of these loops has thickened wall and perivisceral fluid and appears to reduce its caliber. + +## 2.2. Treatment + +The patient was started on conservative management, i.e. nil by mouth, intravenous fluid. Pain was resistant to antispastic and antalgic drugs (included Morphine intramuscular). She did not improve her condition and after 8 h the abdominal pain and the lab exams went worse, so the decision for diagnostic laparoscopy to be performed was taken, after collegial discussion with radiologists. Findings of laparoscopy: the introduction of the first trocar with open technique throught the umbelica allows the discovering of distended small bowel loops, herniated small bowel loop through a http://dx.doi.org/10.1016/j.ijscr.2016.05.003 2210-2612/© 2016 Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons. + +org/licenses/by-nc-nd/4.0/). + +S. et al. / of Case 26 105 + +![1_image_0.png](1_image_0.png) + +![1_image_1.png](1_image_1.png) + +defect in the anterior part of falciform ligament causing a mechanic occlusion, the involved loop appeared to be suffering for venous stasis because of initial strangulation of the vascular axis. It was necessary to introduce other two trocars, both under-mesocolic on the transverse umbelicar line on the two sides laterally to the margin of the rectus muscle. Using the monopolar crochet, the section of fibrous enveloping ring released the loop, which seems to recover with adeguate vascular supply. The falciform ligament was cut and the free margin was ligated with endoloop, in order to reduce the possibility of recurrence. The patient had an uneventful post-operative recovery. She was discharged in the third post-op day (Figs. 1–3 ). + +## 3. **Discussion** + +An internal hernia is a pathological protrusion on an abdominal organ through a normal or abnormal hole within the boundaries of the peritoneal cavity. The incidence of internal hernia is 0.2–2% [3] and among these hernia through the falciform ligament is very rare and accounts for 0.2% [9], caused by malformation and incomplete development of the falciform ligament. Most of the time this defect is discovered during pediatric age. Instead in adult the most frequent cause of abdominal hernia is iatrogenic defect created post laparoscopic surgery (in particular cholecystectomy, bariatric and gastro-esophagus procedures) during insertion of the port cannula + +![2_image_0.png](2_image_0.png) + +106 S. et al. / of Case 26 + +Fig. 3. X ray: gas distension of some intestinal loops in epigastric and right-hypochondriac region with a single air-fluid level in epigastric region. +[10,11]. The first case of falciform hernia was described in 1929 and each decades is characterized by an increasing number of new cases, as such as there is an ever-increasing rate of cases done laparoscopically. This evidence suggests logically the association between the two events [10,15,16]. In the above-described case, the defect did not appear to be iatrogenic because the patient had never undergone abdominal surgical procedures. The nonspecific clinical and laboratory features give to the radiological findings a big role in the diagnosis and the planning of surgicaltreatment[13]. + +Multidetector computed tomography (CT), with its thin-section axial images and high-quality multiplanar reformations currently plays an essential role in preoperative diagnosis of internal hernias. The CT findings include detection of an intestinal closed loop, identification of the hernia orifice (if it is possible)and eventually the abnormal displacement of surrounding structures and vessels around the hernia orifice and hernia sac. Detailed knowledge of the anatomy, etiology, and imaging of the various hernia types is also necessary. Familiarity with the appearances of internal hernias at multidetector CT allows accurate and specific preoperative diagnosis. In the above-described case, emergent CT imaging demonstrated between abdominal wall and liver the presence of some dilated ileal loops. One of these was dilated and edematous in the left paramedian region and reduced its caliber in the right paramedian region; the point of stricture (approximately median region) was the supposed falciform ligament site. Furthermore, that ileal loop had thickened wall, decreased contrast enhancement with perivisceral fluid: these signs are suggestive of vascular suffering. On the base of these findings was performed the suspect of an internal hernia in the site of falciform ligament [17–20]. Nevertheless, because of the preoperative diagnosis is very difficult, laparoscopic approach is mandatory and superior to other diagnostic tools, infact in up to 40% of patients it also can correct an erroneous preoperative diagnosis and exclude other abdominal pathological phenomena; plus the laparoscopic approach allows therapeutic correction of the defect during the same intervention [14]. + +## 4. **Conclusion** + +Internal hernia is a very uncommon pathology and in particular through the falciform ligament is very rare. In the past decades it is increasing constantly associated to the always higher number of laparoscopic procedures. Most often discovered in pediatric age because of congenital abnormalities, it must be included in the differential diagnosis also in adults. Preoperative diagnosis is difficult. The diagnostic laparoscopic approach has shown to be the best associated with a safe resolution and a rapid dismissing of the patient. + +## Conflict Of **Interest** + +There is no conflict of interest to declare. + +Funding None. + +Ethical **approval** +The paper is not a research study. + +## Consent + +Written informed consent was obtained from the patient for publication of this case report. + +## Author **Contributions** + +Simona Macina, ideated the study and drafted the article. Simona Macina, acquisition of data, drafting the article. Caterina Losacco, revising it critically for important intellectual content. + +Tommaso Testa, revising it critically for important intellectual content. Guarantor S. et al. / of Case 26 107 + +## References + +[1] M.A. Meyers, Dynamic Radiology of the Abdomen: Normal and Pathologic Anatomy, 4th ed., Springer Verlag, New York, NY, 1994. + +[2] N.V. Jamieson, The falciform ligament window: an unusual site foran internal hernia causing small bowel obstruction, J. R. Coll. Surg. Edinb. 32 (1978) 374–375. + +[3] D. Gullino, O. Giordano, E. Gullino, Les hernies de l'abdomen. Apropos de 14 cas, J. Chir. 130 (1993) 179–195. + +[4] R.G. Molly, H. Ellamushi, I.S. Smith, Strangulated small bowel hernia of the falciform ligament, Br. J. Surg. 79 (1992) 57. + +[5] A. Blunt, G.F. Rich, Intestinal strangulation through an aperture in the falciform ligament, Aust. N. Z. J. Surg. 37 (1968) 310. + +[6] L.B. Svendsen, T.S. Johansen, P. Kristensen, Intestinal obstruction caused by an aberrant umbilical vain, Acta Chir. Scand. 143 (1977) 191–192. + +[7] D. Mathieu, A. Luciani, Internal abdominal herniation, AJR Am. J. Roentgenol. + +183 (August (2)) (2004) 397–404 (GERMAD Group). + +[8] G.G. Ghahremani, Abdominal and pelvic hernias, in: R.M. Gore, M.S. Levine +(Eds.), Textbook of Gastrointestinal Radiology, 2nd ed., Saunders, Philadelphia, PA, 1994. + +[9] S. Wiseman, Internal herniation through a defect in the falciform ligament: a case report and review of the world literature, Hernia 4 (2) (2000) 117–120. + +[10] A. Charles, A.A. Shaikh, S. Domingo, et al., Falciform ligament hernia after laparoscopic cholecystectomy: a rare case and review of the literature, Am. Surg. 71 (4) (2005) 359–361. + +[11] M.B. Malas, N. Katkhouda, Internal hernia as a complication of laparoscopic nissen fundoplication, Surg. Laparosc. Endosc. Percutan. Tech. 12 (2) (2002) 115–116. + +[12] Y. Janin, A.M. Stone, L. Wise, Mesenteric hernia, Surg. Gynecol. Obstet. 150 +(1980) 747–754. + +[13] C.Y. Fu, W.C. Chang, H.E. Lu, C.J. Su, K.H. Tan, Pericecal hernia of the inferior ileocecal recess: CT findings, Abdom. Imaging 32 (2007) 81–83. + +[14] F. Agresta, I. Michelet, E. Candiotto, N. Bedin, Incarcerated internal hernia of the small intestine through a breach of the broad ligament: two cases and a literature review, JSLS 11 (2007) 255–257. + +[15] S. Sourtzis, C. Canizares, J.F. Thibeau, P. Philippart, N. Damry, An unusual case of small bowel through an iatrogenic defect in the falciform ligament, Eur. Radiol. 12 (2002) 531–533. + +[16] M. Lakdawala, S.R. Chaube, Y. Kazi, A. Bhasker, A. Kanchwala, Internal hernia through an iatrogenic defect in the falciform ligament: a case report, Hernia 13 (2) (2009) 217–219. + +[17] S. Doishita, T. Takeshita, Y. Uchima, et al., Internal hernias in the era of multidetector CT: correlation of imaging and surgical findings, Radiographics 36 (1) (2016) 88–106. + +[18] J. Egle, A. Gupta, V. Mittal, P. Orfanou, S. Silapaswan, Internal hernias through the falciform ligament: a case series and comprehensive literature review of an increasingly common pathology, Hernia 17 (2013) 95–100. + +[19] Hironori Shiozaki, Shintaro Sakurai, Kazuki Sudo, Gen Shimada, Hiroshi Inoue, Seiji Ohigashi, Gautam A Deshpande, Osamu Takahashi, Hisashi Onodera, Pre-operative diagnosis and successful surgery of a strangulated internal hernia through a defect in the falciform ligament: a case report, J. Med. Case Rep. 6 (2012) 206. + +[20] O. Armstrong, Internal hernia through the falciform ligament, Hernia 17 +(2013) 815–816. + +Open Access This article is published Open Access at sciencedirect.com. It is distributed under the IJSCR Supplemental terms and conditions, which permits unrestricted non commercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. \ No newline at end of file diff --git a/medical/md/PMC5078575.md b/medical/md/PMC5078575.md new file mode 100644 index 0000000000000000000000000000000000000000..22ddc56d291a185d9a4f0fa959969af2f80da7e7 --- /dev/null +++ b/medical/md/PMC5078575.md @@ -0,0 +1,264 @@ + +## Description Of Clostridium Phoceensis Sp. Nov., A New Species Within The Genus Clostridium + +M. Hosny, S. Benamar, G. Durand, N. Armstrong, C. Michelle, F. Cadoret, B. La Scola and N. Cassir Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63 CNRS 7278 IRD 198 INSERM U1095, IHU Méditerranée Infection, Pôle des Maladies Infectieuses, Assistance Publique-Hôpitaux de Marseille, Faculté de Médecine, Marseille, France + +## Abstract + +Clostridium phoceensis sp. nov., strain GD3T (= CSUR P1929 = DSM 100334) is the type strain of C. phoceensis sp. nov., a new species within the genus Clostridium. This strain was isolated from the gut microbiota of a 28-year-old healthy French man. C. phoceensis is a Gram-negative, spore-forming, nonmotile, strictly anaerobic bacterium. We describe its complete genome sequence and annotation, together with its phenotypic characteristics. + +© 2016 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. + +Keywords: Anaerobe, Clostridium phoceensis, culturomics, gut microbiota, taxono-genomics Original Submission: 17 June 2016; Revised Submission: 9 September 2016; Accepted: 16 September 2016 Article published online: 25 September 2016 Corresponding author: N. Cassir, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095, Faculté de médecine, AixMarseille Université, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France E-mail: cassirnadim@gmail.com M. Hosny and S. Benamar contributed equally to this article, and both should be considered first author. + +## Introduction + +Human adult gut microbiota is estimated to consist of up to 100 trillion microorganisms, comprising at least 500 different species, mostly anaerobic bacteria [1]. Although the advent of modern molecular microbiological methods has expanded the degree of bacterial detection from stool samples, it does not allow for the phenotypic description of new living species [2]. + +Consequently, there has been renewed interest in culture methods [3]. + +A new taxonomic approach known as taxonogenomics has recently been proposed to describe new isolated bacterial species [4]. This polyphasic strategy combines phenotypic characteristics, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectrum, and the analysis and comparison of the complete genome sequence. + +Since the creation of the genus Clostridium in 1880, more than 200 species have been described [5]. While species belonging to the family Clostridiaceae are mainly associated with the commensal digestive flora of mammals and can be commonly found in the environment, some are major human pathogens, including toxigenic Clostridium botulinum, Peptoclostridium difficile, Clostridium tetani and Clostridium perfringens [6]. + +We propose Clostridium phoceensis sp. nov., strain GD3T (= +CSUR P1929 = DSM 100334) as the type strain of C. phoceensis sp. nov., a new species within the genus Clostridium. This strain was isolated from the gut microbiota of a 28-year-old healthy French man as part of a culturomics study aiming at individually cultivating all bacterial species from a stool sample. Here we describe the characteristics of C. phoceensis sp. nov. strain GD3T, including its phenotype and genome sequence. + +## Materials And Methods Sample Collection + +The stool sample was taken from a 28-year-old healthy French man. The sample was collected as part of a research study on the human gut microbiota. The study was approved by the Institut Fédératif de Recherche 48 (agreement no. 09-022, Marseille, France), and the patient's consent was obtained. The sample was stored at −80°C in La Timone Hospital (Marseille, France). + +## Strain Isolation And Identification (Maldi-Tof Ms And 16S Rrna Sequencing) + +The faecal sample was treated using the concept of culturomics +[7]. The colonies obtained were identified using MALDI-TOF +MS [8,9] (Bruker Daltonics, Leipzig, Germany) and analysed using a Microflex spectrometer (Bruker), leading to the protein spectrum being obtained. A score under 1.7 did not enable any identification. Subsequently, 16S rRNA was sequenced and the sequence was matched using BLAST (Basic Local Alignment Search Tool) against the National Center for Biotechnology Information database [10]. DNA was extracted using the EZ1 Tissue Extraction Kit (Qiagen, Hilden, Germany), and sequences were aligned using ChromasPro 1.6 (Technelysium, South Brisbane, Queensland, Australia). + +## Growth Conditions + +The growth condition of the strain was determined by testing different temperatures and atmospheres. Five growth temperatures (ambient, 28, 37, 45 and 56°C) were tested under anaerobic (GENbag anaer) microaerophilic atmospheres (GENbag microer) (bioMérieux, Marcy l'Étoile, France) and aerobic condition on 5% sheep's blood agar (bioMérieux). Colonies were obtained after thermal shock for 20 minutes at 80°C in an anaerobic blood culture bottle (Bactec Lytic/10 Anaerobic/F) supplemented with 5% sheep's blood at 37°C. + +## Phenotypic, Biochemical And Antibiotic Susceptibility Tests + +Gram staining, motility, catalase and oxidase were determined as described by Lagier et al. [3]. Sporulation was tested using a thermal shock on bacterial colonies (diluted in phosphatebuffered saline) for 10 minutes at 80°C. The biochemical characteristics were tested using API 50CH, API ZYM and API +20A strips (bioMérieux). Antibiotic susceptibility referred to European Committee on Antimicrobial Susceptibility Testing 2015 recommendations. + +## Fatty Acid Methyl Ester (Fame) Analysis By Gas Chromatography/Mass Spectrometry (Gc/Ms) + +FAME analysis was performed by GC/MS. Two samples were prepared with 2 mg of bacterial biomass from several culture plates. Two samples were prepared with approximately 2 mg of bacterial biomass per tube collected from several culture plates. + +FAMEs were prepared as described by Sasser (http://www.midiinc.com/pdf/MIS_Technote_101.pdf). GC/MS analyses were carried out as described by Dione et al. [11]. Briefly, FAMEs were separated using an Elite 5-MS column and monitored by mass spectrometry (Clarus 500-SQ8S; PerkinElmer, Courtaboeuf, France). A spectral database search was performed using MS Search 2.0 operated using the Standard Reference Database 1A (National Institute of Standards and Technology, Gaithersburg, MD, USA) and the FAME mass spectral database (Wiley, Chichester, UK). + +## Microscopy + +Individual cells of C. phoceensis strain GD3T were captured using a Tecnai G20 electron microscopy (FEI Company, LimeilBrevannes, France). A Color Gram 2 Kit (bioMérieux) was used to perform the Gram coloration observed with a 100× oilimmersion objective lens using the DM1000 photonic microscope (Leica, Wetzlar, Germany) [12,13]. + +## Genome Project History + +The organism was selected for sequencing because it had been isolated from a healthy person for the first time and on the basis of its 16S rRNA similarity, phylogenetic position and phenotypic differences with other members of the genus Clostridia. The GenBank accession number is CVUG01000000 and consists of 10 scaffolds with a total of 16 contigs [14]. + +## Genome Sequencing And Assembly + +The genomic DNA of Clostridium phoceensis GD3T was sequenced on the MiSeq sequencer (Illumina, San Diego, CA, +USA) using the mate-pair strategy. The gDNA was barcoded in order to be mixed with 11 other projects using the Nextera mate-pair sample prep kit (Illumina). The mate-pair library was prepared with 1.5 μg of genomic DNA using the Nextera MatePair Illumina guide. The gDNA sample was simultaneously fragmented and tagged using a mate-pair junction adapter. The pattern of the fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) with a DNA 7500 lab chip. The DNA fragments ranged in size from 1 to 10 kb, with an optimal size of 4.490 kb. No size selection was performed, and only 600 ng of tagmented fragments were circularized. The circularized DNA was mechanically sheared to small fragments with an optimal size of 938 bp on the Covaris S2 device in microtubes (Covaris, Woburn, MA, USA). + +The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent Technologies), and the final library concentration was measured at 4.457 nmol/L. The libraries were normalized at 2 nM and pooled. After a denaturation step and dilution at 15 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing run were performed in a single 39-hour run in a 2 × 251 bp; 6.1 Gb NMNI, 14 + +![2_image_0.png](2_image_0.png) + +![2_image_1.png](2_image_1.png) + +of total information was obtained from a 653K/mm2 cluster density, with a cluster passing quality control filters of 96.1% +(12 031 000 clusters). Within this run, the index representation for C. phoceensis GD3T was determined to 9.32%. The 1 121 200 paired reads were filtered according to the read qualities. These reads were trimmed, then assembled by SPDES +software. Finally, the draft genome of C. phoceensis GD3T +consisted of ten scaffolds with 16 contigs and generated a genome size of 3.4 Mb with 59.32% G+C content. Genome annotation Open reading frames (ORFs) were predicted using Prodigal +[15] with default parameters, although the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank database [16] and the Clusters of Orthologous Groups (COGs) databases using BLASTP. The tRNA genes were found using the tRNAScanSE tool [17], while RNAmmer +[18] was used to find ribosomal RNAs and BLASTn against the GenBank database. Lipoprotein signal peptides and the number of transmembrane helices were predicted using SignalP [19] and TMHMM [20] respectively. Artemis [21] was used for data management, and DNA Plotter [22] was used to visualize genomic features. To estimate the mean level of nucleotide sequence similarity at the genome level, we used homemade average genomic identity of orthologous gene sequences +(AGIOS) software [4]. + +Results Strain isolation, identification and phylogeny MALDI-TOF MS failed to identify the strain GD3T, so its mass spectrum was added to the Bruker database (Fig. 1). To improve identification, 16S rRNA sequencing was performed, and the access number in 16S rRNA EMBL-EBI (European Molecular Biology Laboratory–European Bioinformatics Institute) was assigned as LN846907. The highest value of nucleotide sequence similarity was observed with Flavonifractor plautii +(97%), the phylogenetically closest species. + +![2_image_2.png](2_image_2.png) + +## Phenotypic And Biochemical Characterization + +C. phoceensis strain GD3T is a Gram-negative (Fig. 2), sporeforming, nonmotile, strictly anaerobic bacterium that has no catalase and oxidase activities, measuring 1.8 μm in length and 0.5 μm in diameter (Fig. 3). The sporulation test was positive; the organism grows at 45°C in anaerobic conditions. Using the API ZYM gallery, C. phoceensis exhibits esterase (C4), phosphatase acid, naphthol-AS-BI-phosphohydrolase and β-glucosidase activities. When using the API 20A gallery, a positive reaction was only observed for D-glucose (Table 1). + +Of the antibiotics tested, C. phoceensis was found to be sensitive to amoxicillin, amoxicillin–clavulanate, ceftriaxone, ceftazidime, imipenem, doripenem and ciprofloxacin. + +## Predominant Cellular Fatty Acids + +The predominant cellular fatty acids of C. phoceensis strain GD3T are hexadecanoic acid (16:0; 33.1 ± 1.7%), 9octadecenoic acid (18:1n9; 24.3 ± 0.6%), octadecanoic acid (18:0; 20.0 ± 0.1%), 9,12-octadecadienoic acid (18:2n6; 8.6 ± 0.1%), 11-octadecenoic acid (18:1n7; 5.5 ± 0.5%), tetradecanoic acid (14:0; 4.7 ± 0.8%) and trace amounts (less than 1%) of heptadecanoic acid, 9-hexadecenoic acid, pentadecanoic acid, 15-methyl-hexadecanoic acid, 14-methyl-hexadecanoic acid, 10-heptadecenoic acid, 13-methyl-tetradecanoic acid and 12-methyl-tetradecanoic acid (Table 2). + +## Genome Properties + +![3_Image_4.Png](3_Image_4.Png) + +A phylogenetic tree highlighting the position of Clostridium phoceensis GD3T relative to other type strains within the order Clostridiales is provided in Fig. 4. + +The genome of the C. phoceensis strain GD3T is 3 453 562 bp long with 59.32% G+C content. A total of 3320 genes were predicted, of which 3264 were protein-coding + +![3_image_0.png](3_image_0.png) + +TABLE 1. Classification, general features and biochemical tests of Clostridium phoceensis strain GD3T + +Fatty acid Name Mean relative %a + +![3_image_1.png](3_image_1.png) + +![3_image_2.png](3_image_2.png) + +![3_image_3.png](3_image_3.png) + +16:0 Hexadecanoic acid 33.1 ± 1.7 18:1n9 9-Octadecenoic acid 24.3 ± 0.6 18:0 Octadecanoic acid 20.0 ± 0.1 18:2n6 9,12-Octadecadienoic acid 8.6 ± 0.1 18:1n7 11-Octadecenoic acid 5.5 ± 0.5 14:0 Tetradecanoic acid 4.7 ± 0.8 17:0 Heptadecanoic acid TR +16:1n7 9-Hexadecenoic acid TR 15:0 Pentadecanoic acid TR 17:0 iso 15-methyl-Hexadecanoic acid TR 17:0 anteiso 14-methyl-Hexadecanoic acid TR 17:1n7 10-Heptadecenoic acid TR +15:0 iso 13-Methyl-tetradecanoic acid TR +15:0 anteiso 12-Methyl-tetradecanoic acid TR +genes and 56 were RNA genes (three genes are 5S rRNA, one gene is 16S rRNA, one gene is 23S rRNA and 51 genes are TRNA genes). A total of 1967 genes (60.26%) were assigned a putative function. A total of 227 genes (6.95%) were identified as ORFans. The remaining genes (28.92%) were annotated as hypothetical proteins. The properties and statistics of the genome are summarized in Table 3 and Fig. 5. The distribution of genes into COGs functional categories is presented in Table 4 and Fig. 6. + +FIG. 4. Phylogenetic tree high- + +![4_image_0.png](4_image_0.png) + +lighting position of Clostridium phoceensis GD3T relative to other type strains within Clostridiales order. GenBank accession numbers are indicated. Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained using maximum-likelihood method within MEGA 4 software [23]. Numbers at nodes are bootstrap values obtained by repeating analysis 500 times to generate majority consensus tree. + +Scale bar represents 5% nucleotide sequence divergence. Rubidus massiliensis was used as outgroup. + +## Genome Comparison + +We made some brief comparisons against nine genomes: +Intestinimonas butyriciproducens strain ER1 (GenBank accession no. JPJD01000000), Flavonifractor plautii ATCC 29863 +(AGCK01000000), Clostridium leptum DSM 753 +(ABCB02000000), Clostridium cellulosi DG5 (NZ_LM995447), Ethanoligenens harbinense YUAN-3 (NC_01482), Oscillibacter valericigenes Sjm18-20 (NC_016048), Clostridium viride DSM 6836 (NZ_JHZO01000000), Eubacterium siraeum V10Sc8a +(FP929059) and Pseudoflavonifractor capillosus ATCC 29799 +(NZ_AAXG02000000). The draft genome sequence of C. phoceensis is smaller than those of Pseudoflavonifractor TABLE 3. Nucleotide content and gene count levels of + +genome + +![4_image_1.png](4_image_1.png) + +capillosus, Oscillibacter valericigenes, Flavonifractor plautii, Clostridium cellulosi and Intestinimonas butyriciproducens (3.45, 4.24, 4.47, 3.81, 5.68 and 3.57 MB respectively), but larger than those of Clostridium viride, Ethanoligenens harbinense, Clostridium leptum and Eubacterium siraeum (2.41, 3.01, 2.82 and 2.84 MB +respectively). The G+C content of C. phoceensis is smaller than those of Flavonifractor plautii (59.32 and 61.07% respectively) +but larger than those of Pseudoflavonifractor capillosus, Oscillibacter valericigenes, Clostridium viride, Ethanoligenens harbinense, Clostridium leptum, Eubacterium siraeum, Clostridium cellulosi and Intestinimonas butyriciproducens (59.11, 53.19, 49.28, 55.56, 50.25, 45.13, 42.07 and 58.44% respectively). The gene content of C. phoceensis is smaller than those of Pseudoflavonifractor capillosus, Oscillibacter valericigenes, Flavonifractor plautii, Clostridium cellulosi and Intestinimonas butyriciproducens (3264, 4829, 4723, 4278, 5171 and 3529 respectively) but larger than those of Clostridium viride, Ethanoligenens harbinense, Clostridium leptum and Eubacterium siraeum (2321, 2701, 2482 and 2211 respectively). C. phoceensis has a similar distribution of genes into COGs categories with the most of the compared species (Fig. 4). However, Clostridium cellulosi was overrepresented for all the categories, and Clostridium viride was overrepresented for category Z (cytoskeleton). + +In addition, Clostridium phoceensis shared 846, 884, 492, 562, 482, 717, 634, 407 and 842 orthologous genes respectively with Intestinimonas butyriciproducens, Flavonifractor plautii, Clostridium leptum, Clostridium cellulosi, Ethanoligenens harbinense, Oscillibacter valericigenes, Clostridium viride, Eubacterium siraeum and Pseudoflavonifractor capillosus (Table 5). Of these species, the orthologous genes shared ranged from 365 between Eubacte- + +![5_image_0.png](5_image_0.png) + +TABLE 4. Number of genes associated with 25 general COGs functional categories + +![5_image_1.png](5_image_1.png) + +FIG. 5. Graphical circular map of Clostridium phoceensis GD3T genome. + +From outside in, outer two circles show ORFs oriented in forward (colored by COGs categories) and reverse (colored by COGs categories) directions, respectively. Third circle marks tRNA genes (green). + +Fourth circle shows G+C% content plot. Innermost circle shows GC +skew, with purple indicating negative values and olive indicating positive values. COGs, Clusters of Orthologous Groups database; ORF, open reading frame. + +rium siraeum and Clostridium viride to 1079 between Flavonifractor plautii and Intestinimonas butyriciproducens. Compared to other species, C. phoceensis exhibited AGIOS values ranging from 56.64 with Ethanoligenens harbinense to 71.58 with Flavonifractor plautii. + +## Conclusion + +On the basis of taxonogenomic analyses, we propose Clostridium phoceensis sp. nov., strain GD3T (= CSUR P1929 = DSM +100334), as the type strain of C. phoceensis sp. nov., a new species within the genus Clostridium (Fig. 4). This strain was isolated from the gut microbiota of a 28-year-old healthy French man. + +Taxonomic and nomenclatural proposals + +## Description Of Clostridium Phoceensis Sp. Nov. + +Clostridium phoceensis strain GD3T (pho.ce.en'sis, L. fem. adj., +from phoceensis referring to Phocea, the Greek name of the city which founded Marseille, where it was isolated) is a Gramnegative nonmotile bacillus whose individual cell size is FIG. 6. Distribution of functional classes of predicted genes in genomes + +![6_image_0.png](6_image_0.png) + +from Clostridium phoceensis (Cp), +Intestinimonas butyriciproducens strain ER1 (Ib), Flavonifractor plautii ATCC 29863 (Fp), Clostridium leptum DSM 753 (Cl), Clostridium cellulosi DG5 (Cc), Ethanoligenens harbinense YUAN-3 (Eh), Oscillibacter valericigenes Sjm18-20 (Ov), Clostridium viride DSM +6836 (Cv), Eubacterium siraeum V10Sc8a (Es) and Pseudoflavonifractor capillosus ATCC 29799 (PC) genomes according to clusters of orthologous groups of proteins. ATCC, American Type Culture Collection (Manassas, VA, USA). + +![6_image_1.png](6_image_1.png) + +1.8 μm in length and 0.5 μm in diameter. It is a strictly anaerobic and endospore-forming bacterium. Strain GD3T is catalase and oxidase negative, and its optimal growth temperature is 45° C, but it also grows weakly at 37°C. Biochemical analyses showed positive reactions of C. phoceensis for D-glucose and produced esterase (C4), phosphatase acid, naphthol-AS-BIphosphohydrolase and β-glucosidase enzymes. C. phoceensis was sensitive to amoxicillin, amoxicillin–clavulanate, ceftriaxone, ceftazidime, imipenem, doripenem and ciprofloxacin. Its predominant cellular fatty acids are hexadecanoic acid (16:0; 33.1 ± 1.7%), 9-octadecenoic acid (18:1n9; 24.3 ± 0.6%), octadecanoic acid (18:0; 20.0 ± 0.1%), 9,12-octadecadienoic acid +(18:2n6; 8.6 ± 0.1%), 11-octadecenoic acid (18:1n7; 5.5 ± 0.5%) +and tetradecanoic acid (14:0; 4.7 ± 0.8%). Its 16S rRNA sequences were deposited in GenBank under accession numbers CVUG01000000 and LN846907. Strain GD3T, whose CSUR +and DSMZ numbers are respectively CSUR P1929 and DSM +100334, was identified from the stool sample of a 28-year-old healthy French man. + +## Acknowledgements + +The authors thank the Xegen Company (www.xegen.fr) for automating the genomic analysis process and culturomics team in URMITE for the bacteria isolation. This project was funded by the Fondation Méditerranée Infection. + +## Conflict Of Interest + +None declared. + +References +[1] Palmer C, Bik EM, DiGiulio DB, Relman DA, Brown PO. Development of the human infant intestinal microbiota. PLoS Biol 2007;5:e177. + +[2] Lagier JC, Million M, Hugon P, Armougom F, Raoult D. Human gut microbiota: repertoire and variations. Front Cell Infect Microbiol 2012;2:136. + +[3] Lagier JC, Edouard S, Pagnier I, Mediannikov O, Drancourt M, +Raoult D. Current and past strategies for bacterial culture in clinical microbiology. Clin Microbiol Rev 2015;28:208–36. + +[4] Ramasamy D, Mishra AK, Lagier JC, Padhmanabhan R, Rossi M, +Sentausa E, et al. A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species. Int J Syst Evol Microbiol 2014;64:384–91. + +[5] Lawson PA, Rainey FA. Proposal to restrict the genus Clostridium +(Prazmowski) to Clostridium butyricum and related species. Int J Syst Evol Microbiol 2015. + +[6] Popoff MR, Bouvet P. Clostridial toxins. Future Microbiol 2009;4: +1021–64. + +[7] Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. + +Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect 2012;18:1185–93. + +[8] Grosse-Herrenthey A, Maier T, Gessler F, Schaumann R, Böhnel H, +Kostrzewa M, et al. Challenging the problem of clostridial identification with matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry (MALDI-TOF MS). Anaerobe 2008;14:242–9. + +[9] Seng P, Abat C, Rolain JM, Colson P, Lagier JC, Gouriet F, et al. + +Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization–time of flight mass spectrometry. J Clin Microbiol 2013;51:2182–94. + +[10] Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 16S +ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 2000;38:3623–30. + +[11] Dione N, Sankar SA, Lagier JC, Khelaifia S, Michele C, Armstrong N, +et al. Genome sequence and description of Anaerosalibacter massiliensis sp. nov. New Microbes New Infect 2016;10:66–76. + +[12] Mourembou G, Yasir M, Azhar EI, Lagier JC, Bibi F, Jiman-Fatani AA, +et al. Rise of microbial culturomics: noncontiguous finished genome sequence and description of Beduini massiliensis gen. nov., sp. nov. OMICS J Integr Biol 2015;19:766–76. + +[13] Fournier PE, Raoult D, Drancourt M. New species announcement: a new format to prompt the description of new human microbial species. New Microbes New Infect 2016. In press. + +[14] Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. + +Nat Biotechnol 2008;26:541–7. + +[15] Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW, Hauser LJ. + +Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010;11:119. + +[16] Benson DA, Karsch-Mizrachi I, Clark K, Lipman DJ, Ostell J, Sayers EW. + +GenBank. Nucleic Acids Res 2012;40(Database issue):D48–53. + +[17] Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997;25:955–64. + +[18] Lagesen K, Hallin P, Rødland EA, Stærfeldt HH, Rognes T, Ussery DW. + +RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 2007;35:3100–8. + +[19] Dyrløv Bendtsen J, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004;340: 783–95. + +[20] Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden markov model: +application to complete genomes. J Mol Biol 2001;305:567–80. + +[21] Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, +et al. Artemis: sequence visualization and annotation. Bioinformatics 2000;16:944–5. + +[22] Carver T, Thomson N, Bleasby A, Berriman M, Parkhill J. DNAPlotter: +circular and linear interactive genome visualization. Bioinformatics 2009;25:119–20. + +[23] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9. \ No newline at end of file diff --git a/medical/md/PMC5151226.md b/medical/md/PMC5151226.md new file mode 100644 index 0000000000000000000000000000000000000000..9b1796433e27f7a2bf042504c5ccd17c7af82004 --- /dev/null +++ b/medical/md/PMC5151226.md @@ -0,0 +1,69 @@ +of Medicine at the College de France at Paris. + +It may +. Professor causalgia, pains due to vaso-constriction, angina pectoris and inoperable malignant neoplasms. Pain is treated in neuralgia is designated as the 'syndrome of multiple angina pectoris, one encounters the treatment +instruments require their inventors'. It is explained +that is too strictly instrumental. For their proper use, problem of arteritis and vaso-motor disturbances. One of the most fruitful contributions by Professor Leriche iovocainisation '. This is only one of the many notable +, This is, indeed, a book to possess and study. + +P.: N. R.: +MANAGE + +## The Pharmacology And Therapeutics Of The Materia Medica.—By W. J. Dilling, M.B., +Ch.B. (Aberd.), M.P.S. (Hon.). Fifteenth Edition. +1939. Cassell And Company, Limited, London. +Pp. X Plus 600. Price, 10S. 6D. + +THIS book thas gone through 15 editions and 23 reprintings; it has served two generations of medical students and doctors; and there seems every reason of predict that it will serve a third generation equally wll. + +The last edition was published six years ago so that The Addendum to the B.P., was published in 1936, and all the new drugs that appeared in this are dealt been included in the 1933 edition. + +pharmacopceial rank are included, such as nicotinic acid, the sulphanilamide group, and snake poisons. + +The information given is for the most part concise quinine is not a perfect prophylactic in malaria but from the rest of the paragraph one gathers that he has prophylaxis and true causal prophylaxis. The clinical It is a book that we can strongly recommend + +## Your Chest Should Be Flat: The Deep Chest + Makes Better Soil For Tuberculosis. + Lippinoot Company, Philadolphia And London. + Pp. Xvil Plus 145. + +(India), Limited, Calcutta. Price, Rs. 6 THE author, who is Assistant Professor of Medicine, +.. + +fcantly large number of cases from infancy onwards +that the healthy chest is from infancy on a signi­ +70 +chest is flat and wide with an index between 60 to +tuberculous chest is round and deep, while the normal age have been shown to be more prone to disease tuberculous chests and still higher in chronic bronchitis and asthma. + +It has been shown that the thoracie index falls in the case of boys from 72 at the age of 5 years to 67 at the e +of 5 years to 70.4 at the age of 18. Up to the age +however, the as height increases and also as vital capacity increases lt has further been shown that environment has poor type (fof development) +than +'children from environments. + +From the data collected and analysed by the author, especially on tuberculin-tested individuals and on tuberoffers a more favourable soil for the development of ing factors have been generally noticed in association for age, poor socio-economic environment, lower than normal standing in school work and tendency to positive The author considers that the normal development of the chest and the early correction of physical as some of the correctional procedures. +adjustments should be arroutine procedure in the +tubercelosis and the correctional procedures +.. + +simple and mon-technical language, doctors as well as ed main The peruse the book with great interest and profit. The printing and get-up do credit to the publishers. + +A. C. U. + +## Formulary Ormulary Of The Allahabad—Based Jumna Dispensaries, Prmulary Allahabad - Based - On - The Supplement +Pharmacopceia, 1932 Cand - Supplement +1938. Complied By The Staff. 1938. Published By Dr. D. N. For +Allahabad. Pp. 138 + +THE formulary of the Jumna Dispensaries, Allahabad, is based on the British Pharmacopceia 1932 and its is good and there are footnotes and enough space for posological table (B. P. 1932) was also included in the book. The book will serve as an useful guide to M. N. R. C. + +HISTOLOGICAL TECHNIQUE FOR INTRACRANIAL + TUMOURS.—By Dorothy S. Russell, M.D. ( Lond.); +PP. VII +1939. Oxford University Press, London. Pp. Press, Press, Bd. Obtainable +from Oxford University Press, Bombay and Calcutta THIS +book is +.a is specially applicable in the case of brain tumorrs. +is specially applicable in the case of brain tumorrs. +But there must be caution in the case of brain tumorrs. \ No newline at end of file diff --git a/medical/md/PMC5467525.md b/medical/md/PMC5467525.md new file mode 100644 index 0000000000000000000000000000000000000000..2917cd2a0bf4f254347bf1a1e1a0d7412bb92ad5 --- /dev/null +++ b/medical/md/PMC5467525.md @@ -0,0 +1,433 @@ + +## Doi: 10.1002/Ijch.201600110 + +# Synthesis Of The Right-Side Structure Of Type B Physalins + +Masaki Morita,[a, b] Shuntaro Kojima,[a] Megumi Ohkubo, +[a] Hiroyuki Koshino, +[a, c] Daisuke Hashizume, +[d] +Go Hirai,*[a, b, c] Keiji Maruoka,[e] and Mikiko Sodeoka*[a, b, c] +This article is dedicated to Professors K. C. Nicolaou and Stuart L. Schreiber in celebration of their 2016 Wolf Prize. + +Abstract: We present a full account of our synthetic studies on the racemic DEFGH-ring moiety of physalins, featuring domino ring transformation of a tricyclic key intermediate. + +We also report the results of a detailed mechanistic examination of the domino ring transformation, as well as a reoptimization of the 2,3-Wittig rearrangement and methylation steps. Furthermore, we have newly established a method for the preparation of an optically active synthetic intermediate by enzymatic kinetic resolution. Our work provides access to both natural and nonnatural right-side physalin structures. + +Keywords: domino reactions · natural products · optical resolution · physalins · steroids + +## 1. Introduction + +Physalins are the bitter components of Physalis plants, and were first isolated from winter cherry in 1852.[1] A +century later, Matsuura and coworkers determined the structures of two bitter substances isolated from the leaves of Physalis alkekengi var. franchetii and named them physalin A[2] (1) and physalin B +[3] (2) (Figure 1). + +Since then, more than 30 physalins have been identified.[4, 5] Physalins share a unique 13,14-seco-16,24-cycloerostane skeleton, with a highly oxygenated, complex structure. Type B physalins, such as physalin B, have an H-ring with a C14@O@C27 bond and a cage-shaped structure, while Type A physalins do not. Prior to the work of our group, no synthetic study of physalins had been reported, other than derivatization of natural products. + +In addition to the intriguing structure, there is considerable interest in the biological activities of physalins, which include antitumor activity,[6,7] anti-inflammatory activity, [8] and inhibitory activity on NF-kB signaling.[9] Recently, inhibition of the hedgehog signaling pathway[10] +and the ubiquitin proteasome pathway[11] have also been reported. Thus, physalins have the potential to regulate a broad range of biological events. However, the mechanisms of these biological activities at the molecular level remain unknown. The AB-ring of physalins, which is commonly found in plant steroids, has been suggested to be involved in these biological activities. For instance, Ma and coworkers suggested that the A-ring of physalin A could form a covalent bond with cysteine residues of IKKb. + +[12] In contrast, little attention has been paid to the contribution of the cage-shaped right-side structure of Type B physalins. We hypothesized that this unique partial structure would play an important role in the biological activity. + +Figure 1. Structure of physalins 1 and 2. + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +[a] M. Morita, S. Kojima, M. Ohkubo, H. Koshino, G. Hirai, M. Sodeoka Synthetic Organic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan) +Tel.: (+81)48-467-9373, Fax: (+81)48-462-4666 e-mail: gohirai@riken.jp sodeoka@riken.jp +[b] M. Morita, G. Hirai, M. Sodeoka AMED-CREST, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan) +[c] H. Koshino, G. Hirai, M. Sodeoka RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan) +[d] D. Hashizume RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan) +[e] K. Maruoka Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502 (Japan) +Supporting information for this article is available on the WWW +under http://dx.doi.org/10.1002/ijch.201600110. + +T 2016 The Authors. Published by Wiley-VCH Verlag GmbH & +Co. KGaA. 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 noncommercial and no modifications or adaptations are made. + 2017 309 T 2017 The Published by Wiley-VCH GmbH KGaA, Weinheim 309 + +We have already succeeded in synthesizing the cageshaped molecule 3 (Scheme 1),[13,14] and we confirmed that the right-side structure of physalins indeed contributes to NF-kB-inhibitory activity. + +[5] The synthesis of 3 featured precise construction of tricyclic key intermediate 8 through a Diels@Alder reaction and the installation of two C1 units, and its "domino ring transformation", leading to DEFGH-ring compound 3. In this full paper, we present full details of the domino ring transformation to 3, together with the results of reoptimization of the synthesis of 8, and a newly established method to prepare optically pure intermediate 6-M by enzymatic resolution. + +![1_image_1.png](1_image_1.png) + +## 2. Results And Discussion 2.1 Synthesis Of Tricyclic Intermediate + +The preparation of 8 was commenced with the Diels@ +Alder reaction of 4 +[15] with 5 +[16] (Scheme 2A) to afford bicyclic 9 in moderate yield. However, we encountered poor reproducibility (25–65% yield) in this reaction, especially at a gram scale. We speculated that impurities in quinone 5 might be responsible for this, and found that 5 could be obtained asapale yellow solid by washing the crude brown solid with ether. When purified 5 was subjected to a Diels@Alder reaction, 9 was obtained in 77% +yield in a reproducible manner. The two carbonyl groups were reduced with DIBAL-H, and the resulting diol was converted to enone 10 by acid treatment. Protection of OH15 with MOM followed by Luche reduction gave a-allylic alcohol 6-M. + +The next key step is 2,3-Wittig rearrangement to introduce a hydroxymethyl group at C25. In our original report,[13] b-allylic alcohol derivative 11 was utilized as a substrate (Scheme 2B). The stereochemistry of C25 generated by 2,3-Wittig rearrangement should not be critical, because it is lost during the domino ring transformation. + +If homoallylic alcohol 14, which would be obtained by 2,3-Wittig rearrangement of 13, is applicable to the syn- + +![1_image_0.png](1_image_0.png) + +thesis of 3, it would be possible to reduce the number of synthetic steps. + +The precursor 13 was prepared by Williamson ether synthesis with ICH2SnBu3 (Scheme 3A).[17] Unfortunately, treatment of 13 with MeLi +[18] in THF at @5 8C did not give 2,3-Wittig rearrangement product 14 at all. Instead, allylic alcohol 6-M, methyl ether 15, and primary alcohol 16 were obtained, probably owing to stabilization of lithiated intermediate 17 by interaction with the MOM +group, as well as the unfavorable transition state 18 (Scheme 3B). a-Elimination or protonation of 17 leads to the formation of 6-M or 15, respectively, and 1,2-Wittig rearrangement of 19 yielded 16. Although we examined changing the alkyl lithium reagent, solvent, and temperature, 14 was not produced at all, suggesting that introduction of the hydroxymethyl group should be performed from the original precursor 11. + +For 2,3-Wittig rearrangement of 11, OH14 in 6-M was inverted to afford the corresponding b-alcohol 20 by a Mitsunobu reaction, followed by solvolysis. + +[19] Preparation of 11 was accomplished in a similar manner to that described for 13 (Scheme 4). In sharp contrast to the reaction of 13, a 2,3-Wittig rearrangement of 11 proceeded well. When 11 was treated with n-BuLi in THF, the desired primary alcohol 12 was obtained in 61% yield, along with 20 in 7% yield and 24 in 15% yield (Table 1, entry 1). Further optimization using n-BuLi turned out to be ineffective. We then switched to MeLi,[18] and found that the yield of 12 gradually increased as the reaction 2017 309 T 2017 The Published by Wiley-VCH GmbH KGaA, Weinheim www.ijc.wiley-vch.de 310 + +![2_image_0.png](2_image_0.png) + +![2_image_2.png](2_image_2.png) + +temperature was raised, and the formation of 20 was suppressed. Finally, 12 was obtained in 87 % yield as a result of careful control of the reaction temperature at −5°C; +this is a better result than in our original report. [13,20] The higher temperature probably favors the appropriate conformation for the rearrangement. These results clearly indicate that the stereochemistry of the precursor is critical in this case. Since it was difficult to separate the mixture of 12 , 20 , and 24 by normal silica gel chromatography, the mixture was subjected to protection of OH27 with TBS, + +![2_image_1.png](2_image_1.png) + +and deprotection of MPM, to give allylic alcohol 21 in pure form. MnO 2 oxidation and iodination [21] of the α-position of the resulting enone afforded 22. + +The introduction of the C21 methyl group was originally achieved by iron-mediated coupling, [ 22 ] and α -methylenone 7 was obtained in 75% yield. However, the reprodcibility of this reaction was also found to be problematic. Namely, predominant formation of tertiary alcohol 25 was occasionally observed (Table 2, entry 1). Although the formation of 7 is likely to depend on the addition speed of MeMgBr (slow addition of MeMgBr led to significant formation of 25 ), we investigated other transition metal-catalyzed methods for methylation of 22 to obtain + +![2_image_3.png](2_image_3.png) + +Isr. J. Chem. 2017, 57, 309–318 +© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ww.ijc.wiley-vch.de + +the desired 7 more consistently. Although Stille coupling + +![3_image_0.png](3_image_0.png) + +using tetramethyltin[23] or Suzuki@Miyaura coupling using trimethylboroxine[24] gave 7 in moderate yield, the concomitant formation of dimers 26 (entry 2) or 27 and 28 +(entry 3) was observed. Although Negishi coupling with dimethylzinc[25] also generated dimer 26, the desired 7 was obtained in 75% yield, comparable with the best result (entry 1), but in a more reproducible manner (entry 4). + +The diastereoselective addition of an alkyne to 7 was achieved with lithium acetylide in the presence of anhydrous CeCl3, without b-elimination of the oxygen functionality at C15 (Scheme 5). Epoxidation of diene 29 from the convex face with mCPBA was accompanied by tetrahydrofuran formation through attack of OH17 on the right-side epoxide to give 31. Desilylation and Dess@ +Martin oxidation of the secondary alcohol afforded ketone 32. Baeyer@Villiger oxidation of 32 with mCPBA +favored rearrangement of the more electron-rich C14 carbon, and 8 was obtained. + +![3_image_1.png](3_image_1.png) + +## 2.2 Domino Ring Transformation + +We had earlier established a synthetic methodology for the DFGH-ring system 35, +[13] lacking the E-ring. The DFGH-ring core 34 was constructed from a tricyclic framework via domino ring transformation (Scheme 6A). + +By simple LiOH treatment of 33, which was prepared from 8 in 2 steps, +[13] the DFGH-ring compound 34 was obtained in 13% yield, along with hemiacetal 38 in 75% +yield and exo-olefin 36 in 12% yield. Based on the formation of these by-products, we propose the following consecutive mechanism. Treatment with LiOH would lead to b-elimination (33!36), and hydrolysis of the seven-membered lactone would occur (36!37), forming hemiacetal and carboxylate. The resulting carboxylate would intramolecularly attack the epoxide to form the G-ring (37! + +38), and then the hemiacetal would attack the a,b-unsaturated lactone via an oxy-Michael reaction to form the Hring (38!34). We also demonstrated that 34 and 38 are in equilibrium (34 :38= ~1 :6) under the reaction conditions, indicating that 38 is more stable than 34. This thermodynamically controlled process prevented further optimization to improve the yield of 34. Deprotection and oxidation of OH15 afforded 35. + +To obtain the DEFGH-ring system 3, we designed two synthetic routes (Scheme 6B). In Route A, the alkyne moiety of 35 would be directly oxidized to a-ketocarboxylic acid 40, which would be transformed to 3 by acid treatment and reduction of the ketone at C13. In this route, the stability of the DFGH-ring system under oxidative conditions is critical. In Route B, the a-keto or a-hydroxyester moiety should be installed in advance. + +Domino ring transformation of 39 would form the GHring to furnish carboxylic acid 40, as in the case of 33. + +Subsequent acid treatment would afford 3 after deprotection and oxidation of OH15. We expected that the stability advantage of the DEFGH-ring (like Type B physalins) +over the corresponding hemiacetal DEFG-ring (like Type A physalins) would be enhanced, compared with that observed in 34 and 38, because most natural physalins possess Type B structure. + +We first examined Route A. Unfortunately, treatment of 35 with KMnO4 and NaIO4 +[26] in tBuOH-H2O, followed by acidification, did not provide 42 (Scheme 7). Instead, the generation of polar intermediates (probably carboxylic acid) was observed by TLC analysis. Thus, the resulting reaction mixture was treated with TMSCHN2. Unexpect2017 309 T 2017 The Published by Wiley-VCH GmbH KGaA, Weinheim www.ijc.wiley-vch.de 312 + +Full + +![4_image_0.png](4_image_0.png) + +![4_image_1.png](4_image_1.png) + +edly, the only isolated product was C15@O@C27 bridged hemiacetal 41, which does not have a C18 carbon. Although the precise mechanism of formation of 41 remains unclear, we considered that intermediate 43 would be generated by opening of the H-ring and oxidation of C25 and C27. Sequential formation of hemiacetal from OH14, CHO27, and ketone at C15 might give 41. These results indicate that the oxidation of alkyne in the presence of H-ring acetal and/or ketone in RouteAis not a promising strategy for the synthesis of 3. + +Then, we examined Route B (Scheme 6B). Oxidation of alkyne 8 with KMnO4 and NaIO4 proceeded without loss of C18, and the resulting carboxylic acid was esterified with TMSCHN2, affording the desired a-ketoester 44 in 88% yield. Removal of the TBS group and installation of a monochloromethanesulfonyl (Mc) group furnished 45 (Scheme 8A). + +As in the case of 33, 45 was treated with LiOH in THF-H2O. A new spot, probably due to the a-ketoacid species derived from hydrolysis of the a-ketoester, was observed on TLC. After 1.5 h, the reaction mixture was acidified with 1 N HCl aq., and a new, less-polar spot appeared on TLC. Unfortunately, the product was hemiacetal 46, and the target molecule 47 was not detected at all. + +This result indicates that although the E-ring was successfully formed by acid treatment, the H-ring was not formed in the presence of a-ketolactone moiety. This might be due to transient formation of a hemiacetal between OH14 and the ketone at C13, which would block the desired H-ring-forming oxy-Michael reaction. Another possibility is instability of the putative product 47, because it has an sp2carbon at C13, whereas physalins have an sp3carbon at this position. + +Then we prepared 51 bearing an a-hydroxyester group by reduction of the a-ketoester. Although the ketone at C13 is sterically hindered, it is expected to be reduced preferentially, due to its higher electrophilicity, compared with lactone, and ester functionality. Indeed, Luche reduction of 44 afforded the hydroxyester 49 in 99% yield +(a :b=9 : 1, Scheme 8B). Removal of the protecting group at OH27 (P') and introduction of an Mc group provided precursor 51-H. Benzyl-protected precursor 51-Bn was also prepared as follows. Treatment of 49-a with BnBr and NaH in the presence of nBu4NI gave 50, which was then converted to 51-Bn in two steps. + +With the precursors in hand, we examined the domino ring transformation of 51-H. When 51-H was treated with 4 equiv. of LiOH for 1.5 h, almost complete conversion of the substrate, with generation of a carboxylic acid species, was observed on TLC. Pleasingly, subsequent acidification with 1 N HCl aq. predominantly afforded the target DEFGH-ring compound 52-H in 25% yield, along with hemiacetal 53-H-b in 15% yield. This result clearly indi2017 309 T 2017 The Published by Wiley-VCH GmbH KGaA, Weinheim www.ijc.wiley-vch.de 313 + +## Full Paper + +cates that the keto functionality in 45 disturbed H-ring formation. + +We optimized the acidic conditions, because the low mass balance should be attributable to inefficiency of lactone formation (Table 3). Use of concentrated HCl resulted in cleavage of the MOM group, and no desired prod- +Table 3. Optimization of acidic conditions for domino ring transforuct was obtained (entry 2). Employment of an AcOH- +H2O (20:1) system [27] at higher temperature improved the outcome (entries 3–6). When the acid treatment was conducted at 100°C for 1 h, a mixture of 52-H and 53-H +was obtained in 70% total yield (entry 5). Treatment for a shorter or longer time decreased the yield (entries 7 and 8). While the content of water did not affect the reaction efficiency (entries 9 and 10), replacement of acetic acid with dichloroacetic acid resulted in deprotection of the MOM group (entry 11). + +We next examined various basic conditions to improve the ratio of 52/53. As shown in Table 4, the use of 4 equiv. of base at room temperature for 1.5 h was found to be the best, and shorter or longer reaction times resulted in decreased yield of 52. [14] Reduction of the water ratio decreased the formation of 52-H (entry 2). The counter cation also had a significant influence, and LiOH gave the best result (entries 3 and 4). When we applied the optimal conditions (entry 1) to the Bn-protected 51-Bn , the desired 52-Bn was obtained in a better yield (50%, +entry 5), along with hemiacetal 53-Bn (44%). + +In contrast to the domino ring transformation of 33 , +the product ratio of 52 and 53 varied, depending upon the reaction conditions, indicating that the product ratio is probably not determined by the relative stability of 52 and 53. Treatment of 52 or 53 under acidic conditions did Isr. J. Chem. 2017, 57, 309–318 +© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Table 4. Optimization of basic conditions for domino ring transfornot induce isomerization, indicating that H-ring formation or breakage did not proceed upon AcOH treatment +(Scheme 9A). On the other hand, treatment of DEFGH- +ring compound 52 under basic conditions generated a polar intermediate (like 54 and 55) and subsequent acid treatment gave 53 as a major product, regardless of the protecting group on OH 13 . When the same experiment was conducted starting from hemiacetal 53 , DEFGH-ring compound 52 was produced only in up to 11% yield. + +These results clearly indicate that formation of the H-ring from 55 to 54 by oxy-Michael reaction under the basic conditions is an unfavorable process. In other words, the formation of the H-ring occurred via a different process. + +Indeed, when we treated 51-H or 51-Bn with 2 equiv. of + +![5_image_0.png](5_image_0.png) + +53 (α: β ratio) + +![5_image_1.png](5_image_1.png) + +| mation of 51-H. | | | | | | +|---------------------------------------------------------------|---------------------|-------------|--------|------|------| +| 1) LiOH H 2 O (4 equiv) | | | | | | +| (F-H2O (1:1) | | | | | | +| RT, 1.5 h | | | | | | +| 51 H | 2) acidic | | | | | +| conditions | DEFGH ring | | | | | +| P = MOM | Hemiacetal | | | | | +| 52-H | 53-H (α:β ~ 1:5) | | | | | +| entry | acidic conditions | temperature | time | 52-H | 53-H | +| 1 | 1 N HCI aq. | RT | 4 h | 25% | 15% | +| 2 | conc. HCl aq. | RT | 1 h | 0% | 0% | +| ACOH-H 2 O (20:1) | RT | 1 h | 13% | 16% | | +| 3 | | | | | | +| 4 | ACOH-H2O (20:1) | 80 °C | 1 h | 23% | 20% | +| 5 | AcOH - H 2 O (20:1) | 100 °C | 1 h | 3% | 37% | +| ACOH - H 2 O (20:1) | reflux | 1 h | 14% | 26% | | +| 6 | | | | | | +| 7 | AcOH-H 2 O (20:1) | 100 °C | 20 min | 24% | 18% | +| 8 | ACOH-H2O (20:1) | 100 °C | 3 h | 15% | 16% | +| g | ACOH H2O (2:1) | 100 °C | 1 h | 2% | 27% | +| 10 | ACOH | 100 °C | 1 h | 2% | 32% | +| 11 | DCA-H2O (20:1) | 100 °C | 1 h | 0% | 0% | +| Ac = acetyl; conc. = concentrated; DCA = dichloroacetic acid. | | | | | | + +53 H 100% (1:5.0) +ww.ijc.wiley-vch.de + +| mation of 51. | 1) ba | 1.5 h | | | | | +|----------------------------|----------------|---------------|------------|------------|----------------|------------| +| THF-H 2 O | | | | | | | +| 51-Bn (R = Bn) 2) AcOH-H2C | OR | | | | | | +| 100 °C, 1 h | 52-H (R = H) | 5-H (R = H) | | | | | +| P = MOM | 52-Bn (R = Bn) | 5-Bn (R = Bn) | | | | | +| entry | precursor | base | vent ratio | 52 | 53 (α;β ~ 1:5) | | +| 51-H | LiOH H 2 O | 1:1 | 52 H : 33% | 53-H: 37% | | | +| 1 | 2 | 51-H | LiOH H 2 O | 3:1 | 52 H : 19% | 53 H : 30% | +| 52-H: 25% | 53-H: 39% | | | | | | +| 3 | 51-H | NaOH | 1:1 | | | | +| 4 | 51-H | KOH | 1:1 | 52-H: 28% | 53 H : 40% | | +| 5 | 51-Bn | LOH H20 | 1:1 | 52-Bn; 50% | 53-Bn ; 44% | | + +LiOH for 30 min, followed by acidification and esterification, diester 56-H or 56-Bn bearing the H-ring without + +![6_image_0.png](6_image_0.png) + +the G-ring, was isolated as a single isomer in 26% and 43% yield, respectively, along with 52 and 53 +(Scheme 10).[28] Furthermore, treatment of 56 with an excess amount of LiOH, followed by acid treatment, provided 52 and 53, +[28,29] indicating that the corresponding dicarboxylic acid was probably formed as an intermediate during domino ring transformation. + +A plausible mechanism of the domino ring transformation from 51 is shown in Scheme 11. As in the reaction + +![6_image_1.png](6_image_1.png) + +from 33, b-elimination of @OMc and hydrolysis of methylester and seven-membered lactone would give a,b-unsaturated carboxylic acid 58. Then, 7-endo oxy-Michael addition[30] would occur to form the H-ring, followed by diastereoselective protonation at C25 to generate 59. Subsequent formation of d-lactone through ring opening of the epoxide by the carboxylic acid would give 54. Finally, acid treatment would form g-lactone to furnish 52. This proposal is supported by the observation that the process from 55 to 54 is unfavorable, together with the formation of 56. 54 should be kinetically formed and gradually converted to 55 by retro-oxy-Michael reaction, leading to hemiacetal 53. The substituent at C17 favors domino ring transformation, and thus efficient formation of the cageshaped DEFGH-ring compound was achieved. + +## 2.3 Transformation To 3 + +Remaining tasks to complete the synthesis of 3 were removal of the MOM group and oxidation of OH15. For selective oxidation of OH15 over OH13, OH13 was first protected as the acetate to give 60. Treatment of 60 with TMSBr, which had been used for the synthesis of DFGHring 35, gave the desired 61 in 67% yield, along with retro-oxy-Michael reaction product 62 (Scheme 12A, +entry 1). The use of catalytic H2SO4in AcOH gave a similar result to entry 1, with concomitant removal of the acetyl group (entry 2). After further investigations, we found that treatment with AlCl3 and NaI[31] effectively provided 61 in 89% yield (entry 3). Oxidation of 61 to ketone was then examined (Scheme 12B). Dess@Martin oxidation, as used in the synthesis of 35, unexpectedly did not proceed, presumably because of the steric hindrance around OH15 and ring strain of 61. On the other hand, 1- +Me-AZADO +[32] was found to be an effective oxidant, affording the desired ketone 63 quantitatively. + +We then examined removal of the acetyl group. Unfortunately, basic, acidic, or enzymatic conditions did not give 3 at all. The main product was hemiacetal 64, which was formed via retro-oxy-Michael reaction. Surprisingly, even when 63 was dissolved in CD3OD and left at room temperature for 2 days, formation of 64 was observed by 1H NMR. This result suggests that the H-ring is susceptible to even a protic solvent. Therefore, we selected benzyl ether as a protecting group at OH13 instead of acetate. Benzyl-protected 65 was obtained by means of the same procedure established above, without opening of the H-ring (Scheme 12C). As we had hoped, removal of the benzyl group by hydrogenolysis was accomplished with Pd(OH)2/C (PearlmanQs catalyst) in AcOEt, and DEFGH-ring compound 3 was obtained quantitatively. + +## 2.4 Optical Resolution Of Synthetic Intermediate + + ## 1 Introduction The _quantum_ quantum mechanics is a quantum field theory of quantum mechanics. It is a quantum field theory of quantum mechanics. +We performed the synthetic studies using racemic compounds, but it would be better to have optically active compounds for future structure@activity relationship studies. Furthermore, for target identification, comparison of + +![7_image_0.png](7_image_0.png) + +the natural and unnatural enantiomers is valuable. [33] +Therefore, we set out to establish a method to obtain an optically active synthetic intermediate. + +First, we tried optical resolution of (±)-10. The enantiomers were separated by preparative HPLC using CHRALPAK IA to give optically active (–)-10 and +(+)-10 (Scheme 13A). The absolute stereochemistry of (+)-10 was determined by X-ray crystallography analysis of 68 , which was prepared by condensation of (+)-10 with chiral carboxylic acid 67. [ 28 ] As a result, (+)-10 was found to have the absolute stereochemistry leading to the unnatural enantiomer of physalins (Scheme 13B). Hence, +(—)-10 is the enantiomer required for the natural product synthesis. Since HPLC resolution is not suitable for largescale preparation of the optically active synthetic intermediate, we next investigated the enzymatic kinetic resolution of (±)-10. + +The secondary alcohol (±)-10 was treated with lipase in vinyl acetate. We screened various types of lipases, but unfortunately, no reaction occurred. We then tried other synthetic intermediates bearing a secondary alcohol, such Isr. J. Chem. 2017, 57, 309–318 +© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim + +![7_image_1.png](7_image_1.png) + +as (±)-20, (±)-21 and (±)-31, but again almost no conversion of substrates was observed. During the development of the synthetic route to key intermediate 8, we also prepared 15- OBOM -protected compounds 6- B. When (±)-6- +B was used as a substrate, the corresponding acetate 66 was finally detected. Acetylation with lipase SL gave the desired (+)-6-B in 61 % yield and 51 % ee (Table 5, entry 1). After the screening of various enzymes to improve the efficiency of the optical resolution, we eventually found that treatment of ( ± )-6-B with lipase AK for 21 h gave ( − )-66-B in 34% yield as an almost optically pure form (entry 2), although the enantiomeric excess of +(+)-6-B remained at only 46 %. A longer reaction time + +| lipase AMANO | | | | | | | +|-----------------------------------------------------------------|---------|-------------------------|----------------|-------------------|-------------|-------------| +| vinyl acetate | | | | | | | +| OH | | | | | | | +| MPMO | MPMO | O | | | | | +| (±)-6-B | P = BOM | (+)-6-B | P = BOM | | | | +| (±)-6-M | P = MOM | (+)-6-M | P = MOM | (-)-66-M | | | +| entry | 6 | liapse temperature time | yield of (+)-6 | yield of (−)-66 | | | +| 1 | 6-B | s | 70 ° C | 128 h | 61%, 51% ee | 4%, 79% ee | +| 55°C | | | | | | | +| 2 | 6-B | AK | 21 h | 6%, 46% ee | 3%, 99% ee | | +| 3 | 6-B | AK | 55°C | 72 h | 5%, 64% ee | 35%, 95% ee | +| 6-B | AK | 5°C | 154 h | 50%, 87% ee | 5%, 96% ee | | +| 5[9] | 6-B | AK | 55 °C | 170 h 66%, 41% ee | 25%, 98% cc | | +| 6 | 6-M | AK | 55°C | 132 h 50%, 99% ee | 50%, 99% ee | | +| [a] Isopropenyl acetate was used as a solvent. BOM = benzyloxy- | | | | | | | + +ww.ijc.wiley-vch.de + +improved both yield and ee, and (+)-6-B was obtained in 50% yield and 87% ee after 154 h (entry 4). As the reaction proceeded, the generated acetaldehyde might decrease the enzymatic activity due to Schiff base formation.[34] To avoid the formation of acetaldehyde, the reaction in isopropenyl acetate was also examined, but this resulted in low conversion (entry 5). Finally treatment of MOM-protected (:)-6-M with lipase AK in vinyl acetate at 558C afforded the desired (+)-6-M in 50% yield and 99% ee (entry 6). Importantly, the corresponding acetate +(@)-66-M was also formed in 50% yield and 99% ee, and this was converted to (@)-6-M (Scheme 14). The absolute stereochemistry of (+)-6-M was determined to be the one leading to the natural enantiomer of physalin by conversion from (@)-10 using the same procedure as for the racemate synthesis (Scheme 14).[28] + +![8_image_0.png](8_image_0.png) + +Selectivity of optical resolution can be evaluated in terms of E value. + +[35] The E value of optical resolution of 6-B with lipase SL (entry 1) was E= ~15, while with lipase AK (entry 4), the value was E= ~150, clearly indicating that lipase AK was a superior catalyst for this system. The E value of the reaction of 6-M with lipase AK (entry 6) was E>1000, so that both enantiomers were obtained in almost optically pure forms. Thus, the methodology to obtain both the natural and unnatural enantiomers of the right-side structure of physalins was established. + +## 3. Conclusion + +We have further developed and optimized our synthetic methodologies for the complex cage-shaped right-side structure of physalins. We propose a mechanism for the key domino ring transformation employed to construct the DEFGH-ring system on the basis of experimental evidence, including isolation of an intermediate. We have also established an efficient kinetic resolution of a synthetic intermediate. This should enable us to obtain optically active DEFGH-ring compounds. Further work, aimed at the total synthesis of physalins, is underway. + +## Acknowledgements + +We would like to thank Prof. Yoshiharu Iwabuchi +(Tohoku University, Japan) for providing 1-Me-AZADO, +and Amano Enzyme Inc. for providing lipases. We also would like to thank C. J. Henderson for checking part of this manuscript. This work was partially supported by RIKEN project funding and JSPS KAKENHI, Grant Number 16H01167 for the Middle Molecular Strategy and 15K12760. + +## References + +[1] V. Dessaignes, J. Chautard, J. Prakt. Chem. 1852, 55, 323 – +325. + +[2] T. Matsuura, M. Kawai, R. Nakashima, Y. Butsugan, Tetrahedron Lett. 1969, 10, 1083– 1086. + +[3] T. Matsuura, M. Kawai, Tetrahedron Lett. 1969, 10, 1765 – +1766. + +[4] For selected references, see: a) M. Kawai, T. Matsuura, Tetrahedron 1970, 26, 1743– 1745; b) L. R. Row, N. S. Sarma, K. S. Reddy, T. Matsuura, R. Nakashima, Phytochemistry 1978, 17, 1647– 1650; c) L. R. Row, K. S. Reddy, N. S. + +Sarma, T. Matsuura, R. Nakashima, Phytochemistry 1980, 19, 1175– 1181; d) M. Kawai, T. Matsuura, S. Kyuno, H. + +Matsuki, M. Takenaka, T. Katsuoka, Y. Butsugan, K. Saito, Phytochemistry 1987, 26, 3313 - 3317; e) M. Kawai, T. + +Ogura, M. Nakanishi, T. Matsuura, Y. Butsugan, Y. Mori, K. Harada, M. Suzuki, Bull. Chem. Soc. Jpn. 1988, 61, 2696– 2698; f) M. Kawai, T. Ogura, B. Makino, A. Matsumoto, H. Yamamura, Y. Butsugan, M. Hayashi, Phytochemistry 1992, 31, 4299– 4302; g) M. Kawai, A. Matsumoto, B. + +Makino, H. Mori, T. Ogura, Y. Butsugan, K. Ogawa, M. + +Hayashi, Bull. Chem. Soc. Jpn. 1993, 66, 1299– 1300; h) B. + +Makino, M. Kawai, Y. Iwata, H. Yamamura, Y. Butsugan, K. Ogawa, M. Hayashi, Bull. Chem. Soc. Jpn. 1995, 68, 219– 226; i) B. Makino, M. Kawai, T. Ogura, M. Nakanishi, H. Yamamura, Y. Butsugan, J. Nat. Prod. 1995, 58, 1668 – +1674; j) B. Makino, M. Kawai, K. Kito, H. Yamamura, Y. + +Butsugan, Tetrahedron 1995, 51, 12529 - 12538; k) M. Kawai, T. Yamamoto, B. Makino, H. Yamamura, S. Araki, Y. Butsugan, K. Saito, J. Asian Nat. Prod. Res. 2001, 3, 199– 205; l) P.-C. Kuo, T.-H. Kuo, A. G. Damu, C.-R. Su, E.-J. Lee, T.- S. Wu, R. Shu, C.-M. Chen, K. F. Bastow, T.-H. Chen, K.-H. + +Lee, Org. Lett. 2006, 8, 2953– 2956; m) R. Chen, J. Liang, R. Liu, Helv. Chim. Acta 2007, 90, 963– 966; n) L. Qiu, F. + +Zhao, Z.-H. Jiang, L.-X. Chen, Q. Zhao, H.-X. Liu, X.-S. + +Yao, F. Qiu, J. Nat. Prod. 2008, 71, 642 - 646. + +[5] M. Ozawa, M. Morita, G. Hirai, S. Tamura, M. Kawai, A. + +Tsuchiya, K. Oonuma, K. Maruoka, M. Sodeoka, ACS Med. Chem. Lett. 2013, 4, 730 - 735. + +[6] M. D. Antoun, D. Abramson, R. L. Tyson, C. Chang, J. L. + +McLaughlin, G. Peck, J. M. Cassady, J. Nat. Prod. 1981, 44, 579– 585. + 2017 309 T 2017 The Published by Wiley-VCH GmbH KGaA, Weinheim www.ijc.wiley-vch.de 317 + +[7] A. G. Damu, P.-C. Kuo, C.-R. Su, T.-H. Kuo, T.-H. Chen, K. F. Bastow, K.-H. Lee, T.-S. Wu, J. Nat. Prod. 2007, 70, 1146– 1152. + +[8] A. T. Vieira, V. Pinho, L. B. Lepsch, C. Scavone, I. M. Ribeiro, T. Tomassini, R. Ribeiro-dos-Santos, M. B. P. Soares, M. M. Teixeira, D. G. Souza, Br. J. Pharmacol. 2005, 146, 244– 251. + +[9] N. J. Jacobo-Herrera, P. Bremner, N. M\#rquez, M. P. Gupta, S. Gibbons, E. MuÇoz, M. Heinrich, J. Nat. Prod. 2006, 69, 328– 331. + +[10] T. Hosoya, M. A. Arai, T. Koyano, T. Kowithayakorn, M. + +Ishibashi, ChemBioChem 2008, 9, 1082– 1092. + +[11] I. Vandenberghe, L. Cr8ancier, S. Visp8, J.-P. P. Annereau, J.-M. M. Barret, I. Pouny, A. Samson, Y. Aussagues, G. Massiot, F. Ausseil, C. Bailly, A. Kruczynski, Biochem. Pharmacol. 2008, 76, 453– 462. + +[12] L. Ji, Y. Yuan, L. Luo, Z. Chen, X. Ma, Z. Ma, L. Cheng, Steroids 2012, 77, 441 - 447. + +[13] M. Ohkubo, G. Hirai, M. Sodeoka, Angew. Chem. Int. Ed. + +2009, 48, 3862 - 3866. + +[14] M. Morita, G. Hirai, M. Ohkubo, H. Koshino, D. Hashizume, K. Maruoka, M. Sodeoka, Org. Lett. 2012, 14, 3434 – +3437. + +[15] S. D. S. Burke, J. E. Cobb, K. Takeuchi, J. Org. Chem. 1990, 55, 2138– 2151. + +[16] P. A. Wender, D. B. Rawlins, Tetrahedron 1992, 48, 7033 – +7048. + +[17] J. chman, P. Somfai, Synth. Commun. 1994, 24, 1117 - 1120. + +[18] P. A. Caruana, A. J. Frontier, Tetrahedron 2004, 60, 10921 – +10926. + +[19] L. Gentric, I. Hanna, L. Ricard, Org. Lett. 2003, 5, 1139 – +1142. + +[20] The improved yield is attributed to accurate temperature control, using an isothermal bath. Reactions conducted with ice or dry ice baths resulted in a decreased yield of 12. + +[21] C.-K. Sha, S.-J. Huang, Tetrahedron Lett. 1995, 36, 6927 – +6928. + +[22] G. Cahiez, H. Avedissian, Synthesis 1998, 1199– 1205. + +[23] H. Yokoe, C. Mitsuhashi, Y. Matsuoka, T. Yoshimura, M. + +Yoshida, K. Shishido, J. Am. Chem. Soc. 2011, 133, 8854 – +8857. + +[24] T. Taniguchi, G. Tanabe, O. Muraoka, H. Ishibashi, Org. + +Lett. 2008, 10, 197– 199. + +[25] S. I. Ngi, K. Cherry, V. H8ran, L. Commeiras, J. L. Parrain, A. DuchÞne, M. Abarbri, J. Thibonnet, Chem. Eur. J. 2011, 17, 13692 - 13696. + +[26] a) J. H. Tatlock, J. Org. Chem. 1995, 60, 6221 - 6223; b) D. + +Urabe, T. Nishikawa, M. Isobe, Chem. Asian. J. 2006, 1, 125– 135. + +[27] T. Nakajima, D. Yamashita, K. Suzuki, A. Nakazaki, T. + +Suzuki, S. Kobayashi, Org. Lett. 2011, 13, 2980– 2983. + +[28] See Supporting Information. [29] Since hydrolysis was so sluggish, an excess amount of LiOH +was required for complete consumption of 56. + +[30] As far as we know, this is the first example of a simple intramolecular 7-endo oxy-Michael reaction. In most cases, simultaneous 5-exo cyclization occurs. For example, see: S. J. + +Danishefsky, W. H. Pearson, D. F. Harvey, C. J. Maring, J. P. + +Springer, J. Am. Chem. Soc. 1985, 107, 1256 - 1268. + +[31] a) M. Node, K. Nishide, M. Sai, E. Fujita, S. Acids, Tetrahedron Lett. 1978, 19, 5211– 5214; b) E. D. Moher, J. L. Collins, P. A. Grieco, J. Am. Chem. Soc. 1992, 114, 2764– 2765. + +[32] M. Shibuya, M. Tomizawa, I. Suzuki, Y. Iwabuchi, J. Am. + +Chem. Soc. 2006, 128, 8412– 8413. + +[33] Y. Nakamura, R. Miyatake, M. Ueda, Angew. Chem. Int. + +Ed. 2008, 47, 7289 - 7292. + +[34] B. Berger, K. Faber, J. Chem. Soc. Chem. Commun. 1991, 1198– 1200. + +[35] C. S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. + +Chem. Soc. 1982, 104, 7294– 7299. + +Received: September 3, 2016 Published online: November 9, 2016 \ No newline at end of file diff --git a/medical/md/PMC5485066.md b/medical/md/PMC5485066.md new file mode 100644 index 0000000000000000000000000000000000000000..23e91842294892ae60bbebb76237b164d29d602a --- /dev/null +++ b/medical/md/PMC5485066.md @@ -0,0 +1,289 @@ +# Population Pharmacokinetic Modeling Of Secukinumab In Patients With Moderate To Severe Psoriasis + +The Journal of Clinical Pharmacology + +![0_image_0.png](0_image_0.png) + +2017, 57(7) 876–885 +-C 2017 The Authors.*The Journal of Clinical Pharmacology* Published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology DOI: 10.1002/jcph.876 Gerard Bruin, PhD1, Christian Loesche, MD1, Judit Nyirady, MD, MBA2, and Oliver Sander, PhD3 + +## Abstract + +Secukinumab is a human monoclonal antibody with demonstrated efficacy for moderate to severe psoriasis; it binds to and neutralizes interleukin (IL)-17A. The pharmacokinetic (PK) parameters of secukinumab were best described by a 2-compartment model. Only weight was included in the final model, as other covariates did not affect clinical relevance. The estimated serum clearance of secukinumab was 0.19 L/day, with interindividual variability (IIV) of 32% coefficient of variation (CV), and low total volume of distribution (central compartment volume, 3.61 L with IIV of 30% CV; peripheral compartment volume, 2.87 L with IIV of 18% CV). The bioavailability of secukinumab after subcutaneous dosing was approximately 73%, +with an absorption rate of 0.18/day with IIV of 35% CV. The PK profile of secukinumab was linear, with no evidence of a dose dependence of clearance. + +Clearance and volume of secukinumab varied with body weight in an allometric relationship. The time to maximum serum concentration at steady state occurred approximately 6 days after dosing for both secukinumab 300 mg and secukinumab 150 mg. Overall, the PK properties of secukinumab were typical of a 150-kDa human IgG1 antibody interacting with a soluble target. + +Keywords IL-17A, monoclonal antibody, pharmacodynamics, population pharmacokinetics, psoriasis, secukinumab The immunopathogenesis of psoriatic skin inflammation is largely driven by cytokines regulated by T-helper 17 (Th17) cells.1–3 In genetically susceptible individuals, environmental triggers and cutaneous pathogens stimulate dendritic cells to produce interleukin (IL)- +23, which promotes Th17 differentiation. In turn, this leads to elevated levels of serum and cutaneous Th17 cells in patients with psoriasis.3–7 Continued exposure of Th17 cells to IL-23 leads to enhanced production of IL-17A, which has been identified as a central cytokine effector of skin changes associated with psoriasis.1,2,8 Increased IL-17A production promotes keratinocyte activation and production of inflammatory mediators, creating a cycle of inflammation that perpetuates lesion formation. Thus, IL-17A has been identified as a key therapeutic target for the treatment of psoriasis,2,6,8 and biologic agents that target the IL-17 pathway have shown promising efficacy and safety in clinical trials.9–13 One such biologic agent is secukinumab, an immunoglobulin G1κ (IgG1κ) monoclonal antibody that potently and selectively binds to and neutralizes IL-17A. In pivotal phase 3 trials of patients with moderate to severe plaque psoriasis, at least 75% +improvement in the Psoriasis Area and Severity Index +(PASI 75) was achieved by 77% to 82% of patients after 12 weeks of treatment with subcutaneous secukinumab 300 mg, and PASI 75 response rates ranged from 67% to 72% for patients treated with secukinumab 150 mg.9 Investigator's Global Assessment modified 2011 responses of 0 (clear) or 1 (almost clear) at week 12 were reported by 63% to 65% of patients who received secukinumab 300 mg and by 51% who received secukinumab 150 mg.9 In these trials, secukinumab was generally well tolerated, with the most commonly reported adverse events including nasopharyngitis, upper respiratory tract infection, and headache.9,12–14 Findings from a dose-ranging study in patients with psoriasis15 showed that, at therapeutic doses, secukinumab concentration increased in a doseproportional manner. Overall, the pharmacokinetic +(PK) properties of secukinumab are typical of an IgG1 antibody because it exhibits good stability, long persistence in the body, and high selectivity and specificity.16,17 Furthermore, with a novel technique, 1Novartis Pharma AG,Novartis Institutes for BioMedical Research,Basel, Switzerland 2Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA 3Novartis Pharma AG, Basel, Switzerland 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. + +Submitted for publication 14 July 2016; accepted 20 December 2016. Corresponding Author: +Gerard Bruin, PhD, Novartis Institute for Biomedical Research, WSJ386.12.48.03, CH-4002 Basel, Switzerland. + +Email: gerard.bruin@novartis.com open-flow microperfusion, the concentration of secukinumab was reported as 6.8 μg/mL in lesional skin 7 days after a single 300-mg subcutaneous injection of secukinumab.18 In patients with psoriasis, interstitial fluid concentrations of secukinumab in lesional and nonlesional skin were in the range of 28% +to 39% relative to serum concentrations.18 Relevant cytokines and markers were also analyzed directly in the skin through this method. At baseline, levels of IL17A and human β-defensin 2 (hBD-2), a downstream marker of IL-17A signaling and psoriasis disease severity,19–21 were significantly higher in psoriatic skin lesions, and levels of hBD-2 significantly and rapidly decreased following administration of secukinumab.22 The objective of this study was to further characterize the PK properties of secukinumab using pooled results from 6 clinical studies with either intravenous or subcutaneous administration in patients with psoriasis. + +In addition, pharmacodynamic (PD) biomarkers of secukinumab activity are explored based on the measurement of total IL-17A concentrations in the phase 3 JUNCTURE trial.13 + +## Methods + +Some studies were multicenter, and each site complied with the Declaration of Helsinki. All protocols were approved by each site's institutional review board or ethics committee. Informed consent was provided by all patients. Population PK Modeling As mentioned, the relationship between the dose, regimen, and serum concentration of secukinumab was characterized based on pooled data from 6 clinical studies in patients with psoriasis. Included studies (described in Table 1) comprised 5 phase 1 or 2 studies15,23,24 and 1 phase 3 study (ERASURE)9 that assessed a range of subcutaneous (25, 75, 150, and 300 mg) and intravenous (1, 3, and 10 mg/kg) secukinumab dosing regimens. + +Based on exploratory modeling of phase 2 data, it was hypothesized that secukinumab PK parameters could be described using a linear 2-compartmental distribution model with first-order absorption for subcutaneous administration and with zero-order infusion for intravenous administration. Therefore, a linear 2-compartmental distribution model was used as a starting point for this analysis. + +The population PK model included structural, random-effects, covariate, and residual error models. + +The structural model accounted for systematic data trends. Disposition kinetics were modeled using a parameterization involving clearance, central volume, intercompartmental clearance, and peripheral volume. + +Subcutaneous bioavailability between 0 and 1 was modeled on a logit scale, and absorption was modeled as a first-order process for the subcutaneous route of administration. The random-effects model was used to account for between-patient variability in PK parameters using multiplicative exponential random effects, and residual variability was modeled using a combined proportional/additive error model. The residual error model was used to describe discrepancies in the data that were not accounted for by the model, and the covariate model was used to explore the impact of covariates on between-patient variability. The base model included body weight (centered at 90 kg) as a covariate on clearances and volumes of distribution; other covariates explored included age (centered at 45 years), baseline PASI score (centered at 19), sex, geographic region (Europe, United States, Taiwan, East Asia including Japan, East Asia excluding Japan, and Japan), and race (Asian vs non-Asian). Under the assumption that changes to the typical patient parameters of more than 20% are potentially of clinical relevance, only body weight was retained in the population PK +model. The small differences from the influence of age, baseline PASI score, sex, geographic region, and race were not considered clinically relevant. The reference values used for centering of age and baseline PASI score were chosen to approximately reflect the median of these covariates. + +Model components were selected and assembled based on a combination of prior knowledge and data-driven decision making, guided by statistical and heuristic rules. Decision criteria for the model were based on a combination of judgment of model plausibility and robustness, objective function value, goodness-of -fit diagnostics, and simulation-based diagnostics. Predictive performance of the models was assessed in visual predictive checks, which were stratified by treatment group using 400 simulations with 10 time bins, with approximately equal observation counts. + +Data from the phase 3 FIXTURE study9 were used as an external validation data set to provide a rigorous assessment of the predictive performance of the model without reestimation of model parameters. + +The final structural model was used to simulate PK +profiles for dosing regimens of interest (secukinumab 300 and 150 mg subcutaneous regimens used in phase 3 studies). Daily PK concentrations for 1000 patients per dosing regimen were simulated, and derived exposure metrics included minimum, maximum, and average serum drug concentration (Cmin, Cmax, and Cavg), steady-state area under the curve (AUC), time to maximum concentration in serum (tmax), and terminal half -life. The population PK models were fitted using NONMEM 7.2 software. + +| 878 | The Journal of Clinical Pharmacology / Vol 57 No 7 2017 | | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------|----|----| +| Mean Age of Patients Receiving Secukinumab | 43.4–45.7 years | (Continued) | | | +| 42.7–44.5 years | Weeks 12, 24, and 36 39.9–45.0 years | | | | +| 50.7 years | 46.8 years For intravenous: predose and 1, 2, 4, and 8 h; for subcutaneous: predose and 1 and 8 h; and then 1, 3, 4, 7, 9, 14, 21, 28, 29, 31, 32, 35, 38, 42, 49, 56, 70, and 84 days after first dose for both arms | | | | +| Predose, end of infusion, 1 and 2 h after infusion, then at weeks 1, 2, 3, 4, 5, 6, 8, and 12 = 18) Secukinumab 3 mg/kg intravenously (n | Weeks 0, 1, 2, 4, 8, 12, 16, 20, 24, 28, and 32 | infusions at weeks 1, 2, and 4; additional sampling at weeks 1, 6, 8, 10, 12, 16, 20, 24, 28, 32, 40, 48, and 56 | | | +| Predose and 2 and 4 h after | | | | | +| Table 1. Secukinumab clinical studies constituting the PK analysis data set | Study Primary Objective Design Treatment Regimensa PK Sampling | Secukinumab 1 × 150 mg subcutaneously on day 1 followed by 1 × 1 mg/kg intravenously on day 29 (n = 7) | Secukinumab 150 mg subcutaneously at weeks 0, 1, 2, and 4 (n = 133) | Responders: secukinumab 150 mg subcutaneously at weeks 12, 24, or start of relapse Nonresponders and partial responders: open-label secukinumab 150 mg subcutaneously every 4 weeks | +| Secukinumab 1 × 1 mg/kg intravenously on day 1 followed by 1 × 150 mg subcutaneously on day 29 (n = 7) | Secukinumab 150 mg subcutaneously every 4 weeks (n =138) | | | | +| Secukinumab 1 × 150 mg subcutaneously (n = 66) | Secukinumab 1 × 3 mg/kg intravenously (n = 30) Secukinumab 1 × 10 mg/kg intravenously (n = 29) Secukinumab 3 × 10 mg/kg intravenously (n = 31) | | | | +| = 100) Single and multiple doses (n Efficacy Randomized, double-blind, parallel-group, placebo-controlled | | | | | +| = 18) Placebo (n | = 67) Placebo (n | Open-label extension Multiple doses, maintenance | = 10) Placebo (n | | +| = 404) Multiple doses (n Efficacy Randomized, double-blind, parallel-group, placebo-controlled Phase 2 dose-regimen-finding study, Rich et al24 | | | | | +| = 14) Bioavailability Randomized, open-label, crossover Multiple doses (n | | | | | +| = 36) Efficacy Randomized, double-blind, parallel-group, placebo-controlled Single dose (n | Long-term safety, tolerability Extension of dose-regimen-finding study | | | | +| Absolute bioavailability study | Phase 2 dose-finding-study, Reich et al27 | | | | +| Proof of concept, Hueber et al23 | | | | | + +![3_image_0.png](3_image_0.png) + +| Mean Age of Patients Receiving Secukinumab | 45.4–46.3 years Weeks 0, 1, 2, 4, 8, 12, 16, 20, 24, 28, 32, and 36 | | +|----------------------------------------------|-----------------------------------------------------------------------|------------------------------------------| +| Table 1. Continued | Study Primary Objective Design Treatment Regimensa PK Sampling | Phase 2 dose-finding-study, Papp et al15 | + +![3_image_2.png](3_image_2.png) + +Phase 3 FIXTURE study,b Langley et al9 Efficacy superiority vs. placebo +ERASURE, Efficacy of Response and Safety of Two Fixed Secukinumab Regimens in Psoriasis; FIXTURE, Full-Year Investigative Examination of Secukinumab vs. Etanercept Using 2 Dosing Regimens to Determine Efficacy in Psoriasis; PD, pharmacodynamic; PK, pharmacokinetic. aPatient numbers reported in this table are the number of enrolled patients for each study. bResults from FIXTURE were used as an external validation data set. The PK analysis set includes only the patients with evaluable PK data. + +![3_image_1.png](3_image_1.png) + +| 44.5–45.4 years | +|-------------------| +| 44.9 years | + +Bruin et al 879 Pharmacokinetics Serum concentrations of secukinumab were measured in patients from the phase 3 ERASURE trial. In this study, patients in the active-treatment arms received subcutaneous secukinumab 300 mg (n = 245) or 150 mg (n = 245) at baseline; at weeks 1, 2, and 3; and then every 4 weeks from week 4 to week 48.9 The last dose of secukinumab was given at week 48. At week 12, PASI 75 nonresponders receiving placebo were rerandomized to secukinumab 300 mg (n = 105) or 150 mg (n = 109), and 18 patients continued on placebo. Blood samples were taken for PK analysis at baseline; at weeks 4, 12, 24, 52, and 60 (during washout); and during unscheduled visits. Serum levels of secukinumab were determined by an enzyme-linked immunosorbent assay method with a lower limit of quantification of 80 ng/mL. + +## Pharmacodynamics + +The ability of secukinumab to bind and capture circulating IL-17A was formally validated in the phase 3 JUNCTURE trial based on measurement of total serum IL-17A. In this study, patients in the activetreatment arms received subcutaneous secukinumab 300 mg (n = 60) or 150 mg (n = 61) at baseline; at weeks 1, 2, and 3; and then every 4 weeks from week 4 to week 48.13 The last dose of secukinumab was given at week 48. Total IL-17A was defined as free IL-17A plus IL-17A complexed with secukinumab. Blood samples were taken for assessment of total IL-17A at baseline and at weeks 4, 12, 16, 24, and 52. Total serum IL-17A concentrations were determined by an electrochemiluminescence method with a lower limit of quantification of 20 pg/mL. + +## Results Pharmacokinetics + +The population PK model was built based on 10 193 secukinumab serum concentrations from 1233 patients with moderate to severe psoriasis who participated in 5 phase 1 or 2 studies and 1 phase 3 study. The mean ± standard deviation demographic and baseline characteristics used as continuous covariates included body weight (90.7 ± 23.5 kg), PASI score (21.1 ± 8.8), +height (172.7 ± 9.9 cm), body mass index (30.3 ± +7.2 kg/m2), and age (44.6 ± 12.6 years). Categorical covariates included sex, race (Asian vs non-Asian), +and geographic location. The majority of patients were male (897 of 1233 [72.7%]) and non-Asian (1027 of 1233 [83.3%]; of the non-Asian population, 967 [94.2%] +were white). + +The descriptive and predictive capabilities of the population PK model were demonstrated using goodness-of -fit plots (Figure 1) and predictive checks. + +The overall fit of the 2-compartment model to the PK +data was good, with few outlying points. The weighted residuals-versus-time and population predictions showed no evident trends. The predictive performance of the model was confirmed based on goodness-of -fit plots and predictive checks using external validation data from the FIXTURE trial (Figure 2),9 because these results were similar to those of the studies included in the model. The goodness of fit for the external validation data was adequate and similar to the goodness of fit for data that were included in building the PK model. Visual predictive checks for the data used in Figure 1 and Figure 2 are shown in Supplemental Figure 1 and Supplemental Figure 2. + +The PK parameters estimated from the final 2compartment population PK model are shown in Table 2. In a typical patient with psoriasis weighing 90 kg, it was estimated that serum clearance was low +(0.19 L/day with interindividual variability [IIV] of 32% coefficient of variation [CV]), and total volume of distribution was low (central compartment volume, 3.61 L with IIV of 30% CV; peripheral compartment volume, 2.87 L with IIV of 18% CV). Bioavailability after subcutaneous dosing was approximately 73%, +with an absorption rate of 0.18/day with IIV of 35% CV. + +The PK properties of secukinumab were linear, with no evidence of a dose dependence of clearance; there was also no evidence of a time-dependent change in the clearance. Clearance and volume varied with body weight in an allometric relationship. For clearance and central volume of distribution, the allometric exponents were estimated to be between 0.8 and 1.0; in other words, a doubling of body weight could lead to a nearly 2-fold increase in clearance and distribution volume. + +Age, sex, race (Asian vs non-Asian), geographic region, and baseline PASI score did not have a clinically relevant effect on secukinumab clearance after adjusting for body weight. The effects of model covariates are presented in Supplemental Table 1. + +Figure 3 shows simulated concentration profiles, and Table 3 shows simulated PK metrics for the subcutaneous secukinumab dosing regimens used in phase 3 studies (300 or 150 mg at baseline; at weeks 1, 2, and 3; and then every 4 weeks from week 4 to week 48). Based on these simulations, Cavg at steady state was estimated to be 44.5 μg/mL for secukinumab 300 mg and 22.2 μg/mL for secukinumab 150 mg. + +Cmax at steady state was 55.2 μg/mL for secukinumab 300 mg and 27.6 μg/mL for secukinumab 150 mg, and tmax at steady state occurred approximately 6 days after dosing for both regimens. Terminal half -life averaged approximately 27 days for both doses, with an IIV of 27.2% CV. For both dosing regimens, Cmax at steady state was about 2-fold higher than Cmax after the first dose, which was in line with an expected accumulation ratio of 2 with a 4-week dosing interval and a terminal half -life of approximately 4 weeks. + +![5_image_0.png](5_image_0.png) + +Pharmacodynamics Secukinumab binds to human IL-17A and blocks the interaction of this cytokine with its receptor, functionally neutralizing its bioactivity. The complex of secukinumab with IL-17A is eliminated at a slower rate than free IL-17A, resulting in elevated levels of total IL-17A (free + complexed) following secukinumab administration; thus, total IL-17A is a relevant biomarker for secukinumab and is indicative of neutralization of IL-17A by secukinumab. + +Results from the phase 3 JUNCTURE study in patients with psoriasis showed that, at baseline, total +(free) serum IL-17A concentrations were below the total IL-17A assay limit of quantification (20 pg/mL). + +At week 4, after weekly subcutaneous dosing with secukinumab 300 and 150 mg regimens, total IL-17A levels increased to median values ranging from 121 to 142 pg/mL. The duration of increase in total IL-17A +was prolonged with increasing doses of secukinumab, and a plateau was reached at week 24 (Figure 5). It must be noted here that the observed increases in serum total IL-17 were very similar for both dose levels and therefore clearly show target engagement. However, the observed time–concentration profiles for total IL-17A do not allow for conclusions to be made about the level of suppression of free IL-17, neither in the systemic circulation nor in the target tissue. For this purpose, evaluation of secukinumab PK and total serum concentrations of IL-17A in a binding model would be needed. + +882 *The Journal of Clinical Pharmacology /* Vol 57 No 7 2017 + +![6_image_0.png](6_image_0.png) + +## Discussion + +Results from this study confirm that secukinumab displays PK properties that are typical of a 150-kDa human IgG1 antibody interacting with a soluble target +(eg, long half -life, slow serum clearance, and an allometric relationship between body weight and clearance and volume).16 The volume of distribution is low (<9 L) +in patients with psoriasis following intravenous infusion, and clearance is low. At therapeutically relevant doses, exposure increases in a dose-proportional manner. The PK properties of secukinumab are linear, with no evidence of a dose-dependent change in clearance. + +The 2-compartment population PK model adequately describes the observed secukinumab concentration– +time courses, and external validation results suggest that the model will be predictive for PK outcomes of new studies (ie, studies not included in the model). + +When initiating treatment with secukinumab an induction phase allows for a rapid onset of effect.24 The PK parameters of secukinumab allow prescribing physicians to have confidence in obtaining desired exposure. + +For example, a dose of 300 mg results in approximately double the exposure compared with a 150-mg dose. In addition, there is a clear difference in efficacy responses between secukinumab 300 mg and secukinumab 150 mg in patients with psoriasis.9 Secukinumab clearance values allow physicians to predict drug washout; assuming a washout period of 5 half -lives, secukinumab will be washed out approximately 135 days after cessation of therapy. Distribution into skin, the target organ in psoriasis patients, appears to be fast, as skin interstitial fluid level of secukinumab is measurable + +| Table 2. Estimated population parameters of the final 2-compartment PK model Parameter Value | %RSE | Interindividual Variability, %CV | | +|------------------------------------------------------------------------------------------------|--------|------------------------------------|----| +| CL, L/day | 0.19 | 1.9 | 32 | +| V2, L | 3.61 | 2.6 | 30 | +| Q, L/day | 0.39 | 4.6 | NA | +| V3, L | 2.87 | 1.9 | 18 | +| ka, 1/day | 0.18 | 3.6 | 35 | +| F, % | 72.9 | 1.5 | NA | +| Proportional error | 0.17 | 0.8 | | +| Additive error | 371 | 0.5 | | + +![7_image_0.png](7_image_0.png) + +after a single subcutaneous dose of 300 mg, reaching 28%–39% of the serum concentration 7 and 14 days postdose (6–8 μg/mL of secukinumab in the dermal interstitial fluid).18 These dermal concentrations of secukinumab seem largely sufficient to locally neutralize IL-17A in the lesions. Indeed, baseline IL-17A protein concentration was locally elevated in the dermal interstitial fluid of lesional psoriatic skin compared with nonlesional psoriatic skin and skin from healthy volunteers (9.8 vs 0.8 pg/mL), confirming the central role of IL-17A +in the pathogenesis of psoriasis.22 In contrast, IL-17F +(another IL-17 cytokine that shares homology with IL-17A and acts to regulate host defense and inflammatory responses)25 was not significantly increased in the skin of patients with psoriasis compared with healthy controls, and baseline IL-17F levels did not significantly differ between psoriatic lesional and nonlesional skin. Similar to the findings for IL-17A, and confirming its role as a disease marker, dermal baseline hBD-2 concentrations were significantly higher in lesional skin of patients with psoriasis compared with nonlesional skin of patients with psoriasis or the skin of healthy volunteers. Because of the complex formation of IL-17A with secukinumab, the effects of secukinumab on free IL-17A could not be measured.22 Although the accumulation of total IL-17A seems to plateau with the secukinumab 300- and 150-mg dosing regimens, this plateau does not automatically imply a plateau in efficacy, as increasing the dose of monoclonal antibodies has been associated with further reduction of free target concentration and possible inhibition of downstream biomarkers and improved efficacy.26 To understand why the plateau in total target concentration does not imply a plateau in free target suppression and to understand how changes in the dose regimen may impact target engagement, it is useful to characterize the free and bound concentrations for the drug and target using dedicated models, which describe both target-mediated drug disposition and the accumulation of total target during therapy. However, this was outside the scope of this article. Using such models specifically for the secukinumab–IL-17A interaction will be described in an upcoming article. + +These data suggest that secukinumab may act not only systemically but also locally in the dermis at concentrations capable of blocking the effects of IL-17A, which in turn might explain the rapid effect on keratinocyte-driven pathophysiology such as decreases in epidermal thickness and parakeratosis observed after 2 weeks of treatment.27 This rapid distribution of secukinumab to skin tissue may explain the rapid onset of a PD action. hBD-2 has been shown to be upregulated in lesional psoriatic skin and in the serum of patients with psoriasis.28,29 hBD-2 has also been linked to keratinocyte hyperproliferation and chemoattraction + +| Table 3. Simulated PK metrics from phase 3 studies of secukinumab Secukinumab 300-mg Regimen | Secukinumab 150-mg Regimen | | | | | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|------|--------------|---------------|------|--------------| +| Parameter | Mean ± SD | %CV | Range (90%) | Mean ± SD | %CV | Range (90%) | +| Cmin at week 12, μg/mL | 44.2 ± 20.6 | 46.5 | 17.5–83.0 | 22.1 ± 10.3 | 46.5 | 8.7–41.5 | +| Cmin at ss, μg/mL | 32.1 ± 15.4 | 48 | 12.9–62.9 | 16.0 ± 7.7 | 48 | 6.5–31.5 | +| Cavg at ss, μg/mL | 44.5 ± 18.4 | 41.3 | 21.1–77.9 | 22.2 ± 9.2 | 41.3 | 10.5–39.0 | +| AUC at ss, μg·day/mL | 1245.0 ± 514.8 | 41.3 | 590.4–2181.7 | 622.5 ± 257.4 | 41.3 | 295.2–1090.8 | +| Cmax after first dose, μg/mL | 27.3 ± 9.5 | 34.8 | 14.2–44.6 | 13.7 ± 4.8 | 34.8 | 7.1–22.3 | +| tmax after first dose, day | 5.8 ± 0.4 | 7.6 | 5.0–6.0 | 5.8 ± 0.4 | 7.6 | 5.0–6.0 | +| Cmax at ss, μg/mL | 55.2 ± 21.5 | 38.9 | 27.5–94.8 | 27.6 ± 10.7 | 38.9 | 13.7–47.4 | +| tmax at ss, day | 6.0 ± 1.1 | 17.7 | 4.0–8.0 | 6.0 ± 1.1 | 17.7 | 4.0–8.0 | +| Cmax overall, μg/mL | 103.7 ± 33.5 | 32.3 | 59.5–165.5 | 51.8 ± 16.7 | 32.3 | 29.8–82.8 | +| tmax overall, day | 32.3 ± 1.1 | 3.3 | 31.0–34.0 | 32.3 ± 1.1 | 3.3 | 31.0–34.0 | +| Terminal half-life, day | 26.9 ± 7.3 | 27.2 | 16.8–41.0 | 26.9 ± 7.3 | 27.2 | 16.8–41.0 | +| AUC, area under the curve; Cavg, average concentration in serum; Cmax, maximum concentration in serum; Cmin, minimum concentration in serum; CV, coefficient | | | | | | | + +of neutrophils—both important mechanisms in the pathogenesis of psoriasis.30,31 In addition, serum and dermal interstitial fluid levels of hBD-2 were significantly reduced to levels commonly found in healthy individuals 1–2 weeks after subcutaneous administration of a single dose of secukinumab. Results from our analysis indicate that total IL-17A concentrations increase on initiation of secukinumab therapy, and a plateau is reached after 24 weeks for subcutaneous secukinumab doses of 150 mg and higher. + +![8_image_1.png](8_image_1.png) + +![8_image_0.png](8_image_0.png) + +## Conclusions + +In summary, a 2-compartment model can adequately describe secukinumab concentration–time courses; the PK properties of subcutaneous regimens of secukinumab 300 and 150 mg—administered at baseline; at weeks 1, 2, and 3; and then every 4 weeks from week 4 to week 48—are particularly well described by this model in terms of the central trend and variability. + +Secukinumab clearance and volume of distribution vary with body weight in an allometric relationship. + +Serum PK parameters and skin interstitial fluid concentrations of secukinumab contribute to the observed key pharmacodynamic effects (eg, increase of total IL-17A +and decrease of hBD-2 in both serum and skin lesions). + +The fast and efficient distribution of secukinumab into the skin may contribute to the rapid onset of psoriatic lesion clearance. All these findings confirm the key role of IL-17A in the pathogenesis of psoriasis. + +## Acknowledgments + +Editorial assistance was provided by Oxford PharmaGenesis, Inc., and was funded by Novartis Pharmaceuticals Corporation. The authors were fully responsible for all content and editorial decisions for this article. + +## Declaration Of Conflicting Interests + +Gerard Bruin, PhD, Christian Loesche, MD, and Oliver Sander, PhD, are full-time employees of Novartis Pharma AG. Judit Nyirady, MD, is a full-time employee of Novartis Pharmaceuticals Corporation. + +## Funding + +This study was funded by Novartis Pharma AG. + +## References + +1. Blauvelt A. T-helper 17 cells in psoriatic plaques and additional genetic links between IL-23 and psoriasis. *J Invest Dermatol.* +2008;128(5):1064–1067. + +2. Lynde CW, Poulin Y, Vender R, Bourcier M, Khalil S. Interleukin 17A: toward a new understanding of psoriasis pathogenesis. *J Am Acad Dermatol.* 2014;71(1):141–150. + +3. Nestle FO, Kaplan DH, Barker J. Psoriasis. *N Engl J Med.* +2009;361(5):496–509. + +4. Kagami S, Rizzo HL, Lee JJ, Koguchi Y, Blauvelt A. Circulating Th17, Th22, and Th1 cells are increased in psoriasis. J Invest Dermatol. 2010;130(5):1373–1383. + +5. Di Cesare A, Di Meglio P, Nestle FO. The IL-23/Th17 axis in the immunopathogenesis of psoriasis. *J Invest Dermatol.* 2009;129(6):1339–1350. + +6. Nwe SM, Champlain AH, Gordon KB. Rationale and early clinical data on IL-17 blockade in psoriasis. *Expert Rev Clin* Immunol. 2013;9(7):677–682. + +7. Lowes MA, Kikuchi T, Fuentes-Duculan J, et al. Psoriasis vulgaris lesions contain discrete populations of Th1 and Th17 T cells. *J Invest Dermatol.* 2008;128(5):1207–1211. + +8. Krueger JG, Fretzin S, Suarez-Fari ´ nas M, et al. IL-17A is ˜ +essential for cell activation and inflammatory gene circuits in subjects with psoriasis. *J Allergy Clin Immunol.* 2012;130(1):145– +154. + +9. Langley RG, Elewski BE, Lebwohl M, et al. Secukinumab in plaque psoriasis—results of two phase 3 trials. *N Engl J Med.* 2014;371(4):326–338. + +10. Leonardi C, Matheson R, Zachariae C, et al. Anti-interleukin17 monoclonal antibody ixekizumab in chronic plaque psoriasis. + +N Engl J Med. 2012;366(13):1190–1199. + +11. Papp KA, Leonardi C, Menter A, et al. Brodalumab, an antiinterleukin-17-receptor antibody for psoriasis. *N Engl J Med.* +2012;366(13):1181–1189. + +12. Blauvelt A, Prinz JC, Gottlieb AB, et al. Secukinumab administration by pre-filled syringe: efficacy, safety, and usability results from a randomized controlled trial in psoriasis (FEATURE). Br J Dermatol. 2015;172(2):484–493. + +13. Paul C, Lacour JP, Tedremets L, et al. Efficacy, safety and usability of secukinumab administration by autoinjector/pen in psoriasis: a randomized, controlled trial (JUNCTURE). *J Eur* Acad Dermatol Venereol. 2015;29(6):1082–1090. + +14. Mrowietz U, Leonardi CL, Girolomoni G, et al. Secukinumab retreatment-as-needed versus fixed-interval maintenance regimen for moderate to severe plaque psoriasis: A randomized, double-blind, noninferiority trial (SCULPTURE). J Am Acad Dermatol. 2015;73(1):27–36 e21. + +15. Papp KA, Langley RG, Sigurgeirsson B, et al. Efficacy and safety of secukinumab in the treatment of moderate-to-severe plaque psoriasis: a randomized, double-blind, placebo-controlled phase II dose-ranging study. *Br J Dermatol.* 2013;168(2):412–421. + +16. Wang W, Wang EQ, Balthasar JP. Monoclonal antibody pharmacokinetics and pharmacodynamics. *Clin Pharmacol Ther.* +2008;84(5):548–558. + +17. Lobo ED, Hansen RJ, Balthasar JP. Antibody pharmacokinetics and pharmacodynamics. *J Pharm Sci.* 2004;93(11):2645– +2668. + +18. Dragatin C, Polus F, Bodenlenz M, et al. Secukinumab distributes into dermal interstitial fluid of psoriasis patients as demonstrated by open flow microperfusion. *Exp Dermatol.* +2016;25(2):157–159. + +19. Gambichler T, Kobus S, Kobus A, et al. Expression of antimicrobial peptides and proteins in etanercept-treated psoriasis patients. *Regul Pept.* 2011;167(2-3):163–166. + +20. Liang SC, Tan XY, Luxenberg DP, et al. Interleukin (IL)- +22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. *J Exp Med.* 2006;203(10):2271–2279. + +21. Nograles KE, Zaba LC, Guttman-Yassky E, et al. Th17 cytokines interleukin (IL)-17 and IL-22 modulate distinct inflammatory and keratinocyte-response pathways. *Br J Dermatol.* +2008;159(5):1092–1102. + +22. Kolbinger F, Loesche C, Valentin M, et al. β-Defensin-2 is a responsive biomarker of IL-17A-driven skin pathology in psoriasis [published online ahead of print 2016]. J Allergy Clin Immunol. doi: 10.1016/j.jaci.2016.06.038 3 23. Hueber W, Patel DD, Dryja T, et al. Effects of AIN457, a fully human antibody to interleukin-17A, on psoriasis, rheumatoid arthritis, and uveitis. *Sci Transl Med.* 2010;2(52):52ra72. + +24. 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. + +25. Iwakura Y, Ishigame H, Saijo S, Nakae S. Functional specialization of interleukin-17 family members. *Immunity.* 2011;34(2):149–162. + +26. Lowe PJ, Kummel A, Vasalou C, Matsushima S, Skerjanec A. ¨ +Integrated quantitation of biotherapeutic drug–target binding, biomarkers, and clinical response to support rational dose regimen selection. *Pharm Sci Encycl.* 2015;13:1–21. + +27. Reich K, Papp KA, Matheson RT, et al. Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis. *Exp Dermatol.* 2015;24(7):529–535. + +28. Gambichler T, Skrygan M, Tomi NS, et al. Differential mRNA +expression of antimicrobial peptides and proteins in atopic dermatitis as compared to psoriasis vulgaris and healthy skin. + +Int Arch Allergy Immunol. 2008;147(1):17–24. + +29. Jansen PA, Rodijk-Olthuis D, Hollox EJ, et al. β-Defensin-2 protein is a serum biomarker for disease activity in psoriasis and reaches biologically relevant concentrations in lesional skin. PLoS One. 2009;4(3):e4725. + +30. Niyonsaba F, Ushio H, Nakano N, et al. Antimicrobial peptides human beta-defensins stimulate epidermal keratinocyte migration, proliferation and production of proinflammatory cytokines and chemokines. *J Invest Dermatol.* 2007;127(3):594–604. + +31. Niyonsaba F, Ogawa H, Nagaoka I. Human β-defensin-2 functions as a chemotactic agent for tumour necrosis factor-α-treated human neutrophils. *Immunology.* 2004;111(3):273–281. + +## Supporting Information + +Additional Supporting Information may be found in the online version of this article at the publisher's website. \ No newline at end of file diff --git a/medical/md/PMC5536766.md b/medical/md/PMC5536766.md new file mode 100644 index 0000000000000000000000000000000000000000..a9034027ee33e4be158d0e4dde584c542cb2ebdf --- /dev/null +++ b/medical/md/PMC5536766.md @@ -0,0 +1,320 @@ +Journal of International Medical Research + +![0_image_0.png](0_image_0.png) + +2016, Vol. 44(6) 1302–1313 +! The Author(s) 2016 Reprints and permissions: +sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060516664638 imr.sagepub.com Gamma-glutamyl transpeptidase to platelet ratio index is a good noninvasive biomarker for predicting liver fibrosis in Chinese chronic hepatitis B patients Rong-Qi Wang*, Qing-Shan Zhang*, +Su-Xian Zhao, Xue-Min Niu, Jing-Hua Du, Hui-Juan Du and Yue-Min Nan + +## Abstract + +Objective: To evaluate whether gamma-glutamyl transpeptidase to platelet ratio index (GPRI) can diagnose the extent of liver fibrosis in Chinese patients with chronic hepatitis B (CHB) infection. + +Methods: This prospective observational study used liver biopsy results as the gold standard to evaluate the ability of GPRI to predict hepatic fibrosis compared with two other markers, the aspartate aminotransferase (AST) to platelet ratio index (APRI) and fibrosis-4 score (FIB-4). The clinical and demographic factors that affected GPRI, independent of liver fibrosis, were assessed using multivariate linear regression analyses. Results: This study enrolled 312 patients with CHB. GPRI had a significantly positive correlation with liver fibrosis stage and the correlation coefficient was higher than that for APRI and FIB-4. The areas under the receiver operating curves for GPRI for significant fibrosis, bridging fibrosis, and cirrhosis were 0.728, 0.836, and 0.842, respectively. Of the three indices, GPRI had the highest diagnostic accuracy for bridging fibrosis and cirrhosis. Age, elevated AST and elevated total bilirubin levels were independent determinants of increased GPRI. + +Conclusion: GPRI was a more reliable laboratory marker than APRI and FIB-4 for predicting the stage of liver fibrosis in Chinese patients with CHB. + +Department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China +*These authors contributed equally to the work. Corresponding author: Yue-Min Nan, Department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University, 139 Ziqiang Road, Shijiazhuang, 050051, Hebei Province, China. Email: nanyuemin@163.com + +## Introduction Keywords + +Noninvasive biomarker, gamma-glutamyl transpeptidase to platelet ratio index, chronic hepatitis B, +aspartate aminotransferase to platelet ratio index, liver fibrosis, fibrosis-4 score Date received: 30 May 2016; accepted: 26 July 2016 detection of advanced fibrosis and cirrhosis, but they have been mostly studied within relatively small sets of patients with CHB +under somewhat controlled conditions, making the results difficult to generalize to broader patient populations in real-world clinical settings and their ideal cut-offs are unclear.6–8 Thus, new modalities are needed to overcome these problems. Serum gammaglutamyl transpeptidase (GGT) is a microsomal enzyme that can be isolated from hepatocytes and gall bladder epithelium.9 Its levels can increase in many diseases and conditions, for example, alcohol dependency, drug use, viral hepatitis and obesity.10,11 In patients with CHB, it was concluded that an increase in serum GGT +was associated with high ALT and AST levels, low albumin levels, and advanced fibrosis.9 Therefore, it may be considered an indicator of significant fibrosis in CHB +patients. Recently, a study found that the gamma-glutamyl transpeptidase to platelet ratio might be an accurate marker for staging liver fibrosis in patients with CHB +in West Africa,12 but further validation in non-African populations is still required. + +Based on these findings,9,12 this present study evaluated the noninvasive marker, the gamma-glutamyl transpeptidase to platelet ratio index (GPRI) in Chinese patients with CHB. The GPRI is calculated based on the serum GGT value (and the upper limit of normal [ULN] value for the laboratory) and platelet counts using the following formula: [GGT/ULN]/platelet counts [-109/l] - 100. + +The aims of this study were to: (i) investigate a reliable and routine indicator for determining the progression of fibrosis in Chinese patients with CHB, using liver Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV). Worldwide it is estimated that more than 240 million people have suffered from chronic HBV infections and about 780 000 people die every year due to the complications of hepatitis B, including cirrhosis, hepatic failure and hepatocellular carcinoma.1 Patients with significant hepatic inflammation and fibrosis are at the highest risk of these complications. With early diagnosis and the advent of effective antiviral therapies, the prognosis of chronic hepatitis B (CHB) can be improved significantly.2 A precise definition of liver disease severity remains important in predicting prognosis and therapeutic outcomes in patients with CHB. At present, liver biopsy is still regarded as the gold standard for assessing the degree of hepatic inflammation and fibrosis.3 However, it has some limitations such as invasiveness, cost, sampling variability and associated risk for complications. Furthermore, a single biopsy does not measure the dynamic nature of liver fibrosis.4 Therefore, accurate, noninvasive, repeatable, and easily available alternative methods for identifying patients with CHB +are needed urgently. + +To address this unmet need, several serum marker panels thought to be indicators of liver fibrosis have been extensively studied, including the aspartate aminotransferase (AST) to platelet ratio index (APRI), +the fibrosis-4 (FIB-4) score (based on AST, +alanine aminotransferase [ALT], patient age, and platelet count), the AST/ALT +ratio, and Forn's index.5 All of these markers have shown promise for the histology as the gold standard; (ii) compare GPRI with two other biomarker panels; (iii) +explore the influencing factors on GPRI +values. + +## Patients And Methods Patients + +This prospective observational study enrolled consecutive patients with CHB at the Department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China between January 2008 and March 2015. The criterion for a diagnosis of CHB was having serum hepatitis B surface antigen positivity for > 6 months.13 All enrolled patients underwent liver biopsy. + +Patients meeting the following criteria were excluded: (i) co-infection with human immunodeficiency virus, hepatitis A, C or D +virus; (ii) presence of decompensated cirrhosis, hepatocellular carcinoma, hepatic failure, and other causes of chronic liver disease. Written informed consent was obtained from all patients and the study was approved by the Third Hospital of Hebei Medical University Research Ethics Committee and carried out according to the guidelines of the 1975 Declaration of Helsinki. + +## Liver Biopsy + +Ultrasonography-guided percutaneous liver biopsy was performed using a 16 G disposable needle (Bard Biopsy Systems, Tempe, AZ, +USA) under local anaesthesia. All liver biopsies had an adequate specimen of 1.5 cm in length and included at least eight complete portal tracts. The liver specimens were fixed in buffered formalin and embedded in paraffin. + +Fixed hepatic tissues were sectioned and routinely stained with haematoxylin and eosin, and Masson's trichrome. The tissue sections were blindly evaluated by one experienced hepatopathologist, who had no information about the clinical characteristics of the study patients, in order to avoid inter-observer discrepancy. The degree of hepatic inflammation and fibrosis was assessed on the basis of the 2000 Xi'an Viral Hepatitis Management Guidelines recommended by the Chinese Society of Infectious Diseases and Parasitology and the Chinese Society of Hepatology of the Chinese Medical Association.14 Fibrosis was staged from F0 to F4: F0, no fibrosis; F1, mild fibrosis without fibrous septum; F2, fibrosis with a few fibrous septa; F3, numerous septa without cirrhosis; and F4, cirrhosis. Likewise, inflammatory activity was graded from G0 to G4:14 G0, no inflammation; G1, portal inflammation with rare lobular necrosis; G2, mild piecemeal portal necrosis, focal or spotty lobular necrosis; G3, moderate piecemeal portal necrosis, bridging necrosis in lobule; G4, severe piecemeal portal necrosis, multilobular necrosis. The increased numerical value indicated more severe disease. + +## Liver Biochemistry Tests + +Venous blood samples (6 ml) were obtained from overnight fasted patients within 1 week before or after the liver biopsy. Laboratory tests were analysed within 2 h after obtaining the blood samples at room temperature. Serum ALT, AST, total bilirubin (TBIL) +and GGT were measured using an enzymatic method with an automatic biochemistry analyser (Olympus AU2700; Olympus, Tokyo, Japan) according to the manufacturer's instructions. Blood platelet counts were determined using an automated haematology analyser (Sysmex K4500; Sysmex Corporation, Kobe, Japan). From these routine laboratory values, GPRI, APRI and FIB-4 were calculated using the following formulae: +GPRI ¼ GGT levelð Þ =ULN +=platelet count 109=l - 100 ðwhere ULN ¼ upper limit of normal for that laboratoryÞ +APRI ¼ AST levelð Þ =ULN + +=platelet count 109=l - 100 FIB 4 ¼ age years ð Þ- AST Uð Þ =l +=platelet count 109=l + +$$\begin{array}{l}{{\mathrm{/platelet~count}(10^{9},}}\\ {{\times[\mathrm{ALT}(\mathrm{U}/1)]^{1/2}}}\end{array}$$ + +## Statistical Analyses + +All statistical analyses were performed using the SPSS- statistical package, version 16.0 +(SPSS Inc., Chicago, IL, USA) for Windows-. Quantitative variables with a normal distribution were expressed as mean SD, and those with an abnormal distribution as median (25th, 75th percentile). + +The relationship between the noninvasive biomarkers and liver histopathology was determined with Spearman's rank correlation coefficient analysis. The diagnostic performance of all noninvasive markers evaluated was assessed by receiver operating characteristic (ROC) curves using histology as a reference. Optimal cut-off values were chosen based on a maximum sum of sensitivity and specificity. Defining the effect of the clinical and laboratory parameters on GPRI in patients with CHB was undertaken using multivariate linear regression analyses. Qualitative and quantitative differences between subgroups were compared using Mann–Whitney U-test or Student's t-test, respectively. All P-values given are 2-sided and a P-value < 0.05 was considered statistically significant. + +## Results + +From January 2008 to March 2015, 312 subjects who fulfilled the study criteria were enrolled. The mean SD age was 35.26 1.18 years (range 13 - 65 years). + +Of these patients, 227 (72.8%) patients were men and 85 (27.2%) were women. + +The clinical, biological and histological characteristics of the patients are shown in Table 1. The biopsy fibrosis stage distribution was as follows: F0, n ¼ 17 (5.4%); F1, n ¼ 126 (40.4%); F2, n ¼ 76 (24.4%); F3, n ¼ 39 (12.5%); F4, n ¼ 54 (17.3%). + +Significant hepatic fibrosis (F2–F4) was found in 169 (54.2%) patients and significant hepatic inflammatory activity (G3–G4) +was found in 50 (16.0%) patients. + +Box plots of GPRI, APRI and FIB-4 in relation to the fibrosis stage are presented in Figure 1. GPRI had a significant positive correlation with fibrosis stage in patients with CHB (r ¼ 0.516, P < 0.001), with mean values of 0.23, 0.28, 0.33, 0.91, and 1.25 for F0, F1, F2, F3, and F4, respectively. The Spearman's correlation coefficient was Table 1. Baseline clinical and demographic characteristics of the patients with chronic hepatitis B +infection (n ¼ 312) who participated in this study to evaluate a biomarker for the diagnosis of hepatic fibrosis. + +| fibrosis. | Patients with CHB n ¼ 312 | +|------------------------------------------------------------|-----------------------------| +| Age, years | 35.26 1.18 | +| Sex, male/female | 227/85 | +| Alanine transaminase, U/l | 102.46 (82.45–122.47) | +| Aspartate | 69.25 (55.87–82.63) | +| aminotransferase, U/l | | +| Total bilirubin, mmol/l | 21.19 (18.89–24.49) | +| Gamma-glutamyl | 49.57 (44.12–55.03) | +| transpeptidase, U/l | | +| Platelet count, 109 /l | 197.14 71.53 | +| GPRI | 0.82 (0.70–0.93) | +| APRI | 1.06 (0.87–1.26) | +| FIB-4 | 1.52 (1.32–1.72) | +| Fibrosis stage, | 17/126/76/39/54 | +| F0/F1/F2/F3/F4 | | +| Inflammatory activity grade, 0/119/143/47/3 G0/G1/G2/G3/G4 | | + +Data presented as mean SD, median (25th, 75th percentile) or n of patients. + +GPRI, gamma-glutamyl transpeptidase to platelet ratio index; APRI, aspartate aminotransferase to platelet ratio index; FIB-4, fibrosis-4 score. + +![4_image_0.png](4_image_0.png) + +higher than for FIB-4 (r ¼ 0.508, P < 0.001) +or APRI (r ¼ 0.407, P < 0.001). + +The study analysed the data comparing the different biomarkers in relation to different stages of hepatic fibrosis using ROC +curves (Table 2). In discriminating significant fibrosis (F0–F1 versus F2–F4), the area under ROC curve (AUROCs) of GPRI, +APRI and FIB-4 were 0.728 (sensitivity 59%, specificity 78%), 0.686 (sensitivity 70%, specificity 63%) and 0.742 (sensitivity 72%, specificity 67%), respectively +(Figure 2a). For predicting bridging fibrosis +(F0–F2 versus F3–F4), the AUROCs of GPRI, APRI and FIB-4 were 0.836 (sensitivity 76%, specificity 81%), 0.758 (sensitivity 85%, specificity 58%) and 0.803 (sensitivity 69%, specificity 77%), respectively (Figure 2b). For diagnosing cirrhosis +(F0–F3 versus F4), the AUROCs of GPRI, +APRI and FIB-4 were 0.842 (sensitivity 82%, +specificity 77%), 0.710 (sensitivity 85%, specificity 48%) and 0.776 (sensitivity 67%, +specificity 76%), respectively (Figure 2c). + +| and cirrhosis based on optimal cut-off values. Significant fibrosis | Bridging fibrosis | Cirrhosis | | | | | | | | +|-----------------------------------------------------------------------|----------------------|-------------------|-------|-------|-------|-------|-------|-------|-------| +| (F0–F1 versus F2–F4) | (F0–F2 versus F3–F4) | (F0–F3 versus F4) | | | | | | | | +| GPRI | APRI | FIB-4 | GPRI | APRI | FIB-4 | GPRI | APRI | FIB-4 | | +| AUROC | 0.728 | 0.686 | 0.742 | 0.836 | 0.758 | 0.803 | 0.842 | 0.710 | 0.776 | +| 95% CI | 0.67, | 0.60, | 0.69, | 0.78, | 0.70, | 0.75, | 0.79, | 0.64, | 0.71, | +| 0.78 | 0.75 | 0.80 | 0.89 | 0.81 | 0.86 | 0.89 | 0.78 | 0.84 | | +| Cut-off values | 0.46 | 0.42 | 0.86 | 0.53 | 0.43 | 1.19 | 0.65 | 0.41 | 1.34 | +| Sensitivity | 0.59 | 0.70 | 0.72 | 0.76 | 0.85 | 0.69 | 0.82 | 0.85 | 0.67 | +| Specificity | 0.78 | 0.63 | 0.67 | 0.81 | 0.58 | 0.77 | 0.77 | 0.48 | 0.76 | +| PPV, % | 76.34 | 69.00 | 71.86 | 61.02 | 46.20 | 56.14 | 42.31 | 25.58 | 36.08 | +| NPV, % | 61.88 | 63.83 | 66.21 | 89.18 | 90.07 | 85.35 | 95.19 | 92.86 | 91.16 | +| Positive LR | 2.73 | 1.89 | 2.18 | 4.00 | 2.02 | 3.00 | 3.57 | 1.63 | 2.79 | +| Negative LR | 0.52 | 0.48 | 0.42 | 0.29 | 0.26 | 0.40 | 0.23 | 0.31 | 0.43 | +| DA, % | 67.95 | 66.67 | 69.23 | 78.53 | 66.03 | 74.68 | 77.56 | 55.77 | 74.04 | + +AUROC, area under receiver operating characteristic curve; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value; LR, likelihood ratio; DA, diagnostic accuracy. + +Thus, GPRI showed better performances for the diagnosis of bridging fibrosis and cirrhosis than the other two established noninvasive biomarkers in patients with CHB. + +The demographic and clinical characteristics of age, ALT, AST, TBIL, fibrosis stage and inflammatory activity were studied to determine their correlation with GPRI in 312 patients with CHB. According to Spearman's rank correlation coefficient analysis, age, fibrosis stage, inflammatory activity, ALT, AST and TBIL were significantly correlated with GPRI (P < 0.05). + +Multivariate linear regression analyses were undertaken, which showed that in model summary (R multiple ¼ 0.63, adjusted R2¼ 0.40, analysis of variance: +F ¼ 32.15, P < 0.01), regression analyses had statistical significance. As shown in Table 3, the items of age, AST, TBIL, +fibrosis stage and inflammatory activity demonstrated positive correlations with GPRI (P < 0.01). ALT demonstrated no correlation with GPRI. + +All patients were divided into two subgroups according to age (<40 years versus 40 years) as shown in Figure 3a. The GPRI values were significantly higher in patients 40 years of age when patients with all stages of fibrosis were compared +(P < 0.01). + +As shown in Figures 3b and 3c, the 312 patients with CHB were divided into three groups according to the AST and TBIL levels: <1 times ULN, 1–3 times ULN, and >3 times the ULN. The GPRI values progressively increased with increasing AST and TBIL levels, especially in the group with AST or TBIL levels > 3 times the ULN regardless of fibrosis stage (P < 0.001 compared with < 1 times ULN for both). + +## Discussion + +Chronic HBV infection is a prolonged inflammatory disease of the liver that may lead to the progressive development of fibrosis. Because fibrosis and its end-point + +![6_image_0.png](6_image_0.png) + +cirrhosis are the main causes of morbidity and mortality, continued monitoring of fibrosis is a critical determinant for staging, prognosis, as well as therapeutic decisionmaking in CHB patients. 15 Liver biopsy is the current gold standard for staging liver fibrosis, but has some disadvantages and potential complications. 16 Over the last decade, remarkable achievements have been made in the noninvasive diagnosis of fibrosis. 17 However, sensitivity and specificity for diagnosis by these markers are limited, especially in differentiating between adjacent stages of fibrosis. 18 + +| platelet ratio index in patients with chronic hepatitis B infection (n ¼ 312). Standardized Unstandardized coefficient coefficients Constants B Standard error Beta | t | P-value | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------|-----------|-------|-----------|-----------| +| 1.090 | 0.179 | 0.138 | 6.089 | P < 0.001 | | +| Age | 0.020 | 0.004 | 0.220 | 4.887 | P < 0.001 | +| ALT | 0.000 | 0.000 | 0.138 | 0.938 | NS | +| AST | 0.002 | 0.001 | 0.298 | 2.624 | P ¼ 0.009 | +| TBIL | 0.015 | 0.003 | 0.229 | 5.724 | P < 0.001 | +| Fibrosis grade | 0.318 | 0.078 | 0.074 | 4.060 | P < 0.001 | +| Inflammatory activity grade | 0.123 | 0.046 | 0.205 | 2.681 | P ¼ 0.008 | +| ALT, alanine transaminase; AST, aspartate aminotransferase; TBIL, total bilirubin; NS, not significant (P 0.05). | | | | | | + +Recently, serum GGT was reported to be an important parameter in estimating the severity of liver fibrosis.19,20 It is present in several organs, most notably in the liver and is a commonly used diagnostic clinical test for liver function.18,21 GGT levels change in various conditions, such as inflammation, fibrosis, cholestasis and alcohol consumption.22–26 As a marker of oxidative stress, the major function of GGT is to enable the metabolism of glutathione (GSH) and glutathionylated xenobiotics.23 It catalyses the transfer of a g-glutamyl group from glutathione and other g-glutamyl compounds to amino acids or dipeptides.27 Catabolism of GSH by GGT results in pro-oxidant activity, which then leads to downstream cell, tissue, and DNA damage.25,28,29 In mild chronic hepatitis and inactive cirrhosis, GGT is usually not elevated.9 At the precirrhotic chronic hepatitis stage, GGT may increase up to 2-times above the normal range.9 Therefore, increased GGT activity is directly associated with liver injury and predicted fibrosis progression.30 Previous studies have also shown that platelet count is a reflection of disease severity.31,32 There was a negative correlation between significant liver fibrosis and platelet count.33 Worsening of fibrosis and increasing portal pressure are associated with the reduced production of thrombopoietin by hepatocytes and increased platelet sequestration within the spleen.25 Therefore, based on these two routine tests, GGT and platelet count, this present study evaluated the ability of a new serum marker, GGT-to-platelet ratio or GPRI, to determine the degree of fibrosis in chronic HBV-infected patients. + +This present study measured the diagnostic accuracy of GPRI for the noninvasive identification of significant hepatic fibrosis, using liver biopsy as the gold standard reference, compared with two other biomarker indices, APRI and FIB-4. In the 312 Chinese patients with CHB, the GPRI increased with the progressive stages of liver fibrosis and the correlation coefficient +(r ¼ 0.516, P < 0.001) was higher than for APRI (r ¼ 0.407, P < 0.001) and FIB-4 +(r ¼ 0.508, P < 0.001). These results confirmed that GPRI could predict the development of hepatic fibrosis. + +Using ROC curves, the present study demonstrated the good performance of GPRI to diagnose significant fibrosis, bridging fibrosis and cirrhosis, with AUROCs of 0.728, 0.836 and 0.842, respectively. The AUROCs of APRI and Fib-4 to predict significant fibrosis, bridging fibrosis and cirrhosis were 0.686, 0.742, 0.758, 0.803 and 0.710, 0.776 respectively. Thus, for + +![8_image_0.png](8_image_0.png) + +advanced fibrosis (F3–F4), the GPRI +yielded the highest AUROC. This matched with another study that showed that GPRI +was an important predictor of either significant fibrosis or cirrhosis.12 Using optimized cut-off values of GPRI, +significant fibrosis (cut-off value, 0.46) could be accurately diagnosed in 67.95% of patients with CHB and cirrhosis (cut-off value, 0.65) could be accurately diagnosed in 77.56% of patients with CHB. However, the diagnostic accuracy of APRI and FIB-4 for significant fibrosis and cirrhosis in accordance with liver biopsy were 66.67%, 69.23% +and 55.77%, 74.04%, respectively (Table 3). + +The current findings indicated that GPRI +showed a slightly better diagnostic accuracy than FIB-4 for the diagnosis of bridging fibrosis and cirrhosis; and APRI had the lowest diagnostic accuracy for predicting significant fibrosis, bridging fibrosis and cirrhosis compared with GPRI and FIB-4. + +Therefore, although APRI and FIB-4 had previously been shown to be useful to stage liver fibrosis in patients with CHB,34 these current results suggest that GPRI is superior to APRI and FIB-4 in Chinese patients with CHB as demonstrated by higher AUROCs and diagnostic accuracies. + +The present study also evaluated whether individual patient demographic and clinical characteristics, such as age and biochemical parameters, might affect the application of GPRI in the measurement of hepatic fibrosis in patients with CHB. Multivariate linear regression analyses found that age, AST and TBIL were independent significant determinants of GPRI. Patients with more advanced age (40 years) had significantly higher GPRI values than younger patients (<40 years) regardless of the stage of fibrosis. The best explanation of this result was that age might represent the long-term inflammatory and fibrotic processes taking place in many of the Chinese patients who had been infected with HBV at an earlier age. + +It is noteworthy that a chronic inflammatory response drives the progression of liver fibrosiss.35 The role of liver enzymes in the assessment of CHB remains important for the majority of clinical indices estimating the degree of liver fibrosis.3 Studies have reported that the AST level was elevated in the patients with chronic viral hepatitis and the changes in TBIL had an impact on liver cirrhosis.36,37 The present study observed a clear association between the GPRI value and AST and TBIL levels in patients with CHB. The GPRI value increased as the AST +and TBIL levels increased and it might be the significant extrinsic predictor of progressive liver fibrosis. Therefore, caution is advised when interpreting the diagnostic accuracy of GPRI when performed in patients with high AST and TBIL levels +(i.e. > 3 -ULN), as this might result in its overestimation of the severity of liver fibrosis. In such patients, serial measurements of GPRI value are recommended after the resolution of the acute inflammatory phase of hepatitis. In conclusion, this present study demonstrated that GPRI is a reliable method to evaluate the degree of liver fibrosis in Chinese patients with CHB. It showed significantly higher diagnostic accuracy compared with APRI and FIB-4. Age, and elevated AST and TBIL levels (i.e.> 3 - ULN) might affect the diagnostic accuracy of GPRI. Further studies with larger patient populations are needed to corroborate these results. + +## Authors' Contributions + +Y.M.N. designed the study; R.Q.W., Q.S.Z., +S.X.Z., X.M.N., J.H.D. and H.J.D. performed the experiments; R.Q.W. and Q.S.Z. analysed the data and wrote the paper. + +## Declaration Of Conflicting Interests + +The authors declare that there are no conflicts of interest. + +## Funding + +This work was supported by scientific research funds from the Hebei Provincial Health Department (no. ZL20140134), Key Science and Technology Project of Hebei Province (no. + +14277746D), Chinese Foundation for Hepatitis Prevention and Control - 'Wang Bao-en' Liver Fibrosis Research Fund (no. CFHPC20131001) +and Research Project of Hebei Provincial Administration of Traditional Chinese Medicine (no. 20141046). + +## References + +2. Ding H, Wu T, Ma K, et al. Noninvasive measurement of liver fibrosis by transient elastography and influencing factors in patients with chronic hepatitis B - A single center retrospective study of 466 patients. J Huazhong Univ Sci Technolog Med Sci 2012; 32: 69–74. + +3. Gabr SA and Alghadir AH. Prediction of fibrosis in hepatitis C patients: assessment using hydroxyproline and oxidative stress biomarkers. Virusdisease 2014; 25: 91–100. + +4. Jia J, Hou J, Ding H, et al. Transient elastography compared to serum markers to predict liver fibrosis in a cohort of Chinese patients with chronic hepatitis B. + +J Gastroenterol Hepatol 2015; 30: 756–762. + +5. Wan R, Liu H, Wang X, et al. Noninvasive predictive models of liver fibrosis in patients with chronic hepatitis B. Int J Clin Exp Med 2015; 8: 961–971. + +6. Patel K and Shackel NA. Current status of fibrosis markers. Curr Opin Gastroenterol 2014; 30: 253–259. + +7. Xu WS, Qiu XM, Ou QS, et al. Red blood cell distribution width levels correlate with liver fibrosis and inflammation: a noninvasive serum marker panel to predict the severity of fibrosis and inflammation in patients with hepatitis B. Medicine (Baltimore) 2015; 94: e612. + +8. Li J, Gordon SC, Rupp LB, et al. The validity of serum markers for fibrosis staging in chronic hepatitis B and C. J Viral Hepat 2014; 21: 930–937. + +9. Eminler AT, Irak K, Ayyildiz T, et al. The relation between liver histopathology and GGT levels in viral hepatitis: more important in hepatitis B. Turk J Gastroenterol 2014; 25: 411–415. + +10. Dixit S and Singh P. Usefulness of gamma glutamyl transferase as reliable biological marker in objective corroboration of relapse in alcohol dependent patients. J Clin Diagn Res 2015; 9: VC01–VC04. + +11. Whitfield JB. Gamma glutamyl transferase. + +Crit Rev Clin Lab Sci 2001; 38: 263–355. + +12. Lemoine M, Shimakawa Y, Nayagam S, +et al. The gamma-glutamyl transpeptidase to platelet ratio (GPR) predicts significant liver fibrosis and cirrhosis in patients with chronic HBV infection in West Africa. Gut 2016; 65: +1369–1376. + +13. Lok AS and McMahon BJ. Chronic hepatitis B: update 2009. Hepatology 2009; 50: 661–662. + +14. Anonymous. Management scheme of diagnostic and therapy criteria of viral hepatitis. + +Zhonghua Gan Zang Bing Za Zhi (Chinese J +Hepatol) 2000; 6: 324–329. + +15. Asselah T, Marcellin P and Bedossa P. + +Improving performance of liver biopsy in fibrosis assessment. J Hepatol 2014; 61: +193–195. + +16. Yilmaz B, Basar O, Altinbas A, et al. The utility of thrombopoietin in predicting liver fibrosis in chronic hepatitis B. Int J Clin Exp Med 2014; 7: 1430–1434. + +17. Toosi AE. Liver fibrosis: causes and methods of assessment, a review. Rom J Intern Med 2015; 53: 304–314. + +18. Estrabaud E, Vidaud M, Marcellin P, et al. + +Genomics and HCV infection: progression of fibrosis and treatment response. J Hepatol 2012; 57: 1110–1125. + +19. Abdo AA. Laboratory predictors of advanced fibrosis in Saudi patients with chronic hepatitis B and C. Saudi J Gastroenterol 2006; 12: 135–138. + +20. Silva IS, Ferraz ML, Perez RM, et al. Role of gamma-glutamyl transferase activity in patients with chronic hepatitis C virus infection. J Gastroenterol Hepatol 2004; 19: 314–318. + +21. Goldberg DM. Structural, functional, and clinical aspects of gamma-glutamyltransferase. CRC Crit Rev Clin Lab Sci 1980; 12: +1–58. + +22. Zhou K, Gao CF, Zhao YP, et al. Simpler score of routine laboratory tests predicts liver fibrosis in patients with chronic hepatitis B. J Gastroenterol Hepatol 2010; 25: +1569–1577. + +23. Koenig G and Seneff S. Gamma-glutamyltransferase: a predictive biomarker of cellular antioxidant inadequacy and disease risk. + +Dis Markers 2015; 2015: 818570. + +24. Park WY, Kim SH, Kim YO, et al. Serum gamma-glutamyltransferase levels predict mortality in patients with peritoneal dialysis. + +Medicine (Baltimore) 2015; 94: e1249. + +25. Myers RP, Tainturier MH, Ratziu V, et al. + +Prediction of liver histological lesions with biochemical markers in patients with chronic hepatitis B. J Hepatol 2003; 39: 222–230. + +26. Jiang S, Jiang D and Tao Y. Role of gammaglutamyltransferase in cardiovascular diseases. Exp Clin Cardiol 2013; 18: 53–56. + +27. Shaw ML, Pither-Joyce MD and McCallum JA. Purification and cloning of a gammaglutamyl transpeptidase from onion (Allium cepa). Phytochemistry 2005; 66: 515–522. + +28. Stefano GB and Kream RM. Nitric oxide regulation of mitochondrial processes: commonality in medical disorders. Ann Transplant 2015; 20: 402–407. + +29. Sreeram M, Suryakar AN and Dani NH. Is gamma-glutamyl transpeptidase a biomarker for oxidative stress in periodontitis? + +J Indian Soc Periodontol 2015; 19: 150–154. + +30. Everhart JE and Wright EC. Association of g-glutamyl transferase (GGT) activity with treatment and clinical outcomes in chronic hepatitis C (HCV). Hepatology 2013; 57: 1725–1733. + +31. Karasu Z, Tekin F, Ersoz G, et al. Liver fibrosis is associated with decreased peripheral platelet count in patients with chronic hepatitis B and C. Dig Dis Sci 2007; 52: 1535–1539. + +32. Wai CT, Cheng CL, Wee A, et al. Noninvasive models for predicting histology in patients with chronic hepatitis B. Liver Int 2006; 26: 666–672. + +33. Schiavon Lde L, Narciso-Schiavon JL and de Carvalho-Filho RJ. Non-invasive diagnosis of liver fibrosis in chronic hepatitis C. + +World J Gastroenterol 2014; 20: 2854–2866. + +34. Lin CL, Liu CH, Wang CC, et al. Serum biomarkers predictive of significant fibrosis and cirrhosis in chronic hepatitis B. J Clin Gastroenterol 2015; 49: 705–713. + +35. Czaja AJ. Hepatic inflammation and progressive liver fibrosis in chronic liver disease. + +World J Gastroenterol 2014; 20: 2515–2532. + +36. Kim HJ, Lee HK and Cho JH. Factor analysis of the biochemical markers related to liver cirrhosis. Pak J Med Sci 2015; 31: 1043–1046. + +37. Zeng X, Xu C, He D, et al. Influence of hepatic inflammation on FibroScan findings in diagnosing fibrosis in patients with chronic Hepatitis B. Ultrasound Med Biol 2015; 41: 1538–1544. \ No newline at end of file diff --git a/medical/md/PMC5566942.md b/medical/md/PMC5566942.md new file mode 100644 index 0000000000000000000000000000000000000000..1450d791fcb248d496a66158d439a9779475bfd0 --- /dev/null +++ b/medical/md/PMC5566942.md @@ -0,0 +1,179 @@ +# Quality Of Reporting In Abstracts Of Rcts Published In Emergency Medicine Journals: A Protocol For A Systematic Survey Of The **Literature** + +Federico Germini,1,2 Maura Marcucci,3,4 Marta Fedele,5 Maria Giulia Galli,6 Lawrence Mbuagbaw,7,8 Valentina Salvatori,9 Giacomo Veronese,10 Andrew Worster,11 Lehana Thabane7,8,12,13,14 To cite: Germini F, Marcucci M, +Fedele M*, et al*. Quality of reporting in abstracts of RCTs published in emergency medicine journals: a protocol for a systematic survey of the literature. *BMJ Open* 2017;7:e014981. doi:10.1136/ bmjopen-2016-014981 +► Prepublication history and additional material are available. To view these files please visit the journal online (http://dx.doi. org/10.1136/bmjopen-2016014981). Received 2 November 2016 Revised 21 February 2017 Accepted 1 March 2017 For numbered affiliations see + +![0_image_0.png](0_image_0.png) end of article. Correspondence to Dr. Lehana Thabane; thabanl@mcmaster.ca + +## Abstract + +Introduction The quality of reporting of abstracts of randomised controlled trials (RCTs) in major general medical journals and in some category-specific journals was shown to be poor before the publication of the ConsolidatedStandards of ReportingTrials (CONSORT) extension for abstracts in 2008, and an improvement in the quality of reporting of abstracts was observed after its publication. The effect of the publication of the CONSORT extension for abstracts on the quality of reporting of RCTs in emergency medicine journals has not been studied. In this paper, we present the protocol of a systematic survey of the literature, aimed at assessing the quality of reporting in abstracts of RCTs published in emergency medicine journals and at evaluating the effect of the publication of the CONSORT extension for abstracts on the quality of reporting. Methods and analysis The Medline database will be searched for RCTs published in the years 2005–2007 and 2014–2015 in the top 10 emergency medicine journals, according to their impact factor. Candidate studies will be screened for inclusion in the review. Exclusion criteria will be the following: the abstract is not available, they are published only as abstracts, still recruiting, or duplicate publications. The study outcomes will be the overall quality of reporting (number of items reported) according to the CONSORT extension and the compliance with its individual items. Two independent reviewers will screen each article for inclusion and will extract data on the CONSORT items and on other variables, which can possibly affect the quality of reporting. Ethics and dissemination This is a library-based study and therefore exempt from research ethics board review. The review results will be disseminated through abstract submission to conferences and publication in a peerreviewed biomedical journal. + +## Introduction + +Randomised controlled trials (RCTs) are considered the best study design to test interventions in clinical research. Readers often rely on information reported on abstracts when they have to decide whether or not + +## Strengths And Limitations Of This Study + +► This is the first review on the quality of reporting of abstracts of randomised controlled trials in emergency medicine journals. + +► Study selection and quality assessment will be performed in duplicate. + +► This is a comparison of the quality of reporting before and after the publication of Consolidated Standards of Reporting Trials extension for abstracts to assess possible improvement. + +► Possible other factors contributing to the quality of reporting will be investigated. + +► Limited availability of data on explanatory variables could be a limitation. + +to go through the full text of an article, and in many cases abstracts can be the only source of information for physicians and researchers.1 This can be due to lack of access to the full texts, time limitations or inability to understand the technicalities of clinical trials. Moreover, many educational sources (eg, Evidence Update from BMJ and McMasterPLUS)2 provide only the abstracts of selected studies to their users. Hence, despite being encouraged to independently verify any information, readers may be prone to rely solely on abstracts to make their decisions, which is consistent with the process of article selection for systematic reviews, that usually starts with the screening of abstracts.3 For all these reasons, incomplete reporting of studies in their abstracts can lead to inaccurate interpretation of results, missed identification of potential sources of bias, and missed inclusion in systematic reviews. This can translate into an incorrect application of trial results in clinical practice. The Consolidated Standards of Reporting Trials (CONSORT) statement4 was developed by an international group + +## + +of clinical trial methodologists, guideline developers, knowledge translation specialists and journal editors, and consists of a 25-item checklist intended to improve the reporting of RCTs. In an attempt to improve the reporting of abstracts, an extension of the CONSORT statement designed for abstracts was published by the CONSORT +group in 2008.5 The quality of reporting of abstracts in major general medical journals before the publication of CONSORT statement was poor,6 and improved after it was published.7 Still, suboptimal adherence to CONSORT +for abstracts guidelines was observed in general medical journals8 and in some category-specific journals.9–12 Emergency medicine publications are increasing worldwide, and RCTs follow this trend.13 Nevertheless, conducting research in emergency medicine can be challenging, likely more challenging than in other medical areas. The American College of Emergency Physician identified the following as key factors that contribute to making difficult the conduct of research in emergency medicine: +time, money, personnel, disinterest in research, patient mistrust and regulatory burden.14 These factors, although not unique to emergency medicine, are amplified in such a dynamic and intensive clinical setting. This can lead to many methodological issues in the implementation of RCTs, which can eventually translate into suboptimal reporting, even to a larger extent than as already shown in general medicine and other category-specific publications. It is in this context that we planned our methodological systematic survey. To the best of our knowledge, the quality of reporting of abstracts for RCTs published in emergency medicine journals and the impact of the publication of the CONSORT guidelines for abstracts in this field of medicine have not been investigated. We conducted a scoping search in August 2016 and found only one conference abstract exploring the quality of reporting of abstracts in emergency medicine journals.15 + +## Purpose And Objectives + +The purpose of the systematic survey is to inform physicians and researchers in the field of emergency medicine of the quality of reporting in abstracts of RCTs published in emergency medicine journals, and to assess the effect of the publication of the CONSORT extension for abstract on the quality of reporting. Specifically, our primary objective is to assess the compliance with the CONSORT extension items in emergency medicine journals, before comparing with after the publication of the CONSORT extension. Our secondary objective is to explore which factors, other than the year of publication, are associated with the quality of reporting of abstracts. + +## Hypothesis + +We hypothesise that the quality of reporting of abstracts has improved after the publication of the dedicated CONSORT statement extension. + +| Table 1 | Top exclusive emergency medicine journals based | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------| +| on impact factor, according to the Journal Citation Report 2015 provided by Thomson Reuters Impact Journal name factor Resuscitation 5.414 Annals of Emergency Medicine 5.008 Emergencias* 2.917 Academic Emergency Medicine 2.537 Scandinavian Journal of Trauma, Resuscitation 2.310 and Emergency Medicine* Prehospital Emergency Care 2.104 European Journal of Emergency Medicine 2.026 Injury 1.910 Emergency Medicine Journal 1.836 World Journal of Emergency Surgery 1.583 The American Journal of Emergency Medicine 1.504 Emergency Medicine Australasia 1.223 *Not published in 2006. | | + +## Methods Study Design + +This study will be a systematic survey to assess quality of reporting. The Medline database will be searched for RCTs published in the years 2005–2007 and 2014–2015 in the top 10 exclusive emergency medicine journals, ranked on their impact factor according to the Journal Citation Report 2015 provided by Thomson Reuters16 (table 1). + +At first, we decided to take a 2-year period before and after CONSORT for abstracts (2006–2007 and 2014– 2015). After a scoping search, we realised that probably the optimal time was a 3-year period before CONSORT for abstracts (2005–2007) to be able to reach the sample size. Journals created after 2006 will be excluded from the review, otherwise the period before the publication of the CONSORT extension for abstract (ie, before 2008) would not be sufficiently represented for those journals, not allowing a correct before–after comparison. Journals coming next in the Journal Citation Report list will be included to achieve the number of 10. + +The search strategy will include the Cochrane highly sensitive search strategy for identifying randomised trials in Medline (sensitivity-maximising and precision-maximising version, 2008 revision),3 journal names and limits set for the specific time periods of interest (years of publication 2005–2007 and 2014–2015). The search strategy is reported in detail in box 1. + +## Inclusion Criteria + +To be included in the review, the abstract must be a report of primary outcome(s) of an RCT and involves human subjects. Simulation based-studies (eg, study performed using manikins) will be considered for inclusion only if needed to achieve the sample size. + +Box 1 **Search strategy adopted for randomised controlled** trials published in top 10 emergency medicine journals in the years 2005–2007 and 2014–2015 in the Medline database + +![2_image_1.png](2_image_1.png) + +Exclusion criteria Studies will be excluded if the abstract is not available, they are published only as abstracts (eg, conference proceedings), still recruiting, reporting on outcomes other than primary outcome or duplicate publications. + +A flow diagram showing the study screening and selection procedures is reported in figure 1. + +## Outcomes + +The overall quality of reporting (number of items reported) according to the CONSORT extension and the compliance with its individual items will serve as outcomes. The following checklist items will not be included: 'Authors' (contact details for the corresponding author) and 'Recruitment' (trial status), as these items are specific to conference abstracts.5 Thus we will eventually test the compliance with 15 items. + +## Explanatory Variables + +Timing of publication (pre-CONSORT vs post-CONSORT publication) will be the explanatory variable for the primary objective. Explanatory variables for the secondary objective will be the following: CONSORT endorsement by the journal (defined as the presence + +![2_image_0.png](2_image_0.png) + +of the request to comply with the CONSORT statement, among the instructions for authors/authors guidelines section in the journal website), number of centres involved in the study (single-centre vs multicentre), study sample size (≤100 vs >100), type of intervention (pharmacological vs non-pharmacological), significance of results for primary outcome (significant vs non-significant), source of funding (industry-funded vs non-industry-funded) and study setting (out-of-hospital vs in-hospital). + +## Sample Size Calculation + +The primary objective of this systematic survey is to compare the mean number of reported items in pre-publication versus postpublication of the CONSORT +extension for abstracts based on the corresponding checklist.4 A review assessing the quality of abstracts in general medical journals found a mean difference in the number of items reported between after and before the publication of the CONSORT statement for abstracts of 3.05 (95% CI 2.44 to 3.65, p<0.001), going from 9.06 (SD: 2.15) in 2007 to 12.11 (SD: 2.22) in 2012.7 If we expected exactly the same results, the sample size required to show such a difference with a significance of 0.05 and a power of 0.8, considering an SD of about 2, would be eight in each study period (pre-CONSORT vs post-CONSORT). The sample size would be smaller if we consider that it is likely that from 2012 to 2015 a further improvement in the quality of reporting occurred. For example, assuming a difference of four, the sample size would be four in each study period. On the other hand, given that the generalisability of these results to studies in emergency medicine is uncertain, we hypothesise also a worse scenario in which we expect a mean difference of 1.5 items correctly reported between the two periods. The sample size required to show this effect size would be 28 in each study period. Because we are looking at the top 10 journals and so we are including articles published in the same journal, we adjusted the raw estimate in both the scenarios for a possible cluster effect multiplying by a factor of 1.42, obtained assuming an intraclass correlation coefficient of 0.03 and an average number of articles per journal of 15.17 Within the boundaries of the two scenarios, the sample size would range from 6 to 40 articles per period. Furthermore, the effect of the period will be explored in a multivariable model including also the following seven variables: endorsement of CONSORT statement by the journal, number of centres involved in the RCT, type of intervention, sample size, results of the trial, funding status and study setting. Thus, we further adjusted the sample size upward by adding five articles for each variable (35). Hence, we will be able to detect a mean difference as low as 1.5 items correctly reported as long as we find a number of eligible articles of at least 58 per period (116 in total). A smaller number of eligible articles would allow us to detect only larger effect sizes. If we find a number of articles higher than 58 per period, the articles will be randomly selected. If possible, we will select the same number of articles from each journal (6±1 per period). + +## Data Extraction And Synthesis + +For each article, two independent reviewers will screen eligibility criteria and will extract data on the reporting of the 15 items of the CONSORT statement for abstracts that we have selected and data regarding the explanatory variables. When needed, full texts will be reviewed to retrieve those data. The reviewers will use the CONSORT +elaboration and explanation document18 as a guide. + +Disagreements between reviewers will be resolved via discussion or, when necessary, with the intervention of a third reviewer. In order to increase accuracy and consistency between reviewers, an initial trial run involving 10 articles per reviewer will be undertaken. + +## Statistical Analyses + +The characteristics of the included articles will be synthesised using descriptive statistics such as mean (SD) or median (first quartile, third quartile) for continuous variables and count (frequency) for categorical variables. We will describe the frequency with which each item is reported and the mean number of reported items by period of publication (pre-CONSORT vs post-CONSORT). The primary outcome (mean difference of items reported) will be estimated first using a two-sample t-test +(unadjusted mean difference) and then through generalised estimation equations (GEEs) in order to adjust for the prespecified explanatory variables.19 The mean differences and adjusted means will be reported with 95% CIs and p values. The same multivariable GEEs will be used to explore the effect of the explanatory variables other than the period of publication. + +In order to strengthen our confidence in the results, the effect of the period of publication will be explored through two additional approaches. First, we will look at the compliance with each of the 16 items of the CONSORT statement for abstracts for years 2005–2007 versus 2014–2015 as expressed by the binary outcome item reported or not reported. We will perform both unadjusted analyses using χ2 tests and adjusted analyses using GEEs and assuming binomial distributions and unstructured correlation matrices. The results of the GEEs will be reported as adjusted ORs, with 95% CIs and p values. Then, the incidence rate ratios (IRRs) for reporting items for the period 2014–2015 compared with the period 2005–2007 will be estimated using GEEs based on a Poisson distribution and an unstructured correlation matrix. The results will be reported as adjusted IRRs, with 95% CIs and p values. The criterion for statistical significance will be set at alpha=0.05. + +In the multivariable analyses, adjustments will be made for (1) CONSORT endorsement by the journal, (2) number of centres involved in the study (single-centre vs multicentre), (3) study sample size (≤100 vs >100), (4) +type of intervention (pharmacological vs non-pharmacological), (5) significance of results for primary outcome +(significant vs non-significant), (6) source of funding (industry-funded vs non-industry-funded) and (7) study setting (out-of-hospital vs in-hospital), with journal as a grouping factor to adjust for potential clustering or similarity in articles published in the same journal. + +Descriptive data will be presented as counts and percentages. Between-reviewer agreements will be measured using the kappa statistic.20 Data will be analysed using STATA/SE V.12.0. + +## Discussion And Dissemination + +Our systematic survey is aimed at assessing the quality of reporting of abstracts of RCTs published in emergency medicine journals, and the influence on that of the publication of the CONSORT extension for abstracts. Adequate reporting of abstract of RCTs would improve transparency, correct assessment and interpretation of trials, accurate indexing, retrieval, and appropriate inclusion in systematic reviews. We expect the quality of reporting of articles published after the publication of the CONSORT extension for abstract to be better than before. Besides the publication of the CONSORT extension for abstracts, other variables can affect the quality of reporting. That is why we will explore the role of the following: journal's endorsement of the CONSORT statement, number of study centres, sample size, type of intervention, significance of the result of primary outcome, funding of the study and setting (out-of-hospital vs in-hospital). Based on previous experiences, RCTs from journals endorsing CONSORT,21 22 multicentre studies,22 23 studies with larger sample size,22 24 involving pharmacological intervention,22 25 reporting positive results for their primary outcome26 and industry-sponsored studies26 are more prone to fit the CONSORT extension for abstracts. We will also inspect the relationship between quality of reporting and study setting. Indeed, to conduct studies on interventions performed in the out-of-hospital environment is one of the main challenges posed by the emergency medicine to clinical researchers, and we think that this might further affect the quality of reporting compared with studies in the in-hospital setting. + +We decided to include in this review simulation-based RCTs, if they will be needed to achieve the sample size. + +The CONSORT extension for healthcare simulation-based studies has recently been published.27 However, this extension refers to the CONSORT guidelines for full texts, and only one recommendation is given relating to abstracts: '(In abstract or key terms, the MESH or searchable keyword term must have the word 'simulation' or 'simulated')'. Therefore, the CONSORT extension for abstract is still the best tool for evaluating the quality of reporting of simulation-based studies. + +To design the present study, we referred to a previously published protocol of a systematic survey on the reporting quality of abstracts of trials published in pain journals.12 With respect to the previous study, we decided to extend the survey to 10 journals instead of 5, and to consider only 15 of the 17 items of the checklist (as explained in the Outcomes section). + +Ethical committee approval is not required for this review because we will only be dealing with secondary data. + +The review results will be disseminated through publication in a peer-reviewed biomedical journal and abstract submission to conferences. + +In conclusion, with the present review we will investigate the quality of reporting of abstracts of RCTs in emergency medicine journals, and the eventual improvement due to the publication of the CONSORT statement for abstract. Quality of reporting might be found to be inadequate; in that case, we will enlighten factors associated with better quality and try to suggest strategies for its improvement. + +Author affiliations 1Department of Emergency, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Milano, Italy 2Department of Health Sciences, Università degli Studi di Milano, Milano, Italy 3Geriatric Unit, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Milano, Italy 4Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milano, Italy 5Department of Emergency, Area Nord, Azienda Unita Sanitaria Locale di Bologna, Bologna, Italy 6Department of Clinical and Experimental Medicine, Università degli Studi di Parma, Parma, Italy 7Department of Health Research Methods, Evidence, and Impact, McMaster University, Hamilton, Ontario, Canada 8Biostatistics Unit, Father Sean O'Sullivan Research Centre, St Joseph's Healthcare, Hamilton, Ontario, Canada 9General Practitioner Course, Regione Marche - Servizio Sanità, Ancona, Italy 10Department of Emergency Medicine, Ospedale Niguarda Ca' Granda, Milano, Italy 11Division of Emergency Medicine, McMaster University, Hamilton, Ontario, Canada 12Departments of Paediatrics and Anaesthesia, McMaster University, Hamilton, Ontario, Canada 13Centre for Evaluation of Medicine, St Joseph's Healthcare, Hamilton, Ontario, Canada 14Population Health Research Institute, Hamilton Health Sciences, Hamilton, Ontario, Canada Contributors FG is the guarantor of the review. FG, MM, LM, AW and LT drafted the manuscript. FG, MF, MGG, MM, LM, VS, GV, AW and LT contributed to the development of the selection criteria, the risk of bias assessment strategy and data extraction criteria. FG developed the search strategy. MM, LM and LT provided statistical expertise. AW provided expertise on emergency medicine literature. LM and LT provided expertise on quality of reporting. FG, MF, MGG, MM, LM, VS, GV, AW and LT read, provided feedback and approved the final manuscript. Competing interests None declared. Provenance and peer review Not commissioned; externally peer reviewed. 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. Barry HC, Ebell MH, Shaughnessy AF, *et al*. Family physicians' use of medical abstracts to guide decision making: style or substance? J Am Board Fam Pract 2001;14:437–42. + +2. Holland J, Haynes RB, McMaster P. McMaster Premium Literature Service (PLUS): an evidence-based medicine information service delivered on the web. *AMIA Annu Symp Proc* 2005:340–4. + +3. Higgins JPT, Green S. *Cochrane handbook for systematic reviews of* interventions version 5.1.0. The Cochrane Collaboration, 2011. http:// handbook.cochrane.org 4. Schulz KF, Altman DG, Moher D, *et al*. Statement: updated guidelines for reporting parallel group randomised trials. BMJ 2010;340:c332. + +5. Hopewell S, Clarke M, Moher D, *et al*. CONSORT for reporting randomised trials in journal and conference abstracts. *Lancet* 2008;371:281–3. + +6. Berwanger O, Ribeiro RA, Finkelsztejn A, *et al*. The quality of reporting of trial abstracts is suboptimal: survey of Major general medical journals. *J Clin Epidemiol* 2009;62:387–92. + +7. Mbuagbaw L, Thabane M, Vanniyasingam T, *et al*. Improvement in the quality of abstracts in major clinical journals since CONSORT extension for abstracts: a systematic review. *Contemp Clin Trials* 2014;38:245–50. + +8. Ghimire S, Kyung E, Kang W, *et al*. Assessment of adherence to the CONSORT statement for quality of reports on randomized controlled trial abstracts from four high-impact general medical journals. *Trials* 2012;13:77. + +9. Rios LP, Odueyungbo A, Moitri MO, *et al*. Quality of reporting of randomized controlled trials in general endocrinology literature. J Clin Endocrinol Metab 2008;93:3810–6. + +10. Can OS, Yilmaz AA, Hasdogan M, *et al*. Has the quality of abstracts for randomised controlled trials improved since the release of Consolidated Standards of Reporting Trial guideline for abstract reporting? A survey of four high-profile anaesthesia journals. *Eur J* Anaesthesiol 2011;28:485–92. + +11. Fleming PS, Buckley N, Seehra J, *et al*. Reporting quality of abstracts of randomized controlled trials published in leading orthodontic journals from 2006 to 2011. *Am J Orthod Dentofacial Orthop* 2012;142:451–8. + +12. Sriganesh K, Bharadwaj S, Wang M, *et al*. Reporting quality of abstracts of trials published in top five pain journals: a protocol for a systematic survey. *BMJ Open* 2016;6:e012319. + +13. Wilson MP, Itagaki MW. Characteristics and trends of published emergency medicine research. *Acad Emerg Med* 2007;14:635–40. + +14. In: Bebarta VS, Cairns CB, eds. *Emergency care research - a primer*: +American College of Emergency Physicians, 2012. + +15. Varney L, Detweiler B, Chong A, *et al* The quality of research abstracts in emergency medicine. *37th annual meeting of the society* for medical decision making, 2015. + +16. Journal Citation Report 2015. Thomson Reuters 2016. http:// +ipscience.thomsonreuters.com/product/journal-citation-reports/? utm_source=false&utm_medium=false&utm_campaign=false 17. Kerry SM, Bland JM. The intracluster correlation coefficient in cluster randomisation. BMJ 1998;316:1455–60. + +18. Hopewell S, Clarke M, Moher D, *et al*. CONSORT for reporting randomized controlled trials in journal and conference abstracts: explanation and elaboration. *PLoS Med* 2008;5:e20. + +## + +19. Hanley JA, Negassa A, Edwardes MD, *et al*. Statistical analysis of correlated data using generalized estimating equations: an orientation. *Am J Epidemiol* 2003;157:364–75. + +20. Viera AJ, Garrett JM. Understanding interobserver agreement: the kappa statistic. *Fam Med* 2005;37:360–3. + +21. Turner L, Shamseer L, Altman DG, *et al*. Consolidated standards of reporting trials (CONSORT) and the completeness of reporting of randomised controlled trials (RCTs) published in medical journals. Cochrane Database Syst Rev 2012;11:MR000030. + +22. Samaan Z, Mbuagbaw L, Kosa D, *et al*. A systematic scoping review of adherence to reporting guidelines in health care literature. J Multidiscip Healthc 2013;6:169–88. + +23. Balasubramanian SP, Wiener M, Alshameeri Z, *et al*. Standards of reporting of randomized controlled trials in general surgery: can we do better? *Ann Surg* 2006;244:663–7. + +24. Borg Debono V, Zhang S, Ye C, *et al*. The quality of reporting of RCTs used within a postoperative pain management meta-analysis, using the CONSORT statement. *BMC Anesthesiol* 2012;12:13. + +25. Thabane L, Chu R, Cuddy K, *et al*. What is the quality of reporting in weight loss intervention studies? A systematic review of randomized controlled trials. *Int J Obes* 2007;31:1554–9. + +26. Lai R, Chu R, Fraumeni M, *et al*. Quality of randomized controlled trials reporting in the primary treatment of brain tumors. *J Clin Oncol* 2006;24:1136–44. + +27. Cheng A, Kessler D, Mackinnon R, *et al*. Reporting guidelines for Health Care Simulation Research: extensions to the CONSORT and STROBE Statements. *Simul Healthc* 2016;11:238–48. \ No newline at end of file diff --git a/medical/md/PMC5731175.md b/medical/md/PMC5731175.md new file mode 100644 index 0000000000000000000000000000000000000000..1636c615908b4b57b687746b4d143ffd23a30df6 --- /dev/null +++ b/medical/md/PMC5731175.md @@ -0,0 +1,132 @@ + + +![0_image_1.png](0_image_1.png) + +Published online: November 16, 2017 +© 2017 The Author(s) +Published by S. Karger AG, Basel www.karger.com/cop This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). + +Usage and distribution for commercial purposes requires written permission. + +Case Report + +![0_image_0.png](0_image_0.png) + +# Epiretinal Membrane In Dome-Shaped Macula Complicated With Serous Retinal Detachment: Transient Efficacy Of Surgery + +Guillaume Soudiera Alain Gaudricb Vincent Gualinob Mathieu Nardinc Claude Speeg-Schatzc David Gauchera aOphtalmology, Nouvel Hôpital Civil, Strasbourg, France; bUniversity Hospital of Lariboisière, University Paris 7, Paris, France; cOphthalmology, Hôpitaux Universitaires de Strasbourg, Strasbourg, France + +## Keywords + +Myopia · Dome-shaped macula · Epiretinal membrane + +## Abstract + +Dome-shaped macula (DSM) is an entity recently described as a convex anterior protrusion of the macular area within a posterior myopic staphyloma. Specific complications were associated with DSM, like serous retinal detachment (SRD). We describe a woman presenting with a decreased vision at 20/50. SD-OCT scans were performed, showing a macular bulge. SRD +was present and an epiretinal membrane could also be observed. Fluorescein angiography and indocyanin green angiography did not show any leakage nor choroidal neovascularization. Epiretinal membrane peeling was performed, and 3 months after surgery, SRD completely disappeared. However, SRD reappeared 1 year after surgery and enlarged within 2 years following surgery. In conclusion, two mechanisms could be considered for physiopathology of SRD: first, the epiretinal membrane may have exerted traction on the macular retina, second, vitreous body might constitute a tank for cytokines and/or other factors, triggering subretinal fluid accumulation, leading to SRD. © 2017 The Author(s) +Published by S. Karger AG, Basel + + +www.karger.com/cop Soudier et al.: Epiretinal Membrane in Dome-Shaped Macula Complicated with Serous Retinal Detachment: Transient Efficacy of Surgery + +## Introduction + +![1_Image_0.Png](1_Image_0.Png) + +Dome-shaped macula (DSM) is an entity recently described as a convex anterior protrusion of the macular area within a posterior myopic staphyloma [1]. The original description by Gaucher et al. [1] was made out of a group of highly myopic eyes and some visionthreatening complications were specifically described, such as choroidal neovascularization, macular retinal atrophy, flat irregular pigment epithelium deposits, epiretinal membrane, and serous retinal detachment (SRD). SRD physiopathology still remains unclear. Different hypotheses have been proposed. Viola et al. [2] thought that thickened central macular choroid in DSM could lead to choroidal leakage as reported in central serous chorioretinopathy. + +Choroidal vascular changes in the central zone and specific abrupt choroidal thickness changes between central and peripheral macula [3] may alter choroidal blood flow and trigger SRD development [3, 4]. In this clinical case, we describe a patient presenting with DSM associated with epiretinal membrane and SRD. After epiretinal membrane peeling, transient resolution of SRD was noticed, suggesting that vitreous removal had influenced SRD evolution. + +## Case Report + +We report a case of a 63-year-old woman presenting with a decreased vision and metamorphopsia. Her best-corrected visual acuity (BCVA) on the right eye was 20/50. The axial length was 24.4 mm and spherical equivalent was –5. + +The initial OCT horizontal scan showed a macular bulge with a maximal height of 760 µm measured as previously described [5, 6] (Fig. 1). On the vertical scan, there was no macular bulge: the horizontal axis was the most cambered on SD-OCT, indicating that this DSM +had a vertical oval-shaped pattern [6]. An SRD was present and an epiretinal membrane could also be observed on both vertical and horizontal OCT scans on Spectralis HRA OCT® +(Heidelberg Engineering, Heidelberg, Germany) (Fig. 1). + +Fluorescein angiography (FA) and indocyanine green angiography (ICGA) showed hypofluorescent and hypocyanescent perifoveal zones without any leakage or choroidal neovascularization. Infrared image showed multiples epicenters of contraction leading to retinal folds (Fig. 2). + +Epiretinal membrane peeling was performed as vision was decreasing since the last control 6 months before. One month after surgery, we observed a progressive decrease of SRD and a complete resolution of subretinal fluid at 3 months (Fig. 3). However, SRD reappeared 1 year after surgery and enlarged within 2 years following surgery (Fig. 3). + +## Discussion + +Several studies showed the presence of SRD in patients presenting with DSM [1, 2, 6–8]. + +Different hypotheses were advanced to explain SRD appearance. In a robust study, Viola et al. [2] noted that there was more pinpoint leakage on FA and hypercyanescent choroidal points on ICGA in eyes with SRD than without. These findings showed similarities with chronic central serous chorioretinopathy. + +In our clinical case, this leakage on FA and ICGA was not present. The fact that vitrectomy and epiretinal membrane peeling could improve transiently the SRD may suggest two + +| Case Rep Ophthalmol 2017;8:515–520 DOI: 10.1159/000481703 © 2017 The Author(s). Published by S. Karger AG, Basel www.karger.com/cop | +|---------------------------------------------------------------------------------------------------------------------------------------| + +![2_image_1.png](2_image_1.png) + +Soudier et al.: Epiretinal Membrane in Dome-Shaped Macula Complicated with Serous + +![2_image_3.png](2_image_3.png) + +Retinal Detachment: Transient Efficacy of Surgery mechanisms for SRD physiopathology: first, the epiretinal membrane may have exerted traction on the macular retina, favoring the apparition of SRD. Second, the vitreous body might + +![2_image_0.png](2_image_0.png) + +constitute a tank for cytokines and/or other factors, triggering subretinal fluid accumulation and finally leading to SRD formation. + +Some authors noted a variation of choroidal thickness between peripheral and central macular areas in a case series [3]: they suggested that this difference could increase choroid hydrostatic pressure leading to SRD. However, we did not find significant results in SRD +subgroups, in a series comparing choroidal thickness in DSM and no-DSM groups [4]. Furthermore, the different treatments usually used for hematoretinal barrier restoration (i.e., +eplerenone, spironolactone, photodynamic therapy, anti-VEGFs, etc.) seemed inefficient, suggesting other mechanisms than external blood barrier disruption [5, 8]. Even if spontaneous resolution has been observed in a certain proportion of cases [5], the sequence of events in this case strongly suggests a role for vitrectomy in the transient disappearance of SRD. This evolution of fluid movements following vitrectomy has already been observed in macular edema pathology [9]. The removal of the vitreous body may have decreased the level of some factors causing SRD. Oxygen and other factors regulating the external blood retinal barrier may then be implicated. Recently, spironolactone has been shown to have sometimes positive effects on SRD in DSM [10]. Aldosterone or other effectors of aldosterone receptors may be present in the vitreous; their removal during vitrectomy might be responsible for transient decrease of SRD in this case. + +## Conclusion + +SRD is a common complication in DSM, in which treatment management is a difficult task. Vitrectomy seems to play a role for transient resolution of SRD associated with DSM. + +The mechanisms of this effect are unclear; SRD may be due to the surgical removal of factors triggering subretinal fluid accumulation. + +## Statement Of Ethics + +This study adhered to the principles of the declaration of Helsinki. The ethics committee of the French Society of Ophthalmology did not find any challenges to the medical and scientific rules of ethics as accepted in France. + +## Disclosure Statement + +The authors have nothing to disclose. + +## References + +![2_image_2.png](2_image_2.png) + + +www.karger.com/cop Soudier et al.: Epiretinal Membrane in Dome-Shaped Macula Complicated with Serous Retinal Detachment: Transient Efficacy of Surgery 4 Soudier G, et al: Macular choroidal thickness in myopic eyes with and without a dome-shaped macula: a + +![3_image_0.png](3_image_0.png) + +case-control study. Ophthalmologica 2016;236:148–153. + +5 Soudier G, et al: Long-term evolution of dome-shaped macula: increased macular bulge is associated with extended macular atrophy. Retina 2016;36:944–952. + +6 Caillaux V, et al: Morphologic characterization of dome-shaped macula in myopic eyes with serous macular detachment. Am J Ophthalmol 2013;156:958–967 e1. + +7 Errera MH, et al: The extended clinical phenotype of dome-shaped macula. Graefes Arch Clin Exp Ophthalmol 2014;252:499–508. + +8 Fernandez-Vega Sanz A, et al: Serous retinal detachment associated with dome-shaped macula and staphyloma edge in myopic patients before and after treatment with spironolactone. J Ophthalmol 2016;2016:8491320. + +9 Jackson TL, et al: Pars plana vitrectomy for diabetic macular edema: a systematic review, meta-analysis, and synthesis of safety literature. Retina 2017;37:886–895. + +10 Dirani A, et al: Resolution of foveal detachment in dome-shaped macula after treatment by spironolactone: report of two cases and mini-review of the literature. Clin Ophthalmol 2014;8:999– +1002. + +Fig. 1. The initial OCT horizontal scan showed a macular bulge with a maximal height of 760 µm measured +(a). On the vertical scan, there was no macular bulge: the horizontal axis was the most cambered on SDOCT Spectralis HRA OCT® (Heidelberg Engineering, Heidelberg, Germany), indicating that this DSM had a vertical oval-shaped pattern (b). An SRD was present and an epiretinal membrane could also be observed on both vertical and horizontal OCT scans. + + +www.karger.com/cop Soudier et al.: Epiretinal Membrane in Dome-Shaped Macula Complicated with Serous + +![4_image_0.png](4_image_0.png) + +Retinal Detachment: Transient Efficacy of Surgery + + +www.karger.com/cop Soudier et al.: Epiretinal Membrane in Dome-Shaped Macula Complicated with Serous + +![5_image_0.png](5_image_0.png) + +Retinal Detachment: Transient Efficacy of Surgery \ No newline at end of file diff --git a/medical/md/PMC5790714.md b/medical/md/PMC5790714.md new file mode 100644 index 0000000000000000000000000000000000000000..b0f547edc095545628b5c0b0b1490fc50423bfb5 --- /dev/null +++ b/medical/md/PMC5790714.md @@ -0,0 +1,103 @@ +# 【 Case Report 】 + +![0_Image_0.Png](0_Image_0.Png) + +![0_Image_1.Png](0_Image_1.Png) Familial Adenomatous Polyposis With Multiple Helicobacter-Negative Early Gastric Cancers Treated By Endoscopic Submucosal Dissection + +Katsuki Yaguchi 1,2, Makomo Makazu 3, Kingo Hirasawa 3, Makoto Sugimori 1, Ryosuke Kobayashi 3, Chiko Sato 3, Ryosuke Ikeda 3, Takehide Fukuchi 3, Yasuaki Ishii 3, Hiroaki Kaneko 1, Wataru Shibata 1, Shoji Yamanaka 4, Yoshiaki Inayama 5 and Shin Maeda 1 + +## Abstract: + +Stomach cancer rarely develops in patients with familial adenomatous polyposis (FAP), and *Helicobacter* pylori infection may increase the risk of FAP-related gastric cancer. We describe the case of a 64-year-old woman who developed multiple synchronous early gastric cancers without *H. pylori* infection. Nine cancer lesions were successfully treated by endoscopic submucosal dissection. An immunohistochemical analysis revealed that the tumors were positive for mucin (MUC)2, MUC6, and CDX2, but negative for MUC5AC, suggesting that the tumors were gastrointestinal mixed type. Periodical endoscopic surveillance is important for the detection of cancers at an early stage. Key words: familial adenomatous polyposis, endoscopic submucosal dissection, gastric cancer +(Intern Med 56: 3283-3286, 2017) +(DOI: 10.2169/internalmedicine.8735-16) + +## Introduction + +Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary disease caused by adenomatous polyposis coli (APC) mutations (1, 2). FAP patients have multiple colorectal adenomatous polyps. However, extracolonic neoplastic lesions are also reported in FAP patients, especially gastric neoplasms (3, 4). Gastric neoplasms―usually fundic gland polyps (FGP) in the gastric body―are commonly found in FAP patients in Western countries. Gastric adenoma (rarely adenocarcinoma) accounts for approximately 10% of gastric neoplasms in FAP patients (5, 6). In Japan, 39% of FAP patients develop gastric adenoma and the increased incidence of adenoma may be associated with Helicobacter pylori infection (4, 7). Few histological analyses of FAP-associated gastric lesions have been performed. + +FGP and foveolar-type gastric adenoma were frequently identified; the lesions were found to be MUC5AC-positive +(gastric mucin) and MUC2- and CDX2-negative (intestinal markers) (3). Intestinal gastric cancers are closely associated with *H. pylori*-related gastric atrophy and intestinal metaplasia (8). We herein report the case of a patient with FAP who developed nine synchronous early gastric cancers without H. pylori infection who was successfully treated by endoscopic submucosal dissection (ESD). Interestingly, a histological analysis showed that the lesions were gastrointestinal mixedtype tumors. + +## Case Report + +The patient was a 64-year-old woman who had been diagnosed with FAP and who had undergone total colectomy at 24 years of age. She had a family history of FAP (her mother and a brother). At 64 years of age, follow-up endoscopy revealed multiple O-IIa+IIc (Paris classification) le1Department of Gastroenterology, Yokohama City University Graduate School of Medicine, Japan, 2Yokohama City University School of Medicine, Japan, 3Gastroenterological Centre, Yokohama City University Medical Center, Japan, 4Department of Pathology, Yokohama City University Graduate School of Medicine, Japan and 5Department of Diagnostic Pathology, Yokohama City University Medical Center, Japan Received: December 18, 2016; Accepted: April 19, 2017; Advance Publication by J-STAGE: October 11, 2017 Correspondence to Dr. Shin Maeda, shinmaeda2-gi@umin.ac.jp + +![1_image_0.png](1_image_0.png) + +sions in the antrum of the stomach (Fig. 1A). Chromoendoscopy with indigo carmine and narrow band imaging (NBI) +clearly showed a tumor area (Fig. 1B). Magnifying endoscopy with NBI (ME-NBI) indicated that the microsurface pattern of the lesions was slightly irregular and the microvascular pattern was irregular with demarcation lines. + +Thus, early gastric cancer was diagnosed (Fig. 1C) (9). We identified nine lesions, all of which were removed in 3 separate ESD sessions (Fig. 1D). Fig. 1E shows the largest formalin-fixed ESD sample, which included 7 adnocarninomas. The largest cancer lesion was 10 mm × 7 mm in size +(Fig. 1E). + +A pathological analysis revealed that all nine lesions had been removed and that all of the tumors were welldifferentiated intramucosal adenocarcinomas (tub1) (Fig. 2). + +No venous or lymphoid vessel invasion was apparent and the treatment of the cancers was pathologically curative. The surrounding non-tumorous mucosa was almost normal without inflammation or atrophy with a regular arrangement of collecting venules, an endoscopic marker for the *H. pylori*negative gastric mucosa. In accordance with the endoscopic findings, no *H. pylori* infection was identified in the pathological analysis or with serum Hp-IgG. In an immunohistochemical analysis, we used MUC5AC as a marker for foveolar cells, MUC6 for mucous neck or pyloric gland cells, MUC2 for goblet cells, CD10 for small-intestinal-type brush border, and CDX2 for intestinal cells. The tumors were positive for MUC6, but negative for MUC5AC. Interestingly, the tumors were also positive for MUC2, CD10, and CDX2, suggesting that they were the gastrointestinal mixed type (Fig. 2). The proliferative activity and malignant potential were analyzed according to the expression of Ki-67 and TP53, which indicated that the tumors were highly proliferative and had malignant potential. In addition, β-catenin nuclear staining, which is associated with the APC mutation, was strongly positive. To prevent gastric stenosis after large ESD, triamcinolone acetonide (50 mg) was injected into the ESD ulcer bed, and oral steroids were administered for 3 months. After 6 months, the ESD ulcer had healed without stenosis. + +## Discussion + +There are a few reports on the gastric lesions in FAP patients. The reported rate of FAP-associated gastric cancer in Japan is higher than that in Western countries. In Japan, gastric cancer developed in 11 of 261 FAP patients (4.2%) (10), +whereas it was found that the risk of gastric cancer did not differ between FAP and non-FAP patients in Western countries (11). This difference may be due to the higher rate of gastric atrophy or intestinal metaplasia in the Japanese population, which is associated with a higher rate of *H. pylori* infection (4). However, *H. pylori* infection and gastric atrophy were not found in this case. Another reason for this difference is that there are probably differences between Western and Asian FAP patients in the mutation loci in the APC gene (12). We have not yet analyzed the APC mutation locus in the current case. + +The histology of FAP-related gastric cancer has not been clarified. In FAP patients, fundic gland polyps (FGP) are frequently observed and may be precursor lesions of gastric adenocarcinoma (13). In this context, FGP-derived cancer + +![2_image_0.png](2_image_0.png) + +might be positive for MUC5AC, which is a marker for gastric foveolar cells. Hashimoto et al. reported that, in addition to FGP, pyloric gland adenoma (PGA) was frequently observed in FAP patients and was strongly positive for MUC5AC and MUC6 (14). However, FGP and PGA were negative for MUC2 and CDX2 when not accompanied by intestinal metaplasia (15). In our case, the tumors were positive for MUC2, CDX2, and MUC6, but negative for MUC5AC, suggesting that the lesions were gastrointestinal mixed type. The background mucosa did not show inflammation, *H. pylori* infection, or intestinal metaplasia, making the tumors in this case an unusual gastric cancer phenotype. + +Mutations of the APC gene cause the accumulation of βcatenin and might be one of the mechanisms responsible for the development of gastric cancer in FAP patients (14). The overexpression of CDX2 in gastric epithelial cells might induce intestinal metaplasia and eventually cause adenocarcinoma (16), suggesting that, in addition to the accumulation of β-catenin, the overexpression of CDX2 is one of the mechanisms underlying the development of gastric adenocarcinoma. This assumes that additional genetic or epigenetic alterations cause the accumulation of CDX2. Further studies should be performed to analyze the mechanisms of these alterations. + +It is questionable whether the adenocarcinomas in the current case were developed as a result of the adenomacarcinoma sequence or whether they occurred as *de novo* cancer. We tried to analyze a sample of the adenoma, which had been resected by ESD 7 years previously and found that the tumor was positive for MUC2 and CDX2, but negative for MUC5AC and MUC6, suggesting that the adenoma was of the intestinal type. We also found that β-catenin was weakly stained and TP53 nuclear staining was negative +(Supplementary Figure). Taken together, it is likely that the adenocarcinoma in the current case was caused via the adenoma-carcinoma sequence, as is the case of colon cancer. + +Nine synchronous gastric cancers were detected in the current case. The reported frequency of synchronous gastric cancer is approximately 10%; cases involving more than five synchronous lesions are very rare (17). Shibata et al. reported that three of five FAP patients with gastric cancer had synchronous cancers, indicating that the gastric cancers in FAP patients tend to be synchronous. In addition, metachronous gastric cancer has been reported in FAP patients (18); thus, it is important to perform periodic endoscopic surveillance to diagnose cancers at an early stage. + +Our patient had undergone ESD for gastric adenoma 7 years previously, and multiple cancers were found after careful EGD surveillance. + +The authors state that they have no Conflict of Interest (COI). + +## References + +1. Groden J, Thliveris A, Samowitz W, et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66: 589-600, 1991. + +2. Kinzler KW, Nilbert MC, Su L-K, et al. Identification of FAP locus genes from chromosome 5q21. Science 253: 661-665, 1991. + +3. Wood LD, Salaria SN, Cruise MW, et al. Upper GI tract lesions in familial adenomatous polyposis (FAP): enrichment of pyloric gland adenomas and other gastric and duodenal neoplasms. Am J Surg Pathol 38: 389-393, 2014. + +4. Nakamura S, Matsumoto T, Kobori Y, Iida M. Impact of *Helicobacter pylori* infection and mucosal atrophy on gastric lesions in patients with familial adenomatous polyposis. Gut 51: 485-489, 2002. + +5. Sarre RG, Frost AG, Jagelman DG, et al. Gastric and duodenal polyps in familial adenomatous polyposis: a prospective study of the nature and prevalence of upper gastrointestinal polyps. Gut 28: +306-314, 1987. + +6. Spigelman AD, Williams CB, Talbot IC, et al. Upper gastrointestinal cancer in patients with familial adenomatous polyposis. Lancet 2: 783-785, 1989. + +7. Iwama T, Mishima Y, Utsunomiya J. The impact of familial adenomatous polyposis on the tumorigenesis and mortality at the several organs. Its rational treatment. Ann Surg 217: 101-108, 1993. + +8. Uemura N, Okamoto S, Yamamoto S, et al. *Helicobacter pylori* infection and the development of gastric cancer. N Engl J Med 345: 784-789, 2001. + +9. Yao K. The endoscopic diagnosis of early gastric cancer. Ann Gastroenterol 26: 11-22, 2013. + +10. Yamaguchi T, Ishida H, Ueno H, et al. Upper gastrointestinal tumours in Japanese familial adenomatous polyposis patients. Jpn J +Clin Oncol 46: 310-315, 2016. + +11. Offerhaus GJ, Giardiello FM, Krush AJ, et al. The risk of upper gastrointestinal cancer in familial adenomatous polyposis. Gastroenterology 102: 1980-1982, 1992. + +12. Miyaki M, Yamaguchi T, Iijima T, et al. Difference in characteristics of APC mutations between colonic and extracolonic tumors of FAP patients: variations with phenotype. Int J Cancer 122: 24912497, 2008. + +13. Garrean S, Hering J, Saied A, et al. Gastric adenocarcinoma arising from fundic gland polyps in a patient with familial adenomatous polyposis syndrome. Am Surg 74: 79-83, 2008. + +14. Hashimoto T, Ogawa R, Matsubara A, et al. Familial adenomatous polyposis-associated and sporadic pyloric gland adenomas of the upper gastrointestinal tract share common genetic features. Histopathology 67: 689-698, 2015. + +15. Chen ZM, Scudiere JR, Abraham SC, Montgomery E. Pyloric gland adenoma: an entity distinct from gastric foveolar type adenoma. Am J Surg Pathol 33: 186-193, 2009. + +16. Mutoh H, Sakurai S, Satoh K, et al. Development of gastric carcinoma from intestinal metaplasia in Cdx2-transgenic mice. Cancer Res 64: 7740-7747, 2004. + +17. Isobe T, Hashimoto K, Kizaki J, et al. Characteristics and prognosis of synchronous multiple early gastric cancer. World J Gastroenterol 19: 7154-7159, 2013. + +18. Shibata C, Ogawa H, Miura K, et al. Clinical characteristics of gastric cancer in patients with familial adenomatous polyposis. Tohoku J Exp Med 229: 143-146, 2013. + +The Internal Medicine is an Open Access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. To view the details of this license, please visit (https://creativecommons.org/licenses/ +by-nc-nd/4.0/). + +Ⓒ 2017 The Japanese Society of Internal Medicine Intern Med 56: 3283-3286, 2017 \ No newline at end of file diff --git a/medical/md/PMC6133460.md b/medical/md/PMC6133460.md new file mode 100644 index 0000000000000000000000000000000000000000..6710345f7d4622acd01b44e6f52089e1d6b94e7b --- /dev/null +++ b/medical/md/PMC6133460.md @@ -0,0 +1,156 @@ +Clinical Case Report Medicine® + +![0_image_0.png](0_image_0.png) + +OPEN +# Sciatic Neuropathy Caused By A Pressure Ulcer A Case Report Jae Ha Hwang, Md, Phd∗, Dong Wan Kim, Md, Kwang Seog Kim, Md, Phd, Sam Yong Lee, Md, Phd + +Abstract Rationale: Sciatic neuropathy has various causes; however, cases in which a pressure ulcer led to sciatic neuropathy have not been reported to date. Patient concerns: A 33-year-old woman with no pre-existing mobility problems visited our department with the chief complaint of an extensive pressure ulcer and necrosis in her right buttock. She had a medical history of being bedridden for 2 days while in a coma due to a drug overdose 2 months previously. Physical examination revealed loss of sensation and foot drop in the right foot. Diagnosis: Physical examination, magnetic resonance imaging, and nerve conduction studies were conducted; the patient was diagnosed with a common peroneal branch injury of the right sciatic nerve. + +Interventions: The necrotic tissue was debrided and sciatic nerve decompression was performed, followed by frequent dressing changes. In addition, psychiatric treatment and physical therapy were performed simultaneously. + +Outcomes: The pressure ulcer decreased in size and healed to some extent with granulation tissue. However, gait disorders, accompanied by symptoms of sciatic neuropathy, continued. The patient was transferred to the department of gastroenterology for the treatment of toxic hepatitis, which occurred during her inpatient treatment. Lessons: Physicians should be aware that sciatic neuropathy may occur during the treatment of patients with a pressure ulcer who exhibit no symptoms of paraplegia or quadriplegia. To prevent neuropathy, aggressive treatment of the pressure ulcer is necessary. + +Abbreviations: AH = adductor hallucis, CMAP = compound muscle action potential, CV = conduction velocity, DL = distal latency, EDB = extensor digitorum brevis, EMG = electromyography, MRI = magnetic resonance imaging, NE = not evoked, SNAP = sensory nerve action potential, TA = tibialis anterior. + +Keywords: pressure sore, sciatic nerve, sciatic neuropathy 1. Introduction Peripheral neuropathy, a neurological condition, occurs as a result of injury to a peripheral nerve, causing weakness, numbness, and pain. Physicians often encounter patients with neuropathy, and making an accurate diagnosis of the underlying cause is important because the treatment and prognosis of neuropathy are dependent on cause. The most common causes of sciatic neuropathy are hip surgery and trauma, followed by external compression, direct nerve injury, and ischemia. Other known causes include intragluteal injections, compartment syndrome, infection, inflammation, intraneural tumors, vascular Editor: N/A. + +The authors have nothing to disclose and no conflicts of interest. + +Department of Plastic and Reconstructive Surgery, Chonnam National University Medical School, Gwangju, Korea. + +∗Correspondence: Jae Ha Hwang, Department of Plastic and Reconstructive Surgery, Chonnam National University Medical School, 42 Jebong-ro, Dong-gu, Gwangju 61469, Republic of Korea (e-mail: psjhhwang@daum.net). + +Copyright © 2018 the Author(s). Published by Wolters Kluwer Health, Inc. + +This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NCND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. + +Medicine (2018) 97:36(e12254) +Received: 5 May 2018 / Accepted: 14 August 2018 http://dx.doi.org/10.1097/MD.0000000000012254 causes, gynecological causes, piriformis syndrome, and radiotherapy.[1–6] However, no report has described a pressure ulcer causing neuropathy. Herein, the authors report a case of sciatic neuropathy caused by a pressure ulcer in a 33-year-old female patient without pre-existing mobility problems. + +2. Case report A 33-year-old woman presented with a chief complaint of a pressure ulcer measuring 2510 cm that had appeared 1 month previously on her right buttock. The patient received medication to treat paranoid type schizophrenia for 12 years. One month before the occurrence of the pressure ulcer, the patient overdosed and was bedridden for 2 days in a coma, which presumably caused the pressure ulcer to form on her right buttock. The lesion had deteriorated over the following month. At the time of the patient's visit, erythema, pain, and swelling was observed near the lesion, suggesting inflammation, and massive areas of necrotic tissue were found (Fig. 1). According to the National Pressure Ulcer Advisory Panel (NPUAP) staging system, it was classified as unstageable.[7] +Physical examination of the right lower extremity indicated symptoms of sensory deficit in the anterolateral aspect of the calf, dorsomedial and dorsolateral aspect of the foot, first dorsal web space, and great toe. Furthermore, right foot dorsiflexion exhibited significant weakness, with a Medical Research Council scale grade of 1 of 5 (Figs. 2A, B). A common peroneal branch injury in the sciatic nerve was clinically suspected. Magnetic 1 + +resonance imaging (MRI) revealed general edematous changes in the right buttock and inflammatory soft tissue swelling near the sciatic nerve (Fig. 3A, B). Further sensory and motor nerve conduction studies indicated signs of common peroneal branch injury in the sciatic nerve (Tables 1 and 2). It was concluded that the sciatic neuropathy was induced by inflammatory soft tissue swelling near the sciatic nerve, which was caused by the pressure ulcer. + +To treat the necrotic and inflammatory soft tissue swelling, debridement and decompression were performed under general anesthesia. A 3025 cm sized soft tissue defects remained, and greater trochanter, lesser trochanter, and neck of femur were exposed (Fig. 4). Frequent dressing changes were administered on the remaining soft tissue defects and the patient underwent psychiatric treatment with concomitant physical therapy. + +After 6 months of frequent dressing changes and serial debridement, the pressure ulcer exhibited moderate improvement. Soft tissue defects decreased in size from 3025 to 25 x 15 cm and healed with granulation tissue (Fig. 5A, B). Although a gait disorder was still present as a sequela of sciatic neuropathy, the patient's motor function recovered to some extent with MRC +grade 2 of 5. However, valproic acid, which was administered as a psychiatric medication while the patient was admitted to the hospital, caused toxic hepatitis. The patient was transferred to the department of gastroenterology for the treatment of toxic hepatitis. + +The patient provided informed consent for the publication of her clinical and radiological data. This study was approved by the Institutional Review Board of Chonnam National University Hospital (Gwangju, Korea) and was conducted in accordance with the principles of the Helsinki Declaration II. + +![1_image_0.png](1_image_0.png) + +![1_image_1.png](1_image_1.png) + +![2_image_0.png](2_image_0.png) + +3. Discussion The sciatic nerve runs through the lower extremity and is the largest and longest nerve in the human body. The sciatic nerve divides into the tibial nerve and common peroneal nerve branches, and runs to the feet.[8] It is susceptible to injury due to its long length, and sciatic neuropathy can occur due to several causes, including fracture, dislocation, hematoma, and hip replacement surgery,[1–6] leading to neurological deficits. Sciatic neuropathy is diagnosed based on nerve conduction studies, electromyography (EMG), and MRI. One of the most common symptoms of sciatic neuropathy is foot drop; other symptoms include weakened knee flexion, pain and sensory loss in the foot, and decreased ankle jerk.[1,2] It is important to determine the exact cause of sciatic neuropathy before treatment. For example, if a patient has piriformis syndrome, the first-line therapy should be conservative therapy or botulinum toxin injection, and tricyclic antidepressants or anticonvulsants should be administered to control neuropathic pain. In contrast, surgery is the treatment of choice if it is accompanied by compartment syndrome.[1,4] In this study, debridement and decompression were combined with rehabilitation to treat a decubitus ulcer that was accompanied by inflammation and edema, which caused sciatic neuropathy. + +Electrodiagnosis is a very useful method for diagnosing sciatic neuropathy. The following electrodiagnostic findings are characteristic of sciatic neuropathy. Motor nerve conduction studies demonstrate typically decreased amplitudes of compound muscle action potentials in the peroneal and tibial nerves. The F latency of the peroneal and tibial nerves is absent or prolonged, and sensory nerve conduction studies reveal decreased amplitudes of the superficial peroneal sensory and sural sensory nerve action potential.[1,2] In addition, a needle EMG, in addition to a nerve conduction study, is necessary to distinguish between sciatic neuropathy and peroneal mononeuropathy.[1] However, needle EMG was not performed in this case because of the high risk of infection from the soft tissue defects in the right buttock. + +MRI is the optimal medical imaging technique for evaluating pathologies of the sciatic nerve.[1] Depending on the severity of the injury, the nerve exhibits high signal intensity on T2-weighted MRI[9–11]; moreover, MRI can detect the extent of the lesion and identify inflammatory etiology.[1] In this case, we confirmed inflammatory soft tissue swelling using MRI, which was helpful in diagnosing sciatic neuropathy and identifying its cause. + +The currently known complications of pressure ulcers include cellulitis, osteomyelitis, bacteremia, endocarditis, meningitis, septic arthritis, abscesses, and squamous cell skin cancer, among others.[12] The present findings suggest that neuropathy can also occur as a complication of pressure ulcers. + +Table 1 + +Motor nerve conduction studies. + +Nerve Segment Latency, ms Ampulitude, mV CV, m/s F-wave, ms + +Right Peroneal Ankle - knee/EDB ∗NE + +ankle - knee/TA 3.2–7.2 ∗0.1 ∗30.2 + +Tibial Ankle - knee/AH ∗NE + +Left Peroneal Ankle - knee/EDB 4.8–11.2 ∗1.3 41.0 37.9 + +Tibial Ankle - knee/AH 3.1–9.5 20.9 51.5 39.1 + +The compound muscle action potentials (CMAPs) in the right peroneal [tibialis anterior (TA) recording] and left peroneal nerves exhibited a decreased amplitude. A decreased conduction velocity was found for the CMAPs in the right peroneal (TA recording) nerves. CMAPs were not evoked in the right peroneal [extensor digitorum brevis (EDB) recording] and tibial nerves. + +AH=abductor hallucis, CV=conduction velocity, EDB=extensor digitorum brevis, TA=tibialis anterior. + +3 + +Table 2 + +Sensory nerve conduction studies. + +Nerve Segment DL, ms Amplitude, mV + +Right Superficial peroneal Leg/ankle ∗4.4 ∗2.8 Distance: 14 cm + +Sural Midcalf/ankle ∗5.6 ∗5.0 Distance: 14 cm + +Left Superficial peroneal Leg/ankle 2.9 ∗6.8 Distance: 14 cm + +Sural Midcalf/ankle 2.9 ∗5.9 Distance: 14 cm + +| Right | Superficial peroneal | Leg/ankle | ∗ 4.4 | ∗ 2.8 | Distance: 14 cm | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------|-------------|---------|-----------------|-------------------| +| Sural | Midcalf/ankle | ∗ 5.6 | ∗ 5.0 | Distance: 14 cm | | +| Left | Superficial peroneal | Leg/ankle | 2.9 | ∗ 6.8 | Distance: 14 cm | +| Sural | Midcalf/ankle | 2.9 | ∗ 5.9 | Distance: 14 cm | | +| The sensory nerve action potentials (SNAPs) in the right superficial peroneal and sural nerves exhibited decreased peak latency values. The SNAPs in all examined nerves exhibited decreased amplitudes. DL=distal latency, NE=not evoked. ∗ Abnormal finding. | | | | | | + +![3_image_0.png](3_image_0.png) + +Khaykin et al[13] noted that patients with schizophrenia have an increased risk of adverse events including decubitus ulcer and it may be related to medication resulting in oversedation and decreased mobility. Gefen [14] noted that pressure ulcers in subdermal tissues under bony prominences very likely occur approximately between the first hour and 4 to 6 hours after sustained loading. In our case, the patient overdosed on the schizophrenia medication and was bedridden for 2 days in a coma. It is sufficient to cause the pressure ulcer. + +This patient ultimately should have undergone flap surgery to cover the soft tissue defects. However, in general, if a patient's risk factors remained, operative reconstruction must be delayed. Furthermore, if there is a high possibility of a surgery failing or ulcer recurrence, chronic wound care is more effective. In the case study presented here, because of uncontrolled moods or refusal of psychiatric treatment, we anticipated her poor cooperation and prolonged care postoperatively. Moreover, toxic hepatitis occurred during psychopharmacological treatment. The patient was susceptible to hepatic failure, and liver transplantation was a possibility. As a result, we could not perform flap surgery under general anesthesia; instead, the patient received conservative treatment while her general condition improved. Some examples of conservative treatment include debridement, frequent dressing changes, and irrigation of deep areas of the ulcer.[12,15,16] +There are several limitations to our study. First, follow-up nerve conduction studies after 6 months do not exist. Those data provide a better idea of the treatment method and the expected duration of treatment. Second, EMG is helpful to distinguish between sciatic neuropathy and peroneal mononeuropathy, but we could not perform needle EMG because of the high risk of infection. And third, further studies and long-term data are needed to confirm our finding due to small sample size. + +4. Conclusion Because pressure ulcers most commonly occur in patients with paraplegia or quadriplegia, physicians are generally less interested in sensory and motor disorders in the lower extremity caused by pressure ulcers. However, as this case reveals, the possibility of sciatic neuropathy occurring due to a pressure ulcer should be considered when treating pressure ulcer patients who do not have paraplegia or quadriplegia. Sciatic neuropathy should be prevented by aggressive pressure ulcer treatment and, if neurological symptoms occur, an accurate diagnosis of sciatic neuropathy should be made based on a physical examination, nerve conduction studies, and imaging studies to provide the appropriate treatment. + +Author contributions Conceptualization: Jae Ha Hwang. + +Data curation: Dong Wan Kim. Formal analysis: Dong Wan Kim. Resources: Jae Ha Hwang. Supervision: Kwang Seog Kim, Sam Yong Lee. + +Writing - original draft: Jae Ha Hwang. Writing - review & editing: Jae Ha Hwang. + +4 + +![4_image_0.png](4_image_0.png) + +[1] Distad BJ, Weiss MD. Clinical and electrodiagnostic features of sciatic neuropathies. Phys Med Rehab Clin 2013;24:107–20. + +[2] Ghate J, Ghugrare B, Patond KR, et al. The electrophysiological profiles of the footdrop cases: a retrospective study. J MGIMS 2009;14:36–9. + +[3] Altintas¸ A, Gündüz A, Kantarci F, et al. Sciatic neuropathy developed after injection during curettage. Agri 2016; 28:46–48. + +[4] Feinberg J, Sethi S. Sciatic neuropathy: case report and discussion of the literature on postoperative sciatic neuropathy and sciatic nerve tumors. + +HSS J 2006;2:181–7. + +[5] Plewnia C, Wallace C, Zochodne D. Traumatic sciatic neuropathy: a novel cause, local experience, and a review of the literature. J Trauma Acute Care Surg 1999;47:986–91. + +[6] Van Gompel JJ, Griessenauer CJ, Scheithauer BW, et al. Vascular malformations, rare causes of sciatic neuropathy: a case series. + +Neurosurgery 2010;67:1133–42. + +[7] Kwon R, Janis JE. Pressure sores. In: Neligan PC, (eds). Plastic surgery 3rd edn. Philadelphia: Elsevier Saunders; 2013;352–82. + +[8] Prakash BA, Devi MN, Sridevi NS, et al. Sciatic nerve division: a cadaver study in the Indian population and review of the literature. Singapore Med J 2010;51:721–3. + +[9] Chhabra A, Chalian M, Soldatos T, et al. 3-T high-resolution MR +neurography of sciatic neuropathy. J Am Roentgenol 2012;198:W357– +64. + +[10] Ergun T, Lakadamyali H. CT and MRI in the evaluation of extraspinal sciatica. Br J Radiol 2010;83:791–803. + +[11] Maravilla KR, Bowen BC. Imaging of the peripheral nervous system: +evaluation of peripheral neuropathy and plexopathy. J Am Neuroradiol 1998;19:1011–23. + +[12] Liu LQ, Moody J, Traynor M, et al. A systematic review of electrical stimulation for pressure ulcer prevention and treatment in people with spinal cord injuries. J Spinal Cord Med 2014;37:703–18. + +[13] Khaykin E, Ford DE, Pronovost PJ, et al. National estimates of adverse events during nonpsychiatric hospitalizations for persons with schizophrenia. Gen Hosp Psychiatry 2010;32:419–25. + +[14] Gefen A. How much time does it take to get a pressure ulcer? Integrated evidence from human, animal, and in vitro studies. Ostomy Wound Manage 2008;54:26–8. + +[15] Bhattacharya S, Mishra RK. Pressure ulcers: current understanding and newer modalities of treatment. Indian J Plast Surg 2015;48:4–16. + +[16] Pinkney L, Nixon J, Wilson L, et al. Why do patients develop severe pressure ulcers? A retrospective case study. BMJ Open 2014;4: e004303. \ No newline at end of file diff --git a/medical/md/PMC6211938.md b/medical/md/PMC6211938.md new file mode 100644 index 0000000000000000000000000000000000000000..0c3a1309c7eebddd5e40a7eb4ee9a23de329f43f --- /dev/null +++ b/medical/md/PMC6211938.md @@ -0,0 +1,56 @@ + +## Co-Authors: Osman Akdag, Md; Zekeriya Tosun, Md Affiliation: Selcuk University, Konya References: Affiliation: Chosun University, Gwangju + +requiring aesthetic and functional outcome.1,2 Y shape periosteal flap provides acceptable stability and location of the eye lid. Full-thickness defects of the medial canthal area by using Y shape periosteal flap, dacryocystorhinostomy and local flap is an alternative technique in medial canthal reconstruction, with acceptable functional and aesthetic outcomes. + +PURPOSE: Medial canthal area has complex anatomic and functional structures including medial canthal tendon and nasolacrimal duct.1,2 Reconstruction of medial canthal area must be include medial canthal tendon, nasolacrimal duct and soft tissue defect.1,2 In this study, we present functional reconstruction of full thickness medial canthal area with Y shape periosteum flap, dacryocystorhinostomy and local flap. + +1. Panizzo N, Colavitti G, Papa G, Ramella V, Tognetto D, Arnež ZM. Reconstruction after wide excision in medial canthal region: the extended bilobed glabellarpalpebral flap. J Plast Reconstr Aesthet Surg. 2015 Jan;68(1):131–2. + +2. Stewart CM, Norris JH. Reconstruction of extensive medial canthal defects using a single V-Y, island pedicle flap. Orbit. 2018 Jan 15:1–4. + +3. Brunetti B, Tenna S, Aveta A, Segreto F, Persichetti P. + +Angular artery perforator flap for reconstruction of nasal sidewall and medial canthal defects. Plast Reconstr Surg. 2012 Oct;130(4):627e-628e. + +MATERIAL AND METHODS: Between 2014 and 2017 years, 12 patients were included in this study. All of them had extensive medial canthal lession, so the surgical excision included the nasolacrimal duct and the medial canthal tendon. Simultaneous lesion, nasolacrimal duct and the medial canthal tendon was excised and reconstruction was performed for fullthickness defect. The Y shape periosteal flap was horizontally designed from remnant periosteum of nasal bone and frontal process of maxillary bone. + +Upper and lower part of the Y shape periosteal flap was sutured to the upper and lower eyelid. A silicone tube was used for dacryocystorhinostomy. Soft tissues defects were reconstructed with glabellar flap in 5 patient and angular artery perforator propellar flap3 in 7 patients, respectively. The silicon tube was removed at 3th week after surgery. All patients were followed up at least 13 months (13–55). Aesthetic result of surgery, function of nasolacrimal duct and eyelid laxity was evaluated with Satisfaction Evaluation Scale (SES: -1 = not satisfield, 0 = no change, 1 = moderate, 2 = good, 3 = very good), lacrimal duct irrigation test, lower lid distraction test for all patients at twelve months after surgery. + +Simultaneous Breast Augmentation and Rib Cartilage Harvest for Rhinoplasty Using a Single New Inframammary Fold Approach Presenter: Woo Young Choi, MD Co-Authors: Cheol Woo Park, MD, PhD; Myung Ju Lee, MD, PhD +PURPOSE: Rib cartilage provides sufficient volume and strength for stabilizing the cartilage framework of the nose; thus, rib cartilage rhinoplasty is considered ideal for secondary rhinoplasty.1–3 However, an inevitable scar remains on the chest wall when harvesting rib cartilage; thus, scaring is a major complication in rib cartilage rhinoplasty.4 For female patients, the incision is generally made along the inframammary fold (IMF) line.1, 2 Female Asian patients frequently complain of both a low and short nose and small breasts; therefore, simultaneous rib cartilage rhinoplasty and inframammary breast augmentation is increasing in popularity. + +Herein, we introduce a newly developed scar-minimizing method utilizing the predicted new IMF line to harvest rib cartilage and perform inframammary breast augmentation concurrently through one single incision. Moreover, a RESULT: 8 patients were male, 4 female. The mean age of them was 69,5 years. Defects size of the excised soft tissues ranged from 3.8cm2 to 6.9 cm2. There was no intraoperative or postoperative complication such as flap dehiscence or flap necrosis, lid lag deformity, ectropion and ptosis. Epiphora was observed in two patients. One of the was reoperated and problem was resolved, but other one didn't want to reoperation. Tension of the periosteal was good. + +There was no differentiation between glabellar and angular artery perforator flap. All patients except one was very satisfied with the result, other one good satisfied. + +CONCLUSION: Reconstructive surgery of medial canthal area is a challenging surgical prosedure because of subcutaneous flap (called an 'interspace wall flap') is used to isolate the surgical spaces, reducing complications. SURGICAL TECHNIQUE: The interspace wall flap is made between the two surgical spaces and is composed of Scarpa's fascia, deep fat, muscle fascia, and muscle. Because the interspace wall flap is strongly attached to the 5th and 6th rib cartilage and intercostal muscle, the two surgical spaces are completely isolated. + +RESULTS: This method has been evaluated in 31 patients (27 with primary breast augmentation, 4 with revisional breast augmentation) who underwent both operations simultaneously. At the current follow-up duration (ranging from 2 to 12 months), all patients are satisfied with the scar, and there have been no complications. + +CONCLUSION: The innovative method described above addresses two key issues in simultaneous augmentation rhinoplasty and breast augmentation. First, scaring is minimized by using the same incision for harvesting rib cartilage and performing breast augmentation. Second, an interspace wall flap is used to completely isolate the implant pocket space from the harvesting rib cartilage space, preventing potential complications associated with using the same incision line. + +Thus, this method appears to be promising for simultaneous augmentation rhinoplasty and inframammary breast augmentation. + +## References: + +1. Gunter JP, Cochran CS, Marin VP. Dorsal augmentation with autogenous rib cartilage. *Semin Plast Surg*. + +2008;22:74–89. + +2. Marin VP, Landecker A, Gunter JP. Harvesting rib cartilage grafts for secondary rhinoplasty. *Plast Reconstr* Surg. 2008;121:1442–1448. + +3.Toriumi DM, Pero CD. Asian Rhinoplasty. Clin Plast Surg. 2010;37:335–352. + +4. Wee JH, Park MH, Oh S, Jin HR. Complications associated with autologous rib cartilage use in rhinoplasty. + +Facial Plast Surg. 2015;17:49–55. + +Where Does It Hurt? The Distribution of Painful and Non-Painful Sensory Disturbances in Carpal Tunnel Syndrome Presenter: Mitchell Gauger, BS +Co-Authors: Johnny Nelson, MD; James Whaley, MD; Ephraim M. Zinberg, MD + +## Affiliation: Wayne State University School Of Medicine, Detroit, Mi + +PURPOSE: Hand diagrams were first studied in 1990 by Katz et. al. as a diagnostic tool, but the use of the hand diagram as an epidemiologic investigational tool has not been thoroughly explored. The compiling and tracking of patient perceived data can also be used to establish clinically relevant normative data. We explore a novel method of patient reported symptom mapping to establish and compare the normative distribution of painful and non-painful symptomatology in patients diagnosed with median neuropathy. + +METHODS: New patients were given a visual questionnaire consisting of a photo of human hand and wrist and a superimposed numbered grid. Patients were instructed to map their symptoms, painful symptoms noted with an "X" and non-painful sensory disturbance (i.e. numbness or tingling) noted with an "O." Patients were instructed not to put marks where they lacked any symptoms. Grid cells could contain both an "X" and an "O." All patients were then evaluated by a single, hand fellowship trained surgeon, who was blinded to questionnaire results, and a visit diagnosis was made. Questionnaires of patients with an isolated clinical diagnosis of carpal tunnel syndrome were compiled for inclusion in the study. Those with any conflicting or confounding diagnoses (e.g. symptomatic osteoarthritis, tendinosis, recent trauma) in the same limb were excluded. The frequency with which patients marked either painful or non-painful sensory disturbance was tallied and recorded for five discrete anatomic areas: the volar forearm (zone 1), carpal tunnel (zone 2), distal palmar (zone 3), radial three digits (zone 4), and ulnar two digits (zone 5). A frequency score was calculated by dividing the total number of marks in that symptom area across all patients by the total number of marks made by all patients. Photo-editing software was then used to create a visual frequency distribution for both painful sensation and non-painful sensory disturbance. Higher frequencies of marks by the study population were depicted by darker shades for each cell. RESULTS: A total of 34 hands in 26 patients were included in the study. Painful symptoms were clearly centered over the carpal tunnel with a frequency score of 46%. Painful symptoms in the digits were reported less frequently with scores of 23% and 0.3% for the radial and ulnar digits, respectively. Non-painful sensory Posters \ No newline at end of file diff --git a/medical/md/PMC6556065.md b/medical/md/PMC6556065.md new file mode 100644 index 0000000000000000000000000000000000000000..040eac0424a0e0e4f70503ab1259ae8651dfe690 --- /dev/null +++ b/medical/md/PMC6556065.md @@ -0,0 +1,209 @@ +e-ISSN 1643-3750 +© Med Sci Monit, 2019; 25: 3839-3845 DOI: 10.12659/MSM.916472 Received: 2019.03.25 + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +Accepted: 2019.05.01 Published: 2019.05.23 + +# The Effect Of Winter Temperature On Patients With Ischemic Stroke + +| ACDEF 1 | Xiuhui Chen | +|---------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| ACEF 1 | Wenjin Shang | +| ABE 2 | Xinyi Huang | +| BE 2 | Liming Shu | +| BE 1 | Su Xiao | +| E 2 | Qiuhong Jiang | +| AEG 1 | Hua Hong | +| Authors' Contribution: Study Design A Data Collection B Statistical Analysis C Data Interpretation D | | +| Manuscript Preparation E Literature Search F Funds Collection G | 1 Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, P.R. China 2 Department of Neurology, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong, P.R. China | +| Corresponding Author: | Hua Hong, e-mail: hhsums@126.com | +| Source of support: | This study was supported by grants from the Guangzhou Science, Technology and Innovation Commission (2014Y2-00502), Guangdong Provincial Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases (2017B030314103); The Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases (2015B050501003); Guangdong Provincial Engineering Center for Major Neurological Disease Treatment; Guangdong Provincial Translational Medicine Innovation Platform for Diagnosis and Treatment of Major Neurological Disease | +| Background: | The incidence of ischemic stroke increases in winter. This study aimed to explore the effect of winter temperatures on the risk factors, etiology, coagulation, and degree of neurological impairment in patients with ischemic stroke using temperature and rainfall data from the Guangzhou Meteorological Bureau during the winter months of December, January, and February. | +| Material/Methods: | We divided 112 patients with ischemic stroke into low-temperature and non-low-temperature groups. The lowtemperature group experienced an average daily winter temperature of <13°C for five consecutive days within the 14 days before hospital admission and an average temperature of <13°C on admission. The non-low-temperature group experienced an average daily temperature of >13°C in the 14 days before hospital admission and an average daily temperature of >13°C on admission. Neurological deficits were scored and monitored using the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRS) for disability in stroke. Blood pressure and coagulation indices of prothrombin time (PT) and thromboplastin time (TT) were recorded. | +| Results: | Compared with the non-low-temperature group, the low-temperature group showed a significantly increased proportion of patients with hypertension and large artery atherosclerotic stroke, more prolonged PT, and higher NIHSS scores. However, TT was reduced in the low-temperature group (P<0.05). | +| Conclusions: | When the average winter temperature was <13°C, the risk factors, etiology, coagulation factors, and degree of neurological impairment of patients with ischemic stroke were significantly different from patients with ischemic stroke during warmer temperature. | +| MeSH Keywords: | Clinical Outcome - Prothrombin Time - Stroke - Temperature - Weather | +| Full-text PDF: | https://www.medscimonit.com/abstract/index/idArt/916472 3007 3 - 37 | + +![0_image_2.png](0_image_2.png) + +## Background + +Worldwide, hemorrhagic and ischemic stroke results in disability and high mortality. Ischemic stroke accounts for between 60–80% of all cases of stroke [1]. According to a World Health Organization (WHO) report, on average, one person in the United States dies of a stroke every four minutes [2]. In China, stroke has become the leading cause of death among urban residents, and the annual incidence of ischemic stroke is increasing by 8.7% [3]. + +Currently, risk factors for ischemic stroke include increased age, hypertension, diabetes, hyperlipidemia, coronary heart disease, atrial fibrillation, valvular heart disease, obesity, and lack of physical exercise [4]. Hypertension, defined as a blood pressure >140/90 mmHg, is the most important risk factor for ischemic stroke [5]. Epidemiological studies have shown that between 70–80% of patients with ischemic stroke have hypertension [6]. + +Clinically, prothrombin time (PT) and thromboplastin time (TT) +indicate the function of the coagulation and fibrinolytic systems. The PT reflects the activity of exogenous coagulation factors I, II, V, VII, and X, and is the preferred indicator for detecting the effect of oral anticoagulants [7]. A decrease in the PT can be associated with a congenital increase in coagulation factor V, hypercoagulable states, thrombotic disease, and the use of oral contraceptives [8]. The TT is the time required for blood coagulation after the addition of standardized thrombin to the plasma and is mainly used to reflect the function of the fibrinolytic system. The TT is used as a screening test to monitor the function of the fibrinolytic system, and a reduced TT is common in patients with a hypercoagulable state [9]. The incidence of ischemic stroke is significantly increased in winter, especially when the temperature drops sharply in a cold period [10,11]. Currently, the pathogenesis of ischemic stroke remains unclear, but one of the causes may involve a cold-induced increase in blood pressure, hypercoagulation, and fibrinolysis. Currently, studies on the relationship between ischemic stroke and temperature have mainly been epidemiological studies and have not included studies on the effect of temperature. + +Therefore, this study aimed to explore the effect of winter temperatures on the risk factors, etiology, coagulation, and degree of neurological impairment in patients with ischemic stroke at the First Affiliated Hospital of Sun Yat-sen University using temperature and rainfall data from the Guangzhou Meteorological Bureau during the winter months of December, January, and February. + +## Material And Methods Study Inclusion And Exclusion Criteria And Study Groups + +The study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. All patients included in the study provided consent to participate. The inclusion criteria included patients with a confirmed diagnosis of ischemic stroke in accordance with the Fourth National Conference on Cerebrovascular Diseases, 1995 [12], with focal neurological signs confirmed using cranial computed tomography (CT) and/or magnetic resonance imaging (MRI). Patients were admitted to the hospital for no more than 14 days and completed all the required clinical examinations. Neurological deficits were scored and monitored using the National Institutes of Health Stroke Scale (NIHSS) +and the modified Rankin Scale (mRS) for disability in stroke. Patients were excluded from the study if they had a NIHSS +of £1, loss of consciousness, aphasia, or cognitive impairment, a previous history of stroke or transient cerebral ischemic attack (TIA), cerebral arteritis, aneurysm, or vascular malformation, acute angina or myocardial infarction, malignant tumor, chronic renal insufficiency, liver cirrhosis, blood disease, rheumatic disease, or other major systems diseases. Patients who were unwilling to cooperate with the study were excluded. + +There were 112 patients with ischemic stroke who were divided into the low-temperature group and the non-low-temperature group. The low-temperature group experienced an average daily winter temperature of <13°C for five consecutive days within the 14 days before hospital admission and an average temperature of <13°C on admission. The non-low-temperature group experienced an average daily temperature of +>13°C in the 14 days before hospital admission and an average daily temperature of >13°C on admission. + +## Meteorological Data + +Guangzhou Meteorological Bureau provides specific meteorological data for the nine years from December 1, 2006, to February 28, 2015, including the average daily temperature, maximum daily temperature, minimum daily temperature, the difference in daily temperature, precipitation at 20.00 hours, average pressure, average relative humidity, and the number of hours of sunshine. The extent to which the temperature decreased or increased from the previous day and two days was determined according to the differences between the average daily temperature, maximum daily temperature, and minimum daily temperature. Guangzhou is situated in a subtropical coastal area, with the Tropic of Cancer running through its central and southern zones. Based on analysis of the criteria for dividing seasons [13] with winter from December to March and the traditional Chinese winter of the three months of December, January, and February, this study assessed patients during the winter period of the months of December, January, and February. + +Previous studies had shown that the incidence of ischemic stroke increased significantly in autumn and winter when the average daily temperature was <12.79°C [14]. A three-year study conducted between 1998 and 2000 that included 735 patients with ischemic stroke [15] showed that the incidence of stroke in winter and spring was significantly higher than in summer and autumn when the temperature was below the annual average of 12.8°C. Therefore, for this study, 13°C was chosen as the cutoff for defining low temperature, an average daily temperature <13°C was defined as low temperature and an average daily temperature >13°C was defined as a non-low-temperature. + +## Subtypes Of Ischemic Stroke According To The Trial Of Org 10172 In Acute Stroke Treatment (Toast) + +According to the Trial of Org 10172 in Acute Stroke Treatment +(TOAST) classification [16], the patients were classified into five types of ischemic stroke: large-artery atherosclerosis (LAA); cardioembolism (CE); small-vessel occlusion (SVO); stroke of other determined etiology (SOD); stroke of undetermined etiology (SUD). Patients with CE, SAO, SOD, SUD subtypes were combined into a non-large artery atherosclerosis (NLAA) group. + +## Patient Assessment Methods + +The demographic and clinical history of the patients were obtained by reviewing the medical records. Data on the average daily temperature at the onset of ischemic stroke were collected from the meteorological data. The degree of neurological impairment was assessed using the NIHSS, and the modified Rankin Scale (mRS) for disability in stroke were used. The blood pressure of the patients was recorded within eight hours of hospital admission and measurement of coagulation factors thromboplastin time (TT) and prothrombin time (PT) were determined within 24 hours of hospital admission. + +## Statistical Analysis + +Data were analyzed using SPSS version 22.0 software. + +Meteorological data, the NIHSS, TT, and PT of each group did not conform to normal distribution. The median (M) and quartiles (P25, P75) were used to describe the risk factors and the TOAST classification. Meteorological information, NIHSS, TT, PT, +and systolic and diastolic blood pressures were analyzed using the Mann–Whitney U test because these did not conform to normal distribution. Risk factors and TOAST classification were tested using the chi-squared (c2) test. A P-value <0.05 was considered to be statistically significant. + +## Results Characteristics Of The Patients + +Among the 112 patients with ischemic stroke who were admitted to the First Affiliated Hospital of Sun Yat-sen University between January 1, 2007, and February 28, 2015, 72 patients were men, and 40 were women (mean age, 63.59±11.72 years). They were divided into a low-temperature group (85 cases from 67 days) and a non-low-temperature group (27 cases from 23 days). The neurologists of our hospital scored the degree of neurological deficit within eight hours of admission according to the National Institutes of Health Stroke Scale (NIHSS). No significant differences were found in gender or age between the two groups (P>0.05). + +## Clinical And Demographic Findings + +There were no significant differences in the medical history between the patients in the two study groups for hyperlipidemia, diabetes, coronary heart disease, and atrial fibrillation, and no difference in smoking or drinking behavior between the patients, indicating that the two groups of patients in this study were comparable (Table 1). Comparing the daily weather experienced by the different temperature groups, there was no statistical differences in daily temperature, temperature drop from the previous day, temperature drop from the previous two days, temperature rise from the previous day, temperature rise from the previous two days, the precipitation at 20.00 hours, the average air pressure, the average relative humidity, or the number of hours of sunshine. The results are shown in Table 2. + +## Risk Factors, Etiological Types, Coagulation Factors, And Degree Of Neurological Impairment + +According to the Trial of Org 10172 in Acute Stroke Treatment +(TOAST) classification [16], the patients were classified into five types of ischemic stroke: large-artery atherosclerosis (LAA); cardioembolism(CE); small-vessel occlusion (SVO); stroke of other determined etiology (SOD); stroke of undetermined etiology (SUD). The proportion of patients with hypertension in the low-temperature group was 65 (76.5%), which was greater than that in the non-low-temperature group, which was 14 (51.9%). + +The systolic blood pressure of 155 mmHg (140, 169) mmHg in the low-temperature group was higher than in the nonlow-temperature group of 135 mmHg (120, 150) mmHg. The diastolic blood pressure of 89 mmHg (80, 96) mmHg in the low-temperature group was higher than in the non-low-temperature group of 80 mmHg (72, 82) mmHg. The proportion of cases of LAA in the low-temperature group was higher than that in the non-low-temperature group (P<0.05), and the proportion of modified Rankin Scale (mRS) scores for disability in stroke of 0–2 in the low-temperature group was 22 (25.9) compared with 22 (81.5) for the non-low-temperature group +(P<0.05). The thromboplastin time (TT) in the low-temperature group was significantly lower than that in the non-low-temperature group (P<0.05). Compared with the non-low-temperature group, the prothrombin time (PT), NIHSS score, systolic Table 1. Comparison of the clinical information of the patients with ischemic stroke in the low-temperature group and the nonlow-temperature group. + +| Parameters | Low-temperature group (n=85) | Non-low-temperature group (n=27) | P-value | | | +|--------------------------------------------------------------------------------------------------------------------------------------|--------------------------------|------------------------------------|-----------|--------|------| +| Risk factor (n, %) Hypertension | 65 | (76.5) | 14 | (51.9) | 0.02 | +| Diabetes | 29 | (34.1) | 8 | (29.6) | 0.67 | +| Hyperlipidemia | 25 | (29.4) | 7 | (25.9) | 0.73 | +| Coronary heart disease | 7 | (8.2) | 1 | (3.7) | 0.43 | +| Atrial fibrillation | 2 | (2.4) | 2 | (7.4) | 0.22 | +| Smoking | 29 | (34.1) | 14 | (51.9) | 0.10 | +| Drinking | 20 | (23.5) | 6 | (22.2) | 0.85 | +| TOAST classification (n, %) | 0.02 | | | | | +| LAA | 62 | (72.9) | 13 | (48.1) | | +| NLAA | 23 | (27.1) | 14 | (51.9) | | +| mRS scores (n, %) | 0.01 | | | | | +| 0–2 | 22 | (25.9) | 22 | (81.5) | | +| 3–5 | 63 | (74.1) | 5 | (18.5) | | +| Data are expressed as number (%). LAA - large artery atherosclerosis; NLAA - non-large artery atherosclerosis; mRS - modified Rankin | | | | | | + +Data are expressed as number (%). LAA - large artery atherosclerosis; NLAA - non-large artery atherosclerosis; mRS - modified Rankin Scale; TOAST - Trial of Org 10172 in Acute Stroke Treatment classification [16]. + +Table 2. Comparison of the weather information for the low-temperature group and the non-low-temperature group of patients with ischemic stroke. + +| Parameters | Low-temperature group | Non-low-temperature group (n=27) | P-value | | | +|-------------------------------------------------------------------------------------------------------------------------------|-------------------------|-------------------------------------|-----------|----------------|-------| +| (n=85) | | | | | | +| Mean daily temperature on admission (°C) (P25, P75) | 11.00 | (9.20, 12.10) | 19.00 | (16.70, 20.60) | <0.01 | +| Mean daily temperature difference (°C) (P25, P75) | 8.40 | (5.50, 12.55) | 7.10 | (4.90, 10.40) | 0.23 | +| Mean temperature drop difference with the previous day (°C) (P25, P75) | 8.70 | (5.20, 11.50) | 8.70 | (4.70, 9.90) | 0.25 | +| Mean temperature drop difference with the previous two days (°C) (P25, P75) | 8.60 | (4.90, 10.90) | 8.20 | (5.50, 10.00) | 0.73 | +| Mean temperature rise difference with the previous day (°C) (P25, P75) | 8.80 | (5.40, 12.75) | 7.20 | (5.50, 8.60) | 0.09 | +| Mean temperature rise difference with the previous two days (°C) (P25, P75) | 8.40 | (5.50, 12.55) | 7.70 | (6.40, 9.20) | 0.21 | +| Mean rainfall at 20: 00 hrs (0.1 mm) (P25, P75) | 0 | (0, 16) | 0 | (0, 5) | 0.82 | +| Mean pressure (0.1 hPa) (P25, P75) | 10146 | (10121, 10169) | 10123 | (10103, 10161) | 0.07 | +| Mean relative humidity (%) (P25, P75) | 68 | (59, 80) | 69 | (60, 82) | 0.59 | +| Mean hours of sunshine (0.1 h) (P25, P75) | 29.0 | (0.0, 81.5) | 64.0 | (9.0, 86.0) | 0.07 | +| Data are expressed as median (M) and quartiles (P25, P75). Temperature drop difference with the previous day=the previous day | | | | | | + +Data are expressed as median (M) and quartiles (P25, P75). Temperature drop difference with the previous day=the previous day maximum temperature–present day minimum temperature. Temperature drop difference with the previous two days=the previous two day's maximum temperature–present day's minimum temperature. Temperature rise difference with the previous day=present day's maximum temperature–previous day's minimum temperature. Temperature rise difference with the previous two days=present day's maximum temperature–previous two day's minimum temperature. + +| Parameters | Low-temperature group | Non-low-temperature group (n=27) | P-value | | | +|-----------------------------------------------------------------------------------------------------------------------------------------|-------------------------|-------------------------------------|-----------|--------------|-------| +| (n=85) | | | | | | +| Median systolic blood pressure (mmHg) (P25, P75) | 155 | (140, 169) | 135 | (120, 150) | <0.01 | +| Median diastolic blood pressure (mmHg) (P25, P75) | 89 | (80, 96) | 80 | (70, 82) | <0.01 | +| Median NIHSS score (P25, P75) | 6.0 | (4.0, 10.5) | 5.0 | (2.5, 9.0) | 0.04 | +| Median TT (s) (P25, P75) | 17.3 | (16.1, 18.3) | 18.7 | (17.6, 19.5) | <0.01 | +| Median PT (g/L) (P25, P75) | 12.0 | (11.4, 12.6) | 11.3 | (10.5, 12.1) | 0.01 | +| Data are expressed as median (M) and quartile (P25, P75); NIHSS - National Institutes of Health Stroke Scale; TT - thromboplastin time; | | | | | | + +Data are expressed as median (M) and quartile (P25, P75); NIHSS - National Institutes of Health Stroke Scale; TT - thromboplastin time; + +PT - prothrombin time. + +Table 3. Comparison of systolic blood pressure (mmHg), diastolic blood pressure (mmHg), National Institutes of Health Stroke Scale +(NIHSS), thromboplastin time (TT), and prothrombin time (PT) for the low-temperature group and the non-low-temperature group of patients with ischemic stroke. + +blood pressure, and diastolic blood pressure in the low-temperature group were significantly higher (P<0.05). These results are shown in Tables 1 and 3. + +thrombosis [23]. Chronic hypertension is also a cause of cerebral artery atherosclerosis leading to ischemic stroke [24]. + +## Discussion + +Ischemic stroke, also known as cerebral infarction, is mainly caused by insufficient blood and oxygen supply to the brain, and the resulting damage to brain tissues results in structural and functional disorders as well as in neurological impairment. + +The incidence and mortality rates of ischemic stroke increase significantly in cold winter [17,18]. A three-year study conducted in the Biya River, Poland, included 1,173 patients with ischemic stroke and showed that the incidence increased significantly in December and was lowest in August and September [17]. + +In 2007, a review of the incidence of ischemic stroke and meteorological data in Shenzhen, China from 2003 to 2005 showed that cold was the main meteorological association in Shenzhen residents, and with the decrease in temperature, the incidence of ischemic stroke increased, especially among women and patients <65 years [20]. Ischemic stroke has been previously reported to be associated with low temperatures, and every 1°C +fall in temperature below the average daily temperature was associated with a 3.9% increase in incidence [21]. + +The findings of the present study showed that, when compared with the non-low-temperature group, the low-temperature group showed significantly more severe neurological impairment (P<0.05). Also, low temperatures increased the incidence of stroke. However, the pathogenesis of the relationship between low temperature and ischemic stroke remains unclear, but it may be due to vasoconstriction and increased blood pressure vascular congestion and edema that results from the cold [22]. Long-term hypertension can lead to reduced blood coagulation and fibrinolysis and resulting in intravascular There is evidence that low temperature can lead to hypertension. A two-year observational study that included 57,375 people showed that blood pressure in January was significantly higher than that in July (P<0.05), and the average blood pressure increased by 6.9/2.9 mmHg with the decrease of ambient temperature by 10°C [25]. A study of blood pressure in 213 volunteers for three consecutive years showed that systolic blood pressure decreased by 0.4 mmHg and the diastolic blood pressure decreased by 0.28 mmHg for each temperature increase of 1°C when the ambient temperature was >10°C [26]. An animal study showed that blood pressure, heart rate, and blood viscosity of spontaneous hypertensive rats (SHR) increased after cold stimulation (P<0.05) [27]. The findings of the present study showed that the blood pressure of the low-temperature group was higher than that of the non-low-temperature group (P<0.05). [28] There are several possible explanations for these findings. Cold can lead to the upregulation of vasoconstriction-associated molecules, including adrenaline, noradrenaline, and renin, which induce peripheral vasoconstriction, increase peripheral vascular resistance, and increase blood pressure [28]. Also, cold can lead to decreased production of vasodilator molecules, including nitric oxide (NO), which results in increased blood pressure [28]. + +In 1993, the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification [16], identified five types of ischemic stroke, which were used to group the patients in this study as large-artery atherosclerosis (LAA). The TOAST subgroups included cardioembolism (CE); small-vessel occlusion (SVO); +stroke of other determined etiology (SOD); stroke of undetermined etiology (SUD) [16]. The findings from a four-year retrospective study involving 442 patients with ischemic stroke with an average follow-up time of 3.2 years showed that the recurrence rate of stroke at 30 days was significantly different in the LAA group (P<0.001), who had the highest recurrence rate of 18.5% (95% CI, 9.4–27.5%) [29]. A study conducted between 2011 to 2014 showed that the proportion of LAA in patients with ischemic stroke was significantly greater than in other types of stroke (P<0.05), and the incidence of hypertension in patients with LAA was higher than that in other types of stroke [30]. Our results showed that the proportion of patients with LAA in the low-temperature group was significantly higher than that in the non-low-temperature group +(P<0.05), which was consistent with previously published findings. It is possible that low temperature can induce the increase of red blood cells and platelets, promote the increase of blood viscosity, platelet adherence and activation, leading to thrombosis [31]. + +The prothrombin time (PT) not only reflects the activity of exogenous coagulation factors I, II, V, VII, and X but can also reflect the level and activity of prothrombin in the plasma [32,33]. PT is a screening test used to examine the function of the exogenous coagulation system and is also an important detection index for clinical anticoagulation therapy. Ischemic stroke is a thrombotic disease, and PT can be expected to be reduced at low temperatures. However, the increase in PT in patients with ischemic stroke may be explained by several mechanisms. + +First, during thrombosis, a large number of exogenous coagulation factors that included factors I, II, V, VII, and X are consumed and the rate of synthesis of blood coagulation factors is lower than the consumption in the liver. The plasma of patients with exogenous coagulation factors is therefore decreased, resulting in a prolonged PT. Second, there is a fibrinolytic system in the body that opposes the coagulation system, and after the formation of thrombus, the fibrinolytic system plays a protective role. + +Platelets can accumulate at sites of vascular disease, and the activity of the fibrinolytic system releases thrombin releasing into the blood that enhances coagulation activity and promotes thrombosis. In a previously published study of 120 patients with ischemic stroke that compared the blood coagulation indicators with 30 healthy people, the results showed that among the patients with ischemic stroke, the PT was significantly higher than that in the normal control group [34]. These previous findings were consistent with the results of the present study. + +The thromboplastin time (TT) is a screening test that measures the time to convert fibrinogen to fibrin. The TT of patients with cerebral infarction was previously shown to be significantly reduced, and levels of fibrinogen were significantly increased (P<0.01) [35]. In the present study, the TT in the lowtemperature group was significantly lower than that in the non-low-temperature group (P<0.05), and the degree of neurological impairment was significantly greater (P<0.05), indicating that TT could be used as an additional clinical index in ischemic stroke. Timely measurement of coagulation factors in patients with ischemic stroke is helpful in the early identification of a hypercoagulable state, to monitor active treatment, and may be a prognostic marker of disease severity. + +Low temperatures can affect the coagulation and fibrinolysis systems and promote the formation of a hypercoagulable state. A previously published animal study [36] showed that low temperature could significantly reduce the TT in mice and increase platelet aggregation (P<0.05). Other animal experiments [37] have shown that low temperature can alter the regulation of the balance between coagulation and fibrinolysis. + +A change in the levels of the thrombin-antithrombin complex and the balance between fibrinolytic and antifibrinolytic factors may promote the occurrence of stroke. The results of this study showed that the TT in the low-temperature group was significantly reduced compared with the non-low-temperature group (P<0.05), which was consistent with the findings from previous studies. + +This study had several limitations. First, the patient data in this study were obtained from a single center, the First Affiliated Hospital of Sun Yat-sen University, which may have introduced study bias. Second, this study measured coagulation factors in the patients on hospital admission but did not follow-up the changes in coagulation through the course of the disease. Therefore, the findings from this study require validation with large-scale multicenter controlled studies. + +## Conclusions + +The aim of this study was to investigate the effect of winter temperatures on the risk factors, etiology, coagulation, and degree of neurological impairment in patients with ischemic stroke using temperature and rainfall data during the winter months of December, January, and February. The findings showed that when the average winter temperature was <13°C, the risk factors, etiology, coagulation factors, and degree of neurological impairment of patients with ischemic stroke were significantly different from patients with ischemic stroke during warmer temperature. + +Temperature is an important factor to consider in the management of patients with ischemic stroke, and low temperature should be considered as a risk factor associated with reduced prognosis. + +## Acknowledgments + +The authors thank the patients and their families for providing permission to participate in this study. + +## Conflict Of Interest + +None. + +3844Indexed in: [Current Contents/Clinical Medicine] [SCI Expanded] [ISI Alerting System] +[ISI Journals Master List] [Index Medicus/MEDLINE] [EMBASE/Excerpta Medica] [Chemical Abstracts/CAS] + +## References: + +1. Ahn S, Riccio A, Ginty DD: Spatial consideration for stimulus-dependent transcription in neurons. Annu Rev Physiol, 2000; 62: 803–23 2. Jeong HC, Ahn Y, Park KH et al: Effect of statin treatment in patients with acute myocardial infarction and left ventricular systolic dysfunction according to the level of high-sensitivity C-reactive protein. Int Heart J, 2014; 55: +106–12 3. Zhao D, Liu J, Wang W et al: Epidemiological transition of stroke in China: +Twenty-one-year observational study from the Sino-MONICA-Beijing Project. + +Stroke, 2008; 39: 1668–74 4. Circular of the General Office of the Ministry of Health on the Issue of Measures for the Management of Pilot Programs of Screening and Intervention for High-Risk Stroke Populations Trial, 2012 5. Weizhi W: Neurology of cerebrovascular diseases. 4th ed. Beijing: Publication of People's Health, 2001 6. Chen J, Liu J, Ji Z: [Recent progress in multiple risk factors of stroke.] Clinical Rehabilitation in China, 2004; 8: 5330–31 [in Chinese] +7. Xuefeng W: [Clinical application and evaluation of common coagulation tests.] J Clin Hematol, 2014; 2014: 550–54 [in Chinese] +8. Wang H: Thrombosis and hemostasis testing techniques. 1st edition. + +Shanghai: Shanghai Science and Technology Publishing House, 1996 9. Shen Y, Zhang L, Li F et al: Changes in coagulation function and influencing factors in patients with acute cerebral infarction. Chin J Coal Indus Med, 2016; 19: 956–59 10. Jimenez-Conde J, Ois A, Gomis M et al: Weather as a trigger of stroke. Daily meteorological factors and incidence of stroke subtypes. Cerebrovasc Dis, 2008; 26: 348–54 11. Myint PK, Vowler SL, Woodhouse PR et al: Winter excess in hospital admissions, in-patient mortality and length of acute hospital stay in stroke: A hospital database study over six seasonal years in Norfolk, UK. Neuroepidemiology, 2007; 28(2): 79–85 12. Fourth National Conference on Cerebrovascular Diseases of the Chinese Medical Association. Diagnostic Essentials of Cerebral Blood. Chin J Neurol, 1996; 29: 379 13. Jianping H: [It is recommended to clarify the seasonal concept in psychiatric clinical research.] Mental Health in Sichuan, 1995; (3) [in Chinese] +14. Jimenez-Conde J, Ois A, Gomis M et al. Weather as a trigger of stroke. Daily meteorological factors and incidence of stroke subtypes. Cerebrovasc Dis, 2008; 26: 348–54 15. Hong YC, Rha JH, Lee JT et al: Ischemic stroke associated with decrease in temperature. Epidemiology, 2003; 14: 473–78 16. Adams HP Jr., Bendixen BH, Kappelle LJ et al: Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org10172 in Acute Stroke Treatment. Stroke, 1993; 24: +35–41 17. Suzuki K, Kutsuzawa T, Takita K et al: Clinico-epidemiologic study of stroke in Akita, Japan. Stroke, 1987; 18: 402–6 18. Shinkawa A, Ueda K, Hasuo Y et al: Seasonal variation in stroke incidence in Hisayama, Japan. Stroke, 1990; 21: 1262–67 19. Klimaszewska K, Kulak W, Jankowiak B et al: Seasonal variation in ischaemic stroke frequency in Podlaskie Province by season. Adv Med Sci, 2007; 52(Suppl. 1): 112–14 20. Quan CJ, Jianping L, Li ZR et al: [The relationship between stroke incidence and air temperature in Shenzhen.] Chin Public Health, 2007; 23: 970–71 21. Magalhães R, Silva MC, Correia M et al: Are stroke occurrence and outcome related to weather parameters? Results from a population-based study in northern Portugal. Cerebrovasc Dis, 2011; 32: 542–51 22. Khan U, Porteous L, Hassan A et al: Risk factor profile of cerebral small vessel disease and its subtypes. J Neurol Neurosurg Psychiatry, 2007; 7: 702–6 23. Zhang Y, Huang R, Su Z et al: Ultrastructural observation of cerebral microvessels in stroke-prone renovascular hypertensive rats. Chin J Neurol Mental Dis. 1992; 18: 9 24. Zeng J, Huang R: Experimental pathological study of hypertensive arteriosclerotic mixed stroke. Chin J Neuropsychol Dis, 1992; 18: 340–42 25. Su D, Du H, Zhang X et al: Season and outdoor temperature in relation to detection and control of hypertension in a large rural Chinese population. Int J Epidemiol, 2014; 43: 1835–45 26. Hozawa A, Kuriyama S, Shimazu et al: Seasonal variation in home blood pressure measurements and relation to outside temperature in Japan. Clin Exp Hypertens, 2011; 33: 153–58 27. Luo B, Zhang S, Ma S et al: Artificial cold air increases the cardiovascular risks in spontaneously hypertensive rats. Int J Environ Res Public Health, 2012; 9: 3197–208 28. Sun Z, Cade R, Morales C: Role of central angiotensin II receptors in coldinduced hypertension. Am J Hypertens, 2002; 15: 85–92 29. Petty GW, Brown RD Jr., Whisnant JP et al: Ischemic stroke subtypes: A population-based study of functional outcome, survival, and recurrence. Stroke, 2000; 31(5): 1062–68 30. Liu Y, Wang Y, Li WA et al: Validation of the Essen Stroke Risk Score in different subtypes of ischemic stroke. Neurol Res, 2017; 39(6): 504–8 31. Keatinge WR, Coleshaw SR, Cotter F et al: Increases in platelet and red cell counts, blood viscosity, and arterial pressure during mild surface cooling. Br Med J (Clin Res Ed), 1984; 289: 1405 32. Neofotistos D, Oropeza M, Ts'ao CH: Stability of plasma for add-on PT and APTT tests. Am J Clin Pathol, 1998; 109(6): 758–62 33. Gottfried EL, Adachi MM: Prothrombin time and activated partial thromboplastin time can be performed on the first tube. Am J Clin Pathol, 1977; 107: 681–83 34. Chen J, Yuan H, Huang H: Clinical significance of detection of coagulation index in cerebral thrombotic diseases. J Math Phys Med, 2001; 14: 403–4 35. Hao Y, Wu Q, Wang Q: Analysis of blood coagulation test results in patients with cerebral infarction. Chin Med J, 2009; 6: 113–14 36. Zhang F: [Effect of dilong huoluo capsule on platelet aggregation rate and coagulation factor in rats with blood stasis.] China Health Nutrition, 2012; (4): 28 [in Chinese] +37. Lin J, Shi X, Liao S et al: Changes in the function of coagulation and fibrinolysis before stroke in rats induced by artificial cold wave. Thromb Haemost, 2000; 14: 108–12 \ No newline at end of file diff --git a/medical/md/PMC6761452.md b/medical/md/PMC6761452.md new file mode 100644 index 0000000000000000000000000000000000000000..94a46f62dd9b2f03d8c3a9e4cb57f5ab91ec37a5 --- /dev/null +++ b/medical/md/PMC6761452.md @@ -0,0 +1,14 @@ +Eosinophilia has only rarely been a feature of the presentation of Buerger's disease. Its presence does not imply a systemic vasculitis or a related syndrome. Although there is limited evidence for the use of steroids in this condition, inflammatory features such as eosinophilia could strengthen the rationale for using steroids in certain cases by halting the ongoing immune driven inflammatory cascade. The presence of eosinophiliamight suggest alternative pathogenicmechanisms in certain cases ofBuerger's. + +Key learning points: To recognise eosinophilia as a marker in atypical Buerger's disease presentation. Being a disease of exclusion we have to rule out all other possibilities including some rare ones as was done in this caseby seeking advicefrominfectiousdiseaseandhaematology team. Conflicts of interest: The authors have declared no conflicts of interest. + +## 44. Buerger'S Disease With Severe Eosinophilia And Acute Critical Ischemia + +Mansoor ul haq Keen1, Shouvik Dass1, and Aamir Aslam1 1Rheumatology Department, Leeds Teaching Hospitals, Leeds, United Kingdom Introduction: Buerger's disease usually presents with normal blood tests. We present a case with significant eosinophilia, a phenomenon rarely reported. Eosinophilia with acute onset Raynaud's and critical ischemia canbeatypicalearlymanifestationofBuerger'sdisease(thromboangiitisobliterans). + +Case description: A case report of a 24-year-old male, smoker, with no other history, presented acutely, complaining of cool, painful hands with tricolour changes with worsening for 48 hours. On examination, apart fromfeatures ofRaynaud's and tenderfingers, he had good perfusion, as shown by Doppler testing. Blood tests showed eosinophilia; 13x10*9 (Normal 0.04-0.40 x10*9) and mild raised C-reactive protein 24 (normal range <10.0 mg/L). Chest radiograph, ECG, and echocardiography showed no abnormalities. The results of initial screening including Immunoglobulins, complements, ANA, ANCA, RF, APLA and CCP were all negative.As there were no criticalfeatures of ischemia, we considered the patientfor dischargefrom the hospital with a planfor close review.We strongly advised him not to smoke. He re-admitted within a week with worsening of Raynaud's and features of critical ischemia with bluish nail and fingertip and some early features of mild digital necrosis. We began treatment with intravenous heparin, aspirin, and iloprost (prostacyclin) +infusionfor10days.Thepatient'sdigitalischemiaandintermittentclaudication showed some improvement after 1 week, but eosinophilia persisted at 11x10*9 with ruminant pain in digits. The importance of smoking cessation was re-emphasised. Magnetic resonance angiography showed no occlusion of the distal arteries of the upper and lower extremities. Capillaroscopy showed micro haemorrhages (no connective tissue disease/scleroderma pattern abnormality). Investigations for parasitic infections and malignancy were negative. HIV / Hepatitis B/C CMV/EBV/ Parvovirus IGM was all undetected. Schistosoma testing was negative. + +Haematology investigated himfor the rare possibilityof the geneticmutation for hypereosinophilic syndrome (PDGFRA and FIP1L1 genes) which was negative and underwent bone marrow biopsy which was also normal. We started him on oral prednisolone 1 mg/kg body weight with complete resolution of eosinophilia and ischemic symptoms plus Raynaud's.Smoking stoppedwithhelp. + +Discussion:Buerger's disease is a rare disease of exclusion and recognised as oneform of vasculitiswith a strong associationwith smoking and nicotine. There has been no consensus or any significant data for any specific therapies proven a benefit in this condition, more than in alone stopping smokingorevenoral tobaccoproducts.Themaincharacteristic features of this form are present in young smokers with no systemic disease manifestation, normal inflammatory response, and negative immunology. \ No newline at end of file diff --git a/medical/md/PMC6809663.md b/medical/md/PMC6809663.md new file mode 100644 index 0000000000000000000000000000000000000000..970807d58222c59e7cd77f37591b2a0bfb2e37a7 --- /dev/null +++ b/medical/md/PMC6809663.md @@ -0,0 +1,50 @@ +2331. Seroprevalence of Cytomegalovirus in Pregnant Women and Birth Prevalence of Congenital Cytomegalovirus Infection in Henan Province, China Yue Huang, PhD1; Tingdong Li, Doctor's degree1; Qiaoqiao Song, Master's degree1; Tengfei Man2; Han Wang, Master's degree1; Xiaoyi Guo, Doctor's degree1; Huan Yu, Master degree1; Yadong Adam Cui, MD, PhD3; Ting Wu, Doctor's degree1; Morgan Marks, PhD, ScM4; Tong-Ming Fu, PhD MD5; Shengxiang Ge, Doctor's degree1; Ningshao Xia, Prof1; Jun Zhang, Master Degree1; 1Xiamen University, Xiamen, Fujian, China (People's Republic); 2MSD, Beijing, Beijing, China (People's Republic); 3Merck and Co. Inc., North Wales, Pennsylvania; 4Merck and Co Inc., North Wales, Pennsylvania; 5University of Texas Health Science Center at Houston, Philadelphia, Pennsylvania Session: 247. Clinical Virology/Viral Epidemiology Saturday, October 5, 2019: 12:15 PM +Background. Congenital cytomegalovirus infection (cCMVi) is the leading viral cause of birth defects and developmental disabilities in newborns. The epidemiology of cCMVi in settings with high cytomegalovirus (CMV) seroprevalence, such as China, is not well studied. This study sought to describe maternal CMV seroprevalence and cCMVi prevalence at birth in Henan Province, China. + +Methods. A multicenter prospective cohort study was conducted in three counties of Henan Province in China from June 2015 through May 2018. Pregnant women were enrolled early in pregnancy and followed up through delivery. Serum specimens were collected at enrollment for CMV immunoglobulin G serological testing. Saliva and urine specimens were collected in newborns within 72 hours after birth and tested with real-time polymerase chain reaction for CMV DNA. cCMVi was defined as CMV +DNA positive in the infants' urine or saliva specimens. + +Results. A total of 6327 pregnant women underwent CMV serological testing and 6062 were CMV seropositive (95.8%). The maternal age was 26.8 ± 4.3 (mean ± +SD) years. There were 49 (0.7%) newborns identified with cCMVi among 6705 newborns screened. Lower maternal education level (middle school or lower), younger maternal age (<25 years) and twin-pregnancy were associated with higher cCMVi prevalence (P = 0.04, 0.016, and 0.001, respectively). + +Conclusion. Despite a high maternal CMV seroprevalence in this large cohort study from China, the birth prevalence of cCMVi is similar to other studies in settings of high and medium CMV seroprevalence. In settings of high maternal CMV +seroprevalence, additional research is needed to ascertain the relative contribution of non-primary CMV infections during pregnancy to congenital transmission. + +Disclosures. **All authors:** No reported disclosures. + +2332. Low Serum Vitamin D Levels Are Related to Life-Threatening Respiratory Syncytial Virus Infection in Previously Healthy Infants Fausto Martin Ferolla, MD; Eduardo Walter Yfran, MD; Maria Gabriela Ballerini, MSc; Analia Toledano, MSc; Ana Caratozzolo, MSc; Ana Clara Giordano, MD; Barbara Carrizo, MD; Florencia Feldman, MD; Cecilia Anselmino, MD; Agustina Chiormi, MSc; Gabriela Visingardi, MSc; Alejandra Retta, MSc; Mariana Reyero, MSc; Monica Dastugue, MD; Normando Mascardi, MD; Pablo Neira, MD; Gabriela Ropelato, MSc; Maria Marta Contrini, MD; Eduardo Luis Lopez, MD; Hospital de Niños "Dr. Ricardo Gutiérrez," Buenos Aires, Buenos Aires, Argentina Session: 247. Clinical Virology/Viral Epidemiology Saturday, October 5, 2019: 12:15 PM +Background. Serum 25-hydroxy-vitamin D (VD) effects on lung growth and immune system modulation might affect respiratory infections outcomes. Data are controversial regarding the role of VD status in the severity of Respiratory Syncytial Virus +(RSV) infection. The aim of this study was to assess serum VD levels and its association with life-threatening disease (LTD) in previously healthy infants infected with RSV. + +Methods. Prospective cohort study including previously healthy infants +<12 months, hospitalized with a first RSV infection in 2017–2018. Viral load (VL) was assessed by qRT–PCR in nasopharyngeal aspirates and serum VD levels measured by ECLIA, in samples obtained on admission. VD deficiency was defined as levels <20 ng/ mL, VD insufficiency 20–29 ng/mL, and LTD as need of intensive care and mechanical or noninvasive ventilation Results. 98 patients, mean age 4.5 months (±3.1), 55 (56.1%) male. VD status: 18 +(18.4%) with deficiency, 32 (32.6%) with insufficiency; 14 (77.8%) patients with deficiency had not received VD supplementation. There was no relationship between VD +deficiency and anemia (P = 0.28) or age (P = 0.27). LTD was observed in 17 infants, with no significant differences in socioeconomic, pregnancy and infant variables compared with other RSV cases. Patients with LTD had significantly lower levels of VD +(17.5 ng/mL [IQR 15.2–26.3] vs. 31.8 ng/mL [IQR 23.5–52.1, P < 0.001)], Figure 1. 15 patients, 88.2% of all infants with VD levels ≤29 ng/mL developed LTD compared with a study population frequency of LTD of 17.3%. Multivariable regression analysis including breastfeeding confirmed VD deficiency as a risk factor for LTD (aOR +14.3, 95% CI 3.9–51.5, P < 0.001). Normal VD values conferred protection (aOR 0.1, 95% CI 0.02–0.49, P = 0.004). VD levels inversely correlated with days of hypoxemia +(P = 0.007); VD deficiency increased the risk of requiring O2 supplementation >7 days +(aOR 8.5, P < 0.001). VL did not correlate with VD levels (P = 0.696), length of stay (P = 0.378), days of hypoxemia (P = 0.681). VL was not associated with LTD (P = 0.42). + +Conclusion. Vitamin D deficiency was a risk factor for LTD in previously healthy infants with RSV infection. Viral titers did not correlate with VD levels. These findings provide additional evidence for the development of low-cost preventive and therapeutic strategies. + +Disclosures. **All authors:** No reported disclosures. + +![0_image_0.png](0_image_0.png) + +2333. Influenza-Related Neurologic Complications in Hospitalized Children with Underlying Neurologic Disorders Sarah Frankl, BS1; Susan E. Coffin, MD, MPH2; Sanjeev Swami, MD2; Jennifer McGuire, MD MSCE2; 1The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; 2Children's Hospital of Philadelphia, Philadelphia, Pennsylvania Session: 247. Clinical Virology/Viral Epidemiology Saturday, October 5, 2019: 12:15 PM +Background. Children with underlying neurological and neuromuscular conditions are considered "high risk" for developing severe infection due to influenza. + +Prior reports highlighted this population's increased risk for respiratory failure. Little is known about neurological complications experienced by children with pre-existing neurologic disorders (PNDs) when infected with influenza. + +Methods. Retrospective cohort study of children 0.5–18.0 years old hospitalized at a tertiary care pediatric hospital between August 2010 and June 2017 with laboratory-confirmed influenza. Eligible children were identified by electronic medical record query for influenza assay CPT codes with positive results during an admission; cases were confirmed by chart review. Demographics and clinical data were abstracted. + +Results. A total of 1217 immune competent children (median age 5.5 [IQR 2.2– +9.8] years) were hospitalized with laboratory-confirmed influenza during the study period. About 28% (341/1217) had at least one PND, including epilepsy (n = 105), developmental delay or intellectual disability (n = 234), neurogenetic or metabolic disorders (n = 77), neuromuscular disorders (n = 22) and others (n = 253). Compared with previously healthy peers, these children were more often admitted to the intensive care unit (31% vs. 16%, P < 0.001), had a longer length of stay (3 vs. 2 days, P < 0.001), and had a higher incidence of neurologic complications (23% vs. 6%, P < 0.001). Seizures (18% vs. 4%, P < 0.001) and encephalopathy (8% vs. 2%, P < 0.001) in particular were more common in children with PNDs, but other neurologic complications occurred in comparable proportions (3% vs. 1%, P = 0.088). Only 49% of the overall cohort had documented annual influenza vaccine; coverage was slightly better for children with PNDs than those without (55% vs. 48%, P = 0.017). The odds of having a neurologic complication in children with documented vaccination was nearly half that of other children when adjusted for age, influenza strain, and any PND (adjusted OR 0.64, 95% CI 0.44–0.94, P = 0.021). + +Conclusion. The excess risk of neurological complications in children with PNDs highlights the importance of vaccinating this population. Additional consideration should be given to post-exposure prophylaxis for children with PNDs who have not received vaccine. + +![0_image_1.png](0_image_1.png) + +2334. Saliva Screening for Congenital Cytomegalovirus Infection in the Neonatal Intensive Care Unit: Beware! + +Alexandra Medoro, MD1; Masako Shimamura, MD2; Cory T. Hanlon, BS2; Irina Kaptsan2; MD S. Malhotra, MD1; Ursula M. Findlen, PhD2; \ No newline at end of file diff --git a/medical/md/PMC6899795.md b/medical/md/PMC6899795.md new file mode 100644 index 0000000000000000000000000000000000000000..3a53209c890d438fa200920c4475286eec6de0dd --- /dev/null +++ b/medical/md/PMC6899795.md @@ -0,0 +1,330 @@ +DOI: 10.1111/dom.13856 ORIGINAL ARTICLE +Superior efficacy with a fixed-ratio combination of insulin degludec and liraglutide (IDegLira) compared with insulin degludec and liraglutide in insulin-naïve Japanese patients with type 2 diabetes in a phase 3, open-label, randomized trial Kohei Kaku MD1 | Eiichi Araki MD2 | Yukio Tanizawa MD3 | Bue Ross Agner PhD4 | Tomoyuki Nishida MSc5 | Mattis Ranthe MD4 | Nobuya Inagaki MD6 1Department of Internal Medicine, Kawasaki Medical School, Kurashiki, Japan 2Department of Metabolic Medicine, Kumamoto University, Kumamoto, Japan 3Graduate School of Medicine, Yamaguchi University, Ube, Japan 4Novo Nordisk A/S, Søborg, Denmark 5Novo Nordisk Pharma Ltd, Tokyo, Japan 6Department of Diabetes, Endocrinology and Nutrition, Kyoto University Graduate School of Medicine, Kyoto, Japan Correspondence Prof. Kohei Kaku, Department of Internal Medicine, Kawasaki Medical School, Kurashiki, Japan. Email: kka@med.kawasaki-m.ac.jp Funding information The trial was sponsored by Novo Nordisk A/S and is registered with ClinicalTrials.gov (NCT02607306). Peer Review The peer review history for this article is available at https://publons.com/publon/10. 1111/dom.13856. + +## Abstract + +Aims: To investigate the efficacy and safety of insulin degludec/liraglutide (IDegLira) compared with its individual components in Japanese people with type 2 diabetes (T2D) uncontrolled on an oral antidiabetic drug (OAD). + +Materials and methods: This 52-week, open-label, multicentre, treat-to-target trial randomized participants (n = 819) 1:1:1 to IDegLira, liraglutide 1.8 mg or degludec, as add-on to their pre-trial OAD. The maximum IDegLira dose was 50 dose steps (50 U degludec/1.8 mg liraglutide), there was no maximum dose for degludec, and both were titrated based on individual blood glucose measurements. + +Results: After 52 weeks, glycated haemoglobin (HbA1c) decreased by 26 mmol/mol with IDegLira vs 20 mmol/mol with degludec and liraglutide: estimated treatment differences were −6.91 mmol/mol (95% confidence interval [CI] –8.18; −5.64) and −5.30 mmol/mol (95% CI −6.58; −4.03), confirming noninferiority of IDegLira to degludec and superiority of IDegLira to liraglutide (P < .0001 for both [primary endpoint]). Mean body weight changes were 2.9 kg, 4.1 kg and −1.0 kg with IDegLira, degludec and liraglutide, respectively, showing superiority of IDegLira versus degludec (P = .0001), but a significant difference in favour of liraglutide (P < .0001). Rates of severe or blood glucose-confirmed hypoglycaemia for IDegLira were lower versus degludec (rate ratio 0.48 [95% CI 0.35; 0.68]; P < .0001), but higher versus liraglutide (rate ratio 37.58 [95% CI 19.80; 71.31]; P < .0001). Mean daily total insulin dose was lower with IDegLira (27.7 U) versus degludec (34.8 U; P < .0001). Overall adverse event (AE) rates were similar. In total, 34.9%, 22.9% and 41.8% of IDegLira-, degludec- and liraglutide-treated participants experienced gastrointestinal AEs. + +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. © 2019 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd. + +2674 wileyonlinelibrary.com/journal/dom Diabetes Obes Metab. 2019;21:2674–2683. + +Conclusion: IDegLira was superior to degludec and liraglutide in terms of HbA1c reduction and superior to degludec in terms of body weight change and rates of hypoglycaemia in Japanese people with T2D. + +KEYWORDS +basal insulin, liraglutide; hypoglycaemia, randomized trial, type 2 diabetes + +## 1 | Introduction + +The prevalence of diabetes in Japan is expected to reach 8.3% in 2045, up from 7.7% in 2017,1 with the majority of cases being type 2 diabetes (T2D).2-4 This increase is attributable, at least in part, to Japan's ageing population and changes in lifestyle factors, including diet and physical activity.5,6 Impaired insulin secretion is considered more common among Japanese people with T2D compared with white people, probably because of differences in body composition between these populations.7,8 Due to the progressive nature of T2D and associated deterioration of β-cell function, most patients will eventually require treatment intensification to maintain glycaemic targets. Japanese clinical practice guidelines emphasize treatment individualization and intensification with oral antidiabetic drugs (OADs), glucagon-like peptide-1 receptor agonists (GLP-1RAs) and basal or premixed insulin.9 Basal insulin is an effective therapy for reducing fasting plasma glucose (FPG) levels, but it is associated with considerable risk of hypoglycaemia and weight gain.10-12 GLP-1RAs augment glucose-dependent insulin secretion, as well as preserving β-cell function, delaying gastric emptying, promoting weight loss and increasing insulin sensitivity.13 The complementary actions of insulin and GLP-1RAs target multiple pathophysiological defects involved in T2D, and combining both therapies together can reduce glycated haemoglobin (HbA1c) levels with lower risk of hypoglycaemia and weight gain compared with basal insulin alone.14-17 However, this burdens patients with two different treatment regimens, which may limit treatment adherence and intensification.18,19 Insulin degludec/liraglutide (IDegLira) is a fixed-ratio soluble combination of insulin degludec (degludec) and the GLP-1RA liraglutide (100 U and 3.6 mg/mL, respectively), which allows once-daily administration of both active ingredients with a single injection. The efficacy and safety of IDegLira has been investigated in a number of patient populations in the DUAL clinical trial programme.20-28 Based on evidence from these trials, IDegLira received regulatory approval from the European Medicines Agency and the US Food and Drug Administration (FDA).29,30 The global DUAL I trial, which encompassed insulin-naïve participants with T2D, demonstrated that treatment with IDegLira results in improved glycaemic control compared with each component administered separately, whilst mitigating the side effects associated with each, including the gastrointestinal side effects associated with liraglutide.20 The aim of the present study was to confirm the efficacy and safety of IDegLira compared with each of its components in Japanese people with T2D inadequately controlled on one OAD. + +## 2 | Materials And Methods 2.1 | Study Design And Participants + +This 52-week, multicentre, randomized, open-label, three-arm parallel-group trial investigated the efficacy and safety of IDegLira versus each of its components, degludec and liraglutide (Figure 1). The trial consisted of a 2-week screening period and a 52-week treatment period. Participants were Japanese adults (aged ≥20 years) with HbA1c levels of 53–97 mmol/mol (7.0%–11.0%) and a body mass index (BMI) of ≥20 kg/m2, who had been diagnosed with T2D +≥6 months prior to screening and who were on stable therapy with one of six OAD types for at least 60 days prior to screening. Permitted OADs were aligned with Japanese clinical practice guidelines: +α-glucosidase inhibitors; thiazolidinediones; sodium-glucose co-transporter-2 inhibitors; glinides; metformin; or sulphonylureas.9 The protocol was approved by independent ethics committees or institutional review boards at all participating institutions, and the + +![1_image_0.png](1_image_0.png) + +## 2676 Kaku + +study was performed in accordance with the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice guidelines. Written consent was obtained from all participants before enrolment. + +## 2.2 | Treatment + +IDegLira and degludec were administered once daily. Recommended starting doses were 10 dose steps of IDegLira (10 U degludec +0.36 mg liraglutide) or 10 U of degludec. Doses were adjusted twice weekly in increments of ±2 U, aiming for a mean pre-breakfast selfmonitored blood glucose (SMBG; mean from 3 consecutive days) target range of 4.0–5.0 mmol/L (72–90 mg/dL; Table S1). SMBG was assessed using a glucose meter, calibrated to plasma equivalent values. The maximum dose of IDegLira was 50 dose steps, which delivers the maximum licensed liraglutide dose for diabetes (50 U +degludec/1.8 mg liraglutide)31; there was no maximum dose for degludec. + +Liraglutide was administered once daily. Liraglutide was initiated at 0.3 mg and increased by 0.3 mg each week over a 6-week period up to the maximum dose of 1.8 mg. Temporary dose reductions for <1 week were only allowed for safety reasons. OAD treatment continued unchanged at pre-trial doses; however, in case of safety concerns the dose could be reduced at the discretion of the investigator. + +## 2.3 | Randomization And Stratification + +Participants were randomized 1:1:1, via a centralized allocation using an interactive web response system, to receive either IDegLira, degludec or liraglutide, each in combination with pre-trial OAD. Participants were stratified by type of pre-trial OAD treatment to ensure an even distribution of each of the six OAD types across the three treatment arms. + +## 2.4 | Endpoints + +The primary endpoint was change from baseline in HbA1c after 52 weeks of treatment for assessing superiority of IDegLira versus liraglutide and non-inferiority of IDegLira versus degludec. Secondary endpoints included: change from baseline in HbA1c after 52 weeks of treatment for assessing superiority of IDegLira versus degludec; percentages of participants reaching HbA1c <53 mmol/mol (<7.0%) +and ≤48 mmol/mol (≤6.5%) after 52 weeks of treatment; change from baseline in body weight after 52 weeks of treatment; fasting lipid profile; and changes from baseline in FPG, nine-point SMBG profile, prandial plasma glucose increments (from before meal to 90 minutes after for breakfast, lunch and dinner) and blood pressure. Safety endpoints included number of treatment-emergent adverse events (AEs), number of treatment-emergent severe (defined according to the American Diabetes Association classification) or blood glucose-confirmed (plasma glucose <3.1 mmol/L [<56 mg/dL]) symptomatic hypoglycaemic episodes during 52 weeks of treatment, and pulse rate. + +## 2.5 | Statistical Analyses + +The main study objective was jointly confirming the non-inferiority of IDegLira to insulin degludec alone with an upper 95% confidence interval (CI) margin of 0.3%, and the superiority of IDegLira to liraglutide alone with a lower 95% CI margin of 0% (with respect to change in HbA1c at 26 weeks). The sample size was determined using a t-statistic under the assumption of a one-sided test with a type I error rate of 2.5% and a standard deviation of 1.0% for both the superiority and non-inferiority testing. For sample size calculations, the mean difference between treatments in change from baseline in HbA1c after 52 weeks was assumed to be −0.1% for non-inferiority and −0.3% for superiority testing. The per-protocol analysis set (assumed to be 85% of the randomized participant population) +was used for the power calculation for non-inferiority, while the full analysis set was used for superiority. Based on these assumptions, a sample size of 807 participants would provide a non-inferiority power of 98.9% and a superiority power of 93.5%, giving an overall power for meeting the primary objective of 92.5%. A non-inferiority margin for the difference in the change from baseline in HbA1c after 52 weeks of treatment of 0.3% was selected based on existing FDA guidance, and is considered to be the minimal clinically significant change for HbA1c level.32 To ensure that the overall type I error rate was not inflated, a hierarchical testing procedure was used. If the primary hypotheses were confirmed in change in HbA1c (ie, IDegLira superiority to liraglutide and non-inferiority to degludec), the secondary confirmatory tests were performed for superiority of IDegLira versus degludec following a fixed sequence: (a) Change from baseline in body weight after 52 weeks of treatment, (b) Number of treatment-emergent severe or blood glucose-confirmed hypoglycaemic episodes during 52 weeks of treatment, and (c) Change in HbA1c after 52 weeks of treatment. Tests lower down the sequence in the hierarchy were only performed if the preceding test was statistically significant in favour of IDegLira. + +Continuous efficacy endpoints including the primary endpoint were analysed separately using an analysis of covariance (ANCOVA) model including treatment, pre-trial OAD treatment as fixed factors and the baseline value of the parameter as a covariate. For the fasting lipid profile, the endpoint and baseline covariates were log-transformed before the analysis. Insulin dose was analysed using an ANCOVA model including the same fixed factors and baseline HbA1c as a covariate. Last observation carried forward was used to impute missing values for endpoints after 52 weeks of treatment. Attainment of glycaemic targets was analysed using a logistic regression model with treatment and pretrial OAD treatment as fixed factors and baseline HbA1c as covariate. + +Number of treatment-emergent hypoglycaemic episodes was analysed using a negative binominal regression model with a log-link function and the logarithm of the time period in which a hypoglycaemic episode was considered treatment-emergent as offset, with treatment and pre-trial OAD treatment as fixed factors. + +The robustness of the conclusions from the primary and confirmatory secondary analyses were assessed in various sensitivity analyses, including analysis of different analysis sets (per-protocol and completer) +and using a mixed model for repeated measurements and a pattern mixture model approach mimicking an intention-to-treat scenario. + +## 3 | Results 3.1 | Participants + +A total of 819 participants were randomized and treated with either IDegLira (n = 275), degludec (n = 271) or liraglutide (n = 273), all in combination with pre-trial OAD (Figure S1). In total 54 participants withdrew from the trial; 21 (7.6%) in the IDegLira group, 23 (8.5%) in the IDeg group and 10 (3.7%) in the liraglutide group. The number of withdrawals due to AEs was low and similar across treatment groups (eight participants [2.9%] with IDegLira, six [2.2%] with degludec and six [2.2%] with liraglutide). Baseline characteristics were similar in the three treatment groups (Table 1). + +| TABLE 1 | Baseline characteristics of participants IDegLira (N = 275) | Degludec (N = 271) | Liraglutide (N = 273) | +|------------------------------------------------|---------------------------------------------------------------|----------------------|-------------------------| +| Age, y Mean (SD) | 56.9 (10.2) | 57.8 (9.9) | 56.8 (10.1) | +| Median (min.; max.) | 57.0 (29.0; 81.0) | 59.0 (22.0; 80.0) | 57.0 (25.0; 79.0) | +| Sex, n (%) Female | 81 (29.5) | 76 (28.0) | 81 (29.7) | +| Male | 194 (70.5) | 195 (72.0) | 192 (70.3) | +| Body weight, kg Mean (SD) | 70.7 (12.4) | 72.6 (14.5) | 72.2 (15.0) | +| Median (min.; max.) | 68.7 (44.1; 113.3) | 70.5 (44.8; 161.8) | 70.4 (42.3; 142.4) | +| BMI, kg/m2 Mean (SD) | 26.1 (3.7) | 26.6 (4.8) | 26.5 (4.5) | +| Median (min.; max.) | 25.5 (19.9; 36.8) | 25.5 (19.6; 56.0) | 25.8 (19.8; 45.5) | +| HbA1c, mmol/mol Mean (SD) | 69.6 (12.2) | 69.7 (11.5) | 67.4 (10.8) | +| Median (min.; max.) | 67.2 (50.8; 98.9) | 67.2 (51.9; 101.1) | 63.9 (49.7; 101.1) | +| HbA1c, % Mean (SD) | 8.5 (1.1) | 8.5 (1.1) | 8.3 (1.0) | +| Median (min.; max.) | 8.3 (6.8; 11.2) | 8.3 (6.9; 11.4) | 8.0 (6.7; 11.4) | +| FPG, mmol/L Mean (SD) | 9.9 (2.4) | 10.0 (2.3) | 9.7 (2.2)a | +| Median (min.; max.) | 9.4 (5.1; 17.6) | 9.6 (5.5; 17.0) | 9.3 (5.1; 19.3)a | +| Fasting C-peptide, nmol/L Geometric mean (CV%) | 0.49 (42.8) | 0.51 (47.1) | 0.50 (43.0)a | +| Median (min.; max.) | 0.50 (0.20; 1.68) | 0.49 (0.12; 2.19) | 0.49 (0.16; 1.36)a | +| FPG, mg/dL Mean (SD) | 178.7 (43.0) | 179.9 (42.3) | 175.4 (39.8)a | +| Median (min.; max.) | 170.0 (92.0; 318.0) | 173.0 (99.0; 307.0) | 168.0 (92.0; 347.0)a | +| Duration of diabetes, y Mean (SD) | 9.2 (6.2) | 9.7 (6.0) | 9.4 (5.9) | +| Median (min.; max.) | 8.1 (0.6; 45.3) | 8.7 (0.5; 30.7) | 8.4 (0.8; 33.9) | +| OAD use at screening, n (%) Metformin | 47 (17.1) | 46 (17.0) | 47 (17.2) | +| α-glucosidase inhibitors | 41 (14.9) | 40 (14.8) | 41 (15.0) | +| Thiazolidinediones | 43 (15.6) | 43 (15.9) | 42 (15.4) | +| Sulphonylureas | 43 (15.6) | 42 (15.5) | 42 (15.4) | +| SGLT2 inhibitors | 61 (22.2) | 61 (22.5) | 61 (22.3) | +| Glinides | 40 (14.5) | 39 (14.4) | 40 (14.7) | +| Abbreviations: BMI, body mass index; CV, coefficient of variation; FPG, fasting plasma glucose; OAD, oral anti-diabetic drug; SGLT2, sodium-glucose cotransporter-2 inhibitor. a N = 272. | | | | + +## 2678 Kaku 3.2 | Efficacy + +After 52 weeks of treatment, mean HbA1c decreased from 69.6 mmol/mol (8.52%) to 43.1 mmol/mol (6.10%) with IDegLira, 69.7 mmol/mol (8.53%) to 50.1 mmol/mol (6.73%) with degludec, and 67.4 mmol/mol (8.32%) to 47.7 mmol/mol (6.52%) with liraglutide +(Figure 2A). Mean HbA1c was reduced to a significantly greater extent with IDegLira compared with liraglutide (−26.5 mmol/mol [−2.42%] +vs −19.7 mmol/mol [−1.80%], estimated treatment difference [ETD] –5.30 mmol/mol [95% CI –6.58; −4.03; −0.48% {95% CI −0.60; −0.37}]; P < .0001), confirming superiority for IDegLira versus liraglutide. Mean HbA1c was also significantly reduced with IDegLira compared with degludec (−26.5 mmol/mol [−2.42%] vs +−19.6 mmol/mol [−1.80%], ETD −6.91 mmol/mol [95% CI –8.18; +−5.64; −0.63% {95% CI −0.75; −0.52}]; P < .0001) confirming noninferiority of IDegLira versus degludec. The superiority of IDegLira versus degludec was confirmed as a part of confirmatory hierarchical testing procedure (−0.63% [95% CI −0.75; −0.52]; P < .0001). + +The level of glycaemic control demonstrated with IDegLira was supported by the secondary endpoint, achievement of predefined HbA1c targets (Figure S2). The odds of achieving HbA1c +<53 mmol/mol (<7.0%) and ≤ 48 mmol/mol (≤6.5%) were significantly higher with IDegLira compared with either degludec (estimated odds ratios for <53 mmol/mol and ≤48 mmol/mol: 4.70 [95% CI 2.82; 7.82] and 4.76 [95% CI 3.12; 7.25], respectively) or liraglutide (estimated odds ratios for <53 mmol/mol and ≤48 mmol/mol: 3.90 [95% +CI 2.31; 6.59] and 2.94 [95% CI 1.93; 4.50], respectively; P < .0001 for all). + +## 3.3 | Body Weight + +After 52 weeks, there was a significantly smaller increase in body weight with IDegLira (2.9 kg) versus degludec (4.1 kg; Figure 2B) with an ETD of −1.19 kg (95% CI −1.80; −0.59; P = .0001 [P-value for superiority]). The mean change from baseline in body weight was +−1.0 kg with liraglutide versus 2.9 kg with IDegLira (Figure 2B), representing an ETD of 3.89 kg (95% CI 3.29; 4.49; P < .0001). + +An exploratory analysis showed there was no significant interaction between background OAD therapy and treatment for the + +![4_image_0.png](4_image_0.png) + +endpoint change in body weight; there was no effect of pre-trial OAD treatment on the differences between IDegLira and comparator. + +## 3.4 | Hypoglycaemic Episodes + +The cumulative incidence of severe or blood glucose-confirmed hypoglycaemia is shown in Figure 3. After 52 weeks, rates of severe or blood glucose-confirmed hypoglycaemia were 174.3, 331.9 and 4.8 events/100 participant-years of exposure [PYE] for IDegLira, degludec and liraglutide, respectively, with 38.5% of IDegLira-treated participants, 54.6% of degludec-treated participants and 2.2% of liraglutidetreated participants experiencing ≥1 event (Table 2). The treatment rate ratio of severe or blood glucose-confirmed hypoglycaemic episodes was significantly lower for IDegLira versus degludec (0.48 [95% CI 0.35; 0.68]; P < .0001), confirming superiority, and significantly higher for IDegLira versus liraglutide (rate ratio 37.58 [95% CI 19.80; 71.31]; P < .0001). The percentage of participants who experienced severe or blood glucose-confirmed symptomatic hypoglycaemia was 19.3% (IDegLira), 29.5% (degludec) and 1.1% (liraglutide), with rates of 50.4, 138.3 and 1.5 events/100 PYE, respectively (Table 2). + +## 3.5 | Dose + +The mean insulin doses during the first week were similar in the IDegLira (10.5 dose steps) and degludec (10.4 U) groups. After 52 weeks of treatment, the mean daily total insulin dose was significantly lower with IDegLira than degludec (27.7 vs 34.8 U; ETD −7.01 U [95% CI −10.52; −3.50]; P < .0001 [Figure S3A]). Mean liraglutide doses were lower in the IDegLira group compared with the liraglutide group throughout the trial, and at week 52 were 1.0 mg and 1.8 mg, respectively (Figure S3B). Of the participants randomized to IDegLira, 17.1% (n = 47) were on the maximum dose of 50 dose steps, and 74.5% of these participants achieved the target of HbA1c <53 mmol/mol (<7.0%) at end of trial. + +## 3.6 | Fasting Plasma Glucose + +The mean FPG levels over time are shown in Figure 2C. After 52 weeks of treatment, change from baseline in mean FPG was similar for IDegLira FIGURE 3 Cumulative incidence + +![5_image_1.png](5_image_1.png) of severe or blood glucose-confirmed hypoglycaemia (with or without symptoms). Safety analysis set. Observed data. Severe or blood glucose-confirmed: An episode that is severe according to the American Diabetes Association classification or blood glucose-confirmed by a plasma glucose value <3.1 mmol/L (<56 mg/dL) with or without symptoms consistent with hypoglycaemia. IDegLira, insulin degludec/liraglutide + +![5_image_0.png](5_image_0.png) + +and degludec (−4.1 mmol/L [−73.6 mg/dL] and −4.0 mmol/L +[−71.5 mg/dL], respectively) but greater for IDegLira versus liraglutide +(−4.1 mmol/L [−73.6 mg/dL] vs −2.6 mmol/L [−47.1 mg/dL]). + +## 3.7 | Self-Monitored Blood Glucose + +The mean nine-point SMBG profiles decreased across all groups throughout the trial (Figure S4). After 52 weeks, SMBG profiles showed statistically significantly lower pre- and post-prandial glucose concentrations for IDegLira compared with degludec and liraglutide at all timepoints (except for pre-breakfast and at 4:00 AM, which were similar between IDegLira and degludec). + +After 52 weeks of treatment, the reduction from baseline in prandial glucose increments was statistically significantly greater with IDegLira compared with degludec for breakfast (ETD −0.81 mmol/L +[95% CI −1.24; −0.38; −14.58 mg/dL {95% CI −22.27; −6.88}]; P = .0002), dinner (ETD, −0.66 mmol/L [95% CI −1.17; −0.15; −11.94 mg/dL {95% CI −21.15; −2.73}]; P = .0111) and mean of all meals (ETD −0.64 mmol/L [95% CI −0.96; −0.32; −11.51 mg/dL {95% CI −17.26; −5.76}]; P < .0001), but not significant for lunch (ETD −0.49 mmol/L [95% CI −1.02; 0.04; −8.86 mg/dL {95% CI −18.44; 0.72}]; P = .0699). However, although a treatment difference for IDegLira compared with liraglutide was observed for breakfast (ETD 0.48 mmol/L [95% CI 0.06; 0.91]; 8.70 mg/dL {95% CI 1.03; 16.36}]; P = .0263), a significant difference was not observed for dinner (ETD 0.16 mmol/L [95% CI −0.35; 0.67; 2.84 mg/dL {95% CI +−6.33; 12.02}]; P = .5430), lunch (ETD 0.09 mmol/L [95% CI −0.44; 0.61; 1.55 mg/dL {95% CI −7.97; 11.08}]; P = .7488) or mean of all meals (ETD 0.24 mmol/L [95% CI −0.08; 0.56; 4.33 mg/dL {95% CI −1.40; 10.06}]; P = .1385). + +## 3.8 | Vital Signs + +After 52 weeks, there was a statistically significant reduction in systolic blood pressure with IDegLira versus degludec (ETD −2.05 mmHg [95% CI −4.02; −0.08]; P = .0412). However, systolic blood pressure was significantly higher with IDegLira versus liraglutide (ETD 2.88 mmHg [95% CI 0.91; 4.85]; P = .0042). There was no significant + +## 2680 Kaku + +| TABLE 2 | Adverse events | IDegLira(N = 275) | Degludec (N = 271) | Liraglutide (N = 273) | | | | | | | | | +|-------------------------------------------------------------------------------------------------------------|------------------|---------------------|----------------------|-------------------------|-----|------|-----|-------|-----|------|-----|-------| +| Event | n | % | E | R | n | % | E | R | n | % | E | R | +| AE | 229 | 83.3 | 873 | 325.7 | 216 | 79.7 | 829 | 316.6 | 229 | 83.9 | 885 | 325.6 | +| AE possibly or probably related to treatment | 88 | 32.0 | 152 | 56.7 | 55 | 20.3 | 84 | 32.1 | 108 | 39.6 | 210 | 77.3 | +| Most frequent AE (≥5% of participants) Infections and infestations Viral upper respiratory tract infection | 106 | 38.5 | 166 | 61.9 | 91 | 33.6 | 161 | 61.5 | 94 | 34.4 | 155 | 57.0 | +| Pharyngitis | 11 | 4.0 | 13 | 4.9 | 15 | 5.5 | 18 | 6.9 | 9 | 3.3 | 9 | 3.3 | +| Influenza | 15 | 5.5 | 15 | 5.6 | 9 | 3.3 | 9 | 3.4 | 9 | 3.3 | 9 | 3.3 | +| Gastrointestinal disorders Constipation | 27 | 9.8 | 28 | 10.4 | 12 | 4.4 | 12 | 4.6 | 38 | 13.9 | 42 | 15.5 | +| Diarrhoea | 15 | 5.5 | 20 | 7.5 | 12 | 4.4 | 14 | 5.3 | 24 | 8.8 | 31 | 11.4 | +| Nausea | 9 | 3.3 | 13 | 4.9 | 5 | 1.8 | 7 | 2.7 | 23 | 8.4 | 30 | 11.0 | +| Investigations Weight increased | 4 | 1.5 | 4 | 1.5 | 19 | 7.0 | 19 | 7.3 | 1 | 0.4 | 1 | 0.4 | +| Lipase increased | 6 | 2.2 | 8 | 3.0 | 0 | - | - | - | 15 | 5.5 | 15 | 5.5 | +| Eye disorders Diabetic retinopathy | 17 | 6.2 | 17 | 6.3 | 12 | 4.4 | 14 | 5.3 | 11 | 4.0 | 11 | 4.0 | +| Skin and subcutaneous tissue disorders Eczema | 16 | 5.8 | 18 | 6.7 | 6 | 2.2 | 6 | 2.3 | 9 | 3.3 | 10 | 3.7 | +| Nervous system disorders Headache | 7 | 2.5 | 11 | 4.1 | 7 | 2.6 | 12 | 4.6 | 15 | 5.5 | 17 | 6.3 | +| SAE | 17 | 6.2 | 18 | 6.7 | 13 | 4.8 | 16 | 6.1 | 14 | 5.1 | 14 | 5.2 | +| SAE possibly or probably related to trial product | 2 | 0.7 | 2 | 0.7 | 1 | 0.4 | 1 | 0.4 | 1 | 0.4 | 1 | 0.4 | +| Severe or blood glucose-confirmed symptomatic hypoglycaemia Severe or blood glucose-confirmed hypoglycaemia | 106 | 38.5 | 467 | 174.3 | 148 | 54.6 | 869 | 331.9 | 6 | 2.2 | 13 | 4.8 | + +%, percentage of participants with one or more events; AE, adverse event; E, number of adverse events; n, number of participants with one or more events; R, rate (number of AEs divided by participant-years of exposure [365.25 days] multiplied by 100); SAE, serious adverse event. Treatment emergent was defined as onset date on or after the first day of exposure to randomized treatment and no later than 7 days after the last day of randomized treatment. + +difference in diastolic blood pressure between IDegLira and either of its components. After 52 weeks of treatment, mean changes in pulse rate were 3.9, 0.8 and 4.2 beats/min with IDegLira, degludec and liraglutide, respectively; the difference between IDegLira and degludec was statistically significant (ETD IDegLira - degludec: 2.87 beats/min [95% CI 1.43; 4.30]; P < .0001), but a difference was not observed between IDegLira and liraglutide (ETD IDegLira - liraglutide: +−0.43 beats/min [95% CI −1.86; 1.00]; P = .5546). + +## 3.9 | Lipid Profile + +There was a statistically significant difference, in favour of IDegLira, for total cholesterol (vs. degludec and liraglutide), LDL cholesterol (vs. degludec) and free fatty acids (vs. liraglutide), and a statistically significant difference in HDL cholesterol in favour of liraglutide versus IDegLira (Table S2). + +## 3.10 | Adverse Events + +The percentage of participants experiencing at least one AE was similar in each treatment group. The most frequently reported AEs were infections, with viral upper respiratory tract infection experienced by over one-third of participants in each treatment group (Table 2). Gastrointestinal events were reported by 34.9%, 22.9% and 41.8% of participants in the IDegLira, degludec and liraglutide groups, respectively. The overall rate of AEs per 100 PYE was similar between treatments groups. The majority of AEs were nonserious and judged unlikely to be related to trial products. In total, 24 participants experienced 31 AEs that led to dose reductions during the trial; eight events in six participants with IDegLira, 16 events in 13 participants with degludec and seven events in five participants with liraglutide. There were no confirmed events of thyroid disease. The event rate per 100 PYE of elevated lipase or amylase was 4.1, 7.4 and 0.0 with IDegLira, liraglutide and degludec, respectively. There were three events of elevated calcitonin (one with IDegLira and two with liraglutide). + +## 3.11 | Serious Adverse Events + +The percentage of participants experiencing at least one serious adverse event (SAE) was 6.2% in the IDegLira group, 4.8% in the degludec group and 5.1% in the liraglutide group. The majority of SAEs were considered unlikely to be related to trial product (Table 2). The most frequently reported SAEs were in the cardiac disorder System Organ Class: three events in three participants in the IDegLira and degludec groups and one event in the liraglutide group. All other SAEs were single events with no apparent difference in distribution between treatment groups. The event adjudication committee classified five cardiovascular AEs (three with IDegLira and two with degludec) as major adverse cardiovascular events. There was one event adjudication committee-confirmed cardiovascular death in the IDegLira group, which was considered unlikely to be related to the trial product. The event adjudication committee also confirmed one event of mild acute pancreatitis in the IDegLira group, which was considered unlikely to be related to trial product, and 14 neoplasms (four with IDegLira including one malignant [local, gastric intestinal], three with degludec including one malignant [metastatic lung/bronchus] and seven with liraglutide including two malignant [local, breast and skin]). + +## 4 | Discussion + +This open-label, treat-to-target trial investigated the efficacy and safety of IDegLira, in combination with an OAD, in Japanese patients with T2D. After 52 weeks of treatment, the superiority of IDegLira over both liraglutide and degludec in terms of reduction in HbA1c was confirmed. The improvement in HbA1c was reflected by the fact that a significantly higher percentage of participants achieved HbA1c targets (<53 mmol/mol [<7.0%] or ≤ 48 mmol/mol [≤6.5%]) at end of trial with IDegLira compared with degludec or liraglutide, consistent with the results from the global DUAL I trial.21 The −6.91 mmol/mol +(−0.63%) HbA1c treatment difference with IDegLira versus degludec was achieved with a significantly lower daily total insulin dose with IDegLira (27.7 vs 34.8 U, respectively), showing the contribution of the liraglutide component. Only ~17% of the participants randomized to IDegLira reached the maximum allowed dose (50 dose steps) and the majority of these participants (74.5%) achieved the target of HbA1c <53 mmol/mol (<7.0%) after 52 weeks. + +There were significantly fewer severe or blood glucose-confirmed hypoglycaemic episodes with IDegLira compared with degludec, reinforcing the contribution of the liraglutide component of IDegLira to the lower rate of hypoglycaemia seen with IDegLira in comparison with degludec in previous trials.26 There was a significantly lower rate of severe or blood glucose-confirmed hypoglycaemic episodes with liraglutide compared with IDegLira. This outcome was expected, because of the presence of the insulin component and the glucosedependent mode of action of GLP-1RAs.33 A significantly smaller increase in body weight was seen with IDegLira (2.9 kg) compared with degludec (4.1 kg), probably as a result of the weight-reducing effect of liraglutide.20 This significant difference is in alignment with findings from the global trial programme.20 The weight gain associated with IDegLira treatment is in contrast to the modest weight loss (−0.5 kg) observed with IDegLira in the global DUAL I trial,20 which could be attributed to differences in background OAD therapy between the global DUAL I extension trial and the present trial, and/or the difference in the relationship between BMI, insulin resistance and diabetes development in Japanese and white people.7,34-36 When IDegLira was used, the mean daily doses of liraglutide and degludec were both lower compared with using the respective monotherapies after 52 weeks. In the liraglutide group, the daily dose reached the maximum licensed dose for diabetes of 1.8 mg by week 6, whereas in the IDegLira group, the actual daily liraglutide dose remained stable from week 9 onwards at ~1.0 mg. This demonstrates a clinical advantage of using the fixed-dose combination injection compared with the monotherapies alone. + +There were no unexpected safety or tolerability issues identified with IDegLira, and the reported AEs were consistent with those of liraglutide or degludec.37,38 Treatment with IDegLira also resulted in fewer gastrointestinal side effects compared with liraglutide treatment alone. + +The DUAL I Japan trial aimed to compare the efficacy and safety of IDegLira with its components given alone in Japanese patients with T2D. Comparing IDegLira with the free combination of its components was beyond the scope of the present trial and may be perceived as a study limitation. Nonetheless, co-administration of degludec and liraglutide within a single daily injection provides a simpler regimen than administering these components separately, and may help to overcome clinical inertia with respect to intensifying therapy. + +In conclusion, in comparison with the individual components of IDegLira, the fixed-ratio combination offers Japanese participants with T2D who have been on stable OAD therapy a simplified treatment regimen with the benefits of improved glycaemic control, a low risk of hypoglycaemia and less weight gain than insulin treatment alone. + +## Acknowledgments + +The authors are grateful to the people who participated in this study, to Kenichiro Shimizu and Kristina Ranc (Novo Nordisk), for review of and input to the manuscript, and to Bridie Andrews, Victoria Atess and Richard McDonald of Watermeadow Medical (UK), an Ashfield Company, for providing medical writing and editorial support, which was funded by Novo Nordisk A/S, Søborg, Denmark, in accordance with Good Publication Practice (GPP3) guidelines. + +## Conflict Of Interest + +K.K. has received honoraria or consulting fees from Astellas Pharma, AstraZeneca, Kowa Pharmaceutical, Mitsubishi Tanabe Pharma, MSD, + +## 2682 Kaku + +Nippon Boehringer Ingelheim, Novo Nordisk Pharma, Sanwa Kagaku Kenkyusho, Dainippon Sumitomo Pharma, Taisho Toyama Pharmaceutical, Takeda. E.A. has participated on advisory panels for Alcon, Astellas Pharma, Astra Zeneca, Eli Lilly, Kowa Pharmaceutical, Nippon Boehringer Ingelheim, Novo Nordisk Pharma, Sanofi and Terumo Corporation, has received honoraria for lectures from Astellas Pharma, MSD, Ono Pharmaceutical, Novo Nordisk Pharma and Sanofi, and scholarship grants from Astellas Pharma, Daiichi Sankyo, Mitsubishi Tanabe Pharma, Nippon Boehringer Ingelheim, Novo Nordisk Pharma, Ono Pharmaceutical, Sanofi, Shionogi, Sumitomo Dainippon Pharma and Takeda Pharmaceutical. Y.T. has participated on advisory panels for Kowa Pharmaceutical, Novo Nordisk Pharma, and Terumo Corporation, has received honoraria for lectures from Astellas Pharma, MSD, Ono Pharmaceutical, Novo Nordisk Pharma, Sanofi and Takeda Pharmaceutical, and scholarship grants from Astellas Pharma, Eli Lilly, Daiichi Sankyo, Mitsubishi Tanabe Pharma, Nippon Boehringer Ingelheim, Novo Nordisk Pharma, Ono Pharmaceutical, Sanofi, Shionogi, Sumitomo Dainippon Pharma and Takeda Pharmaceutical. B.R.A., T.N. and M.R. are employees and shareholders in Novo Nordisk. N.I. has acted as a speaker for MSD, ARKRAY, Inc., Astellas Pharma, Kissei, Sanofi, Novartis Pharma, Novo Nordisk Pharma, Bayer, Kowa Pharma, Ono Pharma, Kyowa Hakko Kirin, Sumitomo Dainippon Pharma, Daiichi Sankyo, Eli Lilly Japan, Nippon Boehringer Ingelheim, Takeda Pharma, Mitsubishi Tanabe Pharma, Medtronic Japan, Taisho Toyama Pharma, Pfizer Japan, FUJIFILM Pharma, Saishin-igaku, Toyooka Hospital, Terumo, Olympus, Sunstar Foundation, Japan Tobacco, ILSI Japan, University of Occupational and Environmental Health, Japanese Red Cross Wakayama Medical Center, AstraZeneca, Tsumura, Chugai Pharma, Wakayama Medical University, Hokkaido University, Boehringer Ingelheim and MSD Life Science Foundation, and has received grants from Shionogi, Pfizer Japan, Eli Lilly Japan, Tsumura, Taisho Toyama Pharma, Novo Nordisk Pharma, the Japan China Medical Association and Teijin Pharma. + +## Author Contributions + +All authors confirm that they meet the International Committee of Medical Journal Editors (ICJME) uniform requirements for authorship and that they have contributed to: critical analysis and interpretation of the data, drafting/critically revising the article and sharing in the final responsibility for the content of the manuscript and the decision to submit it for publication. K.K. was signatory investigator of this clinical trial, the guarantor of this work and, as such, had full access to all data in the study and takes responsibility for the integrity of the data. + +## Data-Sharing Statement + +The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. + +## References + +1. Federation ID. IDF Atlas, 8th Edition: Japan country report. http:// +reports.instantatlas.com/report/view/846e76122b5f476fa6ef09471 965aedd/JPN. Accessed October, 2018. + +2. Morimoto A, Nishimura R, Tamija N. Trends in the epidemiology of patients with diabetes in Japan. Japan Med Assoc J. 2010;53:36-40. + +3. Kawasaki E, Matsuura N, Eguchi K. Type 1 diabetes in Japan. + +Diabetologia. 2006;49:828-836. + +4. Forouhi NG, Wareham NJ. Epidemiology of diabetes. Medicine +(Abingdon). 2014;42:698-702. + +5. Chan JC, Malik V, Jia W, et al. Diabetes in Asia: epidemiology, risk factors, and pathophysiology. JAMA. 2009;301:2129-2140. + +6. Charvat H, Goto A, Goto M, et al. Impact of population aging on trends in diabetes prevalence: a meta-regression analysis of 160,000 Japanese adults. J Diabetes Investig. 2015;6:533-542. + +7. Moller JB, Pedersen M, Tanaka H, et al. Body composition is the main determinant for the difference in type 2 diabetes pathophysiology between Japanese and Caucasians. Diabetes Care. 2014;37:796-804. + +8. Kuroe A, Fukushima M, Usami M, et al. Impaired beta-cell function and insulin sensitivity in Japanese subjects with normal glucose tolerance. Diabetes Res Clin Pract. 2003;59:71-77. + +9. Haneda M, Noda M, Origasa H, et al. Japanese clinical practice guideline for diabetes 2016. J Diabetes Investig. 2018;9(3):657-697. + +10. DeFronzo RA, Eldor R, Abdul-Ghani M. Pathophysiologic approach to therapy in patients with newly diagnosed type 2 diabetes. Diabetes Care. 2013;36(Suppl 2):S127-S138. + +11. Davies MJ, D'Alessio DA, Fradkin J, et al. Management of hyperglycemia in type 2 diabetes, 2018. A consensus report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care. 2018;41:2669-2701. + +12. Amercian Diabetes Association. 9. Pharmacologic approaches to Glycemic treatment: Standards of Medical Care in Diabetes-2019. Diabetes Care. 2019;42:S90-S102. + +13. Nauck MA. Unraveling the science of incretin biology. Am J Med. + +2009;122:S3-S10. + +14. Buse JB, Bergenstal RM, Glass LC, et al. Use of twice-daily exenatide in basal insulin-treated patients with type 2 diabetes: a randomized, controlled trial. Ann Intern Med. 2011;154:103-112. + +15. Riddle MC, Aronson R, Home P, et al. Adding once-daily lixisenatide for type 2 diabetes inadequately controlled by established basal insulin: a 24-week, randomized, placebo-controlled comparison (GetGoalL). Diabetes Care. 2013;36:2489-2496. + +16. Riddle MC, Forst T, Aronson R, et al. Adding once-daily lixisenatide for type 2 diabetes inadequately controlled with newly initiated and continuously titrated basal insulin glargine: a 24-week, randomized, placebo-controlled study (GetGoal-Duo 1). Diabetes Care. 2013;36: 2497-2503. + +17. Ahmann A, Rodbard HW, Rosenstock J, et al. Efficacy and safety of liraglutide versus placebo added to basal insulin analogues (with or without metformin) in patients with type 2 diabetes: a randomized, placebo-controlled trial. Diabetes Obes Metab. 2015;17:1056-1064. + +18. Russell-Jones D, Pouwer F, Khunti K. Identification of barriers to insulin therapy and approaches to overcoming them. Diabetes Obes Metab. 2018;20:488-496. + +19. Donnelly LA, Morris AD, Evans JM, DARTS/MEMO collaboration. + +Adherence to insulin and its association with glycaemic control in patients with type 2 diabetes. QJM. 2007;100:345-350. + +20. Gough SC, Bode BW, Woo VC, et al. One-year efficacy and safety of a fixed combination of insulin degludec and liraglutide in patients with type 2 diabetes: results of a 26-week extension to a 26-week main trial. Diabetes Obes Metab. 2015;17:965-973. + +21. Gough SC, Bode B, Woo V, et al. Efficacy and safety of a fixed-ratio combination of insulin degludec and liraglutide (IDegLira) compared with its components given alone: results of a phase 3, open-label, randomised, 26-week, treat-to-target trial in insulin-naive patients with type 2 diabetes. Lancet Diabetes Endocrinol. 2014;2: 885-893. + +22. Billings LK, Doshi A, Gouet D, et al. Efficacy and safety of IDegLira versus basal-bolus insulin therapy in patients with type 2 diabetes uncontrolled on metformin and basal insulin: the DUAL VII randomized clinical trial. Diabetes Care. 2018;41:1009-1016. + +23. Lingvay I, Harris S, Jaeckel E, Chandarana K, Ranthe MF, Jodar E. + +Insulin degludec/liraglutide (IDegLira) was effective across a range of dysglycaemia and body mass index categories in the DUAL V randomized trial. Diabetes Obes Metab. 2018;20:200-205. + +24. Harris SB, Kocsis G, Prager R, et al. Safety and efficacy of IDegLira titrated once weekly versus twice weekly in patients with type 2 diabetes uncontrolled on oral antidiabetic drugs: DUAL VI randomized clinical trial. Diabetes Obes Metab. 2017;19:858-865. + +25. Linjawi S, Bode BW, Chaykin LB, et al. The efficacy of IDegLira (insulin degludec/liraglutide combination) in adults with type 2 diabetes inadequately controlled with a GLP-1 receptor agonist and oral therapy: +DUAL III randomized clinical trial. Diabetes Ther. 2017;8:101-114. + +26. Buse JB, Vilsboll T, Thurman J, et al. Contribution of liraglutide in the fixed-ratio combination of insulin degludec and liraglutide (IDegLira). Diabetes Care. 2014;37:2926-2933. + +27. Rodbard HW, Bode BW, Harris SB, et al. Safety and efficacy of IDegLira added to sulphonylureas alone or sulphonylureas and metformin in insulin-naïve people with type 2 diabetes: the DUAL IV trial. + +Diabet Med. 2017;34:189-196. + +28. Philis-Tsimikas A, Billings LK, Busch R, et al. Superior efficacy of insulin Degludec/Liraglutide versus insulin Glargine U100 as add-on to sodium-glucose co-Transporter-2 inhibitor therapy: a randomized clinical trial in patients with uncontrolled type 2 diabetes. Diabetes Obes Metab. 2019;21(6):1399-1408. + +29. Novo Nordisk. Xultophy® Summary of Product Characteristics. 2018. + +https://www.medicines.org.uk/emc/product/3469. Accessed July, 2018. + +30. Novo Nordisk. Xultophy® 100/3.6 Prescribing Information (PI). 2016. + +https://www.accessdata.fda.gov/drugsatfda_docs/label/2016/ 208583s000lbl.pdf. Accessed November, 2018. + +31. Novo Nordisk. Victoza® Summary of Product Characteristics. 2017. + +https://www.medicines.org.uk/emc/product/6585/smpc. Accessed July, 2018. + +32. US Department of Health and Human Services, Food and Drug Administration. Guidance for Industry: Diabetes Mellitus - Evaluating Cardiovascular Risk in New Antidiabetic Therapies to Treat Type 2 Diabetes. Silver Spring, MD: Office of Communications, Division of Drug Information, Center for Drug Evaluation and Research, Food and Drug Administration; 2008. https://www.fda.gov/downloads/Drugs/ Guidances/ucm071627.pdf. Accessed July, 2018. + +33. Norwood P, Chen R, Jaeckel E, et al. Rates of hypoglycaemia are lower in patients treated with insulin degludec/liraglutide (IDegLira) +than with IDeg or insulin glargine, regardless of the hypoglycaemia definition used. Diabetes Obes Metab. 2017;19:1562-1569. + +34. Furukawa Y, Tamura Y, Takeno K, et al. Impaired peripheral insulin sensitivity in non-obese Japanese patients with type 2 diabetes mellitus and fatty liver. J Diabetes Investig. 2018;9(3):529-535. + +35. Ito D, Iuchi T, Kurihara S, Inoue I, Katayama S, Inukai K. Efficacy and clinical characteristics of Liraglutide in Japanese patients with type 2 diabetes. J Clin Med Res. 2015;7:694-699. + +36. Seino Y, Rasmussen MF, Zdravkovic M, Kaku K. Dose-dependent improvement in glycemia with once-daily liraglutide without hypoglycemia or weight gain: a double-blind, randomized, controlled trial in Japanese patients with type 2 diabetes. Diabetes Res Clin Pract. 2008;81:161-168. + +37. Keating GM. Insulin degludec and insulin degludec/insulin aspart: a review of their use in the management of diabetes mellitus. Drugs. 2013;73:575-593. + +38. Bode B. An overview of the pharmacokinetics, efficacy and safety of liraglutide. Diabetes Res Clin Pract. 2012;97:27-42. + +## Supporting Information + +Additional supporting information may be found online in the Supporting Information section at the end of this article. + +How to cite this article: Kaku K, Araki E, Tanizawa Y, et al. + +Superior efficacy with a fixed-ratio combination of insulin degludec and liraglutide (IDegLira) compared with insulin degludec and liraglutide in insulin-naïve Japanese patients with type 2 diabetes in a phase 3, open-label, randomized trial. Diabetes Obes Metab. 2019;21:2674–2683. https://doi.org/ 10.1111/dom.13856 \ No newline at end of file diff --git a/medical/md/PMC7207683.md b/medical/md/PMC7207683.md new file mode 100644 index 0000000000000000000000000000000000000000..cdd2bca1fa19407ef994cfdabc609260f1b38b02 --- /dev/null +++ b/medical/md/PMC7207683.md @@ -0,0 +1,30 @@ +disrupted TR signaling; thus, the specific mechanism has not been cleared. It has been well known that proper motor coordination is deeply related to long term depression (LTD) of synaptic transmission from parallel fiber (PF) to Purkinje cell (PC) in the cerebellum (Ito, 1989). Therefore, we examined the involvement of TR in synaptic plasticity at PF-PC synapses by using transgenic mice (Mf-1 mice) which express dominant-negative TR specifically in PCs. Since Mf-1 display the impairment of motor coordination and motor learning, a decrease in TR signaling in PCs may alter synaptic plasticity and contribute to motor incoordination. A whole-cell patch clamp recording of Mf-1 PCs revealed the inhibition of LTD but instead the induction of long term potentiation (LTP) of the synaptic transmission at PF-PC synapses. This indicates that the intracellular calcium dynamics may be disrupted in Mf-1 PCs since LTD +requires a higher elevation of the intracellular calcium concentration in PCs than LTP does. Indeed, single-PC qPCR +showed that the mRNA levels of some important molecules for the intracellular calcium dynamics in PCs (SERCA2, IP3 R, and P/Q-type calcium channel) are downregulated in Mf-1 PCs. This result suggests those genes as possible TH-target genes. Taken together, the present study suggested a novel possible role of TR in synaptic plasticity at PF-PC synapses by regulating the expression of some important genes for LTD occurrence in the cerebellum. This finding could give a new insight into the mechanism of motor deficits in thyroid diseases. + +## Neuroendocrinology And Pituitary Case Reports In Secretory Pituitary Pathologies, Their Treatments And Outcomes + +Crooke's Cell Adenoma- an Aggressive Form of Cushing's Disease Natasa Radovanovic, MD1, Yuen Alexa, MD2, Vafa Tabatabaie, MD2. 1James J Peters VAMC, The Icahn School of Medicine at Mount Sinai, Bronx, NY, USA, 2Montefiore Medical Center, The University Hospital for Albert Einstein College of Medicine, Bronx, NY, USA. + +## Sat-279 + +Introduction: Cushing's disease is a condition of cortisol overproduction caused by an ACTH-producing tumor. + +Corticotroph cells surrounding an ACTH-producing tumor usually undergo Crooke's hyaline change, where cytokeratin filaments accumulate in the cytoplasm in response to glucocorticoid excess. These changes are thought to be a mechanism of feedback inhibition and thus facilitate a suppression of ACTH. However, in a subtype of ACTH-secreting tumors known as Crooke's cell adenomas +(CCA), the ACTH-producing cells also undergo these hyaline changes. This would be expected to suppress hormone secretion but these cells are still able to release significant amounts of ACTH. + +Case presentation: A 32-year-old woman presented to the hospital after an episode of syncope. On head MRI, she was found to have a 2 cm sellar mass with optic chiasm compression. Labs showed low TSH, free FT4, T3, FSH, and LH. She was also pre-diabetic with an HgbA1c of 6.2%. Her baseline cortisol of 20.6 µg/dL did not suppress after 1 mg of dexamethasone. After receiving 4 mg of dexamethasone, her cortisol suppressed to 5.2 µg/dL. She was diagnosed with hypopituitarism except for cortisol and a likely ACTHproducing pituitary macroadenoma. She completed a transsphenoidal pituitary resection and pathology revealed Crooke's hyaline changes with immunohistochemical stains positive for ACTH. The immunostain for the proliferation marker Ki67 showed a relatively low proliferation index. Her course was complicated by diabetes insipidus. + +She was ultimately discharged on 20 mg hydrocortisone each morning, 10 mg hydrocortisone each afternoon, desmopressin 0.05 µg daily, and levothyroxine 125 µg daily. Two weeks later, the patient was sent to the emergency room by her endocrinologist for hyperglycemia up to 288 mg/dL. She was also found to be newly diabetic with an HgbA1c of 6.5%. A fasting morning cortisol was collected during her admission and showed a cortisol level of <1.0 µg/ dL, proving she was cured of Cushing's disease. However, she will need close endocrinology follow up and MRI imaging of her pituitary for this aggressive type of pituitary adenoma. + +Discussion: We have come across an interesting case of a young woman who presented for syncope and was found to have a pituitary macroadenoma with pathology consistent with CCA. This type of ACTH-producing tumor is known for aggressive patterns including high rates of recurrence with rates of up to 60% reported in literature, persistent disease after surgery, malignant transformation, and metastases. Despite presentation and symptoms similar to those of other ACTH-producing adenomas, the dangerous pattern of Crooke's cell adenomas necessitate long-term follow-up in affected patients. + +## Diabetes Mellitus And Glucose Metabolism Diabetes Technology And Advances In Clinical Trials + +Real-World Minimed™ 670G System Use and Glycemic Outcomes of Pediatric and Adult Individuals Living with Type 1 Diabetes (T1D) in the United States Robert Alan Vigersky, MD, Michael Stone, MS, Pratik Agrawal, MS, Alex Zhong, MS, Kevin Velado, MS, Toni Cordero, PhD, John Shin, PhD. Medtronic Diabetes, Washington, DC, USA. + +## Or30-01 + +Introduction: The MiniMed™ 670G system was FDAapproved in 2016 for adults and adolescents ≥14yrs, and in 2018 for children ages 7-13yrs with T1D. Since then, use of the system has grown to over 180,000 people in the U.S. The glycemic control benefits of real-world MiniMed™ 670G system Auto Mode use in the U.S. were assessed. + +Methods: System data (aggregated five-minute instances of sensor glucose [SG]) uploaded from March 2017 to July 2019 by individuals (N=118,737) with T1D and ≥7yrs of age who enabled Auto Mode were analyzed to determine the mean % of overall time spent <54mg/dL/<70mg/dL (TBR); between 70-180mg/dL (TIR); and >180mg/dL/>250mg/dL (TAR). The impact of Auto Mode was further assessed in a sub-group of individuals (N=51,254) with, at least, 7 days \ No newline at end of file diff --git a/medical/md/PMC7315235.md b/medical/md/PMC7315235.md new file mode 100644 index 0000000000000000000000000000000000000000..09ac5daf67bf8696bf0055a86a5a830fc720680c --- /dev/null +++ b/medical/md/PMC7315235.md @@ -0,0 +1,284 @@ + +## Review Article + +# Therapeutic Angiogenesis With Sound Waves + +## Tomohiko Shindo, Md, Phd And Hiroaki Shimokawa, Md, Phd + +Along with the progress of global aging, the prognosis of severe ischemic heart disease (IHD) remains poor, and thus the development of effective angiogenic therapy remains an important clinical unmet need. We have developed lowenergy extracorporeal cardiac shock wave therapy as an innovative minimally invasive angiogenic therapy and confirmed its efficacy in a porcine chronic myocardial ischemia model in animal experiments as well as in patients with refractory angina. Since ultrasound is more advantageous for clinical application than shock waves, we then aimed to develop ultrasound therapy for IHD. We demonstrated that specific conditions of low-intensity pulsed ultrasound (LIPUS) therapy improve myocardial ischemia in animal models through the enhancement of angiogenesis mediated by endothelial mechanotransduction. To examine the effectiveness of our LIPUS therapy in patients with severe angina pectoris, we are now conducting a prospective multicenter clinical trial in Japan. Furthermore, to overcome the current serious situation of dementia pandemic but with no effective treatments worldwide, we have recently demonstrated that our LIPUS therapy also improves cognitive impairment in mouse models of Alzheimer's disease and vascular dementia. Here, we summarize the progress in our studies to develop angiogenic therapies with sound waves. + +Keywords: shock wave, ultrasound, angiogenesis, mechanotransduction, nitric oxide + +## 1.1 Introduction + +The endeavor of developing "therapeutic angiogenesis" is still halfway. The study on angiogenesis began in the Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan Received: January 20, 2020; Accepted: April 6, 2020 Corresponding author: Hiroaki Shimokawa, MD, PhD. Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan Tel: +81-22-717-7152, Fax: +81-22-717-7156 E-mail: shimo@cardio.med.tohoku.ac.jp +©2020 The Editorial Committee of Annals of Vascular Diseases. This article is distributed under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the credit of the original work, a link to the license, and indication of any change are properly given, and the original work is not used for commercial purposes. Remixed or transformed contributions must be distributed under the same license as the original. + +1800s when pathologists and anatomists made detailed morphological observations of blood vessels and reported the changes in blood vessel morphology. In the 20th century, an active approach to extracting angiogenic factors from cancer was extensively performed, among which basic fibroblast growth factor was first discovered in the 1980s.1) Folkman found in 1971 that cancer growth depends on angiogenesis and suggested that inhibition of angiogenesis leads to cancer control.2) This has led to dramatic progress in angiogenesis research from the perspective of cancer control. In 1989, Ferrara discovered the vascular endothelial growth factor (VEGF), which had a major impact on angiogenesis research.3) In 1994, Isner and colleagues at Tufts University reported successful treatment with VEGF gene therapy in patients with lower limb ischemia.4) In 1997, VEGF gene therapy was performed for patients with severe ischemic heart disease (IHD), which was difficult to treat surgically or medically, and the results were rather favorable.4) Since then, various methods, such as transplantation of stem cells or endothelial progenitor cells, have been developed. Although many clinical trials were performed based on basic studies, none of them has yet reached established standard treatments. Under such circumstances, we aimed to develop a minimally invasive angiogenic therapy with sound waves (SWs) as a novel therapy. Here, we were able to develop innovative treatments with SWs. + +## 2.1 Development Of Extracorporeal Cardiac Sw Therapy For Ihd + +When the expansion velocity of explosions such as volcanic eruptions and lightning exceeds the speed of sound, SWs are generated on the surface of explosions. From such characteristics of a longitudinal wave, SW contains a single short pressure pulse (<1 μs), followed by a tensile portion with a lower amplitude (several μs). Since SW +propagates through water, fat, and soft tissues, extracorporeal SW lithotripsy was clinically applied for the treatment of urolithiasis more than 30 years ago. In the treatment of urolithiasis, high-intensity SW is applied in order to break the urinary stones effectively. In contrast, we confirmed that low-energy SW, which is ∼10% of that used for urolithiasis, was sufficient to enhance the expressions of VEGF and endothelial nitric oxide (NO) +synthase in cultured human umbilical vein endothelial cells (HUVECs).5) Then, we conducted a series of animal experiments using a porcine model of chronic myocardial ischemia and clinical trials in patients with refractory angina to evaluate the efficacy and safety of this low-energy extracorporeal cardiac SW therapy.5–7) +The number of patients with severe angina pectoris without indication of percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) has been increasing. Based on the data of our in vitro studies with HUVECs, we examined the effect on myocardial ischemia of low-energy SW therapy in a porcine model in vivo.5) By placing an ameroid constrictor around the proximal segment of the left circumflex coronary artery (LCx), we made a porcine model of chronic myocardial ischemia. + +The ameroid constrictor gradually induced a total occlusion of the coronary artery with resultant sustained myocardial ischemia in 4 weeks. At 4 weeks after the surgery, low-energy SW therapy was applied 3 times in the first week in the SW-treated group, while the control group received anesthesia 3 times but without the SW therapy +(n=8, each). The extracorporeal cardiac SW therapy has a built-in echo diagnostic device in the SW generator (Storz Medical AG, Kreuzlingen, Switzerland) to specify the irradiation site under the echo guidance, and the SW was irradiated in synchronization with the R wave of electrocardiogram. The low-energy SW was irradiated for a total of 1800 shots for 9 spots (0.09 mJ/mm2, 200 shots/ +spot) to the LCx region of the ischemic site of the left ventricle (LV).5) Reduced LV wall motion in the LCx region was documented in both the control and the SW-treated groups at 4 weeks after the ameroid constrictor implantation. Importantly, the wall motion of the ischemic area was significantly improved in the SW group at 4 weeks after the SW therapy.5) We also found that SW therapy increased myocardial capillary density associated with enhanced VEGF expression and normalized the regional myocardial blood flow in the ischemic myocardium in vivo.5) In this experiment, no myocardial damage, arrhythmias, or LV perforation were detected after the SW irradiation. Altogether, we demonstrated that the low-energy extracorporeal cardiac SW therapy enhances effective angiogenesis in pigs in vivo and may have potential usefulness as a non-invasive angiogenic therapeutic approach for chronic myocardial ischemia.5) +From the promising results in the experimental in vivo study, we moved to the next step and performed the first clinical trial in patients with refractory angina pectoris without indication of PCI or CABG6) (**Fig. 1**). We applied low-energy SW for 4000–8000 shots to 20–40 spots +(0.09 mJ/mm2, 200 shots/spot) of the ischemic region 3 times in the first week with no anesthesia or analgesics. Importantly, we confirmed that the low-energy SW therapy showed significant improvement of symptoms, reduction in nitroglycerin use, and amelioration of myocardial + +![1_image_0.png](1_image_0.png) perfusion in the ischemic area, as assessed by stress scintigraphy (**Fig. 2**). There were no complications or adverse effects associated with SW therapy. We then performed a second clinical trial of SW therapy in a randomized + +Fig. 1 Extracorporeal cardiac sound wave (SW) therapy in action + +![1_image_1.png](1_image_1.png) + +in a patient with refractory angina pectoris. The machine is equipped with a SW generator head and in-line echocardiography. The SW generator is attached to the chest wall of the patient when used. SW pulse is easily focused on the ischemic myocardium under the guidance of echocardiography. There is no need for anesthesia or analgesics. +Fig. 2 Effects of the extracorporeal cardiac sound wave (SW) +therapy on myocardial perfusion in a patient with refractory angina pectoris. Dipyridamole stress thallium-201 single photon emission computed tomography (SPECT) imaging and polar map in a patient with severe 3-vessel coronary artery disease before and after SW therapy. As a result, it was clearly shown that myocardial perfusion was improved only at the myocardial ischemic site where the SW therapy was applied; in the anteroseptal wall after the first treatment (1st Tx) and in the lateral wall after the 2nd Tx (arrows) in a step-wise manner after the staged SW treatment. (from Ref. 6) +and placebo-controlled manner.7) From the results of this second clinical trial, we demonstrated that the low-energy SW therapy improves LV function in severe IHD patients by inducing effective angiogenesis.7) Following our initial clinical studies, clinical trials were subsequently performed worldwide with positive results.8–14) Although it is still not clear whether it improves the long-term prognosis of angina patients, these results indicate that SW therapy improves the quality of life of those patients. + +After the achievement of these positive results, we examined whether SW therapy is effective for treating a porcine model of LV remodeling after myocardial infarction in vivo.15) We found that the LV ejection fraction and LV end-diastolic volume were significantly improved in the SW group compared with the control group at 4 weeks after SW therapy.15) This was the first experiment that demonstrated SW therapy as an effective and noninvasive approach to ameliorate LV remodeling after myocardial infarction.15) In another porcine acute myocardial infarction (AMI) model, which is induced by myocardial ischemia with 90 min coronary occlusion and afterward reperfusion, SW therapy showed significant improvement of the LV ejection fraction in a condition closer to the clinical setting.16) From the promising results of the two AMI +pig models in vivo, we performed the first clinical trial to evaluate the efficacy and safety of SW therapy in AMI patients.17) In this trial, we included 17 patients with AMI +who were successfully treated with PCI (peak creatine kinase<4,000 U/l), and we applied SWs to the border zone of the infarcted LV area as adjunctive therapy at 2, 4, and 6 days after AMI.17) No procedure-related complications or any side effects were reported in this trial. We found that LV functions assessed by magnetic resonance imaging showed no signs of deleterious LV remodeling at 6 and 12 months after AMI.17) + +## 2.2 Additional Indications Of Sw Therapy + +Based on the positive results mentioned previously, thereafter, various clinical trials were conducted to expand the indication of SW therapy. Generally, peripheral arterial disease (PAD), especially in patients with critical limb ischemia, is often associated with IHD, and the prognosis is poor.18–20) Thus, we first examined the effectiveness of SW therapy in a rabbit hindlimb ischemia model induced by the surgical excision of the entire unilateral femoral artery. Low-energy SW was irradiated for a total of 6,000 shots to 30 spots (0.09 mJ/mm2, 200 shots/spot) of the ischemic region 3 times a week for 3 consecutive weeks. Compared with the control group, blood flow and capillary density were significantly increased at 4 weeks after the operation in the SW group.21) Based on these experimental results, to evaluate the effectiveness of SW therapy in PAD patients, we performed the next clinical trial on 12 PAD patients with intermittent claudication (Fontaine stage II).22) Lowenergy SW was irradiated for a total of 8000 shots to 40 spots (0.1 mJ/mm2, 200 shots/spot) of the ischemic region 3 times a week for 3 consecutive weeks. We confirmed that patient symptoms, maximum walking distance, and peripheral perfusion were significantly improved in the SW +group than in the control group.22) A subsequent study confirmed the effectiveness of low-energy SW therapy in PAD patients in Fontaine stages III and IV.23) As a result, by inducing effective angiogenesis, low-energy SW therapy could be a promising novel, non-invasive therapy for PAD patients. + +Moreover, we further applied our low-energy SW +therapy to patients with systemic sclerosis who suffer from Raynaud's phenomenon and digital skin ulcers.24) In the pathogenesis of digital skin ulcers, not only immune activation but also the endothelial damage and persistent vasospasms are considered to be involved.24) Several studies by our and other groups demonstrated that low-energy SW therapy enhances wound healing in rodents25–28) and humans.29,30) Interestingly, we confirmed that the local wound healing of skin ulcers in a mouse model was significantly enhanced by SW therapy, for which endothelial nitric oxide synthase (eNOS), VEGF, and subsequent angiogenesis may be involved in the process of wound repair.27) To examine whether low-energy SW therapy is effective in patients with digital skin ulcers, a clinical trial of 9 patients was conducted, demonstrating that SW therapy may be effective for the treatment of refractory digital ulcers due to systemic sclerosis.31) +In general, some cases of refractory lymphedema cannot be completely cured even by surgery, and such patients have limited treatment options. Thus, we next examined the beneficial effects of low-energy SW therapy on animal models of secondary lymphedema in vivo.32,33) The results showed that SW therapy significantly enhanced the expressions of VEGF-C and basic fibroblast growth factor (bFGF), which improved the lymphatic system and lymphatic density. From these results, low-energy SW therapy may also induce therapeutic lymphangiogenesis in lymphedema through the upregulations of VEGF-C and bFGF, +suggesting that SW therapy is a non-invasive therapy for patients with lymphedema.32,33) +Finally, for the treatment of orthopedic diseases, such as bone non-unions, tendinosis calcarea, epicondylitis, and calcaneal spur, middle levels of SW have been widely used, expecting anti-inflammatory effects.34–37) In the rat spinal cord injury model, we have previously demonstrated that low-energy SW therapy exerts beneficial effects on the locomotor functions in vivo.38) Through the upregulation of VEGF, SW therapy attenuated nerve injury and promoted the recovery of locomotor function. This landmark study demonstrated a new non-invasive therapeutic strategy with low-energy SW for spinal cord injury.38) + +## 3.1 Development Of Low-Intensity Pulsed Ultrasound (Lipus) Therapy For Ihd + +As mentioned above, we reported that low-energy cardiac SW therapy induces neovascularization and effectively ameliorates myocardial dysfunction in animals and humans. Since both SW and ultrasound are SWs and they similarly travel straight through body tissue (fats, muscles, body fluids, etc.), we then examined the potential feasibility of LIPUS for the treatment of IHD as the next generation of non-invasive angiogenic therapy.39) Ultrasound is defined as the SW whose frequency is higher than the basic audible range for humans (>20 kHz), and diagnostic ultrasonography has been widely used for more than 50 years. Recently, ultrasound is also used for therapeutic applications, including tumor ablation, thrombolysis, bone regeneration, and drug delivery system.39) The angiogenic potential of low-intensity ultrasound has been reported in several endothelial cells, chick chorioallantoic membrane, and a rat model of hind limb ischemia.40–42) We examined the various conditions of LIPUS, such as acoustic pressure and the number of cycles (1, 16, 32, 48, and 64), with a needle hydrophone (**Fig. 3**). The number of acoustic waves per 1 pulse is called the number of cycles, and generally, 1 cycle is a condition used for diagnostic devices. + +To prevent the temperature rise of the ultrasound probe, the estimated spatial peak temporal average intensity of LIPUS was controlled to keep it under the upper limit of acoustic output standards (<720 mW/cm2) for diagnostic ultrasound devices (U.S. Food and Drug Administration's Track 3 Limits). We identified that when LIPUS was applied to HUVECs, the maximal effect to upregulate VEGF +messenger ribonucleic acid (mRNA) expression was noted at 32 cycles.43) Although higher intensity ultrasound is used for thrombolysis or tumor ablation (high-intensity focused ultrasound), the intensity of the LIPUS in our study was within the diagnostic range.44) With these findings, we were able to demonstrate that LIPUS therapy induces therapeutic angiogenesis and ameliorates myocardial ischemia in a porcine model of chronic myocardial ischemia.43) In this study, at 8 weeks (post-treatment), left ventricle ejection fraction (LVEF) was normalized in the LIPUS-treated group, whereas it remained unchanged in the control group without the LIPUS (**Fig. 4**). Indeed, the LIPUS therapy normalized global and regional myocardial functions and increased capillary density and regional myocardial blood flow in the chronically ischemic region without any adverse effects, and significantly enhanced protein expressions of VEGF, eNOS, and bFGF in the ischemic myocardium but not in the non-ischemic myocardium in vivo.43) Moreover, in a mouse model of AMI, LIPUS therapy enhanced angiogenesis, ameliorated post-myocardial infarction (MI) LV remodeling, and improved the mortality.45) In this study, we used LIPUS to treat the animals on days 1, 3, and 5 and found enhanced + +![3_image_1.png](3_image_1.png) + +![3_image_0.png](3_image_0.png) + +phosphorylation of ERK1/2 and Akt on day 3 and the upregulation of VEGF and eNOS on day 6.45) Although the LIPUS therapy was performed only in the acute phase of AMI associated with the upregulation of VEGF and eNOS, it enhanced capillary density and ameliorated post-MI LV remodeling in the chronic phase.45) These results indicate that LIPUS therapy is an effective and safe therapeutic strategy for ischemia-induced myocardial dysfunction. Based on these encouraging results, in order to confirm the effectiveness and safety of LIPUS therapy in humans, we started a double blind, placebo-controlled trial in patients with severe angina pectoris at 10 cardiovascular institutes in Japan. We have already completed patient enrollment, and the trial will be completed in 2020. + +## 3.2 Possible Mechanisms For The Beneficial Effects Of Lipus Therapy + +Vascular endothelial cells cover the inner surface of blood vessels and are directly exposed to blood flow-induced mechanical stimuli, including shear stress. These stimuli invoke specific responses within the cells, leading to changes in their intrinsic structure and function.46) Endothelial cells may sense these stimuli and convert them into a sequence of biological responses. Caveolae are flask-like invaginations of the plasma membrane 40–80 nm in diameter organized by caveolins.47,48) One of the important functions of the caveolae is the conversion of mechanical stimuli into chemical signals by flow-sensing organelles transmitted into the cells, called mechanotransduction.47) Caveolins bind to a variety of proteins involved in the signaling pathways such as G-protein subunits, tyrosine kinases, NO synthase, small GTPases, and growth factor receptors.49–52) Caveolar membranes are also enriched in cholesterol, glycosphingolipids, and signaling enzymes such as Src kinase.53) In addition, caveolae are reported to respond to cell stretch, thus contributing to stretchinduced signaling.54) On the other hand, integrins are reported to regulate multiple pathways, including Erk, PI3K, +FAK, Src, and Rho GTPases.55–57) Although caveolin-1 has no extracellular component, it plays an important role in sensing mechanical stress or the distortion of the extracellular membranes through interaction with β1integrin.51,52,58–60) In cultured cell experiments, the degree of the LIPUS-induced upregulation of mRNA was higher in HUVECs than in human cardiomyocytes, suggesting that vascular endothelial cells may be the main player for the LIPUS-induced angiogenesis.45) Microarray analysis showed that not only the VEGF signaling pathway but also the focal adhesion pathway were significantly affected by LIPUS.45) The focal adhesion pathway contains several proteins on the cell membrane, including caveolin-1 and β1-integrin, both of which are known to play key roles in the mechanotransduction process.47,48) Especially, caveolin-1 is required to maintain the structure of caveolae.47,48) The results of our siRNA experiments suggest that caveolin-1, β1-integrin, Fyn, FAK, ERK1/2, and Akt are all involved in the LIPUS-induced upregulation of VEGF.45) In addition, we found that the conformational changes of caveolae by either the knockdown of serum deprivation-response protein with small interfering RNA +or administration of methyl-β-cyclodextrin suppressed the LIPUS-induced upregulation of VEGF.45) Furthermore, we demonstrated that the beneficial effects of LIPUS +therapy on post-MI LV remodeling were blunted in Cav1-knockout (KO) mice.45) LIPUS-induced upregulation of angiogenic molecules was also blunted in the eCav-1-KO mice, suggesting that endothelial cells play pivotal roles in the angiogenic effects of LIPUS therapy.45) Taken together, these results suggest that acoustic streaming by LIPUS induces distortion of caveolae on endothelial cells, which then transmits the mechanical stimuli to intracellular signaling pathways with subsequent phosphorylation of Fyn, FAK, Erk1/2, and Akt and resultant enhanced expression of VEGF and angiogenesis45) (**Fig. 5**). We also confirmed that the same mechanisms are involved in the angiogenic effects of low-energy SW61) (**Fig. 5**). + +The molecules mentioned above, such as Fyn, FAK, β1integrin and Caveolin-1, are also known to play key roles in cell proliferation and angiogenesis induced by mechanical stimuli (e.g., shear stress) on the surface of vascular endothelial cells.51–53) Microarray analysis also suggested that LIPUS exerts biological effects on cell cycles, metabolic pathways, RNA transport, deoxyribonucleic acid +(DNA) replication, mRNA surveillance, mismatch repair, and protein export in addition to its angiogenic effects.45) Ultrasound has been reported to induce sonoporation and subsequent influx of calcium ion, which was correlated to LIPUS-induced bioeffects in cultured cells.62) Thus, LIPUS may exert several biological effects through alteration of intracellular calcium ion levels, and this point remains to be fully elucidated in future studies. Finally, it is reported that the shear stress-induced intracellular signaling is mediated by the activation of β1-integrin and concurrent caveolin-1 phosphorylation.63) β1-integrin-mediated activation of the ERK1/2 and PI3-Akt pathways is mediated by caveolin-1.64) In our study, endothelial expression of caveolin-1 was enhanced in the infarcted area early after AMI in mice and autopsy samples of AMI patients, suggesting that the abrupt reduction in coronary flow and shear stress affects endothelial cells in the ischemic myocardium to upregulate caveolin-1, leading to an increased sensitivity to LIPUS.45) Moreover, we examined whether LIPUS therapy also ameliorates contractile dysfunction in LV pressure-overloaded hearts in vivo.65) Chronic LV pressure overload was induced with transverse aortic con- + +![5_image_0.png](5_image_0.png) + +striction (TAC) in mice, and at 8 weeks after TAC, TACinduced LV dysfunction was significantly ameliorated in the LIPUS-treated group compared with the control group.65) LIPUS therapy may also ameliorate contractile dysfunction in chronically pressure-overloaded hearts through enhanced myocardial angiogenesis and attenuated perivascular fibrosis.65) + +## 3.3 Additional Important Indications Of The Lipus Therapy + +Based on experimental studies of the heart, we aimed to expand the indications of our LIPUS therapy for other disorders, and one of them is dementia. In 2015, approximately 47 million patients worldwide were diagnosed as having dementia, and it is estimated that by 2050, more than 131 million patients will be diagnosed.66) However, at this moment, no curative treatment is available for vascular dementia (VaD) or Alzheimer's disease (AD),67,68) +both of which comprise the most common causes of dementia. It was reported that low-intensity ultrasound (but not pulsed ultrasound like ours) increases the production of a brain-derived neurotrophic factor in astrocytes69) and nerve growth factor in PC12 cells70) and promotes nerve regeneration.71) Recent studies have suggested that cerebral microcirculatory dysfunction, especially reduced NO bioavailability, plays an important role in the pathogenesis of AD in addition to VaD.72,73) Based on our encouraging results in animal models of myocardial ischemia and these recent studies, we performed basic experiments using 2 mouse models of dementia (VaD and AD) to examine our hypothesis that whole-brain LIPUS therapy is useful for the treatment of dementia.74) We used bilateral carotid artery stenosis as a VaD model,74) and we used 5XFAD transgenic mice as an AD model.74) Interestingly, our LIPUS therapy markedly ameliorated cognitive impairments (e.g., Y-maze test and/or passive avoidance test) associated with improved cerebral blood flow in both models.74) In the VaD model, LIPUS therapy significantly increased CD31-positive endothelial cells and Olig2-positive oligodendrocyte precursor cells, while in the AD model, it reduced Iba-1-positive microglias and amyloid-β (Aβ plaque)74) (**Fig. 6**). Mechanistically, in both models, RNA-sequencing showed that endotheliumrelated genes such as eNOS were significantly upregulated. + +Moreover, significant correlations were noted between the increases in glial cells and neurotrophin and eNOS expressions.74) Importantly, these preferable effects of LIPUS were totally absent in eNOS-KO mice.74) These results indicate the effectiveness and safety of the whole- + +![6_image_0.png](6_image_0.png) + +Fig. 6 Low-intensity pulsed ultrasound (LIPUS) therapy reduces Aβ plaques in the Alzheimer's disease model in mice. + +Representative images of coronal sections immunostained for Aβ (stained in brown) on day 86 after LIPUS treatment; the lower 3 panels show magnified views of the cerebral cortex, hippocampus, and thalamus. As compared with the control group, LIPUS therapy markedly reduced the Aβ deposition throughout the brain. The scale bars represent 500 µm (whole-brain) or 50 µm (lower 3 panels). + +(from Ref. 70) +brain LIPUS for dementia and the involvement of eNOS as a common molecular mechanism74) (**Fig. 7**). Based on these encouraging results, we have started a single-center, double blind, placebo-controlled trial with whole-brain LIPUS therapy for patients with AD. The trial will be completed in 2021. + +## 4.1 Conclusion + +Novel physiotherapy applying SW or LIPUS showed a new possibility for angiogenic therapy, and a series of evidence has been emerging. Interestingly, we were able to demonstrate that common intracellular mechanisms and pathways appear to be involved in the angiogenic effects of SW and LIPUS, including β1-integrin/caveolin-1 in the endothelial caveolae, ERK1/2 and Akt phosphorylations, VEGF/eNOS expressions, and eventually, endothelial proliferation.45,61) However, the physical characteristics are different between the two SWs; SW therapy produces strong shear stress, while LIPUS has low shear stress with some thermal effects. Thus, it is important to use the two + +Fig. 7 Schematic diagram showing the proposed mechanisms + +![6_image_1.png](6_image_1.png) + +underlying the beneficial effects of the whole-brain lowintensity pulsed ultrasound (LIPUS) therapy in two mice dementia models. Our findings indicate that activation of endothelial nitric oxide synthase (eNOS) plays a key role for the beneficial effects of LIPUS therapy in both models. eNOS increases proliferation of oligodendrocyte precursor cells (OPCs) in the vascular dementia (VaD) model and decreases microglias in the Alzheimer's disease (AD) model. The proliferation of OPCs leads to an increase in mature oligodendrocytes, ultimately leading to re-myelination. eNOS also inhibits production and accumulation of Aβ. eNOS-induced angiogenesis plays an important role in neurogenesis via a change in cerebral blood flow. Finally, the cognitive dysfunction can be ameliorated by re-myelination, angiogenesis, and neurogenesis in the VaD model, and by reduced microgliosis and Aβ plaques and increased angiogenesis in the AD model. (from Ref. 70) +SWs properly according to the target organ. For example, SW therapy may be more suitable for applying to hard tissues, such as bones and cartilage, while LIPUS may be more suitable for soft tissues, such as the brain and internal organs. Furthermore, although SW therapy has a potential risk of pulmonary hemorrhage, LIPUS has no apparent adverse effects independent of its target organ. The beneficial effects of SW therapy and LIPUS may be commonly mediated by enhanced various intrinsic pathways, where mechanotransduction and its downstream pathways appear to be involved. Although the precise intracellular mechanisms remain to be fully elucidated, low-energy extracorporeal SW and LIPUS therapies are promising as effective, safe, and non-invasive approaches for not only ischemic cardiovascular disorders but also a wide range of ischemic/inflammatory disorders. + +## Acknowledgments + +The authors thank the collaborators at Kyushu University (Takeshita A, Nishida T, Oi K, Uwatoku T, Abe K, Eto M, Matsumoto M, Fukumoto Y, Ito A, Matoba T, Kishi T) and those at Tohoku University (Ito K, Kikuchi Y, Ito Y, Shiroto T, Tsuburaya R, Aizawa K, Hao K, Fukumoto Y, Takahashi J, Takeda M, Nakayama M, Yasuda S, Kuriyama S, Tsuji I, Hanawa K, Hasebe Y, Hasegawa H, Kanai H, Kagaya Y, Nishimiya K, Ogata T, Kurosawa R, Eguchi K, Monma Y, Ichijo S, Hatanaka K, Miyata S, Kasukabe S, Taki H, Watanabe Y, Nishihara A, and Yamashita H). + +## Sources Of Funding + +This study was supported in part by the grants-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology, Tokyo, Japan, the Japanese Ministry of Health, Labor, and Welfare, Tokyo, Japan, and the Japan Agency for Medical Research and Development (AMED), Tokyo, Japan. + +## Disclosure Statements None Author Contributions + +Study conception: HS Data collection: TS Investigation: all authors Writing: all authors Funding acquisition: HS Critical review and revision: all authors Final approval of the article: all authors Accountability for all aspects of the work: all authors + +## References + + 1) Gospodarowicz D, Neufeld G, Schweigerer L. Fibroblast growth factor: structural and biological properties. J Cell Physiol Suppl 1987; 133 **Suppl 5**: 15-26. + + 2) Folkman J. Tumor angiogenesis: therapeutic implications. N +Engl J Med 1971; 285: 1182-6. + + 3) Ferrara N. Vascular endothelial growth factor and the regulation of angiogenesis. Recent Prog Horm Res 2000; 55: 15-35; discussion, 35-6. + + 4) Vale PR, Isner JM, Rosenfield K. Therapeutic angiogenesis in critical limb and myocardial ischemia. J Interv Cardiol 2001; 14: 511-28. + + 5) Nishida T, Shimokawa H, Oi K, et al. Extracorporeal cardiac shock wave therapy markedly ameliorates ischemia-induced myocardial dysfunction in pigs in vivo. Circulation 2004; 110: 3055-61. + + 6) Fukumoto Y, Ito A, Uwatoku T, et al. Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease. Coron Artery Dis 2006; 17: 63-70. + + 7) Kikuchi Y, Ito K, Ito Y, et al. Double-blind and placebo-controlled study of the effectiveness and safety of extracorporeal cardiac shock wave therapy for severe angina pectoris. Circ J 2010; 74: 589-91. + + 8) Khattab AA, Brodersen B, Schuermann-Kuchenbrandt D, +et al. Extracorporeal cardiac shock wave therapy: first experience in the everyday practice for treatment of chronic refractory angina pectoris. Int J Cardiol 2007; 121: 84-5. + + 9) Prinz C, Lindner O, Bitter T, et al. Extracorporeal cardiac shock wave therapy ameliorates clinical symptoms and improves regional myocardial blood flow in a patient with severe coronary artery disease and refractory angina. Case Rep Med 2009; **2009**: 639594. + +10) Vasyuk YA, Hadzegova AB, Shkolnik EL, et al. Initial clinical experience with extracorporeal shock wave therapy in treatment of ischemic heart failure. Congest Heart Fail 2010; 16: +226-30. + +11) Wang Y, Guo T, Cai HY, et al. Cardiac shock wave therapy reduces angina and improves myocardial function in patients with refractory coronary artery disease. Clin Cardiol 2010; 33: 693-9. + +12) Wang Y, Guo T, Ma TK, et al. A modified regimen of extracorporeal cardiac shock wave therapy for treatment of coronary artery disease. Cardiovasc Ultrasound 2012; 10: +35. + +13) Yang P, Guo T, Wang W, et al. Randomized and double-blind controlled clinical trial of extracorporeal cardiac shock wave therapy for coronary heart disease. Heart Vessels 2013; 28: +284-91. + +14) Schmid JP, Capoferri M, Wahl A, et al. Cardiac shock wave therapy for chronic refractory angina pectoris. A prospective placebo-controlled randomized trial. Cardiovasc Ther 2013; 31: e1-6. + +15) Uwatoku T, Ito K, Abe K, et al. Extracorporeal cardiac shock wave therapy improves left ventricular remodeling after acute myocardial infarction in pigs. Coron Artery Dis 2007; 18: 397-404. + +16) Ito Y, Ito K, Shiroto T, et al. Cardiac shock wave therapy ameliorates left ventricular remodeling after myocardial ischemia-reperfusion injury in pigs in vivo. Coron Artery Dis 2010; 21: 304-11. + +17) Kagaya Y, Ito K, Takahashi J, et al. Low-energy cardiac shockwave therapy to suppress left ventricular remodeling in patients with acute myocardial infarction: first-in-human study. Coron Artery Dis 2018; 29: 294-300. + +18) Hiatt WR. Medical treatment of peripheral arterial disease and claudication. N Engl J Med 2001; 344: 1608-21. + +19) Wennberg PW. Approach to the patient with peripheral arterial disease. Circulation 2013; 128: 2241-50. + +20) Jaff MR, White CJ, Hiatt WR, et al. An update on methods for revascularization and expansion of the TASC lesion classification to include below-the-knee arteries: a supplement to the inter-society consensus for the management of peripheral arterial disease (TASC II): The TASC steering committee. Catheter Cardiovasc Interv 2015; 86: 611-25. + +21) Oi K, Fukumoto Y, Ito K, et al. Extracorporeal shock wave therapy ameliorates hindlimb ischemia in rabbits. Tohoku J Exp Med 2008; 214: 151-8. + +22) Serizawa F, Ito K, Kawamura K, et al. Extracorporeal shock wave therapy improves the walking ability of patients with peripheral artery disease and intermittent claudication. Circ J 2012; 76: 1486-93. + +23) Tara S, Miyamoto M, Takagi G, et al. Low-energy extracorporeal shock wave therapy improves microcirculation blood flow of ischemic limbs in patients with peripheral arterial disease: pilot study. J Nippon Med Sch 2014; 81: 19-27. + +24) Abraham DJ, Varga J. Scleroderma: from cell and molecular mechanisms to disease models. Trends Immunol 2005; 26: +587-95. + +25) Stojadinovic A, Elster EA, Anam K, et al. Angiogenic response to extracorporeal shock wave treatment in murine skin isografts. Angiogenesis 2008; 11: 369-80. + +26) Yan X, Zeng B, Chai Y, et al. Improvement of blood flow, expression of nitric oxide, and vascular endothelial growth factor by low-energy shockwave therapy in random-pattern skin flap model. Ann Plast Surg 2008; 61: 646-53. + +27) Hayashi D, Kawakami K, Ito K, et al. Low-energy extracorporeal shock wave therapy enhances skin wound healing in diabetic mice: a critical role of endothelial nitric oxide synthase. Wound Repair Regen 2012; 20: 887-95. + +28) Weihs AM, Fuchs C, Teuschl AH, et al. Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation. J Biol Chem 2014; 289: 27090-104. + +29) Saggini R, Figus A, Troccola A, et al. Extracorporeal shock wave therapy for management of chronic ulcers in the lower extremities. Ultrasound Med Biol 2008; 34: 1261-71. + +30) Moretti B, Notarnicola A, Maggio G, et al. The management of neuropathic ulcers of the foot in diabetes by shock wave therapy. BMC Musculoskelet Disord 2009; 10: 54. + +31) Saito S, Ishii T, Kamogawa Y, et al. Extracorporeal shock wave therapy for digital ulcers of systemic sclerosis: a phase 2 pilot study. Tohoku J Exp Med 2016; 238: 39-47. + +32) Kubo M, Li TS, Kamota T, et al. Extracorporeal shock wave therapy ameliorates secondary lymphedema by promoting lymphangiogenesis. J Vasc Surg 2010; 52: 429-34. + +33) Serizawa F, Ito K, Matsubara M, et al. Extracorporeal shock wave therapy induces therapeutic lymphangiogenesis in a rat model of secondary lymphedema. Eur J Vasc Endovasc Surg 2011; 42: 254-60. + +34) Ogden JA, Alvarez RG, Levitt R, et al. Shock wave therapy +(Orthotripsy) in musculoskeletal disorders. Clin Orthop Relat Res 2001; 387: 22-40. + +35) Birnbaum K, Wirtz DC, Siebert CH, et al. Use of extracorporeal shock-wave therapy (ESWT) in the treatment of nonunions. A review of the literature. Arch Orthop Trauma Surg 2002; 122: 324-30. + +36) Wang CJ. Extracorporeal shockwave therapy in musculoskeletal disorders. J Orthop Surg Res 2012; 7: 11. + +37) Al-Abbad H, Simon JV. The effectiveness of extracorporeal shock wave therapy on chronic Achilles tendinopathy: a systematic review. Foot Ankle Int 2013; 34: 33-41. + +38) Yamaya S, Ozawa H, Kanno H, et al. Low-energy extracorporeal shock wave therapy promotes vascular endothelial growth factor expression and improves locomotor recovery after spinal cord injury. J Neurosurg 2014; 121: 1514-25. + +39) ter Haar G. Therapeutic applications of ultrasound. Prog Biophys Mol Biol 2007; 93: 111-29. + +40) Barzelai S, Sharabani-Yosef O, Holbova R, et al. Lowintensity ultrasound induces angiogenesis in rat hind-limb ischemia. Ultrasound Med Biol 2006; 32: 139-45. + +41) Mizrahi N, Seliktar D, Kimmel E. Ultrasound-induced angiogenic response in endothelial cells. Ultrasound Med Biol 2007; 33: 1818-29. + +42) Ramli R, Reher P, Harris M, et al. The effect of ultrasound on angiogenesis: an in vivo study using the chick chorioallantoic membrane. Int J Oral Maxillofac Implants 2009; 24: +591-6. + +43) Hanawa K, Ito K, Aizawa K, et al. Low-intensity pulsed ultrasound induces angiogenesis and ameliorates left ventricular dysfunction in a porcine model of chronic myocardial ischemia. PLoS One 2014; 9: e104863. + +44) Maloney E, Hwang JH. Emerging HIFU applications in cancer therapy. Int J Hyperthermia 2015; 31: 302-9. 45) Shindo T, Ito K, Ogata T, et al. Low-intensity pulsed ultrasound enhances angiogenesis and ameliorates left ventricular dysfunction in a mouse model of acute myocardial infarction. Arterioscler Thromb Vasc Biol 2016; 36: 1220-9. + +46) Mizrahi N, Seliktar D, Kimmel E. Ultrasound-induced angiogenic response in endothelial cells. Ultrasound Med Biol 2007; 33: 1818-29. + +47) Parton RG. Cell biology-life without caveolae. Science 2001; 293: 2404-5. + +48) Parton RG, del Pozo MA. Caveolae as plasma membrane sensors, protectors and organizers. Nat Rev Mol Cell Biol 2013; 14: 98-112. + +49) Sedding DG, Hermsen J, Seay U, et al. Caveolin-1 facilitates mechanosensitive protein kinase B (Akt) signaling in vitro and in vivo. Circ Res 2005; 96: 635-42. + +50) Sonveaux P, Martinive P, DeWever J, et al. Caveolin-1 expression is critical for vascular endothelial growth factorinduced ischemic hindlimb collateralization and nitric oxidemediated angiogenesis. Circ Res 2004; 95: 154-61. + +51) del Pozo MA, Balasubramanian N, Alderson NB, et al. + +Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization. Nat Cell Biol 2005; 7: 901-8. + +52) Zemljič Jokhadar Š, Majhenc J, Svetina S, et al. Positioning of integrin β1, caveolin-1 and focal adhesion kinase on the adhered membrane of spreading cells. Cell Biol Int 2013; 37: 1276-84. + +53) Wary KK, Mariotti A, Zurzolo C, et al. A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell 1998; 94: 625-34. + +54) Yu J, Bergaya S, Murata T, et al. Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels. J Clin Invest 2006; 116: 1284-91. + +55) Salanueva IJ, Cerezo A, Guadamillas MC, et al. Integrin regulation of caveolin function. J Cell Mol Med 2007; 11: 969-80. + +56) Shyy JY, Chien S. Role of integrins in endothelial mechanosensing of shear stress. Circ Res 2002; 91: 769-75. + +57) Bock-Marquette I, Saxena A, White MD, et al. Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature 2004; 432: 466-72. + +58) Yeo MG, Oh HJ, Cho HS, et al. Phosphorylation of Ser 21 in Fyn regulates its kinase activity, focal adhesion targeting, and is required for cell migration. J Cell Physiol 2011; 226: 236-47. + +59) Gervasio OL, Phillips WD, Cole L, et al. Caveolae respond to cell stretch and contribute to stretch-induced signaling. J Cell Sci 2011; 124: 3581-90. + +60) Stein CK, Qu P, Epstein J, et al. Removing batch effects from purified plasma cell gene expression microarrays with modified ComBat. BMC Bioinformatics 2015; 16: 63. + +61) Hatanaka K, Ito K, Shindo T, et al. Molecular mechanisms of the angiogenic effects of low-energy shock wave therapy: roles of mechanotransduction. Am J Physiol 2016; 311: +C378-85. + +62) Rizzo V, Sung A, Oh P, et al. Rapid mechanotransduction in situ at the luminal cell surface of vascular endothelium and its caveolae. J Biol Chem 1998; 273: 26323-9. + +63) Echarri A, Del Pozo MA. Caveolae internalization regulates integrin-dependent signaling pathways. Cell Cycle 2006; 5: 2179-82. + +64) Jasmin JF, Rengo G, Lymperopoulos A, et al. Caveolin-1 deficiency exacerbates cardiac dysfunction and reduces survival in mice with myocardial infarction. Am J Physiol Heart Circ Physiol 2011; 300: H1274-81. + +65) Ogata T, Ito K, Shindo T, et al. Low-intensity pulsed ultrasound enhances angiogenesis and ameliorates contractile dysfunction of pressure-overloaded heart in mice. PLoS One 2017; 12: e0185555. + +66) Prince M, Adelina CH, Knapp M, et al. World Alzheimer Report 2016. London: Alzheimer's Disease International, 2016. + +67) Scheltens P, Blennow K, Breteler MM, et al. Alzheimer's disease. Lancet 2016; 388: 505-17. + +68) O'Brien JT, Thomas A. Vascular dementia. Lancet 2015; 386: +1698-706. + +69) Liu SH, Lai YL, Chen BL, et al. Ultrasound enhances the expression of brain-derived neurotrophic factor in astrocyte through activation of TrkB-Akt and Calcium-CaMK signaling pathways. Cereb Cortex 2017; 27: 3152-60. + +70) Zhao L, Feng Y, Hu H, et al. Low-intensity pulsed ultrasound enhances nerve growth factor-induced neurite outgrowth through mechanotransduction-mediated ERK1/2-CREB-Trx-1 signaling. Ultrasound Med Biol 2016; 42: 2914-25. + +71) Chang CJ, Hsu SH, Lin FT, et al. Low-intensity-ultrasoundaccelerated nerve regeneration using cell-seeded poly(D,Llactic acid-co-glycolic acid) conduits: an in vivo and in vitro study. J Biomed Mater Res B Appl Biomater 2005; 75B: +99-107. + +72) Katusic ZS, Austin SA. Endothelial nitric oxide: protector of a healthy mind. Eur Heart J 2014; 35: 888-94. + +73) Iadecola C. Untangling neurons with endothelial nitric oxide. Circ Res 2016; 119: 1052-4. + +74) Eguchi K, Shindo T, Ito K, et al. Whole-brain low-intensity pulsed ultrasound therapy markedly improves cognitive dysfunctions in mouse models of dementia—crucial roles of endothelial nitric oxide synthase. Brain Stimul 2018; 11: +959-73. \ No newline at end of file diff --git a/medical/md/PMC7351458.md b/medical/md/PMC7351458.md new file mode 100644 index 0000000000000000000000000000000000000000..737e6ec9eafa395701c4473fe15e34dac3f53e71 --- /dev/null +++ b/medical/md/PMC7351458.md @@ -0,0 +1,201 @@ +# Compassionate Design: Utilizing Design Thinking To Preserve Sanctity, Dignity, And Personhood When Children Die Daniel H. Grossoehme, Dmin*†; Melissa S. Mark, Md‡§; Blake Lane, Phd¶; Allison Rednour, Mdes¶; Rachel Thienprayoon, Md*∥* + +## Abstract + +Introduction: Greater than 70% of children who die in our institution annually die in an intensive care unit (ICU) setting. Family privacy, visitation policies, and an inability to perform religious rituals in the ICU are barriers to provide children with culturally competent, family-centered care when a child dies. The goal of this project was to profoundly understand family and staff experiences surrounding pediatric death in our institution to identify unique opportunities to design improved, novel delivery models of pediatric end of life (EOL) care. Methods: This project utilized a structured process model based on the Vogel and Cagan's 4-phase integrated new product development process model. The 4 phases are identifying, understanding, conceptualizing, and realizing. We utilized an adaptation of this process model that relies on human-centered and design thinking methodologies in 3 phases: research, ideation, and refinement of a process or product opportunity. Results: There were 2 primary results of this project: 5 process and opportunity areas to improve the EOL experience across the hospital, and a set of criteria and considerations for a dedicated EOL space. Discussion: Sometimes, the best outcome we can provide for a child and their family is a peaceful, dignified death. This project utilized human-centered design to create improved process outcomes and to design a dedicated EOL space for children who die in the hospital. Offering grieving families quiet, private time with their child in a beautiful, dignified, peaceful location enables the beginning of improved bereavement outcomes for the family and staff. *(Pediatr Qual Saf 2020;4:e317; doi: 10.1097/pq9.0000000000000317;* Published online 8 July, 2020.) +From the *Haslinger Family Pediatric Palliative Care Center, Akron Children's Hospital, Akron, Ohio; †Rebecca D. Considine Research Institute, Akron Children's Hospital, Akron, Ohio; ‡Eastern Virginia Medical School, Norfolk, Va.; §Children's Hospital of The King's Daughters, Norfolk, Va.; +¶Livewell Collaborative, Cincinnati, Ohio; ∥Department of Anesthesia, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; and *University of Cincinnati College of Medicine, Cincinnati, Ohio Presented at the 2019 Assembly, American Academy of Hospice and Palliative Care, Orlando, FL, March 15, 2019. This study was funded by a Design Thinking Research Award from the University of Cincinnati (UC) Centers for Clinical and Translational Science and Training. Supplemental digital content is available for this article. Clickable URL citations appear in the text. *Corresponding author. Address: Daniel H. Grossoehme, DMin, Haslinger Family Pediatric Palliative Care Center, Akron Children's Hospital, 1 Perkins Square, Akron, OH 44308 PH: 1-330-543-0356 Email: dgrossoehme@akronchildrens.org Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open-access article distributed under the terms of the Creative Commons AttributionNon Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. To cite: Grossoehme DH, Mark MS, Lane B, Rednour A, Thienprayoon R. Compassionate Design: Utilizing Design Thinking to Preserve Sanctity, Dignity, and Personhood When Children Die. Pediatr Qual Saf 2020;4:e317. Received for publication December 23, 2019; Accepted June 2, 2020. Published online 8 July 2020 DOI: 10.1097/pq9.0000000000000317 + +## Introduction + +![0_Image_0.Png](0_Image_0.Png) + +![0_Image_1.Png](0_Image_1.Png) + +Pediatric hospitals' care for children and families usually equates "care" with +"cure."1 When a cure is not possible, care means focusing on compassion and comfort. Children deserve a beautiful, dignified place to die; their families deserve a quiet, peaceful place to say goodbye. + +However, the majority of children who die in children's hospitals do so in an intensive care unit (ICU) setting.2 A total of 445 patients affiliated with this institution died in 2016; 169 died on inpatient units within the hospital, of whom 73% died in an ICU. This fact presents clinicians from numerous disciplines with significant challenges. + +Numerous factors influence parental experience and bereavement outcomes as a child approaches death and immediately afterward in this institution (Fig. 1). Memories of a child and the child's death critically impact the way a family grieves; positive experiences predict better bereavement outcomes.3 Interviews with bereaved mothers identified four end of life (EOL) and postmortem care needs: (1) time with their dying child; (2) space and privacy with their dying child; (3) time with the child's body; and (4) space and privacy with their child's body.3 Mothers reported "peace and serenity" following their child's death when their postmortem care needs were met.4 + +![1_image_0.png](1_image_0.png) + +The high percentage of children who died at this site's ICU reflects national pediatric death trends. 5 Contemporary approaches to pediatric EOL care are generally extrapolated from adult interventions and may not be appropriate for pediatric deaths. 6 This hospital lacks dedicated, quiet, private spaces for pediatric EOL, and spacious, separate areas for their family to gather. The inpatient visitation policy permits 24-hour access to only 4 primary support persons, and only 6 additional people may visit during visiting hours. When a child dies, these policies may be relaxed, but there are no standards for when to make exceptions. The influenza season presents additional complexity. Nevertheless, for many families, "culturally competent" EOL care means allowing the dying child unlimited access to immediate and extended family, and friends. + +After the child has died, removing intravenous and other lines and tubes, laying with and bathing the child, praying, and receiving family, all occur in the room where the child died. Ritual bathing of the body, required by some religions, is challenging to accommodate in a hospital bed, and there are no dedicated areas in the hospital for ritual bathing. Although staff (typically nursing or chaplaincy) tell families that they may remain in the room for "as long as they wish," the best practice is to transfer a child to a morgue or "cool room" within 4–6 hours.7 During high census periods, staff may feel pressure to move a family from the room before the family may be ready. A mother of a child who died in an ICU at this site stated, "Their 'patient' had died…we weren't the patient. We felt that we had to go." The family must then leave the child in that room and return home without them. This transition is emotional for both parents and staff. One bereaved parent stated, "…the hardest thing I ever had to do is walk away from the hospital."3 Some religious/cultural practices dictate that the parents or faith leader accompany the body until burial, including accompanying the child's body to in the morgue. In this institution, the morgue is in the hospital basement, allowing funeral homes access to the loading dock. Depending on the child's location of death, the morgue may be in a separate patient care building. Chaplains described the route to this morgue as "unceremonious" and "stark, uninviting, and utilitarian." Some families have increasingly insisted on accompanying the child to the morgue, frequently related to religious/cultural reasons. + +The goal of this project was to profoundly understand family and staff experiences surrounding pediatric death in our institution and to identify unique opportunities to design improved, novel delivery models of pediatric EOL +care. This project was a collaboration with key stakeholders from this hospital and a nonprofit organization that utilizes a design-thinking process to co-create innovations aimed at improving health.8 + +## Methods + +The hospital's institutional review board reviewed this study and determined it to be quality improvement work, not human subjects research. No institutional review board approval was required to proceed. + +## Participants + +This project setting was a 675-bed academic pediatric medical center in the Midwest. Participants' recruitment proceeded by snowball sampling. Recruitment of bereaved parents proceeded with the recruitment of one bereaved parent well-known to the study team who had previously expressed an interest. After that, other bereaved parents are known to that individual or to study staff members invited to participate. Study staff approached clinical managers to make them aware of the study and asked them for suggestions of clinical staff who might like to be involved. The roles of the clinical staff invited included an array of experiences across the institution's EOL journey. These staff were contacted individually and invited to participate. Knowledge of the project spread by word of mouth and other staff approached the team for participation. + +The ratio of family to the staff participants was limited by our access to families open to participating in a study about what may have been a traumatic experience. The number of clinical staff participants was greater than family participants because the study intended to capture the diverse perspectives of staff experiencing the EOL +journey from various lenses. The final ratio of fewer families to staff is not illustrative of the importance of one voice over another; the experiences and perspectives of the bereaved families were viewed with the utmost respect and concern throughout the study. + +## Procedures + +We used Craig Vogel and John Cagan's structured 4-phase integrated new product development process model that begins with identifying an opportunity and ends with realizing that opportunity into a product.9 The 4 phases are identifying, understanding, conceptualizing, and realizing. This project utilized an adaptation of this process model that relies on human-centered and design thinking methodologies. This process model is a multidisciplinary, 3-phase model focusing on research, ideation, and refinement of a process or product opportunity to develop technologically feasible and cost-effective solutions that address stakeholder needs.10 Stakeholders include all users of a process or product. Design thinking methods include every stakeholder to ensure that solutions meet people's needs and are compatible with daily routines and workflow. + +## Phase 1: Research Preparatory + +The phase's goal was to understand the EOL process through primary and secondary research methods for design members of the study team from the nonprofit organizations to learn about EOL care issues in pediatrics. Two members of the study team (B.L. and A.R.) +researched 24 examples of palliative care and EOL +care from around the world. They conducted a literature search of 35 peer-reviewed articles about death and design, grieving, hospice care, and various cultural understandings of death and dying. The literature review intended to provide a deeper understanding of the current academic and professional discussion, and best practice recommendations or guidelines, occurring within the palliative care and EOL space. This review informed a more empathic approach to interviewing staff and families and influenced later design considerations. + +Study team clinical members provided photographs of EOL spaces at other institutions and solicited additional examples through pediatric chaplaincy and pediatric bereavement e-listserves with national and international membership. The literature review identified 3 key insights: support across the journey, understanding parent priorities, and bonding after death. Pediatric death is not an isolated event; it also includes the parents' entire journey from the emotional loss of their child to the physical separation from their child at the time of death. It consists of the social loss of their roles as parents and their cognitive loss of self-esteem.11 Meyer et al12 identified parent priorities for improving EOL care in the pediatric ICU. These priorities were honest and complete information, ready access to staff, communication/care coordination, staff emotional expression and support, preservation of the parent–child relationship's integrity, and spiritual faith.12 Continued bonding with the child, especially with younger children and infants, is helpful to parents and may be facilitated by encouraging parents to speak to their child even after death.13 + +## Qualitative Data Collection + +Study team members interviewed both hospital staff and bereaved parents during the 16-week project. Hospital staff included hospitalists, nurses, geneticists, palliative care clinicians, intensivists, pathologist, chaplains, child life workers, social workers, bereavement coordinators, protective services (hospital security), and interpreters. A total of 22 hospital staff completed interviews; 5 bereaved parents completed interviews. Interviews were conducted with various critical informants at 3-time points throughout the project. Data were recorded by note-taking during interview sessions. Insights were translated on post-it notes and organized into themes and categories to identify patterns and overlap in the interviews. + +During the interviews, stakeholders explained what they see, think, say, and do during each step of the end of life process. As the stakeholders reported their experiences, the team used a tool called an experience map14 to organize insights with stakeholders during the interview. + +The participants drew from their own experiences to discuss problems that arose during their time with the hospital, identify areas of improvement, and conceptualize potential design interventions. The experience-mapping exercise targets interviewee's emotions by assessing how they felt, what they heard and saw, and what they did during the end of life process. It intends to "uncover, visualize, and better understand the target user's emotional experience."15 + +## Data Analysis + +Transcribed field notes were organized into themes. + +From the themes, the team used qualitative methods of coding to identify patterns, commonalities, and differences across the insights translated from the experience maps.18 These translated insights were the key drivers for the results of the project. We identified 5 key areas of process improvements from the insights and suggestions collected from bereaved parents and clinical staff that the hospital could focus on to improve the EOL experience. Those focus areas were: establish an appropriate level of privacy and communication of privacy for families and patients; transition the mindset from caring for an ICU patient to creating a legacy for the child; provide adequate space and time for staff care; incorporate moments of parent control throughout the EOL process; and to standardize staff communication and care teams during the EOL +process. + +## Phase 2: Ideation + +During ideation, the team's goal was to profoundly understand the 5 key areas of process improvement identified for the hospital. The team interviewed 9 clinicians and 5 bereaved parents. The shift in clinical staff participation from 22 to 9 was based on the clinical staff's role and emphasized their direct engagement in the 5 key areas of process improvements. The 2 clinicians identified to participate in the ideation phase also participated during the research phase. The 5 bereaved parents were the same parents interviewed during the research phase of the study. The interview sessions consisted of interactive exercises, such as mood boards and theme development. + +Mood boards are visual communications of the look and feel of final products or services. They trigger "analogical reconfigurations across the senses that support innovative solutions" during the product design process.16 Mood boarding activity allowed participants to express what the ideal EOL process means to them through abstract images. The selection of images was chosen through an image search utilizing keywords extracted during the experience-mapping exercise (such as tranquility, peaceful, spiritual, intense, etc.) from the research phase. Participants selected images and curated their mood boards through creative expression. While selecting images, the interviewers prompted the participants with questions about what emotions, feelings, and experiences their selections evoked. Participant bias during this process was mitigated by recognizing that the narratives which emerged from the image selections were the most impactful outputs rather than the mood board themselves. The mood boards acted as a vessel to engage participants to create an open dialogue around potential subproducts and services for improving EOL processes. + +After creating mood boards, interviewees and team members co-created potential subproducts and services by using the 5 opportunity areas identified during the research phase as a foundational platform. Participants were asked to describe narratives of difficulty related to the opportunity area, insight into what would improve their experience, and if they had a "magic wand," what would be their ideal fix. The study team used the narratives from the mood board exercise and responses from the co-creation session to inform concepts for opportunity areas. The result of ideation was 12 conceptual ideas that address the 5 key opportunity areas identified in the research phase (see Table 1). + +## Phase 3: Refinement + +During refinement, the team's goal was to create and test prototypes of the 12 concepts created during ideation. Low-resolution prototypes of potential lighting options, privacy indicators, and mental health reminders attached to everyday items were created and evaluated through feedback sessions. The 5 parents and 9 staff who participated in research and ideation also participated in this phase. Other hospital and resource planning departments were also included, to offer logistical perspectives for implementing the concepts. Creating and receiving feedback on prototypes of possible solutions promotes a shared vision of outcomes across stakeholder populations, helps test ideas quickly, and offers methods for iterative improvements.17 Refinement sessions with clinical staff and families elicit ways to improve the prototypes and make the prototypes better fit the hospital workflow and family life. Barriers to consider for implementing the interventions from logistics within the hospital to habit adoption by staff and families were discussed. The final project results (prototype refinement) were driven by staff and family insight. + +## Results + +There were 2 primary results of this project: 5 process/ opportunity areas to improve the EOL experience, and a set of criteria and considerations for a dedicated EOL space. The process and opportunity areas to improve the EOL experience consisted of: + +(1) privacy lights that indicate levels of privacy for families to staff while the families are experiencing a child's death; +(2) a comfort quilt used at time of death for staff to wrap the body in that would offer a softer experience for both staff and family; +(3) graphic "little reminders" around the hospital placed in strategic locations that would empower parents to be advocates for their child and remind staff to take a moment for mindfulness and mental health; +(4) announcement and spiritual lighting to make the walk to the morgue more comfortable for all stakeholders; and +(5) a remembrance garden that would act as a public/ +private space for staff and family to find moments of solace and peace while at the hospital and serve as a memorial space for children who died at the hospital. + +## Privacy Lights + +The notion of privacy lights arose from discussions with parents who strongly felt that they lacked adequate privacy during difficult times (see Figure 1, Supplemental Digital Content 1, *http://links.lww.com/PQ9/A194*). + +These lights would be placed both inside and outside the patient room and set on a timer in 10- to 150minute increments. Different colors could signal different needs: lavender may be used for EOL care, whereas teal might indicate the need for general privacy. This system is superior to the current model, in which a handmade sign is placed on the door when a parent or patient requests privacy, as staff largely ignore these signs. Most staff assume that their needs to enter the room to provide care supersede the family's need for privacy. There is no standardized system for how long a sign stays up or who removes them, and because they are so frequently posted, clinical + +| Conceptual Idea | Purpose | +|-------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Messages and notes given to families or | To remind families and staff to ask important questions or take time for themselves | +| located through the hospital | | +| In-room tablets | To provide a platform for parents to research questions they may have, receive updates on their child's care, or provide them with a schedule for their day with names of clinical staff they may be interacting with throughout the day | +| The use of signals (cards and lighting) and | To communicate family and patient needs of privacy or information | +| infrastructure (windows and doorknobs) | | +| Staff and family discussion prompts | To help facilitate discussions around family needs for staff inexperienced with the EOL process | +| A projector within the patient rooms | To display images or curated scenes to provide a personalized environment for patient and families | +| Memory-making cards with topics and prompts | To engage with families to learn more about each other during this tough time (ie for children of certain ages, prompts may include "Favorite Color" or "Favorite Animal") | +| A Child Life cart | To assist Child Life Services for organizing their memory-making activities | +| An improved fabric cover for the medical gurney | To provide warmth and privacy of the child's body while being transported to the morgue | +| An adjustable feature to the medical gurney | To assist clinical staff when transferring the body from the ICU beds to the gurney for transport to the morgue | +| A patient quilt | To soften the transfer of the body from the ICU bed to the medical gurney before transport to the morgue | +| Symbolic messages and images on the | To comfort families and staff as bodies are transported | +| pathway from the ICUs to the morgue | | +| A lighting system within the staff corridor | To notify staff of an approaching family or staff transporting a body to the morgue | +| leading to the morgue | | + +staff participants described "alarm fatigue" with them. + +Potential barriers for successful implementation of placing privacy lights within line of staff sight; the simplicity of use; appropriate hue to evoke comfort rather than anxiety or confusion for families and staff; ease of maintenance; and habitat adoption by families and staff. + +## Comfort Quilts + +Comfort quilts are a tool to enhance EOL care for staff, and as a memento for families. The current process is that after death, a shroud or plastic body bag contains the child's body for transfer to the morgue. The plastic is cold and sterile, and staff noted how difficult it is to place children inside. The comfort quilt can be given to the family at any time before the child dies or introduced after death if necessary, placed over the shroud for transfer, and transition with the child to the funeral home. Potential barriers for successful implementation of the comfort quilt included a resource sustaining method for creation, logistics for dissemination across the hospital, and planning of when to offer and deliver it to families. + +## Little Reminders + +"Little reminders" may be placed strategically around the hospital. These cards, reading, for example, "Be an advocate for your child," are intended to empower families to regain some control by maintaining self-care and advocating for their child. They also remind staff to maintain self-awareness and mindfulness during stressful times. + +These reminders could take the form of signs in the elevator, at hand-washing stations, cafeteria napkin dispensers, or coffee cup sleeves. Potential barriers for successful implementation of "Little Reminders" included ensuring all messages and designs are approved through hospital administration, the upkeep and changing of signs across the hospital to keep a consistent cycle of new messages and the potential for staff or families spending large amounts of time at the hospital to background messages. + +## Automated Lighting + +The current path from the pediatric ICU setting to the morgue is stark and industrial. Staff entering and exiting the hospital also utilize this hallway. There is no standardized process to announce that a procession is moving a child's body to the morgue. The fourth process improvement was an announcement lighting system that added warmth to the journey and improved staff awareness of a procession. The automated system, placed at the eye level, could be activated by a chaplain's badge or ID tag located on the transport cart such that it only activates at the correct time. The lighting could include a specific color system +(lavender, for example) to ensure consistency with other areas of the hospital, or may consist of images of a spiritual avatar or animal meaningful to the child or family that progresses ahead of or alongside the cart (see Figure 2, Supplemental Digital Content 2, *http://links.lww.com/* +PQ9/A194). This system would make other hospital staff aware of the need to stand aside, respectfully and quietly, as the procession passed. Potential barriers for a successful implementation of this lighting system included the infrastructure of the hallways having the ability to support the lighting system, the cost of the lighting system to install and place, and the habit adoption by staff to use and recognize the meaning of the lighting system. + +## Remembrance Garden + +This institution lacks a remembrance garden to memorialize children who have died and to provide staff and families with a space to grieve. Considerations of this space included public-facing, allowing families to visit at any time, and providing an opportunity for private contributions allowing staff or families to provide a legacy to a decedent child privately. Potential barriers for successful implementation of the garden included cost, location within the institution's campus, and the potential stigma in symbolizing death within the institution. + +## Dedicated Eol Space + + Finally, team members designed a dedicated inpatient EOL space for children and families. Features would include an ample living room for family members to gather, a patient room connected by French doors, large windows allow natural lighting, and outdoor space to enable death or visitation outside when desired (**see** +Figure 3, Supplemental Digital Content 3, *http://links.* +lww.com/PQ9/A194). The area would include amenities for families and a soothing color palette with soft finishes. Parents preferred colors and finish materials that reminded them of nature: soft and soothing light blues, greens, and purples and warm, natural finishes of wood tones, to differentiate the space from clinically-focused areas of the hospital (see Figure 4, Supplemental Digital Content 4, *http://links.lww.com/PQ9/A194*). The design also emphasized access to amenities, such as toothbrushes, water, snacks, and coffee.3 + +## Discussion + +Health "care" normally means "cure." When this is impossible, care means comfort.19 In pediatric healthcare, care of the whole family is normative. Care, as comfort for a dying child and their family, mitigates poor outcomes for the parents and prepares them to grieve well. Perimortem and postmortem space influence parental emotional functioning. We describe the design process undertaken to improve parental and staff perimortem experience in a pediatric hospital. This process led to the description of 5 actionable process improvement opportunities, and the development of a low-fidelity prototype for each. The process additionally resulted in defining the criteria for a dedicated EOL space to inform future renovation or construction planning. This project presents the novel application of Craig and Vogel's design thinking as a method of addressing the need for quality improvement in pediatric EOL care. Their method provided a structured framework with which to gather and understand data. One of the benefits of this process was the breadth of knowledge that was sought. Persons not generally accessed for participation in research studies (eg, bereaved parents, protective services, and interpreters) vetted the intervention prototypes to improve EOL care at this institution. The specific endproducts may not be generalizable. However, the development process is generalizable and replicable in any setting. The results are strengthened by the diversity of participants, which included representation from the numerous roles that influence the EOL process. Future directions include the engagement by hospital development representatives to seek donors to fund individual aspects of this project. + +## Disclosure + +The authors have no financial interest to declare in relation to the content of this article. + +## Acknowledgments + +We publish this in memory of our coauthor, Alison Rednour. May this work, and any future improvements in pediatric care arising from it, continue to honor her life. The authors gratefully acknowledge the extensive contributions to this project of Hannah Eber, Shay Xie, and Max Holden, students in the UC School of Design, Arts, Architecture, and Planning (DAAP). We thank Linda Dunseath, LWC Director, Craig Vogel, Associate Dean of UC DAAP, and Lori Crosby, PhD, for their mentorship and support of this project. The authors gratefully acknowledge Alyssa Greenwell, BS, for her assistance in preparing the manuscript. Finally, we gratefully acknowledge the contributions of all staff and bereaved parents who contributed thoughtfully and selflessly to this work. + +## References + +1. Smith DH. On letting some babies die. *Hastings Center Studies*. + +1974;1:37–46. + +2. Burns JP, Sellers DE, Meyer EC, et al. Epidemiology of death in the PICU at five U.S. teaching hospitals*. *Crit Care Med*. 2014;42:2101–2108. + +3. Davies R. Mothers' stories of loss: their need to be with their dying child and their child's body after death. *J Child Health Care*. 2005;9:288–300. + +4. Milo EM. Maternal responses to the life and death of a child with a developmental disability: a story of hope. *Death Stud*. 1997;21:443–476. + +5. Friebert S, Williams , C. National Hospice and Palliative Care Organization. NHPCO's Facts and Figures: Pediatric Palliative and Hospice Care in America 2015 Edition. http://www.nhpco.org/ sites/default//files/public/quality/Pediatric_Facts-Figures.pdf. 2015. Accessed August 11, 2015. . + +6. Knops RR, Kremer LC, Verhagen AE. Paediatric palliative care: +recommendations for treatment of symptoms in the Netherlands. BMC Palliative Care. 2015;14:57. + +7. Bennett H. Together for short lives: A Guide to End of Life CareCare of children and young people before death, at the time of death and after death. 2012;1–62. + +8. Provost SM, Hoppenjans N. Evaluating Innovation and improvement in the collaborative care network. Paper presented at: Doing Research at the Front Line of Improving Health Care. 2013; Arlington, VA. + +9. Cagan JM, Vogel CM. Creating Breakthrough Products: Revealing the Secrets That Drive Global Innovation. ETC Press; 2012. + +10. Brown T. Change by design. 2009;1–272. 11. Williams C, Munson D, Zupancic J, Kirpalani H. Supporting bereaved parents: practical steps in providing compassionate perinatal and neonatal end-of-life care. *Seminars Fetal Neonatal Med*. + +2008;13:335–3040. + +12. Meyer EC, Ritholz MD, Burns JP, et al. Improving the quality of end-of-life care in the pediatric intensive care unit: parents' priorities and recommendations. *Pediatrics*. 2006;117:649–657. + +13. Forster EM, Windsor C. Speaking to the deceased child: Australian health professional perspectives in paediatric end-of-life care. Int J Palliat Nurs. 2014;20:502–508. + +14. Kumar V. 101 Design Methods: a Structured Approach for Driving Innovation in Your Organization. Hoboken, N.J: Wiley; 2013. + +15. Yayici E. *Design Thinking Methodology Book*. Istanbul, Turkey: +ArtBizTech; 2016. + +16. Endrissat N, Islam G, Noppeney C. Visual organizing: balancing coordination and creative freedom via mood boards. *J Business* Res. 2016;69:2353–2362. + +17. The LUMA Institute. *Innovating for People: Handbook of Humancentered Design Methods*. Pittsburgh, Pa.: The LUMA Institute; 2012. + +18. Creswell JW. Research Design: Qualitative, Quantitative, And Mixed Methods Approaches. 4th ed. Thousand Oaks: SAGE Publications; 2014. + +19. Smith DH. Health and Medicine in the Anglican Tradition: Conscience, Community and Compromise. New York, N.Y.: Crossroad; 1986. \ No newline at end of file diff --git a/medical/md/PMC7485752.md b/medical/md/PMC7485752.md new file mode 100644 index 0000000000000000000000000000000000000000..27f2c9bdff5c652e5b77d0a69e26eb4e08ff0141 --- /dev/null +++ b/medical/md/PMC7485752.md @@ -0,0 +1,13 @@ +MILITARY MEDICINE, 183, 11/12:330, 2018 + +# Videoconferenced Telebehavioral Health Referral Process Adherence: Erratum + +The article, "Videoconferenced Telebehavioral Health Referral Process Adherence," +1 that published online on April 3, 2018 contained errors. The following errors were introduced at the proof stage: On page 93 "Armed Forces Longitudinal Technology Application" should be "Armed Forces Health Longitudinal Technology Application." On page 96 "(3) in their electronically signed note…" is a redundant clause, and instead should be "(3) TBH +consultants were already documenting consent at the time of intake in their electronically signed note." The publisher regrets these errors. + +On page 94 "Institute for Hospital Improvement" should be "Institute for Healthcare Improvement." The author regrets this error. + +## Reference + +1. Jeffrey T: Videoconferenced Telebehavioral Health Referral Process Adherence. Mil Med 2018; 183(suppl_1): 92–8. doi:10.1093/milmed/usx141. \ No newline at end of file diff --git a/medical/md/PMC7571848.md b/medical/md/PMC7571848.md new file mode 100644 index 0000000000000000000000000000000000000000..737c0412484e4c5f6581064eaaf3314378a3ea1b --- /dev/null +++ b/medical/md/PMC7571848.md @@ -0,0 +1,41 @@ +# Editor'S Perspective True Negatives Disseminating Research On Null, Inconclusive, And Confirmatory Findings In Cardiovascular Science + +Science is about filling in the details. + +—Graham Hawkes Publishing research with negative results—that is, null or inconclusive findings—is a critical but often overlooked task of biomedical journals. Without it, the scientific literature relies on highly selected pieces of evidence that viewed in isolation can distort a field. Publishing research with negative results provides a more comprehensive and fuller view of a scientific discipline. It also can mitigate wasted time and resources by other scientists who attempt to duplicate research efforts in vain. By filling in the background, it expedites processes for new discovery and can enhance "positive" research by informing the design of future studies. + +Yet despite its well-recognized and essential necessities for science, significant challenges exist for authors who seek to publish research with negative results. The label itself invokes an undesirable connotation, often dismissing a negative result as a failure or negligible contribution regardless of whether rigorous study design, execution, and analysis occurred. As a result, such work is frequently less cited, and some have even argued that publishing it can undermine early careers.1,2 This can make it unattractive for authors to even take the time to write up and submit results when the findings are null or inconclusive. We have become so accustomed to celebrating successes in science that we forget the crucial role of "failures" in advancing a field. Research with negative results (including inconclusive results) has come to be viewed so poorly that even when well-done, antiscience advocates may use it to undermine faith in the entire scientific process.3,4 The contribution of these data to the research field, however, is critical—its absence indicates publication bias and undermines the whole purpose of systematic review and metaanalyses. + +A few such concerns are playing out recently in the example of hydroxycholoroquine as a treatment for the novel coronavirus disease 2019 (COVID-19).5 Numerous randomized clinical trials with consistent negative findings have now been published given the topic's current importance. However, these trials are competing for attention with less well-designed observational studies with outlier and potentially overhyped results. These studies continue to raise lingering questions—some of which are legitimate—on whether the treatment works and may distract from other promising areas of investigation. Indeed, as researchers the world over address the COVID-19 pandemic, the importance of publishing research with negative results has become clearer as we chase a growing list of novel associations that may have little clinical relevance. Be it basic or clinical, the effort should be to develop and execute rigorous research projects and not just report results that are significant. To move forward, knowing what does not work is just Brahmajee K. + +Nallamothu , MD, MPH +Jonathan Schultz , MA Sandra Petty , MBBS, +PhD +The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. This article is part of the Null Hypothesis Collection, a collaborative effort between CBMRT, AHA Journals, and Wolters Kluwer. For more information, visit https://www.ahajournals.org/ null-hypothesis © 2020 The Authors. *Circulation:* Cardiovascular Quality and Outcomes is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. 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 that the original work is properly cited and is not used for commercial purposes. https://www.ahajournals.org/journal/ circoutcomes as important as knowing what does, especially when resources are limited.6 Admittedly, biomedical journals have a history of diminishing the importance of research with negative results. Most journals explicitly take judgments of novelty and anticipated impact into consideration when selecting manuscripts for publication. This is understandable. For example, associations between most features of any system are unlikely to be significant, which is why discovering new relationships is so exciting. And when a null relationship is reported, it is important to identify whether the absence of an association is a scientific truth or simply a study design flaw (eg, an underpowered experiment). Understanding the difference between research with negative results and unimportant research, therefore, requires careful judgment. But systematically overlooking such research is risky. Already a rare spotting in scientific journals, research with negative results is disappearing from the published literature. + +In a seminal study by Fanelli7 from 2011, the rate of research with positive results in >4600 articles across the published literature grew annually by 22% from 1990 to 2007—a finding that was remarkably consistent across disciplines (including clinical medicine). By the end of the study period, fewer than 15% of published articles were classified as having negative results. + +This clearly needs to change if we want a more comprehensive and honest view of research; however, biomedical journals have rarely offered a clear pathway toward such an advancement. At the American Heart Association, we are interested in changing this culture and are delighted to report our recent efforts to address this issue. The American Heart Association Journals and Wolters Kluwer have entered into an agreement with the Center for Biomedical Research Transparency (CBMRT, http://www.cbmrt.org/) to create an online Null Hypothesis Collection highlighting important research with negative results that includes inconclusive and confirmatory research articles as well. Our goal is to highlight such articles published by the portfolio to raise awareness of their importance to the scientific community. CBMRT has agreed to provide financial support to make the articles freely available online and will help promote the initiative through their channels. Together we can address publication bias by encouraging the write up and publication of all wellperformed studies, including those with negative/null or inconclusive findings. Doing so not only improves research culture and enhances research efficiency but reduces the risk of avoidable harm to patients by ensuring that clinical practice is informed by a more complete and balanced record. + +We hope the Null Hypothesis Collection will be an important vehicle for promoting more balance in our publications. For too long, science has been an unfinished portrait with missing details in its background. + +The subject—the positive discovery—sits flat on the canvas, lacking depth and context. Without crucial details filled in from true negatives in research, it is impossible to understand the full extent of our progress. The Null Hypothesis Collection serves as a tool for showing our conviction to the process of filling in the details and will hopefully encourage authors to continue to submit their rigorous, interesting, innovative research to the American Heart Association portfolio of journals regardless of their reported outcome. Through it, we explicitly recognize that highquality research is ultimately about more than a new and exciting finding but a product of rigorous study design, execution, and analysis. + +## Article Information Correspondence + +Brahmajee K. Nallamothu, MD, MPH, University of Michigan Medical School, Internal Medicine-Cardiovascular Medicine North Campus Research Complex, 2800 Plymouth Rd, Bldg 16, Ann Arbor, MI 48109. Email bnallamo@umich.edu + +## Affiliations + +University of Michigan Medical School, Ann Arbor (B.K.N.). American Heart Association, Dallas, TX (J.S.). Center for Biomedical Research Transparency, New York, NY (S.P.). + +## Sources Of Funding + +None. + +## Disclosures + +Disclosures provided by Dr Nallamothu in compliance with American Heart Association annual Journal Editor Disclosure Questionnaire are available at https:// +www.ahajournals.org/pb-assets/COI_09-2019.pdf. The other authors report no conflicts. + +## References diff --git a/medical/md/PMC7723529.md b/medical/md/PMC7723529.md new file mode 100644 index 0000000000000000000000000000000000000000..ec5124559393a5092618152c831f91d7369c3334 --- /dev/null +++ b/medical/md/PMC7723529.md @@ -0,0 +1,326 @@ + + +![0_image_1.png](0_image_1.png) + +![0_image_0.png](0_image_0.png) + +# Publication Trends Of Research On Uveal Melanoma During 2000– 2020: A 20-Year Bibliometric Study + +## Minyue Xie1,2#, Qianru Wu3#, Yefei Wang1,2, Shengfang Ge1,2, Xianqun Fan1,2 + +1Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; 2Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, China; 3Department of Ophthalmology, Peking University Third Hospital, Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital, Beijing, China Contributions: (I) Conception and design: X Fan, M Xie, Q Wu; (II) Administrative support: X Fan; (III) Provision of study materials or patients: +None; (IV) Collection and assembly of data: M Xie, Q Wu, Y Wang, S Ge; (V) Data analysis and interpretation: M Xie; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. + +\#These authors contributed equally to this work. + +Correspondence to: Xianqun Fan. Department of Ophthalmology, Ninth People's Hospital, Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. Email: fanxq@sjtu.edu.cn. + +Abstract: Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults. Despite a high rate of success in controlling it as a local disease, it is prone to distant metastasis, and its mechanism of metastasis has not been elucidated. This study analyzes trends in UM research and compares contributions from different countries, regions, institutions and authors. We collected all publications related to UM published from 2000 to 2020 from the Web of Science database. GraphPad Prism 6 was used to collect publication data and analyze publication trends. VOSviewer was used for data visualization. A total of 1,710 publications were considered. The United States contributed the most publications [668] and citations (19,605 times) as of 2020 with the highest H-index value [67]. Keywords were classified into three clusters, namely, clinical study, tumor-related study and gene mutation-related study. Average appearing years (AAY) of keywords were calculated. BAP1 (AAY of 2016.3), SF3B1 (AAY of 2015.8) and GNA11 (AAY of 2015.5) were identified as major focuses of this field. We conclude that the United States, Germany, England and the Netherlands have been the most productive regions in terms of UM research over the past two decades. Gene mutations such as GNAQ, GNA11 and BAP1 mutations are identified as potential research focuses. + +Keywords: Bibliometric analysis; publication; uveal melanoma (UM); mutations; metastasis Submitted May 04, 2020. Accepted for publication Sep 18, 2020. doi: 10.21037/atm-20-3700 View this article at: http://dx.doi.org/10.21037/atm-20-3700 + +## Introduction + +Among ocular tumors, uveal melanoma (UM), which is the most prevalent primary intraocular malignancy in adults arising within the uveal tract, iris, or ciliary body (1). It is a rare form of melanoma that accounts for approximately 3% to 5% of all melanoma cases (2). In the United States and Europe, the incidence of UM is five to six per million people, while in Australia, the incidence is seven cases per million people (3). However, the ten-year mortality rate of UM in the United States is 50%, which is markedly higher than those of other melanoma subtypes (4). With the introduction of novel treatment modalities, more patients have been treated with eye-sparing treatments, such as radiation or transpupillary thermotherapy, though the five-year survival rate has remained almost unchanged (4). Studies have shown that intrinsic risk factors for UM include Caucasian race (5); fair hair, skin complexion and iris coloring; UM family history; previous cutaneous malignant melanoma history and specific genetic abnormalities (1). Extrinsic risk factors include artificial UV or blue light exposure and a history of occupational exposure to chemicals (6). + +![1_image_0.png](1_image_0.png) + +Despite a high rate of success in controlling local disease through surgery or radiotherapy, nearly 50% of patients with UM will metastasize within 15 years of the initial diagnosis. The most common initial site of metastasis is the liver (89%), followed by the lungs (29%) and bones (17%) (7-9). However, most patients with metastasized UM survive less than 12 months. This poor prognosis is mainly attributed to the fact that systemic chemotherapy is not highly effective in treating metastatic UM (10,11). In addition to chemotherapy, potential treatments for metastatic UM include immunotherapy and targeted therapy (11). Due to the particular important effects of the immune system on cancer cells, immunotherapy and targeted therapy have drawn extensive attention. However, despite impressive effects of immune-based therapies on metastatic cutaneous melanoma, such therapies appear unsatisfactory in treating UM to date (12). + +Bibliometrics is an optimal means to evaluate specific trends in the research regarding a certain field over time (13). Bibliometric methods are employed to quantitatively and qualitatively analyze publications, including books and journal articles. Meanwhile, such an analysis can characterize and predict the development of a certain field and can be used to compare contributions across countries, institutions and journals. Specifically, a bibliometric analysis has proven important for the formulation of policies and clinical guidelines, making it attractive as an analytical method. + +This work presents a comprehensive analysis of the current state of global UM research based on Web of Science (WOS) data. We identify trends in UM research and predict potential focuses within this field via bibliometrics (14,15). + +## Methods Data Sources And Search Methods + +The Science Citation Index-Expanded (SCI-E) of the WOS has been recognized as the most suitable database for bibliometric analysis. Accordingly, we conducted a literature search for the years 2000 to 2020 using the WOS database. + +All searches were completed on January 17, 2020 to avoid biases introduced by the daily renewal of the open database. The following search terms were used: TI = (uveal melanoma) AND Language = English. Finally, we included all the original articles and reviews in the core database to obtain a total of 1,710 publications meeting the search criteria. Our inclusion and exclusion methods are illustrated in *Figure 1*. + +## Data Collection + +All data (i.e., titles, publication dates, countries and regions, authors, sums of citations and H-indexes) were extracted from the included publications. Microsoft Excel 2010 (Redmond, Washington, USA), GraphPad Prism 6 (GraphPad Prism Software Inc., San Diego, CA) and VOSviewer (Leiden University, Leiden, the Netherlands) were used to input and analyze the data. + +## Bibliometric Analysis + +The characteristics of all publications considered were examined through the Web of Science. Relative research interest (RRI) was measured as the number of publications in a certain field divided by all publications of all fields. The H-index, denoting that a scholar or region has published H papers that have each been cited at least H times, has been widely accepted to reflect the productivity and citation impact of a scholar or region (16). + +The prediction model f(x) = ax 3 +bx 2+cx+d was generated by Microsoft Excel 2010 according to the cumulative publications to analyze time trends and future publication patterns. In this formula, x is time (year) and f (x) denotes the cumulative number of publications produced as of a given year. GraphPad Prism 6 was applied to analyze the quantity and quality of studies published in various regions. VOSviewer, an application used to analyze correlations between highly cited literature and productive authors, was used to construct a co-occurrence network of keywords from titles and abstracts. Moreover, keywords were classified into disparate clusters and simultaneously + +![2_image_0.png](2_image_0.png) + +color-coded by time course. The average appearing year (AAY) was used to evaluate the novelty of a research topic. + +## Results Countries' Contributions To Global Publications + +From 2000 to 2020, the United States produced the most publications (668, 39.1%), followed by Germany (194, 11.4%) and England (188, 11.0%) (*Figure 2A*). Furthermore, for each year over the past two decades, the United States published the most papers (*Figure 2B*). With respect to the number of publications per year, the most studies were published in 2019 (194, 11.3%). A comparison to the number of works published in the whole field based on the RRI value shows that global attention to this field was approximately 0.006% before 2018 and increased to 0.009% in 2019. While it was not until 2006 that China published its first paper in this field, over the past decade, the number of Chinese publications has risen at a surprising rate, and it eventually rose to a rank of fifth. + +After excluding countries and regions that are not connected to other countries, 28 countries and regions + +![3_image_0.png](3_image_0.png) + +are analyzed using VOSviewer (Figure S1). The cooccurrence relations of countries and regions resulted in five clusters: (I) the United States, China, Japan, India, Turkey; (II) England, Germany, Italy, France, Switzerland, Spain, Finland, Austria, Belgium, Poland, Portugal; (III) the Netherlands, Australia, Scotland, Israel, Ireland; (IV) Sweden, Denmark, South Korea, Norway, Hungary; and (V) Canada, Brazil. + +## Citations And H-Index + +From a citation report retrieved from the WOS database, all publications related to UM have been cited 38,353 times since 2000 (21,178 citations without self-citations) with each paper being cited 22.4 times, on average. Papers from the United States were cited significantly more frequently cited than those from other countries, thus accounting for 51.1% of the total citations (19,605 citations and 15,374 citations without self-citations). The United States also achieved the highest paper H-index [67], while Germany ranked second in total citations at 4,982 citations (4,546 citations without self-citations), and England ranked third with 4,729 citations (4,261 citations without selfcitations). Germany's H-index [36], however, was found to be slightly lower than England's [37] (Figure S2A). + +## Publication Growth Trend Predictions + +The growth in UM publication predicted from model fitting curves demonstrates that the cumulative number of publications was significantly related to time (*Figure 3*), +and publication trends for the following five years were estimated based on the cumulative number of works published over the past two decades. It is apparent that the volume of global publications has steadily and slowly increased, echoing trends in several major countries such as B + +![4_image_0.png](4_image_0.png) + +the United States and Germany (*Figures 3B,C*), while China has exhibited significantly faster growth despite its late start in research with respect to this field (*Figure 3F*). + +## Journals Publishing Research On Um + +Nearly half of the publications were published in the 20 journals listed in *Figure 4* (844, 46.7%), with *Investigative* Ophthalmology and Visual Science publishing the most works, specifically, 160 publications. Melanoma research ranks second with 87 publications, while *British Journal* of Ophthalmology, which has published 65 papers in this field, ranks third and ophthalmology ranks fourth with 43 works published over the past 20 years. Other high impact journals, such as *Nature*, published high-quality research in 2009, and *Science* and *the New England Journal of* Medicine published one article each in 2010. + +## Institutions Publishing Research On Um + +With respect to institutions, Leiden University in the Netherlands published the most studies (124, 7.3%) followed by Jefferson University in the United States (97, 5.7%). Of the top 20 institutions identified for their UM research, seven are in the United States, four are German institutions, three are in England, three are in the Netherlands, two are in France and one is in Canada. + +## Authors Publishing Research On Um + +The top ten authors have published a total of 558 publications, thus accounting for 32.6% of all work in this field, but Jager MJ of Leiden University, publishing 97 papers on UM, ranks first in the number of publications +(*Table 1*). Harbour JW of Washington University (WUSTL) + +## Page 6 Of 12 Xie Et Al. Bibliometric Analysis On Um + +| Table 1 Top 10 authors with the most publications in UM research Author Country Affiliation | No. of publications | No. of citations | | | +|-----------------------------------------------------------------------------------------------|-----------------------|-------------------------------|----|-------| +| Jager MJ | Netherlands | Leiden University | 97 | 2,467 | +| Harbour JW | USA | Washington University (WUSTL) | 67 | 4,006 | +| Shields CL | USA | Jefferson University | 65 | 2,095 | +| Shields JA | USA | Jefferson University | 52 | 1,869 | +| Luyten GPM | Netherlands | Leiden University | 51 | 1,010 | +| Singh AD | USA | Cleveland Clinic Foundation | 48 | 1,606 | +| Burnier MN | Canada | Mcgill University | 48 | 606 | +| Coupland SE | England | University of Liverpool | 46 | 1,278 | +| Damato B | England | University of Liverpool | 42 | 1,245 | +| De Klen A | Netherlands | Erasmus University Rotterdam | 36 | 994 | +| Bornfeld N | Germany | University of Duisburg Essen | 36 | 1,274 | +| UM, uveal melanoma. | | | | | + +ranks second, albeit he has the most citations (4,006 times). Following Jager and Harbour is Shield CL of Jefferson University, who has published 65 papers and has been cited 2,095 times. Among the top ten authors, four are from the United States, three are from the Netherlands, two are from England and the remaining two are from Germany and Canada, respectively. Notably, Bastian BC of the University of California, San Francisco and the UCSF Helen Diller Family Comprehensive Cancer Center ranks fourth in the number of citations (1,994 times) even though he is not in the top 50 in terms of total publications with only 12 papers. + +Given that the effective and close cooperation between clinical researchers can improve the quality of observational research and randomized trials, the collaboration network from 2000 to 2020 is presented in Figure S2. The size of the node is proportional to the total strength of the link of each researcher as evidenced by Jager MJ, who has engaged in the greatest number of collaborations with other researchers and has maintained close cooperation with many researchers in recent years. Harbour JW and Shield CL also cooperate with a large number of researchers, but the majority of the cooperation occurred prior to 2012. + +## Analysis Of Keywords And Focuses Of Um Research + +We analyzed the keywords found over 20 times in the titles and abstracts of all 1,710 publications using VOSviewer. After merging words of the same meaning and excluding meaningless words, 89 terms were identified and classified into tumor-related study, clinical study and gene mutationrelated study clusters (*Figure 5A*). With respect to the clinical study cluster, the keywords diameter (159 times), tumor thickness (139 times), plaque brachytherapy (138 times) and chemotherapy (73 times) are among the most frequently mentioned. Regarding the tumor-related study cluster, the primary keywords include cell (572 times), apoptosis (103 times), receptor (101 times) and miRNA (82 times). In the gene mutation-related study cluster, the keywords include BAP1 (170 times), GNAQ (126 times), GNA11 (103 times) and SF3B1 (48 times). + +We then color-coded all keywords based on the AAY of each word (*Figure 5B*). Keywords shown in yellow such as BAP1 (cluster 3, AAY of 2016.3), SF3B1 (cluster 3, AAY of 2015.8), GNA11 (cluster 3, AAY of 2015.5), and miRNA (cluster 2, AAY of 2015.4) appear most recently, and hence, the gene mutation-related study cluster may constitute a new research trend. + +## Discussion Um Research Trends + +Our observations indicate that the United States, Germany and England ranked highest in terms of total number of citations and H-index values in the area of UM research. There is no doubt that the United States has made the greatest contributions. While UM was first described by + +![6_image_0.png](6_image_0.png) + +two Scottish surgeons in the 1800s (17), American scholars focused on this disease earlier than other scholars from other countries. In addition, due to a higher incidence of UM in high latitude areas, the incidence of UM in the United States is 5.1 cases per million people per year (18), while in Asian countries, such as South Korea, the incidence of UM reaches only 0.6 cases per million per year (19). European countries such as the Netherlands, Germany, and Switzerland, which also have higher incidence rates of 4 to 5 cases per million, have also made strong contributions (20). + +Furthermore, basic medical research conditions appear to be superior and clinical trials seem to be more professional and standardized in the United States. + +While China ranks fifth in the total number of publications, its citation and H-index values are ranked eighth. Although China has contributed a large number of publications, the quality of such research must be improved. This may be attributed to the uneven clinical and scientific research competencies of Chinese hospitals. In addition, the inconsistencies and incomplete electronification of + +## Page 8 Of 12 Xie Et Al. Bibliometric Analysis On Um + +| Table 2 Top 10 publications with the most citations in UM research Title Corresponding authors | Journal | Publication year Total citations | | | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------|-------------------------------------|------|-----| +| Frequent somatic mutations of GNAQ in | Bastian, Boris C. | Nature | 2009 | 799 | +| uveal melanoma and blue naevi Mutations in GNA11 in uveal melanoma | Van Raamsdonk, Catherine D. | New England Journal of | 2010 | 700 | +| Medicine | | | | | +| Frequent mutation of BAP1 in metastasizing | Harbour, J. William | Science | 2010 | 660 | +| uveal melanomas Very long-term prognosis of patients with | Kujala, E | Investigative Ophthalmology | 2003 | 414 | +| malignant uveal melanoma | & Visual Science | | | | +| Uveal melanoma: trends in incidence, | Singh, Arun D. | Ophthalmology | 2011 | 390 | +| treatment, and survival Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death Harbour, J. W. | Cancer Research | 2004 | 380 | | +| Incidence of uveal melanoma in the United | Singh, A. D. | Ophthalmology | 2003 | 281 | +| States: 1973–1997 High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL Goldstein, Alisa M. | Cancer Research | 2006 | 240 | | +| Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers | Abdel-Rahman, Mohamed H. | Journal of Medical Genetics | 2011 | 236 | +| Hippo-independent activation of YAP by the | Chen, Qianming | Cancer Cell | 2014 | 222 | +| GNAQ uveal melanoma oncogene through a Trio-regulated Rho GTPase signaling circuitry UM, uveal melanoma. | | | | | + +China's medical records system have created obstacles to the execution of multicenter randomized clinical trials (RCTs). + +As depicted by our model fitted time curve, global research on UM has increased steadily over time. Notably, China's research in this field started late but developed rapidly as China has gradually ascribed importance to research on diseases over the past decade. Due to China's large population, Chinese researchers can examine a relatively large number of cases, which is advantageous for the study of rare diseases such as UM. + +Notably, journals of ophthalmology such as Investigative Ophthalmology and Visual Science, the British Journal of Ophthalmology and Ophthalmology were the primary journals publishing research on UM. Thus, future developments in this field are more likely to be published in these journals. + +Jager MJ of Leiden University, Harbour JW of Washington University (WUSTL), and Shields CL and Shields JA of Jefferson University have published the most research in this field. Jager MJ has mainly focused on the investigation of molecular targets for the prediction and treatment of metastatic UM, while Harbour JW has focused on pathogenic gene mutations. These scholars have been pioneers in the research of UM, and their studies will continue to guide future research and clinical treatment in this field. + +## Um Research Focuses + +Published papers with high academic impact tend to be cited frequently. The ten most cited publications on UM +research are listed in *Table 2*. In recent years, with the discovery of GNAQ, GNA11 and BAP1 mutations, the molecular mechanism of metastasis UM has gradually been revealed with research having identified these metastasis-related mutations using exome sequencing (21-23). A +paper entitled "Frequent somatic mutations of GNAQ +in uveal melanoma and blue naevi" published in Nature in 2009 has been cited 799 times. In 2010, mutations of GNA11 in UM were first reported in the New England Journal of Medicine. In the same year, the mutation of BAP1 in metastasizing UM was also reported for the first time in Science. Immediately afterwards, pathways affected by these three mutations and potential treatments were examined (24,25). There has also been a focus on GNAQ/GNA11 mutations, which constitutively activate Gαq G proteins and initiate oncogenic events in UM. A +study published in Cancer Cell in 2014 reveals that the mutation of GNAQ and GNA11 could stimulate YAP via Trio-Rho/Rac signaling circuitry, thereby identifying YAP as a potential therapeutic target of UM metastasis (26). An article published in Cancer Cell in 2017 also finds that the small GTPase ARF6 mediates oncogenic Gαq signaling, subsequently inducing downstream β-catenin signaling pathways (27). In the same year, it was proved that GNAQ signaling mediates MAPK activation via PKC phosphorylation and RasGRP3 activation, thus potentially creating opportunities for refined therapeutic targeting in UM (28). In 2019, another study published in Cancer Cell demonstrated that focal adhesion kinase (FAK) encoded by PTK2 mediates the phosphorylation of MOB1, which subsequently leads to YAP-dependent tumorigenesis (29). The mutation of CYSLTR2, which exhibits high activation through Gαq, was also defined, thus providing a potential therapeutic strategy for UM (30). Two additional studies that sequenced the exons of primary UM relative to blood samples identified EIF1AX and SF3B1 mutations (31,32). Unlike GNAQ/GNA11, which are considered early events in the development of UM, mutations of BAP1, EIF1AX and SF3B1 were found to be strongly associated with the late development of metastases in most cases (33). + +A retrospective cohort study published in JAMA Ophthalmology in 2015 concluded that germline BAP1 mutations are associated with larger tumors and ciliary body involvement (34). Later, BAP1 was found to regulate cell cycle via E2F1 target genes in UM cells (35). Another retrospective case series published in *Ophthalmology* in 2018 also proved that BAP1 immunoreactivity is significantly lower in primary UM patients treated by enucleation, suggesting that nuclear BAP1 stain is a significant independent predictor of tumor metastasis (36). In targeting BAP1 mutations, researchers have found that histone deacetylase (HDAC) inhibitors can arrest UM cell growth and may have therapeutic potential (37). + +In recent years, miRNA has become another research focus in this field. A latent key effector of p53, microRNA-34a (miR-34a), has been found to be a potential tumor suppressor in UM. The introduction of miR-34a affects the expression of c-Met and subsequently affects the cell cycle (38). Another work concluded that miR-137 acts as a tumor suppressor of UM cell proliferation by downregulating the oncogenic transcription factor MITF and the cell cycle-related protein CDK6 (39). Another p53-dependent miRNA, miR-182, was found to act as a potent tumor suppressor by decreasing expression levels of MITF, BCL2 and cyclin D2 (40). The miR-181 family was also found to be a key negative regulator of cell cycle progression (41). Researchers have thus inserted miRNA +response elements (MREs) of miR-34a, miR-137 and miR-182 into the tumor necrosis factor (TNF)-related apoptosisinducing ligand (TRAIL) coding sequence to generate an MRE-regulated TRAIL expression system via an adenoviral vector. This novel strategy-regulated gene therapy vector has proved to be a promising biological agent for the treatment of UM (42,43). + +While the pathogenesis of cutaneous melanoma is similar to that of UM, UM exhibits different clinical and biological behaviors. Studies have shown that mutations of PTEN and BRAF, which are relatively common in cutaneous melanoma, are not found in UM (44,45). However, mitogen-activated protein kinase/extracellular signalregulated kinase was found to be constitutively activated in UM, indicating that the activation of the MAPK pathway is related to the development of UM (46). Clinical studies published in 2016 indicate that PD-1 and PD-L1 inhibitors, which demonstrate effects in the treatment of advanced cutaneous melanoma, confer durable objective tumor responses in relatively few patients with metastatic UM (47). + +A study published in *Nature Communication* in 2018 claimed that patients with MBD4 germline deleterious mutations exhibit high sensitivity to PD-1 inhibitors, a result that was proven to be related to hypermutated CpG > TpG burden (48). The adoptive transfer of tumor-infiltrating lymphocytes (TIL) has been identified as another efficient treatment of metastatic melanoma (49). An article published in *Lancet Oncology* in 2017 reported a single center phase 2 study found that the transfer of TIL could mediate tumor regression in patients with metastatic UM (50). Future studies should focus on ways to maintain clinical responses completely and durably. + +## Page 10 Of 12 Strengths And Limitations + +We accessed publications on UM extracted from the WOS database and analyzed the data comprehensively and objectively. Despite this, some restrictions were unavoidable as we only considered publications written in English, and thus, we may have overlooked important research published in other languages. Moreover, the more recent papers could not accumulate a large number of citations at the time of this study, which may have affected our conclusions to some degree. + +## Conclusions + +The present study illustrates global trends in UM research. + +The United States has contributed the most research and holds the leading position in this field. The latest progress can be found in *Investigative Ophthalmology and Visual Science*, +the British Journal of Ophthalmology and Ophthalmology. Jager MJ and Harbour JW may be good candidates for academic collaboration in this field. Gene mutation and pathogenesis pathway-related research has not yet been thoroughly studied and is gradually becoming a focus of research in this field. + +## Acknowledgments + +Funding: This work was supported by grants from the National Natural Science Foundation of China (Grants U1932135, 81772875), National Key Research and Development Plan (2018YFC1106100), The Science and Technology Commission of Shanghai (17DZ2260100, 19JC1410200). + +## Footnote + +Peer Review File: Available at http://dx.doi.org/10.21037/ +atm-20-3700 Conflicts of Interest: All authors have completed the ICMJE +uniform disclosure form (available at http://dx.doi. org/10.21037/atm-20-3700). The authors have no conflicts of interest to declare. + +Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. + +Open Access Statement: This is an Open Access article + +## Xie Et Al. Bibliometric Analysis On Um + +distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the noncommercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/. + +## References + +1. Ghazawi FM, Darwich R, Le M, et al. Uveal melanoma incidence trends in Canada: a national comprehensive population-based study. Br J Ophthalmol 2019;103:1872-6. + +2. Komatsubara KM, Carvajal RD. Immunotherapy for the Treatment of Uveal Melanoma: Current Status and Emerging Therapies. Curr Oncol Rep 2017;19:45. + +3. Kaštelan S, Antunica AG, Oresković LB, et al. + +Immunotherapy for uveal melanoma - Current knowledge and perspectives. Curr Med Chem 2020;27:1350-66. + +4. Singh AD, Turell ME, Topham AK. Uveal melanoma: +trends in incidence, treatment, and survival. Ophthalmology 2011;118:1881-5. + +5. Rishi P, Koundanya VV, Shields CL. Using risk factors for detection and prognostication of uveal melanoma. Indian J Ophthalmol 2015;63:110-6. + +6. Di Cesare S, Maloney S, Fernandes BF, et al. The effect of blue light exposure in an ocular melanoma animal model. J Exp Clin Cancer Res 2009;28:48. + +7. Yonekawa Y, Kim IK. Epidemiology and management of uveal melanoma. Hematol Oncol Clin North Am 2012;26:1169-84. + +8. Buder K, Gesierich A, Gelbrich G, et al. Systemic treatment of metastatic uveal melanoma: review of literature and future perspectives. Cancer Med 2013;2:674-86. + +9. Oliva M, Rullan AJ, Piulats JM. Uveal melanoma as a target for immune-therapy. Ann Transl Med 2016;4:172. + +10. Kuk D, Shoushtari AN, Barker CA, et al. Prognosis of Mucosal, Uveal, Acral, Nonacral Cutaneous, and Unknown Primary Melanoma From the Time of First Metastasis. Oncologist 2016;21:848-54. + +11. Dogrusöz M, Jager MJ, Damato B. Uveal Melanoma Treatment and Prognostication. Asia Pac J Ophthalmol (Phila) 2017;6:186-96. + +12. Hutchinson L. Immunotherapy: Cul-TIL-vating uveal melanoma regression. Nat Rev Clin Oncol 2017;14:328-9. + +13. Glanville J, Kendrick T, McNally R, et al. Research output + +## Annals Of Translational Medicine, Vol 8, No 21 November 2020 **Page 11 Of 12** + +on primary care in Australia, Canada, Germany, the Netherlands, the United Kingdom, and the United States: bibliometric analysis. BMJ 2011;342:d1028. + +14. Seriwala HM, Khan MS, Shuaib W, et al. Bibliometric analysis of the top 50 cited respiratory articles. Expert Rev Respir Med 2015;9:817-24. + +15. Khan MS, Ullah W, Riaz IB, et al. Top 100 cited articles in cardiovascular magnetic resonance: a bibliometric analysis. J Cardiovasc Magn Reson 2016;18:87. + +16. Hirsch JE. An index to quantify an individual's scientific research output. Proc Natl Acad Sci USA 2005;102:16569-72. + +17. Kivelä TT. The first description of the complete natural history of uveal melanoma by two Scottish surgeons, Allan Burns and James Wardrop. Acta Ophthalmol 2018;96:203-14. + +18. Kaliki S, Shields CL. Uveal melanoma: relatively rare but deadly cancer. Eye (Lond) 2017;31:241-57. + +19. Park SJ, Oh C-M, Kim BW, et al. Nationwide Incidence of Ocular Melanoma in South Korea by Using the National Cancer Registry Database (1999-2011). Invest Ophthalmol Vis Sci 2015;56:4719-24. + +20. Virgili G, Gatta G, Ciccolallo L, et al. Incidence of uveal melanoma in Europe. Ophthalmology 2007;114:2309-15. + +21. Van Raamsdonk CD, Griewank KG, Crosby MB, et al. + +Mutations in GNA11 in uveal melanoma. N Engl J Med 2010;363:2191-9. + +22. Harbour JW, Onken MD, Roberson EDO, et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 2010;330:1410-3. + +23. Van Raamsdonk CD, Bezrookove V, Green G, et al. + +Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009;457:599-602. + +24. Li Y, He J, Qiu C, et al. The oncolytic virus H101 combined with GNAQ siRNA-mediated knockdown reduces uveal melanoma cell viability. J Cell Biochem 2019;120:5766-76. + +25. Li P, He J, Yang Z, et al. ZNNT1 long noncoding RNA +induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression. Autophagy 2020;16:1186-99. + +26. Feng X, Degese MS, Iglesias-Bartolome R, et al. Hippoindependent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry. Cancer Cell 2014;25:831-45. + +27. Yoo JH, Shi DS, Grossmann AH, et al. ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma. Cancer Cell 2016;29:889-904. + +28. Chen X, Wu Q, Depeille P, et al. RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma. Cancer Cell 2017;31:685-696.e6. + +29. Feng X, Arang N, Rigiracciolo DC, et al. A Platform of Synthetic Lethal Gene Interaction Networks Reveals that the GNAQ Uveal Melanoma Oncogene Controls the Hippo Pathway through FAK. Cancer Cell 2019;35:457-472.e5. + +30. Moore AR, Ceraudo E, Sher JJ, et al. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma. Nat Genet 2016;48:675-80. + +31. Harbour JW, Roberson EDO, Anbunathan H, et al. + +Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat Genet 2013;45:133-5. + +32. Martin M, Maßhöfer L, Temming P, et al. Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat Genet 2013;45:933-6. + +33. Yavuzyigitoglu S, Koopmans AE, Verdijk RM, et al. Uveal Melanomas with SF3B1 Mutations: A Distinct Subclass Associated with Late-Onset Metastases. Ophthalmology 2016;123:1118-28. + +34. Gupta MP, Lane AM, DeAngelis MM, et al. Clinical Characteristics of Uveal Melanoma in Patients With Germline BAP1 Mutations. JAMA Ophthalmol 2015;133:881-7. + +35. Pan H, Jia R, Zhang L, et al. BAP1 regulates cell cycle progression through E2F1 target genes and mediates transcriptional silencing via H2A monoubiquitination in uveal melanoma cells. Int J Biochem Cell Biol 2015;60:176-84. + +36. Szalai E, Wells JR, Ward L, et al. Uveal Melanoma Nuclear BRCA1-Associated Protein-1 Immunoreactivity Is an Indicator of Metastasis. Ophthalmology 2018;125:203-9. + +37. Landreville S, Agapova OA, Matatall KA, et al. Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma. Clin Cancer Res 2012;18:408-16. + +38. Yan D, Zhou X, Chen X, et al. MicroRNA-34a inhibits uveal melanoma cell proliferation and migration through downregulation of c-Met. Invest Ophthalmol Vis Sci 2009;50:1559-65. + +39. Chen X, Wang J, Shen H, et al. Epigenetics, microRNAs, and carcinogenesis: functional role of microRNA-137 in uveal melanoma. Invest Ophthalmol Vis Sci 2011;52:1193-9. + +## Page 12 Of 12 + +40. Yan D, Dong XD, Chen X, et al. Role of microRNA-182 in posterior uveal melanoma: regulation of tumor development through MITF, BCL2 and cyclin D2. PLoS +One 2012;7:e40967. + +41. Zhang L, He X, Li F, et al. The miR-181 family promotes cell cycle by targeting CTDSPL, a phosphatase-like tumor suppressor in uveal melanoma. J Exp Clin Cancer Res 2018;37:15. + +42. Liu J, Ma L, Li C, et al. Tumor-targeting TRAIL +expression mediated by miRNA response elements suppressed growth of uveal melanoma cells. Mol Oncol 2013;7:1043-55. + +43. Zhou Y, Song X, Jia R, et al. Radiation-inducible human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy: a novel treatment for radioresistant uveal melanoma. Pigment Cell Melanoma Res 2010;23:661-74. + +44. Naus NC, Zuidervaart W, Rayman N, et al. Mutation analysis of the PTEN gene in uveal melanoma cell lines. Int J Cancer 2000;87:151-3. + +45. Rimoldi D, Salvi S, Liénard D, et al. Lack of BRAF +mutations in uveal melanoma. Cancer Res 2003;63:5712-5. + +Cite this article as: Xie M, Wu Q, Wang Y, Ge S, Fan X. Publication trends of research on uveal melanoma during 2000–2020: a 20-year bibliometric study. Ann Transl Med 2020;8(21):1463. doi: 10.21037/atm-20-3700 + +## Xie Et Al. Bibliometric Analysis On Um + +46. Zuidervaart W, van Nieuwpoort F, Stark M, et al. + +Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS. Br J Cancer 2005;92:2032-8. + +47. Algazi AP, Tsai KK, Shoushtari AN, et al. Clinical outcomes in metastatic uveal melanoma treated with PD-1 and PD-L1 antibodies. Cancer 2016;122:3344-53. + +48. Rodrigues M, Mobuchon L, Houy A, et al. Outlier response to anti-PD1 in uveal melanoma reveals germline MBD4 mutations in hypermutated tumors. Nat Commun 2018;9:1866. + +49. Forget M-A, Haymaker C, Hess KR, et al. Prospective Analysis of Adoptive TIL Therapy in Patients with Metastatic Melanoma: Response, Impact of Anti-CTLA4, and Biomarkers to Predict Clinical Outcome. Clin Cancer Res 2018;24:4416-28. + +50. Chandran SS, Somerville RPT, Yang JC, et al. Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study. Lancet Oncol 2017;18:792-802. \ No newline at end of file diff --git a/medical/md/PMC7772458.md b/medical/md/PMC7772458.md new file mode 100644 index 0000000000000000000000000000000000000000..8d9ddb06fabc22faeb300583c23bda5350abfff8 --- /dev/null +++ b/medical/md/PMC7772458.md @@ -0,0 +1,221 @@ + + +![0_image_2.png](0_image_2.png) + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +Case report Conversion From Knee Arthrodesis Back to Arthroplasty: A Particular Challenge in Combination With Fungal Periprosthetic Joint Infection Sven Frieler, MD a, b, c, d, *, Emre Yilmaz, MD a, Ryan Goodmanson, DO c, d, Yannik Hanusrichter, MD a, Thomas A. Schildhauer, MD a, Hinnerk Baecker, MD a a Department of Orthopedics and Trauma Surgery, BG University Hospital Bergmannsheil, Ruhr University Bochum - RUB, Bochum, Germany b Hansjorg Wyss Hip and Pelvic Center, Swedish Hospital, Seattle, WA, USA € c Swedish Neuroscience Institute, Swedish Medical Center, Seattle, WA, USA +d Seattle Science Foundation, Seattle, WA, USA +article info + +## Abstract + +| Article history: Received 12 August 2020 Received in revised form 19 October 2020 Accepted 24 October 2020 Available online 5 December 2020 Keywords: Arthroplasty Knee arthrodesis PJI fPJI Fungal periprosthetic joint infection TKA | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +A 58-year-old female treated at an outside facility with knee arthrodesis due to persistent periprosthetic joint infection fulfilled all prerequisites for a conversion back to arthroplasty, as part of a 2-stage revision. + +Owing to the detection of Candida parapsilosis, the treatment concept was converted to a three-stage procedure. A scheduled spacer exchange with additional amphotericin Beloaded polymethylmethacrylate was conducted as an intermediate revision before reimplantation. Conversion in the setting of fungal periprosthetic joint infection presents a challenge, and successful treatment hinges on the use of proper antifungal and antimicrobial protocols, advanced surgical techniques, and a multidisciplinary team approach. At the 3-year follow-up, successful infection eradication as measured by the Delphi-based consensus definition was achieved with a range of motion of 0-100. + +© 2020 The Authors. Published by Elsevier Inc. on behalf of The American Association of Hip and Knee Surgeons. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ +licenses/by-nc-nd/4.0/). + +## Introduction + +Knee arthrodesis is a viable salvage procedure after failed total knee arthroplasty (TKA), providing stability and decreased pain levels, but is also associated with low patient-reported outcome measures and high levels of functional disability [1e3]. Conversion of a fused knee back to arthroplasty can be a challenging procedure and has shown to be associated with high complication rates such as knee stiffness, extensor mechanism insufficiency, instability, BG University Hospital Bergmannsheil, Bochum, Ruhr University Bochum- RUB, +Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany. + +IRB statement: The authors certify that all applicable institutional and governmental regulations concerning the ethical use of clinical data were followed during the course of this research. This study (IRB ID: 18-6538-BR) was approved by the local ethics committee. + +* Corresponding author. 550 17th Avenue, Suite 600, Seattle, WA 98122, USA. Tel.: +þ1 206 475 9514. + +E-mail address: sven.frieler@bergmannsheil.de https://doi.org/10.1016/j.artd.2020.10.007 2352-3441/© 2020 The Authors. Published by Elsevier Inc. on behalf of The American Association of Hip and Knee Surgeons. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). + +Arthroplasty Today 6 (2020) 1038e1044 implant failure, and periprosthetic joint infection (PJI) [1,4e8]. + +Provided adherence to strict indications, a conversion back to knee arthroplasty might be an option for patients wanting to resume normal daily private and professional activities. + +Prerequisites for patients undergoing this procedure are a suf-ficient extensor mechanism and adequate soft-tissue coverage. In addition, realistic patient expectations with regard to the functional outcome along with the willingness and motivation to continuously participate in the postoperative follow-up care are needed for the success of the procedure [1,9,10]. A recent meta-analysis showed good clinical results but a high complication rate of up to 65% [11]. The importance of a thorough patient selection process is emphasized and aids in the outcome of this rare procedure. + +In the setting of PJI, a conversion must be planned meticulously. + +Fungal PJI (fPJI) presents even more of a challenge and has been considered as a difficult-to-treat (DTT) PJI, with high complication and failure rates reported in the literature [12,13]. Owing to its rarity, there is a lack of data for converting a knee arthrodesis back to arthroplasty in a patient with fPJI. + +## Case History + +A 58-year-old Caucasian female without relevant comorbidities in addition to a spinal cord stimulator with spinal instrumentation in her history underwent a one-stage exchange from unicompartmental knee arthroplasty to TKA in June 2016. + +Owing to further postoperative multidirectional instability, a one-stage exchange to a semiconstrained TKA was conducted in October 2016. This included additional metaphyseal augmentation to address relevant bone loss. During the early postoperative course, she developed an acute PJI and underwent multiple revisions with debridement, antibiotics, and implant retention without successful PJI eradication. In December 2016, a one-stage conversion to a cemented, intramedullary knee arthrodesis was carried out because of persistent PJI and large bone defects (Fig. 1a). + +In June 2017, the patient subsequently presented to our total joint outpatient department for further treatment of her chronic PJI. The main objective was to evaluate the possibility of PJI eradication and conversion back to TKA. A thorough workup, following the recommendations of the PRO-IMPLANT Foundation, was initiated, including preoperative joint aspiration for microbial cultures to identify bacterial and fungal pathogens, as well as determination of the synovial white blood cell count. Given the laboratory results of synovial white blood cell count of 3200/ml, draining sinus tract, and recent revision history, she met several major criteria for PJI [14e16]. Serum C-reactive protein and white blood cell were normal, and at this time, cultures were not growing any organisms. Therefore, culture-negative PJI +was suspected. Figure 2 provides a detailed timeline of the patient history. + +## Treatment Concept + +The diagnosis and treatment algorithm is based on the recommendations of the PRO-IMPLANT Foundation [16,17]. It is built on a + +![1_image_0.png](1_image_0.png) + +S. Frieler et al. / Arthroplasty Today 6 (2020) 1038–1044 + +![2_image_0.png](2_image_0.png) + +06/2017: + +![2_image_1.png](2_image_1.png) + +Explantation Figure 2. Timeline: patient history. UKA, unicompartmental knee arthroplasty; DAIR, debridement, antibiotics and implant retention. +multidisciplinary team (MDT) in which different medical specialties including infectologists, microbiologists, and orthopaedic surgeons are represented. + +To determine the true extent of bone loss and to gather multiple tissue samples to identify the causative agent, a two-stage revision was conducted. In addition, evaluation of the extensor mechanism must be an integral part of the initial operation to develop a precise planning of the further treatment. First, the cemented arthrodesis was explanted followed by a radical debridement with removal of all foreign material, cement remnants, and infected tissue. The + +![2_image_2.png](2_image_2.png) + +extensor mechanism appeared to be intact. Tissue biopsies were taken along with sonication of the retrieved implants for microbiology and histologic workup. A fixed temporary arthrodesis with vancomycin- and gentamicin-loaded bone cement for dead-space management and local antimicrobial treatment was implanted +[18,19]. Empirical antimicrobial therapy with ampicillin and sulbactam IV was initiated, after taking biopsies. + +The microbiological analysis showed a polymicrobial infection with Candida parapsilosis and Enterococcus faecium in 2 of 5 samples. Histology of the periprosthetic tissue confirmed the diagnosis of PJI (⩾ 23 granulocytes per 10 high-power fields) according to Krenn and Morawietz [20]. Following the recommendations of the PRO-IMPLANT Foundation [16,17] for dealing with DTT chronic PJI, 3 weeks after explantation, a scheduled spacer exchange to an amphotericin Beloaded, fixed poly(methyl methacrylate) spacer was conducted (Fig. 3). A rotating hinge revision arthroplasty +(NexGen RH Knee, Zimmer Biomet, Warsaw, IN) was implanted another 3 weeks later (Fig. 4). Owing to extensive bone loss (AORI +F2B/T2B), highly porous metaphyseal cones (Trabecular Metal, Zimmer Biomet, Warsaw, IN) were used for tibial and femoral augmentation. Our treatment concept provides consistent antimicrobial and antifungal therapy without drug holiday and joint aspiration before implantation of a new prosthesis. Prerequisites for implantation are satisfactory clinical course and declining inflammatory parameters. + +Owing to the detection of Candida parapsilosis and Enterococcus faecium, antimicrobial treatment with ampicillin and gentamicin in addition to caspofungin for antifungal therapy was started. Antimicrobial and antifungal IV therapy was administered for 10 days and then switched to oral medication with amoxicillin and fluconazole. The total duration of combined antimicrobial and antifungal therapy was 12 weeks with amoxicillin and 6 months with fluconazole (Fig. 5). + +## Outcome + +The patient presented in the outpatient clinic after 3, 6, and 12 + +![3_image_0.png](3_image_0.png) + +months and annually thereafter. At the 3-year follow-up, successful infection eradication was verified because of the Delphi-based consensus definition by Diaz-Ledezma et al. [21]: (1) healed + +![4_image_0.png](4_image_0.png) + +Figure 5. The three-stage exchange procedure. + +![4_image_1.png](4_image_1.png) + +wound without fistula persistens, drainage, or pain and no infection recurrence caused by the same organism; (2) no subsequent surgical intervention for persistent or perioperative infection after revision surgery; and (3) no occurrence of PJI-related mortality. + +The range of motion at the latest follow-up was 0-100 (Fig. 6). + +No extension limitation or weakness, as well as no clinical or radiological signs of loosening, was observed (Fig. 4). No complication or reinfection was documented. The patient showed significant improvements in overall satisfaction and mobility. Her dailylife activity and capability to work have been re-established. The patient went back to work as a full-time geriatric nurse. + +## Discussion + +A standardized antimicrobial and antifungal therapy based on previously published concepts of Zimmerli et al., the PRO-IMPLANT Foundation, and the European Bone and Joint Infection Society +[16,22,23] to improve mobility and achieve infection eradication was conducted. Removal of the implant and any foreign material as part of a one- or two-stage exchange procedure [24] and antimicrobial treatment according to the susceptibility of the microorganism [23] form the core of their current management algorithm in chronic PJI. In severe cases with DTT microorganisms and/or unsatisfactory bone or soft tissue, a three-stage exchange must be considered. + +Owing to the intraoperatively detected DTT pathogen (Candida), +the procedure was adjusted from a two-stage to three-stage revision (Fig. 5) according to the PRO-IMPLANT recommendations. To the best of our knowledge, this is the first report of a patient with an fPJI who successfully underwent a conversion from an intramedullary knee arthrodesis to a rotating hinge prosthesis (Fig. 1a and b). + +fPJI is a rare occurrence and has only been reported in approximately 1% of all PJIs [25]. For fPJI and for conversion from arthrodesis to arthroplasty, the literature provides only a few studies with a small number of patients and various treatment concepts [12]. No gold standard in the management of fPJI has been established. This includes the surgical procedure and the antimicrobial therapy or the duration of treatment, as noticed at the last International Consensus Meeting on Prosthetic Joint Infection [14]. + +Schoof et al. [26] reported heterogeneity in the treatment of fPJIs of the hip. Of 45 cases of fPJIs, 7 (16%) patients received a permanent resection arthroplasty. Two (4%) patients underwent one-stage exchanges and 26 (58%) 2-stage procedures. Furthermore, they noticed delayed arthrodesis in one (2%) patient, 5 (11%) debridements with retention of the prosthesis, and 4 (9%) patients were treated solely with antifungal suppression therapy. In 78% of the cases, the use of local antimycotic agents has not been reported. In 2018, Brown et al. + +[12] showed a similar heterogeneity presenting their fPJI collective, including 31 patients with PJI of the hip and knee, treated with debridement, antibiotics, and implant retention, two-stage exchange, resection arthroplasty, and different antimicrobial therapy. + +Regarding the conversion from knee arthrodesis back to arthroplasty, Jauregui et al. [27] reported in their meta-analysis a good clinical outcome. The overall complication rate was 47% along with a failure rate of 11%. Nevertheless, most patients were satisfied with the procedure. Kernkamp et al. [11] reported in their analysis of 123 converted TKA a mean gain of flexion to be 80 and an improvement of Hospital for Special Surgery score by 20 points. + +However, they reported complications including skin necrosis (25%), infection (11%), revision (11%), and refusion of the knee (4.9%). A complication-free course has only occurred in about 35% of the reported cases and represents the specialized nature of this procedure and the follow-up necessary for its success. + +Issues can arise in instances of infected knee arthrodesis that differ from that of a patient who underwent normal revision arthroplasty. However, owing to advances in total joint arthroplasty techniques, problems that previously posed issues to the orthopaedic surgeon, such as extensive bone defects, insufficiency of extensor apparatus, and persistent PJI, are only relative indications for arthrodesis today. Modern treatment techniques such as highly porous metaphyseal cones to address bone deficiency [28] and extensor mechanism reconstruction by using a particular monofilament polypropylene mesh [29,30] are lessening the instances where knee arthrodesis is needed. Along with technological advancements, new diagnostic and PJI management concepts, realized by an MDT, enable new treatment options for this subset of patients. Thus, the use of knee arthrodesis is limited to a limbsaving procedure and not a general treatment for failed TKA due to PJI. This is in accordance with Gottfriedsen et al. who reported a significant decrease in the 5-year cumulative incidence of knee arthrodesis in a nationwide register-based study [31]. + +Ravikumar et al. [32] showed the successful conversion of an infected fused knee to TKA. In our patient, successful infection eradication as measured by the Delphi-based consensus definition was achieved. The conversion from knee arthrodesis to TKA showed improvement in the range of motion, pain scores, and the overall satisfaction. + +## Conclusion + +MDT approaches are combining modern surgical procedures with advanced antimicrobial therapy strategies, enabling the possibility for successful infection eradication even in PJI with DTT +pathogens and in patients with compromised bone stock and softtissue conditions. + +In the case of fPJI, a three-stage revision, combining the advantages of a second thorough debridement with the benefits of an optimized systemic and local antimicrobial and antifungal therapy, shows promising results. + +## Conflict Of Interests + +The authors declare there are no conflicts of interest. + +## References + +[1] Henkel TR, Boldt JG, Drobny TK, Munzinger UK. Total knee arthroplasty after formal knee fusion using unconstrained and semiconstrained components: a report of 7 cases. J Arthroplasty 2001;16(6):768e76. + +[2] Kuchinad R, Fourman MS, Fragomen AT, Rozbruch SR. Knee arthrodesis as limb salvage for complex failures of total knee arthroplasty. J Arthroplasty 2014;29(11):2150e5. + +## Key Points + +1. Prerequisites for patients to undergo conversion from arthrodesis to total knee arthroplasty are (1) sufficient extensor mechanism, (2) adequate soft-tissue coverage, and +(3) compliance and realistic patient expectations. + +2. A standardized but individually adapted surgical treatment concept in combination with a consistent antimicrobial and antifungal therapy is crucial for successful infection eradication. + +3. Patients need to be treated preoperatively and postoperatively by a multidisciplinary team in specialized departments. +[3] Wood JH, Conway JD. Advanced concepts in knee arthrodesis. World J Orthop 2015;6(2):202e10. + +[4] Cameron HU, Hu C. Results of total knee arthroplasty following takedown of formal knee fusion. J Arthroplasty 1996;11(6):732e7. + +[5] Cho SH, Jeong ST, Park HB, Hwang SC, Kim DH. Two-stage conversion of fused knee to total knee arthroplasty. J Arthroplasty 2008;23(3):476e9. + +[6] Clemens D, Lereim P, Holm I, Reikeras O. Conversion of knee fusion to total arthroplasty: complications in 8 patients. Acta Orthop 2005;76(3):370e4. + +[7] Kim YH, Kim JS, Cho SH. Total knee arthroplasty after spontaneous osseous ankylosis and takedown of formal knee fusion. J Arthroplasty 2000;15(4): +453e60. + +[8] Kim YH, Oh SH, Kim JS. Conversion of a fused knee with use of a posterior stabilized total knee prosthesis. J Bone Joint Surg Am 2003;85(6):1047e50. + +[9] Jamshidi K, Hadi H, Ramezan M, Moslem A. Conversion of fused knee following distal femur tumor surgery to total knee arthroplasty. Iran Red Crescent Med J 2013;15(9):870e2. + +[10] Ruggieri P, Kasimatis G, Errani C, Bosco G, Mercuri M. Desarthrodesis and prosthetic reconstruction of the knee after resection of bone tumors. J Surg Oncol 2010;102(7):832e7. + +[11] Kernkamp WA, Verra WC, Pijls BG, Schoones JW, van der Linden HM, +Nelissen RG. Conversion from knee arthrodesis to arthroplasty: systematic review. Int Orthop 2016;40(10):2069e74. + +[12] Brown TS, Petis SM, Osmon DR, et al. Periprosthetic joint infection with fungal pathogens. J Arthroplasty 2018;33(8):2605e12. + +[13] Theil C, Schmidt-Braekling T, Gosheger G, Idelevich EA, Moellenbeck B, +Dieckmann R. Fungal prosthetic joint infection in total hip or knee arthroplasty: +a retrospective single-centre study of 26 cases. Bone Joint J 2019;101-b(5): +589e95. + +[14] Aalirezaie A, Bauer TW, Fayaz H, et al. Hip and knee section, diagnosis, reimplantation: proceedings of international consensus on orthopedic infections. J Arthroplasty 2019;34(2s):S369e79. + +[15] Parvizi J, Tan TL, Goswami K, et al. The 2018 definition of periprosthetic hip and knee infection: an evidence-based and validated criteria. J Arthroplasty 2018;33(5):1309e1314. e2. + +[16] Renz N, Perka C, Trampuz A. [Management of periprosthetic infections of the knee]. Orthopade 2016;45(1):65e71. + +[17] Izakovicova P, Borens O, Trampuz A. Periprosthetic joint infection: current concepts and outlook. EFORT Open Rev 2019;4(7):482e94. + +[18] Pfitzner T, Preininger B, von Roth P, Perka C. [A novel technique for a stable intramedullary fixation of static spacers in two-stage septic revision TKA with severe bone defects]. Z Orthop Unfall 2015;153(2):203e5. + +[19] Rohner E, Pfitzner T, Preininger B, Zippelius T, Perka C. Temporary arthrodesis using fixator rods in two-stage revision of septic knee prothesis with severe bone and tissue defects. Knee Surg Sports Traumatol Arthrosc 2016;24(1): +84e8. + +[20] Krenn V, Morawietz L, Perino G, et al. Revised histopathological consensus classification of joint implant related pathology. Pathol Res Pract 2014;210(12):779e86. + +[21] Diaz-Ledezma C, Higuera CA, Parvizi J. Success after treatment of periprosthetic joint infection: a Delphi-based international multidisciplinary consensus. Clin Orthop Relat Res 2013;471(7):2374e82. + +[22] Zimmerli W. [Orthopaedic implant-associated infections: update of antimicrobial therapy]. Orthopade 2015;44(12):961e6. + +[23] Zimmerli W, Trampuz A, Ochsner PE. Prosthetic-joint infections. N Engl J Med 2004;351(16):1645e54. + +[24] Zimmerli W, Moser C. Pathogenesis and treatment concepts of orthopaedic biofilm infections. FEMS Immunol Med Microbiol 2012;65(2):158e68. + +[25] Nace J, Siddiqi A, Talmo CT, Chen AF. Diagnosis and management of fungal periprosthetic joint infections. J Am Acad Orthop Surg 2019;27(18):e804e18. + +[26] Schoof B, Jakobs O, Schmidl S, et al. Fungal periprosthetic joint infection of the hip: a systematic review. Orthop Rev (Pavia) 2015;7(1):5748. + +1044 S. Frieler et al. / Arthroplasty Today 6 (2020) 1038e1044 + +[27] Jauregui JJ, Buitrago CA, Pushilin SA, Browning BB, Mulchandani NB, +Maheshwari AV. Conversion of a surgically arthrodesed knee to a total knee arthroplasty-is it Worth it? A meta-analysis. J Arthroplasty 2016;31(8): +1736e41. + +[28] Angerame MR, Jennings JM, Holst DC, Dennis DA. Management of bone defects in revision total knee arthroplasty with use of a stepped, porous-coated metaphyseal sleeve. JBJS Essent Surg Tech 2019;9(2):e14. + +[29] Abdel MP, Fuchs M, von Roth P. [Management of extensor mechanism injuries following total knee arthroplasty]. Orthopade 2016;45(1):47e53. + +[30] Abdel MP, Salib CG, Mara KC, Pagnano MW, Perry KI, Hanssen AD. Extensor mechanism reconstruction with use of Marlex mesh: a series study of 77 total knee Arthroplasties. J Bone Joint Surg Am 2018;100(15):1309e18. + +[31] Gottfriedsen TB, Schroder HM, Odgaard A. Knee arthrodesis after failure of knee arthroplasty: a nationwide register-based study. J Bone Joint Surg Am 2016;98(16):1370e7. + +[32] Ravikumar M, Kendoff D, Citak M, Luck S, Gehrke T, Zahar A. One stage conversion of an infected fused knee to total knee replacement - a surgical challenge. Open Orthop J 2013;7:67e71. \ No newline at end of file diff --git a/medical/md/PMC8072224.md b/medical/md/PMC8072224.md new file mode 100644 index 0000000000000000000000000000000000000000..a7f5a4c237922d902a8adedae40a2581e62e0af0 --- /dev/null +++ b/medical/md/PMC8072224.md @@ -0,0 +1,348 @@ +# Bacterial Detection By Naip/Nlrc4 Elicits Prompt Contractions Of Intestinal Epithelial Cell Layers + +Pilar Samperio Ventayola, Petra Geisera, Maria Letizia Di Martinoa, Alexandra Florbranta, Stefan A. Fattingera,b, Naemi Walderb, Eduardo Simac, Feng Shaod, Nelson O. Gekarae, Magnus Sundbomc, Wolf-Dietrich Hardtb, Dominic-Luc Webbf, Per M. Hellströmf, Jens Erikssona, and Mikael E. Sellina,1 aScience for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden; bInstitute of Microbiology, Department of Biology, ETH Zürich, 8093 Zürich, Switzerland; cDepartment of Surgical Sciences, Uppsala University, 75185 Uppsala, Sweden; dNational Institute of Biological Sciences, 102206 Beijing, China; eDepartment of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm 10691, Sweden; and fDepartment of Medical Sciences, Gastroenterology and Hepatology Unit, Uppsala University, 75185 Uppsala, Sweden Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved February 24, 2021 (received for review July 3, 2020) +The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) +without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (S.Tm) within minutes elicited focal contractions with inward movements of up to ∼1,000 IECs. + +Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D +failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. + +We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin–dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process. + +bacterial infection | epithelium | organoid | inflammasome | contraction Epithelial cells make up barriers that shield the interior of the body from the environment. In the homeostatic intestine, the surface of the epithelium is maximized to facilitate uptake of ingested nutrients, water, and electrolytes (1, 2). This large surface, however, makes the gut epithelium vulnerable to attack by pathogenic microorganisms. Pathogen onslaught and the ensuing inflammation causes death and loss of intestinal epithelial cells +(IECs) (3, 4). This can be beneficial as damaged cells and intracellular pathogens are cleared from the mucosa. At the same time, cell loss may jeopardize epithelial integrity when insufficient IEC +numbers remain to uphold the barrier. One way to prevent such an outcome would be to compact the epithelial layer in affected regions. It is well known that smooth muscle contraction results in shrinkage and compaction of the intestinal wall during inflammation (5), but whether and how the epithelium itself can alter its IEC +packing upon infection appears less clear. + +As a system to sense mucosal intrusions, IECs express pattern recognition receptors (PRRs) [e.g., Toll-like receptors associated with cell membranes (6), and Nod-like receptors (NLRs) that form inflammasomes in the cytosol] (7–9). Epithelial recognition of pathogens through PRRs elicits a panel of countermeasures, including production of proinflammatory cytokines, chemokines, lipids (10–13), secretion of antimicrobial peptides (11, 14), and the death and expulsion of infected IECs into the lumen (12, 15, 16). An intricate cross-talk exists between IECs and immune cells residing in the underlying lamina propria (17). Epithelial defense signaling has in some cases also been shown to engage bystander epithelial cells surrounding a pathogen-infected IEC (18–20). Still, it remains poorly explored how PRR recognition of invading pathogens can instruct tissue-scale epithelial responses. + +Intestinal epithelial organoids (denoted "enteroids" when established from small intestinal crypts) provide a powerful experimental system to assess the behavior of untransformed epithelia in the absence of other mucosal cell types (21). Organoids can be grown as three-dimensional (3D) miniature organs within matrix domes (22, 23) or be disrupted to produce two-dimensional (2D) monolayers atop coated surfaces (24–27). By contrast to tumor cell lines, organoids maintain untransformed properties over time (28) and show reliable PRR expression patterns that mimic the intact gut + +## Significance + +Contractile movements in the mammalian intestine typically rely on dedicated muscle cells. Here, we however show that untransformed intestinal epithelial cell layers initiate immediate focal contractions, affecting hundreds to thousands of epithelial cells, in response to bacterial infection. This epithelial contraction response occurs in the absence of other mucosal cell types. Instead, the epithelium itself senses the pathogen intrusion through a pattern recognition receptor complex— NAIP/NLRC4—and initiates actomyosin contractions that propagate across the epithelial layer. Within minutes, this response densifies the cell packing at infection sites and may prevent tissue disintegration during the subsequent stage of epithelial cell death and expulsion. Our results highlight a previously unappreciated dynamic behavior of intestinal epithelia. + +Author contributions: P.S.V. and M.E.S. conceived the study; P.S.V., P.G., M.L.D.M., A.F., S.A.F., E.S., M.S., W.-D.H., D.-L.W., P.M.H., and M.E.S. designed research; P.S.V., P.G., +M.L.D.M., A.F., S.A.F., N.W., E.S., and J.E. performed research; M.L.D.M., S.A.F., F.S., N.O.G., W.-D.H., and J.E. contributed new reagents/analytic tools; P.S.V., P.G., and A.F. analyzed data; P.S.V. and M.E.S. wrote the paper; and all authors reviewed and edited the paper. The authors declare no competing 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: mikael.sellin@imbim.uu.se. + +This article contains supporting information online at https://www.pnas.org/lookup/suppl/ +doi:10.1073/pnas.2013963118/-/DCSupplemental. + +Published April 12, 2021. + +![0_image_0.png](0_image_0.png) + +epithelium (6, 9). Organoid models have for these reasons become attractive tools for physiological studies of gut infection (25, 29–32). + +In this work, we used time-lapse imaging to follow the tissue dynamicity of enteroid-derived mouse and human intestinal epithelia, placed atop pliable matrix supports. Upon infection with the prototypical enteropathogen Salmonella enterica serovar Typhimurium (S.Tm), we observed prompt and large epithelial contraction foci which preceded and could be uncoupled from IEC death and expulsion. Bacterial type-three secretion system +(TTSS) and/or cytosolic flagellin triggered the epithelial NAIP/ +NLRC4 inflammasome, sublytic Gasdermin D pore formation, ion fluxes, and myosin-dependent contractions spreading from sensing events at focal epicentres. We show that this swift response allows the epithelium to increase its IEC packing at sites of infection, which may minimize the disruptive effects of subsequent cell death and expulsion. + +## Results + +S.Tm Infection Triggers Prompt Focal Contractions in Murine IEC +Monolayers. To explore epithelial tissue dynamics upon infection, we developed enteroid-derived epithelial 2D-monolayers placed atop a flexible hydrogel in live-imaging chambers [informed by (26); see SI Appendix, Materials and Methods]. C57BL/ +6J mouse jejunal enteroids were first kept in 3D-culture containing CHIR99021 (CHIR, a glycogen synthase kinase-3 inhibitor) and valproic acid (VPA) to maximize proliferative potential (33, 34). Enteroids were subsequently fragmented into single cells, seeded out onto collagen-1 hydrogels, and the CHIR/VPA eliminated 24 h postseeding. IECs quickly attached to the hydrogel and extensive proliferation was noted during the first 24 to 48 h (Fig. 1 A–D). By 72 h, a postmitotic confluent epithelial monolayer was established (Fig. 1 A–D), which maintained at 96 h (Fig. 1C), and thereafter gradually deteriorated. + +The mature monolayer expressed low transcript levels of the stem cell marker Lgr5 (Fig. 1E), elevated levels of the differentiated enterocyte markers alkaline phosphatase and Ezrin (Fig. 1E), and featured a standing IEC arrangement with adherence junctions (E-cadherin) and an apical actin brush border +(Fig. 1F). + +To assess the impact of bacterial infection, we challenged mature IEC monolayers atop hydrogels with wild-type (WT) +S.Tm SL1344 ("S.Tmwt") at a multiplicity-of-infection (MOI) of 0.2-2 and imaged them by time-lapse differential interference contrast (DIC) microscopy. Strikingly, within 10 to 20 min postinfection (p.i.), large contraction foci became visible (Movie S1). We employed an optical flow image analysis pipeline to quantify the speed and direction of these epithelial movements +[Movies S2 and S3 and Fig. 1G) (35); see SI Appendix for details]. Speed maps calculated from vector fields showed that each focus involved a 0.21 ± 0.14 mm2 patch of epithelium (i.e., ∼200 to 1700 IECs; Fig. 1 H and I), contracting toward a single epicentre with inward movements typically lasting 1.2 ± 0.3 min and maximal pixel speeds ranging between ∼2 and 8 μm/min +(Fig. 1 G and J). Early upon infection, isolated foci were readily detectable (Fig. 1H, 13 min p.i.), which subsequently coalesced into a patchwork (Fig. 1H, 23 min p.i.). Signs of epithelial cell death +(rounding and dislodging of IECs) became evident thereafter, and contractile movements eventually ceased (Fig. 1H, 83 min p.i.). Infection-elicited contractions were also observed upon inoculation with another S.Tmwt isolate (ATCC 14028s) (SI Appendix, Fig. S1 A +and B) and in monolayers from a donor mouse housed in another facility (SI Appendix, Fig. S1 C and D). Exceedingly rare and small contraction foci could in fact occasionally be observed also prior to infection (Fig. 1 I and J). From this we conclude that murine IEC +monolayers are wired to elicit prompt focal contractions upon S.Tm infection. + +S.Tm TTSS-1 Docking and Invasion Elicits Focal Epithelial Contractions. + +S.Tm employs flagella and TTSS-1 to reach and invade the epithelium (36–38). Using fluorescence-labeled bacteria, we noted that contraction foci emerged in areas with S.Tm bound to the epithelial surface (Fig. 2A). This led us to postulate that S.Tm docking or invasion elicits focal contractions. To test this hypothesis, we infected murine monolayers with S.Tmwt or S.TmΔinvG (lacks a structural component of TTSS-1). As expected, S.Tmwt invaded IECs, while S.TmΔinvG failed to do so (SI +Appendix, Fig. S1 E and F). Live DIC imaging over a range of MOIs (0.2 to 20) showed that epithelial contractions increased in frequency and intensity with S.Tmwt dose (Fig. 2B). By sharp contrast, S.TmΔinvG infection did not elicit epithelial contractions, not even at the highest MOI (Fig. 2B). This points to a central role for TTSS-1 in triggering S.Tm-induced epithelial contractions. + +TTSS-1 docks to the host cell membrane via a SipBC translocon, resulting in the transfer of TTSS-1-effectors into the host cell (39). Among the effectors, SipA stabilizes actin filaments, while SopB, SopE, and SopE2 drive actin filament nucleation through Rho and Arf GTPases (40). These four effectors largely explain TTSS-1–dependent host cell invasion both in vivo and in tissue culture models (38, 41). We used single- and multipleeffector mutant bacteria to assess the relationship between S.Tm IEC docking, invasion, and epithelial contraction. As evident in Fig. 2C, deletion of SipA (S.TmΔsipA), or the combined deletion of SopB, SopE, and SopE2 (S.TmΔsopBEE2), delayed the contraction response. Importantly however, even a strain lacking all four effectors (i.e., ΔsipAsopBEE2; "S.TmΔ4") elicited contractions which exceeded those of S.TmΔinvG-infected monolayers (Fig. 2C). Hence, TTSS-1 docking to the epithelium suffices to elicit focal epithelial contractions, and TTSS-1 invasion effectors augment this effect. + +## Naip/Nlrc4 Sensing Of The Infection At Contraction Focus Epicenters. + +An epithelial NAIP/NLRC4 inflammasome can sense bacterial ligands (TTSS rod and needle proteins, flagellin), trigger IEC death, and restrict bacterial spread to deeper tissues in vivo (12, 16, 42). To examine if the epithelial contraction response might be linked to NAIP/NLRC4, we generated enteroids from Nlrc4−/− (43) and VillinrfpNaip1-6Δ/Δ mice (lacking all NAIP receptors and expressing a red fluorescent protein (RFP) reporter; see SI Appendix, Materials and Methods) and established monolayers atop hydrogels for infection. In side-by-side experiments, WT control monolayers again reacted to S.Tmwt by vigorous contractions (Fig. 3 A–D). This response was, however, completely abolished in both Nlrc4−/− (Fig. 3 A and B) and VillinrfpNaip1-6Δ/Δ +(Fig. 3 C and D) monolayers across all MOIs tested. In fact, NLRC4- or NAIP-deficient monolayers appeared as refractory as WT monolayers infected by S.TmΔinvG (compare Figs. 3 A and C +and 2B). + +The involvement of NAIP/NLRC4 in epithelium contraction +(Fig. 3 A–D) could be explained by several models. In the simplest one, NAIP/NLRC4 activation in an infected IEC initiates the contraction response with that cell at the epicenter. Alternatively, NAIP/NLRC4 might fuel propagation of contraction across the tissue following its initiation by other means. To resolve these possibilities, we established genetically chimeric IEC monolayers. + +3D-enteroids were generated from WT and VillinrfpNaip1-6Δ/Δ mice, the enteroids fragmented, and the fragments mixed in equal proportions before seeding atop hydrogels. This resulted in the outgrowth of confluent monolayers with alternating red Naip1-6Δ/Δ and noncolored WT regions (Fig. 3E). The chimeric monolayers were infected with S.Tmwt, contraction foci tracked by time-lapse DIC +imaging, and the epicenter locations overlaid with the RFPfluorescence channel (Fig. 3E and Movie S4). Strikingly, contraction epicenters mapped virtually exclusively onto noncolored WT +regions (>95% of epicenters; Fig. 3E and Movie S4). Following + +![2_image_0.png](2_image_0.png) + +ICROBIOLOG + +![3_image_0.png](3_image_0.png) + +4 of 11 PNAS +Samperio Ventayol et al. + +![4_image_0.png](4_image_0.png) + +MICROBIOLOGY +their initiation, however, contractions propagated equally across VillinrfpNaip1-6Δ/Δ and WT regions (Fig. 3F). These data support that NAIP/NLRC4 bacterial sensing at invasion sites triggers the focal contraction response. + +We next assessed the impact of specific NAIP ligand(s). Epithelial contractions were elicited also by a S.TmΔfljBfliC strain that lacks flagellin (Fig. 3 G and I; nonmotile but flagellated S.TmΔmotA used as control). Combined with the previous results +(Fig. 2 B and C), this suggests that TTSS-1 is sufficient to elicit contractions. Nevertheless, delivery of Legionella pneumophila flagellin into the IEC cytosol by the N-terminal domain of Bacillus anthracis lethal factor ["FlaTox" (44, 45)] also elicited contractions, while extracellular Fla or the NLRP3 inducer nigericin failed to do so (Fig. 3 H and J). These findings are consistent with the fact that naïve murine IECs express little NLRP3 but significant levels of all NAIP receptors [i.e., NAIP1, 2, 5, and 6 (9)] and are therefore poised to elicit focal contractions upon sensing of either bacterial TTSS or flagellin ligands. + +## Gasdermin D Sublytic Pore Formation Sparks Myosin-Dependent + +Focal Contractions that Precede and Can Be Uncoupled from IEC +Death and Expulsion. Activated NAIP/NLRC4 can in turn activate Caspase-1 or Caspase-8, leading to either Gasdermin D +pore formation and subsequent lytic cell death ["pyroptosis" +(46)] or apoptotic cell death, depending on the cellular context +(12, 47). In the intestinal epithelium, this results in the expulsion of infected IECs into the lumen (12, 15, 16) and the release of proinflammatory mediators (12, 15). We used genetic and pharmacological approaches to probe the relationship between the epithelial contraction response and potential pathways downstream of NAIP/NLRC4. + +The adaptor protein Apoptosis-Associated Speck-Like Protein Containing CARD (ASC) associates with activated NAIP/ +NLRC4 and links this inflammasome to Caspase-8 activation in IECs (12, 47). However, S.Tmwt infection elicited focal contractions in both Asc−/− (43) monolayers and matched controls +(SI Appendix, Fig. S2 A and B), hence excluding a primary involvement of this pathway. By sharp contrast, monolayers established from mice lacking Caspase-1 [Casp1/11−/− (48)] or Gasdermin D [GsdmD−/− (49)] did not exhibit epithelial contractions (Fig. 4 A–D) (i.e., in line with findings in NAIP/ +NLRC4–deficient monolayers). To examine if lytic IEC death downstream of Caspase-1/Gasdermin D might explain the focal contractions, infections were repeated in WT monolayers in the presence of the dye Draq7, which enters lysed cells. Tracing of fluorescence within contraction foci showed that Draq7-positive expelled IECs only became visible in some cases and always later than the focal contraction phase (Fig. 4 E and F). Moreover, pretreatment with the broad-spectrum Caspase-inhibitor Z-VAD-FMK drastically reduced IEC death upon S.Tm infection but did not impact early contractions (Fig. 4 G–J). + +Neighboring epithelial cells form an actin purse string to help expel a dying epithelial cell from a cell layer [a process referred to as "extrusion" (50)]. It remained plausible that this phenomenon could be linked to the large focal contractions noted here. + +However, in the live cell tracing experiments, we only observed IECs expelling from the monolayer subsequent to the contraction phase (exemplified in Fig. 4E). Homeostatic removal of IECs in uninfected monolayers (known to involve a typical extrusion process) were also not accompanied by any contraction foci propagating across the surrounding tissue (SI Appendix, Fig. + +S3A). Most importantly, in Caspase-1(-11)–deficient monolayers, S.Tm still elicited expulsion of infected IECs, but not focal epithelial contractions, hence formally uncoupling these two phenomena from each other (SI Appendix, Fig. S3B). Finally, typical cell extrusion relies on Sphingosine-1-phosphate (S1P) signaling between neighboring IECs (51), but we found that S.Tm-induced focal contractions proceeded normally also in the presence of S1P biosynthesis inhibitors (SI Appendix, Fig. S3C). + +Proinflammatory signaling occurs concomitant with the death of S.Tm-infected IECs and involves interleukin-18 and eicosanoid release (12, 15). Neutralization of IL-18 by saturation with IL-18–binding protein (IL-18BP) had no impact on the epithelial contraction response (SI Appendix, Fig. S4A), neither did preincubation of monolayers with eicosanoid synthesis inhibitors (SI +Appendix, Fig. S4 B and C). Transfer of culture supernatants from infected monolayers to naïve monolayers failed to elicit any contraction foci (SI Appendix, Fig. S4D). We also assessed proinflammatory gene transcription and found that this occurred in both S.Tmwt- and S.TmΔinvG-infected monolayers (SI Appendix, Fig. S4E), whereas exclusively S.Tmwt-infected monolayers produced contraction foci (Fig. 2). Taken together, these results suggest that NAIP/NLRC4–dependent focal epithelial contractions require minute quantities of active Caspase-1 and the poreforming protein Gasdermin D (46), but precede and can be uncoupled from IEC death and lysis, the IEC extrusion process itself, and generalized proinflammatory signaling. + +Work in macrophages has shown that early pore formation by a small number of Gasdermin D molecules causes immediate ion fluxes, which can be blocked by Gd3+ (in the form of Gadolinium chloride) (52). Strikingly, preincubation of WT monolayers with Gd3+ revealed a dose-dependent reduction in the number of S.Tmwt-elicited contraction foci (Fig. 4K). A brief incubation with the Calcium chelator EGTA also markedly attenuated the contraction response (SI Appendix, Fig. S5 A and B). In epithelial cells, nonmuscle myosin II (hereafter simply "myosin") can respond to ion fluxes and is fundamental for force generation, force transmission at adherence junctions, and apical constriction (53). To inhibit myosin activity, blebbistatin was used, as previously reported by others (54). Short-term blebbistatin incubation did not affect the epithelial monolayer morphology nor the numbers of tissue-associated S.Tm or late-stage IEC death +(SI Appendix, Fig. S5 C–F). However, at low dose (10μM), +blebbistatin dramatically reduced the number of S.Tm-induced contraction foci, and at high concentration (50μM), contractile movements ceased entirely (Fig. 4L and SI Appendix, Fig. S5G). From these data, we conclude that early sublytic Gasdermin D +pore formation results in ion fluxes and the swift propagation of focal myosin contraction pulse(s) across the epithelium. + +## Early Focal Contractions Densify Infected Epithelial Regions And May + +Prevent Subsequent Tissue Disintegration. To determine if infection-induced contractions are relevant also for human gut epithelia, human jejunal enteroids were established and 2Dmonolayers cultured atop hydrogels (see SI Appendix, Materials and Methods for details). Imaging of S.Tmwt-infected human monolayers kept in expansion medium revealed some subtle epithelial movements (Fig. 5A, Upper; compare with Fig. 2B). When the medium was exchanged for Wnt3a-deficient medium at the time of confluence, human monolayers adopted a more differentiated appearance (SI Appendix, Fig. S6 A and B). In response to infection, these human monolayers displayed more pronounced focal contractions (Fig. 5A, Bottom and Movie S5). It should be noted that contraction foci were still fewer, and the contraction events less vigorous, than in the murine monolayers. Notably, neither the human transformed cell line Caco-2 C2Bb1 nor the immortalized mouse cell lines m-ICc12 produced contraction foci when placed atop hydrogels and infected with S.Tm +(SI Appendix, Fig. S6 C and D). These results indicate that infection-elicited gut epithelial contraction is a response shared between species but only observed in untransformed epithelial models, and most potent in murine epithelia. + +The appearance of multiple contraction foci, each affecting hundreds to thousands of IECs (Fig. 1 H and I), could have consequences for the compaction of the epithelial layer. We + +![6_image_0.png](6_image_0.png) + +MICROBIOLOGY + +![7_image_0.png](7_image_0.png) + +addressed this possibility, first by measuring the retraction of the murine monolayer edge, after manually detaching it from the chamber wall prior to infection. Infection with S.Tmwt produced an edge retraction in WT monolayers already within the first ∼30 to 60 min p.i. (Fig. 5 B and C). By contrast, only negligible edge retraction was observed in response to noninvasive S.TmΔinvG +(Fig. 5 B and C) or when Nlrc4−/− monolayers were infected with S.Tmwt (Fig. 5D). + +To explore the consequences of the 3D epithelial geometry found in the intact gut, we next microinjected S.Tm into the lumen of murine 3D-enteroids and followed the epithelial behavior by DIC imaging. In line with observations from monolayers, focal contractions were visible in infected 3D-enteroids. + +Strikingly, microinjection of S.Tmwt, but not S.TmΔinvG or mock, resulted in a reduction of the total enteroid size by ∼25 to 50% +during the first 30 to 60 min p.i. (Fig. 5 E and F). This shows that S.Tm infection causes prompt compaction also of 3D-arranged murine epithelia. + +As shown here and previously (12, 15, 16), S.Tm infection causes death and removal of infected IECs. We hypothesized that prompt and early focal contractions may prepare the epithelium to withstand the effects of subsequent IEC loss. Indeed, focal contractions led to an increased IEC packing in the affected regions, as evident from an up to 40% (mean 12.3%) +reduction in the surface area of individual IECs (Fig. 5 G–I). To further test our hypothesis, we exploited the fact that Caspase-1– or Gasdermin D–deficient IECs still undergo NAIP/NLRC4–dependent cell death and expulsion (SI Appendix, Fig. S3B and refs. 12, 47) but that monolayers from these genetic backgrounds fail to elicit focal, epithelium-compacting, contractions (Fig. 4 A–D). WT, Casp1/11−/−, and GsdmD−/− monolayers were infected with S.Tmwt and imaged for a period extending beyond the contraction phase (i.e., for 2 h). As expected, Casp1/11−/− and GsdmD−/− monolayers produced fewer Draq7-positive IECs than WT controls at the end of the imaging series (Fig. 5 J–L), but we still observed expelling (mostly Draq7negative) IECs (Fig. 5J and SI Appendix, Fig. S3B). Remarkably, despite the attenuated IEC death response, both Casp1/11−/− and GsdmD−/− monolayers lost epithelium integrity to a higher degree than WT controls (Fig. 5 M and N). At the end of the imaging series, intact WT monolayer still covered ≥93% of the infected areas, whereas Casp1/11−/− and GsdmD−/− monolayers displayed frequent gaps and covered a mean of 62 to 83% of the surface depending on the MOI (Fig. 5 M and N). + +Taken together, these data associate early NAIP/NLRC4/ +Caspase-1/Gasdermin D/myosin–dependent focal contractions with increased local IEC packing and an enhanced capacity of the epithelium to withstand the potentially disruptive effects of subsequent IEC loss. + +## Discussion + +Defense systems that protect the gut mucosa from infection can be broadly categorized into preexisting homeostatic barriers (e.g., the mucus layer and baseline levels of antimicrobial peptide and soluble IgA secretion) and responses elicited upon pathogen sensing (e.g., proinflammatory cytokine production, immune cell recruitment, and epithelial cell death). The homeostatic barriers are permanently in place but generic in nature. The elicited responses, on the other hand, can be tailored to best counter the invading pathogen but require time—in some cases hours to days—for full effect. This is especially true if they involve communication between multiple cell types. Here, we have uncovered a prompt and autonomous epithelial contraction response, which takes place in the absence of IEC—immune cell cross-talk. + +Occurring already within minutes of bacterial encounter, this may constitute one of the earliest elicited responses to gut mucosa infection. + +Using bacterial and mammalian genetics, we find that sensing of TTSS and/or flagellin at pathogen invasion sites by epithelial NAIP/NLRC4 elicits the focal contractions. This response is still operational in the absence of ASC, which excludes an essential role of this adapter protein and its downstream interaction partners (e.g., Caspase-8 [12, 47]) in the execution. Instead, our data support that minute amounts of activated Caspase-1 and Gasdermin D cause sublytic pore formation, ion fluxes, and the swift propagation of a focal myosin-dependent contraction from the NAIP/NLRC4 sensing event at the epicenter. Importantly, multiple lines of evidence demonstrate that the contraction response precedes and can be uncoupled from previously described effects of IEC inflammasome activation, namely epithelial cell death, the IEC extrusion process, and generalized proinflammatory signaling (12, 13, 15, 16, 55): 1) NAIP/NLRC4– +dependent contraction foci emerge before the expulsion and lysis of infected IECs, 2) broad-spectrum Caspase-inhibitor treatment dramatically reduces epithelial cell death without affecting the contraction response, and 3) we identify at least two conditions +(homeostatic IEC removal in WT epithelia and infectioninduced IEC removal in Casp1/11−/− or GsdmD−/− epithelia) in which IEC death and expulsion occurs in the absence of a contraction response. It is here especially noteworthy that an actin purse string forms in neighboring epithelial cells both during IEC expulsion initiated by NLRC4/Caspase-1/Gasdermin D, by NLRC4/ASC/Caspase-8 (12), and in non-infection–related cell extrusion (50), while the contraction response described here only occurs in the first case and in an earlier time window. + +Moreover, 4) a range of inhibitor, neutralization, and supernatant transfer experiments argue against a central contribution by released mediators. Finally, 5) both TTSS-1–proficient and – +deficient S.Tm trigger generalized proinflammatory gene expression in the epithelium, whereas only TTSS-1–proficient S.Tm elicit contraction foci. Taken together, these observations also distinguish the focal contraction response from previously described modes of cell-to-cell amplification of inflammatory signaling in bacteria-infected epithelia (18, 19). We cannot, however, refute that so far, unknown soluble mediator(s) or other forms of intercellular communication beyond what we report here may contribute to contraction focus propagation. + +Nevertheless, our combined results support a central role for sublytic Gasdermin D pores that allow ion fluxes (including Calcium) to induce swift non-muscle myosin II–driven contractions that propagate from the focal epicenters. The nature of the propagation step remains poorly defined and could involve both chemical and physical transducers. Biophysical propagation of actomyosin contractions, with myosin itself acting as both a force generator and mechanosensor at adherence junctions, has been described in other settings (53, 56, 57). Actomyosin machineries impact many aspects of epithelial function, including tissue growth and morphogenesis (53, 58), which is also true for the gut epithelium. Assemblies of actomyosin, for instance, drive the apical constriction that promotes crypt formation in enteroids (54). In our monolayer experiments, small focal contractions occasionally occurred, although at exceedingly rare frequency, also in the absence of infection. It is appealing to speculate that coupling a PRR pathway to an already existing actomyosin contraction circuit allows the gut epithelium to use a similar machinery for tissue-shaping and in the acute response to infection. In monocytes/macrophages, there is precedence for coupling of inflammasome signaling and regulation of the actin cytoskeleton (59–61), although the molecular wiring appears dissimilar to what we describe here. + +It is noteworthy that epithelial contractions appeared most vigorous in murine epithelia, less pronounced in human epithelia, and were nondetectable in the cell lines Caco-2 C2Bbe1 and m-ICc12 placed atop the same hydrogel. This might be due to the fact that diverse epithelial cells bind unequally to the matrix, which could impact cell–cell junction maturity, mechanosensing, or force generation. However, it appears more likely that differential MICROBIOLOGY +expression of inflammasome components provides the explanation. + +Primary murine IECs express high levels of NAIP1-6, NLRC4, and Caspase-1 (8, 9, 16, 42). Human IECs also express the sole human NAIP (http://www.proteinatlas.org/ENSG00000249437-NAIP), but in addition, a noncanonical Caspase-4 inflammasome impacts the human IEC response to S.Tm (13, 55). The mouse Caspase-4 ortholog, Caspase-11, has a neglectable effect in naïve IECs and requires inflammatory priming for full expression (9, 16, 42, 62). Transformed/ +immortalized epithelial cell lines feature low or even undetectable expression levels of NAIP1-6, NLRC4, and Caspase-1 (9, 13). Hence, high expression of NAIP/NLRC4 inflammasome components permit primary epithelia, and in particular primary murine epithelia, to sense bacterial invasion and elicit epithelial contractions. + +Several beneficial effects of the epithelial contraction response can be pictured. First, monolayers incapable of producing contraction foci but still capable of eliciting IEC death and expulsion +(i.e., Casp1/11−/− and GsdmD−/− backgrounds) lost epithelial layer integrity to a larger extent than WT counterparts. It appears plausible that a denser packing allows the epithelium to better withstand accelerated rates of local IEC loss, thereby preserving overall tissue integrity. Second, the early densification of the epithelium may in fact simplify the IEC expulsion response itself, as local crowding in other contexts has been shown to promote dislodging of both dying and live cells from confluent cell layers (63). Third, we have recently found that S.Tm preferentially invades IECs at apicolateral hotspots close to IEC– +IEC junctions (38). A local densification of the epithelium is likely to decrease pathogen access to such preferred invasion sites following sensing of a pioneer intruder. Finally, we speculate that focal contractions might convey alert signals across larger areas of the gut epithelium. For instance, a subset of enteroendocrine IECs respond to mechanical force through the channel Piezo2 and release serotonin to increase muscular contraction and fluid secretion (64, 65). It is possible that such a responder circuit could couple early local contraction foci in the epithelium to organ-wide changes in gut motility at a later stage of an infection. These possibilities constitute intriguing areas for future inquiry. + +In conclusion, this work has uncovered a prompt and coordinated epithelial contraction response to bacterial invasion, which precedes other established host response mechanisms and has 1. P. R. Kiela, F. K. Ghishan, Physiology of intestinal absorption and secretion. Best Pract. + +Res. Clin. Gastroenterol. 30, 145–159 (2016). + +2. H. F. Helander, L. Fändriks, Surface area of the digestive tract–Revisited. Scand. + +J. Gastroenterol. 49, 681–689 (2014). + +3. A. C. Luissint, C. A. Parkos, A. Nusrat, Inflammation and the intestinal barrier: +Leukocyte-epithelial cell interactions, cell junction remodeling, and mucosal repair. + +Gastroenterology 151, 616–632 (2016). + +4. S. A. Fattinger, M. E. Sellin, W.-D. Hardt, Epithelial inflammasomes in the defense against Salmonella gut infection. Curr. Opin. Microbiol. 59, 86–94 (2021). + +5. W. I. Khan, S. M. Collins, Gut motor function: Immunological control in enteric infection and inflammation. Clin. Exp. Immunol. 143, 389–397 (2006). + +6. A. E. Price et al., A map of Toll-like receptor expression in the intestinal epithelium reveals distinct spatial, cell type-specific, and temporal patterns. Immunity 49, 560–575.e6 (2018). + +7. M. E. Sellin, K. M. Maslowski, K. J. Maloy, W.-D. Hardt, Inflammasomes of the intestinal epithelium. Trends Immunol. 36, 442–450 (2015). + +8. N. Winsor, C. Krustev, J. Bruce, D. J. Philpott, S. E. Girardin, Canonical and noncanonical inflammasomes in intestinal epithelial cells. Cell. Microbiol. 21, e13079 +(2019). + +9. A. Hausmann et al., Germ-free and microbiota-associated mice yield small intestinal epithelial organoids with equivalent and robust transcriptome/proteome expression phenotypes. Cell. Microbiol. 22, e13191 (2020). + +10. B. He et al., Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 26, 812–826 (2007). + +11. S. Hu et al., The DNA sensor AIM2 maintains intestinal homeostasis via regulation of epithelial antimicrobial host defense. Cell Rep. 13, 1922–1936 (2015). + +12. I. Rauch et al., NAIP-NLRC4 inflammasomes coordinate intestinal epithelial cell expulsion with eicosanoid and IL-18 release via activation of caspase-1 and -8. Immunity 46, 649–659 (2017). + +gone unnoticed in cell line infection experiments. We anticipate that the possibilities provided by organoid technology and tissue engineering will pave the way for an improved understanding of this and other complex primary cell and tissue behaviors. + +## Materials And Methods + +Ethical Statement. Human enteroid cultures were established from tissue resected from the proximal small bowel during bariatric surgery. Written informed consent was obtained from each subject. All personal data were pseudonymized before reaching the laboratory. The procedures were approved by the local governing body, Regionala Etikprövningsnämnden, Uppsala, Sweden, license number 2010–157 with addendum 2010–157-1 +(2018-06-13). Procedures for keeping of laboratory mice and tissue harvesting were approved by the Kantonales Veterinäramt Zürich, Switzerland, approval no. 193/2016, or by Umeå Regionala Djurförsöksetiska Nämnd, Sweden, approval no. A25-2019, and were performed in accordance with the legal regulations. + +A detailed description of bacterial strains and plasmids, FlaTox expression and purification, murine and human enteroid culture, 2D monolayer establishment, epithelial cell line culture, RT-qPCR analysis, infection conditions and chemicals, time-lapse microscopy, immunofluorescence, microinjection, and image analysis can be found in SI Appendix. Statistical Analysis. Where applicable, statistical significance was assessed by the Kruskal Wallis with Dunn´s post hoc test and Benjamini-Hochberg correction, the Mann–Whitney U test, the χ2 test, or the two-way ANOVA with Tukey's HSD (Honest Significant Difference), as indicated in the figure legends. Tests were performed in GraphPad Prism (8.4.2) or in R studio. + +Data Availability. All study data are included in the article and/or supporting information. + +ACKNOWLEDGMENTS. We thank members of the Sellin laboratory for helpful discussions. We are grateful to the staff of the Biological Visualization platform, Uppsala University, for instrument access, to the staff at Samariterhemmet surgical unit, Department of Surgery, Uppsala University Hospital, for assistance with human intestinal tissue sampling, and to Hui Jiang, Umeå University, for assistance with murine intestinal tissue sampling. + +Tim Keys is acknowledged for advice on protein purification. W.D.H. acknowledges financial support by the Swiss National Science Foundation +(310030_192567). Work in the Sellin laboratory was supported by the SciLifeLab Fellows program, grants from the Swedish Research Council (201802223), the Swedish Foundation for Strategic Research (ICA16-0031, FFL180165), the Knut and Alice Wallenberg Foundation (2016.0063), and a Lennart Philipson Award to M.E.S. + +13. M. K. Holly et al., Salmonella enterica infection of murine and human enteroidderived monolayers elicits differential activation of epithelium-intrinsic inflammasomes. Infect. Immun. 88, e00017-20 (2020). + +14. K. Brandl, G. Plitas, B. Schnabl, R. P. DeMatteo, E. G. Pamer, MyD88-mediated signals induce the bactericidal lectin RegIII γ and protect mice against intestinal Listeria monocytogenes infection. J. Exp. Med. 204, 1891–1900 (2007). + +15. L. A. Knodler et al., Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proc. Natl. Acad. Sci. U.S.A. 107, 17733–17738 +(2010). + +16. M. E. Sellin et al., Epithelium-intrinsic NAIP/NLRC4 inflammasome drives infected enterocyte expulsion to restrict Salmonella replication in the intestinal mucosa. Cell Host Microbe 16, 237–248 (2014). + +17. L. V. Hooper, Epithelial cell contributions to intestinal immunity. Adv. Immunol. 126, 129–172 (2015). + +18. T. Dolowschiak et al., Potentiation of epithelial innate host responses by intercellular communication. PLoS Pathog. 6, e1001194 (2010). + +19. C. A. Kasper et al., Cell-cell propagation of NF-κB transcription factor and MAP kinase activation amplifies innate immunity against bacterial infection. Immunity 33, 804–816 (2010). + +20. A. L. Chang-Graham et al., Rotavirus induces intercellular calcium waves through ADP +signaling. Science 370, eabc3621 (2020). + +21. K. Kretzschmar, H. Clevers, Organoids: Modeling development and the stem cell niche in a dish. Dev. Cell 38, 590–600 (2016). + +22. T. Sato et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009). + +23. H. Miyoshi, T. S. Stappenbeck, In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat. Protoc. 8, 2471–2482 (2013). + +24. C. Moon, K. L. VanDussen, H. Miyoshi, T. S. Stappenbeck, Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis. Mucosal Immunol. 7, 818–828 (2014). + +25. J. In et al., Enterohemorrhagic Escherichia coli reduce mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cell. Mol. Gastroenterol. Hepatol. 2, 48–62.e3 (2016). + +26. Y. Wang et al., Self-renewing monolayer of primary colonic or rectal epithelial cells. + +Cell. Mol. Gastroenterol. Hepatol. 4, 165–182.e7 (2017). + +27. K. Kozuka et al., Development and characterization of a human and mouse intestinal epithelial cell monolayer platform. Stem Cell Reports 9, 1976–1990 (2017). + +28. T. Sato et al., Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 +(2011). + +29. I. Heo et al., Modelling Cryptosporidium infection in human small intestinal and lung organoids. Nat. Microbiol. 3, 814–823 (2018). + +30. J. Y. Co et al., Controlling epithelial polarity: A human enteroid model for hostpathogen interactions. Cell Rep. 26, 2509–2520.e4 (2019). + +31. J. Zhou et al., Infection of bat and human intestinal organoids by SARS-CoV-2. Nat. + +Med. 26, 1077–1083 (2020). + +32. P. Geiser et al., Salmonella enterica serovar typhimurium exploits cycling through epithelial cells to colonize human and murine enteroids. mBio 12, e02684-20 (2021). + +33. X. Yin et al., Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat. Methods 11, 106–112 (2014). + +34. G. Altay et al., Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function. Sci. Rep. 9, 1–14 (2019). + +35. J. Eriksson, D. Styrström, M. E. Sellin, Cellocity: A Python package for analysis of confluent cell layer dynamics. J. Open Source Softw. 6, 2818 (2021). + +36. J. E. Galán, R. Curtiss III, Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc. Natl. + +Acad. Sci. U.S.A. 86, 6383–6387 (1989). + +37. M. Furter, M. E. Sellin, G. C. Hansson, W.-D. Hardt, Mucus architecture and nearsurface swimming affect distinct Salmonella typhimurium infection patterns along the murine intestinal tract. Cell Rep. 27, 2665–2678.e3 (2019). + +38. S. A. Fattinger et al., Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium. PLoS Pathog. 16, e1008503 (2020). + +39. C. M. Collazo, J. E. Galán, The invasion-associated type III system of Salmonella typhimurium directs the translocation of Sip proteins into the host cell. Mol. Microbiol. + +24, 747–756 (1997). + +40. P. J. Hume, V. Singh, A. C. Davidson, V. Koronakis, Swiss army pathogen: The Salmonella entry toolkit. Front. Cell. Infect. Microbiol. 7, 348 (2017). + +41. K. Zhang et al., Minimal SPI1-T3SS effector requirement for Salmonella enterocyte invasion and intracellular proliferation in vivo. PLoS Pathog. 14, e1006925 (2018). + +42. A. Hausmann et al., Intestinal epithelial NAIP/NLRC4 restricts systemic dissemination of the adapted pathogen Salmonella Typhimurium due to site-specific bacterial PAMP expression. Mucosal Immunol. 13, 530–544 (2020). + +43. S. Mariathasan et al., Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213–218 (2004). + +44. J. von Moltke et al., Rapid induction of inflammatory lipid mediators by the inflammasome in vivo. Nature 490, 107–111 (2012). + +45. I. Rauch et al., NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo. J. Exp. Med. 213, 657–665 (2016). + +46. J. Ding et al., Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111–116 (2016). + +47. N. Van Opdenbosch et al., Caspase-1 engagement and TLR-induced c-FLIP expression suppress ASC/Caspase-8-dependent apoptosis by inflammasome sensors NLRP1b and NLRC4. Cell Rep. 21, 3427–3444 (2017). + +48. P. Li et al., Mice deficient in IL-1 β-converting enzyme are defective in production of mature IL-1 β and resistant to endotoxic shock. Cell 80, 401–411 (1995). + +49. J. Shi et al., Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015). + +50. J. Rosenblatt, M. C. Raff, L. P. Cramer, An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr. Biol. + +11, 1847–1857 (2001). + +51. Y. Gu, T. Forostyan, R. Sabbadini, J. Rosenblatt, Epithelial cell extrusion requires the sphingosine-1-phosphate receptor 2 pathway. J. Cell Biol. 193, 667–676 (2011). + +52. H. M. Russo et al., Active caspase-1 induces plasma membrane pores that precede pyroptotic lysis and are blocked by lanthanides. J. Immunol. 197, 1353–1367 (2016). + +53. A. C. Martin, B. Goldstein, Apical constriction: Themes and variations on a cellular mechanism driving morphogenesis. Development 141, 1987–1998 (2014). + +54. L. Hartl, G. Huelsz-Prince, J. van Zon, S. J. Tans, Apical constriction is necessary for crypt formation in small intestinal organoids. Dev. Biol. 450, 76–81 (2019). + +55. L. A. Knodler et al., Noncanonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens. Cell Host Microbe 16, 249–256 (2014). + +56. M. J. Greenberg, G. Arpag, E. Tüzel, E. M. Ostap, A perspective on the role of myosins ˘ +as mechanosensors. Biophys. J. 110, 2568–2576 (2016). + +57. E. Sadeghipour, M. A. Garcia, W. J. Nelson, B. L. Pruitt, Shear-induced damped oscillations in an epithelium depend on actomyosin contraction and E-cadherin cell adhesion. eLife 7, e39640 (2018). + +58. L. He, X. Wang, H. L. Tang, D. J. Montell, Tissue elongation requires oscillating contractions of a basal actomyosin network. Nat. Cell Biol. 12, 1133–1142 (2010). + +59. A. L. Waite et al., Pyrin and ASC co-localize to cellular sites that are rich in polymerizing actin. Exp. Biol. Med. (Maywood) 234, 40–52 (2009). + +60. A. Akhter et al., Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization. Immunity 37, 35–47 (2012). + +61. S. M. Man et al., Actin polymerization as a key innate immune effector mechanism to control Salmonella infection. Proc. Natl. Acad. Sci. U.S.A. 111, 17588–17593 (2014). + +62. S. M. Crowley et al., Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes. PLoS Pathog. 16, e1008498 (2020). + +63. G. T. Eisenhoffer et al., Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 484, 546–549 (2012). + +64. C. Alcaino et al., A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release. Proc. Natl. Acad. + +Sci. U.S.A. 115, E7632–E7641 (2018). + +65. G. M. Mawe, J. M. Hoffman, Serotonin signalling in the gut–Functions, dysfunctions and therapeutic targets. Nat. Rev. Gastroenterol. Hepatol. 10, 473–486 (2013). \ No newline at end of file diff --git a/medical/md/PMC8134854.md b/medical/md/PMC8134854.md new file mode 100644 index 0000000000000000000000000000000000000000..c85bf3ddfdb3af66f7f433dd8fb4e73ffda1eb79 --- /dev/null +++ b/medical/md/PMC8134854.md @@ -0,0 +1,282 @@ +World Journal of + +![0_image_0.png](0_image_0.png) + +Gastrointestinal W J G E *Endoscopy* +Submit a Manuscript: https://www.f6publishing.com *World J Gastrointest Endosc* 2021 May 16; 13(5): 125-136 DOI: 10.4253/wjge.v13.i5.125 ISSN 1948-5190 (online) +ORIGINAL ARTICLE + +# Observational Study Comparison Of Endoscopic Gastritis Based On Kyoto Classification Between Diffuse And Intestinal Gastric Cancer + +Osamu Toyoshima, Toshihiro Nishizawa, Shuntaro Yoshida, Tomonori Aoki, Fumiko Nagura, Kosuke Sakitani, Yosuke Tsuji, Hayato Nakagawa, Hidekazu Suzuki, Kazuhiko Koike Osamu Toyoshima, Toshihiro Nishizawa, Shuntaro Yoshida, Kosuke **Sakitani,** Department of Gastroenterology, Toyoshima Endoscopy Clinic, Tokyo 157-0066, Japan + +| Osamu Toyoshima, Toshihiro Nishizawa, Shuntaro Yoshida, Kosuke Sakitani, Department of Gastroenterology, Toyoshima Endoscopy Clinic, Tokyo 157-0066, Japan | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| ORCID number: Osamu Toyoshima 0000-0002-6953-6079; Toshihiro Nishizawa 0000-0003-4876-3384; Shuntaro Yoshida 0000-0002-9437- 9132; Tomonori Aoki 0000-0003- 0794-9940; Fumiko Nagura 0000- 0001-5138-7768; Kosuke Sakitani 0000-0002-4537-6023; Yosuke Tsuji 0000-0001-9537-4993; Hayato Nakagawa 0000-0002-6973-5094; Hidekazu Suzuki 0000-0002-8994- 6163; Kazuhiko Koike 0000-0002- 9739-9243. | Toshihiro Nishizawa, Department of Gastroenterology and Hepatology, International University of Health and Welfare, Narita Hospital, Narita 286-8520, Japan Tomonori Aoki, Yosuke Tsuji, Hayato Nakagawa, Kazuhiko Koike, Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan Fumiko Nagura, Internal Medicine, Chitosefunabashi Ekimae Clinic, Tokyo 157-0054, Japan Kosuke Sakitani, Department of Gastroenterology, Sakiatani Endoscopy Clinic, Narashino 275- 0026, Japan | +| Author contributions: Toyoshima O and Nishizawa T designed the study; Toyoshima O analyzed data and wrote the manuscript; Nishizawa T edited the manuscript; Yoshida S, Aoki T, Nagura F, Sakitani K, Tsuji Y, Nakagawa H, Suzuki H, and Koike K revised the manuscript; Suzuki H and Koike K approved the final article. | Hidekazu Suzuki, Department of Gastroenterology and Hepatology, Tokai University School of Medicine, Isehara 259-1193, Japan Corresponding author: Osamu Toyoshima, MD, PhD, Director, Doctor, Department of Gastroenterology, Toyoshima Endoscopy Clinic, 6-17-5 Seijo, Setagaya-ku, Tokyo 157-0066, Japan. t@ichou.com Abstract BACKGROUND Gastric cancers can be categorized into diffuse- and intestinal-type cancers based on the Lauren histopathological classification. These two subtypes show distinct differences in metastasis frequency, treatment application, and prognosis. Therefore, accurately assessing the Lauren classification before treatment is crucial. However, studies on the gastritis endoscopy-based Kyoto classification have recently shown that endoscopic diagnosis has improved. | +| Institutional review board statement: This study was approved by the Certificated Review Board, Hattori Clinic on September 4th, 2020 (approval no. S2009-U04, registration no. UMIN000018541). | AIM To investigate patient characteristics including endoscopic gastritis associated with diffuse- and intestinal-type gastric cancers in Helicobacter pylori (H. pylori)- infected patients. | +| Informed consent statement: Patients were not required to give informed consent to the study because the analysis used | METHODS Patients who underwent esophagogastroduodenoscopy at the Toyoshima | + +| anonymous clinical data that were obtained after each patient agreed to treatment by written consent. For full disclosure, the details of the study are published on the home page of Toyoshima Endoscopy Clinic. Conflict-of-interest statement: All other authors have nothing to disclose. Data sharing statement: Not available. 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 BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: htt p://creativecommons.org/License s/by-nc/4.0/ Manuscript source: Invited manuscript Specialty type: Gastroenterology and hepatology Country/Territory of origin: Japan 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 Received: January 13, 2021 Peer-review started: January 13, 2021 First decision: February 10, 2021 Revised: February 14, 2021 Accepted: April 14, 2021 Article in press: April 14, 2021 | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +Endoscopy Clinic were enrolled. The Kyoto classification included atrophy, intestinal metaplasia, enlarged folds, nodularity, and diffuse redness. The effects of age, sex, and Kyoto classification score on gastric cancer according to the Lauren classification were analyzed. We developed the Lauren predictive background score based on the coefficients of a logistic regression model using variables independently associated with the Lauren classification. Area under the receiver operative characteristic curve and diagnostic accuracy of this score were examined. + +## Results + +A total of 499 H. *pylori*-infected patients (49.6% males; average age: 54.9 years) +were enrolled; 132 patients with gastric cancer (39 diffuse- and 93 intestinal-type cancers) and 367 cancer-free controls were eligible. Gastric cancer was independently associated with age ≥ 65 years, high atrophy score, high intestinal metaplasia score, and low nodularity score when compared to the control. Factors independently associated with intestinal-type cancer were age ≥ 65 years (coefficient: 1.98), male sex (coefficient: 1.02), high intestinal metaplasia score +(coefficient: 0.68), and low enlarged folds score (coefficient: -1.31) when compared to diffuse-type cancer. The Lauren predictive background score was defined as the sum of +2 (age ≥ 65 years), +1 (male sex), +1 (endoscopic intestinal metaplasia), and -1 (endoscopic enlarged folds) points. Area under the receiver operative characteristic curve of the Lauren predictive background score was 0.828 for predicting intestinal-type cancer. With a cut-off value of +2, the sensitivity, specificity, and accuracy of the Lauren predictive background score were 81.7%, 71.8%, and 78.8%, respectively. + +## Conclusion + +Patient backgrounds, such as age, sex, endoscopic intestinal metaplasia, and endoscopic enlarged folds are useful for predicting the Lauren type of gastric cancer. + +Key **Words:** Gastric cancer; Lauren classification; Endoscopy; Pathology; Gastritis; Kyoto classification ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved. + +Core **Tip:** Accurately assessing the Lauren classification before the treatment of gastric cancer is crucial. Factors independently associated with intestinal-type cancer were age +≥ 65 years, male sex, high endoscopic intestinal metaplasia score, and low endoscopic enlarged folds score when compared to diffuse-type cancer. The Lauren predictive background score was defined as the sum of +2 (age ≥ 65 years), +1 (male), +1 (intestinal metaplasia), and -1 (enlarged folds) points. Area under the curve of the Lauren predictive background score was 0.828 (cut-off: +2) for predicting intestinaltype cancer. Age, sex, intestinal metaplasia, and enlarged folds are useful for predicting tumor type. + +Citation: Toyoshima O, Nishizawa T, Yoshida S, Aoki T, Nagura F, Sakitani K, Tsuji Y, +Nakagawa H, Suzuki H, Koike K. Comparison of endoscopic gastritis based on Kyoto classification between diffuse and intestinal gastric cancer. World J Gastrointest *Endosc* 2021; 13(5): 125-136 URL: https://www.wjgnet.com/1948-5190/full/v13/i5/125.htm DOI: https://dx.doi.org/10.4253/wjge.v13.i5.125 + +## Introduction + +The International Agency for Research on Cancer reported in GLOBOCAN 2018 that stomach cancer was the third leading cause of mortality worldwide[1]. Gastric cancers are epidemiologically crucial and can be categorized into two types based on the + +Lauren histopathological classification: diffuse and intestinal-types[2]. Intestinal-type + +cancers are associated with a Helicobacter pylori (H. *pylori)*-induced chronic inflammatory process, known as the Correa pathway, which includes atrophy, metaplasia, + +dysplasia, and cancer[3], whereas diffuse-type gastric cancers directly undergo a highly active inflammation-based carcinogenesis without having to pass through the Correa pathway[4,5]. The two histological subtypes of gastric tumors proposed by + +Lauren exhibit several distinct clinical and molecular characteristics[6-8]. Depending + +on the Lauren type, the frequency of lymph node metastasis[2,9,10] and peritoneal metastasis[11,12], application of endoscopic mucosal dissection[13,14], recommended surgical margin[15], response to chemotherapy[16], and prognosis[2,16,17] differ. The Lauren classification is diagnosed by pathology; however, it would be useful if subtypes could be endoscopically predicted. + +In recent years, advancement in endoscopy has enabled diagnosis that is highly + +consistent with histology[18,19]. In 2013, the endoscopy-based Kyoto classification of gastritis was advocated by the Japan Gastroenterological Endoscopy Society with the aim of unifying the endoscopic diagnosis of gastritis in clinical practice and match it with the pathological diagnosis of gastritis[20]. The Kyoto classification adopted and scored atrophy, intestinal metaplasia, enlarged folds, nodularity, diffuse redness, and the regular arrangement of collecting venules (RAC) as endoscopic findings of gastritis. Among them, the Kyoto score, which is the sum of the scores of these factors, has been vigorously reported to be associated with gastric cancer[21,22], gastric cancer risk[20,23], and H. *pylori* infections[24]. Evaluating the risk of gastric cancer on the basis of endoscopic findings is an important alternative to biopsy. + +Since there are few reports regarding the relationship between the Lauren classification and endoscopic findings based on the Kyoto classification[21,22], we investigated the background patient characteristics and endoscopic gastritis of patients with + +diffuse- and intestinal-type gastric cancers, focusing on H. *pylori* infected patients. Based on these outcomes, a score was created to predict the Lauren classification, and its accuracy was examined. + +| Published online: May 16, 2021 P-Reviewer: Yu CH S-Editor: Zhang L L-Editor: A P-Editor: Yuan YY | +|----------------------------------------------------------------------------------------------------| + +## Materials And Methods Study Design And Oversight + +We conducted a retrospective case-control study at the Toyoshima Endoscopy Clinic, which is an outpatient endoscopy-specialized clinic located in Tokyo, an urban area in Japan. This study was approved by the certificated review board of the Hattori Clinic on September 4, 2020 (approval No. S2009-U04, registration number UMIN000018541). Written informed consent was obtained from all patients. All clinical investigations were conducted in accordance with the ethical guidelines of the Declaration of Helsinki. This study received no financial support. + +## Study Population + +Eligibility criteria included patients with gastric cancer and an H. *pylori* infection who underwent esophagogastroduodenoscopy at the Toyoshima Endoscopy Clinic from September 2008 to February 2020. We excluded patients who did not have H. *pylori* infection, patients in whom H. *pylori* was successfully eradicated, and those whose H. pylori status was unavailable. Patients with gastric cancer and past gastrectomy were also excluded. As control group, patients with H. *pylori*-positive gastritis and without gastric cancer were enrolled. This criterion included patients who underwent esophagogastroduodenoscopy and initial assessments for an H. *pylori* infection from December 2013 to March 2016 and from January 2018 to February 2019. + +## Diagnosis Of Lauren Classification And H. Pylori Infection + +The Lauren classification was diagnosed from resected specimens or, if unresectable, biopsy specimens. + +An H. *pylori* infection was diagnosed using pathology (hematoxylin and eosin staining) or the urea breath test. + +## Endoscopic Gastritis Based On The Kyoto Classification + +The Kyoto score for endoscopic gastritis, which ranges from 0 to 8, is based on the total scores of the following five endoscopic findings: atrophy, intestinal metaplasia, enlarged folds, nodularity, and diffuse redness. A high score represents an increased risk of gastric cancer[20-23] and *H. pylori* infection[24]. + +Endoscopic atrophy was classified based on the extent of mucosal atrophy (the Kimura Takemoto classification)[26]. Non-atrophy and C1 atrophy were scored as atrophy score 0, C2, and C3 atrophies as atrophy score 1, and O1 to O3 atrophies as atrophy score 2. + +Endoscopically, intestinal metaplasia typically appears as grayish-white and slightly elevated plaques surrounded by mixed patchy pink and pale areas of the mucosa, forming an irregular uneven surface. A villous appearance, whitish mucosa, and rough mucosal surface are useful indicators for the endoscopic diagnosis of intestinal metaplasia. Intestinal metaplasia score 0 was defined as the absence of intestinal metaplasia, score 1 as the presence of intestinal metaplasia within the antrum, and score 2 as intestinal metaplasia extending into the corpus. The intestinal metaplasia score was calculated based on the diagnosis of metaplasia using white-light imaging. + +An enlarged fold is defined as ≥ 5 mm width that is not flattened or is only partially flattened by stomach insufflation. The absence and presence of enlarged folds were scored as enlarged fold scores of 0 and 1, respectively. + +Nodularity is a condition in which a miliary pattern similar to "goosebumps" is mainly located in the antrum. The absence and presence of nodularity were scored as nodularity scores of 0 and 1, respectively. + +Diffuse redness refers to uniformly reddish mucosa with continuous expansion located in the non-atrophic mucosa, mainly in the corpus. The RAC is a condition in which collecting venules are arranged in the corpus. From a distance, the venules look like numerous dots; however, up close, the venules appear like a regular pattern of starfish-like shapes. The absence of diffuse redness, presence of mild diffuse redness or diffuse redness with RAC, and severe diffuse redness or diffuse redness without RAC were scored as diffuse redness scores of 0, 1, and 2, respectively. + +## Data Collection And Outcomes + +We obtained data for cancer and participants background information from the endoscopic database of the Toyoshima Endoscopy Clinic from September 2008 to February 2020. Two expert endoscopists reviewed all images and scored them according to the Kyoto classification. + +Clinical data of this study consisted of variables including gastric cancer type according to the Lauren classification, age, sex, and endoscopic gastritis score based on the Kyoto classification (Kyoto score, atrophy score, intestinal metaplasia score, enlarged folds score, nodularity score, and diffuse redness score). + +The main outcome of this study was the differences in patient backgrounds and the endoscopic gastritis between patients with diffuse- and intestinal-type gastric cancers. To predict the Lauren type of cancer, this study developed a Lauren predictive background score using variables associated with the Lauren classification. We assessed the discrimination of the Lauren predictive background score using the receiver operating characteristic (ROC) curve, the corresponding area under the ROC curve (AUC), and the diagnostic accuracy of predicting the Lauren type of tumor. + +We also compared H. *pylori*-infected patients with cancer (whole, diffuse-, and intestinal-type cancers, respectively) and cancer-free *H. pylori*-infected controls. + +## Statistical Analyses + +Univariate and multivariate analyses were conducted using a binomial logistic regression analysis. The multivariate analysis included age, sex, and each score of the Kyoto classification, excluding the Kyoto score. Age was categorized based on the average number of patients with gastric cancer. A multivariate analysis was conducted, using a backward stepwise logistic regression, for variables with P values < 0.1; these values were determined by a univariate analysis. Regarding missing data, we used complete case analysis. + +We developed the Lauren predictive background score based on the coefficients of a logistic regression model, using variables with P values < 0.05 in a multivariate analysis. The AUC for predicting intestinal-type cancer and the sensitivity, specificity, and accuracy of the Lauren predictive background score were measured. The optimal cut-off value of the ROC curve was calculated using the Youden index. + +A two-sided P value of < 0.05 was considered statistically significant. Statistical analyses were performed using Ekuseru-Toukei 2015 (Social Survey Research Information company, Limited, Tokyo, Japan). + +## Results Patient Characteristics + +A total of 132 patients with H. *pylori*-positive gastric cancers (39 diffuse- and 93 intestinal-type, 105 early, and 27 advanced cancers) were included; 11 patients were excluded as they did not have an H. *pylori* infection, 104 due to successful eradication, and 16 due to an unavailable H. *pylori* status. The control group comprised 367 patients with H. *pylori*-positive gastritis (gastric cancer free controls). A total of 499 patients were enrolled in this study. We show patient flowchart in Figure 1. The mean age in this study was 54.9 ± 14.1 (range: 23-89) years, and 49.6% of patients were male; the Kyoto score was 4.93 ± 1.58, (atrophy: 1.53 ± 0.61; intestinal metaplasia: 0.83 ± 0.92; enlarged folds: 0.42 ± 0.49; nodularity: 0.33 ± 0.47; and diffuse redness: 1.83 ± 0.48). + +## H. Pylori-Positive Gastritis With Vs Without Gastric Cancer + +Univariate analysis showed that patients with H. *pylori*-infected cancer patients were older (66.4 vs 50.9 years) and had a higher Kyoto score (5.63 vs 4.69) than H. *pylori*infected non-cancer patients. Among the scores of the items of the Kyoto classification, atrophy and intestinal metaplasia scores for gastric cancer were higher than those for cancer-free gastritis; however, nodularity scores for gastric cancer were lower than those for cancer-free gastritis. There was no significant difference in the enlarged folds and diffuse redness scores. Based on the results of a multivariate analysis, H. *pylori*infected gastric cancer was independently associated with an age of 65 years or more +[odds ratio (OR): 4.01], a high atrophy score (OR: 2.80), high intestinal metaplasia score (OR: 1.57), and a low nodularity score (OR: 0.51, Table 1). + +## H. Pylori-Infected Gastritis With Diffuse-Type Gastric Cancer Vs Without Gastric Cancer + +On comparing H. *pylori*-infected patients with diffuse-type cancer and those without gastric cancer (gastric cancer-free controls), a univariate analysis showed that patients with diffuse-type cancer were older (58.0 vs 50.9 years) and had a higher Kyoto score +(5.33 vs 4.69), higher atrophy score, and higher intestinal metaplasia score than gastric cancer-free patients. In a multivariate analysis, a high atrophy score was independently associated with diffuse-type gastric cancer (Table 2). + +## H. Pylori-Infected Gastritis With Intestinal-Type Gastric Cancer Vs Without Gastric + +cancer H. *pylori*-infected intestinal-type gastric cancer and H. *pylori*-infected non-cancer gastritis were compared. Univariate analysis showed that H. *pylori*-infected patients with intestinal-type gastric cancer were older (69.9 vs 50.9 years), comprised more of males (62.4% vs 47.7%), and had a higher Kyoto score (5.75 vs 4.69), higher atrophy score, higher intestinal metaplasia score, lower enlarged folds score, and lower nodularity score than those with non-cancer gastritis. Similar results were obtained in multivariate analysis (Table 3). + +## H. Pylori-Infected Gastritis With Diffuse- Vs Intestinal-Type Gastric Cancer + +Table 4 shows a comparison of endoscopic background gastritis between H. *pylori*infected patients with diffuse- and intestinal-type cancers. Univariate analysis showed that patients with intestinal-type cancer were older (69.9 vs 58.0 years), comprised more of males (61.5% vs 37.6%), had a higher atrophy score (1.95 vs 1.69), higher intestinal metaplasia score (1.58 vs 0.97), lower enlarged folds score (0.28 vs 0.56), and lower nodularity score (0.10 vs 0.28). There was no significant difference in the Kyoto and diffuse redness scores. In a multivariate analysis, factors independently associated with intestinal-type cancer were an age of 65 years or more (coefficient: 1.98; OR: 7.26), male sex (coefficient: 1.02; OR: 2.78), high intestinal metaplasia score (coefficient: 0.68; OR: 1.97), and low enlarged folds score (coefficient: -1.31; OR: 0.27). + +Based on the coefficients of a multivariate analysis, the equation for the scoring system was calculated based on an assumption that patients receive +2 points if they were aged 65 years or more, +1 point if they were male, +1 point if they had intestinal metaplasia, and -1 point if they had enlarged folds. We defined the Lauren predictive background score as the sum of these points, ranging from -1 to +4. + +The ROC curve based on the Lauren predictive background score in 132 patients with diffuse- or intestinal-type cancer is shown in Figure 2. AUC of the Lauren predictive background score for predicting intestinal-type cancer was 0.828 (95% confidence interval: 0.744-0.912). The optimal cut-off value of the Lauren predictive background score for correlation with intestinal-type gastric cancer was +2, based on + +| Table 1 Endoscopic gastritis based on Kyoto classification of Helicobacter pylori-infected patients with vs without gastric cancer Gastric cancer (+) Cancer (-) Univariate analysis Multivariate analysis Odds ratio 95%CI P value Odds ratio 95%CI P value n 132 367 Age, mean (SD), yr 66.4 (12.4) 50.9 (12.4) 1.099 1.078-1.120 < 0.001 Age ≥ 65 yr, % 60.6 15.3 8.544 5.446-13.405 < 0.001 4.010 2.436-6.603 < 0.001 Male sex, % 55.3 47.7 1.357 0.910-2.024 0.134 Atrophy score, mean (SD) 1.871 (0.336) 1.411 (0.642) 6.173 3.635-10.486 < 0.001 2.800 1.583-4.954 < 0.001 Intestinal metaplasia score, mean (SD) 1.402 (0.809) 0.624 (0.878) 2.570 2.031-3.253 < 0.001 1.567 1.188-2.067 0.001 Enlarged folds score, mean (SD) 0.364 (0.483) 0.441 (0.497) 0.723 0.480-1.090 0.121 Nodularity score, mean (SD) 0.152 (0.360) 0.387 (0.488) 0.283 0.168-0.476 < 0.001 0.508 0.282-0.913 0.024 Diffuse redness score, mean (SD) 1.841 (0.507) 1.823 (0.466) 1.085 0.706-1.667 0.709 Kyoto score, mean (SD) 5.629 (1.149) 4.687 (1.637) 1.568 1.342-1.831 < 0.001 | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +P value was calculated using the binomial logistic regression analysis. CI: Confidence interval; SD: Standard deviation. + +| Table 2 Endoscopic gastritis based on Kyoto classification of Helicobacter pylori-infected patients with diffuse-type gastric cancer vs without gastric cancer Diffuse-type cancer (+) Cancer (-) Univariate analysis Multivariate analysis Odds ratio 95%CI P value Odds ratio 95%CI P value n 39 367 Age, mean (SD), yr 58.00 (13.00) 50.88 (12.41) 1.044 1.018-1.072 0.001 Age ≥ 65 yr, % 28.2 15.3 2.182 1.027-4.634 0.042 1.434 0.633-3.246 0.388 Male sex, % 38.5 47.7 0.686 0.348-1.349 0.275 Atrophy score, mean (SD) 1.692 (0.468) 1.411 (0.642) 2.327 1.223-4.428 0.010 2.327 1.223-4.428 0.010 Intestinal metaplasia score, mean 0.974 (0.903) 0.624 (0.878) 1.516 1.065-2.158 0.021 1.313 0.905-1.906 0.152 (SD) Enlarged folds score, mean (SD) 0.564 (0.502) 0.441 (0.497) 1.638 0.842-3.186 0.146 Nodularity score, mean (SD) 0.282 (0.456) 0.387 (0.488) 0.622 0.300-1.290 0.202 Diffuse redness score, mean (SD) 1.821 (0.556) 1.823 (0.466) 0.990 0.495-1.978 0.976 Kyoto score, mean (SD) 5.333 (1.402) 4.687 (1.637) 1.306 1.044-1.632 0.019 | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +P value was calculated using the binomial logistic regression analysis. CI: Confidence interval; SD: Standard deviation. + +the Youden index. The sensitivity, specificity, and accuracy of the Lauren predictive background score were 81.7%, 71.8%, and 78.8%, respectively. + +## Discussion + +This study showed that old age, male sex, the presence of endoscopic intestinal metaplasia, and the absence of endoscopic enlarged folds were independently associated with intestinal-type gastric cancer compared to diffuse-type cancer among H. *pylori*-infected patients. The Lauren predictive background score created based on these variables was good, with AUC of 0.828, sensitivity of 81.7%, and accuracy of 78.8%. It is well known that old age, male sex[2,27], and endoscopic intestinal metaplasia[28] are indicators of intestinal-type cancers and that endoscopic enlarged folds[5,29] are characteristics of diffuse-type tumors. The strength of this study is that + +| Table 3 Endoscopic gastritis based on Kyoto classification of Helicobacter pylori-infected patients with intestinal-type gastric cancer vs without gastric cancer Intestinal-type cancer (+) Cancer (-) Univariate analysis Multivariate analysis Odds ratio 95%CI P value Odds ratio 95%CI P value n 93 367 Age, mean (SD), yr 69.86 (10.29) 50.88 (12.41) 1.138 1.107-1.169 < 0.001 Age ≥ 65 yr, % 74.2 15.3 15.967 9.261-27.527 < 0.001 6.220 3.394-11.400 < 0.001 Male sex, % 62.4 47.7 1.818 1.140-2.900 0.012 1.794 0.955-3.372 0.069 Atrophy score, mean (SD) 1.946 (0.227) 1.411 (0.642) 15.312 6.147-38.144 < 0.001 6.167 2.321-16.382 < 0.001 Intestinal metaplasia score, 1.581 (0.697) 0.624 (0.878) 3.368 2.499-4.539 < 0.001 1.683 1.166-2.430 0.005 mean (SD) Enlarged folds score, mean (SD) 0.280 (0.451) 0.441 (0.497) 0.491 0.299-0.808 0.005 0.453 0.237-0.867 0.017 Nodularity score, mean (SD) 0.097 (0.297) 0.387 (0.488) 0.170 0.083-0.348 < 0.001 0.323 0.141-0.742 0.008 Diffuse redness score, mean 1.849 (0.488) 1.823 (0.466) 1.135 0.681-1.891 0.626 (SD) Kyoto score, mean (SD) 5.753 (1.007) 4.687 (1.637) 1.696 1.407-2.004 < 0.001 | +|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +P value was calculated using the binomial logistic regression analysis. CI: Confidence interval; SD: Standard deviation. + +independent variables related to cancer type were investigated using the currently vigorously studied endoscopic gastritis evaluation method (Kyoto classification), and Lauren predictive background score was newly created using these variables; moreover, the score was accurate. Predicting cancer types without a biopsy may lead to faster treatment choices. A pathological diagnosis before endoscopic resection, surgery, or chemotherapy is vital to determine the line of treatment of lesions[13-16]. However, cases in which there are differences between the histological diagnoses of biopsy and resected specimens amount to 20%–30% of all cases[30-32]. Biopsy results are supported when the Lauren predictive background score is consistent with the biopsy diagnosis; however, the treatment should be carefully selected when discrepancies are observed. Furthermore, some endoscopic features of cancer are indicated by the Lauren classification. For example, diffuse-type cancers are frequently located in the proximal stomach[33]. The endoscopic gross appearance of an elevated-type cancer predominantly indicated intestinal-type cancer, whereas flat and depressed types of cancers indicated difuse-type cancer[34,35]. In the early stages of gastric cancer, intestinal-type cancer is usually reddish, whereas diffuse-type cancer is pale. While magnifying with narrow-band imaging, a well-demarcated area[36] and a white opaque substance[37] serve as an indicator of intestinal-type cancer, an ill-defined area[36] and a high proportion of the area with an absent microsurface pattern[38] are specific markers for diffuse-type cancer. In contrast, our study is unique in predicting the Lauren classification from background information rather than tumor information. In the future, a combination of both background and tumor information may allow for more accurate predictions, and a diagnosis by artificial intelligence may help. + +We previously showed that corpus-predominant gastritis (5.96) has a higher Kyoto score than pangastritis (5.21)[20]. Corpus-predominant gastritis and pangastritis are risk factors for intestinal- and diffuse-type cancers, respectively[39], and a similar tendency was observed in this study. + +Next, this study demonstrated that the Kyoto score of gastric cancer patients was higher than that of cancer-free patients among H. *pylori*-infected participants, regardless of whether the cancer was diffuse- or intestinal-type. This result is concordant with that of a previous report by Sugimoto *et al*[21]. While examining each item of the Kyoto classification, atrophy and intestinal metaplasia showed a positive association with gastric cancer; however, nodularity was negatively correlated with gastric cancer. This tendency is also the same as that reported in a previous study[21]. + +Nodularity has been reported as a risk factor for stomach cancer in young patients[40]; however, our observation might indicate a negative association since it covers all ages. When we previously investigated the association of the ABC classification, which consisted of a combination of serum *H. pylori* antibody and pepsinogen, + +| Table 4 Endoscopic gastritis based on Kyoto classification of Helicobacter pylori-infected patients with diffuse- vs intestinal-type gastric cancer Diffusetype Intestinaltype Univariate analysis Multivariate analysis Odds ratio 95%CI P value Coefficient 95%CI Odds ratio 95%CI P value n 39 93 Age, mean (SD), yr 58.00 69.86 (10.29) 1.091 1.051- < 0.001 (13.00) 1.132 Age ≥ 65 yr, % 28.2 74.2 7.318 3.166- < 0.001 1.983 1.045, 7.263 2.843- < 0.001 16.917 2.921 18.553 Male sex, % 38.5 62.4 2.651 1.228- 0.013 1.021 0.069, 2.776 1.071- 0.036 5.724 1.973 7.193 Atrophy score, mean (SD) 1.692 1.946 (0.227) 7.822 2.530- < 0.001 0.727 -0.927, 2.069 0.396- 0.389 (0.468) 24.188 2.381 10.816 Intestinal metaplasia score, 0.974 1.581 (0.697) 2.473 1.544- < 0.001 0.678 0.128, 1.970 1.136- 0.016 mean (SD) (0.903) 3.959 1.228 3.413 Enlarged folds score, mean 0.564 0.280 (0.451) 0.300 0.138- 0.002 -1.308 -2.261, - 0.270 0.104- 0.007 (SD) (0.502) 0.653 0.356 0.701 Nodularity score, mean (SD) 0.282 0.097 (0.297) 0.273 0.102- 0.009 -0.237 -1.621, 0.789 0.198- 0.737 (0.456) 0.726 1.147 3.149 Diffuse redness score, mean 1.821 1.849 (0.488) 1.116 0.545- 0.764 (SD) (0.556) 2.288 Kyoto score, mean (SD) 5.333 5.753 (1.007) 1.355 0.987- 0.060 (1.402) 1.860 | +|---| + +P value was calculated using the binomial logistic regression analysis. CI: Confidence interval; SD: Standard deviation. + +![7_image_0.png](7_image_0.png) + +![7_image_1.png](7_image_1.png) + +with endoscopic gastritis, the simplified Kyoto score using only atrophy and intestinal metaplasia scores was more dramatically related to the ABC classifi-cation[27]. Combined with the results of this study, we suggest that nodularity and diffuse redness scores be not included in the gastric cancer risk score. Particularly, enlarged folds scores should be excluded from the risk score for intestinal-type cancer. However, further verifications are required for this matter. + +This study has some limitations. The subjects of our study were limited to H. *pylori*infected patients. Gastric cancer is detected even after H. *pylori* eradication[41]. Take et al[42] described an increased incidence of diffuse-type cancer more than 10 years after H. *pylori* eradication. Studying subjects after H. *pylori* eradication or H. *pylori*uninfected subjects in the future is warranted. The gastric cancer-free control group in this study was extracted from a shorter period than the gastric cancer group. In the future, comparisons between the endoscopic background diagnosis of patients with gastric cancer (especially according to the Lauren classification) and that of non-cancer controls during the same period is desired. In addition, further investigations using prospective study designs are needed to evaluate the accuracy of the Lauren predictive Figure 2 Receiver operating characteristic curve for predicting intestinal-type gastric **cancer.** Receiver operating characteristics curve was based + +![8_image_0.png](8_image_0.png) + +on the Lauren predictive background score in 132 patients with diffuse- or intestinal-type gastric cancer according to Lauren classification. The Lauren predictive background score was defined as a sum of the following points: +2 points for an age of 65 years or older, +1 point for male sex, +1 point for endoscopic intestinal metaplasia, and -1 point for endoscopic enlarged folds. + +background score. The sample size for that study would be 26 (8 patients with diffuse type cancer, and 19 patients with intestinal type cancer). + +## Conclusion + +In conclusion, patient backgrounds, such as age, sex, endoscopic intestinal metaplasia, and endoscopic enlarged folds are useful for predicting tumor type. + +## Article Highlights Research Background + +The accurate diagnosis of gastric cancer using the Lauren classification is crucial. + +## Research Motivation + +The relationship between the Lauren classification and endoscopic findings based on the Kyoto classification is not clear. + +## Research Objectives + +To investigate the background patient characteristics and endoscopic gastritis of patients with diffuse- and intestinal-type gastric cancers, focusing on Helicobacter *pylori* (H. pylori)-infected patients. + +## Research Methods + +This study included participants who underwent esophagogastroduodenoscopy at the Toyoshima Endoscopy Clinic. The endoscopy-based Kyoto classification of gastritis consisted of atrophy, intestinal metaplasia, enlarged folds, nodularity, and diffuse redness. The effects of age, sex, and Kyoto classification score on gastric cancer according to the Lauren classification were analyzed. + +## Research Results + +A total of 499 H. *pylori*-infected patients (49.6% males; average age, 54.9 years) were enrolled. A total of 132 patients with gastric cancer (39 diffuse- and 93 intestinal-type) and 367 cancer-free controls were eligible. Gastric cancer was independently associated with age ≥ 65 years, high atrophy score, high intestinal metaplasia score, and low nodularity score when compared to the control. Factors independently associated with intestinal-type cancer were age ≥ 65 years, male sex, high intestinal metaplasia score, and low enlarged folds score when compared to diffuse-type cancer. The Lauren predictive background score was defined as the sum of the following points: +2 points for an age of ≥ 65 years, +1 point for male sex, +1 point for intestinal metaplasia, and -1 point for enlarged folds. The area under the curve of the Lauren predictive background score was 0.828 for predicting intestinal-type tumors. With a cut-off of +2, the sensitivity and specificity of the Lauren predictive background score were 81.7% and 71.8%, respectively. + +## Research Conclusions + +Patient backgrounds such as age, sex, endoscopic intestinal metaplasia, and endoscopic enlarged folds are useful for predicting tumor type. + +## Research Perspectives + +Studying subjects after H. *pylori* eradication or H. *pylori*-uninfected subjects in the future is warranted. Furthermore, comparisons between the endoscopic background diagnosis of patients with gastric cancer (especially according to Lauren classification) and that of non-cancer controls is desired. + +## References + +Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68: 394-424 [PMID: 30207593 DOI: 10.3322/caac.21492] +1 Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. *Acta Pathol Microbiol Scand* 1965; 64: 31-49 +[PMID: 14320675 DOI: 10.1111/apm.1965.64.1.31] +2 Fox JG, Wang TC. Inflammation, atrophy, and gastric cancer. *J Clin Invest* 2007; 117: 60-69 [PMID: 17200707 DOI: 10.1172/JCI30111] +3 Nardone G, Rocco A, Malfertheiner P. Review article: helicobacter pylori and molecular events in precancerous gastric lesions. *Aliment Pharmacol Ther* 2004; 20: 261-270 [PMID: 15274662 DOI: 10.1111/j.1365-2036.2004.02075.x] +4 5 Watanabe M, Kato J, Inoue I, Yoshimura N, Yoshida T, Mukoubayashi C, Deguchi H, Enomoto S, Ueda K, Maekita T, Iguchi M, Tamai H, Utsunomiya H, Yamamichi N, Fujishiro M, Iwane M, Tekeshita T, Mohara O, Ushijima T, Ichinose M. Development of gastric cancer in nonatrophic stomach with highly active inflammation identified by serum levels of pepsinogen and Helicobacter pylori antibody together with endoscopic rugal hyperplastic gastritis. *Int J Cancer* 2012; 131: 26322642 [PMID: 22383377 DOI: 10.1002/ijc.27514] +6 Study Group of Millennium Genome Project for Cancer, Sakamoto H, Yoshimura K, Saeki N, +Katai H, Shimoda T, Matsuno Y, Saito D, Sugimura H, Tanioka F, Kato S, Matsukura N, Matsuda N, Nakamura T, Hyodo I, Nishina T, Yasui W, Hirose H, Hayashi M, Toshiro E, Ohnami S, Sekine A, Sato Y, Totsuka H, Ando M, Takemura R, Takahashi Y, Ohdaira M, Aoki K, Honmyo I, Chiku S, Aoyagi K, Sasaki H, Yanagihara K, Yoon KA, Kook MC, Lee YS, Park SR, Kim CG, Choi IJ, +Yoshida T, Nakamura Y, Hirohashi S. Genetic variation in PSCA is associated with susceptibility to diffuse-type gastric cancer. *Nat Genet* 2008; 40: 730-740 [PMID: 18488030 DOI: 10.1038/ng.152] +Ma J, Shen H, Kapesa L, Zeng S. Lauren classification and individualized chemotherapy in gastric cancer. *Oncol Lett* 2016; 11: 2959-2964 [PMID: 27123046 DOI: 10.3892/ol.2016.4337] +7 Kim SK, Kim HJ, Park JL, Heo H, Kim SY, Lee SI, Song KS, Kim WH, Kim YS. Identification of a molecular signature of prognostic subtypes in diffuse-type gastric cancer. *Gastric Cancer* 2020; 23: 473-482 [PMID: 31773340 DOI: 10.1007/s10120-019-01029-4] +8 Nakamura R, Omori T, Mayanagi S, Irino T, Wada N, Kawakubo H, Kameyama K, Kitagawa Y. Risk of lymph node metastasis in undifferentiated-type mucosal gastric carcinoma. World J Surg Oncol 2019; 17: 32 [PMID: 30760272 DOI: 10.1186/s12957-019-1571-2] +9 10 Zou Y, Wu L, Yang Y, Shen X, Zhu C. Risk factors of tumor invasion and node metastasis in early gastric cancer with undifferentiated component: a multicenter retrospective study on biopsy specimens and clinical data. *Ann Transl Med* 2020; 8: 360 [PMID: 32355804 DOI: 10.21037/atm.2020.02.42] +Rawicz-Pruszyński K, Mielko J, Pudło K, Lisiecki R, Skoczylas T, Murawa D, Polkowski WP. + +Yield of staging laparoscopy in gastric cancer is influenced by Laurén histologic subtype. J Surg Oncol 2019; 120: 1148-1153 [PMID: 31544969 DOI: 10.1002/jso.25711] +11 12 Dong D, Tang L, Li ZY, Fang MJ, Gao JB, Shan XH, Ying XJ, Sun YS, Fu J, Wang XX, Li LM, Li ZH, Zhang DF, Zhang Y, Li ZM, Shan F, Bu ZD, Tian J, Ji JF. Development and validation of an individualized nomogram to identify occult peritoneal metastasis in patients with advanced gastric cancer. *Ann Oncol* 2019; 30: 431-438 [PMID: 30689702 DOI: 10.1093/annonc/mdz001] +Shim CN, Lee SK. Endoscopic submucosal dissection for undifferentiated-type early gastric cancer: +do we have enough data to support this? *World J Gastroenterol* 2014; 20: 3938-3949 [PMID: 24744583 DOI: 10.3748/wjg.v20.i14.3938] +13 Abdelfatah MM, Barakat M, Lee H, Kim JJ, Uedo N, Grimm I, Othman MO. The incidence of lymph node metastasis in early gastric cancer according to the expanded criteria in comparison with the absolute criteria of the Japanese Gastric Cancer Association: a systematic review of the literature 14 and meta-analysis. *Gastrointest Endosc* 2018; 87: 338-347 [PMID: 28966062 DOI: +10.1016/j.gie.2017.09.025] +Berlth F, Bollschweiler E, Drebber U, Hoelscher AH, Moenig S. Pathohistological classification systems in gastric cancer: diagnostic relevance and prognostic value. *World J Gastroenterol* 2014; 20: 5679-5684 [PMID: 24914328 DOI: 10.3748/wjg.v20.i19.5679] +15 16 Jiménez Fonseca P, Carmona-Bayonas A, Hernández R, Custodio A, Cano JM, Lacalle A, Echavarria I, Macias I, Mangas M, Visa L, Buxo E, Álvarez Manceñido F, Viudez A, Pericay C, Azkarate A, Ramchandani A, López C, Martinez de Castro E, Fernández Montes A, Longo F, Sánchez Bayona R, Limón ML, Diaz-Serrano A, Martin Carnicero A, Arias D, Cerdà P, Rivera F, Vieitez JM, Sánchez Cánovas M, Garrido M, Gallego J. Lauren subtypes of advanced gastric cancer influence survival and response to chemotherapy: real-world data from the AGAMENON National Cancer Registry. *Br J Cancer* 2017; 117: 775-782 [PMID: 28765618 DOI: 10.1038/bjc.2017.245] +17 Petrelli F, Berenato R, Turati L, Mennitto A, Steccanella F, Caporale M, Dallera P, de Braud F, Pezzica E, Di Bartolomeo M, Sgroi G, Mazzaferro V, Pietrantonio F, Barni S. Prognostic value of diffuse vs intestinal histotype in patients with gastric cancer: a systematic review and meta-analysis. J Gastrointest Oncol 2017; 8: 148-163 [PMID: 28280619 DOI: 10.21037/jgo.2017.01.10] +18 Nomura S, Terao S, Adachi K, Kato T, Ida K, Watanabe H, Shimbo T; Research Group for Establishment of Endoscopic Diagnosis of Chronic Gastritis. Endoscopic diagnosis of gastric mucosal activity and inflammation. *Dig Endosc* 2013; 25: 136-146 [PMID: 23362997 DOI: 10.1111/j.1443-1661.2012.01357.x] +19 Pimentel-Nunes P, Libânio D, Marcos-Pinto R, Areia M, Leja M, Esposito G, Garrido M, Kikuste I, Megraud F, Matysiak-Budnik T, Annibale B, Dumonceau JM, Barros R, Fléjou JF, Carneiro F, van Hooft JE, Kuipers EJ, Dinis-Ribeiro M. Management of epithelial precancerous conditions and lesions in the stomach (MAPS II): European Society of Gastrointestinal Endoscopy (ESGE), European Helicobacter and Microbiota Study Group (EHMSG), European Society of Pathology (ESP), and Sociedade Portuguesa de Endoscopia Digestiva (SPED) guideline update 2019. *Endoscopy* 2019; 51: 365-388 [PMID: 30841008 DOI: 10.1055/a-0859-1883] +20 Toyoshima O, Nishizawa T, Yoshida S, Sakaguchi Y, Nakai Y, Watanabe H, Suzuki H, Tanikawa C, Matsuda K, Koike K. Endoscopy-based Kyoto classification score of gastritis related to pathological topography of neutrophil activity. *World J Gastroenterol* 2020; 26: 5146-5155 [PMID: 32982115 DOI: 10.3748/wjg.v26.i34.5146] Sugimoto M, Ban H, Ichikawa H, Sahara S, Otsuka T, Inatomi O, Bamba S, Furuta T, Andoh A. Efficacy of the Kyoto Classification of Gastritis in Identifying Patients at High Risk for Gastric Cancer. *Intern Med* 2017; 56: 579-586 [PMID: 28321054 DOI: 10.2169/internalmedicine.56.7775] +21 Shichijo S, Hirata Y, Niikura R, Hayakawa Y, Yamada A, Koike K. Association between gastric cancer and the Kyoto classification of gastritis. *J Gastroenterol Hepatol* 2017; 32: 1581-1586 [PMID: 28217843 DOI: 10.1111/jgh.13764] +22 23 Nishizawa T, Toyoshima O, Kondo R, Sekiba K, Tsuji Y, Ebinuma H, Suzuki H, Tanikawa C, Matsuda K, Koike K. The simplified Kyoto classification score is consistent with the ABC method of classification as a grading system for endoscopic gastritis. *J Clin Biochem Nutr* 2021; 68: 101-104 [PMID: 33536719 DOI: 10.3164/jcbn.20-41] +24 Toyoshima O, Nishizawa T, Sakitani K, Yamakawa T, Takahashi Y, Yamamichi N, Hata K, Seto Y, +Koike K, Watanabe H, Suzuki H. Serum anti-*Helicobacter pylori* antibody titer and its association with gastric nodularity, atrophy, and age: A cross-sectional study. *World J Gastroenterol* 2018; 24: +4061-4068 [PMID: 30254410 DOI: 10.3748/wjg.v24.i35.4061] +25 Yoshii S, Mabe K, Watano K, Ohno M, Matsumoto M, Ono S, Kudo T, Nojima M, Kato M, Sakamoto N. Validity of endoscopic features for the diagnosis of Helicobacter pylori infection status based on the Kyoto classification of gastritis. *Dig Endosc* 2020; 32: 74-83 [PMID: 31309632 DOI: +10.1111/den.13486] Kimura K, Takemoto T. An endoscopic recognition of the atrophic border and its significance in chronic gastritis. *Endoscopy* 1969; 3: 87-97 [DOI: 10.1055/s-0028-1098086] +26 Kaneko S, Yoshimura T. Time trend analysis of gastric cancer incidence in Japan by histological types, 1975-1989. *Br J Cancer* 2001; 84: 400-405 [PMID: 11161407 DOI: 10.1054/bjoc.2000.1602] +27 28 Chung SJ, Park MJ, Kang SJ, Kang HY, Chung GE, Kim SG, Jung HC. Effect of annual endoscopic screening on clinicopathologic characteristics and treatment modality of gastric cancer in a highincidence region of Korea. *Int J Cancer* 2012; 131: 2376-2384 [PMID: 22362223 DOI: +10.1002/ijc.27501] +29 Nishibayashi H, Kanayama S, Kiyohara T, Yamamoto K, Miyazaki Y, Yasunaga Y, Shinomura Y, Takeshita T, Takeuchi T, Morimoto K, Matsuzawa Y. Helicobacter pylori-induced enlarged-fold gastritis is associated with increased mutagenicity of gastric juice, increased oxidative DNA damage, and an increased risk of gastric carcinoma. *J Gastroenterol Hepatol* 2003; 18: 1384-1391 [PMID: +14675267 DOI: 10.1046/j.1440-1746.2003.03192.x] Jónasson L, Hallgrímsson J, Olafsdóttir G. Gastric carcinoma: correlation of diagnosis based on biopsies and resection specimens with reference to the Laurén classification. *APMIS* 1994; 102: 711715 [PMID: 7946275 DOI: 10.1111/j.1699-0463.1994.tb05224.x] +30 Hansson LE, Lindgren A, Nyrén O. Can endoscopic biopsy specimens be used for reliable Laurén classification of gastric cancer? *Scand J Gastroenterol* 1996; 31: 711-715 [PMID: 8819223 DOI: 10.3109/00365529609009155] +31 32 **Lee JH**, Kim JH, Rhee K, Huh CW, Lee YC, Yoon SO, Youn YH, Park H, Lee SI. Undifferentiated early gastric cancer diagnosed as differentiated histology based on forceps biopsy. *Pathol Res Pract* 2013; 209: 314-318 [PMID: 23598070 DOI: 10.1016/j.prp.2013.02.014] +Waldum HL, Fossmark R. Types of Gastric Carcinomas. *Int J Mol Sci* 2018; 19 [PMID: 30567376 DOI: 10.3390/ijms19124109] +33 Jung DH, Park YM, Kim JH, Lee YC, Youn YH, Park H, Lee SI, Kim JW, Choi SH, Hyung WJ, Noh SH. Clinical implication of endoscopic gross appearance in early gastric cancer: revisited. Surg Endosc 2013; 27: 3690-3695 [PMID: 23588711 DOI: 10.1007/s00464-013-2947-y] +34 Kanesaka T, Nagahama T, Uedo N, Doyama H, Ueo T, Uchita K, Yoshida N, Takeda Y, Imamura K, Wada K, Ishikawa H, Yao K. Clinical predictors of histologic type of gastric cancer. Gastrointest Endosc 2018; 87: 1014-1022 [PMID: 29122604 DOI: 10.1016/j.gie.2017.10.037] +35 Yao K, Oishi T, Matsui T, Yao T, Iwashita A. Novel magnified endoscopic findings of microvascular architecture in intramucosal gastric cancer. *Gastrointest Endosc* 2002; 56: 279-284 [PMID: 12145613 DOI: 10.1016/s0016-5107(02)70194-6] +36 37 Ueo T, Yonemasu H, Yao K, Ishida T, Togo K, Yanai Y, Fukuda M, Motomura M, Narita R, Murakami K. Histologic differentiation and mucin phenotype in white opaque substance-positive gastric neoplasias. *Endosc Int Open* 2015; 3: E597-E604 [PMID: 26716119 DOI: 10.1055/s-0034-1393177] +38 Kanesaka T, Sekikawa A, Tsumura T, Maruo T, Osaki Y, Wakasa T, Shintaku M, Yao K. Absent microsurface pattern is characteristic of early gastric cancer of undifferentiated type: magnifying endoscopy with narrow-band imaging. *Gastrointest Endosc* 2014; 80: 1194-1198. e1 [PMID: 25442093 DOI: 10.1016/j.gie.2014.08.021] Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M, Taniyama K, Sasaki N, Schlemper RJ. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001; 345: 784-789 [PMID: 11556297 DOI: 10.1056/NEJMoa001999] +39 40 Kamada T, Tanaka A, Yamanaka Y, Manabe N, Kusunoki H, Miyamoto M, Tanaka S, Hata J, +Chayama K, Haruma K. Nodular gastritis with helicobacter pylori infection is strongly associated with diffuse-type gastric cancer in young patients. *Dig Endosc* 2007; 19: 180-184 [DOI: 10.1111/j.1443-1661.2007.00750.x] Ford AC, Yuan Y, Moayyedi P. *Helicobacter pylori* eradication therapy to prevent gastric cancer: +systematic review and meta-analysis. Gut 2020; 69: 2113-2121 [PMID: 32205420 DOI: +10.1136/gutjnl-2020-320839] +41 42 Take S, Mizuno M, Ishiki K, Kusumoto C, Imada T, Hamada F, Yoshida T, Yokota K, Mitsuhashi T, Okada H. Risk of gastric cancer in the second decade of follow-up after Helicobacter pylori eradication. *J Gastroenterol* 2020; 55: 281-288 [PMID: 31667586 DOI: +10.1007/s00535-019-01639-w] +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/PMC8358940.md b/medical/md/PMC8358940.md new file mode 100644 index 0000000000000000000000000000000000000000..405989736c3e1fee78c6712bfd5ad8c183332ef9 --- /dev/null +++ b/medical/md/PMC8358940.md @@ -0,0 +1,269 @@ +# High Tensile Strength Regenerated Α‑1,3-Glucan Fiber And Crystal Transition + +Azusa Togo, Shiori Suzuki, Satoshi Kimura, and Tadahisa Iwata* Cite This: ACS Omega 2021, 6, 20361−20368 Read Online ABSTRACT: α-1,3-Glucan is a linear and crystalline polysaccharide which is synthesized by in vitro enzymatic polymerization from sucrose. A previous study reported that regenerated fibers of α-1,3glucan were prepared using a wet-spinning method. However, the mechanical properties were poorer than cellulose regenerated fibers. + +Then, in this study, the mechanical properties of the regenerated α1,3-glucan fiber were improved by the transformation of the crystal structure and stretching. The regenerated fiber stretched in water and dehydrated by heating showed high tensile strength (18 cN/tex) that is comparable with that of viscose rayon. Moreover, the crystal structures of the regenerated fibers were investigated using wide-angle X-ray diffraction (WAXD). To date, four crystal polymorphs of α-1,3glucan from polymorph I to IV have been reported. This study revealed that the regenerated α-1,3-glucan fibers had two different polymorphs, polymorph II (hydrated form) and polymorph III (anhydrous form), depending on post-treatment methods of stretching and annealing procedures. Furthermore, the obtained distinctive 2D-WAXD patterns suggested that polymorph III is identical to polymorph IV. + +ACCESS Metrics & More Article Recommendations *sı Supporting Information + +## 1. Introduction + +Fibers are indispensable for various applications including clothing, medical supplies, and marine products because they are strong and have a large surface area. Many fibers are produced from synthetic polymers such as nylon and polyester, which are inexpensive and strong.1,2 However, the production of synthetic fibers from petroleum resources is problematic because it involves the depletion of fossil resources and contributes to global warming. Therefore, to overcome these environmental problems, fibers produced from sustainable biomass resources have been offered as alternatives. + +Polysaccharides are representative materials of biomass resources.3 They are produced by plants and microorganisms and have various forms depending on their constituent monosaccharides and linkage positions. For instance, potatoes contain starch (α-1,4- and α-1,6-glucan),4 the cell walls of plants contain cellulose (β-1,4-glucan),5,6 and species of the genus Euglena produce paramylon (β-1,3-glucan).7 Recently, our group succeeded in the in vitro enzymatic polymerization of completely linear and pure α-1,3-glucan comprising only glucose units bound through α-1,3-glycosidic linkages.8 Pure α-1,3-glucan can be obtained from sucrose in one-pot under mild conditions without any toxic reagents, and the molecular weights can be adjusted by controlling conditions such as the reaction time and temperature. This enzymatic polymerized α1,3-glucan is highly crystalline.9 Again, there are many types of polysaccharides, and each has different physical characteristics depending on the linkage positions.6 Therefore, the polysaccharides constitute a + +![0_image_1.png](0_image_1.png) + +promising resource for biomass-based fibers. However, there is one significant problem with processing the polysaccharides into fibers: they comprise numerous hydroxyl groups and have strong inter- and intramolecular hydrogen bonds. Therefore, they have little thermoplasticity and cannot be molded by heatmelting methods such as melt-spinning, but they can be useful thermoplastics by esterification. For example, paramylon esters can be melt-spun,10 dextran esters can be used as hot-melt adhesives,11 and nigeran (α-1,3-alt-α-1,4-glucan) esters can be used as melt-press films.12 However, the biodegradability of polysaccharide ester derivatives decreases as the degree of substitution increases.13,14 This decrease in biodegradability is undesirable because it exacerbates environmental pollution from microplastics. + +Wet-spinning is a traditional method to process the chemically unmodified polysaccharides. In this method, the polysaccharide is solubilized in an aqueous alkaline solution, either by temporary derivatization or complex formation.15 The resulting solution is extruded, drawn, and coagulated in an + +![0_image_0.png](0_image_0.png) + +| Received: | May 5, 2021 | +|--------------------------|---------------| +| Accepted: | July 6, 2021 | +| Published: July 27, 2021 | | + +![0_image_2.png](0_image_2.png) + +ACS Omega http://pubs.acs.org/journal/acsodf Article aqueous acidic solution, and then, the polysaccharide is regenerated and processed into fibers. During the regeneration process, the chemical structure of the polysaccharide returns to its original state, and thus, the regenerated fiber retains its inherent biodegradability14 and biocompatibility.16 Conventionally, regenerated cellulose fibers have been commercially produced as viscose, cuprammonium rayon, and lyocell by using various types of cellulose−solvent systems. + +Notably, the crystal structures and the properties of these regenerated celluloses can be changed from those of natural fibers such as cotton.2,17 However, the commercial utilization and research for regenerated fibers have been limited mainly to cellulose, despite the wide variety of polysaccharides. + +Recently, our group has focused on the regeneration fibers of the other polysaccharides than cellulose and succeeded in wetspinning of curdlan (β-1,3-glucan) fibers using an ionic liquid system.18 The resulting regenerated fibers of curdlan showed higher water absorption (∼86%) and elongation capabilities +(20−50%) than conventional regenerated cellulose fibers. + +Moreover, there is some research about α-1,3-glucan regenerated fibers via the viscose method.19,20 Although they show high tensile strength 17−23 cN/tex, the viscose method required harmful reagents such as carbon disulfide. Then, our group developed the wet-spinning system of α-1,3-glucan using an 8 wt % lithium chloride (LiCl)/N,N-dimethylacetamide +(DMAc) solution.21 The mechanical properties and crystal structures of the regenerated α-1,3-glucan fibers were found to depend on the type of used coagulation baths: a fiber coagulated in a water bath has a hydrated crystal form and fragile mechanical properties, whereas a fiber coagulated in an ethanol bath has an anhydrous form and a higher tensile strength of 11 cN/tex.19,20 However, the maximum tensile strength of the regenerated α-1,3-glucan fiber coagulated in ethanol is approximately half that of a regenerated cellulose fiber such as viscose (22 cN/tex). + +Herein, we developed a post-treatment methods to enhance the mechanical properties of regenerated α-1,3-glucan fibers spun using the LiCl/DMAc solution. Generally, it is known that the increases in the orientation of the molecular chains and crystallinity contribute to improve the mechanical strength of fibers.22 However, the regenerated α-1,3-glucan fibers in our previous work could not be subjected to further stretching processes because of the fragility and rigidity. Then, the unique ability of the crystal transition between anhydrous and hydrated forms of α-1,3-glucan was focused on. We assumed that the molecular chains of the α-1,3-glucan could be more movable during the crystal transition induced by a solvent substitution of the anhydrous-form-fiber with water, thus enabling the further drawing process. Furthermore, the resultant hydrate crystal structure of the well-stretched fiber can also be reversible by an additional drying process. Then, in this study, based on the above hypothesis, the effects of the solvent substitution, stretching in the solvents, and drying processes on the mechanical properties of the regenerated α1,3-glucan fibers were investigated, and we successfully developed a post-treatment process, providing a regenerated α-1,3-glucan fiber with sufficiently high mechanical strength +(18 cN/tex). + +## 2. Results And Discussion + +2.1. Wet-Spinning of α-1,3-Glucan and Post-treatment of the Regenerated Fibers. A 10 wt % solution of the α-1,3-glucan in 8 wt % LiCl/DMAc was wet-spun using two types of coagulation baths of EtOH and water as reported + +![1_image_0.png](1_image_0.png) + +previously. It has been confirmed in the previous study that the molecular weight of the fibers was the weight-average molecular weight (Mw) = 0.8−1.0 × 105. + +21 After the wetspinning, Regenerated Fiber (EtOH) was immersed in water, and Regenerated Fiber (Water) was immersed in ethanol to investigate the transitions in the morphologies, crystal structures, and mechanical properties of the fibers before and after solvent substitution. Pictures of the regenerated α-1,3glucan fibers after solvent substitution are shown in Figure 1a,b. There were no significant changes in appearance during solvent substitution. Images of the regenerated α-1,3-glucan fibers after stretching in water and air-drying or oven-drying are shown in Figure 1c,d. The fibers did not change significantly in appearance when they were stretched in water, although their diameters decreased by stretching. + +2.2. Microscopic Observations. Optical microscope images of the four types of regenerated α-1,3-glucan fibers are shown in Figure 2. The images of the fibers after solvent substitution, i.e., Regenerated Fiber 1 (Water → EtOH) and Regenerated Fiber 2 (EtOH → Water), are shown in Figure 2a and b, respectively. There was little significant difference in transparency and appearance between Regenerated Fiber 2 +(EtOH → Water) and Fiber (EtOH) which was examined in our previous study.21 However, the diameter of Regenerated Fiber 2 (EtOH → Water) increased when it was immersed in water owing to swelling, as shown in Table 1. On the other hand, Regenerated Fiber 1 (Water → EtOH) was opaque and white (Figure 2b), whereas Regenerated Fiber (Water) was semitransparent which was examined in our previous study, but the diameter of Regenerated Fiber 1 (Water → EtOH) was not changed by immersion in EtOH.21 Moreover, optical microscope images of the fibers after stretching in wateri.e., +Regenerated Fiber 3 (Stretched) and Regenerated Fiber 4 +(Stretched and Heated)are shown in Figure 2c and d. The transparency of the fibers was not changed by stretching and heating, but the diameters decreased due to stretching from that of Regenerated Fiber (EtOH), as shown in Table 1. + +![2_image_0.png](2_image_0.png) + +| Diameter (μm) | Δn | | | | +|------------------------------------------------------------------------------|----------|----------|----|----| +| (×10−3 ) | fc | Xc | | | +| Fiber | (%) | (%) | | | +| Regenerated Fiber (EtOH)a | 120 ± 16 | 13 ± 5 | 76 | 20 | +| Regenerated Fiber (Water)a | 124 ± 28 | 5 ± 1 | 48 | 44 | +| Regenerated Fiber 1 (EtOH → Water) | 156 ± 8 | 9 ± 0.5 | 74 | 25 | +| Regenerated Fiber 2 (Water → EtOH) | 129 ± 12 | − | − | 49 | +| Regenerated Fiber 3 (Stretched) | 78 ± 7 | 20 ± 1.7 | 87 | 26 | +| 58 ± 8 | 29 ± 4.1 | 80 | 33 | | +| Regenerated Fiber 4 (Stretched and Heated) a Reported by a previous paper.21 | | | | | + +Precisely, the diameter of Regenerated Fiber 4 (Stretched and Heated) was lower than that of Regenerated Fiber 3 +(Stretched), suggesting that the fixed fiber after stretching in water was further stretched during the oven-drying process. + +To investigate the effects of the coagulation baths and solvent substitutions on fiber orientation, the four types of regenerated α-1,3-glucan fibers were examined using a polarized optical microscope (POM). The POM images of the regenerated α-1,3-glucan fibers are also shown in Figure 2. When the fibers were rotated 45 deg in a crossed Nicol arrangement, there was brightness due to molecular orientation along the fiber axis in all of the fibers except for Regenerated Fiber 1 (Water → EtOH), which comprised opaque fibers +(Figure 2a). As shown in Table 1, the birefringence (Δn), which is an analog factor for total orientation, of Regenerated Fiber 2 (EtOH → Water) was lower than that of Regenerated Fiber (EtOH) owing to the swelling by solvent substitution in water. On the other hand, the Δn of Regenerated Fiber 3 (Stretched) was higher than that of Regenerated Fiber (EtOH) owing to stretching. Moreover, the Δn of Regenerated Fiber 4 (Stretched and Heated) increased by 1.5 times than Regenerated Fiber 3 (Stretched), suggesting that heating in a fixed state stretches the fibers because of the shrinkage derived from the dehydration. + +SEM investigations were carried out to reveal the more detailed morphologies of the regenerated fibers, as shown in Figure 3. First, the effect of the type of coagulation bath on the shape of the cross section was investigated. The cross section of Regenerated Fiber 2 (EtOH → Water) was circular (Figure 3b), whereas that of Regenerated Fiber 1 (Water → EtOH) +was irregular and nodulous (Figure 3a). In our previous study,21 Regenerated Fiber (EtOH) had a circular cross section, while Regenerated Fiber (Water) had an irregular and nodulous cross section. This result is consistent with the result of the previous report which suggests that the shape of the cross section depends on the coagulation bath because of the diffusion rate and affinity between α-1,3-glucan and the coagulation bath. After stretching in water, the cross section of Regenerated Fiber 3 (Stretched) remained circular. Furthermore, Regenerated Fiber 4 (Stretched and Heated) also retained almost the same shape of the cross section after stretching and heating. Therefore, it was suggested that the shape of the cross section was determined during regeneration. + +Second, the internal structures and porosities of the fibers were compared. As reported previously, Regenerated Fiber +(EtOH) had a smooth surface and a dense structure,21 and Regenerated Fiber 2 (EtOH → Water) had the same dense structure as Regenerated Fiber (EtOH) after solvent substitution with water, as shown in Figure 3b. Furthermore, Regenerated Fiber 3 (Stretched) and Regenerated Fiber 4 (Stretched and Heated) also had smooth surfaces and dense structures, suggesting that stretching and drying processes did not affect the surface shape and porosity of the fibers. + +Therefore, Regenerated Fiber 3 (Stretched) and Regenerated Fiber 4 (Stretched and Heated) were highly transparent and flexible after stretching and drying. In contrast, Regenerated + +![3_image_0.png](3_image_0.png) + +Fiber 1 (Water → EtOH) had a rough and uneven surface and, therefore, was opaque and white. These results suggest that, in addition to the shape of the cross section, the roughness and porosity of the regenerated fibers were determined during the regeneration process. In summary, the shape of the cross section, the porosity, and the roughness of the regenerated α1,3-glucan fibers were affected by the solvent used as the coagulation bath. These morphological characteristics might be affected by the diffusion rate and the affinity between the α1,3-glucan and the solvent surrounding the fiber during regeneration. + +2.3. WAXD Analysis for Crystal Transition in Posttreatments. The 2D-WAXD patterns of the four types of regenerated α-1,3-glucan fibers are shown in Figure 4a, and 1D-WAXD profiles are shown in Figure 4b. As reported previously,21 the 1D-WAXD profiles of the regenerated α-1,3glucan fibers obtained by wet-spinning differed according to the type of coagulation baths (Figure 4b). Regenerated Fiber (EtOH) showed the anhydrous form, and Regenerated Fiber (Water) showed the hydrated form. The crystal structure of Regenerated Fiber (EtOH) was transformed from the anhydrous form to the hydrated form after substitution of the solvent with water, as demonstrated by the 1D-WAXD +profile of Regenerated Fiber 2 (EtOH → Water). This crystal transformation from the anhydrous form to the hydrated form through hydration is consistent with that in a previous work for investigating the crystal structures of α-1,3-glucan powder from fungal cell walls.23 However, the hydrated form of Regenerated Fiber (Water) retained the hydrated form even after solvent substitution with EtOH, as demonstrated by the 1D-WAXD +profile of Regenerated Fiber 1 (Water → EtOH). This suggests that the hydrated form might be more stable than the anhydrous form, and the crystal transition from hydrated form to anhydrous form did not occur by the solvent substitution. This result is consistent with the fact that no change in diameter was observed with solvent substitution with ethanol. The stability of this hydrated crystal structure is consistent with previous reports.24 The degree of crystalline chain orientation ( fc) was calculated from the 2D-WAXD results. Distinctive arc-shaped reflections were observed in all the fibers (Figure 4a) except for Regenerated Fiber 1 (Water → EtOH) which was a ring pattern owing to a lack of fiber orientation. The fc of Regenerated Fiber 2 (EtOH → Water) was 74%, and that of Regenerated Fiber 3 (Stretched) was 87%, which was higher than that of Regenerated Fiber 2 (EtOH → Water). As described in the previous section, the Δn in Regenerated Fiber 3 (Stretched) increased from that in Regenerated Fiber 2 +(EtOH → Water) (Table 1). These results indicate that stretching effectively increased the degree of orientation of both the amorphous and crystalline regions in the fiber. In contrast, the fc of Regenerated Fiber 4 (Stretched and Heated) +was 80%, which was lower than that of Regenerated Fiber 3 (Stretched), although the Δn of Regenerated Fiber 4 (Stretched and Heated) was higher than that of Regenerated Fiber 3 (Stretched). Considered with the increase in the degree of crystallinity (Xc) after oven-drying (Table 1), it was considered that shrinkage of the fiber occurred during the oven-drying process, especially in the amorphous region, which induced disruption of the orientation and promoted crystallization of some of the amorphous polymer chains. + +According to previous studies, four types of crystal polymorphs have been proposed for α-1,3-glucan.9,23,24 Polymorph I is a hydrated form. This polymorph is a native crystalline form that exists in the cell walls of fungi. Polymorph II is another hydrated form that is observed after extraction of the α-1,3-glucan via dissolution in alkali solution. Polymorph II +is four-chain orthorhombic with unit cell parameters a = +0.9982 nm, b = 1.9026 nm, and c (the fiber axis) = 0.8433 nm.9 Polymorph III is an anhydrous form that is created by the dehydration of polymorph I or polymorph II. Polymorph III is two-chain orthorhombic with unit cell parameters a = 0.9217 nm, b = 1.7158 nm, and c = 0.8417 nm.9 The reversible transformation of polymorph III into polymorph II by hydration and dehydration has also been reported.9,23 Polymorph IV is another anhydrous form. It was reported that this polymorph was observed in α-1,3-glucan when it is an acetylated, stretched and subsequently deacetylated film.24,25 The unit cell of polymorph IV is 2-fold helix orthorhombic with parameters a = 1.65 nm, b = 0.955 nm, and c = 0.844 nm +(the fiber axis).24 The d-spacings of Regenerated Fiber 3 (Stretched) and Regenerated Fiber 4 (Stretched and Heated) are listed in Table S1a and S1b. The d-spacings extracted from Figure 4 were defined as observed d-spacings (dobs), and the d-spacings reported previously9 were defined as calculated d-spacings +(dcal). The dobs values were indexed based on a piece of previous research about polymorphs II and III.9 As shown in Table S1a, the dobs of the regenerated Regenerated Fiber 3 (Stretched) agreed well with the dcal of polymorph II (hydrated form) reported previously. This suggests that Regenerated Fiber 3 (Stretched) existed in the hydrated form of polymorph ACS Omega http://pubs.acs.org/journal/acsodf Article II. In contrast, the dobs of Regenerated Fiber 4 (Stretched and Heated) agreed well with the dcal of polymorph III (anhydrous form, Table S1b). Although the crystal structures of α-1,3glucan polymorphs II and III were revealed by electron microdiffraction and 1D-WAXD patterns in the previous research, their distinctive 2D-WAXD pattern have not yet been reported. Therefore, the 2D-WAXD patterns are meaningful in terms of confirming the structures of polymorphs II and III. + +More precisely, the 2D-WAXD pattern of Regenerated Fiber 4 (Stretched and Heated) (Figure 4a) seemed to be similar to that of polymorph IV reported by Ogawa et al. The dobs of Regenerated Fiber 4 (Stretched and Heated) agreed well with the dcal of polymorph IV (Table S1c).24 Compared to polymorph III (reported by Kobayashi et al.) and polymorph IV (reported by Ogawa et al.), both were 2-fold helix and fourchain orthorhombic. The crystal lattice parameters of polymorph III were reported as a = 0.9217 nm, b = 1.715 nm, and c = 0.8417 nm, and those of polymorph IV were a = 1.646 nm, b = 0.955 nm, and c = 0.844 nm. However, these two crystal unit cells become similar when the lengths of the a axis and b axis are reversed. This similarity has not been properly considered ever, because polymorphs III and IV were investigated using different methodsi.e., 1D-WAXD, electron microdiffraction, and 2D-WAXD. It should be noted that the distinctive 2D-WAXD pattern of the anhydrous and hydrated forms of α-1,3-glucan were first obtained in this study via wet-spinning and stretching in water. The 2D-WAXD pattern of anhydrous form indicate that polymorphs III and IV have similar 2D-WAXD patterns. Therefore, Regenerated Fiber (EtOH) and Regenerated Fiber 4 (Stretched and Heated) might have the same anhydrous unit cell. More details will be obtained by electron diffraction and stereochemical model refinement in the future. + +2.4. Post-treatment's Effect on the Mechanical Properties of the Regenerated Fibers. The mechanical properties of the regenerated α-1,3-glucan fibers were evaluated by tensile tests. The obtained stress−strain curves of the regenerated fibers are shown in Figure 5, and the tensile strength, Young's modulus, and elongation at break values are listed in Table 2. Compared with the mechanical properties of Regenerated Fiber (EtOH) (tensile strength 11 ± 2.9 cN/tex, Table 2. Mechanical Properties of Regenerated α-1,3- +Glucan Fibers + +| Tensile | Young's | | | +|------------------------------------|---------------|-----------|-----------| +| strength (cN/ tex) | Elongation at | Modulus | | +| Fiber | break (%) | (GPa) | | +| Regenerated Fiber 1 | 0.6 ± 0.3 | 5.3 ± 1.9 | 0.5 ± 0.4 | +| (Water → EtOH) Regenerated Fiber 2 | 15 ± 6.2 | 14 ± 3.8 | 1.4 ± 0.1 | +| (EtOH → Water) Regenerated Fiber 3 | 15 ± 3.1 | 9.2 ± 1.8 | 5.7 ± 1.8 | +| (Stretched) Regenerated Fiber 4 | 18 ± 6.8 | 9.5 ± 3.3 | 11 ± 4.3 | +| (Stretched and Heated) | | | | + +strain 12 ± 3.4%, and Young's modulus 3.5 ± 1.0 GPa),21 the tensile strength and strain of Regenerated Fiber 2 (EtOH → +Water) increased, whereas the Young's modulus decreased. As shown in Table 1, the Δn of Regenerated Fiber 2 (EtOH → +Water) was lower than that of Regenerated Fiber 1. It is considered that the decrease of the orientation improved the flexibility of the regenerated fibers. In contrast, Regenerated Fiber 1 (Water → EtOH) had a lower tensile strength (0.6 cN/tex) than Regenerated Fiber (Water) (2.7 ± 0.8 cN/ +tex).21 As described in the previous sections, Regenerated Fiber 1 (Water → EtOH) had a rough and porous structure, which might cause the lowering of the tensile strength and strain values. + +The results in tensile tests of Regenerated Fiber 3 +(Stretched) and Regenerated Fiber 4 (Stretched and Heated) are also shown in Figure 5 and Table 2. The strain in Regenerated Fiber 3 (Stretched) was lower than in Regenerated Fiber 2 (EtOH → Water), but the Young's modulus of Regenerated Fiber 3 (Stretched) was four times higher than that of Regenerated Fiber 2 (EtOH → Water). + +Therefore, it was suggested that increasing fc and Δn improved the Young's modulus while reducing flexibility owing to the oriented amorphous region. This suggests that the mechanical properties of regenerated α-1,3-glucan fibers can be controlled by stretching them and immersing them in water. On the other hand, the Young's modulus of Regenerated Fiber 4 (Stretched and Heated) was significantly increased by drying it at 105 °C, +and its tensile strength increased to 18 cN/tex (Table 3)i.e., +comparable to that of viscose rayon (22 cN/tex)26although fc decreased from 87% to 80% during heating. The enhanced tensile strength may be attributed to the high Δn, the high Xc +(Table 1) and the difference in the elastic modulus in the crystalline regions. This mechanical strength of α-1,3-glucan fiber was still lower than that of lyocell fiber (34−36 cN/tex)2 which is commercial cellulose regenerated fiber. Therefore, the optimization of spinning conditions such as draw ratio and the molecular weight of α-1,3-glucan should be investigated in the future. In addition, the elastic modulus of the crystalline regions of the anhydrous and hydrated forms has not yet been investigated, and it should be researched in the future. + +## 3. Experimental Section + +![4_Image_0.Png](4_Image_0.Png) + +3.1. Materials. α-1,3-Glucan was prepared by in vitro enzymatic polymerization, as described in our previous paper.8 The weight-average molecular weight (Mw) of the resulting polymer was 1.2 × 105 g/mol, and its number-average molecular weight (Mn) was 0.7 × 105 g/mol. The Figure 5. Stress−strain curves of the regenerated α-1,3-glucan fibers in the tensile tests. + +polydispersity (Mw/Mn) was 1.8. The prepared α-1,3-glucan powder was dried at 105 °C in a vacuum oven for 24 h before use. All other reagents were purchased from commercial sources and were used as received. + +3.2. Preparation of Spinning Dope and Wet-Spinning of α-1,3-Glucan. The spinning dope was prepared using the LiCl/DMAc system depicted in Scheme 1. First, a solution of 8 + +![5_image_0.png](5_image_0.png) + +wt % LiCl/DMAc was prepared: LiCl (2 g) was dissolved in DMAc (26 g) by stirring at 100 °C for 20 min. The dried α1,3-glucan (3 g, 10 wt %) was added to the 8 wt % LiCl/DMAc solution (28 g), and the mixture was stirred at 100 °C for 24 h to obtain a clear viscous solution. The resulting α-1,3-glucan solution was transferred to a stainless-steel barrel equipped with a one-hole spinneret (hole-diameter, 1.0 mm), and spun using a lab-scale customized wet-spinning unit (AIKI RIOTECH Co., Ltd., Aichi, Japan) at room temperature. + +The filament was extruded through the spinneret equipped with four types of metal meshes (30, 100, 200, and 300 mesh) +to remove the undissolved substrate. After the fluid filament had passed through an air gap of 30 mm, it was coagulated in distilled water or ethanol (EtOH) at room temperature. The extrusion velocity (Ve) was set to 2 cm3/min (2.5 m/min), and the take-up velocity (Vt) of the godet roller was 17 m/min, resulting in a draw ratio (DR = Vt/Ve) of 6.8. The resulting fibers were washed in water or EtOH at room temperature for 6 days and air-dried for 24 h. + +3.3. Solvent Substitution of the Regenerated α-1,3- +Glucan Fibers. The obtained two types of regenerated fibers are referred to as Regenerated Fiber (EtOH) and Regenerated Fiber (Water). These regenerated fibers were subjected to solvent substitution with either EtOH or water. Precisely, the Regenerated Fiber (EtOH) was replaced with water over 2 days, and the water in Regenerated Fiber (Water) was replaced with EtOH over 2 days. After solvent substitution, the resultant fibers were dried and fixed at room temperature for 24 h and referred to as either Regenerated Fiber 2 (EtOH → Water) or Regenerated Fiber 1 (Water → EtOH) depending on the coagulation bath and substitution solvent. ACS Omega http://pubs.acs.org/journal/acsodf Article 3.4. Stretching the Regenerated α-1,3-Glucan Fibers in Water. Regenerated Fiber (EtOH) was cut into 10 mmlong pieces. The cut fibers were fixed to a stretching machine and immersed in water to allow them to swell for 5 min at room temperature. After immersion in water, the regenerated α-1,3-glucan fibers were stretched to 1.5 times their initial length. The stretched fibers were fixed in the stretching machine in water for 2 days and air-dried at room temperature. + +These stretched and air-dried fibers are referred to as Regenerated Fiber 3 (Stretched). Moreover, some of the Regenerated Fiber 3 (Stretched) was dried in an oven at 105 +°C for 24 h to remove the water, completely. The oven-dried fiber is referred to as Regenerated Fiber 4 (Stretched and Heated). + +3.5. Polarized Optical Microscope (POM). An ECRIPSE +E600 polarized optical microscope (Nikon Co., Tokyo, Japan) equipped with a DFC 450 charge-coupled device camera (Leica Microsystems, Tokyo, Japan) was used to determine the orientation of the regenerated fibers. The polarizer and depolarizer were set at appropriate angles to obtain clear images. The average orientation of both the amorphous and crystalline parts in each fiber was determined by a POM +equipped with a Berek compensator (Nichia Inc., Kyoto, Japan). The birefringence (Δn) values of the fibers were calculated from fiber diameters (nm) which were measured with micrographs. The total orientation factor ( ft) can be determined by dividing Δn by the maximum birefringence +(Δnmax). However, the Δnmax of α-1,3-glucan has never been determined, and the value of Δn was considered as an analog factor for discussing the fiber orientation in this study. + +3.6. Scanning Electron Microscope (SEM). SEM images of the regenerated fibers were obtained using a field emission scanning electron microscope (S-4800, Hitachi Ltd., Tokyo, Japan) operated at an accelerating voltage of 1.0 kV after the fiber samples had been coated with platinum for 30 s using ion sputtering (E-1030, Hitachi Ltd., Tokyo, Japan). + +3.7. Wide-Angle X-ray Diffraction (WAXD). Twodimensional WAXD (2D-WAXD) patterns of the regenerated fibers were obtained using a MicroMax-007HF system (Rigaku Co., Tokyo, Japan) operated at 40 kV and 30 mA with Cu Kα radiation (λ = 0.15418 nm) in transmission geometry mode and in a high vacuum. The X-ray diffraction pattern was recorded on an imaging plate (BAS-SR 127, 2540 × 2540 pixels, 50 × 50 mm2/pixel; Fujifilm Co., Tokyo, Japan) and read with a RAXIA-Di system (Rigaku Co., Tokyo, Japan). The sample-to-detector distance was set at approximately 83 mm. The sample holder was maintained at room temperature, and the measurements were also obtained at room temperature. 2D-WAXD pattern analysis and conversion to onedimensional (1D) profiles were performed using 2DP software +(Rigaku Co. Tokyo, Japan). The degree of crystallinity (Xc) +values of the fibers were determined from the corrected and normalized 1D-WAXD curves. The degree of crystalline chain orientation (fc) was determined by an azimuthal scan of the meridional main interference taken from well-aligned fiber samples in the longitudinal direction. It is defined by (180° − +WH)/180°, where WH represents the half width of the orientation peak from the intensity azimuthal profile. + +3.8. Tensile Tests. The linear density (titer) of the regenerated fibers was determined by a precise gravimetric measurement of the fibers which were cut into 30 mm lengths. + +Tensile tests were performed on the cut fibers using an EZ test instrument (Shimadzu Co., Kyoto, Japan) at a tensile speed of ACS Omega http://pubs.acs.org/journal/acsodf Article 1 mm/min at room temperature. The initial gauge length was set to 10 mm. The tensile test was conducted five times for each sample, and the tensile strength was taken as the average. + +## 4. Conclusions + +The mechanical properties of regenerated α-1,3-glucan fibers were successfully improved by the solvent substitution and the stretching in water. The anhydrous crystalline form of regenerated α-1,3-glucan fibers coagulated in EtOH was transformed to the hydrated form by solvent substitution with water. During this hydration process, the fibers were stretched to 1.5 times the initial length, and the hydrated form was retransformed into the anhydrous form by drying at 105 +°C. The high-oriented regenerated α-1,3-glucan fiber was obtained by these post-treatments, and the fiber had higher tensile strength (18 cN/tex) than that of the regenerated α1,3-glucan fiber without any post-treatment (11 cN/tex). The enhanced tensile strength was comparable to that of viscose rayon. Moreover, distinctive 2D-WAXD patterns of α-1,3glucan polymorph II (the hydrated form) and polymorph III +(the anhydrous form) were first obtained. The 2D-WAXD +pattern of polymorph III was similar to that of another anhydrous form (polymorph IV). This similarity suggests that polymorph III and polymorph IV might have the same anhydrous unit cell. Therefore, this research is meaningful not only in material science but also in crystallography associated with α-1,3-glucan. + +- ASSOCIATED CONTENT + +## *Sı Supporting Information + +The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c02365. + +d-spacings of the regenerated fibers (PDF) +- AUTHOR INFORMATION + +## Corresponding Author + +Tadahisa Iwata − Science of Polymeric Materials, Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; orcid.org/0000-0003-2731-3958; Phone: +81-3-5841-5266; Email: atiwata@g.ecc.utokyo.ac.jp; Fax: +81-3-5841-1304 Authors Azusa Togo − Science of Polymeric Materials, Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Shiori Suzuki − Science of Polymeric Materials, Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Satoshi Kimura − Science of Polymeric Materials, Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; Technology Advancement Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; orcid.org/0000-0002-6383-1923 Complete contact information is available at: https://pubs.acs.org/10.1021/acsomega.1c02365 Notes The authors declare no competing financial interest. + +- ACKNOWLEDGMENTS +This work was supported by the Japan Society for the Promotion of Science (JSPS) DC2 Research Program (grant number 20J11594). + +- REFERENCES +(1) Aizenshtein, E. M. World Production of Textile Raw Materials in 2002. Fibre Chem. 2004, 36 (1), 1−6. + +(2) Shen, L.; Worrell, E.; Patel, M. K. Environmental impact assessment of man-made cellulose fibres. Resour. Conserv. and Recycl. + +2010, 55 (2), 260−274. + +(3) Yu, Y.; Shen, M.; Song, Q.; Xie, J. Biological activities and pharmaceutical applications of polysaccharide from natural resources: +A review. Carbohydr. Polym. 2018, 183, 91−101. + +(4) Dupuis, J. H.; Liu, Q. Potato Starch: a Review of Physicochemical, Functional and Nutritional Properties. Am. J. Potato Res. 2019, 96 (2), 127−138. + +(5) Iwata, T. Biodegradable and Bio-Based Polymers: Future Prospects of Eco-Friendly Plastics. Angew. Chem., Int. Ed. 2015, 54 +(11), 3210−3215. + +(6) Iwata, T.; Gan, H.; Togo, A.; Fukata, Y. Recent developments in microbial polyester fiber and polysaccharide ester derivative research. + +Polym. J. 2021, 53, 221 +(7) Clarke, A. E.; Stone, B. A. Structure of the paramylon from Euglena gracilis. Biochim. Biophys. Acta 1960, 44, 161−163. + +(8) Puanglek, S.; Kimura, S.; Enomoto-Rogers, Y.; Kabe, T.; +Yoshida, M.; Wada, M.; Iwata, T. In vitro synthesis of linear alpha-1,3glucan and chemical modification to ester derivatives exhibiting outstanding thermal properties. Sci. Rep. 2016, 6, 30479. + +(9) Kobayashi, K.; Hasegawa, T.; Kusumi, R.; Kimura, S.; Yoshida, M.; Sugiyama, J.; Wada, M. Characterization of crystalline linear (1→ +3)-α-d-glucan synthesized in vitro. Carbohydr. Polym. 2017, 177, 341−346. + +(10) Gan, H.; Kabe, T.; Iwata, T. Manufacture, Characterization, and Structure Analysis of Melt-Spun Fibers Derived from Paramylon Esters. J. of Fiber Sci. and Technol. 2020, 76 (5), 151−160. + +(11) Togo, A.; Enomoto, Y.; Takemura, A.; Iwata, T. Synthesis and characterization of dextran ester derivatives and their adhesive properties. J. Wood Sci. 2019, 65 (1), 66. + +(12) Togo, A.; Uechi, K.; Mizutani, O.; Kimura, S.; Iwata, T. + +Synthesis and characterization of α-1,3-alt-α-1,4-glucan (nigeran) +ester derivatives. Polymer 2021, 214, 123343. + +(13) Buchanan, C. M.; Gardner, R. M.; Komarek, R. J. Aerobic biodegradation of cellulose acetate. J. Appl. Polym. Sci. 1993, 47 (10), +1709−1719. + +(14) Park, C. H.; Kang, Y. K.; Im, S. S. Biodegradability of cellulose fabrics. J. Appl. Polym. Sci. 2004, 94 (1), 248−253. + +(15) Sayyed, A. J.; Deshmukh, N. A.; Pinjari, D. V. A critical review of manufacturing processes used in regenerated cellulosic fibres: viscose, cellulose acetate, cuprammonium, LiCl/DMAc, ionic liquids, and NMMO based lyocell. Cellulose 2019, 26 (5), 2913−2940. + +(16) SCHMIDT, R. J.; CHUNG, L. Y.; ANDREWS, A. M.; +SPYRATOU, O.; TURNER, T. D. Biocompatibility of Wound Management Products: A Study of the Effects of Various Polysaccharides on Murine L929 Fibroblast Proliferation and Macrophage Respiratory Burst. J. Pharm. Pharmacol. 2011, 45 (6), +508−513. + +(17) Isogai, A.; Atalla, R. H. Dissolution of Cellulose in Aqueous NaOH Solutions. Cellulose 1998, 5 (4), 309−319. + +(18) Suzuki, S.; Togo, A.; Gan, H.; Kimura, S.; Iwata, T. Air-Jet WetSpinning of Curdlan Using Ionic Liquid. ACS Sustainable Chem. Eng. + +2021, 9 (11), 4247−4255. + +(19) Röber, T.; Kaindl, G.; Redlinger, S.; Firgo, H.; Kroner, G. + +POLYSACCHARIDE FIBERS AND METHOD FOR PRODUCING SAME. Nov. 29, 2018. + +(20) Kraft, G.; Kroner, G.; Röder, T.; Firgo, H. POLYSACCHARIDE FIBERS AND METHOD FOR PRODUCING SAME. Dec. 29, 2020. + +(21) Togo, A.; Suzuki, S.; Kimura, S.; Iwata, T. Wet Spinning and Structure Analysis of α-1,3-Glucan Regenerated Fibers. ACS Appl. + +Polym. Mater. 2021, 3 (4), 2063−2069. + +(22) Sixta, H.; Michud, A.; Hauru, L.; Asaadi, S.; Ma, Y.; King, W. T. + +A.; Kilpeläinen, I.; Hummel, M. Ioncell-F: A High-strength regenerated cellulose fibre. Nord. Pulp Pap. Res. J. 2015, 30 (1), +43−57. + +(23) Jelsma, J.; Kreger, D. R. Polymorphism in crystalline (1→3)-αd-glucan from fungal cell-walls. Carbohydr. Res. 1979, 71 (1), 51−64. + +(24) Ogawa, K.; Okamura, K.; Sarko, A. Molecular and crystal structure of the regenerated form of (1→3)-α-d-glucan. Int. J. Biol. + +Macromol. 1981, 3 (1), 31−36. + +(25) Ogawa, K.; Misaki, A.; Oka, S.; Okamura, K. X-Ray diffraction data for (1→3)-α-d-glucan. Carbohydr. Res. 1979, 75, C13−C16. + +(26) Fink, H. P.; Weigel, P.; Purz, H. J.; Ganster, J. Structure formation of regenerated cellulose materials from NMMO-solutions. + +Prog. Polym. Sci. 2001, 26 (9), 1473−1524. \ No newline at end of file diff --git a/medical/md/PMC8444960.md b/medical/md/PMC8444960.md new file mode 100644 index 0000000000000000000000000000000000000000..3dd6cdcc1a2387c1ec529cd584e04b3215822df0 --- /dev/null +++ b/medical/md/PMC8444960.md @@ -0,0 +1,167 @@ +DOI: 10.1002/agm2.12174 ORIGINAL ARTICLE +Influence of oral nutritional agents rich in soluble dietary fiber on intestinal flora of elderly men with malnutrition Dongni Yu1 | Mingyue Cheng2 | Lixin Guo1 | Wenli Liu3 | Ye Liu1 | Kang **Ning2** | Yigang Tong3 | Xuejiao Yan1 | Lei Qiu1 | Haimei Qi1 1Department of Endocrinology/Department of Health Care, The Key Laboratory of Geriatrics, Beijing Institution of Geriatrics, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China 2Key Laboratory of Molecular Biophysics of the Ministry of Education, Hubei Key Laboratory of Bioinformatics and Molecularimaging, Center of AI Biology, Department of Bioinformatics and Systems Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China 3Beijing University of Chemical Technology, Beijing, China Correspondence Qi Haimei, Department of Health Care, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China. Email: qhm68602@126.com Funding information This study was supported by funds from The Sixth Central Health Research Project (No. W2017BJ10). + +## Abstract + +Objective: Observe the influence of oral nutritional agents rich in soluble dietary +(enteral nutritional suspension [TPF-DM]) on intestinal flora of elderly male subjects with malnutrition. + +Method: Seventy-eight subjects with good nutrition were considered as the healthy control group. Twenty-eight male subjects who had malnutrition and were older than 70 years were included and randomly divided into the short-term (3 months) +intervention group (n = 20) and the long-term (12 months) group (n = 8). They were provided with enteral nutritional suspension (TPF-DM) 500 mL/day or maximum tolerance dose, so as to observe the changes in nutrition-related indexes and intestinal flora after the elderly take enteral nutritional suspension (TPF-DM). Results: (1) For elderly male subjects with malnutrition, their body weight, body mass index, hemoglobin, total protein, and albumin were significantly lower than the control group with favorable nutrition. (2) There were obvious differences in intestinal flora between healthy elderly male subjects and those with malnutrition. After the treatment of enteral nutritional suspension (TPF-DM), intestinal flora of the malnourished elderly subjects showed recovery toward the healthy elderly subjects. The obvious gradient changes of the flora were mainly in the bacteroidetes, firmicutes, and proteobacteria phyla, and the relative abundance of CAG2 clusters in the malnourished group was higher than that in the healthy control group, and the relative abundance decreased after long-term treatment, and the change approached the healthy control group. The relative abundance of CAG3 and CAG6 clusters in the malnourished group was lower than that in the healthy control group, and the relative abundance increased after long-term treatment, and the change approached the healthy control group. + +Conclusion: Malnutrition has obvious impact on intestinal flora of the elderly. Enteral nutritional suspension (TPF-DM) not only prevents the further decline in the state of nutrition but also helps the recovery in intestinal flora of the elderly. Long-term application can produce better effects. + +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. © 2021 The Authors. *Aging Medicine* published by Beijing Hospital and John Wiley & Sons Australia, Ltd. + +## 1 | **Introduction** 2.3 | **Criteria Of Exclusion** 2.4 | **Methods Of Research** 2.4.1 | Subjects Of The Control Group With Favorable Nutrition Status 2 | **Research Method** 2.1 | **Objects Of Research** 2.4.2 | Experimental Group With Malnutrition 2.2 | **Criteria Of Inclusion** 2.4.3 | Stool Sample Retain, Disposal And Intestinal Flora 16S Rdna Sequencing + +2. The experimental group with malnutrition: more than 70 years old. Prebiotics/probiotics not taken in the recent 3 months. Favorable nutritional state (MNA-SF score 0 to 7 points). + +Since the end of the last century, our country has become an aging society. Due to health and economic reasons, such as illness, solitude, and declining income, elderly patients are more likely to suffer malnutrition. Nutrition and energy are essential to maintain vitality and organ functions. Malnutrition can severely damage their health and organ functions. It is not conducive to the prognosis of illness and the lifespan of the patients. Modern nutritional therapies are more than just meeting the need of elderly patients for energy. They are also concerned with regulation of blood glucose, protection of intestinal tract barriers, and adjusting intestinal microecology, etc.1 Intestinal microorganism plays an important role in functions like promoting epithelium growth, intestinal tract barrier, immunity stimulation, anti-infection, steady metabolism, and digestive absorption, etc.2 At present, multiple studies and research have proven that intestinal microecology of elderly patients is closely related to metabolism, immunity, sarcopenia, asthenia, and cognitive functions, etc.3,4 As intestinal microecology is connected to old age, nutrition, Subjects who comply with one of the following conditions cannot be included. Contraindications of enteral nutrition treatment: intestinal obstruction, active bleeding of the digestive tract; severe nausea and emesis that cannot be controlled through medication; malabsorption caused by chronic gastrointestinal diseases; congestive heart failure and respiratory failure; severe infection; patients who have used antibiotics within 2 weeks; terminal stages of chronic diseases, chronic kidney disease (CKD) 5 phase, estimated glomerular filtration rate (eGFR) <15 mL/ +min/1.73 m2; chronic liver disease, level C liver function; malignant tumor; severe endocrine diseases, including thyroid dysfunction, but those with normal thyroid functions who also take medicine to cure thyroid gland malfunctions were not included; uncontrolled diabetes, etc. + +and other diseases, intestinal flora regulation may become the new target of nutritional intervention for the elderly. However, the influence of nutrition support on intestinal flora of the elderly who suffer malnutrition. Hence, this study aims to observe the influence of oral nutritional agents rich in soluble dietary fiber on intestinal microecology of elderly male subjects with malnutrition. It is expected to provide more evidence of intestinal nutrition support for the elderly. + +The patient's fasting blood was drawn to test blood routine and biochemical indicators. Personal information and medical history were gathered through questionnaire surveys. Stool samples were retained as well. + +One hundred six subjects, who received treatments in the endocrinology and geriatrics departments of our hospital from October 2017 to June 2020, comply with the inclusion and exclusion criteria. After being approved by the ethics committee, the number of the study was 2017BJYYEC-118-02. It was also registered at the Chinese Clinical Trial Registration Center. The registration number was ChiCTR-OPB-17012750. All subjects took part in the study voluntarily and signed the informed consent form. + +Subjects were given nutritional agents (enteral nutritional suspension [TPF-DM]; Nutricia Corp.) with soluble dietary fiber for nutrition support therapy. They were randomly divided into short-term intervention (3 months) and long-term intervention (12 months) and compared with the changes in nutrition-related blood indexes (blood routine examination and biochemistry) and intestinal flora before and after the use of enteral nutritional suspension (TPF-DM). + +1. The control group with favorable nutritional status: less than 70 years old. Prebiotics/probiotics was not taken in recent 3 months. Favorable nutritional state (Mini-Nutritional Assessment Short-Form [MNA-SF] score 12 to 14 points). + +Approximately 10 g of stool sample with no urine, sanitizer, or sewage, was collected and was placed in the fridge of −80℃. QIAamp Fast DNA Stool Mini Kit (50) was used to extract the total DNA of elderly male subjects, intestinal flora, malnutrition, oral nutritional agents + +## 164  |    Al. + +| TABLE 1 Subjects' baseline in some nutrition indicators Well-nourished <70 Well-nourished ≥70 | Malnourished ≥70 | | | | | +|-------------------------------------------------------------------------------------------------|--------------------|----------------|----------------|--------|-------| +| (n = 41) | (n = 37) | | | | | +| Weight (kg) | 76.89 ± 9.38 | 69.43 ± 11.08 | 64.33 ± 10.52 | 11.316 | 0.000 | +| BMI (kg/m2 ) | 26.52 ± 2.38 | 25.05 ± 3.16 | 22.72 ± 3.43 | 11.382 | 0.000 | +| Hemoglobin (g/L) | 147.64 ± 9.97 | 130.67 ± 17.39 | 121.29 ± 13.58 | 33.529 | 0.000 | +| Lymphocyte (×109 /L) | 2.16 ± 1.71 | 1.88 ± 0.59 | 2.2 ± 3.17 | 0.310 | 0.734 | +| Total protein (g/L) | 72.32 ± 3.11 | 66.86 ± 5.74 | 65.88 ± 6.93 | 16.970 | 0.000 | +| Albumin (g/L) | 45.95 ± 2.61 | 40.11 ± 7.25 | 40.53 ± 7.54 | 12.150 | 0.000 | +| 25(OH)D (ng/mL) | 24.92 ± 11.96 | 21 ± 10.26 | 19.51 ± 8.16 | 2.607 | 0.078 | +| Serum iron (µmol/L) | 21.12 ± 7.64 | 16.11 ± 5.97 | 19.48 ± 39.48 | 0.551 | 0.578 | + +the fecal microbiota. The V3-V4 region of 16S rDNA were amplified with primers of was F_5′-CCTACGGGNGGCWGCAG-3′ and + +| Short-term intervention | Long-term intervention | | | | | | +|---------------------------|--------------------------|----------------|----------|----------------|----------------|-------| +| (n = 20) | (n = 8) | | | | | | +| Baseline | Terminal value | P value | Baseline | Terminal value | P value | | +| Weight (kg) | 69.00 ± 8.85 | 66.08 ± 9.21 | 0.014 | 66.57 ± 7.77 | 66.77 ± 9.03 | 0.867 | +| BMI (kg/m2 ) | 24.2 ± 2.68 | 23.22 ± 3.10 | 0.012 | 23.57 ± 2.37 | 23.42 ± 2.72 | 0.731 | +| Hemoglobin (g/L) | 129.38 ± 9.77 | 121.88 ± 12.26 | 0.037 | 118.05 ± 13.72 | 124.15 ± 16.05 | 0.109 | +| Lymphocyte (×109 /L) | 1.54 ± 0.33 | 1.48 ± 0.48 | 0.434 | 2.47 ± 3.74 | 1.75 ± 1.16 | 0.430 | +| Total protein (g/L) | 68.64 ± 2.96 | 66.01 ± 4.79 | 0.182 | 64.66 ± 8.04 | 66.94 ± 9.37 | 0.436 | +| Albumin (g/L) | 43.5 ± 2.19 | 39.53 ± 3.05 | 0.004 | 39.34 ± 8.59 | 38.02 ± 4.77 | 0.375 | +| 25(OH)D (ng/mL) | 20.97 ± 10.63 | 15.18 ± 6.22 | 0.082 | 18.56 ± 7.17 | 20.57 ± 5.28 | 0.143 | +| Serum iron (µmol/L) | 17.22 ± 4.74 | 10.47 ± 2.62 | 0.106 | 10.09 ± 4.73 | 10.35 ± 4.10 | 0.380 | + +R_5′-GGACTACHVGGGTATCTAATCC-3′. The 16S rDNA library of 600 bp was sequenced on the Miseq sequencing platform. + +## 2.4.4 | Bioinformatic Methods + +QIIME 2 (version 2020.11.0) was used for quality control of the sequencing data, including sequencing separation (was "qiime cutadapt demux-paired" was parameter: -p-error-rate 0), eliminating primer sequence ("qiime cutadapt trim-paired" parameter: --p-match-adapter-wildcards --p-match-read-wildcards +--p-discard-untrimmed), denoising ("qiime dada2 denoise-paired," was parameter:-p-trunc-len-f 270 --p-trunc-len-r 228 --p-n-threads 20 --p-min-fold-parent-over-abundance 4), and driving the feature sequences ("qiime feature-table rarefy" parameter:--p-sampling-depth 4000). A total of about 4000 sequences was obtained for each sample; Amplicon Sequence Variants (ASVs) feature table was obtained; and was finally annotated by comparing it to the Silva database. The α diversity was estimated by observed ASVs, Chao1 index, Shannon index, and Simpson index; the β diversity of principle coordinates analysis (PCoA) was analyzed based on was unweighted UniFrac distance. + +Analysis of the co-abundance groups (CAG) was performed as follows: (1) calculating the Pearson correlation coefficients based on ASVs relative abundance; and (2) hierarchical clustering was based on the Ward-linkage method and the Euclidean distance. Pearson correlation coefficients were calculated based on category levels. The threshold value of correlation coefficient was 0.4. This experiment conducts significantly different analysis among levels, categories, and classification grade of ASVs. For the Pearson correlation, an P value < 0.05 was considered statistically significant after the adjustment with the Benjamini-Hochberg method. + +## 2.4.5 | Statistical Methods + +Continuous variables were described as mean value with standard deviation, or median value with interquartile range (IQR). Categorial variables were descript by the frequency and percentage. For categorical variables, the Chi-square or the Fisher exact test were carried out to test the group differences; for continuous variables, variance, or analysis of covariance, or nonparametric methods were used. All the tests were two-tailed and a P value < 0.05 was considered statistically significant. + +## 3 | **Research Results** 3.1 | **The Baseline Of Subjects** + +The average age of the 116 male subjects who were involved in this experiment was 75.7 ± 14.9 years. They were divided into three groups by nutritional status and age - group 1 had 41 people who were well-nourished and under 70 years old, group 2 had 37 cases who were well-nourished and above or equal to 70 years old, and group 3 were 28 people who were malnourished and above or equal to 70 years old. The comparing baseline was the relevant indicators of nutrition (see Table 1). This research finds that the subjects in group 3 have significant poorer performance than the other two groups in weight, body mass index (BMI), total protein, and albumin. The difference among the three groups was statistically significant +(P < 0.05). + +## 3.2 | **Effect Of Tpf-Dm On Relevant Indicators Of The** Malnourished Elderly + +The subjects in group 3 were treated with TPF-DM from small amounts to 500 mL per day or the maximum dose (<500 mL). Twenty + +![3_image_0.png](3_image_0.png) + +people were short-term (3 months) subjects of TPF-DM treatment, and eight people were long-term (12 months) subjects. Then, some nutrition indicators of these patients before and after treatment were compared. The results are shown in Table 2. + +The results demonstrate that, compared with the baseline, the indicators like weight, BMI, hemoglobin, and albumin still decrease after short-term TPF-DM intervention. The difference between groups was statistically significant (P < 0.05). However, there was no significant difference in subjects treated long term. + +## 3.3 | **Effect Of Malnutrition On Intestinal Flora In** Elderly Men + +PCoA analysis (Figure 1A) shows that the first and second principal components contributed to 15.45% and 8.2% of the variations, respectively, between the malnourished and control subjects. The performance between the two groups was significantly different. The indicators of the malnourished elderly (group 3) after treatment approached the control group (group 2), and the intestinal flora of some healthy elderly men also reaches those dystrophic aged people. At the level of ASVs, the heatmap (Figure 1B) shows that the dystrophic and healthy groups can be, to a large extent, clustered. After the treatment of TPF-DM, the malnourished people's flora becomes more similar to the healthy ones, and the phyla had a gradient change among the groups. There were significant differences in bacteroidetes, firmicutes, and proteobacteria phyla between healthy control group, malnutrition group, short-term treatment, and longterm treatment groups (P < 0.05; Table S1). + +## 3.4 | **Changes Of Intestinal Microflora In** Malnourished Old Man After Tpf-Dm Treatment + +Figure 2 shows the changes of gut microbiota at the genus level before and after TPF-DM treatment in malnourished old men. Similar with the results obtained at ASVs, the genus of microbiota was clearly clustered for the malnutrition and healthy subjects, respectively. By the horizontal clustering, the genera were divided into six clusters, namely, CAG1, CAG2, CAG3, CAG4, CAG5, and CAG6. Bacteroidota and firmicutes were the two mostly affected phyla; among the six CAGs, CAG2 was the largest group containing 15 genera and six families (Table S2). Microbial network + +166  |    al. + +![4_image_0.png](4_image_0.png) + +FIGURE 2 Changes of intestinal microflora in malnourished old man after enteral nutritional suspension TPF-DM treatment [Colour figure can be viewed at wileyonlinelibrary.com] + +## 4 | **Discussion** + +In the important part of the human body, intestinal microecology, +   | al.  **167** +lifestyle, diet structure, antibiotics, nonsteroidal anti-inflammatory drugs, and other drugs, pathogenic bacteria infection, etc., can change the intestinal microecology,7 and reduce intestinal microbial diversity. With the decrease of intestinal probiotics, conditional pathogenic bacteria increased and intestinal probiotic metabolite short-chain fatty acids (SCFAs) decreased, also. In addition, the changes in intestinal microecology are closely related to diabetes, obesity, cardiovascular diseases, autoimmune diseases, Alzheimer's disease, etc.8,9 How to improve the intestinal microecology of the elderly, especially the malnourished, deserves the attention of medical staff. + +This study found that the nutrition-related indicators of the malnourished elderly, including body weight, BMI, hemoglobin, albumin, and flora structure, are significantly different from those of the healthy subjects, and the flora of some elderly subjects over 70 years also began to develop toward the direction of malnutrition. Therefore, this study suggests that nutritional status is an important factor affecting intestinal flora in the elderly. Due to the loss of appetite, chewing, and digestion ability, the elderly, especially the senile, show a significant decrease in the intake of protein and dietary fiber.10 Both nutritional deficiency and decreased intake of specific nutrients can lead to changes in the composition and function of intestinal flora in the elderly, including the decrease of microbial diversity, butyric acid-producing bacteria, and SCFA level of intestinal flora, whereas harmful substances, such as lactic acid, methane, and branched-chain fatty acid increased significantly.11 Through nutrition intervention on the malnourished elderly, the results showed that, compared with the baseline, the nutritionrelated clinical indexes of the elderly, including body weight, BMI, hemoglobin, and albumin, still decreased after short-term TPF-DM intervention. However, there was no obvious change in nutritionrelated indexes after long-term TPF-DM intervention. TPF-DM is rich in nutrients, including protein, fat, carbohydrate, dietary fiber, vitamins, minerals, and trace elements. However, due to the low energy density (0.75 kcal/mL), TPF-DM was mostly used in patients with diabetes in previous studies. Some research results have found that, after 3 months of treatment of TPF-DM, the albumin, blood sugar, and blood lipid metabolism of patients with diabetes over the age of 60 years with malnutrition or high risk of malnutrition (MNA-SF score of approximately 0 to 11) can be effectively improved. The reason for the inconsistent results is considered to be related to the worse nutritional status of the subjects of all elderly subjects over the age of 70 years in this study (MNA-SF score was approximately 0 to 7). We had only observed changes in the flora, which may be due to the shorter intervention time in this study, and it may take longer to see clinical changes. + +In addition, this study also found that after short-term treatment of TPF-DM, the intestinal flora of malnourished elderly men shows a recovery tendency to the state of healthy elderly people over the age of 70 years. After long-term treatment, the intestinal flora may become healthier, mainly manifested in obvious flora gradient changes in phyla B, F, and P, and abundance changes in CAG2/3/6 clusters. This study suggests that the mechanism of intestinal flora improvement may be related to the rich dietary fiber and monounsaturated fatty acids of TPF-DM. Different from other nutritional preparations, TPF-DM features a high content of monounsaturated fatty acids (70% of total fat) and rich dietary fiber. As the nutritional preparation with the highest soluble fiber content today, TPF-DM contains three soluble dietary fibers and three insoluble dietary fibers, and the content of the former one is as high as 80%. Due to failing to be decomposed by human enzymes, dietary fibers can strengthen the barrier of the intestinal tract against pathogens, accelerate the intestinal movement, and reduce potential inflammatory state, thus improving the health state.13 In addition, soluble dietary fiber is an indigestible carbohydrate rich in polysaccharides and oligosaccharides, which can be fermented by one or a few intestinal florae in the intestinal tract to produce nutrient substrates (mainly SCFAs) of probiotics, thus stimulating the growth and activity of probiotics.14 Meanwhile, monounsaturated fatty acids can improve the biodiversity of intestinal microorganisms, increase the content of bacteroides and bifido bacteria, and decrease the content of sclerenchyma. The increase of probiotics is related to the increase of intestinal SCFA by mono-unsaturated fatty acids.15 To sum up, this study suggests that attention should be paid to the assessment and nutritional support of nutritional status and intestinal flora of the elderly, especially the senile. Nutritional supplements for the malnourished senile elderly using TPF-DM can not only prevent the declined nutritional status, but improve the intestinal flora. Moreover, the long-term use effect is better. However, the shortcomings of this study lie in the small cases as well as the single-center study. There still needs to be a larger sample size and a longer time for further verification in the later period. + +## Conflicts Of Interest Nothing To Disclose. Author Contributions + +Y.D.N. and Q.H.M. supervised the project and designed the workflow and performed the statistical analysis. C.M.Y., G.L.X., L.W.L., L.Y., N.K., T.Y.G., Y.X.J., and Q.L. performed material preparation and data collection. Yu wrote the first draft. C.M.Y. prepared the figures and tables. All authors commented on the manuscript. All authors read and approved the final manuscript. + +## Orcid + +Dongni Yu https://orcid.org/0000-0002-5472-5715 + +## References + +1. Corrigendum. *JPEN J Parenter Enteral Nutr*. 2016;40(8):1200. + +2. Falony G, Joossens M, Vieira-Silva S, et al. Population-level analysis of gut microbiome variation. *Science*. 2016;352(6285):560-564. + +3. Jeffery IB, Lynch DB, O'Toole PW. Composition and temporal stability of the gut microbiota in older persons. *ISME J*. + +2016;10(1):170-182. + +4. Collino S, Montoliu I, Martin FP, et al. Metabolic signatures of extreme longevity in northern Italian centenarians reveal a complex +168  |    al. + +remodeling of lipids, amino acids, and gut microbiota metabolism. PLoS One. 2013;8(3):e56564. + +5. Lynch SV, Pedersen O. The human intestinal microbiome in health and disease. *N Engl J Med*. 2016;375(24):2369-2379. + +6. Yatsunenko T, Rey FE, Manary MJ, et al. Human gut microbiome viewed across age and geography. *Nature*. 2012;486(7402):222-227. + +7. Marchesi JR, Adams DH, Fava F, et al. The gut microbiota and host health: a new clinical frontier. Gut. 2016;65(2):330-339. + +8. Salgaco MK, Oliveira LGS, Costa GN, Bianchi F, Sivieri K. Relationship between gut microbiota, probiotics, and type 2 diabetes mellitus. Appl Microbiol Biotechnol. 2019;103(23–24):9229-9238. + +9. Pluta R, Ulamek-Koziol M, Januszewski S, Czuczwar SJ. Gut microbiota and pro/prebiotics in Alzheimer's disease. *Aging*. 2020;12(6):5539-5550. + +10. Corish CA, Bardon LA. Malnutrition in older adults: screening and determinants. *Proc Nutr Soc*. 2019;78(3):372-379. + +11. Salazar N, Valdes-Varela L, Gonzalez S, Gueimonde M, de Los Reyes-Gavilan CG. Nutrition and the gut microbiome in the elderly. Gut Microbes. 2017;8(2):82-97. + +12. Li YX, Pan SH, Zeng JB, et al. The effect of enteral nutritional suspension (diabetes) (TPF-DM) on blood glucose, serum insulin and lipids in patients with type 2 diabetes. *Zhonghua Nei Ke Za Zhi*. 2007;46(3):193-196. + +13. Barber TM, Kabisch S, Pfeiffer AFH, Weickert MO. The health benefits of dietary fibre. *Nutrients*. 2020;12(10):3209. + +14. Danneskiold-Samsoe NB, de Freitas D, Queiroz Barros H, et al. + +Interplay between food and gut microbiota in health and disease. Food Res Int. 2019;115:23-31. + +15. Machate DJ, Figueiredo PS, Marcelino G, et al. Fatty acid diets: regulation of gut microbiota composition and obesity and its related metabolic dysbiosis. *Int J Mol Sci*. 2020;21(11):4093. + +## Supporting Information + +Additional supporting information may be found online in the Supporting Information section. + +How to cite this article: Yu D, Cheng M, Guo L, et al. Influence of oral nutritional agents rich in soluble dietary fiber on intestinal flora of elderly men with malnutrition. *Aging Med*. 2021;4:162–168. https://doi.org/10.1002/agm2.12174 \ No newline at end of file diff --git a/medical/md/PMC8558693.md b/medical/md/PMC8558693.md new file mode 100644 index 0000000000000000000000000000000000000000..685aa78e2f820411258cb50c8e74f65b0c24a6d0 --- /dev/null +++ b/medical/md/PMC8558693.md @@ -0,0 +1,130 @@ + + +![0_image_0.png](0_image_0.png) + +# Experimental Laboratory Modeling Of Choroidal Vasculature: A Study Of The Dynamics Of Intraoperative Choroidal Hemorrhage During Pars Plana Vitrectomy + + Mahmut Doğramacı*, Funda Dikkaya**, Fevzi Şentürk**, Cengiz Aras** +*Princess Alexandra Hospital, Clinic of Ophthalmology, Essex, United Kingdom **Medipol University Faculty of Medicine, Department of Ophthalmology, İstanbul, Turkey + +## Abstract + +Objectives: Choroidal hemorrhages (CH) result from rupture of choroidal vessels leading to extravasation of blood into the suprachoroidal space. In this study, we aimed to understand the hemodynamics of CH by developing a purpose-built scale model of the choroidal vasculature and calculating stress levels in the model under different conditions. Materials and Methods: We modeled the choroidal vasculature using a rubber tube 10 cm in length and 1 cm in diameter that was wrapped with conductive thread to enable the measurement of stress at the walls of the tube. Stress levels across the tube were continuously measured under different systemic intravascular blood pressure levels (IVP), intraocular pressure (IOP) levels, and distortion. Results: Stress values across the choroidal vessel model correlated negatively with IOP and positively with IVP and distortion. All correlations were statistically significant (p<0.05) and were stronger when the model was filled with expansile tamponade compared to non-expansile tamponades. Distortion showed the strongest correlation in terms of increasing stress across the model, while IVP showed stronger correlation compared to IOP. Raising IOP to counteract the stress in the model was effective when the stress in the model was secondary to increased IVP, but this approach was not effective when the stress in the model was caused by distortion. Conclusion: Excessive distortion of the globe during surgical maneuvers could be the primary reason for the rarely observed intraoperative CH. Non-expansile ocular tamponade provides better support for the vascular bed against CH and should be the recommended choice of tamponade in patients with existing CH. Increasing IOP excessively is of limited effect in preventing CH in vessels that are under stress as a result of distorting surgical maneuvers. Keywords: Choroid hemorrhage, vitrectomy, choroid, suprachoroidal space + +## Introduction + +Choroidal hemorrhages (CH) are relatively uncommon but can lead to devastating outcomes. They result from rupture of arterioles, venules, and capillaries which initially lead to extravasation of blood into the choroid and subsequently into the suprachoroidal space.1 Possible mechanisms for vascular ruptures are believed to be drop in intraocular pressure (IOP), increase in systemic blood pressure, distortion of the vascular architecture, and direct injury to the blood vessels. However, these suppositions have not been supported by experimental data. Therefore, research to understand the hemodynamics of CH is important to enable surgeons to take effective measures to reduce the risks. Surgeons are generally advised to avoid hypotony during surgery, refrain from operating on patients with high systemic blood pressure, and minimize the use of expansile tamponades in high-risk patients. However, the role of avoiding inadvertent intraoperative distortion of ocular tissues in prevention of CH is generally underemphasized. Nevertheless, mathematical models showed that globe distortion from mechanical strain plays a more important Address for Correspondence: Funda Dikkaya, Medipol University Faculty of Medicine, Department of Ophthalmology, İstanbul, Turkey E-mail: fundadikkaya@hotmail.com **ORCID-ID**: orcid.org/0000-0003-2312-2521 Received: 24.07.2020 **Accepted:** 29.12.2020 Cite this article as: Doğramacı M, Dikkaya F, Şentürk F, Aras C. Experimental Laboratory Modeling of Choroidal Vasculature: A Study of the Dynamics of Intraoperative Choroidal Hemorrhage during Pars Plana Vitrectomy. Turk J Ophthalmol 2021;51:294-300 role in causing CH compared to hypotony or increased systemic blood pressure.2 Therefore, in this study we aimed to understand the hemodynamics of CH by creating a purposebuilt scale model of the choroidal vasculature and calculating stress levels in the model under different conditions. + +## Materials And Methods + +The choroidal vasculature was modeled using a rubber tube 10 cm in length and 1 cm in diameter wrapped with conductive thread to enable the measurement of stress at the walls of the tube. The tube was filled with clear water and pressurized to various levels using an electric pump to simulate systemic intravascular blood pressure (IVP) and placed into a sealed container which was also pressurized to various levels to simulate variable IOP. A hydraulic actuator was also used to apply various longitudinal strain on the tube to simulate ocular distortion during surgical maneuvers. The stress levels in the tube were continuously monitored under different IVP, IOP, and distortion levels. IOP and IVP were measured in mmHg and stress was measured in Pascals (Pa), while distortion was measured as percentage of change in the length of the model (e.g., stretching the 10-cm rubber tube to 11 cm correlated to 10% distortion) (Figure 1, 2). + +The experiments were carried out in two phases. Phase 1 consisted of 3 sets of experiments that aimed to observe changes in stress levels in the choroidal vasculature in relation to changes in IOP, systemic blood pressure, and longitudinal strain. Phase 2 consisted of 2 sets of experiments that aimed to observe the benefits of increasing the IOP to counteract high stress levels in the choroidal vasculature secondary to high systemic blood pressure and ocular distortion. + +During phase 1, initially stress measurements were taken while dropping the IOP from 35 mmHg to 0 mmHg while maintaining IVP at 120 mmHg and distortion at 0%. These conditions reflected the effect of hypotony on the choroidal vasculature. Subsequently, stress measurements were taken while increasing IVP from 120 mmHg to 200 mmHg while maintaining IOP at 35 mmHg and distortion at 0%. These conditions reflected the effect of high systemic blood pressure on the choroidal vasculature. Finally, stress measurements were taken while gradually distorting the model up to 12% of its length while maintaining IOP at 35 mmHg and IVP at 120 mmHg. These conditions reflected the effect of peroperative ocular distortion on the choroidal vasculature. + +In phase 2, stress levels in the choroidal vasculature were first increased up to 6 Pa by raising the IVP followed by a gradual increase in IOP from 35 mmHg to 80 mmHg to counteract the stress. These conditions reflected the role of increasing IOP during surgery to prevent CH in patients with high systemic blood pressure. Subsequently, the tests were repeated but this time distortion was used to raise the stress levels in the choroidal vasculature to 6 Pa while the IVP remained at 120 mmHg followed by a gradual increase in IOP from 35 mmHg to 80 mmHg to counteract the stress. These conditions reflected the role of increasing IOP during surgery to prevent CH in eyes subjected to distortion during surgery. + +## Statistical Analysis + +All tests were performed separately while the container was filled with water and with air to reflect eyes filled with non-expansile tamponades and with expansile tamponades, respectively. To enhance accuracy and enable statistical analysis, each test was repeated 10 times. Spearman's correlation was used to assess the statistical significance of the observations. Correlations were considered significant when p value was ≤0.05. Statistical analyses were performed using SPSS software version 18 (SPSS Inc., Chicago, IL, USA) (Table 1). + +## Results + +The first set of experiments in phase 1 showed a statistically significant negative correlation between stress in the choroidal vasculature and IOP while IVP was fixed at 120 mmHg and distortion was 0%. The correlation was stronger when the model was filled with expansile tamponade. Spearman's correlation rho was -0.504 (p<0.001) and stress levels reached 1.7 Pa when the model was filled with expansile tamponade compared to Spearman's rho of -0.190 (p<0.001) and stress levels up to 0.1 Pa when the model was filled with nonexpansile tamponade. The second set of experiments in phase 1 also showed a statistically significant but positive correlation between stress in the choroidal vasculature and IVP while IOP was fixed at 35 mmHg and distortion was 0%. The correlation was stronger when the model was filled with expansile tamponade, as Spearman's correlation rho was -0.771 +(p<0.001) and stress levels reached a maximum of 61 Pa when the model was filled with expansile tamponade compared to Spearman's rho of 0.570 (p<0.001) and stress levels up to 9.9 Pa when the model was filled with non-expansile tamponade. The third set of experiments in phase 1 also showed a statistically significant positive correlation between stress in the choroidal vasculature and the extent of distortion applied on the tubular structure while IOP was fixed at 35 mmHg and IVP was fixed at 120 mmHg. However, the strength of the correlation was almost the same both when the container was filled with expansile tamponade (Spearman's rho 0.624, p<0.001) and with non-expansile tamponade (Spearman's rho 0.629, p<0.001) (Figure 3). + +When the role of increasing IOP in counteracting stress in the choroidal vasculature was tested in phase 2, the first set of experiments showed a statistically significant negative correlation between IOP and stress in the choroidal vasculature when the stress was due to increased IVP. This correlation was marginally stronger when the container was filled with expansile tamponade (Spearman's rho -0.670, p<0.001) compared to non-expansile tamponade (Spearman's rho -0.580, p<0.001). However, the second set of experiments in phase 2 surprisingly showed a statistically significant positive correlation between IOP and stress in the choroidal vasculature when the stress was caused by distortion. This correlation was almost the same both when the container was filled with expansile tamponade (Spearman's rho 0.419, p<0.001) and non-expansile tamponade (Spearman's rho 0.413, p<0.001) (Figure 4). + +## Discussion + +Hemorrhages in highly vascular choroid can arise from ciliary arteries, arterioles, venules, and capillaries. Hypotony3,4,5,6 and high systemic blood pressure7,8 are regarded as common contributing factors in addition to glaucoma7,9, aphakia10, and advanced age.3,9 It is believed that hypotony (e.g., caused by incising the eyeball) induces a vascular pressure gradient that leaves the vascular bed unsupported. Blood pressure can rise in the unsupported vascular bed, leading to a rupture, particularly in the presence of other contributing factors like arteriolar necrosis of the short and long posterior ciliary arteries.7,11 Alternatively, increased IVP (e.g., from obstructing the outflow of the vortex veins) can also increase pressure in the long and short posterior ciliary arteries, leading to rupture.8,12 Nevertheless, pressure in the choroidal vasculature is also susceptible to direct or indirect distortion, such as when the eyeball is inadvertently deformed during surgical maneuvers. Ocular distortion can force the outer and middle ocular coats to slide over each other, as these coats are only separated by a thin layer of fluid and sheets of fine collagen fibrils. Therefore, the vasculature around the suprachoroidal space must be competent enough to withstand not only the transmural pressure gradient but also the relative motion between ocular coats to avoid suprachoroidal hemorrhage.13,14 Several reports in the literature have described experimental rabbit eye models of expulsive CH. In all these models, high IVP was induced by obstructing the venous flow through blocking or ligating the vortex veins or by inducing proptosis using latex bands, while hypotony in these models was induced by incising the globe or removing the corneal button.12,13,14 In contrast to earlier studies, we used a novel in vitro model of the choroidal vasculature, this approach enabled us to precisely calculate stress levels in the choroidal vasculature and study their relation with gradual changes in IOP, IVP, and distortion levels both when the model was filled with non-expansile and expansile tamponades. + +| Table 1. Initial and final values for IOP, IVP, distortion, and stress in each experiment set in phase 1 and phase 2 while the model was filled with expansile and non-expansile tamponade. Note the final stress values are the average of 10 values measured at the end of each experiment Phase Set Aim Tamponade No of IOP (mmHg) IVP (mmHg) Distortion (%) Stress (Pa) tests Initial Final Initial Final Initial Final Initial Final To investigate the effect of low 1 IOP on stress in choroidal vessels Non-expansile 10 35 0 120 120 0 0 0 0.1 Expansile 10 35 0 120 120 0 0 0 1.7 To investigate the effect of high 2 IVP on stress in choroidal vessels Non-expansile 10 35 35 120 200 0 0 0 9.9 One Expansile 10 35 35 120 200 0 0 0 61 To investigate the effect of distortion 3 on stress in choroidal vessels Non-expansile 10 35 35 120 120 0 12 0 23 Expansile 10 35 35 120 120 0 12 0 64 To investigate the role of increasing IOP in counteracting stress in choroidal vessels caused by high IVP Non-expansile 10 35 80 180 180 0 0 6 0.6 1 Expansile 10 35 80 180 180 0 0 6 0.4 Two To investigate the role of increasing IOP in counteracting stress in choroidal vessels caused by distortion Non-expansile 10 35 80 120 120 12 12 6 6.5 2 Expansile 10 35 80 120 120 12 12 6 6 | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +![3_image_0.png](3_image_0.png) + +![3_image_1.png](3_image_1.png) + +Our model suggested that distorting the globe plays a more important role in inducing stress in the choroidal vasculature compared to increased IVP, which in turn has a greater effect than hypotony. Based on this, traumatic CH in patients with closed globe injuries and intraoperative CH during certain surgical maneuvers like indenting and tightening sutures over an external buckle could primarily be explained by distortion of the choroidal vasculature.15,16 It also explains the limited effect of hypotony in experimental induction of CH reported in previous studies.17,18 In fact, the reasons for CH in hypotonic eyes are believed to be indirectly related to distortion of the choroidal vasculature. It has been postulated that hypotony increases the filtration rate by magnifying the transmural pressure differential, resulting in choroidal effusion which in turn stretches and distorts the long and short ciliary blood vessels, leading to their rupture and hemorrhage.6,12,13,19,20,21,22,23,24 The current model also showed that the risk of CH due to high systemic IVP and hypotony is higher when the eye is filled with expansile tamponades. This is most likely due to the extra support for the vascular bed provided by the density of non-expansile tamponades compared to expansile tamponades.7,11 This explains why it is advisable to use nonexpansile tamponades if CH is detected or suspected during surgery.2 The model also revealed that increasing IOP to counteract high stress in the choroidal vasculature is only effective when the stress in the vasculature is generated by high IVP. High IOP levels are not effective in reducing stress in the choroidal vascular if the stress was primarily generated by distortion. This is an important finding, as increasing IOP is the most + +![4_image_0.png](4_image_0.png) + +common strategy used to counteract intraoperative CH, while efforts to minimize surgical distortions are often ignored. + +Study Limitations One of the shortfalls of the study was that the model was a scaled up model of the choroidal vasculature. Although this method provided more precise measurement of the changes in stress levels, the actual stress levels in the choroidal vasculature could be different from those measured in our model. + +## Conclusion + +Inadvertent excessive distortion of the globe during surgical maneuvers could be the primary reason for the rarely observed intra-operative CH. Therefore, care must be exercised to avoid unnecessary distortion of ocular tissues during surgery. Non-expansile ocular tamponade provides better support for the vascular bed against CH and should be the recommended choice of tamponade in patients with existing CH. Hypotony should be avoided during surgery, particularly when the eye is filled with expansile tamponade, and maximum effort should be made to avoid distorting a hypotonic globe filled with expansile tamponade. Finally, raising IOP excessively during intraocular surgery has limited effect in preventing CH that may arise from distortion of the delicate ocular tissues. + +![5_image_0.png](5_image_0.png) + +Figure 4. The results of phase 2 experiments when the model was filled with non-expansile tamponade (left column) and expansile tamponade (right column), showing the role of increasing intraocular pressure in counteracting stress in the choroidal vasculature produced by high systemic intravascular blood pressure (top row) and produced by distortion (bottom row) +Acknowledgments We would like to thank Selin Doğramacı for her help in drawing the diagram. + +Ethics Ethics Committee Approval: Ethics committee approval was not obtained because our study was conducted with an experimental model in the laboratory without including human and animal elements. + +## Informed Consent: + +Peer-review: Externally peer reviewed. Authorship Contributions Concept: M.D., F.Ş., Design: M.D., C.A., Data Collection or Processing: F.D., Analysis or Interpretation: M.D., C.A., Literature Search: M.D., F.Ş., Writing: M.D., F.D. + +Conflict of Interest: No conflict of interest was declared by the authors. + +Financial Disclosure: The authors declared that this study received no financial support. + +## References + +1. Wolter JR. Expulsive hemorrhage: a study of histopathological details. Graefes Arch Clin Exp Ophthalmol. 1982;219:155-158. + +2. Williamson, T.H., Intraocular Surgery: A Basic Surgical Guide. Switzerland; Springer International Publishing; 2016:87-110. + +3. Gressel MG, Parrish RK, Heuer DK. Delayed nonexpulsive suprachoroidal hemorrhage. Arch Ophthalmol. 1984;102:1757-1760. + +4. Purcell JJ Jr, Krachmer JH, Doughman DJ, Bourne WM. Expulsive Hemorrhage in Penetrating Keratoplasty. Ophthalmology. 1982;89:41-43. + +5. Manschot WA. The pathology of expulsive hemorrhage. Am J Ophthalmol. + +1955;40:15-24. + +6. Maumenee AE, Schwartz MF. Acute intraoperative choroidal effusion. Am J +Ophthalmol. 1985;100:147-154. + +7. Taylor DM. Expulsive hemorrhage. Am J Ophthalmol. 1974;78:961-966. 8. Zauberman H. Expulsive choroidal haemorrhage: an experimental study. Br J +Ophthalmol. 1982;66:43-45. + +9. Samuels B. Postoperative nonexpulsive subchoroidal hemorrhage. Arch Ophthalmol. 1931;6:840-851. + +10. Ruderman JM, Harbin TS Jr, Campbell DG. Postoperative suprachoroidal hemorrhage following filtration procedures. Arch Ophthalmol. 1986;104:201205. + +11. Taylor DM. Expulsive hemorrhage: some observations and comments. Trans Am Ophthalmol Soc. 1974;72:157-169. + +12. Beyer CF, Peyman GA, Hill JM. Expulsive choroidal hemorrhage in rabbits: a histopathologic study. Arch Ophthalmol. 1989;107:1648-1653. + +13. Fuchs E. Ablösung der aderhaut nach staaroperation. Albrecht von Graefes Arch Ophthalmol. 1900;51:199-224. + +14. Lakhanpal V. Experimental and clinical observations on massive suprachoroidal hemorrhage. Trans Am Ophthalmol Soc. 1993;91:545-652. + +15. Wolter JR. Expulsive Hemorrhage During Retinal Detachment Surgery: A +case with survival of the eye after Verhoeff sclerotomy. Am J Ophthalmol. 1961;51:264-266. + +16. Chandra A, Jazayeri F, Williamson TH. Warfarin in vitreoretinal surgery: a case controlled series. Br J Ophthalmol. 2011;95:976-978. + +17. Capper SA, Leopold IH. Mechanism of serous choroidal detachment: A review and experimental study. AMA Arch Ophthalmol. 1956;55:101-113. + +18. Collins ET. An experimental investigation as to some of the effects of hypotony in rabbits' eyes. Tr Ophthalmol Soc UK. 1918;38:217-227. + +19. Wolter JR, Garfinkel RA. Ciliochoroidal effusion as precursor of suprachoroidal hemorrhage: a pathologic study. Ophthalmic Surg. 1988;19:344-349. + +20. Pederson JE, Gaasterland DE, MacLellan HM. Experimental ciliochoroidal detachment: effect on intraocular pressure and aqueous humor flow. Arch Ophthalmol. 1979;97:536-541. + +21. Verhoeff FH, Waite JH. Separation of the choroid, with report of a spontaneous case. Trans Am Ophthalmol Soc. 1925;23:120-139. + +22. Chylack LT Jr, Bellows AR. Molecular sieving in suprachoroidal fluid formation in man. Invest Ophthalmol Vis Sci. 1978;17:420-427. + +23. Brubaker RF. Intraocular surgery and choroidal hemorrhage. Arch Ophthalmol. + +1984;102:1753-1754. + +24. Campbell J. Expulsive choroidal hemorrhage and effusion: A reappraisal. Ann Ophthalmol. 1980;12:332-340. \ No newline at end of file diff --git a/medical/md/PMC8637585.md b/medical/md/PMC8637585.md new file mode 100644 index 0000000000000000000000000000000000000000..9424d46a34eee1b16fddce61300cd11a9c9af63a --- /dev/null +++ b/medical/md/PMC8637585.md @@ -0,0 +1,383 @@ +http://pubs.acs.org/journal/acsodf Mini-Review + +![0_image_0.png](0_image_0.png) + +# Application Of Cavitation In Oil Processing: An Overview Of Mechanisms And Results Of Treatment Olesya P. Stebeleva* And Andrey V. Minakov + +Cite This: ACS Omega 2021, 6, 31411−31420 Read Online + +![0_image_1.png](0_image_1.png) + +ABSTRACT: The integrated effect on homogeneous and heterophase liquids that can be used for technological purposes has drawn the attention of researchers in various sciences. Cavitation impact on oil is among the efficient methods of intensifying chemical−technological, hydromechanical, and mass-exchange processes and the destruction of substances. This work reviews in detail and analyzes the mechanisms of impact and application of cavitation in various processes in the petroleum industry, including the refining processes, that are associated with crude oil and petroleum waste, such as reduction of viscosity, demulsification, desulfurization, and improvement of quality of heavy oil and petroleum refinery products, including oil sludge and waste oil-containing water. + +- INTRODUCTION +Use of liquid fuel has forced mankind to process and use heavy and ultraheavy oil to produce special chemicals and dispose oil industry waste. Existing conventional processing technologies are unable to economically process ultraheavy oil stock without contaminating the environment. Technologies based on ultrasonic or hydrodynamic cavitation effects could be among the breakthrough technologies to address the challenges of oil production and refining. + +Cavitation is the formation, growth, and collapse of bubbles filled with gas, vapor, or their mixture when pressure in the liquid drops to the saturated vapor pressure of the liquid. Irrespective of its nature, cavitation is a multicomponent process1−5 accompanied by local increase of temperature and pressure inside the exploding bubble, turbulent mixing and micromixing by formation of microjets during bubble explosion, and thermal dissociation of water molecules forming hydroxyl radicals (Figure 1). In the case of hydrodynamic cavitation, the turbulent mixing of the processed volume of liquid is more intensive and accompanied by rapid mass transfer. The rapid bubble collapse occurs in an adiabatic state because the extremely short time of collapse denies heat exchange inside and outside the bubble. The totality of physical and chemical processes during cavitation changes the properties of processed liquids. The results of cavitation processing can conditionally be divided into the increase of oxidative reactions, the increase of dispersion of components of heterogeneous liquids (emulsions or suspensions), and variation of colloid stability. + +ACCESS Metrics & More Article Recommendations Figure 1. Processes in blue bubbles and phenomena in pink bubbles during cavitation. +Hydrodynamic cavitation is observed when a high pressure drop occurs inside the moving liquid, for example, passage through constriction such as a throttle valve or aperture. The hydrodynamic cavitation arises in various mechanical devices, such as a Venturi tube, nozzle, or rotor-stator units when the local pressure in the liquid drops to threshold value−liquid vapor pressure at an operational temperature. Then, when the Received: October 19, 2021 + +![0_image_2.png](0_image_2.png) Accepted: November 4, 2021 Published: November 16, 2021 ACS Omega http://pubs.acs.org/journal/acsodf Mini-Review liquid jet expands, the pressure recovers, resulting in the explosion of cavities (cavitation bubbles, voids). The acoustic cavitation is mainly initiated by piezoelectric transformers to generate sound of high amplitude. The frequency range of ultrasound used in sonochemistry is about 20 kHz to 10 MHz.5 Ultrasound is a mechanical wave with pressure oscillations capable of propagating through gaseous, liquid, and solid media. Under ultrasonic irradiation, the medium is exposed to an expansion and contraction cycle under pressure. + +In the field of oscillating pressure when the bubble reaches an unstable size, it explodes to generate shock waves and microjets. + +The integral cavitation effect on homogeneous and heterophase liquids, which can be used for technological purposes, has widespread application in various fields of human activities. Cavitation is considered a green technology because it avoids excessive use of toxic compounds by replacing them with environmentally safer solvents and, in some cases, avoids the use of chemical additives at all. Technological processes involving cavitation are specified by improved reaction conditions and selectivity, lack of toxic settlement, and reduced consumption of energy for chemical transformations. + +Ultrasonic activation at room temperature makes if possible to esterify and transesterify brown greasea mixture of oils, fats, solid substances, and detergents from food-industry waste captured in grease-removal tanks to produce biodiesel fuel in several minutes. Ultrasonic cavitation improves not only the efficiency but also the safety of the process compared to a conventional thermal activation method. These results offer challenges to develop efficient, inexpensive, and clean production of biodiesel.6 Ultrasound is a technology with great potential for the food industry. Extraction of polysaccharides of plant or animal origin by ultrasound increases the yield of the product and reduces extraction time and modifies their physical−chemical and biological properties. Physical processes caused by acoustic cavitation decrease the size of milk component particles, modify protein structures (casein, lactalbumin, and lactoglobulin), and increase activity of enzymes to improve the texture and increase their shelf life. The mechanical impact of the collapsing bubble on bacterial cells, formation of radicals, and activation of sonosensitizers caused by cavitation were recommended as antibacterial mechanisms alternative to thermal treatment. The benefits of ultrasound in the treatment of food products over conventional methods include more efficient mixing and micromixing, faster transfer of energy and mass, reduced temperature in the reactor, compact size of equipment, and faster reaction in extraction process control.7 For years, ultrasonic technologies have been used to improve the efficiency of mineral and coal flotation.8 Longitudinal sonic waves increase the probability of collision of ultrafine particles with flotation-sized bubbles, impact the oxidation films on minerals, and make their surface less hydrophilic by increasing adhesion and reducing detachment. Carryover of clay or waste rock deteriorates the quality of ore concentrate which can be reduced by cavitation devices in mineral flotation processes. + +To prepare coal−water fuels, which are a suspension of coal in water, it is common to use hydrodynamic cavitation; this affects the basic properties of liquid fuel (the size of particles of the disperse phase of the slurry, thermal−physical, rheological, and sedimentation properties) and provides for intense mass transfer and turbulent stirring. In ref 9, cavitation treatment of the coal−water slurry and its components was shown to reduce harmful emissions during its combustion. Time-stable coal− +water mixtures produced by cavitation are attributed to a sophisticated mechanism of interaction of coal particles, modifying additives and humic acids, which are also a part of coal and the variation of pH of the disperse medium. + +The method of cavitation treatment of suspensions, emulsions, and other liquids is successfully used in the following cases: to prepare cementing slurry and drilling fluids for drilling rig systems; to prepare concrete compositions with improved performance; to control radioactive scales in gas and oil wells; to dispose of radioactive waste of atomic power plants; to prepare nanoemulsions for use in pharmacology and medicine. Among the most amazing applications of cavitation effects is the sonochemical synthesis of nanoparticles. Shorttime localized hot points of explosions of the cavitation bubbles at extremely high temperatures and pressures provide conditions to synthesize nanostructured metals, alloys, oxides, sulfides, carbides and carbon, polymers, and biomaterials.10 Even though the cavitation technologies, as evident, have widespread application in various industries, for the challenges of the petroleum industry, they have been used relatively recently. Currently, intense research is carried out to intensify individual technological processes in oil recovery to recover oil from oil-bearing sand, to demulsify oil emulsion at the stages of desalination, to reduce viscosity of crude oil, to oxidatively desulfurize and demetallize, and to improve the quality of crude oil for cracking11 and decontaminating oil-containing waste. + +Figure 2 shows diagrams produced by analytical instrument SciVal data of Scopus based on the number of publications + +![1_image_0.png](1_image_0.png) + +dedicated to cavitation studies, including fundamental problems of single cavitation bubble motion and application of cavitation technologies in oil extraction and oil refining. As is evident, the subject of these publications is relevant in recent several years. + +The cavitation method is in agreement with principles of sustainable development of "green science" because it operates under soft conditions and is not associated with the production of hazardous chemical reagents. Complex multiple-parameter physical and chemical effects of cavitation make the mechanism of powerful ultrasound or intense hydrodynamic cavitation flow for different applications in the petroleum industry that are necessary to optimize operational parameters and improve efficiency difficult to understand. Major obstacles for the broad application of cavitation include scaling and energy efficiency. Although the laboratory equipment used in ultrasonic reactors is commercially available, large-scale equipment is still rare. The equipment for the hydrodynamic cavitation effect is practically unavailable for sale; to develop it is a challenge for independent solutions of individual research teams.12 This work analyzes the mechanisms of the cavitation effect on different oil liquids conditioned mostly by variation of frequency and intensity of ultrasound, treatment time, temperature of the process, and the nature of the medium processed. Here, we present a detailed review of applying cavitation in different processes of the petroleum industry, including oil refining processes, that are associated with crude oil and oil waste, such as reduction of viscosity, demulsification, desulfurization, and demetallization, and improvement of the quality of crude oil and petroleum refinery products. + +- REDUCTION OF CRUDE OIL VISCOSITY +Exploration of heavy crude oil and bitumen faces engineering problems at every stage from extraction (extraction from reservoir) to transportation and processing. Transport of heavy crude oil and bitumen by pipes to storage or oil refineries for processing is a difficult problem because of its high density and viscosity. In addition, deposition of asphaltenes and paraffins on the pipe walls hinders its operation and is among the major engineering challenges of beneficial use of crude heavy and ultraheavy oil. + +There are a number of methods to reduce the density of petroleum products based on increased temperature (hot piping), addition of surfactants, and dilution with lighter oil compositions, gas condensate, or water. Diluted heavy oil can change the stability of asphaltenes, promoting their flocculation and deposition, which can also clog the pipes. To avoid failure of the equipment and transportation systems, it is necessary to understand the reasons and mechanisms of aggregation of flocculation of asphaltenes and crystallization and deposition of paraffins. + +Heavy oil comprises a large fraction of asphaltenes, whose molecular weight is difficult to evaluate because of their selfaggregation and coagulation. The asphaltenes can be present in the oil in dissolved form but also can form a disperse system. In a colloidal disperse system of crude oil, the asphaltenes are in the form of micellar cores stabilized by adsorbed resins and are alkane-insoluble. Therefore, stable asphaltene colloid formation in the oil depends on the structure and component composition of disperse medium and the disperse phase of oilthe ratio of asphaltenes and resins, aromaticity, etc. + +The authors of a previous study13 found the viscosity of heavy oil to increase exponentially with asphaltene content: from trace amounts in the light oil to 20% in highly viscous oil, even though they are not components prevailing in the composition. It is also known that the size of asphaltene formation determines the rheological behavior of oil. Thus, prevention of serious agglomeration of asphaltenes is a key to reduce the viscosity of crude oil, especially of heavy and ultraheavy oil. + +In crude heavy oil, cavitation effects are possible with transmission of ultrasound energy or hydrodynamic movement into the energy of the collapsing bubble. This process can promote transfer of large molecules of ultraheavy oil into molecules of light hydrocarbons of smaller size without additional heating of the processed system, as the cavitation process is accompanied by an increase of temperature. The efficiency of ultrasonic processing on various oils has been shown by many authors. In ref 14, the effect of ultrasonic hydrodynamic treatment of oil was studied, and its advantage over acoustic treatment was shown. Intense treatment of the medium flow caused an instantaneous (1−100 μs) pressure drop with instantaneous emission of gas in the drastic pressure drop zone. Pressure pulsations arising in the constrained zone of the flow at the nozzle exit and interacting with the obstacle cause a discontinuity in the processed oil (Figure 3). + +![2_image_0.png](2_image_0.png) + +Moreoever, the pass over of the medium movement mode from supersonic to subsonic flow makes the pressure surge (8− +50 MPa at the reactor inlet, 0.05−0.09 MPA in the reactor to 3.5−8 MPa at the reactor exist). In the efficient zone of the pressure surge, the gas-vapor bubbles collapse to mechanically impact the liquid. + +This treatment results in the destruction of micellar connections and partial cracking of high-molecule compounds to increase the yield of light fraction in oil refining. Actually the authors observed that the fraction composition changed and the oil viscosity reduced by more than 30%. Combined use of chemical reactant (30% butyl acetate, 60% toluene, 10% xylene) reduced the viscosity by 72%. The authors note that the time between the treatment and monitoring of viscosity is irrelevant, and the difference between the viscosity index is less than 1%. However, another study15 showed considerable reversibility of viscosity change caused by ultrasonic exposure, and it is important to measure the viscosity within several seconds after treatment. + +Reversibility of viscosity after exposure of oil to irradiation is associated with thermal effects of ultrasonic irradiation. In ref 16, the relationship between parameters of ultrasonic treatment (irradiation time, output power, frequency), viscosity, and thermal behavior of crude oil with high asphaltene content was established. The optimal irradiation time at which the viscosity of cooled irradiated product is reduced to minimal values is shown to decrease due to an increase of power and/or frequency of ultrasonic irradiation. Reduction of viscosity caused by ultrasonic irradiation is connected to induced emission of heat, cavitation in the liquid, and changes in oil composition and molecular structure, including destruction of asphaltenes and heavy molecules into lighter ones. Qualitative evaluation of the effect of thermal destruction on viscosity of oil illustrates the results of a simple increase of temperature and ultrasound from the viewpoint of viscosity and amount of residual oil at the end of pyrolysis and oxidation stages. Even though the values of viscosity at equal temperatures in these + +![3_image_0.png](3_image_0.png) processes are comparable, the ultrasound results in a constant decrease of viscosity (more than by 10%) and does not return to the values before the treatment (Figure 4, experimental data are taken from ref 16). Thermogravimetric measurements prove that the ultrasonic irradiation decreases the heavy components of oil by distillation and thermal cracking mechanisms, reducing the total amount of asphaltenes. Analogous results were produced in ref 17 for crude oil with an initial asphaltene content of 1.76 wt %. + +The treatment time has been found to be the governing parameter in reduced oil viscosity, and the optimal treatment time increases with asphaltene content;18 this is in agreement with the calculated data.19 On the basis of experiments,20 it can be argued that ultrasonic irradiation can, within optimal time, reduce asphaltene content in crude oil; after that, with an increase of time, the small molecules and free radicals reintegrate to form large oil molecules to increase its viscosity.21 Within this optimal irradiation time, the decomposition rate of asphaltenes is equal to the rate of asphaltene formation by the mechanism of free radicals generated by ultrasonic cavitation.12 To fix and reinforce the reduction of oil viscosity, the treated oil can be added to various reactants. To estimate the expedience of the addition of a reactant after or during ultrasonic treatment and conversion of heavy asphaltenes and resins requires additional experimental studies. + +- DEMULSIFICATION OF OIL EMULSION +The presence of asphaltenes in oil not only determines the rheological properties of liquid but also affects the processes of demulsification (dehydration, desalting) of heavy oil. Within the framework of this section, we consider the formation of an emulsion as a negative factor affecting the properties of heavy and ultraheavy oil during its production and the wear of equipment during transportation and distillation and do not touch upon the increase of oil production by emulsification. + +In traditional development of oil fields, water is most frequently injected in the presence of surfactants. The produced crude oil is the emulsion of "water-in-oil" type, in which the water droplets are dispersed in the oil, and "oil-inwater" type and multiple emulsions. The presence of surfactants in the oil emulsion and the presence of chlorine, sodium, calcium, and magnesium salts in demulsified water, asphaltenes, and resins make the emulsion stable for several hours or days. Therefore, stability of the emulsion during crude oil production entails challenges in further demulsification, desalting, and dehydration. + +The demulsification methods used under industrial and laboratory conditions include physical, chemical, and biological demulsification. The demulsification process chain can be constructed as follows: aggregation of water (or oil) droplets and flocculation, creaming or sedimentation with stratification to be followed by coalescence and/or Ostwald ripening. The stability of the emulsion is governed by the strength of interfacial tension (water−oil boundary), and the differences in crude oil composition, presence, and component ratio of asphaltenes and resins, in particular, promote variability in the processes of emulsification and demulsification. + +Stability of oil or water droplets can be explained by the presence of repelling electric charges on its surface. This stability is initiated by the formation of interfacial films encapsulating the droplets of oil or water, while destabilization or destruction of emulsions is directly connected with elimination of this interfacial film. The stability of an emulsion can be evaluated by taking into account the interaction forces between the droplets: coagulation of particles is described by Deryagin−Landau−Fairway−Overbeck theory, to be more specific, by the sum of energies of van der Waals attraction and electrostatic repulsion arising because of the presence of a double electric layer on the surface of the droplet.22 When the repulsion forces are dominating, the system is in a stable state. On the contrary, when the interaction (attraction) force is increased by addition of an emulsifier or application of a physical field, the droplets flocculate and emulsions destabilize. + +The feasibility of producing stable or unstable emulsions can be precisely estimated by zeta-potential measurements. + +To rationally manage resources and reduce environment pollution, energy consumption and waste flow during demulsification and dehydration must be reduced. Ultrasonic treatment energy avoids the use of chemical reactants and deemulsifiers; however, the synergistic effect of their combined use can demonstrate an increased stratification effect. + +The effect of ultrasonic waves on suspended particles, droplets, and bubbles has been thoroughly studied. As stated previously, the emulsion is destroyed as a result of creaming, coagulation, and coalescence followed by sedimentation +(separation) processes. With the demulsification method with ultrasound, the droplets coalesce in the standing wave of the acoustic field generated between the transducer and reflector when the distance between them is a multiple of the half-wavelength. When water droplets in the oil get into the pressure nodes of the standing wave field, they agglomerate +(aggregate) into strips to increase the probability of collision and coalescence of droplets. Because of the difference of density and compressibility of oil and water, the droplets of water in oil move toward pressure nodes of the standing wave (Figure 5) and the droplets of oil in water move toward the antinodes.23,24 In addition, several droplets can be exposed to interparticle forces. When the droplets move either to the node or to the antinode of the standing wave, forces emerge, acting for short distances, called "secondary acoustic forces" or "Bjerkness forces", caused by sound scattering by neighboring droplets. These forces tend to attract the droplets within the plane. + +ACS Omega http://pubs.acs.org/journal/acsodf Mini-Review + +![4_image_0.png](4_image_0.png) + +First among the acoustic parameters affecting efficiency of demulsification are frequency, intensity of sound, and irradiation time. The lower frequency results in higher efficiency of the demulsification of crude oil. In ref 25, the water yield is shown to increase when the initial concentration of water in the emulsion is 15%, with use of ultrasonic horn at frequency 20 kHz and a power range from 80 to 1000 W and exposure time of 2 min. At higher initial water concentrations of 20 and 25%, the separation is less efficient, as well as when the irradiation time is increased to 5 min. It is noted that the higher the salt content in the crude oil, the more efficient the ultrasonic fields in separating water emulsion droplets from the crude oil and desalting the crude oil samples (Figure 6). + +![4_image_1.png](4_image_1.png) + +Efficiency of demulsification at different frequencies is + +![4_image_2.png](4_image_2.png) + +shown in ref 26. Powerful ultrasound (20−100 kHz) can transfer into the medium high specific density of energy and cause strong mechanical oscillations. In this case, the droplets of different sizes oscillate at different speeds and collide with each other to promote destruction of the interfacial boundary and coalescence of the droplets (Figure 7). Hence, the lowfrequency ultrasound can separate the droplets from the carrying (disperse) phase, which is connected by mechanical oscillations. In high-frequency acoustic fields, the primary acoustic force makes the dispersed droplets unite into strips. However, the interfacial boundary cannot be destroyed and prevents coalescence of the droplets, reducing the efficiency of demulsification. + +High-frequency ultrasound helps avoid strong mechanical effects of acoustic cavitation at low frequencies. As shown in ref 27, when a suitable frequency (>1 MHz) is chosen, the mechanical effects of cavitation can be restricted by chemical effects represented by dissociation of water. This technological difficulty of controlling bubbles in the turbulent flow restricts using hydrodynamic cavitation treatment for demulsification applications. The most known efficient demulsification of crude oil up to 65% without use of demulsifers was achieved in the frequency range of 25−45 kHz in 15 min.28 Demulsification in the MHz range can be achieved by use of two independent transformers operating in resonance.29 Transverse arrangement of piezoelectric transducers to the direction of flow monitors the resonance frequency to produce a standing wave in the emulsion, even when composition and temperature of emulsion changes in a short irradiation time. + +The effect of standing wave field combined with demulsifier, including factors affecting dehydration under the effect of ultrasound (power of ultrasound, operational temperature, demulsifier, and voltage in the desalting tank), demonstrates better results as compared to each method individually.12,30 +- OIL SLUDGE TREATMENT +Demand for petroleum products is permanently increasing. + +Residue after crude oil distillation adversely affects systems of the oil refinery and environment because of its high viscosity and asphaltene content. Processing of oil sludge not only resolves the problem of adverse contamination but also can be a source of lubricants, fuels, etc. + +The oil industry at the stages of production, transportation, storage, and processing is specified by production of oil sludge. + +The sources of oil sludge are bottoms of crude oil tanks, oil emulsion, contaminated effluents, oil-contaminated soil, and settling ponds. Worldwide production of oil-bearing sludge is more than 60 million tons.31 Each ton of produced crude oil is thought to have up to 2 kg of oil sludge.32 Oil sludge is a complex mixture of oil sediments containing different amounts of petroleum products (oil as it is) up to ACS Omega http://pubs.acs.org/journal/acsodf Mini-Review 30%, from 30 to 40% of water, and solid inclusions (sand, soil, mineral components, heavy metals). Chemical composition of oil sludge is not standard and depends mostly on the composition of initial crude oil. The critical reasons of oil sludge formation can be changes in external conditions (temperature, pressure, humidity), cooling, evaporation of light factions, mixing with incompatible materials, and addition of water-forming emulsions. Toxicity, carcinogenicity, and mutagenicity of hydrocarbon compounds make them the most hazardous types of waste in the oil industry. The high viscosity of oil sludge and sophisticated mechanism of interaction with water, soil, and atmosphere, makes oil or its production waste spill remediation in the world's oceans or on land a great challenge. + +Reduction of oil sludge amount can conditionally be divided into three states: reduction of sediment formation by technologies impacting oil at the stage of its production or processing, extraction of oil from oil sludge, and disposal of nonextractable oil sludge. Studies show that the profit from oil extracted from oil sediment exceeds the cleaning expenditures.33 However, physical−chemical characteristics of oilbearing sludge, produced quantities, availability, and feasibility of using the equipment are important parameters to define the technologies to treat oil sludge formed at a chosen oil refinery or deposit or burial site. + +To characterize oil-containing sediment (sludge) requires evaluation of such quantitative parameters as pH, content of solid matter, phosphorus, nitrogen, carbon, sulfur, metals (As, Pb, Cd, Co, Cu, Cr, Ni, Zn, V, Hg), and polycyclic aromatic hydrocarbons (PAHs) and quantity of oil hydrocarbons, benzene, toluene, ethylbenzene, and xylenes. These parameters are most important; they should be correlated with specifics of each processing technology. Most of these components are difficult to process because of their strong molecular bonds, high molecular weight, hydrophobicity, and low solubility in water. The oil-containing sludges containing high concentration of sulfur, Cr, Pb, Hg, and PAH cannot be treated by thermal methods, such as combustion, combined treatment in clinkering kilns, microwave liquefaction, or destructive distillation.34 Treatment of oil sludge is the most preferable version in the oil industry as it makes the production potential more complete. Moreover, processing of the oil-containing residue can reduce the volume of hazardous waste, prevent contamination, and reduce use of nonrenewable energy sources. The known extraction methods are by dilutant, centrifugation, addition of surfactants, freezing/melting, pyrolysis of sediment, microwave irradiation, electrokinetic exposure, and creaming flotation. + +High efficiency and minimal impact on the environment from ultrasonic technology are important as it is widely used in oil processing. Reduction of viscosity when treated with low power density is caused by disaggregation of the colloidal system. Processing with high power density can cause both disruption of the chemical bond and disaggregation of the colloidal system.35 In oil sludge processing, ultrasonic waves are used to change the physical properties and condition of the oil-containing sediment, containing solid particles of different sizes. The effect of cavitation and mechanical vibration, caused by acoustic irradiation, removes droplets of crude oil initially fixed to the surface of solid particles by disrupting hydrogen bonds between oil and solid particles to desorb the oil. This decreases the amount of crude oil on the surface of solid + +![5_image_0.png](5_image_0.png) + +particles36 (Figure 8). + +Impulse waves of sound pressure make the water droplets and solid particles in sludge vibrate to destabilize it. Ultrasound can penetrate each phase of oil-containing sludge and completely disintegrate it. Under continuous irradiation by ultrasonic waves, the viscosity of water−oil emulsions continuously decreases. Small droplets in the emulsion mixture increase their motion, collide, and coalesce; in this manner, the water phase and oil separate. When oil sludge disintegrates into smaller parts, the efficient area between the cavitation bubbles and emulsion increases, and this makes removal of oil from solid surfaces deeper. The effect of cavitation bubbles on removal of oil from the oil-containing sludge includes not only mechanical disintegration but also oxidative degradation. + +The ultrasonic waves create and uniformly distribute explosions of cavitation bubbles in liquid medium. The energy released can reach and penetrate the pores, closed holes, and areas generally inaccessible for other cleaning methods. This can substantially simplify further disposal of oil sludge. + +Efficient use of ultrasound to separate water−oil sludge emulsion depends on several factors (described in the demulsification section), including frequency of the sound, intensity, treatment time, temperature properties of sludge, additives, and salts or other impurities. In ref 37, the power of ultrasound was shown to be the most important factor in treatment; its increase in totality with the least content of asphaltene oil extraction from the oil sludge amounted to 92%. Some model studies show that the light oil components (PAH, light hydrocarbons, kerosene, gasoline) can be relatively easily removed from solid substances, whereas heavy hydrocarbons +(asphaltenes) remain fixed to solid surfaces.38 Additional mixing of emulsion during acoustic impact also increases the amount of separated oil. Combined use of ultrasound and thermochemical treatment made extraction of more than 99% of oil under ultrasonic treatment parameters of 28 kHz, 15 min, 400 W, and 60 °C possible; an increase of treatment time to 400 min did not further increase oil recovery.39 Complex use of acoustic impact with surfactants decreases the average size of solid particles in oil sludge; on the whole, this is useful to extract oil but heavily depends on the treatment time and can increase the probability of recombination of oil and solid matter with time.40 Ultrasonic irradiation is a processing method that makes it possible to treat oil-containing sludges in a relatively short time. High efficiency of oil recovery and lack of secondary contamination notwithstanding, ultrasonic irradiation to recover oil from sludge is most frequently reported for laboratory systems and small volumes of treated media. To use a large tank for ultrasonic treatment can be more prospective from the viewpoint of processing large amounts of oil containing sludge, but low intensity of ultrasound can reduce its performance; implementation of this method is associated with special tanks or structures.41 The high cost of equipment and maintenance also prevent commercial use of this technology. + +- OIL DESULFURIZATION +Organic compounds of sulfur are difficult (most commonly impossible) to separate from crude oil and its derivatives, such as gasoline and diesel fuel, which account for a high share in total production of oil refineries. These compounds include sulfides, thiols, thiophenes, substituted benzo- and dibenzothiophenes, benzonaphthothiophenes, and numerous other more complex molecules. + +Organic sulfur compounds during combustion in fuel form SOx compounds. They react in the atmosphere to form H2SO4, generating acid rain. Furthermore, SO2 is associated with adverse respiratory effects in humans. Moreover, the content of pollutants (sulfur, nitrogen, oxygen, and metals) adversely affects equipment, catalysts, and the quality of finished product. + +High environmental protection standards restrict sulfur content in fuel, demanding deep desulfurization of both fuel and petroleum products. The conventional method of removing sulfur from oil includes hydrotreatment entailing capital-intensive investments into hydrodesulfurization equipment at high pressure and high temperature. Furthermore, persistent aromatic sulfur compounds (e.g., 4,6-dimethyldibenzothiophene) cannot be removed by desulfurization because of their low reactivity. Oxidative desulfurization by ultrasound (UAOD) forms useful alternatives to conventional methods and makes deeper desulfurization under less aggressive conditions of the process possible with operating temperatures of 40−100 °C and pressures of 1−2 atm.42 The effect of ultrasound on desulfurization efficiency is technically associated with its chemical and physical mechanism. The chemical mechanism of ultrasonic treatment involves acceleration of the reaction by formation of radicals due to short-time collapse of cavitation bubbles, whereas the physical mechanism is formation of thin oil-in-water emulsion or water-in-oil emulsion, which increases mass transfer by increasing the inertial field between the oil and the oxidizing phase. In the oxidative desulfurization process, the ultrasonic cavitation operates by forming microturbulence, which increases the oxidation reaction between the hydrocarbons and H2O2, forming or added into the water. + +Studies investigating oxidative desulfurization with use of ultrasound are aimed to select optimal, rational, and efficient operating parameters of the process to increase extraction of sulfur compounds. In ref 43, conversion of sulfur amounted to 98% at a temperature of 52 °C; the ultrasonic treatment time was 15 min, and ultrasound power was 70 W. Design of experimental techniques made it possible to determine the optimal UAOD parameter values for gas−oil volume of the oxidant, mass of the catalyst, and phase transfer agent and amplitude of ultrasonic waves for maximum conversion of sulfur at 95.92%. UAOD employing ionic liquids at room temperature (the organic salts which form liquid phase at temperatures below 100 °C) as a catalyst for phase transfer and extractive solvent achieved 99.9% desulfurization in diesel fuel samples exposed to 10 min long ultrasonic impact.44 It should be noted that to make the UAOD process most efficient, the combination of oxidant, catalyst, and phase transfer agent should be selected for each oil fuel and its specific sulfur compounds. Ultrasonic treatment helps desulfurize oil, so oil and petroleum products can be expected to demetalize. + +The complexity of scaling ultrasonic cavitation to a commercial volume is by restricting the volume of the ultrasound source. Hydrodynamic cavitation can form an alternative to ultrasonic (acoustic) cavitation. Hydrodynamic movement of liquid through a cavitation device creates cavitation over the entire volume of treated liquid and, consequently, can be efficiently used in large-scale deep and complete desulfurization operation. + +- TREATMENT OF OIL-CONTAINING WASTEWATER +The amount of used oil increases with industrial development; the engineering solutions are, however, imperfect, and large quantities of oil get into water and pollute it. Moreover, the tertiary production processes, such as alkaline water flooding, polymer water flooding, and flooding with alkaline surfactants, form large amounts of oil-containing wastewater (stratal) that is difficult to process compared to conventional wastewater. To treat oil-containing wastewater, the oil industry (oil refining, oil storage, transport, and petroleum industry) has developed various methods, such as flotation, membrane filtration, electrochemical methods, centrifugation, and chemical oxidation. + +Ultrasonic impact on oil-containing wastewater treatment processes is usually used with other methods to improve oil removal or compensate for their shortcomings. Ultrafiltration is efficient to treat complex oil-containing wastewater, as it does not need additions of chemical reactants. This reduces the cost of treatment and improves the quality of produced permeate. Ultrafiltration, however, has its own restrictions, resulting in reduction of permeate flow with time. Adsorption and accumulation of oil and particles on the membrane surface contaminate it and reduce service life of the membrane because of frequent chemical and physical cleaning. Ultrasound is among the most efficient methods of cleaning of ultrafiltration membranes.45 Low irradiation frequency has been shown to be able to decelerate deposit formation, and higher frequency can improve removal of organic substances. On the other hand, cleaning using ultrasonic cavitation makes it possible to clean while filtration is still going on. There is no need to stop the filtration process or to pass over the pulsing mode, as this can reduce the cost of membrane cleaning. + +Therefore, combination of ultrasound with membrane ultrafiltration can reduce the rate of membrane contamination with high flow through the membrane and lower frequency. + +In ref 46, the authors studied the effect of ultrasound in insignificant volumes of chemical reagents for flotation of oilcontaining water to intensify the treatment process. After ultrasonic treatment, the flaky particles reduce their size and increase their number, and the capability to form conglomerates decreases. Experimental data speak in favor of increasing the number of coagulation kernels, this results in an improved degree of cleaning of water contaminated with petroleum products with similar consumption of the reactant. Solutions of alumosilicic flocculant coagulant contain both aluminum salts and active silicic acid, providing for coagulation and flocculation activities of the solutions. The authors of ref 47 showed a maximum degree of cleaning oil-contaminated water at a temperature of 6 °C and an ultrasound impact time of 60 s, with commercial flocculant "Praestol" 853 (2 mg/L) to 99.2%. + +Combined use of ultrasound and chemical or electrical flocculation makes the organic substances agglomerate and separate in water. Droplets of oil in wastewater feature high negative zeta-potential and high electrostatic repulsion among themselves, resulting in a stable disperse system (as noted in the demulsification section), and cationic flocculants play the role of an adsorption bridge and neutralize the charge on the surface of oil droplets. + +Improved oxidation methods reinforced with ultrasound can be used to decompose organic matter in water: OH radicals promote a high rate of accelerated oxidation reaction +(degradation rate of about 106−109 l mol−1 s +−1), reacting with organic molecules of contaminants.48 It is common knowledge that, during cavitation, adiabatic collapse of microscopic bubbles takes place, and this creates localized high pressure and high temperature for reaction behavior. H2O +thermally dissociates to form various highly active particles, such as radicals •OH, •OOH, •H, H2O2, and O2 +−. These circumstances promote oxidation reaction and recovery of the contaminant molecules, resulting in complete degradation. + +Sonophotolysis is a combined improved oxidation method in which ultrasound and ultraviolet (UV) waves are simultaneously used to irradiate wastewater.49 Treatment of wastewater by sonophotolysis forms highly active •OH radicals during themolysis of water molecules inside the cavitation microbubble (eqs 1−3): +H O (irradiation with ultrasound) +H OH(thermolysis) +2 + +→ •+• (1) +HH H •+ •→ 2 (2) H O HO •+ →2 2• (3) +and by decomposition of H2O2 under UV effect (eq 4): +H O ( 254)(irradiation with ultraviolet) OH h OH +2 2 = < →• ν + +- (4) +Hydrogen peroxide also forms during implosive collapse of the cavitation bubble or reacts with contaminant to destroy it or decomposes into the •OH radical under the effect of UV +irradiation. Excess •OH radicals recombine to produce on site hydrogen peroxide, which further decomposes under UV light to form free radicals (eqs 5−7): +HO HO H O O 2 2 22 2 •+ •→ + (5) + +$\mathcal{L}_{\phi}$ +$\mathbf{a}$ +$\tau$ . +$$\mathrm{H}\bullet\,+\,\mathrm{HO}_{2}\bullet\,\to\,\mathrm{H}_{2}\mathrm{O}_{2}$$ +H HO H O •+ •→ 2 22 (6) +- +• → OH OH H O2 2 (7) +These oxidizing radicals attack organic contaminants to decompose them to their intermediate products (eq 8): +•+ + + + OH pollutants intermediate products CO H 2 2O +(8) +Excess concentration of H2O2 also results in the capture of radicals in the medium (eqs 9−11): + +| - | + | → | + | - | +|-----|-----|-----|-----|------| +| OH | H O | H O | HO | | +| 2 | 2 | 2 | 2 | (9) | +| H | H O | H | HO | | +| •+ | → | + | | | +| 2 | 2 | 2 | 2• | (10) | + +$$\bullet\mathrm{OH}\,+\,\mathrm{HO}_{2}\bullet\,\rightarrow\,\mathrm{H}_{2}\mathrm{O}\,+\,\mathrm{O}_{\mathrm{x}}$$ +- + •→ + OH HO H O O 2 22 (11) +Thermal decomposition and mineralization of intermediate products takes place at the gas−liquid interface of the bubbles. + +Hence, the ultrasound combined with UV light can provide for a synergistic effect in the degradation/mineralization of persistent organic contaminants by evaporation and thermal and photolytic dissociation of hydrogen peroxide caused by the sonoluminescence light emitted by short-living cavitation bubbles. Ultrasonic waves create microturbulence and intense mixing in the medium by shock waves. These physical properties of cavitation also accelerate the hydroxylation +(oxidation) reaction and increase decomposition of intermediate products of UV radiation and reduce toxicity of contaminants. + +The presence of dissolved oxygen in liquid medium can also intensify additional chemical reactions to form additional oxidizing radicals (eq 12): +- + → •+ OH O HO O 2 2 - (12) + +$\left(1\right)$. +$\eqref{eq:walpha}$. +Therefore, combinations of ultrasound and ultraviolet waves demonstrate their ability to provide for considerable synergistic effect. + +Hybrid technologies including cavitation treatment are of much interest among research communities working on environmental remediation because sonohybridization methods are capable of destroying persistent organic contaminants. + +For instance, the combined use of hydrodynamic cavitation and the Fenton reaction improve the process of dewatering waste from activated sludge.50 +- CONCLUSION +Cavitation caused by ultrasonic waves or variation of hydrodynamic states draws has drawn the attention of the oil industry for the development of environmentally benign technology. Decreasing efficiency of crude oil production, the high cost of energy for oil transportation, and processing and increasing the scale of the environment pollution by oil waste has stimulated researchers to continuously search for alternative solutions. This promotes the investigation and implementation of cavitation treatment as a promising technology in various processes of the oil business in all countries associated with oil production in the past 30 years. + +The reduction of viscosity of ultraheavy oil is one of the most promising directions of using wave technologies in oil industrydestruction of micelles and partial cracking of high molecular compounds increases the yield of light fractions in oil distillation without additional heating. Hydrodynamic cavitation is more efficient than acoustic methods due to the intensity of flow processing and the powerful mechanical impact on liquid. However, the technological difficulty of controlling bubbles in the turbulent flow restricts the application of hydrodynamic cavitation treatment for demulsi-fication tasks. Ultrasonic demulsification equipment should provide for the possibility of varying acoustic parameters and stable output power to create the standing wave field. Combined use of ultrasound with a demulsifier to dehydrate the oil emulsion, including oil-containing waste, demonstrates better results than those for each method separately. + +$\left(6\right)$. +The ultrasonic effect is commonly used in cleaning of waste or stratal oil-containing water in combination with other methods to improve oil removal or compensate their shortcomings. Persistent organic contaminants have been found to be sensitive to hybrid technologies including ultrasonic treatment; this has a positive effect on environmental remediation. In a general case, the trend is to use low irradiation frequency and high power with cavitation treatment for a short duration of seldom more than 1 h. To scale laboratory results, it is preferable to use equipment with several piezoelectric sources operating at certain frequencies. Oxidative processes used to treat waste and stratal water and in oxidative desulfurization by ultrasound play key role. High temperature (about 5000 °C) and high pressure (up to 5000 atm) within the scale of a microbubble along with slightly varying basic temperature and pressure of the liquid (at the environment level) efficiently help the oxidizing processes. + +The cavitation technology is a technology relatively young for the petroleum industry; this calls for an investigation of its effects rather at the experimental stage of refining different oil compositions (particularly heavy and ultraheavy with high asphaltene content) and petroleum refinery products; this requires more time for numeric studies and cavitation aggregate simulation. Finally, achievement of stable output yield of oil production and oil refining and strict control of all parameters under the cavitation effect will allow mankind to prolong the efficient use of fossil energy. + +- AUTHOR INFORMATION + +## Corresponding Author + +Olesya P. Stebeleva − Siberian Federal University, 660041 Krasnoyarsk, Russian Federation; orcid.org/0000-00029559-1522; Phone: 89233175840; Email: opstebeleva@ mail.ru + +## Author + +Andrey V. Minakov − Siberian Federal University, 660041 Krasnoyarsk, Russian Federation; Kutateladze Institute of Thermophysics, SB RAS, 630090 Novosibirsk, Russia; orcid.org/0000-0003-1956-5506 Complete contact information is available at: https://pubs.acs.org/10.1021/acsomega.1c05858 + +## Notes + +The authors declare no competing financial interest. + +Biographies Dr. Olesya P. Stebeleva is an associate professor of the Department of Heat Engineering and Hydrodynamics of the Siberian Federal University. She received her Ph.D. in 2011 in powder metallurgy and composite materials from the Siberian Federal University. She is a researcher at the Laboratory of Physical and Chemical technologies for the development of hard-to-recover hydrocarbon reserves of the Siberian Federal University. Her research interests cover creation of new types of environmentally friendly fuels and high-energy treatment of liquid fuels, including oil and coal−water suspension. + +Dr. Andrey V. Minakov is the director of the Institute of Engineering Physics and Radio Electronics and the head of the Laboratory of Physical and Chemical technologies for the development of hard-torecover hydrocarbon reserves of the Siberian Federal University. He received his Ph.D. in 2008 in the field of mathematical modeling, numerical methods. and software packages. His research focuses on the processes of heat and mass transfer with nanofluids and numerical modeling of thermophysical processes in liquids of the oil industry. He is the author of more than 170 publications. He is a member of editorial and scientific advisory boards and professional societies. + +- ACKNOWLEDGMENTS +The study was performed within the framework of the state assignment for science (SFU, FSRZ-2020-0012). + +- REFERENCES +(1) Flint, E. B.; Suslick, K. S. The temperature of cavitation. Science 1991, 253, 1397−1399. + +(2) Shamseddini, A.; Mowla, D.; Esmaeilzadeh, F. Continuous treatment of petroleum products in a tailor-made flow-through sonoreactor. J. Pet. Sci. Eng. 2019, 173, 1149−1162. + +(3) Peng, C.; Tian, S.; Li, G.; Sukop, M. C. Simulation of laserproduced single cavitation bubbles with hybrid thermal Lattice Boltzmann method. Int. J. Heat Mass Transfer 2020, 149, 119136. + +(4) Patidar, R.; Srivastava, V. C. Mechanistic and kinetic insights of synergistic mineralization of ofloxacin using a sono-photo hybrid process. Chem. Eng. J. 2021, 403, 125736. + +(5) Klima, J. Application of ultrasound in electrochemistry. An overview of mechanisms and design of experimental arrangement. + +Ultrasonics 2011, 51 (2), 202−209. + +(6) Savun-Hekimoğlu, B. A. Review on sonochemistry and its environmental applications. Acoustics 2020, 2 (4), 766−775. + +(7) Chemat, F.; Zill-e-Huma; Khan, M. K. Applications of ultrasound in food technology: Processing, preservation and extraction. Ultrason. Sonochem. 2011, 18 (4), 813−835. + +(8) Mao, Y.; Xia, W.; Peng, Y.; Xie, G. Ultrasonic-assisted flotation of fine coal: A review. Fuel Process. Technol. 2019, 195, 106150. + +(9) Stebeleva, O. P.; Kashkina, L. V.; Petrakovskaya, E. A.; Nikitin, S. + +L.; Valiullin, T. R. The impact of cavitation-activated water on combustion dynamics and environmental characteristics of coal-water slurry fuel. IOP Conf. Ser.: Mater. Sci. Eng. 2019, 537, 062056. + +(10) Bang, J. H.; Suslick, K. S. Applications of ultrasound to the synthesis of nanostructured materials. Adv. Mater. 2010, 22 (10), +1039−1059. + +(11) Liu, D.; Li, Z.; Fu, Y.; Zhang, Y.; Gao, P.; Dai, C.; Zheng, K. + +Investigation on asphaltene structures during Venezuela heavy oil hydrocracking under various hydrogen pressures. Energy Fuels 2013, 27 (7), 3692−3698. + +(12) Avvaru, B.; Venkateswaran, N.; Uppara, P.; Iyengar, S. B.; Katti, S. S. Current knowledge and potential applications of cavitation technologies for the petroleum industry. Ultrason. Sonochem. 2018, 42, 493−507. + +(13) Ghanavati, M.; Shojaei, M.-J.; Ramazani, S. A. A. Effects of asphaltene content and temperature on viscosity of Iranian heavy crude oil: experimental and modeling study. Energy Fuels 2013, 27 +(12), 7217−7232. + +(14) Abramov, V. O.; Abramova, A. V.; Bayazitov, V. M.; Mullakaev, M. S.; Marnosov, A. V.; Ildiyakov, A. V. Acoustic and sonochemical methods for altering the viscosity of oil during recovery and pipeline transportation. Ultrason. Sonochem. 2017, 35, 389−396. + +(15) Gizem Gunal, O.; Islam, M. Alteration of asphaltic crude rheology with electromagnetic and ultrasonic irradiation. J. Pet. Sci. + +Eng. 2000, 26 (1−4), 263−272. + +(16) Razavifar, M.; Qajar, J. Experimental investigation of the ultrasonic wave effects on the viscosity and thermal behaviour of an asphaltenic crude oil. Chem. Eng. Process. 2020, 153, 107964. + +(17) Shedid, S. A.; Attallah, S. R. Influences of ultrasonic radiation on asphaltene behavior with and without solvent effects. SPE International Symposium and Exhibition on Formation Damage Control 2004, SPE-86473-MS. + +(18) Amani, M.; Najafi, I. Asphaltene flocculation inhibition with ultrasonic wave radiation: a detailed experimental study of the governing mechanisms. Adv. Pet. Explor. Dev. 2011, 2 (2), 32−36. + +(19) Doust, A. M.; Rahimi, M.; Feyzi, M. Effects of solvent addition and ultrasound waves on viscosity reduction of residue fuel oil. Chem. + +Eng. Process. 2015, 95, 353−361. + +(20) Salehzadeh, M.; Akherati, A.; Ameli, F.; Dabir, B. Experimental study of ultrasonic radiation on growth kinetic of asphaltene aggregation and deposition. Can. J. Chem. Eng. 2016, 94 (11), +2202−2209. + +(21) Luo, X.; Gong, H.; He, Z.; Zhang, P.; He, L. Recent advances in applications of power ultrasound for petroleum industry. Ultrason. Sonochem. 2021, 70, 105337. + +(22) Shehzad, F.; Hussein, I. A.; Kamal, M. S.; Ahmad, W.; Sultan, A. S.; Nasser, M. S. Polymeric surfactants and emerging alternatives used in the demulsification of produced water: a review. Polym. Rev. + +2018, 58 (1), 63−101. + +(23) Trujillo, F. J.; Juliano, P.; Barbosa-Cánovas, G.; Knoerzer, K. + +Separation of suspensions and emulsions via ultrasonic standing waves +- A review. Ultrason. Sonochem. 2014, 21 (6), 2151−2164. + +(24) Evander, M.; Nilsson, J. Acoustofluidics 20: Applications in acoustic trapping. Lab Chip 2012, 12 (22), 4667. + +(25) Sadatshojaie, A.; Wood, D. A.; Jokar, S. M.; Rahimpour, M. R. + +Applying ultrasonic fields to separate water contained in mediumgravity crude oil emulsions and determining crude oil adhesion coefficients. Ultrason. Sonochem. 2021, 70, 105303. + +(26) Luo, X.; Gong, H.; Yin, H.; He, Z.; He, L. Optimization of acoustic parameters for ultrasonic separation of emulsions with different physical properties. Ultrason. Sonochem. 2020, 68, 105221. + +(27) Lavigne, O.; Takeda, Y.; Shoji, T.; Sakaguchi, K. Water irradiation by high-frequency ultrasonic wave: Effects on properties of passive film formed on stainless steel. Ultrason. Sonochem. 2011, 18 +(6), 1287−1294. + +(28) Antes, F. G.; Diehl, L. O.; Pereira, J. S. F.; Guimaraes, R. C. L.; ̃ +Guarnieri, R. A.; Ferreira, B. M. S.; Flores, E. M. M. Effect of ultrasonic frequency on separation of water from heavy crude oil emulsion using ultrasonic baths. Ultrason. Sonochem. 2017, 35, 541− +546. + +(29) Atehortua, C. M. G.; Pérez, N.; Andrade, M. A. B.; Pereira, L. ́ +O. V.; Adamowski, J. C. Water-in-oil emulsions separation using an ultrasonic standing wave coalescence chamber. Ultrason. Sonochem. + +2019, 57, 57−61. + +(30) Saad, M. A.; Kamil, M.; Abdurahman, N. H.; Yunus, R. M.; +Awad, O. I. An overview of recent advances in state-of-the-art techniques in the demulsification of crude oil emulsions. Processes 2019, 7 (7), 470. + +(31) Tian, Y.; Li, J.; Whitcombe, T. W.; McGill, W. B.; Thring, R. + +Application of oily sludge-derived char for lead and cadmium removal from aqueous solution. Chem. Eng. J. 2020, 384, 123386. + +(32) Hu, G.; Li, J.; Zeng, G. Recent development in the treatment of oily sludge from petroleum industry: A review. J. Hazard. Mater. + +2013, 261, 470−490. + +(33) Chrysalidis, A.; Kyzas, G. Z. Applied cleaning methods of oil residues from industrial tanks. Processes 2020, 8 (5), 569. + +(34) Da Silva, L. J.; Alves, F. C.; de Franca, F. P. A review of the ̧ +technological solutions for the treatment of oily sludges from petroleum refineries. Waste Manage. Res. 2012, 30 (10), 1016−1030. + +(35) Qiao, J.; Zuo, K.; Sun, Y.; Song, W.; Zhang, X.; Dai, L.; Wang, W.; Jian, C. Experimental studies on the effect of ultrasonic treatment and hydrogen donors on residual oil characteristics. Ultrason. + +Sonochem. 2020, 69, 105266. + +(36) Luo, X.; Gong, H.; He, Z.; Zhang, P.; He, L. Research on mechanism and characteristics of oil recovery from oily sludge in ultrasonic fields. J. Hazard. Mater. 2020, 399, 123137. + +(37) He, S.; Tan, X.; Hu, X.; Gao, Y. Effect of ultrasound on oil recovery from crude oil containing sludge. Environ. Technol. 2019, 40 +(11), 1401. + +(38) Adams, J. J. Asphaltene adsorption, a literature review. Energy Fuels 2014, 28 (5), 2831−2856. + +(39) Jin, Y.; Zheng, X.; Chu, X.; Chi, Y.; Yan, J.; Cen, K. Oil recovery from oil sludge through combined ultrasound and thermochemical cleaning treatment. Ind. Eng. Chem. Res. 2012, 51 (27), 9213−9217. + +ACS Omega http://pubs.acs.org/journal/acsodf Mini-Review +(40) Lin, Z.; Xu, F.; Wang, L.; Hu, L.; Zhu, L.; Tan, J.; Li, Z.; Zhang, T. Characterization of oil component and solid particle of oily sludge treated by surfactant-assisted ultrasonication. Chin. J. Chem. Eng. 2021, 34, 53. + +(41) Ronchi, R. P.; Negris, L.; Melo, B. N.; Pereira, L. S. F.; Vicente, M. A.; Flores, E. M. M.; Santos, M. de F. P. Removal of oil from synthetic heavy crude oil-in-water emulsions by the association of glass raschig rings and ultrasound. J. Dispersion Sci. Technol. 2020, 1− +11. + +(42) Choi, A. E. S.; Roces, S.; Dugos, N.; Wan, M.-W. Oxidation by H2O2 of bezothiophene and dibenzothiophene over different polyoxometalate catalysts in the frame of ultrasound and mixing assisted oxidative desulfurization. Fuel 2016, 180, 127−136. + +(43) Karami, E.; Sobati, M. A.; Khodaei, B.; Abdi, K. An experimental investigation on the ultrasound-assisted oxidation of benzothiophene in model fuel: Application of response surface methodology. Appl. Therm. Eng. 2017, 118, 691−702. + +(44) Cheng, S.-S.; Yen, T. F. Use of Ionic Liquids as Phase-Transfer Catalysis for Deep Oxygenative Desulfurization. Energy Fuels 2008, 22 +(2), 1400−1401. + +(45) Zhang, R.; Huang, Y.; Sun, C.; Xiaozhen, L.; Bentian, X.; Wang, Z. Study on ultrasonic techniques for enhancing the separation process of membrane. Ultrason. Sonochem. 2019, 55, 341−347. + +(46) Abramov, O. V.; Abramov, V. O.; Veksler, G. B.; Kulov, N. N.; +Zabotina, E. V.; Kashirskaya, O. A.; Shkol'nikov, A. V.; Mullakaev, M. + +S. Ultrasonic activation of reagent purification of surface wastewaters from oil products. Theor. Found. Chem. Eng. 2009, 43 (4), 568−574. + +(47) Keremetin, P. P.; Parilov, P. S.; Mullakaev, M. S.; Veksler, G. B.; +Kruchinina, N. E.; Abramov, V. O. Determination of the technological parameters for the sonochemical purification of oily water. Theor. + +Found. Chem. Eng. 2011, 45 (4), 568−574. + +(48) Medel, A.; Treviño-Reséndez, J.; Brillas, E.; Meas, Y.; Sirés, I. + +Contribution of cathodic hydroxyl radical generation to the enhancement of electro-oxidation process for water decontamination. Electrochim. Acta 2020, 331, 135382. + +(49) Patidar, R.; Srivastava, V. C. Evaluation of the sono-assisted photolysis method for the mineralization of toxic pollutants. Sep. Purif. + +Technol. 2021, 258 (1), 117903. + +(50) Cai, M.; Hu, J.; Lian, G.; Xiao, R.; Song, Z.; Jin, M.; Dong, C.; +Wang, Q.; Luo, D.; Wei, Z. Synergetic pretreatment of waste activated sludge by hydrodynamic cavitation combined with Fenton reaction for enhanced dewatering. Ultrason. Sonochem. 2018, 42, 609−618. \ No newline at end of file diff --git a/medical/md/PMC8702903.md b/medical/md/PMC8702903.md new file mode 100644 index 0000000000000000000000000000000000000000..fccf76e77d3fe310334b1ab758a4216f09d0665e --- /dev/null +++ b/medical/md/PMC8702903.md @@ -0,0 +1,10 @@ +Fresh Cadaveric Study of the Distances between the Deep Plantar Arch and the Lesser Metatarsals at Risk During Osteotomy of the Lesser Metatarsals Ichiro Tonogai, MD, PhD; Ichiro Tonogai, MD, PhD +Category: Midfoot/Forefoot Keywords: Midfoot; Metatarsals; Osteotomies Introduction/Purpose: Osteotomy of the lesser metatarsals is useful to treat forefoot deformity. Although there is the possibility that some arteries supplying the lesser metatarsals are injured during osteotomy, there are few informations about the distances from the deep plantar arch to the lesser metatarsals. This study aimed to identify the distances from the deep plantar arch to the lesser metatarsals and to reveal how osteotomy of the lesser metatarsals might injure the deep plantar arch. + +Methods: Twenty fresh cadaveric feet were injected with barium through the external iliac artery, and enhanced computed tomography images were assessed. The distance between the deep plantar arch and each lesser metatarsal was measured on both axial and sagittal images. Results: The distances between the deep plantar arch and the second, third, and fourth metatarsals on the axial plane were 0.5, 2.2, and 2.8 mm, respectively. The distances from the distal epiphysis to the line passing through the deep plantar arch perpendicular to the longitudinal axis of the lesser metatarsal on the sagittal plane were 47.0, 45.7, and 46.4 mm, respectively, and those from the proximal epiphysis were 23.0, 21.0, and 18.6 mm, respectively. The deep plantar arch coursed at the level of the middle third, proximal to this third in 11/20 (55.0%), 7/20 (35.0%), and 5/16 (31.2%) feet, respectively. Conclusion: This study suggested that overpenetration into the medial-plantar direction of the second metatarsal or the + +![0_image_0.png](0_image_0.png) + +proximal-plantar direction of the fourth metatarsal during the shaft or proximal osteotomy might injure the deep plantar arch easily. This study also suggested that on the plantar aspect, the shaft or proximal osteotomy about 45-47 mm proximal to the distal epiphysis of the lesser metatarsal or 18-23 mm distal to the proximal epiphysis might damage blood flow of the deep plantar arch. + +Foot & Ankle Orthopaedics, 5(4) DOI: 10.1177/2473011420S00467 ©The Author(s) 2020 \ No newline at end of file diff --git a/medical/md/PMC9039408.md b/medical/md/PMC9039408.md new file mode 100644 index 0000000000000000000000000000000000000000..861340822fda8f3a8cfd4d94d1f1b2fc68b296ef --- /dev/null +++ b/medical/md/PMC9039408.md @@ -0,0 +1,245 @@ +# Exercise Dose–Response Relationship + +with heart rate variability in individuals with overweight and obesity: protocol for a systematic review and metaanalysis of randomised controlled **trials** +Mukesh Kumar Sinha ,1 G Arun Maiya,1 Ana Maria Moga,2 Shivashankar K N,3 Ravi Shankar N,4 Vaishali K 1 To cite: Sinha MK, Maiya GA, +Moga AM, *et al*. Exercise dose– +response relationship with heart rate variability in individuals with overweight and obesity: protocol for a systematic review and meta-analysis of randomised controlled trials. *BMJ Open* 2022;12:e047821. doi:10.1136/ bmjopen-2020-047821 +► Prepublication history for this paper is available online. To view these files, please visit the journal online (http://dx.doi. org/10.1136/bmjopen-2020047821). Received 14 December 2020 Accepted 26 January 2022 © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. For numbered affiliations see end of article. Correspondence to Dr Vaishali K; vaishali.kh@manipal.edu + +## Abstract + +Introduction Obesity is a chronic relapsing disease process and serious public health concern that can lead to chronic diseases, medical complications and a higher risk of disability. Another significant feature of obesity is dysfunction in cardiac autonomic function, which leads to changes in parasympathetic and sympathetic regulation, which can be measured using heart rate variability (HRV). The objective of this review is to estimate the extent to which exercise doses impacts on HRV among individuals living with overweight and obesity class I and II. + +Methods and analysis A systematic literature search will be performed using PubMed/Medline, Scopus, EMBASE, ProQuest, CINAHL, Web of Science and the Cochrane Library for articles dating from 1965 to December 2021. + +Inclusion criteria include studies designed as parallelarm randomised trials, enrolling adolescent and adult individuals with overweight (body mass index, BMI≥25 to ≤29.9) and obesity (class I BMI: 30–34.9 and class II BMI: 35–39.9) undergoing aerobic or resistance training or concurrent exercise training. For data synthesis, sensitivity analysis, subgroup analysis and risk of bias assessment, Stata V.13.0 software will be used. Ethics and dissemination Formal ethical approval is not required. This systematic review will be submitted to a peer-reviewed journal. PROSPERO registration number CRD42019104154. + +## Introduction + +Over the past 35 years, the global prevalence of obesity has tripled and current trends, if extrapolated would lead to approximately over one billion people by 2030.1 2 Individuals living with obesity have a significantly higher risk of developing cardiovascular disease, diabetes, hypertension, cancer, stroke and chronic disease, including osteoarthritis.1 Obesity has also been linked to alteration in cardiac autonomic activity as seen when measuring heart rate variability (HRV).3 4 HRV is a non-invasive technique for analysing autonomic function by measuring beat-to-beat + +## Strengths And Limitations Of This Study + +► Strength of the design is the focus on dose–response analysis of intervention. + +► Two reviewers will perform data extraction and risk of bias evaluation separately. + +► Only English language article will be included. + +changes in R-R intervals of ECG signals.5 Low HRV is associated with higher skinfold thickness, higher body mass index (BMI), higher body fat percentages and is an autonomous predictor of cardiovascular mortality and sudden cardiac death.6–8 In contrast, higher HRV is found to be associated with reduced morbidity, mortality, improved quality of life and psychological well-being.9–11 Earlier studies have reported that obese individuals are relatively more susceptible to ventricular arrhythmias, which has been found to be a powerful indicator of sudden death.12–15 Several researchers have shown decreased HRV in obese people (BMI ≥30) +and this suggests that autonomic disturbances could be involved in the processes stimulating arrhythmia in such people.16–18 Weight loss by exercise training and dietary intervention, on the other hand, has been shown to reverse the detrimental impact of weight gain on autonomic function.6 7 18 19 Benefits of exercise training are documented as a possible non-pharmacological weight-loss approach.20 21 All forms of exercise, whether aerobic, resistance or combination of aerobic and resistance (concurrent), +are effective methods of improving anthropometric indicators of adiposity.22–24 These exercise types are characterised by multiple subdivisions such as frequency, intensity and volume of exercise that may be considered to constitute the exercise 'dosage'. The effectiveness of the exercise intervention in reducing body weight is documented as dose-dependent and it is mediated by autonomic control.25–29 Current evidence on the influence of long-term exercise training on HRV in healthy or obese individuals is inconsistent, with several studies showing significant increase in the HRV following an exercise training with varying dose ranging from 3 weeks to 12 months of exercise training in healthy and obese individuals7 19 30–33 while other studies did not show such an effect.34–36 Such differences in effect may be due to either participant attributes, a technique of measurement to estimate HRV, study design, exercise types and/or exercise dose parameter.36 A meta-analysis done using data from studies carried out in healthy people suggested that aerobic exercise training can make substantial improvements in the R-R interval, and the effect size for changes in the R-R interval recorded in this study was significantly higher in long exercise interventions (>12 weeks) than in shorter treatments (<12 weeks).29 Meta-analysis including studies done in the elderly37 suggested endurance-type exercise is effective for increasing HRV, and exercise frequency appears to be a powerful component of training that leads to HRV improvement. + +A recent meta-analysis19 reported improvement in HRV following weight-loss strategies such as dietary approaches, aerobic training, strength training and exercise programmes coupled with dietary approaches. Also, this study suggested that the impact of weight loss on the autonomic nervous system (ANS) might depend primarily on the amount of weight loss. Differences in the dosage of exercise, such as the duration, frequency and strength of exercise training, are considered to be responsible for the degree of improvement in autonomic cardiac function and the change in body weight.19 The exercise-based weight loss programmes are known to be a key part of therapy for obesity and evaluating its impact on HRV would add value to current assessments of the evidence base. In addition, no studies to date have comprehensively analysed and examined the evidence of exercise dose-response on HRV in people with overweight or obesity. Therefore, the objective of this review is to estimate the extent to which exercise-dose increases HRV in individuals living with overweight and obesity class I and II. + +## Methods Patient And Public Involvement + +No patient involved as it is a systematic review. The results will be disseminated by the publication of the manuscript in a peer-reviewed journal. + +The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Protocols guidelines are used for the current study. PRISMA will be used to assist reporting of the SR, once completed.38 This systematic review will consider only randomised controlled trials +(RCTs). Any amendments to this study protocol will be reported. + +## Electronic Search + +Seven databases will be searched; PubMed/Medline, Scopus, EMBASE, ProQuest, CINAHL, Web of Science and the Cochrane Library, for articles dating from 1965 to December 2021.5 We will also refer to ClinicalTrials. + +gov, the WHO's registry platform ICTRP, the reference lists of key articles identified via Scopus and articles that cited the included articles. Also, authors will be contacted to obtain for studies that have been completed but not published. If more than one publication describes the same study, the one that provides the most data will be included in the meta-analysis. Studies will be limited to publications in the English language. The search will be carried out by the first author and a medical librarian. Box 1 Shows the search strategy for PubMed. + +## Eligibility Criteria And Study Selection + +The titles and abstracts screening will be done for eligibility and the article considered appropriate will be reviewed in full-text papers. This process will be conducted using Covidence (www.covidence.org)39 and it is expected to be completed by December 2021. + +## Inclusion Criteria + +Studies will be included if they report data from (1) parallel-arm RCTs, (2) enrolled adolescent (age ≥10 years) and adult individuals with overweight (BMI≥25 to ≤29.9) and obesity (class I BMI: 30–34.9 and class II BMI: 35–39.9) undergoing aerobic or resistance exercise training or concurrent exercise training (table 1) +27 and had an outcome of interest as HRV (3) exercise intervention is reported in terms of frequency, intensity, time and type, and (4) measurement of at least one variable of HRV before and after the training intervention is reported. + +## Exclusion Criteria + +Exclusion criteria will be (1) observational studies, (2) studies measuring acute exercise effects, (3) obesity class III (BMI ≥40) as it has been found that individuals living with severe obesity may have impaired autonomic function and this would confound the outcome of interest, and (4) studies including individuals with cardiac, neurodegenerative, kidney or metabolic disease as they have an impact on autonomic function.37 40 41 + +## Study Selection + +Following different database searches, retrieved articles will be imported to the Covidence platform39 where the results will be combined and duplicates will be removed. As a large number of papers are expected to require screening, four authors will be involved in screening. + +These authors will also perform pilot-testing of eligibility criteria on the first 10% of titles and abstracts. To harmonise the screening process, a training session will be provided to all reviewers. During this session, + +## Box 1 **Shows The Search Strategy For Pubmed** + +((((((((((((((((((("exercise"[MeSH Terms]) OR exercise) OR exercise[Text Word]) OR exercise[Title]) OR exercise[Title/Abstract])) OR ((((aerobic exercise) OR aerobic exercise[Text Word]) OR aerobic exercise[Title]) OR +aerobic exercise[Title/Abstract])) OR ((((resistance exercise) OR resistance exercise[Text Word]) OR resistance exercise[Title]) OR resistance exercise[Title/Abstract])) OR ((((concurrent exercise) OR concurrent exercise[Text Word]) OR concurrent exercise[Title]) OR concurrent exercise[Title/Abstract])) OR ((((combination exercise) OR combination exercise[Text Word]) OR combination exercise[Title]) OR combination exercise[Title/Abstract])) OR ((((("resistance training"[MeSH Terms]) OR resistance training) OR resistance training[Text Word]) OR resistance training[Title]) OR resistance training[Title/Abstract])) OR ((((aerobic training) OR aerobic training[Text Word]) OR aerobic training[Title]) OR aerobic training[Title/Abstract])) exercise dose OR dose response OR +aerobic dose OR resistance dose OR concurrent dose OR combination dose OR ((((("cardiorespiratory fitness"[MeSH Terms]) OR cardiorespiratory fitness[Text Word]) OR cardiorespiratory fitness[Title]) OR +cardiorespiratory fitness[Title/Abstract]) OR cardiorespiratory fitness)) OR ((((physical activity) OR physical activity[Text Word]) OR physical activity[Title]) OR physical activity[Title/Abstract])) OR ((((cardiorespiratory endurence) OR cardiorespiratory endurence[Text Word]) OR cardiorespiratory endurence[Title]) OR cardiorespiratory endurence[Title/ +Abstract])) OR ((((strength training) OR strength training[Text Word]) OR strength training[Title]) OR strength training[Title/Abstract])) OR ((((strengthening) OR strengthening[Text Word]) OR strengthening[Title]) OR strengthening[Title/Abstract]))) AND (((((((("overweight"[MeSH Terms]) OR overweight) OR overweight[- Text Word]) OR overweight[Title]) OR overweight[Title/Abstract])) OR ((((("obesity"[MeSH Terms]) OR obesity) OR obesity[Text Word]) OR obesity[Title]) OR obesity[Title/Abstract])) OR ((((healthy individuals[- Text Word]) OR healthy individuals[Title]) OR healthy individuals[Title/ Abstract]) OR healthy individuals))) AND (((((((((heart rate variability) OR heart rate variability[Text Word]) OR heart rate variability[Title]) OR heart rate variability[Title/Abstract])) OR ((((autonomic function) OR autonomic function[Text Word]) OR autonomic function[Title]) OR autonomic function[Title/Abstract])) OR ((((sympathetic function) OR sympathetic function[Text Word]) OR sympathetic function[Title]) OR sympathetic function[Title/Abstract])) OR ((((parasympathetic function) OR parasympathetic function[Text Word]) OR +parasympathetic function[Title]) OR parasympathetic function[Title/ Abstract])) OR ((((vagal function) OR vagal function[Text Word]) OR vagal function[Title]) OR vagal function[Title/Abstract])) +reviewer will be asked to pilot-screen 15 titles/abstracts to prompt clarifications and screening decisions will be taken in compliance with inclusion/exclusion criteria. After scanning for titles and abstracts, articles that do not meet the inclusion requirements will be excluded and the remaining articles will have their full-text versions retrieved. The full-text screening will be done by two lead members of the synthesis team using the level of agreement between reviewers. Kappa statistics will be used to test the agreement (ie, thresholds: <0.20 slight agreement, 0.21–0.40 fair agreement, 0.41–0.60 moderate agreement, 0.61–0.80 substantial agreement and >0.80 almost perfect agreement).42 Disputes will be settled by + +| Table 1 | Operational definitions of exercises type used for | +|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------| +| the current systematic review according to the American College of sports Medicine27 Exercise type Operational definition Aerobic/ endurance exercise training Aerobic exercise as any activity that uses large muscle groups, can be maintained continuously and is rhythmic in nature Resistance/ strength exercise training Strength training that involves the performance of physical exercises which are designed to improve muscle strength and endurance Concurrent The combination of muscle strength and exercise training aerobic exercise during the same session or training programme | | + +agreement with the reviewers or by contacting an adjudicator. In a PRISMA flow chart, the study selection process is displayed(figure 1).43 + +## Data Extraction And Analysis + +Outcomes In this study, the primary outcome of interest is the time domain (SDNN, SDANN, RMSSD, pNN50) and frequency domain variables of HRV (total power, VLF, LF, HF, LF/ HF ratio) (table 2). + +Secondary outcomes include cardiorespiratory endurance, muscular strength, adiposity/anthropometric measures. These outcomes are chosen based on the previous research.7 19 30–33 If data are available in qualifying studies, the relationship of exercise training with other endpoints, such as time effect and interaction effect with sociodemographic variables, anthropometric measures, presence of cardiovascular risk factors, diet, exercise adherence and life stress, will also be analysed. + +## Data Extraction + +A data extraction form will be adopted from published literature.44–46 Data extraction process consists of manuscript title, author, time of publication, source of literature, characteristics of the trial (author, conducted/ +publication year, duration, place of the trial conducted, number of participating centres, study design), the participants (sample size, participants randomised and patients analysed in each group, age, sex, socioeconomic status, height, weight, BMI, waist circumference, waist-hip ratio, waist-height ratio and body fat per cent), intervention (aerobic, resistance and concurrent exercise dose in terms of frequency, intensity, number of sessions, duration and progression), control group treatment, method of randomisation, method of allocation, blinding process, outcome time point and follow-up period, lack of follow-up or withdrawal, any incidental findings reported and the main outcome measurement HRV reported either in absolute or log transformed or both. Two independent reviewers will pilot test the data extraction form and to resolve any disagreements team meetings will be conducted to refine the form. The two reviewers will perform data extraction separately. A training session + +![3_image_0.png](3_image_0.png) + +Systematic Reviews and Meta-Analyses. + +| Table 2 | Heart rate variability (HRV) domains5 | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------| +| Time domain measures of HRV: variable (units) and description SDNN (ms) SD of all NN intervals SDANN (ms) SD of the averages of NN intervals RMSSD (ms) The square root of the mean of the sum of the squares of differences between adjacent NN intervals pNN50 % NN50 count divided by the total no of all NN intervals Frequency domain measures of HRV: variable (units) and description Total power ms2 The variation of NN intervals over the temporal segment VLF (ms2 ) Power in very low frequency range LF (ms2 ) Power in low frequency range HF (ms2 ) Power in high frequency range LF/HF ratio Ratio of LF and HF | | + +4 *et al. BMJ Open*12 + +will be held to harmonise the extraction of data, and at least two pilot extractions will be carried out to ensure accuracy. A written 'Data Extraction Guide' with detailed instructions will also be provided to reviewers. To assure accuracy, one lead member of the systematic review team will extract data from each article. An impartial third reviewer will cross-check the data extracted in duplicate. Inconsistencies in the data obtained will be resolved by agreement between the reviewers after reviewing the fulltext article. When discrepancies occur, an adjudicator will be contacted. If the data published is incomplete or vague, the authors of the research will be contacted. Data extraction will be independently cross-checked. + +## Quality And Risk Of Bias Assessment + +Two reviewers will independently review each selected article to eliminate bias. All selected articles will be evaluated for their quality based on the Cochrane Collaboration's Risk of Bias Tool 2.047 for risk of bias assessment across five domains. Assessments will be carried out using an iterative online form available.48 The domain of missing outcome data will be evaluated, as per Akobeng and Ebrahim *et al*. + +49 50 For each domain, the probability of bias will be evaluated as 'low risk', 'some concerns' or 'high risk'. If at least one area is listed as 'high risk', studies will be deemed to have an overall high risk of bias. Quality of evidence will be measured using the GRADE +(Grading of Recommendations, Assessment, Development and Evaluations) rating system.51 Publication bias will be evaluated using visual inspection of funnel plot asymmetry.52 + +## Data Synthesis Strategy: Meta-Analysis + +We will primarily examine the training effect (aerobic, resistance and concurrent exercise training) on HRV. We will also explore possible sources of heterogeneity among studies by examining aerobic, resistance and concurrent exercise impact with time point. To attain the standardised mean difference and 95% CI, the data of interest given as continuous will be used for meta-analysis. The Q-statistic and I2 tests will be used to test for heterogeneity between the included studies. Heterogeneity will be considered low if I² is ≤40%, and high if I² is ≥75%. We will use a randomeffects model for meta-analysis If substantial heterogeneity (I²>40 %) or fixed effects for homogeneous effects +(I²<40%).53 Aggregate data obtained from the included studies will be used for quantitative synthesis. By plotting the data on a forest plot, heterogeneity will be evaluated visually.54 + +## Analysis Of Subgroups Or Subsets + +The subanalysis will include baseline participant characteristics and exercise intervention characteristics. Interaction effects between variables will be identified for subgroup analysis.54 + +## Significance + +Due to modernisation and mechanisation of lifestyle, there is an increase in overweight and obesity globally. Exercise is a key element to prevent lifestyle disease, therefore, it is important to explore dose–response benefits specifically towards HRV to maximise the physiological benefits. The study would help to understand the autonomic response of the heart (ie, HRV) at different doses of exercise training. Also can help to recommend the training regimen for overweight and obese people for optimum gain in HRV. + +## Ethics And Dissemination + +This review will not require an ethical authorisation, since participant privacy issues do not exist. Our results will provide data on the various forms of exercise doseresponse on the HRV in overweight and obese people. The results of this study will be published in a peerreviewed international journal, displayed at relevant conferences and disseminated to obesity-focused public organisations. + +Author affiliations 1Department of Physiotherapy, Manipal College of Health Professions, Manipal Academy of Higher Education, Manipal, Karnataka, India 2School of Physical and Occupational Therapy, Faculty of Medicine and Health Sciences, McGill University, Montreal, Quebec, Canada 3Department of Medicine, Kasturba Medical college, Manipal Academy of Higher Education, Manipal, Karnataka, India 4Department of Biostatistics, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India Contributors MKS, VK, AMM and GAM conceived of the study and provided guidance for drafting the protocol. AMM and MKS designed the search strategy. MKS, VK, AMM, SKN, RSN and GAM drafted and reviewed the final version systematic review protocol manuscript for submission. All the contributors read and approved the final manuscript. Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. Patient consent for publication Not applicable. Provenance and peer review Not commissioned; externally peer reviewed. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iDs Mukesh Kumar Sinha http://orcid.org/0000-0002-7407-7153 Vaishali K http://orcid.org/0000-0001-5746-1682 + +## References + +1 O'Donoghue G, Blake C, Cunningham C, *et al*. What exercise prescription is optimal to improve body composition and cardiorespiratory fitness in adults living with obesity? A network meta-analysis. *Obes Rev* 2021;22:e13137. + +2 World Health Organization. Obesity and overweight fact sheet, 2014. + +Available: http://www.who.int/mediacentre/factsheets/fs311/en/ [Accessed 21 Jul 2020]. + +3 Fidan-Yaylali G, Yaylali YT, Erdogan Çağdaş, *et al*. The association between central adiposity and autonomic dysfunction in obesity. Med Princ Pract 2016;25:442–8. + +4 Das D, Mondal H. Evaluation of cardiac autonomic function in overweight males: a cross-sectional study. *Adv Hum Biol* 2017;7:23–6. + +5 Heart rate variability: standards of measurement, physiological interpretation and clinical use. Task force of the European Society of cardiology and the North American Society of pacing and electrophysiology. *Circulation* 1996;93:1043–65. + +6 Chen LY, Zmora R, Duval S. Cardiorespiratory fitness, adiposity, and heart rate variability: the cardia study. *Med Sci Sports Exerc* 2019;51:509. + +7 Tian Y, Huang C, He Z, *et al*. Autonomic function responses to training: correlation with body composition changes. *Physiol Behav* 2015;151:308–13. + +8 Goldenberg I, Goldkorn R, Shlomo N, *et al*. Heart rate variability for risk assessment of myocardial ischemia in patients without known coronary artery disease: the HRV-DETECT (heart rate variability for the detection of myocardial ischemia) study. *J Am Heart Assoc* 2019;8:e014540. + +9 Schmid K, Schönlebe J, Drexler H, *et al*. Associations between being overweight, variability in heart rate, and well-being in the young men. Cardiol Young 2010;20:54–9. + +10 Gutin B, Howe C, Johnson MH, *et al*. Heart rate variability in adolescents: relations to physical activity, fitness, and adiposity. *Med* Sci Sports Exerc 2005;37:1856–63. + +11 Young HA, Benton D. Heart-rate variability: a biomarker to study the influence of nutrition on physiological and psychological health? Behav Pharmacol 2018;29:140–51. + +12 Csige I, Ujvárosy D, Szabó Z, *et al*. The impact of obesity on the cardiovascular system. *J Diabetes Res* 2018;2018:1–12. + +13 Ashraf MJ, Baweja P. Obesity: the 'huge' problem in cardiovascular diseases. *Mo Med* 2013;110:499. + +14 Poirier P, Giles TD, Bray GA, *et al*. Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight loss: an update of the 1997 American heart association scientific statement on obesity and heart disease from the obesity Committee of the Council on nutrition, physical activity, and metabolism. *Circulation* 2006;113:898–918. + +15 Avsar A, Acarturk G, Melek M, *et al*. Cardiac autonomic function evaluated by the heart rate turbulence method was not changed in obese patients without co-morbidities. *J Korean Med Sci* 2007;22:629–32. + +16 Karason K, Mølgaard H, Wikstrand J, *et al*. Heart rate variability in obesity and the effect of weight loss. *Am J Cardiol* 1999;83:1242–7. + +17 Adam M, Imboden M, Schaffner E, *et al*. The adverse impact of obesity on heart rate variability is modified by a NFE2L2 gene variant: the SAPALDIA cohort. *Int J Cardiol* 2017;228:341–6. + +18 Lambert EA, Esler MD, Schlaich MP. Obesity-Associated organ damage and sympathetic nervous activity: a target for treatment? Hypertension 2019;73:1150–9. + +19 Costa J, Moreira A, Moreira P, *et al*. Effects of weight changes in the autonomic nervous system: A systematic review and meta-analysis. Clin Nutr 2019;38:110–26. + +20 Vina J, Sanchis-Gomar F, Martinez-Bello V, *et al*. Exercise acts as a drug; the pharmacological benefits of exercise. *Br J Pharmacol* 2012;167:1–12. + +21 Higuera-Hernández MF, Reyes-Cuapio E, Gutiérrez-Mendoza M, +et al. Fighting obesity: non-pharmacological interventions. *Clin Nutr* ESPEN 2018;25:50–5. + +22 García-Hermoso A, Ramírez-Vélez R, Ramírez-Campillo R, *et al*. + +Concurrent aerobic plus resistance exercise versus aerobic exercise alone to improve health outcomes in paediatric obesity: a systematic review and meta-analysis. *Br J Sports Med* 2018;52:161–6. + +23 Schwingshackl L, Dias S, Strasser B, *et al*. Impact of different training modalities on anthropometric and metabolic characteristics in overweight/obese subjects: a systematic review and network meta-analysis. *PLoS One* 2013;8:e82853. + +24 Monteiro PA, Chen KY, Lira FS, *et al*. Concurrent and aerobic exercise training promote similar benefits in body composition and metabolic profiles in obese adolescents. *Lipids Health Dis* 2015;14:153. + +25 Chaput J-P, Klingenberg L, Rosenkilde M, *et al*. Physical activity plays an important role in body weight regulation. *J Obes* 2011;2011:360257 doi:10.1155/2011/360257 26 Batrakoulis A, Fatouros IG, Chatzinikolaou A, *et al*. Dose-Response effects of high-intensity interval neuromuscular exercise training on weight loss, performance, health and quality of life in inactive obese adults: study rationale, design and methods of the DoIT trial. Contemp Clin Trials Commun 2019;15:100386. + +27 American College of Sports Medicine. *ACSM's exercise testing and* prescription. Lippincott williams & wilkins, 2017. + +28 Gifford RM, Boos CJ, Reynolds RM, *et al*. Recovery time and heart rate variability following extreme endurance exercise in healthy women. *Physiol Rep* 2018;6:e13905. + +29 Sandercock GRH, Bromley PD, Brodie DA. Effects of exercise on heart rate variability: inferences from meta-analysis. Med Sci Sports Exerc 2005;37:433–9. + +30 Facchini M, Malfatto G, Sala L, *et al*. Changes of autonomic cardiac profile after a 3-week integrated body weight reduction program in severely obese patients. *J Endocrinol Invest* 2003;26:138–42. + +31 Phoemsapthawee J, Prasertsri P, Leelayuwat N. Heart rate variability responses to a combined exercise training program: correlation with adiposity and cardiorespiratory fitness changes in obese young men. J Exerc Rehabil 2019;15:114–22. + +32 Goulopoulou S, Baynard T, Franklin RM, *et al*. Exercise training improves cardiovascular autonomic modulation in response to glucose ingestion in obese adults with and without type 2 diabetes mellitus. *Metabolism* 2010;59:901–10. + +33 Routledge FS, Campbell TS, McFetridge-Durdle JA, *et al*. + +Improvements in heart rate variability with exercise therapy. *Can J* Cardiol 2010;26:303–12. + +34 Boutcher SH, Stein P. Association between heart rate variability and training response in sedentary middle-aged men. *Eur J Appl Physiol* Occup Physiol 1995;70:75–80. + +35 Davy KP, Willis WL, Seals DR. Influence of exercise training on heart rate variability in post-menopausal women with elevated arterial blood pressure. *Clin Physiol* 1997;17:31–40. + +36 Bhati P, Moiz JA, Menon GR, *et al*. Does resistance training modulate cardiac autonomic control? A systematic review and meta-analysis. Clin Auton Res 2019;29:75–103. + +37 Raffin J, Barthélémy J-C, Dupré C, *et al*. Exercise frequency determines heart rate variability gains in older people: a metaanalysis and meta-regression. *Sports Med* 2019;49:719–29. + +38 Moher D, Shamseer L, Clarke M, *et al*. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. *Syst Rev* 2015;4. + +39 Covidence systematic review software, veritas health innovation, Melbourne, Australia. Available: www.covidence.org [Accessed 29 Sep 2020]. + +40 Buccelletti E, Gilardi E, Scaini E, *et al*. Heart rate variability and myocardial infarction: systematic literature review and metanalysis. Eur Rev Med Pharmacol Sci 2009;13:299–307. + +41 Christensen JH. Cardiac autonomic dysfunction in hemodialysis patients assessed by heart rate variability. *Miner Urol Nefrol* 2012;64:191–8. + +42 Landis JR, Koch GG. The measurement of observer agreement for categorical data. *Biometrics* 1977;33:159–74. + +43 Prisma flow diagram. Available: http://www.prisma-statement.org +[Accessed 14 Dec 2020]. + +44 Centre for Reviews and Dissemination (CRD). Systematic Reviews: +CRD's Guidance for Undertaking Reviews in Health Care. 3 edition. York, UK: CRD, Univeristy of York, 2009. Available: http://www.york. ac.uk/inst/crd/pdf/ Systematic_Reviews.pdf 45 Higgins J, Green S. Cochrane Handbook for systematic reviews of interventions, version 5.1.0. The Cochrane Collaboration, 2011. + +46 Shaw KA, Gennat HC, O'Rourke P, *et al*. Exercise for overweight or obesity. *Cochrane Database Syst Rev* 2006;73:CD003817. + +47 Sterne JAC, Savović J, Page MJ, *et al*. Rob 2: a revised tool for assessing risk of bias in randomised trials. BMJ 2019;366:l4898. + +48 Risk of bias tools. Available: https://www.riskofbias.info 49 Akobeng AK. Understanding systematic reviews and meta-analysis. + +Arch Dis Child 2005;90:845–8. + +50 Ebrahim S, Akl EA, Mustafa RA, *et al*. Addressing continuous data for participants excluded from trial analysis: a guide for systematic reviewers. *J Clin Epidemiol* 2013;66:1014–21. + +51 Guyatt GH, Oxman AD, Vist GE, *et al*. Grade: an emerging consensus on rating quality of evidence and strength of recommendations. BMJ 2008;336:924–6. + +52 Sterne JAC, Sutton AJ, Ioannidis JPA, *et al*. Recommendations for examining and interpreting funnel plot asymmetry in meta-analyses of randomised controlled trials. BMJ 2011;343:d4002. + +53 Ho M, Garnett SP, Baur L, *et al*. Effectiveness of lifestyle interventions in child obesity: systematic review with meta-analysis. *Pediatrics* 2012;130:e1647–71. + +54 Heterogeneity and subgroup analyses in Cochrane consumers and communication review group reviews: planning the analysis at protocol stage, 2020. Available: https://cccrg.cochrane.org 55 Moher D, Liberati A, Tetzlaff J, *et al*. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. *PLoS* Med 2009;6:e1000097. \ No newline at end of file diff --git a/medical/md/PMC9064423.md b/medical/md/PMC9064423.md new file mode 100644 index 0000000000000000000000000000000000000000..ea779373084eff650246315738de848f9baccac2 --- /dev/null +++ b/medical/md/PMC9064423.md @@ -0,0 +1,275 @@ +# + +Cite this: RSC Adv., 2019, 9, 16900 + +# Global Ab Initio Exploration Of Potential Energy + +![0_Image_0.Png](0_Image_0.Png) Surfaces For Radical Generation In The Initial Stage Of Benzene Oxidation† + +## Hai-Bei Li * And Qingqing Jia + +The potential energy surfaces (PESs) of benzene oxidation by molecular oxygen were explored using the anharmonic downward distortion following (ADDF) and artificial force induced reaction (AFIR) methods of the global reaction route mapping (GRRM) strategy. The reaction mechanism of benzene activation by initial molecular oxygen depends on the combustion temperature. At high temperature, the benzene molecule could be oxidized by abstracting hydrogen atoms and form the radical fragments, C6H5 and OOH. However, before reaching its auto-ignition point, the formation of a singlet bridging peroxide molecule C6H6O2 from the triplet reactants via electronic non-adiabatic transition will play a critical role in the increase of the combustion temperature by the generation of initial free radicals. Bridging peroxide C6H6O2 could isomerize to other stable isomers by a consecutive series of oxygen and hydrogen atom transfers. Importantly, these C6H6O2 isomers are vital sources of free radical generation in the initial stage of benzene oxidation. Free radicals, such as OOH, O, and OH, could be generated during the further oxidation of these oxygenated hydrocarbon species C6H6O2 due to the presence of active groups or sp3-C–H bonds. + +Received 24th April 2019 Accepted 16th May 2019 DOI: 10.1039/c9ra03048d rsc.li/rsc-advances + +## 1. Introduction + +The pervasive use and importance of aromatic compounds in fuels contrast strikingly with uncertainties in their major reaction pathways and limited chemical kinetic and thermodynamic data. Benzene is one of the simplest aromatic hydrocarbons and its chemical kinetics of combustion have long been of interest as a mirror to the study of the chemical and physical processes of the formation of polycyclic aromatic hydrocarbons (PAH) and soot. + +Many efforts have been made experimentally and theoretically in benzene combustion over the last three decades to understand and evaluate the important reaction pathways and to validate the mechanisms.1–15 It is well known that hydrocarbon combustion follows a chain reaction mechanism, where free radicals play a dominant role in the destruction of reactant molecules. Free radicals such as OOH, OH, O, and H, are well known to be active in the benzene ame10,16–18 as well as in other hydrocarbon combustion.14,19–21 In near-sooting premixed lowpressure ames of benzene, Bittner and Howard2 have observed an abundance of the free radicals OH, O, OOH, and H +in the benzene ame and suggested that the activation and decomposition of benzene are initiated by the oxidation of atomic O, while that by molecular oxygen and other free radicals would be too slow to account for their experimental observations, which is consistent with the observations by Ko et al.22 While, Madronich and Felder17 found that the hydrogen abstraction channel by OH radical plays a critical role in benzene consumption, giving an important intermediate phenyl radial. In contrast, in hydrogen-rich environments, hydrogen abstraction by atomic H is predicted to be important in the benzene oxidation, due to its low activation barrier.8 These benzene destruction mechanisms by free radical species have been well proven by a large number of kinetic modeling studies, which are used to present a quantitative description of the combustion processes.6–9,16–18,22 In addition to these combustion models, investigations of the reaction pathways of benzene activation by OH and H radicals have been undertaken using high-level ab initio methods,23,24 which provide evidence that hydrogen abstractions from benzene by these radicals are favorable with a low activation energy of around 10 kcal mol1. All of these investigations indicated that free radicals, such as OH, O and atomic H, play crucial roles in benzene decomposition and subsequent PAH formation. A +chain reaction mechanism consists of several steps, which are the initiation of radicals, then their propagation and branching, followed by a termination process. It is obvious that, until now, almost all investigations of benzene combustion have focused mainly on the reactions between free radicals and benzene or its derivatives, that is, on the propagation and branching reactions. + +School of Ocean, Shandong University, Weihai 264209, People's Republic of China. + +E-mail: lihaibei@sdu.edu.cn +† Electronic supplementary information (ESI) available: The Gibbs free energy surfaces for all of the corresponding PESs. See DOI: 10.1039/c9ra03048d The signicance of these free radicals in benzene combustion makes it reasonable to raise questions such as how these free radicals are generated in the initial stage of benzene oxidation. The proposed mechanism for the initiation step of benzene oxidation by molecular oxygen is the generation of a phenyl radical through hydrogen abstraction.4,25 Westbrook and coworkers16 using chemical kinetic modeling have proposed that in benzene combustion, hydrogen abstraction by molecular oxygen is the principal initiation reaction as well as that by various free radicals. However, theoretical studies of benzene oxidation, that is C6H6 + O2, are still rare, except for recent work,26 where the barrier to hydrogen abstraction by molecular oxygen was calculated to be around 250 kJ mol1 by density functional theory calculations. The higher activation barrier implies that it is difficult to generate the free radicals via direct hydrogen abstraction by molecular oxygen in the initial stage of benzene oxidation. Despite many experimental and modeling efforts, a large number of uncertainties still exist regarding the main benzene combustion kinetics and reaction pathways, such as the initial chemical reactions of benzene oxidation, C6H6 + O2, the chemistry of free radical generation, and the fate of the oxygenated C6 and C5 species. For instance, it was reported that an oxygenated hydrocarbon compound C6H6O2 achieves a certain population in an early stage of benzene combustion.2 However, its generation mechanism and how this species decomposes into free radicals have not yet been explained well. The importance of the generation of free radicals in the initial stage of combustion could be imagined, which will induce and simultaneously determine the subsequent ignition and combustion processes. Therefore, it is necessary to systematically investigate the elementary processes of free radical production in the initial stage of benzene oxidation. + +Global reaction route mapping (GRRM)27 has been conrmed as nding all local minima and transition states on single potential energy surfaces and providing a systematic elucidation of complex chemical reaction mechanisms. Thus, for free radical generation in benzene oxidation, GRRM is a powerful tool to globally explore its reaction mechanism. The present work focuses on systematically elucidating the fundamental chemical and physical processes taking place in the initial stage of benzene oxidation, with special attention on free radical generation. The lowest reaction routines for benzene activation and the generation of free radicals will be presented by systematically exploring all the possible chemical processes which involve a series of competitive reaction paths. This detailed theoretical study could provide deeper insights into the chemical kinetics and important reaction pathways involved in benzene oxidation. + +## 2. Computational Methods + +All the potential energy surfaces (PESs) were explored by the global reaction route mapping (GRRM) program28 in which energies, gradient, and Hessian were calculated using the Gaussian 09 program29 as an external subroutine. The reaction pathways between two molecules were searched using the articial force induced reaction (AFIR) method.27 The isomerization and dissociation pathways starting from stable intermediates were explored by the GRRM method,30,31 where the lADDF option of the GRRM method, which searches for the 10 lowest barrier paths around each intermediate, was adopted in this study.32 These automated searches, performed at the UB3LYP/6-31+G(d) level, gave several reaction pathways starting from each intermediate. The pathways with barriers higher than Elowest + 50 kJ mol1 were omitted, where Elowest is the barrier height for the most preferred path among them. All obtained structures were re-optimized at the UB3LYP/cc-pVTZ level, and frequency analysis calculations were performed to conrm the local minima (with zero imaginary frequency) and the transition states (TSs, possessing one and only one imaginary frequency) +on the potential energy surfaces. The intrinsic reaction coordinate (IRC) method33 was also performed to ensure that the TS +connects two appropriate local minima in the reaction pathway. Zero-point energy (ZPE) corrections were taken into account during the calculation of barrier heights. The Gibbs free energies, which are presented in the ESI,† were calculated including ZPE and thermal correction at 1000.0 K. Some minimum energy structures on seam-of-crossing hyperspaces (MESXs) were searched as critical points of non-adiabatic transitions between the ground singlet and triplet PESs at the same level. The UB3LYP method might result in signicant error due to the absence of dispersion or signicant spin contamination. For MESXs searching, Maeda and coworkers26 have conrmed that, for the oxidation of hydrocarbon, the UB3LYP method in combination with the aug-cc-pVDZ basis set could give similar results and correct energy trends which were obtained at CASPT2/aug-cc-pVDZ. Thus, considering the high requirement for computing resources in this work, we applied UB3LYP/ccpVTZ to investigate the potential energy surfaces of benzene oxidation. + +Based on the thermodynamic parameters obtained from the above calculation, we calculated the reaction rate constant for the rst step of benzene oxidation, utilizing the well-known Rice–Ramsperger–Kassel–Marcus (RRKM) theory with the expression as follows: + +$$k(E)={\frac{W(E-E_{0})}{h\rho(E)}}$$ + +hrðEÞ (1) +where W(E E0) is the rovibrational sum of states at the geometry of the transition state, E0 is the reaction activation energy, r(E) is the density of rovibrational states of the reactant at energy E, and h is Planck's constant. Based on microcanonical nonadiabatic transition state theory,34 we also calculated the rate constant for spin-hopping processes using the Landau–Zener spin-hopping model.35 + +$$\left(1\right)$$ +$$k(E)={\frac{W_{\mathrm{mESX}}(E)}{h\rho(E)}}$$ +$$\mathbf{\mu}$$ +$$\mathbf{\Sigma}$$ +$\downarrow$ . + +$$W_{\mathrm{MESX}}(E)=\int_{0}^{E}\rho^{\mathrm{MESX}}(E-E_{\mathrm{H}})P_{\mathrm{SH}}(E)\mathrm{d}E_{\mathrm{H}}$$ + +$$P_{\mathrm{SH}}(E_{\mathrm{H}})=(1+P)(1-P)$$ +PSH(EH) ¼ (1 + P)(1 P) (4) +$\mathfrak{AXP}$ +P ¼ exp-2pH122 + +![2_image_0.png](2_image_0.png) + +r (5) +where WMESX(E) is the convolution of the density of states at the geometry of MESX, r(E) has the same meaning as in formula (1), +rMESX(E EH) is the density of rovibrational states for the degrees of freedom within the crossing seam at the MESX, EH is the part of the available energy which is in coordinates orthogonal to the seam, and PSH(E) is the spin-forbidden hopping probabilities. In formula (5), H12 is the matrix element for coupling between the two surfaces, m is the reduced mass for movement along the vector orthogonal to the singlet/ +triplet crossing seam, and DF is the slope of two surfaces at the crossing seam. All of the computational details are described elsewhere.34 We calculated the rate constants using the open-source code, MESMER.36 + +## 3. Results And Discussion 3.1 Activation Of Benzene + +Fig. 1 shows all of the optimized structures of intermediates, transition states, and MESXs with their energies for the considered reaction pathways during benzene interacting with the rst molecular oxygen. The singlet and triplet PESs for all possible reaction pathways of benzene activation are presented in Fig. 2. The corresponding Gibbs free energies are supplied in the ESI (Fig. S1–S4†). In the initial stage of benzene oxidation, ground state triplet molecular oxygen instead of excited singlet state would have a high population. Interestingly, for the triplet reaction pathways, two well-known radicals, phenyl and phenoxy, are generated through the reaction pathways 10 / TS10/90 +/ 90 / C6H5 + OOH, and 10 / TS10/110 / 110 / TS110/120 / +120/ C6H5O + OH, respectively. The generation of a phenyl radical is directly by the hydrogen abstraction process (C6H6 + 3O2/ C6H5 + OOH). This hydrogen abstraction mechanism has been proposed for the activation of benzene in previous studies.4,25 Direct hydrogen abstraction from benzene by triplet molecular oxygen is quite endothermic, requiring around 260.0 kJ mol1 in order to generate the radical fragments C6H5 and OOH. This high barrier makes it difficult to activate benzene through direct hydrogen abstraction by initial molecular oxygen at a low temperature. This is consistent with a theoretical study concerning the initial chemical events of benzene oxidation by molecular dynamic simulation using a reactive force eld,37 where there is no C–H bond cleavage by molecular oxygen. Compared to hydrogen abstraction, the generation of a phenoxy radical is much more difficult. The initial molecular oxygen inserts into the C–H bond forming phenyl hydroperoxide, followed by the dissociation of O–O +peroxide group generating phenoxy, C6H5O and OH. It has been proposed from kinetic modeling that species C6H5O is highly abundant and is considered to play critical roles in the subsequent formation of small fragments.18 While here in the former insertion process, transition state TS10/110 lies 332.1 kJ mol1 higher than the reactants. This higher barrier indicates that species C6H5O is not produced by initial molecular oxygen, but might be by atomic oxygen, as suggested by Richter et al.18 On + +$\square$ +the other hand, the higher barrier of this path also demonstrates that the formation of phenyl hydroperoxide is highly unlikely by the addition of initial molecular oxygen. + +The other likely pathway for benzene activation, i.e., +production of 2, 8, 9, or 10, involves a singlet molecular oxygen generated by the non-adiabatic transition from triplet to singlet state PES through the lowest MESX1 (see Fig. 2). The generation of singlet molecular oxygen has been discussed in detail in ref. + +26. The spin-orbital coupling for MESX1 is calculated with a value of 159.9 cm1 using the state-averaged CASSCF method and the cc-pVTZ basis set with an active space including 10 electrons and 8 orbitals. Unsurprisingly, the generated singlet molecular oxygen could react further with another benzene molecule to produce intermediates 8, 9, or 10 as well as intermediate 2. Thermodynamically, intermediate 8 with one trans- +$C–C–O–O$ group is not stable. Instead of transforming into phenyl hydroperoxide via a high barrier transition state TS8/11, it could quickly reverse to the reactants, C6H6 + 1O2, through TS1/8 or C6H6 + 3O2 via MESX4. Correspondingly, the molecular oxygen could also attack one carbon atom, generating a cis-$C– +C–O–O$ group in TS1/10, producing intermediate 10 with 4/6membered rings. The barrier for this path is comparable to the direct hydrogen abstraction process of benzene on the singlet state PES, 1 / TS1/9 / 9 / C6H5 + OOH, both requiring around 205 kJ mol1 210 kJ mol1. In contrast to all these reaction paths starting from reactants C6H6 + 1O2 ( +3O2), +the reaction pathway, 10 / MESX1 / TS1/2 / 2, is the most favorable way in benzene activation, which requires around 209.6 kJ mol1 to produce the bridging peroxide molecule 2. + +This is in good agreement with the experimental results,2 where oxygenated hydrocarbon compound C6H6O2 has been observed early in the benzene ame. In order to assess the relative importance of reaction channels under different temperature conditions, we performed a calculation of the reaction rate constant (k) based on the well-known RRKM theory, as shown in Fig. 3. At a lower temperature, the production of bridging peroxide molecule 2 is predominant due to the easier nonadiabatic transition from triplet to singlet molecular oxygen. + +With an increase in combustion temperature, the hydrogen abstraction processes 1 (10) / TS1/9(TS10/90) / 9(90) / C6H5 + +OOH become more important. At a temperature higher than 1200 K, which is much higher than its auto-ignition temperature, the reaction rate of hydrogen abstraction by molecular oxygen has exceeded that of the formation of the bridging peroxide molecule 2. This illustrates that at high temperature, the benzene molecule could be easily oxidized by abstracting the hydrogen atoms and forming radical fragments C6H5 and OOH. However, before it reaches its auto-ignition point, the production of bridging peroxide molecule 2 will play a critical role in the increase of the combustion temperature by the generation of initial free radicals. + +Obviously, the bridging peroxide structure of intermediate 2 is not stable kinetically and thermodynamically, especially at high temperature. The unstable feature of structure 2 is characterized by the approach of another triplet molecular oxygen, which results in the scission of the peroxy O–O bond forming complex 3 + O2 with a barrier height of 97.8 kcal mol1 (the IRC + + + +![3_image_0.png](3_image_0.png) + +calculation with the TS and product are presented in Fig. S5†). This indicates that the entropy effect at high temperature has a signicant inuence on the stability of intermediate 2. This characteristic of intermediate 2 might be the reason why some research groups preferred the hydrogen abstraction mechanism in the initiation step,4,25 instead of the formation of a bridging peroxide molecule. We stepwisely explored the global minimum isomerization processes for the oxygenated hydrocarbon compound C6H6O2 based on the structure of intermediate 2. + +The rst isomerization step 2 / 3, the scission of the bridging O–O peroxy bond and the formation of two sp3-C atoms and two radical sites C–O$, was found to have a barrier height of 98.0 kJ mol1, and this reaction is endothermic by 28.2 kJ mol1. Compared to bridging peroxide structure 2, intermediate 3 is more active with two radical sites. It is liable to transform into structure 4 by oxygen transfer, forming one epoxide group. Step 3 / 4 has a barrier height of only 35.5 kJ mol1 and is quite exothermic by 325.6 kJ mol1 (see Fig. 2). The active epoxide group in structure 4 induces further isomerization, 4 / TS4/5 / 5 / TS5/6 / 6, corresponding to oxygen and hydrogen transfer, respectively, generating more stable intermediates 6. The high stability of 4 and 6 provides direct evidence to explain the long-term existence of the species C6H6O2 early in the benzene ame.2 Further carbonyl-phenol tautomerization from 6 to 7 requires a higher barrier height with a value of 236.0 kJ mol1, which implies that this isomerization has a low probability of occurrence. The small mole fraction of species C6H6O2 in the benzene ame, which is shown in Fig. 7 in ref. 2, indicates that, in addition to these consecutive isomerization processes, 2 / 3 / 4 / 5 / 6, it is + +![4_image_0.png](4_image_0.png) + +![4_image_1.png](4_image_1.png) + +highly likely there will be other competitive or more favorable reaction pathways for the consumption of the species C6H6O2. + +Therefore, we will consider the further oxidation of intermediates 3, 4, and 6 by adding another molecular oxygen in the following section. + +## 3.2 Generation Of Radicals + +The computed PESs for the interactions between intermediates 3, 4, 6 and molecular oxygen are depicted in Fig. 4–6. As discussed above, bridging peroxide intermediate 2 could automatically transform into isomer 3 following the approach of the second molecular oxygen. Thus, instead of considering the oxidation of intermediate 2, we present the PES for the oxidation of intermediate 3 in Fig. 4. Structure 3 has two H–C–O$ +groups which nd it easier to form C]O carbonyl group by losing the hydrogen atoms. Compared to the isomerization step 3 / 4, the hydrogen abstraction process by the second molecular oxygen is more favorable kinetically for reaction path 13 / 14 with a calculated barrier of only 7.3 kJ mol1. It was found that the interaction between triplet intermediate 30 and molecular oxygen has almost the same barrier height as that in reaction 13 / 14. Step 13 / 14 is found to be 205.7 kJ mol1 exothermic and product 14 is a complex with one carbonyl group hydrogen-bonded to an OOH radical. Aer this, two possible pathways could happen, (1) the OOH radical moves away from species 15 aer absorbing heat of 42.0 kJ mol1 and forms free radicals, which could play pivotal roles with other radicals in the following ignition and combustion processes, or (2) the OOH radical, roaming to the other side of fragment 15, abstracts hydrogen from the other sp3-C atom with only a small barrier of 27.9 kJ mol1, and produces complex 19 consisting of H2O2 and C6H4O2 fragments. The production of H2O2 is another source of free radical generation. It has been found that the decomposition of H2O2 could produce OH or OOH radicals under different experimental conditions.38 Although the barriers for these two processes are similar, the OOH radical is likely to dissociate before nding the H atom to be abstracted, especially at high temperature because the migration length of the OOH in the isomerization from 14 to 18 is pretty long and also because the dissociation energy (15 + OOH) is 18.1 kJ mol1 lower than that of TS18/19. In this case, the rst free radical in the initiation step of benzene oxidation should be OOH. If there are many free radicals such as OOH around 15, it will + +![5_image_0.png](5_image_0.png) + +immediately be dehydrogenated into 1,4-benzoquinone through TS like TS18/19. The produced intermediate 15, C 6 H 5 O 2 , has three possibilities for isomerization to structures 16, 20, and 21 by oxygen and hydrogen transfer, respectively. Thermodynamically, reaction 15 → 21 is more exothermic with an energy of 194.6 kJ mol-¹, while this reaction has a higher barrier of 58.1 kJ mol¯¹, compared to reactions 15 → 16 and 15 +-> 20 with only 30.5 kJ mol-' and 37.6 kJ mol-', respectively. + +Intermediate 16 is easier to generate kinetically, which is quickly followed by the formation of a seven-membered ring + +![5_image_1.png](5_image_1.png) + +![6_image_0.png](6_image_0.png) + +isomer 17 via C–C bond cleavage of the epoxide group. The structures and their energies for isomers 15–21 have been studied by Mebel and coworkers,39 to elucidate the reaction mechanisms of the fundamental reaction, C6H5 + O2, which produces atomic hydrogen or oxygen. It could be imagined that the isomers 15 and 20 could immediately lose a hydrogen atom to form a stable conjugated structure 1,4-benzoquinone by another molecular oxygen or other radicals in a later stage of combustion. + +In addition to isomerization to 5, there are two alternative reaction pathways for intermediate 4, which are further oxidation by another molecular oxygen, as illustrated in Fig. 5. Unlike the active structure 3 with its radical sites, intermediate 4 has one epoxy group and two sp3-C–H bonds, which could result in +(1) a ring-opening in the epoxy group and the subsequent production of a chain structure with a terminal peroxide radical, 22 / TS22/23 / 23 / 24, or (2) hydrogen atom abstraction from an sp3-C atom, generating an OOH radical and intermediate 16, 22 / TS22/34 / 34 / 16. The ring-opening exhibits a barrier of 157.8 kJ mol1, which is close to the corresponding values of 160.2 kJ mol1 (TS22/34) and 159.4 kJ mol1 (TS4/5) for the parallel hydrogen abstraction and isomerization processes, respectively, indicating that these three reaction pathways for intermediate 4 are competitive. On the one hand, hydrogen abstraction of intermediate 4 with molecular oxygen could produce an OOH radical. On the other hand, chain intermediate 24 is not stable due to its active peroxide radical site, which is liable to lose the terminal atomic 3O, forming a stable intermediate 31 with double-ended aldehyde groups, as shown in Fig. 5. This process requires only 50.0 kJ mol1. In addition, the generated atomic 3O atom could also actively abstract an adjacent hydrogen atom with only 11.9 kJ mol1 and produce stable complex 26 with an OH +radical, 25 / TS25/26 / 26. Complex 26 is likely to lose an OH +radical, and then chain structure 27 proceeds through several rearrangements to stable conjugated structure 30 with one radical site. A further reaction between the OH radical and the remaining fragment in 26 forming 32 has a higher barrier (TS26/32) than OH radical dissociation, and thus is a minor channel. In short, the further oxidation of intermediate 4, to some extent, could result in the generation of OOH, O and OH +radicals. + +As mentioned above, an alternative reaction pathway for intermediate 4 is isomerization to 6 via the sequential migration of oxygen and hydrogen atoms. In Fig. 2, intermediate 6 is located in a deep well on the PES of C6H5O2. It needs to overcome a barrier height of 236.0 kJ mol1 to isomerize into structure 7 through hydrogen transfer. However, structure 6 has two sp3-C atoms with two sp3-C–H bonds, respectively, which has a higher possibility for further oxidization by another molecular oxygen. The PES for the further oxidation of intermediate 6 is shown in Fig. 6. Two hydrogen atoms were abstracted from two sp3-C atoms with reaction barriers of 120.9 kJ mol1 and 152.9 kJ mol1, respectively, giving complexes 36 and 41 with an OOH radical through hydrogenbonding interactions. Obviously, complex 36 is more stable due to the higher delocalization of the lone electron within the p network of the oxygenated hydrocarbon fragment. Thus, the reaction 35 / TS35/36 / 36 has a higher possibility of proceeding kinetically and thermodynamically, due to its lower barrier height and being less endothermic. Complex 36 could produce intermediate 37 aer absorbing heat of 40.6 kJ mol1, giving free radical OOH. Alternatively, the generated OOH +radical, wandering around 37, might abstract the second hydrogen atom from the sp3-C atom, giving intermediate 40 and an active H2O2 molecule, 36 / 38 / TS38/39 / 39 / 40. + +Undoubtedly, the latter path requires a higher barrier than the former one, which prefers to happen in the following ignition and combustion step. + +It is worth noting that the oxygenated hydrocarbon species C6H6O2 can be vital sources of the initial free radical generation, such as OOH, OH, and atomic 3O. Free radical OOH is likely to be generated by the further oxidation of C6H6O2 through TS13/ +14. The presence of a terminal peroxide radical group in chain structure 24 could easily split into stable aldehyde and atomic 3O, which subsequently abstracts hydrogen atom, giving radical OH through TS25/26. Up to now, this is the rst time the reaction mechanisms for the initial generation of free radicals in benzene oxidation have been systematically studied. + +## 3.3 Role Of Radicals On Pah Growth And Small Fragment Dissociation + +As mentioned in the above discussion, free radicals OOH, OH, +and O could be produced from the oxidation of the species C6H6O2. The importance of these free radicals including H2O2 in the combustion chemistry has been studied in detail earlier,16,18–21,40 which mainly focused on small aliphatic hydrocarbons. They could abstract hydrogen atoms from fuel and generate hydrocarbon radical species. Importantly, they could induce chain reactions and introduce more and more free radicals into the combustion system.18 These chain-propagation processes in the combustion account to a considerable extent for the combustion kinetics, including not only the oxidation of hydrocarbon fuels to nal products, such as CO2, H2O, and CO, +but also the growth of PAH fragments. The oxidation of benzene by free radicals has also been studied,6,22,24,39 and was found to be much easier compared with oxidation by molecular oxygen. + +It only requires around 40 kJ mol1 to abstract a hydrogen atom by OH24 or atomic H,23 generating the hydrocarbon radical C6H5. In this way, free radicals will play a leading role in the activation of most benzene molecules by abstracting hydrogen atoms, which could explain why the mole fraction of C6H6O2 is not large in experiments.2 In addition to the radical C6H5, species C6H6O and C5H6 were also observed as having large mole fractions early in the ame,2 but their formation mechanisms have not yet been investigated. It would be interesting to study thoroughly how these free radicals, such as OH, O, OOH, +and H, oxidize benzene molecules using ADDF and AFIR +methods and nd the most favorable reaction pathway for benzene oxidation in combustion and explain the experimental observations. Furthermore, the generated species C6H5 could not only be further oxidized41 by molecular or atomic oxygen via radical–molecule or radical–radical reaction mechanisms producing CO2, and CO, but also could react with other hydrocarbon radical or benzene molecules forming PAH fragments. It is well known that PAH fragments are widespread organic pollutants, while their formation mechanisms are still in a period of exploration.42,43 Therefore, one of the most urgent tasks is to demonstrate clearly the reaction mechanism for PAH +growth. Using ADDF and AFIR methods could be expected to supply deeper insights into the formation mechanism of PAH +fragments in the near future. These works are currently in progress within our group. + +## 4. Conclusions + +We have explored the various potential energy surfaces (PESs) of the oxidation of benzene by molecular oxygen using the powerful anharmonic downward distortion following (ADDF) +and articial force induced reaction (AFIR) methods of the global reaction route mapping (GRRM) strategy. Before reaching the auto-ignition point, the most favorable reaction route of benzene activation is the addition of singlet molecular oxygen, forming a singlet bridging peroxide molecule C6H6O2. The system undergoes non-adiabatic transition from triplet to singlet PES during the initiation step of benzene oxidation. The formed bridging peroxide molecule could isomerize to three stable isomers by stepwise oxygen and hydrogen transfer. The further oxidations of these oxygenated hydrocarbon species were studied in detail by the addition of another triplet molecular oxygen. Interestingly, it was found that species C6H6O2 are important sources for free radical generation in the initial stage of benzene oxidation. Free radicals, such as OH, OOH and O, could be generated when the above isomers C6H6O2 interact with molecular oxygen. The molecular oxygen could easily abstract hydrogen atoms from the sp3-C atom of the isomers of C6H6O2, generating OOH radicals. Alternatively, it could also attack one C atom, resulting in ring opening and the formation of a ring structure with a terminal peroxide radical group, which could further dissociate into atomic O. These initial free radical species could propagate and branch into more free radicals through chain-propagation processes. They would play critical roles in the following PAH growth and production of small molecules, such as CO2, CO and H2O. + +In the present study, the reaction mechanism of free radical generation in the initial stage of benzene oxidation have been systematically studied theoretically. Due to the high requirement for computing resources, we have to admit that the UB3LYP/cc-pVTZ level applied in this work is not the best computational method to study the non-adiabatic transitions and the chemical kinetics, although it has been conrmed to give the correct energy trends compared with the CASPT2/augcc-pVDZ level [J. Phys. Chem. Lett., 2011, 2, 852]. A higher computational level is needed for a further and more accurate description of the reaction kinetics. + +## Conflicts Of Interest + +There are no conicts to declare. + +## Acknowledgements + +We thank the National Natural Science Foundation of China +(21403127) and the Natural Science Foundation of Shandong Province, China (ZR2014BQ015). Hai-Bei Li thanks the fund of Young Scholars Program of Shandong University (Weihai), +YSPSDUWH, and the supercomputing system at the Supercomputing Center, Shandong University, Weihai. + +## References + +1 N. Fujii and T. Asaba, Symposium (International) on Combustion, 1973, 14, 433–442. + +2 J. D. Bittner and J. B. Howard, Symposium (International) on Combustion, 1981, 18, 1105–1116. + +3 R. D. Kern, H. J. Singh, M. A. Esslinger and P. W. Winkeler, Symposium (International) on Combustion, 1982, 19, 1351– +1358. + +4 C. Venkat, K. Brezinsky and I. Glassman, Symposium +(International) on Combustion, 1982, 19, 143–152. + +5 H. J. Singh and R. D. Kern, Combust. Flame, 1983, 54, 49–59. + +6 J. H. Kiefer, L. J. Mizerka, M. R. Patel and H. C. Wei, J. Phys. + +Chem., 1985, 89, 2013–2019. + +7 R. P. Lindstedt and G. Skevis, Combust. Flame, 1994, 99, 551– +561. + +8 R. A. Shandross, J. P. Longwell and J. B. Howard, Symposium +(International) on Combustion, 1996, 26, 711–719. + +9 Y. Tan and P. Frank, Symposium (International) on Combustion, 1996, 26, 677–684. + +10 A. Sch¨obel, A. G. Class, L. Krebs, M. Braun-Unkhoff, C. Wahl and P. Frank, Chemosphere, 2001, 42, 591–599. + +11 F. Defoeux, V. Dias, C. Renard, P. J. Van Tiggelen and J. Vandooren, Proc. Combust. Inst., 2005, 30, 1407–1415. + +12 H. Richter, S. Granata, W. H. Green and J. B. Howard, Proc. + +Combust. Inst., 2005, 30, 1397–1405. + +13 S.-I. Shih, T.-C. Lin and M. Shih, J. Hazard. Mater., 2005, 117, 149–159. + +14 J. M. Simmie, Prog. Energy Combust. Sci., 2003, 29, 599–634. + +15 M. Frenklach, Z. Liu, R. I. Singh, G. R. Galimova, V. N. Azyazov and A. M. Mebel, Combust. Flame, 2018, 188, 284–306. + +16 C. K. Westbrook and F. L. Dryer, Prog. Energy Combust. Sci., +1984, 10, 1–57. + +17 S. Madronich and W. Felder, J. Phys. Chem., 1985, 89, 3556– +3561. + +18 H. Richter and J. B. Howard, Phys. Chem. Chem. Phys., 2002, 4, 2038–2055. + +19 D. E. Hoare and M. Patel, Trans. Faraday Soc., 1969, 65, 1325– +1333. + +20 V. N. Kondratiev and V. V. Azatyan, Symposium (International) +on Combustion, 1973, 14, 37–44. + +21 X. Zhong and J. W. Bozzelli, J. Phys. Chem. A, 1998, 102, 3537– +3555. + +22 T. Ko, G. Y. Adusei and A. Fontijn, J. Phys. Chem., 1991, 95, 8745–8748. + +23 A. M. Mebel, M. C. Lin, T. Yu and K. Morokuma, J. Phys. + +Chem. A, 1997, 101, 3189–3196. + +24 D. S. Hollman, A. C. Simmonett and H. F. Schaefer, Phys. + +Chem. Chem. Phys., 2011, 13, 2214–2221. + +25 G. Bermudez and L. Pfefferle, Combust. Flame, 1995, 100, 41– +51. + +26 S. Maeda, R. Saito and K. Morokuma, J. Phys. Chem. Lett., +2011, 2, 852–857. + +27 S. Maeda and K. Morokuma, J. Chem. Phys., 2010, 132, 241102–241104. + +28 S. Maeda, Y. Osada, K. Morokuma and K. Ohno, GRRM, +a developmental version, 2011. + +29 G. W. T. M. J. Frisch, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. ratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2009. + +30 K. Ohno and S. Maeda, Chem. Phys. Lett., 2004, 384, 277–282. 31 S. Maeda and K. Ohno, J. Phys. Chem. A, 2005, 109, 5742– +5753. + +32 S. Maeda and K. Ohno, J. Phys. Chem. A, 2007, 111, 4527– +4534. + +33 K. Fukui, Acc. Chem. Res., 1981, 14, 363–368. 34 J. N. Harvey, Phys. Chem. Chem. Phys., 2007, 9, 331–343. + +35 A. W. Jasper, C. Zhu, S. Nangia and D. G. Truhlar, Faraday Discuss., 2004, 127, 1–22. + +36 D. R. Glowacki, C.-H. Liang, C. Morley, M. J. Pilling and S. H. Robertson, J. Phys. Chem. A, 2012, 116, 9545–9560. + +37 K. Chenoweth, A. C. T. van Duin and W. A. Goddard, J. Phys. + +Chem. A, 2008, 112, 1040–1053. + +38 H. Kijewski and J. Troe, Int. J. Chem. Kinet., 1971, 3, 223–235. + +39 A. M. Mebel and M. C. Lin, J. Am. Chem. Soc., 1994, 116, 9577–9584. + +40 R. W. Walker, Symposium (International) on Combustion, 1989, 22, 883–892. + +41 P. Frank, J. Herzler, T. Just and C. Wahl, Symposium +(International) on Combustion, 1994, 25, 833–840. + +42 H. Richter and J. B. Howard, Prog. Energy Combust. Sci., 2000, 26, 565–608. + +43 D. Golea, Y. Rezgui, M. Guemini and S. Hamdane, J. Phys. + +Chem. A, 2012, 116, 3625–3642. \ No newline at end of file diff --git a/medical/md/PMC9298267.md b/medical/md/PMC9298267.md new file mode 100644 index 0000000000000000000000000000000000000000..18130c9e44c62d31690937d50b1a62e99c1ed371 --- /dev/null +++ b/medical/md/PMC9298267.md @@ -0,0 +1,297 @@ +# Fluorination Reactions At A Platinum Carbene **Complex:** Reaction Routes To Sf3, S(=O)F And Fluorido **Complexes** + +Dilcan Dirican,[a] Maria Talavera,[a] and Thomas Braun*[a] +Abstract: The electron-rich Pt complex [Pt(IMes)2] (IMes: [1,3bis(2,4,6-trimethylphenyl)-2-imidazolinylidine]) can be used as precursor for the syntheses of a variety of fluorido ligand containing compounds. The sulfur fluoride SF4 undergoes a rapid oxidative addition at Pt0 to yield *trans*-[Pt(F)(SF3)(IMes)2]. + +A photolytic reaction of SF6 at [Pt(IMes)2] in the presence of IMes gave the fluorido complexes *trans*-[Pt(F)2(IMes)2] and trans-[Pt(F)(SF3)(IMes)2] along with *trans*-[Pt(F)(SOF)(IMes)2] +and *trans*-[Pt(F)(IMes')(IMes)] (IMes': cyclometalated IMes ligand), the latter being products produced by reaction with adventitious water. *trans*-[Pt(F)(SOF)(IMes)2] and *trans*-[Pt- (F)2(IMes)2] were synthesized independently by treatment of [Pt(IMes)2] with SOF2 or XeF2. A reaction of [Pt(IMes)2] with a HF source gave *trans*-[Pt(H)(F)(IMes)2], and an intermediate bifluorido complex *trans*-[Pt(H)(FHF)(IMes)2] was identified. + +Compound *trans*-[Pt(H)(F)(IMes)2] converts in the presence of CsF into *trans*-[Pt(F)(IMes')(IMes)]. + +## Introduction + +Transition metal fluorido complexes often display unique properties when compared to other halogenido analogues.[1] +Reaction pathways include metal-mediated fluorination reactions, but transition metal fluorides can also play a role in catalytic reaction cycles, for instance in borylation reactions. + +Such transformations can impart C F bond activation reactions of highly fluorinated organic compounds to access fluorinated building blocks.[2] Catalytic fluorination reactions can occur via transition metal fluorination with nucleophilic or electrophilic fluorinating agents.[1f,i,k,3] However, other strategies to synthesize particular fluorido complexes are based on oxidative addition reactions, for instance with XeF2, or on ligand exchange reactions involving fluoride sources like AgF and HF.[1j,4] +At Pt, various Pt fluorido complexes were prepared by treatment of iodo complexes with AgF by the research groups of Vigalok, Wendt, Beyzavi, Shahsavari and Beweries.[5] Seppelt et al. and Perutz et al. described routes to access fluorido complexes by treatment of methyl or hydrido compounds with HF sources.[6] Other conversions include the generation of fluorido complexes by treatment of Pt precursors with NFSI +(NFSI: N-fluorobenzenesulfonimide), or Selectfluor (Nchloromethyl-N'-fluorotriethylenediammonium bis +(tetrafluoroborate).[3b,7] Love and co-workers reported on C F +bond activation reactions to result in Pt(IV) fluorido complexes.[2g,u,3c,8] Another method to access Pt fluorido complexes imparts fluorination with XeF2. Thus, oxidative addition reactions of XeF2 at Pt(II) yielded Pt(IV) difluorido compounds.[9] +Treatment of monoaryl Pt(IV) complexes with electrophilic fluorinating agents like XeF2 can led to C F bond formation as shown by Gagné, Vigalok and Vedernikov.[7,10] Similar reactions with diaryl ligand containing Pt resulted in difluorination at the metal along with reductive elimination of biaryl compounds.[5a,11] Alternatively, it was demonstrated that metalbound alkyne ligands at Pt(0) are fluorinated by XeF2 yielding βfluorovinyl Pt(II) complexes, which can undergo further fluorination processes to give *ortho*-metalated Pt(IV) fluorido compounds.[3d] Platinum fluorido complexes can generally play certain roles in metal-mediated fluorination and defluorination reactions.[1a,m,2a,c,e,g,m,p,u,3c,10] +SF4 and its derivatives are applied for deoxyfluorination reactions, for instance of ketones or alcohols.[12] At rhodium, iridium and platinum centers, SF4 converts by oxidative addition into SF3 fluorido complexes.[13] SF6, on the other hand, is fairly inert and applied as dielectric in high-voltage power devices. + +Reactivity studies of SF6 at transition metal complexes are rare. + +They led to the generation of a variety of metal fluorido complexes as it was shown by Ernst, Limberg, or Braun et al.[14] +Remarkably, the reaction of SF6 at the platinum phosphine complexes [Pt(PR3)2] (R=Cy, iPr) in the presence of free phosphine enabled the generation of λ4-trifluoro sulfanyl ligands to yield *trans*-[Pt(F)(SF3)(PR3)2].[13d,15] It was additionally shown that the sulfur(VI) fluorides SF5X (X=Cl, CF3) react with iron, rhodium, iridium or platinum complexes to result in a complete defluorination at sulfur by fluorination of the metal centres.[16] Peacock and co-workers proposed the formation of a SF5 complex by reaction of [Pt(E-PhC=CPh)(PPh3)2] with SF5Cl, but this report was not confirmed further.[16c,17] However, the generation of SF5 anions was observed at rhodium complexes Chem. *Eur.* J. **2021**, 27, 17707–17712 17707 © 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH +when [Rh(X)(CO)(IMes)2] (X=F, Cl) was reacted with SF5Cl.[18] +Note that Pt(0) complexes are generally suitable for the element-fluorine bond activation as it was shown for instance by Braunschweig and Finze et al. with BF3 and PF5 as substrates.[19] +In this contribution we report on the reactivity of the electron-rich carbene complex [Pt(IMes)2] (1, IMes: (1,3-bis(2,4,6trimethylphenyl)-2-imidazolinylidine]) towards the sulfur fluorides SF4, SOF2 and SF6. The generation of sulfanyl ligands and fluorido complexes was observed. The latter were synthesized independently by investigating the reactivity of [Pt(IMes)2] +towards HF sources and XeF2. + +## Results And **Discussion** + +Treatment of [Pt(IMes)2] (1) with SF4 gave the trifluorosulfanyl fluorido complex *trans*-[Pt(F)(SF3)(IMes)2] (2) (Scheme 1). The conversion resembles the reactions of [Pt(PR3)2] (R=Cy, iPr) +with SF4, which led to the formation of *trans*-[Pt(F)(SF3)(PR3)2].[13d] +The 19F NMR data of 2 are comparable to those of the phosphine analogues *trans*-[Pt(F)(SF3)(PR3)2] (PR3=PCy3, PiPr3). + +The 19F NMR spectrum shows a doublet with Pt satellites ( +2JF,Pt= +86 Hz) at δ=60.9 ppm for the two axial fluorine atoms of the sulfanyl ligand of a trigonal-bipyramidal configuration at sulfur, when taking the electron lone pair at sulfur into account +(Figure 1). The signal exhibits a coupling of 2JF,F=71 Hz to the equatorial fluorine atom at the SF3 group. The resonance for the latter appears at higher field at δ= 67.9 ppm as a triplet with Pt satellites ( +2JF,Pt=297 Hz). The signal for the metal-bound fluorido ligand is found as a singlet with Pt satellites ( +1JF,Pt= +Scheme 1. S F oxidative addition reactions at [Pt(IMes)2] (1). + +![1_image_0.png](1_image_0.png) + +![1_image_2.png](1_image_2.png) + +227 Hz) at δ= 318.7 ppm, which is a typical chemical shift for transition metal bound fluorido ligands.[1e,i,2h,3b,d,5a,6b,8d,11b,13d,15,20] The two IMes ligands give one set of signals in the 1H and 13C +{ +1H} NMR spectra. On 13C labeling of the carbene carbon atom at IMes the 13C{1H} NMR spectrum of *trans*-[Pt(F)(SF3)(13IMes)2] +(2') revealed a doublet at δ=169.5 ppm with a coupling to the metal bound fluoride ( +2JC,F=6 Hz) and Pt satellites ( +1JC,Pt= +1123 Hz). + +The above mentioned trigonal-bipyramidal geometry at the sulfur atom was also confirmed by DFT calculations of 2 (BP86/ +def2-SVP).[13d–f] The frontier orbital analysis revealed that the electron pair is reflected by the HOMO (Figure 2). Hence, the λ4trifluoro sulfanyl ligand can be considered as an one-electron donor or two-electron donor, when the sulfanyl group is considered to be anionic.[21] +A reaction of [Pt(IMes)2] (1) with SF6 for 4 h at 60°C in a PFA +tube did result in a depletion of the sulfur fluoride, but only a mixture of platinum containing products was observed consisting of *trans*-[Pt(F)(SOF)(IMes)2] (3), *trans*-[Pt(H)(F)(IMes)2] (4) and trans-[Pt(F)(IMes')(IMes)] (5) (IMes'=cyclometalated IMes ligand) +in a ratio of 1:1.2:1.6. In addition, the complexes *trans*- +[Pt(F)2(IMes)2] (6) and cis-[Pt(H)2(IMes)2] (cis-7) were identified in traces. Apparently, adventitious water hampered the generation of a SF3 ligand containing complex. The reaction was then performed in the presence of additional IMes, which could trap generated HF formed by hydrolysis, but also act as an acceptor for fluorine atoms during the course of the SF6 activation. As mentioned above, the conversion of SF6 at [Pt(PR3)2] (R=Cy, iPr) +is promoted by phosphine to give *trans*-[Pt(F)(SF3)(PR3)2] and F2PR3. + +[13d,15] Yet, reactions of [Pt(IMes)2] (1) with SF6 in the presence of IMes at higher temperatures in toluene-d8 were again not selective, but after 8 h small amounts of the sulfanyl complex *trans*-[Pt(F)(SF3)(IMes)2] (2) were detected. A better conversion was found upon irradiation of SF6 with 1 and IMes for 40 min at 311 nm in toluene-d8 to result in the generation of trans-[Pt(F)(SF3)(IMes)2] (2), *trans*-[Pt(F)(SOF)(IMes)2] (3), *trans*- [Pt(F)2(IMes)2] (6) and *trans*-[Pt(F)(IMes')(IMes)] (5) in a ratio of 1:0.9:0.4:1.7 (Scheme 2), as well as small amounts of the fluorinated carbene IMes-F2 (IMes-F2=C2H2(NMes)2CF2). An addi- + +![1_image_1.png](1_image_1.png) + +![2_image_0.png](2_image_0.png) + +tional platinum compound was observed in traces, which could not be characterized further (see Supporting Information). The added IMes acts mainly as base, giving upon reaction with HF +[IMesH][FHF], which precipitated out of the reaction solution.[22] +When 13C labeled 13IMes was added instead of IMes, +[ +13IMesH][FHF] was furnished. + +Mechanistically, it was suggested in the literature that the activation of SF6 can be initiated by a SET from the metal to SF6 to give the SF6 +· radical anion.[13g,14a–c,23] This process might be facilitated by irradiation; and photocatalytic conversions of SF6 were described.[23b,c,f,g,i] Fast decomposition of SF6 +· can then lead either to SF5 and F· or SF5 +· and F depending on the electron excess energy.[24] Other assumptions include a nucleophilic attack of strong nucleophiles at SF6. + +[25] +The SF6 activation at [Pt(IMes)2] (1) to yield *trans*-[Pt- +(F)(SF3)(IMes)2] (2) is accompanied by the generation of *trans*- [Pt(F)2(IMes)2] (6). Thus, 1 seems to act as additional acceptor of fluorine atoms. Note also that a photolytic SF6 activation at 311 nm with IMes has been reported to produce IMes-F2 and the thiourea derivative IMes=S, but a much longer reaction time was required to achieve a considerable conversion.[23b] +However, such a background reaction seems not to occur as no thiourea derivative was detected, even when 13C labeled 13IMes was added to the reaction mixture instead of IMes. Only traces of 13IMes-F2 were then found in solution. + +A partial reaction of 2 with adventitious water would yield trans-[Pt(F)(SOF)(IMes)2] (3) and HF. In turn, the presence of HF +can lead by reaction with 1 to the formation of *trans*- +[Pt(H)(F)(IMes)2] (4), which then can produce *trans*-[Pt- +(F)(IMes')(IMes)] (5) by cyclometalation (see below). An independent experiment showed that *trans*-[Pt(F)2(IMes)2] (6) is not prone for a cyclometalation process. In another independent reaction it was shown, that the irradiation of *trans*-[Pt- +(H)(F)(IMes)2] (4) in the presence of free IMes at 311 nm leads to the formation of [IMesH][FHF], which also explains the absence of 4 in the product mixture. + +To verify the identity of the complexes 3–6 reaction routes for their independent preparations were developed. Thus, complex *trans*-[Pt(F)(SOF)(IMes)2] (3) was synthesized by treatment of [Pt(IMes)2] (1) with SOF2 (Scheme 1). An oxidative addition of SOF2 at a metal center under preservation of the S(=O)F function is to the best of our knowledge unprecedented. + +Reactions of SOF2 at fine powdered Ag(0), Cu(0) and Zn(0) as well as laser-ablated U(0) atoms led to fluorination and sulfurization of the metals, and in the latter case U(O)(S)(F)2 was furnished.[26] Treatment of the metal oxides Ag2O, CuO and ZnO +with SOF2 resulted in the fluorination of the metal and release of SO2. + +[26a] The sulfur-bound fluorine atom in 3 reveals a resonance in the 19F NMR spectrum at low field at δ= 7.3 ppm as a singlet with Pt satellites ( +2JF,Pt=140 Hz). Comparable data were described for other metal bound S(=O)F groups.[13d,15] The resonance for the fluorido ligand appears at δ= 289.2 ppm as a singlet ( +1JF,Pt=826 Hz) at a typical range for metal bound fluorides.[1e,i,2h,3b,d,5a,6b,8d,11b,13d,15,20,27] The 13C{1H} NMR resonance for the 13C labeled carbene complex *trans*-[Pt(F)(SOF)(13IMes)2] (3') appears at δ=174.2 ppm as a doublet with a coupling of 2JC,F= +5 Hz to the fluorido ligand ( +1JC,Pt=1199 Hz). The distorted tetrahedral geometry at the sulfur atom was confirmed by DFT calculations of 3 (BP86/def2-SVP). The frontier orbital analysis revealed that the electron pair resembles the HOMO (see Supporting Information). + +In order to investigate the possible generation of the cyclometalated complex *trans*-[Pt(F)(IMes')(IMes)] (5) from the hydrido fluorido compound *trans*-[Pt(H)(F)(IMes)2] (4), the reactivity of Pt(IMes)2] (1) towards a HF source was studied to access 4. Treatment of 1 with an excess of the hydrogen fluoride source PVP·HF (PVP·HF=Poly-[4-vinylpyridiniumpoly +(hydrogenfluoride)]) led to oxidative addition of HF followed by the formation of a bifluorido ligand in the coordination sphere of Pt (Scheme 3). Removal of the solvent from the product solution under reduced pressure afforded the bifluorido complex *trans*-[Pt(H)(FHF)(IMes)2] (8). Transition metal complexes with hydrido bifluorido ligands are described in the literature and often result from reaction of the corresponding hydrido compounds with an excess HF or coordination of HF at fluorido ligands.[1j,3b,5d,6b,9a,27] A subsequent conversion of 8 into the fluorido complex *trans*-[Pt(H)(F)(IMes)2] (4) was successful by removal of HF from the ligand sphere by addition of CsF to a solution of 8 in C6H6 (Scheme 3). + +Compound *trans*-[Pt(H)(FHF)(IMes)2] (8) exhibits in the 1H +NMR spectrum a resonance for the proton in the FHF entity at δ=12.91 ppm as a broad doublet with a coupling constant of 372 Hz to the distal fluorine. The hydrido ligand gives a doublet at -=27.37 ppm with Pt satellites ( +2JH,F=130, 1JH,Pt=1623 Hz) +and 1H{19F} NMR measurements confirm the coupling to the + +![2_image_1.png](2_image_1.png) + +proximal fluoride in the *trans* position. In the 19F NMR spectrum two resonances of equal intensity were found. One signal appears at δ= 179.1 ppm as a broadened doublet with a coupling constant of 1JF,H=373 Hz to the FHF proton and is assigned to the distal fluorine.[1j,3b,6b,14d,28] The signal for the proximal fluorine atom was detected at δ= 269.5 ppm as a broadened multiplet with a coupling to Pt of 1JF,Pt=714 Hz. The 13C{1H} NMR spectrum of the 13C labeled isotopologue 8' revealed a broadened 13C signal at 180.6 ppm with Pt satellites +( +1JC,Pt=1070 Hz). The ATR IR spectrum of 8 shows vibrational bands at ~v=420 and 445 cm 1, which can be assigned to Pt-F +vibrations.[3b,d,5b,13d,15,29] Strong absorption bands for the stretching vibration of Pt-H appear at ~v=2314 cm 1, which has been confirmed by DFT calculations. Additionally, two broad vibrational bands for the FHF unit are present in the IR spectrum of 8 at ~v=2453 cm 1 and ~v=1906 cm 1. For comparison, the vibrational modes for the FHF unit in *trans*-[Pt(H)(FHF)(PiPr3)2] +appear at ~v=2273 and 1903 cm 1 and for [Pt(PhC=CFPh)(FHF)- +{k2(P,N)-iPrPC2H4NMe2}] at ~v=2495 and 1807 cm 1. + +[3b,6b] The vibration bands for the FHF ligand in 8 appear at higher wavenumbers than the data for bifluorido salts (~v=1284– +1372 cm 1), which demonstrates that the hydrogen bonding interaction in the FHF ligand is asymmetric.[1j,30] +The hydrido ligand in 4 appears as a doublet with satellites in the 1H NMR spectrum at δ= 24.50 ppm ( +2JH,F=102 Hz, 1JH,Pt=1361 Hz). It couples to the fluoride, the signal of which is observed in the 19F NMR spectrum as a doublet with satellites at δ= 281.5 ppm ( +1JF,Pt=521 Hz, PtF). Note that for the bifluorido complex 8 a larger Pt,F coupling was found when compared to the one in 4. Analogous observations were reported for *trans-* +[Pt(H)(X)(PR3)2] (X=F, FHF; R=Cy, iPr).[6b,15] The IR spectrum of 4 exhibits a vibrational band for Pt-H at ~v=2188 cm 1 and for PtF at ~v=413 cm 1, which is in accordance with DFT +calculations.[3b,d,5b,13d,15,29] +As speculated, once the fluorido ligand in 4 is not stabilized by HF coordination, further reactivity was observed, which was monitored by 19F NMR spectroscopy. A decrease of the intensities of the resonances for 4 were observed along with the increasing intensity of a compound for which we propose the cyclometalated structure *trans*-[Pt(F)(IMes')(IMes)] (5, Scheme 3). However, the reaction was very slow and not selective, and several unknown hydrido species were detected. The latter remain unidentified (see Supporting Information). Nevertheless, this outcome indicates that such a reaction step might play a role during the SF6 activation process, although reaction times are not consistent. Cyclometalation was observed before at complexes comprising the {Pt(CH3)(NHC)} +entity to yield the cyclometalated product and methane.[31] The signal for the fluorido ligand in 5 is found at δ= 292.6 ppm in the 19F NMR spectrum as a singlet with Pt satellites ( +1JF,Pt= +414 Hz). Furthermore, two signals for the metal-bound carbon atoms in the 13C{1H} NMR spectrum of the isotopologue *trans*- +[Pt(F)(13IMes')(13IMes)] (5') confirm the presence of two inequivalent carbene ligands. They appear at δ=184.3 and 180.2 ppm as doublets of doublets ( +2JC,F=4, 2JC,C=48 Hz) and both reveal a cis-coupling to the fluorido ligand. + +Chem. *Eur.* J. **2021**, 27, 17707–17712 **www.chemeurj.org** 17710 © 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH +Another effort included the generation of dihydrido complexes, as cis-[Pt(H)2(IMes)2] (*cis-*7) was identified in traces in one + +![3_image_0.png](3_image_0.png) + +of the reactions of SF6 with [Pt(IMes)2] (1). They might be generated from 1 and small amounts of H2, the latter being formed in the cyclometallation process of 4 to yield 5, as mentioned above. Treatment of 1 with H2 resulted in the formation of the dihydrido complexes *cis/trans*-[Pt(H)2(IMes)2] +(*cis/trans-*7) (Scheme 4). A conversion of approximately 30% was observed after four weeks to give 7 in a *cis/trans* ratio of 1:16. DFT calculations confirm that the *trans* isomer is by 42.1 kJ/mol more stable than the cis derivative (see Supporting Information). The IR spectrum for *trans*-[Pt(H)2(IMes)2] (*trans-*7) +shows an absorption band at v˜=1652 cm 1 for the PtH2 unit.[32] +Finally, the difluorido complex *trans*-[Pt(F)2(IMes)2] (6) was synthesized independently by treatment of 1 with XeF2 in the presence of CsF at 30°C (Scheme 4). Since chlorinated solvents like CH2Cl2 undergo rapid oxidative addition at 1 to give *trans-*[Pt(Cl)(CH2Cl)(IMes)2] +[33] (9, see Supporting Information), toluene was used as solvent, although this can result in the fluorination of toluene by XeF2 to give benzyl fluoride and HF, which can be trapped by the CsF.[34] +The 19F NMR spectrum of 6 shows a singlet with Pt satellites +( +2JF,Pt=883 Hz) at δ= 463.9 ppm. Consistent to the literature, the mutually *trans*-standing fluorido ligands cause a resonance at higher field.[5a] On 13C{1H} labeling of the carbene carbon atom the corresponding signal in the 13C NMR spectrum of 6' at δ=174.2 ppm appears as a triplet with a coupling to the fluorido ligands of 2JC,F=4 Hz and to Pt of 1JC,Pt=1086 Hz. The ATR IR spectrum of 6 exhibits a vibrational band for Pt-F at ~v= +513 cm 1. + +## Conclusion + +In conclusion, we demonstrated that the electron-rich zerovalent Pt complex [Pt(IMes)2] (1) is highly reactive and can be readily converted into various fluorinated compounds under mild conditions. At room temperature reactions take place with SF4 and SOF2 via oxidative addition, as well as activation of SF6. + +The {Pt(IMes)2} entity enables the stabilization of SF3 and S(=O)F +ligands in the coordination sphere of Pt(II). Such complexes are still rare and their properties have hardly been investigated. An oxidative addition of SOF2 to a transition metal complex to yield a S(=O)F compound is unprecedented. The independent syntheses of fluorido ligand containing complexes by fluorination with a HF source or XeF2 gave stable products. + +Scheme 4. Oxidative addition reactions at [Pt(IMes)2] (1). + +![3_image_1.png](3_image_1.png) + +## Experimental **Section** + +Details of experimental procedures, characterization of the complexes, NMR and IR data, as well as computational details can be found in the Supporting Information. + +## Acknowledgements + +We acknowledge the DFG (Deutsche Forschungsgemeinschaft) for financial support (BR 2065/10-2). The CRC 1349 funded by the Deutsche Forschungsgemeinschaft is also gratefully acknowledged (DFG, German Research Foundation; Gefördert durch die Deutsche Forschungsgemeinschaft - Projektnummer 387284271 - SFB 1349). We thank the Solvay Fluor GmbH for a gift of SF6. We would like to thank Dr. N. Pfister and R. Jaeger for the synthesis of SOF2. Open Access funding enabled and organized by Projekt DEAL. + +## Conflict Of **Interest** + +The authors declare no conflict of interest. + +| Keywords: fluorido | complexes | · | fluorine | · | platinum | · | S F | +|-------------------------------|-------------|-----|------------|-----|------------|-----|-------| +| activation · sulfur fluorides | | | | | | | | + +[1] a) N. M. Doherty, N. W. Hoffmann, Chem. *Rev.* **1991**, 91, 553–573; b) E. F. + +Murphy, R. Murugavel, H. W. Roesky, Chem. *Rev.* **1997**, 97, 3425–3468; c) A. Mezzetti, C. Becker, Helv. Chim. *Acta* **2002**, 85, 2686–2703; d) S. Libri, N. A. Jasim, R. N. Perutz, L. Brammer, J. Am. Chem. *Soc.* **2008**, 130, 7842–7844; e) V. V. Grushin, Acc. Chem. *Res.* **2010**, 43, 160–171; f) A. + +Vigalok, *Organometallics* **2011**, 30, 4802–4810; g) M. Crespo, *Organometallics* **2012**, 31, 1216–1234; h) D. A. Smith, T. Beweries, C. Blasius, N. + +Jasim, R. Nazir, S. Nazir, C. C. Robertson, A. C. Whitwood, C. A. Hunter, L. Brammer, R. N. Perutz, J. Am. Chem. *Soc.* **2015**, 137, 11820–11831; i) A. Vigalok, Acc. Chem. *Res.* **2015**, 48, 238–247; j) F. Nahra, M. Brill, A. Gómez-Herrera, C. S. J. Cazin, S. P. Nolan, Coord. Chem. *Rev.* **2016**, 307, 65–80; k) R. Szpera, D. F. J. Moseley, L. B. Smith, A. J. Sterling, V. + +Gouverneur, Angew. Chem. Int. Ed. **2019**, 58, 14824–14848; Angew. Chem. **2019**, 131, 14966–14991; l) K. Fagnou, M. Lautens, Angew. *Chem.* Int. Ed. **2002**, 41, 26–47; Angew. *Chem.* **2002**, 114, 26–49; m) A. Vigalok, Chem. *Eur.* J. **2008**, 14, 5102–5108; n) J. Miró, C. del Pozo, Chem. *Rev.* 2016, 116, 11924–11966. + +[2] a) J. L. Kiplinger, T. G. Richmond, C. E. Osterberg, Chem. *Rev.* **1994**, 94, 373–431; b) W. D. Jones, Dalton *Trans.* **2003**, 3991–3995; c) H. Torrens, Coord. Chem. *Rev.* **2005**, 249, 1957–1985; d) T. Schaub, P. Fischer, A. Steffen, T. Braun, U. Radius, A. Mix, J. Am. Chem. *Soc.* **2008**, 130, 9304– 9317; e) H. Amii, K. Uneyama, Chem. *Rev.* **2009**, 109, 2119–2183; f) R. P. Hughes, Eur. J. Inorg. *Chem.* **2009**, *2009*, 4591–4606; g) A. D. Sun, J. A. Love, Dalton *Trans.* **2010**, 39, 10362–10374; h) E. Clot, O. Eisenstein, N. Jasim, S. A. Macgregor, J. E. McGrady, R. N. Perutz, Acc. Chem. *Res.* **2011**, 44, 333–348; i) S. A. Johnson, J. A. Hatnean, M. E. Doster, in *Progress* in Inorganic *Chemistry*, **2011**, pp. 255–352; j) P. Fischer, K. Götz, A. Eichhorn, U. Radius, *Organometallics* **2012**, 31, 1374–1383; k) M. F. Kuehnel, D. Lentz, T. Braun, Angew. Chem. Int. Ed. **2013**, 52, 3328–3348; Angew. *Chem.* **2013**, 125, 3412–3433; l) J. Weaver, S. Senaweera, Tetrahedron **2014**, 70, 7413–7428; m) T. Ahrens, J. Kohlmann, M. Ahrens, T. Braun, Chem. *Rev.* **2015**, 115, 931–972; n) Q. Shen, Y.-G. Huang, C. Liu, J.-C. Xiao, Q.-Y. Chen, Y. Guo, J. Fluorine *Chem.* **2015**, 179, 14–22; o) W. Chen, C. Bakewell, M. R. Crimmin, *Synthesis* **2017**, 49, 810–821; p) O. Eisenstein, J. Milani, R. N. Perutz, Chem. *Rev.* **2017**, 117, 8710–8753; q) T. Fujita, K. Fuchibe, J. Ichikawa, Angew. Chem. Int. Ed. **2019**, 58, 390–402; Angew. *Chem.* **2019**, 131, 396–408; r) M. W. Kuntze-Fechner, C. Kerpen, D. Schmidt, M. Häring, U. Radius, Eur. J. Inorg. *Chem.* **2019**, *2019*, 1767– +1775; s) M. W. Kuntze-Fechner, H. Verplancke, L. Tendera, M. Diefenbach, I. Krummenacher, H. Braunschweig, T. B. Marder, M. C. + +![4_image_0.png](4_image_0.png) + +Holthausen, U. Radius, Chem. *Sci.* **2020**, 11, 11009–11023; t) S. A. + +Macgregor, Chem. Soc. *Rev.* **2007**, 36, 67–76; u) L. Keyes, J. A. Love, in CH and C–X Bond Functionalization: Transition Metal *Mediation*, The Royal Society of Chemistry, **2013**, pp. 159–192. + +[3] a) J. M. Brown, V. Gouverneur, Angew. Chem. Int. Ed. **2009**, 48, 8610– +8614; Angew. *Chem.* **2009**, 121, 8762–8766; b) J. Berger, T. Braun, R. Herrmann, B. Braun, Dalton *Trans.* **2015**, 44, 19553–19565; c) N. A. LaBerge, J. A. Love, in Organometallic Fluorine *Chemistry* (Eds.: T. Braun, R. P. Hughes), Springer International Publishing, Cham, **2015**, pp. 55– +111; d) J. Berger, T. Braun, T. Ahrens, P. Kläring, R. Laubenstein, B. BraunCula, Chem. *Eur.* J. **2017**, 23, 8886–8900. + +[4] R. W. Cockman, E. A. V. Ebsworth, J. H. Holloway, H. Murdoch, N. + +Robertson, P. G. Watson, in Inorganic Fluorine Chemistry, Vol. 555, American Chemical Society, **1994**, pp. 326–337. + +[5] a) A. Yahav, I. Goldberg, A. Vigalok, Inorg. *Chem.* **2005**, 44, 1547–1553; b) P. Nilsson, F. Plamper, O. F. Wendt, *Organometallics* **2003**, 22, 5235– 5242; c) J. Hu, M. Nikravesh, H. R. Shahsavari, R. Babadi Aghakhanpour, A. L. Rheingold, M. Alshami, Y. Sakamaki, H. Beyzavi, Inorg. *Chem.* **2020**, +59, 16319–16327; d) M. Joksch, J. Haak, A. Spannenberg, T. Beweries, Eur. J. Inorg. *Chem.* **2017**, *2017*, 3815–3822. + +[6] a) R. Friedemann, K. Seppelt, Eur. J. Inorg. *Chem.* **2013**, *2013*, 1197–1206; b) N. A. Jasim, R. N. Perutz, J. Am. Chem. *Soc.* **2000**, 122, 8685–8693. + +[7] S.-B. Zhao, R.-Y. Wang, H. Nguyen, J. J. Becker, M. R. Gagne, *Chem.* +Commun. **2012**, 48, 443–445. + +[8] a) H. L. Buckley, T. Wang, O. Tran, J. A. Love, *Organometallics* **2009**, 28, 2356–2359; b) T. Wang, J. A. Love, *Organometallics* **2008**, 27, 3290–3296; c) T. Wang, B. J. Alfonso, J. A. Love, Org. *Lett.* **2007**, 9, 5629–5631; d) T. Wang, L. Keyes, B. O. Patrick, J. A. Love, *Organometallics* **2012**, 31, 1397– 1407; e) L. Keyes, A. D. Sun, J. A. Love, Eur. J. Org. *Chem.* **2011**, *2011*, 3985–3994. + +[9] a) I. S. Dubinsky-Davidchik, I. Goldberg, A. Vigalok, A. N. Vedernikov, Chem. *Commun.* **2013**, 49, 3446–3448; b) A. W. Kaspi, I. Goldberg, A. Vigalok, J. Am. Chem. *Soc.* **2010**, 132, 10626–10627. + +[10] I. Dubinsky-Davidchik, I. Goldberg, A. Vigalok, A. N. Vedernikov, Angew. + +Chem. Int. Ed. **2015**, 54, 12447–12451; Angew. *Chem.* **2015**, 127, 12624– 12628. + +[11] a) I. S. Dubinsky-Davidchik, S. Potash, I. Goldberg, A. Vigalok, A. N. + +Vedernikov, J. Am. Chem. *Soc.* **2012**, 134, 14027–14032; b) A. Yahav, I. Goldberg, A. Vigalok, J. Am. Chem. *Soc.* **2003**, 125, 13634–13635; c) E. Sarkissian, M. Golbon Haghighi, Inorg. *Chem.* **2021**, 60, 1016–1020. + +[12] a) C.-L. J. Wang, in Organic Reactions, *Vol.* 34 (Ed.: A. S. Kende), John Wiley & Sons, Inc., **1985**, pp. 319–400; b) G. A. Boswell, Jr., W. C. Ripka, R. M. Scribner, C. W. Tullock, in Organic Reactions, *Vol.* 21, John Wiley & Sons, Inc., **1974**, pp. 1–124; c) W. C. Smith, C. W. Tullock, R. D. Smith, V. A. Engelhardt, J. Am. Chem. *Soc.* **1960**, 82, 551–555; d) A. L. Oppegard, W. C. Smith, E. L. Muetterties, V. A. Engelhardt, J. Am. Chem. *Soc.* **1960**, 82, 3835–3838; e) W. R. Hasek, W. C. Smith, V. A. Engelhardt, J. Am. Chem. *Soc.* **1960**, 82, 543–551; f) P. Kirsch, Modern Fluoroorganic Chemistry: Synthesis, Reactivity, Application, *Vol.* 1, Wiley-VCH, Weinheim, 2004; g) K. Matsumoto, M. Gerken, Dalton *Trans.* **2021**, 50, 12791– 12799. + +[13] a) R. W. Cockman, E. A. V. Ebsworth, J. H. Holloway, J. Am. Chem. *Soc.* +1987, 109, 2194–2195; b) P. Watson, University of Edinburgh **1990**; c) H. M. Murdoch, University of Edinburgh **1991**; d) C. Berg, T. Braun, M. Ahrens, P. Wittwer, R. Herrmann, Angew. Chem. Int. Ed. **2017**, 56, 4300– 4304; Angew. *Chem.* **2017**, 129, 4364–4368; e) N. Pfister, T. Braun, P. Wittwer, M. Ahrens, Z. Anorg. Allg. *Chem.* **2018**, 644, 1064–1070; f) N. Pfister, M. Bui, T. Braun, P. Wittwer, M. Ahrens, Z. Anorg. Allg. *Chem.* 2020, 646, 808–815; g) D. Dirican, N. Pfister, M. Wozniak, T. Braun, *Chem.* Eur. J. **2020**, 26, 6945–6963. + +[14] a) P. Holze, B. Horn, C. Limberg, C. Matlachowski, S. Mebs, Angew. Chem. + +Int. Ed. **2014**, 53, 2750–2753; Angew. *Chem.* **2013**, 125, 8195–8198; b) R. Basta, B. G. Harvey, A. M. Arif, R. D. Ernst, J. Am. Chem. *Soc.* **2005**, 127, 11924–11925; c) B. G. Harvey, A. M. Arif, A. Glöckner, R. D. Ernst, *Organometallics* **2007**, 26, 2872–2879; d) M. Wozniak, T. Braun, M. Ahrens, B. + +Braun-Cula, P. Wittwer, R. Herrmann, R. Laubenstein, *Organometallics* 2018, 37, 821–828. + +[15] C. Berg, N. Pfister, T. Braun, B. Braun-Cula, Chem. *Eur.* J. **2018**, 24, 7985– +7990. + +[16] a) L. Zámostná, T. Braun, B. Braun, Angew. Chem. Int. Ed. **2014**, 53, 2745– +2749; Angew. *Chem.* **2014**, 126, 2783–2787; b) A. G. Massey, K. J. Packer, J. Chem. *Soc.* **1961**, 5554–5555; c) J. Stocks, University of Leicester **1969**. + +Chem. *Eur.* J. **2021**, 27, 17707–17712 **www.chemeurj.org** 17711 © 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH +[17] R. D. W. Kemmitt, R. D. Peacock, J. Stocks, J. Chem. *Soc.* D **1969**, 554a– +554a. + +[18] R. Jaeger, M. Talavera, T. Braun, J. Fluorine *Chem.* **2021**, 247, 109803. [19] a) J. Bauer, H. Braunschweig, K. Kraft, K. Radacki, Angew. Chem. Int. Ed. + +2011, 50, 10457–10460; Angew. *Chem.* **2011**, 123, 10641–10644; b) N. Arnold, R. Bertermann, F. M. Bickelhaupt, H. Braunschweig, M. Drisch, M. Finze, F. Hupp, J. Poater, J. A. P. Sprenger, Chem. *Eur.* J. **2017**, 23, 5948– 5952. + +[20] a) H. C. S. Clark, J. Fawcett, J. H. Holloway, E. G. Hope, L. A. Peck, D. R. + +Russell, J. Chem. Soc. Dalton *Trans.* **1998**, 1249–1252; b) V. V. Grushin, W. J. Marshall, Angew. Chem. Int. Ed. **2002**, 41, 4476–4479; Angew. *Chem.* 2002, 114, 4656–4659; c) C. Berg, T. Braun, R. Laubenstein, B. Braun, Chem. *Commun.* **2016**, 52, 3931–3934; d) N. D. Ball, J. W. Kampf, M. S. Sanford, Dalton *Trans.* **2010**, 39, 632–640; e) T. Beweries, L. Brammer, N. A. Jasim, J. E. McGrady, R. N. Perutz, A. C. Whitwood, J. Am. Chem. Soc. **2011**, 133, 14338–14348. + +[21] J. Deng, C. Wang, Q.-s. Li, Y. Xie, R. B. King, H. F. Schaefer, Inorg. *Chem.* +2011, 50, 2824–2835. + +[22] B. Alič, G. Tavčar, J. Fluorine *Chem.* **2016**, 192, 141–146. [23] a) H. L. Deubner, F. Kraus, *Inorganics* **2017**, 5, 68; b) P. Tomar, T. Braun, E. Kemnitz, Chem. *Commun.* **2018**, 54, 9753–9756; c) D. Rombach, H.-A. + +Wagenknecht, *ChemCatChem* **2018**, 10, 2955–2961; d) P. Kirsch, G.-V. Röschenthaler, D. Sevenard, A. A. Kolomeitsev, Vol. *DE10321114* A1 (Ed.: M. P. GmbH), **2003**; e) M. Rueping, P. Nikolaienko, Y. Lebedev, A. Adams, Green *Chem.* **2017**, 19, 2571–2575; f) T. A. McTeague, T. F. Jamison, Angew. Chem. Int. Ed. **2016**, 55, 15072–15075; Angew. *Chem.* **2016**, 128, 15296–15299; g) G. Iakobson, M. Pošta, P. Beier, J. Fluorine *Chem.* **2018**, 213, 51–55; h) R. F. Weitkamp, B. Neumann, H.-G. Stammler, B. Hoge, Chem. *Eur.* J. **2021**, 27, 6460–6464; i) D. Rombach, H.-A. Wagenknecht, Angew. Chem. Int. Ed. **2020**, 59, 300–303; Angew. *Chem.* **2020**, 132, 306– 310. + +[24] A. Akhgarnusch, R. F. Höckendorf, M. K. Beyer, J. Phys. *Chem.* A **2015**, +119, 9978–9985. + +[25] a) D. J. Sheldon, M. R. Crimmin, Chem. *Commun.* **2021**, 57, 7096–7099; b) F. Buß, C. Mück-Lichtenfeld, P. Mehlmann, F. Dielmann, Angew. *Chem.* Int. Ed. **2018**, 57, 4951–4955; Angew. *Chem.* **2018**, 130, 5045–5049; c) B. S. N. Huchenski, A. W. H. Speed, Chem. *Commun.* **2021**, 57, 7128– 7131; d) S. Huang, Y. Wang, C. Hu, X. Yan, Chem. *Asian* J. **2021**, 16, 2687–2693. + +[26] a) D. K. Padma, V. Subrahmanya Bhat, A. R. Vasudeva Murthy, J. *Inorg.* +Nucl. *Chem.* **1981**, 43, 3099–3101; b) R. Wei, X. Chen, Y. Gong, Chem. Commun. **2020**, 56, 6782–6785. + +[27] T. Braun, S. Sander, R. Müller, M. Ahrens, M. Kaupp, Chem. *Eur.* J. https:// +doi.org/10.1002/chem.202102451. + +[28] a) D. Noveski, T. Braun, S. Krückemeier, J. Fluorine *Chem.* **2004**, 125, 959– +966; b) P. Kläring, A.-K. Jungton, T. Braun, C. Müller, Eur. J. Inorg. *Chem.* +2012, *2012*, 1430–1436; c) H. Baumgarth, G. Meier, T. Braun, B. BraunCula, Eur. J. Inorg. *Chem.* **2016**, *2016*, 4533–4533; d) J. Kohlmann, T. + +Braun, J. Fluorine *Chem.* **2017**, 203, 140–145. + +[29] a) S. S. Rudel, T. G. Müller, F. Kraus, Z. Anorg. Allg. *Chem.* **2015**, 641, 298– +303; b) F. Brosi, T. Vent-Schmidt, S. Kieninger, T. Schlöder, H. Beckers, S. Riedel, Chem. *Eur.* J. **2015**, 21, 16455–16462. + +[30] a) J. Emsley, Chem. Soc. *Rev.* **1980**, 9, 91–124; b) B. S. Ault, Acc. Chem. + +Res. **1982**, 15, 103–109. + +[31] a) G. C. Fortman, N. M. Scott, A. Linden, E. D. Stevens, R. Dorta, S. P. + +Nolan, Chem. *Commun.* **2010**, 46, 1050–1052; b) O. Rivada-Wheelaghan, B. Donnadieu, C. Maya, S. Conejero, Chem. *Eur.* J. **2010**, 16, 10323– 10326; c) O. Rivada-Wheelaghan, M. A. Ortuño, J. Díez, A. Lledós, S. Conejero, Angew. Chem. Int. Ed. **2012**, 51, 3936–3939; Angew. *Chem.* 2012, 124, 4002–4005; d) M. Roselló-Merino, O. Rivada-Wheelaghan, M. A. Ortuño, P. Vidossich, J. Díez, A. Lledós, S. Conejero, *Organometallics* **2014**, 33, 3746–3756; e) K. M. Altus, E. G. Bowes, D. D. Beattie, J. A. Love, *Organometallics* **2019**, 38, 2273–2277. + +[32] a) P. G. Leviston, M. G. H. Wallbridge, J. Organomet. *Chem.* **1976**, 110, 271–279; b) H. C. Clark, A. B. Goel, C. S. Wong, J. Organomet. *Chem.* +1978, 152, C45-C47; c) R. G. Goel, W. O. Ogini, R. C. Srivastava, *Organometallics* **1982**, 1, 819–824; d) R. S. Paonessa, W. C. Trogler, J. Am. *Chem.* +Soc. **1982**, 104, 1138–1140; e) D. L. Packett, W. C. Trogler, Inorg. *Chem.* 1988, 27, 1768–1775; f) A.-K. Jungton, C. Herwig, T. Braun, C. Limberg, Chem. *Eur.* J. **2012**, 18, 10009–10013; g) A.-K. Jungton, A. Meltzer, C. Präsang, T. Braun, M. Driess, A. Penner, Dalton *Trans.* **2010**, 39, 5436– 5438; h) M. A. Salomon, T. Braun, I. Krossing, Dalton *Trans.* **2008**, 5197– 5206; i) T. Braun, D. Noveski, M. Ahijado, F. Wehmeier, Dalton *Trans.* 2007, 3820–3825; j) D. Noveski, T. Braun, M. Schulte, B. Neumann, H.-G. Stammler, Dalton *Trans.* **2003**, 4075–4083. + +[33] F. Hering, U. Radius, *Organometallics* **2015**, 34, 3236–3245. [34] a) H. H. Hyman, M. J. Shaw, R. Filler, J. Am. Chem. *Soc.* **1970**, 92, 6498– +6502; b) H. H. Hyman, M. J. Shaw, R. Filler, J. Org. *Chem.* **1971**, 36, 2917– 2922. + +Manuscript received: September 12, 2021 Accepted manuscript online: October 11, 2021 Version of record online: October 29, 2021 \ No newline at end of file diff --git a/medical/md/PMC9528538.md b/medical/md/PMC9528538.md new file mode 100644 index 0000000000000000000000000000000000000000..d44cfd877dc1d80177e379aa7ee9aede374b46f6 --- /dev/null +++ b/medical/md/PMC9528538.md @@ -0,0 +1,136 @@ + + +![0_image_0.png](0_image_0.png) + +# Detection Of A Weaker Subgroup Of A In Abo Blood Group System + +Gurika Chopra, Manpreet Kataria, Arshpreet Kaur Batra, Gurkiran kaur, Rajesh Kumar + +## Abstract: + +Testing to detect ABO incompatibility between a donor and potential transfusion recipient is the foundation on which all other pretransfusion testing is based. Sometimes, weak agglutination reactions may be obtained with reagent antibodies due to weak expression of A and B antigens on red blood cell (RBC) surface which may cause a discrepancy in blood group typing. Here, we report a patient showing discrepancy between RBCs (forward) and serum (reverse) typing. After doing detailed analysis, the blood type as a variant of blood Group A revealed. Subgroups of A are very rare phenotype of blood. Weaker subgroups of A blood group reported so far are mainly A3 +, Aend, Ax +, +Am, Ay +, and Ael. We are reporting a case of a 35‑year‑old patient whose RBCs showed a discrepancy between cell and serum grouping during initial testing. Serological investigation included absorption elution tests and saliva tests after performing initial blood grouping. The serological characteristics of the patient's red cells were similar to A3 subtype. The patient was a secretor and A and H substance was present in the saliva. Serum did not show any presence of anti‑A1. The weak A phenotype identified had serological characteristics similar to A3 +. + +Keywords: A3 subgroup, blood grouping, discrepancy, forward typing, reverse typing + +## Introduction + +The ABO blood group continues to be the most important blood group in transfusion and transplantation due to immunogenicity.[1] It is the only blood group system in which individuals already have antibodies in their serum to antigens that are absent from their red blood cells (RBCs) without any prior exposure to RBCs through transfusion or pregnancy. Due to the presence of these antibodies, transfusion of an incompatible ABO type may result in immediate lysis of donor RBCs. This produces a very severe, if not fatal, transfusion reaction in the patient. ABO antigens are widely expressed on human tissues and fluids, leading to This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. + +| For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com | +|------------------------------------------------------------------| + +their designation as histo‑blood group antigens. The inheritance of ABO genes follows simple Mendelian genetics. ABO, like most other blood group systems, is codominant in expression.[2] One position, or locus, on each chromosome 9 is occupied by an A, B, or O gene.[3,4] The expression of A and B antigens on the RBCs is fully developed by 2–4 years of age and remains constant throughout life.[2] The ABO +antibodies are predominantly IgM, activate complement, and react at room temperature or colder.[5] ABO antibodies produce strong direct agglutination reactions during ABO testing. The ABO system contains several weak subtypes. All are characterized by decreased A/B and a parallel increase in H‑antigen expression. Weak subtypes can be associated with ABO typing discrepancies during serum grouping due to the presence of unexpected anti‑A or anti‑B activity. In some instances, A/B expression is so weak that red cells in forward typing can How to cite this article: Chopra G, Kataria M, Batra AK, +Kaur G, Kumar R. Detection of a weaker subgroup of A in ABO blood group system. Asian J Transfus Sci 2022;16:132-4. + +Department of Immuno‑Hematology and Blood Transfusion, Dayanand Medical College and Hospital, Ludhiana, Punjab, India Address for correspondence: +Dr. Gurika Chopra, Department of Immuno-Hematology and Blood Transfusion, Dayanand Medical College and Hospital, Ludhiana, Punjab, India. + +E-mail: gurikachopra@ +gmail.com Submitted: 31-05-2021 Revised: 20-06-2021 Accepted: 18-07-2021 Published: 26-05-2022 type as Group O. Subgroups weaker than A2 occur infrequently and are most often recognized through ABO discrepancy with unexpected reaction in the forward and reverse grouping. These subgroups of A make up 1% of those encountered in the laboratory and therefore academic interest.[6] They are wrongly mistyped as Group O because of decreased number of A antigen sites per RBC membrane thus resulting in weak or no agglutination with human polyclonal anti‑A. + +A2 and other week A subgroup red cell subtypes are serologically distinguished from A1 red cells by their lack of reactivity with the lectin *Dolichos biflorus*. Weak A/B subtypes have increased reactivity with *Ulex europaeus*, +an anti‑H lectin. These weak phenotypes, in majority of cases, result from expression of a variant A allele present at the ABO loci. These can be divided into two categories depending on whether the cells are agglutinated with anti‑A, antisera‑A3 +, Aend, and Ax are agglutinated, while Am, Ay +, and Ael cells are not. The above weaker phenotypes can be serologically differentiated from each other using the following techniques:[7] +- Forward grouping of A and H antigens with anti‑A, +anti‑A, B, and anti‑H +- Reverse grouping of ABO isoagglutinins and the presence of anti‑A1 +- Testing with different batches of anti‑A reagent - Adsorption‑elution experiments with polyclonal anti‑A and anti‑A + B from Group B and Group O individuals +- Secretor status for the presence of H and A antigen in saliva +- Molecular genotyping of ABO gene, especially exons 6 and 7 as they encode for 77% of glycosyltransferase activity. + +The weak A phenotype identified in the present case had serological characteristics similar to A3 blood type. + +## Case Report + +A 35‑year‑old female visited obstetrics and gynecology OPD in our hospital. Blood sample was sent to the IHBT department for ABO grouping and Rh typing. Discrepancy was observed between cell and serum grouping. The patient's red cells were nonreactive with anti‑B and give mixed field pattern of agglutination with anti‑A and 1 + with anti‑A1. However, it showed 2 + pattern of agglutination with anti‑A, B and strong agglutination pattern with anti‑H (4+). The patient's serum showed the presence of anti‑B antibody. Blood grouping pattern observed was suggestive of the presence of weaker subgroup of A, indicating Type II +discrepancy.[8] +The results are summarized in Table 1. + +## Blood Grouping + +Blood grouping was again repeated with the same sample and technical errors were ruled out. To detect the presence of weak A antigen, heat elution was performed on the patient's red cells. The elute showed microscopic agglutination with three different A group cells [results summarized in Table 2]. To detect the presence of soluble substances, secretor status was determined using patient's saliva. The patient was found to be a secretor, having A and H substance detectable in the saliva [results summarized in Table 3]. These serological reactions obtained were consistent with reactivity pattern of A3 subtype. + +## Discussion + +The risk of hemolytic transfusion reaction due to transfusion of ABO‑incompatible blood is 100–1000 times higher than the risk of transfusion‑transmitted infections, and such a reaction may lead to serious consequences in the recipient.[9] More than 250 ABO alleles have been identified by molecular investigation.[10,11] The ABO gene, located on chromosome 9, consists of seven exons.[3] The last two exons (6 and 7) encode for the catalytic domain of the ABO glycosyltransferases.[12] Exons 6 and 7 constitute 77% of the gene, with most of the coding sequence lying in exon 7.[13,14] Amino acid substitutions, resulting primarily from deletions, mutations, or gene recombination's within these two exons of the coding DNA of variant ABO glycosyltransferases, are responsible for the less efficient transfer of the immunodominant sugar to H substance, resulting in weak serologic reactions observed in ABO subgroups.[12] Weaker variants of A and B arise due to inheritance and expression of variant alleles at the ABO locus. Hemagglutination‑based methods are used to identify these subgroups. However, due to variation in reagents and techniques used, these weaker phenotypes are often mistyped as O group. Sometimes, weaker subgroups of A may present practical problems, as if donor mistyped as Group O and transfused to a Group O recipient. This is potentially dangerous because the Group O patient possesses anti‑A, B, which agglutinates and lyses RBCs, causing rapid intravascular hemolysis. Hence, it is important to characterize these weaker + +| Group | Forward | Reverse | | | | | | | | | +|----------------|-----------|-----------|-----------|--------|--------|---------|----------|---------|---------|----| +| Anti-A | Anti-A1 | Anti-B | Anti-A, B | Anti-H | Anti-D | A cells | A1 cells | B cells | O cells | | +| A3 | mf | 1+ | 0 | 2+ | 4+ | 4+ | 0 | 0 | 4+ | 0 | +| Mf=Mixed field | | | | | | | | | | | + + 133 + +| Table 2: Adsorption-elution Anti-A Anti-B | Anti-A+B | | +|-------------------------------------------------|------------|----| +| 2+ | 0 | 3+ | +| Table 3: Secretor status A, H present in saliva | | | + +subgroups as accurate determination of the ABO type would help in better management of "O" group RBCs and "AB" plasma for transfusion in discrepant cases.[15] +In this case study, the patient's blood group was serologically identified as A3 type. The red cells of A3 individuals distinguish themselves by giving mixed field by anti‑A sera, negative reactions with anti‑B sera, but remarkably strong reactions with anti‑A, B sera. + +Weak subgroups of "A" are rare and require advanced techniques like "Adsorption and Elution" for their detection. Molecular testing is usually required for confirmation and exact typing of the subgroups (not available in our institute). + +## Conclusion + +If any ABO discrepancy found in any donor or recipient between forward and reverse typing, that should be informed both to the laboratory staff and physicians due to the high probability of weak expression of blood group antigens. The absence of a disease process should be confirmed before subgroup investigation because ABH antigens are altered in various malignancies and other hematologic disorders. Additional special procedures such as molecular testing for mutations or serum glycosyltransferase studies for detecting the A enzyme can be performed for differentiation of weak subgroups.[6,16] Patient should be transfused with Group +"O" red cell components and should receive group matched/compatible plasma and platelet components. + +Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/ have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. + +Acknowledgments The authors take full responsibility for the content of this manuscript. We gratefully acknowledge the support of Marksman Healthcare Communications for editorial services. + +Financial support and sponsorship Nil. + +Conflicts of interest There are no conflicts of interest. + +## References + +1. Dean L. The ABO blood group. In: Blood Groups and Red Cell Antigens. Ch. 5. Bethesda, MD: National Center for Biotechnology Information (US); 2005. Available from: https://www.ncbi.nlm. nih.gov/books/NBK2267/. [Last accessed on 2021 Sep 21]. + +2. Klein HG, Anstee DJ. Mollison's Blood Transfusion in Clinical Medicine. 12th ed. Somerset, NJ: Wiley‑Blackwell Publishing; 2014. + +3. Olsson ML, Chester MA. Polymorphism and recombination events at the ABO locus: A major challenge for genomic ABO blood grouping strategies. Transfus Med 2001;11:295‑313. + +4. Chester MA, Olsson ML. The ABO blood group gene: A locus of considerable genetic diversity. Transfus Med Rev 2001;15:177‑200. + +5. Daniels G, Bromilow I. Essential Guide to Blood Groups. Somerset, NJ: Wiley‑Blackwell Publishing; 2013. + +6. Mizuno N, Ohmori T, Sekiguchi K, Kato T, Fujii T, Fujii K, et al. + +Alleles responsible for ABO phenotype‑genotype discrepancy and alleles in individuals with a weak expression of A or B antigens. J Forensic Sci 2004;49:21‑8. + +7. Heier HE, Namork E, Calkovská Z, Sandin R, Kornstad L. + +Expression of A antigens on erythrocytes of weak blood group A subgroups. Vox Sang 1994;66:231‑6. + +8. Harmening DM, Forneris G, Tubby BJ. The ABO blood group system. In: Harmening D, editor. Modern Blood Banking and Transfusion Practices. PA: F.A Davies Company; 2012. + +9. Kaur G, Kaur P, Basu S, Kaur R. Blood group discrepancies at a tertiary care centre‑analysis and resolution. Int J Lab Hematol 2014;36:481‑7. + +10. Yamamoto F, Cid E, Yamamoto M, Saitou N, Bertranpetit J, +Blancher A. An integrative evolution theory of histo‑blood group ABO and related genes. Sci Rep 2014;4:6601. + +11. Yamamoto F, Cid E, Yamamoto M, Blancher A. ABO research in the modern era of genomics. Transfus Med Rev 2012;26:103‑18. + +12. Lee AH, Reid ME. ABO blood group system: A review of molecular aspects. Immunohematology 2000;16:1‑6. + +13. Yamamoto F. Cloning and regulation of the ABO genes. Transfus Med 2001;11:281‑94. + +14. Seltsam A, Hallensleben M, Kollmann A, Burkhart J, Blasczyk R. + +Systematic analysis of the ABO gene diversity within exons 6 and 7 by PCR screening reveals new ABO alleles. Transfusion 2003;43:428‑39. + +15. Kulkarni S, Gogri H. Molecular concepts in immunohematology. + +In: Choudhury N, editor. A Textbook on Laboratory and Clinical Transfusion Medicine: Basics of Blood Bank Practices (Process Control). Vol. 2. New York: Nova Science Publishers, Inc.; 2017. + +16. Seltsam A, Blasczyk R. Missense mutations outside the catalytic domain of the ABO glycosyltransferase can cause weak blood group A and B phenotypes. Transfusion 2005;45:1663‑9. \ No newline at end of file diff --git a/medical/md/PMC9577576.md b/medical/md/PMC9577576.md new file mode 100644 index 0000000000000000000000000000000000000000..ae00e18b40ba2e103c39e502ce1df5161df2c492 --- /dev/null +++ b/medical/md/PMC9577576.md @@ -0,0 +1,409 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +# Infantile Colic And The Subsequent Development Of The Irritable Bowel Syndrome + +Ju Hee Kim,1 Seung Won Lee,2 Yoowon Kwon,3 Eun Kyo Ha,4 Jaewoo An,5 Hye Ryeong Cha,6 **Su Jin Jeong,5* and Man Yong Han5*** +1 Department of Pediatrics, Hallym University Kangdong Sacred Heart Hospital, Seoul, Korea; 2Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do, Korea; 3Department of Pediatrics, Chungnam National University Sejong Hospital, Sejong, Korea; 4Department of Pediatrics, Hallym University Kangnam Sacred Heart Hospital, Seoul, Korea; 5Department of Pediatrics, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam, Gyeonggi-do, Korea; and 6Department of Comuputer Science and Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do, Korea + +## Background/Aims + +Little is known about the association between infantile colic and the later onset of irritable bowel syndrome (IBS). + +## Methods + +This study examined all 917 707 children who were born in Korea between 2007 and 2008. Infantile colic was defined with 1 or more diagnoses of ICD-10 code R10.4 or R68.1 at the age of 5 weeks to 4 months, and infants with a diagnosis of infantile colic and without were allocated into the infantile colic group and the control group. IBS was defined as 2 or more diagnoses of ICD-10 code K58.X after 4 years of age. Each child was traced until 2017. The risk of IBS with infantile colic was evaluated using a Cox proportional hazards model with propensity score inverse probability of treatment weighting (IPTW). + +## Results + +After IPTW, 363 528 and 359 842 children were allocated to the control group and the infantile colic group, respectively. The infantile colic group had a higher risk of developing IBS in childhood (hazard ratio [95% CI], 1.12 [1.10 to 1.13]) than the control group. Moreover, the subgroup analyses according to the feeding status, birth weight, sex, or economic status, showed that the risk of IBS with former infantile colic remained statistically significant. + +## Conclusions + +Children with a diagnosis of infantile colic during the infant period had a significant risk of developing IBS after 4 years of age. Understanding the pathogenesis of infantile colic in the neonatal period may reduce the prevalence and severity of functional gastrointestinal disorders from childhood to adolescence to adulthood. + +(J Neurogastroenterol Motil 2022;28:618-629) + +## Key Words + +Colic; Gastrointestinal diseases; Irritable bowel syndrome Received: September 9, 2021 Revised: December 3, 2021 Accepted: May 7, 2022 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. + +*Correspondence: Man Yong Han and Su Jin Jeong are equally responsible for this work. + +Man Yong Han, MD +Department of Pediatrics, CHA University School of Medicine, 59, Yatap-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 13496, Korea Tel: +82-31-780-6262, Fax: +82-31-780-5239, E-mail: drmesh@gmail.com Su Jin Jeong, MD, PhD +Department of Pediatrics, CHA University School of Medicine, 59, Yatap-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 13496, Korea Tel: +82-31-780-5349, Fax: +82-31-780-5011, E-mail: jinped@chamc.co.kr Ju Hee Kim and Seung Won Lee contributed equally to this work. + +## Introduction + +Infantile colic is a common disease in the infant period, affecting between 4% and 28% of all infants.1 Its most prominent feature is excessive crying, which peaks at 6 weeks and disappears at approximately 4 to 5 months of age.2 Although the pathogenesis of infantile colic is not well understood, it has been generally viewed as a temporary disorder with a benign prognosis. However, it has been recently reported that infantile colic is associated with a low grade systemic inflammation caused by a pathogenic microbiota composition.3 Therefore, infantile colic may be considered as a potential early manifestation of later onset disorders including the irritable bowel syndrome (IBS). IBS is a common and chronic functional gastrointestinal disorder (FGID) at childhood that affects the quality of life, with a prevalence of 2% to 24% worldwide.4 A multinational group of experts at the Rome IV Conference in December 2014 established the most recent definition of IBS.5 A characteristic of IBS is abdominal pain or discomfort with changes in bowel habits, including the frequency of defecation and stool consistency, in the absence of an organic disease.6 The pathophysiology of IBS is not understood completely.7 Various central and peripheral mechanisms have been described in the pathophysiology of IBS, including the dysregulation of the brain-gut axis,8 altered gastrointestinal motility, visceral hypersensitivity,9 alterations in the intestinal microbiota,10 low-grade immune activation, and intestinal inflammation,7 which may be associated with increased intestinal permeability to induce exposure to antigens. Gut dysbiosis is thought to be the most convincing factor, as some factors may be derived directly from the gut dysbiosis.11 Some points of pathogenesis show an association between infantile colic and IBS. + +Few previous studies have reported an association between infantile colic and the subsequent development of IBS,12,13 but there is a paucity of large cohort studies reporting the association between infantile colic in infants and IBS. Therefore, our purpose is to evaluate the association of infantile colic diagnosis at 5 weeks to 4 months of age with the onset of IBS after 4 years of age, using an analysis of an administrative nationwide cohort database in South Korea. + +## Materials And Methods Study Design And Setting + +This study was conducted using data from the National Investigation of Birth Cohort in Korea study 2008 (NICKs-2008) dataset, which comprised 917707 children born between 2008 and 2009 in Korea.14,15 The National Health Screening Program for Infants and Children (NHSPIC) is a population surveillance program that provides seven rounds of screening services to all health insurance subscribers from 4 months to 72 months of age. The use of de-identified individual data for research purposes was authorized under the current National Health Insurance Act. The study protocol was reviewed and approved by the Institutional Review Board of the Korea National Institute for Bioethics Policy (P01-20160321-005). The study followed recommended guidelines for observational studies that use routinely collected health data (Supplementary Table 1). + +## Data Sources + +Patient demographic characteristics, health care utilization (type of visit, medical cost, drug classification code, and disease information [International Classification of Diseases 10th revision {ICD10 codes}] were obtained from the database of the NICKs-2008 cohort. The patients' health care utilization data were collected during the process of claiming for healthcare services. The residential status at birth was divided into Seoul, metropolitan (Busan, Daegu, Incheon, Gwangju, Daejeon, and Ulsan), urban, and rural. The socio-economic status was classified according to quintiles and determined by the amount of the insurance co-payment. Additional information on the databases, variable definitions, and administrative codes are shown in Supplementary Table 2. The cohort was traced to the time when participants lost their eligibility for health care services due to death or immigration, or until 2017. + +## Data Sharing Statement + +This study was based on the National Health Claims Database +(National Health Insurance Service [NHIS]-2019-1-560) established by the NHIS of the Republic of Korea. Applications for using NHIS data are reviewed by the Inquiry Committee of Research Support; if the application is approved, raw data is provided to the applicant for a fee. We cannot provide access to the data, analytic methods, and research materials to other researchers because of the intellectual property rights of this database that is owned by the National Health Insurance Corporation. However, investigators who wish to reproduce our results or replicate the procedure can be used in the database, which is open for research purposes (https://nhiss. nhis.or.kr/). + +## Study Population + +The cohort included children who underwent the first round of the NHSPIC at 4 months to 6 months of age and with properly recorded birth weight information during the first round of the NHSPIC. In addition, children who were born before 37 weeks of gestational age, born with a birth weight of less than 2.5 kg or more than 4.5 kg, who had been admitted to the intensive care unit within the first 1 month of birth, or who were diagnosed with congenital malformations, deformations, and chromosomal abnormalities, were excluded. A total of 368 556 children met the inclusion and exclusion criteria. Among these children, 359769 who had not been diagnosed with infantile colic from the age of 5 weeks to 4 months were allocated to the control group and 8787 who had been diagnosed with infantile colic between the ages of 5 weeks and 4 months were allocated to an infantile colic group (Fig. 1). + +## Exposure + +The exposure of interest was a diagnosis of infantile colic from the age of 5 weeks to 4 months, because this time of life is known to be the most prevalent period of infantile colic.3,4 The diagnosis of infantile colic was defined as at least 1 diagnosis of ICD-10 code R10.4 (other and unspecified abdominal pain) or R68.1 (nonspecific symptoms peculiar to infancy) from the age of 5 weeks to 4 months (Supplementary Table 3). Validation data for this definition of infantile colic are presented in the Supplementary material, and the positive predictive value (PPV) was 88% (95% CI, 83-92%). + +## Outcomes + +All outcomes were pre-specified. The outcome was the risk of IBS after 48 months of age. IBS was defined as a diagnosis of ICD-10 codes K58.X (IBS), more than twice, after 4 years of age.16 To assess the robustness of the results, we analyzed the association of IBS with infantile colic using the different definitions of IBS. The stricter IBS was defined as at least 1 diagnosis of ICD-10 code K58.X (IBS) and R10.X (abdominal pain) and at least 1 of ICD-10 code K59.1 (functional diarrhea), K52.2 (allergic and dietetic gastroenteritis and colitis), K52.8 (other specific noninfective gastroenteritis), or K59.0 (constipation) after the age of 4 years. A previous study showed that this definition had a PPV of 83% (95% +CI, 75-91%).17 In addition, we defined the IBS sub-type by further considering the symptomatic diagnostic ICD-10 codes. Diarrheapredominant IBS (IBS-D) was defined as having at least 1 diagnosis of ICD-10 code K58.X (IBS) and R10.X (abdominal pain) and at least 1 of the ICD-10 codes K59.1 (functional diarrhea) after the age of 4 years. Constipation-predominant IBS (IBS-C) was defined as having at least 1 diagnosis of ICD-10 code K58.X (IBS) and R10.X (abdominal pain) and at least 1 of ICD-10 code K59.0 (constipation) after the age of 4 years. To apply stricter diagnostic criteria for the IBS sub-types, the strict IBS-D was defined as using + +![2_image_0.png](2_image_0.png) + +least 1 prescription of drug classification 237 (antidiarrheal drug) as well as the IBS-D definition, and the strict IBS-C was defined as using at least 1 prescription of drug classification 238 (laxative drug) as well as IBS-C definition, which showed PPV 76% (95% +CI, 67-85%) in a previous study.17 + +## Covariates + +Propensity score (PS) matching was performed using the 97 covariates shown in Supplementary Table 2 in order to create a balance between the control group and the colic group. The children's demographic variables included age, sex, income quintile (by insurance premium), and residence at birth. Nutritional assessments, including the status of breastfeeding within 4 months of age and the additional complementary feeding status were acquired from the first round of the NHSPIC questionnaire (4 to 6 months of age) given to children's parents. In addition, the results of general physical examinations from head to extremity by physicians in the first round of the NHSPIC were assessed. The perinatal conditions were evaluated using the ICD-10 codes. To evaluate their clinical conditions within the first 6 months of age, the most prevalent 43 diseases, including respiratory infections, gastrointestinal infections, and dermatitis, were identified using the ICD-10 codes. In addition, 12 types of drugs, including the drugs used commonly in infants, were defined using the drug classification codes. Hospitalization records, emergency room visits, and visits to pediatricians within the first 6 months of age were recorded. + +## Statistical Methods + +The inverse probability of treatment weighting (IPTW) on the PS was used to balance the comparison groups on the 97 covariates that were chosen previously (as defined in the Supplementary Table 2). The PS was estimated using a multivariable logistic regression with the covariates chosen a priori. Participants in the reference group were weighted (PS/[1-PS]),18,19 which produced a weighted pseudosample of patients in the reference group with the same distribution of measured covariates as the exposed group.19 The between-group differences in baseline characteristics were compared using standardized differences in both the unweighted and weighted samples (differences > 10% were considered meaningful).20 A Cox proportional hazards model was used to assess the relationship between the infantile colic at 5 weeks to 4 months of age and the development of IBS. Hazard ratios (HRs) and 95% CIs were calculated for each outcome of interest. + +To minimize the type 1 error by the inflated number from IPTW, we additionally analyzed using stabilized IPTW and weight trimming. In addition, a 4:1 PS matching was performed instead of IPTW, based on the Mahalanobis algorithm with a caliper of 0.01 and multivariable regression with covariates. + +In addition, prespecified subgroup analyses by type of feeding (grouped into 2 categories: breast fed, or no breast fed), birth weight (grouped into 2 categories: lower half (< 3.26 kg), or up-per half (≥ 3.26 kg), sex (male or female), and income status (low or high). Furthermore, the number of episodes of infantile colic (grouped into 2 categories: once or more than once) was assessed to determine whether the higher the number of infantile colic episodes led to a higher the risk of IBS. + +Two-tailed P-values of less than 0.001 were interpreted as being statistically significant. All statistical analyses were performed using SAS version 9.4 (SAS Institute Inc, Cary, NC, USA). + +## Results Participants + +The basic sociodemographic characteristics of the children are presented in Table 1. In the weighted data, both the control and infantile colic groups were balanced for all variables. In addition to the results that comprised the results of the physical examinations by physicians and questionnaires, including visual and auditory senses at 4-6 months of age, there was no imbalance between the 2 groups (Supplementary Table 4). The basic clinical characteristics of the children in both groups are shown in Table 2. Although there were imbalances between the 2 groups in the number of visits to pediatricians and emergency rooms (standardized differences = 22.5% +and 32.3%, respectively) before weighting, all data on the hospital utilization was balanced between the 2 groups after weighting. With regard to the perinatal conditions, the prevalence of diagnosed diseases within the first 6 months of age, and the use of major drugs, and all standardized differences between the 2 groups, were less than 10% after weighting. The full set of clinical characteristics is shown in Supplementary Table 5. + +After each application of the stabilized and trimmed IPTW +and the 4:1 PS matching, all standardized differences between the infantile colic and control group were less than 10% (data not shown). + +## Association Of Infantile Colic With Diagnosis Of Irritable Bowel Syndrome After 4 Years Of Age + +Table 3 shows the associations of infantile colic with IBS. In the weighted data, 61 759 (17.2%) children with IBS were in the infan- + +| aUnless otherwise specified, baseline characteristics were assessed on the birth date of the patient. bWeighted using inverse probability of exposure weighting based on the propensity score. The propensity score was estimated using multivariable logistic regression with 97 previously covariates, as defined in Supplementary Table 2. Participants in the reference group were weighted as (propensity score/[1-propensity score]). This method produces a weighted pseudo sample of participants in the reference group with the same distribution of measured covariates as the exposure group. cResults are reported as n (%) unless otherwise indicated. dAs the reference group, the control group consisted of children who had not been diagnosed with infantile colic at 5 weeks to 4 months of age. eThe infantile colic group consists of children who have been diagnosed with infantile colic at least once between 5 weeks and 4 months of age. fDifferences greater than 10% were interpreted as meaningful differences. All standardized differences in the cohort values were < 0.05. gMetropolitan areas were defined as 6 metropolitan cities (Busan, Incheon, Gwangju, Daejeon, Daegu, and Ulsan), urban areas as cities, and rural areas as non-city areas. Missing data of observed data: control group = 3220, infantile colic group = 102; missing data of weighted data: control group = 3281, infantile group = 3157. hObtained from the first National Health Screening Program of Infants and Children (NHSPIC) at 4-6 months of birth. iObtained from the first NHSPIC at 4-6 months of birth. Missing data of all data: control group = 1506, infantile colic group = 33. jIncome status was categorized into the quintile of insurance premium at birth. Missing data of all data: control group = 12 204, infantile colic group = 288; missing data of weighted data: control group = 12 338, infantile colic group = 12 109. | | | | | | | | | | | | | | +|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------|----------------------------------------------------------------------|----------------------------------------------------------------------|----------------------------------------------------------|----------------------------------------------------------|---------|---------|---------|---------|-----|-----|---------|-----| +| | | | | | | | | | | | | | | +| difference (%)f | | | | | | | | | | | | | | +| Standardized | | | | | | | | | | | | | | +| n (%)c | 0.3 | 0.1 | 0.1 | 0.0 0.1 | 0.1 | 0.2 | 0.2 | 0.1 | 0.1 | 0.2 | 0.1 | 0.0 0.1 | 0.1 | +| Infantile colic groupe (n = 359 842) | | | | | | | | | | | | | | +| Weighted data (n = 723 370)b | 175 873 (48.9) 183 969 (51.1) | 98 793 (27.5) 76 547 (21.3) 142 079 (39.5) 39 266 (10.9) 3.24 (0.34) | 165 291 (45.9) 124 049 (34.5) 68 877 (19.1) | 54 083 (15.0) 96 592 (26.8) 113 306 (31.5) 56 961 (15.8) | | | | | | | | | | +| 1581 (0.4) | 26 791 (7.4) | | | | | | | | | | | | | +| Control groupd (n = 363 528) | 178 831 (49.2) 184 696 (50.8) | 90 259 (24.8) 83 809 (23.1) 144 522 (39.8) 41 658 (11.5) 3.26 (0.36) | 165 601 (45.6) 126 706 (34.9) 69 773 (19.2) | 53 143 (14.6) 96 534 (26.6) 114 922 (31.6) 58 688 (16.1) | | | | | | | | | | +| 1401 (0.4) | 27 903 (7.7) | | | | | | | | | | | | | +| Standardized difference (%)f n (%)c | 0.2 | 5.6 | 10.6 | 12.7 0.5 | 4.6 | 5.7 7.1 | 2.5 5.8 | 0.4 1.6 | 0.5 0.4 | 0.2 | | | | +| Observed data (n = 368 556) | Infantile colic groupe (n =8787) | 4331 (49.3) 4456 (50.7) | 1977 (22.5) 1656 (18.8) 4031 (45.9) 1021 (11.6) 3.24 (0.36) | 3745 (42.6) 3344 (38.1) 1593 (18.1) | 1334 (15.2) 2354 (26.8) 2792 (31.8) 1336 (15.2) | | | | | | | | | +| 72 (0.8) | 683 (7.8) | | | | | | | | | | | | | +| Table 1. Basic Sociodemographic Characteristics of the Participantsa | Sociodemographic characteristics Control groupd (n = 359 769) | 176 967 (49.2) 182 802 (50.8) | 89 501 (24.9) 83 324 (23.2) 142 540 (39.6) 41 184 (11.4) 3.26 (0.36) | 163 567 (45.5) 124 583 (34.6) 68 775 (19.1) | 52 574 (14.6) 95 514 (26.5) 113 679 (31.6) 58 181 (16.2) | | | | | | | | | +| 1338 (0.4) | 27 617 (7.7) | | | | | | | | | | | | | +| Birth weighth (mean [SD], kg) Type of feedingi | | | | | | | | | | | | | | +| Only breastfeeding | | | | | | | | | | | | | | +| Only formula milk | | | | | | | | | | | | | | +| Residence at birthg | | | | | | | | | | | | | | +| Seoul | Income quintilej | 1 (Lowest) | | | | | | | | | | | | +| | | | | | | | | | | | | | | +| Metropolitan | | | | | | | | | | | | | | +| Special milk | 3 (Middle) | 5 (Highest) | | | | | | | | | | | | +| Sex | Female | Urban | Mixed | | | | | | | | | | | +| | | | | | | | | | | | | | | +| Male | Rural | 2 | 4 | | | | | | | | | | | + +Ju Hee Kim, et al 622 + +| Standardized difference (%)f | | | | | | | | | | | | | | | | | | | | | | | | | | | +|-----------------------------------------------------------------------|----------------------------------------------------------------------------|--------------------------------------------------------------------|------------------------------|-----------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|-----|----------|-----|-----|-----|-----|--------------|-----|-----|-----|-----|---------|---------|-----|----------|-----|-----|-----|-----|-----|-----| +| n (%)c | 2.2 | 1.2 | 0.1 | 0.2 | 0.1 | 0.0 | 0.2 | 0.1 | 0.6 | 0.5 | 0.6 | 0.7 | 0.7 | 0.6 | 0.6 | 0.4 | 0.2 | 0.8 | 0.7 | 0.0 | 0.1 | 0.1 | 0.0 | 0.1 | 0.5 | 0.1 | +| Weighted data (n = 723 370)b | Infantile colic groupe (n = 359 842) Control groupd (n = 363 528) | 342 290 (95.1) | 51 833 (14.4) 112 868 (31.4) | 107 273 (29.8) 39 901 (11.1) 84 377 (23.4) | 66 559 (18.5) 50 601 (14.1) | | | | | | | | | | | | | | | | | | | | | | +| 18 281 (5.1) | 14 480 (4.0) | 10 410 (2.9) | 7869 (2.2) | 10 044 (2.8) | 11 772 (3.3) 5995 (1.7) 8590 (2.4) 8289 (2.3) 18 306 (5.1) 16 272 (4.5) 5375 (1.5) 10 274 (2.9) 15 785 (4.4) 4498 (1.3) 8302 (2.3) 11 869 (3.3) 21 194 (5.9) | | | | | | | | | | | | | | | | | | | | | | +| 345 435 (95.0) | 50 342 (13.8) 113 338 (31.2) | 105 974 (29.2) 38 483 (10.6) 82 711 (22.8) | 66 995 (18.4) 49 176 (13.5) | | | | | | | | | | | | | | | | | | | | | | | | +| 16 843 (4.6) | 14 845 (4.1) | 10 283 (2.8) | 8058 (2.2) | 9569 (2.6) | 10 064 (2.8) 5524 (1.5) 8617 (2.4) 7834 (2.2) 16 709 (4.6) 15 699 (4.3) 5360 (1.5) 10 248 (2.8) 15 630 (4.3) 4820 (1.3) 7861 (2.2) 10 699 (2.9) 18 507 (5.1) | | | | | | | | | | | | | | | | | | | | | | +| Standardized difference (%)f | | | | | | | | | | | | | | | | | | | | | | | | | | | +| n (%)c | 22.5 | 32.3 | 4.0 | 5.3 | 0.2 | 5.4 | 0.4 12.5 | 6.4 | 5.1 | 0.1 | 0.6 | 2.2 12.5 5.2 | 8.6 | 8.7 | 8.2 | 9.9 | 6.1 7.3 | 5.6 1.1 | 3.1 | 13.6 4.1 | | | | | | | +| Observed data (n = 368 556) | Infantile colic groupe (n =8787) Control groupd (n = 359 769) | 8688 (98.9) 1174 (13.4) | 1373 (15.6) 2755 (31.4) | 3056 (34.8) 1103 (12.6) 2198 (25.0) | 1634 (18.6) 1129 (12.8) | | | | | | | | | | | | | | | | | | | | | | +| 427 (4.9) | 331 (3.8) | 204 (2.3) | 225 (2.6) | 459 (5.2) 192 (2.2) 333 (3.8) 315 (3.6) 561 (6.4) 574 (6.5) 205 (2.3) 357 (4.1) 480 (5.5) 131 (1.5) 231 (2.6) 495 (5.6) 524 (6.0) | | | | | | | | | | | | | | | | | | | | | | | +| 341 551 (94.9) | 49 707 (13.8) 112 149 (31.2) | 104 381 (29.0) 37 920 (10.5) 81 665 (22.7) | 66 304 (18.4) 48 798 (13.6) | | | | | | | | | | | | | | | | | | | | | | | | +| 15 903 (4.4) | 14 605 (4.1) | 10 079 (2.8) | 7989 (2.2) | 9496 (2.6) | 9741 (2.7) 5416 (1.5) 8416 (2.3) 7635 (2.1) 16 372 (4.6) 15 365 (4.3) 5223 (1.5) 10 060 (2.8) 15 342 (4.3) 4750 (1.3) 7731 (2.1) 10 334 (2.9) 18 237 (5.1) | | | | | | | | | | | | | | | | | | | | | | +| Certain conditions (ICD-10 codes) originating in the perinatal period | Respiratory and cardiovascular disorders specific to the perinatal period | Prevalent diseases (ICD-10 codes) diagnosed within 6 months of age | | | | | | | | | | | | | | | | | | | | | | | | | +| Otitis media, unspecified | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Table 2. Basic Clinical Caracteristics of the Participantsa | Clinical characteristics | Hemorrhagic and hematological disorders of fetus | and newborn | Infectious gastroenteritis and colitis of infectious origin | | | | | | | | | | | | | | | | | | | | | | | +| Gastroesophageal reflux disease without esophagitis | Noninfective gastroenteritis and colitis, unspecified | | | | | | | | | | | | | | | | | | | | | | | | | | +| Digestive system disorders of fetus and newborn | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Acute upper respiratory infection, unspecified | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Hospital utilization within 6 months of age | Number of visits to pediatricians | Functional intestinal disorder, unspecified | | | | | | | | | | | | | | | | | | | | | | | | | +| Infections specific to the perinatal period | Urinary tract infection, site not specified | | | | | | | | | | | | | | | | | | | | | | | | | | +| Viral intestinal infection, unspecified | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Pneumonia, unspecified | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Number of visiting ER | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Acute nasopharyngitis | Functional dyspepsia | Nausea and vomiting | | | | | | | | | | | | | | | | | | | | | | | | | +| Functional diarrhea | Pyogenic granuloma | | | | | | | | | | | | | | | | | | | | | | | | | | +| | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Acute bronchiolitis | Fever, unspecified | | | | | | | | | | | | | | | | | | | | | | | | | | +| | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Acute pharyngitis | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Acute bronchitis | | | | | | | | | | | | | | | | | | | | | | | | | | | +| Constipation | Dermatitis | | | | | | | | | | | | | | | | | | | | | | | | | | + +Infantile Colic and IBS +Ju Hee Kim, et al + +| aUnless otherwise specified, all baseline characteristics were assessed in the first 6 months of age of the participants. bWeighted using inverse probability of exposure weighting based on the propensity score. The propensity score was estimated using multivariable logistic regression with 97 previously covariates, as defined in Supplementary Table 2 Participants in the reference group were weighted as (propensity score/[1-propensity score]). This method produces a weighted pseudo sample of participants in the reference group with the same distribution of measured covariates as the exposure group. cResults are reported as n (%) unless otherwise indicated. dAs the reference group, the control group consisted of children who had not been diagnosed with infantile colic at 5 weeks to 4 months of age. eThe infantile colic group consists of children who have been diagnosed with infantile colic at least once between 5 weeks and 4 months of age. fDifferences greater than 10% were interpreted as meaningful differences. All standardized differences in the cohort values were < 0.05. ICD, International Classification of Diseases; ER, emergency room. | | | | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|----------------|----------------------------------------------|------|-----| +| Standardized difference (%)f | | | | | | +| n (%)c | 0.5 4.4 | 0.5 | 0.7 2.8 | 0.8 | 0.6 | +| Weighted data (n = 723 370)b | Infantile colic groupe (n = 359 842) Control groupd (n = 363 528) | 134 985 (37.5) | 168 537 (46.8) 198 425 (55.1) 191 892 (53.3) | | | +| 11 444 (3.2) | 9205 (2.6) | 16 764 (4.7) | | | | +| 127 985 (35.2) | 179 449 (49.4) 197 134 (54.2) 191 926 (52.8) | | | | | +| 11 172 (3.1) | 8741 (2.4) | 17 530 (4.8) | | | | +| Standardized difference (%)f | | | | | | +| n (%)c | 14.3 7.5 | 23.1 | 2.4 3.3 | 30.3 | 0.5 | +| Observed data (n = 368 556) | Infantile colic groupe (n =8787) Control groupd (n = 359 769) | 3410 (38.8) | 4231 (48.2) 4905 (55.8) 5899 (67.1) | | | +| 524 (6.0) | 638 (7.3) | 432 (4.9) | | | | +| 126 399 (35.1) | 168 646 (46.9) 195 021 (54.2) 188 764 (52.5) | | | | | +| 10 825 (3.0) | 8222 (2.3) | 17 352 (4.8) | | | | +| Drug (drug classification code) used within 6 months of age | | | | | | +| Sedative-hypnotics | | | | | | +| Clinical characteristics | Respiratory system drugs | | | | | +| Digestive system drugs | | | | | | +| Table 2. Continued | Hormone drugs | | | | | +| | | | | | | +| Antispasmodics | | | | | | +| Antihistamines | | | | | | +| | | | | | | +| Antipyretics | | | | | | + +624 tile colic group and 56547 (15.6%) in the control group (HR [95% CI], 1.12 [1.10 to 1.13]). In the stabilized and trimmed weighted data and the 4:1 matched data, the associations between infantile colic and IBS were still statistically significant and consistent with the results in the weighted data. + +In addition, we evaluated whether the risk of IBS increased according to the number of infantile colic episodes (Table 4). Children with both a single diagnosis and more than 1 diagnosis of infantile colic had a significant risk of IBS (HR [95% CI], 1.11 [1.10 to 1.13] and 1.11 [1.07 to 1.15], respectively). + +| Table 3. Risk of Irritable Bowel Syndrome With Infantile Colica | Unweighted data | | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------|---------------|---------------------| +| Control groupb (n = 359 769) | Infantile colic groupc (n = 8787) | HR (95% CI)d | | +| IBSe (n [%]) | 55 887 (15.5) | 1533 (17.5) | 1.14 (1.08 to 1.20) | +| Weighted dataf | | | | +| Control group (n = 363 528) | Infantile colic group (n = 359 842) | HR (95% CI) | | +| IBSe (n [%]) | 56 547 (15.6) | 61 759 (17.2) | 1.12 (1.10 to 1.13) | +| Sensitivity analysis | | | | +| Stabilized and trimmed weighted data | | | | +| Control group (n = 363 528) | Infantile colic group (n = 85 854) | HR (95% CI) | | +| IBSe (n [%]) | 56 547 (15.5) | 14 929 (17.4) | 1.13 (1.11 to 1.15) | +| 4:1 Matched data | | | | +| Control group (n = 34 408) | Infantile colic group (n = 8637) | HR (95% CI) | | +| IBSe (n [%]) | 5561 (16.2) | 1504 (17.4) | 1.09 (1.03 to 1.15) | +| a The cohort consisted of the infantile colic group who had experienced infantile colic from 5 weeks to 4 months of age and the control group without infantile colic histories during the same time period. | | | | + +aThe cohort consisted of the infantile colic group who had experienced infantile colic from 5 weeks to 4 months of age and the control group without infantile colic histories during the same time period. + +bAs the reference group, the control group comprises children who had not been diagnosed with infantile colic at 5 weeks to 4 months of age. cInfantile colic group consists of children who have been diagnosed with infantile colic at least once between 5 weeks and 4 months of age. dThe hazard ratios (HRs) were assessed using a Cox proportional hazards model to examine the relationship between infantile colic histories and the risk of irritable bowel syndrome (IBS) development in the cohort. + +eIBS was defined as having the diagnosis of International Classification of Diseases 10th version (ICD-10) codes K58.X (IBS) more than twice, after 4 years of age. fWeighted using inverse probability of exposure weighting based on the propensity score. The propensity score was estimated using multivariable logistic regression with 97 previously covariates, as defined in Supplementary Table 2. Participants in the reference group were weighted as (propensity score/[1-propensity score]). This method produces a weighted pseudo sample of participants in the reference group with the same distribution of measured covariates as the exposure group. + +| Table 4. Risk of the Irritable Bowel Syndrome According to the Number of Infantile Colica Unweighted data (n = 368 556) | Weighted data (n = 723 370)b | | | | | | +|---------------------------------------------------------------------------------------------------------------------------|--------------------------------|-----------------------|------------------------------------|--------------|---------------|---------------------| +| The number of infantile colic history | Subjects (n) | Events of IBS (n [%]) | Subjects (n) Events of IBS (n [%]) | HR (95% CI)c | | | +| IBS Referent | Control groupd | 359 769 | 55 887 (15.5) | 363 528 | 56 547 (15.6) | Ref | +| Exposure | 1 e | 7756 | 1349 (17.4) | 319 475 | 54 833 (17.2) | 1.11 (1.10 to 1.13) | +| Exposure | ≥ 2e | 1031 | 184 (17.9) | 40 367 | 6925 (17.2) | 1.11 (1.07 to 1.15) | +| a The cohort consists of the infantile colic group who had experienced infantile colic from 5 weeks to 4 months of age and the control group without infantile colic history during the same time period. | | | | | | | + +IBS + + Referent Control groupd 359 769 55 887 (15.5) 363 528 56 547 (15.6) Ref + + Exposure 1 + +e 7756 1349 (17.4) 319 475 54 833 (17.2) 1.11 (1.10 to 1.13) + + Exposure ≥ 2e 1031 184 (17.9) 40 367 6925 (17.2) 1.11 (1.07 to 1.15) + +aThe cohort consists of the infantile colic group who had experienced infantile colic from 5 weeks to 4 months of age and the control group without infantile colic history during the same time period. + +bWeighted using inverse probability of exposure weighting based on the propensity score. The propensity score was estimated using multivariable logistic regression + +with 97 previously covariates, as defined in Supplementary Table 2. Participants in the reference group were weighted as (propensity score/[1-propensity score]). + +This method produces a weighted pseudo sample of participants in the reference group with the same distribution of measured covariates as the exposure group. cHazard ratios (HRs) were assessed using a Cox proportional hazards model to examine the relationship between infantile colic history and the risk of irritable bowel + +syndrome (IBS) in the cohort. + +dAs the reference group, the control group comprised children who had not been diagnosed with infantile colic at 5 weeks to 4 months of age. eInfantile colic group consists of children who have been diagnosed with infantile colic at least once between 5 weeks and 4 months of age. + +## Sensitivity Analysis Using Various Definitions Of Irritable Bowel Syndrome + +The sensitivity analyses using various definitions of IBS are shown in Table 5. The risk for the stricter IBS was significant (HR [95% CI], 1.25 [1.23 to 1.28]) in the infantile colic group. For the IBS subtypes for symptoms of diarrhea and constipation, the risks of IBS-D and IBS-C in the infantile colic group were significantly high (HR [95% CI], 1.20 [1.17 to 1.23] and 1.22 [1.1.19 to 1.26], respectively). The risk remained statistically significant for the strict IBS-D and the strict IBS-C in the infantile colic group (HR [95% CI], 1.20 [1.17 to 1.23], and 1.17 [1.07 to 1.28], respectively), +which were more stringent defined by their use of drug prescriptions. + +## Subgroup Analysis + +The subgroup sensitivity analyses were conducted to assess the robustness of the results (Fig. 2 and Supplementary Table 6). First, when divided into subgroups by the type of feeding during the 4 to 6 months age range, children with former infantile colic had a higher risk of IBS regardless of the type of feeding (HR [95% CI], 1.15 +[1.12 to 1.17] in the breastfeeding group and 1.09 [1.06 to 1.11] +in the no breastfeeding group), than children without infantile colic history, which was consistent with the main results. Second, all children with birth weights under 3.26 kg (median birth weight) and not less than 3.26 kg in the infantile colic group had a significant risk of IBS (HR [95% CI], 1.14 [1.11 to 1.16] and 1.09 [1.01 to 1.11], respectively). Third, the risk of IBS remained consistently significant in the infantile colic group even after dividing the subgroup according to sex (HR [95% CI], 1.15 [1.12 to 1.17] in male and 1.08 [1.05 to 1.10] in female), and economic status (HR [95% CI], 1.08 [1.06 to 1.11] in the low economic group and 1.16 [1.13 to 1.19] in the high economic group). + +In addition, we performed subgroup analyses using the strict IBS definition (Supplementary Table 7). The results regarding the risk of strict IBS with infantile colic were consistently significant in all subgroups. + +| Table 5. Risk of Irritable Bowel Syndrome With Several Definitions of Infantile Colica Unweighted data (n = 368 556) Weighted data (n = 723 370)b | HR (95% CI) | | | | | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------|----------------|------------------------|---------------|---------------------| +| n (%) | n (%) | e | | | | +| Control groupc | Infantile colic groupd | Control groupc | Infantile colic groupd | | | +| (n = 359 769) | (n = 8787) | (n = 363 528) | (n = 359 842) | | | +| Strict IBSf | 34 476 (9.6) | 1099 (12.5) | 34 926 (9.6) | 42 246 (11.7) | 1.25 (1.23 to 1.28) | +| IBS-Dg | 21 203 (5.9) | 699 (7.6) | 21 492 (5.9) | 25 256 (7.0) | 1.20 (1.17 to 1.23) | +| IBS-Ch | 19 184 (5.3) | 625 (7.1) | 19 438 (5.4) | 23 205 (6.5) | 1.22 (1.19 to 1.26) | +| Strict IBS-Di | 21 176 (5.9) | 668 (7.6) | 21 465 (5.9) | 25 207 (7.0) | 1.20 (1.17 to 1.23) | +| Strict IBS-Cj | 1689 (0.5) | 58 (0.7) | 1715 (0.5) | 1958 (0.5) | 1.17 (1.07 to 1.28) | +| a The cohort consisted of the infantile colic group who had experienced infantile colic from 5 weeks to 4 months of age and the control group without infantile colic histories during the same time period. | | | | | | + +HR (95% CI) n (%) n (%) e Control groupc +(n = 359 769) +Infantile colic groupd +(n = 8787) +Control groupc +(n = 363 528) +Infantile colic groupd +(n = 359 842) +Strict IBSf 34 476 (9.6) 1099 (12.5) 34 926 (9.6) 42 246 (11.7) 1.25 (1.23 to 1.28) +IBS-Dg 21 203 (5.9) 699 (7.6) 21 492 (5.9) 25 256 (7.0) 1.20 (1.17 to 1.23) +IBS-Ch 19 184 (5.3) 625 (7.1) 19 438 (5.4) 23 205 (6.5) 1.22 (1.19 to 1.26) Strict IBS-Di 21 176 (5.9) 668 (7.6) 21 465 (5.9) 25 207 (7.0) 1.20 (1.17 to 1.23) +Strict IBS-Cj 1689 (0.5) 58 (0.7) 1715 (0.5) 1958 (0.5) 1.17 (1.07 to 1.28) +aThe cohort consisted of the infantile colic group who had experienced infantile colic from 5 weeks to 4 months of age and the control group without infantile colic histories during the same time period. + +bWeighted using inverse probability of exposure weighting based on the propensity score. The propensity score was estimated using multivariable logistic regression with 97 previously covariates, as defined in Supplementary Table 2. Participants in the reference group were weighted as (propensity score/[1-propensity score]). + +This method produces a weighted pseudo sample of participants in the reference group with the same distribution of measured covariates as the exposure group. cAs the reference group, the control group comprises children who had not been diagnosed with infantile colic at 5 weeks to 4 months of age. dInfantile colic group consists of children who have been diagnosed with infantile colic at least once between 5 weeks and 4 months of age. eThe hazard ratios (HRs) were assessed using a Cox proportional hazards model to examine the relationship between infantile colic histories and the risk of irritable bowel syndrome (IBS) development in the cohort. + +fDefined with at least 1 diagnosis of International Classification of Diseases 10th version (ICD-10) code K58.X (IBS) and R10.X (abdominal pain) and at least 1 of ICD-10 code K59.1 (functional diarrhea), K52.2 (allergic and dietetic gastroenteritis and colitis), K52.8 (other specific noninfective gastroenteritis), or K59.0 (constipation) after the age of 4 years. + +gDefined with at least 1 diagnosis of ICD-10 code K58.X (IBS) and R10.X (abdominal pain) and at least 1 diagnosis of ICD code K59.1 (functional diarrhea) after 4 years of age. + +hDefined with at least 1 diagnosis of ICD-10 code K58.X (IBS) and R10.X (abdominal pain) and at least 1 diagnosis of ICD code K59.0 (constipation) after 4 years of age. + +iDefined with at least 1 diagnosis of ICD-10 code K58.X (IBS) and R10.X (abdominal pain) and at least 1 diagnosis of ICD code K59.1 (functional diarrhea) and at least once of drug classification 237 (antidiarrheal drug) after 4 years of age. + +jDefined with at least 1 diagnosis of ICD-10 code K58.X (IBS) and R10.X (abdominal pain) and at least 1 diagnosis of ICD code K59.0 (constipation) and at least once of drug classification 238 (laxative drug) after 4 years of age. IBS-D, diarrhea-predominant IBS; IBS-C, constipation-predominant IBS. + +626 + +![9_image_0.png](9_image_0.png) + +## Discussion + +Our large national administrative cohort showed that infantile colic in young infants may be an early clinical manifestation of functional gastrointestinal disease. Children with a history of infantile colic at the age of 4 weeks to 5 months had a statistically significant risk for the development of IBS after 4 years of age. In the subgroup analyses according to the type of feeding, birth weight, sex, and economic status, the association of infantile colic with IBS, remained consistent. Furthermore, in the sensitivity analyses using the different definitions of IBS, the associations between infantile colic and IBS remain significant. + +A few studies have reported the association of infantile colic with the onset of IBS. In a randomized, double-blind, prospective study in Finland (n = 74), children with former colic-type crying during the seventh week of life had more developed functional gastrointestinal diseases by the age of 13 years, than children without former colictype crying (28.0% versus 5.6%, respectively, P-value = 0.05).21 In Italy (n = 96), children diagnosed with functional gastrointestinal diseases had more histories of infantile colic than healthy children +(odds ratio, 2.81; P-value, < 0.001).22 However, these previous studies included a small number of participants. As our results are consistent with those of previous studies, this large-scale general population design supports the association of infantile colic with the development of IBS in childhood. + +Although our results do not prove that the 2 diseases have common pathogeneses, several studies support our conclusions. Aberrant gut microbiota and low-grade inflammation may explain the link between infantile colic and IBS. Intestinal microbiota of infants with infantile colic were shown to consist of a lower abundance of Bifidobacteria and a higher abundance of Proteobacteria.2,23 Children with IBS had a 1.5-fold higher concentration of Ruminococcus and Clostridium spp. in the stool compared with healthy controls.24 In addition, fecal calprotectin, a marker for intestinal inflammation, was elevated in infants with infantile colic25 and in children with IBS.26,27 Low-grade inflammation in the intestinal mucosa and immune alterations have been found in infants with infantile colic2,28 and children with IBS.29 In an animal study, rats exposed to colonic inflammation during the infant period had a chronic visceral hypersensitivity during the adult period, which is considered the major pathophysiology of IBS.30-32 Psychosocial factors play a role in the development of IBS and infantile colic. Children's personalities, with difficult temperaments such as irritability and hypersensitivities and who experience parental anxieties or depression, have been thought to be risk factors for development of infantile colic.33 In addition, poor emotion regulation and parental concerns about the child were associated with a steeper increase in the prevalence of abdominal pain.34 We suggest that children with these risk personalities may develop clinical manifestations of infantile colic in young infants and IBS in children. + +However, there were some limitations to our study. First, we relied on the definitions of infantile colic and IBS according to the ICD-10 codes and the drug classification codes from the administrative data. Therefore, we attempted to use the definition of IBS +that validated the diagnostic yield according to previous studies16,17 and we performed additional subgroup and sensitivity analyses. + +Second, this was an administrative cohort that may have had unmeasured confounders. Although IPTW was used for the demographic/clinical characteristics as well as the results of the physical examinations to match the 2 groups to baseline health status and reduce potential confounding factors, the inflated numbers in the weighted data could increase the type 1 error, and our results cannot infer any causal relationship. Therefore, we tried to perform the stabilized and trimmed IPTW and 4:1 PS matching to confirm the consistency of results and minimize the type 1 error. + +An important strength of this study was that it enrolled all children born between 2008 and 2009, and the data in this study had representative characteristics and the results can be generalized. Furthermore, to reduce the various potential confounding factors between the 2 groups, including demographic characteristics and clinical baseline conditions, 97 covariates were matched using PS +matching. In addition, we accessed the results of the physical examinations by the physicians and the nutritional status to balance the clinical baseline conditions between the 2 groups. Finally, to verify our results, several subgroup and sensitivity analyses were performed, and all results remained consistently significant. + +In conclusion, we found that children with infantile colic histories in the early infant period had a significant risk for later IBS development after 4 years of age. In particular, IBS is a representative form of a functional gastrointestinal disease that extends into the adult period. Efforts to uncover and solve the pathogenesis of infantile colic in the neonatal period may reduce the prevalence and severity of FGID from childhood to adolescence to adulthood. + +## Supplementary Materials + +Note: To access the supplementary material and tables mentioned in this article, visit the online version of Journal of Neurogastroenterology and Motility at http://www.jnmjournal.org/, and at https://doi.org/10.5056/jnm21181. + +Financial support: This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (NRF2020R1F1A1076452). The funders had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. + +## Conflicts Of Interest: None. + +Author contributions: Ju Hee Kim, Seung Won Lee, Su Jin Jeong, and Man Yong Han conceptualized and designed this study; Hye Ryeong Cha, Seung Won Lee, Jaewoo An, and Man Yong Han were involved in data collection, data analysis, and interpretation; Ju Hee Kim, Eun Kyo Ha, and Yoowon Kwon were involved in manuscript writing and revision; and Ju Hee Kim, Yoowon Kwon, Eun Kyo Ha, and Su Jin Jeong supervised the data interpretation and critically reviewed the manuscript for important intellectual content. All authors read and approved the final manuscript. + +## References + +1. Hjern A, Lindblom K, Reuter A, Silfverdal SA. A systematic review of prevention and treatment of infantile colic. Acta Paediatr 2020;109:17331744. + +2. Mai T, Fatheree NY, Gleason W, Liu Y, Rhoads JM. Infantile colic: new insights into an old problem. Gastroenterol Clin North Am 2018;47:829844. + +3. Shirazinia R, Golabchifar AA, Fazeli MR. Efficacy of probiotics for managing infantile colic due to their anti-inflammatory properties: a metaanalysis and systematic review. Clin Exp Pediatr 2021;64:642-651. + +4. Ong TG, Gordon M, Banks SS, Thomas MR, Akobeng AK. + +Probiotics to prevent infantile colic. Cochrane Database Syst Rev 2019;3:CD012473. + +5. Mearin F, Lacy BE, Chang L, et al. Bowel disorders. Gastroenterology 2016;150:1393-1405, e5. + +6. Camilleri M. Diagnosis and treatment of irritable bowel syndrome: a review. JAMA 2021;325:865-877. + +7. Ford AC, Sperber AD, Corsetti M, Camilleri M. Irritable bowel syndrome. Lancet 2020;396:1675-1688. + +8. Koloski NA, Jones M, Kalantar J, Weltman M, Zaguirre J, Talley NJ. + +The brain–gut pathway in functional gastrointestinal disorders is bidirectional: a 12-year prospective population-based study. Gut 2012;61:12841290. + +9. Houghton LA, Foster JM, Whorwell PJ. Alosetron, a 5-HT3 receptor antagonist, delays colonic transit in patients with irritable bowel syndrome and healthy volunteers. Aliment Pharmacol Ther 2000;14:775-782. + +10. Zhao L, Yang W, Chen Y, et al. A Clostridia-rich microbiota enhances bile acid excretion in diarrhea-predominant irritable bowel syndrome. J Clin Invest 2020;130:438-450. + +11. Principi N, Cozzali R, Farinelli E, Brusaferro A, Esposito S. Gut dysbiosis and irritable bowel syndrome: the potential role of probiotics. J Infect 2018;76:111-120. + +12. Savino F, Castagno E, Bretto R, Brondello C, Palumeri E, Oggero R. A +prospective 10-year study on children who had severe infantile colic. Acta Paediatr Suppl 2005;94:129-132. + +13. Croffie JM, Barbour B, Gupta SK, Fitzgerald JF. A study of the relationship between infatile colic and some chronic gastrointestinal (GI) disorder od childhood. J Pediatr Gastroenterol Nutr 1998;27:483. + +14. Kim JH, Lee JE, Shim SM, et al. Cohort profile: national investigation of birth cohort in Korea study 2008 (NICKs-2008). Clin Exp Pediatr 2021;64:480-488. + +15. Chung HJ, Yang D, Kim GH, et al. Development of the Korean developmental screening test for infants and children (K-DST). Clin Exp Pediatr 2020;63:438-446. + +16. Tan TK, Chen AC, Lin CL, Shen TC, Li TC, Wei CC. Preschoolers with allergic diseases have an increased risk of irritable bowel syndrome when reaching school age. J Pediatr Gastroenterol Nutr 2017;64:26-30. + +17. Goff SL, Feld A, Andrade SE, et al. Administrative data used to identify patients with irritable bowel syndrome. J Clin Epidemiol 2008;61:617621. + +18. Sato T, Matsuyama Y. Marginal structural models as a tool for standardization. Epidemiology 2003;14:680-686. + +19. Brookhart MA, Wyss R, Layton JB, Stürmer T. Propensity score methods for confounding control in nonexperimental research. Circ Cardiovasc Qual Outcomes 2013;6:604-611. + +20. Austin PC, Stuart EA. Moving towards best practice when using inverse probability of treatment weighting (IPTW) using the propensity score to estimate causal treatment effects in observational studies. Stat Med 2015;34:3661-3679. + +21. Partty A, Kalliomaki M, Salminen S, Isolauri E. Infant distress and development of functional gastrointestinal disorders in childhood: is there a connection? JAMA Pediatr 2013;167:977-978. + +22. Indrio F, Di Mauro A, Riezzo G, Cavallo L, Francavilla R. Infantile colic, regurgitation, and constipation: an early traumatic insult in the development of functional gastrointestinal disorders in children? Eur J Pediatr 2015;174:841-842. + +23. Dubois NE, Gregory KE. Characterizing the intestinal microbiome in infantile colic: findings based on an integrative review of the literature. Biol Res Nurs 2016;18:307-315. + +24. Rajilić–Stojanović M, Biagi E, Heilig HG, et al. Global and deep molecular analysis of microbiota signatures in fecal samples from patients with irritable bowel syndrome. Gastroenterology 2011;141:1792-1801. + +25. Rhoads JM, Fatheree NY, Norori J, et al. Altered fecal microflora and increased fecal calprotectin in infants with colic. J Pediatr 2009;155:823828, e1. + +26. Choi YJ, Jeong SJ. Is fecal calprotectin always normal in children with irritable bowel syndrome? Intest Res 2019;17:546-553. + +27. Jeong SJ. The role of fecal calprotectin in pediatric disease. Korean J Pediatr 2019;62:287-291. + +28. Korpela K, Renko M, Paalanne N, et al. Microbiome of the first stool after birth and infantile colic. Pediatr Res 2020;88:776-783. + +29. Akiho H, Ihara E, Nakamura K. Low-grade inflammation plays a pivotal role in gastrointestinal dysfunction in irritable bowel syndrome. World J Gastrointest Pathophysiol 2010;1:97-105. + +30. Al–Chaer ED, Kawasaki M, Pasricha PJ. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development. Gastroenterology 2000;119:1276-1285. + +31. Kanazawa M, Hongo M, Fukudo S. Visceral hypersensitivity in irritable bowel syndrome. J Gastroenterol Hepatol 2011;26:119-121. + +32. Stark D, Van Hal S, Marriott D, Ellis J, Harkness J. Irritable bowel syndrome: a review on the role of intestinal protozoa and the importance of their detection and diagnosis. Int J Parasitol 2007;37:11-20. + +33. Hall B, Chesters J, Robinson A. Infantile colic: a systematic review of medical and conventional therapies. J Paediatr Child Health 2012;48:128-137. + +34. Jones MP, Faresjö Å, Beath A, Faresjö T, Ludvigsson J. Abdominal pain in children develops with age and increases with psychosocial factors. Clin Gastroenterol Hepatol 2020;18:360-367, e1. \ No newline at end of file diff --git a/medical/md/PMC9582421.md b/medical/md/PMC9582421.md new file mode 100644 index 0000000000000000000000000000000000000000..0c9f494227b0f03df9ee277ed365ea065305283b --- /dev/null +++ b/medical/md/PMC9582421.md @@ -0,0 +1,279 @@ +eISSN: 2671-9525 + +![0_image_0.png](0_image_0.png) + +![0_image_1.png](0_image_1.png) + +Original Article + +![0_image_2.png](0_image_2.png) + +![0_image_3.png](0_image_3.png) + +# Use Of Dna Adduct And Histopathological Defects As Indications For Bio-Persistence Potency Of Zinc Oxide Nanoparticles In Gastropod, Monacha Cartusiana (Mǜller) After Short-Term Exposure + +Khaled Yassin Abdel-Halim 1 ,* , Safaa Ramadan Osman 1, Atef Mohamed Khedr Nassar 2, Alaa Khozimy 2, Heba Mohamed El-Danasoury 3 1 Mammalian & Aquatic Toxicology Department, Central Agricultural Pesticides Laboratory (CAPL), Agricultural Research Center (ARC), 12618-Dokki, Giza, Egypt 2 Department of Plant Protection, Faculty of Agriculture, Damanhour University, PO Box 59, Damanhour, El-Beheira, Egypt 3 Department of Plant Protection, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt *Correspondence: khaled_yassen68@yahoo.com Received: June 7, 2022 **Accepted**: August 22, 2022 + +## Abstract + +The cytotoxic effects of metallic nanoparticles (MNPs) might be revealed in genomic and histopathological defects. Therefore current study aimed to assess the bio-persistence and adverse effects potency of zinc oxide nanoparticles (ZnONPs) in the gastropod, *Monacha cartusiana.* Gastropods were exposed to 74 μg/mL for 14 d then the DNA adduct and histopathological defect profiles were assessed. The results demonstrated significant decline in the estimated genomic template stability (GTS%) in haemolymph and digestive gland ranging from 10.0 to 42.9% in treated animals compared to controls. In the treated and recovered snails, randomly amplified polymorphic (RAPD)-DNA showed the appearance and/or disappearance of DNA bands, indicating DNA damage due to the cytotoxicity of ZnONPs on gastropods. Significant defects in microvilli (MV), nucleus (N), mitochondria (M), and execratory glands (EXG) were noticed in the treated individuals with respect to controls. The remaining live animals were subjected to a recovery period (14 d, without treatment) and slight recovery profiles were reported for both measures compared to the control group. The recovery pattern was recognized in the nucleus/cytoplasm ratio with 0.186 and 0.428 in the treated and recovered groups concerning their control (0.176). The studied parameters reported herein might be reliable tools to assess accumulation and bio-persistence patterns of NPs in the organisms for short-term exposure indicating the cytotoxic and genotoxic effects. Also, gastropods may provide simple models for evaluating the ecotoxicological effects of nanomaterials. + +Keywords: zinc oxide nanoparticles, gastropods, DNA adduct, histopathological defects, short-term exposure + +## Introduction + +Nanotechnology has attained an exceptional position, especially in biotechnology, which has an unlimited character to support life superiority [1]. Metallic nanoparticles (MNPs), such as zinc oxide nanoparticles (ZnONPs), are useful in a variety of agricultural (pesticide manufacturing and antimicrobial agents), industrial, and medical applications (including sun protection creams and ointments to treat skin disorders and bioimaging [2-6]. Moreover, NMs could interfere with the aquatic ecosystems triggering a prospective risk to the aquatic creatures. As a result, legalizing the safe limits of NMs for both occupational and environmental models is crucial. Also, it is vital to develop effective and sensitive analytical methods for the environmental monitoring of NMs [7,8]. A diversity of bioindicators might be used to study the potential toxicity of NMs. For example, ZnONPs showed ecotoxicological effects on zooplankton [9], phytoplankton [10], amphibians [11], fish [12], reptiles [13], mammals [14,15], snails [16] and plants [17]. Specifically, the exposure of guppy (*Poecilia reticulata*) to zinc sulfate (at 10, 50, and 100 μg/L) and ZnONPs increased DNA damage after 48 h and antioxidant enzymes after 21 d, suggesting that NMs including the ZnNPs' have genotoxic impacts [18]. It could incidentally stimulate oxidative damage through the production of reactive oxygen species (ROS). + +Oxidative stress (OXS) is a useful indicator for determining the ecotoxicity of ZnONPs against various organisms +[21-23]. The OXS is the primary mechanism proposed to illustrate NM's toxicity [24]. ROS levels in haemocytes have been used to quantify OXS in terrestrial environments [25] and marine [26]. Additionally, the generation of ROS might induce DNA breakage [27], protein carbonylation [28], lipid peroxidation [26,29], reaction with oxidative organelles, e.g., mitochondria [30], and apoptosis [31]. + +The genotoxicity was evaluated by examining variations in DNA profiles independent of band shifts and intensity +[32,33] and the gain and loss of bands [34,35]. According to several studies, DNA fingerprints could be used as a functional biomarker assay in toxicology. Therefore, several aquatic and terrestrial models were employed to screen xenobiotic toxicity, owing to their ability to bioaccumulate metals, adapt to numerous environments, and ease of rearing in diverse climatic conditions. Similarly, the gastropods were used as biomarkers for DNA damage. Genotoxicity and cytotoxicity were induced by different substances including NMs (ZnNPs) in different species of gastropods, e.g., *Nerita chanaeleon* [36], Morula granulata [37], *Lymnaea luteola* [38], and *M. cartusiana* [39]. Consequently, it was an excellent bioindicator class [40]. Therefore, the current study aimed to look at DNA adducts and histopathological changes in the land snail (*M. cartusiana*) caused by short-term exposure to sub-lethal levels of ZnONPs. + +## Materials And Methods Preparation Of Zinc Oxide Nanoparticles (Znonps) + +The ZnONPs were produced in an alcoholic medium through the condensation and hydrolysis of zinc acetate dehydrates at low-temperature conditions by potassium hydroxide. The precipitate was formed and distributed in a combination of methanol and chloroform, cleaned with deionized water, and dried at 100 C for 15 min [41]. + +## Characterization Of Znonps + +The size and shape of ZnO powder's aliquot deposited on a copper grid were investigated using the JOEL +Scanning Electron Microscopy (SEM, JSM5300). To determine the purity of the prepared NPs, an X-ray Oxford detector unit (model 6647, England) was used in conjunction with the SEM (JOEL, JSM5300) and Oxford detector unit of X-ray (model 6647, England). A double beam UV-VIS spectrophotometer (HEIOS α v 4.6 Serial No 111518 Thermo Spectronic) was used to analyze the ZnONPs suspension's optical absorption at room temperature in the range of 200 to 800 nm. + +## Experimental Design Examined Land Snail + +The snail, *M. cartusiana* (Müller), was chosen as the subject of the experiments. Healthy individuals weighing 2.5 +±0.7 g were obtained from Delta Egypt's clover farms. Every 100 individuals were kept in an aerated wooden cage with dimensions of 40×40×40 cm for 14 d under laboratory conditions (25±2 C; 12:12 h light/dark and 63±2% relative humidity). + +Animals were fed leaves of lettuce *ad libitum.* + +## Toxicity Test + +The animals were categorized into three replicates, each of 20 individuals. As a control group, land snails were fed lettuce leaves (toxicant free). Treated animals were fed lettuce leaves that were soaked in 74 μg/mL for two weeks, representing 0.1 of the LC50 value reported by Abdel-Halim et al. [16]. They were monitored daily. Some of the living animals were collected for toxicological analyses. A 40 mm needle connected with a sterilized 10 mL syringe was cautiously inserted below the shell from the hemocoel beside the right side of the skull to collect the haemolymph. The fluid was collected in anticoagulant vials and kept at -20 C until it was analyzed. The digestive glands were separated from the animals and kept at -20 C until being analyzed. + +## Recovery Experiment + +Following the toxicity experiment, the live animals have nourished with lettuce leaves (toxicant-free) for 14 d and animals were monitored daily. Then samples were taken in the manner above-mentioned. + +## Fragmentation Of Dna Extraction Of Dna + +A 50 mg of the digestive glands or 100 μL of the haemolymph fluid were aliquoted and mixed with 940 μL of lysis buffer [100 mM EDTA, pH 8.0; 200 mM Tris-HCl, pH 8.0; 250 mM NaCl), 30 μl proteinase K (10 mg/mL) and 30 μL 20% +SDS]. In a water bath, the contents were kept at 48 C for 45-50 min. Phenol, chloroform, and isoamyl were added with a ratio of 25:24:1 (v/v), gently inverted for 10 min and centrifuged at 9200×g for 10 min. Supernatants were transferred into new 1.5 mL Eppendorf tubes. After that, DNA was precipitated by adding equivalent volumes of isopropanol to 0.2 volume of 10 M ammonium acetate, tenderly inverting several times, and centrifuging at 13200 g for 10 min. The pellet was quickly cleaned in 500 μL of 70% ethanol, air-dried, and re-suspended in 200 μL of sterilized water/TE buffer [42]. + +The purity factor (A260/A280 nm) was quantified spectrophotometrically after assuring the complete solubility of DNA. Its reliability was confirmed by loading 10 μL of the prepared DNA (2 μL extracted DNA, 2 μL dye, and 6 μL sterilized water) on a 0.7 percent agarose gel and staining with ethidium bromide and the quantity and quality compared to 0.2 μL of DNA standard marker. + +## Issr Conditions And Preparations + +The PCR amplification was performed using six Inter Simple Sequence Repeats (ISSRs) primers to differentiate and fingerprint six samples: (1) control haemolymph, (2) control tissue, (3) treatment haemolymph, (4) treatment tissue, (5) recovered individuals' haemolymph, and (6) recovered individual tissue (Table 1). A total volume (25 μL) was used for the amplification, including 2 μL DNA, 2 μL primer, 2.5 μL 10X buffer, 2.5 μL MgCl2 (50 mM), 2.5 μL dNTPs (4 mM), and 0.2 μL (5 units/μL) Taq DNA polymerase (Promega Germany). It was carried out using an automated thermal cycler with the following PCR conditions: one cycle at 94 C for 5 min, followed by 40 cycles [1 min at 94 C, 75 seconds of temperature annealing for each primer as itemized in Table 1 and 2 min at 72 C], and finally a final extension for 10 min at 72 C. Gel electrophoresis in a 1 percent (w/v) agarose gel in TBE buffer at 120 V for 30 min separated the PCR products. The bands were then noticed with ethidium bromide staining and photographed with a gel documentation system (Bio-Rad Gel Doc., 2000). + +| Primer | Primer sequence (5′-3′) | Annealing temperature (C) | +|---------------------------------------|---------------------------|------------------------------| +| HB-09 | GTGTGTGTGTGTGG | 53 | +| HB-12 HB-14 UBC811 UBC868 UBC840 | CACCACCACGC | | +| GTGTGTGTGTGTGC | | | +| GAGAGAGAGAGAGAGC | | | +| GAAGAAGAAGAAGAAGAA GAGAGAGAGAGAGAGATT | 55 57 57 51 55 | | + +Table 1. List of used ISSR primers, their sequences, and annealing temperatures. + +## Treatments' Phylogenetic Relationships + +To calculate the pairwise difference matrix among the treatments, the Gene-Profiler-Eval computer application was performed to record variations in separated bands with each primer of ISSRs markers. Nei Li's coefficient, which is included in the Multivariate Statistical Package (MVSP) Version 3.1 computer program, was used to perform genetic comparisons based on ISSR fingerprints among treatments. The Unweighted Pair Group Method with Arithmetic Means (UPGMA) used Nei Li's similarity coefficient to cluster the data matrix [43]. + +## Genomic Template Stability + +The following formula was used to estimate genomic template stability (GTS percent): + +## Gts% = 1 − A N × 100 (1) + +where; a represents the average number of polymorphic bands discovered in each treated sample and n represents the total number of bands in control. Compared to the control profile, polymorphism in the RAPD profile comprises the disappearance of a regular band and the introduction of a new band [44]. + +## Examination Of The Ultrastructure + +The individuals from both treatments and control groups were dissected, and the digestive glands were taken. + +Small fragments were fixed as quickly as possible with 2 mL of 2.5% glutaraldehyde diluted in 0.1 M phosphate buffer, pH +7.2, and kept at 4 C until the analysis. The fixed tissues were rinsed in a phosphate buffer solution of 0.1 M, pH 7.2. After that, it was washed with buffer for 2 min before being submerged in 1% osmium tetraoxide (OsO4) for 1-2 h at 4 C. The samples were dehydrated for 5 min with an increasing concentration of acetone (25, 50, 75, and 100%). The tissues were infiltrated with propylene oxide after being dehydrated in 100% acetone. The specimen was embedded in Epon Araldite for 48 h at room temperature. Capsulated samples were sectioned at 20-30 nm thickness using an Ultratome machine. The sections were obtained on metal mesh (grids) and stained with toluidine blue for orientation. The grids were dyed for 5 min with 4% uranyl acetate and then rinsed with pure water. After rinsing, the grids were stained for 5 min with 1% lead acetate, rinsed again in water, and stored in a grid box until they were examined [45]. The grids were viewed using a Transmission Electron Microscope (TEM) (JOEL 1400 Plus, Japan) to understand the cell changes. As necessary, a sequence of bright–field imaging was used. + +## Statistical Analysis + +All data were presented as mean ± standard deviation (SD), underwent analysis of variance (ANOVA), and means were assessed to significance using the least significant difference (LSD) method at a probability of 0.05 [46]. Furthermore, according to the fisher distribution, line regression with * and ** is significant with 5% and 1% probability levels for DNA gel pictures, respectively (Statist XL v 2.1, 2003). The principal component analysis (PCA) was used to examine multilinear regression between ZnONPs-treated and recovered snails and DNA fingerprints in the haemolymph and digestive tract [47]. To determine the diversity of histopathological investigation between treatments and the control, box plot graph analysis was made using SPSS-Version 16. + +## Results + +![3_Image_0.Png](3_Image_0.Png) Nanoparticles Characterization + +The UV-VIS absorbance showed an absorption band at nearly 360 nm (Figure 1a). The ZnONPs exhibited nearly spherical shapes with sizes ranging from 22 to 45 nm (Figure 1b). In addition, the elemental analysis using EDA pattern was plotted in Figure 1c exhibited domination of Zn (96.0%) of the total content of minerals in the sample. + +Figure 1. Characterized patterns of ZnONPs: (a) UV-VIS spectrum of ZnONPs suspension; (b) SEM image at 35.000X; (c) XAD pattern of ZN ions percent in the prepared NPs. + +## Genomic Dna Diversity + +Different profiles were obtained when the genomic DNA of *M. cartusiana* animals that were exposed to 74 μg/mL +of ZnONPs (Figure 2). These profiles among genetic diversities of *M. cartusiana* were noticed using Nei Li's coefficient embedded in the MVSP program, where a pairwise difference matrix was stated (Figure 3). Six primers were employed to amplify genomic DNA successfully. The RAPD fingerprints with varied bands ranging from 200 to 1000 bp were obtained, regulated by the state of treated and recovered animals and the used primer. + +The results in Figure 4 show the relationship between treated haemolymph and digestive g. with recovery ratios. + +It was found that recovered haemolymph positively high associated with treated haemolymph and partially associated with treated digestive g. In the same way, recovered digestive g. positively associated with treated digestive gland. The HB-14 primer produced 5-6 bands with sizes ranging from 300 to 1000 bp (Figure 2a) with significant declined GST 20.0% (r2=0.796*) (p<0.05) for the treated haemolymph and increased to the same value in the recovered individuals (r2=-1.377*), respect to control (Figure 4). Also, it was declined to 14.3% for the treated digestive glands (r2=0.185) and significantly declined to 42.9% for the recovered individuals (r2=0.957*). In case of the primer HB-09, the number of bands ranged from 5 to 7 bands with sizes from 200 to 700 bp in the digestive glands and haemolymph samples with GST values 50.0 (r2=0.818*) and 25.0% (r2=0.413) in haemolymph and 28.0 (r2=0.55*) and 00.0% in digestive glands for the treatment and recovery, respectively + +![4_image_0.png](4_image_0.png) (Figure 2b). The increased pattern was recognized in the generated bands for primer HB-12. The number of bands ranged from 8-10 with sizes ranging from 200 to 1000 bp (Figure 2c) with GST values 12.5 (r2=0.407) and 25.0% (r2=0.521*) greater than haemolymph's of control animals. HB-12 declined to 11.1% (r2=0.167) for the treatment of digestive glands, followed by increased GST in the recovered individuals. + +The primer UBC811 revealed a slight generation of bands. The number of bands was 4-7 bands in the size of 400800 bp (Figure 2d) with increased GST% (16.6%) (r2= -0.266) compared to control in haemolymph and a significant decline to 40.0% in the digestive glands (r2= 0.655*), respect to control. However, significant abundant bands were generated with the primer UBC868 (Figure 2e). The number of bands was 10-12 in size of 300-1000 bp, with GST having values similar in haemolymph and increased in the recovered animals to 10.0% (r2=-0.107). Also, the GST value increased to 10.0% in the treated digestive glands and declined to a steady state (0 level) in the recovered samples. Finally, slight generation was obtained using the primer UBC840, resulting in the range of 5-7 bands with sizes of 300-1000 bp (Figure 2f). Declines in GST% were conducted in the treated and recovered haemolymph with 28.6% (r2=-0.240) but increased to 33.4% (r2=0.663*) in the treated and recovered digestive glands. + +The calculated similarity coefficient among groups of individuals ranged from 0.76 to 0.89 (Figure 3). The greatest value (0.89) was observed in the control's haemolymph and digestive glands samples and between the digestive glands of the control and its treatment. A similar finding with a value (0.88) was recorded between the treatment for both haemolymph and digestive gland, with respect to the recovered haemolymph, and between the treated haemolymph and digestive g. (0.88). The least value (0.76) was noticed between the treated digestive gland and its recovered counterpart sample. + +![5_image_0.png](5_image_0.png) + +![5_image_1.png](5_image_1.png) + +## Ultrastructural Investigations + +The electron microscope images of the digestive gland sections from control snails (untreated) showed a firm structure of the organ at 4000X (Figures 5A and 6A). It was noticed that the completed microvilli (MV) had a regular brush border and surface area of 13.27 μm2. The cells were characterized by the nucleus (N) with a regular nuclear membrane, mitochondria (M), execratory vacuoles (EXV), execratory gland (EXG), digestive cells (DC), Golgi bodies (G), and smooth endoplasmic reticulum (SER). However, in the tissue of ZnONPs-treated snail (74.0 μg/mL) (Figures 5B and 6B) at the same magnification, it was noticed a disturbed brush border of MV arising surface area with a mean value of 2.96 μm2(4.49-folds) lower than the control. Also, ZnONPs caused a destructed cell membrane, rough endoplasmic reticulum (RER), destructed and irregular execratory glands (EXG), execratory vacuoles (EXV), nucleus (N) containing eccentric nuclei (Nu), migrated and aggregated chromatin, irregular and dilated nuclear membrane, and vacuoles (V). On the other hand, sections of the digestive glands of the recovered individuals for 14 d displayed a regular brush border of MV with a mean surface area of 7.66 μm2, representing 1.73-folds of the control sections at the same magnification power (Figures 5C and 6C). It was noticed significant forming of digestive cells (DC), regular cellular membranes with diluted intercellular spaces, mitochondria (M), smooth endoplasmic reticulum (SER), numerous shapes of the nucleus (N), concentric chromatin, lipid droplets (L), and slight firming of Golgi bodies (G) (Figure 6C). + +![6_image_0.png](6_image_0.png) + +![6_image_1.png](6_image_1.png) + +Figure 6. Electron micrographs illustrate [A] section of digestive glands (control) of snail, *Monacha cartusiana* +(Mǜller) showing nucleus (N), regular nuclear membrane (head arrow), mitochondria (M), execratory vacuoles +(EXV), execratory gland (EXG) (arrow), digestive cells (DC) and Golgi bodies (G); [B] section in digestive gland of ZnONPs-treated snail, *H. aspersa* for 14 d showing destructed cell membrane (arrow), rough endoplasmic reticulum (RER), destructed and irregular execratory glands (EXG), execratory vacuoles (EXV), eccentric nuclei (Nu) with irregular and dilated nuclear membrane (head arrow), nucleus (N) and vacuoles (V) and [C] section in digestive gland of recovered individuals for 14 d showing significant forming of digestive cells (DC) (head arrow), regular cellular membranes (arrow), diluted intercellular spaces, mitochondria (M), smooth endoplasmic reticulum (SER), Nemours shapes of nucleus (N) with irregular nuclear membrane, lipid droplets (L) and slight Golgi bodies (G) [Glutaraldehyde-OsO4 fixed uranyl acetate lead citrate stained preparation 4000X]. + +In magnified sections at 10000X, normal mitochondria (M) with lamellar cristae, Golgi bodies (G), and smooth + +![7_image_0.png](7_image_0.png) + +endoplasmic reticulum (SER) were noticed in the control section (Figure 7A). In ZnONPs-treated snails, NPs accumulation in the lysosome was noticed. Also, a lack of mitochondria (M) and rough endoplasmic reticulum (SER) were identified (Figure 7B). On the other hand, the sections of the digestive glands of the recovered individuals showed mitochondria (M) forming with concentric cristae and disrupted mitochondrial membrane. Nucleus (N) with migration and chromatin aggregation is likely to be detectable in the nucleus (Figure 7C). + +The mitochondrial defects in the treated snails appeared as a slight decrease in surface area (mean; 0.161 μm2) +(0.95-folds) compared to control. A significant decrease in the surface area of mitochondria of recovered individuals was noticed (mean; 0.107 μm2) (1.43-folds) (Figure 8). Also, the integrated density of mitochondria declined with 2.86-folds in the treated group, followed by 3.21-folds in the recovered one, with respect to their controls. A similar pattern was noticed in the mitochondria/cytoplasm ratio, where the treated individuals exhibited significant declines (0.0121), followed by (0.0127) in the recovered ones. On the other hand, a significant increase in surface area of the nucleus (N) with a mean of 2.333 μm2(4.11-folds) was noticed compared to control. The recovered individuals exhibited the mean surface area value of the nucleus of 3.489 μm2(6.14-folds). The same pattern was recognized in the nucleus/cytoplasm ratio with values of 0.186 and 0.428 in the treated and recovered groups with respect to their control (0.176). + +Figure 7. Electron micrographs illustrate [A] section of digestive g. of snail, *Monacha cartusiana* (Mǜller) (control) +showing normal mitochondria (M) with lamellar cristae (head arrow), smooth endoplasmic reticulum (SER), cell wall (arrow), and Golgi bodies (G); [B] magnified field to obtain NPs precipitation in the lysosome (arrow), lack of mitochondria (M) and rough endoplasmic reticulum (RER), and [C] section in the digestive gland of recovered individuals showing forming of mitochondria (M) with concentric cristae and disrupted mitochondrial membranes (double arrow) and nucleus (N) with migration & aggregation of chromatin is likely to be detectable in the nucleus (head arrow) [Glutaraldehyde-OsO4 fixed uranyl acetate lead citrate stained preparation 10000X]. + +![8_image_0.png](8_image_0.png) + +## Discussion + +The obtained data showed genotoxicity (DNA adduct) and cytotoxicity of ZnONPs in studied animals. Also, based on reported results, the *M. cartusiana* could provide a reliable model to study the impacts of such NMs on non-target organisms. As documented previously in the literature, OXS is the primary mechanism of action for NPs and ultrafine particles. The findings herein showed damage in cell components: organelles and nucleic acids ending in organ dysfunction and/or apoptosis [48,49]. Such modifications in the structural and functional integrity of adjusted biomolecules across the process of OXS of NMs might initiate a range of downstream functional consequences associated with cellular damage and organ dysfunction [50]. The toxicity of ZnONPs is regulated by the released rate of ions in the solution (indirect effect). Ions +(Zn2+) may be augmented with the increasing concentrations of NMs, where ZnONPs toxicity and bioavailability were controlled by the release of Zn2+ [21,51]. Also, ZnONPs might interact with the tissues and organelles (direct effect) [2,52], which were reported in current study. + +## Genomic Alterations + +Variations in ISSR-PCR patterns stimulated by chemicals can be interpreted due to differences in the stability of genomic DNA template. The present findings reported different patterns of polymorphism for the six studied primers in the amplification of DNA bands in samples from the treated and recovered snails, either as loss and/or gain [53]. Exposure to ZnONPs could provoke genetic diseases owing to their reported genotoxicity in the current study. Other studies reported similar results on amphibians [54] and fish [18]. Also, other organisms were employed to investigate the genotoxicity of NPs. For example, exposing tadpoles, *Pleurodeles waltl,* to CeO2NPs (1 and 10 mg/L) for 12 d showed an increase in micronucleus (MN) [55]. Also, when zebrafish were imperiled by ZnONPs and zinc ions (1-100 mg/L) for 144 h showed modification of gene expression related to oxidizing enzymes and increased DNA damage [12]. Similarly, AgNPs at 55 mg/L induced genomic damage by DNA breaks and OXS in *Labeo rohita* after 14d exposure [56]. ZnONPs at 0.1, 1.0, and 10 mg/L exhibited DNA damage in tadpoles (*Lithobates cutesbeianus*) compared to the control group. It was revealed that the 10 mg/L had provoked a high incidence of MNs in erythrocytes and nucleated cells, indicating probable mutagenic and genotoxic consequences of ZnONPs for amphibians [57]. Other studies have also shown that NPs might cause mitochondria damage, DNA adducts, and DNA oxidation [58]. DNA damage was associated with the excessive generation of ROS, which might cause the deterioration of DNA [59]. In parallel, the digestive glands are a vital organ in detoxifying, absorbing, and metabolizing toxicants. Toxicants were carried out via haemolymph to the digestive glands [60]. As a result, it might be useful to utilize this organ to assess biochemical responses to nanomaterials. + +## Histopathological Defects + +Snail, *H. aspersa* exposed to different concentrations of ZnONPs had altered digestive glands through increased secretory cells, vacuolation, and hemocytic infiltration [61]. Also, Ali et al. [62] documented an increase in the excretory cells and flattened digestive glands in AgNPs-treated snail, *Eobania vermiculata*. Sawasdec et al. [63] reported damage to tubule, vacuoles, enlarged tubule lumen, and destructiveness of digestive glands in copper and lithium-treated snail, Marisa cornuarietis. Moreover, dilation and vacuolation in the crayfish hepatopancreas were induced after exposure to CuONPs [64]. TiO2NPs caused histopathological differences in the red swamp crayfish organs, where the 25 mg/L dose triggered minor changes in hepatopancreatic tubules, such as the emergence of colored pyknotic nuclei and epithelial cell lysis in certain tubules. However, at high concentrations (250 mg/L) of TiO2NPs, epithelial cells lysed, cellular atrophy occurred, and significant vacuolation occurred [64]. Also, TiO2NPs caused significant deterioration of the cellular organelles, including ruptured microvilli, distorted mitochondria, vacuolated cytoplasm, pyknotic nuclei, and fragmented endoplasmic reticulum [65]. Therefore, the interaction of NMs with enzymes might cause OXS and the production of ROS, which could lead to cell necrosis [66,67]. ZnONPs initiated histopathological alterations in bivalves' DG and gills of *C. aegptiaca* [68,69]. The pathological reactions to ZnONPs were cell hypertrophy and hyperplasia; the organs demonstrated gradual alterations in glandular activity. In addition, the lamellae of the gills were uneven, and the filaments were swollen [70]. + +The measurement of mitochondrial swelling can be explained by the activation of membrane permeability in the amended mitochondria. This discovery involves necrosis or death of apoptotic cells as a function of the involved mitochondria number [71]. Hödl et al. [72] state that these tissues can bind Zn2+, and others trigger high levels of metallothioneins. Zinc ions toxicity is intensified, leading to OXS, lipofuscin accumulation, and membrane damage. This result is in accordance with the existing data in the recovered animals, where they could not be completely recovered. Biopersistent potency of ZnONPs displayed this concept through outcome profiles of DNA adducts in the haemolymph and digestive g. and the histopathological defects in sections of digestive glands. + +## Conclusions + +The present findings showed that ZnONPs were bio-persistent in the biological system of snail *M. cartusiana*, and the treated animals couldn't completely be recovered after 14 d. This was clear in the reported patterns of DNA adducts and histopathological defects data. Such changes may represent the outcome of NMs' stress in organisms. Moreover, the present study showed the utility of land snail *M. cartusiana* as a bioindicator for NPs toxicity. + +## Funding + +The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. + +## Conflict Of Interest + +The authors have no relevant financial or non-financial interests to disclose. + +## Credit Author Statement + +KYAH: Supervision, Writing-Reviewing & Editing; SRO: Conceplualization, Methodology; AMKN: Conceplualization, Methodology; AK: Data Curation, Writing-Orginal draft Preparation; HMED: Visualization, Investigation. + +## Orcid + +Khaled Yassin Abdel-Halim: 0000-0003-3389-2693 Atef Mohamed Khedr Nassar: 0000-0002-0394-1530 Alaa Khozimy: 0000-0001-5297-5959 Heba Mohamed El-Danasoury: 0000-0001-6462-3440 + +## References + +[1] Singh A, Mohan Prasad S, Nanotechnology in medical world: a futuristic planning. Int J Sci Res ISSN (Online) +2013;4(6):2319-7064 https://www.ijsr.net/get_abstract.php?paper_id=SUB155517 +[2] Almansour MI, Alferah MA, Shraideh ZA, Jarrar BM. Zinc oxide nanoparticles hepatotoxicity: histological and histochemical study. Environ Toxicol Pharmacol 2017;51:124-130. https://doi.org/10.1016/j.etap.2017.02.015 +[3] Xiong HM. ZnO nanoparticles applied to bioimaging and drug delivery. Adv Mater 2013;25(37):5329-5335. + +https://doi.org/10.1002/adma.201301732 Halim et al. | Bio-persistence potency of zinc oxide nanoparticles in the gastropods +[4] Alkaladi A, El-Deen NAN, Afifi M, Zinadah OAA. Hematological and biochemical investigations on the effect of vitamin E and C on *Oreochromis niloticus* exposed to zinc oxide nanoparticles. Saudi J Biol Sci 2015;22(5):556-563. https://doi.org/10.1016/j.sjbs.2015.02.012 +[5] Husen A. Natural product-based fabrication of zinc-oxide nanoparticles and their applications. Nanomaterals and Plant Potential. Springer, Cham 2019;193-219. https://doi.org/10.1007/978-3-030-05569-1_7 +[6] Kumar CSSR. In: Nanotachnologies for the life sciences 5 1 st ed. Weinheim, Wiley-VHC 2006. + +[7] Mueller NC, Nowack B. Exposure modelling of engineered nanoparticles in the environment. Environ Sci Technol 2008;42(12):4447-4453. https://doi.org/10.1021/es7029637 +[8] Miranda RR, Damaso da Silveira ALR, de Jesus IP, Grötzner SR, Voigt CL, Campos SX, et al. Effects of realistic concentrations of TiO2 and ZnO nanoparticles in *Prochilodus lineatus* juvenile fish. Environ Sci Poll Res 2016;23(6):51795188. https://doi.org/10.1007/s11356-015-5732-8 +[9] Adam N, Schmitt C, Galceran J, Companys E, Vakurov A, Wallace R, et al. The chronic toxicity of ZnO nanoparticles and ZnCl2 to *Daphnia magna* and the use of different methods to assess nanoparticle aggregation and dissolution. Nanotoxicol 2014;8(7):709-717. https://doi.org/10.3109/17435390.2013.822594 +[10] Miao AJ, Zhang XY, Luo Z, Chen CS, Chin WC, Santschi PH, et al. Zinc oxide–engineered nanoparticles: dissolution and toxicity to marine phytoplankton. Environ Toxicol Chem 2010;29(12):2814-2822. https://doi.org/10.1002/etc.340 +[11] Falfushynska H, Gnatyshyna L, Horyn O, Shulgai A, Stoliar O. A calcium channel blocker nifedipine distorts the effects of nano-zinc oxide on metal metabolism in the marsh frog *Pelophylax ridibundus*. Saudi J Biol Sci 2019;26(3):481-489. https://doi.org/10.1016/j.sjbs.2017.10.004 +[12] Zhao X, Wang S, Wu Y, You H, Lv L. Acute ZnO nanoparticles exposure induces developmental toxicity, oxidative stress and DNA damage in embryo-larval zebrafish. Aquat Toxicol 2013;136:49-59. https://doi.org/10.1016/j.aquatox.2013.03.019 +[13] Da Costa Araújo AP, Gomes AR, Malafaia G. Hepatotoxicity of pristine polyethylene microplastics in neotropical Physalaemus cuvieri tadpoles (Fitzinger, 1826). J Hazard Mater 2020;386:121992. https://doi.org/10.1016/j.jhazmat.2019.121992 +[14] Vandebriel RJ, De Jong WH. A review of mammalian toxicity of ZnO nanoparticles. Nanotechnol Sci Appl 2012;5:6171. https://doi.org/10.2147/NSA.S23932 +[15] de Souza JM, de Oliveira Mendes B, Guimar˜aes ATB, de Lima Rodrigues AS, Chagas TQ, Rocha TL, et al. Zinc oxide nanoparticles in predicted environmentally relevant concentrations leading to behavioral impairments in male swiss mice. Sci Total Environ 2018;613:653-662. https://doi.org/10.1016/j.scitotenv.2017.09.051 +[16] Abdel-Halim KY, Osman SR, Abdou GY. In vivo evaluation of oxidative stress and biochemical alteration as biomarkers in glass clover snail, *Monacha cartusiana* exposed to zinc oxide nanoparticles. Environ Poll 2020;257:113120. https://doi.org/10.1016/j.envpol.2019.113120 +[17] Lin D, Xing B. Phytotoxicity of nanoparticles: inhibition of seed germination and root growth. Environ Poll 2007;150(2):243-250. https://doi.org/10.1016/j.envpol.2007.01.016 +[18] Qualhato G, Rocha TL, de Oliveira Lima EC, de Silva DM, Cardoso JR, Grisolia CK, et al. Genotoxic and mutagenic assessment of iron oxide (maghemite-γ-Fe2O3) nanoparticle in the guppy *Poecilia reticulata*. Chemosphere 2017;183:305314. https://doi.org/10.1016/j.chemosphere.2017.05.061 +[19] Petros RA, Ropp PA, DeSimone JM. Reductively labile PRINT particles for the delivery of doxorubicin to HeLa cells. J +Am Chem Soc 2008;130(5):5008-5009. https://doi.org/10.1021/ja801436j +[20] Dendukuri D, Doyle PS. The synthesis and assembly of polymeric microparticles using microfluidics. Adv Mater 2009;21(41):4071-4086. https://doi.org/10.1002/adma.200803386 +[21] Ali D, Alarifi S, Kumar S, Ahamed M, Siddiqui MA. Oxidative stress and genotoxic effect of zinc oxide nanoparticles in freshwater snail *Lymnaea luteola* L. Aquat Toxicol 2012;124-125:83-90. https://doi.org/10.1016/j.aquatox.2012.07.012 +[22] Fahmy SR, Abdel-Ghaffar F, Bakry FA, Sayed DA. Ecotoxocological effect of sublethal exposure to zinc oxide nanoparticles on freshwater *Biomphalaria alexandrina*. Arch Environ Contam Toxicol 2014;67(2):192-202. https://doi.org/10.1007/s00244-014-0020-z +[23] Trevisan R, Delapedra G, Mello DF, Arl M, Schmidt EC, Meder F, et al. Gills are an initial target of zinc oxide nanoparticles in oysters *Crassostrea gigas*, leading to mitochondrial disruption and oxidative stress. Aquat Toxicol 2014;153:27-38. https://doi.org/10.1016/j.aquatox.2014.03.018 +[24] Mocan T, Clichici S, Agoşton-Coldea L, Mocan L, Şimon Ş, Ilie I, et al. Implications of oxidative stress mechanisms in toxicity of nanoparticles (review). Acta Physiol Hung 2010;97(3):247-255. https://doi.org/10.1556/aphysiol.97.2010.3.1 +[25] Itziou A, Kaloyianni M, Dimitriadis VK. Effects of organic contaminants in reactive oxygen species, protein carbonylation and DNA damage on digestive gland and haemolymph of land snails. Chemosphere 2011;85(6):1101-1107. https://doi.org/10.1016/j.chemosphere.2011.07.043 +[26] Taze C, Panetas I, Kalogiannis S, Feidantsis K, Gallios GP, Kastrinaki G, et al. Toxicity assessment and comparison between two types of iron oxide nanoparticles in *Mytilus galloprovincialis*. Aquat Toxicol 2016;172:9-20. https://doi.org/10.1016/j.aquatox.2015.12.013 +[27] McKelvey-Martin VJ, Green MHL, Schmezer P, Pool-Zobel BL, De Méo MP, Collins A. The single cell gel electrophoresis assay (comet assay): a European review. Mutat Res Fundam Mol Mech Mutagen 1993;288(1):47-63. https://doi.org/10.1016/0027-5107(93)90207-V +[28] Mohanty JG, Bhamidipaty S, Evans MK, Rifkind JM. A fluorimetric semi-microplate format assay of protein carbonyls in blood plasma. Anal Biochem 2010;400(2):289-294. https://doi.org/10.1016/j.ab.2010.01.032 +[29] Kaloyianni M, Feidantsis K, Nteli I, Stergiou P, Tsoulia T, Dimitriadi A, et al. Biochemical and molecular responses of cyprinids in two Mediterranean lacustrine ecosystems: Opportunities for ecological assessment and biomonitoring. Aquat Toxicol 2019;211:105-115. https://doi.org/10.1016/j.aquatox.2019.03.021 +[30] Zhang DX, Gutterman DD. Mitochondrial reactive oxygen species-mediated signaling in endothelial cells. Am J Physiol Heart Circ Physiol 2007;292(5):H2023-H2031. https://doi.org/10.1152/ajpheart.01283.2006 +[31] Gomes T, Chora S, Pereira CG, Cardoso C, Bebianno MJ. Proteomic response of mussels Mytilus galloprovincialis exposed to CuO NPs and Cu2+: an exploratory biomarker discovery. Aquat Toxicol 2014;155:327-336. https://doi.org/10.1016/j.aquatox.2014.07.015 +[32] Atienzar FA, Jha AN. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. Mutat Res 2006;613(2-3):76-102. https://doi.org/10.1016/j.mrrev.2006.06.001 +[33] Cenkci S, Yildiz M, Cigerci IH, Konuk M, Bozag A. Toxic chemicals-induced gentoxicity detected by random amplified polymorphic DNA (RAPD) in bean (*Phaseolus vulgaris* L.) seedlings. Chemosphere 2009;76(7):900-906. https://doi.org/10.1016/j.chemosphere.2009.05.001 +[34] Kekec G, Sakcali MS, Uzonur I. Assessment of genotoxic effects of Boron on wheat (*Triticum aestivum* L.) and bean +(*Phaseolus vulgaris* L.) by using RAPD analysis. Bull Environ Contamin Toxicol 2010;84(6):759-764. https://doi.org/10.1007/s00128-010-0031-z +[35] Yildirim N, Agar G. Determination of genotoxic effects of fipronil in *Vicia faba* using random amplified polymorphic DNA analysis. Toxicol Ind Health 2016;32(8):1450-1455. https://doi.org/10.1177/0748233714564416 +[36] Sarkar A, Bhagat J, Ingole BS, Rao DP, Markad VL. Genotoxicity of cadmium chloride in the marine gastropod Nerita chamaeleon using Comet assay and alkaline unwinding assay. Environ Toxicol 2015;30(2):177-187 https://doi.org/10.1002/tox.21883 +[37] Sarkar A, Bhagat J, Sarker S. Evaluation of impairment of DNA in marine gastropod, *Morula granulata* as a biomarker of marine pollution. Ecotoxicol Environ Saf 2014;106:253-261. https://doi.org/10.1016/j.ecoenv.2014.04.023 +[38] Ali D, Ali H, Alarifi S, Al-Amer A, Kumar PG, Kumar S, et al. Genotoxicity in the freshwater gastropod *Lymnaea luteola* L: assessment of cell type sensitivities to lead nitrate. Chem Ecol 2017;33(2):171-179. https://doi.org/10.1080/02757540.2016.1275587 +[39] Helmy ET, Ali MA, Ayyad MA, Mohamedbakr HG, Vama RS, Pan JH. Molluscicidal and biochemical effects of greensynthesized F-doped ZnO nanoparticles against land snail *Monacha cartusiana* under laboratory and field conditions. + +Environ Poll 2022;308:119691. https://doi.org/10.1016/j.envpol.2022.119691 +[40] Viard B, Maul A, Pihan JC. Standard use conditions of terrestrial gastropods in active biomonitoring of soil contamination. J Environ Monit 2004;6(2):103-107. https://doi.org/10.1039/B307484F +Halim et al. | Bio-persistence potency of zinc oxide nanoparticles in the gastropods +[41] Seow ZLS, Wong ASW, Thavasi V, Jose R, Ranrakrishna S, Ho GW. Controlled synthesis and application of ZnO +nanoparticles, nanorods and nanospheres in dye-sensitized solar cells. Nanotechnol 2008;20(4):045604. + +[42] Kumar A, Sahoo B, Montpetit A, Behera S, Lockey RF, Mohapatra SS, Development of hyaluronic acid-Fe2O3 hybrid magnetic nanoparticles for targeted delivery of peptides. Nanomed 2007;3(2):132-137. https://doi.org/10.1016/j.nano.2007.03.001 +[43] Sneath PHA, Sokal RR. Numerical Taxonomy: The Principles and Practice of Numerical Classification. WF Freeman & +Co., San Francisco 1973;573. + +[44] Atienzar FA, Conradi M, Evenden AJ. Jha AN, Depledge MH. Qualitative assessment of genotoxicity using random amplified polymorphic DNA: Comparison of genomic templete stability with key fitness parameters in *Daphina magna* exposed to benzo[a]pyrene. Environ Toxicol Chem 1999;18(10):2275-2282. https://doi.org/10.1002/etc.5620181023 +[45] Reynolds ES. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 1963;17(1):208-212. https://doi.org/10.1083/jcb.17.1.208 +[46] Cohort Software Inc, Costat User Manual, Version 3. Cohort Tucson, Arizona, USA, 1985. [47] Hotelling H. Analysis of a complex of statistical variables into principal components. J Educ Psychol 1933;24(6):417-441. + +https://doi.org/10.1037/h0071325 +[48] El-Demerdash FM. Lambda-cyhalothrin-induced changes in oxidative stress biomarkers in rabbit erythrocytes and alleviation effect of some antioxidant. Toxicol In Vitro 2007;21(3):392-397. https://doi.org/10.1016/j.tiv.2006.09.019 +[49] Ma H, Diamond SA. Phototoxicity of TiO2 nanoparticles to zebrafish (*Damio rerio*) is dependent on life stage. Environ Toxicol Contam 2013;32(9):2139-2143. https://doi.org/10.1002/etc.2298 +[50] Pamplona R. Membrane phospholipids lipoxidative damage and molecular integrity: a causal role in aging and longevity. Biochim Biophys Acta Bioenerg 2008;1777(10):1249-1262. https://doi.org/10.1016/j.bbabio.2008.07.003 +[51] Xiong D, Fang T, Yu L, Sima X, Zhu W. Effects of nano-scale TiO2, ZnO and their bulk counterparts on zebra fish: acute toxicity, oxidative stress, and oxidative damage. Sci Total Environ 2011;409(8):1444-1452. https://doi.org/10.1016/j.scitotenv.2011.01.015 +[52] Siddiqi KS, Husen A. Properties of zinc oxide nanoparticles and their activity against microbes. Nanosci Res Lett 2018;13(1):1-13. https://doi.org/10.1186/s11671-018-2532-3 +[53] Arya SK, Ghosh I, Banerjee R, Mukherjee A. Malathion and dithane induce DNA damage in *Vicia faba.* Toxicol Ind Health 2017;33(11):843-854. https://doi.org/10.1177/0748233717726877 +[54] Al Mahrouqi D, Al Riyami S, Barry MJ. Effects of Zn and Ti nanoparticles on the survival and growth of sclerophrys arabica tadpoles in a two level trophic system. Environ Contam Toxicol 2018;101(5):592-597. https://doi.org/10.1007/s00128-018-2449-7 +[55] Bour A, Mouchet F, Cadarsi S, Silvestre J, Baqu'e D, Gauthier L, et al. CeO2 nanoparticle fate in environmental conditions and toxicity on a freshwater predator species: a microcosm study. Environ Sci Poll Res 2017;24(20):17081-17089. https://doi.org/10.1007/s11356-017-9346-1 +[56] Khan MS, Qureshi NA, Jabeen F. Assessment of toxicity in fresh water fish *Labeo rohita* treated with silver nanoparticles. + +Appl Nanosci 2017;7(5):167-179. https://doi.org/10.1007/s13204-017-0559-x +[57] Motta AGC, do Amaral DF, Benvindo-Souza M, Rocha TL, e Silva DDM. Genotoxic and mutagenic effects of zinc oxide nanoparticles and zinc chloride on tadpoles of Lithobates catesbeianus (Anura: Ranidae). Environmental Nanotechnology, Monitoring & Management 2020;14:100356. https://doi.org/10.1016/j.enmm.2020.100356 +[58] Zhang Q, Zhang K, Xu D, Yang G, Huang H, Nie F, et al. CuO nanostructures: Synthesis, characterization, growth mechanisms, fundamental properties, and applications. Prog Mater Sci 2014;60:208-337. https://doi.org/10.1016/j.pmatsci.2013.09.003 +[59] Ozkan F, Gündüz SG, Berkóz M, Hunt AO, Yalin S. The protective role of ascorbic acid (vitamin C) against chlorpyrifosinduced oxidative stress in *Oreochromis niloticus*. Fish Phys Biochem 2012;38(3):635-643. https://doi.org/10.1007/s10695011-9544-6 Environmental Analysis Health and Toxicology **2022**, 37(3):e2022025 [60] Regoli F, Gorbi S, Machella N, Tedesco S, Benedetti M, Bocchetti R, et al. Pro-oxidant effects of extremely low frequency electromagnetic fields in the land snail *Helix aspersa*. Free Radic Biol Med 2005;39(12):1620-1628. https://doi.org/10.1016/j.freeradbiomed.2005.08.004 +[61] Abdel-Azeem1 HH, Osman GY. Oxidative stress and histopathological effect of zinc oxide nanoparticles on the garden snail *Helix aspersa*. Environ Sci Poll Res 2021;28(8):9913-9920. https://doi.org/10.1007/s11356-020-11438-z +[62] Ali SM, Yousef NMH, Nafady NA. Application of biosynthesized silver nanoparticles for the control of land snail Eobania vermiculata and some plant pathogenic fungi. J Nanomater 2015. http://dx.doi.org/10.1155/2015/218904 +[63] Sawasdee BH, Köhler HR, Triebskorn R. Histopathological effects of copper and lithium in the ramshorn snail, *Marisa* cornuarietis (Gastropoda, Prosobranchia). Chemosphere 2011;85(6):1033-1039. https://doi.org/10.1016/j.chemosphere.2011.07.030 +[64] El-Atti A, Desouky M, Mohamadien A, Said R. Impact of Copper Oxide Nanoparticles on freshwater crayfish, Procambarus clarkia. A combined histopathological, Biochemical and genotoxicological study. Journal of Egyptian Academic Society for Environmental Development. D, Environmental Studies 2019;20(1):1-18. + +[65] Roncero V, Duran E, Soler F, Masot J, Gomez L. Morphometric structural and ultrastructural studies of tench (Tinca tinca L.) hepatocytes after copper sulphate administration. Environ Res 1992;57(1):45-58. https://doi.org/10.1016/S00139351(05)80018-0 +[66] Chio CP, Chen WY, Chou WC, Hsieh NH, Ling MP, Liao CM. Assessing the potential risks to zebrafish posed by environmentally relevant copper and silver nanoparticles. Sci Total Environ 2012;420:111-118. https://doi.org/10.1016/j.scitotenv.2012.01.023 +[67] Braunbeck T, Burkhardt-Holm P, Storch V. Liver pathology in eels (*Anguilla anguilla*) from Rhine River exposed to the chemical spill at Basle in November 1986. Limnologie ak1ucll 1990. + +[68] Koehler A, Marx U, Broeg K, et al. Effects of nanoparticles in *Mytilus edulis* gills and hepatopancreas-a new threat to marine life. Mar Environ Res 2008;66(1):12-14. https://doi.org/10.1016/j.marenvres.2008.02.009 +[69] Hu S, Han J, Yang L, Li S, Guo Y, Zhou B, et al. Impact of co-exposure to titanium dioxide nanoparticles and Pb on zebrafish embryos. Chemosphere 2019;233:579-589. https://doi.org/10.1016/j.chemosphere.2019.06.009 +[70] Fahmy SR, Sayed DA. Toxicological perturbations of zinc oxide nanoparticles in the *Coelatura aegyptiaca* mussel. Toxicol Ind Health 2017;33(7):564-575. https://doi.org/10.1177/0748233716687927 +[71] Salmi A, Rouabhi R, Toualbia N, Taib C, Chenikher H, Gasmi S, et al. Subcellular study of the cadmium selenide nanoparticles (CdSe-NPs) toxicity on a terrestrial bioindicator model *Helix aspersa*. Toxicol Environ Healh Sci 2017;9(2):135-140. https://doi.org/10.1007/s13530-017-0314-4 +[72] Hödl E, Felder E, Chabicovsky M, Dallinger R. Cadmium stress stimulates tissue turnover in *Helix pomatia*: increasing cell proliferation from metal tolerance to exhaustion in molluscan midgut gland. Cell Tissue Res 2010;341(1):159-171. https://doi.org/10.1007/s00441-010-0980-x \ No newline at end of file diff --git a/medical/md/PMC9679747.md b/medical/md/PMC9679747.md new file mode 100644 index 0000000000000000000000000000000000000000..272b2f040cebbb583f262d6d86c69880e9c4b49f --- /dev/null +++ b/medical/md/PMC9679747.md @@ -0,0 +1,225 @@ +Data Article + +![0_image_0.png](0_image_0.png) + +Contents lists available at ScienceDirect + +![0_image_1.png](0_image_1.png) + +![0_image_2.png](0_image_2.png) + +![0_image_4.png](0_image_4.png) + +![0_image_3.png](0_image_3.png) + +Plasmid sequence dataset of multidrug-resistant Enterobacterales isolated from hospital effluents and wastewater treatment plant Christopher Mutukua, Barbara Kutasy b, Peter Urbanc, Szilvia Meleghd, Robert Herczegc, Zoltan Gazdaga, Eszter Viráge,∗ +a Department of General and Environmental Microbiology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary b Department of Plant Physiology and Plant Ecology, Hungarian University of Agriculture and Life *Sciences,* +Georgikon Campus Keszthely, 8360 Keszthely, Festetics Gy. u.7., Hungary c Bioinformatics Research Group, Szentágothai Research Centre, 7624 Pécs, Hungary d Department of Medical Microbiology and Immunology, Medical School, University of Pécs, 7622, Pécs, Hungary e Department of Molecular Biotechnology and Microbiology, Institute of Biotechnology, Faculty of Science and Technology, University of Debrecen, Egyetem Square 1, 4032 Debrecen, *Hungary* + +## A R T I C L E I N F O + +Article *history:* Received 28 September 2022 Revised 2 November 2022 Accepted 4 November 2022 Available online 9 November 2022 Dataset link: Enterobacterales plasmids - +raw sequence reads (Original data) Keywords: +Hospital effluents Wastewater treatment plant Enterobacterales β-lactamases Resistance genes Plasmid + +## A B S T R A C T + +We present plasmid sequences of 21 multidrug resistant isolates of Enterobacterales belonging to Escherichia *coli* (n=10), +Klebsiella *pneumoniae* (n=9), Klebsiella *oxytoca* (n=1), and Citrobacter *freundii* (n=1). The isolates originated from effluent collected from hospital sewer pipes and from a wastewater treatment plant (WWTP) in a southwestern Hungarian city. Isolation was carried out using eosin methylene blue agar supplemented with ceftriaxone and the isolates were identified with MALDI-TOF MS. Screening for multidrug resistance was conducted by determining susceptibility to four chemical classes namely, beta-lactams, aminoglycoside, fluoroquinolone, and sulfonamide. Plasmid DNA was isolated by alkaline lysis method using the Monarch plasmid DNA +miniprep kit from freshly grown pure colonies. Molecular https://doi.org/10.1016/j.dib.2022.108736 2352-3409/© 2022 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/) +2 C. B. and P. et al. in 45 + +| typing | and | Illumina | sequencing | of | plasmid | DNA | of | mul | +|-------------|---------|------------|--------------|-------|-----------|----------|------|---| +| tiresistant | strains | were | performed. | After | the | assembly | of | | +| contigs, genes localized on plasmid sequences were determined and functionally annotated. These reconstructed plasmid sequences supplemented with gene functional annotations were deposited in the Mendeley data. Using these datasets different plasmid incompatibility groups were identified. These conjugative plasmids appear to play a key role in the transmission of multiple resistance genes in enteric bacteria via wastewater. The presented data may provide useful insight on the correlations between environmental antibiotic contamination and the development of bacterial resistance, which poses a serious public health threat. © 2022 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/) | | | | | | | | | + +## Specifications **Table** + +| Specifications Table Subject | Microbiology: Applied Microbiology | +|--------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Specific subject area | Wastewater from human sources may serve as an important reservoir of multiresistant Enterobacterales. The bacterial strains were cultured with an aim to isolate and sequence plasmids harboring antibiotic resistance genes. | +| Type of data | Tables Database record Figure | +| How the NGS data were | Illumina sequencing of plasmid DNA of 21 bacterial strains was performed on | +| acquired | the NovaSeq 6000 instrument (Illumina, San Diego, CA, USA) with a 2 × 151 run configuration. Quality control (QC), trimming, and filtering of 150 bp paired-end raw reads were performed by using FastQC and Trimmomatic software programs. For plasmid identification, genes characteristically encoded on plasmids for each strain were determined based on literature and by aligning them to the contigs using locally the Blast+ program. Prokaryotic gene finding was performed by Glimmer and their functional annotation and Gene Ontology (GO) analysis were carried out using OmixBox.Biobam software package. | +| Data format | Raw Analyzed Filtered | +| Description of data collection | The multirestistant enteric bacterial isolates were collected from the effluent of hospital sewer pipes and from a wastewater treatment plant (WWTP) in a southwest Hungarian city. Illumina sequencing was performed for the plasmids isolated from the multiresistant strains. The reconstructed plasmid sequences of 21 resistant strains are reported here. | +| Data source location | - Institution: Department of General and Environmental Microbiology, Faculty of Sciences, University of Pécs. - City/Town/Region: Pécs, Baranya, 7622 - Country: Hungary | +| Data accessibility | The BioProject and sequence reads are available in National Center for Biotechnology Information (NCBI) database under the accessions: Repository name: Enterobacterales plasmids - raw sequence reads Data identification number: PRJNA877861 Direct link to dataset: https://www.ncbi.nlm.nih.gov/sra/PRJNA877861 Repository name: Enterobacterales Plasmids, Mendeley Data, V1 Data identification number: doi:10.17632/j3mkwhzh84.1 Direct URL to data: https://data.mendeley.com/datasets/j3mkwhzh84/1 (continued on next page) | + +Instructions for accessing these data: The contigs of identified plasmid sequences of 21 samples can be found in the file: Plasmid_sequences (1).rar. + +Annotation of these contigs can be found in the file: Omicsbox_annot_table.zip Related research article Christopher Mutuku, Szilvia Melegh, Krisztina Kovacs et al. Characterization of β-Lactamases and Multidrug Resistance Mechanisms in Enterobacterales from Hospital Effluents and Wastewater Treatment Plant (2022) Antibiotics 11, 776. 6. https://doi.org/10.3390/antibiotics11060776 + +## Value Of The **Data** + +- These data shed light on the current and developing trends in acquired antimicrobial resistance in clinically important bacteria including E. coli, Klebsiella spp, and Enterobacter *spp,* +which are of human origin and enter the environment via wastewater. It can form a basis for comparison with clinical surveillance data, which is useful for epidemiological studies. + +- The presence of plasmid mediated resistance among enteric bacteria in wastewater, which primarily form part of human and animal gut microbiota, is subject to further investigation to ascertain whether (1) the opportunistic pathogens isolated already carried these genes from the source population of the wastewater or (2) horizontal gene transfer in the environment lead to acquisition of resistance genes by these human associated microbes from the environmental microbes. + +- Pharmaceutical antimicrobials in untreated hospital effluents whose presence is likely to create selection pressure leading to the emergence of multidrug resistant bacteria (ARB) demonstrated in our data require to be investigated to give an insight into the possible correlation between environmental antibiotic contamination and the development and spreading of resistance. + +- The selection of isolates for next generation sequencing was based on the following considerations: (1) number of antimicrobials that the isolate was resistant to (higher number being preferred) and, (2) the frequency of isolation (higher occurrence being preferred). In this study, E. *coli* (n=58), K. *pneumoniae* (n=26), K. *oxytoca* (n=17) and C. *freundii* (n=14) were the most frequently identified species. The selected E. *coli* (n=10), K. *pneumoniae* (n=9), K. + +oxytoca (n=1) and C. *freundii* (n=1) isolates that demonstrated outstanding multiresistance, were considered to be the most suitable candidates for NGS as shown in Table 1. + +## 1. Data **Description** + +The origin of the 21 resistant enteric bacterial strains is detailed in Table 1. The reconstructed plasmid sequences of these samples are deposited to the Mendeley Data, Plasmid_sequences (1).rar. Prokaryotic genes presented on these plasmids with GO annotation are deposited on Mendeley Data, Omicsbox_annot_table.zip. Sequences in .fasta format and an annotation table in .xlsx format under the doi number: 10.17632/j3mkwhzh84.1. + +Different plasmid incompatibility groups, including IncB/O/K/Z, IncFIA, IncFIB, and IncFII, carrying multiresistant genes where identified, suggesting a key role in the transmission of multiple resistance genes in enteric bacteria via wastewater. The isolates were clustered in accordance with the resistance genes harbored (Fig. 1). + +| Table 1 Isolates, collection date and sources of samples. Serial number Strain Isolate | Sampling location | Collection date | | | +|------------------------------------------------------------------------------------------------------------|---------------------|-----------------------|------|------------| +| 1 | CF102 | Citrobacter freundii | DGSL | 17-7-2019 | +| 2 | EC10 | Escherichia coli | H3 | 10-4-2019 | +| 3 | EC11 | Escherichia coli | H3 | 31-10-2019 | +| 4 | EC14 | Escherichia coli | H3 | 31-10-2019 | +| 5 | EC19 | Escherichia coli | ACSL | 17-7-2019 | +| 6 | EC20 | Escherichia coli | ACSL | 17-7-2019 | +| 7 | EC66 | Escherichia coli | H4 | 16-5-2019 | +| 8 | EC74 | Escherichia coli | NH | 15-10-2019 | +| 9 | EC75 | Escherichia coli | NH | 15-10-2019 | +| 10 | EC81 | Escherichia coli | H2 | 18-6-2019 | +| 11 | EC92 | Escherichia coli | H1 | 13-6-2019 | +| 12 | KO54 | Klebsiella oxytoca | H4 | 16-5-2019 | +| 13 | KP2 | Klebsiella pneumoniae | H1 | 13-6-2019 | +| 14 | KP4 | Klebsiella pneumoniae | INFL | 12-11-2019 | +| 15 | KP45 | Klebsiella pneumoniae | H2 | 18-6-2019 | +| 16 | KP57 | Klebsiella pneumoniae | H4 | 28-10-2019 | +| 17 | KP76 | Klebsiella pneumoniae | H4 | 28-10-2019 | +| 18 | KP79 | Klebsiella pneumoniae | H4 | 28-10-2019 | +| 19 | KP93 | Klebsiella pneumoniae | H2 | 23-10-2019 | +| 20 | KP96 | Klebsiella pneumoniae | H2 | 23-10-2019 | +| 21 | KP6 | Klebsiella pneumoniae | H1 | 8-10-2019 | +| H1-H4; hospital effluents, NH: nursing home, INFL: influent, ACSL: activated sludge, DGSL: digested sludge | | | | | + +![3_image_0.png](3_image_0.png) + +## 2. Wastewater Treatment Plant Operational **Parameters** + +The WWTP located in a southwest Hungarian city serves a population equivalent to slightly over 200,000 inhabitants. The wastewater treatment involves three stages (primary clarification, secondary-activated sludge system, and tertiary-UV treatment), with the final effluent discharged into the nearby surface stream. The wastewater quality data is shown in Tables 2 and 3. In addition, anaerobic digestion occurs in large tanks designed to operate with continuous input of untreated sludge and removal of the final, stabilized sludge product. The digesters operate in the mesophilic range, with the regulated heating kept at 36.5 - 37.5°C. The technology is codigestion fermentation, i.e. in addition to the sludge produced at the sewage treatment plant, other organic wastes with a high organic matter content that are easily biodegradable for biogas production are used. Typical wastes treated at the plant include: sludge from other sewage plants, fatty flotation from the dairy industry, whey, kitchen and restaurant waste (swill), sludge from grease trap, slaughterhouse waste and sludge from sewage treatment plants in slaughterhouses. + +At the initial step, the raw (primary sludge settled in the pre-settler) and excess sludge (secondary sludge produced during biological treatment) generated at the sewage plant are mixed and gravity-compressed in a 1500 m3 dorr-type stick sedimentation basin. The raw sludge with a dry matter content of 1.5% is removed from the pre-settler, as well as the redundant sludge with a dry matter content of 0.85%. After their mixing and gravity compression, a mixed sludge with an average dry matter content of 2.6% is obtained. In the biogas plant, this sludge is further thickened to a dry matter content of 5.5 - 6 % with the addition of polyelectrolyte using pre-dewatering tables. The pre-dewatered sludge goes into a 70 m3 homogenizing tank, where it is mixed with the received crushed waste. The waste is pre-treated with a specially developed device. The technology has a 30 m3 receiving container where the waste is emptied. A hardox plate with 8 mm perforation is placed at the bottom of the receiving bunker, above which two grinding wheel like devices with a diameter of 1 m rotate. The waste is shredded and pulped with this equipment, the required 5.5-6% dry matter content is adjusted by diluting with water and the resultant mixture flowing through the perforation is pumped into the homogenizing + +| Table 2 Water quality data on physico-chemical parameters during the sampling period. Biochemical oxygen Chemical oxygen demand demand Ammonium-nitrogen | Kjeldahl nitrogen | | | | | | | | | | | | +|------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------|------------------|------|------------------------|-------|------|-------|------|-------|-------|------|-----| +| mg/l | mg/l | mg/l | mg/l | | | | | | | | | | +| 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | | +| Mean | 928.8 | 534.5 | 28.4 | 483.5 | 325.9 | 5.2 | 60.45 | 64.5 | 0.53 | 82.6 | 79.4 | 2.9 | +| Nitrite | Nitrate | Total phosphorus | pH | Total suspended solids | | | | | | | | | +| mg/l | mg/l | mg/l | mg/l | mg/l | | | | | | | | | +| 3 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 | | | +| Mean | 0.58 | 39.59 | 9.6 | 8.6 | 0.44 | 7.74 | 7.77 | 7.62 | 366.8 | 139.9 | 4.57 | | +| 1 = incoming raw sewage sample, 2 = sample taken after mechanical cleaning, before biological cleaning, 3 = treated wastewater sample. | | | | | | | | | | | | | + +| Table 3 Other WWTP operational parameters Average amount of incoming wastewater | 24.600 m3/d. | +|-----------------------------------------------------------------------------------|-----------------------------------------------------------------------| +| Average wastewater treatment plant total residence time | 17 hours | +| Biological reactor residence time | 4.5 hours | +| Sludge age (summer - winter) | 15 - 19 days | +| The proportion of recirculation sludge at the biological cleaning stage | 1.5 times the incoming wastewater (the recirculation circle is 150%). | + +tank. The homogenized sewage sludge and waste mixture is pumped into the 2 piece 4000 m3 mesophilic fermentation towers. + +SRT: not interpretable in these mesophilic digesters. + +HRT: varies between 19 and 24 days, depending on the amount of externally delivered waste. + +## 3. Materials And **Methods** 3.1. Sampling Strategy + +Wastewater samples were drawn from four hospital wastewater discharge points, H1 (387 beds), H2 (106 beds), H3 (127 beds), and H4 (348 beds), discharge points of a nursing home for the elderly (NH, 490 beds), and from a wastewater treatment plant (WWTP). Effluent samples from the healthcare facilities were collected directly from two or three separate generation points serving different buildings before joining the main sewer pipe. Samples from the WWTP +were collected from the influent directly behind the grating screen. A 30 mL sample was collected every 15 minutes by lowering a flask into the wastewater flow over a period of 4 h and the aliquots pooled to constitute 480 mL composite sample in sterile 500 mL glass bottles. One grab sample was drawn from the activated sludge reactor and the final product of the mesophilic digested sludge. The study was aimed at comparing the change in the presence of antibiotic resistance genes during wastewater treatment. Therefore, the samples were drawn from influent wastewater, the activated sludge reactor, and mesophilic digested sludge because the activated sludge reactor harbors the bacterial cells for hours, and they last for days in mesophilic fermentation towers. In both spaces, the metabolism of microbes is most intense, and there is a chance for gene exchange. Samples were transported on ice to the laboratory and stored at 4°C before assaying within 6 hours. The study was conducted in 2019. + +## 3.2. Isolation Of Multiresistant Enterobacterales + +Isolation was carried out using eosin methylene blue agar supplemented with ceftriaxone +(2.0 μg ml−1) [1] to select for the lactose fermenting colonies characteristic of the fecal coliform bacteria, including E. coli, Klebsiella pneumoniae, *Enterobacter* spp., and *Citrobacter* spp. + +The isolates were identified with MALDI-TOF MS (MALDI Biotyper, Bruker) according to the instructions by the manufacturer. Score value ≥2.0 was considered as species level identification. + +If the score value was less than 2.0, the measurement was repeated until species level identification was achieved. Screening for multidrug resistance was based on disk diffusion testing with four chemical classes namely, beta-lactams (ceftriaxone, CRO, 30 μg, ceftazidime, CAZ, +10 μg, cefotaxime, CTX, 30 μg, cefpodoxime, CPD, 10 μg, cefoxitin, FOX, 30 μg, imipenem, IMP, 10 μg, and meropenem, MEM, 10 μg), aminoglycoside (gentamicin, GN, 10 μg), fluoroquinolone (ciprofloxacin, CIP, 5 μg), and sulfonamide (sulfamethoxazole/trimethoprim, SXT, 1.25/23.75 μg) Oxoid, Wesel, Germany). Quality control was performed using known characterized strains. + +Source of the isolates selected for NGS are presented in Table 1. + +## 3.3. Plasmid Dna Isolation + +Fresh pure colonies grown on Mueller Hinton agar were transferred into Luria Bertani broth and incubated in an orbital shaker at 35 °C and 200 rpm for 12–16 h. All the centrifugation steps were carried out at 16,000 × g. Plasmid DNA was isolated using the Monarch plasmid DNA miniprep kit according to the manufacturer's instructions (New England Biolabs T1010, Ipswich, Massachusetts, USA). DNA concentration and purity were determined using a Nanodrop spectrophotometer (NanoDrop 2000, Thermo Scientific, Wilmington, NC, USA), and stored at –20 +°C. + +## 3.4. Sequencing Library Preparation + +The sequencing library preparation of selected isolates (Table 1) was prepared using Swift 2S Turbo DNA Library Kits (Swift Biosciences, Ann Arbor, Michigan, United States). 100 ng genomic DNA was fragmented, end prepped, and adapter ligated. Magnetic bead size selection was performed to select 250–300 bp insert size fragments, followed by the library amplification according to the manufacturer's instructions. The quality of the library was checked on the 4200 TapeSation System using D1000 Screen Tape (Agilent Technologies, Palo Alto, CA, USA) and the quantity was measured on Qubit 3.0. (Thermo Scientific, Waltham, MA, USA). Illumina sequencing was performed on the NovaSeq 6000 instrument (Illumina, San Diego, CA, USA) with a 2 × 151 run configuration. + +## 3.5. Pre-Processing And Assembly Of Reads + +The QC analysis was performed using FastQC software (v0.11.9) [2]. Phred-like quality scores +(Qscores) were set to >30. Poor quality reads, adapters at the ends of reads, limited skewing at the ends of reads were eliminated by using Trimmomatic [3]. De novo assembly of cleaned reads was performed with SPAdes (v3.15.3) [4]. Since the data contained genomic DNA debris, identification of plasmid- derived contigs was performed after this step as described in Mutuku et al., 2022 [5]. Number, total length, and N50 of assembled contigs ranged between 17 and 64, 182 477–547 810 bp, and 11 403–62 343 bp, respectively. + +## 3.6. Gene Prediction + +Prokaryotic gene identification was performed using Glimmer (v3.0) based on an interpolated Markov model [6]. Functional annotation and Gene Ontology (GO) analysis of identified genes were performed using OmixBox.Biobam [7] as described by Mutuku et al., 2022 in detail [5]. + +For detection of antimicrobial resistance genes and the identification of plasmid incompatibility groups, ResFinder (v4.1) and PlasmidFinder (v2.1) were used [8,9]. + +## 3.7. Clustering Of Isolates + +MUM index (MUMi) was calculated as described by Marc Deloger et al. [10] Briefly nucmer +(mummer-4.0.0) was used to search for all matches between plasmid sequences pairwise +(dataset1 and dataset2) with the '–mum' option. MUMs were filtered to remove overlaping sequences. First MUMs completely included in larger MUMs were excluded; next MUMs completely included in adjacent MUMs were removed; at last MUMs with partial overlaps were trimmed for both plasmid sequence datasets by shifting the corresponding coordinates and recalculating MUM length in both datasets. The three-step filtering was done for each of the plasmid sequence pairs (first for dataset1 and then for dataset2). MUM indices were calculated as: +MUMi = 1 - Lmum / Lav where Lmum is the total of the length of all nonoverlapping MUMs in the plasmid sequence dataset and Lav stands for the average length for the two plasmid sequence datasets to be compared. The MUMi distance matrix was imported into SplitsTree4 and neighbor joining method was used for clustering of plasmid sequences. + +According to MUM indices, the isolates were clustered into six groups, designated as G1–G6. + +The two main groups contained the majority of E. *coli* (G1) and were shown to harbor *blaCTX-M-27* type ESBL gene, aminogylcoside (aadA5, aph(3)-Ib, *aph(6)-Id*), folate inhibitor (dfrA17, *sul1,* +sul2), tetracycline (*tet(A)*), macrolide (*mph(A*)), and quaternary ammonium compound (*qacE*-) +resistance genes. Cluster G2 enclosed eight, closely related K. *pneumoniae* isolates. The presence of multiple β-lactamase genes (Extended spectrum β-lactamse-ESBL: *blaCTX-M-27*, non- +ESBL: blaTEM-1, *blaOXA-1*), aminoglycoside (aac(6)-Ib-cr, aph(3)-Ib, and *aph(6)-Id)*, chloramphenicol (*catB3*), folate inhibitor (dfrA14, *sul2*), and quinolone (qnrB1, aac(6*)-Ib-cr*) resistance genes. + +Four unrelated isolates, namely C. freundii, K. pneumoniae, K. *oxytoca*, and E. *coli*, were shown to harbor either blaCTX-M-15, blaSHV-12, blaCTX-M-30, or blaCTX-M-1 ESBL genes, respectively. The clustering is visualized in Fig. 1. + +## 4. Limitation Of The **Study** + +Due to limited resources, a small number of multiresistant isolates were selected for NGS to provide representative information. + +## Ethics **Statements** + +Not relevant for the data. + +## 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. + +## Data **Availability** + +Enterobacterales plasmids - raw sequence reads (Original data) (Mendeley Data). + +## Credit Author **Statement** + +Christopher **Mutuku:** Investigation, Data curation, Methodology, Writing - original draft; Barbara **Kutasy:** Validation; Peter **Urban:** Methodology; Szilvia **Melegh:** Writing - review & +editing, Data curation; Robert **Herczeg:** Software; Zoltan **Gazdag:** Conceptualization, Methodology, Resources, Writing - review & editing; Eszter **Virág:** Software, Writing - review & editing. + +## Acknowledgments + +The research was performed in collaboration with Bioinformatics Research Group, Genomics and Bioinformatics Core Facility at the Szentágothai Research Centre of the University of Pécs. + +We acknowledge all the personnel who guided the collection of wastewater samples including László Bicsák, Viktória Fébó in the wastewater treatment plant, Ferenc Illés, Gyula Tóth, József Zsámboki, Tamás Hrakovszky in the hospitals and the nursing home. Further, we acknowledge Csabane Boros for technical laboratory assistance. + +## References + +[5] C. S. Melegh, K. Kovacs, P. Urban, E. Virág, R. Heninger, R. Herczeg, Á. Sonnevend, A. Gyenesei, C. Fekete, Z. Gazdag, Characterization of β-lactamases and multidrug resistance mechanisms in enterobacterales from hospital effluents and wastewater treatment plant, Antibiotics 11 (6) (2022) 776. + +[6] S.L. Salzberg, A.L. Delcher, S. Kasif, O. White, Microbial gene identification using interpolated Markov models, Nucl. + +Acid. Res. 26 (2) (1998) 544–548. + +[7] B. Bioinformatics, S. Valencia, OmicsBox-Bioinformatics made easy, March 3 (2019) 2019. + +[8] A. Carattoli, E. Zankari, A. García-Fernández, M. Voldby Larsen, O. Lund, L. Villa, F. Møller Aarestrup, H. Hasman, In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing, Antimicrob. Agent. Chemother. 58 (7) (2014) 3895–3903. + +[9] V. Bortolaia, R.S. Kaas, E. Ruppe, M.C. Roberts, S. Schwarz, V. Cattoir, A. Philippon, R.L. Allesoe, A.R. Rebelo, A.F. Florensa, ResFinder 4.0 for predictions of phenotypes from genotypes, J. Antimicrob. Chemother. 75 (12) (2020) +3491–3500. + +[10] M. Deloger, M.E. Karoui, M-A Petit, A genomic distance based on MUM indicates discontinuity between most bacterial species and genera, J. Bacteriol. 191 (1) (2009) 91–99. \ No newline at end of file diff --git a/medical/md/PMC9806436.md b/medical/md/PMC9806436.md new file mode 100644 index 0000000000000000000000000000000000000000..8d9dab91b3e9eea1c085822b8d17faa2de41433f --- /dev/null +++ b/medical/md/PMC9806436.md @@ -0,0 +1,424 @@ +# Microvessels Derived From Hipscs Are A Novel Source For Angiogenesis And Tissue Regeneration + +Journal of Tissue Engineering Volume 13: 1–21 +© The Author(s) 2022 Article reuse guidelines: +sagepub.com/journals-permissions DOI: 10.1177/20417314221143240 journals.sagepub.com/home/tej https://doi.org/10.1177/20417314221143240 Xin Gao\#, Shixing Ma\#, Xiaotao Xing\#, Jian Yang, Xun Xu, Cheng Liang, Yejia Yu, Lei Liu, Li Liao and Weidong Tian + +## Abstract + +The establishment of effective vascularization represents a key challenge in regenerative medicine. Adequate sources of vascular cells and intact vessel fragments have not yet been explored. We herein examined the potential application of microvessels induced from hiPSCs for rapid angiogenesis and tissue regeneration. Microvessels were generated from human pluripotent stem cells (iMVs) under a defined induction protocol and compared with human adipose tissue-derived microvessels (ad-MVs) to illustrate the similarity and differences of the alternative source. Then, the therapeutic effect of iMVs was detected by transplantation in vivo. The renal ischemia-reperfusion model and skin damage model were applied to explore the potential effect of vascular cells derived from iMVs (iMVs-VCs). Besides, the subcutaneous transplantation model and muscle injury model were established to explore the ability of iMVs for angiogenesis and tissue regeneration. The results revealed that iMVs had remarkable similarities to natural blood vessels in structure and cellular composition, and were potent for vascular formation and self-organization. The infusion of iMVs-VCs promoted tissue repair in the renal and skin damage model through direct contribution to the reconstruction of blood vessels and modulation of the immune microenvironment. Moreover, the transplantation of intact iMVs could form a massive perfused blood vessel and promote muscle regeneration at the early stage. The infusion of iMVs-VCs could facilitate the reconstruction and regeneration of blood vessels and modulation of the immune microenvironment to restore structures and functions of damaged tissues. Meanwhile, the intact iMVs could rapidly form perfused vessels and promote muscle regeneration. With the advantages of abundant sources and high angiogenesis potency, iMVs could be a candidate source for vascularization units for regenerative medicine. + +## Keywords + +Angiogenesis, vascular cells, stem cells, vascular regeneration Date received:: 12 July 2022; accepted: 18 November 2022 + +## Introduction + +Angiogenesis is an essential process in tissue repair and regeneration. Blood vessels supply oxygen and nutrients to ensure the survival and biological function of migrated or transplanted tissue cells.1 The impact of vascularization on regenerative outcomes has been demonstrated.2,3 Vascular instability and slow infiltration of vessels can prevent adequate vascularization, leading to graft failure.4 Recently, emerging studies have demonstrated that engraftments containing vessel fragments efficiently promote cell survival and reconstitution of graft function.5,6 Human adipose tissue-derived microvessels (ad-MVs) have represented a high angiogenic activity after their seeding on scaffolds and subsequent in vivo implantation.5,7 Ad-MVs can rapidly State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Engineering Research Center of Oral Translational Medicine, Ministry of Education & National Engineering Laboratory for Oral Regenerative Medicine & Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, China +\#means co-first author Corresponding authors: Li Liao, State Key Laboratory of Oral Disease, West China School of Stomatology, Sichuan University, 14\# South Renmin Road, Chengdu, Sichuan 610018, China. Email: lliao@scu.edu.cn Lei Liu, Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, No.14, 3rd Section, Renmin South Road, Chengdu, Sichuan 610041, China. Email: liulei@scu.edu.cn Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). + +interconnect and inosculate with the host vasculature within days of transplantation.8 However, these approaches face the challenge of the limited source of adipose tissues. Finding out the optimal source of microvessels is crucial for rapid angiogenesis. + +Several strategies, including 3-dimensional printing, microfluidic techniques, cell-laden microfibers, and cell infusion in piping systems, have been applied to fabricate microvessels.9–11 However, two challenges limited the application of these strategies. First, is the lack of vascular cell sources. Studies showed that ad-MVs contain the major subgroups of vascular cells including endothelial cells and pericytes.12 Due to the limit of cell proliferation in culture, it remains difficult to harvest large amounts of these vascular cells.13 Second, is the lack of a well-defined culture system to support the regenerate functional microvessels. The suitable scaffold, cytokines, and culture system remain to be established. + +Human induced pluripotent stem cells (hiPSCs) +allow the harvesting of abundant cells of different lineages and generate microtissues with complicated cell components and structures. The discoveries that hiPSCs could differentiate into vascular cells in defined induction conditions were breakthroughs in microvessel engineering.14–16 However, these protocols could not generate physiological vessel structures containing essential cell types. To facilitate the translation to the clinic, a more effective method should be developed to harvest different vascular cells for the generation of entire blood vessels. Recently, a pilot study by Wimmer et al. found that pluripotent stem cells could be induced to form self-organizing three-dimensional human blood microvessels.17 These microvessels are initiated via mesoderm induction of hiPSCs aggregates and further differentiated into pericytes, and a few vascular smooth muscle cells (vSMCs), suggesting a possibility to generate microvessels via hiPSCs for tissue regeneration.18 In this study, we successfully generated microvessels from iPSCs (iMVs) and comprehensively compared iMVs with ad-MVs. Furthermore, we evaluated the angiogenic potential of vascular cells derived from iMVs (iMVs-VCs) in the animal model of renal ischemia-reperfusion (IR) and skin wounds. At last, we investigated the potential of iMVs for angiogenesis and tissue repair in the subcutaneous tissue regeneration model and muscle injury model. Our results demonstrated that iMVs had similar cell components, gene expression patterns, and structures to ad-MVs. The iMVs-VCs could directly be involved in the reconstruction and regeneration of blood vessels to facilitate tissue repair. Moreover, the transplantation of iMVs could regenerate perfused blood vessels in the early period and promote tissue regeneration. + +These findings suggest that iMVs could be applied for tissue repair and regeneration. + +## Methods Animals + +The animal experiments in this study were approved by the Experimental Animal Ethics Committee of West China Hospital of Stomatology, Sichuan University. Four-week-old male Balb/c nude mice were purchased from GemPhamatech Co, Ltd (Nanjing, China). All experimental procedures were performed under relevant guidelines and regulations. + +## Induction Of Hipscs To Microvessels Differentiation To Generate Vascular Cells + +Human-induced pluripotent stem cell line urine cell \#1 (uc01) was kindly provided by Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science. The passage numbers of the UC cells used here ranged from 22 to 35. Undifferentiated hiPSCs were maintained in mTeSR medium (Stemcell Technologies, 85850) on Growth Factor Reduced Matrigel (Corning, 354320)— +coated feeder-free plates. To coat the plates, 100μL +Matrigel from storage was added into 12mL of prechilled, pure DMEM/F12 (Gibco, 21041025) to a 15-mL Falcon tube on ice. The prechilled six well plates were added 1mL +cold medium per well for 30min at 37°C. + +Protocol for blood vessel organoid generation was as described previously.18 Briefly, for differentiation, hiPSCs were dissociated by 0.5mM EDTA (Gibco, AM9260G) for 5min and resuspended in 3mL differentiation media (DMEM: F12 medium,12660012, 20% KOSR, 10828010, Glutamax, 35050079, NEAA, 1030021; all from Gibco) +including 50μM Y-27632 (Selleck, S6390). A total of 1×106 cells were plated into the ultra-low attachment surface six well plate (Corning, 3471) and incubated the cells for at least 1d until the cell aggregates with smooth borders +(50–100μM diameter). Then, cell aggregates were treated with 12μM CHIR99021 (Selleck, S2924) and BMP-4 +(30 ng/mL, Peprotech) on days 3–6 to induce mesoderm, followed by VEGF-A (100 ng/mL, Peprotech, 120-05) +and 2μM forskolin (Selleck, S2449) treatment to trigger vascular development on days 7–9. Gentle treatment of aggregates was important. Always collect aggregates by gravitation. The resulting cell aggregates were embedded in a collagen I-Matrigel (3:1) matrix and overlaid with prewarmed StemPro-34 SFM (Gibco, 10639011) complete medium including 15% FBS, VEGF-A (100ng/mL, +Peprotech, 100-20), and FGF-2 (100 ng/mL, Peprotech, 100-26) for at least 5days. A full six-well plate of aggregates was embedded in an entire 12-well plate, corresponding to 40–60 aggregates per well. + +After vascular network formation, microvessels were extracted from gels by 1mL needles, washed three times in PBS, and dissociated in Accutase (Stemcell Technologies, 07920) containing 0.2UμL−1 Dnase I (Roche, 10104159001) +for 45min at 37°C, followed by filtering through 40μm diameter strainers (Corning, 431750) to create iMVs-VCs suspension. Add an equal volume of PBS containing 2% FBS to stop the digestion. Cell viability was assessed by Trypan blue staining (Gibco, 15250016) and counted using an automatic cell counter (Countess, ThermoFisher). + +## Flow Cytometric Analysis + +Flow cytometric analysis was conducted to detect the major cell populations presented in iMVs-VCs. Microvessels were dissociated into single cells according to the above method. The cell mixture was washed and resuspended in 2%FBS/PBS containing the following antibodies for 30min at 4°C: Isotype control (Thermo, PA5-33198), PE +Mouse anti-Human CD31 (Biolegend, 303105, 5μL/test), and PE Mouse anti-Human CD140b (BD, 558821, 5μL/ +test). Then, cells were washed three times and resuspended in 2%FBS/PBS for analysis by an Accuri C6 flow cytometer (BD). The control group was used to exclude debris and doublets and yield the total cell population. Gating the negative cells was critical in this assay. Subsequently, CD31positive and CD140b-positive cells were gated according to the isotype control. The data were analyzed using FlowJo V10 software. + +## Tube Formation Assay + +The tube formation assay was used to investigate the angiogenic activity of iMVs-VCs. First, allow the Matrigel +(Corning, 354234) to thaw at 4°C, and coated u-Slide +(iBide, 81506) with 10μL per well of Matrigel. Let the plates sit at room temperature for at least 15min to allow gelling of the Matrigel. Then, plate 50μL of iMVs-VCs, which contained 8000–10,000 cells, into each Matrigelcoated well. The dish was incubated at 37°C for at least 6 h. Pictures were taken at 12 and 24 h to observe the tube structures. + +## Isolation Of Ad-Mvs + +Human adipose tissues were obtained from patients conducting cosmetic liposuction at Sichuan Huamei Zixin Medical Aesthetic Hospital. The ad-MVs were isolated as described previously.19 Briefly, wash 5mL adipose tissues with 15mL α -MEM containing 2% penicillinstreptomycin. After washing, the tissues were cut up with scissors. Centrifuge the tissues at 300g for 5min and aspirate the medium. The 5mL adipose tissues were digested for 15min with 10mL collagenase I (1mg/mL, Sigma, SCR103) at 37°C. The digestion was neutralized with an equal volume of α—MEM supplemented with 20% FBS. + +Aspirate the fat supernatant. The remaining suspension was filtered using a 500μm strainer (pluriSelect, 435000199) +to remove undigested adipose tissues and a 20μm strainer +(pluriSelect, 435000199) to harvest ad-MVs. Then, adMVs were washed from the 20μm strainers and collected by centrifugation at 5min, 600g. + +## Library Construction And Rna-Sequencing + +Three ad-MVs samples, as the control group, and three iMVs-VCs samples were separately treated with TRIzol Reagent (Invitrogen, 10296028). RNA was collected from each sample according to the manufacturer's instructions and genomic DNA was removed using DNase I (TaKara, 2270A). Then RNA quality was determined by 2100 BIoanalyser (Agilent) and quantified using the ND-2000 +(NanoDrop Technologies). Only a high-quality RNA sample (OD260/280=1.8–2.2, OD260/230⩾2.0, RIN⩾6.5, 28S:18S⩾1.0, >1μg) was used to construct the sequencing library. RNA-seq transcriptome library was prepared following the TruSeq™ RNA sample preparation Kit from Illumina using 1μg of total RNA. Shortly, messenger RNA +was isolated according to the polyA selection method by oligo(dT) beads, and the RNA was fragmented and reversetranscribed into single-stranded cDNA. Then, doublestranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen) with random hexamer primers (Illumina). After PCR amplification and purification, the library was paired-end sequenced at Shanghai Majorbio Bio-pharm Technology Co., Ltd using the Illumina HiSeq Xten/NovaSeq 6000 sequencer +(2×150bp read length). + +## Expression Analysis + +The expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. RSEM (http://deweylab.biostat.wisc.edu/rsem/) +was used to quantify gene abundances. Essentially, differential expression analysis was performed using the DESeq2 with Q value ⩽ 0.05, DEGs with |log2FC|>1 were considered to be significantly differentially expressed genes). In addition, functional-enrichment analyses including GO and KEGG were performed to identify which DEGs were significantly enriched in GO terms and metabolic pathways at Bonferroni-corrected p-value⩽0.05 compared with the whole-transcriptome background. GO functional enrichment, KEGG pathway analysis, GSEA analysis were carried out by Goatools (https://github.com/ tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku. edu.cn/home.do). + +## Renal Ischemia-Reperfusion Injury Model + +The renal ischemia-reperfusion (IR) injury model was performed as previously described with modifications.20 In brief, nude mice were anesthetized by an intraperitoneal injection of pentobarbital (10%, Sigma, 57330), and unilateral/bilateral renal pedicle occlusions were induced by vascular clamping for 45min. Ischemia was confirmed by the color change of the kidney. With the removal of the clamp, the color of the kidney quickly changed from purple-black to bright red, which indicated proper reperfusion. After the layered suture, the iMVs-VCs (2×105 cells/mouse) in 0.1mL PBS were injected by tail intravenous injection (iMVs-VCs group). Meanwhile, mice injected with 0.1mL PBS (as vehicle control) as the IR group, and normal mice without any treatment were the normal group. At 5- or 30-days post-injection, the mice were sacrificed. The kidneys were collected. The blood samples from mice with bilateral injuries were collected on day 5. + +## Skin Damage Model + +Nude mice were anesthetized by an intraperitoneal injection of pentobarbital (10%, Sigma), and then a full-thickness wound with a length of 2cm on both sides of the mouse's back. After aligning the suture, the iMVs-VCs +(2×105 cells/mouse) in 0.1mL PBS were injected intracutaneously on the left side (VCs side) and 0.1mL PBS was injected on the right side (PBS side) from the incision about 1cm. Wounds were collected at 3- or 30days for analysis. + +## Angiogenesis In Vivo Assay + +To explore the angiogenic ability of iMVs in vivo, a custom-designed silicone tube was used to load 4–6 iMVs in 100μL collagen I-Matrigel (3:1) matrix and implanted into the back of nude mice. The silicone tube containing collagen I-Matrigel (3:1) matrix only was used as the control group. Samples were collected at 2 and 4weeks. + +## Muscle Injury Model + +BALB/c-Nude mice (4weeks of age; approx. 20g; GemPharmatech, Nanjing; China) were used for the muscle injury model. Unilateral muscle defects measuring 1.5cm in length, 0.5 cm in width, and 0.5 cm in height were created by removing the middle portion of the tibialis anterior muscle of recipient mice. Skin and fascia were separated from the underlying muscle using blunt dissection. The muscle was excised by two blades fixed at a 0.5cm distance. Constructs were tightly placed into the defect site. The constructs were divided into three groups: collagen I-Matrigel (3:1) matrix as the control group, collagen I-Matrigel (3:1) matrix containing 1×105 endothelial cells as the cell group, and collagen I-Matrigel (3:1) matrix containing 2–3 iMVs as the organoid group. The wound was closed in layers. At 30-days post-surgery, the mice were sacrificed. The middle portion of the tibialis anterior muscles was collected. + +## Histologic And Immunofluorescence Analysis + +Frozen sections of formalin-fixed tissues (7μm thick) +were stained with hematoxylin and eosin (HE), Periodic acid Schiff (PAS), and Masson trichrome according to the manufacturer's recommended protocols to assess the histological injury, kidney tubular necrosis, and fibrosis. + +Immunofluorescence staining of primary antibodies was performed at 4°C overnight and labeled with secondary antigens for 30min at 37°C. Nuclei were marked with 4′ +6-diamidino-2-phenylindole (DAPI) for 5min at room temperature. The primary antibodies of CD31 (Abcam, ab24590, 1:1000), CD140b (Zen, 345674, 1:100), and αSMA (Abcam, ab5694, 1:100) were used to label vascular cells. Primary antibodies of CD163 (Abcam, ab182422, 1:200), and CD68 (Abcam, ab201340, 1:1000) were used to label macrophages. Human-derived cells were identified with human mitochondria (Abcam, ab92824, 1:800) +and STEM101 (Takara, Y40400, 1:50). Regenerated myofibers were indicated by Myosin IIa antibody (CST, 3403, 1:50). The secondary antibodies of Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen, A11001, 1:400) and Alexa Fluor 555-conjugated goat anti-rabbit (Invitrogen, A21428, 1:400) were used. Each group selected 4–6 random fields of view for analysis. + +## Biodistribution Of The Imvs-Vcs In Vivo + +iMVs-VCs (2×105 cells/mouse) were labeled with fluorescent lipophilic tracer DiR (Invitrogen) according to the manufacturer's instructions. Briefly, the iMVs-VCs were incubated with labeling solutions for 30min at 37°C. After labeling, the cells were spun, rinsed, and resuspended in 0.1mL PBS. And then, the labeled iMVs-VCs were injected intravenously or intracutaneously into animal models. The biodistribution of the iMVs-VCs was imaged using the IVIS Spectrum (PerkinElmer) for the first 3 days. + +## Blood Urea Nitrogen Analysis + +Blood samples were collected to test the kidney functional biomarker blood urea nitrogen (BUN, BOXBIO) according to the manufacturer's instructions. + +## Western Blot Analysis + +The total proteins were extracted by RIPA lysis buffer containing protease inhibitors PMSF (100:1) (Millipore, USA), and BCA Protein Assay Kit (Bio-Rad, USA) was applied to quantify the proteins. 25μg total proteins were loaded into 12% or 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis to separate proteins of different molecular. Then, proteins were transferred onto blocked polyvinylidene difluoride (PVDF) membranes (Millipore, USA) at room temperature, and incubated with primary antibodies: CD146 (Zen, 380835, 1:1000), VE-Cadherin (Abcam, ab33168, 1:1000), VWF (Abcam, ab154193, 1:1000) and β-actin (Zen,380624, 1:5000) at 4°C overnight. After washing, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Zen,1:5000) at room temperature for 60min, and visualized by a western lightning chemiluminescence detection kit (PerkinElmer, N0775330). + +Relative signal intensities were determined using the ImageJ software. + +## Statistical Analysis + +We performed statistical analyses with GraphPad Prism 9(GraphPad Software, 9.1.1, California USA). For the quantification of the images, four different fields of images are selected for statistics. One-way analysis of variance, a two-tailed unpaired t-test was utilized to identify significant differences, which were accepted when the p-value was less than 0.05. + +## Data Availability + +The data that support the findings of this study are available from the corresponding author upon reasonable request. + +## Results Generation Of Imvs From Hipscs + +To generate iMVs, hiPSCs were induced with sequential cocktails of small molecules and growth factors First, hiPSCs were treated with ROCK inhibitor to induce the autonomous formation of aggregates. Stimulation of CHIR99021 and BMP-4 induced mesodermal differentiation of iPSCs. VEGF-A and FGF-2 were then used for vascular lineage induction. Subsequently, the cell aggregates were embedded in collagen I-Matrigel gels, which provide a 3D scaffold to facilitate the vessel sprouting (Figure 1(a)).21 During vessel sprouting, cells gradually migrated outward from the aggregates and formed microvessel-like structures (Figure 1(b)). The immunofluorescence analysis of CD31 and CD 140b showed that CD31+ endothelial cells and CD140b+ pericytes were located around the wall of microvessels and formed a 3-dimensional network structure. (Figure 1(c) and (d)). Taken together, we successfully generated iMVs from hiPSCs. + +## Comparisons Between Imvs And Ad-Mvs + +To further evaluate the resemblances between iMVs and natural blood vessels, we compared the iMVs with as-MVs. As shown in Figure 2(a), immunofluorescence staining revealed a microvessel-like luminal structure with tight interaction between CD140b+ pericytes and CD31+ +ECs, which was very similar to natural microvessels derived from adipose tissues. + +To gain insight into the identity of iMVs, RNA-seq was conducted to compare gene expression between iMVs and ad-MVs. The Venn diagram showed that the iMVs expressed 16,530 genes, while the ad-MVs expressed 17,519 genes (Figure 2(b)). A total of 14,366 (72.99%) genes were co-expressed in iMVs and ad-MVs. There were 2164 genes (13.09%) only expressed in the iMVs and 3153 genes (18.00%) only expressed in the ad-MVs. The composition of the transcriptome is mostly common. Furthermore, a correlation analysis was performed to reveal the relevance between iMVs and ad-MVs from the statistical level. The average of Spearman's rank correlation coefficient between the two groups was 0.795, suggesting that iMVs and ad-MVs were genetically similar +(Figure 2(c)). + +We then compared prototypic marker genes of ECs, pericytes, and hiPSCs in two groups. The heatmap showed that both iMVs and ad-MVs expressed EC markers (PECAM1, ICAM2, and PTPRB) and pericytes markers +(PDGFRB, ACTA2, and MYH11), albeit the slight difference in levels of expression (Figure 2(d)). It should be mentioned that the marker genes of hiPSCs could rarely be detected in iMVs (Figure 2(d)), suggesting the high efficiency of angiogenic differentiation. Western blot was also performed to show the maturation of iMVs. Similar to ad-MVs, iMVs expressed VE-Cadherin, CD146, and less VWF, which indicated the maturation of ECs and pericytes in iMVs (Figure 2(e)). Although the quantification of VE-Cadherin and VWF was statistically significant, the expression difference between the two groups was less than one-fold, suggesting the similarity. + +To confirm the vascular functions of iMVs, GO analysis was employed. Figure 2(f) presented that the genes expressed in iMVs were involved not only in angiogenesis but also in the regulation of blood pressure, coagulation, and vascular permeability. Moreover, genes involved in the wound healing pathways, as well as organ development pathways, were enriched in iMVs. + +We further identified the pathways of iMVs involved in angiogenesis and healing. As shown in Figure 2(g), the iMVs could activate the homology direct repair and involve the transcriptional regulation of stem cells. Meanwhile, the iMVs played a positive role in regulating the migration of endothelial and epidermal cells. In addition to regulating muscle organization, the iMVs also promoted epithelial keratinization. Taken together, these results revealed that iMVs shared fairly similar genetic signatures with natural microvessels and possessed potential capacities for angiogenesis and tissue regeneration. + +## Identification Of Vcs From Imvs + +To further clarify the cellular composition of iMVs, immunofluorescence and flow cytometry assays were performed to detect ECs, pericytes, and vSMCs in the cells isolated from iMVs. Immunofluorescence staining revealed that + +6 *Journal of Tissue Engineering* + +![5_image_0.png](5_image_0.png) + +CD140b+ pericytes and CD31+ ECs accounted for a large proportion of the cells isolated from iMVs, while only a few cells expressed α-SMA (Figure 3(a)–(c)). Meanwhile, flow cytometry defined that 64.4% of iMVs-VCs expressed pericyte markers CD140b. 22.9% of iMVs-VCs expressed CD31 (Figure 3(d)). + +We further explored the potency of iMVs-VCs for angiogenesis in vitro through tube formation assay. iMVs-VCs were digested from iMVs into single-cell suspension and then seeded on Matrigel. After 12h, the iMVs-VCs formed a complex cell-cell network with close contact between ECs and pericytes (Figure 3(f) and (h)). Notably, the iMVsVCs re-self-organized into cell aggregates after 24h (Figure 3(g) and (i)). Recent data had indicated that cell aggregates could enhance cellular retention and angiogenic activity after transplantation.22 The ability of iMVs-VCs in self-organization, which involved embryonic development, regeneration, and formation of complex structures, 23 suggests the advantages of iMVs-VCs in tissue regeneration. + +Taken together, these findings revealed the excellent angiogenesis and self-organization capabilities of iMVs-VCs. + +## Treatment Effects Of The Imvs-Vcs On Ir-Induced Injury In Mice + +To explore whether iMVs-VCs had therapeutic effects, we established a mouse model of renal ischemia-reperfusion + +![6_image_0.png](6_image_0.png) + +![7_image_0.png](7_image_0.png) + +(IR) injury, which induces sustained vascular damage. Transplantation of endothelial cells has been demonstrated to accelerate the repair of ischemia-reperfusion injury by attenuating endothelial damage.24 Therefore, we administrated the iMVs-VCs intravenously into the IR mouse model 45min after IR surgery (Figure 4(a)). Five days after surgery, the ischemic condition of the kidneys was alleviated in the iMVs-VCs treatment group (Figure 4(b)). Treatment of iMVs-VCs obviously decreased the blood urea nitrogen (BUN), a marker of glomerular filtration function to a level slightly higher than that of the normal group. HE staining showed administration of iMVs-VCs alleviated the disorganized structures, dilation of renal tubules, and atrophy of glomeruli of renal cortex at 5days post-IR. (Figure 4(d)). Similarly, PAS staining revealed that the number of damaged tubules was reduced in the iMVs-VCs group compared with the IR group (Figure 4(e) and (f)). Moreover, Masson trichrome staining confirmed that the interstitial fibrosis area in the iMVs-VCs group was less than that in the IR group (Figure 4(g) and (h)). These results indicated that iMVs-VCs could preserve the structures of the kidney in the early stage of IR injury. + +To investigate the long-term effects of iMVs-VCs on kidneys, renal samples were collected 30days after IR surgery. There was no apparent difference in the color of the kidneys in each group (Figure 4(b)). HE staining showed ischemic necrosis in the IR group, while no obvious structural changes were observed in normal and iMVs-VCs groups (Figure 4(i)). Consistent with the results of 5days after surgery, treatment of iMVs-VCs reduced the number + +![8_image_0.png](8_image_0.png) + +10 *Journal of Tissue Engineering* + +![9_image_0.png](9_image_0.png) + +Gao et al. 11 + +![10_image_0.png](10_image_0.png) + +of damaged tubules (Figure 4(j) and (k)) and attenuated the fibrosis (Figure 4(l) and (m)) after 30days. + +The angiogenic ability of iMVs-VCs was also detected by staining renal vessels after treatment for 5 days. + +Co-staining of CD31 and VWF were clearly observed in the renal glomerulus of iMVs-VCs groups, and many costained cells accumulated around the perfused vessels. + +However, the fluorescence intensity of glomerulus was very low in the untreated group, although co-stained vessels were presented (Figure 4(n)). Taken together, the administration of iMVs-VCs could restore renal functions, inhibit interstitial fibrosis, and positively regulate angiogenesis. + +## Imvs-Vcs Promote The Healing Of Skin Wound + +Rapid regeneration and reconstruction of blood vessels can stimulate wound healing.25 To further evaluate the therapeutic effect of iMVs-VCs, a mice model of fullthickness skin wounds was established. iMVs-VCs were injected intracutaneously into the wound on one side (iMVs-VCs side) of the back of nude mice, and PBS was injected into the other side (PBS side). Nude mice with skin wounds on both sides untreated for spontaneous healing served as the blank control (damage group) (Figure 5(a)). After 3 days after surgery, both iMVs-VCs and PBS sides achieved skin healing (Figure 5(b)). HE and Masson trichrome staining were performed to assess the healing pathology of wounds treated by iMVs-VCs. As shown in Figure 5(c) to (e), unlike the damage group which exhibited a rare well-structured neoepidermis, complete skin repair was observed on the iMVs-VCs side on day 3. It should be noted that the PBS side also healed better than the damage group. There was no significant difference in the epithelial length between the iMVs-VCs and PBS +sides (Figure 5(c)), suggesting that the locally transplanted iMVs-VCs could promote repair of the distant wound sites. We then observed the angiogenesis in damage and iMVs-VCs treated groups. The edges of the wound exhibited a large number of blood vessels (stained with VWF and CD31) in iMVs-VCs treated groups, whereas this was not the case for damage groups (Figure 5(f)). The infusion of iMVs-VCs promoted vascular reconstruction and regeneration. + +## Imvs-Vcs Possess Capacities For Migration, Angiogenesis, And Macrophage Regulation + +Angiogenesis is a dynamic process closely related to the migration of vascular cells.26 To determine the migration of iMVs-VCs, we labeled iMVs-VCs by DiR and monitored them after local or systemic infusion by an in vivo imaging system (Figure 6(a)). In the IR model, the iMVsVCs injected intravenously mainly migrated to the liver and kidney on day 1 after administration. Higher fluorescence intensity was detected at the IR-side kidney than on the normal side until day 3 (Figure 6(c)). In the skin wounds model, the iMVs-VCs locally injected around the subcutaneous skin wound were mainly located around the wound. Interestingly, a few Dir-labeled cells were also detected around the wound on the side which received only PBS injection (Figure 6(d)). The fluorescence signals were accumulated at the PBS side after 2days of injection (Figure 6(e)), suggesting the chemotaxis of the iMVs-VCs to damaged tissues. To further clarify the location of the iMVs-VCs in damaged tissues, we marked the transplanted cells with human mitochondrial protein (MITO) +for immunofluorescence staining of tissue sections. In the IR-side kidney, iMVs-VCs-derived ECs (CD31+MITO+) and pericytes (CD140b+MITO+) were detected around the microvessels. More iMVs-VCs were located around blood vessels of the IR kidney compared with the normal side (Figure 6(f)). Consistent with the biodistribution in the wound edges, the iMVs-VCs were aggregated around the blood vessels of the bilateral skin wounds (Figure 6(g)). The evidence indicated that the iMVs-VCs could migrate toward injured tissues and aggregate around the vessels to participate in repair. + +To further study the destiny of the injected iMVs-VCs, we collected samples of kidney and skin treated with the iMVs-VCs on day 30 for immunofluorescence staining. A decrease in MITO-positive cells could be clearly observed. + +The transplanted ECs (CD31+ MITO+) and pericytes (CD140b+ MITO+) were directly involved in the vascular regeneration of damaged kidneys (Figure 7(a)), as well as in skin wounds (Figure 7(b)), suggesting high cell retention of iMVs-VCs in the ischemic and inflammatory conditions. Particularly, reconstructed vessels in healing skin exhibited significant perfusion. Notably, we could also observe a few MITO+ cells on the normal side of the IR +model, suggesting that iMVs-VCs were involved in the metabolism of normal blood vessels. + +Endothelial cells have been reported to suppress the classical activation of macrophage (M1 phenotype) and promote alternative activation of macrophage (M2 phenotype), which may have anti-inflammatory and woundhealing effects.27 To confirm whether iMVs-VCs could regulate macrophage subtypes, we examined the expression of CD68 (M1 phenotype marker) and CD163 (M2 phenotype marker) in post-IR kidneys and skin wounds. + +Immunofluorescence staining revealed that the accumulation of CD68+ cells was increased in the IR group +(Figure 8(a)) and skin damage model (Figure 8(c)). The infiltration of M1 macrophages was reduced by half after the infusion of iMVs-VCs (Figure 8(e) and (f)). In contrast, the accumulation of CD163-positive cells was tripled in the iMVs-VCs-treated IR model (Figure 8(e)). The increase was also statistically significant in the skin damage model (Figure 8(f)). The above results were further proved by GSEA analysis of gene expression in iMVs-VCs, which showed that iMVs-VCs expressed high levels of gene sets that positively regulate the activation of M2 phenotype (Figure 8(g)). + +Taken together, these results indicate the iMVs-VCs could directly contribute to angiogenesis during tissue repair and attenuate the inflammatory response by regulating macrophage subtypes. + +Gao et al. 13 + +![12_image_0.png](12_image_0.png) + +14 *Journal of Tissue Engineering* + +![13_image_0.png](13_image_0.png) + +## The Imvs Rapidly Form Perfused Blood Vessels In Vivo + +The above experiments confirmed that the cells derived from iMVs have a strong potential for angiogenesis. We then investigated whether the iMVs could be applied for angiogenesis and tissue regeneration. The ability of iMVs for angiogenesis in vivo was explored by subcutaneous transplantation into a custom-designed silicone tube. Fourteen days after transplantation, the histologic analysis showed that cells fully infiltrated the iMVs grafts, compared to the matrix-only group. Moreover, more perfused blood vessels formed in the iMVs group, especially in the areas close to the skin (Figure 9(a)). At 1month of transplantation, the matrix group formed large volume cavities in the transplants. Cells and blood vessels were rarely observed on the distal side of the skin. Whereas, distinct blood vessels containing blood cells in the vessel lumen could be observed throughout the iMVs grafts (Figure 9(b)). + +To clarify whether the formed vessels were derived from iMVs, STEM101 and CD31 were co-stained. Co-expressed vessels were observed at both time points. (Figure 9(c)). The results suggested that the transplantation of iMVs could rapidly form perfused blood vessels in vivo, which facilitated cell infiltration and tissue vascularization. + +## The Imvs Promote Muscle Regeneration + +Rapid angiogenesis is necessary to bring nutrients, immune cells, and oxygen to healing sites.28 Skeletal muscle is highly vascularized with a dense microvasculature. An increase in angiogenesis can lead to the success of muscle regeneration.29,30 The muscle injury model was established to investigate the effects of iMVs on muscle regeneration. 4weeks after surgery, HE staining demonstrated that there was little evidence of cellular infiltration in collagen matrix grafts. Muscle defect with HUVECs implantation had a small amount of immature fibers formation near the + +![14_image_0.png](14_image_0.png) + +16 *Journal of Tissue Engineering* + +![15_image_0.png](15_image_0.png) + +interface. Whereas, a notable presence of muscle fibers was clearly observed in the iMVs group (Figure 10(a)). Notably, perfused microvessels located in disordered myofibers were observed in the iMVs group. Regenerated myofibers were indicated by the presence of Myosin IIa +(MF20) in defects.6 The MF20 was rarely expressed in the collagen matrix group. Small amounts of MF20 were expressed at the edge of the defect in the group that received HUVECs transplantation, suggesting regeneration of myofibers. In the iMVs group, the ability to regenerate myofibers performed better with more notable expressions of MF20 (Figure 10(b)). + +To explore whether muscle regeneration relied on angiogenesis, the microvessels were detected by the expression of CD31 in the defect sites. In the collagen matrix group, fewer blood vessels were formed than those in HUVECs and iMVs groups. Some of the microvessels co-expressed STEM101, representing that HUVECs and iMVs were involved in the angiogenic process in the defect area (Figure 10(c)). The results showed that the transplantation + +Gao et al. 17 + +![16_image_0.png](16_image_0.png) + +of iMVs increased the formation of blood vessels, which promoted the regeneration of muscles. + +## Transplantation Of Imvs-Vcs Does Not Lead To Tumor Formation + +Tumorigenesis by the undifferentiated pluripotent stem cells is a critical risk in the application of seed cells derived from iPSCs. To confirm whether the iMVs-VCs were tumorigenic, kidney samples of normal mice injected with iMVs-VCs were collected (Supplemental Figure S1(a)). There was no abnormal cell hyperplasia or oncogenesis in the renal according to HE and Masson staining +(Supplemental Figure S1(b)). Moreover, the damaged skin with local injection of iMVs-VCs was collected on day 30 (Supplemental Figure S1(c)). The skin wound achieved full-thickness healing, and there was no tumorlike tissue formed after injection (Supplemental Figure S1(d)). Moreover, transplantation of iMVs in the subcutaneous and muscle of nude mice did not generate dysplastic tissue. Taken together, the results indicated that the iMVs-VCs were not tumorigenic. + +## Discussion + +This study has revealed that the iMVs have similar gene expressions and structures to ad-MVs. the administration of the vascular cells derived from iMVs could rescue the renal ischemia-reperfusion and also promote earlier healing of skin wounds. In the early stages of tissue damage, the exogenous iMVs-VCs aggregate around blood vessels and attenuates the inflammation. The iMVs-VCs are directly involved in vascular reconstruction during late tissue repair. + +Furthermore, the transplantation of iMVs can form perfused blood vessels in the early period and promote muscle regeneration. These findings suggest that iMVs can be vascularization units for tissue repair and regeneration. + +Several studies have demonstrated that microvessels isolated from the adipose tissue (ad-MVs) retained the native structure and cell composition of natural blood vessels.5,31,32 Meanwhile, ad-MVs have the outstanding angiogenic potential of enhancing the vascularization of implants and grafts at defect sites.7,12 However, the method of adipose tissue extraction is invasive, and its quantity is limited. In the present study, we explored the applications of hiPSCs-derived microvessels. Compared with microvessels derived from adipose tissue, hiPSCs have excellent proliferation capabilities, allowing for a large output of microvessels. The results revealed the similarities between iMVs and natural blood vessels and suggested promising prospects of iMVs for tissue regeneration. Moreover, we used iMVs-derived cells for the treatment of ischemic disease and also achieved good therapeutic effects. We provide not only a new vascularization strategy for tissue engineering but also a novel cell source for cell therapy. + +hiPSCs can offer outstanding advantages in the treatment of disease because they can derive from patients' somatic cells, and have maximum immune compatibility.33 Meanwhile, hiPSCs can differentiate into a sufficient number of various types of cells.34 The microvessels derived from hiPSCs (iMVs) have been well established, and ECs, pericytes, and vSMCs can be efficiently differentiated from hiPSCs in one procedure, which is more practical than other protocols.18 Especially in the present study, we first illustrated that the gene expressions of iMVs and adMVs are similar according to the results of RNA-seq. + +Moreover, the iMVs have a more prominent role in regulating endothelial cell migration and vascular differentiation, which is essential to angiogenesis.35 To explore the application potential of iMVs in vascularization strategy, different types of in vivo models were used. + +Ischemia/reperfusion contributes to increased mortalities during various pathological conditions such as stroke, kidney injury, and cardiac failure.36 Cell therapy has achieved promising results. In our study, vascular cells derived from iMVs have been shown to have anti-inflammatory, antifibrotic, and pro-angiogenic therapeutic effects in the renal IR injury model. Moreover, local injection of iMVs-VCs could promote early healing of skin damage. The above results suggest that iMVs could be a new source of cells for the treatment of injury and ischemic diseases. The intact microvessels derived from adipose tissues have been applied to construct vascularized grafts due to their remarkable angiogenic activity.37 Therefore, we investigated the angiogenic capacity of intact iMVs after transplantation. Subcutaneous transplantation and muscle defect models well demonstrated the potential of iMVs in tissue engineering. + +Pericytes are the mural cell types that surround the endothelial layer to form integral functional vessels through interactive communication.38 Previous studies have demonstrated that pericyte-endothelial cell attachment is essential in the vascular repair process.39 In the tube formation assay of iMVs-VCs, pericytes aggregated around endothelial cells and formed complex networks. + +Furthermore, the iMVs-VCs still had a strong selforganization ability to reassemble after dissociation. Selforganization is not restricted to developmental processes. + +In adult organisms, tissue regeneration is also an emerging self-organizing feature of cells.23 In the iMVs-VCs, tight cell-cell interactions could positively drive collective behaviors during the angiogenesis processes.40 Ischemia-reperfusion injury is a common complication of ischemic disease, which usually leads to further damage. In renal IR injury, pericytes play a central role in the development of interstitial fibrosis through differentiation into myofibroblasts.41,42 However, some studies have revealed that pericytes therapy could improve vascularization and reduce interstitial fibrosis in a swine model of reperfused acute myocardial infarction.43 In addition to its angiogenesis, the exogenous pericytes can ameliorate the inflammation and attract circulating reparative cells to promote the healing process.44 In the present study, we examined the effects of the iMVs-VCs on renal IR injury. Our data suggested that the iMVs-VCs could suppress the inflammatory response, which might attenuate renal fibrosis and preserve the functions. + +One of the challenges of cell therapy is transplantation efficiency. Transplanted cells display a very high death percentage.45,46 Many strategies have been used to enhance cell survival, including combined administration with supporting cells.47 The results of biodistribution and IF +showed considerable cell survival of the iMVs-VCs in vivo. Meanwhile, the iMVs-VCs could target the damaged tissues and aggregate around the blood vessels, which might be guided by a graded distribution of VEGF-A levels in a damaged environment.48 During tissue repair, the iMVs-VCs were directly involved in the reconstruction of blood vessels. The regulation of the iMVs-VCs on the microenvironment might play a leading role in successful transplantation and restoration. + +Adipose tissue-derived microvessels (ad-MVs) have served as natural vascularization units in angiogenesis research and tissue regeneration due to their outstanding ability to rapidly reassemble into microvascular networks.37 The transplantation experiment showed that iMVs formed functional microvascular networks during the early period in our study. Our data also demonstrated the ability of iMVs to promote muscle regeneration by improving the vascularization of defect sites. Compared to ad-MVs, iMVs are derived from hiPSCs and can be massively induced, which is more promising for tissue engineering. + +The present study provided a new source of cells for the treatment of ischemic diseases; besides preserving the functions after renal IR, the iMVs-VCs can promote earlier healing of skin wounds. We also found the administration of the iMVs-VCs could aggregate around blood vessels of damaged tissues and ameliorate the inflammation. In the process of tissue repair, the iMVs-VCs maintained a certain survival rate and participated in neovascularization. Moreover, iMVs could serve as a new kind of vascularization unit to promote tissue regeneration. However, there were still some limitations. Although we illustrated that the iMVs-VCs could attenuate the inflammation, more studies should be done to clarify the proteomics during the repair process. Besides, we should use more models to validate the application of iMVs in the future. + +The field of regenerative medicine has been looking for a pluripotent stem cell able to be safely employed in therapy. The potential clinical applications of the hiPSCs have presented several problems, including genomic instability, and the risk of teratoma formation.49 Highly efficient differentiation prior to cell transplantation is one way to eliminate the teratogenic potential of hiPSCs.50 In the present study, we efficiently induced differentiation of hiPSCs toward the vascular lineage with no expressions of tumorigenic genes. No tumor-like tissue formed when iMV-derived vascular cells or intact iMVs were applied. However, further animal studies, including large animal models, to evaluate the safety and efficacy of iMVs are crucial before their translation into the clinic. + +## Conclusion + +In the present study, we generated microvessels from pluripotent stem cells. The iMVs possess similar cell components and gene expression patterns to microvessels derived from adipose tissue. The cells derived from iMVs possess the capacities of migration, angiogenesis, and immune regulation, and could be directly involved in the regeneration of microvascular networks. The iMVs-VCs derived from microvessels could reduce renal injury after IR and promote skin wound healing. Besides, the transplantation of iMVs could form perfused blood vessels during the early period and promote tissue regeneration in a muscle damage model. Taken together, this study demonstrates that microvessels generated by iPSCs are a new source of microvessels for tissue repair and regeneration. + +## Author Contribution + +Xin Gao and Shixing Ma participated in the design of this study, and they both carried out the study and wrote the manuscript. + +Xiaotao Xing participated animal experiment and revised the manuscript. Jian Yang and Yejia Yu performed the statistical analysis. Xun Xu and Cheng Liang collected important background information. Lei Liu, Li Liao, and Weidong Tian carried out the design, data acquisition, and manuscript editing. All authors gave final approval and agree to be accountable for all aspects of the work. + +## Declaration Of Conflicting Interests + +The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. + +## Funding + +The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Key Research and Development Program of China (2021YFA1100603), the National Natural Science Foundation of China (82071092, U21A20369), and the Fundamental Research Funds for the Central Universities (YJ201878). + +## Orcid Id + +Li Liao https://orcid.org/0000-0002-1937-9212 + +## Supplemental Material + +Supplemental material for this article is available online. + +## References + +1. Ding MH, Lozoya EG, Rico RN, et al. The role of angiogenesis-inducing microRNAs in vascular tissue engineering. *Tissue Eng Part A* 2020; 26(23-24): 1283–1302. +2. Linn T, Schmitz J, Hauck-Schmalenberger I, et al. Ischaemia is linked to inflammation and induction of angiogenesis in pancreatic islets. *Clin Exp Immunol* 2006; 144(2): 179–187. + +3. Roux BM, Cheng MH and Brey EM. Engineering clinically relevant volumes of vascularized bone. *J Cell Mol Med* 2015; 19(5): 903–914. + +4. Santos MI and Reis RL. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. *Macromol Biosci* 2010; 10(1): 12–27. + +5. Aghazadeh Y, Poon F, Sarangi F, et al. Microvessels support engraftment and functionality of human islets and hESC-derived pancreatic progenitors in diabetes models. Cell Stem Cell 2021; 28(11): 1936–1949.e8. + +6. Pilia M, McDaniel J, Guda T, et al. Transplantation and perfusion of microvascular fragments in a rodent model of volumetric muscle loss injury. *Eur Cell Mater* 2014; 28: 11–24. + +7. Xu X, Liang C, Gao X, et al. Adipose tissue-derived microvascular fragments as vascularization units for dental pulp regeneration. *J Endod* 2021; 47(7): 1092–1100. + +8. Sun X, Wu J, Qiang B, et al. Transplanted microvessels improve pluripotent stem cell-derived cardiomyocyte engraftment and cardiac function after infarction in rats. *Sci* Transl Med 2020; 12(562): eng1015050861946. + +9. Song HG, Rumma RT, Ozaki CK, et al. Vascular tissue engineering: progress, challenges, and clinical promise. Cell Stem Cell 2018; 22(3): 340–354. + +10. Rickel AP, Deng X, Engebretson D, et al. Electrospun nanofiber scaffold for vascular tissue engineering. Mater Sci Eng C Mater Biol Appl 2021; 129: 112373. + +11. Saberianpour S, Heidarzadeh M, Geranmayeh MH, et al. + +Tissue engineering strategies for the induction of angiogenesis using biomaterials. *J Biol Eng* 2018; 12: 36. + +12. Kamat P, Frueh FS, McLuckie M, et al. Adipose tissue and the vascularization of biomaterials: stem cells, microvascular fragments and nanofat-a review. *Cytotherapy* 2020; 22(8): 400–411. + +13. Chen SG, Ugwu F, Li WC, et al. Vascular tissue engineering: advanced techniques and gene editing in stem cells for graft generation. *Tissue Eng Part B Rev* 2021; 27(1): 14–28. + +14. Orlova VV, van den Hil FE, Petrus-Reurer S, et al. + +Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. *Nat Protoc* 2014; 9(6): 1514–1531. + +15. Cheung C, Bernardo AS, Trotter MW, et al. Generation of human vascular smooth muscle subtypes provides insight into embryological origin-dependent disease susceptibility. Nat Biotechnol 2012; 30(2): 165–173. + +16. Patsch C, Challet-Meylan L, Thoma EC, et al. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. *Nat Cell Biol* 2015; 17(8): 994–1003. + +17. Wimmer RA, Leopoldi A, Aichinger M, et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 2019; 565(7740): 505–510. + +18. Wimmer RA, Leopoldi A, Aichinger M, et al. Generation of blood vessel organoids from human pluripotent stem cells. Nat Protoc 2019; 14(11): 3082–3100. + +19. Frueh FS, Später T, Scheuer C, et al. Isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering. *J Vis Exp* 2017; (122): +eng1013132521940. + +20. Ozbilgin S, Ozkardesler S, Akan M, et al. Renal ischemia/ +reperfusion injury in diabetic rats: the role of local ischemic preconditioning. *Biomed Res Int* 2016; 2016: 8580475. + +21. McCoy MG, Seo BR, Choi S, et al. Collagen I hydrogel microstructure and composition conjointly regulate vascular network formation. *Acta Biomater* 2016; 44: 200–208. + +22. Wolint P, Bopp A, Woloszyk A, et al. Cellular self-assembly into 3D microtissues enhances the angiogenic activity and functional neovascularization capacity of human cardiopoietic stem cells. *Angiogenesis* 2019; 22(1): 37–52. + +23. Xavier da Silveira Dos Santos A and Liberali P. From single cells to tissue self-organization. *FEBS J* 2019; 286(8): +1495–1513. + +24. Herrler T, Wang H, Tischer A, et al. Decompression of inflammatory edema along with endothelial cell therapy expedites regeneration after renal ischemia-reperfusion injury. *Cell Transplant* 2013; 22(11): 2091–2103. + +25. Ishihara J, Ishihara A, Starke RD, et al. The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing. *Blood* 2019; 133(24): 2559–2569. + +26. Lee HW, Xu Y, He L, et al. Role of venous endothelial cells in developmental and pathologic angiogenesis. *Circulation* 2021; 144(16): 1308–1322. + +27. Chawla A, Nguyen KD and Goh YP. Macrophage-mediated inflammation in metabolic disease. *Nat Rev Immunol* 2011; 11(11): 738–749. + +28. DiPietro LA. Angiogenesis and wound repair: when enough is enough. *J Leukoc Biol* 2016; 100(5): 979–984. + +29. Reddy LVK, Murugan D, Mullick M, et al. Recent approaches for angiogenesis in search of successful tissue engineering and regeneration. *Curr Stem Cell Res Ther* 2020; 15(2): 111–134. + +30. Latroche C, Weiss-Gayet M, Muller L, et al. Coupling between myogenesis and angiogenesis during skeletal muscle regeneration is stimulated by restorative macrophages. + +Stem Cell Reports 2017; 9(6): 2018–2033. + +31. Strobel HA, Gerton T and Hoying JB. Vascularized adipocyte organoid model using isolated human microvessel fragments. *Biofabrication* 2021; 13(3): eng1015219641758. + +32. Acosta FM, Stojkova K, Brey EM, et al. A straightforward approach to engineer vascularized adipose tissue using microvascular fragments. *Tissue Eng Part A* 2020; 26(15–16): +905–914. + +33. Yamanaka S. Pluripotent stem cell-based cell therapy-promise and challenges. *Cell Stem Cell* 2020; 27(4): 523–531. + +34. Picanço-Castro V, Moreira LF, Kashima S, et al. Can pluripotent stem cells be used in cell-based therapy? *Cell* Reprogram 2014; 16(2): 98–107. + +35. Lamalice L, Le Boeuf F and Huot J. Endothelial cell migration during angiogenesis. *Circ Res* 2007; 100(6): 782–794. + +36. Shiva N, Sharma N, Kulkarni YA, et al. Renal ischemia/ +reperfusion injury: an insight on in vitro and in vivo models. Life Sci 2020; 256: 117860. + +37. Laschke MW, Später T and Menger MD. Microvascular fragments: more than just natural vascularization units. Trends Biotechnol 2021; 39(1): 24–33. + +38. Liu C, Ge HM, Liu BH, et al. Targeting pericyte-endothelial cell crosstalk by circular RNA-cPWWP2A inhibition aggravates diabetes-induced microvascular dysfunction. *Proc* Natl Acad Sci U S A 2019; 116(15): 7455–7464. + +39. Wang M, Xu H, Li Y, et al. Exogenous bone marrow derived-putative endothelial progenitor cells attenuate ischemia reperfusion-induced vascular injury and renal fibrosis in mice dependent on pericytes. *Theranostics* 2020; 10(26): 12144–12157. + +40. Wong SP, Rowley JE, Redpath AN, et al. Pericytes, mesenchymal stem cells and their contributions to tissue repair. + +Pharmacol Ther 2015; 151: 107–120. + +41. Castellano G, Franzin R, Stasi A, et al. Complement activation during ischemia/reperfusion injury induces pericyteto-myofibroblast transdifferentiation regulating peritubular capillary lumen reduction through pERK signaling. Front Immunol 2018; 9: 1002. + +42. Chou YH, Pan SY, Shao YH, et al. Methylation in pericytes after acute injury promotes chronic kidney disease. *J Clin* Investig 2020; 130(9): 4845–4857. + +43. Ellison-Hughes GM and Madeddu P. Exploring pericyte and cardiac stem cell secretome unveils new tactics for drug discovery. *Pharmacol Ther* 2017; 171: 1–12. + +44. Avolio E, Meloni M, Spencer HL, et al. Combined intramyocardial delivery of human pericytes and cardiac stem cells additively improves the healing of mouse infarcted hearts through stimulation of vascular and muscular repair. Circ Res 2015; 116(10): e81–e94. + +45. Abdelwahid E, Kalvelyte A, Stulpinas A, et al. Stem cell death and survival in heart regeneration and repair. + +Apoptosis 2016; 21(3): 252–268. + +46. Geng YJ. Molecular mechanisms for cardiovascular stem cell apoptosis and growth in the hearts with atherosclerotic coronary disease and ischemic heart failure. *Ann N Y Acad* Sci 2003; 1010: 687–697. + +47. Recchia FA and Sharp TE. Combination cell therapy for ischemic cardiomyopathy: is the whole greater than sum of its parts? *J Am Coll Cardiol* 2017; 70(20): 2516–2518. + +48. Gerhardt H and Betsholtz C. How do endothelial cells orientate? EXS 2005; (94): 3–15. + +49. Suman S, Domingues A, Ratajczak J, et al. Potential clinical applications of stem cells in regenerative medicine. *Adv Exp* Med Biol 2019; 1201: 1–22. + +50. Gorecka J, Kostiuk V, Fereydooni A, et al. The potential and limitations of induced pluripotent stem cells to achieve wound healing. *Stem Cell Res Ther* 2019; 10(1): 87. \ No newline at end of file diff --git a/medical/md/PMC9974085.md b/medical/md/PMC9974085.md new file mode 100644 index 0000000000000000000000000000000000000000..7a8c17fe1886af40a75e7b258092270e4c9d4fac --- /dev/null +++ b/medical/md/PMC9974085.md @@ -0,0 +1,230 @@ +# Measures Of Admission Immunocoagulopathy As An Indicator For In-Hospital Mortality In Patients With Necrotizing Fasciitis + +A Retrospective Study + +![0_image_0.png](0_image_0.png) + +Samuel R. Johnson, BS, Teresa Benvenuti, BA, Hui Nian, PhD, Isaac P. Thomson, MD, MSCI, Keith Baldwin, MD, MPH, MSPT, +William T. Obremskey, MD, MPH, MMHC, Jonathan G. Schoenecker, MD, PhD, and Stephanie N. Moore-Lotridge, PhD +Background: Necrotizing fasciitis is a rapidly progressive infection with a high mortality rate. Pathogens evade the host containment and bactericidal mechanisms by hijacking the coagulation and inflammation signaling pathways, leading to their rapid dissemination, thrombosis, organ dysfunction, and death. This study examines the hypothesis that measures of immunocoagulopathy upon admission could aid in the identification of patients with necrotizing fasciitis at high risk for in-hospital mortality. + +Methods: Demographic data, infection characteristics, and laboratory values from 389 confirmed necrotizing fasciitis cases from a single institution were analyzed. A multivariable logistic regression model was built on admission immunocoagulopathy measures (absolute neutrophil, absolute lymphocyte, and platelet counts) and patient age to predict in-hospital mortality. + +Results: The overall in-hospital mortality rate was 19.8% for the 389 cases and 14.6% for the 261 cases with complete measures of immunocoagulopathy on admission. A multivariable logistic regression model indicated that platelet count was the most important predictor of mortality, followed by age and absolute neutrophil count. Greater age, higher neutrophil count, and lower platelet count led to significantly higher risk of mortality. The model discriminated well between survivors and non-survivors, with an overfitting-corrected C-index of 0.806. + +Conclusions: This study determined that measures of immunocoagulopathy and patient age at admission effectively prognosticated the in-hospital mortality risk of patients with necrotizing fasciitis. Given the accessibility of neutrophil-tolymphocyte ratio and platelet count measurements determined from a simple complete blood-cell count with differential, future prospective studies examining the utility of these measures are warranted. + +Level of Evidence: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence. + +Necrotizing fasciitis is a rapidly evolving and destructive infection that is one of the most potentially lethal infections of the musculoskeletal system. Causative pathogens evade the host's protective containment and bactericidal mechanisms by hijacking the coagulation and inflammation signaling pathways during the survival acute phase response (APR). This leads to the rapid dissemination of the infection, eliciting both sepsis-induced coagulopathy (SIC) and systemic inflammatory response syndrome (SIRS)1. Together, these 2 pathologic states are principal causes of complications, such as multiple organ dysfunction syndrome (MODS) and death. + +The APR is initiated in proportion to the degree of tissue damage, directing a coordinated response between coagulation factors and the survival inflammatory response to temporarily seal off affected tissue regions with a fibrin and platelet seal2-4. In addition to achieving hemostasis, this sealant promotes the ingress of inflammatory cells, such as neutrophils and lymphocytes, which help to contain and combat the infection4,5. Once survival is ensured, the APR then transitions to a reparative inflammatory Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJSOA/ +A476). + +Copyright 2023 The Authors. Published by The Journal of Bone and Joint Surgery, Incorporated. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. + +response that paves the way for the regeneration of the damaged tissues2,4,6. In cases of necrotizing fasciitis, pathogens have evolved virulence factors that hijack components of the APR, evading containment by fibrin-platelet networks and allowing the pathogen to rapidly disseminate throughout the body1,3,4. As the infection progresses, the recurrent damage to surrounding tissues continually activates the APR, driving inflammation and coagulation to pathologic levels that can lead to SIC, SIRS, +MODS, and death1,7. + +Given that an exuberant APR is central to this pathologic response, the purpose of this study was to examine if coagulation and inflammation at admission, assessed together as measures of immunocoagulopathy, are predictive of the prognosis, specifically in-hospital mortality, in patients with necrotizing fasciitis. Specifically, this study examined if admission measures of inflammation, assessed by the white blood-cell +(WBC) count, absolute neutrophil count, absolute lymphocyte count, and/or neutrophil-to-lymphocyte ratio (NLR), and measures of coagulation, assessed by the platelet count, were predictive of in-hospital mortality. + +## Materials And Methods Patient Identification + +After institutional review board approval (\#171361), this retrospective study exclusively utilized de-identified information extracted from the medical records in the Vanderbilt University Medical Center "Synthetic Derivative" (SD) database. + +All patients from February 1982 to December 2020 with the International Classification of Diseases, 9th Revision (ICD-9) code 728.86 or the ICD-10 code M72.6, indicating necrotizing fasciitis, were reviewed by the research team, to ensure rigor in the selection criteria (n = 1,213 patients). Patients were included if they were +‡19 years of age and met the criteria outlined in Figure 1. Patients who were admitted for an alternative cause and developed necrotizing fasciitis during hospitalization were excluded from this study. These criteria resulted in the inclusion of 389 cases of necrotizing fasciitis across 383 patients in the study. + +Data Collection + +![1_image_0.png](1_image_0.png) + +A database modeled after past large retrospective cohort studies on necrotizing fasciitis was developed8-13. Demographic data Fig. 1 CONSORT diagram showing the retrospective identification and validation of patients with necrotizing fasciitis. Utilizing the deidentified synthetic derivative database, all patients with an ICD-9 or 10 code for necrotizing fasciitis were identified. All charts were individually reviewed to confirm the diagnosis of necrotizing fasciitis by surgeon notes, operative findings, and/or pathology reports. + +## D 3 + +including age, sex, race, and comorbidities were collected from the SD. Comorbidities were confirmed through individual chart review and/or the presence of the following diseaseassociated ICD-9 and 10 codes listed in Appendix Supplemental Index 1. An aggregate comorbidity score of 0 to 7 was generated for each patient; 1 point each was given for confirmed diabetes, hypertension, peripheral vascular disease, kidney disease, history of cancer, cirrhosis, and heart disease. + +Characteristics of the infection, including its initial location and cause, were collected. Operational definitions for the location and mechanism of infection are noted in Appendix Supplemental Index 2. + +The primary outcome of this study was in-hospital mortality. Laboratory values assessing inflammation and coagulation were collected from the complete blood-cell count (CBC) with differential, both at the time of admission to the tertiary care center and throughout the course of disease. Inflammation was assessed by the WBC count, absolute neutrophil count, absolute lymphocyte count, and neutrophil-to-lymphocyte ratio (NLR). Coagulation was assessed by the platelet count. All of the laboratory values analyzed in the study were the first values obtained in the emergency department or on the floor upon admission. + +## Statistical Analysis + +Descriptive statistics for demographics and infection sequelae were presented using the frequency and proportion. A multivariable logistic regression model was built on immunocoagulopathy measures (absolute neutrophil, absolute lymphocyte, and platelet counts) and patient age to predict in-hospital mortality in cases of confirmed necrotizing fasciitis. Platelet count was natural-logarithm-transformed to be included in the model. We initially assumed a nonlinear relationship for all predictors by including restricted cubic spline terms with 3 knots. The nonlinear term was removed if a Wald test on it gave a p value of +>0.2. In the final model, the nonlinear term was only included for the natural-logarithm-transformed platelet count. Predictor importance was measured by degrees-of-freedom-penalized Wald statistics. The model performance (discrimination and calibration) in future patients was evaluated using a bootstrap approach. A sensitivity analysis was performed using multiple imputation for missing immunocoagulopathy measures. Patient demographics, medical history, and other laboratory results were included in the imputation model. Multiple imputation was done using the aregImpute function from the Hmisc package in R (R Foundation for Statistical Computing). Ten imputed data sets were generated, logistic regression models were fitted on each of the data sets, and coefficients and standard errors from the 10 models were pooled using the Rubin rules14. The threshold for significance was set at p < 0.05, and all statistical analyses were performed using IBM SPSS Statistics version 27 or R version 4.1.0. + +## Source Of Funding + +Funding was provided by the Caitlin Lovejoy Fund, the Vanderbilt University Medical Center Department of Orthopaedics, and the Vanderbilt School of Medicine Research Immersion program. Creation of the retrospective database utilized in this study was supported by the Clinical and Translational Science Award (CTSA) number UL1 TR002243 from the National Center for Advancing Translational Sciences. Its contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health. + +| TABLE I Demographics of the 389 Retrospectively Identified Cases of Necrotizing Fasciitis Gender (no. [%]) Male 214 (55.0) Female 175 (45.0) Median age (range) (yr) 51 (19-85) Race (no. [%]) Caucasian 302 (77.6) African American 52 (13.4) Asian/Pacific 5 (1.3) Native American/other 12 (3.1) Unknown 18 (4.6) Mechanism of infection (no. [%]) Idiopathic 130 (33.4) Infected wound 131 (33.7) Postoperative infection 24 (6.2) Puncture wound 31 (8.0) Trauma 32 (8.2) Other 3 (0.8) History unattainable 38 (9.8) Infection origin (no. [%]) Abdomen 41 (10.5) Back/flank 6 (1.5) Chest 5 (1.3) Extremity 136 (35.0) Head/neck 31 (7.9) Pelvis 170 (43.7) Comorbidities (no. [%]) Diabetes 221 (56.8) Obesity 238 (61.1) Hypertension 229 (58.9) Peripheral vascular disease 29 (7.5) Kidney disease 59 (15.2) History of cancer 55 (14.1) Cirrhosis 21 (5.4) Heart disease 93 (23.9) Median comorbidity score 2 (0-6) (range) Amputation (no. [%]) 41 (10.5) Multiorgan dysfunction (no. [%]) 61 (15.7) | +|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| + +![3_image_0.png](3_image_0.png) + +## Results Patient Demographics + +The retrospective review identified 389 verified cases of necrotizing fasciitis infections treated at our tertiary care center (Fig. 1). The primary mechanisms of infection among these cases were infected wounds (n = 131) and idiopathic +(n = 130), accounting for ;67% of all cases (Table I, Fig. 2-A). + +The pelvis (n = 170) and extremities (n = 136) were the most common locations of the infection, accounting for ;79% of all cases (Table I, Fig. 2-B). The median age of the patients was 51 years, and comorbidities including diabetes, obesity, and hypertension were common among the study cohort (Table I). The overall in-hospital mortality rate in this population was 19.8% (Fig. 2-C). + +Of the 389 cases of confirmed necrotizing fasciitis, 261 +(67.1%) had complete study data obtained from a CBC with differential measured near the time of admission, including absolute neutrophil, absolute lymphocyte, and platelet counts, allowing for assessment of immunocoagulopathy. Univariate correlation between mortality status and each immunocoagulopathy measure is presented in Fig. 3. + +## Utilizing Measures Of Immunocoagulopathy To Predict In-Hospital Mortality + +Guided by the above results, a multivariable logistic regression model was built including the measures of immunocoagulopathy collected from a CBC with differential, specifically absolute neutrophil count and absolute lymphocyte count evaluated as an NLR and a transformed platelet count, along with patient age, to predict in-hospital mortality in cases of confirmed necrotizing fasciitis. Measures from the 261 cases with complete data led to the generation of the following model: + +Odds of in 2 hospital mortality = rmortality + +![3_image_1.png](3_image_1.png) + +where Transformed platelet = e 2:23·lnðplateletÞ 2 0:76·ðlnðplateletÞ 2 4:64Þ +3 1 11:76·ðlnðplateletÞ 2 5:45Þ +3 1 2 1:00·ðlnðplateletÞ 2 6:05Þ +3 1 and ðxÞ1 = x if x > 0; 0 otherwise This model differentiated between survivors and nonsurvivors well, with an original area under the receiver operating characteristic curve (AUC) of 0.829 (Fig. 4-A). In internal validation, the model has an overfitting-corrected AUC of 0.806 (Fig. 4-B). To facilitate clinical implementation of this prediction model, a user-friendly online risk calculator was developed (https://statcomp2.app.vumc.org/NF_Mortality/). + +The most important predictor in this model was the platelet count, which accounted for 66.7% of the total variance explained by the model, followed by age (27.1%) and + +![4_image_0.png](4_image_0.png) + +neutrophil count (14.7%) (Fig. 4-C). The ratio of NLR to platelet count (i.e., a measure of immunocoagulopathy) accounted for 69.9% of the total variance. The mortality risk increased with a decreasing level of platelets (p < 0.001), and the adjusted odds ratio for mortality was estimated to be 5.43 +(95% confidence interval [CI], 2.50 to 11.77) for a patient with a platelet count of 100 · 103/mL compared with a patient with a platelet count of 300 · 103. A high level of neutrophils also increased mortality risk (p = 0.020), with an adjusted odds ratio of 2.34 (95% CI, 1.15 to 4.76) for 30 · 103 /mL compared with 10 · 103/mL neutrophils. Older patients were more likely to die in the hospital (p = 0.002), and the odds of mortality for a 60-year-old were 170% (95% CI, 42% to 413%), or 2.7 times, higher than those for a 40-year-old (Figs. 4-D and 4-E). + +Because of the retrospective nature of this study, neutrophil and lymphocyte counts were available for only 67.4% of the cases, and the mortality rate was much higher in cases with missing neutrophil and/or lymphocyte counts (29.9%) than those with available neutrophil and lymphocyte counts (14.6%). In the model with multiple imputation, the effects of platelet count and age on in-hospital mortality remained similar, but the association between neutrophil count and mortality was no longer significant (see Appendix Supplemental Figs. 1-A, 1-B, +and 1-C), and the predicted mortality risks in the complete-case model agreed with those in the model with multiple imputation (see Appendix Supplemental Fig. 1-D). + +## Discussion + +This retrospective study found that measures of immunocoagulopathy at admission effectively prognosticated inhospital mortality risk in patients with necrotizing fasciitis and complete data. Immunocoagulopathy can be sensitively quantified openaccess.jbjs.org + +![5_image_0.png](5_image_0.png) + +in real time through serial measures of coagulation and inflammation. For example, coagulation activation can be assessed by the platelet count. In addition to forming a physical barrier with fibrin, platelets are capable of directly interacting with bacteria15,16, engulfing bacteria17, and releasing bactericidal molecules from their granules. For these reasons, elevated platelet counts have long been examined as a prognostic indicator for infection18. Conversely, an uncontrolled APR may result in thrombocytopenia due to consumption (formation of a fibrin-platelet complex), sequestration (binding to cells in a tissue depot), or clearance (by macrophages in the spleen) of platelets from circulation. Therefore, platelet counts have a bimodal relationship with outcomes, in which both abnormally high as well as abnormally low platelet counts are associated with adverse outcomes following infection3,19,20. In the present study, we observed that non-survivors had lower platelet counts and greater proportion of patients had thrombocytopenia at admission compared with survivors, aligning with recent findings by Chen et al.21. + +WBC counts are commonly utilized to assess the presence of an infection. While the total WBC count can be nonspecific 3, analysis of specific subtypes of leukocytes, such neutrophils, lymphocytes, and their proportions relative to each other, has been useful for assessing the presence and/or severity of infection4. Neutrophils, in cooperation with the host's coagulation response, work to trap bacteria in neutrophil extracellular traps (NETs) composed of DNA and in fibrin-platelet webs before releasing chemotoxins to kill the pathogens3,5,22. An elevated neutrophil count has long been utilized as a prognostic indicator of infection; however, the neutrophil count alone does not predict infection severity23, or the risk of inpatient mortality from necrotizing fasciitis as seen in this present study. For these reasons, studies have examined cellular ratios, such as the NLR, as more sensitive predictors of disease severity and prognosticators of patient outcomes4,24. While frequently utilized as a prognostic indicator for patient outcomes in the fields of cardiology25-27, oncology28-30, and infectious disease4,23,31,32, fewer studies to date have examined the utility of the NLR in cases of musculoskeletal infection or emergency general surgery33,34. In a recent study by Ravindhran et al., a preoperative NLR of >7.5 was reported to be a reliable predictor of poor outcomes of necrotizing fasciitis35. A study by Yim et al. illustrated that high NLR (‡8) upon admission positively predicted inhospital mortality in patients with Fournier gangrene34. In the present study, while the NLR was predictive of in-hospital mortality, a model that also included the patient's age and platelet count at admission outperformed the simple NLR at predicting in-hospital mortality for necrotizing fasciitis. + +## Clinical Algorithms + +Prior studies have examined the utility of clinical algorithms to aid in the diagnosis and prognostication of patients with suspected necrotizing fasciitis. One of the most utilized systems for aiding in the diagnosis of necrotizing fasciitis, LRINEC +(Laboratory Risk Indicator for Necrotizing Fasciitis), considers multiple circulating humoral markers of the APR, including Creactive protein (CRP), hemoglobin, sodium, creatinine, glucose, and WBC count1,36,37. Thus, to fully calculate a LRINEC score, patients must have a CBC, a complete metabolic panel (CMP), +and CRP measurement. CRP, a measure of inflammation, has been demonstrated to be a powerful predictor of disease severity and patient prognosis in a variety of musculoskeletal infection studies3,4,38. While the original LRINEC score places marked weight on CRP values, subsequent versions of this scoring system have lessened the impact of CRP in exchange for a greater focus on cellular changes and patient factors39. In this current retrospective database study, one limitation encountered was that only 36.9% (115) of the 312 survivors and 28.6% (22) of the 77 nonsurvivors had the required CRP values at admission to fully calculate a LRINEC score retrospectively. With the limited data, no difference in the LRINEC score was observed between survivors and non-survivors. Therefore, while a study by El-Menyar et al. suggests that the LRINEC score may likewise be useful as a prognostic measure to identify high-risk patients40, this could not be confirmed in the present retrospective study population. + +The lack of available data illustrates a potential limitation to applying the LRINEC score retrospectively. A greater proportion of patients in the present study possessed a CBC +with platelet counts and leukocyte differentials, allowing for the assessment of immunocoagulopathy retrospectively: +94.6% (295) of the 312 survivors and 94.8% (73) of the 77 nonsurvivors had platelets assessed at the time of admission, while 71.5% (223) of the 312 survivors and 50.6% (39) of the 77 non-survivors additionally had the leukocyte differential assessed. This single test (the CBC with differential) can obtain all necessary values to calculate a measure of immunocoagulopathy at admission, thus representing a sensitive, time- and cost-effective prognostic indicator of in-hospital mortality for patients with necrotizing fasciitis. + +Given the limited LRINEC scores available retrospectively, the present study could conclude that measures of immunocoagulopathy are equivalent or superior to the LRINEC score; however, the ease and availability of assessing immunocoagulopathy make this measure advantageous. Thus, future prospective studies are warranted. + +## Strength And Limitations + +This study analyzed one of the largest retrospective cohorts of necrotizing fasciitis cases from a single center. As a tertiary referral center, our population is likely biased toward severe cases and conditions such as necrotizing fasciitis, thus providing ample patients to be assessed and analyzed. Given the retrospective nature of the study, there were limitations in our ability to regulate the timing and availability of laboratory blood draws, and to assess the impact of the causative pathogen(s) or medication(s) administered, such as anticoagulants, on laboratory values and mortality from necrotizing fasciitis. Furthermore, the missingness of neutrophil and lymphocyte counts cannot be fully accounted for by other variables; therefore, the missing-at-random assumption may not be valid and the model with multiple imputation could introduce bias41. We suggest that our model d 8 +(with a complete-case approach) should only be applied to the patients with neutrophil and lymphocyte counts measured under a situation similar to our current clinical settings. Finally, while all cases of necrotizing fasciitis were confirmed through evaluation of the medical and surgical records, we likely excluded some positive cases because insufficient records were available retrospectively, and we potentially included some cases involving other forms of necrotizing soft-tissue infections (NSTI). + +As part of this study, NLR and the platelet count were evaluated together as a measure of immunocoagulopathy. However, alternative clinical laboratory values that can accurately depict immunocoagulopathy likely exist. Alternative measures of inflammation include CRP, procalcitonin, and cytokines such as interleukin (IL)-6, and coagulopathy can be assessed by the prothrombin time or fibrinogen level. Given the retrospective nature of our data set, alternative measures were not reliably available to determine which measure of immunocoagulopathy is superior. Future prospective studies would be required to determine the most sensitive measures of immunocoagulopathy for predicting patient morbidity and mortality. + +## Conclusions + +This study determined that, in patients with necrotizing fasciitis, age and measures of immunocoagulopathy at admission, specifically the NLR and platelet count assessed from the CBC with differential, can accurately prognosticate a patient's in-hospital mortality risk, with an overfitting-corrected AUC of 0.806. Paralleling these findings, numerous recent studies have illustrated the utility of similar measures for predicting severe outcomes and death in cases of COVID-194,42-45. Given the accessibility of measures of immunocoagulopathy determined from a simple CBC with differential, future prospective studies examining the utility of these measures in cases of necrotizing fasciitis and other serious musculoskeletal infections are warranted. + +## Appendix + +Supporting material provided by the authors is posted with the online version of this article as a data supplement at jbjs.org (http://links.lww.com/JBJSOA/A477). n NOTE: The authors thank members of the Schoenecker Laboratory, in particular Drs. Andrew Rees and Jacob Schultz, for their review of this work. Additionally, we thank all of the experts who have provided guidance for this study, including Drs. Jackie Pennings and Frank Harrell. Finally, we thank our families and friends for their continued support and understanding. + +Samuel R. Johnson, BS1 Teresa Benvenuti, BA2 Hui Nian, PhD3 Isaac P. Thomson, MD, MSCI4,5 Keith Baldwin, MD, MPH, MSPT6 William T. Obremskey, MD, MPH, MMHC2 Jonathan G. Schoenecker, MD, PhD2,5,7,8,9,10 Stephanie N. Moore-Lotridge, PhD2,8,10 1Vanderbilt University School of Medicine, Nashville, Tennessee 2Department of Orthopaedics, Vanderbilt University Medical Center, Nashville, Tennessee 3Department of Biostatistics, Vanderbilt University Medical Center, Nashville, Tennessee 4Division of Infectious Disease, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, Tennessee 5Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 6Department of Orthopaedics, The Children's Hospital of Pennsylvania, Philadelphia, Pennsylvania 7Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 8Division of Pediatric Orthopaedics, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, Tennessee 9Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 10Center for Bone Biology, Vanderbilt University Medical Center, Nashville, Tennessee Email for corresponding author: jon.schoenecker@vumc.org + +## References + +1. Hysong AA, Posey SL, Blum DM, Benvenuti MA, Benvenuti TA, Johnson SR, An TJ, +Devin JK, Obremskey WT, Martus JE, Moore-Lotridge SN, Schoenecker JG. Necrotizing Fasciitis: Pillaging the Acute Phase Response. J Bone Joint Surg Am. 2020; 102(6):526-37. 2. Benvenuti M, An T, Amaro E, Lovejoy S, Mencio G, Martus J, Mignemi M, Schoenecker JG. Double-edged sword: musculoskeletal infection provoked acute phase response in children. Orthop Clin North Am. 2017;48(2):181-97. + +3. An TJ, Benvenuti MA, Mignemi ME, Thomsen IP, Schoenecker JG. Pediatric musculoskeletal infection: Hijacking the acute-phase response. JBJS Rev. 2016 Sep 27;4(9):e4. + +4. Moore-Lotridge SN, Gibson BH, Duvernay MT, Martus JE, Thomsen IP, Schoenecker JG. Pediatric Musculoskeletal Infection. J Pediatric Orthopaedic Society North America. 2020;2(2). 5. Kobayashi SD, Voyich JM, Burlak C, DeLeo FR. Neutrophils in the innate immune response. Arch Immunol Ther Exp (Warsz). 2005;53(6):505-517. + +6. Baker CE, Moore-Lotridge SN, Hysong AA, Posey SL, Robinette JP, Blum DM, +Benvenuti MA, Cole HA, Egawa S, Okawa A, Saito M, McCarthy JR, Nyman JS, Yuasa M, Schoenecker JG. Bone Fracture Acute Phase Response-A Unifying Theory of Fracture Repair: Clinical and Scientific Implications. Clin Rev Bone Miner Metab. + +2018;16(4):142-58. + +7. Lee YT, Chou TD, Peng MY, Chang FY. Rapidly progressive necrotizing fasciitis caused by Staphylococcus aureus. J Microbiol Immunol Infect. 2005 Oct;38(5):361-4. + +8. Wall DB, de Virgilio C, Black S, Klein SR. Objective criteria may assist in distinguishing necrotizing fasciitis from nonnecrotizing soft tissue infection. Am J Surg. + +2000 Jan;179(1):17-21. 9. Wall DB, Klein SR, Black S, de Virgilio C. A simple model to help distinguish necrotizing fasciitis from nonnecrotizing soft tissue infection. J Am Coll Surg. 2000 Sep;191(3):227-31. + +10. Childers BJ, Potyondy LD, Nachreiner R, Rogers FR, Childers ER, Oberg KC, +Hendricks DL, Hardesty RA. Necrotizing fasciitis: a fourteen-year retrospective study of 163 consecutive patients. Am Surg. 2002 Feb;68(2):109-16. + +11. Wong CH, Chang HC, Pasupathy S, Khin LW, Tan JL, Low CO. Necrotizing fasciitis: clinical presentation, microbiology, and determinants of mortality. J Bone Joint Surg Am. 2003 Aug;85(8):1454-60. + +12. Sarani B, Strong M, Pascual J, Schwab CW. Necrotizing fasciitis: current concepts and review of the literature. J Am Coll Surg. 2009 Feb;208(2):279-88. + +13. Huang KF, Hung MH, Lin YS, Lu CL, Liu C, Chen CC, Lee YH. Independent predictors of mortality for necrotizing fasciitis: a retrospective analysis in a single institution. J Trauma. 2011 Aug;71(2):467-73, discussion 473. + +14. Rubin DB. Multiple Imputation for Nonresponse in Surveys. New York: John Wiley and Sons; 2004. + +15. Kerrigan SW. The expanding field of platelet-bacterial interconnections. Platelets. 2015;26(4):293-301. + +16. Ali RA, Wuescher LM, Dona KR, Worth RG. Platelets mediate host defense against Staphylococcus aureus through direct bactericidal activity and by enhancing macrophage activities. J Immunol. 2017 Jan 1;198(1):344-51. + +17. McNicol A, Israels SJ. Beyond hemostasis: the role of platelets in inflammation, malignancy and infection. Cardiovasc Hematol Disord Drug Targets. 2008 Jun;8(2): 99-117. 18. Riise ØR, Kirkhus E, Handeland KS, Flatø B, Reiseter T, Cvancarova M, Nakstad B, Wathne KO. Childhood osteomyelitis-incidence and differentiation from other acute onset musculoskeletal features in a population-based study. BMC Pediatr. + +2008 Oct 20;8(1):45. 19. Assinger A, Schrottmaier WC, Salzmann M, Rayes J. Platelets in sepsis: an update on experimental models and clinical data. Front Immunol. 2019 Jul 17;10:1687. + +20. Gafter-Gvili A, Mansur N, Bivas A, Zemer-Wassercug N, Bishara J, Leibovici L, +Paul M. Thrombocytopenia in Staphylococcus aureus bacteremia: risk factors and prognostic importance. Mayo Clin Proc. 2011 May;86(5):389-96. + +21. Chen YC, Liou YT, Tsai WH, Chen LW. Prognostic Role of Subsequent Thrombocytopenia in Necrotizing Fasciitis Without Liver Disease. Ann Plast Surg. 2022 Mar 1;88(1s)(Suppl 1):S99-105. + +22. Kobayashi SD, Malachowa N, DeLeo FR. Influence of microbes on neutrophil life and death. Front Cell Infect Microbiol. 2017 May 1;7:159. + +23. de Jager CP, van Wijk PT, Mathoera RB, de Jongh-Leuvenink J, van der Poll T, +Wever PC. Lymphocytopenia and neutrophil-lymphocyte count ratio predict bacteremia better than conventional infection markers in an emergency care unit. Crit Care. + +2010;14(5):R192. + +24. Schwab P, Varady N, Chen A, editors. Novel marker for septic hip and knee arthritis: neutrophil-to-lymphocyte ratio is a strong predictor of treatment failure and postoperative 90-day mortality. Orthopaedic Proceedings. 2019;101-B: No. SUPP_4. 25. Bhat T, Teli S, Rijal J, Bhat H, Raza M, Khoueiry G, Meghani M, Akhtar M, Costantino T. Neutrophil to lymphocyte ratio and cardiovascular diseases: a review. Expert Rev Cardiovasc Ther. 2013 Jan;11(1):55-9. + +26. Tamhane UU, Aneja S, Montgomery D, Rogers EK, Eagle KA, Gurm HS. Association between admission neutrophil to lymphocyte ratio and outcomes in patients with acute coronary syndrome. Am J Cardiol. 2008 Sep 15;102(6):653-7. + +27. N´uñez J, N´uñez E, Bod´ı V, Sanchis J, Miñana G, Mainar L, Santas E, Merlos P, +Rumiz E, Darmofal H, Heatta AM, Ll`acer A. Usefulness of the neutrophil to lymphocyte ratio in predicting long-term mortality in ST segment elevation myocardial infarction. Am J Cardiol. 2008 Mar 15;101(6):747-52. + +28. Templeton AJ, McNamara MG, Seruga B, Vera-Badillo FE, Aneja P, Ocaña A, ˇ +Leibowitz-Amit R, Sonpavde G, Knox JJ, Tran B, Tannock IF, Amir E. Prognostic role of neutrophil-to-lymphocyte ratio in solid tumors: a systematic review and meta-analysis. J Natl Cancer Inst. 2014 May 29;106(6):dju124. + +29. Azab B, Bhatt VR, Phookan J, Murukutla S, Kohn N, Terjanian T, Widmann WD. + +Usefulness of the neutrophil-to-lymphocyte ratio in predicting short- and long-term mortality in breast cancer patients. Ann Surg Oncol. 2012 Jan;19(1):217-24. + +30. Proctor MJ, McMillan DC, Morrison DS, Fletcher CD, Horgan PG, Clarke SJ. A derived neutrophil to lymphocyte ratio predicts survival in patients with cancer. Br J Cancer. 2012 Aug 7;107(4):695-9. 31. Naess A, Nilssen SS, Mo R, Eide GE, Sjursen H. Role of neutrophil to lymphocyte and monocyte to lymphocyte ratios in the diagnosis of bacterial infection in patients with fever. Infection. 2017 Jun;45(3):299-307. + +32. Farah R, Khamisy-Farah R. Association of neutrophil to lymphocyte ratio with presence and severity of gastritis due to Helicobacter pylori infection. J Clin Lab Anal. 2014 May;28(3):219-23. + +33. Yapıcı O, Berk H, Oztoprak N, Seyman D, Tahmaz A, Merdin A. Can Ratio of ¨ +Neutrophil-to-Lymphocyte Count and Erythrocyte Sedimentation Rate in Diabetic Foot Infecti on Predict Osteomyelitis and/or Amputation? Hematol Rep. 2017 Feb 23;9(1):6981. 34. Yim SU, Kim SW, Ahn JH, Cho YH, Chung H, Hwang EC, Yu HS, Oh KJ, Kim SO, Jung SI, Kang TW, Kwon DD, Park K. Neutrophil to Lymphocyte and Platelet to Lymphocyte Ratios Are More Effective than the Fournier's Gangrene Severity Index for Predicting Poor Prognosis in Fournier's Gangrene. Surg Infect (Larchmt). 2016 Apr;17(2):217-23. + +35. Ravindhran B, Rajan S, Kerketta D, Balachandran G, Mohan LN. Neutrophil to Lymphocyte Ratio (NLR) and Platelet to Lymphocyte Ratio (PLR) Versus Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) as Predictors of Outcome in Necrotising Fasciitis. Indian J Surgery. 2020;82(3):325-30. + +36. Wong CH, Khin LW, Heng KS, Tan KC, Low CO. The LRINEC (Laboratory Risk Indicator for Necrotizing Fasciitis) score: a tool for distinguishing necrotizing fasciitis from other soft tissue infections. Crit Care Med. 2004 Jul;32(7):1535-41. + +37. Bechar J, Sepehripour S, Hardwicke J, Filobbos G. Laboratory risk indicator for necrotising fasciitis (LRINEC) score for the assessment of early necrotising fasciitis: a systematic review of the literature. Ann R Coll Surg Engl. 2017 May;99(5): +341-6. + +38. Amaro E, Marvi TK, Posey SL, Benvenuti MA, An TJ, Dale KM, Lovejoy SA, Martus JE, Johnson ME, Mencio GA, Moore-Lotridge SN, Thomsen IP, Schoenecker JG. CReactive Protein Predicts Risk of Venous Thromboembolism in Pediatric Musculoskeletal Infection. J Pediatr Orthop. 2019 Jan;39(1):e62-7. + +39. Borschitz T, Schlicht S, Siegel E, Hanke E, von Stebut E. Improvement of a Clinical Score for Necrotizing Fasciitis: 'Pain Out of Proportion' and High CRP Levels Aid the Diagnosis. PLoS One. 2015 Jul 21;10(7):e0132775. + +40. El-Menyar A, Asim M, Mudali IN, Mekkodathil A, Latifi R, Al-Thani H. The laboratory risk indicator for necrotizing fasciitis (LRINEC) scoring: the diagnostic and potential prognostic role. Scand J Trauma Resusc Emerg Med. 2017 Mar 7;25(1): 28. 41. Baldwin KD, Ohman-Strickland P. Missing data in orthopaedic research. U Pennsylvania Orthopaedic Journal. 2009;19. + +42. Lagunas-Rangel FA. Neutrophil-to-lymphocyte ratio and lymphocyte-to-C-reactive protein ratio in patients with severe coronavirus disease 2019 (COVID-19): A metaanalysis. J Med Virol. 2020 Oct;92(10):1733-4. + +43. Li X, Liu C, Mao Z, Xiao M, Wang L, Qi S, Zhou F. Predictive values of neutrophilto-lymphocyte ratio on disease severity and mortality in COVID-19 patients: a systematic review and meta-analysis. Crit Care. 2020 Nov 16;24(1):647. + +44. Fu J, Kong J, Wang W, Wu M, Yao L, Wang Z, Jin J, Wu D, Yu X. The clinical implication of dynamic neutrophil to lymphocyte ratio and D-dimer in COVID-19: A retrospective study in Suzhou China. Thromb Res. 2020 Aug;192:3-8. + +45. Simadibrata DM, Calvin J, Wijaya AD, Ibrahim NAA. Neutrophil-to-lymphocyte ratio on admission to predict the severity and mortality of COVID-19 patients: A metaanalysis. Am J Emerg Med. 2021 Apr;42:60-9. \ No newline at end of file diff --git a/medical/md_images/PMC10249382/0_image_1.png b/medical/md_images/PMC10249382/0_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..bc155e284e7cbca64d582eabe8be82325d7337c1 --- /dev/null +++ b/medical/md_images/PMC10249382/0_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:a7c63defd560dcd50f10a9b522f218d20e923fda0f2120e9ab81cadd8063a78a +size 43725 diff --git a/medical/md_images/PMC10249382/9_image_2.png b/medical/md_images/PMC10249382/9_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..1086a9e35f8b5a9ce3658870dc4bc6e79c2621fd --- /dev/null +++ b/medical/md_images/PMC10249382/9_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:4bff21e17ce73ad1896f10f001dc005ff9ca73b188c425df6f57b662fd0cbc37 +size 30064 diff --git a/medical/md_images/PMC10332048/32_image_0.png b/medical/md_images/PMC10332048/32_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..7ef08f221b750f6ddd8ed1a949051dc7cfd99381 --- /dev/null +++ b/medical/md_images/PMC10332048/32_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:1717257736655c05f7922a72e3c8d87396b2d23b4706c1988cc777c7469b8c78 +size 46727 diff --git a/medical/md_images/PMC10349221/PMC10349221.md b/medical/md_images/PMC10349221/PMC10349221.md new file mode 100644 index 0000000000000000000000000000000000000000..e470bfcfc2f1ef92f0f512200767ae98ee9e7831 --- /dev/null +++ b/medical/md_images/PMC10349221/PMC10349221.md @@ -0,0 +1,518 @@ +ACR Open Rheumatology Vol. 5, No. 7, July 2023, pp 333–344 DOI 10.1002/acr2.11555 +© 2023 The Authors. ACR Open Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology. + +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. + +REVIEW ARTICLE + +# Human Leukocyte Antigen B27-Negative Axial Spondyloarthritis: What Do We Know? + +Atul Deodhar,1 Tejpal Gill,1 and Marina Magrey2 Axial spondyloarthritis (axSpA) is a chronic, immune-mediated disease characterized by inflammatory axial skeleton involvement and extra-musculoskeletal manifestations. The continuum of axSpA ranges from nonradiographic axSpA +(nr-axSpA) to ankylosing spondylitis, also known as radiographic axSpA; the latter is defined by definitive radiographic sacroiliitis. Human leukocyte antigen B27 (HLA-B27) is a genetic marker strongly associated with axSpA; it aids in the diagnosis of axSpA, and its absence leads to delay in diagnosis. For HLA-B27-negative patients, disease pathogenesis is poorly understood, signs and symptoms are frequently underrecognized, and diagnosis and treatment are commonly delayed. The proportion of HLA-B27-negative patients may be higher among non-White patients and those with nr-axSpA, who can face additional diagnostic challenges related to lack of definitive radiographic sacroiliitis. In this narrative review, we discuss the role of HLA-B27 in the diagnosis and pathogenesis of axSpA and highlight various pathways and genes that may be related to axSpA pathogenesis in HLA-B27-negative patients. We also emphasize the need to characterize gut microbial communities in these patients. Adequate understanding of clinical and pathological features underlying HLA-B27-negative patients with axSpA will improve diagnosis, treatment, and outcomes for this complex inflammatory disease. + +## Introduction + +Axial spondyloarthritis (axSpA) is a chronic, polygenic, immune-mediated inflammatory disease affecting the sacroiliac +(SI) joints and the spine and is characterized by back pain, inflammation in peripheral joints and entheses, extra-musculoskeletal involvement, and new bone formation in patients with advanced disease (1,2). According to the 2009-2010 National Health and Nutrition Examination Survey, the prevalence of axSpA in the US +adult population is estimated at 0.9% to 1.4% (3). axSpA consists of a continuum of disease presentations ranging from nonradiographic axSpA (nr-axSpA) to ankylosing spondylitis (AS), also known as radiographic axSpA. nr-axSpA is differentiated from AS based on the absence of definitive sacroiliitis on radiographs +(4,5). AS, the best known and most studied form of axSpA, is characterized by sacroiliitis on radiographs as described in the 1984 modified New York classification criteria (6,7). + +The genetic marker human leukocyte antigen B27 (HLA-B27) +is strongly associated with axSpA and plays a critical role in its diagnosis (1). The prevalence of HLA-B27 varies globally across populations, and the incidence of spondyloarthritis (SpA)-related diseases generally mirrors HLA-B27 prevalence locally (8–10). + +Although HLA-B27 is commonly relied upon for diagnosis of axSpA, a substantial proportion of patients with axSpA do not carry the HLA-B27 allele, including 37.5% of Black patients and 40.0% +of women (40%) (11–13). However, women are more frequently diagnosed with nr-axSpA, which has a lower prevalence of HLAB27 than AS (14–16). axSpA is an underrecognized disease, in which there is a delay of several years between symptom onset and diagnosis (17). A recent survey conducted in 2652 patients from 13 European countries reported a more than 7-year diagnostic delay in patients with axSpA (Figure 1) (18). The diagnostic delay may be as much as 3 years longer for HLA-B27-negative patients than for patients who are HLA-B27 positive (19,20). + +This review aims to update clinicians on the available data regarding the prevalence, clinical phenotype, and underlying pathogenic mechanisms of HLA-B27-negative axSpA. We also discuss the differential response to therapy between HLA-B27-negative and -positive patients with axSpA. + +## Statement Of Literature Search + +A series of PubMed and Ovid searches were conducted to identify gaps in knowledge related to HLA-B27-negative axSpA and potentially relevant manuscripts. Example searches included terms such as "nonradiographic axial spondyloarthritis" OR "nr-axSpA" OR "axSpA" AND "HLA-B27 negative" OR "HLA-B27 status" and relevant variations of "pathogenesis," +"gut microbiome," "microbial dysbiosis," "diagnosis," or +"treatment." Publications mentioning clinical presentations, classification criteria, axSpA pathogenesis, disease progression, diagnostic delay, and treatment response were considered for inclusion; publications deemed irrelevant (author replies, errata, etc) were excluded. The authors also included studies that were previously known to them as well as references cited within any relevant article. + +## Discovery Of Hla-B27 And Its Worldwide Prevalence + +The discovery of the relationship between the genetic marker HLA-B27 and AS was reported independently by two research groups in 1973 and is among the pivotal advances in rheumatology over the last century (10,21). The association between HLA-B27 and AS is the strongest known genetic relationship between a major histocompatibility complex (MHC) antigen and disease (22). HLA-B27 has since been shown to be associated with other SpA-related diseases, including acute anterior uveitis, reactive arthritis, and psoriatic arthritis (PsA), although to a lesser degree (23–25). The global distribution of the almost 170 HLA-B27 subtypes is widely influenced by race and ethnicity, with varying strengths of association with AS (9,22,26–32). + +However, the association between axSpA and other HLA allotypes is not known due to their low prevalence and because many have not been extensively studied (33). In the US, the ageadjusted prevalence of HLA-B27 was found to be 6.1% (95% CI: 4.6%-8.2%): 7.5% (95% CI: 5.3%-10.4%) in nonHispanic Whites, 4.6% (95% CI: 3.4-6.1) in Mexican Americans, + +![1_image_0.png](1_image_0.png) + +and 3.5% (95% CI: 2.5%-4.8%) in all other US races and ethnicities besides non-Hispanic Whites combined (which includes Mexican Americans) (34). The most common disease-associated subtypes are B*2702 (Mediterranean population), B*2705 (White and Native American populations), and B*2704 (Asian population) (9,35,36). Other HLA-B27 subtypes are either weakly associated with AS due to differential binding of ligands (B*2709) or potentially provide a protective effect against the development of AS +(B*2706) (37–39). + +Since its discovery in families with multiple affected members, HLA-B27 has been established as an important genetic risk factor for axSpA (1,2,40–44). However, the genetic risk associated with HLA-B27 status does not completely explain axSpA +pathogenesis in the entire population. Pooled data from a Canadian cohort together with three other local, national, and worldwide cohorts have shown that approximately 25% of patients with axSpA are HLA-B27 negative (45,46). Other reports state that the number of patients with nr-axSpA who are HLA-B27 negative could be as high as 40% (47). Furthermore, HLA-B27 may account for only approximately 20% of the overall contribution to heritability, with more than 100 MHC and nonMHC genetic loci contributing less than 10% to the known heritability (48–51). Although AS is a highly heritable disease, the majority of the genetic risk associated with disease pathogenesis remains unknown. + +## Pathogenesis Of Axspa In Hla-B27-Positive Patients + +Although the pathogenesis of axSpA is complex and is likely to be polygenic, more is currently known about the role of HLA-B27 than of other factors in the development of axSpA (1). Since the discovery of the association between HLA-B27 and AS, several hypotheses have been developed relating to the role of HLA-B27 in axSpA pathogenesis. These hypotheses include (i) cytotoxic CD8+ T-lymphocyte autoreactivity against HLA-B27–peptide complexes (also known as the arthritogenic peptide hypothesis); (ii) activation of inhibitory natural killer cell receptor KIR3DL2-expressing CD4+ T cells by inappropriate expression of disulfide-linked HLA-B27 heavy-chain homodimers on the cell surface, leading to increased interleukin (IL) 17 production; and (iii) accumulation of misfolded HLA-B27 in the endoplasmic reticulum that triggers the unfolded protein response and activates the IL-23/IL-17 proinflammatory pathway +(52–57); however, none of these hypotheses fully explain the disease pathogenesis (58,59). Recent studies have highlighted an alteration of the gut microbial community in the pathogenesis of many HLA-B27-associated SpAs, including axSpA (60–64). Disease development in animal models of SpA has also been found to be dependent on the gut microbiome (65,66). This would suggest that HLA-B27 itself may influence gut dysbiosis. Taken together, although the mechanism by which HLA-B27 contributes to axSpA remains elusive, interactions between host immune response and gut microbiome are thought to play a key role in disease pathogenesis. + +## Pathogenesis Of Axspa In Hla-B27-Negative Patients + +The pathogenic mechanisms driving axSpA development in HLA-B27-negative patients are even less well understood and are likely more varied than in HLA-B27-positive patients with axSpA (Figure 2) (50,64–100). Much of our understanding of the genetic risk factors for axSpA comes from genome-wide association studies in patients with AS, which have demonstrated and confirmed strong associations with ERAP1, IL-23R, RUNX3, TNFR1/LTBR, IL-12B, PTGER4, TBKBP1, ANTXR2, CARD9, IL-1R2, TRADD, STAT3, and KIF21B (23,101–105). However, most non-HLA-B27 pathways were identified in studies that included mostly patients who were HLA-B27 positive. + +These risk loci included genes associated with T-cell differentiation, immune-cell signaling, and innate immune response pathways as well as cytokines and chemokines. Although genome-wide association studies in patients with AS have illustrated a number of key genes and pathways involved in disease pathogenesis (103,104,106,107), these studies have also highlighted the fact that known genetic risk loci only explain a minority (<30%) of AS heritability (108). One model of disease heritability suggests that major genetic risk factors for disease come from common disease alleles that appear with appreciable frequency in the general population but individually have relatively low disease penetrance (108,109). These interacting disease alleles would have additive effects on disease susceptibility and, in combination with environmental risk factors, would need to reach a "threshold" for axSpA disease development. A combination of a range of different genes would be sufficient to cross this disease "threshold," which could explain why HLA-B27 is not required for axSpA pathogenesis in HLA-B27-negative patients. + +This model of disease inheritance could also explain the significant overlap in immune-mediated inflammatory disease genetic risk loci, as each gene or pathway would contribute an increased risk of developing disease and the interactions between different risk factors would lead to disease development. + +Because most of the alternative mechanisms of axSpA +development were identified in studies that included patients who were HLA-B27 positive, it remains unclear the extent to which these genes and pathways contribute to disease development in patients who are HLA-B27 negative. The lack of experimental animal models of axSpA independent of HLA-B27 further limits our ability to experimentally examine these alternative mechanisms. Therefore, the contribution of these alternative pathways to the development of HLA-B27-negative disease remains largely hypothetical and requires further investigation. Below, we outline some of the potential alternative mechanisms that have been implicated or hypothesized in axSpA development. + +Other HLA-B allotypes. HLA-B27 subtypes share several structural features, including the B pocket of the peptide binding site, which interacts and binds with a conserved Arg 2 (R2) motif (110). It was previously believed that few allotypes other than HLA-B27 could bind peptides with R2. However, other allotypes such as HLA-B39 have been shown to also accept peptides with this motif, suggesting that these allotypes could contribute to disease development in HLA-B27-negative patients (67). Importantly, several HLA-B allotypes, including B7, B14, B39, and B60, have been discovered to be significantly + +![2_image_0.png](2_image_0.png) + +associated with AS among HLA-B27-negative individuals +(67–70). Like most other genes, two HLA-B alleles are carried by each individual. In a cohort of patients with axSpA, we observed that individuals who are HLA-B27 negative presented with a wide range of HLA-B alleles (eg, B8, B14, B15, B35, B40, B44, B47, B49, B51, B57, and B58) in various combinations (unpublished data). Interestingly, most of these alleles are also seen in combination with HLA-B27 in patients with axSpA who are HLA-B27 positive, further emphasizing the need to characterize these alleles and study their relationship to disease development and heterogeneity. The importance of other HLA-B alleles in axSpA pathogenesis is further supported by studies that found associations between the inflammatory disease PsA and several HLA alleles +(eg, B8, B18, B38, B39, B55, and C06), in addition to the association between PsA and HLA-B27 (111,112). In line with this, HLA-B*1403, an allotype only reported in the African populations of Cameroon and Togo, was discovered to be associated with AS in these populations, which is especially significant because AS and HLA-B27 positivity are rare (70). Among eight Togolese patients with AS identified in a large epidemiologic analysis of 9065 West African rheumatology patients, AS was strongly associated with the B*1403 antigen, and the B27 antigen was found in only one patient (71). The authors of this study concluded that HLA-B*1403 shows the B27 "super-type" motif and may have an effect on AS susceptibility according to the arthritogenic peptide hypothesis. Another pooled analysis of African patients from three studies found that 24 of 26 patients were male, a ratio that is consistent with Western populations, suggesting that a male predominance still exists in countries where HLA-B27 positivity is low (113,114). Additionally, the interaction between HLA-B60 and HLA-B27 was identified as a better marker for AS risk than either allotype individually (69), suggesting that other HLA-B allotypes could contribute to disease development even in HLA-B27-positive patients. Further investigation of axSpA pathogenesis in these populations in which SpA disease is more strongly associated with allotypes other than HLA-B27 could significantly add to our understanding of disease pathogenesis in HLA-B27-negative individuals. + +Factors beyond HLA-B alleles in axSpA +pathogenesis. More than 110 additional genes and genetic regions have been identified that are associated with axSpA irrespective of HLA-B27 involvement and contribute approximately 10% of the axSpA heritability, and many of these genetic regions are outside of HLA-B alleles (48–51). Additionally, only a minority of individuals positive for HLA-B27 (approximately 5%-10%) will develop axSpA and clinical manifestations of HLA-B27-positive and -negative axSpA overlap, suggesting these other factors besides HLA-B alleles are most likely contributing to axSpA pathogenesis. These regions also share some genetic and clinical overlap with other inflammatory diseases, such as inflammatory bowel disease (IBD) (115). Taken together, these factors highlight the multigenic nature of axSpA. Some of these additional genes that likely contribute to axSpA pathogenesis are discussed below. + +CARD9. Mutations in caspase recruitment domain-containing protein 9 (CARD9) are associated with a number of inflammatory diseases, including Crohn's disease, ulcerative colitis, and AS +(116). CARD9 has been implicated in both axSpA and IBD pathogenesis (117,118). In response to innate pattern recognition receptor activation, CARD9 initiates an inflammatory cytokine cascade through induction of transcription factors (116). Mutations in CARD9 are associated with increased susceptibility to fungal infections, highlighting a critical role for CARD9 in the initiation of immune responses (116,119). CARD9 signaling leads to dendritic cell and macrophage activation and secretion of proinflammatory cytokines, including IL-23, that ultimately drive differentiation of IL-17–producing T helper (Th) 17 cells (72,73). Single nucleotide polymorphisms (SNPs) in CARD9 were associated with AS in a Han Chinese patient population (74) and in patients in the UK of British White origin (101). A CARD9 SNP was also identified that conferred protection against AS in Iranian patients who were HLA-B27 negative, potentially through decreased CARD9 expression leading to altered IL-23/IL-17 signaling (75,76), suggesting that this pathway could be important for axSpA development independent of HLA-B27. + +NOD2. Another important protein implicated in axSpA and IBD pathogenesis and in IL-23/IL-17 signaling is nucleotide binding oligomerization domain containing 2 (NOD2). In response to intracellular bacterial cell wall components, NOD2 signaling activates transcription factors to increase transcription of proinflammatory cytokines and growth factors (120). Peptidoglycan motifs on bacterial cell walls induce NOD2 to form a complex with CARD9 that regulates immune response through mitogenactivated protein kinase–mediated transcriptional changes, such as differentiation of Th1 and Th17 cells and proinflammatory cytokine secretion (77,116). A NOD2 polymorphism that was associated with Crohn's disease was found at a higher frequency in patients with SpA who had chronic intestinal inflammation than in patients with SpA without intestinal inflammation, suggesting that NOD2 can contribute to disease pathogenesis in a variety of inflammatory-mediated diseases (78). In a rodent model of autoimmune arthritis, genetic deletion of NOD2 in SKG mice increased the severity of arthritis in part through increased numbers of IL17–producing CD4+ T cells and an exaggerated release of IL-17 from stimulated T cells (79). Similarly, blood mononuclear cells from patients with AS treated with a NOD2 ligand showed a greater increase in proinflammatory gene expression +(including IL-23, IL-17, IL-1β, and tumor necrosis factor [TNF] α) +than cells from healthy controls, suggesting that NOD2 could be a significant driver of inflammatory-mediated disease development in axSpA (80). However, these findings have yet to be confirmed in patients with axSpA who are HLA-B27 negative. + +MIF and CD74. Another protein implicated in inflammatory disease pathogenesis is macrophage migration inhibitory factor +(MIF). MIF binding to the cell surface ligand-binding protein CD74 alters transcription factor activity to increase proinflammatory gene expression, including increased TNF, IL-1, and IL-17 expression (121). MIF/CD74 signaling is known to play an important role as a regulator of inflammation in the pathogenesis of inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and IBD (122–124). Additionally, genetic deletion of MIF in curdlan-treated SKG mice (a model of SpA) significantly reduced SpA disease severity, suggesting a role for MIF +in SpA pathogenesis as well (81). Patients with AS, nr-axSpA, and PsA with axial involvement all had higher concentrations of antiCD74 autoantibodies than healthy controls, suggesting higher circulating levels of CD74 (82). Patients with AS also had higher serum and synovial fluid concentrations of MIF than healthy controls, with higher serum MIF levels predictive of AS progression +(83). Neutrophils isolated from patients with SpA and from a mouse model of SpA secreted higher concentrations of MIF, suggesting that MIF/CD74 signaling could be a major contributor to disease pathogenesis (81). An elevation in the number of MIFproducing macrophages and Paneth cells was observed in the ileum of patients with AS compared with healthy controls, which could provide a mechanistic link between SpA and intestinal inflammation (83). A majority of patients with HLA-B27-negative axSpA were positive for CD74 autoantibodies (88%), and the presence of CD74 antibodies was associated with disease activity as measured by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), suggesting that MIF could play a significant role in axSpA pathogenesis in the absence of HLA-B27 (84,85). + +The gut microbiome and mycobiome. Studies in the last decades have demonstrated alterations in gut microbial communities (known as "dysbiosis") in patients with all SpA subtypes, adding gut microbiota as a central environmental component to SpA pathogenesis (60,61,64,86). Furthermore, experimental animal models of SpA require the gut microbiota for the development of disease; however, this evidence is derived from models with either endogenous or elevated HLA-B27 expression and may not be generalizable to HLA-B27-negative patients (65,66,125). + +Intestinal inflammation was found in almost 50% of patients with AS and was associated with the extent of bone marrow edema in the SI joints on magnetic resonance imaging (MRI) (87,88). Because HLA-B27-positive and -negative axSpA share many clinical manifestations, we can hypothesize that there are microbial perturbations associated with HLA-B27-negative axSpA. + +Several studies have described a relationship between gut inflammation and axSpA, supporting the gut dysbiosis hypothesis as a possible explanation of axSpA pathogenesis. Gut dysbiosis in patients with AS and rodent models of AS is also associated with inflammasome activation, ileitis, and increased IL-1β release and IL23A expression (89). In addition to gut bacteria, disruption of the normal gut resident fungi or bacterial fungal-interactome could also contribute to gastrointestinal dysbiosis associated with inflammatory diseases through altered fungi-bacterial interactions, nutrient metabolism, and translocation of fungal antigens across the epithelial barrier (90). When colonic fungal species were surveyed in patients with IBD, an increase in fungal diversity and altered composition of fungal microbiota was observed compared with healthy controls. However, whether this increase in fungal diversity is disease associated or secondary to microbial dysbiosis needs to be investigated (91). A polymorphism in the gene encoding Dectin-1, an innate immune receptor involved in antifungal immunity, is strongly associated with severe ulcerative colitis, and genetic deletion of Dectin-1 in mice increased the severity of chemically induced colitis through altered cellular responses to fungi (92). Patients with AS also had alterations in gut fungal microbiota, including increased and decreased abundance of different fungal species as well as altered bacterial-fungal interactions, leading to an overall decrease in the gut microbiota diversity of patients with AS compared with healthy control patients (93). The change in gut fungal composition observed in patients with AS was strongly correlated with disease severity, as measured by BASDAI and C-reactive protein (CRP) levels, and it was associated with the degree of radiographic damage (93). However, to our knowledge, no studies have been performed to characterize alterations in gut bacterial or fungal diversity in patients with HLA-B27-negative axSpA. + +These studies that show a strong association between bacterial and fungal dysbiosis in inflammatory diseases highlight the need for a thorough analysis of the gut microbiota in HLA-B27-negative patients with axSpA. + +Interleukins. The IL-23/IL-17 and IL-1 signaling pathways have a well-established role in the pathogenesis of axSpA +(50,77,94–100). Recently, an SNP in IL1R2 was discovered to confer mild protection from AS in a population of patients who were HLA-B27 negative (75), suggesting a role for IL-1 signaling in axSpA disease development independent of HLA-B27. However, further research is needed to fully understand the potential contributions of IL-1 and IL-23/IL-17 signaling to axSpA development in patients who are HLA-B27 negative. + +## Challenges In Diagnosis Of Axspa In Hla-B27-Negative Patients + +Diagnostic criteria for daily clinical practice have not been developed for any form of axSpA. Diagnosis of axSpA relies on summation of available evidence by an expert rheumatologist. A method has been proposed for quantitatively estimating the probability of axSpA in patients with chronic back pain, age at onset under 45 years, and no definitive radiographic changes in the SI joints (126). The primary aim of this method was to increase physician confidence in the diagnosis of early axSpA in patients with chronic back pain according to the absence or presence of certain clinical features, laboratory test findings, or findings on skeletal imaging, and it was ultimately shown that HLAB27 positivity has a likelihood ratio of 9 for diagnosing AS (127). + +The negative predictive value for HLA-B27 is between 55% and 81% for axSpA depending on HLA-B27 prevalence in the population (84,128–130), reinforcing the clinical utility of HLA-B27 status in diagnosing axSpA. + +Despite advancements in diagnosis, challenges exist regarding improvement of the delayed diagnosis of axSpA, with better identification needed particularly in HLA-B27-negative patients who may experience greater diagnostic delay than HLA-B27-positive patients (19). Several cross-sectional studies of diagnostic biomarkers have been performed in patients with axSpA. One potentially alternative diagnostic test for axSpA could be the presence of circulating CD74 antibodies (131). These antibodies are found in a majority of patients with axSpA (85%-88%), +including Lebanese patients who come from a population with low HLA-B27 prevalence, and are found in few patients without axSpA (8%-13%) (84,132). Antibodies for CD74 were detected in 88% of patients with axSpA who were HLA-B27 negative (84). However, the diagnostic utility of anti-CD74 antibodies for axSpA may be limited due to low specificity (133). In addition to anti-CD74 antibodies, a number of other targets have shown potential as future diagnostic biomarkers, including adipokines, cytokines, and microRNAs (134). Despite these advancements, biomarkers that can identify axSpA early after disease onset remain a major unmet need (131). + +Polygenic risk scores, which are quantitative measures that use findings from up to thousands of genetic variants to assess the risk of disease, could have greater discriminatory capacity in axSpA (135). Using polygenic risk scores captures a higher proportion of the disease heritability and performs better overall than CRP levels, MRI, or HLA-B27 alone, allowing for a more accurate diagnosis of axSpA and distinguishing it from other diseases that appear clinically similar but have different etiologies (136). However, more research is needed to confirm the utility of polygenic risk scores specifically in patients with who are HLA-B27 negative. This integration of different omics technologies has greater potential to enable early and accurate diagnosis of patients with axSpA, ultimately leading to better treatment (134). + +## Clinical Features Of Hla-B27-Positive And -Negative Patients + +Some clinical phenotypes of axSpA may differ between HLA-B27-positive and -negative patients (Figure 3) (19,46, 47,137–144). HLA-B27-positive patients with axSpA have earlier disease onset and are diagnosed earlier than HLA-B27-negative patients, regardless of sex (19,46,47). In a comparative, crosssectional study reviewing data from the REGISPONSER database, which includes more than 1000 patients with AS (of whom approximately 20% are HLA-B27 negative), the presence of HLA-B27 was associated with an earlier disease onset and higher family aggregation (140). + +Studies have reportedthat HLA-B27 positivity is associated with a higher prevalence of uveitis and cardiac involvement in patients with AS (141,142). Several studies in a Korean population demonstrated a higher frequency of hip joint involvement in HLA-B27-positive AS than in HLA-B27-negative AS (138,143,145). One study assessing the influence of HLA-B27 status on axSpA in the largest cohort of Chinese patients to date revealed that HLA-B27 positivity is associated with male sex, younger age, longer disease duration (possibly due to earlier disease recognition), greater familial aggregation, and higher frequency of uveitis (137). HLA-B27-negative patients experienced a higher frequency of peripheral arthritis, dactylitis, psoriasis, IBD, and extra-musculoskeletal manifestations (140). However, because the prevalence of HLA-B27 is lower in patients with PsA or IBD-associated arthritis than in patients with AS, some of these + +![5_image_0.png](5_image_0.png) + +patients may have had PsA and enteropathic arthritis but fulfilled the 1984 modified New York criteria due to involvement of the SI joints. + +This association confirmed previous findings among patients from the DESIR cohort, in which HLA-B27 positivity was also found to associate with earlier disease onset and earlier diagnosis (47). A more recent study explored whether axial involvement in psoriasis and AS with or without psoriasis were different clinical presentations of the same disease, axSpA, or whether they were in fact separate diseases with overlapping features. The study hypothesized that HLA-B27 status was key, proposing that HLA-B27-negative patients present with less radiographic severity, less involvement of the SI joints and spine, less symmetry of syndesmophytes, and unusually shaped, bulkier, nonmarginal syndesmophytes. HLA-B27-positive patients exhibited more severe radiographic damage, more bilateral fused SI joints, more typical marginal syndesmophytes, and more symmetry in the spine. No difference was shown in symmetry of either SI involvement or of nonmarginal syndesmophytes according to HLA-B27 status (146). + +Current evidence suggests that HLA-B27-positive patients with axSpA might suffer from a worse disease prognosis than HLA-B27-negative patients, although this is not a universal finding. Patients with axSpA who were HLA-B27 positive were shown to have higher disease activity as measured by CRP and the Ankylosing Spondylitis Disease Activity Score (ASDAS)–CRP, +more frequent MRI inflammation of the SI joints and the spine, and a higher frequency of radiographic sacroiliitis compared with HLA-B27-negative patients (47,137). Male patients with AS who were HLA-B27 positive more frequently experienced radiographic progression compared with HLA-B27-negative men, although HLA-B27 status was not associated with radiographic progression in women (144,147). Patients with undifferentiated SpA who were HLA-B27 positive were also more likely to progress to AS than patients who were HLA-B27 negative (148). However, other studies in patients with axSpA found that HLA-B27-positive patients had less active SpA as determined by BASDAI score and more mild back pain than HLA-B27-negative patients, although these HLA-B27-positive patients typically received an earlier diagnosis and more frequently received nonsteroidal antiinflammatory drug treatment (148,149). + +## Treatment Response Among Hla-B27-Positive And -Negative Patients + +HLA-B27 status may predict the likelihood of response to treatment for patients with axSpA, although the extent of this association remains unclear. Results of clinical trials exploring treatment efficacy in HLA-B27-positive and -negative patients have demonstrated that HLA-B27 status is a predictor of response to treatment (150,151). An open-label phase 3b trial evaluating the effectiveness and safety of the TNF inhibitor adalimumab in a large cohort of patients with AS identified that various factors—including younger age, high CRP levels, and HLA-B27 positivity—were strongly associated with BASDAI50, Assessment of SpondyloArthritis international Society (ASAS) 40, and partial remission responses (151). A post hoc analysis of data from two clinical trials found that HLA-B27 positivity was associated with large improvements in clinical measures and achievement of disease state targets (ASDAS major improvement, ASAS partial remission, ASDAS inactive disease) in response to TNF inhibitor treatment (150). A pooled analysis of patients with AS from the MEASURE 1-5 studies stratified by HLA-B27 status found that HLA-B27-positive patients experienced increased therapeutic benefit from treatment with the IL-17 inhibitor secukinumab compared with HLA-B27-negative patients (152). Data from COAST-X, which evaluated the efficacy of the IL-17 inhibitor ixekizumab in patients with nr-axSpA with or without baseline HLA-B27 positivity, demonstrated that patients exhibited at least a numerical treatment response regardless of HLA-B27 status, but the responses were significant over placebo for the HLAB27-positive patients (P < 0.05) (153). + +A post hoc analysis of the phase 3 Speed of Onset of SecuKinumab-Induced Relief From Pain in Patients With AxIal SpoNdyloarthritis (SKIPPAIN) trial (NCT03136861) explored the potential association of HLA-B27 subtypes with the effect of secukinumab treatment in patients with axSpA (154). The results demonstrated that, when compared with the HLA-B27 heterozygous group, a higher proportion of patients in the HLA-B27 homozygous group had uveitis, peripheral arthritis, enthesitis, and a family history of SpA. The study also found an apparent relationship between HLA-B27 zygosity and secukinumab treatment efficacy: patients who were homozygous for HLA-B27 had greater improvements in spinal pain scores with secukinumab treatment than patients who were HLA-B27 heterozygous compared with their respective placebo controls. Both groups of HLA-B27-positive patients receiving secukinumab showed greater improvements following treatment than HLA-B27-negative patients, with HLA-B27 homozygous patients experiencing the greatest improvements from baseline. These differences in treatment response underscore the importance of identifying the varied pathogenic mechanisms that lead to axSpA disease, both in patients with HLA-B27-positive and -negative axSpA. + +## Conclusions And Future Directions + +HLA-B27 is strongly associated with axSpA and is commonly relied upon for diagnosis. Prevalence typically varies among ethnic groups; however, a large subset of patients with axSpA, particularly non-White patients and patients with nr-axSpA, lack HLA-B27. The pathogenesis of axSpA has been more extensively characterized in HLA-B27-positive patients and is less well defined in HLA-B27-negative patients. Genome-wide association studies have helped uncover shared pathogenic mechanisms among AS, +IBD, and psoriasis that are independent of HLA-B27. The exploration of other HLA allotypes that have been described in patients with HLA-B27-negative axSpA may offer additional insight into axSpA pathogenesis among patients with or without HLA-B27. + +Further research into the relative contributions of gut dysbiosis, proinflammatory cytokine signaling, and cellular antigen presentation to HLA-B27-negative axSpA pathogenesis is needed. Patients with axSpA who are HLA-B27 negative often experience increased diagnostic delays; more frequently present with peripheral arthritis, IBD, and extra-musculoskeletal manifestations; and potentially face worse disease prognosis and reduced therapeutic responses, reinforcing the need for early and accurate diagnosis in these patients. Increased awareness of the clinical phenotype of HLA-B27-negative axSpA among health care providers and health care systems is vital to preventing delays in diagnosis and improving patient outcomes in axSpA disease, and equal attention must be paid to the substantial group of patients whose symptoms thus far cannot be traced to a genetic marker. + +## Acknowledgments + +This work was supported by Novartis Pharmaceuticals Corporation, East Hanover, New Jersey. Medical writing support was provided by Prasanthi Mandalay, PhD, of Articulate Science, LLC, and Ken Gresham, PhD, of Health Interactions, Inc, and was funded by Novartis Pharmaceuticals Corporation. This manuscript was developed in accordance with Good Publication Practice (GPP 2022) guidelines (http:// +www.ismpp.org/gpp3). Authors had full control of the content and made the final decision on all aspects of this publication. + +## Author Contributions + +All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. + +## Role Of The Study Sponsor + +Novartis Pharmaceuticals Corporation had no role in the study design or in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication. Publication of this article was not contingent upon approval by Novartis Pharmaceuticals Corporation. + +## References + +1. De Koning A, Schoones JW, van der Heijde D, et al. Pathophysiology of axial spondyloarthritis: consensus and controversies. Eur J Clin Invest 2018;48:e12913. + +2. Sieper J, Poddubnyy D. Axial spondyloarthritis. Lancet 2017;390: +73–84. + +3. Reveille JD, Witter JP, Weisman MH. Prevalence of axial spondylarthritis in the United States: estimates from a cross-sectional survey. + +Arthritis Care Res (Hoboken) 2012;64:905–10. + +4. Rudwaleit M, Khan MA, Sieper J. The challenge of diagnosis and classification in early ankylosing spondylitis: do we need new criteria? Arthritis Res Ther 2005;52:1000–8. + +5. Sieper J, Rudwaleit M, Baraliakos X, et al. The Assessment of SpondyloArthritis international Society (ASAS) handbook: a guide to assess spondyloarthritis. Ann Rheum Dis 2009;68 Suppl 2:ii1–44. + +6. Rudwaleit M, van der Heijde D, Landewé R, et al. The development of Assessment of SpondyloArthritis international Society classification criteria for axial spondyloarthritis (part II): validation and final selection. Ann Rheum Dis 2009;68:777–83. + +7. Van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum 1984;27:361–8. + +8. Reveille JD, Weisman MH. The epidemiology of back pain, axial spondyloarthritis and HLA-B27 in the United States. Am J Med Sci 2013;345:431–6. + +9. Khan MA. An update on the genetic polymorphism of HLA-B*27 with 213 alleles encompassing 160 subtypes (and still counting). Curr Rheumatol Rep 2017;19:9. + +10. Schlosstein L, Terasaki PI, Bluestone R, et al. High association of an HL-A antigen, W27, with ankylosing spondylitis. N Engl J Med 1973; 288:704–6. + +11. Akassou A, Bakri Y. Does HLA-B27 status influence ankylosing spondylitis phenotype? Clin Med Insights Arthritis Musculoskelet Disord 2018;11:1179544117751627. + +12. Ortolan A, van Lunteren M, Ramiro S, et al. Are gender-specific approaches needed in diagnosing early axial spondyloarthritis? Data from the SPondyloArthritis Caught Early cohort. Arthritis Res Ther 2018;20:218. + +13. Jamalyaria F, Ward MM, Assassi S, et al. Ethnicity and disease severity in ankylosing spondylitis a cross-sectional analysis of three ethnic groups. Clin Rheumatol 2017;36:2359–64. + +14. Glintborg B, Sørensen IJ, Østergaard M, et al. Ankylosing spondylitis versus nonradiographic axial spondyloarthritis: comparison of tumor necrosis factor inhibitor effectiveness and effect of HLA-B27 status. An observational cohort study from the nationwide DANBIO registry. + +J Rheumatol 2017;44:59–69. + +15. Hong C, Kwan YH, Leung YY, et al. Comparison of ankylosing spondylitis and non-radiographic axial spondyloarthritis in a multi-ethnic Asian population of Singapore. Int J Rheum Dis 2019;22:1506–11. + +16. Wright GC, Kaine J, Deodhar A. Understanding differences between men and women with axial spondyloarthritis. Semin Arthritis Rheum 2020;50:687–94. + +17. Magrey MN, Danve AS, Ermann J, et al. Recognizing axial spondyloarthritis: a guide for primary care. Mayo Clin Proc 2020;95: +2499–508. + +18. Garrido-Cumbrera M, Navarro-Compan V, Bundy C, et al. Identifying parameters associated with delayed diagnosis in axial spondyloarthritis: data from the European map of axial spondyloarthritis. + +Rheumatology (Oxford) 2021;61:705–12. + +19. Feldtkeller E, Khan MA, van der Heijde D, et al. Age at disease onset and diagnosis delay in HLA-B27 negative vs. positive patients with ankylosing spondylitis. Rheumatol Int 2003;23:61–6. + +20. Fallahi S, Jamshidi AR. Diagnostic delay in ankylosing spondylitis: +related factors and prognostic outcomes. Arch Rheumatol 2016; 31:24–30. + +21. Brewerton DA, Hart FD, Nicholls A, et al. Ankylosing spondylitis and HL-A 27. Lancet 1973;1:904–7. + +22. Sheehan NJ. The ramifications of HLA-B27. J R Soc Med 2004;97: +10–4. + +23. Brewerton DA, Caffrey M, Nicholls A, et al. HL-A 27 and arthropathies associated with ulcerative colitis and psoriasis. Lancet 1974;1: +956–58. + +24. Aho K, Ahvonen P, Lassus A, et al. HL-A 27 in reactive arthritis. + +A study of Yersinia arthritis and Reiter's disease. Arthritis Rheum 1974;17:521–6. + +25. Brewerton DA, Caffrey M, Nicholls A, et al. Acute anterior uveitis and HL-A 27. Lancet 1973;302:994–6. + +26. Bhatia K, Prasad ML, Barnish G, et al. Antigen and haplotype frequencies at three human leucocyte antigen loci (HLA-A, -B, -C) in the Pawaia of Papua New Guinea. Am J Phys Anthropol 1988;75: +329–40. + +27. Gofton JP, Chalmers A, Price GE, et al. HL-A 27 and ankylosing spondylitis in B.C. Indians. J Rheumatol 1984;11:572–3. + +28. Benevolenskaia LI, Erdes S, Krylov MI, et al. The epidemiology of spondyloarthropathies among the native inhabitants of Chukotka. + +2. The prevalence of HLA-B27 in the population and among spondyloarthropathy patients. Ter Arkh 1994;66:41–4. + +29. Khan MA. HLA-B27 and its subtypes in world populations. Curr Opin Rheumatol 1995;7:263–9. + +30. Mustafa KN, Hammoudeh M, Khan MA. HLA-B27 prevalence in Arab populations and among patients with ankylosing spondylitis. + +J Rheumatol 2012;39:1675–7. + +31. Hukuda S, Minami M, Saito T, et al. Spondyloarthropathies in Japan: +nationwide questionnaire survey performed by the Japan Ankylosing Spondylitis Society. J Rheumatol 2001;28:554–9. + +32. Slimani S, Hamdi W, Nassar K, et al. Spondyloarthritis in North Africa: an update. Clin Rheumatol 2021;61:705–12. + +33. Robinson PC, Brown MA. Genetics of ankylosing spondylitis. Mol Immunol 2014;57:2–11. + +34. Reveille JD, Hirsch R, Dillon CF, et al. The prevalence of HLA-B27 in the US: data from the US National Health and Nutrition Examination Survey, 2009. Arthritis Rheum 2012;64:1407–11. + +35. Reveille JD. Major histocompatibility genes and ankylosing spondylitis. Best Pract Res Clin Rheumatol 2006;20:601–9. + +36. Bowness P. HLA-B27. Annu Rev Immunol 2015;33:29–48. + +37. Ramos M, Paradela A, Vazquez M, et al. Differential association of HLA-B*2705 and B*2709 to ankylosing spondylitis correlates with limited peptide subsets but not with altered cell surface stability. + +J Biol Chem 2002;277:28749–56. + +38. Van Gaalen FA. Does HLA-B*2706 protect against ankylosing spondylitis? A meta-analysis. Int J Rheum Dis 2012;15:8–12. 39. Ren EC, Koh WH, Sim D, et al. Possible protective role of HLAB*2706 for ankylosing spondylitis. Tissue Antigens 1997;49:67–9. + +40. Van Lunteren M, Sepriano A, Landewé R, et al. Do ethnicity, degree of family relationship, and the spondyloarthritis subtype in affected relatives influence the association between a positive family history for spondyloarthritis and HLA-B27 carriership? Results from the worldwide ASAS cohort. Arthritis Res Ther 2018;20:166. + +41. Brown MA, Kennedy LG, MacGregor AJ, et al. Susceptibility to ankylosing spondylitis in twins: the role of genes, HLA, and the environment. Arthritis Rheum 1997;40:1823–8. + +42. Reveille JD. Biomarkers for diagnosis, monitoring of progression, and treatment responses in ankylosing spondylitis and axial spondyloarthritis. Clin Rheumatol 2015;34:1009–18. + +43. Geirsson AJ, Kristjansson K, Gudbjornsson B. A strong familiality of ankylosing spondylitis through several generations. Ann Rheum Dis 2010;69:1346–8. + +44. Van der Linden SM, Valkenburg HA, de Jongh BM, et al. The risk of developing ankylosing spondylitis in HLA-B27 positive individuals. A comparison of relatives of spondylitis patients with the general population. Arthritis Rheum 1984;27:241–9. + +45. Wallis D, Haroon N, Ayearst R, et al. Ankylosing spondylitis and nonradiographic axial spondyloarthritis: part of a common spectrum or distinct diseases? J Rheumatol 2013;40:2038–41. + +46. Baraliakos X, Braun J. Non-radiographic axial spondyloarthritis and ankylosing spondylitis: what are the similarities and differences? RMD Open 2015;1:e000053 47. Chung HY, Machado P, van der Heijde D, et al. HLA-B27 positive patients differ from HLA-B27 negative patients in clinical presentation and imaging: results from the DESIR cohort of patients with recent onset axial spondyloarthritis. Ann Rheum Dis 2011;70: +1930–6. + +48. McHugh K, Bowness P. The link between HLA-B27 and SpA—new ideas on an old problem. Rheumatology (Oxford) 2012;51:1529–39. + +49. Ellinghaus D, Jostins L, Spain SL, et al. Analysis of five chronic inflammatory diseases identifies 27 new associations and highlights disease-specific patterns at shared loci. Nat Genet 2016;48:510–8. + +50. Reveille JD. Genetics of spondyloarthritis—beyond the MHC. Nat Rev Rheumatol 2012;8:296–304. + +51. Ranganathan V, Gracey E, Brown MA, et al. Pathogenesis of ankylosing spondylitis: recent advances and future directions. Nat Rev Rheumatol 2017;13:359–67. + +52. Benjamin R, Parham P. Guilt by association: HLA-B27 and ankylosing spondylitis. Immunol Today 1990;11:137–42. + +53. Kollnberger S, Chan A, Sun MY, et al. Interaction of HLA-B27 homodimers with KIR3DL1 and KIR3DL2, unlike HLA-B27 heterotrimers, is independent of the sequence of bound peptide. Eur J Immunol 2007;37:1313–22. + +54. Wong-Baeza I, Ridley A, Shaw J, et al. KIR3DL2 binds to HLA-B27 dimers and free H chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis. + +J Immunol 2013;190:3216–24. + +55. Bowness P, Ridley A, Shaw J, et al. Th17 cells expressing KIR3DL2+ +and responsive to HLA-B27 homodimers are increased in ankylosing spondylitis. J Immunol 2011;186:2672–80. + +56. Nakamura A, Boroojeni SF, Haroon N. Aberrant antigen processing and presentation: key pathogenic factors leading to immune activation in ankylosing spondylitis. Semin Immunopathol 2021;43: +245–53. + +57. Bettigole SE, Glimcher LH. Endoplasmic reticulum stress in immunity. Annu Rev Immunol 2015;33:107–38. + +58. Taurog JD, Dorris ML, Satumtira N, et al. Spondylarthritis in HLAB27/human beta2-microglobulin-transgenic rats is not prevented by lack of CD8. Arthritis Rheum 2009;60:1977–84. + +59. Jansen DT, Hameetman M, van Bergen J, et al. IL-17-producing CD4+ T cells are increased in early, active axial spondyloarthritis including patients without imaging abnormalities. Rheumatology +(Oxford) 2015;54:728–35. + +60. Manasson J, Shen N, Garcia Ferrer HR, et al. Gut microbiota perturbations in reactive arthritis and postinfectious spondyloarthritis. + +Arthritis Rheumatol 2018;70:242–54. + +61. Scher JU, Ubeda C, Artacho A, et al. Decreased bacterial diversity characterizes the altered gut microbiota in patients with psoriatic arthritis, resembling dysbiosis in inflammatory bowel disease. Arthritis Rheumatol 2015;67:128–39. + +62. Breban M, Tap J, Leboime A, et al. Faecal microbiota study reveals specific dysbiosis in spondyloarthritis. Ann Rheum Dis 2017;76: +1614–22. + +63. Gill T, Asquith M, Rosenbaum JT, et al. The intestinal microbiome in spondyloarthritis. Curr Opin Rheumatol 2015;27:319–25. + +64. Costello ME, Ciccia F, Willner D, et al. Brief report: intestinal dysbiosis in ankylosing spondylitis. Arthritis Rheumatol 2015;67:686–91. + +65. Taurog JD, Richardson JA, Croft JT, et al. The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats. J Exp Med 1994;180:2359–64. + +66. Rath HC, Herfarth HH, Ikeda JS, et al. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats. + +J Clin Invest 1996;98:945–53. + +67. Yamaguchi A, Tsuchiya N, Mitsui H, et al. Association of HLA-B39 with HLA-B27-negative ankylosing spondylitis and pauciarticular juvenile rheumatoid arthritis in Japanese patients. Evidence for a role of the peptide-anchoring B pocket. Arthritis Rheum 1995;38: +1672–7. + +68. Khan MA, Kushner I, Braun WE. A subgroup of ankylosing spondylitis associated with HLA-B7 in American blacks. Arthritis Rheum 1978;21:528–30. + +69. Wei JC, Tsai WC, Lin HS, et al. HLA-B60 and B61 are strongly associated with ankylosing spondylitis in HLA-B27-negative Taiwan Chinese patients. Rheumatology (Oxford) 2004;43:839–42. + +70. Merino E, Montserrat V, Paradela A, et al. Two HLA-B14 subtypes +(B*1402 and B*1403) differentially associated with ankylosing spondylitis differ substantially in peptide specificity but have limited peptide and T-cell epitope sharing with HLA-B27. J Biol Chem 2005; 280:35868–80. + +71. Lopez-Larrea C, Mijiyawa M, Gonz alez S, et al. Association of ankylosing spondylitis with HLA-B*1403 in a West African population. + +Arthritis Rheum 2002;46:2968–71. + +72. LeibundGut-Landmann S, Gross O, Robinson MJ, et al. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17. Nat Immunol 2007;8: +630–8. + +73. Hara H, Ishihara C, Takeuchi A, et al. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat Immunol 2007;8:619–29. + +74. Ma X, Liu Y, Zhang H, et al. Evidence for genetic association of CARD9 and SNAPC4 with ankylosing spondylitis in a Chinese Han population. J Rheumatol 2014;41:318–24. + +75. Momenzadeh P, Mahmoudi M, Beigy M, et al. Determination of IL1 R2, ANTXR2, CARD9, and SNAPC4 single nucleotide polymorphisms in Iranian patients with ankylosing spondylitis. Rheumatol Int 2016;36:429–35. + +76. Ji C, Yang Z, Zhong X, et al. The role and mechanism of CARD9 gene polymorphism in diseases. Biomed J 2021;44:560–66. + +77. Sharif K, Bridgewood C, Dubash S, et al. Intestinal and enthesis innate immunity in early axial spondyloarthropathy. Rheumatology +(Oxford) 2020;59:iv67–78. + +78. Laukens D, Peeters H, Marichal D, et al. CARD15 gene polymorphisms in patients with spondyloarthropathies identify a specific phenotype previously related to Crohn's disease. Ann Rheum Dis 2005;64:930–5. + +79. Napier RJ, Lee EJ, Vance EE, et al. Nod2 deficiency augments Th17 responses and exacerbates autoimmune arthritis. J Immunol 2018; 201:1889–98. + +80. Vanaki N, Golmohammadi T, Jamshidi A, et al. Increased inflammatory responsiveness of peripheral blood mononuclear cells +(PBMCs) to in vitro NOD2 ligand stimulation in patients with ankylosing spondylitis. Immunopharmacol Immunotoxicol 2018;40: +393–400. + +81. Nakamura A, Zeng F, Nakamura S, et al. Macrophage migration inhibitory factor drives pathology in a mouse model of spondyloarthritis and is associated with human disease. Sci Transl Med 2021; 13:eabg1210. + +82. Gaydukova IZ, Rebrov A, Maslyanskiy A, et al. Diagnostic value of anti-CD74 autoantibodies in axial spondyloarthritis and axial psoriatic arthritis. Results of open-label, cross-sectional, controlled, multicenter progress study. Ann Rheum Dis 2018;77:348. + +83. Ranganathan V, Ciccia F, Zeng F, et al. Macrophage migration inhibitory factor induces inflammation and predicts spinal progression in ankylosing spondylitis. Arthritis Rheumatol 2017;69:1796–806. + +84. Ziade NR, Mallak I, Merheb G, et al. Added value of anti-CD74 autoantibodies in axial spondyloarthritis in a population with low HLA-B27 prevalence. Front Immunol 2019;10:574. + +85. Abdelaziz MM, Gamal RM, Ismail NM, et al. Diagnostic value of antiCD74 antibodies in early and late axial spondyloarthritis and its relationship to disease activity. Rheumatology (Oxford) 2021;60:263–8. + +86. Viladomiu M, Kivolowitz C, Abdulhamid A, et al. IgA-coated E. coli enriched in Crohn's disease spondyloarthritis promote TH17dependent inflammation. Sci Transl Med 2017;9:eaaf9655. + +87. Van Praet L, Van den Bosch FE, Jacques P, et al. Microscopic gut inflammation in axial spondyloarthritis: a multiparametric predictive model. Ann Rheum Dis 2013;72:414–7. + +88. Van Praet L, Jans L, Carron P, et al. Degree of bone marrow oedema in sacroiliac joints of patients with axial spondyloarthritis is linked to gut inflammation and male sex: results from the GIANT cohort. Ann Rheum Dis 2014;73:1186–9. + +89. Guggino G, Mauro D, Rizzo A, et al. Inflammasome activation in ankylosing spondylitis is associated with gut dysbiosis. Arthritis Rheumatol 2021;73:1189–99. + +90. Berthelot JM, Darrieutort-Laffite C, Trang C, et al. Contribution of mycobiota to the pathogenesis of spondyloarthritis. Joint Bone Spine 2021;88:105245. + +91. Ott SJ, Kühbacher T, Musfeldt M, et al. Fungi and inflammatory bowel diseases: alterations of composition and diversity. Scand J +Gastroenterol 2008;43:831–41. + +92. Iliev ID, Funari VA, Taylor KD, et al. Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science 2012;336:1314–7. + +93. Li M, Dai B, Tang Y, et al. Altered bacterial-fungal interkingdom networks in the guts of ankylosing spondylitis patients. mSystems 2019;4:e00176-18. + +94. Pedersen SJ, Maksymowych WP. The pathogenesis of ankylosing spondylitis: an update. Curr Rheumatol Rep 2019;21:58. + +95. Smith JA, Colbert RA. Review: The interleukin-23/interleukin-17 axis in spondyloarthritis pathogenesis: Th17 and beyond. Arthritis Rheumatol 2014;66:231–41. + +96. DeLay ML, Turner MJ, Klenk EI, et al. HLA-B27 misfolding and the unfolded protein response augment interleukin-23 production and are associated with Th17 activation in transgenic rats. Arthritis Rheum 2009;60:2633–43. + +97. Sherlock JP, Joyce-Shaikh B, Turner SP, et al. IL-23 induces spondyloarthropathy by acting on ROR-γt+ CD3+CD4-CD8- entheseal resident T cells. Nat Med 2012;18:1069–76. + +98. Ciccia F, Accardo-Palumbo A, Rizzo A, et al. Evidence that autophagy, but not the unfolded protein response, regulates the expression of IL-23 in the gut of patients with ankylosing spondylitis and subclinical gut inflammation. Ann Rheum Dis 2014;73:1566–74. + +99. Akhtari M, Zargar SJ, Vojdanian M, et al. Monocyte-derived and M1 macrophages from ankylosing spondylitis patients released higher TNF-α and expressed more IL1B in response to BzATP than macrophages from healthy subjects. Sci Rep 2021;11:17842. + +100. Reveille JD. The genetic basis of spondyloarthritis. Ann Rheum Dis 2011;70 Suppl 1:i44–50. + +101. Pointon JJ, Harvey D, Karaderi T, et al. Elucidating the chromosome 9 association with AS; CARD9 is a candidate gene. Genes Immun 2010;11:490–6. + +102. Pointon JJ, Harvey D, Karaderi T, et al. The chromosome 16q region associated with ankylosing spondylitis includes the candidate gene tumour necrosis factor receptor type 1-associated death domain +(TRADD). Ann Rheum Dis 2010;69:1243–6. + +103. Burton PR, Clayton DG, Cardon LR, et al. Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants. Nat Genet 2007;39:1329–37. + +104. Reveille JD, Sims AM, Danoy P, et al. Genome-wide association study of ankylosing spondylitis identifies non-MHC susceptibility loci. + +Nat Genet 2010;42:123–7. + +105. Evans DM, Spencer CC, Pointon JJ, et al. Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility. + +Nat Genet 2011;43:761–7. + +106. Costantino F, Breban M, Garchon HJ. Genetics and functional genomics of spondyloarthritis. Front Immunol 2018;9:2933. + +107. Li Z, Brown MA. Progress of genome-wide association studies of ankylosing spondylitis. Clin Transl Immunology 2017;6:e163. + +108. Brown MA, Laval SH, Brophy S, et al. Recurrence risk modelling of the genetic susceptibility to ankylosing spondylitis. Ann Rheum Dis 2000;59:883–6. + +109. Schork NJ, Murray SS, Frazer KA, et al. Common vs. rare allele hypotheses for complex diseases. Curr Opin Genet Dev 2009;19: +212–9. + +110. Rojo S, García F, Villadangos JA, et al. Changes in the repertoire of peptides bound to HLA-B27 subtypes and to site-specific mutants inside and outside pocket B. J Exp Med 1993;177:613–20. + +111. Winchester R, Giles J, Jadon D, et al. Implications of the diversity of class I HLA associations in psoriatic arthritis. Clin Immunol 2016; 172:29–33. + +112. FitzGerald O, Haroon M, Giles JT, et al. Concepts of pathogenesis in psoriatic arthritis: genotype determines clinical phenotype. Arthritis Res Ther 2015;17:115. + +113. Mijiyawa M, Oniankitan O, Khan MA. Spondyloarthropathies in subSaharan Africa. Curr Opin Rheumatol 2000;12:281–6. + +114. Belachew DA, Sandu N, Schaller B, et al. Ankylosing spondylitis in sub-Saharan Africa. Postgrad Med J 2009;85:353–7. + +115. Robinson PC, Leo PJ, Pointon JJ, et al. Exome-wide study of ankylosing spondylitis demonstrates additional shared genetic background with inflammatory bowel disease. NPJ Genom Med 2016; 1:16008. + +116. Zhong X, Chen B, Yang L, et al. Molecular and physiological roles of the adaptor protein CARD9 in immunity. Cell Death Dis 2018;9:52. + +117. Furst DE, Louie JS. Targeting inflammatory pathways in axial spondyloarthritis. Arthritis Res Ther 2019;21:135. + +118. Luo P, Yang Z, Chen B, et al. The multifaceted role of CARD9 in inflammatory bowel disease. J Cell Mol Med 2020;24:34–9. + +119. Drummond RA, Franco LM, Lionakis MS. Human CARD9: a critical molecule of fungal immune surveillance. Front Immunol 2018;9: 1836. + +120. Negroni A, Pierdomenico M, Cucchiara S, et al. NOD2 and inflammation: current insights. J Inflamm Res 2018;11:49–60. + +121. Kang I, Bucala R. The immunobiology of MIF: function, genetics and prospects for precision medicine. Nat Rev Rheumatol 2019;15: +427–37. + +122. Wallace DJ, Figueras F, Wegener WA, et al. Experience with milatuzumab, an anti-CD74 antibody against immunomodulatory macrophage migration inhibitory factor (MIF) receptor, for systemic lupus erythematosus (SLE). Ann Rheum Dis 2021;80:954–5. + +123. Ayoub S, Hickey MJ, Morand EF. Mechanisms of disease: macrophage migration inhibitory factor in SLE, RA and atherosclerosis. + +Nat Clin Pract Rheumatol 2008;4:98–105. + +124. Nishihira J, Mitsuyama K. Overview of the role of macrophage migration inhibitory factor (MIF) in inflammatory bowel disease. Curr Pharm Des 2009;15:2104–9. + +125. Rehaume LM, Mondot S, Aguirre de Carcer D, et al. ZAP-70 genotype disrupts the relationship between microbiota and host, leading to spondyloarthritis and ileitis in SKG mice. Arthritis Rheumatol 2014;66:2780–92. + +126. Rudwaleit M, van der Heijde D, Khan MA, et al. How to diagnose axial spondyloarthritis early. Ann Rheum Dis 2004;63:535–43. + +127. Rudwaleit M, Khan MA, Sieper J. The challenge of diagnosis and classification in early ankylosing spondylitis: do we need new criteria? Arthritis Rheum 2005;52:1000–8. + +128. García-Salinas R, Ruta S, Chichande JT, et al. The role of HLA-B27 in Argentinian axial spondyloarthritis patients. J Clin Rheumatol 2021; 28:e619–22. + +129. Ziade N, Abi Karam G, Merheb G, et al. HLA-B27 prevalence in axial spondyloarthritis patients and in blood donors in a Lebanese population: results from a nationwide study. Int J Rheum Dis 2019;22: +708–14. + +130. Juanola X, Fernandez-Sueiro JL, Torre-Alonso JC, et al. Comparison of 2 referral strategies for the diagnosis of axial spondyloarthritis in Spain. The RADAR study. Reumatol Clin 2013;9:348–52. + +131. Maksymowych WP. Biomarkers for diagnosis of axial spondyloarthritis, disease activity, prognosis, and prediction of response to therapy. Front Immunol 2019;10:305. + +132. Baraliakos X, Baerlecken N, Witte T, et al. High prevalence of antiCD74 antibodies specific for the HLA class II-associated invariant chain peptide (CLIP) in patients with axial spondyloarthritis. Ann Rheum Dis 2014;73:1079–82. + +133. De Winter JJ, van de Sande MG, Baerlecken N, et al. Anti-CD74 antibodies have no diagnostic value in early axial spondyloarthritis: data from the spondyloarthritis caught early (SPACE) cohort. Arthritis Res Ther 2018;20:38. + +134. Brown MA, Li Z, Cao KL. Biomarker development for axial spondyloarthritis. Nat Rev Rheumatol 2020;16:448–63. + +135. Brown MA, Xu H, Li Z. Genetics and the axial spondyloarthritis spectrum. Rheumatology (Oxford) 2020;59:iv58–66. + +136. Li Z, Wu X, Leo PJ, et al. Polygenic risk scores have high diagnostic capacity in ankylosing spondylitis. Ann Rheum Dis 2021;80: +1168–74. + +137. Zhang S, Wang Y, Peng L, et al. Comparison of clinical features in HLA-B27 positive and negative patients with axial spondyloarthritis: results from a cohort of 4,131 patients. Front Med (Lausanne) 2020;7:609562. + +138. Kim TJ, Kim TH. Clinical spectrum of ankylosing spondylitis in Korea. + +Joint Bone Spine 2010;77:235–40. + +139. Rudwaleit M, Claudepierre P, Wordsworth P, et al. Effectiveness, safety, and predictors of good clinical response in 1250 patients treated with adalimumab for active ankylosing spondylitis. + +J Rheumatol 2009;36:801–8. + +140. Arévalo M, Gratacos Masmitjà J, Moreno M, et al. In fluence of HLAB27 on the ankylosing spondylitis phenotype: results from the REGISPONSER database. Arthritis Res Ther 2018;20:221. + +141. Lautermann D, Braun J. Ankylosing spondylitis—cardiac manifestations. Clin Exp Rheumatol 2002;20:S11–5. + +142. Khan MA, Kushner I, Braun WE. Comparison of clinical features in HLA-B27 positive and negative patients with ankylosing spondylitis. + +Arthritis Rheum 1977;20:909–12. + +143. Yang M, Xu M, Pan X, et al. Epidemiological comparison of clinical manifestations according to HLA-B*27 carrier status of Chinese ankylosing spondylitis patients. Tissue Antigens 2013;82:338–43. + +144. Atagunduz P, Aydin SZ, Bahadir C, et al. Determinants of early radiographic progression in ankylosing spondylitis. J Rheumatol 2010;37: +2356–61. + +145. Kim TJ, Na KS, Lee HJ, et al. HLA-B27 homozygosity has no influence on clinical manifestations and functional disability in ankylosing spondylitis. Clin Exp Rheumatol 2009;27:574–9. + +146. Coates LC, Baraliakos X, Blanco FJ, et al. The phenotype of axial spondyloarthritis: is it dependent on HLA-B27 status? Arthritis Care Res (Hoboken) 2021;73:856–60. + +147. Ramiro S, Stolwijk C, van Tubergen A, et al. Evolution of radiographic damage in ankylosing spondylitis: a 12 year prospective follow-up of the OASIS study. Ann Rheum Dis 2015;74:52–9. + +148. Lim CS, Sengupta R, Gaffney K. The clinical utility of human leucocyte antigen B27 in axial spondyloarthritis. Rheumatology (Oxford) +2018;57:959–68. + +149. Rosenbaum JT, Weisman MH, Hamilton H, et al. HLA-B27 is associated with reduced disease activity in axial spondyloarthritis. Sci Rep 2021;11:12331. + +150. Vastesaeger N, van der Heijde D, Inman RD, et al. Predicting the outcome of ankylosing spondylitis therapy. Ann Rheum Dis 2011;70: +973–81. + +151. Rudwaleit M, Claudepierre P, Wordsworth P, et al. Effectiveness, safety, and predictors of good clinical response in 1250 patients treated with adalimumab for active ankylosing spondylitis. + +J Rheumatol 2009;36:801–8. + +152. Deodhar A, Mease P, Poddubnyy D, et al. Impact of HLA-B27 status on clinical outcomes among patients with ankylosing spondylitis treated with secukinumab. Ann Rheum Dis 2020;79:721. + +153. Deodhar A, van der Heijde D, Gensler LS, et al. Ixekizumab for patients with non-radiographic axial spondyloarthritis (COASTX): a randomised, placebo-controlled trial. Lancet 2020;395: +53–64. + +154. Marzo-Ortega H, Poddubnyy D, Pournara E, et al. Efficacy of secukinumab and HLA-B27 subtypes: results from a phase IIIb randomised controlled trial in axial SpA [Abstract]. Arthritis Rheumatol 2022;61:Suppl 1. \ No newline at end of file diff --git a/medical/md_images/PMC10349221/PMC10349221_meta.json b/medical/md_images/PMC10349221/PMC10349221_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..bb3cae10cd21f4f2c20c996bd2d62c88e1ef59af --- /dev/null +++ b/medical/md_images/PMC10349221/PMC10349221_meta.json @@ -0,0 +1,331 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Human Leukocyte Antigen B27-Negative Axial Spondyloarthritis: What Do We Know?", + "level": 0, + "is_closed": true, + "n_kids": 13, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 673.0, + 612.0, + 653.0 + ] + }, + { + "title": "Introduction", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 408.0, + 399.0, + 395.0 + ] + }, + { + "title": "Statement of literature search", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 169.0, + 612.0, + 156.0 + ] + }, + { + "title": "Discovery of HLA-B27 and its worldwide prevalence", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 306.0, + 399.0, + 293.0 + ] + }, + { + "title": "Pathogenesis of axSpA in HLA-B27-positive patients", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 366.0, + 612.0, + 353.0 + ] + }, + { + "title": "Pathogenesis of axSpA in HLA-B27-negative patients", + "level": 1, + "is_closed": true, + "n_kids": 1, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 652.0, + 399.0, + 639.0 + ] + }, + { + "title": "Outline placeholder", + "level": 2, + "is_closed": true, + "n_kids": 2, + "page_index": null, + "view_mode": "?", + "view_pos": [] + }, + { + "title": "Other HLA-B allotypes", + "level": 3, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 424.0, + 612.0, + 411.0 + ] + }, + { + "title": "Factors beyond HLA-B alleles in axSpA pathogenesis", + "level": 3, + "is_closed": true, + "n_kids": 5, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 203.0, + 399.0, + 190.0 + ] + }, + { + "title": "CARD9", + "level": 4, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 684.0, + 612.0, + 671.0 + ] + }, + { + "title": "NOD2", + "level": 4, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 411.0, + 612.0, + 398.0 + ] + }, + { + "title": "MIF and CD74", + "level": 4, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 60.0, + 612.0, + 47.0 + ] + }, + { + "title": "The gut microbiome and mycobiome", + "level": 4, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 346.0, + 399.0, + 333.0 + ] + }, + { + "title": "Interleukins", + "level": 4, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 359.0, + 612.0, + 346.0 + ] + }, + { + "title": "Challenges in diagnosis of axSpA in HLA-B27-negative patients", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 223.0, + 612.0, + 210.0 + ] + }, + { + "title": "Clinical features of HLA-B27-positive and -negative patients", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 5, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 639.0, + 612.0, + 626.0 + ] + }, + { + "title": "Treatment response among HLA-B27-positive and -negative patients", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 184.0, + 399.0, + 171.0 + ] + }, + { + "title": "Conclusions and future directions", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "FitR", + "view_pos": [ + 213.0, + 184.0, + 612.0, + 171.0 + ] + }, + { + "title": "ACKNOWLEDGMENTS", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 7, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 483.0, + 399.0, + 470.0 + ] + }, + { + "title": "AUTHOR CONTRIBUTIONS", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 7, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 364.0, + 399.0, + 351.0 + ] + }, + { + "title": "ROLE OF THE STUDY SPONSOR", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 7, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 299.0, + 399.0, + 286.0 + ] + }, + { + "title": "REFERENCES", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 7, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 213.0, + 399.0, + 200.0 + ] + } + ], + "pages": 12, + "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/PMC10440390/PMC10440390.md b/medical/md_images/PMC10440390/PMC10440390.md new file mode 100644 index 0000000000000000000000000000000000000000..836d59303d3ccf3a2bf0a6b63c5a12dbea440d4a --- /dev/null +++ b/medical/md_images/PMC10440390/PMC10440390.md @@ -0,0 +1,345 @@ +Contents lists available at ScienceDirect + +![0_image_0.png](0_image_0.png) + +# European Journal Of Radiology Open + +journal homepage: www.elsevier.com/locate/ejro Review + +![0_image_1.png](0_image_1.png) + +MRI features of intraductal papillary mucinous neoplasm of the bile ducts, +"The myth about the cyst": A systematic review Matan Kraus a,b,*, Eyal Klang a,b, Shelly Soffer c,d, Yael Inbar a,b, Eli Konen a,b, Tamer Sobeh a,b, Sara Apter a,b a Department of Diagnostic Imaging, Sheba Medical Center, Ramat Gan, Israel b Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel c Internal Medicine B, Assuta Medical Center, Ashdod, Israel d *Ben-Gurion University of the Negev, Be'er Sheva, Israel* HIGHLIGHTS +- The entity of IPMN-B, including its MRI features are poorly known. - Distinguishing signs between IPMN-B and other hepatic or biliary cystic lesions or intraductal cholangiocarcinoma lack consensus. - Radiologists should recognize IPMN-B, as early detection of its malignant transformation improves prognosis. + +ARTICLE INFO + +| Keywords: Biliary tract IPMN IPNB Cystic tumors Mucinous tumors | +|------------------------------------------------------------------------| + +## Abstract + +Rationale and objectives: Intraductal papillary mucinous neoplasm of the bile ducts (IPMN-B) is a true precancerous lesion, which shares common features with pancreatic IPMN (IPMN-P). While IPMN-P is a well described entity for which guidelines were formulated and revised, IPMN-B is a poorly described entity. We carried out a systematic review to evaluate the existing literature, emphasizing the role of MRI in IPMN-B depiction. + +Materials and methods: PubMed database was used to identify original studies and case series that reported MR +Imaging features of IPMN-B. The search keywords were "IPMN OR intraductal papillary mucinous neoplasm OR +IPNB OR intraductal papillary neoplasm of the bile duct AND Biliary OR biliary cancer OR hepatic cystic lesions". + +Risk of bias and applicability were evaluated using the QUADAS-2 tool. + +Results: 884 Records were Identified through database searching. 12 studies satisfied the inclusion criteria, resulting in MR features of 288 patients. All the studies were retrospective. Classic features of IPMN-B are underdescribed. Few studies note worrisome features, concerning for an underlying malignancy. 50 % of the studies had a high risk of bias and concerns regarding applicability. + +Conclusions: The MRI features of IPMN-B are not well elaborated and need to be further studied. Worrisome features and guidelines regarding reporting the imaging findings should be established and published. Radiologists should be aware of IPMN-B, since malignancy diagnosis in an early stage will yield improved prognosis 1. **Introduction** +Intraductal papillary mucinous neoplasm of the bile ducts (IPMN-B) +is a mucin producing cystic epithelial tumor. IPMN-B is characterized by intraluminal papillary ductal growth, communication with the biliary ducts, as well as intrahepatic and extrahepatic biliary ducts dilatation Abbreviations: IPMN-B, Intraductal papillary mucinous neoplasm of the bile ducts; IPMN-P, Intraductal papillary mucinous neoplasm of the pancreas; PRISMA, +Preferred Reporting Items for Systematic Reviews and Meta-Analyses. + +* Correspondence to: Department of Diagnostic Imaging, Sheba Medical Center, 2 Sheba Road, Ramat Gan ZIP 5262000, Israel. + +E-mail addresses: Matan.Kraus@gmail.com, Matan.Kraus@sheba.health.gov.il (M. Kraus), Eyal.Klang@sheba.health.gov.il (E. Klang), Shellyso@assuta.co.il +(S. Soffer), Yael.Inbar@sheba.health.gov.il (Y. Inbar), Eli.Konen@sheba.health.gov.il (E. Konen), Tamer.Sobeh@gmail.com (T. Sobeh), Sara.Apter@sheba.health. + +gov.il (S. Apter). https://doi.org/10.1016/j.ejro.2023.100515 Received 17 May 2023; Received in revised form 3 July 2023; Accepted 7 August 2023 Available online 12 August 2023 2352-0477/© 2023 The Authors. 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/). + +## 2 + +due to mucin hypersecretion [1–3]. The imaging appearance of IPMN-B +varies, and depends on the equilibrium between the degree of intraductal proliferation and mucin hypersecretion. Predominant intraductal proliferation would appear on imaging as an intraductal mass, accompanied by a mostly upstream biliary dilatation. Substantial mucin production would manifest on imaging as diffuse dilatation of the bile ducts with no detectable mass [3]. + +On T1-weighted MRI, IPMN-B appears as hypointense or isointense, and on T2-weighted MR images as hyperintense. IPMN-B commonly presents as an intraductal mass, characterized by proximal and, at times, distal biliary dilatation [3]. At MRCP, in some cases, the intraluminal mucin appears as stringlike filling defects, known as the "Thread sign" +[4,5]. Usual enhancement pattern consists of isointensity or hyperintensity during the late arterial phase, with no remaining hyperintensity in the portal venous and late phases [6]. Persistent and progressive enhancement pattern in the portal venous and late phases is suspicious malignant intraductal cholangiocarcinoma [7]. + +Malignant IPMN-B typically present a clearly visible mass within the duct on imaging [8]. Approximately 28 % of cases exhibit evident invasion beyond the duct, while involvement of neighboring organs is observed in up to 8 % of cases [9]. Diffusion-weighted MRI enhances the visibility of tumors and has the potential to reveal their invasiveness, but it does not accurately depict the full extent of the tumor [10]. + +IPMN-B is considered to be analogous to the intraductal papillary mucinous neoplasm of the pancreas (IPMN-P); since both entities manifest with intraductal growth and excessive mucin production [3,7,11, 12]. IPMN-P is well described and diagnostic criteria and guidelines have been formulated and revised. Moreover, "high-risk stigmata" and "worrisome features" have been published and widely used in clinical practice to detect malignant transformation and guide treatment [13, 14]. + +IPMN-P is widely recognized while IPMN-B is still relatively an under-described entity [15,16]. It has been argued that invasive carcinoma and malignancy are more frequently associated with IPMN-B than with IPMN-P [17]. Hence, it is important to diagnose IPMN-B early since it is a precursor lesion to a worse-prognosis biliary cholangiocarcinoma [16,18,19]. Unfortunately, currently, no guidelines exist regarding IPMN-B malignancy prediction or structured radiologic reporting. + +In this study, we carried out a systematic review to evaluate the literature emphasizing the role of MRI in IPMN-B depiction and assessment. + +## 2. **Materials And Methods** 2.1. Literature Search And Eligibility Criteria + +The systematic review was conducted with adherence to the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [20]. + +We searched the PubMed database for relevant studies. The search keywords were "IPMN OR intraductal papillary mucinous neoplasm OR +IPNB OR intraductal papillary neoplasm of the bile duct AND Biliary OR +biliary cancer OR hepatic cystic lesion". Bibliographies of included studies were searched for additional relevant studies. The search included studies published from August 1975 up to September 23, 2022. Inclusion criteria were studies that (a) evaluated MR Imaging features of IPMN-B, (b) were published in English, and (c) were Original Articles or Case Series. Abstracts and case reports were excluded. We reviewed the bibliographies of the included studies for relevant studies as well. + +The study was registered with PROSPERO, the prospective international register of systematic reviews (identification number CRD42022300610). + +2.2. *Data extraction and analysis* Data of all included studies were collected into a standardized data extraction sheet. The data sheet included: year of publication, journal name, journal's medical specialty, study design, size of the database. + +Data from all included studies were collected into a standardized data extraction sheet as well. The data sheet included: Biliary Dilatation (intrahepatic/extrahepatic, downstream/upstream), Intraluminal mass, the presence of the "Thread sign", cystic communication with the biliary tract, biliary stones, pancreatic duct dilatation, Intrahepatic cystic lesion, worrisome features, MRI diffusion series. + +The Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) +criteria were used to assess the risk of bias and applicability [21]. + +## 3. **Results** 3.1. Included Studies And Reported Mr Features + +Of 884 retrieved studies, 12 studies [4,5,10,22–30] satisfied the inclusion criteria, and included MR features of 288 patients. Flow diagram of the search and inclusion process is presented in Fig. 1. + +The studies were all retrospective, and published between 2008 and 2020. The studies were published in journals with different affiliations, most prevalent was Radiology (8/12, 66.7 %), followed by Gastroenterology (3/12, 25.0 %) and Surgery (1/12, 8.3 %). + +Characteristics of the included studies are summarized in Table 1. Biliary dilatation was described in 8/12 (66.7 %) of the studies [4, 10,23,26–30], and only 2 studies [27,28] distinguished between intrahepatic or extrahepatic biliary dilatation. + +The presence or absence of an intra-ductal luminal mass was described in 8/12 (66.7 %) of the studies [4,5,23–27,30]. The "Thread Sign" was described in only one study [5]. + +Differentiation of cyst morphology was described in 3/12 (25.0 %) of the studies [22,24,28]. + +Cystic communication with the biliary tract was mentioned in 3/12 +(25.0 %) [22,23,26]. + +The presence of biliary stones or pancreatic dilatation were not mentioned. + +The included studies report use of different field strengths and various MRI sequences. + +4 Studies (33.3 %) [10,23,27,30] used a 3.0-Tesla system. Yet, Lim et al. [30] reported that the MRI technique for 1 patient was unknown, being performed outside of the author's institution. + +4 Studies (33.3 %) [4,5,25,28] used a 1.5-Tesla system, and 1 study +[24] reported that scans were made in either 1.5-Tesla or 3.0-Tesla system. + +3 studies (25 %) [22,26,29] did not elaborate regarding the MRI field strength. + +Description of using MR diffusion series in the scanning technique was described in 6 studies [5,10,23–25,28]. + +4 studies [10,23–25] reported signs differentiating IPMN-B from other hepatic or biliary cystic lesions or from intraductal cholangiocarcinoma. + +One study [22] described that all (4/4, 100 %) IPMN-B cases were misdiagnosed prior to the pathology result. The other 11 studies did not mention if IPMN-B was diagnosed using imaging prior to the pathology result [4,5,10,23–30]. + +The radiological features are summarized in Table 2. + +## 3.2. Quality Assessment + +High risk of bias was ascribed to studies in which the interpreting radiologists were not blinded to the pathological diagnosis of IPMN-B. + +Studies that did not have MRI scans for all pathologically confirmed IPMN-B, therefore effectively excluding patients, were also deemed high risk of bias. + +The QUADAS-2 tool identified high risk of bias and concerns regarding applicability 6/12 (50.0 %) of the studies, respectively. + +Complete QUADAS-2 result are presented in Table 3. + +![2_image_0.png](2_image_0.png) + +Fig. 1. Flow diagram of the search and inclusion process. The study was reported according to the Preferred Reporting Items for Systematic Reviews and MetaAnalyses guidelines. + +| Characteristics of the studies included in the qualitative synthesis. Study Publication year Journal's medical affiliation | Study design | Database size | Pathological correlation | | | +|-------------------------------------------------------------------------------------------------------------------------------|----------------|------------------|----------------------------|-----|-----| +| Frezin et al. [22] | 2020 | Surgery | Retrospective | 4 | Yes | +| Siripongsakun et al. [23] | 2019 | Radiology | Retrospective | 8 | Yes | +| Lee et al. [24] | 2019 | Gastroenterology | Retrospective | 120 | Yes | +| Jin et al. [25] | 2019 | Radiology | Retrospective | 52 | Yes | +| Wu et al. [4] | 2017 | Radiology | Retrospective | 15 | Yes | +| Mondal et al. [26] | 2016 | Radiology | Retrospective | 6 | Yes | +| Hong et al. [5] | 2016 | Radiology | Retrospective | 38 | Yes | +| Ying et al. [27] | 2015 | Gastroenterology | Retrospective | 5 | Yes | +| Yoon et al. [10] | 2013 | Radiology | Retrospective | 23 | Yes | +| Lim et al. [28] | 2011 | Radiology | Retrospective | 3 | Yes | +| Li et al. [29] | 2009 | Gastroenterology | Retrospective | 10 | Yes | +| Lim et al. [30] | 2008 | Radiology | Retrospective | 4 | Yes | + +| Table 2 MR radiological features reported in the included studies. Study Biliary dilatation | Biliary dilatation | Thread Sign | Intra-luminal | | | | | +|-------------------------------------------------------------------------------------------------|----------------------|-------------------------|-----------------|------------|----------------|----------------------|---------| +| Yes | Intraheaptic | Extraheaptic | Upstream | Downstream | Mass | | | +| Frezin et al. [22] | NR | NR | NR | NR | NR | NR | NR | +| Siripongsakun et al. [23] | 87.5 % | NR | NR | NR | NR | NR | 62.5 % | +| Lee et al. [24] | NR | NR | NR | NR | NR | NR | 85.0 % | +| Jin et al. [25] | NR | NR | NR | NR | NR | NR | 26.8 % | +| Wu et al. [4] | NR | NR | NR | 100.0 % | 60.00 % | NR | 100.0 % | +| Mondal et al. [26] | 66.7 % | NR | NR | NR | NR | NR | 66.7 % | +| Hong et al. [5] | NR | NR | NR | NR | NR | 44.7–52.6 % | 50.0 % | +| Ying et al. [27] | 80.0 % | 80.0 % | 80.0 % | 100.0 % | 100.0 % | NR | 100.0 % | +| Yoon et al. [10] | 100.0 % | NR | NR | NR | NR | NR | NR | +| Lim et al. [28] | 100.0 % | 100.0 % | 66.7 % | NR | 66.7 % | NR | NR | +| Li et al. [29] | 70.0 % | NR | NR | NR | NR | NR | NR | +| Lim et al. [30] | 100.0 % | NR | NR | NR | NR | NR | 0.0 % | +| Study | Single cyst | Single cyst | Multiple cysts | Septations | Calcifications | Cystic communication | | +| Unilocular | Multilocular | with the biliary tracts | | | | | | +| Frezin et al. [22] | 75.0 % | NR | 25.0 % | 50.0 % | 25.0 % | 50.0 % | | +| Siripongsakun et al. [23] | NR | NR | NR | NR | NR | 75.0 % | | +| Lee et al. [24] | NR | NR | 40.8 % | NR | NR | NR | | +| Jin et al. [25] | NR | NR | NR | NR | NR | NR | | +| Wu et al. [4] | NR | NR | NR | NR | 0.0 % | NR | | +| Mondal et al. [26] | NR | NR | NR | NR | NR | 16.7 % | | +| Hong et al. [5] | NR | NR | NR | NR | NR | NR | | +| Ying et al. [27] | NR | NR | NR | NR | NR | NR | | +| Yoon et al. [10] | NR | NR | NR | NR | NR | NR | | +| Lim et al. [28] | NR | 33.3 % | 33.3 % | NR | NR | NR | | +| Li et al. [29] | NR | NR | NR | NR | NR | NR | | +| Lim et al. [30] | NR | NR | NR | NR | NR | NR | | +| NR - Not Reported. | | | | | | | | + +| Table 3 Methodological analysis of the included studies based on the QUADAS-2 tool. Study Risk of bias | Applicability | | | | | | | +|------------------------------------------------------------------------------------------------------------|-----------------|--------------------|-----------------|-------------------|------------|--------------------|-----| +| Patient selection | Index test | Reference standard | Flow and timing | Patient selection | Index test | Reference standard | | +| Frezin et al. [22] | Low | Low | Unclear | Low | Low | High | Low | +| Siripongsakun et al. [23] | Low | Low | Unclear | Low | High | Low | Low | +| Lee et al. [24] | Low | Low | High | Low | Low | Low | Low | +| Jin et al. [25] | Low | Low | Low | Low | Low | Low | Low | +| Wu et al. [4] | Low | Low | Low | Low | Low | Unclear | Low | +| Mondal et al. [26] | High | Low | High | Low | High | High | Low | +| Hong et al. [5] | Low | Low | Low | Low | Low | Low | Low | +| Ying et al. [27] | Low | Low | Unclear | Low | Low | Low | Low | +| Yoon et al. [10] | Low | Low | High | Low | Low | Low | Low | +| Lim et al. [28] | High | Low | High | Low | High | Unclear | Low | +| Li et al. [29] | High | Low | Unclear | Low | Low | High | Low | +| Lim et al. [30] | High | Low | Unclear | Low | High | Unclear | Low | + +## 4. **Discussion** 4.1. Classic Mri Features + +In this systematic review we have found that there are only several original articles or case series that inquired about the role of MR imaging in IPMN-B diagnosis and assessment. The available data lacks description of the classic MR findings of IPMN-B, including presence of an intraluminal biliary mass, biliary dilatation or differentiation between intrahepatic or extra-hepatic biliary duct involvement. Additionally, the +"Thread-sign", which is almost pathognomonic to IPMN-B alone, is not mentioned in the majority of studies. + +We speculate that the diversity in reporting derives from decreased familiarity, which in turn leads to a non-uniform report. + +Signs differentiating IPMN-B from other hepatic or biliary cystic lesions or were not well elaborated. Alongside, the majority of the studies did not go into detail regarding the assumed diagnosis of the IPMN-B +cases prior to the final pathology result. The only study that reported the assumed diagnosis based on imaging had a misdiagnosis rate of 100 % [22]. In our opinion, this further highlights the need to increase the awareness of radiologists to this important entity. + +## 4.2. Invasiveness And Worrisome Features + +Some studies did address the issue of IPMN-B which is suspicious for malignant transformation, or the presence of intraductal cholangiocarcinoma. Siripongsakun et al. [23] summarized that findings such as intraductal nodules and connection to the biliary tracts may help differentiate between IPMN-B and biliary intraepithelial neoplasia. + +Lee et al. [24] described intraductal visible mass, tumor size +≥ 2.5 cm, multiplicity of the tumor, bile duct wall thickening, adjacent organ invasion, as significant MRI findings, suspicious for IPMN-B with an associated invasive carcinoma. + +Jin et al. [25] suggested that using ADC histogram analysis provides a fair competence for prediction of IPMN-B invasiveness. Yoon et al. + +[10] reported that the diffusion series may be beneficial in improving tumor conspicuity and therefore helpful in defining IPMN-B invasiveness. + +These features may serve as the basis for establishing the "high-risk stigmata" and "worrisome features" for IPMN-B. + +IPMN-B is treated mainly by hepatic resection when possible, as this neoplasm commonly harbors high-grade dysplasia and invasive carcinoma components [31]. + +Also, the prognosis is better following complete resection [32]. + +Luvira et al. [33] examined the long-term outcomes and survival of IPMN-B patients that underwent curative-intent hepatic resection surgery. They found that lesser level of invasiveness was associated with better patient prognosis. + +We believe this further validates the importance of IPMN-B early detection. + +## 4.3. Limitations + +Our review has several limitations. We focused on including case series with a minimum of 3 patients, although there were more individual case reports available. We think that case series offer a standardized approach to patient management and outcome assessment, which leads to data robustness and consistency. While individual case reports may exhibit greater variability in their approach and data presentation, despite their larger cumulative number. + +We reviewed the bibliographies of the included studies, to ensure that we captured the most directly relevant sources of evidence. + +Eventually, only a small cohort of studies were suitable to analysis, due to the scarce literature available and the exclusion criteria. + +Furthermore, all studies included were retrospective, and were accompanied by a high risk of suspected bias and concerns regarding applicability. These limitations may hazard both precision and pertinence of the results. + +## 4.4. Conclusions + +The existing literature describes MRI features of IPMN-B, but without adequate elaboration or consensus on a reporting template. Certain MRI characteristics could potentially define the "high-risk stigmata" and +"worrisome features" for IPMN-B, yet their application remains limited. + +We advocate for more comprehensive studies on IPMN-B's MRI features. + +Concurrently, it's crucial to establish and disseminate definitive guidelines on reporting these imaging findings. Familiarity with IPMN-B +among radiologists can facilitate early diagnosis and improved prognosis. We propose a structured report template for IPMN-B MRI findings, available in our Supplementary material. + +## Funding Statement + +This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. + +## Ethical Statement Not Applicable. Credit Authorship Contribution Statement + +Sobeh Tamer: Writing - review & editing. **Sara Apter:** Writing - +review & editing, Methodology, Formal analysis, Conceptualization. Yael Inbar: Writing - review & editing. **Eli Konen:** Writing - review & editing. **Eyal Klang:** Writing - review & editing, Methodology, Formal analysis, Conceptualization. **Shelly Soffer:** Writing - review & editing, Visualization, Validation, Formal analysis. **Matan Kraus:** Writing - +original draft, Methodology, Investigation, Data curation, Conceptualization. + +## 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. + +## Appendix A. **Supporting Information** + +Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ejro.2023.100515. + +## References + +[1] T.C. Chen, Y. Nakanuma, Y. Zen, et al., Intraductal papillary neoplasia of the liver associated with hepatolithiasis, Hepatology 34 (4 Pt 1) (2001) 651–658. + +[2] Y. Zen, F. Pedica, V.R. Patcha, et al., Mucinous cystic neoplasms of the liver: a clinicopathological study and comparison with intraductal papillary neoplasms of the bile duct, Mod. Pathol. 24 (2011) 1079–1089. + +[3] A. Chatterjee, C. Lopes Vendrami, P. Nikolaidis, et al., Uncommon intraluminal tumors of the gallbladder and biliary tract: spectrum of imaging appearances, Radiographics 39 (2019) 388–412. + +[4] C.-H. Wu, Y.-C. Yeh, Y.-C. Tsuei, et al., Comparative radiological pathological study of biliary intraductal tubulopapillary neoplasm and biliary intraductal papillary mucinous neoplasm, Abdom. Radiol. 42 (2017) 2460–2469. + +[5] G.-S. Hong, J.H. Byun, J.H. Kim, et al., Thread sign in biliary intraductal papillary mucinous neoplasm: a novel specific finding for MRI, Eur. Radiol. 26 (2016) +3112–3120. + +[6] H. Ogawa, S. Itoh, T. Nagasaka, K. Suzuki, T. Ota, S. Naganawa, CT findings of intraductal papillary neoplasm of the bile duct: assessment with multiphase contrast-enhanced examination using multi-detector CT, Clin. Radiol. 67 (2012) +224–231. + +[7] X.-S. Wan, Y.-Y. Xu, J.-Y. Qian, et al., Intraductal papillary neoplasm of the bile duct, World J. Gastroenterol. 19 (2013) 8595–8604. + +[8] K. Takanami, T. Yamada, M. Tsuda, et al., Intraductal papillary mucininous neoplasm of the bile ducts: multimodality assessment with pathologic correlation, Abdom. Imaging 36 (2011) 447–456. + +[9] A.N. Gordon-Weeks, K. Jones, E. Harriss, A. Smith, M. Silva, Systematic review and meta-analysis of current experience in treating IPNB: clinical and pathological correlates, Ann. Surg. 263 (2016) 656–663. + +[10] H.J. Yoon, Y.K. Kim, K.-T. Jang, et al., Intraductal papillary neoplasm of the bile ducts: description of MRI and added value of diffusion-weighted MRI, Abdom. + +Imaging 38 (2013) 1082–1090. + +[11] S. Ainechi, H. Lee, Updates on precancerous lesions of the biliary tract: biliary precancerous lesion, Arch. Pathol. Lab. Med. 140 (2016) 1285–1289. + +[12] S. Bennett, E.C. Marginean, M. Paquin-Gobeil, et al., Clinical and pathological features of intraductal papillary neoplasm of the biliary tract and gallbladder, HPB +17 (2015) 811–818. + +[13] M. Tanaka, International consensus on the management of intraductal papillary mucinous neoplasm of the pancreas, Ann. Transl. Med. 3 (2015) 286. + +[14] M. Tanaka, C. Fern´andez-Del Castillo, T. Kamisawa, et al., Revisions of international consensus Fukuoka guidelines for the management of IPMN of the pancreas, Pancreatology 17 (2017) 738–753. + +[15] Y. Sakai, M. Ohtsuka, H. Sugiyama, et al., Current status of diagnosis and therapy for intraductal papillary neoplasm of the bile duct, World J. Gastroenterol. 27 +(2021) 1569–1577. + +[16] A. Adibi, N. Shabanikia, A. Taheri, Intraductal papillary mucinous neoplasm of biliary ducts: literature review and a case report with emphasis on radiological manifestation, J. Res. Med. Sci. 25 (2020) 114. + +[17] N. Minagawa, N. Sato, Y. Mori, T. Tamura, A. Higure, K. Yamaguchi, A comparison between intraductal papillary neoplasms of the biliary tract (BT-IPMNs) and intraductal papillary mucinous neoplasms of the pancreas (P-IPMNs) reveals distinct clinical manifestations and outcomes, Eur. J. Surg. Oncol. 39 (2013) +554–558. + +[18] S.S. Lee, M.-H. Kim, S.K. Lee, et al., Clinicopathologic review of 58 patients with biliary papillomatosis, Cancer 100 (2004) 783–793. + +[19] Y.P. Yeung, K. AhChong, C.K. Chung, A.Y.W. Chun, Biliary papillomatosis: report of seven cases and review of English literature, J. Hepatobiliary Pancreat. Surg. 10 +(2003) 390–395. + +[20] D. Moher, A. Liberati, J. Tetzlaff, D.G. Altman, Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement, Ann. Intern. Med. + +151 (2009) 264–269. W64. + +[21] P.F. Whiting, A.W.S. Rutjes, M.E. Westwood, et al., QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy studies, Ann. Intern. Med. 155 +(2011) 529–536. + +[22] J. Frezin, M. Komuta, F. Zech, et al., Mucin-producing hepatic cystic neoplasms: an uncommon but challenging disease often misdiagnosed and mismanaged, Acta Chir. Belg. 120 (2020) 6–15. + +[23] S. Siripongsakun, W. Sapthanakorn, P. Mekraksakit, et al., Premalignant lesions of cholangiocarcinoma: characteristics on ultrasonography and MRI, Abdom. Radiol. + +44 (2019) 2133–2146. + +5 + +## + +[24] S. Lee, M.-J. Kim, S. Kim, D. Choi, K.-T. Jang, Y.N. Park, Intraductal papillary neoplasm of the bile duct: assessment of invasive carcinoma and long-term outcomes using MRI, J. Hepatol. 70 (2019) 692–699. + +[25] K.-P. Jin, S.-X. Rao, R.-F. Sheng, M.-S. Zeng, Skewness of apparent diffusion coefficient (ADC) histogram helps predict the invasive potential of intraductal papillary neoplasms of the bile ducts (IPNBs), Abdom. Radiol. 44 (2019) 95–103. + +[26] D. Mondal, M.A. Silva, Z. Soonawalla, L.M. Wang, H.K. Bungay, Intraductal papillary neoplasm of the bile duct (IPN-B): also a disease of western Caucasian patients. A literature review and case series, Clin. Radiol. 71 (2016) e79–e87. + +[27] S.-H. Ying, X.-D. Teng, Z.-M. Wang, et al., Gd-EOB-DTPA-enhanced magnetic resonance imaging for bile duct intraductal papillary mucinous neoplasms, World J. Gastroenterol. 21 (2015) 7824–7833. + +[28] J.H. Lim, Y. Zen, K.T. Jang, Y.K. Kim, Y. Nakanuma, Cyst-forming intraductal papillary neoplasm of the bile ducts: description of imaging and pathologic aspects, AJR Am. J. Roentgenol. 197 (2011) 1111–1120. + +[29] T. Li, Y. Ji, X.-T. Zhi, et al., A comparison of hepatic mucinous cystic neoplasms with biliary intraductal papillary neoplasms, Clin. Gastroenterol. Hepatol. 7 (2009) +586–593. + +[30] J.H. Lim, K.-T. Jang, D. Choi, Biliary intraductal papillary-mucinous neoplasm manifesting only as dilatation of the hepatic lobar or segmental bile ducts: imaging features in six patients, AJR Am. J. Roentgenol. 191 (2008) 778–782. + +[31] K. Ozcan, D.S. Klimstra, A review of mucinous cystic and intraductal neoplasms of the pancreatobiliary tract, Arch. Pathol. Lab. Med. 146 (2022) 298–311. + +[32] F.G. Rocha, H. Lee, N. Katabi, et al., Intraductal papillary neoplasm of the bile duct: +a biliary equivalent to intraductal papillary mucinous neoplasm of the pancreas? + +Hepatology 56 (2012) 1352–1360. + +[33] V. Luvira, A. Pugkhem, V. Bhudhisawasdi, et al., Long-term outcome of surgical resection for intraductal papillary neoplasm of the bile duct, J. Gastroenterol. + +Hepatol. 32 (2017) 527–533. \ No newline at end of file diff --git a/medical/md_images/PMC10440390/PMC10440390_meta.json b/medical/md_images/PMC10440390/PMC10440390_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..8ebd528d4887325bdbce0fa5815273f92add45b9 --- /dev/null +++ b/medical/md_images/PMC10440390/PMC10440390_meta.json @@ -0,0 +1,275 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "MRI features of intraductal papillary mucinous neoplasm of the bile ducts, \u201cThe myth about the cyst\u201d: A systematic review", + "level": 0, + "is_closed": false, + "n_kids": 18, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 629.5350341796875, + 0.0 + ] + }, + { + "title": "1 Introduction", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 218.03399658203125, + 0.0 + ] + }, + { + "title": "2 Materials and methods", + "level": 1, + "is_closed": false, + "n_kids": 2, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 301.2359924316406, + 0.0 + ] + }, + { + "title": "2.1 Literature search and eligibility criteria", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 280.31500244140625, + 0.0 + ] + }, + { + "title": "2.2 Data extraction and analysis", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 81.04199981689453, + 0.0 + ] + }, + { + "title": "3 Results", + "level": 1, + "is_closed": false, + "n_kids": 2, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 306.60400390625, + 625.5180053710938, + 0.0 + ] + }, + { + "title": "3.1 Included studies and reported MR features", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 306.60400390625, + 604.5960083007812, + 0.0 + ] + }, + { + "title": "3.2 Quality assessment", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "XYZ", + "view_pos": [ + 306.60400390625, + 154.26699829101562, + 0.0 + ] + }, + { + "title": "4 Discussion", + "level": 1, + "is_closed": false, + "n_kids": 4, + "page_index": 3, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 255.3070068359375, + 0.0 + ] + }, + { + "title": "4.1 Classic MRI features", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 234.38600158691406, + 0.0 + ] + }, + { + "title": "4.2 Invasiveness and worrisome features", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "XYZ", + "view_pos": [ + 306.60400390625, + 217.031005859375, + 0.0 + ] + }, + { + "title": "4.3 Limitations", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 592.9080200195312, + 0.0 + ] + }, + { + "title": "4.4 Conclusions", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 392.803955078125, + 0.0 + ] + }, + { + "title": "Funding Statement", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 254.01400756835938, + 0.0 + ] + }, + { + "title": "Ethical Statement", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 198.9099884033203, + 0.0 + ] + }, + { + "title": "CRediT authorship contribution statement", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 37.61289596557617, + 154.26699829101562, + 0.0 + ] + }, + { + "title": "Declaration of Competing Interest", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 306.60400390625, + 740.708984375, + 0.0 + ] + }, + { + "title": "Appendix A Supporting information", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 306.60400390625, + 677.9450073242188, + 0.0 + ] + }, + { + "title": "References", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "XYZ", + "view_pos": [ + 306.60400390625, + 625.6409301757812, + 0.0 + ] + } + ], + "pages": 6, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 11, + "code": 0, + "table": 4, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC10514721/PMC10514721.md b/medical/md_images/PMC10514721/PMC10514721.md new file mode 100644 index 0000000000000000000000000000000000000000..8ae9f05498fa80442de01842310880ae41e857fb --- /dev/null +++ b/medical/md_images/PMC10514721/PMC10514721.md @@ -0,0 +1,299 @@ +World Journal of + +![0_image_0.png](0_image_0.png) + +Gastrointestinal W J G O *Oncology* +Submit a Manuscript: https://www.f6publishing.com *World J Gastrointest Oncol* 2023 September 15; 15(9): 1595-1604 DOI: 10.4251/wjgo.v15.i9.1595 ISSN 1948-5204 (online) +ORIGINAL ARTICLE + +![0_image_1.png](0_image_1.png) + +Case Control Study Comparison of ethanol-soaked gelatin sponge and microspheres for hepatic arterioportal fistulas embolization in hepatic cellular carcinoma Guang-Sheng Yuan, Li-Li Zhang, Zi-Tong Chen, Cun-Jing Zhang, Shu-Hui Tian, Ming-Xia Gong, Peng Wang, Lei Guo, Nan Shao, Bin Liu + +| Guang-Sheng Yuan, Ming-Xia Gong, Nan Shao, Institute of Interventional Oncology, Shandong University, Jinan 250033, Shandong Province, China; Department of Radiology, Dongying Traditional Chinese Medicine Hospital, Dongying 257055, Shandong Province, China | | +|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| +| Specialty type: Oncology Provenance and peer review: Unsolicited article; Externally peer reviewed. | Li-Li Zhang, Department of Gastroenterology, People's Hospital of Qihe County, Dezhou 251100, Shandong Province, China | +| Peer-review model: Single-blind | Zi-Tong Chen, Shu-Hui Tian, Bin Liu, Department of Interventional Medicine, The Second Hospital of Shandong University, Jinan 250033, Shandong Province, China; Institute of Interventional Oncology, Shandong University, Jinan 250033, Shandong Province, China | +| Peer-review report's scientific quality classification Grade A (Excellent): A Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 | Cun-Jing Zhang, Dean's Office, Jinan Vocational College of Nursing, Jinan 250100, Shandong Province, China Peng Wang, Department of Interventional Medicine, People's Hospital of Zouping City, Binzhou 256299, Shandong Province, China | +| P-Reviewer: Lykoudis PM, United Kingdom; Ueda H, Japan | Lei Guo, Department of Vascular Anomalies and Interventional Radiology, Qilu Children's Hospital of Shandong University, Jinan 250022, Shandong Province, China | +| Received: June 1, 2023 Peer-review started: June 1, 2023 First decision: July 17, 2023 Revised: July 24, 2023 Accepted: August 18, 2023 Article in press: August 18, 2023 Published online: September 15, 2023 | Corresponding author: Bin Liu, MD, Professor, Researcher, Department of Interventional Medicine, The Second Hospital of Shandong University, No. 247 Beiyuan Street, Jinan 250033, Shandong Province, China. gordon0221@sdu.edu.cn Abstract BACKGROUND Hepatic arterioportal fistulas (APFs) are common in hepatocellular carcinoma (HCC). Moreover, correlated with poor prognosis, APFs often complicate antitumor treatments, including transarterial chemoembolization (TACE). AIM To compare the efficacy of ethanol-soaked gelatin sponges (ESG) and microspheres in the management of APFs and their impact on the prognosis of HCC. METHODS | + +Data from patients diagnosed with HCC or hepatic APFs between June 2016 and December 2019 were retrospectively analyzed. Furthermore, APFs were embolized with ESG (group E) or microspheres (group M) during TACE. The primary outcomes were disease control rate (DCR) and objective response rate (ORR). The secondary outcomes included immediate and first follow-up APF improvement, overall survival (OS), and progression-free survival (PFS). + +## Results + +Altogether, 91 participants were enrolled in the study, comprising 46 in group E and 45 in group M. The DCR was 93.5% and 91.1% in groups E and M, respectively (P = 0.714). The ORRs were 91.3% and 66.7% in groups E and M, respectively (P = 0.004). The APFs improved immediately after the procedure in 43 (93.5%) patients in group E and 40 (88.9%) patients in group M (P = 0.485). After 2 mo, APF improvement was achieved in 37 (80.4%) and 33 +(73.3%) participants in groups E and M, respectively (P = 0.421). The OS was 26.2 ± 1.4 and 20.6 ± 1.1 mo in groups E and M, respectively (P = 0.004), whereas the PFS was 16.6 ± 1.0 and 13.8 ± 0.7 mo in groups E and M, respectively (P = 0.012). + +## Conclusion + +Compared with microspheres, ESG embolization demonstrated a higher ORR and longer OS and PFS in patients of HCC with hepatic APFs. + +Key Words: Hepatocellular carcinoma; Arterioportal fistula; Ethanol; Gelatin sponge; Microsphere; Embolization +©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. + +Core **Tip:** Hepatocellular carcinoma (HCC) was considered the seventh most common cancer and the second leading cause of cancer-related deaths worldwide in 2020. Hepatic arterioportal fistulas (APFs) are common in HCC and often complicate anti-tumor treatments, including transarterial chemoembolization. The ethanol-soaked gelatin sponge combined the advantages of alcohol and gelatin sponges, contributed to better local control of hepatic APFs, and improved the survival of patients with HCC. + +Citation: Yuan GS, Zhang LL, Chen ZT, Zhang CJ, Tian SH, Gong MX, Wang P, Guo L, Shao N, Liu B. Comparison of ethanolsoaked gelatin sponge and microspheres for hepatic arterioportal fistulas embolization in hepatic cellular carcinoma. World J +Gastrointest Oncol 2023; 15(9): 1595-1604 URL: https://www.wjgnet.com/1948-5204/full/v15/i9/1595.htm DOI: https://dx.doi.org/10.4251/wjgo.v15.i9.1595 + +## Introduction + +Hepatocellular carcinoma (HCC) was the seventh most common cancer and the second leading cause of cancer-related deaths worldwide in 2020, with 905677 new cases and 830180 deaths recorded annually[1]. Hepatic arterioportal fistulas (APFs), defined as fistulas between the hepatic artery and the neighboring portal vein[2,3], are common in HCC owing to tumor infiltration, vascular damage[4], or remodeling of the cirrhotic parenchyma. + +Hepatic APFs may cause portal hypertension, ascites, and varices[5], which are strongly associated with poor prognosis[6]. The presence of hepatic APFs often complicates anti-tumor treatments, including transarterial chemoembolization (TACE). Chemotherapeutic agents and embolic materials run off through the fistulas, and tumor cells may detach from the hepatic artery, resulting in portal vein thrombosis[7]. + +Many materials have been used to treat hepatic APFs, including gelatin sponges[8], microspheres[9], coils[10], +histoacryl[10], absolute ethanol[10], polyvinyl alcohol particles[10], and ethanol-soaked gelatin sponges (ESG)[11,12]. Additionally, ESG combines the advantages of alcohol and gelatin sponges and provides convincing results at different APF stages[12]. However, to the best of our knowledge, no study has compared the efficacies of ESG and microspheres. We conducted a retrospective study to evaluate the efficacy of ESGs and microspheres for the treatment of HCC with hepatic APF. + +## Materials And Methods + +Patients with HCC and hepatic APF treated with TACE and ESG (group E) or microspheres (group M) were enrolled between June 2016 and December 2019. The study protocol was approved by the ethics committee of the leading center. The requirement for written informed consent was waived owing to the retrospective nature of the study. All the experiments were performed in compliance with the Ethical Principles for Medical Research Involving Human Subjects outlined in the 1975 Declaration of Helsinki (revised in 2000). + +The inclusion criteria were as follows: (1) Confirmed diagnosis of HCC based on the American Association for the Study of Liver Diseases practice guidelines[13]; (2) Hypervascular tumor with Barcelona Clinic Liver Cancer (BCLC) Staging A-C; (3) Hepatic APF confirmed by angiography; (4) Predicted life span ≥ 1 year; and (5) Karnofsky score > 70. + +The exclusion criteria were as follows: (1) Other malignancies within 5 years; (2) Child-Pugh score ≤ 10; and (3) Severe coagulopathy (prothrombin time > 17 s and/or platelet count ≤ 60 × 109/L). + +## Treatment Of Apf + +For group E, an appropriate-sized gelatin sponge (Alicon Inc., Hangzhou, China) was mixed with 10 mL of iodixanol +(Hengrui Co. Ltd, Lianyungang, China) and 10 mL of ethanol (Lingfeng Inc, Shanghai, China). For group M, appropriatesized microspheres (Embosphere, Merit Medical, UT, United States) were mixed with 10 mL of iodixanol. Digital subtraction angiography (DSA) was performed after catheterization of the celiac or superior mesenteric artery to validate the location and size of the hepatic APFs (Figure 1). APFs were classified according to a previous study by Zhou et al[12] (Table 1). Each APF feeding artery was superselected using a 2.7-F microcatheter. ESG or the microspheres were injected under fluoroscopic guidance until the fistula was blocked. Coils were used if the fistula was not completely blocked. DSA was repeated to confirm the complete embolization of the APFs (Figure 2). + +## Tace Procedure + +After APF embolization, a microcatheter was advanced into each feeding artery of the HCC. An emulsion of poppy Lipiodol (Hengrui Co. Ltd., Lianyungang, China) and epirubicin (Qilu Co. Ltd., Jinan, China) was injected via a microcatheter until complete embolization of the tumor was achieved (Figure 3)[14]. + +## Follow-Up + +Follow-up was conducted every 2 mo and included standard blood count, liver functional tests, alpha-fetoprotein (AFP), and abdominal contrast-enhanced computed tomography (CECT) or magnetic resonance imaging (MRI). The images were interpreted based on the consensus of three skilled interventional radiologists. + +In case the tumor recurrence was detected on CECT or MRI, TACE was repeated. If APF recurrence with a grade ≥ 2 was observed, ESG or microsphere APF embolization was repeated; however, if APFs did not recur, TACE was the only procedure performed. Follow-up intervention was determined based on the tumor condition and general status. + +## Outcome Measures + +The modified Response Evaluation Criteria in Solid Tumors for HCC[15] were applied to assess tumor response after 4 mo. The primary outcomes were disease control rate (DCR) and objective response rate (ORR), and the secondary outcomes included immediate and first-time follow-up of APF improvement, overall survival (OS), and progression-free survival (PFS). + +Immediate APF improvement was defined as a decrease in grade to 1 or 0. First-time follow-up APF improvement was defined as a decrease in at least two grades confirmed by angiography in the second session, whereas APF progression was defined as an increased grade on the first-time follow-up angiography. If the grade remained the same or decreased by one, the APFs were not considered to improve. Moreover, OS was defined as the time interval between the initial TACE and death or the last follow-up. Furthermore, PFS was defined as the time interval between initial TACE and disease progression or death. + +## Statistical Analysis + +Continuous variables were analyzed using Student's t-test to determine whether the variables were normally distributed; otherwise, the Mann–Whitney U test was used. Categorical variables were analyzed using the χ 2 or Fisher's exact tests. + +Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test. Statistical significance was defined as a two-tailed P < 0.05. All statistical analyses were conducted using the SPSS software (version 24.0; IBM Inc., Armonk, NY, United States). + +## Results Participant Characteristics + +A consecutive series of 91 patients were enrolled in the study. During TACE, APFs were embolized using ESG in 46 participants and microspheres in 45 participants. The ratios of men to women were 33/13 in group E and 33/12 in group M (χ 2 = 0.029, P = 0.865), with a mean age of 63.4 ± 8.5 and 58.4 ± 10.1 years (P = 0.092), respectively. The etiologies included hepatitis B virus (HBV) (38/46, 82.6% in group E; 39/45, 86.7% in group M), hepatitis C (4/46, 8.7% in group E; and 2/45, 4.4% in group M), HBV + hepatitis C virus (2/46, 4.3% in group E; 2/45, 4.4% in group M), and alcohol consumption (2/46, 4.3% in group E; 2/45, 4.4% in group M) (P = 0.952). No significant differences in the Child-Pugh stage, BCLC stage, or tumor location were observed between the two groups. The mean tumor diameters were 6.8 ± 2.9 mm and 7.1 ± 1.6 mm in groups E and M (P = 0.765), respectively. Portal vein thrombi were identified in 24 participants (24/46, 52.2%) in group E and 22 participants (22/45, 48.9%) in group M (χ 2 = 0.098, P = 0.754), respectively. The treatments administered before TACE included surgery, microwave ablation (MWA), radiofrequency ablation (RFA), TACE, radiation, and TACE + MWA/RFA. We observed no significant differences in previous treatments between the + +| Yuan GS et al. ESG vs microsphere for hepatic APFs Table 1 Grading of arterioportal fistula Grade Definition | Class | | +|----------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------|----------| +| 0 | APFs were not observed | - | +| 1 | APFs flow to the subsegmental portal branch | Mild | +| 2 | APFs flow to the segmental portal branch | Moderate | +| 3 | APFs flow into the main portal branch of the ipsilateral lobe | Moderate | +| 4 | APFs flow into the main portal branch of the contralateral lobe and/or the main portal vein | Severe | +| 5 | APFs flow into the main portal vein presenting with hepatofugal portal venous flow | Severe | + +![3_image_0.png](3_image_0.png) + +two groups (P = 0.925). The median levels of AFP were 137 [interquartile range (IQR): 9.8, 970.1] and 114.9 (IQR: 3.7, 725.7) ng/mL in groups E and M, respectively (P = 0.734). APF grades 1, 2, 3, 4, and 5 were recorded in 5 (5/46, 10.9%) and 6 (6/45, 13.3%); 15 (15/46, 32.6%) and 16 (16/45, 35.6%); 11 (11/46, 23.9%) and 14 (14/45, 31.1%); 9 (9/46, 19.6%), and 7 (7/45, 15.6%); and 6 (6/46, 13%) and 2 (2/45, 4.4%) participants in groups E and M, respectively (P = 0.636) (Table 2). + +The mean follow-up period was 35.3 ± 2.7 mo in group E and 30.9 ± 3.8 mo in group M (P = 0.195). After 4 mo, complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) were achieved in 18 (18/ 46, 39.1%) and 8 (8/45, 17.8%) patients; 21 (21/46, 45.7%) and 18 (18/45, 40%) patients; 4 (4/46, 8.7%) and 15 (15/45, 33.3%) patients; and 3 (3/46, 6.5%) and 4 (4/45, 8.9%) participants in groups E and M, respectively (P = 0.014). The DCR + +| Yuan GS et al. ESG vs microsphere for hepatic APFs | | | | | +|--------------------------------------------------------------------------------------------------------------------------------------------------|------------------|--------------------|---------|-------| +| Table 2 Demographic and baseline characteristics Characteristics E group (n = 46) | M group (n = 45) | χ 2 | P value | | +| Sex, n (%) | 0.029 | 0.865 | | | +| Male | 33 (71.7) | 33 (73.3) | | | +| Female | 13 (28.3) | 12 (26.7) | | | +| Age (yr) | 63.4 ± 8.5 | 58.4 ± 10.1 | - | 0.092 | +| Etiology, n (%) | 0.909 | 0.952 | | | +| HBV | 38 (82.7) | 39 (86.8) | | | +| HCV | 4 (8.7) | 2 (4.4) | | | +| HBV + HCV | 2 (4.3) | 2 (4.4) | | | +| Alcohol | 2 (4.3) | 2 (4.4) | | | +| Child-Pugh stage, n (%) | 0.297 | 0.586 | | | +| A | 25 (54.3) | 27 (60) | | | +| B | 21 (45.7) | 18 (40) | | | +| BCLC stage, n (%) | 0.271 | 0.873 | | | +| A | 7 (15.2) | 6 (13.3) | | | +| B | 19 (41.3) | 21 (45.7) | | | +| C | 20 (43.5) | 18 (40) | | | +| Tumor location | 0.837 | 0.658 | | | +| Right lobe | 30 (65.2) | 28 (62.2) | | | +| Left lobe | 9 (19.6) | 10 (22.2) | | | +| Right and left lobes | 7 (15.2) | 7.1 ± 1.6 | | | +| Mean tumor diameter (cm) | 6.8 ± 2.9 | - | 0.765 | | +| Portal vein thrombus | 0.098 | 0.754 | | | +| Present | 24 (52.2) | 22 (48.9) | | | +| Absent | 22 (47.8) | 23 (51.1) | | | +| Previous treatment | 1.639 | 0.925 | | | +| Surgery | 7 (15.2) | 6 (13.3) | | | +| MWA/RFA | 9 (19.6) | 7 (15.6) | | | +| TACE | 4 (8.7) | 5 (11.1) | | | +| Radiation | 4 (8.7) | 6 (13.3) | | | +| TACE + MWA/RFA | 2 (4.3) | 3 (6.7) | | | +| None | 20 (43.5) | 18 (40) | | | +| AFP [ng/mL, median (IQR)] | 137 (9.8, 970.1) | 114.9 (3.7, 725.7) | - | 0.734 | +| APF grade, n (%) | 2.689 | 0.636 | | | +| 1 | 5 (10.9) | 6 (13.3) | | | +| 2 | 15 (32.6) | 16 (35.6) | | | +| 3 | 11 (23.9) | 14 (31.1) | | | +| 4 | 9 (19.6) | 7 (15.6) | | | +| 5 | 6 (13) | 2 (4.4) | | | +| HCV: Hepatitis C virus; BCLC: Barcelona Clinic Liver Cancer; MWA: Microwave ablation; RFA: Radiofrequency ablation; TACE: Transcatheter arterial | | | | | + +HCV: Hepatitis C virus; BCLC: Barcelona Clinic Liver Cancer; MWA: Microwave ablation; RFA: Radiofrequency ablation; TACE: Transcatheter arterial chemoembolization; AFP: Alpha-fetoprotein; IQR: Interquartile range; APFs: Arterioportal fistulas. + +![5_image_0.png](5_image_0.png) + +![5_image_1.png](5_image_1.png) + +was 93.5% (43/46) in group E and 91.1% (41/45) in group M (P = 0.714). The ORRs were 91.3% (42/46) and 66.7% (30/45) in groups E and M, respectively (P = 0.004). + +The APFs immediately improved after the procedure in 43 (43/46, 93.5%) and 40 (40/45, 88.9%) participants in groups E and M, respectively (P = 0.485). After 2 mo, APF improvement was achieved in 37 (37/46, 80.4%) and 33 (33/45, 73.3%) participants in groups E and M, respectively (P = 0.421). The median AFP levels at 4 mo after the procedure were 28.48 (IQR: 4, 257.9) and 45.25 (IQR: 4.43, 359.5) ng/mL in groups E and M, respectively (P = 0.045). After 4 mo, the difference in Child-Pugh class distribution between the two groups was not significant (P = 0.083) (Table 3). + +The OS was 26.2 ± 1.4 and 20.6 ± 1.1 mo in groups E and M, respectively (χ 2 = 10.3, P = 0.004; Figure 4A) (Table 3). The PFS was 16.6 ± 1.0 and 13.8 ± 0.7 mo in groups E and M, respectively (P = 0.012; Figure 4B) (Table 3). + +## Discussion + +According to the updated BCLC prognosis and treatment strategy[16], TACE is recommended for intermediate-stage B HCC. With its tendency to infiltrate the portal and hepatic venous structures, HCC is often accompanied by APFs, which may reduce the therapeutic benefits of TACE[7]. Our study focused on comparing ESG and microspheres for the treatment of hepatic APFs. The DCRs were 93.5% (43/46) in group E and 91.1% (41/45) in group M (P = 0.714). The ORRs were 91.3% and 66.7% in groups E and M, respectively (P = 0.004). The OS was 26.2 ± 1.4 and 20.6 ± 1.1 mo in groups E and M, respectively (P = 0.004). The PFS was 16.6 ± 1.0 and 13.8 ± 0.7 mo in groups E and M, respectively (P = 0.012; Figure 4B) (Table 3). + +| Table 3 Outcome characteristics Characteristics | E group (n = 46) | M group (n = 45) | χ 2 | P value | +|---------------------------------------------------|--------------------|---------------------|-------|-----------| +| Tumor response after four months (%) | 10.578 | 0.014 | | | +| CR | 18 (39.1) | 8 (17.8) | | | +| PR | 21 (45.7) | 18 (40) | | | +| SD | 4 (8.7) | 15 (33.3) | | | +| PD | 3 (6.5) | 4 (8.9) | | | +| DCR | 43 (93.5) | 41 (91.1) | 0.714 | | +| ORR | 42 (91.3) | 30 (66.7) | 8.358 | 0.004 | +| Immediate improvement of APF (%) | - | 0.485 | | | +| Yes | 43 (93.5) | 40 (88.9) | - | | +| No | 3 (6.5) | 5 (11.1) | | | +| First-time follow-up APF improvement (%) | 0.646 | 0.421 | | | +| Improved | 37 (80.4) | 33 (73.3) | | | +| Not improved | 9 (19.6) | 12 (26.7) | | | +| AFP after 4 mo [ng/mL, median (IQR)] | 28.48 (4, 257.9) | 45.25 (4.43, 359.5) | 0.045 | | +| Child-Pugh score after 4 mo (%) | 5.321 | 0.083 | | | +| A | 33 (71.7) | 23 (51.1) | | | +| B | 10 (21.7) | 20 (44.4) | | | +| C | 3 (6.6) | 2 (4.5) | | | +| OS, months (mean ± SD) | 26.2 ± 1.4 | 20.6 ± 1.1 | 10.3 | 0.004 | +| PFS, months (mean ± SD) | 16.6 ± 1.0 | 13.8 ± 0.7 | 6.3 | 0.012 | + +CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease; DCR: Disease control rate; ORR: Objective response rate; APFs: + +![6_image_0.png](6_image_0.png) + +![6_image_1.png](6_image_1.png) Arterioportal fistulas; AFP: Alpha-fetoprotein; AP: Arterioportal; IQR: Interquartile range; OS: Overall survival; PFS: Progression-free survival. + +Gelatin sponges and microspheres have several disadvantages in the treatment of hepatic APF. Gelatin sponges are absorbed 2-3 wk after the procedure, and APFs can be recanalized. Microspheres exerted a physical embolic effect without causing protein degradation in the vascular wall. Ethanol has been widely used in the embolization of arteriovenous malformations[17], which can denature blood proteins, dehydrate vascular endothelial cells, and cause segment fractures in the vascular wall[18-20]. Compared to gelatin sponges alone, ethanol demonstrated an improved long-term effect on hepatic APFs[21]. However, because of its liquid properties, ethanol alone is not suitable for shunts with high blood flow. ESG combines the advantages of ethanol and gelatin sponges, promoting local control of hepatic APFs and liver tumors[12]. + +In our study, the immediate improvement and first-time follow-up rates of APFs in group E were not significantly higher than those in group M (93.5% and 88.9%, P = 0.485, 80.4% and 73.3%, P = 0.421, respectively). Thus, ESG and microspheres may have similar short-term effects on the treatment of hepatic APFs. The immediate improvement rate in group E was comparable to the 97% reported by Zhou et al[12], whereas the first follow-up APF improvement rate was higher in both groups than that reported by Zhou et al[12] (54%). This discrepancy may be attributed to the higher proportion of patients with grades 1–3 APFs in our study. + +Our study investigated tumor response 4 mo after the procedure and revealed that the CR, PR, SD, and PD rates were 39.1% and 17.8%, 45.7% and 40%, 8.7% and 33.3%, and 6.5% and 8.9% in groups E and M, respectively (P = 0.014). Moreover, the ORR was 84.8% and 57.8% in groups E and M, respectively (P = 0.004). Compared with microspheres, ESG led to complete long-term control of hepatic APF, including physical blockade and chemical destruction and yielded a significantly better local tumor response. Both the DCRs (93.5%) and ORRs (84.8%) in group E patients were higher than those reported in Zhou et al's study (81.9% and 42.6%, respectively)[12]. This has three possible reasons. First, the tumor response in our study was evaluated 4 mo after the procedure, which provided an additional opportunity for tumor control. Second, the percentage of participants with portal vein thrombus (52.5%) was lower than that reported by Zhou et al's study[12]. Third, the proportion of grade 1-3 APFs in our study was higher, resulting in a better embolic response. + +The OS, PFS, and median AFP levels at 4 mo after the procedure in group E were significantly better than those in group M. The aforementioned outcome may be attributed to the complete blockage of hepatic APFs and well-controlled tumors. Compared with microspheres, ESG embolization demonstrated complete long-term blockade of hepatic APFs and therefore improved the local control of HCC and survival of patients with HCC. + +Nevertheless, the study had some limitations. As this was a retrospective study, selection bias may have reduced the value of the results. However, further prospective studies are required to validate the findings. + +## Conclusion + +Compared to microsphere embolization, ESG embolization resulted in a higher ORR and longer OS and PFS. The findings may contribute to the selection of embolic agents for treating hepatic APFs in patients with HCC. + +## Article Highlights Research Background + +Hepatic arterioportal fistulas (APFs) are common in hepatocellular carcinoma (HCC) because of tumor infiltration, vascular damage, and remodeling of the cirrhotic parenchyma. The presence of hepatic APFs often complicates antitumor treatments, including transarterial chemoembolization (TACE). + +## Research Motivation + +Ethanol-soaked gelatin sponges (ESG) combine the advantages of alcohol and gelatin sponges, demonstrating a convincing effect at different stages of hepatic APFs. However, to date, no study has compared the efficacy of ESG and microspheres. + +## Research Objectives + +This retrospective study aimed to compare the efficacy of ESG and microspheres in the management of APFs, and their impact on the prognosis of HCC. + +## Research Methods + +The APFs were embolized using ESG (group E) or microspheres (group M) during TACE. The disease control rate (DCR) and objective response rate (ORR) were considered the primary outcomes. The secondary outcomes included immediate and first follow-up APF improvement, overall survival (OS), and progression-free survival (PFS). + +## Research Results + +The DCR was 93.5% and 91.1% in groups E and M, respectively (P = 0.714). The ORRs were 91.3% and 66.7% in groups E and M, respectively (P = 0.004). In 43 (93.5%) patients in group E and 40 (88.9%) patients in group M. the APFs improved immediately after the procedure (P = 0.485). After 2 mo, APF improvement was achieved in 37 (80.4%) and 33 (73.3%) +participants in groups E and M, respectively (P = 0.421). The OS was 26.2 ± 1.4 and 20.6 ± 1.1 mo in groups E and M, +respectively (P = 0.004). The PFS was 16.6 ± 1.0 and 13.8 ± 0.7 mo in groups E and M, respectively (P = 0.012). + +## Research Conclusions + +Compared with microspheres, ESG embolization demonstrated a higher ORR and longer OS and PFS in patients with HCC with hepatic APFs. + +## Research Perspectives + +The findings may aid the selection of embolic agents for the treatment of hepatic APFs in patients with HCC. + +## Footnotes + +Author contributions: Yuan GS and Zhang LL have contributed equally to this work; Yuan GS and Liu B performed the conception and design; Guo L and Liu B contributed to the administrative support; Zhang LL, Chen ZT, and Zhang CJ performed the provision of study materials and patients; Chen ZT, Tian SH, Gong MX, Wang P, Guo L, and Shao N performed the data collection and assembly; Chen ZT and Zhang CJ contributed to the data analysis and interpretation; All authors wrote the manuscript and performed the final approval of manuscript. + +Supported by National Natural Science Foundation of China, No. 12171285 and No. 11971269; Program for Integrated Traditional Chinese and Western Medicine in Shandong Province of China, No. YXH2019ZXY007; and Jinan New Support Projects for Universities (Talent Development Special Fund), No. 20228118. + +Institutional review board statement: The study was reviewed and approved by the Ethics Committee of the Second Hospital of Shandong University (KYLL-2020 [LW] 23). + +Informed consent statement: All study participants or their legal guardian provided informed written consent about personal and medical data collection prior to study enrolment. + +Conflict-of-interest statement: The authors declare no competing interests. + +Data sharing statement: No additional data are available. + +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. + +Moreover, the article 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: https://creativecommons.org/Licenses/by-nc/ 4.0/ + +## Country/Territory Of Origin: China + +ORCID number: Guang-Sheng Yuan 0009-0008-1787-1781; Li-Li Zhang 0009-0001-3906-3565; Zi-Tong Chen 0000-0002-9606-1654; Cun-Jing Zhang 0000-0003-1856-0543; Shu-Hui Tian 0009-0003-2712-9568; Ming-Xia Gong 0009-0005-2178-6246; Peng Wang 0009-0006-1343-7887; Lei Guo 0000-0002-7986-3786; Nan Shao 0009-0001-2916-6384; Bin Liu 0000-0003-1686-1553. + +S-Editor: Fan JR +L-Editor: A +P-Editor: Zhao S + +## References + +1 **Sung H**, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. *CA Cancer J Clin* 2021; 71: 209-249 [PMID: 33538338 DOI: 10.3322/caac.21660] +2 **Gülberg V**, Haag K, Rössle M, Gerbes AL. Hepatic arterial buffer response in patients with advanced cirrhosis. *Hepatology* 2002; 35: 630-634 +[PMID: 11870377 DOI: 10.1053/jhep.2002.31722] +3 **Richter S**, Mücke I, Menger MD, Vollmar B. Impact of intrinsic blood flow regulation in cirrhosis: maintenance of hepatic arterial buffer response. *Am J Physiol Gastrointest Liver Physiol* 2000; 279: G454-G462 [PMID: 10915656 DOI: 10.1152/ajpgi.2000.279.2.G454] +4 **Choi BI**, Lee KH, Han JK, Lee JM. Hepatic arterioportal shunts: dynamic CT and MR features. *Korean J Radiol* 2002; 3: 1-15 [PMID: +11919473 DOI: 10.3348/kjr.2002.3.1.1] +5 **Kakati BR**, Pedersen MR, Chen SY, Hirsch KS, Berggreen PJ, Seetharam AB. Hepatic arterioportal fistula presenting as gastric variceal hemorrhage. *J Gastrointestin Liver Dis* 2014; 23: 211-214 [PMID: 24949615 DOI: 10.15403/jgld.2014.1121.232.brk1] +6 **Wakamatsu T**, Ogasawara S, Chiba T, Yokoyama M, Inoue M, Kanogawa N, Saito T, Suzuki E, Ooka Y, Tawada A, Yokosuka O. Impact of Radiofrequency Ablation-Induced Glisson's Capsule-Associated Complications in Patients with Hepatocellular Carcinoma. *PLoS One* 2017; 12: e0170153 [PMID: 28099460 DOI: 10.1371/journal.pone.0170153] +7 **Bruix J**, Sherman M; American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma: an update. *Hepatology* 2011; 53: 1020-1022 [PMID: 21374666 DOI: 10.1002/hep.24199] +8 **Kanogawa N**, Chiba T, Ogasawara S, Ooka Y, Suzuki E, Motoyama T, Saito T, Sekimoto T, Tawada A, Maruyama H, Yoshikawa M, +Yokosuka O. Successful interventional treatment for arterioportal fistula caused by radiofrequency ablation for hepatocellular carcinoma. *Case* Rep Oncol 2014; 7: 833-839 [PMID: 25685134 DOI: 10.1159/000370305] +9 **Cai L**, Li H, Guo J, Zhao W, Duan Y, Hou X, Cheng L, Du H, Shao X, Diao Z, Hao Y, Zheng X, Li C, Li W. Treatment efficacy and safety of drug-eluting beads transarterial chemoembolization versus conventional transarterial chemoembolization in hepatocellular carcinoma patients with arterioportal fistula. *Cancer Biol Ther* 2022; 23: 89-95 [PMID: 35230928 DOI: 10.1080/15384047.2021.2020059] +10 **Chan WS**, Poon WL, Cho DH, Chiu SS, Luk SH. Transcatheter embolisation of intrahepatic arteriovenous shunts in patients with hepatocellular carcinoma. *Hong Kong Med J* 2010; 16: 48-55 [PMID: 20124574] +11 **Li J**, Kang X, Guo L, Xiao J, Cheng J. Embolization of hepatic arterioportal shunt with ethanol-soaked gelatin sponge. *J Cancer Res Ther* 2019; 15: 336-340 [PMID: 30964107 DOI: 10.4103/jcrt.JCRT_825_17] +12 **Zhou WZ**, Shi HB, Liu S, Yang ZQ, Zhou CG, Xia JG, Zhao LB, Li LS. Arterioportal shunts in patients with hepatocellular carcinoma treated using ethanol-soaked gelatin sponge: therapeutic effects and prognostic factors. *J Vasc Interv Radiol* 2015; 26: 223-230 [PMID: 25645411 DOI: 10.1016/j.jvir.2014.11.002] +13 **Heimbach JK**, Kulik LM, Finn RS, Sirlin CB, Abecassis MM, Roberts LR, Zhu AX, Murad MH, Marrero JA. AASLD guidelines for the treatment of hepatocellular carcinoma. *Hepatology* 2018; 67: 358-380 [PMID: 28130846 DOI: 10.1002/hep.29086] +14 **Xie DY**, Ren ZG, Zhou J, Fan J, Gao Q. 2019 Chinese clinical guidelines for the management of hepatocellular carcinoma: updates and insights. *Hepatobiliary Surg Nutr* 2020; 9: 452-463 [PMID: 32832496 DOI: 10.21037/hbsn-20-480] +15 **Lencioni R**, Llovet JM. Modified RECIST (mRECIST) assessment for hepatocellular carcinoma. *Semin Liver Dis* 2010; 30: 52-60 [PMID: +20175033 DOI: 10.1055/s-0030-1247132] +16 **Reig M**, Forner A, Rimola J, Ferrer-Fábrega J, Burrel M, Garcia-Criado A, Kelley RK, Galle PR, Mazzaferro V, Salem R, Sangro B, Singal AG, Vogel A, Fuster J, Ayuso C, Bruix J. BCLC strategy for prognosis prediction and treatment recommendation Barcelona Clinic Liver Cancer (BCLC) staging system. The 2022 update. *J Hepatol* 2021 [DOI: 10.1016/j.jhep.2021.11.018] +17 **Do YS**, Yakes WF, Shin SW, Lee BB, Kim DI, Liu WC, Shin BS, Kim DK, Choo SW, Choo IW. Ethanol embolization of arteriovenous malformations: interim results. *Radiology* 2005; 235: 674-682 [PMID: 15858106 DOI: 10.1148/radiol.2352040449] +18 **Buchta K**, Sands J, Rosenkrantz H, Roche WD. Early mechanism of action of arterially infused alcohol U.S.P. in renal devitalization. + +Radiology 1982; 145: 45-48 [PMID: 7122894 DOI: 10.1148/radiology.145.1.7122894] +19 **Hammer FD**, Boon LM, Mathurin P, Vanwijck RR. Ethanol sclerotherapy of venous malformations: evaluation of systemic ethanol contamination. *J Vasc Interv Radiol* 2001; 12: 595-600 [PMID: 11340138 DOI: 10.1016/s1051-0443(07)61482-1] +20 **Takebayashi S**, Hosaka M, Kubota Y, Ishizuka E, Iwasaki A, Matsubara S. Transarterial embolization and ablation of renal arteriovenous malformations: efficacy and damages in 30 patients with long-term followup. *J Urol* 1998; 159: 696-701 [PMID: 9474128 DOI: 10.1016/s0022-5347(01)63703-0] +21 **Huang MS**, Lin Q, Jiang ZB, Zhu KS, Guan SH, Li ZR, Shan H. Comparison of long-term effects between intra-arterially delivered ethanol and Gelfoam for the treatment of severe arterioportal shunt in patients with hepatocellular carcinoma. *World J Gastroenterol* 2004; 10: 825-829 [PMID: 15040025 DOI: 10.3748/wjg.v10.i6.825] +Published by **Baishideng Publishing Group Inc** + +![10_image_0.png](10_image_0.png) + +![10_image_1.png](10_image_1.png) + +7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA +Telephone: +1-925-3991568 E-mail: bpgoffice@wjgnet.com Help Desk: https://www.f6publishing.com/helpdesk https://www.wjgnet.com \ No newline at end of file diff --git a/medical/md_images/PMC10751439/0_image_0.png b/medical/md_images/PMC10751439/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..367939331a62f49e112e920f1b8de042cb33ff9c --- /dev/null +++ b/medical/md_images/PMC10751439/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f25fa259b93c014954003e07704a92bbc63d6689e48c962515bd7159c1bb4de9 +size 460 diff --git a/medical/md_images/PMC10751439/PMC10751439.md b/medical/md_images/PMC10751439/PMC10751439.md new file mode 100644 index 0000000000000000000000000000000000000000..a3f49409c9d9055811d3df9ce9a25fc93e95a4b8 --- /dev/null +++ b/medical/md_images/PMC10751439/PMC10751439.md @@ -0,0 +1,49 @@ + + +![0_image_0.png](0_image_0.png) + +Journal of Cachexia, Sarcopenia and Muscle 2023; 14: 2993–2994 Published online 21 September 2023 in Wiley Online Library (wileyonlinelibrary.com) **DOI:** 10.1002/jcsm.13338 + +# Comment On: "Triceps Skinfold-Albumin Index + +significantly predicts the prognosis of cancer cachexia: +A multicentre cohort study" by Yin et al. - the authors reply Liangyu Yin1 , Jiuwei Cui2, Xin Lin1, Long Li1, Na Li1, Yang Fan1, Ling Zhang1, Jie Liu1, Feifei Chong1, Zongliang Lu1, Chang Wang2, Tingting Liang2, Xiangliang Liu2, Li Deng2, Mei Yang3, Jiami Yu3, Xiaojie Wang3, Minghua Cong4, Zengning Li5, Min Weng6, Qinghua Yao7, Pingping Jia8, Zengqing Guo3, Wei Li2, Chunhua Song9*, Hanping Shi8,10* & Hongxia Xu1* 1Department of Clinical Nutrition, Daping Hospital, Army Medical University (Third Military Medical University), Chongqing, China; 2*Cancer Center of the First Hospital of Jilin* University, Changchun, China; 3Department of Medical Oncology, Fujian Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou, China; 4Department of Comprehensive Oncology, National Cancer Center or Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; 5Department of Clinical Nutrition, The First Hospital of Hebei Medical University, Shijiazhuang, China; 6Department of Clinical Nutrition, The First Affi*liated Hospital of Kunming Medical* University, Kunming, China; 7*Department of Integrated Chinese and Western Medicine, Cancer Hospital of the University of Chinese Academy of Science (Zhejiang Cancer* Hospital), Hangzhou, China; 8Department of Gastrointestinal Surgery and Department of Clinical Nutrition, Beijing Shijitan Hospital, Capital Medical University, Beijing, China; 9Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou, China; 10*Key Laboratory of Cancer FSMP for State Market Regulation,* +Beijing, China Received: 8 August 2023; Accepted: 24 August 2023 *Correspondence to: Hongxia Xu, Department of Clinical Nutrition, Daping Hospital, Army Medical University (Third Military Medical University), Chongqing 400042*, China.* Email: hx_xu2015@163*.com;* Hanping Shi, Department of Gastrointestinal Surgery and Department of Clinical Nutrition, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038*, China. Email:* shihp@ccmu.edu.cn; Chunhua Song, Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou, Henan 450001, China. Email: sch16*@zzu.edu.cn* + +## Dear Editor, + +We would like to express our sincere gratitude for the valuable comments and interesting findings by Ding et al.1 regarding the triceps skinfold-albumin index (TA) proposed in our research article.2 Based on data from multiple cohorts of locally advanced gastric cancer (LAGC), the authors found that a higher TA was associated with prolonged overall survival (OS) and disease-free survival (DFS) in LAGC patients with cachexia. They further performed subgroup survival analyses based on tumour pTNM staging, which demonstrated similar results in stage II and stage III patients, respectively. + +Ding et al. provided new insights in addition to the original research, the leading one among them being that they extended the prognostic value of TA to the realm of DFS. This was meaningful because DFS is widely used as a primary endpoint in phase III trials of adjuvant therapies for gastric cancer.3,4 Therefore, their findings imply the potential of TA +as a decision support tool for the anti-cancer treatment of gastric cancer. Secondly, our original research found that the association of TA with OS was literally attenuated in stage I-II tumours. The authors further found that this association sustained in stage II gastric cancer, implying that this attenuation might be mostly concentrated in patients with stage I tumours, at least for gastric cancer. These findings are consistent with our understanding because depletion of adipose tissue is one of the earliest and most common manifestations of cancer cachexia,5,6 and cancer cachexia is more prevalent in advanced lethal cancers.7 Therefore, patients with stage I tumours might be least affected by cachexia-induced fat depletion that reaches the threshold to cause a significant survival difference. + +The findings by Ding et al. align well with the original research, further supporting the use of TA as a practical prognostic tool for the risk stratification of cancer cachexia. The authors also raised several interesting points that might be addressed by upcoming studies. Looking ahead, we believe that future studies in this field should continue to explore the potential of TA as a clinically meaningful and promising biomarker in a wider spectrum of diseases and clinical scenarios. Further investigations could focus on validating the performance of TA across different cancer types and stages, or other subgroups with specific concerns, as well as evaluating its utility in guiding targeted interventions for the management of cachexia or malnutrition in oncology patients. Additionally, monitoring the dynamic changes of the TA index over time and investigating the impact of such changes on the treatment and prognosis in patients with cancer cachexia might be an interesting research direction. + +Once again, we were delighted that our research has generated peer interest, and we sincerely appreciate Ding et al. for their kind attention to our research and for highlighting the significance of TA in predicting the survival of LAGC patients with cancer cachexia. The authors' insights contribute to the advancement of this important area of study. + +## References Acknowledgements + +This work was supported by the Natural Science Foundation of Chongqing, China (CSTB2022NSCQ-MSX1069) and the National Key Research and Development Program of China (2017YFC1309200). The authors would like to thank the INSCOC project members for their substantial work on data collection and patient follow-up. + +## Conflict Of Interest + +The authors declare that they have no conflict of interest. + +1. Ding P, Wu H, Wu J, Sun C, Meng M, Yang P, +et al. Comment on: 'Triceps skinfold-albumin index significantly predicts the prognosis of cancer cachexia: a multicentre cohort study' by Yin et al. *J Cachexia Sarcopenia* Muscle 2023. https://doi.org/10.1002/jcsm. 13304 (online ahead of print) +2. Yin L, Cui J, Lin X, Li L, Li N, Fan Y, et al. + +Triceps skinfold-albumin index significantly predicts the prognosis of cancer cachexia: a multicentre cohort study. *J Cachexia Sarcopenia Muscle* 2023;14:517–533. + +3. Tsuburaya A, Yoshida K, Kobayashi M, +Yoshino S, Takahashi M, Takiguchi N, et al. Sequential paclitaxel followed by tegafur and uracil (UFT) or S-1 versus UFT or S-1 monotherapy as adjuvant chemotherapy for T4a/b gastric cancer (SAMIT): a phase 3 factorial randomised controlled trial. Lancet Oncol 2014;15:886–893. + +4. Zhang X, Liang H, Li Z, Xue Y,Wang Y, Zhou Z, +et al. Perioperative or postoperative adjuvant oxaliplatin with S-1 versus adjuvant oxaliplatin with capecitabine in patients with locally advanced gastric or gastro-oesophageal junction adenocarcinoma undergoing D2 gastrectomy (RESOLVE): an open-label, superiority and noninferiority, phase 3 randomised controlled trial. *Lancet Oncol* 2021;22:1081–1092. + +5. Argiles JM, Busquets S, Stemmler B, LopezSoriano FJ. Cancer cachexia: understanding the molecular basis. *Nat Rev Cancer* 2014; 14:754–762. + +6. Poisson J, Martinez-Tapia C, Heitz D, Geiss R, +Albrand G, Falandry C, et al. Prevalence and prognostic impact of cachexia among older patients with cancer: a nationwide cross-sectional survey (NutriAgeCancer). J Cachexia Sarcopenia Muscle 2021;12: 1477–1488. + +7. von Haehling S, Anker MS, Anker SD. Prevalence and clinical impact of cachexia in chronic illness in Europe, USA, and Japan: facts and numbers update 2016. *J Cachexia* Sarcopenia Muscle 2016;7:507–509. \ No newline at end of file diff --git a/medical/md_images/PMC10751439/PMC10751439_meta.json b/medical/md_images/PMC10751439/PMC10751439_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..ef8627446b66eacef1974f765899f5cf0436b0dc --- /dev/null +++ b/medical/md_images/PMC10751439/PMC10751439_meta.json @@ -0,0 +1,70 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Comment on: “Triceps skinfold‐albumin index significantly predicts the prognosis of cancer cachexia: A multicentre cohort study” by Yin et al. ‐ the authors reply", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 596.0, + 783.0 + ] + }, + { + "title": "Conflict of interest", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 596.0, + 783.0 + ] + }, + { + "title": "References", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 596.0, + 783.0 + ] + } + ], + "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/PMC10906307/0_image_1.png b/medical/md_images/PMC10906307/0_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..135873dcd22db5763551a29a63bfbf161f937377 --- /dev/null +++ b/medical/md_images/PMC10906307/0_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:ba99f1fe4d048deb997ed9d53aa953080b81945a104b7c3e535da69aee6ee1a5 +size 1883 diff --git a/medical/md_images/PMC10908222/1_image_0.png b/medical/md_images/PMC10908222/1_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..5a23645e20988438b884ed1cb405195b288686b1 --- /dev/null +++ b/medical/md_images/PMC10908222/1_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:aa6bb3558cc0209c0e52b643068eb27bd67e8bd8107e0bcf54506664a4ac0cfb +size 38412 diff --git a/medical/md_images/PMC2238873/2_image_0.png b/medical/md_images/PMC2238873/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..ea278d1182cc492612aa5eb2fcb3570e893f0f86 --- /dev/null +++ b/medical/md_images/PMC2238873/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:77c7a57d09cd01fa5841cf94e8f1c635f0e8f01857cfb57f586aab21cf3a420b +size 90173 diff --git a/medical/md_images/PMC2238873/PMC2238873.md b/medical/md_images/PMC2238873/PMC2238873.md new file mode 100644 index 0000000000000000000000000000000000000000..20c314a49c50c319309dec429583bacbf73955ee --- /dev/null +++ b/medical/md_images/PMC2238873/PMC2238873.md @@ -0,0 +1,205 @@ +# The Biogrid Interaction Database: 2008 Update + +Bobby-Joe Breitkreutz1, Chris Stark1, Teresa Reguly1, Lorrie Boucher1,2, Ashton Breitkreutz1, Michael Livstone3, Rose Oughtred3, Daniel H. Lackner4, Ju¨ rg Ba¨ hler4, Valerie Wood4, Kara Dolinski3 and Mike Tyers1,2,* +1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, 2Department of Molecular Genetics, University of Toronto, Toronto, M5S 1A8, Canada, 3Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA and 4Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK +Received September 20, 2007; Revised October 19, 2007; Accepted October 22, 2007 + +## Abstract + +The Biological General Repository for Interaction Datasets (BioGRID) database (http://www. + +thebiogrid.org) was developed to house and distribute collections of protein and genetic interactions from major model organism species. + +BioGRID currently contains over 198 000 interactions from six different species, as derived from both high-throughput studies and conventional focused studies. Through comprehensive curation efforts, BioGRID now includes a virtually complete set of interactions reported to date in the primary literature for both the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. A number of new features have been added to the BioGRID including an improved user interface to display interactions based on different attributes, a mirror site and a dedicated interaction management system to coordinate curation across different locations. The BioGRID provides interaction data with monthly updates to Saccharomyces Genome Database, Flybase and Entrez Gene. + +Source code for the BioGRID and the linked Osprey network visualization system is now freely available without restriction. + +## Introduction + +Protein interactions underlie cell structure, biochemical activity and dynamic behavior; in turn, myriad genetic interactions reflect the vast functional interconnectivities of the protein network (1). High-throughput technologies now generate large datasets of protein and genetic interactions, which compliment more conventional detailed investigations of cellular processes (2). The collation of various types of interaction data is essential for interrogation of system-level attributes (3), and to this end a number of important interaction databases have been developed (4–8). Previously, we described a database called 'Biological General Repository for Interaction Datasets' (BioGRID) (www.thebiogrid.org) to archive and distribute comprehensive collections of physical and genetic interactions (9). + +The BioGRID has grown into a general resource for the research community with an average of 80 000 queries per month and millions of interactions downloaded per year. + +The 1 October 2007 version of BioGRID (v2.0.33) contains 198 791 (129 584 non-redundant) interaction records comprised of 137 834 (90 577 non-redundant) protein interactions and 60 957 (39 007 non-redundant) genetic interactions (Table 1). BioGRID provides full annotation support for 13 major model organism species (9), and currently houses interactions for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens. Gene annotation tables for all supported species are routinely updated to prevent ambiguous search results. Sources of data in BioGRID include publications that report high-throughput interaction datasets and many focused individual studies curated from inspection of the primary literature (2). Each interaction record in BioGRID contains experimental evidence codes and is linked to the supporting publication. In addition to the BioGRID website, all interactions in BioGRID are available through the dynamically linked Osprey visualization system, which can be used to query network organization in a user-defined fashion (10). BioGRID currently holds observer status + +| Table 1. Interactions in BioGRID Species Set | Nodes | Interactions (redundant) | Interactions (non-redundant) | Publication sources | | +|----------------------------------------------------------------------------------------------------------------------------------------------|---------|----------------------------|--------------------------------|-----------------------|--------| +| S. cerevisiae | P | 4936 | 64 799 | 38 298 | 4017 | +| G | 3746 | 47 116 | 33 354 | 4339 | | +| P+ G | 5196 | 111 915 | 71652 | 7193 | | +| S. pombe | P | 843 | 2631 | 1209 | 595 | +| G | 689 | 2275 | 1769 | 733 | | +| P+ G | 1057 | 4906 | 2978 | 1077 | | +| D. melanogaster | P | 7091 | 22 834 | 22 479 | 140 | +| G | 1199 | 10 689 | 3020 | 1515 | | +| P+ G | 7548 | 33 523 | 25 499 | 1626 | | +| C. elegans | P | 2816 | 4670 | 4557 | 8 | +| G | 654 | 861 | 857 | 3 | | +| P+ G | 3267 | 5531 | 5414 | 10 | | +| M. musculus | P | 335 | 532 | 499 | 49 | +| G | 3 | 2 | 2 | 2 | | +| P+ G | 337 | 534 | 501 | 51 | | +| H. sapiens | P | 8062 | 42 871 | 24 011 | 12 288 | +| G | 29 | 20 | 11 | 17 | | +| P+ G | 8069 | 42 891 | 24 022 | 12 296 | | +| All interactions | P | 23 767 | 137 834 | 90 577 | 16 972 | +| G | 6309 | 60 957 | 39 007 | 6599 | | +| P+ G | 25 148 | 198 791 | 129 584 | 22 108 | | +| Redundant interactions include self edges, multiple sources and multiple experimental evidence types; non-redundant interactions exclude all | | | | | | + +Redundant interactions include self edges, multiple sources and multiple experimental evidence types; non-redundant interactions exclude all + +duplicates. P, protein interactions; G, genetic interactions. + +in the IMEx consortium of interaction databases (http:// +imex.sourceforge.net/). + +## Literature Curation + +Comprehensive manual curation of the entire S. cerevisiae literature for protein and genetic interactions yielded 35 224 (21 281 non-redundant) protein interactions and 19 172 (13 963 non-redundant) genetic interactions (2), +all of which are accessible through BioGRID. Comparison of the literature-curated protein interaction dataset to recent high-throughput studies (11–13) reveals a considerable degree of non-overlap, suggesting that many interactions remain to be validated and discovered in this yeast (Figure 1). We have since continued to curate the current S. cerevisiae literature and have added 29 575 +(17 017 non-redundant) protein and 27 994 (19 391 nonredundant) genetic interactions to BioGRID since the original curation effort. Updates to BioGRID are made on the first of every month; each release of BioGRID is date stamped and archived for comparative purposes. + +All S. cerevisiae interactions deposited in BioGRID are immediately imported into the Saccharomyces Genome Database (SGD) with associated citations and evidence codes (14). Additional interaction attributes, including post-translational modifications associated with protein interactions and specific phenotypes associated with each genetic interaction, are currently being annotated and will be released for the entire S. cerevisiae dataset in the near future. + +To complement the comprehensive S. cerevisiae dataset, we have recently completed exhaustive manual curation of the S. pombe literature. Interactions were classified based on the same experimental evidence codes for protein and genetic interactions used previously (2). This effort yielded 2631 (1209 non-redundant) protein interactions and 2275 (1769 non-redundant) genetic interactions, as derived from 1077 publications. This new dataset has recently been deposited in BioGRID and, as for S. cerevisiae, will be updated on a monthly basis and provided to the S. pombe genome database (GeneDB) currently hosted by the Sanger Institute at www.genedb. + +org/genedb/pombe/ (15). Comparison of orthologous interactions between these evolutionary distant yeasts should prove informative for biological network structure and function. Imminent high-throughput studies in S. pombe should rapidly elaborate the cellular interaction network in this organism (16–18). + +In addition to systematic yeast curation, we have also undertaken partial interaction curation for higher species, for example, D. melanogaster and H. sapiens (Table 1). These curation efforts are often focused on specific aspects of biology and are in part guided by Gene Ontology inference codes for protein and genetic interactions (19) +and the Textpresso text-mining algorithm (20). Other species interactions are added to BioGRID on an ongoing basis and, when available, released in monthly BioGRID +updates. Contributions of curated interaction datasets from any species for deposition in the BioGRID are welcomed (www.thebiogrid.org). + +## Database Improvements + +We have expanded accessibility to interaction data in BioGRID via a primary mirror site at the SGD colony in Princeton (http://grid.princeton.edu/). In addition, source code for BioGRID and Osprey has been made available without restriction at SourceForge. BioGRID data files are currently linked to SGD, Flybase and NCBI, to which we provide automatic monthly updates. + +![2_image_0.png](2_image_0.png) + +Analogous relationships are underway with the Arabidopsis Information Resource (TAIR) and S. pombe GeneDB (15,21). We will endeavor to fulfill all requests for custom datasets for export to other model organism databases; the download page at BioGRID contains examples of existing datasets created for export. + +The dataset download system for the BioGRID is now powered by Asynchronous JavaScript and XML (AJAX), such that downloads are available for every search result page and publication in the database. Users may thus download interactions associated with any gene or all interactions reported by a single publication. Further data subsets organized by experimental system or by organism, including a recently described multivalidated yeast– protein interaction dataset (22), are also available on the BioGRID download page. + +The tabular user interface of BioGRID has been improved through implementation of AJAX techniques. The interface now provides the option to narrow search results to quantitative datasets; this feature will soon be elaborated to enable user-defined search criteria according to data type, evidence codes and data source. The ability to expand hidden fields with a single mouse click to provide greater detail, such as for Gene Ontology classifications (23), has also been added. Search results now include bait and hit designations to indicate the directionality of interactions. Additional annotation features including phenotype, post-translational modification, domains and motifs are currently under construction. + +## The Interaction Management System + +We have implemented an interaction management system (IMS) to support multiple simultaneous curators for each species supported by BioGRID. The IMS is a multiuser web-based application written in PHP that interfaces directly with the BioGRID. An intuitive graphical interface allows curators to quickly record interactions from an automatically updated list of publications. All interactions added via the IMS are verified against current annotation tables to eliminate errors and ambiguity in gene nomenclature. The IMS instantly commits new interactions to the BioGRID update pipeline, unless specified otherwise; interactions are collated each month and released as updates to the primary BioGRID and mirror sites, as well as model organism collaboration sites. Interactions may also be removed or modified in each monthly build, for example, in response to community feedback. + +All retired datasets are archived on the BioGRID downloads page in case the need for back-comparison arises. + +## Future Development + +We will continue to curate interactions from major model organism species, with a view to comprehensive backcuration, as we have done for S. cerevisiae and S. pombe. + +Further refinement of tools and display features in the BioGRID graphical user interface based on a flexible record tag structure will enable greater control over data views and downloadable datasets by the user. New plugins for data visualization are under development for Osprey, Cytoscape (24) and the Edinburgh Pathway Editor (25), +which will allow more sophisticated interrogation of interaction networks. In order to facilitate dissemination of our open source software tools, we will strive for compatibility with the Generic Model Organism Database +(GMOD) project (26). Finally, we will continue to develop our record structures in compliance with the Proteomics Standards Initiative Molecular Interactions (PSI-MI) +standard (27,28). + +## Acknowledgements + +We thank Gary Bader, Sue Rhee, Michael Cherry and David Botstein for helpful discussions; Eurie Hong and Benjamin Hitz for support at SGD; Rachel Drysdale and Don Gilbert for assistance in parsing interactions from Flybase; and Nevan Krogan, Jef Boeke, Tim Hughes and Charlie Boone for pre-publication release of largescale datasets. M.T. was supported by a Canada Research Chair in Functional Genomics and Bioinformatics, a Howard Hughes Medical Institute International Scholar Award and a Royal Society Wolfson Research Merit Award. D.H.L. and J.B. were supported by Cancer Research UK and V.W. was supported by the Wellcome Trust. This work was supported by a Canadian Institutes of Health Research grant (GSP-36651 to M.T.) and a NIH +National Center for Research Resources grant (1R01RR024031-01 to M.T. and K.D.). Funding to pay the Open Access publication charges for this article was provided by the NIH. + +Conflict of interest statement. None declared. + +## References + +1. Jorgensen,P., Breitkreutz,B.J., Breitkreutz,K., Stark,C., Liu,G., +Cook,M., Sharom,J., Nishikawa,J.L., Ketela,T. et al. (2003) Harvesting the genome's bounty: integrative genomics. Cold Spring Harb. Symp. Quant. Biol., 68, 431–443. + +2. Reguly,T., Breitkreutz,A., Boucher,L., Breitkreutz,B.J., Hon,G.C., +Myers,C.L., Parsons,A., Friesen,H., Oughtred,R. et al. (2006) +Comprehensive curation and analysis of global interaction networks in Saccharomyces cerevisiae. J. Biol., 5, 11. + +3. Batada,N.N., Reguly,T., Breitkreutz,A., Boucher,L., +Breitkreutz,B.J., Hurst,L.D. and Tyers,M. (2006) Stratus not altocumulus: a new view of the yeast protein interaction network. + +PLoS Biol., 4, e317. + +4. Mishra,G.R., Suresh,M., Kumaran,K., Kannabiran,N., Suresh,S., +Bala,P., Shivakumar,K., Anuradha,N., Reddy,R. et al. (2006) Human protein reference database–2006 update. Nucleic Acids Res., +34, D411–D414. + +5. Salwinski,L., Miller,C.S., Smith,A.J., Pettit,F.K., Bowie,J.U. and Eisenberg,D. (2004) The database of interacting proteins: 2004 update. Nucleic Acids Res., 32, D449–D451. + +6. Mewes,H.W., Frishman,D., Mayer,K.F., Munsterkotter,M., +Noubibou,O., Pagel,P., Rattei,T., Oesterheld,M., Ruepp,A. et al. (2006) MIPS: analysis and annotation of proteins from whole genomes in 2005. Nucleic Acids Res., 34, D169–D172. + +7. Kerrien,S., Alam-Faruque,Y., Aranda,B., Bancarz,I., Bridge,A., +Derow,C., Dimmer,E., Feuermann,M., Friedrichsen,A. et al. (2007) IntAct–open source resource for molecular interaction data. + +Nucleic Acids Res., 35, D561–D565. + +8. Chatr-Aryamontri,A., Ceol,A., Palazzi,L.M., Nardelli,G., +Schneider,M.V., Castagnoli,L. and Cesareni,G. (2007) MINT: the Molecular INTeraction database. Nucleic Acids Res., 35, D572–D574. + +9. Stark,C., Breitkreutz,B.J., Reguly,T., Boucher,L., Breitkreutz,A. + +and Tyers,M. (2006) BioGRID: a general repository for interaction datasets. Nucleic Acids Res., 34, D535–D539. + +10. Breitkreutz,B.J., Stark,C. and Tyers,M. (2003) Osprey: a network visualization system. Genome Biol., 4, R22. + +11. Gavin,A.C., Aloy,P., Grandi,P., Krause,R., Boesche,M., +Marzioch,M., Rau,C., Jensen,L.J., Bastuck,S. et al. (2006) Proteome survey reveals modularity of the yeast cell machinery. + +Nature, 440, 631–636. + +12. Ho,Y., Gruhler,A., Heilbut,A., Bader,G.D., Moore,L., Adams,S.L., +Millar,A., Taylor,P., Bennett,K. et al. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature, 415, 180–183. + +13. Krogan,N.J., Cagney,G., Yu,H., Zhong,G., Guo,X., +Ignatchenko,A., Li,J., Pu,S., Datta,N. et al. (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature, 440, 637–643. + +14. Nash,R., Weng,S., Hitz,B., Balakrishnan,R., Christie,K.R., +Costanzo,M.C., Dwight,S.S., Engel,S.R., Fisk,D.G. et al. (2007) Expanded protein information at SGD: new pages and proteome browser. Nucleic Acids Res., 35, D468–D471. + +15. Wixon,J. and Wood,V. (2006) Tools and resources for Sz. pombe: a report from the 2006 European Fission Yeast Meeting. Yeast, 23, 901–903. + +16. Gould,K.L., Ren,L., Feoktistova,A.S., Jennings,J.L. and Link,A.J. + +(2004) Tandem affinity purification and identification of protein complex components. Methods, 33, 239–244. + +17. Matsuyama,A., Arai,R., Yashiroda,Y., Shirai,A., Kamata,A., +Sekido,S., Kobayashi,Y., Hashimoto,A., Hamamoto,M. et al. (2006) ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat. Biotechnol., +24, 841–847. + +18. Roguev,A., Wiren,M., Weissman,J.S. and Krogan,N.J. (2007) +High-throughput genetic interaction mapping in the fission yeast Schizosaccharomyces pombe. Nature Methods, 4, 861–866. + +19. Drabkin,H.J., Hollenbeck,C., Hill,D.P. and Blake,J.A. (2005) +Ontological visualization of protein–protein interactions. + +BMC Bioinformatics, 6, 29. + +20. Muller,H.M., Kenny,E.E. and Sternberg,P.W. (2004) Textpresso: an ontology-based information retrieval and extraction system for biological literature. PLoS Biol., 2, e309. + +21. Rhee,S.Y., Beavis,W., Berardini,T.Z., Chen,G., Dixon,D., +Doyle,A., Garcia-Hernandez,M., Huala,E., Lander,G. et al. (2003) The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res., 31, 224–228. + +22. Batada,N.N., Reguly,T., Breitkreutz,A., Boucher,L., +Breitkreutz,B.J., Hurst,L.D. and Tyers,M. (2007) Still stratus not altocumulus: further evidence against the date/party hub distinction. PLoS Biol., 5, e154. + +23. Harris,M.A., Clark,J., Ireland,A., Lomax,J., Ashburner,M., +Foulger,R., Eilbeck,K., Lewis,S., Marshall,B. et al. (2004) +The Gene Ontology (GO) database and informatics resource. + +Nucleic Acids Res., 32, D258–D261. + +24. Shannon,P., Markiel,A., Ozier,O., Baliga,N.S., Wang,J.T., +Ramage,D., Amin,N., Schwikowski,B. and Ideker,T. (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res., 13, 2498–2504. + +25. Sorokin,A., Paliy,K., Selkov,A., Demin,O.V., Dronov,S., Ghazal,P. + +and Goryanin,I. (2006) The Pathway Editor: a tool for managing complex biological networks. IBM J. Res. Dev., 6, 561–575. + +26. Stein,L.D., Mungall,C., Shu,S., Caudy,M., Mangone,M., Day,A., +Nickerson,E., Stajich,J.E., Harris,T.W. et al. (2002) The generic genome browser: a building block for a model organism system database. Genome Res., 12, 1599–1610. + +27. Orchard,S., Hermjakob,H. and Apweiler,R. (2003) The proteomics standards initiative. Proteomics, 3, 1374–1376. + +28. Orchard,S., Salwinski,L., Kerrien,S., Montecchi-Palazzi,L., +Oesterheld,M., Stumpflen,V., Ceol,A., Chatr-Aryamontri,A., Armstrong,J. et al. (2007) The minimum information required for reporting a molecular interaction experiment (MIMIx). + +Nat. Biotechnol., 25, 894–898. \ No newline at end of file diff --git a/medical/md_images/PMC3171077/1_image_1.png b/medical/md_images/PMC3171077/1_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..7cb661364a651f06141d883218f3225ebec25df5 --- /dev/null +++ b/medical/md_images/PMC3171077/1_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f0958b89d86ab23e38380bde3a9606077295c64c2e024e42ab2d335879a05800 +size 19796 diff --git a/medical/md_images/PMC3171077/2_image_0.png b/medical/md_images/PMC3171077/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..926c9792d3824692f67763093d28cfecb1705b27 --- /dev/null +++ b/medical/md_images/PMC3171077/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:88ad2bef6df489033a3bee0a8738bf997ae1cf39ba9d3635bfec08fa21acdebf +size 64420 diff --git a/medical/md_images/PMC3276013/2_image_0.png b/medical/md_images/PMC3276013/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..54ca147d0c5afe445acc77cc99659aef3460ca84 --- /dev/null +++ b/medical/md_images/PMC3276013/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:679ecd2c8c82adbcd4167170fe5fc21f99000c867ea35a3d84a4e6df80bd804f +size 110567 diff --git a/medical/md_images/PMC3276013/2_image_2.png b/medical/md_images/PMC3276013/2_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..11fb1f35573d1b767a054bc8b077807f93d545f5 --- /dev/null +++ b/medical/md_images/PMC3276013/2_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:96079d5427de3900cf62019c30e10515e01b82b295eea1bc637129dc940b6f34 +size 99534 diff --git a/medical/md_images/PMC3465144/2_image_2.png b/medical/md_images/PMC3465144/2_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..3d0791f13a45deb9d0aa1f673c99401b87304930 --- /dev/null +++ b/medical/md_images/PMC3465144/2_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:7143764fc3087841ad7ffddfd878702dceb863bfb9cd36199463f97aa7f7ea2f +size 52914 diff --git a/medical/md_images/PMC3639050/2_image_0.png b/medical/md_images/PMC3639050/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..64deafe43282fd663deef12a13c4fd240678dfe1 --- /dev/null +++ b/medical/md_images/PMC3639050/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:9f2077439b01ab5d1f5aeb2ea54ad50ada77014b63910e0c1fe33a41fc86b9d3 +size 89707 diff --git a/medical/md_images/PMC3639050/5_image_0.png b/medical/md_images/PMC3639050/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..ba8918132f65d5d475e35d3312d1f499b1ef89c6 --- /dev/null +++ b/medical/md_images/PMC3639050/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:54889ba2f5b57a2df1d2a6597a09f0b43b283b6ce09942a51cde84655466fa81 +size 138574 diff --git a/medical/md_images/PMC4142939/2_image_1.png b/medical/md_images/PMC4142939/2_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..d84961f09bff0dc5d0391815da65c72c91037ee3 --- /dev/null +++ b/medical/md_images/PMC4142939/2_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:55514027bdfb95466145a731c9ce23dd8ef247ff6432377c62434019511d731a +size 99544 diff --git a/medical/md_images/PMC4142939/4_image_1.png b/medical/md_images/PMC4142939/4_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..1403e3eaf1e643dd1c896e74525985998d1c7344 --- /dev/null +++ b/medical/md_images/PMC4142939/4_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:2bbf74006d2beba16afc17fc115b5ef21621016dca239f7e03d5ed87da1764cb +size 85337 diff --git a/medical/md_images/PMC4203998/2_image_0.png b/medical/md_images/PMC4203998/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..aa8228773133851ff8aba1cfb59e35e848e04a2d --- /dev/null +++ b/medical/md_images/PMC4203998/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:bd2b5c7a0e759abffae51fb456cb69bdfb9d3bb11e56f44ff22c352627660930 +size 8015 diff --git a/medical/md_images/PMC4203998/3_image_0.png b/medical/md_images/PMC4203998/3_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..b62dedfed2dab2dbf0c1f037566683b3c155a3a2 --- /dev/null +++ b/medical/md_images/PMC4203998/3_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:3e873639fe76fd8e062d73abf8a3f258402505aabd7c1097cad4b31c682d7e3c +size 24957 diff --git a/medical/md_images/PMC4203998/PMC4203998.md b/medical/md_images/PMC4203998/PMC4203998.md new file mode 100644 index 0000000000000000000000000000000000000000..9cc6f5ab503cd9f5d76cc6d54bc58099c1c9cf55 --- /dev/null +++ b/medical/md_images/PMC4203998/PMC4203998.md @@ -0,0 +1,220 @@ +# Common Misconceptions About Data Analysis And Statistics + +Harvey J. Motulsky Received: 8 August 2014 /Accepted: 14 August 2014 /Published online: 12 September 2014 \# The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Ideally, any experienced investigator with the right tools should be able to reproduce a finding published in a peerreviewed biomedical science journal. In fact, the reproducibility of a large percentage of published findings has been questioned. Undoubtedly, there are many reasons for this, but one reason maybe that investigators fool themselves due to a poor understanding of statistical concepts. In particular, investigators often make these mistakes: 1. P-Hacking. This is when you reanalyze a data set in many different ways, or perhaps reanalyze with additional replicates, until you get the result you want. 2. Overemphasis on P values rather than on the actual size of the observed effect. 3. Overuse of statistical hypothesis testing, and being seduced by the word "significant". 4. Overreliance on standard errors, which are often misunderstood. + +## Introduction + +Ideally, any experienced investigator with the right tools should be able to reproduce a finding published in a peerreviewed biomedical science journal. In fact, the reproducibility of a large percentage of published findings has been questioned. Investigators at Bayer Healthcare were reportedly This commentary evolved from multiple conversations between the author and editors of several pharmacology journals. This work represents solely the opinions of the author. Publication of this article does not represent an endorsement of GraphPad Software. This article is being simultaneously published in Br J Pharmacol, J Pharmacol Exp Ther, NaunynSchmiedeberg's Arch Pharmacol, and Pharmacol Res Perspect in a collaborative effort to help investigators and readers to appropriately use and interpret statistical analyses in pharmacological research studies. This work is licensed under a Creative Commons Attribution-NoDerivs 4.0 International (CC BY-ND 4.0) license (https://creativecommons.org/ +licenses/by-nd/4.0/legalcode). doi: 10.1007/s00210-014-1037-6. + +H. J. Motulsky (*) +GraphPad Software Inc., La Jolla, California, USA e-mail: hmotulsky@graphpad.com able to reproduce only 20–25 % of 67 preclinical studies +(Prinz et al. 2011), and investigators at Amgen were able to reproduce only 6 of 53 studies in basic cancer biology despite often cooperating with the original investigators (Begley and Ellis 2012). This problem has been featured in a cover story in The Economist (Anonymous 2013) and has attracted the attention of the NIH leaders (Collins and Tabak 2014). + +Why can so few findings be reproduced? Undoubtedly, there are many reasons. But in many cases, I suspect that investigators fooled themselves due to a poor understanding of statistical concepts (see Marino 2014, for a good review of this topic). Here, I identify five common misconceptions about statistics and data analysis, and explain how to avoid them. My recommendations are written for pharmacologists and other biologists publishing experimental research using commonly used statistical methods. They would need to be expanded for analyses of clinical or observational studies and for Bayesian analyses. This editorial is about analyzing and displaying data, so it does not address issues of experimental design. + +My experience comes from basic pharmacology research conducted decades ago, followed by 25 years of answering email questions from scientists needing help analyzing data with GraphPad Prism,1 and authoring three editions of the text Intuitive Biostatistics (Motulsky 2014a). + +## Misconception 1: P-Hacking Is Ok + +Statistical results can only be interpreted at face value when every choice in data analysis was performed exactly as planned and documented as part of the experimental design. + +From my conversations with scientists, it seems that this rule is commonly broken in reports of basic research. Instead, analyses are often done as shown in Fig. 1. Collect and analyze 1 http://www.graphpad.com/prism + +![1_image_0.png](1_image_0.png) + +some data. If the results are not statistically significant but show a difference or trend in the direction you expected, collect some more data and reanalyze. Or try a different way to analyze the data: remove a few outliers; transform to logarithms; try a nonparametric test; redefine the outcome by normalizing (say, dividing by each animal's weight); use a method to compare one variable while adjusting for differences in another; the list of possibilities is endless. Keep trying until you obtain a statistically significant result or until you run out of money, time, or curiosity. + +The results from data collected this way cannot be interpreted at face value. Even if there really is no difference +(or no effect), the chance of finding a "statistically significant" result exceeds 5 %. The problem is that you introduce bias when you choose to collect more data (or analyze the data differently) only when the P value is greater than 0.05. If the P +value was less than 0.05 in the first analyses, it might be larger than 0.05 after collecting more data or using an alternative analysis. But you would never see this if you only collected more data or tried different data analysis strategies when the first P value was greater than 0.05. + +Exploring your data can be a very useful way to generate hypotheses and make preliminary conclusions. But all such analyses need to be clearly labeled, and then retested with new data. + +There are three related terms that describe this problem: +& Ad hoc sample size selection. This is when you did not choose a sample size in advance, but just kept going until you liked the results. Figure 2 demonstrates the problem with ad hoc sample size determination. Distinguish unplanned ad hoc sample size decisions from planned "adaptive" sample size methods that make you "pay" for the increased versatility in sample size collection by requiring a stronger effect to reach "significance" (Kairalla et al. 2012; FDA 2010). + +& Hypothesizing after the result is known (HARKing; Kerr 1998). This is when you analyze the data in many different ways (say different subgroups), discover an intriguing relationship, and then publish the data so it appears that the hypothesis was stated before the data were collected (Fig. 3). This is a form of multiple comparisons (Berry 2007). Kriegeskorte et al. (2009) call this double dipping, + +![2_image_0.png](2_image_0.png) + +![2_image_1.png](2_image_1.png) + +as you are using the same data both to generate a hypothesis and to test it. + +& P-hacking. This is a general term that encompasses dynamic sample size collection, HARKing, and more. It was coined by Simmons et al. (2011) who also use the phrase, "too many investigator degrees of freedom." P-hacking is especially misleading when it involves changing the actual values analyzed. + +Examples include ad hoc sample size selection (see above), +switching to an alternate control group (if you do not like the first results and your experiment involved two or more control groups), trying various combinations of independent variables to include in a multiple regression (whether the selection is manual or automatic), and analyzing various subgroups of the data. Reanalyzing a single data set in various ways is also Phacking but will not usually mislead you quite as much. + +## My Suggestion For Authors: + +& For each figure or table, clearly state whether or not the sample size was chosen in advance, and whether every step used to process and analyze the data was planned as part of the experimental protocol. + +& If you use any form of P-hacking, label the conclusions as +"preliminary." + +![2_image_2.png](2_image_2.png) + +Misconception 2: P values convey information about effect size To compute a P value, you first must clearly define a null hypothesis, usually that two means (or proportions or EC50's…) are identical. Given some assumptions, the P values are the probability of seeing an effect as large as or larger than you observed in the current experiment if in fact the null hypothesis was true. But note that the P value gives you no information about how large the difference (or effect) is. Figure 4 demonstrates this point by plotting the P values that result from comparing two samples in experiments with different sample sizes. Even though the means and standard deviations are identical for each simulated experiment, the P values are far from identical. With n=3 in each group, the P value is 0.65. When n=300 in each group, the P value is 0.000001. + +The dependence of P values on sample size can lead to two problems. + +## A Large P Value Is Not Proof Of No (Or Little) Effect + +The top two rows of Table 1 presents the results of two simulated experiments. The two P values are both about 0.6, but the two experiments lead to very different conclusions. + +In experiment A (from Table 1), the difference between means in the experimental sample is 10, so the difference equals 1 % of the mean of treatment 1. Assuming random sampling from Gaussian populations, the 95 % confidence interval for the difference between the two population means ranges from −30 to 50. In other words, the data are consistent (with 95 % confidence) with a decrease of 3 %, an increase of 5 %, or anything in between. The interpretation depends on the scientific context and the goals of the experiment, but in most contexts, these results can be + +![3_image_0.png](3_image_0.png) + +summarized simply: The data are consistent with a tiny decrease, no change, or a tiny increase. These are solid negative data. + +Experiment B is very different. The difference between means is larger, and the confidence interval is much wider (because the sample size is so small). Assuming random sampling from Gaussian populations, the data are consistent +(with 95 % confidence) with anything between a decrease of 18 % and an increase of 28 %. The data are consistent with a large decrease, a small decrease, no difference, a small increase, or a large increase. These data lead to no useful conclusion at all! An experiment like this should not be published. + +## A Small P Value Is Not Proof Of A Large Effect + +The bottom two rows of Table 1 presents the results of two simulated experiments where both P values are 0.001, but again two experiments lead to very different conclusions. + +In experiment C (from Table 1), the difference between means in the experimental sample is only 2 (so the difference equals 2 % of the mean of treatment 1). Assuming random sampling from Gaussian populations, the 95 % confidence interval for the difference between the two population means ranges from 0.8 to 3.2. In other words, the data are consistent +(with 95 % confidence) with anything between an increase of 0.8 % and an increase of 3.2 %. How to interpret that depends on the scientific context and the goals of the experiment, but in most contexts, this can be summarized simply: The data clearly demonstrate an increase, but that increase is tiny. + +Experiment D is very different. The difference between means is 35 (so 35 % of the control mean), and the confidence interval extends from an increase of 23.7 % to an increase of 46.3 %. The data clearly demonstrate that there is an increase that is (with 95 % confidence) substantial. + +My suggestions for authors: + +& Always show and emphasize the effect size (as difference, percent difference, ratio, or correlation coefficient) along with its confidence interval. + +& Consider omitting reporting of P values. + +## Misconception 3: Statistical Hypothesis Testing And Reports Of "Statistical Significance" Are Necessary In Experimental Research + +Statistical hypothesis testing is a way to make a crisp decision from one analysis. If the P value is less than a preset value +(usually 0.05), the result is deemed "statistically significant" and you make one decision. Otherwise, the result is deemed "not statistically significant" and you make the other decision. This is helpful in quality control and some clinical studies. It also is useful when you rigorously compare the fits of two scientifically + +## Table 1 Identical P Values With Very Different Interpretations + +Treatment 1 (mean±SD, n) Treatment 2 + +(mean±SD, n) + +Difference between means P value 95 % CI of the difference + +between means + +| Table 1 Identical P values with very different interpretations Treatment 1 (mean±SD, n) Treatment 2 (mean±SD, n) Experiment A 1,000±100, n=50 990.0±100, n=50 | 10 | 0.6 | −30 to 50 | | | +|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|----------------|-------------|-------|-------------| +| Experiment B | 1,000±100, n=3 | 950.0±100, n=3 | 50 | 0.6 | −177 to 277 | +| Experiment C | 100±5.0, n=135 | 102±5.0, n=135 | 2 | 0.001 | 0.8 to 3.2 | +| Experiment D | 100±5.0, n=3 | 135±5.0, n=3 | 35 | 0.001 | 24 to 46 | + +Experiments A and B have identical P values, but the scientific conclusion is very different. The interpretation depends upon the scientific context, but in most fields experiment A would be solid negative data proving that there either is no effect or that the effect is tiny. In contrast, experiment B has such a wide confidence interval as to be consistent with nearly any hypothesis. Those data simply do not help answer your scientific question Similarly, experiments C and D have identical P values, but should be interpreted differently. In most experimental contexts, experiment C demonstrates convincingly that while the difference is not zero, it is quite small. Experiment D provides convincing evidence that the effect is large sensible models to your data, and choose one to guide your interpretation of the data and to plan future experiments. + +Here are five reasons to avoid use of statistical hypothesis testing in experimental research: +& The need to make a crisp decision based on one analysis is rare in basic research. A decision about whether or not to place an asterisk on a figure does not count! If you are not planning to make a crisp decision, the whole idea of statistical hypothesis testing is not helpful. + +& Statistical hypothesis testing "does not tell us what we want to know, and we so much want to know what we want to know that, out of desperation, we nevertheless believe that it does!" (Cohen 1994). Statistical hypothesis testing has even been called a cult (Ziliak and McCloskey 2008). The question we want to answer is: Given these data, how likely is the null hypothesis? The question that a P values answers is: Assuming the null hypothesis is true, how unlikely are these data? These two questions are distinct, and so have distinct answers. + +& Scientists who intend to use statistical hypothesis testing often end up not using it. If the P value is just a bit larger than 0.05, scientists often avoid the strict use of hypothesis testing and instead apply the "time-honored tactic of circumlocution to disguise the nonsignificant result as something more interesting" (Hankins 2013). They do this by using terms such as "almost significant," "bordered on being statistically significant," "a statistical trend toward significance," or "approaching significance." Hankins lists 468 such phrases he found in published papers! + +& The 5 % significance threshold is often misunderstood. If you use a P value to make a decision, of course it is possible that you will make the wrong decision. In some cases, the P value will be tiny just by chance, even though the null hypothesis of no difference is actually true. In these cases, a conclusion that a finding is statistically significant is a false positive and you will have made what is called a type I error. + +2 Many scientists 2 In contrast, a type II, or false negative, error is when there really is a difference but the result in your experiment is not statistically significant. + +mistakenly believe that the chance of making a false positive conclusion is 5 %. In fact, in many situations, the chance of making a type I false positive conclusion is much higher than 5 % (Colquhoun 2014). For example, in a situation where you expect the null hypothesis to be true 90 % of the time (say you are screening lightly prescreened compounds, so expect 10 % to work), you have chosen a sample size large enough to ensure 80 % +power, and you use the traditional 5 % significance level, the false discovery rate is not 5 % but rather is 36 % (the calculations are shown in Table 2). If you only look at experiments where the P value is just a tiny bit less than Table 2 The false discovery rate when P<0.05 This table tabulates the theoretical results of 1,000 experiments where the prior probability that the null hypothesis is false is 10 %, the sample size is large enough so that the power is 80 %, and the significance level is the traditional 5 %. In 100 of the experiments (10 %), there really is an effect +(the null hypothesis is false), and you will obtain a "statistically significant" result (P<0.05) in 80 of these (because the power is 80 %). In 900 experiments, the null hypothesis is true but you will obtain a statistically significant result in 45 of them (because the significance threshold is 5 and 5 % of 900 is 45). In total, you will obtain 80+45=125 statistically significant results, but 45/125=36 % of these will be false positive. The proportion of conclusions of "statistical significance" that are false discoveries or false positives depends on the context of the experiment, as expressed by the prior probability (here 10 %). If you do obtain a small P value and reject the null hypothesis, you will conclude that the values in the two groups were sampled from different distributions. As noted above, there may be a high chance that you made a false positive conclusion due to random sampling. But even if the conclusion is "true" from a statistical point of view and not a false positive due to random sampling, the effect may have occurred for a reason different than the one you hypothesized. When thinking about why an effect occurred, ignore the statistical calculations, and instead think about blinding, randomization, positive controls, negative controls, calibration, biases, and other aspects of experimental design. + +| P<0.05 | P>0.05 | Total | | +|----------------------------------|----------|---------|-------| +| Really is an effect | 80 | 20 | 100 | +| No effect (null hypothesis true) | 45 | 855 | 900 | +| Total | 125 | 875 | 1,000 | + +0.050, the probability of a false positive rises to 79 % +(Motulsky 2014b). Ioannidis (2005) used calculations like these (and other considerations) to argue that most published research findings are probably false. + +& The word "significant" is often misunderstood. The problem is that "significant" has two distinct meanings in science (Motulsky 2014c). One meaning is that a P value is less than a preset threshold (usually 0.05). The other meaning of "significant" is that an effect is large enough to have a substantial physiological or clinical impact. These two meanings are completely different, but are often confused. + +My suggestions for authors: +& Only report statistical hypothesis testing (and place significance asterisks on figures) when you will make a decision based on that one analysis. + +& Never use the word "significant" in a scientific paper. If you use statistical hypothesis testing to make a decision, state the P value, your preset P value threshold, and your decision. When discussing the possible physiological or clinical impacts of a finding, use other words. + +## Misconception 4: The Standard Error Of The Mean Quantifies Variability + +Pharmacology journals are full of graphs and tables showing the mean and the standard error of the mean (SEM). + +A quick review. The standard deviation (SD) quantifies variation among a set of values, but the SEM does not. The SEM is computed by dividing the SD by the square root of sample size. With large samples, the SEM will be tiny even if there is a lot of variability. + +One problem with plotting or displaying the mean±SEM is that some people viewing the graph or table may mistakenly think that the error bars show the variability of the data. A second problem with reporting means with SEM is that the range mean±SEM cannot be rigorously interpreted. The SEM +gives information about how precisely you have determined the population mean. So the range mean±SEM is a confidence interval, but the confidence level depends on sample size. With large samples, that range is a 68 % CI of the mean. + +When n=3, that range is only a 58 % CI.3 My suggestions for authors: +& If you want to display the variability among the values, show raw data (which is not done often enough in my opinion). If 3 Computed using this Excel formula: = (1-T.DIST.2T (1.0,2)). The first argument (1.0) is the number of SEMs (in each direction) included in the confidence interval, and the second argument (2) is the number of degrees of freedom, which equals n-1. + +showing the raw data would make the graph hard to read, show instead a box-whisker plot, a frequency distribution, or the mean and SD. + +& If you want readers to see how precisely you have determined the mean, report a confidence interval (95 % confidence intervals are standard). Figure 5 shows a dataset plotted using all of these methods. + +& When reporting results from regression, show the 95 % +confidence interval of each parameter rather than standard errors. + +## Misconception 5: You Do Not Need To Report The Details + +The methods section of every paper should report the methods with enough detail that someone else could reproduce your work. This applies to statistical methods just as it does to experimental methods. + +My suggestions for authors: +& When reporting a sample size, explain exactly what you counted. Did you count replicates in one experiment + +![5_image_0.png](5_image_0.png) + +(technical replicates), repeat experiments, the number of studies pooled in a meta-analysis, or something else? + +& If you eliminated any outliers, state how many outliers you eliminated, the rule used to identify them, and a statement whether this rule was chosen before collecting data. + +& If you normalized data, explain exactly how you defined 100 and 0 %. + +& When possible, report the P value up to at least a few digits of precision, rather than just stating whether the P +value is less than or greater than an arbitrary threshold. For each P value, state the null hypothesis it tests if there is any possible ambiguity. + +& When reporting a P value that compares two groups, state whether the P value is one- or two-tailed. If you report a one-tailed P value, state that you recorded a prediction for the direction of the effect (for example increase or decrease) before you collected any data and what this prediction was. If you did not record such a prediction, report a two-tailed P value. + +& Explain the details of the statistical methods you used. For example, if you fit a curve using nonlinear regression, explain precisely which model you fit to the data and whether (and how) data were weighted. + +Also state the full version number and platform of the software you use. + +& Consider posting files containing both the raw data and the analyses so other investigators can see the details. + +## Summary + +The physicist E. Rutherford supposedly said, "If your experiment needs statistics, you ought to have done a better experiment4." There is a lot of truth to that statement when you are working in a field with a very high signal-to-noise ratio. In these fields, statistical analysis may not be necessary. But if you work in a field with a lower signal-to-noise ratio, or are trying to compare the fits of alternative models that do not differ all that much, you need statistical analyses to properly quantify your confidence in your conclusions. + +I suspect that one of the reasons that the results reported in many papers cannot be reproduced is that statistical analyses are often done as a quick afterthought, with the goal to sprinkle asterisks on figures and the word "significant" on conclusions. The suggestions I propose in this commentary can all be summarized simply: If you are going to analyze your data using statistical methods, then plan the methods carefully, do the analyses seriously, and report the data, methods, and results completely. + +Open Access This article is distributed under the terms of the Creative Commons Attribution NoDerivatives 4.0 International License which permits re-use and distribution of the original version in any medium provided that inter alia the original author(s) and the source are credited. The license does not allow the distribution of modified versions of the article. + +## References + +Anonymous (2013). Trouble at the lab. The Economist 409:23–27, 2013 Begley CG, Ellis LM (2012) Raise standards for preclinical cancer research. Nature 483:533 Berry DA (2007) The difficult and ubiquitous problems of multiplicities. + +Pharm Stat 6:155–160 Cohen J (1994) The earth is round (p<0.05). Am Psychol 49:997-1003 Collins FS, Tabak LA (2014) Policy: NIH plans to enhance reproducibility. Nature 505:612–613 Colquhoun D (2014). An investigation of the false discovery rate and the misinterpretation of P values. http://arxiv.org/abs/1407.5296 FDA (2010) Guidance for Industry: Adaptive Design Clinical Trials for Drugs and Biologics. Accessed July 29, 2014 at http://www.fda.gov/ +downloads/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/UCM201790.pdf Hankins, M. Still not significant. Psychologically Flawed April 21, 2013. + +Accessed July 28, 2013 at mchankins.wordpress.com/2013/04/21/ +still-not-significant-2/ +Ioannidis JPA (2005) Why most published research findings are false. + +PLoS Med 2:e124 Kairalla JA, Coffey CS, Thomann MA, Muller KE (2012) Adaptive trial designs: a review of barriers and opportunities. Trials 13:145–145 Kerr NL (1998) HARKing: hypothesizing after the results are known. + +Pers Soc Psychol Rev 2:196–217 Kriegeskorte N, Simmons WK, Bellgowan PSF, Baker CI (2009) +Circular analysis in systems neuroscience: the dangers of double dipping. Nat Neurosci 12:535–540 Marino MJ (2014) The use and misuse of statistical methodologies in pharmacology research. Biochem Pharmacol 87:78–92 Motulsky, HJ (2014a) Intuitive Biostatistics 3rd edition, Oxford University Press Motulsky HJ (2014b) Using simulations to calculate the false discovery rate. Accessed June 1, 2014 at http://www.graphpad.com/support/ faqid/1923/ +Motulsky, HJ (2014c). Opinion: Never use the word 'significant' in a scientific paper. Advances in Regenerative Biology 2014, 1: 25155. http://dx.doi.org/10.3402/arb.v1.25155 (in press) +Prinz F, Schlange T, Asadullah K (2011) Believe it or not: how much can we rely on published data on potential drug targets? Nat Rev Drug Discovery 10:712–713. DOI 10.1038/ +Simmons J, Nelson L, Simonsohn U (2011) False-positive psychology: +undisclosed flexibility in data collection and analysis allow presenting anything as significant. Psychol Sci 22:1359–1366 Ziliak, S, & McCloskey, DN (2008). The cult of statistical significance: +How the standard error costs us jobs, justice, and lives. Ann Arbor: +University of Michigan Press. ISBN=0472050079. + +4 The quotation is widely attributed to this famous physicist, but I cannot find an actual citation. \ No newline at end of file diff --git a/medical/md_images/PMC4203998/PMC4203998_meta.json b/medical/md_images/PMC4203998/PMC4203998_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..6a3dc19073bf720ee74db8b012e396a15a9b3504 --- /dev/null +++ b/medical/md_images/PMC4203998/PMC4203998_meta.json @@ -0,0 +1,196 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Common misconceptions about data analysis and statistics", + "level": 0, + "is_closed": false, + "n_kids": 11, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 791.0, + 596.0, + 791.0 + ] + }, + { + "title": "Abstract", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 52.0, + 548.0, + 545.0, + 548.0 + ] + }, + { + "title": "Introduction", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 52.0, + 327.0, + 290.0, + 327.0 + ] + }, + { + "title": "Misconception 1: P-Hacking is OK", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 307.0, + 202.0, + 545.0, + 202.0 + ] + }, + { + "title": "Misconception 2: P values convey information about effect size", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 52.0, + 725.0, + 290.0, + 725.0 + ] + }, + { + "title": "A large P value is not proof of no (or little) effect", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 52.0, + 488.0, + 290.0, + 488.0 + ] + }, + { + "title": "A small P value is not proof of a large effect", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 307.0, + 576.0, + 545.0, + 576.0 + ] + }, + { + "title": "Misconception 3: statistical hypothesis testing and reports of \u201cstatistical significance\u201d are necessary in experimental research", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 307.0, + 190.0, + 545.0, + 190.0 + ] + }, + { + "title": "Misconception 4: the standard error of the mean quantifies variability", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 5, + "view_mode": "FitR", + "view_pos": [ + 52.0, + 426.0, + 290.0, + 426.0 + ] + }, + { + "title": "Misconception 5: you do not need to report the details", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 5, + "view_mode": "FitR", + "view_pos": [ + 307.0, + 588.0, + 545.0, + 588.0 + ] + }, + { + "title": "Summary", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "FitR", + "view_pos": [ + 52.0, + 376.0, + 290.0, + 376.0 + ] + }, + { + "title": "References", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "FitR", + "view_pos": [ + 307.0, + 592.0, + 545.0, + 592.0 + ] + } + ], + "pages": 7, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 0, + "code": 0, + "table": 2, + "equations": { + "successful_ocr": 0, + "unsuccessful_ocr": 0, + "equations": 0 + } + }, + "postprocess_stats": { + "edit": {} + } +} \ No newline at end of file diff --git a/medical/md_images/PMC4616385/4_image_1.png b/medical/md_images/PMC4616385/4_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..7d244b8beafd6af5ed2203a6d87b3e5cbcea313b --- /dev/null +++ b/medical/md_images/PMC4616385/4_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f3eacaac149d52489489135c6e1a68b6ab6ac728b7c9091535b86dc145857052 +size 19914 diff --git a/medical/md_images/PMC4616385/PMC4616385.md b/medical/md_images/PMC4616385/PMC4616385.md new file mode 100644 index 0000000000000000000000000000000000000000..1a99e2143dd4d1d34e57137434c0ca3ef846e604 --- /dev/null +++ b/medical/md_images/PMC4616385/PMC4616385.md @@ -0,0 +1,364 @@ +# Evaluation Of A Novel Functional Single-Nucleotide Polymorphism (Rs35010275 G>C) In Mir196A2 Promoter Region As A Risk Factor Of Gastric Cancer In A Chinese Population + +Ming Xu, PhD, Fulin Qiang, MD, Yan Gao, MD, Meiyun Kang, MD, Meilin Wang, PhD, +Guoquan Tao, PhD, Weida Gong, PhD, Haixia Zhu, MD, Dongmei Wu, PhD, +Zhengdong Zhang, PhD, and Qinghong Zhao, PhD +Abstract: Single-nucleotide polymorphisms (SNPs) in microRNAs +(miRNAs) have been suggested to influence the occurrence and progression of cancer through altering the expression and biological function of miRNAs. The aim of this study was to investigate whether the potential functional SNPs in MIR196A2 promoter had effect on the susceptibility to gastric cancer (GC) in a Chinese population. + +We conducted a 2-stage case–control study (753 cases and 854 controls in testing set; 940 cases and 1061 controls in validation set) to evaluate the association between 2 potential functional SNPs in MIR196A2 promoter (rs12304647 A>C and rs35010275 G>C) and GC risk. The luciferase reporter assay and electrophoretic mobility shift assay were used to examine the functionality of the important polymorphism. + +We found that the rs35010275 C allele was significantly associated with the decreased risk of GC (adjusted odds ratio ¼ 0.85, 95% confidence interval¼ 0.77–0.94) in the combined case–control studies. + +The miR-196a expression levels in GC tissues were significantly higher than that in corresponding adjacent normal tissues (P < 0.001). Besides, Editor: Bulent Kantarceken. Received: June 15, 2014; revised: September 9, 2014; accepted: September 13, 2014. From the Department of Environmental Genomics (MX, YG, MK, MW, +HZ, DW, ZZ), Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, Nanjing; Department of Genetic Toxicology (MX, YG, MK, MW, DW, ZZ), The Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing; Core Laboratory of Nantong Tumor Hospital (FQ, HZ), Nantong; Department of General Surgery (GT), Huai-An First People's Hospital Affiliated to Nanjing Medical University, Huai-an; Department of General Surgery (WG), Yixing Cancer Hospital, Yixing; and Department of General Surgery (QZ), The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China. + +Correspondence: Zhengdong Zhang, Department of Environmental Genomics, School of Public Health, Nanjing Medical University, 818 East Tianyuan Road, Jiangning District, Nanjing 211166, China (e-mail: drzdzhang@gmail.com). + +MX, FQ, and YG contributed equally to this work. This study was partly supported by the National Natural Science Foundation of China (81230068, 81302490, 81373091, and 81473049), the Natural Science Foundation of Jiangsu Province (BK2011194 and BK2012842), the Key Program for Basic Research of Jiangsu Provincial Department of Education (12KJA330002), Jiangsu Provincial Science and Technology Innovation Team, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine). + +The authors have no funding and conflicts of interest to disclose. + +Copyright \# 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. + +This is an open access article distributed under the Creative Commons Attribution-NonCommercial License, where it is permissible to download, share and reproduce the work in any medium, provided it is properly cited. The work cannot be used commercially. ISSN: 0025-7974 DOI: 10.1097/MD.0000000000000173 each allele of rs35010275 displayed completely opposite effects to influence the transcription activity of MIR196A2 promoter via recruiting different transcription factors or complexes. + +The functional rs35010275 G>C polymorphism in MIR196A2 promoter was significantly associated with miR-196a expression and influenced the genetic susceptibility to GC. (Medicine 93(26):e173) +Abbreviations: 30-UTR = 30-untranslated region, EMSA = +electrophoretic mobility shift assay, GC = gastric cancer, HWE = Hardy–Weinberg equilibrium, MAF = minor allele frequency, SNPs = single-nucleotide polymorphisms. + +## Introduction + +Gastric cancer (GC) remains the fourth most common cancer worldwide with a huge number of new diagnosed cases every year, especially in Asian countries.1,2 In recent decades, GC mortality has been reported to decline; however, it is still the second fatal cause in China, following lung cancer.2 Clinical data have shown that most GC patients were diagnosed with an advanced stage, which means these patients have missed their best treatment period and will confront a poor survival. A better understanding of the molecular mechanisms underlying gastric carcinogenesis may help to identify more accurate diagnostic markers and even more effective treatment strategies for this lethal disease.3–5 Classic genetic studies have implicated that aberrant expressions of tumor-suppressor genes or oncogenes are crucial factors for GC occurrence. With the development of epigenomics, alterations of CpG islands and whole genomic methylation were proved to be involved in gastric carcinogenesis.6–8 Besides, accumulating evidences have indicated that microRNAs (miRNAs), as important factors, also participate in the etiology of GC.9–11 MiRNAs are a sort of endogenous small noncoding RNAs, which cleaved from 70 to 100 nucleotides hairpin miRNA precursors, and are about 19 to 25 nucleotides long in mature form. In eukaryotes, mature miRNAs mainly affect the expressions of diverse protein-coding genes by targeting their mRNA in 30-untranslated regions +(30-UTRs), and then leading to a post-transcriptional retardation by either inhibiting mRNAs' translation or accelerating their degradation.12 It has been reported that miRNAs, including miR-196a, were noticed for their aberrant behaviors on various target mRNAs covering almost all of the important signal pathways.13–16 Recently, single-nucleotide polymorphisms (SNPs) in several miRNAs genes have aroused researchers' concern. The potential role of these SNPs have been identified in the cancer development, such as nasopharyngeal carcinoma,17 breast cancer,18,19 hepatocellular carcinoma,20,21 lung cancer,22,23 head and neck cancer,24 and GC.25,26 The MIR196A2 rs11614913, as a prestigious biomarker in cancers, including GC, was reported in our previous study.27 The variant of rs11614913 could strongly influence the binding ability of mature miR-196a to its target mRNAs.28,29 Overexpression of miR-196a was common in colon cancer and GC.16,30–32 Considering functional polymorphisms in promoter region could influence gene expression,33–35 it is rational to hypothesize that there exist some SNPs in MIR196A2 promoter region that might directly trigger the transcription of MIR196A2 and finally affect GC susceptibility. In our previous report, rs11614913 SNP was associated with the risk of GC.36 However, as a variant existing in mature miRNA, it was still difficulty to affect the transcription of miR-196a-2. As an intergenic miRNA gene, MIR196A2 has recently been reported to possess its own promoter that might alter miR-196a expression level and closely associate with the incidence of GC.36To test this hypothesis, we initiated to screen MIR196A2 promoter region and then focused on 2 potentially functional SNPs (rs12304647 A>C and rs35010275 G>C). + +Through further functional study, we testified their probable mechanisms and evaluated the possibility of these SNPs as biomarkers for GC susceptibility and clinical outcomes. + +## Materials And Methods Study Population + +This study was approved by the Institutional Review Board of Nanjing Medical University, Nanjing, China, and all subjects participated in this 2-stage study signed a written informed consent form. Seven hundred fifty-three GC cases and 854 cancer-free controls in testing set and additional subjects consisted of 1022 cases and 1061 cancer-free participants in validation set were periodical recruited from the Cancer Clinical Research Base of Nanjing Medical University during March 2006 to January 2010 and January 1999 and December 2006, respectively, which had been described previously in details.37 In the set for validation, 78 patients (7.6%) with incomplete follow-up information and 4 patients with nonadenocarcinoma were excluded, and finally 940 patients with gastric adenocarcinoma were enrolled. All patients involved in our study were newly diagnosed and histopathologically identified, without previous history of cancer or previous chemotherapy or radiotherapy. Collecting data on patients mainly included age, sex, and tumor site histological type, depth of invasion, lymph node metastasis, distant metastasis, as well as clinical tumor node metastasis (TNM) stage. Histopathology of tumor was classified to diffuse type and intestinal type according to Lauren criteria.38 The tumor invasion, lymph nodes metastasis, distant metastasis, and clinical TNM stage were recorded according to TNM classification (American Joint Commission on Cancer Staging, 6th).39 All controls were enrolled in the same period with no genetic relationship to the cases. Besides, the controls were frequency matched to cases by sex and age (-5 years) (Table 1). Each subject donated 5 mL peripheral blood after interviews. + +## Snp Selection + +SNPs in MIR196A2 promoter region were selected based on HapMap data (http://hapmap.ncbi.nlm.nih.gov/) and dbSNP +data (http://www.ncbi.nlm.nih.gov/projects/SNP/). The potentially functional polymorphisms were identified following the criteria: located in the MIR196A2 50-flanking region; and Minor allele frequency (MAF) >0.05 in Chinese population. According to the criteria, 2 SNPs (rs12304647 A>C and rs35010275 G>C) remained in our study. + +## Dna Isolation And Genotyping + +Genomic DNA was extracted from peripheral blood and paraffin sections of tissues for testing set and validation set, respectively. After proteinase K digestion and phenol–chloroform extraction, approximately 10% of DNA samples were randomly selected for agarose electrophoresis as quality control. Genotyping was carried out by fluorescent-based TaqMan SNP Genotyping Assay using ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Quality control was performed with the quality criteria as our previous study.37 + +## Rna Isolation And Qrt-Pcr + +Total RNA were extracted by TRIzol reagent (Invitrogen, Carlsbad, CA) according to the protocol, which were from 66 pairs of patients' tumor and adjacent normal tissues including 753 cases in our testing set. RNA was measured by Nanodrop ND-2000 spectrophotometer (Thermo, Waltham, MA) for its quality and quantity, and then stored at 808C. + +Quantitative reverse transcriptase PCR was carried out by ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). The PCR of each sample was normalized against an U6 internal control. The primer sequences were mentioned in supplementary data (see supplementary Digital Content 1, http://links.lww.com/MD/A80). All PCR reactions were conducted in triplicate. + +## Construction Of Promoter–Reporter Plasmids The 50-Flanking Region Sequence Of Human Mir196A2 + +gene was obtained from the Homo sapiens chromosome 12 (NC_000012.11) after a blast search, starting from the first base of pri-miRNA. Luciferase reporter plasmids and corresponding variants were constructed by PCR amplification of nucleotides 1000, 700, 500 to transcriptional start site of MIR196A2 promoter from human genomic DNA. All amplified fragments were cloned into pGL3-basic vectors (Promega, Madison, WI) and sequenced to confirm the orientation and integrity of each construct's inserts. + +## Cell Culture + +Gastric epithelium cell line (GES-1) and SGC-7901 are both cultured in Dulbecco's Modified Egale medium/high glucose (Invitrogen) culture medium, supplementing with 10% heat-inactivated fetal bovine serum obtained from GIBCO (Burlington, ON, Canada), 10 mmol/L 4-(2-hydroxyethyl)-1piperazineethanesulfonicaci, 2 mmol/L L-glutamine, 1 mmol/L +pyruvate sodium, 100 U/mL penicillin, and 100mg/mL streptomycin at 378C in a humidified atmosphere containing 5% +CO2. + +## Transfection And Luciferase Assay + +For transfection, 1 106 cells were seeded in individual well of a 24-well culture plate. Cells were transfected via lipofectamine-2000 transfection reagent with 0.5mg constructed luciferase reporter plasmids mentioned above. The pRL-SV40 (as internal control) was transiently cotransfected into cells. Twenty-four hours after transfection, all cells were + +| TABLE 1. Demographic Characteristics and Clinical Features Testing Set | Validation Set | P | | | | | +|--------------------------------------------------------------------------|--------------------|-----------------|---------------------|------------|------------|-------| +| Cases (n ¼ 753) | Controls (n ¼ 854) | Cases (n ¼ 940) | Controls (n ¼ 1061) | | | | +| n (%) | n (%) | n (%) | n (%) | | | | +| P | | | | | | | +| Variables Age, y 65 | 432 (57.4) | 473 (55.4) | 0.424 | 604 (64.3) | 660 (62.2) | 0.343 | +| >65 | 321 (42.6) | 381 (44.6) | 336 (35.7) | 401 (37.8) | | | +| Sex Male | 512 (68.0) | 564 (66.0) | 0.406 | 724 (77.0) | 805 (75.9) | 0.546 | +| Female | 241 (32.0) | 290 (34.0) | 216 (23.0) | 256 (24.1) | | | +| Tumor sites Cardia | 295 (39.2) | 318 (33.8) | | | | | +| Noncardia | 458 (60.8) | 622 (66.2) | | | | | +| Histological types Diffuse | 437 (58.0) | 541 (57.6) | | | | | +| Intestinal | 316 (42.0) | 399 (42.4) | | | | | +| Depth of invasiony T1 | 130 (17.3) | 148 (15.7) | | | | | +| T2 | 130 (17.3) | 200 (21.3) | | | | | +| T3 | 381 (50.6) | 545 (58.0) | | | | | +| T4 | 112 (14.8) | 45 (4.8) | | | | | +| Lymph node metastasis N0 297 (39.4) | 374 (39.8) | | | | | | +| N1/N2/N3 | 456 (60.6) | 566 (60.2) | | | | | +| Distant metastasis M0 | 655 (87.0) | 882 (93.8) | | | | | +| M1 | 98 (13.0) | 58 (6.2) | | | | | +| TNM stages I | 202 (26.8) | 261 (27.8) | | | | | +| II | 165 (21.9) | 186 (19.8) | | | | | +| III | 266 (35.3) | 396 (42.1) | | | | | +| IV | 120 (16.0) | 97 (10.3) | | | | | +| TNM ¼ tumor node metastasis. Two-sided x2 test for the frequency distributions of selected variables. y Information of depth of invasion was not available for 2 patients in the validation set. | | | | | | | + +washed with Phosphate Buffered Saline and lysed with 1 passive lysis buffer. Luciferase activity was determined with a dual luciferase report assay system (Promega) following the manufacturer's protocol. + +detected by Student t test or x2 test. Unconditional univariate and multivariate logistic regression analyses were done to estimate adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs), which were used to evaluate the importance of genetic variants in different clinical features of GC. + +## Electrophoretic Mobility Shift Assay (Emsa) + +SGC-7901 cells (2–4 106) were collected to prepare nuclear extracts by the NE-PER kit (Pierce; Rockford, IL). Biotin-labeled oligonucleotides probes were annealed at 50 fmol. Reactions were applied onto 5% polyacrylamide gels, and then transferred to nylon membranes. Biotin-labeled DNA was detected by the LightShift Chemiluminescent EMSA Kit (Pierce). + +## Statistical Analysis + +In this study, statistical analyses were performed by SAS +software (version 9.13; SAS Institute Inc, Cary, NC). All P values were 2-sided with P < 0.05. Goodness-of-fit x2 test was used to test for each SNP in control subjects to meet the Hardy–Weinberg equilibrium (HWE). Differences in the distributions of demographic characteristics, selected variables, and frequencies of genotypes between cases and controls were + +## Results Expression Of Mir-196A In Gastric Tumor And Adjacent Normal Tissues + +MiR-196a expression levels were detected in 66 new diagnostic gastric tumor and adjacent normal tissue samples, which were chosen to keep the same genetic background. As shown in Figure 1, the average miR-196a level in tumor tissues was significantly higher than that in adjacent normal tissues (53.89 - 3.991 vs 22.68 - 2.568, P < 0.001). + +## Identification Of Snps In Mir196A2 Gene Promoter Region + +With regard to higher levels of miR-196a in GC tissues, we attempted to investigate the regulatory mechanism of miR-196a-2 expression. After screening the promoter region + +![3_image_0.png](3_image_0.png) + +of MIR196A2 gene with SNP database (http://www.ncbi.nlm. nih.gov/snp), 2 SNPs within MIR196A2 gene reveal their presence in Chinese population with MAF >5%, named rs12304647 A>C and rs35010275 G>C (Figure 2A). + +## Effects Of Snps In Mir196A2 Promoter Region On Gene Transcriptional Regulation + +To identify whether these 2 variations have influence on the transcription activity of MIR196A2, we constructed fulllength luciferase reporter plasmid and its variants by using pGL3-basic vector, named pGL3–1000 wt, 1000mt1, 1000mt2, and 1000mt3, which were transfected into SGC-7901 and normal GES-1. As shown in Figure 2B, comparing with pGL3-1000 wt and pGL3-1000mt1, the transcription activity of pGL3-1000mt2 and pGL3-1000mt3 were significantly lower in SGC-7901 and GES-1, which suggested these SNPs in MIR196A2 promoter region took effect on its transcription. To further confirm the individual functions of rs12304647 and rs35010275, 3 plasmids containing shorter MIR196A promoter regions (pGL3-700 wt, 700mt, and 500) were transfected in SGC-7901 cell line. All of these reporter plasmid assays displayed higher luciferase activities than pGL3-basic vector. + +pGL3-1000 wt and pGL3-1000mt1 showed significant increases in promoter activity than pGL3-700 wt and pGL3700mt1. However, opposite trends displayed in pGL3-1000mt2 and pGL3-1000mt3 (Figure 2C). These results suggested that different allele of rs35010275 might possess different function in miR-196a expression. + +Based on the above results, we further investigated potential regulatory role of rs35010275 G>C. EMSA was performed with probes carrying the SNP major (G) or minor (C) allele. + +![3_image_1.png](3_image_1.png) + +Proteins or protein complexes binding to G-probe have larger molecular weight than those bind to C-probe, suggesting that they might be different (Figure 2D). Through competition assays between G and C-probes, it was obvious that both of the protein complexes displayed less intense bands as the decreasing concentration of both probes. These results suggested that these different protein complexes might contain sequence-specific DNA-binding domain that could recognize different alleles of rs35010275. + +## Characteristics Of Gc Patients And Controls + +The frequency distributions of cases and controls in 2 stages are summarized in Table 1. The cases and controls appeared to be adequately matched on age (P ¼ 0.424 and 0.343) and sex (P ¼ 0.406 and 0.546). In testing set, 26.8%, 21.9%, 35.3%, and 16.0% of patients were in stage I, II, III, and IV disease, respectively, whereas, 27.8%, 19.8%, 42.1%, and 10.3% of the patients in validation set were diagnosed in stage I, II, III, and IV, respectively. + +## Association Of Rs35010275 G>C Polymorphism With Mir-196A-2 Levels In Gc Tissues + +Next, we performed genotyping analysis for rs35010275 in above 66 patients' tissues from which we successfully obtained both genomic DNA and RNA. Data were showed as following: GG, 36 cases (54.6%); GC, 24 cases (36.3%); and CC, 6 cases (9.1%). As presented in Figure 3, patients carrying GG genotype had higher miR-196a-2 expression levels than those with GC or CC genotype in both cancer tissues (P ¼ 0.032 for GG vs GC and P ¼ 0.001 for GG vs CC) and adjacent normal tissues (P ¼ 0.048 for GG vs GC and P ¼ 0.049 for GG vs CC). + +## Overall Effects Of Mir196A2 Promoter Rs35010275 G>C On Risk Of Gc In 2-Stage Case–Control Studies + +We further performed a 2-stage case–control study to evaluate the overall associations between rs35010275 G>C +polymorphism and GC risk. Genotype frequencies in the combined controls was conformed to HWE (x2¼ 0.885, P ¼ 0.347). + +As presented in Table 2, we found that GC/CC genotypes were associated with a significantly decreased risk of GC compared with GG genotype in both stage of the study (adjusted OR ¼ 0.82, 95% CI ¼ 0.67–0.99 in testing set and adjusted OR ¼ 0.82, 95% CI ¼ 0.68–0.97 in validation set). Moreover, with the expansion of samples in combined analysis, more significant protective effects displayed in GC, CC, and GC/ CC genotypes (adjusted OR ¼ 0.82, 95% CI ¼ 0.72–0.95 for GC; adjusted OR ¼ 0.76, 95% CI ¼ 0.59–0.99 for CC; adjusted OR ¼ 0.81, 95% CI ¼ 0.71–0.93 for GC/CC) compared to GG genotype. Simultaneously, difference between the subjects carrying C allele and G allele was observed in allele comparison with adjusted OR ¼ 0.85, 95% CI ¼ 0.73–1.00 in testing set, adjusted OR ¼ 0.85, 95% CI ¼ 0.74–0.98 in validation set, and adjusted OR ¼ 0.85, 95% CI ¼ 0.77–0.94 in combined set. + +## Associations Between Rs35010275 G>C Polymorphism And Clinical Features Of Gc + +Next, we evaluated the effects of rs35010275 G>C on the progression of GC by comparing the rs35010275 G>C polymorphism with different clinical features. As shown in Table 3, with polymorphism in genetic dominant model of combined analysis, GC/CC genotypes have a statistical significantly decreased risk of GC compared with wild homozygous GG genotype in patients with cardiac cancer (adjusted OR ¼ 0.71, 95% CI ¼ 0.59–0.85), diffuse type (0.85, 0.73–1.00), intestinal type (0.77, 0.64–0.92), T3 depth invasion (0.74, 0.63–0.87), + +Noncancerous tissue Cancerous tissue + +![4_image_1.png](4_image_1.png) + +![4_image_0.png](4_image_0.png) + +| TABLE 2. Genotype frequencies and overall effects of MIR196A2 rs35010275 G>C polymorphism on gastric cancer risk Testing set Validation set Combined set Cases/Controls (753/854) P Adjusted OR Cases/Controls (940/1061) P Adjusted OR Cases/Controls (1693/1915) P Adjusted OR (95% CI)y (95% CI)y (95% CI)y Genotypes GG 436/452 1.00 (ref.) 532/548 1.00 (ref.) 968/1000 1.00 (ref.) GC 264/331 0.069 0.82 (0.67-1.02) 348/432 0.043 0.83 (0.69-0.99) 612/763 0.006 0.82 (0.72-0.95) CC 53/71 0.179 0.77 (0.53-1.13) 60/81 0.119 0.75 (0.53-1.08) 113/152 0.040 0.76 (0.59-0.99) GC/CC 317/402 0.042 0.82 (0.67-0.99) 408/513 0.023 0.82 (0.68-0.97) 725/915 0.002 0.81 (0.71-0.93) G allele 1136/1235 1.00 (ref.) 1412/1528 1.00 (ref.) 2548/2763 1.00 (ref.) C allele 370/473 0.046 0.85 (0.73-1.00) 468/594 0.023 0.85 (0.74-0.98) 838/1067 0.002 0.85 (0.77-0.94) Ptrend 0.049 0.027 0.003 | +|---| + +CI ¼ confidence interval, OR ¼ odds ratio. Two-sided x2 test for genotype frequency distributions between cases and controls. yAdjusted for age and sex in logistic regression model. + +positive lymph node metastasis (0.73, 0.62–0.85), negative distant metastasis (0.82, 0.72–0.94), and advanced stage (0.72, 0.62–0.85). + +## Discussion + +In the present study, miR-196a acted as an oncogenic miRNA, which expressed significantly higher in cancerous tissues than in noncancerous tissues. Moreover, a notable SNP (rs35010275 G>C) in our study displayed potential transcript regulating function on MIR196A2 and was associated with the susceptibility of GC in the Chinese population. These findings suggested that MIR196A2 rs35010275 G>C might be regarded as a potential biomarker for GC prediction. + +It has been reported that the miR-196a may display different effects as an oncogenic miRNA in the pathology of + +| dominant model Variables | Testing set | Validation set | Combined set | | | | | | | +|----------------------------------|---------------|-----------------------|------------------|-------------|-----------------------|------------------|---------|-----------------------|------------------| +| GG/(GC/CC) | P | Adjusted OR (95% CI)y | GG/(GC/CC) | P | Adjusted OR (95% CI)y | GG/(GC/CC) | P | Adjusted OR (95% CI)y | | +| Controls Total | 452/402 | 1.00 (ref.) | 548/513 | 1.00 (ref.) | 1000/915 | 1.00 (ref.) | | | | +| Cases Tumor sites Cardia | 184/111 | 0.005 | 0.68 (0.52-0.89) | 188/130 | 0.015 | 0.73 (0.56-0.94) | 372/241 | <0.001 | 0.71 (0.59-0.85) | +| Non-cardia | 252/206 | 0.400 | 0.91 (0.72-1.14) | 344/278 | 0.136 | 0.86 (0.70-1.05) | 596/484 | 0.096 | 0.88 (0.76-1.02) | +| Histological types Diffuse | 250/187 | 0.148 | 0.84 (0.67-1.06) | 299/242 | 0.157 | 0.86 (0.70-1.06) | 549/429 | 0.043 | 0.85 (0.73-1.00) | +| Intestinal | 186/130 | 0.065 | 0.78 (0.60-1.02) | 233/166 | 0.020 | 0.76 (0.60-0.96) | 419/296 | 0.003 | 0.77 (0.64-0.92) | +| Depth of invasionz T1 | 72/58 | 0.560 | 0.90 (0.62-1.30) | 80/68 | 0.685 | 0.93 (0.66-1.31) | 152/126 | 0.482 | 0.91 (0.71-1.18) | +| T2 | 69/61 | 0.992 | 1.00 (0.69-1.44) | 107/93 | 0.696 | 0.94 (0.70-1.27) | 176/154 | 0.782 | 0.97 (0.77-1.22) | +| T3 | 231/150 | 0.010 | 0.72 (0.56-0.92) | 319/226 | 0.009 | 0.76 (0.62-0.93) | 550/376 | <0.001 | 0.74 (0.63-0.87) | +| T4 | 64/48 | 0.369 | 0.83 (0.56-1.24) | 26/19 | 0.635 | 0.87 (0.48-1.56) | 90/67 | 0.291 | 0.84 (0.60-1.16) | +| Lymph node metastasis N0 161/136 | 0.684 | 0.95 (0.73-1.23) | 195/179 | 0.829 | 0.97 (0.77-1.23) | 356/315 | 0.664 | 0.96 (0.81-1.15) | | +| N1/N2/N3 | 275/181 | 0.009 | 0.74 (0.58-0.93) | 337/229 | 0.002 | 0.72 (0.59-0.89) | 612/410 | <0.001 | 0.73 (0.62-0.85) | +| Distant metastasis M0 | 382/273 | 0.034 | 0.80 (0.65-0.98) | 493/389 | 0.053 | 0.84 (0.70-1.00) | 875/662 | 0.004 | 0.82 (0.72-0.94) | +| M1 | 54/44 | 0.635 | 0.90 (0.59-1.38) | 39/19 | 0.027 | 0.53 (0.30-0.93) | 93/63 | 0.079 | 0.74 (0.53-1.04) | +| TNM stages IþII | 203/164 | 0.429 | 0.90 (0.71-1.16) | 237/210 | 0.546 | 0.93 (0.75-1.17) | 440/374 | 0.330 | 0.92 (0.78-1.09) | +| IIIþIV | 233/153 | 0.013 | 0.73 (0.57-0.94) | 295/198 | 0.003 | 0.72 (0.58-0.89) | 528/351 | <0.001 | 0.72 (0.62-0.85) | + +CI ¼ confidence interval, OR ¼ odds ratio, TNM ¼ tumor node metastasis. Two-sided x2 test for the frequency distributions of selected variables between cases and controls. yAdjusted for age and sex in logistic regression model. + +zInformation was not available for 2 patients in validation set and combined set. + +GC, after quantitatively assessing the levels of miR-196a in patients.16,30 Our study also confirmed that miR-196a unduly existed in GC tissues, which were consistent with the results reported by Sun et al.40 Besides, the genotyping of the 66 patients also manifested that patients carrying rs35010275 C allele were correlated with lower expression level of miR-196a in both tumor and normal tissues. These evidences suggested that the increase of miR-196a expression in rs35010275 GG genotype was a crucial event in gastric carcinogenesis; while some other transcriptional or post-transcriptional mechanism might exist in normal tissues to protect the abnormal expression of miR-196a. + +It was proposed that various genomic and epigenomic mechanisms were involved in dysregulation of miRNA genes in cancers. The study by Croce41 had concretely demonstrated these different causes such as deletions, amplifications, mutations, epigenetic silencing, and even the aberrant expression of transcription factors targeting miRNAs. To date, compared with thorough disclosure of miR-196a functions, the culprits of abnormal expression of miR-196a have been involved seldom. Derived from 2 different loci, nominated MIR196A1 and MIR196A2 gene, detailed mechanism of miR-196a expression may be diverse and complicated. With regard to affecting the mature process of miR-196a-2 transcription,23,42,43 rs11614913 SNP was one exploration raising the concerns of researchers.20,42,44,45 Recent studies have identified this polymorphism as a risk factor for several diseases, including hepatocellular carcinoma,20 leukemia,46 lung cancer,22,23 breast cancer,44,47 colorectal cancer,32 and pancreatic adenocarcinoma.48,49 Apart from MIR196A1, MIR196A2 possesses its own promoter in structure, in which the variations might also alter miR-196a expression and then influence the susceptibility of GC. In this study, we identified 2 SNPs (rs12304647 A>C and rs35010275 G>C) in MIR196A2 promoter region of 1000 bp away from the transcription starting site. Taken functional analyses together, these observations supported the idea that MIR196A2 rs35010275 G>C resulted in different recruits of transcription factors or complexes to MIR196A2 gene promoter region, which modulated the miR-196a expression. To our knowledge, this is the first study providing direct evidence that MIR196A2 promoter polymorphism may influence individuals' susceptibility to GC through affecting miRNA +biogenesis. + +MiRNAs potentially display their distinct effects in various biologic processes via influencing their target genes. As dysregulation of miR-196a may contribute to tumor detachment, migration, invasion, and proliferation13,16,32,40,50,51 through regulating its fundamental target genes, it is biologically plausible that rs35010275 G>C influence the development of GC. Our 2-stage epidemiological study indicated that rs35010275 C allele displayed protective effect on GC with T3 depth of invasion, positive lymph node metastasis, negative distant metastasis, and advanced TNM stage. Therefore, MIR196A2 may participate in the invasion and migration pathways of gastric carcinogenesis, and rs35010275 C allele may act as a repressor in hazardous biological processes. In previous studies, the members of homeobox Gene (HOX) family15,50,52 and annexin A113,53 have been reported as target genes of miR-196a in vivo. Schimanski et al32 identified that through regulating HOX family genes, miR-196a could activate the v-akt murine thymoma viral oncogene (AKT) signaling pathway by increasing phosphorylation of AKT. These results suggested that MIR196A2 rs35010275 could trigger the expression of miR-196a and finally induce the AKT pathway to influence susceptibility and processes of gastric carcinogenesis. + +Considering rs35010275 G>C as an molecular biomarker to predict individuals' susceptibility to GC, our study also showed that the protective effects of rs35010275 C allele were more predominant in cardia patients (adjust OR ¼ 0.71, 95% CI ¼ 0.59–0.85, P < 0.001) and stages III and VI patients +(adjust OR ¼ 0.72, 95% CI ¼ 0.62–0.85, P < 0.001), suggesting that there exists associations of rs35010275 G>C with tumor site and advanced TNM stage. Further investigation to validate this association is necessary, and it would be interesting to clarify whether the carcinogenesis mechanism in cancer with low TNM stage might be distinct from that in cancer with advanced TNM stage. Besides, MIR196A2 rs35010275 G>C might majorly take its contribution on precluding the onset of cancer in cardia. + +Several limitations exist in our study. First, small sample size may limit the statistical power of our study, especially for subgroup analyses. Second, our study was retrospective hospital-based case–control studies; the inherent selection bias and information bias were unavoidable. Under this circumstance, we applied a rigorous epidemiological design in selecting study subjects and conducted statistical adjustment for known risk factors to minimize the potential biases. Finally, our study lacks some information on GC risk factors, such as Helicobacter pylori infection status, diet habit, tobacco smoking, and alcohol consumption status. Therefore, our results need to be validated in population-based studies with larger sample size and more detailed information. + +In conclusion, our study revealed that miR-196a was dysregulated in GC. Moreover, a functional SNP rs35010275 G>C in the promoter region of MIR196A2 gene was significantly associated with miR-196a-2 expression. Further insights into functional and clinical investigation of MIR196A2 gene may contribute to the diagnosis and prognosis of GC. + +ACKNOWLEDGMENT +The authors would like to appreciate the technical help of Biolight Tech Company, Nanjing, China. + +## References + +1. Danaei G, Vander Hoorn S, Lopez AD, et al. Causes of cancer in the world: comparative risk assessment of nine behavioural and environmental risk factors. Lancet. 2005;366:1784–1793. + +2. Ferlay J, Shin HR, Bray F, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010;127:2893– +2917. + +3. Ajani JA. Optimizing docetaxel chemotherapy in patients with cancer of the gastric and gastroesophageal junction: evolution of the docetaxel, cisplatin, and 5-fluorouracil regimen. Cancer. + +2008;113:945–955. + +4. Yoong J, Michael M, Leong T. Targeted therapies for gastric cancer: +current status. Drugs. 2011;71:1367–1384. + +5. Field K, Michael M, Leong T. Locally advanced and metastatic gastric cancer: current management and new treatment developments. Drugs. 2008;68:299–317. + +6. Matsusaka K, Kaneda A, Nagae G, et al. Classification of EpsteinBarr virus-positive gastric cancers by definition of DNA methylation epigenotypes. Cancer Res. 2011;71:7187–7197. + +7. Rusiecki JA, Al-Nabhani M, Tarantini L, et al. Global DNA +methylation and tumor suppressor gene promoter methylation and gastric cancer risk in an Omani Arab population. Epigenomics. + +2011;3:417–429. +8. Xu L, Li X, Chu ES, et al. Epigenetic inactivation of BCL6B, a novel functional tumour suppressor for gastric cancer, is associated with poor survival. Gut. 2012;61:977–985. + +9. Li X, Zhang Y, Ding J, et al. Survival prediction of gastric cancer by a seven-microRNA signature. Gut. 2010;59:579–585. + +10. Gao C, Zhang Z, Liu W, et al. Reduced microRNA-218 expression is associated with high nuclear factor kappa B activation in gastric cancer. Cancer. 2010;116:41–49. + +11. Craig VJ, Cogliatti SB, Rehrauer H, et al. Epigenetic silencing of microRNA-203 dysregulates ABL1 expression and drives Helicobacter-associated gastric lymphomagenesis. Cancer Res. 2011;71:3616– +3624. + +12. Ambros V. The functions of animal microRNAs. Nature. + +2004;431:350–355. + +13. Luthra R, Singh RR, Luthra MG, et al. MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers. Oncogene. 2008;27:6667–6678. + +14. Mansfield JH, Harfe BD, Nissen R, et al. MicroRNA-responsive +''sensor'' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat Genet. + +2004;36:1079–1083. + +15. Niinuma T, Suzuki H, Nojima M, et al. Upregulation of miR-196a and HOTAIR drive malignant character in gastrointestinal stromal tumors. Cancer Res. 2012;72:1126–1136. + +16. Tsai KW, Liao YL, Wu CW, et al. Aberrant expression of miR-196a in gastric cancers and correlation with recurrence. Genes, Chromosomes Cancer. 2012;51:394–401. + +17. Lung RW, Wang X, Tong JH, et al. A single nucleotide polymorphism in microRNA-146a is associated with the risk for nasopharyngeal carcinoma. Mol Carcinog. 2013;52:E28–E38. + +18. Smith RA, Jedlinski DJ, Gabrovska PN, et al. A genetic variant located in miR-423 is associated with reduced breast cancer risk. + +Cancer Genomics Proteomics. 2012;9:115–118. + +19. Zhang M, Jin M, Yu Y, et al. Associations of miRNA polymorphisms and female physiological characteristics with breast cancer risk in Chinese population. Eur J Cancer Care. 2012;21:274–280. + +20. Li XD, Li ZG, Song XX, et al. A variant in microRNA-196a2 is associated with susceptibility to hepatocellular carcinoma in Chinese patients with cirrhosis. Pathology. 2010;42:669–673. + +21. Xu Y, Liu L, Liu J, et al. A potentially functional polymorphism in the promoter region of miR-34b/c is associated with an increased risk for primary hepatocellular carcinoma. Int J Cancer. + +2011;128:412–417. + +22. Hu Z, Chen J, Tian T, et al. Genetic variants of miRNA sequences and non-small cell lung cancer survival. J Clin Investig. + +2008;118:2600–2608. + +23. Tian T, Shu Y, Chen J, et al. A functional genetic variant in microRNA-196a2 is associated with increased susceptibility of lung cancer in Chinese. Cancer Epidemiol, Biomarkers Prevention. + +2009;18:1183–1187. + +24. Christensen BC, Avissar-Whiting M, Ouellet LG, et al. Mature microRNA sequence polymorphism in MIR196A2 is associated with risk and prognosis of head and neck cancer. Clin Cancer Res. + +2010;16:3713–3720. + +25. Mu YP, Su XL. Polymorphism in pre-miR-30c contributes to gastric cancer risk in a Chinese population. Med Oncol. 2012;29:1723– +1732. + +26. Kogo R, Mimori K, Tanaka F, et al. Clinical significance of miR146a in gastric cancer cases. Clin Cancer Res. 2011;17:4277–4284. + +27. Wang S, Tao G, Wu D, et al. A functional polymorphism in MIR196A2 is associated with risk and prognosis of gastric cancer. + +Mol Carcinog. 2013;52(Suppl 1):E87–E95. + +28. Okubo M, Tahara T, Shibata T, et al. Association between common genetic variants in pre-microRNAs and gastric cancer risk in Japanese population. Helicobacter. 2010;15:524–531. + +29. Peng S, Kuang Z, Sheng C, et al. Association of microRNA-196a-2 gene polymorphism with gastric cancer risk in a Chinese population. + +Dig Dis Sci. 2010;55:2288–2293. + +30. Inoue T, Iinuma H, Ogawa E, et al. Clinicopathological and prognostic significance of microRNA-107 and its relationship to DICER1 mRNA expression in gastric cancer. Oncol Rep. + +2012;27:1759–1764. + +31. Ahmed FE, Ahmed NC, Vos PW, et al. Diagnostic microRNA +markers to screen for sporadic human colon cancer in stool: I. Proof of principle. Cancer Genomics Proteomics. 2013;10:93–113. + +32. Schimanski CC, Frerichs K, Rahman F, et al. High miR-196a levels promote the oncogenic phenotype of colorectal cancer cells. World J +Gastroenterol. 2009;15:2089–2096. + +33. Liu G, Gramling S, Munoz D, et al. Two novel BRM insertion promoter sequence variants are associated with loss of BRM +expression and lung cancer risk. Oncogene. 2011;30:3295–3304. + +34. Hao B, Miao X, Li Y, et al. A novel T-77C polymorphism in DNA +repair gene XRCC1 contributes to diminished promoter activity and increased risk of non-small cell lung cancer. Oncogene. + +2006;25:3613–3620. + +35. Wang M, Yuan L, Wu D, et al. A novel XPF 357A>C polymorphism predicts risk and recurrence of bladder cancer. Oncogene. + +2010;29:1920–1928. + +36. Ozsolak F, Poling LL, Wang Z, et al. Chromatin structure analyses identify miRNA promoters. Genes Dev. 2008;22:3172–3183. + +37. Wang M, Bai J, Tan Y, et al. Genetic variant in PSCA predicts survival of diffuse-type gastric cancer in a Chinese population. Int J +Cancer. 2011;129:1207–1213. + +38. Lauren P. The two histological main types of gastric carcinoma: +diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathologica Microbiologica Scandinavica. 1965;64:31–49. + +39. Green FL, Page DL, Fleming ID, et al. Stomach of the digestive system. AJCC Cancer Staging Handbook. 6th ed. New York: +Springer Press; 2002:111–118. + +40. Sun M, Liu XH, Li JH, et al. MiR-196a is upregulated in gastric cancer and promotes cell proliferation by downregulating p27(kip1). Mol Cancer Therap. 2012;11:842–852. + +41. Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet. 2009;10:704–714. + +42. Xu J, Hu Z, Xu Z, et al. Functional variant in microRNA-196a2 contributes to the susceptibility of congenital heart disease in a Chinese population. Hum Mutat. 2009;30:1231–1236. + +43. Hu Z, Liang J, Wang Z, et al. Common genetic variants in premicroRNAs were associated with increased risk of breast cancer in Chinese women. Hum Mutat. 2009;30:79–84. + +44. Hoffman AE, Zheng T, Yi C, et al. MicroRNA miR-196a-2 and breast cancer: a genetic and epigenetic association study and functional analysis. Cancer Res. 2009;69:5970–5977. + +45. Catucci I, Yang R, Verderio P, et al. Evaluation of SNPs in miR146a, miR196a2 and miR-499 as low-penetrance alleles in German and Italian familial breast cancer cases. Hum Mutat. 2010;31:E1052– +E1057. + +46. Coskun E, von der Heide EK, Schlee C, et al. The role of microRNA-196a and microRNA-196b as ERG regulators in acute myeloid leukemia and acute T-lymphoblastic leukemia. Leuk Res. + +2011;35:208–213. + +47. Hui AB, Shi W, Boutros PC, et al. Robust global micro-RNA +profiling with formalin-fixed paraffin-embedded breast cancer tissues. Lab Investig. 2009;89:597–606. + +48. Kong X, Du Y, Wang G, et al. Detection of differentially expressed microRNAs in serum of pancreatic ductal adenocarcinoma patients: +miR-196a could be a potential marker for poor prognosis. Dig Dis Sci. 2011;56:602–609. + +49. Szafranska-Schwarzbach AE, Adai AT, Lee LS, et al. Development of a miRNA-based diagnostic assay for pancreatic ductal adenocarcinoma. Expert Rev Mol Diagn. 2011;11:249–257. + +50. Braig S, Mueller DW, Rothhammer T, et al. MicroRNA miR-196a is a central regulator of HOX-B7 and BMP4 expression in malignant melanoma. Cell Mol Life Sci. 2010;67:3535–3548. + +51. Kim YJ, Bae SW, Yu SS, et al. MiR-196a regulates proliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue. J Bone Miner Res. 2009;24:816–825. + +52. Mueller DW, Bosserhoff AK. MicroRNA miR-196a controls melanoma-associated genes by regulating HOX-C8 expression. Int J +Cancer. 2011;129:1064–1074. + +53. Kan T, Meltzer SJ. MicroRNAs in Barrett's esophagus and esophageal adenocarcinoma. Curr Opin Pharmacol. 2009;9:727–732. \ No newline at end of file diff --git a/medical/md_images/PMC4616385/PMC4616385_meta.json b/medical/md_images/PMC4616385/PMC4616385_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..5eecff79e2efc91334d88f52120e99ccf99da03c --- /dev/null +++ b/medical/md_images/PMC4616385/PMC4616385_meta.json @@ -0,0 +1,336 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "Evaluation of a Novel Functional Single-Nucleotide Polymorphism (rs35010275 G>C) in MIR196A2 Promoter Region as a Risk Factor of Gastric Cancer in a Chinese\u2122Population", + "level": 0, + "is_closed": true, + "n_kids": 5, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 44.0, + 843.0, + 517.0, + 740.0 + ] + }, + { + "title": "INTRODUCTION", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 11.0, + 470.0, + 558.0, + 459.0 + ] + }, + { + "title": "MATERIALS AND METHODS", + "level": 1, + "is_closed": true, + "n_kids": 9, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 489.0, + 509.0, + 478.0 + ] + }, + { + "title": "Study Population", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 472.0, + 509.0, + 461.0 + ] + }, + { + "title": "SNP Selection", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 170.0, + 509.0, + 159.0 + ] + }, + { + "title": "DNA Isolation and Genotyping", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 8.0, + 702.0, + 558.0, + 691.0 + ] + }, + { + "title": "RNA Isolation and qRT-PCR", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 8.0, + 591.0, + 558.0, + 580.0 + ] + }, + { + "title": "Construction of Promoter-Reporter Plasmids", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 8.0, + 441.0, + 558.0, + 430.0 + ] + }, + { + "title": "Cell Culture", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 8.0, + 318.0, + 558.0, + 308.0 + ] + }, + { + "title": "Transfection and Luciferase Assay", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 8.0, + 206.0, + 558.0, + 195.0 + ] + }, + { + "title": "Electrophoretic Mobility Shift Assay (EMSA)", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 312.0, + 511.0, + 301.0 + ] + }, + { + "title": "Statistical Analysis", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 219.0, + 511.0, + 208.0 + ] + }, + { + "title": "RESULTS", + "level": 1, + "is_closed": true, + "n_kids": 7, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 11.0, + 300.0, + 558.0, + 289.0 + ] + }, + { + "title": "Expression of miR-196a in Gastric Tumor and Adjacent Normal Tissues", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 11.0, + 283.0, + 558.0, + 272.0 + ] + }, + { + "title": "Identification of SNPs in MIR196A2 Gene Promoter Region", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 2, + "view_mode": "FitR", + "view_pos": [ + 11.0, + 189.0, + 558.0, + 178.0 + ] + }, + { + "title": "Effects of SNPs in MIR196A2 Promoter Region on Gene Transcriptional Regulation", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 3, + "view_mode": "FitR", + "view_pos": [ + 8.0, + 771.0, + 558.0, + 760.0 + ] + }, + { + "title": "Characteristics of GC Patients and Controls", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 670.0, + 511.0, + 659.0 + ] + }, + { + "title": "Association of rs35010275 G>C Polymorphism With MiR-196a-2 Levels in GC Tissues", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 557.0, + 511.0, + 546.0 + ] + }, + { + "title": "Overall Effects of MIR196A2 Promoter rs35010275 G>C on Risk of GC in 2-Stage Case-Control Studies", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 11.0, + 771.0, + 558.0, + 760.0 + ] + }, + { + "title": "Associations Between rs35010275 G>C Polymorphism and Clinical Features of GC", + "level": 2, + "is_closed": null, + "n_kids": 0, + "page_index": 4, + "view_mode": "FitR", + "view_pos": [ + 11.0, + 537.0, + 558.0, + 526.0 + ] + }, + { + "title": "DISCUSSION", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 5, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 545.0, + 509.0, + 534.0 + ] + }, + { + "title": "Acknowledgment", + "level": 1, + "is_closed": null, + "n_kids": 0, + "page_index": 6, + "view_mode": "FitR", + "view_pos": [ + 11.0, + 429.0, + 558.0, + 419.0 + ] + } + ], + "pages": 9, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 33, + "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/PMC4665231/11_image_0.png b/medical/md_images/PMC4665231/11_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..dab471788a51a063c065ba155cc9682220f7a65a --- /dev/null +++ b/medical/md_images/PMC4665231/11_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:52c54b9dd96e68e215977e0debdd935975a02022d7662cb4b0562e427ae52b41 +size 156451 diff --git a/medical/md_images/PMC4665231/9_image_0.png b/medical/md_images/PMC4665231/9_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..a86d587288350474e61fc5ee0e8a732852305776 --- /dev/null +++ b/medical/md_images/PMC4665231/9_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:4b5b8283fdffa50aa402d52f3de6f9d76ab686c0279ded124bc933ef361e247e +size 25987 diff --git a/medical/md_images/PMC4665231/9_image_1.png b/medical/md_images/PMC4665231/9_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..c11fdf0dca2c0ffc20e4baf631db2f45a241eac2 --- /dev/null +++ b/medical/md_images/PMC4665231/9_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:ab45c381d24f17146a2d6d56608a8e34d99be2a6b76ef88a0f0f1bfb80010607 +size 1060 diff --git a/medical/md_images/PMC5136409/0_image_0.png b/medical/md_images/PMC5136409/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..264ee72bc555bb9d40ff147d4bb7d5c99a71ff2c --- /dev/null +++ b/medical/md_images/PMC5136409/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f1159340f28bb5679fc4b1a61255179c8678ebc366e8061be945d849288069b1 +size 21494 diff --git a/medical/md_images/PMC5136409/4_image_1.png b/medical/md_images/PMC5136409/4_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..0adad3087c83adc901df40c90ed72763be4e8c21 --- /dev/null +++ b/medical/md_images/PMC5136409/4_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:7ba8bc84980867918706081552785f7f95327cbb764667d143d039c75f66399b +size 41113 diff --git a/medical/md_images/PMC5136409/5_image_0.png b/medical/md_images/PMC5136409/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..b689e55deead71723be46449ab92a27207e240f8 --- /dev/null +++ b/medical/md_images/PMC5136409/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:92128ab9f439c9a57d0fb3c360529bf2e805c8dfb46f617dd6ed12c6402d596d +size 44058 diff --git a/medical/md_images/PMC5152344/PMC5152344.md b/medical/md_images/PMC5152344/PMC5152344.md new file mode 100644 index 0000000000000000000000000000000000000000..0a410a747bf662732fa6efd05956c8dd03cfe0de --- /dev/null +++ b/medical/md_images/PMC5152344/PMC5152344.md @@ -0,0 +1,9 @@ + +## London Letter. The Mid Wives Bill. + +The Bill now before Parliament has for its object the protection of the public by insuring, as far as that is possible, that women who undertake the management of natural labour shall be competent and qualified, and that such persons shall not undertake responsibilities and duties, such as attending "cases of abnormal labour, or of disease, or of danger to the mother or the child, for which they are not qualified." There are three interests which are mainly affected by this measure, namely, the interests of the public which are paramount, of medical practitioners, whose occupation and perhaps income may be diminished by the legal certification, registration, and licensing of a class which has hitherto held the title and status of nurse, and has worked mostly in subordination to the qualified man midwife; and of the midwives, who in recent yeai'3 have sought to fit themselves for their work by attending courses of instruction and training, and obtain-ing certificates of proficiency and competence. + +The present Bill proposes to constitute a Central Midwives Board, similar in some respects to the General Medical Council, for the purpose of supervising the education, examination, certi-fication and registration of midwives. + +The rights of midwives already in practice are protected, and the local supervision of all midwives is placed in the hands of county and borough councils, who are empowered by the act to issue licences and to suspend from practice under certain circumstances; also to investigate charges of malpractice, negligence or misconduct and report the same, if established, to the Central Midwifes Board, to which the power is given to remove names from the roll on account of sundry offences. Penalties are prescribed by the act for obtaining a certificate or license by false representation, or acting without a certificate or license, or for " wilful falsification of the roll." +This bill, which there is every reason to believe will become law substantially in the present form, is greatly- exercising the mind of the medical profession which is divided as to the need of any legislation on the subject at all, and as to the merit of this particular scheme of legisla-tion. The principal objection taken is that the midwife under the new law will assume an independent and antagonistic position as re-gards the medical practitioner, and will not incline as often as she ought to call him in when the labour is not " natural," or when any disease or danger threatens mother or child. It is sought to append to the act a schedule defining the circumstances under which medical assist-ance should be sought; but apart from the difficulty of making these definitions exhaustive, or rendering penal the failure to recognise the need of help in time to be of service, it- will probably not be considered proper to load an Act of Parliament with technicality of this kind, and the upshot will probably be to leave such matters to the conscience and common sense of all concerned, guided by the general principle above quoted. It is also urged as a grave imperfection in the act that no penalty is prescribed for inidwives, who fail to call in medical aid when circumstances arise, which the law will give her no right or title to deal with. \ No newline at end of file diff --git a/medical/md_images/PMC5152344/PMC5152344_meta.json b/medical/md_images/PMC5152344/PMC5152344_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..256b4e25471f1e60c72715f3fd765c25afb0fbc6 --- /dev/null +++ b/medical/md_images/PMC5152344/PMC5152344_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 1, + "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/PMC5469383/2_image_0.png b/medical/md_images/PMC5469383/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..cc3214de53b90e1a8600a3cbca40e968c760f439 --- /dev/null +++ b/medical/md_images/PMC5469383/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8f405d9d45b4781ebcdbcaf7ab18d691e746212de4e9915b83f9486e5d182123 +size 177778 diff --git a/medical/md_images/PMC5469383/4_image_0.png b/medical/md_images/PMC5469383/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..661c50710470f8cf6f0a0e4498ea6fc3b17ad8e8 --- /dev/null +++ b/medical/md_images/PMC5469383/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:6afb3047e224ed28fa2e57db699f37e88956711b15787ae09231cc131e6eb6c7 +size 24872 diff --git a/medical/md_images/PMC5944538/0_image_0.png b/medical/md_images/PMC5944538/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..705156760b0846a86e90883ef6e35f0c094d6d99 --- /dev/null +++ b/medical/md_images/PMC5944538/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:97a3a93f89710cd0c9de80fbaf510ae6f48779b0bf64324650312789c05816fd +size 7808 diff --git a/medical/md_images/PMC5944538/2_image_0.png b/medical/md_images/PMC5944538/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..e09565fab5ebc869648b575fdaac66af2efc4426 --- /dev/null +++ b/medical/md_images/PMC5944538/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:b31fba89190af60bfcf26af79cfd576e96503af36ec6772113d83d1d35c148a9 +size 54367 diff --git a/medical/md_images/PMC6148981/4_image_1.png b/medical/md_images/PMC6148981/4_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..fba6ec78ee6f60cbfd0bf462aeb5fa646d5c7260 --- /dev/null +++ b/medical/md_images/PMC6148981/4_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:4a582a0fe84817920c6ec4a54761ac407e1b72233142c9698395c6a69c9f6f83 +size 78656 diff --git a/medical/md_images/PMC6250473/1_image_0.png b/medical/md_images/PMC6250473/1_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..b106594db5eec17008091aad55470f907079ea30 --- /dev/null +++ b/medical/md_images/PMC6250473/1_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:a300e064170331ea1395c3c87deafffc3ad64cc295702c6225c90d365efb64d6 +size 211542 diff --git a/medical/md_images/PMC6250473/2_image_2.png b/medical/md_images/PMC6250473/2_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..ca83e58efffb2e3dcf783eb86f902a632c708f3d --- /dev/null +++ b/medical/md_images/PMC6250473/2_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:d92d6b7b5a488fba6b261d4c38b78df8d46d3ca169863e89ff53f021d77320bf +size 113582 diff --git a/medical/md_images/PMC6250473/PMC6250473.md b/medical/md_images/PMC6250473/PMC6250473.md new file mode 100644 index 0000000000000000000000000000000000000000..dd4312ccebbceac4b81e4b78ad7cf08d54a91882 --- /dev/null +++ b/medical/md_images/PMC6250473/PMC6250473.md @@ -0,0 +1,108 @@ +# Original Article + +![0_Image_0.Png](0_Image_0.Png) Hand/Peripheral Nerve Rationalizing The Vascular Anatomy For Oblique Osteotomy Of The Metatarsal Head During Toe/Joint Transfers Of The Metatarsophalangeal Joint + +Yu-Te Lin, MD, MS +Shih-Heng Chen, MD +Charles Yuen Yung Loh, MBBS, +MSc Chung-Cheng Hsu, MD +Cheng-Hung Lin, MD +From the Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Keelung & Taoyuan, Taiwan. + +Background: The metatarsophalangeal joint (MTPJ) of the lessor toe has been used to reconstruct the metacarpophalangeal joint. When an oblique osteotomy of the metatarsal head is performed, the orientation of the vascular pedicle is crucial to preserve the blood supply to the MTPJ. This study was conducted to identify the path of the nutrient artery to the MTPJ. Methods: We reviewed our operative records in consecutive series of 45 patients during toe or joint harvest. Retrograde dissection of the vascular pedicle was performed starting from the distal communicating artery at the first webspace toward the first dorsal metatarsal and first plantar metatarsal arteries. All the vessels related to the MTPJ were explored and ligated if the MTPJ was not included in the flap. + +Results: The condylar branches to the proximal phalanx and the metaphyseal branches to the second metatarsus were barely identifiable during the dissections. The articular branch running perpendicularly from the plantar artery toward the plantar surface of MTPJ was verified in all cases. The articular branches originated either from the first plantar metatarsal artery (92.1%) or from the tibial plantar digital artery (7.9%). The external diameter of the articular branches was around 0.5–1mm. When the articular branch was included and preserved, the metatarsal heads oozed immediately after the flap was reperfused. Conclusions: The study demonstrated the constant and sizable articular branch of the MTPJ that originates from the plantar artery system. The consistency of the vascular anatomy enables oblique osteotomies of the metatarsal head to be performed without fear of injury to the pedicle. (Plast Reconstr Surg Glob Open 2018;6:e1805; doi: 10.1097/GOX.0000000000001805; Published online 3 October 2018.) + +## Introduction + +Transfer of the vascularized metatarsophalangeal joint +(MTPJ) is not as common as proximal interphalangeal toe joint transfers because of its limited indication. MTPJ +transfers are indicated in metacarpophalangeal joint +(MCPJ) reconstruction mainly. To transfer the MTPJ of a toe to the hand, differences in arc of motion between the MTPJ and the MCPJ are usually corrected. The MTPJ +tends to be hyperextended, while the MCPJ requires flexion for function. Two techniques have been described in Received for publication November 12, 2017; accepted April 11, 2018. Copyright © 2018 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. + +Supplemental digital content is available for this article. Clickable URL citations appear in the text. DOI: 10.1097/GOX.0000000000001805 transferring the MTPJ for the MCPJ reconstruction. One method is the turning over of the MTPJ along its longitudinal axis during the inset.1–4 The other method involves performing an oblique osteotomy at the metatarsal head to neutralize the hyperextension of the MTPJ after the transfer.5 Additionally, when MTPJ is included during lessor toe transfers, an oblique osteotomy of the metatarsal head is recommended. + +Even though the procedure has been applied for several decades, the vascular anatomy of the MTPJ transfer is still not clear. During the MTPJ harvest, the flap is based on either the first dorsal metatarsal artery (FDMA) or the first plantar metatarsal artery (FPMA). Anatomically, the FDMA and FPMA connect to each other at the distal communicating artery, which is adjacent to the MTPJ. The ori- +Disclosure: *The authors have no financial interest to declare in relation to the content of this article. The Article Processing Charge was paid for by the authors.* + +2 + +![1_image_0.png](1_image_0.png) + +entation of the articular branches to the MTPJ becomes very critical, especially when an oblique osteotomy at the metatarsal head is performed. There is a paucity of literatures regarding the vascular anatomy related to the MTPJ. + +In 1984, Kuo et al.1 described 3 patterns of nutrition vessels that supplies the MTP joint. Nonetheless, there were different reports from China regarding the typical number and diameter of the condylar, articular, and metaphyseal branches that nourish the MTPJ.6–9 They describe the condylar branches being distributed usually between 5 and 12mm proximal to the MTPJ, and the metaphyseal branches located around 5mm distal to the joint (Fig. 1). + +According to these articles, the orientation of the nourishing vessels is not predictable. Hence, performing an oblique osteotomy of the metatarsal head could endanger the blood supply of the MTPJ. + +To avoid devascularization of the MTPJ, it is imperative to clarify the vascular pedicle of the MTPJ. The second MTPJ is the most common joint transferred for MCPJ +reconstruction. Instead of identifying the vessels around the MTPJs, this study attempts to report the crucial vessels that could be preserved when performing the oblique osteotomy of the metatarsal head for the toe/joint transfers including the MTP joint. + +## Materials And Methods + +When performing toe or the joint transfers in the clinical practices, the arterial pedicle usually has to be dissected from the toe to the FDMA and FPMA. When harvesting second toes and vascularized joints, a retrograde dissection of the vascular pedicle was performed starting from the distal communicating artery at the first webspace toward the FPMA and the FDMA. All the vessels related to the MTPJ were explored and ligated if the MTPJ was not included in the flap (see video, Supplemental Digital Content 1, which displays the video demonstrating the articular branch during a dissection for the PIP joint transfer, *http://links.lww.com/PRSGO/A831*). The orientation of the vascular branches around the MTPJ were observed. Operation records were reviewed retrospectively in 16 second toes, 25 proximal interphalangeal toe joint, and 4 MTPJ transfers all performed by the senior author. There were 34 males and 4 females. The average age was 32 years. None of the patients had a history of trauma to the second toe. + +## Results + +The condylar branches to the proximal phalanx and the metaphyseal branches to the second metatarsus were barely identifiable during the dissections. The articular branch running perpendicularly from the plantar artery toward the plantar surface of MTPJ was verified in all cases. The articular branches originated either from the FPMA (92.1%, before the distal communicating branch between the FDMA and FPMA; Fig. 1) or from the tibial plantar digital artery (7.9%, after the distal communicating branch; Fig. 2). The external diameter of the articular branches was around 0.5–1mm. When the oblique osteotomy of the metatarsal head was performed, the articular branch could be retracted and protected from injury. + +With inclusion of this articular branch, oozing from the obliquely osteotomized surface of the metatarsal head could be observed immediately with reperfusion of the flap in 1 toe transferred at the MTPJ level (Fig. 3). All the toes and joints were transferred without complications, + +![1_image_1.png](1_image_1.png) + +![2_image_0.png](2_image_0.png) + +![2_image_2.png](2_image_2.png) + +## Discussion + +Although the concept of oblique osteotomy has been described to correct the hyperextension of the MTPJ, only a very limited number of cases have been reported in English literature. Several authors from China have reported their series of MTPJ transfers using rotational osteotomy techniques in the Chinese literature. In 2004, Fang et al.11 obliquely resected the plantar surface of the metatarsal head, rotated the head 90°, and osteosynthesized the resected surface to the metacarpal neck. Eight MTPJs were transferred, and 45–70° (average 66°) of motion was observed. Intracapsular wedge osteotomy of the metatarsal head was published as a technique from a group in China.12,13 They reported that an average of 65° of active motion was obtained in their series. Nonetheless, performing an osteotomy greater than 5–12mm proximal to the MTPJ +was suggested to prevent injury of the vascular pedicle.8,13 When performing an oblique osteotomy of the metatarsal head, the plane of osteotomy has to be within 1cm + +![2_image_1.png](2_image_1.png) + +from the articular surface (Fig. 4). If the osteotomy is too proximal to make the metatarsal too long, the MTPJ will protrude into the palm after inset. This would in turn have a knock-on effect on the flexor and extensor tendons, which would have distorted dynamics. The protruding metatarsal head would be a point of tenderness of which patients often complain of postoperatively. However, when the oblique osteotomy is performed within 1cm from the articular surface, the condylar branches would be endangered and may be compromised, leading to an avascular joint. Furthermore, the condylar branches could not be identified during our clinical dissections. The blood supply to the metatarsal head might then be compromised with this approach. + +Up to now, not as many studies were reported to describe the vascular anatomy in English literature. Kuo et al.1 reported their observation of the articular branch nourishing the MTPJ in 1984. However, their results were not consistent with the findings stated by other articles reported from China.6–9 The information presented in the literatures is thus confusing, and clarification of the vascular anatomy is needed to safely transfer the MTPJ, especially when an oblique osteotomy of the metatarsal head is indicated. This retrospective series is thus important from a surgical technique point of view. + +From our intraoperative observations, constant articular branches running perpendicularly from either the FPMA or the tibial plantar digital artery toward the plantar surface of the MTPJ could be identified. The location and the diameter of these branches were constant and sizable, which were unlike the condylar branches to the proximal phalanx or the metaphyseal branches to the metatarsus. The metatarsal head with oblique osteotomy could be reperfused well with inclusion of the articular branches in 1 case. During the oblique osteotomy of the metatarsal head, the articular branches could be retracted and protected. + +There are some limitations of this study. Although the articular branches from the plantar artery system could be well recognized in our dissections, we could not confirm that they are the only vessels that supply the MTPJ. Direct correlation between the inclusion of these branches and the functional outcomes could not be determined from our limited clinical experience. Nonetheless, by know4 ing the orientation of these articular braches, the MTPJ can be transferred with more confidence and certainty as demonstrated in our series. + +This study was supported with the grant CMRPG3D0801 from Chang Gung Memorial Hospital, Linkou, Taiwan. + +Yu-Te Lin, MD, MS +Department of Plastic and Reconstructive Surgery Chang Gung Memorial Hospital Chang Gung University College of Medicine 222, Mai-Chin Road Keelung City 204, Taiwan linutcgmh@gmail.com + +## References + +1. Kuo ET, Ji ZL, Zhao YC, et al. Reconstruction of metacarpophalangeal joint by free vascularized autogenous metatarsophalangeal joint transplant. *J Reconstr Microsurg*. 1984;1:65–74. + +2. Chen SH, Wei FC, Chen HC, et al. Vascularized toe joint transfer to the hand. *Plast Reconstr Surg*. 1996;98:1275–1284. + +3. Kanaya K, Wada T, Usui M, et al. Fourteen-year results of a reversed vascularized second metatarsophalangeal joint transfer: a case report. *J Hand Surg Am*. 2005;30:120–124. + +4. Kanaya K, Wada T, Kitajima K, et al. Vascularized metatarsophalangeal joint transfer for giant cell tumor of the proximal phalanx of the hand. *Plast Reconstr Surg*. 2008;121:354–355. + +5. Pei GX, Ren GH, Ren YJ, et al. Reconstruction of phalangeal articulations of the hand with vascularised phalangeal articulations of foot. *Injury*. 2008;39:S109–S115. + +6. Ding ZH XC, Pei GX, et al. Applied anatomty of the toe joint for digital joint reconstruction. *Chinese J Clin Anat*. 1995;13:241–244. + +7. Wei CY RZ, Wang H, et al. Arterial supply to the second, third metatarsophalangeal joints and proximal interphalangeal joints of the toe and its clinical significance. *Chinese J Pract Hand Surg*. + +2004;18:158–161. + +8. Yang T WL, Chen CH, et al. Anatomy and clinical application of transplanting modified the second metatarsophalangeal joint for metacarpophalangeal joint reconstruction. *Chinese J Clin* Anat. 2011;29:105–108. + +9. Zhang XY WS, Xiao YF, et al. The replacement of damaged metacarpophalangeal joint with metatarsophalangeal joint: microdissection and clinical application. *Chinese J Clin Anat*. 2009;27:723–727. + +10. Spanio S, Wei FC, Coskunfirat OK, et al. Symmetry of vascular pedicle anatomy in the first web space of the foot related to toe harvest: clinical observations in 85 simultaneous bilateral second-toe transfer patients. *Plast Reconstr Surg*. + +2005;115:1325–1327. + +11. Fang GD XH, Qu ZG, Jiang K, et al. A modified toe-to-hand transfer: ante-rotation of metatarso-phalangeal joint. Chinese J Microsurg. 2004;27:241–242. + +12. Lao KC LZ, Chang CJ, et al. Wedged osteotomy of metatarsal head to improve reconstructed finger function in toe-to-hand transfer. *Chinese J Hand Surg*. 2005;21:288–289. + +13. Li XY LX, Wang CQ, et al. Repair of traumatic metacarpophalangeal joit defect by metatarsophalangeal joint composite tissue flap autograft. *Chinese J Reparative Reconstr Surg*. 2011;25:1043– 1046. \ No newline at end of file diff --git a/medical/md_images/PMC6250473/PMC6250473_meta.json b/medical/md_images/PMC6250473/PMC6250473_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..de55296233b3029b44d4d788277e172a88a1e2a2 --- /dev/null +++ b/medical/md_images/PMC6250473/PMC6250473_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 4, + "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/PMC6410970/3_image_0.png b/medical/md_images/PMC6410970/3_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..992dce3456cd1bbf918a954eaedfc27422431593 --- /dev/null +++ b/medical/md_images/PMC6410970/3_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:6748b3ab54cf998d8026acf30d11fb74501c6b4e8d5b5d8a588f16c1df1a3530 +size 50786 diff --git a/medical/md_images/PMC6410970/5_image_1.png b/medical/md_images/PMC6410970/5_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..21b15b29f8a6ca7039c576440e3bf39aabf9c387 --- /dev/null +++ b/medical/md_images/PMC6410970/5_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f168f46891cd0fbe99961ec42632c1760225ab0ea313e40ca89e2f73905e1b9e +size 27487 diff --git a/medical/md_images/PMC7056307/PMC7056307.md b/medical/md_images/PMC7056307/PMC7056307.md new file mode 100644 index 0000000000000000000000000000000000000000..114f9d77bc892d17e276287aa251012efa24fdf4 --- /dev/null +++ b/medical/md_images/PMC7056307/PMC7056307.md @@ -0,0 +1,177 @@ +BIOPHYSICS + +# A Molecular Rack And Pinion Actuates A **Cell-Surface** Adhesin And **Enables Bacterial Gliding Motility** + +Abhishek Shrivastava1,2,3* and Howard C. **Berg1,4*** +The gliding bacterium *Flavobacterium johnsoniae* **is known to have an adhesin, SprB, that moves along the cell** surface on a spiral track. Following viscous shear, cells can be tethered by the addition of an anti-SprB antibody, causing spinning at 3 Hz. Labeling the type 9 secretion system (T9SS) with a YFP fusion of GldL showed a yellow fluorescent spot near the rotation axis, indicating that the motor driving the motion is associated with the T9SS. The distance between the rotation axis and the track (90 nm) was determined after adding a Cy3 label for SprB. A rotary motor spinning a pinion of radius 90 nm at 3 Hz would cause a spot on its periphery to move at 1.5 μm/s, the gliding speed. We suggest the pinion drives a flexible tread that carries SprB along a track fixed to the cell surface. Cells glide when this adhesin adheres to the solid substratum. + +Copyright © 2020 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 + +Rod-shaped gliding bacteria move in a manner similar to a selfpropelled screw; i.e., they roll along their long axis as they move forward on an external surface (1, 2). Bacteria related to *Flavobacterium* johnsoniae, which is the fastest of all known gliders, are present in diverse environments such as the human oral microbiome (3), fish scales (4), water bodies (5), and plant rhizosphere (6), where they glide over and colonize their preferred surfaces. How the molecular players form the machinery that actuates this motion is not clearly understood. *F. johnsoniae* is now the organism of choice for studies of this process because rates of movement are high, and much of the genetics is known (7, 8). A mobile cell-surface adhesin, SprB, has been identified, which plays a central role in gliding (9, 10). Tracking of SprB in three-dimensional space has revealed the presence of a spiral cell-surface track on which SprB moves (1). Cells subjected to viscous shear stop gliding, but they can be tethered to an external surface using anti-SprB antibody. Tethered cells pinwheel around a fixed axis, suggesting that a rotary motor that generates high torque is a part of the gliding machinery (11). + +Bacterial motility machines have external components and are thus coupled with protein secretion systems. The flagellar motor is associated with the type 3 secretion system (12, 13), and the type 4 pili motor is associated with the type 2 secretion system (14–16). In Flavobacterium, the type 9 secretion system (T9SS) is required for the secretion of SprB, and cells lacking the T9SS are nonmotile (17). T9SS spans the inner and outer membranes and the periplasmic region. The T9SS has an outer-membrane barrel made up of a protein SprA. It is one of the largest transmembrane barrels known in biology (18). Structural data suggest that in the periplasmic region, the T9SS forms at least one ring-shaped structure that contains GldK, while GldM spans the periplasmic region (19, 20). GldL is a cytoplasmic membrane protein and is one of the core T9SS proteins (17). Figures that summarize the current proposed macromolecular arrangement of T9SS can be found in (18, 19). Our results suggest that T9SS is associated with the rotary component of the gliding motor and drives a tread carrying the adhesin SprB along a track fastened to the rigid framework of the cell wall. + +## Results The Axis Ofrotation Is Localized Near Gldl + +GldK, GldL, GldM, GldN, SprA, SprE, SprF, and SprT are the core T9SS proteins required for the transport of SprB to the external surface. ∆*gldL* mutants do not have SprB on the cell surface. They are not able to stick to a glass surface and do not display motility (17). We found that tethered cells of ∆*gldL* mutants complemented by GldL– +yellow fluorescent protein (YFP) were able to bind to glass and exhibited wild-type levels of cell rotation (movie S1). This suggests that the rotary motor is fully functional in this strain. + +Fluorescent GldL-YFP appeared as spots that were fixed in the frame of reference of a cell (Fig. 1A and movie S2). Using ImageJ and custom MATLAB codes C1 to C3 that fit a Gaussian to each GldL spot, the positions of GldL foci from 42 cells were determined. Custom MATLAB codes used in this article are freely available on GitHub (https://github.com/Abhishek935/Molecular-Rack-andPinion). The GldL spots appeared to be localized at random within a cell, with the average distance between two neighboring spots of about 1.5 m (fig. S1). The peak of a Gaussian fit to a frequency distribution of number of foci per cell as measured from 42 cells is 3.47 with an SD of 0.87 (Fig. 1B). The total internal reflection fluorescence (TIRF) microscope used for these measurements strongly illuminates only the cylindrical face of the cell adjacent to the glass with a penetration depth around 100 nm. A similar distribution of GldL is expected on the opposite cylindrical face. Hence, the total number of GldL foci per cell is about seven. + +Using tethered cells of ∆*gldL* background complemented with GldL-YFP, the position of the axis of rotation was determined by adding successive images of an image stack of a rotating cell and fitting the new matrix that contains the sum of images with a Gaussian that has the X-Y coordinates as position and the Z coordinate as intensity. Code C4 was used to add successive images. Given that the axis of rotation remains fixed over all image frames of a rotating cell, for a matrix that contains the sum of stacks, the intensity is highest at the axis of rotation. All GldL foci in a rotating cell were + +![1_image_0.png](1_image_0.png) + +tracked over several frames. All but one GldL spot per tethered cell showed a circular trajectory (Fig. 1A). The foci that did not move with an apparent circular trajectory were within a distance range of few hundred nanometers from the axis of rotation (Fig. 1C). This spot was named the "nearest neighbor" GldL. + +Similar measurements were made for each image frame from image stacks of eight rotating tethered cells with 60 frames on average per image stack. This provided a total of 484 measurements. The SDs for the Gaussian fits to the images of the rotation axes and the nearest-neighbor GldL foci bloom up to the resolving power of the microscope. The position of nearest-neighbor GldL relative to the axis of rotation is shown for all eight cells in Fig. 2A, with the axis of rotation normalized to be at the origin. The position of nearestneighbor GldL relative to the axis of rotation, one cell at a time, is shown in fig. S2. The peak of a Gaussian fit to a frequency distribution of distance between the nearest-neighbor GldL and the axis of rotation for all 484 measurements was 109.7 nm with an SD of 68.5 nm +(Fig. 2B). These measurements tell us that the axis of rotation was found near a GldL spot, as expected if GldL is part of the rotary motor. However, we do not know whether GldL is centered on the rotation axis. + +## Cell Rotation And **Sprb Motion Use The** Same Power Source + +A preparation of tethered cells was exposed to 10 M carbonyl cyanide-m-chlorophenylhydrazone (CCCP), which is known to abolish the proton motive force that acts across the inner cell membrane (21). The cells stopped rotating after the addition of CCCP and started rotating again after its removal (figs. S3 and S4 and movie S3). The loss of rotation was rapid and the recovery was gradual, taking about four times longer, as expected if time was required to wash CCCP out of the cells. These results are consistent with previous observations where motion of SprB was stopped after the addition of CCCP and was restored after washing it away (10). This suggests that both cell rotation and SprB translocation use the same fuel. + +MreB, a protein that helps to build the cell wall, is involved in the gliding motility of a rod-shaped bacterium *Myxococcus xanthus* (22). A22, a drug that alters the motion of MreB and stops the gliding of M. xanthus (22), did not affect the gliding of *F. johnsoniae* (fig. S5). As in *F. johnsoniae*, binding of a cell-surface adhesin to an external surface results in gliding of *M. xanthus*. However, there are obvious differences between the gliding of the two types of bacteria. *M. xanthus* uses different motility proteins and glides about 60 times more slowly than *F. johnsoniae*. + +## The Track On Which Sprb Moves Is Close To The **Gliding Motor** While The Motor Labeled By Gldl-Yfp Is Fixed On The Cell Surface, + +the SprB adhesin moves along the length of the cell. How rotation might be coupled to linear motion is a puzzle. The ∆*gldL* strain is complemented for rotary motor function by the addition of GldLYFP (movie S1). However, the long-distance motion of the cell and the adhesin SprB is not fully restored; i.e., some cells and beads attached to SprB move very short distances (movie S1). This suggests that the C-terminal region of GldL might play a role in coupling the rotary motor with the motion of SprB. To study the long-distance movement of SprB in a *gldL-yfp* strain, a *gldL-yfp*–containing plasmid was added to wild-type cells to form a hybrid motor containing both GldL and GldL-YFP subunits. Western blot analysis suggests that GldL expressed from the chromosome and pCP23 is of similar cellular concentration (17). A Cy3 tag was attached to SprB via an antibody, and the positions of both SprB and GldL were determined within same cells (Fig. 3A, fig. S6, and movie S4). The shortest distance between the trajectory of SprB labeled with Cy3 and motors labeled with GldL-YFP was measured for 42 motors from 10 cells. The average positions of SprB labeled with Cy3 and GldL-YFP (motor) were determined by fitting Gaussians to respective foci from all frames of an image stack. The positions of motors were at a distance (d) of 90.9 nm with an SD of 63.7 nm from the SprB trajectory (Fig. 3B). + +As a control, a simulation in which 42 motors were randomly localized in one cell was performed, together with the SprB trajectory from one cell. This simulation was carried out using a random number generator in MATLAB and similar boundaries as that of a cell in an image plane were used. The shortest distances between the motors and the track were measured for the simulation. The distribution for these distances was broad and did not show a discernible peak near 90 nm (Fig. 3, C and D). + +6 + +![2_image_0.png](2_image_0.png) + +![2_image_1.png](2_image_1.png) + +3 of 6 Tracks carrying SprB did not loop around GldL spots. This argues against a model in which chains carrying SprB are driven by sprockets (7). A viable alternative, explained below, involves racks and pinions. + +## A Model For Gliding Is Suggested In Which A **Pinion Attached** + +![3_Image_0.Png](3_Image_0.Png) To The Rotary Motor'S Drive Shaft Engages A **Rack (A Tread)** That Slides Along A Track Fixed To The **Rigid Framework** Ofthe **Cell Wall** + +A cartoon illustrating this idea is shown in Fig. 4A. A combination of a pinion (a circular gear) and a rack (a straight element with teeth matching those on the gear) is a common device for converting rotary motion into linear motion. If the motor rotates at frequency f and the radius of the pinion is r, then the rack will move at velocity v = 2rf, which is the gliding speed (about 1.5 m/s). Given the maximum f that the rotary motor exhibits is about 3 Hz (11), we find r = v/2f = +80 nm, which is on the mark. + +The rotary motor, although it runs at constant speed (11), could be similar in architecture to the flagellar rotary motor, with a rotor embedded in the inner cell membrane surrounded by stator elements (the stationary part of a motor) anchored to the peptidoglycan layer and a drive shaft that passes through the peptidoglycan with the aid of a bushing. The pinion would be mounted on the drive shaft but could rotate under or within the outer cell membrane, driving the tread along a track anchored to the peptidoglycan. Only SprB needs to extend beyond the outer cell membrane. An animation of the behavior of our model is shown in Fig. 4B and movie S5. + +## Discussion + +It appears that the presence of multiple SprBs ensures smooth gliding; i.e., if several SprBs are mounted on a given tread, a cell can glide continuously. An electron microscope image suggests that there are about 40 SprBs on a given cell (10). So, there will be about 20 on one cylindrical face of a cell. An SprB arriving at the back of a cell can peel off the substrate to be replaced by new SprB adsorbed to the substrate at the front of the cell. As a result, the cell can glide smoothly over many body lengths. However, if a single SprB is mounted on a given tread, then that SprB will remain fixed and the free end of the cell will lift off the surface and flip over, allowing the cell to continue to glide in the same direction but without any net progress. Gliding cells often display flips; i.e., the lagging pole of a cell gliding in the x-y image plane gets stuck to glass, and the leading pole rises up along the z axis and moves rapidly, forming an arc such that it moves behind the lagging pole (fig. S7 and movie S5). The flips are illustrated in fig. 1 and movie S1 of (7) and in fig. 4 of (23). After a flip, the direction along which a cell glides changes. + +At the time of the early work (23), no gliding motility proteins were known; all that could be surmised was that sites able to adhere to the substrate moved the length of a cell along tracks fixed to the rigid framework of the cell wall. Fluorescent labeling of proteins that enable gliding motility has improved our knowledge of the mechanism. Future experiments will be aimed toward functional characterization of proteins that form this machinery. Several *F. johnsoniae* mutants are deficient in proteins that either form the T9SS or are involved in the stability of the T9SS proteins. In these mutants, SprB is not secreted to the cell surface and the cells do not glide. Of more interest are mutants that do secrete SprB to the cell surface but, nonetheless, are not able to glide. Cells that lack the C-terminal region of GldJ (24), a protein that is not a part of the T9SS, are an example. Recently, it was found that when the C-terminal 8 to 13 amino acid regions of GldJ are deleted, secretion via the T9SS is fine, while motion of SprB on the cell surface is significantly reduced. GldJ localizes helically, is an outermembrane lipoprotein (25), and might be associated with the tread/track complex. Identification of additional components of the rack and pinion assembly will enhance our understanding of bacterial gliding. + +## Materials And **Methods** Strains And **Plasmids** + +A GldL-YFP fusion was generated by amplifying eYFP from pHL55 (26) using primers P65 and P67 (for primers, see the Supplementary Materials) and cloning it into the vector pCP23. *gldL* was amplified using primers P63 and P64 and was cloned into pCP23 with YFP to generate the plasmid pAS6 such that the C terminus of GldL was fused with the N terminus of YFP. Strains were grown at 25°C with shaking, as described previously (1). + +## Tirf Imaging Ofgldl In **Tethered Cells** + +Cells were tethered using a protocol described previously (11). To image rotating tethered bacteria with GldL-YFP, a microscope (Nikon Eclipse Ti-U; Nikon, Melville, NJ) with an Apo TIRF 60× 1.49 numerical aperture (NA) objective was used in TIRF mode. A 515-nm, 25-mW laser was used as an excitation source. Fluorescent images were recorded with an Andor Ixon Camera. Phase-contrast images were recorded at the same time in the near infrared using a ThorLabs DCC1545M-GL camera. Images were analyzed using the ImageJ plugin Trackmate (27) and custom MATLAB codes. + +## Simultaneous Tirf Imaging Of **Sprb Andgldl** + +SprB was labeled with Cy3 as follows: 2 l of 1:10 diluted purified anti-SpB antibody (1) and 2 l of Cy3 conjugated Goat Anti-Rabbit IgG Polyclonal Antibody (VWR R611-104-122) were added to 40 l of 0.4 Optical Density bacterial culture and incubated for 10 min at 25°C. After incubation, the preparation was centrifuged at 12,000g for 5 min. The supernatant fraction was discarded, and the pellet was resuspended in 40 l of motility medium [MM; per liter: 1.1 g of Bacto Casitone, 0.55 g of yeast extract, and 1.1 mM tris (pH 7.5)]. A +tunnel slide was prepared, and 40 l of bovine serum albumin +(10 mg/ml) was added to the tunnel and allowed to stand for 1 min, after which the Cy3-labeled cells were flowed into one end of the tunnel, and a Kimwipe was used to wick the excess fluid from the other end. This preparation was allowed to stand for 10 min and then was washed twice in the tunnel slide with 40 l of MM. + +To image YFP and Cy3 signals simultaneously, the TIRF system described above was used with a Photometrics DV2 attachment with yellow and red filters (Chroma ET 535/30 and ET 630/75, respectively) to image YFP on one side of the screen and Cy3 on the other. Using a custom MATLAB code, Gaussians with the position along the x-y axis and intensity along the z axis were fit to the Cy3 and YFP spots in each image frame. The position of the peak of each Gaussian fit was recorded as the average position of each spot. + +## Addition Of Cccp And A22 + +Wild-type *F. johnsoniae* cells were sheared and tethered to a glass coverslip and attached to a flow cell, and rotation speed was measured as described previously (11). A CCCP stock was prepared (10 M in MM and added at a rate of 50 l/min using a syringe pump, Harvard Apparatus 22). To remove the drug, MM was pumped into the flow cell. + +A22 (50 g/ml; Millipore Sigma, catalog no. 475951) was added to cells gliding on glass in a tunnel slide, as described previously. Cells were imaged using a phase-contrast microscope with a Nikon Plan 40× BM NA 0.65 objective (Nikon, Melville, NY) and a Thorlabs DCC1545M-GL (Thorlabs, Newton, NJ) camera. Images were analyzed using a custom MATLAB code. + +## Supplementary Materials Supplementary Material For This Article Is Available At Http://Advances.Sciencemag.Org/Cgi/ + +content/full/6/10/eaay6616/DC1 Primers used in the study Computer scripts Fig. S1. A frequency distribution of nearest-neighbor distances between 42 GldL foci found on 10 cells. Fig. S2. Position of the nearest-neighbor GldL foci from the axis of rotation of eight tethered cells shown for the individual cells. Fig. S3. Rotation speed of a cell plotted as a function of time. Fig. S4. The rate for the "drop" in rotation speed after addition of CCCP (red linear regression fit) is four times faster than the rate of increase following removal of CCCP. Fig. S5. Speed of a smooth gliding cell measured by tracking the center of mass does not change after the addition of A22. Fig. S6. A cell with SprB-Cy3 and GldL-YFP labels. + +Fig. S7. Tracking the flips displayed by gliding cells. + +Movie S1. A phase-contrast image stack where GldL-YFP strain displays wild-type levels of rotation. Similar to sheared and tethered wild-type cells, two cells in the field of view display rotation. Other cells attach to glass and beads coated with anti-SprB antibody. Some cells and beads display motion over short distances. Movie S2. A tethered cell with five fluorescent GldL spots. The cell appears to rotate around a fixed axis. + +Movie S3. A phase-contrast image stack showing that addition of CCCP stops rotation of a tethered cell. Movie S4. A Cy3-tagged SprB moving along the length of a cell. For this movie, a filter that allows only Cy3 emission to pass was used. Movie S5. An animation of the model suggesting how a molecular rack and pinion could drive gliding. The pinion is blue, the tread is green, and the adhesin SprB is magenta. Movie S6. A cell gliding smoothly over a glass surface. Movie S7. A gliding cell displaying flips. + +View/request a protocol for this paper from *Bio-protocol*. + +## References And Notes + +1. A. Shrivastava, T. Roland, H. C. Berg, The screw-like movement of a gliding bacterium is powered by spiral motion of cell-surface adhesins. *Biophys. J.* 111, 1008–1013 (2016). + +2. L. M. Faure, J. B. Fiche, L. Espinosa, A. Ducret, V. Anantharaman, J. Luciano, S. Lhospice, S. T. Islam, J. Tréguier, M. Sotes, E. Kuru, M. S. Van Nieuwenhze, Y. V. Brun, O. Théodoly, L. Aravind, M. Nollmann, T. Mignot, The mechanism of force transmission at bacterial focal adhesion complexes. *Nature* 539, 530–535 (2016). + +3. A. Shrivastava, V. K. Patel, Y. Tang, S. C. Yost, F. E. Dewhirst, H. C. Berg, Cargo transport shapes the spatial organization of a microbial community. *Proc. Natl. Acad. Sci. U.S.A.* 115, 8633–8638 (2018). + +4. E. Duchaud, T. Rochat, C. Habib, P. Barbier, V. Loux, C. Guérin, I. Dalsgaard, L. Madsen, H. Nilsen, K. Sundell, T. Wiklund, N. Strepparava, T. Wahli, G. Caburlotto, A. Manfrin, G. D. Wiens, E. Fujiwara-Nagata, R. Avendaño-Herrera, J. F. Bernardet, P. Nicolas, Genomic diversity and evolution of the fish pathogen *Flavobacterium psychrophilum*. + +Front. Microbiol. 9, 138 (2018). + +5. A. Eiler, S. Bertilsson, Flavobacteria blooms in four eutrophic lakes: Linking population dynamics of freshwater bacterioplankton to resource availability. *Appl. Environ. Microbiol.* +73, 3511–3518 (2007). + +6. S. B. Peterson, A. K. Dunn, A. K. Klimowicz, J. Handelsman, Peptidoglycan from *Bacillus cereus* mediates commensalism with rhizosphere bacteria from the cytophaga-flavobacterium group. *Appl. Environ. Microbiol.* 72, 5421–5427 (2006). + +7. A. Shrivastava, H. C. Berg, Towards a model for flavobacterium gliding. Curr. Opin. + +Microbiol. 28, 93–97 (2015). + +8. R. G. Rhodes, H. G. Pucker, M. J. McBride, Development and use of a gene deletion strategy for *Flavobacterium johnsoniae* to identify the redundant gliding motility genes remF, remG, remH, and remI. J. *Bacteriol.* 193, 2418–2428 (2011). + +9. S. S. Nelson, S. Bollampalli, M. J. McBride, SprB is a cell surface component of the Flavobacterium johnsoniae gliding motility machinery. J. *Bacteriol.* 190, 2851–2857 +(2008). + +10. D. Nakane, K. Sato, H. Wada, M. J. McBride, K. Nakayama, Helical flow of surface protein required for bacterial gliding motility. *Proc. Natl. Acad. Sci. U.S.A.* 110, 11145–11150 (2013). + +11. A. Shrivastava, P. P. Lele, H. C. Berg, A rotary motor drives flavobacterium gliding. + +Curr. Biol. 25, 338–341 (2015). + +12. M. Erhardt, N. Namba, K. T. Hughes, Bacterial nanomachines: The flagellum and type III +injectisome. *Cold Spring Harb. Perspect. Biol.* 2, a000299 (2010). + +13. A. Blocker, K. Komoriya, S. I. Aizawa, Type III secretion systems and bacterial flagella: +Insights into their function from structural similarities. *Proc. Natl. Acad. Sci. U.S.A.* 100, 3027–3030 (2003). + +14. Y. W. Chang, L. A. Rettberg, A. Treuner-Lange, J. Iwasa, L. Søgaard-Andersen, G. J. Jensen, Architecture of the type IVa pilus machine. *Science* 351, aad2001 (2016). + +15. J. M. Skerker, H. C. Berg, Direct observation of extension and retraction of type IV pili. + +Proc. Natl. Acad. Sci. U.S.A. 98, 6901–6904 (2001). + +16. D. A. Cisneros, G. Pehau-Arnaudet, O. Francetic, Heterologous assembly of type IV pili by a type II secretion system reveals the role ofminor pilins inassembly initiation. *Mol. Microbiol.* 86, 805–818 (2012). + +17. A. Shrivastava, J. J. Johnston, J. M. van Baaren, M. J. McBride, *Flavobacterium johnsoniae* GldK, GldL, GldM, and SprA are required forsecretion of the cell surface gliding motility adhesins SprB and RemA. J. *Bacteriol.* 195, 3201–3212 (2013). + +18. F. Lauber, J. C. Deme, S. M. Lea, B. C. Berks, Type 9 secretion system structures reveal a new protein transport mechanism. *Nature* 564, 77–82 (2018). + +19. P. Leone, J. Roche, M. S. Vincent, Q. H. Tran, A. Desmyter, E. Cascales, C. Kellenberger, C. Cambillau, A. Roussel, Type IX secretion system PorM and gliding machinery GldM +form arches spanning the periplasmic space. *Nat. Commun.* 9, 429 (2018). + +20. D. G. Gorasia, P. D. Veith, E. G. Hanssen, M. D. Glew, K. Sato, H. Yukitake, K. Nakayama, E. C. Reynolds, Structural insights into the PorK and PorN components of the Porphyromonas gingivalis Type IX secretion system. *PLOS Pathog.* 12, e1005820 (2016). + +21. F. M. Harold, J. R. Baarda, Inhibition of membrane transport in *Streptococcus faecalis* by uncouplers of oxidative phosphorylation and its relationship to proton conduction. + +J. *Bacteriol.* 96, 2025–2034 (1968). + +22. E. M. Mauriello, F. Mouhamar, B. Nan, A. Ducret, D. Dai, D. R. Zusman, T. Mignot, Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA. + +EMBO J. 29, 315–326 (2010). + +23. I. R. Lapidus, H. C. Berg, Gliding motility of *cytophaga* sp. strain U67. J. *Bacteriol.* 151, 384–398 (1982). + +24. J. J. Johnston, A. Shrivastava, M. J. McBride, Untangling *Flavobacterium johnsoniae* gliding motility and protein secretion. J. *Bacteriol.* 200, (2018). + +25. T. F. Braun, M. J. McBride, *Flavobacterium johnsoniae* GldJ is a lipoprotein that is required for gliding motility. J. *Bacteriol.* 187, 2628–2637 (2005). + +26. A. Boehm, M. Kaiser, H. Li, C. Spangler, C. A. Kasper, M. Ackermann, V. Kaever, V. Sourjik, V. Roth, U. Jenal, Second messenger-mediated adjustment of bacterial swimming velocity. *Cell* 141, 107–116 (2010). + +27. J. Y. Tinevez, N. Perry, J. Schindelin, G. M. Hoopes, G. D. Reynolds, E. Laplantine, S. Y. Bednarek, S. L. Shorte, K. W. Eliceiri, TrackMate: An open and extensible platform forsingle-particle tracking. *Methods* 115, 80–90 (2017). + +## Acknowledgments + +Funding: This work was supported by National Institutes of Health K99/R00 grant DE026826 to A.S. and an R01 grant AI016478 to H.C.B. **Author contributions:** A.S. and H.C.B. designed the experiments and wrote the paper. A.S. performed the experiments. **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. Additional data related to this paper may be requested from the authors. Submitted 8 July 2019 Accepted 10 December 2019 Published 4 March 2020 10.1126/sciadv.aay6616 Citation: A. Shrivastava, H. C. Berg, A molecular rack and pinion actuates a cell-surface adhesin and enables bacterial gliding motility. *Sci. Adv.* 6, eaay6616 (2020). \ No newline at end of file diff --git a/medical/md_images/PMC7056307/PMC7056307_meta.json b/medical/md_images/PMC7056307/PMC7056307_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..2483d041da9435bc311ce4f24ddf68854229a269 --- /dev/null +++ b/medical/md_images/PMC7056307/PMC7056307_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 6, + "ocr_stats": { + "ocr_pages": 1, + "ocr_failed": 0, + "ocr_success": 1, + "ocr_engine": "surya" + }, + "block_stats": { + "header_footer": 30, + "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/PMC7216764/2_image_0.png b/medical/md_images/PMC7216764/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..d3a695c59cccbc50fc6f6e6952773dccf8eecaca --- /dev/null +++ b/medical/md_images/PMC7216764/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0b553b192def742e4f288df4d41f3fdb23e7e9e7b630c9cccd7681188ed71c56 +size 51268 diff --git a/medical/md_images/PMC7216764/4_image_0.png b/medical/md_images/PMC7216764/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..1b97c2a3043adb9b7a120db33bbe190ec669d41e --- /dev/null +++ b/medical/md_images/PMC7216764/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:660184546071e8c3458c546c0968d32e91d6bcce60862ef102594682c69c52e0 +size 31761 diff --git a/medical/md_images/PMC7216764/5_image_0.png b/medical/md_images/PMC7216764/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..f00feeb34ee4ab26e5d8b823a45fa48e0c68f8ea --- /dev/null +++ b/medical/md_images/PMC7216764/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:aec51490d275c2579c659f741f57ab5b350351f9adae8d013d114d1622acc2cf +size 56880 diff --git a/medical/md_images/PMC7359379/2_image_0.png b/medical/md_images/PMC7359379/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..90b903fc6f092adfe36db72e2fdc45cc42b02cd5 --- /dev/null +++ b/medical/md_images/PMC7359379/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:507c876c0ef1aaabe16abbea2fe59042efd616338c4e35d8d02c970df0f04a70 +size 86908 diff --git a/medical/md_images/PMC7359379/3_image_0.png b/medical/md_images/PMC7359379/3_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..80ce7676f0377b4496b7fc53dcb814b4720ce168 --- /dev/null +++ b/medical/md_images/PMC7359379/3_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:fd3b3a0c83303200eed595f6107c7952260b597f3e426c96f895c6f5b45430b0 +size 354229 diff --git a/medical/md_images/PMC7359379/4_image_0.png b/medical/md_images/PMC7359379/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..714820179f4e1974ab4dbc2b67d2a8fc6181228c --- /dev/null +++ b/medical/md_images/PMC7359379/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:4a503c0a72664df5438f9eccb4ea49712b0a77e1bd2ab8a24ce286d43882c3f2 +size 2054 diff --git a/medical/md_images/PMC7359379/4_image_1.png b/medical/md_images/PMC7359379/4_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..07bb55689befa11a9120b17a3b6b952f4f14ad07 --- /dev/null +++ b/medical/md_images/PMC7359379/4_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f0e8f9a0783ddf091e78e7373f1ae4b0b7389a9096b1d542389b201e6433e829 +size 52750 diff --git a/medical/md_images/PMC7359379/4_image_2.png b/medical/md_images/PMC7359379/4_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..9555782c9f0151380d129c368b15a110b5a5cd90 --- /dev/null +++ b/medical/md_images/PMC7359379/4_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:109147db2449c0378a4b809344aaf1962352e522a7ecfa35f0ecc24eb25935e4 +size 3206 diff --git a/medical/md_images/PMC7359379/PMC7359379.md b/medical/md_images/PMC7359379/PMC7359379.md new file mode 100644 index 0000000000000000000000000000000000000000..5c42365ba4fbae857aa8eb1105112e5af57ffea9 --- /dev/null +++ b/medical/md_images/PMC7359379/PMC7359379.md @@ -0,0 +1,185 @@ +# Novel Education-Based Intervention To + +reduce inappropriate antibiotic prescribing for treatment of gonorrhoea in China: protocol for a cluster randomised controlled **trial** +Ting-Ting Jiang ,1,2 Yun-Qing Yang,3 Ning-Xiao Cao,1,2 Yue-Ping Yin,1,2 Xiang-Sheng Chen 1,2 To cite: Jiang T-T, Yang YQ, Cao N-X, *et al*. Novel education-based intervention to reduce inappropriate antibiotic prescribing for treatment of gonorrhoea in China: protocol for a cluster randomised controlled trial. *BMJ Open* 2020;10:e037549. doi:10.1136/ bmjopen-2020-037549 +► 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-2020037549). Received 06 February 2020 Revised 11 May 2020 Accepted 13 May 2020 © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. + +1Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing, China 2National Center for STD Control, Chinese Center for Disease Control and Prevention, Nanjing, China 3Department of prevention and health care, Guangzhou Institute of Dermatology, Guangzhou, China Correspondence to Dr Xiang-Sheng Chen; chenxs@ncstdlc.org + +## Abstract + +Introduction Inappropriate use of antibiotics to treat gonorrhoea can lead to antibiotic resistance. Education programmes may be helpful for improving physician prescribing behaviours in accordance with treatment guidelines. As traditional education based on printed materials may have limited effect on guideline-based treatment, innovative education strategies are needed. The current trial aims to assess the effectiveness of a novel education intervention to increase guideline-based treatment of gonorrhoea in China. Methods and analysis We will conduct a two-arm cluster randomised control trial at 144 hospitals (clusters) in eight Chinese provinces. The intervention will include an online training video developed on the WenJuanXing platform that covers workflows and requirements for managing a patient with uncomplicated gonorrhoea. Outpatient physicians in dermatology (dermatovenerology), urology, andrology and gynaecology will be given access to the video via a quick response code. In hospitals allocated to the control arm, physicians will continue to participate in their standard of care training programme. The primary outcome is the proportion of gonorrhoea antibiotic prescriptions adherent to Chinese national guidelines at the cluster level. In addition, to understand the reasons of physician's non-adherence to the intervention by conducting a questionnaire survey will be considered as the secondary outcome of the study. Ethics and dissemination Ethical approval was obtained from the Medical Ethics Committee of the Chinese Academy of Medical Sciences Institute of Dermatology (2020-LS-004). All physicians will provide an informed consent prior to participating in the study. Findings of the trial will be disseminated through conferences and peerreviewed journals, and will be used to develop training programmes for physicians. Trial registration number ChiCTR2000029591. + +## Introduction + +Gonorrhoea, caused by *Neisseria gonorrhoeae*, +remains one of the most common sexually transmitted diseases (STDs) worldwide. + +## Strengths And Limitations Of This Study + +► The study design (cluster randomised controlled trial) is a robust methodology to assess the effectiveness of behavioural interventions. + +► The study will be carried out in eight different provinces in China, thus will provide insights on whether the intervention can be implemented in different local contexts. + +► The questionnaire administered to physicians prior to the intervention may influence the prescribing behaviours of physicians from hospitals in the control arm, which may undermine the effect of intervention. + +According to the most recent WHO report in 2016, there were 86.9 million incident cases (15–49 years of age) globally, which were more than 10% higher than that in 2012.1 Left untreated, the infection can cause serious medical problems, particularly for women, including chronic pelvic pain, ectopic pregnancy and even infertility.2 In addition to the behavioural interventions to prevent infection of gonorrhoea, timely detection of the infection followed by effective treatment with antibiotics remains the mainstream strategy to control this infection. Unfortunately, N. gonorrhoeae has developed resistance to nearly every antibiotic ever used to effectively treat it since sulphonamides were introduced as the first drugs for this treatment in 1940s.3 Due to widespread emergence of the resistant strains, only the extended-spectrum cephalosporin (ESC) and azithromycin which is usually used with ceftriaxone in dual therapy are left to be recommended as first-line regimen for treatment of gonorrhoea in most countries.4–7 However, in recent years, gonococcal strains with reduced susceptibility or resistance to ESC and resistance to high-level azithromycin have been identified from many countries including China.8 9 Treatment failures with ceftriaxone have been reported in Japan, Australia, European countries, Canada and South Africa.10–13 Data from the China Gonococcal Resistance Surveillance Programme indicated that 18.6% of clinical isolates were resistant to azithromycin and 9.7%–12.2% of clinical isolates were less susceptible to ceftriaxone over the years of 2013 and 2016.14 It has been recognised that much of the antimicrobial resistance (AMR) of gonorrhoea originates in Asia and then transmitted to the rest of world.15 16 Although many factors contribute to the emergence of gonococcal AMR, inappropriate use (misuse or overuse) of antibiotics for treatment of gonorrhoea in clinical practice in many countries in Asia may be one of drivers for AMR due to antibiotic selective pressure. A recent study in China indicated less than 1% of patients with uncomplicated gonorrhoea were treated following the ceftriaxone regimen recommended by the national guidelines (ceftriaxone 250 mg intramuscular as a single dose).17 More than 70% of the patients received a dosage of more than 1 g for their treatment.17 Several factors affect physicians' prescribing behaviours, including knowledge about the antibiotics, awareness of national guidelines, previous experience in treatment and trust to the antibiotics. A nationwide survey in China revealed a higher adherence to the national guidelines for treatment of uncomplicated gonorrhoea among physicians who were aware of the guidelines than those who were not. Additionally, participation in training courses did not significantly improve the prescribing behaviours of physicians,18 indicating that more innovative interventions are needed to address the issues of inappropriate antibiotic use in treatment of gonorrhoea in China. Exploring innovative strategies to ensure appropriate use of antibiotics for treatment of gonorrhoea has been listed as one of the priorities in a comprehensive research plan called ROADMAP (resistance surveillance, outcomes due to AMR, antibiotic stewardship and application, diagnostic tools, mechanisms of AMR, antimicrobial assessment and population pharmacokinetics and pharmacodynamics) to address research needs for gonococcal AMR in China.19 Mobile health (mHealth), defined as interventions and programmes designed to support medical and public health through the use of mobile technology, has been used as a tool to deliver continuous training and education to healthcare providers.20 21 Previous randomised controlled trials (RCTs) have shown success of mHealth in optimising antibiotic prescribing.22–24 However, there has been no studies to shine a light on whether such training programme is beneficial to improve antibiotic prescribing behaviours among physicians who provide treatment to patients with STDs. For this purpose, we will conduct a cluster randomised trial to evaluate whether the proposed intervention (online video-based training programme) could significantly improve physicians' prescribing behaviours to adhere to the regimens for treatment of uncomplicated gonorrhoea (1 g intramuscular ceftriaxone single dose) recommended by the National STD Treatment Guidelines25 (hereinafter referred to the National Guidelines). Our secondary objective is to understand the reasons of physicians' non-adherence to the online video-based training programme. + +## Methods **And Analysis** Study Design And Setting + +We will use a parallel-group, cluster-RCT with one intervention and one control arm, using a 1:1 allocation ratio, to evaluate whether the intervention is superior to the control treatment (figure 1). Selection of provinces for the study is based on the reported incidence of gonorrhoea in 2018. The eight provinces (Jiangsu, Shanghai, Zhejiang, Fujian, Guangdong, Guangxi, Hainan and Yunnan) with the highest incidence of reported gonorrhoea cases (figure 2) in China were selected. Among the eight selected provinces, Jiangsu, Shanghai, Zhejiang, Fujian, Guangdong and Hainan are in the eastern areas, while Guangxi and Yunnan are in the western areas of China. According to geographic location and level of economic development, mainland China was classified into three areas—eastern areas (high level of economic development), middle areas (middle level of economic development) and western areas (low level of economic development). In China, healthcare workers (especially highly educated physicians) usually prefer to work in more economically developed areas, which results in a severe inequality in the quantity and quality of healthcare workforce. Training is considered an important way to enhance professional skills of healthcare workers, but the effects of training may vary from area to area. Therefore, by evaluating the intervention effects in Chinese hospitals in multiple provinces, the current study may provide insights on whether the intervention can be effective across different local contexts. + +Within each study province, we will invite the city-level hospitals with the highest volume of reported gonorrhoea cases to participate in the study. Hospitals at national or province-level will be excluded from the study, because there are such a small number of these hospitals that will make it difficult to ensure comparable assignments into the intervention and control arms. Hospitals will be randomised into the intervention or the control arm, stratified by province, considering the potential variation in outcomes between provinces. Core elements of the intervention are online training on antibiotic prescribing for treatment of uncomplicated gonorrhoea among physicians working in the department of dermatology (dermatovenerology), urology, andrology or gynaecology in the intervention arm hospitals. A clustered design is used to avoid the risk of contamination between different physicians within hospitals. + +The study will be divided into two subtrials, that is, a pilot trial and a main trial. The pilot trial will be conducted in one province randomly selected from the eight study provinces. Data will be collected simultaneously in nine + +![2_image_0.png](2_image_0.png) + +intervention and nine control hospitals for a month to examine the feasibility and acceptability of the intervention. If more than 60% of physicians are willing to use the online training, the internal pilot hospitals and their outcome data will then become part of the main trial, and will be followed up for another 5 months (figure 1). The remaining hospitals will be enrolled into the main trial and followed up for 6 months (in other words, data collection in the pilot and main trial hospitals will finish at different times). An independent advisory group consisting of key investigators, a programme manager, hospital representatives and a statistician will be established to oversee the trial implementation, review the study progress and provide the corresponding advices. + +## Participants, Recruitment And Consent + +The study is planned to start in October 2020 and end by August 2021. The hospital selection and project coordinator recruitment will take place in October 2020. Baseline assessments will be carried out before randomisation between November and December 2020. The internal pilot trial will be conducted in January 2021. The intervention programme will be administered from February to July 2021 followed by a questionnaire survey in intervention hospitals in August 2021. + +City-level hospitals with highest volume of reported gonorrhoea cases are eligible to participate in the study. Our investigators will prepare a list of eligible hospitals to invite to participate in each of the eight study provinces. The objectives and processes of the study will be explained to the hospital administrations and the in-hospital project coordinators. If a hospital agrees to participate in the study, the in-hospital project coordinator will work together with the study investigators to invite physicians to participate in the study. Physicians who work in the outpatient departments of dermatology (dermatovenerology), urology, andrology or gynaecology and provide informed consent (see online supplementary file 1) will be eligible to participate in the study. Prescriptions of these physicians for treatment of uncomplicated gonorrhoea will be evaluated for their adherence to the National Guidelines. Prescriptions will be excluded from data analysis if they are prescribed to the patients who meet any of the following criteria: (1) younger than 18 years; (2) being pregnant or lactating woman; (3) being allergic to cephalosporin or penicillin or having contraindication to cephalosporin; (4) being treated with antibiotics for other infections or (5) have been diagnosed as complicated gonorrhoea, such as disseminated gonococcal infection and pelvic inflammatory disease. + +![3_image_0.png](3_image_0.png) + +## Intervention And Control + +The proposed intervention to be evaluated in the study is aimed to improve the physicians' adherence to the National Guidelines by reducing inappropriate antibiotic prescribing for treatment of patients infected with N. gonorrhoeae. Our research team have designed an information card (figure 3) to be distributed to all participating physicians in the intervention arm hospitals by surface mail. By scanning a quick response (QR) code on the information card (figure 3), physicians will have access to an online training video developed on the internet platform of WenJuanXing (https://www.wjx.cn/). The video covers the workflows and requirements to manage a patient with uncomplicated gonorrhoea, including diagnostic criteria and treatment recommendations in accordance with the National Guidelines, and provides the contact information for inquiry related to the diagnosis and treatment. + +In the intervention arm hospitals, project coordinators will provide a less than 30-minute interactive training session in each of the relevant departments (integrated within monthly department meeting) prior to the intervention to explain the information card and the way to use the QR code for accessing the online training video. The intervention will last for 6 months. During the 6 months, the project coordinators will send reminders for the video playback to participating physicians through monthly Wechat messages. + +The frequency of visiting the online training video from each intervention hospital will be recorded in real-time on the WenJuanXing platform. The project manager will evaluate whether or not the intervention is delivered as intended from the server logs. + +In hospitals allocated to the control arm, physicians will continue to participate in their training programme if they conventionally do it and to prescribe antibiotics for treatment of gonorrhoea cases according to their own knowledge and beliefs. + +## Outcomes + +The primary outcome is the proportion of prescriptions adherent to the regimens for treatment of uncomplicated gonorrhoea recommended by the National Guidelines at the cluster level, which will be documented at baseline and at final evaluation. + +The secondary outcome is a physician-level binary indicator of non-adherence (defined as having at least 25% of prescriptions non-adherent to the National Guidelines). Sample size estimation The minimum sample size for this study was obtained using the formula outlined below26: + +![4_image_0.png](4_image_0.png) + +![4_image_1.png](4_image_1.png) + +Figure 3 The information card with a quick response (QR) code (hosted by WenJuanXing) to be distributed to physicians in the intervention arm hospitals. + +## C = 1+(Zα/2 + Zβ)2[Π0(1 − Π0)/N+ Π1(1 − Π1)/N + K2(Π02 + Π21 )]/(Π0 − Π1)2 + +where Zα/2 is the critical value of the normal distribution at α/2 (for a confidence level of 95%, α is 0.05 and the critical value is 1.96), Zβ is the critical value of the normal distribution at β (for a power of 90%, β is 0.1 and the critical value is 1.28), π0 and π1 are the proportions in the control and intervention arms, respectively, n is the number of samples needed in each cluster and k is the between-cluster coefficient of variation of the proportions between clusters within each arm. + +We used the following parameters and assumptions to estimate the sample size: Zα/2=1.96, Zβ +=1.28, π0 +=37.8% +(based on a nationwide survey18 in China), π1 +=41.6% +(based on a consultation of STD clinical experts and control programmers, a minimum 10% increase in adherence to the National Guidelines is deemed clinically relevant in the intervention arm), n=100 (minimum number of uncomplicated gonorrhoea patients per hospital within 6 months) and k=0.1 (based on a nationwide survey18 in China). + +By entering these parameter and assumptions into the above formula, it is indicated that a total of 60 hospitals per arm are needed. Considering a possibility to have 10% missing data for analysis and a necessity to do stratified randomisation, a total of 144 hospitals will be recruited + +![4_image_2.png](4_image_2.png) in the eight provinces for the study (18 hospitals in each province). + +## Cluster Randomisation + +Within each province, we will stratify the hospitals (clusters) into two groups (provincial capital city and other cities), and 4 hospitals from capital city and 14 hospitals from other cities will be purposively selected in consideration of the highest volume of reported cases. In each stratification, a simple randomisation process will be used to assign the hospitals into the intervention or the control arm. Thus, we will have 72 hospitals assigned to the control group and 72 to the intervention group, 9 and 9, respectively for each of the eight participating provinces. After all 144 hospitals were randomised, one province will be randomly selected from the eight participating provinces to become the internal pilot area using closed and opaque envelopes by an independent person from the research team, thus the 18 participating hospitals (nine from each arm) in this selected province will become the internal pilot clusters. The remaining 126 hospitals (63 from each arm) will, therefore, participate in the main trial, along with the 18 hospitals involved in the internal pilot (figure 1). Randomisation is conducted by an independent statistical analyst using a computer program written in R (V.3.5.3). + +It will not be possible to blind either in-hospital project coordinators or participating physicians on the arm assignment, given the explicit nature of the intervention components (eg, physicians in the intervention hospitals will be trained by in-hospital project coordinators). + +However, the prescription assessors and the data analyst will not be aware of the assignments. Moreover, the patients will not be informed of the physician's status in the control or intervention arm. + +## Data Collection + +Physician participating in the study will be assigned a 4-digit working number by the project coordinators, and this number will be used as an identification code in the questionnaire surveys and prescription audits. + +This number will only be known to the project coordinator and be used to match the questionnaire surveys and prescription audits. + +Prior to implementation of the intervention, we will have a baseline survey to collect background information of the participating hospitals (see online supplementary file 2) and another baseline questionnaire survey to collect background information of the participating physicians (see online supplementary file 3) in both intervention and control groups for evaluating the appropriateness of randomisation. A specific questionnaire survey (see online supplementary file 4) will be conducted among the physicians in the intervention hospitals after implementing the intervention to collect data for measuring the secondary outcome. + +Based on the Hospital Information System, our investigators (prescription assessors) will screen the prescription to obtain the most recent 100 eligible prescriptions from each participating hospital prior to intervention implementation period and 6 months after the implementation, and extract patient's characteristics, diagnosis and treatment, using a data collection form (see online supplementary files 5 and 6). + +## Data Analysis + +The data analysis methods are described in full detail in the accompanying data collection and analysis plan (see online supplementary file 7) and are, therefore, only outlined in brief here. We will conduct a complete case analysis of outcomes unless there is an indication that data may be missing not at random, in which multilevel multiple imputation methods to deal with missingness will be employed as sensitivity analysis in addition to the complete case analysis.27 The characteristics of the participating hospitals +(hospital level, hospital category) and the participating physicians (including sex, age, education level, working year, department and previous training) will be summarised using frequencies (plus sample sizes) and means (plus SDs) as appropriate for each group. + +For the primary outcome, data at the hospital level will be used to calculate weighted rate ratios and 95% CIs (accounting for between-cluster variance and stratification), and formal hypothesis testing with stratified t-tests will be conducted.28 29 To adjust for potentially important covariates, including individual and contextual factors, a two-stage adjusted analysis will be conducted.29 We will fit a logistic regression model to the individuallevel binary outcome data including all covariates of interest as fixed effects, but without adjusting for the treatment effect. The covariate-adjusted cluster-level ratio residuals will then be calculated and be used in place of raw cluster-level outcome data to conduct stratified t-test and calculate 95% CI using the above methods. Statistical significance will be based on the (two-sided) p value estimated for the primary outcome obtained from the covariate-adjusted analysis, considering adjusting increases power and reduces the effect of imbalances between arms with typically minimal risks. We will also conduct a subgroup analysis on the primary outcome to indicate the intervention effect across the subgroups in terms of hospitals and physicians. For the secondary outcome, we will define the physicians who have at least 25% prescriptions non-adherent to the National Guidelines as the physician with non-adherence, and then use multivariate logistic regression model to identify the independent variables associated with the non-adherence at a significant level of p≤0.05 and estimate adjusted OR and its 95% CIs by adjusting for potential confounding factors. No interim analyses are planned. + +## Process Evaluation + +Process evaluation will be conducted by collecting real-time summary data on access to the intervention (watching the video) from the WenJuanXing platform. Based on the summary data, hospitals with poor access to the intervention will be selected to conduct qualitative interviews of the public health staff, physicians and administrative staff to discuss the factors related to the poor access at cluster level. The qualitative data will be analysed using a simple thematic approach. + +## Patient And Public Involvement + +Patients and the public were not involved in the development of the research question or outcome measures. + +Physicians will be invited to give feedback on their barriers to comply with the intervention during the pilot trial in order to improve the intervention. + +## Ethics **And Dissemination** + +The study protocol has been reviewed and approved by the Medical Ethics Committee of the Chinese Academy of Medical Sciences Institute of Dermatology in Nanjing (2020-LS-004). An amendment will be submitted to the Committee for review and approval if any changes are made to the study protocol. All questionnaires, prescription audits and other data sources will be kept securely and available to authorised individuals for data analysis and reporting purposes only. Data will be entered into the database in an anonymised form to ensure confidentiality. Only principal investigator and other authorised personnel will have access to the final study database. The findings of the trial will be disseminated through reports at the national and international conferences, at least two publications in peer-reviewed journals, and provision of evidence for developing the on-the-job training programmes for physicians who provide services to patients with STDs. + +Acknowledgements The authors wish to acknowledge Ms Jingting Li for her assistance in English editing. Contributors X-SC: conceptualised the study. T-TJ, Y-QY and X-SC: designed the study. N-XC and Y-PY: participated in the study design. T-TJ: wrote the first draft of this protocol. X-SC: made critical revision of the study protocol. Y-QY, N-XC and Y-PY: contributed to the revision of the study protocol. All authors reviewed and approved the final manuscript. Funding This work was funded by research funding to X-SC from the National Science and Technology Major Project (2018ZX10101001-001), the Chinese Academy Medical Sciences Initiative for Innovative Medicine (2016-I2M-3021) and the Nanjing Incubation Program for National Clinical Research Center (2019060001). Map disclaimer The depiction of boundaries on this map does not imply the expression of any opinion whatsoever on the part of BMJ (or any member of its group) concerning the legal status of any country, territory, jurisdiction or area or of its authorities. This map is provided without any warranty of any kind, either express or implied. Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. ORCID iDs Ting-Ting Jiang http://orcid.org/0000-0002-0789-8816 Xiang-Sheng Chen http://orcid.org/0000-0003-3927-2065 + +## References + +1 Rowley J, Vander Hoorn S, Korenromp E, *et al*. Chlamydia, gonorrhoea, trichomoniasis and syphilis: global prevalence and incidence estimates, 2016. *Bull World Health Organ* 2019;97:548–62. + +2 Centers for Disease Control and Prevention. *Antibiotic resistance* threats in the United States, 2013. US Department of Health and Human Services, 2013: 1–114. + +3 Wi T, Lahra MM, Ndowa F, *et al*. Antimicrobial resistance in Neisseria gonorrhoeae: global surveillance and a call for international collaborative action. *PLoS Med* 2017;14:e1002344. + +4 Centers for Disease Control and Prevention (CDC). Update to CDC's sexually transmitted diseases treatment guidelines, 2010: oral cephalosporins no longer a recommended treatment for gonococcal infections. *MMWR Morb Mortal Wkly Rep* 2012;61:590–4. + +5 Workowski KA, Bolan GA, Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. *MMWR Recomm Rep* 2015;64:1–137. + +6 Bignell C, Unemo M, European STI Guidelines Editorial Board. 2012 European guideline on the diagnosis and treatment of gonorrhoea in adults. *Int J STD AIDS* 2013;24:85–92. + +7 Wang QQ, Liu QZ, JH X, eds. *Guidelines of Clinical Management* of Sexually Transmitted Diseases [in Chinese]. Shanghai: Shanghai Science and Technology Press, 2014. + +8 Chen S-C, Yin Y-P, Dai X-Q, *et al*. First nationwide study regarding ceftriaxone resistance and molecular epidemiology of Neisseria gonorrhoeae in China. *J Antimicrob Chemother* 2016;71:92–9. + +9 Ni C, Xue J, Zhang C, *et al*. High prevalence of Neisseria gonorrhoeae with high-level resistance to azithromycin in Hangzhou, China. *J Antimicrob Chemother* 2016;71:2355–7. + +10 Unemo M, Ison C. *Laboratory diagnosis of sexually transmitted* infections, including human immunodeficiency virus. Gonorrhoea: World Health Organization (WHO), 2013. + +11 Unemo M, Shafer WM. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future. Clin Microbiol Rev 2014;27:587–613. + +12 Golparian D, Natarajan U, Alexander S, *et al*. First global case of gonorrhea treatment failure with dual antimicrobial therapy (ceftriaxone [500 mg] and azithromycin [1 g]) - the continuously evolving superbug. Neisseria gonorrhoeae escapes last resort of treatment. *(Submitted)*. + +13 Unemo M. Current and future antimicrobial treatment of gonorrhoea +- the rapidly evolving Neisseria gonorrhoeae continues to challenge. BMC Infect Dis 2015;15:364. + +14 Yin Y-P, Han Y, Dai X-Q, *et al*. Susceptibility of Neisseria gonorrhoeae to azithromycin and ceftriaxone in China: a retrospective study of national surveillance data from 2013 to 2016. *PLoS Med* 2018;15:e1002499. + +15 Centers for Disease Control and Prevention (CDC). Cephalosporin susceptibility among *Neisseria gonorrhoeae* isolates--United States, 2000-2010. *MMWR Morb Mortal Wkly Rep* 2011;60:873–7. + +16 Frost I, Van Boeckel TP, Pires J, *et al*. Global geographic trends in antimicrobial resistance: the role of international travel. *J Travel Med* 2019;26:taz036. + +17 Han Y, Yin Y, Dai X, *et al*. Widespread use of high-dose ceftriaxone therapy for uncomplicated gonorrhea without reported ceftriaxone treatment failure: results from 5 years of multicenter surveillance data in China. *Clin Infect Dis* 2020;70:99–105. + +18 Han Y, Yin Y-P, Zhou Y, *et al*. Nonadherence to national guidelines for antibiotic treatment of uncomplicated gonorrhea in China: results from a nationwide survey. *Sex Transm Dis* 2018;45:600–6. + +19 Chen X-S, Yin Y-P, Li X-Y. A roadmap plan to address research needs for gonococcal antimicrobial resistance in China. *Clin Infect Dis* 2019;68:505–10. + +20 Labrique AB, Vasudevan L, Kochi E, *et al*. mHealth innovations as health system strengthening tools: 12 common applications and a visual framework. *Glob Health Sci Pract* 2013;1:160–71. + +21 O'Donovan J, Bersin A, O'Donovan C. The effectiveness of mobile health (mHealth) technologies to train healthcare professionals in developing countries: a review of the literature. *BMJ Innov* 2015;1:33–6. + +22 Cohen SM, Volandes AE, Shaffer ML, *et al*. Concordance between proxy level of care preference and advance directives among nursing home residents with advanced dementia: a cluster randomized clinical trial. *J Pain Symptom Manage* 2019;57:37–46. + +23 O'Donovan J, Kabali K, Taylor C, *et al*. The use of low-cost android tablets to train community health workers in Mukono, Uganda, in the recognition, treatment and prevention of pneumonia in children under five: a pilot randomised controlled trial. *Hum Resour Health* 2018;16:49. + +24 Yardley L, Douglas E, Anthierens S, *et al*. Evaluation of a web-based intervention to reduce antibiotic prescribing for LRTI in six European countries: quantitative process analysis of the GRACE/INTRO randomised controlled trial. *Implement Sci* 2013;8:134. + +25 Wang QQ, Liu QZ, JH X, eds. Guidelines of clinical management of sexually transmitted diseases [in Chinese]. Shanghai: Shanghai Science and Technology Press, 2020. + +26 Hayes RJ, Bennett S. Simple sample size calculation for clusterrandomized trials. *Int J Epidemiol* 1999;28:319–26. + +27 DiazOrdaz K, Kenward MG, Gomes M, *et al*. Multiple imputation methods for bivariate outcomes in cluster randomised trials. *Stat* Med 2016;35:3482–96. + +28 Zou G, Wei X, Hicks JP, *et al*. Protocol for a pragmatic cluster randomised controlled trial for reducing irrational antibiotic prescribing among children with upper respiratory infections in rural China. *BMJ Open* 2016;6:e010544. + +29 Hayes RJ, Moulton LH. *Cluster randomised trials*. CRC Press, 2009. \ No newline at end of file diff --git a/medical/md_images/PMC7448026/20_image_0.png b/medical/md_images/PMC7448026/20_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..5a527aaa5a7d6705408a73c6e8982365925a7f35 --- /dev/null +++ b/medical/md_images/PMC7448026/20_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:ec375407afbe82fa4ecb4d5ba5d92c7c22eacc0747d989d1a48e4ff045ecafae +size 15275 diff --git a/medical/md_images/PMC7448026/31_image_0.png b/medical/md_images/PMC7448026/31_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..fd9e7aa6ea9413fdf9957a7cf2c200e0139a7e14 --- /dev/null +++ b/medical/md_images/PMC7448026/31_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:4c597656ec742530c7ac2843e33130859f44c1ed55cb5d43f9e08272732977a9 +size 14937 diff --git a/medical/md_images/PMC7448026/4_image_0.png b/medical/md_images/PMC7448026/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..76b6ed0581479934df7e14d182680fb2f0708acd --- /dev/null +++ b/medical/md_images/PMC7448026/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:516354b1d845dfcf1d77f7f9101796a95d25b28ea25c2ac49536f1af0c684be3 +size 15295 diff --git a/medical/md_images/PMC7467701/4_image_0.png b/medical/md_images/PMC7467701/4_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..4971dd5bb13031442b4d3138389ba7d35478f483 --- /dev/null +++ b/medical/md_images/PMC7467701/4_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f580b588bd31624cc38875bb32930d3f2eec9798ea8001007b97094cbf837661 +size 108077 diff --git a/medical/md_images/PMC7879814/0_image_1.png b/medical/md_images/PMC7879814/0_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..8a484e6e75c834007a5e309615616d8aec6e7b30 --- /dev/null +++ b/medical/md_images/PMC7879814/0_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e62728ebf804130eed8516716635cda86cd8bc8c68f58136ee749e0cff995535 +size 2142 diff --git a/medical/md_images/PMC7879814/1_image_0.png b/medical/md_images/PMC7879814/1_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..42cb7adc514cad8b591c49540504682bd1d854e5 --- /dev/null +++ b/medical/md_images/PMC7879814/1_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:75ee9c425397d704ebb3893e48744b34abdfe2effce69d7ca18917ac78efb09c +size 3697 diff --git a/medical/md_images/PMC7944174/0_image_0.png b/medical/md_images/PMC7944174/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..8befb5ff4a2cf5e06d9f2802bb95637357cd5a7e --- /dev/null +++ b/medical/md_images/PMC7944174/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:617ed4d274a7b4310ed25169667385de04f53dd0abb79a834859b54ede425170 +size 1066 diff --git a/medical/md_images/PMC7944174/2_image_0.png b/medical/md_images/PMC7944174/2_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..bd95d184b3a4fbea7d149a2217feb23a0e5af59c --- /dev/null +++ b/medical/md_images/PMC7944174/2_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0731ff70b54d87acf6947295d4a393776fe0f5d61f5b841fd5e604b40ae0931b +size 32848 diff --git a/medical/md_images/PMC7944174/2_image_1.png b/medical/md_images/PMC7944174/2_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..1b560112ffd9f61ae140d3034500845f33833c7c --- /dev/null +++ b/medical/md_images/PMC7944174/2_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e1bb3dc2d01dc0f960db4f39fe8664c48ae96c02ade577207b276de4f9e1b63b +size 16732 diff --git a/medical/md_images/PMC7944174/8_image_0.png b/medical/md_images/PMC7944174/8_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..b451eec85c5bf767c70581eeaa3d6abb4485344f --- /dev/null +++ b/medical/md_images/PMC7944174/8_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e30ac214af7ba7943b1ea68c414a7d5976dfd9636eceab026cc5c77f86d54cec +size 35339 diff --git a/medical/md_images/PMC8240778/0_image_0.png b/medical/md_images/PMC8240778/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..9b61ad88798b5120adae1d22c1576bfa83a472ce --- /dev/null +++ b/medical/md_images/PMC8240778/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:2f6865ace6b2c399aefd2ad13dcfe8352fae91df3b49c073fb744ef5dde142f9 +size 2357 diff --git a/medical/md_images/PMC8463271/3_image_0.png b/medical/md_images/PMC8463271/3_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..57eb46a1fa9544264149c26e7fb5ff6952a804f3 --- /dev/null +++ b/medical/md_images/PMC8463271/3_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:7c29ae5171d16086dce3255125ff02b47c2f4f4b7fe48e0ba59c1bb0ff203683 +size 57832 diff --git a/medical/md_images/PMC8463271/3_image_1.png b/medical/md_images/PMC8463271/3_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..24043325216ccb76af5dffcaca0553346fbbb650 --- /dev/null +++ b/medical/md_images/PMC8463271/3_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:6fb609d292ba2b3f5241d307cc2a5d81298a67ff4f1e301da311d90f34c1fbec +size 99561 diff --git a/medical/md_images/PMC8720718/PMC8720718_meta.json b/medical/md_images/PMC8720718/PMC8720718_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..993d338adcc6deeb3a1a1f2154ae1b5ed693eb89 --- /dev/null +++ b/medical/md_images/PMC8720718/PMC8720718_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 4, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 12, + "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/PMC8930376/0_image_0.png b/medical/md_images/PMC8930376/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..8276114105656370f2f272c5ac501f98e6482c3b --- /dev/null +++ b/medical/md_images/PMC8930376/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:880aef7a46cd8a71b8d535368e86b69ac489c0c4f538d447ba110e13f00cd74e +size 72052 diff --git a/medical/md_images/PMC9120064/0_image_0.png b/medical/md_images/PMC9120064/0_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..d031867ef316b984ccb139831fd74b3a90491ea2 --- /dev/null +++ b/medical/md_images/PMC9120064/0_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:8f3bbec244be741f492b85fa1dc6668954c9ea69bb5cabbe149377818e8c2cb4 +size 10598 diff --git a/medical/md_images/PMC9120064/5_image_0.png b/medical/md_images/PMC9120064/5_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..8e006435af55c04aa0087f604f95f9cb2e492506 --- /dev/null +++ b/medical/md_images/PMC9120064/5_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e186615e3a24c308342642aca91f5d8c4947c0753751446293d7a535fc7879e9 +size 53705 diff --git a/medical/md_images/PMC9120064/PMC9120064.md b/medical/md_images/PMC9120064/PMC9120064.md new file mode 100644 index 0000000000000000000000000000000000000000..a25292dbbd57ddcb75d2250e5b53aa37880b5d01 --- /dev/null +++ b/medical/md_images/PMC9120064/PMC9120064.md @@ -0,0 +1,439 @@ + + +![0_image_0.png](0_image_0.png) + +![0_image_3.png](0_image_3.png) + +![0_image_6.png](0_image_6.png) + +# Contents Lists Available At Sciencedirect + +![0_Image_1.Png](0_Image_1.Png) + +![0_Image_2.Png](0_Image_2.Png) + +![0_Image_4.Png](0_Image_4.Png) + +![0_Image_5.Png](0_Image_5.Png) International Journal Of Cardiology Cardiovascular Risk And Prevention Journal Homepage: Www.Journals.Elsevier.Com/International-Journal-Of-Cardiologycardiovascular-Risk-And-Prevention + +Effects of moderate ethanol consumption as a function of n-6:n-3 dietary ratio on lipid profile, inflammation, and liver function in mice Kristine Giltvedt a, Luciano S. Voutour b, Brianna Tursellino b, Alexandra Zella b, Susan M. Brasser b, Mee Young Hong a,* +a School of Exercise and Nutritional Sciences, San Diego State University, 5500 Campanile Drive, San Diego, CA, 92182, USA b *Department of Psychology, San Diego State University, 5500 Campanile Drive, San Diego, CA, 92182, USA* +ARTICLE INFO + +| Keywords: Moderate ethanol n-3 fatty acids Lipid profiles Inflammation Liver function markers | +|------------------------------------------------------------------------------------------------------| + +Objective: It is critical to understand how moderate ethanol exposure interacts with dietary components such as essential fatty acids to influence inflammatory processes underlying CVD pathogenesis. The purpose of this study was to examine the effects of moderate ethanol consumption and dietary n-6:n-3 fatty acid composition on markers associated with CVD in mice. Methods: Twenty-three C57BL/6J mice consumed an 18% ethanol solution or 26.9% maltose dextrin solution +(isocaloric control) for 12 weeks. Within each group, the mice were fed either a high n-6 (n-6:n-3 = 50:1) diet or a balanced n-3 (n-6:n-3 = 1:1) diet *ad libitum*. Following the exposure period, serum samples were analyzed to assess lipid profile, inflammatory markers, antioxidant capacity, DNA damage, and liver function enzyme activity. + +Results: The control group gained more weight than the ethanol group (P = 0.020). In ethanol-exposed mice, HDL was significantly increased (P = 0.009). C-reactive protein (CRP; P < 0.001), high mobility group box 1 protein +(HMGB1; P = 0.011), 8-oxo-deoxyguanosine (8-oxo-dG; P = 0.019), ALT (P = 0.002) and AP (P = 0.021) were lower in the ethanol group. There was a significant main effect of the n-3 diet on total antioxidant capacity (TAC; +P < 0.001) and 8-oxo-dG (P = 0.047). + +Conclusion: These findings indicate that moderate ethanol consumption and a balanced n-6:n-3 diet improve several inflammatory and lipid markers associated with CVD. Observed differences in weight gain between groups should be considered when interpreting these results. + +| ABSTRACT | +|------------| + +![0_image_7.png](0_image_7.png) + +## 1. **Introduction** + +Cardiovascular disease (CVD) is the leading cause of death in the United States, accounting for over 655,000 deaths and $219 billion in healthcare costs annually [1]. Epidemiological studies in humans and experimental data from rodent models indicate that there is a non-linear J-shaped relationship between alcohol consumption and CVD risk, with low-to-moderate levels of alcohol exposure conferring cardioprotective effects, including lower risk of mortality following acute myocardial infarction, while excessive alcohol consumption conversely increases the risk of cardiovascular disease and adverse coronary events [2–7]. + +Possible mechanisms by which low-to-moderate ethanol intake may reduce CVD risk include improving lipid profile, reducing body fat, modulating gene expression involved in cholesterol synthesis, reducing platelet aggregation, and having anti-inflammatory effects [4,8–14]. + +Despite well-established dose-dependent associations between alcohol consumption and cardiovascular risk, the interaction between moderate ethanol consumption and other dietary factors in regulating physiological processes underlying CVD risk is largely unexplored in controlled preclinical models. In particular, the combined interaction between moderate levels of ethanol exposure and the ratio of dietary omega-6 (n-6) to omega-3 (n-3) fatty acids is an important understudied area of research because n-3 fatty acids appear to provide cardioprotective benefits that overlap with those of moderate ethanol consumption [15,16]. Experimental studies indicate that supplementation of n-3 fatty acids may lower cardiovascular risk by improving lipid profile, blood pressure, endothelial function, and inflammatory markers +[17]. Epidemiological studies reveal similar effects of dietary consumption of n-3 fatty acids, reporting lower total cardiovascular mortality and reduction of secondary cardiovascular outcomes [15]. By +* Corresponding author. School of Exercise and Nutritional Sciences, San Diego State University, 5500 Campanile Drive, San Diego, CA, 92182-7251. + +E-mail address: mhong2@sdsu.edu (M.Y. Hong). + +https://doi.org/10.1016/j.ijcrp.2022.200132 Received 28 December 2021; Received in revised form 1 April 2022; Accepted 19 April 2022 Available online 14 May 2022 2772-4875/© 2022 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/). + +## 2 + +contrast, increases in n-6 to n-3 ratios facilitate a proinflammatory state in the body and are associated with progression of coronary atherosclerosis [18,19]. Metabolically, an increased n-6:n-3 ratio may lead to lipogenesis and hepatic steatosis, thereby increasing CVD mortality +[19]. In addition, high levels of n-6 fatty acids are associated with unfavorable changes in lipid profile, including increased total cholesterol, LDL, and triglycerides [20]. The ratio of n-6:n-3 dietary fatty acids in Western diets has markedly increased over the last century, which has been associated with increased inflammation and risk of many chronic diseases [21]. Dietary lipids are known to play a role in modulating disease course related to excessive alcohol consumption (e.g., alcoholic liver disease) [22]. However, little to no controlled experimental data has addressed how dietary fatty acid composition may serve to differentially modulate the effects of moderate alcohol intake on cardiovascular risk. + +The objective of this study was to examine the effects of moderate ethanol consumption and dietary n-6:n-3 fatty acid composition on markers associated with cardiovascular disease and liver dysfunction in mice. It was hypothesized that moderate ethanol consumption in combination with a balanced (1:1) dietary ratio of n-6:n-3 fatty acids may result in potential additive protective effects on lipid and inflammatory markers associated with CVD, whereas a high (50:1) n-6:n-3 diet may by contrast antagonize any beneficial effects of moderate ethanol exposure on physiological indicators of CVD risk. + +## 2. **Methods** 2.1. Animals + +Twenty-three adult C57BL/6J mice (12 male, 11 female; The Jackson Laboratory, Bar Harbor, ME) were used. Mice were 20 weeks of age at the start of the experiment (i.e., ~20–30 years human equivalent). + +Throughout the experiment, mice were housed individually in standard plastic bottom cages for precise measurement of fluid and food intake, within a testing room maintained on a 12:12 h light/dark cycle and at an ambient temperature of approximately 23 ◦C. Fluids and chow diets were available to mice *ad libitum*. Mice were randomly assigned to one of four experimental groups (detailed in section 2.2), with each group balanced as equally as possible by gender and initial body weight. All study procedures were approved by the Institutional Animal Care and Use Committee (Animal protocol \#:18-12-015B) at San Diego State University and were in accordance with National Institutes of Health guidelines. + +## 2.2. Ethanol And Diet Exposure + +All animals were initially acclimated to housing and testing procedures for 14 days during which they received free access to standard chow (LabDiet, St. Louis, MO) and water, and body weights were measured every 48 h. Following the initial acclimation period, mice were exposed for 15 weeks to either ethanol or isocaloric maltose dextrin (control) in the drinking water and were concurrently fed a compositional defined diet (LabDiet, St. Louis, MO) enriched with either corn oil or fish oil, comprising a high (50:1) or balanced (1:1) n-6:n-3 ratio, respectively [Fluid (2; ethanol or control) x Diet (2; high or balanced n-6:n-3) factorial design]. Chow diets were matched for all other macro- and micronutrients (Supplement Table 1). Ethanolexposed mice were given graduating ethanol concentrations of 2.5%, +5%, and 10% during the first three weeks of exposure to acclimate to drinking ethanol and were maintained on an 18% ethanol solution during the remaining 12 weeks of exposure. The control group received isocalorically matched maltose dextrin mixed in their drinking water during corresponding weeks at concentrations of 3.7%, 7.5%, and 14.9% +during the first three weeks and a maintenance level of 26.9% for the remaining 12 weeks. This model of moderate ethanol consumption in C57BL/6J mice has been demonstrated in previous studies to produce mean blood ethanol concentrations (BECs) of 24–67 mg/dl [23–25]. The latter blood alcohol levels approximate 0.02–0.06% in humans, or that normally resulting from 1 to 2 standard drinks [26], corresponding with the current definition of moderate or 'low-risk' drinking as defined by the National Institute on Alcohol Abuse and Alcoholism and U.S. Department of Health and Human Services Dietary Guidelines for Americans 2015–2020 [27]. Body weights and bottle weights (to assess fluid intake) were measured every 48 h and food intake (g) was measured once per week. + +## 2.3. Blood Glucose And Lipids + +At the end of the exposure period, blood samples were collected via cardiac puncture under terminal isoflurane anesthesia. After centrifugation for 10 min at 1200×g at 4 ◦C, serum was collected and stored at +− 80 ◦C until analysis. Blood glucose was measured using the Glucose LiquiColor assay kit (EKF Diagnostics, Cardiff, UK). Serum triglycerides, total cholesterol, and high-density lipoprotein (HDL) were determined using assay kits (EKF Diagnostics). Non-HDL cholesterol was calculated using the formula: Non-HDL Cholesterol = TC - HDL - (TG/5). + +## 2.4. C-Reactive Protein + +C-reactive protein (CRP) levels were measured using a double antibody sandwich ELISA kit (Crystal Chem, Elk Grove Village, IL). Serum samples were incubated with anti-CRP antibodies and then with HRPconjugated anti-CRP antibodies. TMB substrate was added to measure CRP concentration. Absorbance was read by spectrophotometer at 450 nm. + +## 2.5. Hmgb1 + +High-mobility group box1 protein (HMGB1) was measured by competitive ELISA (MyBioSource, San Diego, CA). Serum samples were incubated with HMGB1-HRP conjugate. After washing, samples were incubated with an HRP enzyme substrate, forming a blue-colored complex. Stop solution was added and absorbance was read at 450 nm. + +## 2.6. Total Antioxidant Capacity + +Serum total antioxidant capacity (TAC) was measured using an antioxidant assay kit (Cayman Chemical Company, Ann Arbor, MI). The capacity to which the antioxidants in the serum sample prevent oxidation of 2,2′-azino-di-[3-ethylbenthiazoline sulphonate] (ABTS) was measured. Trolox was used as a standard and absorbance was read at 405 nm. + +## 2.7. 8-Oxo-Deoxyguanosine + +Levels of 8-oxo-deoxyguanosine (8-oxo-dG) were measured to assess DNA damage using HT 8-oxo-dG ELISA kit II (Trevigen, Gaithersburg, MD). Following the manufacturer's protocol, serum samples were incubated with monoclonal and then anti-Mouse IgG-HRP conjugate. TACS-Sapphire substrate was added and incubated in the dark. Stop solution was added, and absorbance was read at 450 nm. + +## 2.8. Liver Function Enzymes + +Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using Liqui-UV assay kits (EKF Diagnostics) +and alkaline phosphatase (AP) was measured using the LiquiColor kit +(EKF Diagnostics) according to the manufacturer's protocols. For AST +and ALT assays, absorbance was read by spectrophotometer at 340 nm and absorbance per minute was calculated for AST and ALT. To determine AP enzyme activity, the rate of hydrolysis of 4-nitrophenyl phosphate to 4-nitrophenol was measured and the absorbance was read at 405 nm. + +## 2.9. Statistical Analysis + +Data were analyzed using SPSS Version 26.0 (IBM, Armonk, New York). The primary analyses were 2 (Fluid: ethanol or control) x 2 (Diet: +50:1 or 1:1 n-6:n-3) factorial ANOVAs. If there was an interaction, Bonferroni post-hoc tests were used for multiple comparisons. ANOVAs were run for the following dependent variables: body weight, body weight gain, fluid intake, food intake, glucose, triglycerides, total cholesterol, HDL, LDL, ALT, AST, AP, AST:ALT ratio, CRP, HMGB1, TAC, +and 8-oxo-dG. For all main effects or interactions from the overall analyses, a p-value of <0.05 was considered statistically significant. Spearman's correlations were conducted to analyze relationships between the intake of total PUFAs, n-3 PUFAs, n-6 PUFAs and alcohol, with metabolic parameters. + +## 3. Results + +3.1. Body weight gain, food intake, and fluid intake There were no significant differences in either initial body weight or + +![2_image_0.png](2_image_0.png) + +Fig. 1. Mean (±SEM) body weight, fluid intake, and food intake over time in control and ethanol-exposed mice consuming the high n-6 or balanced n-3 diet. (A, +B) Two-way Fluid (2) x Diet (2) factorial ANOVAs revealed no significant differences in either initial body weight or final body weight between groups. + +However, analysis of weight gain indicated that regardless of diet, control mice gained more weight than ethanol-treated mice over the course of the experiment (main effect of Fluid: F 1 , 19 = 6.03, P = +0.02). Day 0 represents initial pretreatment body weight and Day 108 final body weight at the end of the treatment period. (C,D) Fluid intake was greater in control mice than ethanol-treated mice during all weeks except Week 1 (main effect of Fluid: F1,19 = +22.34, P < 0.001; main effect of Week: F14,266 = +59.98, P < 0.001; Fluid x Week interaction: F 14 , 266 = +37.52, P < 0.001). Mice were given ascending concentrations of either ethanol or maltose dextrin during the first 3 weeks of exposure and were then maintained on 18% ethanol or 26.9% maltose dextrin during the remaining 12 weeks (dotted line denotes start of maintenance phase). (E,F) Control mice consumed less food than ethanol-exposed mice during Weeks 3–15 (main effect of Fluid: F 1.19 = 121.86, P < +0.001; main effect of Week: F 14 , 266 = 29.21, P < +0.001; Fluid x Week interaction: F14, 266 = 16.16, P < +0.001). Fluid and food intake did not significantly differ by diet. + +final body weight between groups (Fig. 1 and Supplemental Table 2). However, analysis of weight gain indicated that regardless of diet, control mice gained significantly more weight than those in the ethanol group (main effect of Fluid: F1,19 = 6.03, P = 0.020). Control mice also consumed significantly more fluid and less food compared to ethanolexposed mice (main effect of Fluid: F1,19 = 222.34, P < 0.001; main effect of Fluid: F1,19 = 121.86, P < 0.001, respectively). Body weight, fluid intake, and food intake were not significantly influenced by diet. + +Energy intake was lower in the ethanol group than the control group (Supplemental Table 2). Total PUFA, n-6 and n-3 fatty acid intakes were greater in ethanol groups (main effect of Fluid: F1,19 = 193.32, P < 0.001; main effect of Fluid: F1,19 = 104.32, P < 0.001; main effect of Fluid: F1,19 = 212.35, P < 0.001, respectively) due to their greater food intakes. + +## 3.2. Blood Glucose And Lipid Profile + +Glucose levels were significantly lower in the control group compared to the ethanol group, regardless of diet (main effect of Fluid: +F1,19 = 6.69, P = 0.018; Table 1). Compared to controls, there was a trend toward lower triglycerides in ethanol-exposed mice (P = 0.066). + +Total cholesterol did not differ between the control group and the ethanol group overall; however, there was a trend toward lower LDL (P += 0.062) in mice fed the balanced n-3 vs high n-6 diet. HDL cholesterol was significantly higher among ethanol-exposed mice compared to control mice (main effect of Fluid: F1,19 = 8.52, P = 0.009), but was not affected by diet composition. + +## 3.3. Crp, Hmgb1, Tac, And 8-Oxo-Dg + +C-reactive protein was significantly lower in ethanol-consuming mice compared to control mice (main effect of Fluid: F1,19 = 25.16, P +< 0.001; Fig. 2A). HMGB1 was also significantly lower in the ethanol group compared to the control group (main effect of Fluid: F1,19 = 7.95, P = 0.011; Fig. 2B). The balanced n-3 diet increased TAC relative to the high n-6 diet in both control and ethanol-treated mice (main effect of Diet: F1,19 = 32.88, P < 0.001), with no significant effect of fluid type on TAC (Fig. 2C). The oxidative marker 8-oxo-dG was significantly lower in ethanol-exposed mice compared to controls (main effect of Fluid: F1,19 += 6.61, P = 0.019) and in mice consuming the n-3 diet compared to the n-6 diet (main effect of Diet: F1,19 = 4.50, P = 0.047; Fig. 2D). Ethanol in combination with the n-3 diet showed the lowest 8-oxo-dG levels among all groups, suggesting beneficial effects of these dietary factors. + +Table 1 + +Glucose, triglycerides, total cholesterol, high-density lipoprotein, and lowdensity lipoprotein. + +Control Ethanol + +n-6 n-3 n-6 n-3 + +Glucose (mmol/ + +L) + +14.22 ± + +2.09a + +13.68 ± + +1.37a + +18.12 ± + +0.59b + +17.08 ± + +0.94b + +TG (mmol/L) 1.54 ± 0.16 1.63 ± 0.22 1.41 ± 0.18 1.02 ± 0.19 TC (mmol/L) 2.81 ± 0.45 2.80 ± 0.35 3.24 ± 0.46 2.43 ± 0.35 + +HDL (mmol/L) 0.95 ± 0.13a 1.16 ± 0.09a 1.55 ± 0.23b 1.57 ± 0.20b + +LDL (mmol/L) 1.44 ± 0.22 1.19 ± 0.10 1.33 ± 0.23 0.84 ± 0.12 + +Data are represented as mean ± standard error. TG, triglyceride; TC, total + +cholesterol; HDL, high density lipoprotein; LDL, low density lipoprotein. Twoway Fluid (2) x Diet (2) factorial ANOVAs were used for analysis. Data within + +rows with varying superscript letters are statistically significant (P ≤ 0.05). + +There was a significant main effect of fluid on glucose level (P = 0.018; NS effect of diet, P = 0.583; NS interaction: P = 0.864) and HDL cholesterol (P = 0.009; NS effect of diet, P = 0.494; NS interaction: P = 0.585). There was a trend toward + +lower TG in the ethanol group compared to controls (P = 0.066) and a trend + +toward lower LDL within the ethanol group in mice consuming the n-3 vs n-6 + +diet (P = 0.062). + +## 3.4. Ast, Alp, And Ap + +There was an interaction of fluid and diet on AST levels (main effect of Fluid: F1,19 = 9.40, P = 0.006; fluid x diet interaction: F1,19 = 6.91, P += 0.017). AST levels were lower in ethanol-exposed mice fed either diet and in controls fed the balanced n-3 diet (Fig. 3A). Regardless of diet, both ALT and AP were significantly lower in ethanol-consuming mice compared to controls (main effect of Fluid: F1,19 = 13.60, P = 0.002; main effect of Fluid: F1,19 = 6.34, P = 0.021, respectively; Fig. 3B and C). + +AST:ALT ratio was significantly greater in ethanol-exposed mice relative to controls (main effect of Fluid: F1,19 = 5.10, P = 0.036; Fig. 3D). + +There was an inverse correlation (rho = − 0.703, P = 0.011) between n-3 and AST in the control groups. Within ethanol groups, there were a trend or significant positive correlation between ethanol intake and AST +(rho = 0.583, P = 0.060) and AP (rho = 0.656, P = 0.028). + +## 4. **Discussion** + +The present study was conducted to examine the effects of moderate ethanol consumption in conjunction with varied dietary n-6:n-3 fatty acid composition on risk factors for cardiovascular disease. It was found that moderate levels of ethanol consumption decreased key inflammatory markers CRP and HMGB1, reduced oxidative DNA damage as assessed via 8-oxo-dG, increased HDL cholesterol, and lowered both ALT +and AP liver function enzyme activity. The balanced n-3 diet independently increased TAC and decreased 8-oxo-dG relative to a high n-6 diet. + +In combination, concurrent intake of moderate levels of ethanol together with the n-3 diet resulted in further decreases in 8-oxo-dG, as well as trends toward lower levels of triglycerides and LDL. + +Previous data have documented that excessive ethanol consumption and heavy "binge" drinking episodes are associated with increased oxidative stress and inflammation [8,28] leading to detrimental effects on many organ systems and increased risk for adverse coronary events [29,30]. By contrast, several lines of evidence indicate that low-to-moderate ethanol exposure is associated with reduced incidence of cardiovascular disease [2,3] and may provide cardioprotective effects through modulation of inflammatory markers and gene expression and a reduction in oxidative damage [8,12]. Controlled preclinical studies in rats have demonstrated that moderate ethanol intake results in decreased oxidative stress by upregulation in the expression of *Pon-1* and *Aldh2* and lowered inflammation by decreased expression in *Cox-2* and *Rela* [12,31]. Human observational studies have demonstrated that moderate alcohol consumption, and in particular red wine, which contains higher amounts of polyphenols compared to other types of alcohol, may prevent the activation of the proinflammatory NF-κB pathway as well as decrease CRP and increase TAC [32–34]. It is possible that moderate levels of ethanol may reduce inflammation and oxidative damage by acting as a hormetic stressor. Ethanol generates reactive oxygen species and aldehydes in the body, which can cause oxidative injury; however, in acute conditions, low levels of reactive oxygen species may induce cytoprotective effects [35]. As such, low-to-moderate levels of alcohol may engage cellular defense mechanisms and stimulate compensatory protective effects [12]. A previous study found that moderate ethanol consumption also reduced DNA +damage and inflammation in markers associated with colon cancer risk [31]. In the present study, mice exposed to moderate ethanol had significantly lower CRP, HMGB1, and DNA damage as measured by 8-oxo-dG, but no significant differences in TAC. It is important to note that tissues in the current study were collected one day after the final alcohol exposure session at a time when ethanol was absent from blood in order to assess the effects of long-term low-to-moderate ethanol exposure as opposed to direct effects of ethanol in the system. It would be interesting for subsequent studies to determine the long-term persistence of these protective effects during a period of sustained abstinence. + +Consumption of ethanol increases HDL cholesterol in a dose- + +![4_image_0.png](4_image_0.png) + +Fig. 2. Mean values of C-reactive protein, HMGB1, and total antioxidant capacity. (A) There was a significant main effect of ethanol treatment on CRP +(main effect of fluid, P < 0.001; NS effect of diet, P = +0.239; NS interaction, P = 0.678). (B) HMGB1 was significantly lower in ethanol-exposed animals compared to controls (main effect of fluid, P = 0.011; NS effect of diet, P = 0.537; NS interaction, P = +0.666). (C) The n-3 diet resulted in significantly higher TAC compared to the n-6 diet (main effect of diet, P < 0.001; NS effect of fluid, P = 0.310; NS +interaction, P = 0.407). (D) There was a significant main effect of ethanol on 8-oxo-dG (main effect of fluid, P = 0.019) and of n-3 diet on 8-oxo-dG (main effect of diet, P = 0.047). Two-way Fluid (2) x Diet (2) +factorial ANOVAs were used for primary analysis. + +Since there were no significant interactions, no-posthoc tests were performed. Lines for each group represent mean ± standard error. Varying superscripts are significantly different (P ≤ 0.05). Each group was comprised of 3 male and 3 female mice, except the Ethanol n-3 group which contained 3 male and 2 female mice. Closed circles (•) indicate male mice and open circles (o) female mice. 8-oxo-dG: 8oxo-deoxyguanosine; CRP, c-reactive protein; HMGB1: high mobility group box 1; NS: not significant; TAC: total antioxidant capacity. + +dependent manner [8]. The results of the current study were consistent with this finding, demonstrating significantly higher HDL levels in ethanol-exposed mice, which was evident regardless of n-6:n-3 diet composition. We also observed a borderline trend for lower triglycerides in the ethanol group, which was most pronounced in ethanol-exposed mice also consuming the balanced n-3 diet. Alcohol in high doses is known to increase plasma triglycerides; however, the effects of moderate ethanol on triglycerides has been inconclusive and may depend on the type of alcohol consumed [28,32,36]. + +The liver is the primary site of ethanol metabolism, and as such, liver function becomes impaired with heavy drinking [37]. Chronic excessive consumption of alcohol can lead to alcoholic liver disease (ALD) and cirrhosis [37]. Interestingly, prior rodent studies employing paradigms that result in low-to-moderate levels of ethanol drinking have shown improved liver function as indexed by reductions in both ALT and LDH (lactate dehydrogenase) [31,38]. Potential mechanisms for this improvement in liver function may be related to a reduction in serum ammonia [38]. The present study found reductions in both ALT and AP +resulting from moderate alcohol consumption; however, an increase in AST:ALT ratio was also observed. An AST:ALT ratio >1 is suggestive of alcoholic liver disease, observed in 92% of patients with ALD [39]. In this study, control mice displayed AST:ALT ratios <1, while both ethanol groups had ratios >1. These findings indicate that there may be adverse consequences of moderate ethanol intake on the liver, despite observed overall reductions in ALT and AP. Additional research should be conducted in this area to further elucidate these paradoxical consequences of moderate alcohol consumption on liver function. Conflicting data also exist with regard to the effects of moderate alcohol exposure on the development and progression of nonalcoholic liver disease, with several reports indicating reduced incidence of fatty liver development and liver fibrosis, while other data report increased progression of fibrosis and hepatocellular carcinoma [40–42]. These diverse findings may be influenced by multiple factors including age, metabolic status, existing pathology, and methodological issues such as inaccuracies in self-reported alcohol intake and heterogeneity in defining moderate consumption levels [40–43]. + +Heavy ethanol consumption and binge drinking are associated with an increase in blood glucose and consequently an increased risk of type 2 diabetes and metabolic syndrome [44,45]. High doses of ethanol may result in an increase in glucose levels by inducing insulin resistance [44, 45]. It has been previously shown that binge drinking impairs insulin signaling in the hypothalamus, inducing systemic insulin resistance [45]. The current study found increased plasma glucose in ethanol-exposed mice, although prior studies have indicated possible benefits in glucose metabolism and lowered blood glucose from moderate alcohol consumption [12,46]. Proposed mechanisms by which moderate ethanol exposure may decrease fasting glucose are through increased insulin secretion or the inhibition of gluconeogenesis via alcohol metabolism [47]. Studies directly measuring effects of ethanol on insulin sensitivity have produced conflicting results, showing slight benefits or no difference [46]. There are several potential reasons for the observed variation in blood glucose among moderate alcohol + +![5_image_0.png](5_image_0.png) + +Fig. 3. Mean values of liver enzymes AST, ALT, and AP (U/L). (A) There was an interaction of fluid and diet on AST levels (main effect of fluid, P = 0.006; fluid x diet interaction: P = 0.017; NS effect of diet, P += 0.304). AST was significantly lower in ethanolexposed mice fed either diet and in controls fed the n-3 diet only. Regardless of diet, ALT (B) and AP (C) +were significantly lower in ethanol-consuming mice compared to controls (main effects of fluid: P = 0.002 and P = 0.021, respectively). (D) AST/ALT ratio was significantly greater in ethanol-exposed mice compared to control mice (main effect of fluid, P = 0.036; NS effect of diet, P = 0.408; NS interaction: P = 0.868). Two-way Fluid (2) x Diet (2) factorial ANOVAs were used for analysis. If there was an interaction, Bonferroni post-hoc tests were used for multiple comparisons. Lines for each group represent mean ± standard error. Varying superscripts are significantly different (P ≤ 0.05). Each group was comprised of 3 male and 3 female mice, except the Ethanol n-3 group which contained 3 male and 2 female mice. Closed circles (•) indicate male mice and open circles (o) female mice. AST, aspartate aminotransferase; ALT, alanine transaminase; AP, alkaline phosphatase; NS: not significant. + +consumers, including the type of alcohol consumed, whether it was consumed concurrently with food, and if glucose measurements were taken in a fasted state or not [48]. There were no significant differences in blood glucose in the present study as a function of n-6:n-3 dietary ratio. + +Previous studies have shown that n-3 fatty acids can lower triglycerides; however, current research does not strongly support beneficial effects of n-3 fatty acids on total cholesterol, LDL, or HDL [15,49]. + +While the present study did not find any main effects of n-3 diet alone on lipid profile, several trends were observed for the balanced n-3 diet to reduce triglycerides, total cholesterol, and LDL in mice also consuming ethanol. It is well known that n-3 fatty acids promote anti-inflammatory effects in the body, likely by reducing the production of pro-inflammatory cytokines as well as attenuating oxidative stress and improving endothelial function [15]. Additionally, when n-3 fatty acids bind with peroxisome proliferated-activated receptors (PPAR) transcription factors, they are activated for cytoprotective responses [50]. Recent research indicates that n-3 fatty acids may decrease inflammation by modulating the HMGB1/NF-κB pathway [51]. In addition, several studies have reported an increase in total antioxidant capacity and decreased DNA damage from n-3 fatty acids [52–55]. One mechanism by which n-3 fatty acids may improve antioxidant status is through upregulation of the transcription factor NRF2, a key regulator of antioxidant gene expression [53,54]. The results of this study are in line with these findings, showing a significant reduction in 8-oxo-dG, an increase in total antioxidant capacity, as well as a trend toward lower CRP in the balanced n-3 diet group. Concurrent intake of ethanol together with the n-3 diet resulted in further reductions in 8-oxo-dG, potentially suggesting beneficial effects in combination. It should be noted that while ethanol combined with the n-3 diet resulted in apparent facilitative effects on multiple measures, the main effects of ethanol in this study were strong, which may have overshadowed more robust interactions of these dietary factors in combination. + +Finally, the results of the present study should be interpreted within the context of observed differences in fluid and food intake between ethanol-exposed and control mice which resulted in a significantly greater body weight gain in the control group compared to the ethanol group. Although control mice were provided with a standard maltose dextrin control solution that was isocalorically matched to ethanol, greater preference for the maltose dextrin solution led to increased fluid consumption and higher body weight gain compared to ethanolconsuming animals. An alternate feeding model employing pair feeding of fluids according to the rate-limiting group and/or an alternative control fluid may be useful in future studies in order to better equate levels of fluid consumption and body weight gain between groups. Further studies are also needed to better understand the effects of moderate alcohol consumption on liver function including histology and additional parameters of oxidative stress, as well as blood glucose and insulin sensitivity including a glucose tolerance test with assessment of glucose stimulated insulin secretion, given across-study variation in these measures. Additionally, future studies that systematically evaluate alcohol dose-related effects, including an experimental group + +## 7 + +administered a higher dose of alcohol, will allow for within-study comparison of the effects of moderate vs. excessive alcohol exposure on key measures of interest. + +Alcohol consumption can affect the gastrointestinal tract by direct exposure to ethanol and/or its metabolite acetaldehyde. Alcohol abuse can damage the mucous cells in the gastrointestinal tract, induce inflammation and lesions and/or alter gastric acid output [56]. Many studies have examined effects of excessive or heavy ethanol drinking on the gastrointestinal tract, however, effects of moderate ethanol consumption have been less well studied. Moderate ethanol consumption has been shown to increase glutathione-S-transferase M1 expression and aldehyde dehydrogenase 2 expression in the colon, which may reduce mucosal colonic damage [31]. More research is necessary to investigate the effect of moderate ethanol consumption on the gastrointestinal tract. + +## 5. **Conclusion** + +Taken together, the present findings indicate independent beneficial effects of moderate alcohol consumption and a balanced n-6:n-3 diet on certain inflammatory and lipid profile markers associated with cardiovascular disease in mice. These findings add to a growing body of literature exploring the protective effects of moderate ethanol consumption on CVD risk and underlying modulatory mechanisms. Nevertheless, it is critical to recognize that dose-dependent effects of ethanol are complex, and beneficial effects of moderate ethanol are not ubiquitous across all disease processes, and further vary by gender, genetic background, and other inter-individual differences. The mechanisms by which moderate alcohol drinking promotes or protects against disease and how this is modulated by other interacting dietary factors is an important area for continued research. + +## Funding Disclosure + +This work was supported by the National Institutes of Health [NIH +AA023291]. + +## Credit Authorship Contribution Statement + +Kristine Giltvedt: Data curation, Investigation, Visualization, Writing - original draft. **Luciano S. Voutour:** Data curation, Investigation. **Brianna Tursellino:** Data curation. **Alexandra Zella:** Data curation. **Susan M. Brasser:** Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing - review & +editing, Funding acquisition. **Mee Young Hong:** Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing - review & editing. + +## Declaration Of Competing Interest + +The authors have no conflicts of interest to declare. + +## Acknowledgments + +The authors thank Trisha Molina, Lani Morales, Yuko Murase, Sydney Sagisi, and Nicole Wells for their help with tissue harvest. + +## Appendix A. **Supplementary Data** + +Supplementary data related to this article can be found at https:// +doi.org/10.1016/j.ijcrp.2022.200132. + +## References + +[1] Center for Disease Control and Prevention, Heart Disease Facts, 2021. https:// +www.cdc.gov/heartdisease/facts.htm. (Accessed 20 September 2021). + +[2] M.A. Collins, et al., Alcohol in moderation, cardioprotection, and neuroprotection: +epidemiological considerations and mechanistic studies, Alcohol Clin, Exp. Res. 33 +(2009) 206–219. https://doi:10.1111/j.1530-0277.2008.00828.x. + +[3] M. Krenz, R.J. Korthuis, Moderate ethanol ingestion and cardiovascular protection: +from epidemiologic associations to cellular mechanisms, J. Mol. Cell. Cardiol. 52 +(2012) 93–104. https://doi:10.1016/j.yjmcc.2011.10.011. + +[4] S. Minzer, R.A. Losno, R. Casas, The effect of alcohol on cardiovascular risk factors: +is there new information? Nutrients 12 (2020) 912. https://doi:10.3390/n u12040912. + +[5] K.J. Mukamal, et al., Moderate alcohol consumption and chronic disease: the case for a long-term trial, Alcohol Clin. Exp. Res. 40 (2016) 2283–2291. https://doi +:10.1111/acer.13231. + +[6] P.L. Thompson, J-curve revisited: cardiovascular benefits of moderate alcohol use cannot be dismissed, Med. J. Aust. 198 (2013) 419–422. https://doi:10.5694/mj a12.10922. + +[7] Y. Yang, et al., Alcohol consumption and risk of coronary artery disease: a doseresponse meta-analysis of prospective studies, Nutridate 32 (2016) 637–644. + +https://doi:10.1016/j.nut.2015.11.013. + +[8] G. Chiva-Blanch, L. Badimon, Benefits and risks of moderate alcohol consumption on cardiovascular disease: current findings and controversies, Nutrients 12 (2019) 108. https://doi:10.3390/nu12010108. + +[9] J.P. Cullen, et al., Ethanol inhibits monocyte chemotactic protein-1 expression in interleukin-1{beta}-activated human endothelial cells, Am. J. Physiol. Heart Circ. + +Physiol. 289 (2005) H1669–H1675. https://doi:10.1152/ajpheart.00106.2005. + +[10] J. Godfrey, et al., Chronic voluntary ethanol consumption induces favorable ceramide profiles in selectively bred alcohol-preferring (P) rats, PLoS One 10 +(2015), e0139012. https://doi:10.1371/journal.pone.0139012. + +[11] A. Imhof, W. Koenig, Alcohol inflammation and coronary heart disease, Addict. + +Biol.iol 8 (2003) 271–277. https://doi:10.1080/13556210310001602176. + +[12] M. Justice, et al., Effects of moderate ethanol consumption on lipid metabolism and inflammation through regulation of gene expression in rats, Alcohol Alcohol 54 +(2019) 5–12. https://doi:10.1093/alcalc/agy079. + +[13] J.D. Klein, et al., A snapshot of the hepatic transcriptome: ad libitum alcohol intake suppresses expression of cholesterol synthesis genes in alcohol-preferring (P) rats, PLoS One 9 (2014), e110501. https://doi:10.1371/journal.pone.0110501. + +[14] J. Rehm, C.T. Sempos, M. Trevisan, Alcohol and cardiovascular disease–more than one paradox to consider. Average volume of alcohol consumption, patterns of drinking and risk of coronary heart disease–a review, J. Cardiovasc. Risk 10 (2003) +15–20. https://doi:10.1097/01.hjr.0000051961.68260.30. + +[15] T.A. Mori, Dietary n-3 PUFA and CVD: a review of the evidence, Proc. Nutr. Soc. 73 +(2014) 57–64. https://doi:10.1017/S0029665113003583. + +[16] J.K. Innes, P.C. Calder, Marine omega-3 (n-3) fatty acids for cardiovascular health: +an update for 2020, Int. J. Mol. Sci. 21 (2020) 1362. https://doi:10.3390/ijms21 041362. + +[17] Q. Wang, et al., Effect of omega-3 fatty acids supplementation on endothelial function: a meta-analysis of randomized controlled trials, Atherosclerosis 221 +(2012) 536–543. https://doi:10.1016/j.atherosclerosis.2012.01.006. + +[18] T. Nozue, et al., Effects of serum n-3 to n-6 polyunsaturated fatty acids ratios on coronary atherosclerosis in statin-treated patients with coronary artery disease, Am. J. Cardiol. 111 (2013) 6–11. https://doi:10.1016/j.amjcard.2012.08.038. + +[19] R. Valenzuela, L.A. Videla, The importance of the long-chain polyunsaturated fatty acid n-6/n-3 ratio in development of non-alcoholic fatty liver associated with obesity, Food Funct. 2 (2011) 644–648. + +[20] L.Z. Liao, et al., Exploring the causal pathway from omega-6 levels to coronary heart disease: a network Mendelian randomization study, Nutr. Metabol. + +Cardiovasc. Dis. 30 (2020) 233–240. https://doi:10.1016/j.numecd.2019.09.013. + +[21] A.P. Simopoulos, An increase in the omega-6/omega-3 fatty acid ratio increases the risk for obesity, Nutrients 8 (2016) 128. https://doi:10.3390/nu8030128. + +[22] S. Barve, S.Y. Chen, I. Kirpich, W.H. Watson, C. McClain, Development, prevention, and treatment of alcohol-induced organ injury: the role of nutrition, Alcohol. Res. + +38 (2017) 289–302. + +[23] Y. Chen, G. Davis-Gorman, R.R. Watson, P.F. McDonagh, Chronic ethanol consumption modulates myocardial ischaemia-reperfusion injury in murine AIDS, +Alcohol, Alcohol 38 (2003) 18–24. https://doi:10.1093/alcalc/agg014. + +[24] F. Fan, et al., Impact of chronic low to moderate alcohol consumption on blood lipid and heart energy profile in acetaldehyde dehydrogenase 2-deficient mice, Acta Pharmacol. Sin. 35 (2014) 1015–1022. https://doi:10.1038/aps.2014.46. + +[25] H.Z. Zhou, J.S. Karliner, M.O. Gray, Moderate alcohol consumption induces sustained cardiac protection by activating PKC-epsilon and Akt, Am. J. Physiol. + +Heart Circ. Physiol. 283 (2002) H165–H174. https://doi:10.1152/ajpheart.0040 8.2001. + +[26] S.M. Brasser, M.E. McCaul, E.J. Houtsmuller, Alcohol effects during acamprosate treatment: a dose-response study in humans, Alcohol Clin. Exp. Res. 28 (2004) +1074–1083. https://doi:10.1097/01.alc.0000130802.07692.29. + +[27] National Institute on Alcohol Abuse and Alcoholism, Drinking Levels Defined, 2020. https://www.niaaa.nih.gov/alcohol-health/overview-alcohol-consumptio n/moderate-binge-drinking. + +[28] M.R. Piano, Alcohol's effects on the cardiovascular system, Alcohol. Res. 38 (2017) +219–241. + +[29] J.B. Standridge, R.G. Zylstra, S.M. Adams, Alcohol consumption: an overview of benefits and risks, South. Med. J. 97 (2004) 664–672. https://doi:10.1097/00007 611-200407000-00012. + +[30] Y. Zhou, et al., Alcoholic beverage consumption and chronic diseases, Int. J. + +Environ. Res. Publ. Health 13 (2016) 522. https://doi:10.3390/ijerph13060522. + +[31] D.S. Klarich, et al., Effects of moderate alcohol consumption on gene expression related to colonic inflammation and antioxidant enzymes in rats, Alcohol 61 +(2017) 25–31. https://doi:10.1016/j.alcohol.2017.02.179. + +[32] S. Minzer, R. Estruch, R. Casas, Wine intake in the framework of a mediterranean diet and chronic non-communicable diseases: a short literature review of the last 5 years, Molecules 25 (2020) 5045. https://doi:10.3390/molecules25215045. + +[33] P.L. da Luz, et al., Red wine consumption, coronary calcification, and long-term clinical evolution, Braz, J. Med. Biol. Res. 51 (2018), e7703. https://doi:10.1590/1 414-431X20187703. + +[34] M.I. Covas, P. Gambert, M. Fito, ´ R. de la Torre, Wine and oxidative stress: up-todate evidence of the effects of moderate wine consumption on oxidative damage in humans, Atherosclerosis 208 (2010) 297–304. https://doi:10.1016/j.atherosclero sis.2009.06.031. + +[35] L.A. Videla, R. Valenzuela, Perspectives in liver redox imbalance: toxicological and pharmacological aspects underlying iron overloading, nonalcoholic fatty liver disease, and thyroid hormone action, Biofactors (2021). https://doi:10.1002/bio f.1797. + +[36] B. Klop, A.T. do Rego, M.C. Cabezas, Alcohol and plasma triglycerides, Curr. Opin. + +Lipidol. 24 (2013) 321–326. https://doi:10.1097/MOL.0b013e3283606845. + +[37] A. Rocco, D. Compare, D. Angrisani, M. Sanduzzi Zamparelli, G. Nardone, Alcoholic disease: liver and beyond, World, J. Gastroenterol. 20 (2014) +14652–14659. https://doi:10.3748/wjg.v20.i40.14652. + +[38] A. Osaki, Y. Okazaki, A. Kimoto, H. Izu, N. Kato, Beneficial effect of a low dose of ethanol on liver function and serum urate in rats fed a high-fat diet, J. Nutr. Sci. Vitaminol. 60 (2014) 408–412. https://doi:10.3177/jnsv.60.408. + +[39] P. Hall, J. Cash, What is the real function of the liver 'function' tests? Ulster Med. J. + +81 (2012) 30–36. + +[40] A.A. Protopapas, E. Cholongitas, L. Chrysavgis, K. Tziomalos, Alcohol consumption in patients with nonalcoholic fatty liver disease: yes, or no? Ann. Gastroenterol. 34 +(2021) 476–486, https://doi.org/10.20524/aog.2021.0641. + +[41] J.H. Choi, W. Sohn, Y.K. Cho, The effect of moderate alcohol drinking in nonalcoholic fatty liver disease, Clin. Mol. Hepatol. 26 (2020) 662–669, https:// +doi.org/10.3350/cmh.2020.0163. + +[42] I. Kwon, D.W. Jun, J.H. Moon, Effects of moderate alcohol drinking in patients with nonalcoholic fatty liver disease, Gut Liver 13 (2019) 308–314, https://doi. + +org/10.5009/gnl18175. + +[43] R.H. McMahan, et al., A novel murine model of multi-day moderate ethanol exposure reveals increased intestinal dysfunction and liver inflammation with age, Immun. Ageing 18 (2021) 37, https://doi.org/10.1186/s12979-021-00247-8. + +[44] K. Nygren K, A. Hammarstrom, ¨ O. Rolandsson, Binge drinking and total alcohol consumption from 16 to 43 years of age are associated with elevated fasting plasma glucose in women: results from the northern Swedish cohort study, BMC Publ. Health 17 (2017) 509. https://doi:10.1186/s12889-017-4437-y. + +[45] C. Lindtner, et al., Binge drinking induces whole-body insulin resistance by impairing hypothalamic insulin action, Sci. Transl. Med. 5 (2013), 170ra14, https +://doi:10.1126/scitranslmed.3005123. + +[46] G. Chiva-Blanch, S. Arranz, R.M. Lamuela-Raventos, R. Estruch, Effects of wine, alcohol and polyphenols on cardiovascular disease risk factors: evidences from human studies, Alcohol Alcohol 48 (2013) 270–277. https://doi:10.1093/alcalc/a gt007. + +[47] I. Shai, et al., Glycemic effects of moderate alcohol intake among patients with type 2 diabetes: a multicenter, randomized, clinical intervention trial, Diabetes Care 30 +(2007) 3011–3016. https://doi:10.2337/dc07-1103. + +[48] P.A. Engler, S.E. Ramsey, R.J. Smith, Alcohol use of diabetes patients: the need for assessment and intervention, Acta Diabetol. 50 (2013) 93–99. https://doi:10.100 7/s00592-010-0200-x. + +[49] Z.S. Natto, W. Yaghmoor, H.K. Alshaeri, T.E. Van Dyke, Omega-3 fatty acids effects on inflammatory biomarkers and lipid profiles among diabetic and cardiovascular disease patients: a systematic review and meta-analysis, Sci. Rep. 9 (2019) 18867. + +https://doi:10.1038/s41598-019-54535-x. + +[50] F. Echeverria, M. Ortiz, R. Valenzuela, L.A. Videla, Long-chain polyunsaturated fatty acids regulation of PPARs, signaling: relationship to tissue development and aging, Prostaglandins Leukot. Essent. Fatty Acids 114 (2016) 28–34. https://doi:10 +.1016/j.plefa.2016.10.001. + +[51] X. Chen, et al., Omega-3 polyunsaturated fatty acid attenuates the inflammatory response by modulating microglia polarization through SIRT1-mediated deacetylation of the HMGB1/NF-κB pathway following experimental traumatic brain injury, J. Neuroinflammation 15 (2018) 116. https://doi:10.1186/s12974-0 18-1151-3. + +[52] J. Heshmati, et al., Omega-3 fatty acids supplementation and oxidative stress parameters: a systematic review and meta-analysis of clinical trials, Pharmacol. Res. 149 (2019) 104462. https://doi:10.1016/j.phrs.2019.104462. + +[53] P. Golpour, et al., Improvement of NRF2 gene expression and antioxidant status in patients with type 2 diabetes mellitus after supplementation with omega-3 polyunsaturated fatty acids: a double-blind randomised placebo-controlled clinical trial, Diabetes Res. Clin. Pract. 162 (2020) 108120. https://doi:10.1016/j.diabres. 2020.108120. + +[54] C. Sakai, et al., Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells, PLoS One 12 (2017), +e0187934. https://doi:10.1371/journal.pone.0187934. + +[55] M. Carvalho-Silva, et al., Omega-3 fatty acid supplementation decreases DNA +damage in brain of rats subjected to a chemically induced chronic model of Tyrosinemia type II, Metab. Brain Dis. 32 (2017) 1043–1050. https://doi:10.1007/ +s11011-017-9994-3. + +[56] A. Rocco, et al., Alcoholic disease: liver and beyond, World J. Gastroenterol. 28 +(2014) 14652–14659. https://doi:10.3748/wjg.v20.i40.14652. \ No newline at end of file diff --git a/medical/md_images/PMC9574760/0_image_2.png b/medical/md_images/PMC9574760/0_image_2.png new file mode 100644 index 0000000000000000000000000000000000000000..0276d41e15956dcc02838f4709473e30e05e1953 --- /dev/null +++ b/medical/md_images/PMC9574760/0_image_2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:88da4c2b5ee81f5077d552bd67d0a98571e8ea85d610b15213113fa525cce27b +size 1890 diff --git a/medical/md_images/PMC9605188/PMC9605188.md b/medical/md_images/PMC9605188/PMC9605188.md new file mode 100644 index 0000000000000000000000000000000000000000..ceeba73e7b127a4f22aaabcb500bf184c2478825 --- /dev/null +++ b/medical/md_images/PMC9605188/PMC9605188.md @@ -0,0 +1,182 @@ +# The Association Between Temperature, Heart Rate, And Respiratory Rate In Children Aged Under 16 **Years Attending** + +urgent and emergency care settings Calvin Heala, Anna Harveyb, Stephen Brownc, Andrew Graeme Rowlandb,d and Damian Rolande,f Background and importance **Body temperature is** considered an independent determinant of respiratory rate and heart rate; however, there is limited scientific evidence regarding the association. This study aimed to assess the association between temperature, and heart rate and respiratory rate in children. + +Objective **The objective of this study was to validate** +earlier findings that body temperature causes an increase of approximately 10 **bpm rise in heart rate per 1 °C rise in** temperature, in children aged under 16 **years old.** Design **A prospective study using anonymised** prospectively collected patient data of 188 **635 attendances,** retrospectively extracted from electronic patient records. Settings and participants **Four Emergency or** Urgent Care Departments in the North West of England. Participants were children and young people aged 0–16 **years old who attended one of the four sites over a** period of 3 **years.** Outcome measures and analysis **Multiple linear** regression models, adjusted for prespecified confounders (including oxygen saturation, heart rate, respiratory rate, site of attendance, age), were used to examine the influence of various variables on heart rate and respiratory rate. + +Main results Among the 235 **909 patient visits (median** +age 5) included, the mean temperature was 37.0 (SD, 0.8). + +## Introduction + +It is widely accepted that an increase in body temperature will be associated with an increase in heart rate +[1,2]. In children, this increase has been suggested to be 10 beats per minute (bpm) per 1 °C rise in temperature [1,3]. There is currently limited scientific evidence regarding the size of this increase, and there is a need for these earlier findings to be validated in a separate group of children, from a different centre. Between 10 and 20% of attendances to the children's emergency department (ED) involve pyrexia [4–6]. + +This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBYNC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. + +Mean heart rate and respiratory rate were 115.6 (SD, 29.0) and 26.9 (SD, 8.3), respectively. For every 1 °C increase in temperature, heart rate will on average be 12.3 **bpm** higher (95% CI, 12.2–12.4), after accounting for oxygen saturation, location of attendance, and age. For every 1 °C increase in temperature, there is on average a 0.3% +decrease (95% CI, 0.2–0.4%) in respiratory rate. + +Conclusion **In this study on children attending urgent** and emergency care settings, there was an independent association between temperature and heart rate but not between temperature and respiratory rate. *European* Journal of Emergency Medicine **29: 413–416 Copyright ©** 2022 The Author(s). Published by Wolters Kluwer Health, Inc. + +European Journal of Emergency Medicine 2022, 29:413–416 Keywords: association, child, emergency medicine, heart rate, physiological parameters, physiology, respiratory rate, temperature, urgent care aCentre for Biostatistics, The University of Manchester, bNorth Manchester General Hospital, Manchester University NHS Foundation Trust, Manchester, cThe Northern Care Alliance NHS Foundation Trust, dSchool of Health and Society, The University of Salford, Salford, ePaediatric Emergency Medicine Leicester Academic (PEMLA) Group, Children's Emergency Department, Leicester Royal Infirmary and fSAPPHIRE Group, Health Sciences, Leicester University, Leicester, UK Correspondence to Andrew Graeme Rowland, PhD, FRCEM, FRCPCH, School of Health and Society, The University of Salford, Salford, M6 6PU, UK Tel: +0161 720 2672; e-mail: a.rowland@salford.ac.uk Received 31 January 2022 **Accepted** 10 May 2022 Pyrexia, or fever, can be defined as a rise in core body temperature above the normal daily variation. Pyrexia is, most usually, secondary to the body's response to infection, both self-limiting infection and significant illness. With this pyrexia is an associated increase in heart rate, likely due to an increase in metabolic rate [7]. There are, however, other physiological reasons for tachycardia in children presenting to the ED, such as pain, anxiety, or agitation. Body temperature has also previously been found to be an independent determinant of respiratory rate [1]. In a clinical setting, observations are taken to allow clinicians to identify the most unwell children and allow the prompt assessment, investigation, and treatment of their illness or injury. A patient with a raised temperature and a tachycardia could be suffering from a significant 0969-9546 Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. DOI: 10.1097/MEJ.0000000000000951 infection or sepsis; however, they could also be tachycardic due to the fever itself, the distress of a fever, or pain from a self-limiting condition such as otitis media. + +Understanding the association between a rise in temperature and heart rate will aid clinicians in their interpretation of a patient's observations, in the context of each individual's attendance to an emergency or urgent care setting. For example, a patient with a significant infection is likely to have a temperature but a raised heart rate as a compensatory mechanism to ensure adequate oxygen delivery to potentially poorly perfused organs. If temperature's impact on heart rate is removed, it may be possible to work out what the true baseline heart rate is and whether there is a serious underlying condition. + +This study aimed to validate earlier findings that body temperature is an independent determinant of heart rate and causes an increase of approximately 10 bpm per 1 °C rise in temperature [1]. Previous research looked at data from 21 033 patients from two sites over a 3-year period, with data on children not sent home from the ED removed, to exclude any patient with haemodynamic shock [1]. + +The goal of this study was to aid clinicians in understanding whether an apparent tachycardia is likely to be an actual tachycardia, and therefore, the authors considered that any association between heart rate and temperature should apply to an undifferentiated cohort. If such an association is evident, then this acts as a cognitive prompt to establish the cause of the raised heart rate - especially because of the link between tachycardia and other serious illness described in national guidance [8]. + +Our primary aim was to determine the association between temperature and heart rate in children aged under 16 years old. Our secondary aims were to determine the association between temperature and respiratory rate, the association between oxygen saturation and respiratory rate, and the association between oxygen saturation and heart rate. + +## Methods Study Design And Setting + +The sample included patients seen consecutively from three ED and one Urgent Care Centre in the North West of England, UK. Eligible cases were children and young people 0–16 years old who attended one of four hospital sites within one National Health Service (NHS) organisation in Greater Manchester, UK. Study design and protocol was prospective over the whole 3 years (01 October 2017–30 September 2020) to avoid potential bias from seasonal variability. Approval was given by both the data controller's research and innovation department and Caldicott Guardian to proceed with this study without seeking formal research ethics committee approval. + +## Data Collection + +The data were collected prospectively and then retrospectively extracted from the electronic patient records for analysis purposes. Following approval by the NHS organisation's Caldicott Guardian and Data Protection Officer, access was granted to anonymised data from the ED and Urgent Centre electronic patient records, to extract the physiological data necessary for this study (as well as the sex, date of birth, and date of attendance, from which the exact age of the child could be calculated). + +## Data Processing + +Data were cleaned and anonymised by a data manager in the Research and Innovation Department before passing the data onto the study statistician. As it was the intention to summarise the characteristics from the set of hospitals collectively, as opposed to describing them individually, the data from all four hospitals were combined into one dataset. Values deemed recording errors due to their incompatibility with life were removed. + +## Data Analysis + +Descriptive statistics were initially used to examine the distribution of the variables being considered. For each of the research questions, a separate model was used, adjusting for prespecified confounders, though age and location of attendance were included in all. To answer the primary research question, a multiple linear regression model was used with heart rate as the dependent variable. Temperature was included as the independent variable of interest and oxygen saturation, location of attendance, and age were included as prespecified confounders. + +For each of the secondary research questions, this methodology was repeated, replacing the variables as appropriate. It is not appropriate to report P-values which, given the very large sample size, would be deemed significant regardless of whether the effect size was at all meaningful. Assumptions for linear regression were assessed and for the secondary research questions, respiratory rate was log-transformed to meet the required assumptions. This necessitates that the results for these aims are interpreted in terms of a percentage change in the outcome rather than a raw unit change. Where the association was of a clinically meaningful magnitude, the strength of interaction between age and the independent variable was also investigated. This investigation was achieved by first fitting the previously described regression model with an added interaction term between age and the independent variable of interest, and then by examining the marginal means at each age. Analysis was conducted in Stata MP 14 (StataCorp LLC, College Station, Texas., USA) + +## Results + +Data on 235 909 patient visits were recorded. The median age was 5 years (interquartile range, 1–10 years) and the sample was 45% female and 55% male. The four sites provided differing number of records (35.7, 34.2, 17.0, and 13.1%). Limited demographic characteristics are shown in Table 1. The number of respondents included in each analysis was determined by how many had data recorded for all of the variables in that model. + +For the primary analysis, n = 188 635 patients had information on the necessary variables. This and all other results to the research questions are shown in Table 2. The coefficients for the confounder variables are not reported in this table as these are not of interest in answering the research questions. For every 1 °C increase in temperature, heart rate will on average be 12.3 bpm higher [95% confidence interval +(CI), 12.2–12.4], after accounting for oxygen saturation, location of attendance, and age. There is evidence of some limited interaction between age and temperature, meaning the estimate differs depending on the age of the child. The marginal means are reported in Table 3, which gives the amount of change in heart rate with a 1 °C increase in temperature while holding age constant at different values. The marginal means suggest that the influence of temperature on heart rate ranges from a 13.7 bpm increase for a 1 °C temperature increase for the youngest children down to an 8.7 bpm increase for a 1 °C temperature increase for the oldest children. After accounting for heart rate, location of attendance, and age, there is essentially no influence of temperature on respiratory rate. For every 1 °C increase in temperature, there is on average a 0.3% decrease (95% CI, 0.2– +0.4%) in respiratory rate. + +Similarly, for every one-percentage-point increase in oxygen saturation, respiratory rate will on average be 1.8% +lower (95% CI, 1.8–1.9%), after accounting for heart rate, location of attendance, and age. For every 1%-point increase in oxygen saturation, heart rate is on average 0.9 bpm lower (95% CI, 0.9–1.0), after accounting for respiratory rate, location of attendance, and age. As these secondary outcomes did not exhibit clinically meaningful differences, interaction terms were not explored. + +## Discussion + +This study is in keeping with previous research in this area suggesting a rise of 10 bpm increase in heart rate for every degree rise in temperature. At the time of this study's results being analysed, this is the largest examination (188 635 patient attendances) of the association between temperature and heart rate in a UK nonspecialist setting of a cohort of undifferentiated children attending for urgent or emergency care. + +The CI of the results in this study suggests a slightly higher change in heart rate (by 2 bpm) for every 1 °C rise in temperature, than that which was reported in a previous study involving 21 033 patients [1]. The clinical significance of this slightly different finding is questionable; however, it has been possible to investigate the earlier-reported association in a much larger sample. + +This study's results from a very large cohort of patients, a decade on from the previous work by Davies and Maconochie [1], and from different sites in a different part of the UK, would suggest that the utilisation of this principle should become more widespread. Current guidance in England and Wales for the identification of serious bacterial illness in children (National Institute for Health and Care Excellence Guidance 51: Sepsis and 143: Feverish illness in children) suggests a direct association between heart rate and risk of infection as part of screening and identification processes. However, studies have demonstrated that this guidance is unspecific and leads to the potential overtreatment with the risks of increased antimicrobial resistance [9,10]. The addition of a correcting variable for fever may improve the specificity of the guidance, and while it is noted that a previous study didn't demonstrate such an association [11] between serious illness and corrected heart rates, the demographics of + +| Table 1. | Data availability from the four sites in this study | | | +|-------------------------------------------------------------------|-------------------------------------------------------|----------------|--------| +| Variable | Summary | Min–Max Number | | +| Age Median age in years (IQR) | 5 (1–10) | 0–15 | 235909 | +| Sex Male | 129703 (55.0%) | N/A | 235899 | +| Female | 106196 (45.0%) | | | +| Attendances at each site: number (%) Site 1: Emergency Department | 30894 (13.1%) | N/A | 235909 | +| Site 2: Emergency Department | 84104 (35.7%) | | | +| Site 3: Urgent Care Centre | 40179 (17.0%) | | | +| Site 4: Emergency Department | 80732 (34.2%) | | | +| Heart rate (beats per minute): mean (SD) | 115.6 (29.0) | 50–255 191292 | | +| Temperature (o C): median (IQR) | 36.8 (36.5–37.3) 25.2–45 192325 | | | +| Respiratory rate (breaths per minute): | 25 (22–30) | 9–90 | 192147 | +| median (IQR) | | | | +| Oxygen saturation (%): median (IQR) | 99 (98–100) | 40–100 191256 | | +| IQR, interquartile range. | | | | + +| Table 2. | Coefficients of interest from regression models, stratified by research question | | | | +|------------------------------------|------------------------------------------------------------------------------------|---------------------------|------------------------------|--------| +| Dependent variable | Independent variable | Coefficient (95% CI) | Adjusted for | Number | +| Heart rate | Temperature | 12.33 (12.24–12.43) | Oxygen saturation, Site, Age | 188635 | +| Respiratory rate (log-transformed) | Temperature | −0.003 (−0.003 to −0.002) | Heart rate, Site, Age | 188670 | +| Respiratory rate (log-transformed) | Oxygen saturation | −0.019 (−0.019 to −0.018) | Heart rate, Site, Age | 189957 | +| Heart rate | Oxygen saturation | −0.91 (−0.96 to −0.86) | Respiratory rate, Site, Age | 189957 | +| CI, confidence interval. | | | | | + +| Table 3. | Marginal means and temperature coefficient | +|--------------------------|----------------------------------------------| +| Age (years) | Temperature coefficient (95% CI) | +| 0 | 13.71 (13.58–13.85) | +| 1 | 13.38 (13.26–13.50) | +| 2 | 13.04 (12.93–13.15) | +| 3 | 12.71 (12.61–12.80) | +| 4 | 12.37 (12.27–12.46) | +| 5 | 12.03 (11.94–12.13) | +| 6 | 11.70 (11.59–11.80) | +| 7 | 11.36 (11.25–11.48) | +| 8 | 11.03 (10.89–11.16) | +| 9 | 10.69 (10.54–10.84) | +| 10 | 10.35 (10.19–10.52) | +| 11 | 10.02 (9.83–10.20) | +| 12 | 9.68 (9.48–9.89) | +| 13 | 9.35 (9.12–9.57) | +| 14 | 9.01 (8.76–9.26) | +| 15 | 8.67 (8.40–8.94) | +| CI, confidence interval. | | + +attendances and vaccination schedules have altered significantly in the 10 years since that work was published. + +The persistence of a general trend of a 10–12 bpm rise in heart rate for each 1 °C rise in temperature with tight CIs suggests a new lens with which to look at heart rate in relation to illness identification. The fact that the association doesn't hold for respiratory rate may indicate either a difference in the accuracy of the methods of taking the observation (digital probes for heart rate versus manual counting for respiratory rate) or that heart rate is a less dynamic variable than originally thought. + +## Limitations + +This study has several limitations. First, as with all studies of this type, there are certain to be several confounders we do not have data on and so cannot adjust for, potentially biasing the results. Second, these measures, especially heart and respiratory rate, are known for their potential to vary considerably from minute to minute, which introduces an unaccountable element of randomness to the data. Third, respiratory rates were calculated manually, which is known to be less accurate than other methods, though this is internationally standard practice. Fourth, results in this study are only applicable for nonextreme values. Taking temperature as an example, less than 1% of the dataset had a temperature below 35.8 °C. Below this temperature, it is therefore not possible to determine whether the association between temperature and heart rate is linear; therefore, it cannot be suggested what influence a temperature lower than 35.8 °C may have on heart rate. However, such patients are typically identified as being potentially critically unwell from the outset. + +## Conclusion + +After accounting for appropriate confounders, there is a clinically significant association between heart rate and temperature, but no clinically significant influence of temperature on respiratory rate, oxygen saturation on heart rate, or oxygen saturation on respiratory rate. + +## What Is Already Known On This Topic + +- Body temperature is an independent determinant of respiratory rate and heart rate. + +- Previous studies suggest a rise in heart rate of 10 bpm for every 1 °C increase in temperature. + +- There is currently limited scientific evidence regarding the size of this increase. + +## What This Study Adds + +- For every 1 °C increase in temperature, heart rate will on average be 12.3 bpm higher. + +- The influence of temperature on heart rate is age-dependent and ranges from 8.7 to 13.7 bpm increase for a 1 °C temperature increase. + +- After accounting for age, heart rate, and emergency care location, there is essentially no influence of temperature on respiratory rate. + +## Acknowledgements + +C.H., A.R., and D.R. devised the study and sought the necessary information governance approvals. S.B. extracted and cleaned the data. C.H. analysed the data. + +All authors contributed to the preparation of the manuscript. All authors approved the final manuscript before submission. + +## Conflicts Of Interest + +There are no conflicts of interest. + +## References + +1 Davies P, Maconochie I. The relationship between body temperature, heart rate and respiratory rate in children. *Emer Med J* 2009;26:641–643. + +2 Daymont C, Bonafide CP, Brady PW. Heart rates in hospitalized children by age and body temperature. *Pediatrics* 2015;135:e1173–e1181. + +3 Kirschen GW, Singer DD, Thode HC Jr, Singer AJ. Relationship between body temperature and heart rate in adults and children: a local and national study. *Am J Emerg Med* 2020;38:929–933. + +4 Sands R, Shanmugavadivel D, Stephenson T, Wood D. Medical problems presenting to paediatric emergency departments: 10 years on. *Emerg Med* J 2012;29:379–382. + +5 Armon, K. Audit: determining the common medical presenting problems to an accident and emergency department. *Arch Dis Childhood* 2011;84:390–392. + +6 Leigh S, Robinson J, Yeung S, Coenen F, Carrol ED, Niessen LW. + +What matters when managing childhood fever in the emergency department? A discrete-choice experiment comparing the preferences of parents and healthcare professionals in the UK. *Arch Dis Child* 2020;105:765–771. + +7 van den Anker JN. Optimising the management of fever and pain in children. + +Int J Clin Pract 2012;67:26–32. + +8 National Institute for Health and Care Excellence (NICE). Sepsis: +recognition, diagnosis and early management. 13 July 2016 (revised 13 September 2017). NICE Guideline [NG51]. https://www.nice.org.uk/ guidance/ng51/resources. [accessed 8 November 2021] +9 Lim E, Van Dam CJ, Agbeko RS, Emonts M. Suspected sepsis: +summary of NICE guidance and the potential impact to paediatrics. BMJ 2016;354:i4030. + +10 Nijman RG, Jorgensen R, Levin M, Herberg J, Maconochie IK. Management of children with fever at risk for pediatric sepsis: a prospective study in pediatric emergency care. *Front Pediatr* 2020;8:548154. + +11 Brent AJ, Lakhanpaul M, Ninis N, Levin M, MacFaul R, Thompson M. Evaluation of temperature-pulse centile charts in identifying serious bacterial illness: observational cohort study. *Arch Dis Child* 2011;96:368–373. \ No newline at end of file diff --git a/medical/md_images/PMC9605188/PMC9605188_meta.json b/medical/md_images/PMC9605188/PMC9605188_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..ad33d56f3c837d98882dd1fc43c57fed914556de --- /dev/null +++ b/medical/md_images/PMC9605188/PMC9605188_meta.json @@ -0,0 +1,27 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [], + "pages": 4, + "ocr_stats": { + "ocr_pages": 0, + "ocr_failed": 0, + "ocr_success": 0, + "ocr_engine": "none" + }, + "block_stats": { + "header_footer": 0, + "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/PMC9624911/PMC9624911.md b/medical/md_images/PMC9624911/PMC9624911.md new file mode 100644 index 0000000000000000000000000000000000000000..9158fcaa8af03a64d8b0eb072e48c403267f8018 --- /dev/null +++ b/medical/md_images/PMC9624911/PMC9624911.md @@ -0,0 +1,17 @@ +Abstract citation ID: bvac150.231 + +## Adrenal Psat073 + +The Human Gonads Do Not Contribute to the Circulating Pool of 11-Oxygenated Androgens Amira Rabbah, Angela Davio, BS, Haiping Liu, PhD, Xhorlina Marko, MD, William Sherk, MD, Juilee Rege, PhD, William Rainey, PhD, and Adina Turcu, MD, MS +A113 J Endocrine Soc, Volume 6, Issue Supplement_1, November–December 2022 Background: Androstenedione (A4) and testosterone (T) are produced by both the adrenal glands and the gonads. The adrenal enzyme steroid 11β-hydroxylase (CYP11B1) executes the last step in cortisol synthesis; CYP11B1 also uses A4 and T as substrates, generating 11-hydroxyadrostenedione (11OHA4) and 11-hydroxytestosterone +(11OHT), respectively. 11OHA4 and 11OHT are further metabolized, primarily in periphery, to 11-ketoandrostenedione and 11-ketotestosterone. It has been suggested that CYP11B1 is expressed in the gonads, yet the circulating levels of all 11-oxygenated C19 steroids (11-oxyandrogens) +are similar in males and females of reproductive ages, despite enormous differences in T. Objective: To assess the gonadal contribution to the circulating pool of 11-oxyandrogens. + +Methods: We used liquid chromatography-tandem mass spectrometry to measure 13 steroids, including traditional and 11-oxy-androgens in: (I) paired peripheral and gonadal vein serum samples from 11 patients (7 women), median age 37 years (range 31-51); and (II) 17 women, median age 57 years (range 41-81) before and after bilateral salpingo-oophorectomy (BSO). We also performed CYP11B1, 17αhydroxylase/17,20-lyase, and cytochrome b5 immunohistochemistry in formalin-fixed paraffin-embedded ovaries and testes (10 each). + +Results: A4, T, estradiol, estrone, progesterone, 17α- and 16α-hydroxyprogesterone were all higher in gonadal veins than in periphery (p<0.001 for all), while all 4 11-oxyandrogens were similar between matched peripheral and gonadal vein samples. Similarly, in women who underwent BSO, A4 (median [interquartile range]: 33 [24.5-49] ng/dL vs. 60 +[47.5-71] ng/dL, p=0.0008) and T (15 [12.5-20] vs. 24 [15.5-33.5], p=0.0005) were lower after vs. before surgery, while the four 11-oxyandrogens were not significantly different. Immunohistochemistry staining for CYP11B1 was negative in both ovarian and testicular tissue. + +Conclusion: Our results indicate that despite producing substantial amounts of both A4 and T, the human gonads are not important sources of 11-oxyandrogens. + +Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m. \ No newline at end of file diff --git a/medical/md_images/PMC9624911/PMC9624911_meta.json b/medical/md_images/PMC9624911/PMC9624911_meta.json new file mode 100644 index 0000000000000000000000000000000000000000..24d721a6de32d49f57cfdc4e0a993dc1346b6ba0 --- /dev/null +++ b/medical/md_images/PMC9624911/PMC9624911_meta.json @@ -0,0 +1,84 @@ +{ + "languages": [ + "en" + ], + "filetype": "pdf", + "toc": [ + { + "title": "PSAT072 Clinical Features of Patients with 21-Hydroxylase Deficiency and Testicular Adrenal Rest Tumors", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 594.0, + 0.0 + ] + }, + { + "title": "PSAT073 The Human Gonads Do Not Contribute to the Circulating Pool of 11-Oxygenated Androgens", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 0, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 594.0, + 0.0 + ] + }, + { + "title": "PSAT074 Patient-Centered Research in Adrenal Insufficiency for Adults and Children Using a Registry and Longitudinal Cohort", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 594.0, + 0.0 + ] + }, + { + "title": "PSAT082 A Survey of Patients Living with CAH Assessing Their Perceptions on Glucocorticoid Regimen and Glucocorticoid-Associated Side Effects", + "level": 0, + "is_closed": null, + "n_kids": 0, + "page_index": 1, + "view_mode": "FitR", + "view_pos": [ + 0.0, + 783.0, + 594.0, + 0.0 + ] + } + ], + "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/PMC9641775/0_image_1.png b/medical/md_images/PMC9641775/0_image_1.png new file mode 100644 index 0000000000000000000000000000000000000000..e75637d7a98647452f1b1e28009212b1a7c1a436 --- /dev/null +++ b/medical/md_images/PMC9641775/0_image_1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:f62e7def400216df4c0e466cab2d2881e90d7e8ff4d4d33066e7cd14959dde54 +size 4401 diff --git a/medical/md_images/PMC9641775/10_image_0.png b/medical/md_images/PMC9641775/10_image_0.png new file mode 100644 index 0000000000000000000000000000000000000000..9cfcc88af865ef8e5e2146f2dc557ceb28050b25 --- /dev/null +++ b/medical/md_images/PMC9641775/10_image_0.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:5ffd45621f2a57e8b07e14a0e4f8a6497df7826e197e0657641227eba1f822b5 +size 4462