This issue of the Journal contains papers of great interest for the Reader. These highlights attempt to provide an overview of the papers and to quickly introduce them to our audience. The Reader will go through papers written by authors from various countries, including Greece, India, Iran, Iraq, Israel, Malta, and Turkey. The international reputation of the Journal increases, its visibility accelerates, attracting papers of high calibre considerably contributing to the field of rheumatology and the affiliated specialties.1The review paper adds to the current knowledge about the syndrome. The updated major and minor diagnostic criteria of the syndrome are presented. We also learn that currently more than 4000 documented cases suffering from ASIA have been reported, making it not as rare as considered before. More importantly, the Authors underline basic concepts, which must be known by physicians to facilitate early and prompt diagnosis of the syndrome.Prof. Yehuda Shoenfeld’s review paper is an update on the current understanding of what he firstly described as ASIA (Autoimmune/inflammatory Syndrome Induced by Adjuvants) and the rest of us usually call Shoenfeld’s syndrome.2As the Authors underline, at present, the rehabilitation programs should be designed as patient-centered using a multi-disciplinary approach and individual goals of therapy should also be clearly defined before the rehabilitation intervention. New data emerge suggesting that rehabilitation of specific patient groups may be more beneficial in warm climate settings such as the Mediterranean region, providing the rationale for better designing of studies in these countries. In another review paper, Prof. Sakkas provides an overview of the current knowledge of role of regulatory B cells, a relatively new subset of suppressor cells, in autoimmune rheumatic diseases.3The Author’s contribution to the field has been decisive, as his group was the first to describe the functional impairment of regulatory B cells in systemic sclerosis and more recently in psoriatic arthritis patients. From the overview, it appears that these cells not only play a role in the pathogenesis of these diseases, but also their state at baseline influences’ response to treatment and progression of the diseases over time. Tabrizi et al.4investigated the expression levels of microRNA expression profiles of the Drosha, Dicer and DGCR8 maturing microprocessor components in patients with ankylosing spondylitis. Recent data suggest that various microRNAs could be involved in the pathogenesis of spondylarthtitides including ankylosing spondylitis. Since Drosha, Dicer and DGCR8 are major microRNA biogenesis components, the Authors hypothesized that they could be involved in disease pathogenesis. Though their data do not show correlation between expression levels of the microRNAs with disease activity, they are suggestive of dysregulated expression levels which requires further investigation.Prof. Aysen Akinci and Dr. Gamze Kilic review the available data assessing rehabilitation interventions for rheumatic diseases in the Mediterranean region.5investigated the manifestations of peripheral arterial disease in 100 patients with rheumatoid arthritis. The investigators noted that the majority of the patients had normal ankle brachial tibial artery index; however, waveform analysis was biphasic  in approximately one-third of those. This finding has prompted them to go one step further suggesting that current recommendations about physiological testing of peripheral perfusion in rheumatoid arthritis should consider including waveforms as part of the assessment.Grech and collaborators6The Author’s goal is to emphasize the urgent need for the Greek rheumatological community to embrace the new perception for the economic evaluation of medical procedures, to consent to the gradual change from the medical-oriented to a society-oriented model, to synergize efforts with state authorities, and finally, to organize a health system based on actual needs and social priorities.Dr. Trontzas, the ex-president of the Greek Rheumatology Society and Professional Association of Rheumatologists, position paper tackles the difficulties facing the rheumatologists practising in Greece, especially during the last few years of the extended monetary crisis.7is addressing the challenges that journals of Rheumatology are currently facing. The Authors introduce the Indian Journal of Rheumatology, the official Journal of Indian Rheumatology Association and the challenges the Editors and the members of the Editorial Board face to increase the visibility of the Journal. Commonalities and differences amongst their Journal and ours are highlighted.The paper by Ravindran et al.8The Author underlines the need for CDS to be considered in the differential diagnosis of acute neck pain, particularly in older patients and provide informative information regarding the clinical features of the syndrome, and the need for CT to assist accurate diagnosis which can be helpful in order to prevent misdiagnosis and unnecessary diagnostic procedures such as lumbar puncture and biopsy, as well as inappropriate treatment with antibiotics or antiviral drugs.Prompted from an individual case is presenting, Dr. Ali Younis is reviewing the literature regarding the Crowned dens syndrome (CDS), a rare clinical entity characterized by acute neck pain due to calcification around the odontoid process of the axis in “crown-like” configuration.9The Authors report on the history of Severus Iatrosophista, Theophilus the Philosopher, and Jacobus Psychrestos, who were the first healers to use the plant Colchicum for the treatment of gout, long before others had implemented this plant in the management of podagra. According to the Authors, Rheumatology owes these three ‘forgotten’ Byzantine physicians an honorable citation, who still seek their fair place in the history of medicine.In their historical paper, Profs. Tsoucalas and Sgantzos reveal the decisive role that three Greek Byzantine physicians, ‘unknown’ to most of us, have played in the prompt management of gout.We hope that the papers will be of interest for the Reader and we are committed to reiterate our focus on stimulating papers in our next issue.Enjoy the reading.


Electives are perceived by medical students as a valuable, highly regarded experience, allowing them to customize learning experiences and enabling them to early differentiate during medical training. The present work aims to uncover students’ major determinants of satisfaction and how they interfere with their future elective choices in order to identify the best approach to implement electives in medical curricula.nd to the 5th year. Students’ satisfaction was assessed through a validated questionnaire with eight sentences expressing opinions related to electives global satisfaction. Data from 538 inquiries from 229 students were analyzed quantitatively using regression and correlation models, and qualitatively through phenomenography.A cross-sectional study was conducted through a written evaluation survey concerning the electives available in the academic year 2015–2016. Our institution provides 106 electives to students from the 2Agreement with teaching and learning methodologies, followed by Agreement with assessment methodologies employed, Perception of the workload demanded and Requirement for continuous work and 2) The predictors of students preferences in the following years, namely, Agreement with assessment methodologies employed, Classes attendance and Ranking of the allocated elective established in the previous year. Qualitative analysis of questionnaires revealed that students consider electives as being innovative and interesting, claiming that some, for their relevant content, could be integrated into the medical core curriculum.Quantitative analysis of the questionnaires allowed to establish both: 1) The determinants of students’ satisfaction with electives, which were Our work raises awareness on the best practices when it comes to electives’ organization to meet students’ satisfaction. We can conclude that medical schools should measure students satisfaction as a tool to organize and predict future needs of electives and placements when designing and implementing this alternative student-centred curriculum or even to improve the existing practices regarding electives in medical courses. The participants were 229 students from 3number of students enrolling an elective, we found a negative correlation between the number of students allocated to an elective and age of the enrolled students. We also observed a positive association with the class attendance, showing that, as the number of students enrolling in an elective increased, so did the class attendance to that elective.Regarding the order of preference established by students was positively associated with both age and classes attendance and negatively associated with the agreement with both assessment and teaching methodologies, meaning that the less desired electives had higher classes attendance and older students. Contrarily, the more students agreed with the assessment and teaching methodologies, the more they preferred the elective  was positively associated with 1) the grade the student achieved in the elective, 2) the agreement with assessment methodologies employed, 3) the agreement with teaching methodology and 4) with the number of students assigned to the elective. Nevertheless, neither gender, age, classes attendance, requirement for continuous work nor the ranking of the allocated elective in the list of preferences were significant determinants of students’ global satisfaction with electives. Furthermore, there was a negative association with the perception of the workload demanded, and with the estimated workload of the elective , we verified that both d. Table .Overall the analysis of the questionnaires demonstrates that: 1) For each position that the elective decreased in students order of preferences, the global satisfaction increased 0.537 points; 2) For each additional standard deviation above the average grade of the elective, their global satisfaction increased 1.325 points; 3) If students had to study continuously during the class period (period of exams not included), the global satisfaction increased 2.217 points; 4) For each additional standard deviation in the perception of workload demanded, the global satisfaction of students decreased 2.531 points; 5) If students agreed with the assessment methodologies employed in an elective, their global satisfaction increased in 4.850 points, compared with students who do not agree and 6) If students agreed with the teaching methodologies of an elective, their global satisfaction increased in 8.297 points, compared with students who do not agree (Table p < 0.001) Regarding the content of the open-ended answers, the free text was typically short, ranging from single words to shortlists or simple sentences. Table the innovative (24.2%) and interesting (27.5%) character of the electives, and their relevant content (8.8%), perceived by students. Determinants such as the adequacy of the previous acquaintances (5.5%), the involvement of both teachers (4.4%) and students (9.9%), as well as the acquisition of transversal competences (12.1%) were also acknowledged by students. The statement “Allows for unique experiences and competencies that would not have been otherwise acquired during the course” (id 181) was one of the statements that supported the innovative character of the electives offered while “With crucial components, this is an elective that should be part of the medical curriculum as core curricular unit.” (id 194) and “I loved this elective. It made me learn much more in practical terms than any other core curricular unit, besides making me feel realized.” (id 227) reinforced their relevance for the medical curriculum.Open-ended answers provided the opportunity to enlighten about additional features not mentioned in close-ended questions, such as Excess of contents/Workload inadequate) overlapped simultaneously with the Perception of the workload demanded and the estimated workload, both already included in the close-ended questions of the survey. Another example of overlapped content was topic 2 (Inadequate Assessment), which was the one which registered the highest occurrence (27.5%) and, even though it was already assessed in the survey through questioning about the Agreement with assessment methodologies employed, the open-ended answer provided students the opportunity to further enlighten on the reason for their disagreement. In this case, the subjective assessment and the absence of full insight into the assessment criteria were some of the mentioned reasons that made 27.5% of students declare that the elective assessment was inadequate. The same occurred with topic 4 (Typology of classes inadequate), which overlapped the disagreement with teaching methodology, having 12.1% of the students identifying their reasons for disagreeing and mentioning opportunities for improvement.However, some answers mentioned in open-ended questions had already been evidenced by the close-ended topics. For instance, topic 11  classes attendance and 3) the ranking of the allocated elective established in the previous year. This means that students prefer electives with which assessment methodology they agree, followed by the electives in which students have to attend classes and which students have already identified as being the most requested in previous years  rs Table . These tWhen it comes to teaching-learning effectiveness, there is no single approach that best fits all students. Thus, there is a pressing need to adjust teaching/learning experience to each student , which jLately, academic institutions have devoted an increasing amount of time and effort in developing tools that collect students feedback about their educational satisfaction. This feedback is twofold providing both the opportunity for students to express satisfaction with their educative experience and the possibility for the organization to use and process this information for improving the teaching/learning experience .The existing literature states that the higher the satisfaction of students with their educative experience, the higher their learning engagement and, subsequently, the greater the ability to report an improvement in learning effectiveness . TherefoAgreement with teaching methodology, followed by the Agreement with assessment methodologies employed, the Perception of the workload demanded and the Requirement for continuous work throughout the semester. These results are similar even after standardization of the coefficients.In this study, through quantitative assessment, we were able to demonstrate that the major determinant of students’ global satisfaction was the These findings are in accordance with the already reported teaching and learning interference on students’ satisfaction , justifyGrade achieved in the electives neither the Number of students enrolled in the same elective were positioned as one of the main determinants, as we would expect by the causal relationship between grades and student satisfaction, and by the likelihood of large classes resulting in dissatisfaction , secondly, the electives in which students were expected to have higher attendance to classes during the semester [Classes attendance] and, lastly, the electives that had been already in their personal preference list, but which they had not been assigned previously [Ranking of the allocated elective established in the previous year]. In this context, we demonstrated a strong positive relationship between the order of preferences in two consecutive years, evidence that could additionally support us to predict what students’ preferences will be.Moreover, as there are organizational and financial restrictions that prevent universities from providing unlimited placements for each elective, developing a method that would allow predicting which electives will students select in the following years, is of extreme utility. Therefore, another important result of the present study was to help foreseeing the behavior of students when selecting their electives’ preferences. We concluded that students tended to choose electives whose assessment methodology they agreed with [Number of students, caution should be taken when interpreting these data, as we should take into account that this variable depends on the number of places available for each elective, which are determined by the coordinator of the elective, not depending on students’ selection options. Regarding our result that states that the global satisfaction increases 0.537 points as the electives are less prefered, we should consider a bias introduced by the possibility that the lower the expectations for the elective are, the higher the likelihood of the student to be positively surprised. Lastly, concerning the relationship between the attendance demanded by an elective and the students placing the same elective in their preference list, we should stress out that in the particular case of FMUP, these results might be twisted by an elective that simultaneously has the highest number of placements available and one of the highest demands on class attendance.Possible weaknesses of this quantitative analysis derive from the distinct environment in which electives are managed at FMUP. For example, regarding the inference that the less desired electives had higher classes attendance and older students, one should consider that older students are mainly student-workers, that might have other demands besides medical classes. Moreover, concerning the relations identified with the different ways in which people experience, conceptualise, perceive and understand various aspects” of the world [We complemented our quantitative analysis with a qualitative study of the open-ended question as we expected it would aid in deepening our understanding of students’ perceptions and in the generation of additional thoughts, to be used for the improvement of electives. We used phenomenography as it is recognized as a method that identifies the “he world , explorihe world . This quhe world , has beehe world . A bottohe world .With the answers to the open-ended question, students were no longer confronted with a set of expected answers, having the opportunity to further elaborate on their close-answers, and to convey their opinions regardless of what the team of investigators perceptions and beliefs of what a complete range of answer options should be. Besides, students might intend to emphasize a feature that is of greater importance to them or to further elaborate on the answers they had provided in the close-ended questions . TherefoConcerning the methodology used, phenomenography provided a way to improve the understanding of the data obtained by the close-ended answers . Three cA more objective approach for determining the adequate number of topics addressed by the open-ended questions than the bottom-up approach used for the analytical analysis of the free text generated might be desirable. However, the coherence of the derived topics from human ratings does not necessarily line up with the standard quantitative measures of model fit based on the likelihood that help to determine the adequate number of topics , which jRepresentativeness was pursued through triangulation , which rThe reference of the innovative, interesting and relevant character of electives as well as the ability to acquire transversal competences, show one of the advantages of bottom-up data analysis, which is the provision of hints about topics that are important to respondents but were not included in the close-ended questions of the survey. Moreover, even in the case of overlap, the derived topics reassured that all relevant issues had been covered up, upholding the answers to closed questions, as well as providing further information beyond the close-ended questions , 21.One limitation of this qualitative analysis is not assessing the relationship between respondents’ characteristics – gender and age – and the likelihood to answer open-ended questions, as well as the lack of testing on whether there is a relationship between global satisfaction assessed in close-ended questions and the likelihood to answer the open-ended question. Therefore, further development of the present work might be the evaluation of the relationship between answers patterns in open- and close-ended questions, allowing for the identification of which students make use of the open-ended questions and elaborating on how free texts are related to respondents’ characteristics. Moreover, pursuing a correlation analysis and developing a topic-level study should be helpful to identify topics that potentially distinguish between respondents which are more or less satisfied with electives , identifAdditionally, although studying the interference of electives in future career was not a goal of the present study, it would be interesting to follow the career choices of these students in order to understand the long-term contributions of electives experience to students’ future professional activities and choices, and maybe relate with different levels of satisfaction achieved throughout the undergraduate curriculum.Organization of electives in the medical curriculum should be done through a student-centered approach, in order to maximize educational, personal and professional development , thus, sElectives allow for a more in depth development of the syllabus, contributing to the diversity of experiences and scientific knowledge, while simultaneously instilling the responsibility on the learner to build its own educational path. This has been supported by some students’ statements describing electives as being innovative, interesting and offering relevant content, adding competencies to the core curricula and making students feel realized with their learning experience, which reinforces their relevance in medical curriculum.In order to engage students, we should aim to enhance their satisfaction and atenuate factors that create dissatisfaction. Therefore, our work heightened electives’ potential in undergraduate medical curricula by unhiding the main determinants of students’ satisfaction with electives, making it possible to improve their features in order to meet their full potential and students’ expectations, adding to the current literature clear structural recommendations for electives design that may lead to students’ satisfaction and engagement.The first step in creating a successful elective is to determine whether there is student interest in the topic. Then, the ideal course structure should be assessed . The resAdditionally, our work provides predictors of students’ electives preferences, being their first choice the electives whose assessment methodology they agreed with, followed by the electives in which students were expected to have higher attendance to classes during the semester and, lastly, the electives that they had previously selected in their preferences. In fact, our work reinforces the usefulness of analyzing the records of the students’ order of preferences from previous years, to accurately foresee the needs of the following year, besides providing the foundations to identify patterns of choices from students, which will also contribute to better meet their needs and expectations.Summing up, the present study allowed us to identify the most important determinants on students’ choices and satisfaction with electives, some of which allow us to foresee which electives will be selected in the future, which had not been done yet. It also distinguishes the features that make students prefer one specific elective over another, providing the foundation for future electives and curriculum design, at the same time that reveals opportunities to improve already existing practices with electives. Furthermore, it might even provide opportunities to improve current medical core curricula, namely by shifting current assessment methodologies and teaching and learning methodologies towards the ones most preferred by medical students.Moreover, our data strongly reinforces the need to closely monitor the implementation of electives in medical curricula as crucial dynamic elements of the medical curricula, opposing the tendency of maintaining the electives untouched in the curricula of medical schools, compromising their potential .Finally, we point out potential opportunities for improving the organization of the medical curricular reform ongoing at FMUP. These improvements includes, among others, the introduction of electives as a crucial dynamic part of the curricula, that is, to develop a comprehensive and adaptive model of medical education so that FMUP medical students become referenced professionals, equipped with the enhanced skills to better improve people’s health, through excellence in clinical practice, research, innovation and leadership.Additional file 1. Assessment of the quality of Electives in the 1st/2nd semester. The Additional File 1 represents the written evaluation survey handed once to students who attended electives when they were in the 2nd and 3rd years, in the academic year of 2015/2016.


Involving and engaging vulnerable communities from the very beginning is important if we wish to enhance general well-being. With a focus on equal partnership with low-socioeconomic status (SES) families, a Trading Shop in Vaals was developed as a community engagement initiative. In the current study, we focused on the participation process, from preparation to sustaining the Trading Shop, in order to define whether the Trading Shop can be successful in engaging families through focusing specially on their needs and perceived positive health. A formative case study design was carried out to monitor, evaluate, and timely adjust the developments within the Trading Shop by using participatory action research. The Trading Shop was monitored from its preparation to its opening, as well as during the start and the steps taken towards continuation in the form of municipal policy. The results showed one central theme during all phases: the optimal navigation between top-down support from professionals and bottom-up developments among the volunteers in the Trading Shop. With the input from both approaches, it was possible to create an optimal environment for the volunteers to achieve personal development. The inclusivity and accessibility of the Trading Shop as a community engagement initiative offered the opportunity to volunteers to enhance their needs, realizing personal growth and development of their talents in several positive health domains. This famous quote grasps the essence of community engagement. Community engagement is defined as the process of working collaboratively with and through specified groups of people (communities) affiliated by geographic proximity, special interest, or similar situations to address issues affecting their well-being . CreatinCommunity engagement practices require the finest navigation of top-down approaches, led by the expertise of professionals, as well as bottom-up approaches. The community is in charge, since they hold important local knowledge that is assumed to enhance the effectiveness and sustainability of health promotion programs . The ultIn the past years, several projects were carried out that made use of community participation activities as a key element of reaching health-promotion goals, ensuring that the needs of community members are addressed ,13,14,15Although community engagement projects showed promising results, low-SES families remain a hard-to-engage group. This became obvious when vulnerable groups were asked to participate in projects with predefined health-promotion themes . With thA formative case study design was carried out to monitor, evaluate, and timely adjust the developments within the Trading Shop through participatory action research (PAR) . Mixed-mIn 2015, individuals from low-SES families were invited to take part in a photovoice study enabling them to define their needs . In JanuThe Trading Shop is located in the Municipality of Vaals, the Netherlands. The municipality has approximately 10,000 citizens and is defined as a moderate-to-low SES region. Although the majority of citizens have a Dutch origin, the location near the border with Germany and Belgium ensures a relatively large migration towards the Municipality of Vaals. Additionally, its citizens have an unhealthier lifestyle, more mental health issues, a poorer health status, and a shorter life expectancy compared with the average Dutch population ,27. DuriWithin the Trading Shop, several activities were undertaken to support the engagement of citizens and create a flourishing “trading community”. Basically, in the Trading Shop, products and services can be traded. The trading transactions work through points earned for products and services. The transactions take place without any money changing hands, with the idea of making it accessible especially for citizens in vulnerable positions. For the number of points that members of the Trading Shop had earned, they could “buy” products or services in the Trading Shop, enabling them to gain something extra that some societal groups normally cannot afford. In addition to the trading aspect, members of the Trading Shop are welcome to drink a free cup of coffee or tea at the communal table situated in the center of the shop. The community engagement activities within the Trading Shop were intended to contribute to various social objectives, such as combating the waste of products and materials, reducing poverty, and activating societal groups to participate. To this end, the targets of the Trading Shop were based on the concept of positive health , as it sWith top-down support from professionals and local partners and bottom-up expertise of low-SES families and other citizens in vulnerable positions, the preparation, opening, starting, and sustainability phases of the Trading Shop were cocreated. Commitment from the families but, also, from broader community partners and citizens, was an important aspect to making the Trading Shop a success. A key factor in this process was the collaborative partnership on the level of practice, policy, and research. Under the supervision of three professionals closely involved from the beginning of the project , low-SES families, together with other (vulnerable) citizens and local formal and informal partners, prepared, opened, started up, and eventually, sustained the Trading Shop for at least five more years. Over time, this collaborative partnership was put in place to monitor, evaluate, and timely adjust the development of the Trading Shop before and after the opening.n = 48). Over time, a distinction became evident in a stable group of volunteers, who were retired or permanently unfit for work and wanted to stay active (n = 25) and a more unstable group of citizens receiving a social welfare benefit who needed activating for employment. For this so-called “exchange” group, the Trading Shop has about 10 places available, where it serves a societal goal as a next step towards employment. A close collaboration between the Municipal Social Service and the Trading Shop was in place to activate social welfare recipients.To start up the Trading Shop and make it sustainable following its starting phase, the recruitment of more low-SES families, other volunteers (mainly vulnerable groups), and formal and informal partners was needed. Solid professional support was put in place to support volunteers in managing the Trading Shop. First, this was done by the existing collaboration between the policy, practice, and research professionals. Later, with the focus on achieving policy support for sustaining the Trading Shop, a paid manager and an independent board took over (about one year after the opening). During the whole study period, the practice intermediary, a so-called activation broker , played To make the Trading Shop a sustainable initiative over time, and to assess whether municipal subsidies could be granted and become part of the local policy, the Trading Shop was carefully monitored and evaluated using feedback loops. Insights in the community engagement processes, its outputs, and outcomes were collected in interim reports for the municipality. These reports were used with the aim of making the Trading Shop part of the municipal policy with some financing.The data collected for this study had a mixed-methods nature, with the phases being monitored and evaluated: preparation (July 2015–16 March 2018) with opening (17 March 2018), starting (18 March 2018–July 2019), and continuation for municipal support in policy plans (an ongoing process). The quantitative data within this study concerned were retrieved from the monitoring system of the Trading Shop, such as number of Trading Shop members, their characteristics, and number of trading transactions of products and services of members. Next to providing an overview of members of the Trading Shop, this information was seen valuable to estimate how many low-SES families, defined as our main target group, were supported by the Trading Shop activities. Besides, these numbers gave an indication of the intensity with which the Trading Shop was used by members, gaining insight in how the Trading Shop engaged the community.n = 213) of the Trading Shop were carried out four times per year to explore the reasons for visiting the Trading Shop, how long and how often the shop was visited, what they liked about the shop, and if they had points for improvement  over a period of 15 months . A focusrovement . The resrovement . The sixrovement . At the All quantitative data was able to be extracted from the Trading Shop database “Cyclos” and exported into Excel sheets. These Excel sheets were then imported into SPSS so that the background characteristics of all members and volunteers of the Trading Shop could be calculated with descriptive statistics and frequencies. Information about the number of trading transactions was able to be retrieved directly from the database by predesigned application programming interfaces (APIs). Next, the qualitative data gathered from notes of observations and minutes of meetings were written down in Word files. Individual interviews and group interviews were recorded and transcribed verbatim afterwards. All data, almost 700 written pages in Word files, were organized together using NVivo software to gain insight in the process, output, and outcomes of the Trading Shop for each phase. Since an abductive process was used to analyze the data, we did not start completely inductive or deductive but worked somewhat in the middle. Indicating that our previous experiences and the current state of research in the field made us sensitive for some themes in the data—for example, the positive health domains. However, these themes were not strictly defined from the beginning, enabling us to start with a rather open view towards the data . First, n = 18) were committed to supporting the Trading Shop in terms of products or money, and sufficient numbers of volunteers (vulnerable citizens/families) (n = 30) were available to do chores in the Trading Shop in preparation for the opening. Although the deadline for the opening was set top-down, the preparatory actions developed more bottom-up, with the festive opening as a success  managed the paperwork. The researcher collected the “best practice” through observations and interviews with key stakeholders in two other existing Trading Shops. This was helpful in defining the fit that the Trading Shop would have with the targets of Vaals Meets and those of the participating families. It also provided some first-hand input for the adaptations that we needed to create a better fit. After the principle agreement from the municipality and the commitment of the location, the families and other, mainly vulnerable, volunteers were included from the start of the preparations for the festive opening of the shop. Sufficient numbers of community partners (cess See .n = 15), having different volunteer work (n = 4), and having physical and/or mental complaints hindering their work (n = 7). The main purpose for volunteering was to become or stay socially active, have something meaningful to do, and gain some work experience. For some volunteers, the provision of structure to their day/life was important, just as the possibility to learn to deal with others and have more personal contacts. Other volunteers needed to be at the Trading Shop to stay vital and active.A total of 63 volunteers contributed actively to the Trading Shop. Thirty volunteers assisted during the preparation of the Trading Shop—of whom, 12 continued volunteer work in the Trading Shop after the opening and were still active after the first 15 months. The Trading Shop has—and needs—about 35 active volunteers to keep it going. From preparation to the starting phase, 28 volunteers stopped volunteering due to multiple reasons, such as having a paid job or starting a procedure to get this job  informing each other, including sharing advice and local possibilities to, for example, getting support, (2) undertaking activities together, such as a local hiking group, and (3) providing support, such as offering transportation. We observed that these contributions mostly arose spontaneously. Points for improvements mentioned by visitors were mainly focused on practical things such as more exchanges of products in the shop, fewer intake stops on specific products, a better focus on the quality of products that is accepted, a loading and unloading place for transport nearby the shop to prevent dragging, and more extended opening hours to give employed citizens a chance to visit.Fifteen months after the festive opening, the Trading Shop had 1139 members. About 75% of all members were females. The senior group of above 66 years was the largest, with almost 40%, and the smallest group was aged up to 35 years, with just over 10%. About 15% of all members (170 members) were expected to be a poorer/low-SES family, with children under the age of eighteen. Most members lived in the Netherlands (72.4%), followed by Germany (21.4%) and Belgium (6.2%). Of all members, 60% lived in the Municipality of Vaals. A variety of 28 other Dutch municipalities were represented in the additional 12% of Dutch members. In Germany, Aachen, described as the Big Sister of the Municipality of Vaals, contributed 18.4% of all members.During the opening hours (four hours per day), the average number of visitors was 62—of whom, about 10 were low-SES families. On a busy day, the Trading Shop had 85 visitors, and the lowest number of daily visitors observed was 45. Every day, about 20 visitors brought products to the shop, and about 25 visitors “bought” one or more items. Together, members traded products and services for a total of 133,126 points in 8757 trading receipts, indicating that over 20,000 products and services were traded. For citizens in a vulnerable position who were not able to trade but would have been supported by the shop, a support fund was set up by the Trading Shop. Members/citizens could also donate products, and the points earned were included in this fund for people in need. They have already donated 15,078 points and used about 2373 points to support citizens in need and societal initiatives. Two to three new visitors became a member of the Trading Shop during every day that was observed. When visitors became members, they earned 15 points for free to immediately start trading. Of all members, 164 (14.4%) still had 15 points on their account as at July 2019, indicating that they probably visited the Trading Shop just once. The other members made use of the Trading Shop to some extent. The mean number of visitors drinking a cup of coffee at the communal table was, on average, 18 visitors per day.The Trading Shop received support from two structural partners, who provided the groceries needed for the communal table. In addition to these partners, four professionals providing support to citizens in need were available in the Trading Shop at least once a week to support members.After founding the Trading Shop, monitoring cycles were installed to timely support the management and volunteers in running the shop. Many small moments of adjustment took place in the first 15 months. These were mainly practical enhancements that were noticed by volunteers, visitors/members, the researcher, and the broker and discussed together with the daily management. Enhancements focused on reducing the number of products that a member could bring in at one time, rearranging the shop, adding product labels to the shop, obtaining more garden tools, putting additional advertisements on social media, and introducing more rotations in the cleaning scheme. During the first 12 months, these small adjustments were mainly registered by voluntary coordinators and during the interviews/observations of the researcher. All possible adaptations were discussed together with the policy-maker and activation broker before making a decision. An overarching theme in this process was the daily management of the shop, where the lack of unambiguous and correct communication among the voluntary coordinators was described as being problematic. To get the daily management on track, a monitoring cycle was put in place. In addition to all the small adaptations achieved using short feedback loops, several feedback loops had to be passed to enhance this daily management. This adaptation towards efficient and satisfactory daily management was perceived by all partners involved as being the most difficult to get on track to enhance the continuation . In the The current study aimed to define the value of a Trading Shop as a community engagement approach for low-SES inhabitants of Vaals. The process, output, and outcome of the Trading Shop were monitored from the preparation phase to the steps needed to create a more sustained municipal support, with special attention for community participation. Finally, the value of the Trading Shop was explored in terms of perceived needs and positive health. After the first 15 months, the Trading Shop had engaged 62 volunteers; it had 1139 members and an average of 62 visitors per four-hour workday. This indicates that the shop occupied an important position in the Municipality of Vaals and beyond. The results showed that the Trading Shop focused on the target population’s basic needs of meeting each other and helping each other . ActivitAlthough the bottom-up defined idea of preparing and initiating a Trading Shop occurred in the partnership activities with low-SES families, the actual preparation phase was shaped with relatively much top-down input. Some activities had to be carried out within a certain time frame, such as the paperwork but, also, keeping track of the actual organization of the chores that had to be done in the Trading Shop before the festive opening. The deadline was set very strictly by the Municipal Aldermen, indicating only six weeks to get the shop ready. During these “chore weeks”, at least one of the three professionals—the policy-maker, activation broker, or researcher—was always present in the shop to coordinate tasks and do chores. Although the professionals felt they had to push this (vulnerable) group of volunteers too hard to get things done, still, a feeling of ownership and belongingness arose among most volunteers, which became visible after the opening. We learned that, for this preparation phase, an “economic” top-down-oriented approach was successful in achieving quick results , making Following the festive opening, a focus on “community development” navigating towards a more participatory bottom-up-oriented approach was considered important to achieve community engagement goals . ProfessVarious societal objectives came together in participatory activities of the Trading Shop. These included facilitating opportunities to meet and help yourself and others, increasing and retaining independence and resilience, combating waste, and alleviating financial problems. Constant feedback loops were in place to facilitate this process, enhancing the navigation between the top-down and bottom-up inputs. In particular, social participation and activation was mentioned by volunteers as being the most important contribution of the Trading Shop to their own lives and to the lives of visitors. During interviews, the enhancements in their social network and having a sense of belongingness were said to be the most important personal additions of the Trading Shop. Volunteers called the Trading Shop a “home”, showing the importance of a strong social network . AdditioA major strength of this study was the participatory design that provided the opportunity for the researcher to develop a close collaboration with everyone involved in the Trading Shop, such as the volunteers, members, and partners. This was found helpful in gaining insight into the engagement of the community at a specific point in time, making timely adaptations possible. Second, the inclusion of the process, output, and outcome data using various quantitative and qualitative sources were helpful in creating an overview of the added value of the Trading Shop to the community. Although we had some privacy constrictions regarding gathering data about the reach of our target group as members of the Trading Shop, in conjunction with the observations, we were able to estimate the number of families supported by the Trading Shop.This study also had some limitations. First, due to the dynamic context and the open research design, it was hard to define the actual contributions of the Trading Shop to the volunteers and members. We used the positive health tool  to obtaiMany community engagement initiatives are undertaken with the aim of creating equal partnerships. More insight in these actual partnerships within the broader community are required to better understand the processes that underly successes and failures in community engagement initiatives . We encoIn practice, we encourage professionals working in community engagement activities to take steps towards collaboration and empowerment . In ordeThe Trading Shop seemed to be a promising community engagement initiative in satisfying various societal needs of low-SES families. For the vulnerable community in the present study, the Trading Shop was found to be particularly successful in facilitating opportunities for social participation and activation. A key promoting factor for the community engagement activities of the Trading Shop is the optimal navigation between top-down steering from professional coordinators and bottom-up-oriented activities from citizens. Overall, the Trading Shop can be seen as a sustainable connecting factor within the Municipality of Vaals.


The development of the modern hospital is usually dated to the nineteenth century. During this time, many municipal and sectarian hospitals were established and developed, and Jewish hospitals were no exception. Such developments also occurred in the Netherlands. This essay describes the different histories of the Jewish hospitals in Rotterdam and The Hague during the nineteenth century. The Rotterdam institution lasted for more than 130 years (until it was closed by the Nazis during the Second World War), whereas the one in The Hague existed for only 31 years. This study will suggest a number of possible explanations for the relatively long and successful history of the Jewish hospital in Rotterdam and the contrastingly brief duration of the Jewish hospital in The Hague. The nineteenth century marked the beginning of the development of the modern hospital. In the capital cities of many European countries, municipal hospitals served the needs of the poor, but there were also private, charitable institutions , usually founded by special groups, such as religious organizations, which cared for their needy ill. Jewish communities were no exception. During this time, the first Jewish hospitals were established in Austria Vienna, 1793),793,1 Eng3Throughout their history in the Netherlands, most Jews (>50%) lived in Amsterdam. The cities with the next largest Jewish population numbers were Rotterdam, one of the most important Dutch ports and the second most populous city, and The Hague, the seat of the national government. A comparison of the Jewish population trends in these three cities during the nineteenth century is shown in A brief history of the Jews in these cities is essential to understanding why the Jewish hospitals in these cities developed so differently. In the early decades of the seventeenth century, Conversos from Antwerp were the first Jewish settlers in Rotterdam, and by 1647 the city fathers granted the Portuguese Jews the freedom to practice their religion, carry out wholesale and foreign trade, and to practice medicine.4(e)Parnassim were responsible for the care of their communal poor and ensured that the poor were supported in part by voluntary charitable donations and a tax on kosher meat. In 1836, the community established the Dutch Israelite (Jewish) Poor Administration of The Hague  whose responsibilities included poor aid relief, an old age home, and an orphanage.As Parliament and the palace of the stadtholder  were situated in The Hague, the city attracted a number of wealthy Portuguese Jewish diplomats, merchants, and physicians during the first half of the seventeenth century. However, not until the end of the century did the Jewish Portuguese community formally organize and found two synagogues. These two congregations merged about 50 years later in 1743 and lasted until the Second World War. Until the beginning of the nineteenth century, this Portuguese community was quite influential due to its wealth, as well as its national (including the House of Orange), governmental, and international connections. The rich Portuguese lived in sumptuous houses in the best neighborhood.th century a Dutch hospital was not a place where one would go in order to be made well: rather it was a place to die.”12In contrast to today, hospitals in the eighteenth and early nineteenth centuries were intended exclusively for the sick poor, as all others who were afflicted by illness would request a physician to come to their home in order to be diagnosed and treated there. Hospitals were considered a “last resort” for “patients who could no longer care for themselves and whose relatives could no longer care for them extramurally” so that “In the early 19Coolsingel Ziekenhuis,Gasthuis [almshouse] was founded in 1576 and functioned not only as a hospital where the sick poor were treated but also as a poorhouse that tended to the poor and aged.)During the nineteenth century, Rotterdam experienced a 6-fold increase in its population .Poor relief was always a concern for the leadership of every Jewish community, and medical care for the indigent sick was an important aspect of the Jewish communal poor relief effort. Usually, the local Jewish community, as stated in their bylaws, would be responsible for providing (and remunerating) the services of either a physician or surgeon assigned specifically to care for the indigent patients. Though this practice continued during the nineteenth century, some Jewish patients were also treated in municipal hospitals. For example, this occurred in The Hague (see below) in 1823 and in Leeuwarden in 1865.3Gasthuis was inadequate. As early as 1798, a committee from the Jewish community requested permission from the municipal authorities to establish a Jewish hospital, but the request was denied.gasthuis, the Gesticht voor Israelitische Oude Lieden en Zieken (Institution for the Jewish Aged and Sick) was actually the second hospital that was established in Rotterdam.Parnassim. The hospital seems to have had a precarious financial existence, as it had to be totally supported by contributions of the synagogue members, who were mostly not wealthy. Not much is known about this institution, but in 1811 the administration of the institution included six directors, a secretary, and several matrons. However, financial problems constantly threatened its survival, and in 1820 the hospital went bankrupt. Within 7 years, the Jewish community rented another house Parnassim turned to the municipal authorities to approve the establishment within the municipal hospital of a separate facility to prepare ritually permitted food. In 1832, this request was denied, but the hospital was permitted to supply Jewish patients with kosher food prepared elsewhere.At the end of the eighteenth century, the Rotterdam Jewish community had increased sufficiently so that a Jewish hospital was urgently needed, as about half of the 2,500 Jews residing in the city were paupers.Mechon Hatsedek (The Abode of Righteousness) was established on the Hoogstraat, wijk 10 (district 10), No. 107, at the instigation of Mr L.S. de Sterke with the support of the Parnassim and Chief Rabbi E.J. Lowenstam.Israelitische gemeente (Jewish congregation) as well as private donations from both Jewish and non-Jewish residents.Parnassim, with the medical care managed by Dr L. Levie, physician, and Dr P.J. van Wageningen, surgeon. However, the situation changed in January 1838.This situation remained until 1837 when a new Jewish Hospital and Old Age Home, named Parnassim lost most of their power outside the synagogue, including supervision of all Jewish poor relief activities. The management of these relief activities, including medical care for indigent Jews, was assumed by the newly established (1838) Rotterdam Dutch Israelite (Jewish) Poor Administration . As a result, Messrs Ezechiels, Levisson, and van Raalte, governors of the NIA, were assigned in 1838 to manage the Mechon Hatsedek.Mechon Hatsedek became the Israelite (Jewish) Home for the Elderly and Sick. In the early decades of the nineteenth century, the national government mandated that the Dutch Jewish community be reorganized whereby the Mechon Hatsedek had to be expanded, and in 1864 a suitable opportunity presented itself whereby a house with suitable adjacent plots of land located on the Houtlaan (wijk 14), No. 728 was acquired.Mechon Hatsedek itself did not have sufficient financial resources. The building opened its doors on April 15, 1866, and a picture of this institution is shown in An increase of the number of Jews in Rotterdam  was accompanied by a substantial rise in the number of destitute Jews .Mechon Hatsedek was intended to care for the indigent Jewish sick,Mechon Hatsedek hospital during three key periods of its history: from 1840 to 1856 at the Hoogstraat location, from 1890 to 1898 at the Houtlaan site and from 1901 to 1914 at the Schietbaanlaan.Mechon Hatsedek hospital, with an entrance on the Schietbaanlaan No. 42 and a separate Jewish Old Age home located around the corner on the Claes de Vrieslaan. These institutions were inaugurated on October 22, 1900. The hospital contained a large as well as a small operating room, four large wards, and rooms for private patients.Despite the fact that part of gasthuis for the indigent ill with non-contagious diseases in The Hague dates from 1355, and it is unknown how long it survived.gasthuizen which either focused on the prevention of communicable diseases (pest- and leper-houses) or the treatment or housing of special groups such as soldiers or the mentally ill. Not until 1815 was a house on the Koediefstraat purchased to treat the sick city poor. However, it soon became apparent that this building was much too small to meet the requirements of the local needy. A much larger residence, located on the Zuidwal, with a substantial garden, was bought in 1821 and opened its doors two years later as the municipal Gasthuis. The first floor was for male patients and included an operating room and wards for surgical patients and those suffering from venereal diseases or scabies, whereas the second floor was designed for female patients.25(b)The only report of a Gasthuis was that Jewish patients were admitted right after its opening. As early as October 1823, special permission was obtained from the municipal magistrates so that a Jewish patient was allowed to receive ritually prepared food (prepared elsewhere).Gasthuis. Thus, it was agreed to let the Parnassim provide ritually prepared food to the Jewish patients, to allow Jewish representatives to be present during operations, and to permit the removal of Jewish corpses for burial in the Jewish cemetery. All these changes were codified as six added Articles to the official Regulations of the Municipal Gasthuis.Gasthuis  despite the fact that during this period (from 1830 to 1850) the number of Jews in The Hague increased from 2,268 to 3,319. In 1861, these separate Jewish wards were abolished as their space was needed for the increasing numbers of admitted (non-Jewish) patients. In the early 1860s, the hospital was treating between 700 and 900 patients.25(e)An unusual aspect of The Hague municipal Hoogduitse) and Sefardi (Portuguese) communities. However, opinions in the Jewish community regarding the need for such a hospital were quite divided. The opponents argued that the municipal Gasthuis was willing to accommodate Jewish patients  and that a Jewish hospital would represent the idea of Jewish separateness, isolation, and intolerance.f 30,000,Gasthuis in wards designated exclusively for Jewish patients, where they would always have a Jewish nurse available and where the patients would receive ritually prepared food that would be supervised by the Rabbinical authorities.25(f)The lack of separate Jewish wards seems to have been a major impetus to consider establishing a Jewish hospital in The Hague, since many indigent ill Jews reportedly preferred to be poorly cared for, even in an attic room, than to go to a municipal hospital which would not arrange for the appropriate religious ceremonies in case (s)he died.In view of the above discussion, there are several possible rationalizations for the different developments of the Jewish hospitals in Rotterdam and The Hague.Gasthuis did not adequately meet the needs, especially the religious requirements, of the destitute Jewish ill. Furthermore, the idea of a Jewish hospital in Rotterdam seemed to have preceded similar considerations made in 1802 in the Amsterdam Jewish community. From this early concern onwards, there seemed to always have been a core group of members within the Rotterdam Jewish community who created and maintained a strong tradition to ensure that their city always had a Jewish hospital. Overt opposition was not evident. As a result, very few Jews opted to use the municipal hospitals during the nineteenth century.During the eighteenth and first half of the nineteenth century, most of the Rotterdammers were poor, and the situation was even worse for the Jewish inhabitants of the city since they were banned from most trades. Hence it is not surprising that this situation, coupled with the typical insularity of most Jewish communities of the time, resulted in a community that was totally dependent on its own resources. Since caring for the sick Jewish indigent was an important religious obligation, treating them in a Jewish hospital had many advantages such as providing the patients with kosher food and religious ceremonies and being among their own people during these hard times. It seems that as early as 1798 several community members felt that there was an urgent need for a Jewish hospital. This suggests that at the end of the eighteenth century the existing municipal Parnassim.Gasthuis shortly after it opened in 1823.Gasthuis were abolished (see above), this undoubtedly changed the relations between the hospital and the Jewish community. This in turn impelled a group of individuals, including both Chief Rabbis, to try and establish a Jewish hospital in The Hague. However, there was strong opposition within the Jewish community to this suggestion. These opponents argued that such an institution was unnecessary since the municipal Gasthuis would be willing and able to meet the religious needs of the Jewish patients. Even though the Jewish hospital opened in 1873, its financial support was probably weaker than it could have been due to this intra-communal dispute; this ultimately led to the closure of The Hague Jewish hospital after only 31 years of existence.The situation in The Hague was different. It seems that the Jews in The Hague were probably more influential than their co-religionists in Rotterdam. This may have contributed to the fact that the relations between the municipal authorities and the Jewish communal leadership were quite satisfactory. For example, when the Regents of the Surgeon’s Guild objected to allowing a Jew to become an apprentice to a Guild member, the Mayor and councilors overruled these objections at the urging of the community’s Gasthuis authorities was also very important in attracting Jewish patients to this institution. Finally, the refusal of the municipal poor administration to provide medical care and free medicines to patients in the Jewish hospital also played into the hands of the opponents.30Why was there such strong support within the Jewish community for using the municipal hospital facilities for Jewish patients? First of all, the financial angle cannot be dismissed, as it was probably much less expensive to provide support for Jewish patients in an established municipal institution than to maintain an entire Jewish hospital, its staff, instruments, upkeep, etc. When it became essential to upgrade its aged facilities around the beginning of the twentieth century to meet the increasing technical demands for an up-to-date hospital, the lack of sufficient funds and receptive support doomed this institution. Second, to maintain an “all-Jewish” environment  seemed less important to a substantial part of the community (in apparent contrast to their charitable co-religionist benefactors in Rotterdam) so that they did not provide monetary support for this charitable institution. Third, it seems that the friendly attitude of both the municipal and Bikur Cholim (visiting the sick) society in The Hague. The primary function of a communal Bikur Cholim charitable association was to visit the sick  and, if necessary, to provide help in obtaining medical care. Bikur Cholim societies have been very common in nearly all Jewish communities throughout the ages.Bikur Cholim charities came from donations and legacies. In Rotterdam, a Bikur Cholim society that helped in the care of the needy ill in their homes had been reported during the eighteenth century, but very little is known about it. However, this group had ceased to function by the middle of the nineteenth century as it no longer was included in the list of Jewish charitable organizations in Rotterdam.18(d)Another group possibly opposed to establishing a Jewish hospital might have been the Bikur Cholim association in The Hague during the eighteenth century,Gasthuis.Bikur Cholim, and this would result in a greatly diminished influence of the charity within the Jewish community. It would not have been surprising if the leaders of this society may have tried to undermine the hospital project. This might explain why the communal leaders (perhaps to circumvent this presumed “opposition”) decided to grant the Bikur Cholim organization two representatives on the governing Board of the Jewish hospital.Bikur Cholim society also agreed to pay the hospital one-third of the annual patient care.There is already a mention of a Bikur Cholim society, especially in the municipal Gasthuis, and the restoration of its influence certainly would have been a very welcome development.The closure of the hospital in 1904 led to increased responsibilities for the It is noteworthy that the founding of the Jewish hospital in The Hague (though not in Rotterdam) coincided with the rapid rise in religious-based hospitals in the second half of the nineteenth century in Holland. For example, six such institutions were established in Rotterdam and two in The Hague.This history of the Jewish hospitals in Rotterdam and The Hague is a nineteenth-century cautionary tale, especially in terms of longevity. The Rotterdam Jewish hospital, without major controversy, lasted for nearly a century and a half, but dissensions within The Hague Jewish community were a major reason for the barely 30-year existence of its hospital.


Although protocols concerning the establishment and maintenance of GF mouse models have previously been reported, the establishment, maintenance and monitoring of GF rodents are labor-intensive, tedious and take experience and skills. The aim of our study was to establish a GF rat model for the following microbiota-related researches and provide an easy-to-use protocol for the establishment and maintenance of GF rat model in detail, including steps to set up the isolator, sterilize the flexible isolator bubble, import food, water and other supplies, and methods to acquire newborn GF rats, hand rearing of suckling GF rats and reproduction of GF offspring. During the hand feeding period, the body weight of suckling GF rats was weighed once a day to ascertain the amount of artificial milk was given. Based on our results, the body weight of suckling GF rats decreased 1 week after birth and then began to increase. Methods for verifying the quality of the model like assessing the sterile status of the rat colony are also described. Moreover, possible difficulties and challenges, especially during gavage, and suggestions to avoid contamination will be discussed. The protocol presented will facilitate the establishment of GF rat models and downstream microbiota-related researches.Numerous studies have demonstrated that the gut microbiota plays a vital role in human health and disease development. Although the number of studies on host–microbiota interactions have increased in recent years, the underlying pathogenesis of dysbiosis-related diseases are still largely unknown. Germ-free (GF) rodent models, with the animals housed in sterile isolators and completely free of microbiota, are useful tools to advance our understanding of host–microbiota relationship  Accumul process . The str process . Additio process , diabete process , cardiov process , inflamm process , asthma  process , and all process . HoweverExperimental models utilizing germ-free (GF) animals are invaluable tools for understanding how the gut microbiota may affect host physiology and metabolism, or for exploring interactions between the host and its microbiota . The terThe main principle behind the establishment of GF animal models is the theory that the fetus in the uterus is sterile . TherefoProtocols for establishing GF mouse models have previously been published . HoweverA scheme summarizing the methodology for the establishment of a GF rat model is shown in The GF rats should be kept in specialized isolators. A complete set of equipment for setting up the incubator was purchased from Suzhou Fengshi Laboratory Animal Equipment Co., Ltd. , including the frame, air pump, removable metal air intake and outflow chambers (two per isolator), metal connector ring, flexible PVC bubble, plastic sheet (for covering the bottom of the bubble), internal and external port cover with rubber band or plugs, and other accessories such as PVC film, polypropylene (PP) filter paper, and stainless cylinder. The protocol for the assembly of the isolator will be described below.A 2% solution of peracetic acid was used as a sterilizing agent for sterilization and maintenance of the housing environment . Because60Co-γ radiation   . Water wciences) . Beddingciences) . The ste60Co-γ radiation for sterilization. The water used to dissolve the milk powder was sterilized by autoclaving for 1 h. After sterilization, the components of the formula were imported into the isolator through the transfer port.It is recommended to freshly prepare the artificial milk before use to decrease nutrient loss. The artificial milk used during gavage was prepared by using a simplified formulation based on a previously published formula . The speSpecific-pathogen-free (SPF) pregnant Sprague-Dawley (SD) rats were purchased from Zhejiang Academy of Medical Science. All the animal experiments were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Medical college of Zhejiang University (permit number: 2017-400-3) and were performed in accordance with criteria of “Guide for the Care and Use of Laboratory Animals” (NIH publication).Preparation of the isolator entails isolator set up, sterilization and importation of previously sterilized water, food, beddings, and other supplies necessary for the rearing of the GF rats. Methods for assembling and sterilizing the isolator and how to import supplies into the isolator are shown in The first part of assembling the isolator was to install the transfer port . The traSecondly, the gloves were installed . The gloLastly, the air filter apparatus was installed . The airAfter ensuring that the isolator was air tight, that the autoclaved air intake and outflow chamber were connected to the bubble and fixed on the isolator frame and that the top air port of the air filter apparatus was sealed with vinyl tape, the bubble was sterilized . First, To facilitate work on experiments in the isolator, food, water, beddings, and other supplies should be imported into the isolator bubble in advance. As shown in Unlike water and food, supplies were imported through the sterilizing cylinder into the isolator bubble through a plastic sleeve. Firstly, the sterilizing cylinder and transfer port were connected with a plastic sleeve, the vinyl tape was used to seal it tightly, the sterilizing agent was sprayed through the port on the sleeve, then the port was blocked with plugs and the plugs were sealed with vinyl tape. A total of 2 h after sterilization, the rubber band was taken off, the internal port cover was opened, the PVC film was punctured , the beddings and other supplies in the cylinder were taken out and transferred into the isolator bubble. Lastly, the internal port cover was closed and strengthened with the rubber band, the vinyl tape was torn off, the cylinder and sleeve were removed, then the external port cover was closed and the transfer port was sterilized as described above.To prevent contamination, every time the external port cover was open, the internal port cover should be closed, and the transfer port should be sterilized with the sterilizing agent. Additionally, the empty bottles and dirty beddings should be transferred out through the transfer port from time to time to keep the interior of the isolator bubble orderly.in utero  must be kept in the sterile isolator to avoid contamination. Since they were not capable of sucking milk by themselves, the newborn GF rats were hand fed by gavage until weaning. Due to the fragility of neonates, the metallic gavage needle was inappropriate. A micro medical tubing  was connected to the needle and then to the 1 mL syringe with calibration , which wThe rat’s head was held, and the rat’s body was kept upright, then the tube was inserted slowly through the mouth into the stomach, and the artificial milk was injected. In general, the suckling GF rats were fed every 4 h, six times a day, and the equal dose of milk was given each time. The amount of milk they drink in one day is ∼20% of its body weight . To know what amount of milk to give to the rats, the rats were weighed under fast once a day.During gavage, made sure that there was no air flow into the stomach of the rat. The times of gavage and the amount of milk fed to the rat were increased or decreased according to the digestive status of rat. Since suckling rats are hairless, it is possible to discern whether there is milk in the stomach or not by visible inspection. Moreover, it is important to stimulate suckling rats to defecate and urinate by using a cotton swab. The temperature and humidity in the isolator during the feeding process should be kept at 31–33°C and 50–65%, respectively. When the suckling rats’ eyes were completely open and their teeth were long enough, the gavage was stopped and the rats were instead given soft food soaked in milk to train their ability to eat by themselves. According to the different growth status of the suckling GF rats in each experiment, at which day to stop hand-rearing was different. In our experiments, the suckling GF rats began to eat soft food at day 22 in test 1, at day 19 in test 2, and at day 18 in test 3. When the rats could drink water by themselves, they were weaned and instead supplied with normal, solid food and water. Additionally, the temperature in the isolator was adjusted to about 26°C.As mentioned above, the weaning GF rats should be kept in the sterilized isolator at all times. When the rats were old enough to eat and drink by themselves, sterilized food and water needs to be regularly provided, and the dirty beddings need to be replaced to maintain an orderly environment in the isolator. Male and female rats should be caged separately until they were old enough and ready to reproduce. In general, SD rats about 90 days old were of a suitable age to reproduce. To facilitate reproduction, put the male and female rat in the same cage and separate them when the female rat was certain to be pregnant. Abdominal palpation at about 14 days after mating was used to judge whether the rat was pregnant or not. After approximately 21 days of gestation, the pregnant female rat will give birth to GF neonates. When the newborn GF rats grow up and can reproduce, the population of GF rats can remain stable, the GF rat colony has been successfully established.Additionally, the female rat which has just given birth can be used as a GF foster mother to nurse the new strains of newborn GF rats  until they can eat and drink by themselves. As the new strain of newborn GF rats grew up and can reproduce, the new strain of GF rat colony was successfully rederivated. It should be noted that foster nursing can only be applied to closely related strains of rats.Assessment of the GF status is an essential aspect in establishing and maintaining a GF rat model. Indeed, cecum enlargement is the most striking anomaly in GF rodents. This phenomenon is due to the accumulation of mucus, a mixture of macromolecular, sulfate-containing glycoproteins that normally are degraded by the microflora of the lower gut . HoweverThree typical methods for assessing the GF status were used for validation in the current study and are described in detail below. They consist of cultivation of stool samples under aerobic and anaerobic conditions, Gram staining of fecal specimen followed by light microscopy and PCR-based detection using universal primers targeting the bacterial 16S rRNA genes . HoweverCulturing is the most used method to detect whether there is fecal contamination of bacteria or not. The culture medium and specific culturing method were selected according to earlier studies . Fecal pBoth the results of the cultivation in broth and on agar should be evaluated. It is recommended to do these tests weekly. Importantly, not all bacteria are cultivable by these culturing methods, and so negative results does not conclusively prove that the samples are free from contamination. As such, it is important to complement cultivation methods with other methods to ascertain the sterility of the GF rat model.Staining-based methods are convenient methods used to detect the presence of bacteria in samples. In addition to bacteria detectable by culturing, staining-based detection methods can also detect non-culturable bacteria. In contrast to culturing, staining methods also give information on size and morphology of the contaminant. Although various single step staining methods are available , Gram staining is a particularly useful staining method since it additionally can provide broad bacterial typing information  on detected contaminants.Gram staining was performed according to a previously published protocol . In shorThe bacterial 16S rRNA gene contains regions conserved through most eubacterial species. By using primers complementary and specific to these regions, bacteria can be detected with high sensitivity and specificity with PCR-based methodology. In the current study, DNA was extracted from the fecal samples by using a fecal DNA extraction kit . A PCR reaction mixture consisting of the extracted DNA as template, dNTPs and DNA polymerase  in a PCR reaction buffer with bacterial universal primers  targeting the 16S rRNA gene , was putIn addition to the template DNA, a positive control using DNA extracted from SPF rat feces as template DNA and a no template control using distilled water instead of template DNA were run simultaneously to the PCR for the GF rat feces template DNA to ensure the quality of the results. The PCR products were subsequently analyzed by using agarose (1%) gel electrophoresis. A notable shortcoming of this method is, however, that PCR will detect DNA from dead bacterial cells in addition to DNA from living cells, which can yield false positive results.P < 0.05, P < 0.05, respectively), whereas there is no significant differences between Test 1 and Test 2 (P > 0.05). After the hand-feeding period, a total of 18 GF rats survived, nine of which were male and female rats. The body weight of weaned GF rats in Test 1 has significant differences compared to Test 2 and Test 3 , whereas there is no significant differences between Test 2 and Test 3 (P > 0.05)  were obtained; in Test 2, 11 GF neonates  were obtained; and in Test 3, 10 GF neonates  were acquired . The bod > 0.05) .Due to the time it needed to prepare sterile isolator bubble, food, water and other supplies, and about a month of artificial feeding cycle, we did the experiment almost once in 45 days. The results of three of the experiments  are shown below to illustrate the growth status of GF rats . Our resAfter weaning, the adolescent GF rats were given sterile food and water. Their body weight and shape were like normal rats. At about age 90 day, the female and male rats were caged together to facilitate reproduction. The offspring rats feeding naturally from their biological mother grew better than the rats hand fed through gavage. This results is consistent with previous reports . The difTo ensure that immediate action can be taken in case of contamination, regular monitoring of the environment in the isolator is necessary and sterility tests were done weekly. Culturing, Gram staining and PCR were used to test the GF status of the rats. The fecal samples of the newborn rats acquired by hysterectomy were the first samples to be tested. After that, the stools of rats were collected once a week to assess the sterility. The BHI, FT, and TSB broths supplemented with fecal samples showed no visible growth of bacteria after 7 days of incubation, and the blood agar plates showed no growth of colonies after 2 days of cultivation. No visible bacterial cells could be discerned in the Gram stained preparation of the fecal sample under a light microscope. The PCR assay could not detect any 16S rRNA genes in the extracted DNA from the fecal samples as indicated by the absence of bands in the gel electrophoresis.Germ-free rats are attractive for research use because they do not carry any microbes in their guts, therefore, well-suited to study host–microbial interactions in health and disease. Comparison of GF and conventional animals and colonizing of GF animals with commensal bacteria are the most effective methods to directly and indirectly prove correlations between microbiota and disease phenotypes . HoweverAcquisition of GF rats entail generation of newborn GF rats, hand-rearing to acquire adolescent GF rats and subsequent GF offspring rats. The initial delivery of newborn GF rats through sterile cesarean section should be carefully performed to avoid contamination. In addition, the dogma that the fetus is sterile in the uterus, which is the basic principle in the establishment of GF animal models, has been challenged. Studies from the recent decade have indicated that diverse microbial communities may exist in human semen and in the womb . And yetHand-rearing of suckling GF rats is another challenging and critical step. Successful rearing is influenced by various factors, including the body weight of newborn GF rats, proficiency in gastric gavage and changes in temperature and humidity of the environment. During the periods in which artificial feeding is necessary, the neonates are small and fragile, and care should be taken during gavage to avoid inserting the gavage needle into the trachea. Furthermore, the pups have low immunity and the lack of microbes in their gut complicate digestion  of milk.Regarding adult rats, the low nutrition of repeatedly sterilized food and poor nutrient absorption of GF rats may contribute to infertility. For irradiation sterilization, we recommend the use of food with higher nutritional value.After successful acquisition of GF rats, maintaining a sterile environment is a continuous challenge. Repeated importation of food, water and supplies, transfer of rats between different isolators and overall isolator management may lead to microbial contamination. Unlike many other forms of experiments, any mistake risks contaminating the entire experimental model, necessitating expensive and time-consuming re-establishment of a sterile GF rat colony. Hence, every step in establishing the model and all work performed in the isolator should be carefully performed and quality control is essential to carry out often. Furthermore, we recommend to regularly sterilize the isolator bubble and to replace the filter paper of the air filter apparatus and sterilizing cylinder. Additionally, the gloves are the most vulnerable parts, which should be inspected for any damage before each use to prevent possible contamination and changed before it is too late.As mentioned above, quality control of the GF status is essential; however, ascertaining the sterile status of the GI tract is difficult. The currently used methods to assess the quality of GF animals, aerobic and anaerobic culturing of fresh fecal samples, microscopic examination of stained fecal sample preparations and PCR methods based on the 16S rRNA gene  all haveRat and mouse belong to the rodent and they are very similar in many ways. In general, protocols for establishment of GF rat colony are applicable to establish the GF mouse colony. Methods for assembling and sterilizing isolator, importing supplies, performing the hysterectomy, hand feeding the neonates by gavage and assessing the microbiological status by culture, Gram-staining and PCR based on 16S rRNA gene are the same. However, it should be noted that during the hand feeding period, the amount of artificial milk which was given to the suckling GF mouse is less than the rat due to their difference in the body size. The depth of the insertion of the gavage needle is different as well. Moreover, the day of giving soft food and weaning are different too. According to our experience, since mouse are much smaller than the rat, hand feeding of the GF suckling mouse by gavage might be more difficult than the suckling GF rat. Proficiency in gavage operation of experimenters, environmental conditions and the status of the neonates are the main factors to determine whether the experiment is successful or not. Therefore, the survival rate of weaned GF mouse is difficult to predict. Moreover, there is no need to worry too much about the cost of this experiment, the isolator bubble is reusable and other reagents are commonly used.We present an easy-to-use protocol for the establishment and maintenance of a GF rat model in an isolated environment. The validity of this protocol was confirmed by evaluation of bacterial contamination testing using three different methods. The most challenging parts in establishing and maintaining a GF rat model is, in our experience, the handfeeding of suckling rats until weaning and maintaining a GF environment. Repeated assessment of the GF status of the model is essential and improved methods for detecting contamination with increased sensitivity and accuracy are needed. GF rat models are ideal for investigating the interaction between the host and its microbiota relating to disease and the protocol presented herein will facilitate the establishment of experimental models aimed for microbiota-related research.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Laboratory Animal Ethics Committee of the First Affiliated Hospital of Medical College of Zhejiang University.LQ, SM, YL, and JZ performed all the experiments together. LQ and ZY designed the manuscript. LQ drafted the manuscript and prepared all the figures and tables. LQ, ZY, MY, and LL revised the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.