Many challenges emerged during completion of a study to examine radiation dose and acute leukemia among children in areas of the former Soviet Union. In an era of globalization, our experiences might benefit others involved in multinational investigations. This paper identifies the major challenges faced and the lessons learned in addressing them by the collaborative research groups involved in developing and conducting a large, multi-national case-control study of acute leukemia among children in areas of the former Soviet Union (FSU) that were most heavily exposed to radioactive fallout as a result of the April 1986 accident in reactor vessel #4 of the Chernobyl Nuclear Power Plant.131I), cesium , and strontium (90Sr), were released from the damaged reactor vessel contaminating soil, vegetation, and groundwater [In this accident, a variety of radioisotopes including iodine 31I, cesiAcute external exposures to ionizing radiation have been etiologically linked with observed increases in the risk of all types of leukemia, except chronic lymphocytic leukemia; the risk is greatest for acute myeloid leukemia -12. The Collaborative studies are challenging at best; international consortia efforts are even more so. The study of border-crossing disasters such as that in Chernobyl need be investigated as a single scientific challenge to take advantage of standardizing the investigative process across national boundaries, and to afford adequate sample size and the associated statistical power to reach meaningful conclusions. This paper will not recount the events of the Chernobyl disaster or specific results of the investigation. Rather, it will share the lessons learned while conducting a collaborative, multinational study for the benefit of other investigators.One of our first challenges was to establish an understanding of and a presence in each of the affected areas of the FSU. Multinational/multi-site studies are labor intensive and difficult to coordinate from distant locations. Therefore, the identification of a strong, on-site research team is essential. Each group must be treated as a full-member of the larger study team and be fully engaged. The local team is primarily responsible for study implementation. It is imperative that local teams with relevant expertise be formed. Our teams employed physicians, epidemiologists, statisticians, and dosimetrists. Local teams participated in study design, as well as study management in the field and troubleshooting problems. This required teams that were extremely knowledgeable about geopolitical boundaries, location of cities/small villages, governmental structures, and the health care delivery system. The latter area was particularly important given that the entire study hinged on the identification of acute leukemias and pair matched healthy controls. The health care systems in Belarus, the Russian Federation, and Ukraine have been adapted from the FSU system. Each Oblast  has 1–2, and in rare cases 4, large cancer treatment and diagnostic facilities (termed oncodispensaries). There are separate facilities for childhood  and adult patients. In addition to providing nearly all cancer-related medical treatment, oncodispensaries house records of all reported cancer patients within the Oblast. These treatment centers contained critical source records vital to the success of our study.On-site research teams knew how to identify cases , where to identify controls , and what agency permission was needed to obtain access to these data. This would have been impossible for an "outsider" to do. The identification of committed and skilled collaborators from each of the Republics facilitated project coordination and monitoring from outside the FSU. However, not surprisingly, there have been some difficulties related to language and culture. Although English language proficiency is progressing rapidly within the FSU, many of the individual researchers were unable to effectively communicate via English. Moreover, the collaborators based in the USA were even more limited in their Russian language fluency. This issue of language was addressed early in this collaboration through a consensus that English would be used as the common language, which was endorsed by our FSU collaborators. Certain investigators were well trained in English and the Consortium relied on simultaneous interpretation for initial meetings of collaborators, and then switched to having an interpreter present to provide general and personal assistance as needed. All printed materials, such as study protocols and survey instruments were produced in dual language versions (Russian and English) and verified via back translation.Since the late 1990s, key project staff members in the USA and each FSU Republic maintained frequent linkages through e-mail communications at intervals between site visits and periodic project reviews. However, it should be noted that prior to the late 1990s, e-mail was scarce in the FSU and somewhat unreliable. International communications were first established via facsimile transmissions then evolved to an electronic platform (e-mail) as Internet coverage in the FSU expanded.This study was supported by the International Consortium for Research on the Health Effects of Radiation (ICRHER), based in the United States.  Our reThe ICRHER was incorporated in June 1993. It was born of the insight and enthusiasm of one man, the late Admiral Elmo Zumwalt, Jr., United State Navy (retired), who was concerned about environmental exposures and cancer risk; particularly, how such exposures might affect military personnel. Admiral Zumwalt was also concerned about the humanitarian aspects of accidental exposure to toxic agents, including ionizing radiation. The Chernobyl reactor accident provided an opportunity to pursue both these interests.Initially, the ICRHER (the "Consortium") was comprised of collaborative groups at institutions in the United States of America (USA) and FSU republics, specifically, the Baylor College of Medicine, Houston, Texas; the Research Center for Radiation Medicine, Kiev, Ukraine; the Fred Hutchinson Cancer Research Center (FHCRC), Seattle, Washington; the National Center for Hematology, Moscow, Russia; Hadassah Medical Organization, Jerusalem and Haifa, Israel; and the National Marrow Donor Program, Minneapolis, Minnesota. The Bridgeport Hospital/Yale University and the Research Institute of Radiation Medicine and Endocrinology, Minsk, Belarus, joined the Consortium later. The final research team for the multinational Leukemia case-control study includes the FHCRC in collaboration with the Medical Radiological Research Center in Russia and two groups from the Roswell Park Cancer Institute .The Consortium helped to bring structure and organization in a variety of areas, from assembling the principal investigators to creating working groups to empanelling external advisory boards. A corporate entity or central office was formed to coordinate these activities, and to manage common logistic requirements, to draw up contracts for U.S. and Israeli institutions, to support the FSU collaborators, and to provide an administrative office of record. Satellite offices were established in Kiev, Minsk, Moscow, Obninsk, Bryansk and Jerusalem to provide administrative, communication and logistical support to the research activities in the FSU and Israel. FSU offices, as will be seen later, were critical to the success of this study.Given the changes in leadership and make up of the ICRHER since its inception, it was imperative to maintain flexibility and responsiveness to change. Modifications in research partners may occur for many reasons. Therefore, it is critical that some type of "corporate entity" exist to aid in these transitions through detailed documentation and to provide continuity at the executive/advisory level.Consortium investigators developed a pilot study to test the feasibility of initiating a full-scale epidemiologic investigation, and to determine whether needed collaborations could be established. The primary goals were to assess radiation dose, to identify individuals at risk for radiation-related illness (including émigrés to Israel), to develop data collection instruments and common study protocols, to elucidate mechanisms of radiation damage, and to establish core support through the central office. The original group invested considerable time in reviewing exposure data, the populations involved, and possible health outcomes. Following these critical discussions, a decision was made to launch a pilot investigation of acute leukemia. This study would prove challenging, but it was facilitated by the knowledge and experiences gleaned from the feasibility study.Based on the feasibility and pilot studies, a consensus decision was made to begin a multinational leukemia case-control study in Belarus, the Russian Federation, and the Ukraine, including the Israeli component (the Israeli studies concluded in August 2000). The focus has been on the most radiosensitive malignancy in the most radiosensitive at-risk age group: occurrence of acute leukemia in those 0 to <6 years at the time of the accident .Initially, the aim of the Consortium was to investigate the occurrence of acute leukemia among individuals who were 0 to age 20 years at the time of the Chernobyl accident. The original population was selected due to early indications of high radiation dose exposures and easily achievable sample size estimates based on power calculations. When doses were not found to be as high as predicted, the sample size requirements became exceedingly large. Therefore, the researchers decided to narrow their focus to individuals aged 0 to <6 years at the time of the accident, since they represented a much more radiosensitive subpopulation. Hence, there is the need for well-designed feasibility studies to provide critical information in study planning and decision-making.in utero to <6 years of age at the time of the Chernobyl accident in April 1986.The final study population included 421 confirmed cases of acute leukemia and 842 population controls pair-matched to cases on age, gender, and type of residence. All participants were Case identification was often difficult, particularly when compared to studies conducted in the U.S. that typically rely on either population-based cancer registries or hospital-based rapid case identification. Many regions studied did not have an up-to-date, population-based cancer registry. And, since nearly all medical records in the FSU exist in paper form, exhaustive manual reviews of hard copy records were often required. This task was somewhat simplified by the local custom of referring cancer cases to regional oncodispensaries, thereby, restricting record reviews to a finite number of facilities. Moreover, it was also necessary to review death files to ascertain cases not previously brought to medical attention. Trained physicians reviewed the records at the oncodispensaries in Belarus, the Russian Federation, and the Ukraine. Records were retrieved for all potential patients . After verifying eligibility, trained interviewers contacted parents of these patients and scheduled an interview.Control selection was equally vexing. In the US, researchers typically use neighborhood controls, random digit telephone dialing methods, and/or computerized databases -16. In tThe research teams visited the polyclinic and randomly selected 20 potential controls fitting the inclusion criteria. Interviews were generally scheduled and completed for the first two potential controls on the list. Other names on the list were contacted and scheduled for interviews as needed. Over 90% of potential controls contacted by the research teams agreed to participate. These high rates of case and control participation may be attributed to the dedication and resourcefulness and commitment of the research teams and the use of modest incentives (food baskets).As formidable as the above tasks were, they paled in comparison to actual data collection. During the feasibility study, investigator meetings were held in the US to develop common data collection instruments. These instruments were structured to address issues of cultural and language compatibility for use in the three Republics. While Russian was adopted as the standard language for data collection, study instruments contained both Russian and English text. Instruments were back translated into English to ensure accuracy of translation.Next, a team of interviewers needed to be recruited and trained. We relied almost exclusively on recruiting physicians who were familiar with the disease process, were credible representatives, were respected by study participants, and who could be depended on to provide accurate and verifiable data. The mix of urban areas and rural settlements presented logistical challenges in terms of tracking participants, arranging appointments, and completing face-to-face interviews. To overcome this challenge, some interviewers mailed letters to introduce the project, and to request that the participant contact the interviewer. Others traveled to local communities to personally discuss participation and to schedule interviews. Still others used local contacts to identify participant places of employment for either telephone or direct contact.Traveling to interview study participants was another challenge. In contrast to conducting research in the US where there are widespread and reliable telecommunication systems, well-developed highway systems, and accurate maps, FSU travel was more problematic. For example, not all roads to small villages in the FSU are paved and most are limited to a single lane in either direction. Roads can be particularly treacherous in the winter and rainy seasons. Maps do not always show the precise location of small villages. Villages have neither street signs nor house numbers. Further, the collapse of the Soviet Union resulted in changes in the names of many streets, settlements, and villages. This arduous process of locating and interviewing controls took considerable effort and time. Where interviewers in the U.S. are generally out and back in the same day, FSU colleagues were out in the field for days at a time and would be fortunate if they could complete a handful of interviews each day. These differences in operations must also be factored in to the overall cost of the study. Finally, local residents tended to be wary of strangers and special introductions were often necessary to gain entry.One cannot overstate the importance of having a well-trained and dedicated on-site research team that is familiar with cultural norms and local "maps". The two-hour, face-to-face interviews were generally completed with the mothers of cases and controls. Interview items were developed by US and FSU scientists and included sections addressing demographics, general health status, maternal and paternal occupational history, and a detailed questionnaire for obtaining information necessary for developing estimates of individualized internal and external radiation exposures, including questions regarding consumption of locally produced milk, meats, and vegetables; residence history and type of housing structure; use of protective measures immediately after the accident; and time spent outdoors.Those involved in multinational or multi-site investigations may consider the creation of Working Groups to monitor the various components of the study. As has been utilized in other international studies , the leaA common methodology to assess individual absorbed radiation doses and corresponding uncertainties was developed and tested by the dosimetry intercomparison working group (DIWG) for all subjects based on interview data and available exposure data. The interview collected detailed exposure data for each subject from the time of the Chernobyl accident until the reference date. It should be noted that interview data alone were not sufficient to determine individual dose estimations. For retrospective dose estimation specialized radioecological data were necessary. These resources provided information concerning local soil types, food contamination with different radionuclides, dates of radioactive cloud arrivals to each local community, etcetera. These data were collected by the members of DIWG based on information published within the FSU.Dosimetrists are a good example of the specialized personnel that need to be a full-time, on-site presence. They have critical knowledge of the local area as well as access to primary data essential in quantifying exposure. Individual dose estimates reflected local conditions  at each location where a particular subject lived during the appropriate time interval. The subject's residence history and other important personal information, such as milk consumption and food sources, were collected during the standardized interviews by trained examiners. Fieldwork was performed jointly by physicians and dosimetrists.A Data Analysis Working Group was essential for oversight of the analytic phase. Composition included representatives from each site with a strong chair to maintain focus and momentum. Also, a Data Audit Working Group was established at study inception to conduct periodic reviews to assure adherence to study protocols. And finally, a centralized Data Coordination Office served as a repository of the common dataset and oversaw periodic computerized checks for quality and completeness.Our experiences in the organization and successful implementation of a multinational, retrospective study of acute leukemia in regions impacted by the Chernobyl disaster have been highlighted. Issues identified during the implementation of our multi-national epidemiology study, along with strategies for resolution are summarized in table The breakup of the former Soviet Union in 1991 created national autonomy in Belarus, the Russian Federation, and Ukraine. Although the study area for this project included selected regions of the FSU, the intent was to study acute leukemia without regard to specific national boundaries, while recognizing the requirement to bring together investigators from these three Republics in a common effort. Proprietary concerns and country-specific restrictions on the sharing of scientific information were thoroughly addressed to gain agreement and to facilitate the pooling of analytic information.The Consortium played an integral role in providing infrastructure support for this project through the appointment of project support administrators at each research site to oversee communications, equipment procurement/maintenance, and compensation. The fiscal aspects of supporting research are unique to each country. A careful examination of collaborating scientific institutions and the financial regulations of each participating country prior to setting up any support mechanisms is critical, and may result in country-specific arrangements. Essential computer, laboratory, and communication equipment was supplied. Computer software, which was compatible across the three research settings, was installed and upgraded periodically. Equipment was segregated and secured to insure exclusive use by project staff.It should be emphasized that this investigation represents the largest retrospective study examining the relationship between Chernobyl radiation exposures and risk of acute leukemia conducted to date and the only research effort to bring together data from the most exposed areas into a single study; results of the multinational case-control study are presented in a separate paper . The conFSU, former Soviet UnionUSSR, Union of Soviet Socialist RepublicsUSA, United States of AmericaICRHER, International Consortium for Research on the Health Effects of Radiation; the "Consortium"FHCRC, Fred Hutchinson Cancer Research CenterDIWG, dosimetry intercomparison working groupThe author(s) declare that they have no competing interests.MCM and AMM were responsible for the study concept. MCM and AMM drafted the manuscript; KMM, PLM, RCM, VFS and RWD provided critical review and input. MCM, KMM, PLM, RCM, VFS, RWD and AMM participated in interpretation, as well as in data acquisition efforts. All authors read and approved this manuscript.The pre-publication history for this paper can be accessed here:


Global health research is essential for development. A major issue is the inequitable distribution of research efforts and funds directed towards populations suffering the world's greatest health problems. This imbalance is fostering major attempts at redirecting research to the health problems of low and middle income countries. Following the creation of the Coalition for Global Health Research – Canada (CGHRC) in 2001, the Canadian Society for International Health (CSIH) decided to review the role of non-governmental organizations (NGOs) in global health research. This paper highlights some of the prevalent thinking and is intended to encourage new thinking on how NGOs can further this role.This paper was prepared by members of the Research Committee of the CSIH, with input from other members of the Society. Persons working in various international NGOs participated in individual interviews or group discussions on their involvement in different types of research activities. Case studies illustrate the roles of NGOs in global health research, their perceived strengths and weaknesses, and the constraints and opportunities to build capacity and develop partnerships for research.NGOs are contributing at all stages of the research cycle, fostering the relevance and effectiveness of the research, priority setting, and knowledge translation to action. They have a key role in stewardship , resource mobilization for research, the generation, utilization and management of knowledge, and capacity development. Yet, typically, the involvement of NGOs in research is downstream from knowledge production and it usually takes the form of a partnership with universities or dedicated research agencies.There is a need to more effectively include NGOs in all aspects of health research in order to maximize the potential benefits of research. NGOs, moreover, can and should play an instrumental role in coalitions for global health research, such as the CGHRC. With a renewed sense of purpose and a common goal, NGOs and their partners intend to make strong and lasting inroads into reducing the disease burden of the world's most affected populations through effective research action. Each country needs to be able to generate knowledge relevant to its own situation, to allow it to determine its particular health problems, appraise the measures available for dealing with them, and choose the actions likely to produce the greatest improvement in health. This should not be seen as the exclusive preserve of universities or research councils, but equally of health/public services, non-governmental organizations, etc." ) and for the development of tools to assess the quality of nutrition interventions leading to adoption of relevant strategies . HKI has in-house expertise in research. There are several full-time research positions. In addition, it works with research partners at the local level, as well as with universities in Canada and USA.Forum Afrique Canada , for instance, it is studying Canadian government trade and aid policy after G-8. It has in-house research expertise, particularly in sociology, although there is no research position as such. It usually works with partners, as it is a small NGO. Inter Pares uses research findings mainly for education and advocacy.Inter Pares was created in 1975 to support NGOs from the South and to provide international development education in Canada. Inter Pares uses its own funds to conduct social research on political and economic issues, primarily action research. For instance, it carried out collaborative research with NGOs in the Philippines and of Bangladesh on family planning policy, and in Africa it has carried out research on economic issues. With AMREF has been active since 1957 in the field of applied health research and has an extensive bibliography documenting research results in the form of peer-reviewed publications, theses, manuals, reports, abstracts and conference presentations. The focus of AMREF's research activities has been primarily in the operational and applied domains. Many have addressed the important disease burden caused by communicable diseases such as malaria , an NGO affiliated with WHO, specifically provides support for research among its numerous activities. In particular, it supports the research work of the 10/66 Dementia Research Group . The viMédecins sans Frontières (MSF) was the first NGO to both provide emergency medical assistance and publicly bear witness to the plight of the populations they served. MSF is at the forefront of emergency health care as well as care for populations suffering from endemic diseases and neglect. MSF has undertaken an initiative on drugs for neglected infectious disease which combines advocacy, research and capacity development, and networking. In contrast with private sector research, it is need-driven rather than profit-driven. Five pilot projects are currently underway focusing on capacity building and technology transfer. This initiative started with a review of pharmaceutical research and development outcomes over the last 25 years and of current private and public initiatives. Highlights of the findings and conclusions, published in Lancet , are proResearch for International Tobacco Control (RITC) is an International Secretariat based at IDRC headquarters (Ottawa) that funds multidisciplinary tobacco control research projects in developing countries. Its mission is to create a strong research, funding and knowledge base for the development of effective tobacco control policies and programs, through a combination of research, dissemination, strengthening of capacity and coordination. RITC concentrates on research on psycho-social correlates of tobacco use. It provides support to research projects conducted by NGOs, such as, the Youth and Tobacco Survey conducted in Russia by the Russian Public Health Association, with the technical assistance of the CPHA table .CPHA has also provided technical and financial support through its various international initiatives funded by CIDA to its public health association partners to carry out of the GYTS in Burkina Faso, Niger, Haiti, and Cuba; and in partnership with Institutes of Public Health in Bosnia & Herzegovina, Serbia & Montenegro, and in the UN-administered province of Kosovo. The results from the surveys (carried out in collaboration with the Centers for Disease Control and Prevention [CDC] of USA) are being used to develop tobacco control policy and youth-focused smoking prevention and cessation programs.The experience of CSIH in global health research is described under 5.2.1.The following summary of perceived NGO strengths (and weaknesses) in health research is based primarily on the data from individual and group interviews that were specifically conducted as inputs to this discussion paper.NGOs give a human face to research, and they are in a good position also to build on indigenous capacity" . Additionally, they may be more compelled to complete the research cycle and apply the findings. Their involvement in research is perceived as a motivation to use the research to design, develop and respond to circumstances affecting development. Evaluation research, in which they are frequently involved, tells them whether or not they have an impact.It is a common view that NGOs are in a good position to participate in health research because of their knowledge of, and their presence in, local communities. Furthermore, their involvement increases the relevance of research to communities. "Since they are closely connected with communities, they have the ability to see the application of their research results. For this reason, NGO-initiated research is often more likely to be translated into practice in a timely manner as it is almost always directly related to practice. The NGO structure brings concreteness and a style which is guided by values and beliefs with an action orientation.Another major strength of some NGOs is their international networks which give them access to technical information and support. Finally, NGO values of ethics, solidarity and dialogue are important for health research to contribute to reducing inequities and for empowerment.Many reasons that account for the reluctance of NGOs to become (more) involved in research activities pertain to NGO perceptions on research . In the past, research was an academia-driven and based, elitist and theoretical exercise, the results of which are of little use to NGOs and the communities with which they work. Traditional research strategies and approaches were seen as top-down, non-participative and controlled by external actors. Research activities were regarded as requiring special technical expertise, much time and effort, access to professional journals and research literature, and substantial human and financial resources, characteristics not typically found in NGOs. Finally, the scientific rigor demanded by researchers was believed to be difficult to achieve in field-based situations, where unpredictability and subjectivity are the norm.What NGOs perceive as research and as their role in this respect varies widely. For instance, there is some hesitation and even reluctance in including baseline studies or project evaluation under 'research'.Donors do not want to hear that NGOs are doing research as they are implementation organizations" .Another obstacle is the fact that some NGOs that raise funds from the public are afraid to go against the expectations of the donors if the money is reallocated for research, and particularly for policy research in Canada: "Among other barriers, interview respondents mentioned lack of training opportunities, lack of funding owing to their (limited) mandate, priorities of funding agencies, and time constraints.Because of lack of training or of specialized researchers, NGOs may not be in a position to conduct top quality research, and scientific rigor may be lacking in certain instances. Lack of access to scientific literature when in the field can also be a major shortfall.It is often difficult to secure research funding from certain donors. Many Canadian NGOs rely heavily on negotiated contracts with CIDA, which leaves little time and place for research. However, CIDA does fund some research (in a broad sense), particularly formative and summative evaluations. These are encouraging trends, but the aim should be for bilateral agencies to openly fund some research, like in the UK and some Scandinavian countries.NGOs do not have a research mandate, and therefore we do not foresee developing research expertise in-house. Linking for instance with universities is feasible for development-driven research" . NGOs may have to change their structures and priorities in order to support autonomous research.Lack of interest or of a clear view of the whole research process can also be considered as impediments. As several NGOs do not see research as part of their mandate, they may not be willing to get involved in research: "Because NGO research is often conducted on a small scale and is usually of a qualitative nature, it often goes unrecognized by governments, and even by research organizations and funding agencies, which tend to favour large scale quantitative research.NGOs interested in pursuing a research profile require a type of mentorship in terms of standard performance indicators in the research domain. For example, publication has traditionally not been a strength and much NGO research does not reach beyond the gray literature or report level. Because scientific publications are an important means of transfer or dissemination of research results, NGO capacity to publish their findings needs to be strengthened.There is not enough networking and collaboration between NGOs and the international research community, including academia. This has traditionally been due to a dichotomy in the interests of NGOs and the academic community, in that NGOs are more oriented towards a development agenda, while academics tend towards special research interests.In light of the above issues and concerns, and in order to foster greater interest and participation of NGOs in research, the barriers of lack of interest, lack of funds, lack of training and lack of recognition, among others, need to be addressed. We discuss some strategies below.Substantial global health academic capacity has developed within NGOs both in the U.S. and the U.K. For example, Family Health International (FHI) has integrated research, training, and development capacity on an evidence-based foundation. It also embodies many of the competitive aspects of private sector-led initiatives that can allow creativity and innovation.As emphasized by Harrison and Neufeld , howeverA peer-learning process is among the strategies for NGO capacity building in research. NGOs can draw on expertise already developed in research-based NGOs. The learning process should be shared with NGOs from the North and from the South. Additionally, NGOs should consider taking the initiative in organizing scientific activities  on global health research topics, which could serve as a catalyst in bringing together different stakeholders.The creation of networks which have the common goal of supporting global health research is one way to strengthen partnerships and to consolidate valuable resources from each partner. Leadership and governance issues are necessary hurdles which can be overcome by focusing on the ultimate gains in terms of supporting and conducting successful research activities. NGOs can assist in the establishment and functioning of these networks, particularly by providing stable infrastructure support. One of the greatest challenges is in making the network function effectively through different leadership turnovers in the different partner organizations.CSIH has had global health research as part of its mandate since its formation. CSIH is an active member of the Canadian Coalition for Global Health Research (CCGHR), and a key challenge in this capacity is to develop a strong foundation of understanding and mutual respect amongst all players in global health research, including NGOs. CSIH experience in global health research is described in table Partnerships with universities and other research institutions is one means of strengthening the research capacity of NGOs, and also of academia. NGOs and research organizations each have a unique 'value-added' contribution to make to global health research and therefore, partnering among them amplifies their individual strengths. Such partnering may be a real challenge for NGOs, however, as their institutional culture is so different. NGOs may be invited by universities to partner, but plans are often already laid out, so that the NGOs may only be involved in executing the plans. What NGOs want is to be part of the research process from the start. An interesting initiative to document and promote South-Canada health research partnerships is currently underway .NGOs are open to partnerships with academia, but the goal has to be development-oriented. Experience suggests that it is often difficult to reconcile the academic and development framework, for instance when integrating MSc or PhD students in development projects. Nonetheless, integrating graduate students in NGO projects should be a good strategy for balanced and equal partnerships., the prospects of university-NGO research links are constrained by funding. Nevertheless, the recent Global Health Research Initiative of the coalition of Canadian institutions funding global health research is promising as it opens new avenues for research collaboration between the North and the South, and hopefully also between universities and NGOs.As less than 1% of university-based health research in Canada is directed towards the problems of global health according to a Canadian Consultation on Global Health Research held in 2001 During the 1990s, there were several attempts to bridge the NGO/academic gap with respect to health research in developing countries. Save the Children UK, Oxfam and some US-based institutions supported workshops and symposia that aimed at bringing together representatives from both communities as a means of building links and forging partnerships in support of increasing the scale, scope and relevancy of health research in developing countries . CPHA, through the CIDA-funded Canada's International Immunization Program – Phase 2 (CIIP2), supported applied research carried out by Laval University, Université de Montreal and University of British Columbia. At the time, these were quite innovative approaches to applied health research, linking the universities with local NGOs. In 1995 CPHA also organized a Symposium on NGO/University Linkages for Health Research .One mechanism to expand the use of research generated by NGOs is to improve the linkage between NGOs and universities. Each complements the other in the area of health research. NGOs offer proximity to people and situations, reality-based and context-specific research environments, opportunities to develop and assess innovative strategies and research methods, a means of disseminating and popularizing the results of research projects, and credibility outside of academia. Universities and other research organizations offer expertise in research design and application, an environment for reflection, access to and knowledge about most recent literature, a tradition of scientific rigor and interest in new, innovative research methods and approaches, and a high degree of credibility. Academics can also provide guidance and advice on how to prepare research proposals and to carry out research studies, guidance in the preparation of reports and publishing of research results, and training for NGO staff in research methods.The participants of the CPHA Symposium on NGO/University Linkages for Health Research in Developing Countries  identifiThe development of innovative North-South research partnerships is the focus of a working paper prepared for the CCGHR by Neufeld et al . As emphFinally, as mentioned earlier, stronger partnerships between NGOs and social science researchers in particular should be sought in resource-poor countries. Lessons learned from these partnerships, in the areas of action and indeed policy and legislation  show how evidence can be transformed into action with the right partnerships between researchers and NGOs.NGOs may benefit in various ways from developing a global health research network. First, many NGOs already operate at national and international levels and understand the challenges of coordination and communication which this entails. There is a need to identify overarching principles of NGO contribution to health research. Second, NGOs must be both proactive and interactive within the framework of the health research agenda. Roles must be clearly understood by each partner. In any case, advocacy for relevant research and use of results would be a critical function of the network (see training modules on advocacy ). Such nIn the framework of an NGO network of this sort, the following discrete activities could be envisaged by the lead NGO:• To invite NGOs to post on a selected website success stories, as well as their experience/opinions/needs/priority research issues, using a template adapted from the one developed for this purpose in the UK• To organize workshops for NGOs who are, or who wish to become, involved in research, with research organizations where deemed appropriate.The purpose would be:• To link these NGOs in order for them to interact on research issues;• To share lessons learned and success stories of research involvement of NGOs and their partners• To enhance understanding of, and collaboration with, potential research partners;• To set-up a core group of NGOs involved in global health research to convey NGO views to global health research fora and organizations.The following are a few key questions that could be addressed by an NGO network:1. How can NGOs contribute to the framing of the research questions if we were to support the necessary equity-based research for improving the overall performance of the health system?2. How do we balance this with the necessity for research which documents and monitors sustained and emerging inequities which may have a greater impact on the health and well being of individuals than the health (care) system will ever have?3. How can we ensure that NGOs influence research priorities so that they are reflective and evaluative of overseas development assistance (ODA) direction and priorities such as national poverty reduction strategies. For example what is the impact of PRSPs on equity and health? How will researchers monitor this? What could be the potential role of NGOs in partnership with researchers to begin to monitor and evaluate this new direction in overall aid policy?4. How can NGOs be best represented within the international research community?The new millennium offers many challenges. In order to maximize the potential benefits of health research, all partners including NGOs must share a common vision and recognize and appreciate the strengths of each. Participation in health research needs to be a coordinated effort. One key challenge will be to establish better communication among all partners in health research. This can only be achieved by a willingness to share in leadership, ownership and in the conduct of health research activities. Another key challenge will be to explore ways in which funding for health research can be strengthened. Leveraging must be seen as a strategic tool of NGOs to maximize dollars allocated to health research.Several NGOs have had impressive track records in global health research. Other NGOs have expressed an interest in becoming more involved in global health research. Their contribution to more equitable, ethical, relevant and effective research is crucial and needs to be strengthened. Research has to be regarded as a broad loop system rather than restricted narrowly to the production of knowledge. This is particularly critical for global health research whose primary goal should be to improve health and its determinants in low and middle income countries. NGOs principal roles in the process pertain to shaping research priorities, advocacy for more relevant research, translating and using research findings, in addition to generating new knowledge in areas where they may have a comparative advantage, notably qualitative, social, action, evaluative, and policy research. NGO partnerships with research organizations should be seen as means of a mutual enhancement of health research capacity and contribution to development. NGOs should be instrumental in building with other stakeholders coalitions for global health research with the aim of closing the 10/90 health research gap.ADI Alzheimer's Disease InternationalAFRO-NETS African Networks for Health Research and DevelopmentAIDS Acquired immunodeficiency syndromeAMREF African Medical and Research FoundationARCH Applied Research on Child Health (ARCH) ProjectCCIC Canadian Council for International CooperationCCISD Centre de coopération internationale en santé et développementCCIH Canadian Conference on International Health (hosted by CSIH)CECI Centre canadien d'étude et de coopération internationaleCCGHR Canadian Coalition for Global Health ResearchCDC Centers for Disease Control and PreventionCHW Community health workerCIDA Canadian International Development AgencyCIHR Canadian Institutes of Health ResearchCIIP2 Canada's International Immunization Program – Phase 2COHRED Council on Health Research for DevelopmentCPHA Canadian Public Health AssociationCSIH Canadian Society for International HealthDFID Department for International Development (UK)DSA Development Studies AssociationENHR Essential National Health ResearchFCTC Framework Convention on Tobacco Control (WHO)FHI Family Health InternationalFRAT Fortification Rapid Assessment TechniqueGYTS Global Youth and Tobacco Survey (WHO and CDC)HKI Helen Keller InternationalIDRC International Development Research CentreIHPP International Health Policy ProgramINTRAC International NGO Training and Research CentreKFPE Commission for Research Partnerships with Developing CountriesLMIC Low and middle-income countriesMICAH Micronutrients and Health in Africa (WV project)MSF Médecins sans frontièresNEPAD New Partnership for Africa's DevelopmentNGO Non-governmental organizationOECD Organization for Economic Co-Operation and DevelopmentODA Overseas Development AssistancePRSP Poverty Reduction Strategy PapersRITC Research for International Tobacco ControlRPHA Russian Public Health AssociationSHARED Scientists for Health and Research for DevelopmentTRIPS Trade-Related Aspects of Intellectual Property RightsUNDP United Nations Development ProgrammeWHO World Health OrganizationWV World Vision (Canada)HD designed the outline of the paper, conducted interviews with NGO representatives, wrote the first complete draft, and coordinated the review process within the CSIH. JHR drafted some sections and provided comments on the successive versions of the papers. MM provided international NGO and a field based perspectives to the paper, in addition to conducting group discussions with NGO personnel. LJ designed the figures, and edited and formatted the text. TG contributed to the conceptualisation and content of the manuscript. She wrote several sections and edited others, and she provided case studies.


Dendritic cells (DCs) are professional antigen-presenting cells that play important roles during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 derived lentiviral vectors (LVs) transduce DCs at high efficiency but their effects on DC functions have not been carefully studied. Modification of DCs using LVs may lead to important applications in transplantation, treatment of cancer, autoimmune and infectious diseases.Using DCs prepared from multiple blood donors, we report that LV transduction of DCs resulted in altered DC phenotypes and functions. Lentiviral transduction of DCs resulted in down-regulation of cell surface molecules including CD1a, co-stimulatory molecules CD80, CD86, ICAM-1, and DC-SIGN. DCs transduced with LVs displayed a diminished capacity to polarize naive T cells to differentiate into Th1 effectors. This impaired Th1 response could be fully corrected by co-transduction of DCs with LVs encoding interleukin-12 (IL-12), interferon-gamma (IFN-γ), or small interfering RNA (siRNA) targeting IL-10.DCs transduced with LVs in vitro displayed diminished Th1 functions due to altered DC phenotypes. Our study addresses an important issue concerning lentiviral infection and modification of DC functions, and provides a rational approach using LVs for immunotherapy. During HIV-1 infection, an increase in DC-SIGN and CD40 has been reported, as has a decrease in the expression of CD80 and CD86 in dendritic cells (DCs) of lymphoid tissue  others hLVs are useful gene transfer tools that can efficiently target many types of cells including DCs. As important immune modulating cells for immunotherapy and vaccine applications, DCs play critical roles in activating the host immune response. DCs can capture, process, and present foreign antigens, migrate to lymphoid-rich tissues, and stimulate antigen-specific immune responses . DCs pre+ T cells toward Th1 function, which is essential for the clearance of intracellular pathogens. IL10, on the other hand, suppresses IL-12 production from DCs and diminishes the commitment of Th1 differentiation. Besides cytokine signaling, there is accumulating evidence that co-stimulatory molecules and adhesion molecules such as CD80, CD86, and ICAM-1 not only engage in T-cell stimulation, but also direct the differentiation of naive T cells [Engagement between DCs and T cells not only stimulates T-cell proliferation, but also polarizes differentiation of naïve T helper (Th) cells into IFN-γ-producing Th1 or IL-4-producing Th2 effector cells ,7. ProduEfficient gene transfer into DCs without cytotoxicity has always been difficult ,12. LVs 2. On Day 3, 1 ml of fresh AIM-V medium containing 560 u/ml of GM-CSF and 25 ng/ml of IL-4 was added to the culture. On Day 5, the non-adherent cells were harvested by gentle pipetting. After washing, the DCs were frozen for later use or used immediately.Peripheral blood mononuclear cells (PBMCs) from healthy donors  were isolated from buffy coats by gradient density centrifugation in Ficoll-Hypaque  as previously described . DCs werMLV and LVs were constructed as described previously ,20. The The LVs were produced and concentrated as described previously . Lentivi5 per well in a 24-well plate containing 200 μl of medium supplemented with GM-CSF (560 u/ml) and IL-4 (25 ng/ml). DC infection was carried out by adding concentrated LVs to the cells at a multiplicity of infection (MOI) of 50–100 (~105–106 transducing units/ng of p24) as previously described [We plated Day-5 immature DCs at 5 × 10and IL-4  ng/ml. DFor analysis of cell-surface marker expression by flow cytometry, we incubated DCs for 10 min with normal mouse serum and then 30 min with fluorochrome-conjugated anti-human monoclonal antibodies. In different experiments, these antibodies included HLA-ABC ; HLA-DR ; CD1a ; CD80 ;CD86 ; ICAM-1 ; DC-SIGN ; CD11c ; CD40 ; CD123 ; and CD83 . We included the corresponding isotype control antibody in each staining condition. After two washes, the cells were resuspended and fixed in 1% paraformaldehyde in PBS and analyzed using a FACSCalibur flow cytometer and the CELLQUEST program (Becton Dickinson). The live cells were gated by forward- and side-light scatter characteristics and the percentage of positive cells and the mean fluorescence intensity (MFI) of the population were determined.2O to make an 8:1:1 ddH2O/DMSO/ethanol solution. The cells were incubated in 37°C water bath for 1–1.5 min, and diluted with 10-fold volume of cold medium and kept on ice until FACS sorting.The lentiviral siRNA vector-transduced cells co-expressing nuclear lacZ gene were separated from un-transduced cells by staining with fluorescent LacZ substrate and sorted by FACS. To label the lacZ-positive cells, we resuspended cells in 100 ul medium and added 100 ul of FDG  working solution (2 mM) which was diluted from a 10 × stock FDG solution (20 mM). The stock solution was made by dissolving 5 mg FDG  in a 1:1 mixture of DMSO/ethanol and mixing with ice-cold ddH+ T cells from PBMCs were collected by negative selection, using a CD4+ T cell isolation Rosette cocktail  according to the manufacturer's instructions. Briefly, we centrifuged 45 ml of buffy coat (approximately 5 × 108 PBMCs) in a sterile 200-ml centrifuge tube with 2.25 ml of the CD4+ T cell-enrichment Rosette cocktail at 25°C for 25 min. Thereafter, 45 ml of PBS containing 2% FBS was added to dilute the buffy coat. After gentle mixing, we layered 30 ml of the diluted buffy coat on top of 15 ml of Ficoll Hypaque in a 50-ml centrifuge tube and centrifuged for 25 min at 1,200 g. Non-rosetting cells were harvested at the Ficoll interface and washed twice with PBS (2% FBS), counted, and cryopreserved in aliquots in liquid nitrogen for future use. The purity of the isolated CD4+ T cells was consistently above 95%. The CD4+CD45RA naïve T cells were purified based on negative selection of CD45RO- cells using the MACS  magnetic affinity column according to the manufacturer's instructions.The CD4in vitro DC:T cell co-culture method was modified based on Caron et al[The ron et al. Briefly5 allogeneic CD4 T cells in a 96-well U-bottomed plate in a total volume of 200 μl for 5 days. The proliferation of T cells was monitored by adding 20 μl of the CellTiter96 solution to each well according to the manufacturer's instruction (Promega). The cells were further cultured for 4 hr before reading the OD490 value using a microplate reader .We co-cultured serial dilutions of DCs, from 10,000 cells per well to 313 cells per well, with 1 × 10To investigate the effects of lentiviral vectors (LVs) on DCs, we transduced monocyte-derived DCs with LVs encoding different reporter genes. The efficiency of LV transduction of DCs is illustrated by a reporter gene assay Fig. . The DCsTo see if LVs affected DC surface marker expression, we examined the expression profile of surface molecules on DCs by antibody immunostaining. We transduced PBMC-derived imDC with mock (control 293 supernatants) vectors, empty LV particles, LV, and MLV carrying a reporter gene. After induction of maturation with LPS plus TNF-α, we harvested the DCs for antibody staining and FACS. The results are shown in Fig. in vitro immunity assay using co-culture of human DCs and naïve T cells.It has been reported that retroviral infection induces up-regulation of Th2 cytokines including IL-10 and impairs DC maturation ,28. Beca+ T cells from healthy donors' blood and co-cultured the T cells with allogeneic monocyte-derived DCs treated with TNFα and LPS to induce maturation, as illustrated in Fig. We generated DCs from PBMCs and infected the d5 DCs with LV carrying a reporter gene. To characterize the function of DCs, we purified naïve CD4in vivo study [Because T cell co-stimulatory molecules are important mediators of DC functions, the down-regulation of CD80 and CD86 in DCs after LV transduction might contribute to the observed Th1 impairment. To examine this possibility, CD80 and CD86 were up-regulated in DCs using LVs encoding these two genes to see if the impaired Th1 response could be corrected. The LVs encoding human CD80 and CD86 were constructed as shown in Fig. vo study . DCs werTo see if the up-regulation of the T-cell co-stimulatory molecules in DCs could restore the Th1 response, we co-cultured naïve CD4 T cells with DCs transduced with mock, LV-PLAP, LV-PLAP plus LV-CD80, or LV-PLAP plus LV-CD86. After 8 days, the T cells were reactivated and analyzed by ICCS and FACS using anti-IL-4 and anti-IFN-γ antibodies as described earlier. We found that after LV transduction the Th1 population was reduced from 24% to 13%. Moreover, this impairment could not be corrected by the up-regulation of CD80 and CD86 in DCs  has been shown to enhance IL-12 production in DCs . To overIL-10 is a critical immune modulatory gene and modulation of IL-10 gene expression may alter DC function. To test this, we constructed LVs encoding siRNA targeting IL-10. We chose two regions in the IL-10 mRNA as the siRNA target sites Fig. . The siRThe effect of the IL-10 LV siRNAs was then examined in DCs by co-transduction using a reporter LV and the IL-10 LV-siRNAs. The transduced DCs were then treated with LPS and analyzed for IL-10 expression as described above. Again, the empty LV had no effect and LV transduction alone up-regulated IL-10 expression. However, co-transduction with LV-siRNA targeting IL-10 down-regulated IL-10 expression Fig. ; the lowTo examine whether co-transduction of DCs with LVs expressing the IL-10 siRNA could promote a Th1 response, we transduced DCs with LV alone or together with either an LV-siRNA (#2) or a control LV-siRNA (GFPi). For positive control, we incubated DCs with soluble IFN-γ as previously described. After the DCs were co-cultured with naïve T cells for 10 days, the T cells were reactivated and analyzed for Th functions by ICCS to determine intracellular expression of IFN-γ and IL-4. The results clearly demonstrated that the IL-10 LV-siRNA vector, but not the GFPi LV-siRNA vector, enhanced Th1 response at levels comparable to that of the positive control  is a 44-kDa type I membrane protein with an external mannose-binding, C-type lectin domain . It has Polarization of naïve Th cells into Th1 cells is critical for the induction of cellular immunity against intracellular pathogens and cancer cells. The observed impairment of the Th1 response by LV-transduced DCs raises a potential issue with LV-based immunotherapy. We illustrated that co-transduction with LV encoding IL-12 or IFN-γ, but not CD80, CD86, or ICAM-1, in DCs effectively restored Th1 immunity. In addition, co-transduction with LVs expressing small interfering RNA targeting IL-10 could also promote DC-mediated Th1 immunity. In a step toward future generation of vaccines, LVs encoding IL-12 and IL-10-siRNA as potent Th1 adjuvants may be used to enhance the cellular immune response in the prime-and-boost vaccination regimen. In summary, our study has addressed an important immune suppression effect of LVs and presented a solution that is important for future LV-based DC immunotherapy applications.HIV-1 human immunodeficiency virus type 1LV lentiviral vectorDC dendritic cellTh T helperMLV murine leukemia virusRT-PCR reverse transcription-polymerase chain reactionFACS fluorescence activated cell sorterICCS intracellular cytokine stainingIL interleukinPLAP placenta alkaline phosphatasesiRNA small interfering RNAPBMC peripheral blood mononuclear cellsMOI multiplicity of infectionLPS lipopolysaccharideTNFα tumor necrosis factor alphaINF-γ interferon-gammaThe author(s) declare that they have no competing interests.The study was conceived by LJC; XC and LJC participated in designing and coordinating the study; JH carried out some of the lentiviral constructions, siRNA designs and participated in result discussion; XC performed the statistical analysis; LJC and XC carried out detailed analysis of the results and XC drafted and LJC finalized the manuscript. All authors read and approved the final manuscript.


By combining ontologies from different sources the authors developed a novel approach to describing phenotypes of mutant mice in a standard, structured manner. The mouse is an important model of human genetic disease. Describing phenotypes of mutant mice in a standard, structured manner that will facilitate data mining is a major challenge for bioinformatics. Here we describe a novel, compositional approach to this problem which combines core ontologies from a variety of sources. This produces a framework with greater flexibility, power and economy than previous approaches. We discuss some of the issues this approach raises. N-ethyl-N-nitrosourea (ENU) mutagenesis ." The authors also describe the assays used to record these observations: "Nesting Patterns: six cages of wild-type and six cages of mutant mice (N = 4 mice per cage) were used to evaluate nesting patterns. A 5 × 5 cm piece of cotton nesting material  was placed in each cage. After 45 min, photographs were taken of each nest and the nest depth was measured. Nest height data were analyzed using the Student's t test."In this section we describe an example of the application of the compositional schema. We chose a phenotype example at random from the MP database: 'nest building' [MP:0001447]. Several descriptions of nest-building patterns can be found in the corresponding reference . For exaFor some users/applications the compound term 'abnormal nest building' might be a sufficient description of this particular phenotypic instance, but this would result in information loss. A human would have to retrieve and read the reference to extract further information. Our schema allows the expression of this information in a machine and human readable manner. In Table Nest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [well-formedPatterns ) {returnNest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [poorly-formedPatterns ) {returnNest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [fluffyPatterns ) {returnWe note here that had the value 'fluffy' not been included in the standard values for a quality assay, it could be captured in the free-text field provided by the schema. To express a nest of 50 mm depth or significantly shallower:Nest building {has_attribute} attribute:absolute_depth {characterized_by} undefined_ absolute_depth_assay {returns_value} 50 mmNest building {has_attribute} attribute: relative_depth {characterized_by} undefined_ relative_depth_assay {returns_value} shallow {has_qualifier} significantWith this information one could go back to a higher level and still be able to express a more general characterization of this phenotype as 'abnormal nest building' but obviously the opposite is not possible.An important unresolved issue concerning the use of ontologies to describe phenotypes arises from the fact that all the ontological structures developed so far are designed to describe individual mice. Mutagenesis experiments usually characterize a number of mutant mice to take into account variable penetrance of the mutation and other stochastic effects. A strategy will therefore need to be developed to describe the generalized phenotypic properties of a cohort of mice. This may involve the use of more sophisticated relations such as {usually characterized by} or even quantitative relations such as {80% characterized by}.The assay plays a central role in our schema Figure . Assays eye {has_attribute} attribute:color {characterized_by} visual inspection {returned_value} pinkOn a practical level, assays can add specificity and functionality to the relationship between entities, their attributes and the corresponding values. Most important, an assay vocabulary allows the entire schema to be dynamic by including new assays and capturing explicit differences between assays in different laboratories. The assay will also allow standardization and definition of values for a given phenotypic character, for example, how abnormal is defined in relation to body position.Our schema can be expressed using a variety of modeling tools and knowledge representation (KR) languages . We chosThe schema was designed to be easily populated using extant core ontologies, such as anatomy, and defining attributes related to each entity. The assay vocabulary can be constructed as required. Permitted values are defined in the range of different assay attributes in part devised in the form of a general scheme and in part built from the output of particular assays. Although we include for demonstration purposes three core ontologies, namely behavior, anatomy, and developmental anatomy Figures  and 4, wFigure et al. [Since most of the ontologies we are planning to use were generated using the DAG-edit  format, et al. , with miDecisions will inevitably have to be made to combine a core ontology with its attributes and then define facets of that relationship, for example, cardinality, attribute value type and attribute range. In our schema, the class hierarchy of all ontologies employed represents an 'is-a' relation. So, mouse social behavior 'is-a' mouse behavior, or mouse social behavior is a 'kind-of' mouse behavior and so forth. All other relationships, including PATO and 'part-of' relationships, are modeled as attributes. However, we note here that efforts are currently being made by the GO consortium to define and formalize the 'part-of' relationship, which is considered vital and special in bio-ontologies, especially anatomy .Because our phenotype ontology and PATO need to be the result of a collaborative effort within the communities, we feel that it is important to set out the basic modeling concepts that need to be applied upon allocating attributes to the core ontologies. Deciding whether to introduce a new attribute or represent this functionality through an entity is often quite difficult. Several things need to be considered in order to make the best decision, although it should be noted that there are no clear distinction as to what is a right or wrong decision.The first thing to take into account is that subclasses of a class inherit all properties of the parent and could have additional properties and different restrictions from the latter. PATO should remain as general as possible, and, when possible, care should be taken to avoid making PATO domain specific. For example, in the behavior ontology there is a class named 'reflexes' that contains children such as 'blinking reflex', 'Preyer reflex' and 'righting reflex'. It might be worth considering having one 'attribute of reflex' available in PATO rather than creating a separate attribute 'of' for each individual reflex, such as 'attribute of blinking reflex', 'attribute of Preyer reflex', and so on. Then again, if one wishes to assign different functionalities to these properties, creating separate attributes might be useful. As a rule though, one should consider that PATO needs to be consistent, usable and interoperable if it is to be applied to the general domain of phenotypes. Repetition between core ontologies and PATO should be avoided where possible.What is also often not clear is whether one should add a new class to represent functionality or assign attributes to already existent classes. For example, think of the entity 'body position'. There are several ways to model this entity in the mouse behavior phenotype ontology. One could declare 'body position' as a child of a class called 'posture'. An 'attribute of body position' could then be assigned to this class with a range of values that might be specific to an assay, for example SHIRPA  allows t'Body position' could also be split into an entity of 'body' and an attribute of 'position'. Again, a new class 'body position' should be assigned, if one considers the objects with different attributes as different kind of object and this distinction important in the domain. As a general rule, before assigning new classes and attributes one should consider the functionality and their role in the domain, creating more distinctions as the depth of knowledge that is required to be expressed in the ontology increases.Classes in the hierarchy should not necessarily have to introduce new properties . AlthougWe have presented here an approach to the use of ontologies in describing mouse phenotypes that could provide a platform for the consistent representation of mouse phenotypic data. We have also described in detail a possible methodology to construct applications of this schema across different domains. We have dealt with modeling issues and provide guidelines to deal with semantic and practical problems.We maintain that such modeling efforts in any domain should be done in a collaborative fashion in the community. Repetition between different parts of the mouse phenotype ontologies is unavoidable. However, the use of consistent IDs, synonyms and records for associated annotations could allow seamless integration of ontology products. The nature of the schema proposed, as well as its components, is extremely dynamic; therefore coordination of efforts is vital.The structure allows extensibility and interoperability. Although an ontology should not cover all possible information about a domain, the main idea behind this concept is to allow the phenotype ontology to cope with novel and unpredictable phenotypes and account for new assays, serving scientific autonomy and information validity and integrity. We have built a software system  which in


An increase in the number of attendees due to acute gastroenteritis and fever was noted at one hospital emergency room in Taiwan over a seven-day period from July to August, 2001. Molecular and epidemiological surveys were performed to trace the possible source of infection.An epidemiological investigation was undertaken to determine the cause of the outbreak. Stool and blood samples were collected according to standard protocols per Center for Disease Control, Taiwan. Typing of the Salmonella isolates from stool, blood, and food samples was performed with serotyping, antibiotypes, and pulsed field gel electrophoresis (PFGE) following XbaI restriction enzyme digestion.Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was isolated from the stool samples of 28 of 32 individuals and from a recalled bread sample. All S. Enteritidis isolates were of the same antibiogram. PFGE typing revealed that all except two of the clinical isolates and the bread isolates were of the same DNA macrorestriction pattern.Comparison of the number of patients with and without acute gastroenteritis  during the six weeks before the outbreak week revealed a significant increase in the number of patients during the outbreak week  (relative risk (RR): 1.44, 95% confidence interval (CI): 1.22–1.70, P value < 0.001). During the week of the outbreak, 34 of 162 patients with gastroenteritis were positive for Salmonella, and 28 of these 34 cases reported eating the same kind of bread. In total, 28 of 34 patients who ate this bread were positive for salmonella compared to only 6 of 128 people who did not eat this bread . These breads were produced by the same bakery and were distributed to six different traditional Chinese markets., S. Enteritidis was confirmed as the vehicle of infection. Alertness in the emergency room, surveillance by the microbiology laboratory, prompt and thorough investigation to trace the source of outbreaks, and institution of appropriate control measures provide effective control of community outbreaks.The egg-covered bread contaminated with  Salmonellosis, resulting from the ingestion of contaminated poultry, beef, pork, eggs, and milk , is an iSalmonella species are relatively uncommon in Taiwan [Salmonella food-borne disease outbreaks but serovar Enteritidis has emerged as a new serovar in Taiwan [Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) infections [Food-borne disease outbreaks due to n Taiwan  comparedn Taiwan  and Japan Taiwan . Thirty-n Taiwan . Serovarn Taiwan , consistfections ,24,25.S. Enteritidis that was performed after notification of the rapid increase of the number of patients with febrile gastroenteritis in an emergency room and an unusually high percentage of Group D Salmonella isolated since it comprised only 7.4% of the various Salmonella groups in the hospital one year before the outbreak.We report the findings of the investigation of an outbreak of Salmonella infection. A cohort study of all patients attending the ER with acute gastroenteritis between July 28 and August 3, 2001 was performed in order to test the hypothesis that illness was caused by a specific food.A rapid increase in the number of attendees due to acute gastroenteritis and fever was noted to have begun on July 28, 2001 at the emergency room (ER) of Kaohsiung Municipal Hsiao-Kang Hospital, and this increase continued for the following six days. Clinical and demographic features and the food reported to have been consumed three days before development of gastrointestinal symptoms for all patients with acute gastroenteritis at the ER from July 28 to August 3, 2001 were reviewed by ER personnel and the infection control team. The infection control team conducted patient interviews and reviewed charts using a standardized case record form. Stool and blood cultures were performed once specimens were available. These specimens were further analyzed by the hospital's clinical microbiology laboratory and the laboratory of the Center for Disease Control, Taiwan  in Kaohsiung, Taiwan. Charts of all patients with and without acute gastroenteritis who visited the ER from six weeks before until two weeks after July 28, 2001 were reviewed by infection control nurses for the baseline data of the ER. An outbreak-associated case was defined as a patient visiting the ER with the diagnosis of acute gastroenteritis and having Methods for sample collection, cultivation and isolation were conducted according to the standard protocol of the CDC, Taiwan for food-borne disease outbreaks as described previously .Salmonella serotypes were determined with the use of antiserum  according to the manufacturer's instructions. Serogrouping and serotyping were performed by the slide agglutination method and tube agglutination method to identify the somatic O antigen and flagellar H antigen, respectively [ectively .Escherichia coli ATCC 25922 was used as the quality control organism.Antimicrobial susceptibility was determined by the disk diffusion method according to the National Committee for Clinical Laboratory Standards . The antXbaI  was used at the manufacturer's suggested temperature. Restriction fragments were separated by PFGE in 1% agarose gel  in 0.5X TBE buffer  for 25 h at 200 V at a temperature of 14°C, with ramped times of 2 to 40 s using the Bio-Rad CHEF-DRII apparatus . Gels were then stained with ethidium bromide and photographed under ultraviolet light. The resulting genomic-DNA profiles, or "fingerprints," were interpreted according to established guidelines [Total DNA was prepared and PFGE was performed as described previously ,13. The Salmonella infection were suspected as the vector of infection. A sample of the implicated food was collected from a patient's home within six hours of the patient having symptoms of febrile gastroenteritis and it was stored at 4°C. Further tracing the source of the suspected food and the subsequent environmental investigation were undertaken by the Bureau of Health, Kaohsiung County and CDC, Taiwan.Food items having a significant relationship with gastroenteritis cases with t-test. Significance was considered at a P value < 0.05 (Epi Info. Version 3.2.2 Centers for Disease Control and Prevention (CDC), USA).Differences between groups were compared using Chi-Square analysis. Relative risk and 95% confidence intervals were also calculated when Chi-Square analysis was used. Continuous variables were analyzed with Student's An increase in the number of patients admitted due to acute gastroenteritis and fever was noticed beginning from the early morning of July 28, 2001 with a return to normal levels one week later. Reviewing daily case-visits to the ER due to all causes and due to gastroenteritis six weeks before this outbreak period revealed significantly more cases due to all causes visiting the ER on Saturday and Sunday than on weekdays (From Monday to Friday) (mean ± standard deviation (S.D.): 140.3 ± 38.3 compared with 109.6 ± 16.7, P = 0.004). The number of patients visiting due to gastroenteritis on Saturday and Sunday were also higher than those visiting on weekdays   of 942 patients visiting the ER during the outbreak period had acute gastroenteritis. Compared with 85 (9.7%) of 872 patients in the week before the outbreak, significantly higher percentages of patients with gastroenteritis were observed in the outbreak week (Relative risk (RR): 1.92, 95% confidence interval (CI): 1.38 – 2.26, P < 0.001). A comparison of the number of patients with and without acute gastroenteritis  during the six weeks before the outbreak week and during it also revealed a significant increase in the number of patients of gastroenteritis during the outbreak week .Salmonella compared to only 6 of 128 people who did not eat this bread . The association of consuming the kind of bread and having Salmonella infection was significant . No other identified food or restaurant exposure was significantly associated with the outbreak.Investigation of food consumed by gastroenteritis patients during the outbreak period revealed 34 (21%) had consumed the same kind of egg covered bread decorated with mayonnaise and fried seasoned pork fiber from six traditional Chinese markets (three located in Kaohsiung City and three in Kaohsiung County). There were 2, 2, 2, 3, 3, and 22 gastroenteritis patients that consumed the bread purchased from 6 markets, respectively. In total, 28 of 34 patients who ate this bread were positive for Salmonella cases that consumed the implicated bread, 12 were male. Their age ranged from three to 71 years old. Eleven cases (39.3%) were under 18 years old and one case was over 65. The incubation periods ranged from 4 to 17  hours after consumption of the bread. Twenty-seven of the 28 patients were hospitalized. The clinical symptoms among the 28 cases included abdominal pain (100%), fever (100%), diarrhea (100%), vomiting (85.7%), chills (35.7%), and headache (7.1%). The laboratory data showed eight (28.6%) cases had white blood cell counts higher than 10000 /mm3. Positive occult blood reaction was found in stool specimens of 24 of 27 (88.9%) patients tested. Colitis was found in all four patients who received colonoscopy examination. The two bacteremia patients' fever subsided one day and two days after admission, respectively, without antimicrobial therapy. No mortality or sequellae occurred among these cases during hospitalization or in the three months' follow-up by infection control nurses.Regarding the 28 Salmonella and surveillance culture of the workplace surfaces were not performed.Further investigation by the city public health administration found the incriminated bread from the six markets was all produced by the same bakery that was prohibited from production of all kinds of bread on August 1, 2001. Because the bakery had stopped their production one day before the official prohibition, no further samples of the components of bread, such as the egg and mayonnaise, were available. Investigation of the bakery staff with stool cultures for Salmonella isolates. Twenty-eight Salmonella isolates were cultured from 32 available stool specimens from 34 patients who consumed the implicated food. These isolates were all of group D and were further identified as S. Enteritidis. This pathogen grew in two of the 34 patients' blood cultures. A Salmonella isolate of the same serovar was cultured from bread provided by a patient. The sample had been stored in the refrigerator because the patient had not finished eating it.In the week of this outbreak, there was an extraordinarily high percentage (94.1%) of group D of Salmonella was isolated from six of the 25 patients' stool specimens. For the six Salmonella isolates from cases not consuming the implicated food during the outbreak period, two were of group B and four were of group D. Further serovar analysis and PFGE analysis of the four group D isolates was not performed. Among the four group D isolates, two were resistant to ampicillin and trimethoprim/sulfamethoxazole, which was different to the antibiogram of the isolates from cases consuming the implicated bread.A total of 30 stool specimens from 25 patients not consuming the implicated bread were collected and All 28 stool isolates, two blood isolates and one food isolate were susceptible to the nine antimicrobial agents tested. Only two stool isolates showed unrelated PFGE patterns with more than six band differences compared to the epidemic PFGE pattern of the other isolates. All the other stool, blood, and food isolates had the same PFGE pattern indicating a clonal relationship  had not consumed the implicated bread, Fig.  suggestiS. Enteritidis [S. Enteritidis [Salmonella isolates.Compared with phage typing  and plaseritidis ,19 thougeritidis . AnalytiSalmonella groups and knowledge that the percentage of group D Salmonella patients was low before. Thus, the unusually high percentage of group D Salmonella seen within the week of the outbreak led to the investigation. The implicated vehicle of infection in the outbreak was an egg-covered bun topped with mayonnaise and fried seasoned pork fiber which had been distributed to different markets after production at the same bakery. Epidemiological study, antibiograms, and serologic and molecular typing patterns revealed that almost all cases of S. Enteritidis infection during the period were the same as that of the bread isolate. Only the implicated baked good from the same bakery caused Salmonella infections while other items from the bakery were not found to be epidemiologically related with the outbreak. This finding suggests contamination of the pathogen did not occur during the common routes of production, transportation, or selling of goods from the bakery. Although the bread sold in the traditional Chinese markets frequently does not conform to sanitary requirements, and although the staff involved in distribution and selling are not subject to the routine hygiene inspections, in food stores, the possibility of contamination of a specific bakery product simultaneously at six markets in the same time period is very low, suggesting that the contamination occurred during the production process. Food contamination in the outbreak was traced to the same bakery but an investigation of infection among the bakery staff and sources of the contents of the bread was not conducted. Although the baking process involves high temperatures sufficient to kill pathogens, the manual addition of toppings or flavors, such as mayonnaise, eggs, and meat products provide possibilities for contamination with food-borne pathogens. In addition, insufficient baking may be a risk factor for human health because it may not destroy microbial contamination.Outbreaks can be missed easily if the increase in case number is not noticed due to the wide distribution of outbreak sources in different areas or if a relatively small outbreak occurs among a large population and cases are exposed to the outbreak source at different times. Recognition of the outbreak reported in this study was aided by the routine surveillance of Salmonella serovars have been related specifically with some foods. S. Enteritidis is particularly related to eggs [S. Enteritidis outbreaks related to baked goods[Salmonella isolated from the bread had the same serovar, antibiogram, and PFGE pattern as isolates from patients and the significant relationship between consumption of this baked good and the isolation of Salmonella implicated this food product as the vehicle of contamination.Different  to eggs -28. In tked goods,5,7,8, aSalmonella outbreaks [This outbreak had a relatively short incubation compared to the 24–72 hour range reported for other utbreaks ,30. WhetSalmonella Enteritidis outbreaks.For many food-borne outbreaks, the pathogens and transmission vehicles are often not identified, usually because of delayed collection of epidemiologic and microbiologic information . InitiatThe author(s) declare that they have no competing interest.Po-Liang Lu and L. K. Siu were in charge of the investigation, data handling and writing of the manuscript. Po-Liang Lu and Shang- Jyh Hwang participated in the design of the study. Po-Liang Lu, In-Jane Hwang, Ya-Lina Tung and Shang-Jyh Hwang were in the investigation team for data collecting and data analysis. Po-Liang Lu performed the statistical analysis. Chun-Lu Lin and L. K. Siu carried out microbiological assays. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In a murine model of early systemic inflammation, we have explored the association between the sinusoidal HbsO2 detected with a remission spectroscopy system and 1.) the NAD(P)H autofluorescence  and 2.) the markers of hepatocellular injury.During the early stages of systemic inflammation, the liver integrity is compromised by microcirculatory disturbances and subsequent hepatocellular injury. Little is known about the relationship between the hemoglobin oxygen saturation  and the continuous infusion of endothelin-1 (ET-1) further aggravated the hypoxia in HbsO2. The detected NAD(P)H autofluorescence correlated with the detected HbsO2 values and showed the same developing. Three hours I/R resulted in elevated NAD(P)H autofluorescence compared with sham animals. Animals after 6.0 h I/R and continuous infusion of ET-1 revealed higher NAD(P)H autofluorescence compared with 3.0 h I/R animals. Overall the analysed HbsO2 values correlated with all markers of hepatocellular injury.Animals submitted to 1 hour bilateral hindlimb ischemia (I) and 3 hours of reperfusion (R) (3.0 h I/R) exhibited lower HbsO2. In parallel, we detected an increasing NAD(P)H autofluorescence representing an intracellular inadequate oxygen supply. Both changes are accompanied by increasing markers of liver cell injury. Therefore, remission spectroscopy in combination with NAD(P)H autofluorescence provides information on the oxygen distribution, the metabolic state and the mitochondrial redox potential, within the mouse liver.During the early stages of systemic inflammation, there is a significant decrease in hepatic sinusoidal HbsO Hepatic microcirculatory failure is a major prerequisite for the development of hepatocellular dysfunction in a number of conditions like trauma/hemorrhage, liver transplantation and systemic inflammation. In various inflammatory states, the degree of lethal hepatocyte necrosis can be predicted from the extent of hepatic microcirculatory failure , possibl2 at specific points; but the technique is invasive and consumes oxygen. Electron paramagnetic resonance oximetry techniques or nuclear MRI approaches allow the detection of changes in tissue pO2; however, their resolution is too low [et al. [in vivo visualization of the tissue pO2. This technique allows the visualization of oxygen distribution on tissue surfaces, but this method comprised some technical limitations. The oxygen-sensitive membrane has to be used under gastight and watertight conditions during microscopy and the fluorescent membrane shows a photobleaching effect. Paxian et al. [2. A disadvantage of this method, especially when used in small rodents like mice, is that it requires changing the continuous intravenous infusion rates of the dye to provide stable plasma concentrations. With mice  there is a growing need for a method able to reliably detect tissue oxygenation or, at least, hemoglobin oxygen saturation (HbsO2) in capillaries of small animals.Several methods have been reported in the past couple of years directly quantifying the oxygen distribution in tissues; however, their applicability in tissues, especially in small rodents like mice, is limited due to technical reasons. For instance, microelectrodes measure tissue pO too low . A fluor [et al.  on the bn et al.  recentlyin vivo detection of liver sinusoidal HbsO2. In a mouse model of early systemic inflammation, we examined whether the detected changes in hepatic HbsO2 correlated with the established method of NAD(P)H autofluorescence and hepatocellular injury.The aim of the present study was to investigate whether the utility of a new and simple remission spectroscopy system allows reliable Consistent with previous reports ,11, mean2 for all groups ) when compared with sham (68.4 (14.1); p < 0.01). No statistically significant differences were observed between 3.0 h I/R and 6.0 h I/R treated animals. However, an obvious shift of hepatic HbsO2 towards a lower oxygenation was observed when compared with 3.0 h I/R treated animals. Animals treated with 6.0 h I/R and a continuous infusion of endothelin-1 (ET-1) showed significant reduced HbsO2 values (44.8 (14.7)) when compared with 3.0 h I/R treated animals (56.2 (13.2); p < 0.006). More than half of the measured data from these animals revealed HbsO2 values lower than 50%. There was no apparent difference in the local tissue hemoglobin (Hb) content detected (data not shown).Hepatic sinusoidal HbsO2 detected in the same animal , as depicted in Figure Animals subjected to 3.0 h I/R revealed significantly higher NAD(P)H autofluorescence (141.6 12.8)); therefore, a significant decline in hepatic tissue oxygenation was observed when compared with sham (100.0 (6.7)) ; ther2 revealed a significant correlation , as shown in Figure Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels are summarized in Table in vivo measurement of murine hepatic sinusoidal HbsO2 that showed a significant correlation with the established method of NAD(P)H autofluorescence, as well as with the extent of hepatic tissue injury.In the present study, we demonstrate the utility of a remission spectroscopy system for the In vitro bench analysis capabilities have spurred the desire to accomplish accurate in vivo measurement through various techniques. The 1930's and 1940's were a particularly active period for oximetry advances culminating in the development of pulse oximeters in the 1970's [2 in the tissue. Previously, it has been shown that bilateral hindlimb I/R results in the deterioration of liver microcirculation [2.Oximetry relies on the detection of the spectral properties of oxygenated and reduced Hb. e 1970's . Remissiculation . Since t+, NAD(P)H is highly fluorescent [2 obtained by the remission spectroscopy system under pathophysiological conditions. Whether induced by I/R or by the combination of I/R and infusion of ET-1, both analytical methods showed a decrease in hepatic oxygen supply, either as an elevation in NAD(P)H autofluorescence or as a diminution in hepatic HbsO2. The significant correlation between remission spectroscopy and NAD(P)H fluorescence indicates that after 3.0 h I/R, 6.0 h I/R and 6.0 h I/R+ET-1, hepatic oxygen supply was compromised. This is further emphasized by the statistical relationship found between hepatic HbsO2 and the extent of subsequent hepatocyte death.In the past, we have shown that bilateral hindlimb I/R results in a systemic inflammation with hepatic microcirculatory disturbances, in terms of reduced sinusoidal diameters and sinusoidal volumetric blood flow accompanied by elevated levels of sinusoidal leukocytes ,5. Theseorescent . Therefo2 in the sinusoids, whereas NAD(P)H autofluorescence depends upon the mitochondrial redox state and the activity of the mitochondrial electron transport chain. It was previously proposed that during systemic inflammation the NADH/NAD+ redox potential may increase, and oxygen utilization may be altered [2. Obviously, the observed hypoxia did not occur through altered oxygen utilization, but rather through a reduced oxygen supply induced by sinusoidal microcirculatory disturbances. This corroborates our previous contention that the simultaneous use of remission spectroscopy, and that of NAD(P)H autofluorescence, provides additional information regarding the underlying pathophysiological mechanisms. That technical approach allows the correlation between disturbances in oxygen supply and those of oxygen utilization.Both remission spectroscopy and NAD(P)H autofluorescence provide information on the metabolic state of the murine liver. Remission spectroscopy is directly dependent on the HbsO altered . The pre2 during the early stages of systemic inflammation. In parallel, we detected an increasing NAD(P)H autofluorescence representing an intracellular inadequate oxygen supply. Both changes are accompanied by increasing markers of liver cell injury. Future therapeutic interventions should focus on the amelioration of sinusoidal HbsO2 followed by an improvement in mitochondrial redox state. Remission spectroscopy represents a simple and reliable method for hepatic sinusoidal HbsO2 determination in small rodents. In combination with NAD(P)H autofluorescence, it provides information on the oxygen distribution, the metabolic state and the mitochondrial redox potential within the hepatic tissue.There is a significant reduction in hepatic sinusoidal HbsOMale C57/BL6 mice  g) were used for all experiments. The experimental protocols were in compliance with the guidelines of the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council as well as those of Germany. Animals were maintained under controlled conditions  with free access to tap water and a standard laboratory chow.Mice  were randomly assigned to either a Sham or a hindlimb ischemia/reperfusion (I/R) group. Animals of the I/R groups were treated with 60 minutes bilateral hindlimb ischemia induced by tightening a tourniquet above the greater trochanter of each leg while under anaesthesia. Sham animals were not subjected to ischemia, but remained anaesthetized for the same period of time. Tourniquets were removed just prior to recovery from anaesthesia. The animals were awake during the 3 hours (3.0 h I/R) or the 6 hours (6.0 h I/R) reperfusion periods, and re-anaesthetized for the intravital microscopy procedure.To further induce liver microcirculatory disturbances and contribute towards a reduction in liver oxygen supply 6.0 h I/R, mice were further randomized to a group treated with a continuous infusion of ET-1  starting 15 minutes prior to microscopy. This dose of ET-1 was chosen because it produced alteration in the oxygen distribution, along with derangements in the hepatic tissue perfusion .Animals received anaesthesia, by inhalation, for all procedures. As previously described , anaesthin vivo with an i.v. bolus of the vital dye PI (0.05 mg/100 g). As previously stated [-1 mm3.Details of this technique have been described elsewhere ,21. For 2 was measured using the remission spectroscopy system Oxygen-to-See (O2C-ATS) supplied with the micro probe VM-3 . White light was continuously emitted via one channel of the micro probe light-guide and was continuously detected via another channel (channel diameter 70 μm). The backscattered light was analyzed in steps of 1 nm (500–650 nm). Each HbsO2 value was defined by specific Hb spectra. The local tissue light absorbance depends on the total local tissue content of Hb. The local content of Hb was calculated from the local light absorbance and emission. The flexible VM-3 micro probe allowed the detection of oxygen saturation of the left liver lobe placed on the glass slide of the inverted microscope. A special clamping system fixed the micro probe close to the surface of the glass slide and permitted contact-free systematic scanning of the liver lobe .p < 0.05.Data in text and Tables is given as: Mean (SD). Statistical differences between groups and from baseline within each group were determined by ANOVA, followed by the Tukey post-hoc test. The Kolmogorov-Smirnov test was previously used to confirm the normal distribution of data. For checking the nature and extend of the relationship between two variables linear regression analysis was performed. All figures were generated with Sigma Plot (Ver. 8.0) and statistical analyses were performed using Sigma Stat software . Differences were considered significant for CW conceived the design of the study and conducted the laboratory experiments; RB drafted the manuscript and coordinated the study; AK assisted in technical questions. NR participated in design and coordination and OE participated in animal procedures and in drafting the paper. All authors approved and read the final manuscript.