Hepatitis delta frequently leads to liver cirrhosis and hepatic decompensation. As treatment options are limited, there is a need for biomarkers to determine disease activity and to predict the risk of disease progression. We hypothesized that anti-HDV IgM could represent such a marker.Samples of 120 HDV-infected patients recruited in an international multicenter treatment trial (HIDIT-2) were studied. Anti-HDV IgM testing was performed using ETI-DELTA-IGMK-2-assay (DiaSorin). In addition, fifty cytokines, chemokines and angiogenetic factors were measured using multiplex technology (Bio-Plex System). A second independent cohort of 78 patients was studied for the development of liver-related clinical endpoints .Anti-HDV IgM serum levels were negative in 18 (15%), low (OD<0.5) in 76 (63%), and high in 26 (22%) patients of the HIDIT-2 cohort. Anti-HDV IgM were significantly associated with histological inflammatory (p<0.01) and biochemical disease activity . HDV replication was independent from anti-HDV IgM, however, low HBV-DNA levels were observed in groups with higher anti-HDV IgM levels (p<0.01). While high IP-10 (CXCL10) levels were seen in greater groups of anti-HDV IgM levels, various other antiviral cytokines were negatively associated with anti-HDV IgM. Associations between anti-HDV IgM and ALT, AST, HBV-DNA were confirmed in the independent cohort. Clinical endpoints occurred in 26 anti-HDV IgM positive patients (39%) but in only one anti-HDV IgM negative individual .Serum anti-HDV IgM is a robust, easy-to-apply and relatively cheap marker to determine disease activity in hepatitis delta which has prognostic implications. High anti-HDV IgM levels may indicate an activated interferon system but exhausted antiviral immunity. Hepatitis delta is caused by infection with the hepatitis D virus (HDV) and represents the most severe form of chronic viral hepatitis There is currently no reliable non-invasive marker associated with disease activity in hepatitis delta. Quantitative HDV-RNA levels do not correlate with grade or stage of liver disease in HDV-infected patients Anti-HDV Immunoglobulin M (IgM) testing was used to diagnose hepatitis delta infection before HDV-RNA assays became available Our primary aim was, therefore, to investigate possible associations of anti-HDV IgM with grade and stage of liver disease in hepatitis delta in a cross-sectional approach testing very well characterized samples from a large multicenter study. In a second step, we investigated whether or not anti-HDV IgM activity can predict the clinical long-term outcome in hepatitis delta. Finally, we questioned if specific cytokines, chemokines and angiogentic factors were associated with anti-HDV IgM to understand possible mechanisms regulating humoral immunity against HDV.www.clinicaltrials.gov; NCT00932971; EudraCT-No.: 2008-005560-13). Patients with compensated liver disease (Child A) and absence of HIV or HCV coinfection were eligible. Liver biopsies were performed within one year prior baseline.Two independent cohorts of patients were studied. First, we analyzed baseline data of the Hep-Net-International-Delta-Hepatitis-Intervention Trial-2 (HIDIT-2) an prospective international, multicentre trial, investigating the efficacy of PEG-IFN alfa-2a plus tenofovir or placebo for 96 weeks in 121 patients chronically infected with HDV . Grading and staging was performed according to the Ishak score A total activity index of 0–7 was considered as minimal or mild hepatitis, a score of 8–18 indicated severe hepatitis.As the HIDIT-2 trial allowed only cross sectional investigation we recruited an additional independent cohort with patients seen at the Hannover medical school from 1995 to 2012 (“MHH cohort”). Of all anti-HDV IgM positive patients a cohort of 78 chronic hepatitis delta patients were screened to correlate liver-related endpoints with the anti-HDV IgM status. Liver-related endpoints were defined as defined as hepatic decompensation , liver transplantation, HCC or liver-related death. Patients were included in this study if serum samples or anti-HDV IgM testing were available in addition to the following inclusion criteria. Patients were required to have detectable HBsAg and either anti-HDV IgM antibodies or HDV-RNA for at least six months. Only patients with an available follow up of at least 6 months with a minimum of 2 visits and no longer than 2 years between these visits were included. Patients were excluded if they had undergone liver transplantation, or suffered from HCC before the first observation. Virological parameters for hepatitis B and delta were measured as previously described The MHH cohort was recruited from a real world setting and thus patients received various medications during further follow up including nucleoside and nucleotide analogues (n = 16), PEG- IFN alpha (n = 3) and combinations of interferon and HBV polymerase inhibitors (n = 13). 46 patients did not receive any antiviral therapy during follow-up (n = 46).The HIDIT-2 study was obtained by the approving institutional review board of the Hannover Medical School (Ethikkommission der Medizinischen Hochschule Hannover) and the local ethic committees  . All of the named ethic committees approved this study. For all of the patients informed consent paperwork was written.For the measurement of cytokines, chemokines and angiogenetic factors the Ethikkommission der Medizinischen Hochschule Hannover had reviewed the experiments and approved this study design (Nr. 5258).The internal use of data of the MHH cohort was reviewed by the Ethikkommission der Medizinischen Hochschule Hannover and considered that no informed consent was necessary, given the retrospective and noninterventional nature of the study, and that patient's data were analyzed anonymously.Anti-HDV IgM testing was performed by using ETI-DELTA-IGMK-2 assay  according to manufacturer's instructions. In the first step samples were attached to monoclonal IgG directed at human IgM. After one hour of incubation and washing, HDAg was added. Staining was achieved by adding anti-HD horseradish peroxidase and in a last step chromogen to the sample dilution. The optical density (OD) 450/620 values were ascertained by using photometry .All samples with an anti-HDV IgM OD 450/620 above the mean extinction of the negative control plus 0.1 were considered to be positive. Samples with optical density values of 10% below or above this cut-off were retested.To investigate to what extent quantitative values of the assay could be used for further analyses we evaluated the performance of the assay with different sera and plasma samples. Sera and plasma samples obtained in parallel from same individuals revealed similar IgM in all individuals tested (data not shown).We next investigated different incubation periods. While samples with low IgM levels (<0.5 OD) and very high levels (>2.5) gave similar values irrespective of the incubation period, a high variability was observed for samples with intermediate and high values (0.5–2.5) considering different incubation periods .We therefore did not further consider evaluation of OD as linear variables for further investigations but rather grouped IgM levels into four categories .The concentrations of fifty cytokines, chemokines and angiogenetic factors were measured in sera using the Luminex-base multiplex technology.Bio-Plex assays  contain standard concentrations of each analyte and the calculation of respective standard curves allows a precise definition of the concentrations of the protein of interest. The assay was performed according to the manufacturer's protocol. In brief, lyophilized cytokine standard was resuspended in standard diluent. Serial dilution series to generate standard curves for each cytokine, chemokine and growth factor of interest were performed. The bead mixture, specific for cytokines, chemokines or growth factors was incubated for 30 min at RT with 50 µl standard or serum samples that were diluted in sample diluent (1∶2 dilution). Several washing steps were performed with 100 µl wash buffer/well, using the automated washer for magnetic beads. After addition of secondary biotinylated antibody mix for 30 min at RT and three more washing steps, Streptavidin-conjugated R-phycoerythrin (SAPE) was added for 10 min at RT (1∶100 dilution). After three final washing steps, beads were resuspended with 125 µl assay buffer, acquired and analyzed by the BioPlex Manager 6.0 software.Statistical analyses were performed by using SPSS software .All parameters were described as median. P-values <0.05 were considered as significant factors.Parametric values were analysed by T-Test. For non-parametric (distribution-free) parameters Mann-Whitney-U-tests were used. A Fisher exact test was calculated for the comparison of discrete variables.The association of IgM with liver related clinical endpoints was additionally identified by Cox-regression models and Kaplan- Meier analysis.To investigate the role of anti-HDV IgM testing in hepatitis delta, two independent patient cohorts were studied. Baseline characteristics of patients in both cohorts are shown in Anti-HDV IgM tested positive in 102 patients (85%) of the HIDIT-2 cohort and in 67 patients (85.9%) of the MHH cohort. 63% of patients in the HIDIT-2 cohort and 49% of patients in the MHH cohort presented with low anti-HDV IgM levels. More patients in the MHH cohort showed intermediate IgM values (32%) or even high IgM levels (5%) than in the HIDIT-2 cohort  .We next investigated if different categories of anti-HDV IgM levels were associated with clinical, biochemical or virological markers of liver disease in hepatitis delta. As shown in HDV-RNA levels, analyzed both as linear log-transformed values as well as grouped into low, median or high viremia, did not show any association with anti-HDV IgM categories in either of the cohorts . SimilarThus, high anti-HDV IgM groups were associated with biochemical and histological activity of liver disease and with lower HBV-DNA levels but were independent from HDV replication.To investigate if certain serum inflammatory patterns are associated with anti-HDV IgM levels, a large set of cytokines, chemokines and angiogenic factors was determined in plasma by multipex protein arrays . PatientTo illustrate the univariate ANOVA analysis of all measured parameters we developed a figure indicating which parameters were associated with the three anti-HDV IgM groups in the HIDIT-2 cohort and marked parameters that were high in the intermediate IgM group or low in the low IgM group .Clinical long-term outcome was investigated in 78 patients who were followed for up to 14 years . A liver-related clinical decompensation occurred only in one anti-HDV IgM negative patient (9%) but in 26 patients (39%) with positive IgM levels (p = 0.05). In addition to the Fisher exact test , endpoinThus, anti-HDV IgM testing may identify a subgroup of hepatitis delta patients with a very mild clinical course.Hepatitis delta is the most severe form of chronic viral hepatitis. Considering the limited efficacy and the significant side effect profile of PEG-IFNα therapy, biomarkers are needed to distinguish patients with a more benign natural course of liver disease from individuals who have a higher risk to experience clinical decompensation. We here suggest that anti-HDV IgM may represent such a marker. IgM levels were associated with histological disease activity and, maybe even more importantly, the absence of anti-HDV IgM identified a subgroup of patients with a very mild course of liver disease that may not require immediate antiviral therapy.The presence of IgM antibodies against a viral pathogen is usually considered to be associated with early acute infections. However, it is well established for various viral infections including hepatitis B that virus-specific IgM immunogloublins can be detected during all phases of the infection. E.g., anti-HBc IgM antibodies have been observed during flares of chronic hepatitis B The clinical value of anti-HDV IgM testing in patients with chronic hepatitis delta may be to identify patients with more advanced disease activity. Indeed, ALT and AST levels were significantly higher in patients with intermediate and high anti-HDV IgM levels. More importantly, patients with intermediate anti-HDV IgM levels had more frequently a high inflammatory activity than patients with low levels of or negative anti-HDV IgM. Remarkably, this association with disease activity also translated in differences in the clinical long-term outcome. Only one anti-HDV IgM-negative patient developed a clinical decompensation in the MHH cohort after a follow-up time of up to 12 years. Thus, in clinical practice, the decision to start a potentially toxic treatment based on PEG-IFNα could possibly be delayed in this subgroup of patients which represented around 15% of the cohorts analyzed in the current study. We do not believe that delaying therapy in anti-HDV IgM negative cases would reduce the chance to respond to PEG-IFNα therapy as our preliminary data from another international treatment trial indicated that lower anti-HDV IgM levels maybe associated with weaker but not better treatment responses Even though the specificity of a negative test to predict a benign clinical course was very high, the sensitivity of anti-HDV IgM testing concerning this particular read-out is still very limited. Grouping patients into low and intermediate anti-HDV IgM levels did not allow to further distinguish different clinical courses of hepatitis delta and thus additional biomarkers would be needed to identify more patients in which treatment may be safely deferred.Anti-HDV IgM levels were strongly associated with serum CXCL10 (IP-10) levels. CXCL10 has recently been investigated in other hepatitis virus infections and was proposed as a serum marker indicating the activation status of the interferon system. A possible link between serum CXCL10 and anti-HDV IgM could be that IFNα induces CXCL10 and stimulates plasma cells which, thereby, possible leads to an increase anti-HDV IgM levels In contrast to serum CXCL10, all other proinflammatory cytokines investigated in this study showed higher levels when anti-HDV IgM was negative. Thus, controlled HDV infection as indicated by lower disease activity and absence of anti-HDV IgM is associated with a functional broad proinflammatory immune response, indicated by high levels of these cytokines, while more severe disease seems to lead to exhaustion of the antiviral immune defence. Indeed, HDV-specific T cell responses are rather weak in chronic hepatitis delta with active disease Our study has obvious limitations. The clinical course of liver disease in the MHH cohort may have been altered by treatment interventions even though antiviral therapies were not associated with clinical long-term outcome. Moreover, future studies need to investigate anti-HDV IgM dynamics over time, which may even increase the value of IgM testing to predict the clinical outcome. Furthermore, anti-HDV IgM should be investigated in more detail during antiviral therapy. The anti-HDV IgM assay used here allowed reliable determination of semi-quantitative IgM levels; however, improved assays with a broader linear range would be needed to analyse anti-HDV IgM as a continuous linear variable. Finally, although a large cohort of HDV patients was studied, the absolute number of IgM negative patients was still limited. On the other hand, particular strengths of the study are the large sample size, the very well defined cohort of patients recruited in an international multicenter trial, and validation of key findings in an independent cohort.In summary, our date show that anti-HDV IgM testing is a relatively easy and robust marker which can provide important clinical information as IgM values were associated with disease activity and clinical long-term outcome. We suggest that IgM testing should be introduced in the routine clinical workup of hepatitis delta patients.Figure S1Evaluation of the anti-HDV IgM assay indicated deviations of the optical density values based on variation in time.(TIF)Click here for additional data file.Table S1All measured cytokines, chemokines and angiogenic factors associated with the groups of anti-HDV IgM based on ANOVA analysis.(DOCX)Click here for additional data file.


Microgrants are a mechanism for providing funding to community organizations or groups to support health initiatives. Little research to date has examined the use of microgrants in promoting physical activity (PA), and no studies have explored how microgrants may support PA promotion among adolescents. The purpose of this study was to explore the role of microgrants in enhancing PA opportunities for Canadian adolescents.M = 16.3, SD = 2.6) and 17 adult organizers/instructors . Interview transcripts were inductively and deductively coded to identify concepts and create a hierarchy of themes.Employing a case study approach, nine community organizations from across Canada were selected as cases providing sports and physical activities with the support of microgrant funding. Researchers visited each organization and conducted semi-structured interviews with 40 program participants . Opportunities for PA programming would not have been possible without the microgrant funding. Microgrant funding was valuable in promoting PA for adolescents, and they afforded opportunities for adolescents to engage in new and/or nontraditional activities. In addition to promoting PA, the microgrants had benefits for participants and the community organizations including improved organizational capacity.Analysis produced themes regarding participants’ perceptions of the Microgrants appear to be an effective mechanism for enhancing community capacity to provide PA opportunities for Canadian adolescents by helping to reduce financial barriers and empowering adolescents to take an active role in identifying and hosting new and creative PA events within their communities. The majority of Canadian children and adolescents are insufficiently physically active to achieve health benefits . FurtherReducing the upfront costs of participation or access to services, and alleviating the burden of the associated expenses through tax deductions are two suggested approaches for addressing financial barriers to PA –14. In tMicrogrants are a mechanism for providing funding through subsidies to community organizations or groups to encourage public participation in health initiatives . They apFurthermore, there is no research examining microgrant funding for programs targeting children or adolescents. Thus, the introduction of a microgrants program in Canada to encourage adolescents ages 13 to 19 years to get physically active provides a unique opportunity to examine the potential role of such subsidies in promoting and facilitating PA. Specifically, ParticipACTION, a national not-for-profit Canadian organization dedicated to increasing PA in the Canadian population , supporthttp://www.participaction.com/teen-challenge/). Given limited research in this area, we adopted a qualitative approach in our investigation. This type of research comprises a wide range of approaches but it is usually characterized by rich description and narrative and is used to more closely represent the experience of participants. In particular, our goal was to develop a holistic and contextualized account of a purposefully sampled range of microgrant programs operating across Canada, and case study research is particularly well suited for such a goal  have really allowed it to happen from a grass roots place, organically is really great … having faith that people have great ideas and know what their community needs.” (YK organizer P5)Community organization representatives identified that one of the major benefits of the microgrants was the ability to use the money flexibly within their organization: “they give you a lot of flexibility in what you want to spend it on to promote your idea to get kids active” (MB organizer P1). The microgrants allowed organizers to develop programs which were responsive to their community’s needs:“It gives you that ability to tweak things. And so for us we realized realistically 10 weeks with holidays is too long of a challenge … So that’s something I would not have known until we actually delivered the program … I think that’s what’s cool about [the microgrants] because they will help fund the same [program] again, and they will help, in a sense tweak it.” (ON organizer P2)Organizers also spoke about the value of the microgrants as opportunities to try out new programs or activities, or to refine the programs or events within the organization:Organizers in Québec said that in addition to being able to try out new activities, they were also happy that they could use the funding to maintain programs that had been previously developed: “there is a reality, do not reinvent the wheel. There were two skiing [programs]; even if they were similar, this is not serious. If something works, it isn’t necessary to always innovate. We must do what young people like” (QC organizer P6). The microgrants were unique funding opportunities because they allowed community organizations to sustain previously-established programs/events, or organizations could develop a completely new PA program/event based upon the teen participants’ interests. This process of having the opportunity to pilot programs helped organizers to determine which programs were going to be viable and sustainable within their organization and to develop ‘best practices’ for running programs in the future. Thus, the flexible use of grant funds was considered a major strength of the microgrants.Many of the community organizations that received funding served as the main providers of PA opportunities for adolescents in the community. For example, the leader of a teen group said: “there’s nothing here for them so I just felt bad about it” (NL organizer P7). In some communities the local high schools served as a provider of PA opportunities for adolescents, particularly for adolescents from low socioeconomic households: “I think that money is kind of a part of it because I know that dance is really expensive, and there are a lot of sports that are really expensive so a lot of kids don’t have enough money to do those things” (YK organizer P5). As a result, a common theme throughout the interviews was that sport and PA was often “available but not accessible” to adolescents (QC organizer P8). The idea of activities being available but not accessible was particularly evident for families of adolescents with disabilities in small communities in Newfoundland & Labrador. Program organizers said that a swimming program served as one of the only sources of PA for the adolescents: “a lot of kids don’t feel comfortable going into the regular stream programming. But there’s nothing else…a lack of funding available for these families for recreation is big as well” (NL organizer P9).Consequently, the microgrants enabled community organizations to make PA affordable and accessible within the community: “By giving these young people a wide range of activities, the resulting impact provides accessibility” (QC organizer P6). A teen from Manitoba said that without the funding to subsidize the school’s program, “it would have been more of a financial strain on my parents because they don’t really have the most optimal jobs and I don’t work either so I wouldn’t have been able to pay for it” (MB participant P14).“I’ve applied for a lot of grants for a lot of things that require you to write so much and they’re looking for something very specific … I really like the process for this one a lot better.” (NS organizer P4)Program organizers identified two key facilitators for running their programs/events. First, all the program organizers discussed the ease of applying for the Teen Physical Activity Grants and said the application process helped them run the program or event:“In terms of, like ParticipACTION [Teen Challenge], the biggest takeaway I have was that because of the challenge, the way they worded challenge, it’s helped us on the organizational side get organized. ‘Cause we’re a new charity, we have all these ideas but we didn’t know how to make it most effective, and also we didn’t know how to measure. So we were just writing a program every Saturday with no real focus. The challenges have actually helped us focus and they’ve actually helped us in terms of making it a little bit better.” (ON organizer P2)The grant application process was also useful for new organizations that did not have much experience applying for grants:“This is going to sound kind of silly but what helps to run the programs is to help keep the people running the programs from burning out … just making sure that anybody who is running these things has that kind of underlying support so that they don’t run out of energy to keep these activities going for the kids.” (MB organizer P8)Another facilitator was having motivated, supportive leaders and instructors as the ‘champions’ for running programs/events: “I think that once you get an outside instructor locked down it becomes easy. A regular outside person that has good rapport with the kids” (NS organizer P4). It was also important to ensure the organizers and instructors had the support necessary to continue running the programs or planning events:The community organizers reported a number of general challenges to running PA programs, including a lack of space or infrastructure to host events/programs, difficulties in recruiting volunteers  to run events/programs, and insufficient government funding for PA programming. First and foremost, the organizers indicated that PA programming was expensive and they lacked money to run programs or events, which was one reason they were appreciative of the Teen Physical Activity Grants: “One of our biggest complaints is cost obviously. Yeah I think if I had to put my finger on the biggest challenge it’s trying to keep the costs affordable” (BC organizer P1). For example, hiring instructors to lead teen programs was often expensive: “I’d love to have more money to be able to pay the artists who are coming in to teach the kids what they’re worth as opposed to what the going cheapy rate is, so I always feel bad about that” (NS organizer P4). Another organizer in Manitoba said that cost was a challenge in trying to provide PA programming to large groups of teens: “it does get expensive especially when you’re working with large numbers of people so we have been getting, trying to be very creative with our funding and got used equipment and so on. I think the more we remove those barriers I think the easier it is for kids to participate” (MB organizer P1).Some community organizations also reported difficulty in recruiting and retaining teens for programs/events: “One of the challenges is getting kids. You know like if you start something and you have 50 people sign up, after 3 or 4 months you only have 10 people left, and that’s what always happens with me” (NS organizer P4). Organizers identified that increasing teen recruitment was one of their main objectives for the future: “we have been trying to recruit more, that’s always been our priority really so we would just continue to do that” (NL organizer P3). Conversely, other community organizations reported that running programs or events with the Teen Physical Activity Grants increased enrollment in their programs (see below: Sustainability and legacy). Organizers who reported strong enrollment said that they used targeted approaches to advertising the program/event through word of mouth, media such as radio announcements, social media including Facebook and Twitter accounts updated by youth participating in the program, and through advertisements in local newsletters. Community organizers said that engaging youth in the initial development and advertising of the programs/events was also a key strategy to overcome the challenge of recruiting and retaining teens.The microgrants were valuable in promoting PA opportunities for adolescents in communities across Canada. However, the microgrants had value beyond providing opportunities for PA: we also identified ways in which the microgrants had an impact for the organization, for the individuals participating in the programs, and for the participants’ families and communities.“Definitely it allowed us to strengthen [partnerships with other organizations]. Minor hockey has way more money than we do. And they can offer so many more things than we can and normally we would not be able to participate in that kind of day with them because our costs are much higher.” (BC organizer P1)The microgrants allowed community organizations to build and strengthen partnerships with other groups in the community. For example, a skating club in British Columbia partnered with a minor hockey organization to run a ‘try it free’ skating day. The organizer said that without the grant funding, the skating club would not have been able to participate with other organizations to run the event:“I can know a sponsor-partner but if I don’t have any money to get an advantage from him, then there’s no point … Previously, I was working at Sport Expert and the boss, I know him well … so he gave us some discounts. So, we had a bit more. For example, for five hundred dollars, you can usually buy a pair of skis if you are lucky, so if it isn’t too expensive and on special … Anyway, we got a lot more than what we could with regular prices so that helped a lot. The money helped our ski club a lot.” (QC organizer P8)The microgrants were used to maintain existing partnerships. For example, a community organizer from a fitness centre in Prince Edward Island sought funding to allow youth in grade nine to participate in a weekly fitness class to familiarize them with the fitness centre and teach them about PA and working out at a gym, which addressed the strength-training curriculum in the students’ physical education classes. The microgrant funding was also used to work with existing community partners to maximize the impact it could have. For example, organizers purchasing ski equipment had existing relationships with the manager of a sporting goods store, and they were able to purchase ski equipment and maximize their relationship with the store:“… in terms of the granting world how it works is when you go to apply for these grants they will only give you what you’ve managed. So these little grants over time are helping us show we manage our money responsibly, we’re able to deliver these things with the money. It helps build credibility with these other granters. And now this is our 3rd year and we just got approved for a $10,000 grant from a church, and it looks like we might get some other funding from [other granting agencies]. I would say it does help in terms of building credibility.” (ON organizer P2)The microgrants helped community organizations to build credibility that they were competent and able to secure and use grant funding successfully. One organizer said:“I think success in grants definitely creates more buzz and success in grants. I think people who offer grant money want to see that it's be used. They want to see it's going to be used the way you said it was going to be used. They like to see a product of some kind …there's definitely a buzz about grants and getting out there as long as you know you can do it. Then I think that one grant turns into another, turns into another, turns into another.” (NS organizer P4)This organizer also said that the microgrant funding could be used as leverage to obtain additional funding from other organizations: “if we’re able to get $500 from ParticipACTION [Teen Challenge] and $500 from the police, now we have $1,000 budget, the program is going to grow” (ON organizer P2). Similarly, organizers in Nova Scotia said that by getting smaller grants such as the microgrants, it generated more grant funding opportunities for community organizations:Thus, another benefit of the ParticipACTION Teen Challenge Grant Funding was the ability for the community organizations to apply for further grants. If a Teen Challenge Grant was used to successfully implement a PA program or event, the organizers were appreciative of the option to apply for additional Teen Challenge Grants to support the program over time.Community organizers in Québec also noted the value in obtaining funding from the ParticipACTION Teen Challenge program and the importance of associating themselves with organizations with the same values and priority on PA: “We attempted to associate ourselves, like with ParticipACTION, to promote sports, PA” (QC organizer P6). The director of a skating program said that when advertising the ‘try it free’ days and a skating scholarship program, “we actually put this article in the paper stating that it was in partnership with Coca-Cola and ParticipACTION” (BC organizer P1). Thus, by obtaining funding from the ParticipACTION Teen Challenge program, community organizations built credibility around their mission and their ability to use funding to support PA programs for adolescents within the community.In some cases the microgrants were used to sustain programs and created a lasting legacy beyond the duration of the program or event. One of the grants was used to hold a ski and snowboard event in Québec and the money was used in part to purchase ski equipment for the event; the equipment was then used by the community organization’s ski club. The ski club had been a tradition in the community for nearly 30 years, although it had suffered from low attendance and high costs. “We want to keep it here because it’s a tradition. So we work very hard to keep it running. So, I think without the equipment maybe it wouldn’t be, it wouldn’t have been possible to keep it … So this allowed the club to continue” (QC organizer P8).One high school received funding to purchase new basketball jerseys, which improved athletes’ enjoyment while participating on the team and instilled a sense of school pride: “the uniforms offered us a way to have a legacy to continue using them to actually give the kids a set that they’d be proud to wear, represent our school in other buildings and you know I just thought there was great value in that” (MB organizer P1). One teen spoke about how wearing ‘real’ basketball jerseys helped to improve others’ perceptions of the inner-city school: “Well everyone around the school and other schools they basically say that our school’s really ghetto, so the [new] uniforms don’t make us look really ghetto” (MB organizer P3).Several community organizations also said that running the programs or events with the microgrants increased teen enrollment in their programs. For example, one organizer said, “it increased our membership which increased our income, which then increased our staffing and our equipment, and just overall just everything got better for everyone” (BC organizer P1).“I’ve been watching [breakdancing] forever and I’ve never gotten a chance to learn it, now I do. And it was a big bonus that I got in shape, but it wasn’t really a factor that pushed me, because I wasn’t like ‘man I really need to get in shape.’ I was just kind of like, I really like this, I want to do this, and I happened to get in shape because of it.” (NS participant P5)The programs or events offered with the microgrant funding allowed adolescents opportunities to develop existing physical abilities or to be exposed to new physical activities: “I never used to be able to swim on my own without anybody really close to me, but now I can swim on my own and not have to worry that I’m going to drown” (NL participant P7). The majority of adolescents said that by trying new activities, they also improved their physical fitness:One community organizer said that exposing youth to various activities helped to reinforce the message that “you don’t have to be an athlete to be active” (PEI organizer P4). Furthermore, adolescents said that the programs funded by the microgrants enabled them to discover a passion for an activity they had never experienced before: “It gave me something to do that I like enjoy doing, like I never had anything else that I liked so much before. Does that make sense? I finally found something that I think I would be doing for a really long time” (YK participant P1).“I’ve gotten noticeably thinner so … people are just asking if I had gone on a diet, if I had been exercising more. I say yeah, I just got more active … I kind of feel proud that I’m not the person I used to be and I’m proud that they are noticing that I’m making an effort to change. So it’s a kind of confidence boost there.”As a result of adolescents developing their physical abilities and trying new activities, the programs made it possible for adolescents to become more physically active and develop healthy lifestyles: “I’ve definitely become more physically active, and that’s a big difference in a person’s life … it’s made me healthier, it’s made me more aware, so I make better choices, and that’s definitely changed my life by doing things like that” (PEI participant P2). A teen in Ontario said that without the boxing program, “I would be way less active, I would come home, play [video] games, do nothing” (ON participant P6). In Québec, a teen said that before playing sports at the youth centre, “I was not very energetic at all. I hardly did anything, only walking with my friends and we would talk. That was it. I did not do any sports, nothing. But, now things are different. A couple of my friends practice sport a lot, and we are active. It’s fun like that” (QC participant P2). Another teen in Nova Scotia said that as a result of the fitness program offered through the high school, “I got a lot more active and just said I’ll stay away from the couch, I’ll stay away from the computer, all that stuff, just other lifestyle choices that I made” (NS participant P2). He continued and said:“With sports I definitely lacked a lot of confidence. I mean even last year when I was in the sports I didn’t want to try a lot of things, different moves and things, tried to play it really safe … [Now] I will try new things, more difficult things. Yeah, [I] feel more confident on the team.” (PEI participant P3)The adolescents’ development of their physical abilities also changed the way they viewed themselves and felt about themselves. For example, some adolescents said that being more physically active helped them to feel better about their weight and their abilities:“If I don’t do this program, I don’t think I would ever knew how to protect myself, and if I got bullied or raped or anything I wouldn’t know how to protect myself … So boxing give me a lot, and my body changed … before I was a little big and when I came to boxing, my body changed, so [did] my personality, I became better person.” (ON participant P3)Another teen said that through his participation in the program he learned “it’s OK to be different, like being fat and skinny, or tall and short, ‘cause it doesn’t really measure your talent. You can be tall but not good at dribbling, or short and good at rebounding” (MB participant P5). A girl participating in a boxing/life skills program said,Thus, the development of physical abilities helped the adolescents to develop a positive self-image as a result of participating in the community organizations’ programs or events.“I feel I have more confidence … Whereas before I had a massive inferiority complex. I used to be incredibly hard on myself, I would push myself to be better than other people just to feel like I could be more successful in ways that they were or successful in ways that they weren’t. But now I’ve stopped comparing myself to others and it was about a month after I started [the program] that so I think there is a correlation there.” (NS participant P2)Adolescents participating in the PA challenges also reported that they gained self-esteem, confidence, leadership, and social skills:He continued, saying “I’m much more confident with myself now, I would say that breakdancing has been a big part of that.” The adolescents’ personal growth was corroborated by the adult organizers and instructors: “their self-esteem changes and they become very proud of what they’re doing and ‘look at me, look at what I can do’” (BC participant P1).The adolescents’ positive growth and social skills also transferred into other areas of their lives. For example, Michael (pseudonym) was a teen with a disability who participated in a swimming program, and his aunt reported that prior to engaging in the swimming program he would not have felt comfortable even participating in an interview about his experiences: “he’s not a loner anymore … this is different too, for him to talk to a stranger and answer questions … And I said how do you feel about that? And he said ‘that’s OK Aunt Susan’” (NL participant P6). In Michael’s case, participating in a social activity such as a swimming program helped him to become more comfortable interacting with strangers in everyday life. Another teen who participated in a dance program and was able to go on a trip with his classmates said “Coming from a different country to Canada where things are a lot different, at first I didn’t really know how to engage in some of this stuff but once I started joining … I kind of gained the confidence that I know I need to live in this foreign country so I kind of developed a lot of confidence and self-esteem” (MB participant P12).“The ability that [the grants] allow us, like I say, to offer a free program is a big plus because a lot of our families are financially strapped so this gives them an opportunity to partake, and not only our participants partake but other kids that they’re financially strapped to keep active. ‘Cause keeping active is not cheap, to be involved in sports and other recreational programs are not cheap, so the fact that we can allow this not only for our participants, for their family, is a big thing.” (NL organizer P9)The microgrants had a broader impact on the communities beyond the adolescents or the organizations that benefitted from the funding. A breakdance program served as an opportunity to develop stronger community relationships with new Canadians: “creating stronger partnerships with the international community right, like with the Filipino community … I think we have the same opportunity around dance to do that [develop partnerships] because that’s another thing that we’re very, very interested in” (YK organizer P5). Some of the PA programs or events offered opportunities for families to engage in affordable PA together. Organizers in Newfoundland & Labrador said:Similarly, organizers running a teen boxing program hoped to have an impact on PA and healthy lifestyles for adolescents’ families: “It’s all about inclusion and even if you’re like, ‘you know what, I want to bring my big sister, I want to bring my mom,’ bring your mom, she can work out too, we don’t care, right, it’s all about creating that blueprint for life, it’s like a whole family working out together” (ON organizer P2). Adolescents also said that their increased PA had a positive impact on their family members: “it kind of passed over to my family … we've all been kind of getting more physically active. So it's kind of like a domino effect, like this starts and then the rest follows” (PEI participant P2).“The whole goal is for kids in the program to later come back and become mentors. So for example one of the programs we ran with ParticipACTION is called ‘my turn to coach.’ The coach designs a workout plan, they assign tasks, they do a report, they learn to invoice, and then they get an honorarium of $20. So they’re actually getting real life skills as well as sometimes cash.” (ON organizer P2)There were several examples of community organizations and events/programs supported by the microgrants contributing to the development of volunteers and teen leaders, which in turn benefitted the organizations as well as the wider community. In some cases, adolescents were informal peer leaders when teaching one another skills, such as in the case of the breakdancing programs in some communities. In other cases, adolescents had more formal volunteer and leadership roles, such as helping with fundraising and event organization. For example, organizers in Québec engaged their teen advisory board to provide input for PA programming and to run the ski and snowboard day at the youth centre. A community organization in Ontario involved adolescents in the process of leading boxing classes and also in writing grant reports following the completion of events or programs:“You learn how to volunteer, you learn how to organize things, you learn how to say ‘OK this time I need this,’ and you learn how to schedule, you learn penmanship, people are like ‘I can’t read your writing,’ you’re like ‘oh I better fix that.’ You learn typing skills, e-mail skills, language skills, you get more opportunities ’cause you’re talking to parents and you have to talk respectfully, but you have to assert yourself as a person of authority, so you learn that kind of thing.” (BC participant P2)Teen volunteers reported that they learned a variety of skills through volunteering with the community organizations:“It really helped me realize how much I enjoy being active in school, active in the community and extracurricular activities … I started joining other programs that would help out with things because I enjoy being part of something, so it helped me realize that I enjoy being part of something big really and helping and meeting new people.” (NS participant P6)Adolescents also expressed that they enjoyed becoming more involved in their community through volunteering:The objective of this study was to explore the role of microgrants for enhancing PA opportunities for Canadian adolescents. Overall, there was considerable consistency among participants across the cases in describing the positive impact of the grants in their local context. In several cases, opportunities for PA programming would simply not have been possible without funding. How organizations ‘stretched’ the relatively modest grants to achieve their goals and the creativity with which many grants were put to use were both remarkable and innovative.There are clear socioeconomic disparities in PA among children in Canada , 6. The The microgrants had impacts both at an organizational level with community partners and an individual level for adolescents themselves. Any increases in PA at an individual level are unlikely if organizational capacity to support PA opportunities is not enhanced , 33. AccOne strength of this qualitative research was that it sampled a broad range of cases across Canada, and a qualitative approach was valuable in highlighting the range of benefits and positive impacts that the microgrant funding had for organizations, participants, and communities/families, beyond promoting PA for adolescents. Many of the adolescents reported that they were more active as a result of the event or program funded by the microgrants, however caution is required in determining the impact of the microgrants on actual PA levels of participating adolescents due to our qualitative research design. Building on this qualitative study, objective measurement of PA is likely needed to accurately assess such impact, although an appropriate research design to test such effects is likely challenging for two main reasons: first, to objectively assess the impact of microgrant-funded programs on adolescents’ PA, it would be necessary to track adolescents’ activity before and after the implementation of a PA program supported by a microgrant, which would be logistically challenging considering it is not known which adolescents might participate in any given program; second, the cost associated with such an approach would likely be substantial. What can be clearly supported is the role of the grants in exposing adolescents to a range of new activities and opportunities. Such experience might increase the chances of those adolescents finding an activity that best suits their interests and abilities which is more likely to lead to sustained engagement. A critique of traditional physical education curricula and school based sport programs is an unbalanced emphasis upon competition and team games which are taught using a limited range of didactic pedagogic approaches . The majThe importance of a ‘champion’ in the community that seeks out funding, motivates participation, and engages adolescents was seen as a key factor in the success of the cases. However the sustainability of programs does become less assured if there is reliance on one individual. Despite the value of the microgrants, community organizers still faced challenges in terms of infrastructure, funding and volunteers to help run programs. Importantly, retaining adolescent participation was a challenge at some sites. Most organizations were attempting to examine how best to engage youth earlier in the development of the programmes and in reaching out to other youth using modes of communication such as social media which may be more resonant with adolescents. Participants provided a range of recommendations for other organizations in seeking funding and running programs including: 1) offering programs on a regular and consistent basis, such as running programs in the same location on a weekly basis instead of monthly; 2) engaging adolescents in program development by creating a teen advisory committee to discuss how funding should be used and to volunteer in running programs and preparing reports after the progam/event; 3) providing introductory sport and PA programs for beginners; 4) using funding to partner with local stores and businesses to provide discounted equipment or supplies for running programs/events; and 5) using funding to purchase resources that can last beyond the event or program and be re-purposed for future programs or physical activities within the organization.Given calls for further research examining the use of microgrants in the promotion of PA , the curFuture evaluations of the ParticipACTION Teen Challenge should examine the extent to which the programs that receive Teen Physical Activity Grants are sustained over the long term. Thus, questions relating to infrastructure, programming, and impact on the PA of adolescents could be addressed. Along these lines, it would also be useful to examine more thoroughly the context in which the Teen Physical Activity Grants are more or less effective, for example based on location [rural/urban], within isolated communities, the population being served, and the programming of new or unique activities. Finally, gaining insight on the economic impact and organizations’ leveraging of other funding by offering such microgrants would be a valuable contribution to the literature and practice.Based on this analysis of nine case studies, modestly scaled microgrants appear to be an effective mechanism for enhancing community capacity to provide opportunities for Canadian adolescents to engage in PA. Microgrants can help reduce financial barriers and empower adolescents to take an active role in identifying and hosting new and creative PA events within their communities. These types of funding programs may play an important role in promoting and enabling PA across populations, and the Teen Physical Activity Grants were found to have positive impacts for the organizations, participants, and for the broader community. Thus, microgrants merit further consideration as a mechanism for PA promotion at a population level.aThree participants ages 21, 22, and 25 took part in a swimming program for individuals with disabilities. The program focused on adolescents and their families, but organizers did not prevent older individuals from participating. We chose to include their experiences to represent the diversity of cases supported by the microgrant funding.KT is an assistant professor in the Faculty of Kinesiology and Physical Education at the University of Toronto. GF is a professor in the Faculty of Kinesiology and Physical Education at the University of Toronto and a Canadian Institutes of Health Research-Public Health Agency of Canada (CIHR-PHAC) Chair in Applied Public Health. CW is an assistant professor in the Faculty of Health Sciences at the University of Lethbridge. JS is a professor at in the Faculty of Physical Education and Recreation at the University of Alberta.


The paper titled “Process Evaluation of a Positive Youth Development Program in Hong Kong Based on Different Cohorts” , publish


Re-biopsy for resistant non-small cell lung cancer (NSCLC) after treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is important for selection of better therapy, but there have been no reports about the utility of endobronchial ultrasound (EBUS)-guided procedures for such purpose. The aim of this study was to evaluate the utility of EBUS-guided re-biopsy for resistant NSCLC after treatment with EGFR-TKIs.From January 2013 to December 2015, 53 consecutive patients who underwent EBUS-guided re-biopsy for mutation analysis of NSCLC after EGFR-TKI treatment were assessed.n = 6) or insufficient specimen (n = 5). There were no related severe complications.Nine patients underwent EBUS-guided transbronchial needle aspiration (EBUS-TBNA) and 44 patients underwent EBUS with a guide sheath (EBUS-GS) transbronchial biopsy. The technical success rates were 100 %. As for mutation analysis, all 9 specimens (100 %) from EBUS-TBNA and 33 specimens (75.0 %) from EBUS-GS were adequate for gene profiling. The remaining 11 specimens from EBUS-GS procedures were inadequate for mutation analysis owing to the absence of tumor component in the sample (Re-biopsy by both EBUS-TBNA and EBUS-GS were useful and safe sampling procedures for mutation analysis of EGFR-TKI resistant NSCLC. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been demonstrated to be effective in treating non-small cell lung cancer (NSCLC) patients with EGFR mutations –3. EGFR-Until now, several studies have uncovered several mechanisms of acquired resistance, such as secondary EGFR mutations (T790M mutation), mesenchymal-to-epithelial transition factor receptor (MET) amplification, and human epidermal growth factor receptor 2 (HER2) gene amplification . Among tIn November 2015, a third generation EGFR-TKI  has been approved by the US Food and Drug Administration (FDA) to treat patients with a type of advanced NSCLC that has a specific EGFR mutation, called T790M, and which has become worse after treatment with other EGFR-TKIs. Osimertinib has shown clinical effectiveness and tolerability in NSCLC patients with T790M mutation of EGFR .Examining T790M is very important in treatment selection. Because EGFR-TKI treatment is indicated for advanced and unresectable NSCLC, examination for T790M mutation is usually performed on small biopsy specimens.So far, most reports on re-biopsy procedures after EGFR-TKI resistance have been on computed tomography-guided transthoracic needle biopsy (CTNB), which is also the common first method of choice for the initial diagnosis of lung cancer . HoweverAnother biopsy approach is by bronchoscopy, but its use has been limited by a lower diagnostic accuracy compared with CTNB. Recently, endobronchial ultrasound (EBUS) guidance during bronchoscopy procedures, such as EBUS guided-transbronchial needle aspiration (EBUS-TBNA) and EBUS with a guide sheath (EBUS-GS) transbronchial biopsy, have been introduced and improved the diagnostic accuracy of bronchoscopy , 11. HowIn this study, we evaluated the efficacy and safety of EBUS procedures for re-biopsy and mutation analysis of EGFR-TKI resistant NSCLC.This study was approved by the National Cancer Center Institutional Review Board. Written informed consent for the procedure was obtained from all patients. Consecutive patients who underwent re-biopsy by EBUS procedures for mutation analysis of NSCLC after EGFR-TKI treatment at the hospital between January 2013 and December 2015 were enrolled. There were 2907 patients in whom EBUS-guided bronchoscopy procedures were performed during the study period. Among these, 89 patients were for re-biopsy purposes. Among these re-biopsy patients, we selected NSCLC patients who were treated with at least one EGFR-TKI regimen. Disease progression during chemotherapy, based on the RECIST ver1.1 definition, was a criterion for inclusion; 36 patients were ineligible because of the absence of prior EGFR-TKI treatment.All bronchoscopies were performed via the oral route under local anesthesia with mild sedation by intravenous administration of midazolam.EBUS-GS was performed for patients with peripheral pulmonary lesions (PPLs) without mediastinal and hilar lymphadenopathy; a GS kit  and a radial EBUS (R-EBUS) probe  were used. The bronchial route was planned by reviewing the chest CT images before EBUS-GS. Virtual bronchoscopic navigation/simulation systems  were used to detect the bronchial route to the target PPLs. A PPL was defined as an abnormal growth surrounded by normal lung parenchyma and was bronchoscopically invisible. Upon reaching the target bronchus, the GS together with the R-EBUS probe was inserted through the working channel of the bronchovideoscope and was advanced towards the PPL under fluoroscopic guidance . Ultrasound scanning was performed while manipulating the R-EBUS probe until the lesion was localized by the corresponding EBUS image. After confirming the location of the PPLs, the R-EBUS probe was removed while keeping the GS in place for the usual transbronchial sampling using forceps, brush, and/or transbronchial needle aspiration under fluoroscopic guidance. Lesion location was assigned based on a study of Baaklini et al. , but witEBUS-TBNA was performed for patients who had mediastinal and hilar lymphadenopathy. EBUS–TBNA was performed using either one of the following 22G needles: Vizishot  or SonoTip EBUS Pro with stainless steel . The convex probe EBUS  was inserted through the oral route in the same way as during usual bronchoscopy. The ultrasound images were generated using a dedicated ultrasound processor . When the target lesions were visualized by CP-EBUS, the TBNA needle was inserted through the working channel of the bronchoscope and was advanced to penetrate the tracheobronchial wall into the target lesion under real-time EBUS guidance. Aspiration was done by moving the needle back and forth inside the target lesion for 20–30 times, under negative pressure. After sampling, suction was released before retracting the needle from the scope.Cytology and histology specimens were sent for pathologic examination. Positive diagnostic criteria suitable for molecular analysis were defined by the presence of malignant cells based on histologic features or based on class IV/V on cytology examination by Papanicolaou stain. Overall detection rate was based on a positive diagnosis by histology and/or cytology.EGFR mutation analyses of the genomic DNA extracted from tumor samples were performed using the Scorpion ARMS method.www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html), a graphical user interface for R  and a modified version of R commander (version 1.8-4).Descriptive statistics were presented as frequency, percentage, and median (range). Univariate analyses were performed using Fisher’s exact test. Multivariate analysis using logistic regression was performed to determine the factors associated with the yield. Variables selected via univariate analyses  were evaluated using multivariate logistic regression analysis. A two-tailed P value <0.05 was considered to indicate statistical significance. Correlation of study variables were performed with EZR  or insufficient specimen (n = 5).For re-biopsy, EBUS–TBNA was performed in 9 patients, whereas EBUS-GS was performed in 44 patients. The technical success rates of the re-biopsy procedures were 100 %. All 9 specimens (100 %) from EBUS-TBNA and 33 specimens (75.0 %) from EBUS-GS were adequate for gene profiling and mutation analysis. The remaining 11 specimens from EBUS-GS procedures were inadequate for mutation analysis owing to the absence of tumor component in the sample (The baseline characteristics of the study population are summarized in Table The adequacy of the re-biopsy specimens for mutation analysis is described in Table The factors affecting re-biopsy by EBUS-GS are shown in Table There were no severe complications after both EBUS-TBNA and EBUS-GS re-biopsy procedures.Currently, the significance of re-biopsy for mutation analysis of NSCLC has been increasing because of a wider range of therapeutic options. The standard cytotoxic chemotherapy for NSCLC patients has limited therapeutic response . MoreoveTo our best knowledge, this study was the first to demonstrate the utility of bronchoscopic procedures, especially with EBUS guidance, for mutation analysis of NSCLC after EGFR-TKI therapy. EBUS is a very important procedure to determine and collect samples from target sites in the mediastinal, hilar, and peripheral locations under real-time ultrasound , 18. AdeEBUS-TBNA is an established minimally invasive procedure for proper staging and diagnosis of lung cancer , 21. In In this study, EBUS-GS was able to obtain samples for mutation analysis of NSCLC after EGFR-TKI. However, when compared with EBUS-TBNA, the detection rate for malignancy was only 75 %. The yield of EBUS-GS for primary diagnosis of PPLs has been reported to be about 70–80 % , which iCTNB is another diagnostic procedure for PPLs, with a relatively high rate of both diagnostic accuracy and complications, such as pneumothorax. On the contrary, EBUS-GS was a safe method . HoweverAnother alternative approach, specifically liquid biopsy, now present as a crucial point in the field . Liquid The limitations of this study were its retrospective and single-institution design. Prospective, multi-center trials are ideal and recommended in the future.EBUS procedures for re-biopsy were useful sampling methods for mutation analysis of NSCLC after EGFR-TKI treatment. This could play an important role in the choice of better targeted therapy and for the development of a novel treatment for advanced lung cancer patients.CP-EBUS, convex probe endobronchial ultrasound; CTNB, computed tomography-guided transthoracic needle biopsy; EBUS, endobronchial ultrasound; EBUS-GS, endobronchial ultrasound with a guide sheath; EBUS-TBNA, endobronchial ultrasound guided-transbronchial needle aspiration; EGFR-TKIs, epidermal growth factor receptor-tyrosine kinase inhibitors; NSCLC, non-small cell lung cancer; FDA, Food and Drug Administration; HER2, human epidermal growth factor receptor 2; MET, mesenchymal-to-epithelial transition factor receptor; PPLs, peripheral pulmonary lesions; R-EBUS, radial endobronchial ultrasound


Acute liver injury is a clinically important pathology and results in the release of Danger Associated Molecular Patterns, which initiate an immune response. Withdrawal of the injurious agent and curtailing any pathogenic secondary immune response may allow spontaneous resolution of injury. The role B cells and Immunoglobulin M (IgM) play in acute liver injury is largely unknown and it was proposed that B cells and/or IgM would play a significant role in its pathogenesis.Tissue from 3 models of experimental liver injury  and patients transplanted following paracetamol overdose were stained for evidence of IgM deposition. Mice deficient in B cells (and IgM) were used to dissect out the role B cells and/or IgM played in the development or resolution of injury. Serum transfer into mice lacking IgM was used to establish the role IgM plays in injury.Significant deposition of IgM was seen in the explanted livers of patients transplanted following paracetamol overdose as well as in 3 experimental models of acute liver injury . Serum transfer into IgM-deficient mice failed to reconstitute injury (p = 0.66), despite successful engraftment of IgM. Mice deficient in both T and B cells (RAG1-/-) mice (p<0.001), but not B cell deficient (μMT) mice (p = 0.93), were significantly protected from injury. Further interrogation with T cell deficient (CD3εKO) mice confirmed that the T cell component is a key mediator of sterile liver injury. Mice deficient in B cells and IgM mice did not have a significant delay in resolution following acute liver injury.IgM deposition appears to be common feature of both human and murine sterile liver injury. However, neither IgM nor B cells, play a significant role in the development of or resolution from acute liver injury. T cells appear to be key mediators of injury. In conclusion, the therapeutic targeting of IgM or B cells (e.g. with Rituximab) would have limited benefit in protecting patients from acute liver injury. The term acute liver injury (ALI) encompasses a spectrum of sterile or infective hepatocellular insults characterised by acute inflammation within the liver. Injury results in the release of Danger Associated Molecular Patterns (DAMPs), which initiate an immune response. Withdrawal of the injurious agent and curtailing any pathogenic secondary immune response may allow spontaneous resolution of injury , 2.ALI may progress to acute liver failure, which is associated with a mortality of up to 50% , 4. In tIRI results from the interruption then reinstatement of an organ’s blood supply. It limits access to donor organs and has been linked to early graft failure, as well as both acute and chronic rejection , 8. IRI B cells are capable of shaping the nature of an immune response through their ability to present antigen and via their ability to produce both cytokines and antibodies. This may have a pro-inflammatory or regulatory influence on the resulting immune response . B cellsImmunoglobulin M (IgM) is produced by B cells and is a normal component of serum. Natural IgM is thought to be produced as a response to self-antigen; it is polyreactive and has a role in the clearance of apoptotic cells . IgM wasThe role IgM and B cells play in acute liver injury is largely unknown and it was proposed that IgM and/or B cells would play a significant role in its pathogenesis.Following local ethical approval at the University of Edinburgh, animal work was carried out according to UK Home Office regulations  under licenses 60/4045 and 60/2605. Mice were housed under specific pathogen-free conditions at the University of Edinburgh. General anaesthesia (GA) was induced via inhaled isoflurane and post-operatively subcutaneous opioid analgesia (buprenorphine) was administered. Animals were sacrificed under GA by terminal exsanguination by way of cardiac puncture.The Tissue Governance Unit (South East Scotland SAHSC BioResource) provided ethical approval for the human tissue used in this study.All wild type (WT), knockout and transgenic mice were on a C57BL/6J background and matched for age and sex. RAG1-/- lack IgM and both mature B and T cells . μMT micIn this model of warm partial hepatic IRI, an atraumatic clamp was applied to the vascular pedicle supplying the left lobe for 40 minutes; the liver was then allowed to reperfuse for between 1 hour and 7 days. Care was taken to maintain intraoperative core body temperature at 36°C to minimise the masking effects of hypothermia on liver IRI . In seruParacetamol-induced liver injury and concanavalin A (con A) hepatitis models were used as alternative models of sterile liver inflammation. Briefly, following a period of 18–24 hours of fasting, mice were given a 200mg/kg intraperitoneal injection of paracetamol . Con A hepatitis is a model of immune-mediated hepatitis  and was The extent of any liver injury was assessed in terms of the serum level of alanine aminotransfersase (ALT), a biochemical marker of liver injury . ALT wasThe Tissue Governance Unit (South East Scotland SAHSC BioResource) kindly provided formalin-fixed sections from the explanted livers of patients requiring transplantation following paracetamol overdose; tissue blocks were specifically chosen by a consultant pathologist with a special interest in transplantation to include areas of both injured and uninjured liver. Antigen retrieval on these sections was performed by heating samples with Borg Decloaker . Tissue from the models of murine liver injury were fixed in methacarn and did not require a specific stage of antigen retrieval.Sections were deparaffinised and rehydrated before endogenous peroxidase and avidin/biotin activity were quenched, prior to incubating with a goat anti-mouse IgM antibody  at a dilution of 1 in 5000 or goat anti-human IgM antibody  at a dilution of 1 in 1000. Slides were subsequently incubated at room temperature with a polyclonal rabbit anti-goat biotinylated secondary antibody  at 1 in 400 dilution for 40 minutes. Sections were then developed with VectaStain RTU Elite (Vector Laboratories) followed by diaminobenzidine, before being counterstained.IgM deposition was quantified from 6 randomly taken images at 200x magnification from each sample using ImageJ software ; it is expressed as percentage of total area.Single cell preparations from the liver were generated by a combination of mechanical  and enzymatic  digestion. This was then passed through a 70μm filter and centrifuged at 50g for 5 minutes to remove hepatocytes, clumps of cells and debris. Red cells were lysed . Immune cells were isolated by positive selection using a CD45+ MicroBead AutoMACS separation (Miltenyi Biotec) and then stained with a fixable Live-Dead marker  and a multi-colour panel of antibodies, including CD3, CD4, CD8, CD19 and NK1.1 . Samples were then run on a BD LSR II Fortessa  and analysed with FlowJo software . B cells were defined as CD3-CD19+ cells, within a forward-side scatter defined lymphocyte gate.Groups were analysed with the aid of Prism 5 for Mac OSX ; statistical methods are referred to specifically in the results section.To investigate whether IgM was deposited in areas of acute liver injury, sections from the explanted livers of patients transplanted for fulminant liver failure following paracetamol overdose were stained for IgM. Significant staining for IgM was seen in areas of injury . This deRAG1-/- are deficient in mature T cells, B cells and IgM . RAG1-/-Given the protection seen in RAG1-/- mice was not attributable to deficiency in immunoglobulins normally present in serum , it was Following reperfusion, there was a rapid accumulation of immune cells within the injured liver ; these wB cell deficient (μMT) mice were subjected to hepatic IRI and no significant protection was seen compared to wild type controls . This isDeposition of IgM occurred early after injury and disappeared with injury resolution . To testThis implies that neither B cells nor IgM were important in the resolution of tissue injury in the post-ischemic liver.Paracetomol toxicity and hypoxic liver injury typically cause zone 3 necrosis. In this study, the pattern of IgM staining in experimental models of these injuries, as well as in sections of liver from patients transplanted as a result of liver failure induced by paracetomol toxicity, mirrored this zone 3 distribution of injury . DepositGiven the time-frame in which this occurs (hours), the IgM “painting” injured cells is likely to represent polyreactive natural antibody (IgM) produced by B cells and found naturally in serum . This “pWe also found that RAG1-/- mice, which also lack both B and T cells, were significantly protected from injury ; this coThis is the first study, to look directly at the role T cells play in hepatic IRI with the use of a specific T cell knockout mice (CD3εKO). Work using TCR-/- mice has been performed in models of renal IRI, where T cells were also found to be key mediators of injury , 39. TheBased on our data, therapeutic intervention targeting IgM or B cells (such as with Rituximab) would have no significant benefit to patients or impact on their recovery following liver injury.