VERMONT COBRE: PROJECT 2: INNATE IMMUNE RESPONSES TO CRYPTOSPORIDIUM PARVUM

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aim 3: ¿¿ T cells in childhood cryptosporidiosis. This aim examined peripheral lymphocytes from children with acute Cryptosporidium infection to determine if peripheral ¿¿ T cells, and particularly V¿1 cells, are increased peripherally in this infection. Cell surface markers from the infected children were additionally evaluted to determine if they were enriched in human gut homing markers. We successfully initiated the clinical arm of this aim in year 1. Under approved IRB-protocols, specimens of peripheral blood mononuclear lymphocytes (PBML) were obtained from Bangladeshi children under the age of 2 during acute Cryptosporidium infection (cases), as well as from children with diarrhea not due to Cryptosporidium infection (controls). After shipment to the University of Vermont, specimens from 10 case children and 10 control children remained viable after thawing. All specimens were evaluated by multi-color flow cytometry for the following markers: ¿¿ and ¿¿ lymphocytes, V¿1 (a tissue-specific subset of ¿¿ lymphocytes), and human homing markers ¿4, ¿E/CD103 and CCR9; IL-15RA and NKG2D. The analysis of this data demonstrates that all V¿1 ¿¿ lymphocytes express the ¿4 integrin. However, although ¿4 was expressed on all V¿1 ¿¿ T cells, neither CD103 nor NKG2D expression was found in this peripheral cell population. Interestingly, the frequency of ¿¿ T cells in this population was found to be markedly lower than that described in the literature for this age range. The functional characteristics of ¿¿ cells have not yet been examined in this work. Unfortunately, as shown on Table 1, we were unable to further demonstrate that the percentage of ¿¿ or V¿1 lymphocytes differed between case and control children. Sample sizes limit these findings. Additionally, the distribution of homing markers, although higher for ¿4 and CCR9 in the case population, was not significantly different between cases and controls. As has been seen with other infectious diseases, the frequency of the studied population in the peripheral blood can be quite small (1%), yet still significant to human disease. Currently, we are re-evaluating this data using the integrated mean fluroscence intensity (iMFI) to better determine the significance of differences in these small peripheral populations. Using these same samples, ¿¿ cell lines have been established from three case children and two control children. If differences between cases and controls are determined in the the iMFI analysis, then the established lines will be compared in year 3 for functional differences (cytokine expression, cytotoxicity). Aim 4: Cryptosporidium-specific antigen identification. In year 2, we published data demonstrating that individuals expressing specific MHC class I and/or class II alleles are more susceptible to Cryptosporidium infection. Genetic susceptibility had not previously been shown in human Cryptosporidium infections. Using a well-established cohort of children with and without Cryptosporidium infection (n=226), PCR-sequence specific oligonucleotide data was evaluated to determine if differences in HLA class I and II alleles were discernable between groups. As shown in Table 2, a significance difference was demonstrated with both the HLA class I allele, B15 and the HLA class II allele DQB1 *0301. Both of these alleles were more common in children with Cryptosporidium infection, particularly children with symptomatic and asymptomatic infections. Mentoring Summaries: Elizabeth Bonney I currently meet periodically with all trainees, and discuss science as well as help with reports, presentations, and publications. I moreover participate in general career advising. Beth Kirkpatrick, a COBRE Junior Faculty and I share an interest in human mucosal immunology and infectious disease, and I have met with her and provided consultation on her projects. My goal is to help her focus her project Ralph Budd I have met with Beth Kirkpatrick on a regular basis to discuss the two directions that her research is taking. These are Beth's interest in mucosal immunology and specifically gd T cells, and her growing expertise in vaccine trials. Beth had submitted an application this past year for be a vaccine center for NIAID and was a semifinalist in this competition. This spoke volumes about her ability to organize a sound program and to quickly put in place the multiple pieces. Although Beth was not chosen in the final group, she definitely got her name known in this arena. Partly as a result of this, she was asked by the infectious diseases group at Johns Hopkins to replace another collaborator in the renewal application of their vaccine trial grant. She is now preparing this with the Hopkins group. The second area is Beth's studies on gd T cells in the gut and their potential response to Cryptosporidium. This has been a greater challenge. Although she has been able to culture bulk populations of gd T cells, deriving clones has been a considerable challenge. Since the subset in the gut (Vd1) is the same as the joint, which our lab studies, we have tried to perform parallel experiments. I am helping Beth to put together a manuscript with the initial characterization of a gd T cell response to Cryptosporidium. I think that Beth's real interest and expertise lies in building the vaccine trial center. But this would be a considerable assistance to the COBRE goals. I have discussed this with the EAC and they agree that this would be a worthy route for her to pursue. ***Please see paper copy for figures and tables (would not reproduce here)***