The bursa of Fabricius has been a durable model of B lymphocyte development. Yet there are unique aspects of B- lymphocyte development in the bursa that remain to be elucidated, and these may reveal important functional differences in the avian system and distinct evolutionary mechanisms from the canonical murine and human models of B- lymphocyte development. Our laboratory has been interested in the function of the chB6 alloantigen. ChB6 has three defined alleles and is present on B lymphocytes in chicken from their earliest development at ED 12. ChB6 continues to be expressed through B- lymphocyte ontogeny as well as on a subset of macrophages. We have shown that chB6 ligation by antibody leads to rapid apoptosis. Transfection of cDNA- encoding chB6 replicates this in mammalians cells, suggesting a common signaling pathway, but there remain no clear mammalian homologues. Structurally, the extracellular domain of chB6 is similar to mammalian SLAM (signaling lymphocyte activation molecules) proteins and chB6 partitions into lipid rafts in close proximity to the B- cell receptor. The lack of homology within the intracellular domain remains puzzling. Utilizing genomic resources, we have found a number of similar molecules in both birds and reptiles; however, they show greater conservation in the intracellular domain, including an SH3 motif that we have shown to be critical in inducing apoptosis.

Education in the health professions occurs at the graduate level, increasing the barriers to underrepresented and first-generation students. Some programs attempt to alleviate these barriers by streamlining passage from the undergraduate campus to the medical campus within a university or by accelerating entry to the professions by bypassing the bachelor's degree. These solutions each have their own limitations for students, including added pressure to maintain grades and a limited ability to explore multiple professions. The Alliance for Health Sciences, a partnership between DePaul University and Rosalind Franklin University of Medicine and Science (RFUMS), was formed to address these issues and has created the Pathways Honors program. Through this program, current DePaul students may apply for an early admission decision to one of 6 of RFUMS's programs and, if qualified, pursue an accelerated entry. They have the option to apply for early decision at any time during their undergraduate career to encourage exploration of career options and to allow students to adjust to college rigor with lower stakes. In addition, collaborative, innovative advising contributes to creating well-rounded students and admissions committee decisions not dictated solely by test metrics. The Pathways Honors program has continued to attract a diverse student pool to DePaul, and early indications are that many of these students are making a successful transition to professional programs. Currently, we have data on our first cohort of students. Continued development of this program will be necessary to ensure that it is preparing students for rigorous graduate programs and helping to diversify the health care workforce.

In chickens, B cells develop in the bursa of Fabricius, a unique organ for B cell development. Most B cells will die within the bursa, mirroring cell losses seen in mammalian bone marrow as central tolerance is enforced at the transition to mature cells. B cell responses are shaped by a complex interplay of signals. Signals in addition to BCR that impact central tolerance have recently been described. We have been interested in chB6, a novel alloantigen on B cells in the chicken. chB6 is found in close proximity to the BCR and can trigger apoptosis after cross-linking by antibody. chB6 has two Ig domains, placing it within the CD2/SLAM family of molecules, but its cytoplasmic domain is unique. We have used a site-specific mutagenesis approach to show that an SH3 binding site in chB6 is required for the induction of apoptosis, suggesting parallels to CD2 signaling.

It is well established that central tolerance of B cells involves the deletion of large numbers of B cell clones. However, the mechanism of this deletion remains unclear. Chickens are one of the classic models of vertebrate immunity and bursa of Fabricius presents model system to study this process. Our lab has been interested in the role of the chB6 alloantigen (formerly called Bu1) in the developing of B cells within the bursa. We have shown that chB6 can trigger apoptosis when stimulated by anti-chB6 antibody and this apoptosis uses intermediates commonly found in death receptor signaling pathways. Recently, chB6 has been found in close proximity to the BCR on DT40 cells. Others have noted that BCR signals in DT40 lead to apoptosis. Homology analysis places chB6 within the larger CD2/SLAM family of proteins based on the presence of two extracellular Ig domains. CD2/SLAM family members serve to modulate signals in immune cells. How chB6 initiates signals, and how those signals might be coordinated with other components of the BCR complex, remains unclear. Previous work indicated a 15 amino acid stretch in the cytoplasmic domain and critical in initiating apoptosis. Within this region is the PXXP motif indicating a binding site for SH3 domain containing proteins. We have mutated the proline codons within this region and transfected these mutated chB6 cDNAs into BK3a cells to test the hypothesis that this SH3 binding site is important in death signaling via chB6. Preliminary evidence suggests that this region is in fact critical in transducing a death signal. This project furthers our efforts to understand the biology of chB6. This work supported by a grant from the DePaul University Research Council.

In the vertebrate immune system, each B-lymphocyte expresses a surface IgM-class B cell receptor (BCR). When cross-linked by antigen or anti-IgM antibody, the BCR accumulates with other proteins into distinct surface clusters that activate cell signaling, division, or apoptosis. However, the molecular composition of these clusters is not well defined. Here we describe a quantitative assay we call selective proteomic proximity labeling using tyramide (SPPLAT). It allows proteins in the immediate vicinity of a target to be selectively biotinylated, and hence isolated for mass spectrometry analysis. Using the chicken B cell line DT40 as a model, we use SPPLAT to provide the first proteomic analysis of any BCR cluster using proximity labeling. We detect known components of the BCR cluster, including integrins, together with proteins not previously thought to be BCR-associated. In particular, we identify the chicken B-lymphocyte allotypic marker chB6. We show that chB6 moves to within about 30 - 40 nm of the BCR following BCR cross-linking, and we show that cross-linking chB6 activates cell binding to integrin substrates laminin and gelatin. Our work provides new insights into the nature and composition of the BCR cluster, and confirms SPPLAT as a useful research tool in molecular and cellular proteomics.

Chickens are one of the classic models of vertebrate immunity. We have been interested in the role of the alloantigen chB6 (formerly called BU1) in the development of B cells within the bursa and have presented evidence that chB6 can trigger apoptosis. Furthermore, the pathway that triggers apoptosis is similar to that triggered by FAS, with caspase 8 as an initiator, caspases 9 and 3 as later caspases, and bcl-xl functioning as an inhibitor of apoptosis. We have been unable to demonstrate a steady state association of chB6 and caspase 8 in DT40, so the mechanism of caspase 8 activation remains unclear. Here we show that chB6 localizes in distinct clusters on the surface of DT40 cells, reminiscent of the SPOTS others have described in Fas signaling. These clusters co-localize with cholera toxin labeled lipid rafts and chB6 appears to be internalized by the B cells. Western blots indicate that chB6 is present in both detergent soluble and insoluble membrane fractions on resting DT 40 cells. However, cross-linking chB6 with a secondary antibody shows that chB6 is internalized selectively from detergent insoluble (lipid raft) fractions of the cell membrane. This suggests an active distribution of chB6 in the membrane and an internal trafficking that may be functionally important. This work offers a new insight into the signaling via chB6. This work supported by a grant from the DePaul University Research Council.

We present a laboratory-based exercise that is used to teach basic lab skills (e. g., aseptic technique and enumeration) using naturally occurring microbial communities in a real biological context. Students examine the colonization by microbial communities of leaves that fall into streams. Leaf decomposition reflects enzymatic activity by microorganisms such as aquatic fungi and bacteria and maceration by invertebrate shredders. The microorganisms help facilitate the cycling of nutrients and energy in the stream's ecosystem. This exercise effectively teaches students to use lab skills to quantify microorganisms found in nature, investigates groups of microorganisms involved in leaf degradation in streams, and stimulates interest in both microbiology and ecology.

Chickens are one of the classic models of vertebrate immunity. We have been interested in the role of the alloantigen chB6 (formerly called BU1) in the development of B cells within the bursa and have presented evidence that chB6 can trigger apoptosis. Furthermore, the pathway that triggers apoptosis is similar to that triggered by FAS, with caspase 8 as an initiator, caspases 9 and 3 as later caspases, and bcl-xl functioning as an inhibitor of apoptosis. We have been unable to demonstrate a steady state association of chB6 and caspase 8 in DT40, so the mechanism of caspase 8 activation remains unclear. Other investigators have shown that FAS segregates into lipid rafts in type I cells and this is correlated with death signaling. In addition, FAS ligation leads to the formation of distinct signaling complexes termed SPOTS. Using immunofluorescence, we observed that chB6, when cross-linked by secondary antibodies, is found in patchy areas on DT40 cells reminiscent of SPOTS. By double labeling with fluorescent cholera toxin B, we show that chB6 co-localizes with lipid raft areas of the DT40 membrane and will be internalized by the cells. This suggests an active distribution of chB6 in the membrane and it may be preferentially loaded into lipid rafts along with BCR in avian cells. This work offers a new insight into the signaling via chB6. This work supported by a grant from the DePaul University Research Council.

Chickens are one of the classic models of vertebrate immunity. We have been interested in the role of the alloantigen chB6 (formerly called BU1) in the development of B cells within the bursa and have presented evidence that chB6 can trigger apoptosis. chB6 is an alloantigen and in the original report of its cloning three alleles were reported, chB6.1 in RPL line 6 flocks, chB6.2 in RPL line 7, and chB6.3 in Light Sussex birds. However, no further search for other chB6 alleles has been reported. Using the chicken EST database we have identified five potential new alleles of chB6, although some of these EST clones are not full length cDNAs.. We have resequenced these EST clones to confirm the base sequence and translation. These results suggest greater genetic diversity of the chB6 locus than previously appreciated. Furthermore, rt-PCR was used to clone the turkey homologue of chB6. The results presented here are the beginnings of a larger study to examine chB6 and can provide novel insights into the evolution of immune related genes in Aves. This work supported by a summer grant from the DePaul College of Liberal Arts and Sciences.

The chicken is a foundational model for immunology research and continues to be a valuable animal for insights into immune function. In particular, the bursa of Fabricius can provide a useful experimental model of the development of B lymphocytes. Furthermore, an understanding of avian immunity has direct practical application since chickens are a vital food source. Recent work has revealed some of the molecular interactions necessary to allow proper repertoire diversification in the bursa while enforcing quality control of the lymphocytes produced, ensuring that functional cells without self-reactive immunoglobulin receptors populate the peripheral immune organs. Our laboratory has focused on the function of chB6, a novel molecule capable of inducing rapid apoptosis in bursal B cells. Our recent work on chB6 will be presented and placed in the context of other recent studies of B cell development in the bursa.

Many developing B lymphocytes are deleted by apoptosis. However, the mechanism signaling their demise remains poorly understood. Like mammals, chicken B cells are selected during their development; >95% of the cells in the bursa of Fabricius die without entering the secondary immune system. The molecule chB6 (Bu-1) has been used as a marker to identify B cells in the chicken. ChB6 is a type I transmembrane glycoprotein whose function is enigmatic. We have provided evidence that chB6 can induce a rapid form of cell death exhibiting characteristics of apoptosis. Here we further examine cell death induced by chB6 in a transfected mouse cell line. ChB6 is shown to cause apoptosis in this cell line as detected by a TUNEL assay for DNA fragmentation. This apoptosis is subject to regulation by signals from growth factor or by Bcl-x(L). Furthermore, we show that Ab binding to chB6 leads to cleavage of caspase 8, caspase 3, and poly(ADP ribose) polymerase. Overall, these data support the hypothesis that chB6 is a novel death receptor on avian B cells.