Sepsis is the overwhelming inflammatory response to infection, especially bacterial infection and associated bacterial products. It has major healthcare impacts, affecting an estimated 19-30 million persons/year worldwide with a mortality of 30-70%. Despite intense research, no specific therapy has been established for sepsis and in addition to the high mortality, the associated economic costs are very high. For example, recent data shows that the annual cost of patients with sepsis is more than $20 billion in the USA, and £2.5 billion in the UK. Therefore, novel targets and new therapies for sepsis are required which will have an important impact on both mortality and economic benefits. Recent work has demonstrated that the phopsholipid modifying enzyme, LPCAT, has a role in the regulation of inflammatory responses to bacterial infections. However, the mechanism of action in this regard is not well understood. This project aimed to identify the role of LPCAT-2 in inflammatory response to infections. This project has utilized the RAW264.7 murine macrophage cell line as an experimental model and LPS or Pam3CSK4 as infectious stimuli to investigate the role of overexpressing LPCAT-2 as well as silencing the over-expressed LPCAT-2 using siRNA technique. RAW264.7 cells transiently or stably transfected with the LPCAT-2 gene were used to study the role of LPCAT-2 in the inflammatory responses of macrophages. LPCAT-2 was successfully over-expressed in RAW264.7 cells and the overexpression was successfully confirmed with real time polymerase chain reaction (RT-PCR) and western blotting. The overexpression of LPCAT-2 significantly upregulated the pro-inflammatory cytokines TNF-α and IL-6, at both gene expression, and protein level, while the anti-inflammatory cytokine, IL-10, was down regulated in these cells. Moreover, overexpression of LPCAT-2 significantly decreased the expression of TLR4, peroxisome proliferator-activated receptors –gamma (PPARγ) and CD206 (a marker of M2 macrophages) while it significantly increased CD14, TLR2, COX-2 and iNOS (M1 markers). LPCAT-2 gene expression was also increased when PPARγ was blocked with the selective (PPAR-γ) antagonist T0070907. Importantly, silencing the transiently over-expressed murine LPCAT-2 resulted in a significant reduction in TNF-alpha and a significant increase in IL-10 gene expression. Both the transient and stably transfected RAW264.7 cells have been used to study the role of LPCAT-2 in regulating inflammatory responses in macrophages. The results have significantly added to knowledge of the role of LPCAT-2 in the inflammatory response and will aid in the development of novel therapies for inflammatory conditions such as sepsis.

Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP) to CD8 + single positive (SP) transition, but regulation of Notch activation is not well understood. In this thesis, I explored the potential of p53, endocytosis, and Cbl-b to regulate Notch activation. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. One aim of this thesis was to determine if p53 regulates Notch activity during T cell development. I found that Notch1 expression and activation were negatively regulated by p53 in several thymoma lines. Additionally, NIC was elevated in Trp53 −/− thymocytes as compared to Trp53 +/+ thymocytes. To determine if elevated Notch1 activation in Trp53−/− thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP:CD8+ SP T cell ratio was decreased in Trp53 −/− splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8 + SP T cells in Trp53−/− mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, I suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation. In the second aim of my thesis I present preliminary data showing that endocytosis does not appear to be involved in mammalian Notch activation although there is evidence in Drosophila for a positive endocytic role in Notch activation. The ability of Cbl-b to regulate Notch activation was the final aim of this thesis. Cbl-b, like Notch, has been shown to play a role in regulating the T cell signaling threshold. With this aim, I wanted to address the possibility of Cbl-b regulating T cell signaling via regulating Notch activation. Due to technical difficulties I was only able to obtain preliminary data suggesting that Cbl-b does positively regulate Notch activation in peripheral T cells. In this dissertation I have shown that Notch activation is regulated by controlling the expression of cellular components needed for cleavage, not just by encountering ligand on neighboring cells.

Methods for engineering mice to express polyclonal repertoires of human antibodies are well established and their use to produce human monoclonal antibodies of predefined specificity has been widely demonstrated (Ishida, et al., 2002; Lonberg, et al., 1994; Mendez, et al., 1997; Nicholson, et al., 1999). Although such engineered mice do expresses diverse repertoires of human antibodies and are immunophysiologically similar to humans; due to their small size, they are not suitable for the production of significant quantities of human polyclonal antibodies (hPAbs). Currently, hPAbs are in wide clinical use for prophylaxis and therapy in immunocompetent and immunodeficient patients [Keller, et al., 2000). Because these antibodies are obtained from human sources their supply is limited and their titers are often low because immunization protocols to raise pathogen-specific antibodies in donors are optimized for safety rather than for magnitude and duration of antibody response. Given these limitations, a technology for the production of antigen-specific hPAbs in large nonhuman hosts is novel and has significant biomedical and biodefense interest. Considering the differences in the mechanisms and strategies used by bovines and humans to diversify their antibody repertoires (Butler, 1998; Flajnik, 2002; Reynaud, et al., 1991; Meyer, et al. , 1997), questions arise about the capacity of HACs to sponsor the generation of functional human Ig repertoires. This prompted the following critical questions to be addressed: Can a large and viable population of human Ig-producing cloned HAC-Tc cattle be produced? Does human Ig synthesis persist as the animals mature? Is any portion of the human Ig assembled as fully human antibodies free of bovine heavy or light chains? Do rearranged human heavy chain loci undergo class switching in bovine cells? Does the HAC construct encode a broad diversity of human immunoglobulins in cattle? Most importantly, does immunization induce any fully human, antigen-specific polyclonal antibodies in cloned, HAC-Tc cattle, and effect protective functions? Resolving these questions is necessary to determine if the immunological divergence of bovines and humans prevent the use of HAC-bovines as suitable bioreactors for production of human antibodies for therapy. The availability of cloned HAC-Tc cattle that are imrnunologically mature has enabled the conduct of studies to address these questions, and the following results have been obtained. Biochemical and serological studies determine that fully human Ig isolated from HAC-Tc cattle is polyclonal and is comprised of both human μ and γ isotypes, demonstrating that the HAC-borne human IgH locus undergoes class switching within bovine cells. (Abstract shortened by UMI.)

The objective of this dissertation was to identify and characterize diversity within the expressed primary immunoglobulin heavy chain (IgH) repertoire in cattle. Determinations relied heavily upon different comparative analysis strategies focussing on both germline and expressed IgH gene segment sequences. We determined that the early fetal expressed IgH repertoire is constituted by as few as 2–3 VH genes and a single JH gene (multiple D). VH gene use increases with age, though IgH expression is restricted to members of a single VH family and primarily one JH gene (>300 sequences analyzed). All isolated germline VH genes also belong to a single VH family, corresponding to that in the expressed repertoire. Therefore, germline-encoded VH (and JH) sequence polymorphism is low, making limited contributions to overall IgH sequence diversity. In sharp contrast, bovine CDR3 regions exhibit extremely high levels of heterogeneity both in terms o f sequence and hypervariable lengths. A major fraction of IgH diversification occurs after rearrangement, most likely via untemplated somatic hypermutation. Nucleotide substitutions within the JH-Cμ intron, which does not support gene conversion due to a lack of known donor sequences, were consistent with USH on multiple levels, including hotspot targeting, the ratio of transitions to transversions, preferential nucleotide substitutions and potential strand bias. Sequence diversity levels varied with time among immunologically relevant tissues. Early fetal spleen and late fetal ileum appear to be two important sites of B cell diversification during their respective developmental stages. Patterns of IgH expression suggest that spleen is the early site of substantial gene rearrangement, and source of B cell emigrants, which subsequently populate other peripheral tissues including liver, ileum, and bone marrow.

Human B lymphocytes are notoriously difficult to culture. Two to three days after plating, a sharp decline in viability and cell number can be observed. The objective of this study was to evaluate the effect of support cells on B cell viability in an in vitro human immune system construct. B cells were combined with dendritic cells (DCs) and cultured for various periods of time in the presence of one of three types of support cells: EA cells, HS-5 cells, and HS-27 A cells. The B cells were either in physical contact with the support cells, or allowed to interact through soluble factors in the media in order to determine if the effect on viability was contact dependent or independent. Viability was assessed using flow cytometry. Finally, two functional assays were performed to evaluate the ability of the cultured B cells to respond to an immune challenge. Both recall and nai've antigens were used. The B lymphocytes were then assessed for viability, proliferation and activation using flow cytometry. ELISPOT was also employed to determine if any antigen specific antibodies were produced by the B cells. It was found that while the support cells did improve viability, they did not produce consistent or reliable results. Additionally, B lymphocytes cultured in the presence of support cells or support cell conditioned media had no antigen specific tetanus response and reduced proliferation. Therefore, even though the support cells did under some conditions enhance lymphocyte viability, the lack of a positive functional response negates the value of using them in an experimental system.