This thesis presents genetic studies of atopic dermatitis (AD) and IgA deficiency in dogs. AD is a chronic inflammatory and pruritic skin disorder caused by allergic reactions against environmental allergens. Both genetic and environmental factors are involved in the development of Canine AD (CAD) and human AD. In Paper I, we performed genome-wide association studies (GWAS) and identified a locus on chromosome 27 significantly associated with CAD in German shepherd dogs (GSDs). The locus contains several genes and fine-mapping indicated strongest association close to the candidate gene PKP2. In Paper II, we performed additional fine-mapping and identified four highly associated SNPs located in regions with transcriptional regulatory potential in epithelial and immune cells. The risk alleles were associated with increased transcriptional activity and the effect on expression was cell-type dependent. These data indicate that multiple cell-type specific enhancers regulate the expression of PKP2, and/or the neighboring genes YARS2, DNM1L and FGD4, and predispose GSDs to CAD. IgA deficiency is the most common primary immune deficiency disorder in both humans and dogs, characterized by a higher risk of recurrent mucosal tract infections, allergic and other immune-mediated diseases. In Paper III, we performed the widest screening (to date) of serum IgA levels in dog breeds (Ndogs=1267, Nbreeds=22) and defined eight breeds as predisposed to low IgA levels. In Paper IV, we performed GWAS in four of the breeds defined as prone to low IgA levels. We used a novel percentile groups-approach to establish breed-specific cut-offs to perform analyses in a close to continuous manner. In total, 35 genomic loci were suggestively associated (p<0.0005) to IgA levels, and three genomic regions (including the genes KIRREL3 and SERPINA9) were genome-wide significantly associated with IgA levels in GSDs. A ~20kb long haplotype on chromosome 28, significantly associated to IgA levels in Shar-Pei dogs, was positioned within the first intron of the gene SLIT1 overlapping with a possible dog domestication sweep. This thesis suggests novel candidate genes involved in two immune-mediated disorders in the dog. Hopefully, these results will become an important resource for the genetic research of the corresponding human diseases.

The mucosal antibody repertoire of the cervical mucosa in commercial sex workers from Nairobi, Kenya, who are highly sexually exposed to human immune deficiency virus type-1 (HIV-1) but remain persistently IgG seronegative (HEPS), may represent a novel source of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1. Mucosal IgA specific for HIV-1 envelope (Env) subunit gp41 has been suggested as a correlate of protection in HEPS individuals. The in depth studies at both the gene and function level required to confirm their role in HIV-1 resistance are possible only using recombinant monoclonal IgAs. Human mAbs have traditionally been selected from libraries displayed on the surface of microorganisms (phage, yeast). However, due to inherent limitations, such techniques may not be optimal for isolating such rare mAbs from a pool of cervical B cells. We have developed an antibody selection system based on surface display on mammalian cells and used this technology to isolate four novel monoclonal antibodies, against linear epitopes on gp41, from the IgA repertoire of the cervical mucosa in Kenyan HEPS. Furthermore, three of the four mAbs were shown to bind with surface expressed consensus clade B and clade C Env on mammalian cells. Characterization of the variable region cDNA of the two strongest binding mAbs reveals extensive somatic mutations with a bias of replacement mutations clustering in the complementary determining regions (CDR) indicating antigen-driven affinity maturation had occurred. Affinity matured monoclonal IgAs, such as these, may play a role in the identification of new, vulnerable epitopes on HIV-1, or act as a component in a topical microbicide.

Heterosexual intercourse now accounts for the majority of HIV transmission within sub-Saharan Africa. The generation of microbicides and vaccines, therefore, requires a better understanding of the mucosal correlates of protection, including the role of HIV-specific IgA. It is now accepted that not all individuals are equally susceptible to HIV-1 infection, as exemplified by the HIV Exposed Seronegative (HESN) women of the Pumwani Cohort in Nairobi, Kenya. To assess whether mucosal IgA responses contribute to this protection, 3 novel IgA variable genes were cloned from HESN cervical B-cell cDNA. Nine monoclonal IgA Abs were produced, two of which were properly produced from cell culture. The HESN-derived A6/30L and A9/30L variants had a greater specificity for gp120IIIB than their A6/4L and A9/4L counterparts, while the A6 variant recognizes a distinct gp120 epitope compared to the broadly neutralizing antibody IgGb12. Further characterization of these IgA chains may suggest their suitability for use in microbicides or mucosal vaccines.

The mucosal antibody repertoire of the cervical mucosa in commercial sex workers from Nairobi, Kenya, who are highly sexually exposed to human immune deficiency virus type-1 (HIV-1) but remain persistently IgG seronegative (HEPS), may represent a novel source of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1. Mucosal IgA specific for HIV-1 envelope (Env) subunit gp41 has been suggested as a correlate of protection in HEPS individuals. The in depth studies at both the gene and function level required to confirm their role in HIV-1 resistance are possible only using recombinant monoclonal IgAs. Human mAbs have traditionally been selected from libraries displayed on the surface of microorganisms (phage, yeast). However, due to inherent limitations, such techniques may not be optimal for isolating such rare mAbs from a pool of cervical B cells. We have developed an antibody selection system based on surface display on mammalian cells and used this technology to isolate four novel monoclonal antibodies, against linear epitopes on gp41, from the IgA repertoire of the cervical mucosa in Kenyan HEPS. Furthermore, three of the four mAbs were shown to bind with surface expressed consensus clade B and clade C Env on mammalian cells. Characterization of the variable region cDNA of the two strongest binding mAbs reveals extensive somatic mutations with a bias of replacement mutations clustering in the complementary determining regions (CDR) indicating antigen-driven affinity maturation had occurred. Affinity matured monoclonal IgAs, such as these, may play a role in the identification of new, vulnerable epitopes on HIV-1, or act as a component in a topical microbicide.

Heterosexual intercourse now accounts for the majority of HIV transmission within sub-Saharan Africa. The generation of microbicides and vaccines, therefore, requires a better understanding of the mucosal correlates of protection, including the role of HIV-specific IgA. It is now accepted that not all individuals are equally susceptible to HIV-1 infection, as exemplified by the HIV Exposed Seronegative (HESN) women of the Pumwani Cohort in Nairobi, Kenya. To assess whether mucosal IgA responses contribute to this protection, 3 novel IgA variable genes were cloned from HESN cervical B-cell cDNA. Nine monoclonal IgA Abs were produced, two of which were properly produced from cell culture. The HESN-derived A6/30L and A9/30L variants had a greater specificity for gp120IIIB than their A6/4L and A9/4L counterparts, while the A6 variant recognizes a distinct gp120 epitope compared to the broadly neutralizing antibody IgGb12. Further characterization of these IgA chains may suggest their suitability for use in microbicides or mucosal vaccines.

Glycosylation is one of the most abundant post-translational modifications of proteins. Glycoproteins participate in virtually all aspects of cellular functions. ST6Gal I is a glycosyltransferase highly expressed by B and T cells. Here, we interrogated the role of ST6Gal I in viral specific B and T cell immune responses, as well as examined how loss of this enzyme impacted viral pathogenesis. First, to understand how loss of ST6Gal I expression impacted viral specific humoral responses, we infected ST6Gal I-/- mice with influenza virus. We discovered that loss of ST6Gal I expression results in both reduced influenza specific antibodies levels and decreased viral-specific antibody secreting cells numbers. Following influenza infection, mice that received ST6Gal I-/- B cells showed reduced influenza-specific IgM responses compared to mice that received wild-type B cells. These experiments demonstrated that the expression of ST6Gal I by B cells is required for optimal viral-specific humoral response. We further examined how loss of ST6Gal I expression impacted the anti-influenza IgA response. We observed that immune ST6Gal I-/- mice displayed higher viral specific IgA levels and altered sialylation of IgG and IgA, which have been implicated in a human disease, IgA nephropathy. Moreover, ST6Gal I-/- mice exhibited increased immunoglobulin deposition in kidney glomeruli following influenza infection. These data suggest that ST6Gal I deficiency, together with influenza infection, may result in the initiation of a kidney disease. Finally, we examined how ST6Gal I expression regulated CD8 T cell responses. We discovered that ST6Gal I is differentially expressed during CD8 T cell activation. To understand its relevance, we infected ST6Gal I-/- mice and demonstrated that the early expansion of effector T cells was impaired in a cell intrinsic manner. Moreover, in the absence of ST6Gal I, the differentiation of CD8 T cells skewed towards memory precursor cells, whereas terminal effector cell expansion was impaired. Mechanistically, we identified delayed surface expression of IL-2Ralpha on ST6Gal I-/- CD8 T cells due to impaired IL-2/IL-2R signaling. These studies implicate that ST6Gal I expression enhances early proliferation of terminal effector CD8 T cells by promoting the rapid surface expression of IL2Ralpha during acute viral infection. 

No description available.

The lungs and upper airways are mucosal surfaces that are common site for infection with an enormous variety of inhaled pathogens. Therefore, induction of immune responses in the respiratory tract is crucial for protection against respiratory diseases. One of the pathogens infecting the host via the respiratory tract is Mycobacterium Tuberculosis. The reported efficacy of the currently used Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis is highly variable, ranging from 50% against pulmonary tuberculosis to 80% against disseminated tuberculosis. Recently, the current route of vaccination (intradermal) has been considered as a possible factor influencing the protective capacity of the BCG vaccine. In this regard, intradermal route most likely induces protective systemic responses while it fails to induce optimal responses in the lungs. Therefore, our working hypothesis is that vaccination should be directed towards the respiratory mucosal immunity in order to improve the degree of host protection in the lungs. In this thesis we studied the effect of the route of immunization as well as of different mucosal adjuvants on the induction of mucosal immune responses against the mycobacterial surface antigen PstS-1. We found that, the intranasal (i.n.) route of immunization was a more favorable route inducing strong local immune responses, than intraperitoneal (i.p.) route. Indeed, i.n. route immunization, unlike the i.p. route, elicited strong IgA responses in the lungs accompanied by a major influx of CD4+ T cells and a significant local production of IFN-gamma. IgA, being the predominant Ig isotype at mucosal tissues, is considered a major effector molecule involved in defense mechanisms against viral and bacterial pathogens at these sites. Therefore, we investigated the possible role of IgA in the protection of the respiratory mucosa against mycobacterial infections, using mice deficient in IgA and in the polymeric Ig receptor. We show that, deficient mice are more susceptible to mycobacterial infections than wild type mice, thereby demonstrating a role for IgA in protection against mycobacteria. Importantly, our studies revealed a reduced production of protective factors, such as INF-gamma and TNF-alpha in the lungs of deficient mice that was associated with the higher susceptibility seen in these mice compared to wild-type mice. We also conducted challenge experiments against another respiratory pathogen, Chlamydia pneumoniae, using IgA deficient mice. Likewise to mycobacteria, our data support a role for IgA in the protection of the respiratory tract against C. pneumoniae infection. Finally, we investigated the possible mechanisms explaining the reduced pro-inflammatory responses in IgA deficient mice. Our data indicated that IgA deficient mice present a defective response to stimulation with LPS or 19kDa which appears to be both, essentially due to suboptimal stimulation of macrophages and restricted to the lungs.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 121-143). Also available in print.

Made available in DSpace on 2014-12-17T14:13:51Z (GMT). No. of bitstreams: 1 MariaLSP_DISSERT_1-12.pdf: 307341 bytes, checksum: 501d00488698b866628dca8bc24c0c6e (MD5) Previous issue date: 2010-03-31 / Trata-se de um coorte prospectivo com amostras de leite de 28 m?es da zona rural da Para?ba, durante diferentes dias de amamenta??o exclusiva, com objetivo de avaliar atrav?s do ensaio imunoenzim?tico a presen?a de imunoglobulina A secretora (sIgA) total e espec?fica contra ant?genos de Escherichia coli enteropatog?nica (EPEC) e Shigella flexneri. A reatividade dos anticorpos foi analisada pelo Western blot . Os resultados mostram presen?a da sIgA em todas as amostras, com medianas no colostro de 8,092 g/L(4,546-17,252) e leite de 0,695g/L (0,020-2,830). As medianas nos t?tulos de colostro de IgA anti-EPEC foi 41 (1-659) e anti-Shigella flexneri de 18 (1-4727) enquanto no leite anti-EPEC foi de 8 (1-288) e anti-Shigella flexneri de 6 (1-450). Houve grandes varia??es entre as m?es e entre os dias de amamenta??o. No Western blot os anticorpos sIgA reagiram com prote?nas de EPEC e Shigella flexneri, destacando-se a fra??o antig?nica de 94kDa, correspondente a intimina. Os resultados mostram que a presen?a de sIgA total e de anticorpos IgA contra EPEC e Shigella flexneri no colostro e leite de m?es residentes em zona rural, com prec?rias condi??es s?cioecon?mica e sanit?rias, n?o diferem de estudos realizados com popula??es de ?rea urbana e refor?am a import?ncia do leite materno na defesa contra infec??es ent?ricas. Apesar da aus?ncia na literatura de estudos avaliando o perfil de anticorpos sIgA no leite de m?es residentes em zona rural do Brasil, os resultados demonstraram que a presen?a de sIgA total e de anticorpos IgA contra EPEC e Shigella flexneri no colostro e leite de m?es residentes em zona rural, com prec?rias condi??es s?cio-econ?mica e sanit?rias, n?o diferem de estudos realizados com popula??es de ?rea urbana e refor?am a import?ncia do leite materno na defesa contra infec??es ent?ricas