Thesis (doctoral)--Mainz, 1983.

University, Diss., 2004--Kiel.

Indiana University-Purdue University Indianapolis (IUPUI) / Though the elimination of carbohydrate xenoantigens has reduced the antibody barrier to clinical xenotransplantation, identification of additional targets of rejection could further increase the immunologic compatibility of pig tissues with humans. Many patients in need of organ transplantation have antibodies to proteins encoded by the human major histocompatibility complex (MHC) which have high similarity to their swine homologs. The goal of this thesis was to determine if the class II genes of the swine MHC can bind human antibodies. To characterize antibody binding effect to class II swine leukocyte antigens (SLA), a constitutively positive SLA class II cell was created through transfection with the human class II transactivator (CIITA). Cells expressing only SLA-DR or SLA-DQ were also created using the CRISPR/Cas9 gene knockout tools. These various lines were incubated with human sera and tested for binding to IgM and IgG in a flow cytometry crossmatch (FCXM). The results demonstrate reliable antibody binding to each of the SLA class II –DR and –DQ derivatives. A two-way paired t-test revealed statistical difference in total sera binding between to the DR(+)DQ(+) and DR(-)DQ(-) clones for IgG (p = 0.0059) but not IgM (p = 0.2460). Looking at the subset of individuals with and without anti-HLA class II sensitization, statistical difference was noted for IgG (p = 0.0229) but not IgM (p = 0.3045). Examining further the role of DR(+) vs DQ(+), statistical analysis revealed difference in the DR(+)DQ(-) vs. the DR(-)DQ(+) FCXM (p = 0.0099), the DR(+)DQ(-) vs. the DR(+)DQ(+) FCXM (p = 0.0192), and the DR(-)DQ(-) parent vs. DR(+)DQ(+) FCXM (p = 0.0329). No difference was found in the DR(-)DQ(+) vs. DR(+)DQ(+) FCXM (p = 0.1601). The results of this project suggest that SLA class II, specifically SLA-DQ, could be a target of antibody binding and cross-reactive anti-HLA class II antibodies may be capable of binding SLA class II.

Um dos maiores desafios em estudos de genética de populações é a diferenciação entre os sinais de diversidade genética resultantes de efeitos estocásticos dos sinais de seleção natural. Neste trabalho, o objetivo central foi inferir a história evolutiva, em termos de demografia e seleção natural, do MHC e alguns de seus genes HLA de classe II (HLA-DRB1, -DQB1 e -DPB1). A primeira estratégia usada foi sequenciar os referidos genes em 14 populações ameríndias e, em conjunto com dados já publicados em outras populações ameríndias e não ameríndias, contrastou-se sua distribuição de alelos, diversidade genética intra e interpopulacional e testes de neutralidade com as mesmas estatísticas obtidas a partir de 54 STR genômicos genotipados para estas mesmas populações. Em nossos resultados verificamos: a) a maior variabilidade dos genes HLA em relação aos STR, b) a menor variabilidade de ameríndios se comparados com não ameríndios e c) correlação das estimativas de FST obtidas de STR com as obtidas dos genes HLA. Esses resultados indicam a ação da demografia como principal responsável pelos padrões encontrados. No entanto, outras evidências sugerem um padrão de diversidade genética que não pode ser explicado por mera ação do acaso, como por exemplo: a) o grande número e dispersão geográfica de alelos privados e/ou mais prevalentes em ameríndios (vinte vezes mais frequentes) para HLA-DRB1, b) a não correlação das estimativas de diversidade genética intrapopulacional obtidas de genes HLA com as obtidas de STR, c) os maiores valores de FST para genes HLA em comparação com o FST de STR e a d) ausência de correlação dos valores de D de Tajima (para HLA) com a estimativa &beta; (para STR). Assim, apesar da grande influência de fatores estocásticos, os sinais de seleção natural são perceptíveis. A segunda abordagem investigou sinais de seleção recente na região do MHC na população miscigenada de Belém do Pará (n=202), utilizando como ferramenta a comparação entre os componentes de ancestralidade estimados para a região do MHC (com sete STR) com os observados no restante do genoma (54 STR) para detecção de desvios na ancestralidade das populações parentais. As proporções de ancestralidade média para os STR em Belém (africana= 0,19, europeia= 0,51 e ameríndia=0,30), foram diferentes das estimadas para os marcadores do MHC (africana= 0,23, europeia= 0,67 e ameríndia=0,11), sendo as diferenças entre as estimativas ameríndia e europeia significativa (Wilcoxon; p<0,0001). Além disso, os valores de FST observados eram maiores entre Belém e Ameríndios, considerando-se os STR do MHC. É possível concluir, portanto, que foi observada uma perda de componente genético ameríndio na região do MHC durante o processo de miscigenação, quando contrastarmos com as estimativas genômicas, sugerindo ação de seleção natural recente. Em conjunto, nossos resultados mostram que tanto a seleção natural como a demografia moldaram a variação genética em genes HLA, e que usando métodos apropriados, incluindo o mapeamento por miscigenação, eventos de seleção natural que ocorreram em escalas de tempo recentes podem ser detectados / One of the greatest challenges on the Genetics Populations Studies is the differentiation among the resulting genetic signs of diversity from the stochastic signs of natural selection. On the present study, the main objective was inferring the evolutive history, in terms of demography and natural selection, from the MHC and some of its HLA class II genes (HLA-DRB1, -DQB1 e -DPB1). The first strategy used was sequentiating the referred genes in 14 Amerindian populations and, in conjunction with data already published in other Amerindian and non-Amerindian populations, we have contrasted its distribution in alleles, intra and inter-populational genetic diversity and neutrality tests with the same statistics obtained from 54 STR genomics genotyped for these same populations. In our results we have verified: a) greater variability of the HLA genes in relation to the STR, b) greater variability of Amerindians, if compared to non-Amerindians and c) correlation of the estimates of FST obtained from STR with the ones obtained from the HLA genes. These results indicate the demography action as the main responsible for the found patterns. However, other evidences suggest a pattern of genetic diversity that can\'t be explained only by chance, as for example: a) the great number and geographic dispersion of the private alleles and/or more prevalent in the Amerindians (twenty times more frequent) for HLA-DRB1, b) a non-correlation of the estimates on interpopulational genetic diversity obtained from HLA genes with the ones obtained from STR, c) greater values of FST for the HLA genes, in comparison with the FST of STR and d) absence of correlation on the D values of Tajima (for HLA) with &beta; estimates (for STR). So, despite the great influence of stochastic factors, the signs of natural selection are perceptible. The second approach investigated signs of recent selection in the MHC region of the admixture population of Belém do Pará (n=202), using as tool the comparison between the ancestrality components estimated for the MHC region (with seven STR) with the ones observed on the remaining genome (54 STR) for the detection of deviations on the ancestrality of the parental populations. The medium ancestrality proportions for the STR in Belém (African=0,19, European=0,51 and Amerindian=0,30), was different from the estimates for the MHC markers (African=0,23, European=0,67 and Amerindian=0,11), being significative the differences between Amerindian and European estimates (Wilcoxon; p<0,0001). Furthermore, the FST values observed were greater between Belém and the Amerindians, considering the STR from the MHC. It\'s then possible to get to the conclusion that a loss in Amerindian genetic component was observed in the MHC region during the miscegenation process, when contrasted with the genomic estimates, suggesting recent action of natural selection. In whole, our data show that even natural selection as demography have molded the genetic variation in the HLA genes, and by using the appropriated methods, including the miscegenation mapping, natural selection events occurred in recent time scales can be detected

Um dos maiores desafios em estudos de genética de populações é a diferenciação entre os sinais de diversidade genética resultantes de efeitos estocásticos dos sinais de seleção natural. Neste trabalho, o objetivo central foi inferir a história evolutiva, em termos de demografia e seleção natural, do MHC e alguns de seus genes HLA de classe II (HLA-DRB1, -DQB1 e -DPB1). A primeira estratégia usada foi sequenciar os referidos genes em 14 populações ameríndias e, em conjunto com dados já publicados em outras populações ameríndias e não ameríndias, contrastou-se sua distribuição de alelos, diversidade genética intra e interpopulacional e testes de neutralidade com as mesmas estatísticas obtidas a partir de 54 STR genômicos genotipados para estas mesmas populações. Em nossos resultados verificamos: a) a maior variabilidade dos genes HLA em relação aos STR, b) a menor variabilidade de ameríndios se comparados com não ameríndios e c) correlação das estimativas de FST obtidas de STR com as obtidas dos genes HLA. Esses resultados indicam a ação da demografia como principal responsável pelos padrões encontrados. No entanto, outras evidências sugerem um padrão de diversidade genética que não pode ser explicado por mera ação do acaso, como por exemplo: a) o grande número e dispersão geográfica de alelos privados e/ou mais prevalentes em ameríndios (vinte vezes mais frequentes) para HLA-DRB1, b) a não correlação das estimativas de diversidade genética intrapopulacional obtidas de genes HLA com as obtidas de STR, c) os maiores valores de FST para genes HLA em comparação com o FST de STR e a d) ausência de correlação dos valores de D de Tajima (para HLA) com a estimativa &beta; (para STR). Assim, apesar da grande influência de fatores estocásticos, os sinais de seleção natural são perceptíveis. A segunda abordagem investigou sinais de seleção recente na região do MHC na população miscigenada de Belém do Pará (n=202), utilizando como ferramenta a comparação entre os componentes de ancestralidade estimados para a região do MHC (com sete STR) com os observados no restante do genoma (54 STR) para detecção de desvios na ancestralidade das populações parentais. As proporções de ancestralidade média para os STR em Belém (africana= 0,19, europeia= 0,51 e ameríndia=0,30), foram diferentes das estimadas para os marcadores do MHC (africana= 0,23, europeia= 0,67 e ameríndia=0,11), sendo as diferenças entre as estimativas ameríndia e europeia significativa (Wilcoxon; p<0,0001). Além disso, os valores de FST observados eram maiores entre Belém e Ameríndios, considerando-se os STR do MHC. É possível concluir, portanto, que foi observada uma perda de componente genético ameríndio na região do MHC durante o processo de miscigenação, quando contrastarmos com as estimativas genômicas, sugerindo ação de seleção natural recente. Em conjunto, nossos resultados mostram que tanto a seleção natural como a demografia moldaram a variação genética em genes HLA, e que usando métodos apropriados, incluindo o mapeamento por miscigenação, eventos de seleção natural que ocorreram em escalas de tempo recentes podem ser detectados / One of the greatest challenges on the Genetics Populations Studies is the differentiation among the resulting genetic signs of diversity from the stochastic signs of natural selection. On the present study, the main objective was inferring the evolutive history, in terms of demography and natural selection, from the MHC and some of its HLA class II genes (HLA-DRB1, -DQB1 e -DPB1). The first strategy used was sequentiating the referred genes in 14 Amerindian populations and, in conjunction with data already published in other Amerindian and non-Amerindian populations, we have contrasted its distribution in alleles, intra and inter-populational genetic diversity and neutrality tests with the same statistics obtained from 54 STR genomics genotyped for these same populations. In our results we have verified: a) greater variability of the HLA genes in relation to the STR, b) greater variability of Amerindians, if compared to non-Amerindians and c) correlation of the estimates of FST obtained from STR with the ones obtained from the HLA genes. These results indicate the demography action as the main responsible for the found patterns. However, other evidences suggest a pattern of genetic diversity that can\'t be explained only by chance, as for example: a) the great number and geographic dispersion of the private alleles and/or more prevalent in the Amerindians (twenty times more frequent) for HLA-DRB1, b) a non-correlation of the estimates on interpopulational genetic diversity obtained from HLA genes with the ones obtained from STR, c) greater values of FST for the HLA genes, in comparison with the FST of STR and d) absence of correlation on the D values of Tajima (for HLA) with &beta; estimates (for STR). So, despite the great influence of stochastic factors, the signs of natural selection are perceptible. The second approach investigated signs of recent selection in the MHC region of the admixture population of Belém do Pará (n=202), using as tool the comparison between the ancestrality components estimated for the MHC region (with seven STR) with the ones observed on the remaining genome (54 STR) for the detection of deviations on the ancestrality of the parental populations. The medium ancestrality proportions for the STR in Belém (African=0,19, European=0,51 and Amerindian=0,30), was different from the estimates for the MHC markers (African=0,23, European=0,67 and Amerindian=0,11), being significative the differences between Amerindian and European estimates (Wilcoxon; p<0,0001). Furthermore, the FST values observed were greater between Belém and the Amerindians, considering the STR from the MHC. It\'s then possible to get to the conclusion that a loss in Amerindian genetic component was observed in the MHC region during the miscegenation process, when contrasted with the genomic estimates, suggesting recent action of natural selection. In whole, our data show that even natural selection as demography have molded the genetic variation in the HLA genes, and by using the appropriated methods, including the miscegenation mapping, natural selection events occurred in recent time scales can be detected

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) has been used as a vaccine, yet safety concerns remain due to its viral immunoevasive properties. Among these, VV infection of antigen presentation cells (APC) perturbs MHC class II-mediated antigen (Ag) presentation. The goals of this project include: 1) to define mechanisms by which VV disrupts class II presentation; and 2) to examine whether disruption of the class II pathway by VV alters T cell responses in vitro and in vivo. A significant reduction in the expression of the class II chaperone, invariant chain (Ii), was observed during the late stage of VV infection. Yet surface expression of MHC class II molecules was maintained along with cell viability. To examine whether VV acts solely to disrupt host protein synthesis, B cells were treated with an inhibitor of translation-cycloheximide (CHX). Like VV, CHX negatively regulated Ii protein expression and class II presentation. Ii proteolysis also contributed in part to reduce Ii expression in VV infected and CHX treated APC. Yet only VV infection altered lysosomal protease expression, potentially influencing Ii degradation. Over-expression or ectopic-expression of Ii partially protected cells from VV-induced class II dysfunction. These studies suggest VV destabilizes class II molecules by disrupting Ii expression. To examine the presentation of viral Ags by class II, CD4 T cells from VV-primed mice were used. Viral proteins were presented by class II shortly after APC exposure to low concentrations of VV. The presentation of VV Ags correlated temporally with reductions in exogenous peptide presentation. At higher MOI (≥ 1), class II presentation of VV Ags was reduced. To examine the in vivo effects of VV on Ag presentation, a mouse model of ovalbumin-induced airway hypersensitivity was used. Th2 cytokine production was reduced, while a novel inflammatory cytokine Interleukin-17 (IL-17) production was enhanced in asthmatic VV-infected mice. In health mice, repeated VV infections lead to enhanced CD8 T cell production of Interferon-γ (IFN-γ) and IL-17. Finally, antibodies to a viral protein H3 were generated and shown to preserve class II presentation. Together these studies suggest VV disruption of the class II pathway may blunt T cell responses to VV.

No description available.

Kin-recognition abilities, first demonstrated 25 years ago in toad tadpoles, now appear to be widespread among amphibians. In some vertebrates kin recognition is based, at least in part, on highly polymorphic major histocompatibility complex (MHC) genes. Besides protecting animals from disease resistance, MHC genes regulate social behaviour. They allow relatives to recognise one another so that they can cooperate for mutual benefit. These two seemingly distinct functions of MHC genes may be integrally related, because animals need to outbreed to optimise the immune systems of their offspring. The ability to discriminate MHC-type is therefore likely to facilitate kin discrimination in tadpoles. I tested association preferences of African clawed-frog (Xenopus laevis) tadpoles in a laboratory choice apparatus. As in other anuran species, I found that tadpoles at earlier developmental stages preferentially associate with unfamiliar siblings over unfamiliar non-siblings but that this preference reverses during development. Tadpoles approaching metamorphosis demonstrated a reversal in their preference; they preferentially school with non-kin rather than kin. The ontogenetic switch in larval schooling preferences coincides with the onset of thyroid hormone (TH) controlled development and may be indicative of decreased fitness benefits associated with schooling with kin at later developmental stages. These may result from an increase in intraspecific competition, predation, or disease susceptibilities of prometamorphic individuals. Alternatively, the kin avoidance behaviours observed at later larval stages might reflect disassociative behaviour that facilitates inbreeding avoidance at reproductive maturity. This is the first study to find a shift from an association preference for kin to non-kin during amphibian larval development. Using allele-specific PCR techniques to MHC-type tadpoles, I tested association preferences among siblings based on shared MHC haplotypes. By using only full siblings in experimental tests, I controlled for genetic variation elsewhere in the genome that might influence schooling preferences. I found that X. laevis tadpoles discriminate among familiar full siblings based on differences at MHC genes. Subjects from four families preferentially schooled with MHC-identical siblings over those with which they shared no or one haplotype. Furthermore, the strength of tadpoles’ MHC-assortative schooling preferences significantly correlated with amino acid differences in the peptide-binding region (PBR) of both the MHC class I and II loci. Since MHC-PBR polymorphisms determine the pool of peptides that can serve as ligands for MHC molecules, these findings support the hypothesis that MHC peptide ligands mediate MHC type discrimination. As test subjects were equally familiar with all stimulus groups, tadpole discrimination appears to involve a self-referent genetic recognition mechanism whereby individuals compare their own MHC type with those of conspecifics. I also found that non-MHC-linked genetic differences contribute to tadpole association preferences in tests that contrast MHC and kinship. Tadpoles did not discriminate between MHC-similar non-siblings and MHC-dissimilar siblings and preferentially associated with MHC-dissimilar non-siblings rather than MHC-similar non-siblings. Although the MHC may be not solely responsible for the genetically determined cues that direct tadpole association preferences, it certainly is important in facilitating discrimination among conspecifics in X. laevis tadpoles. MHC-based discrimination may be retained through ontogeny and serve to maintain MHC-polymorphisms by facilitating disassortative mating.

No description available.

No description available.