Genome-wide association studies have very successfully found highly significant disease associations with single nucleotide polymorphisms (SNP) in the Major Histocompatibility Complex for adverse drug reactions, autoimmune diseases and infectious diseases. However, the extensive linkage disequilibrium in the region has made it difficult to unravel the HLA alleles underlying these diseases. Here I present two methods to comprehensively predict 4-digit HLA types from the two types of experimental genome data widely available. The Virtual SNP Imputation approach was developed for genome scan data and demonstrated a high precision and recall (96% and 97% respectively) for the prediction of HLA genotypes. A reanalysis of 6 genome-wide association studies using the HLA imputation method identified 18 significant HLA allele associations for 6 autoimmune diseases: 2 in ankylosing spondylitis, 2 in autoimmune thyroid disease, 2 in Crohn's disease, 3 in multiple sclerosis, 2 in psoriasis and 7 in rheumatoid arthritis. The EPIGEN consortium also used the Virtual SNP Imputation approach to detect a novel association of HLA-A*31:01 with adverse reactions to carbamazepine. For the prediction of HLA genotypes from next generation sequencing data, I developed a novel approach using a naïve Bayes algorithm called HLA-Genotyper. The validation results covered whole genome, whole exome and RNA-Seq experimental designs in the European and Yoruba population samples available from the 1000 Genomes Project. The RNA-Seq data gave the best results with an overall precision and recall near 0.99 for Europeans and 0.98 for the Yoruba population. I then successfully used the method on targeted sequencing data to detect significant associations of idiopathic membranous nephropathy with HLA-DRB1*03:01 and HLA-DQA1*05:01 using the 1000 Genomes European subjects as controls. Using the results reported here, researchers may now readily unravel the association of HLA alleles with many diseases from genome scans and next generation sequencing experiments without the expensive and laborious HLA typing of thousands of subjects. Both algorithms enable the analysis of diverse populations to help researchers pinpoint HLA loci with biological roles in infection, inflammation, autoimmunity, aging, mental illness and adverse drug reactions.

Tsang Kam Sze, Kent. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 155-174). / Abstract --- p.i / Acknowledgements --- p.v / List of Abbreviations --- p.viii / Table of Contents --- p.x / List of Figures --- p.xvi / List of Tables --- p.ixx / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Idiotype Network --- p.2 / Chapter 1.2. --- Anti-idiotype Classification --- p.8 / Chapter 1.3. --- Blood Transfusion Effect --- p.11 / Chapter 1.4. --- Transfusion Protocol --- p.12 / Chapter 1.5. --- Mechanism of Beneficial Transfusion Effect --- p.15 / Chapter 1.5.1. --- Donor Selection --- p.15 / Chapter 1.5.2. --- Clonal Deletion --- p.16 / Chapter 1.5.3. --- Suppressor Cells Induction --- p.18 / Chapter 1.5.4. --- Prostaglandins Mediation --- p.19 / Chapter 1.5.5. --- Mixed Chimerism Motivation --- p.20 / Chapter 1.5.6. --- Fc-receptor Blocking Antibodies Stimulation --- p.22 / Chapter 1.5.7. --- Anti-idiotypic Antibodies Instigation --- p.23 / Chapter 1.6. --- Study Aims --- p.25 / Chapter 1.7. --- Technical Strategy --- p.26 / Chapter Chapter 2. --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.31 / Chapter 2.1.1. --- Patient Population --- p.31 / Chapter 2.1.2. --- Normal Control Group --- p.31 / Chapter 2.1.3. --- Serum Samples --- p.32 / Chapter 2.1.4. --- Additional Specimens --- p.32 / Chapter 2.1.5. --- Chemicals --- p.32 / Chapter 2.1.6. --- Antisera --- p.34 / Chapter 2.1.7. --- Buffers --- p.35 / Chapter 2.1.8. --- Consumables --- p.38 / Chapter 2.1.9. --- Apparatus and Equipment --- p.39 / Chapter 2.2. --- Methods --- p.40 / Chapter 2.2.1. --- Purification of Human Polyclonal Anti-HLA Antisera --- p.40 / Chapter 2.2.1.1. --- Affinity Chromatography --- p.41 / Chapter 2.2.1.2. --- Dialysis --- p.41 / Chapter 2.2.1.3. --- Concentration --- p.42 / Chapter 2.2.1.4. --- Quantitation --- p.42 / Chapter 2.2.2. --- Generation of F(ab')2 fragments from the Purified Human Anti-HLA Antibodies --- p.42 / Chapter 2.2.2.1. --- Buffer Exchange --- p.43 / Chapter 2.2.2.2. --- Pepsin Digestion --- p.43 / Chapter 2.2.2.3. --- Purification of (ab')2、 --- p.43 / Chapter 2.2.3. --- Enzyme-Linked Immunosorbent Assay for anti-Idiotypes against anti-HLA antibodies --- p.44 / Chapter 2.2.3.1. --- Optimization --- p.44 / Chapter 2.2.3.2. --- Quality Control --- p.45 / Chapter 2.2.3.2.1. --- F(ab')2 Specificity --- p.45 / Chapter 2.2.3.2.2. --- Fc Contamination --- p.46 / Chapter 2.2.3.2.3. --- Precision Test --- p.47 / Chapter 2.2.4. --- Anti-Casein Interference --- p.47 / Chapter 2.2.5. --- Test Protocol --- p.48 / Chapter 2.3. --- Statistical Analysis --- p.48 / Chapter Chapter 3. --- Purification of Anti-HLA IgG and F(ab')2 --- p.50 / Chapter 3.1. --- Immunoglobulin Concentration --- p.51 / Chapter 3.2. --- F(ab')2 Specificity --- p.51 / Chapter 3.3. --- Fc-fragments Contamination --- p.53 / Chapter 3.4. --- Discussion --- p.56 / Chapter Chapter 4. --- ELISA Optimization --- p.57 / Chapter 4.1. --- Coating F(ab')2 Quantitation --- p.58 / Chapter 4.2. --- Blocking and Diluting Agent Concentration --- p.61 / Chapter 4.3. --- Serum Analyte Dilution --- p.61 / Chapter 4.4. --- Conjugated Detector Antibody Titration --- p.64 / Chapter 4.5. --- Discussion --- p.66 / Chapter Chapter 5. --- Quality Control --- p.70 / Chapter 5.1. --- Avoidance of Prozone Phenomenon --- p.71 / Chapter 5.2. --- Inter-assay and Intra-assay Precision --- p.71 / Chapter 5.3. --- Discussion --- p.74 / Chapter Chapter 6. --- Adjustment of Anti-casein Interference --- p.77 / Chapter 6.1. --- Casein Allergy --- p.78 / Chapter 6.2. --- Prevalence of Anti-casein --- p.80 / Chapter 6.3. --- Discussion --- p.81 / Chapter Chapter 7. --- Prevalence of Anti-idiotypic Antibodies --- p.86 / Chapter 7.1. --- Formation Kinetics --- p.87 / Chapter 7.2. --- Occurrence in Transplant Patients --- p.87 / Chapter 7.3. --- Transfusion Effect --- p.101 / Chapter 7.3.1. --- Comparison between Transfused Transplant Patients and Normal Controls --- p.103 / Chapter 7.3.2. --- Comparison between Transfused Transplant Patients and Non-transfused Transplant Patients --- p.116 / Chapter 7.3.3. --- Association with Graft Survival --- p.117 / Chapter 7.4. --- Discussion --- p.128 / Chapter Chapter 8. --- Correlation of Transfusion with the Outcome of Transplant --- p.137 / Chapter 8.1. --- Rejection Episode --- p.138 / Chapter 8.2. --- Graft Survival --- p.139 / Chapter 8.3. --- Discussion --- p.142 / Chapter Chapter 9. --- General Conclusions --- p.149 / References --- p.153

O câncer de próstata (CaP) e a hiperplasia prostática benigna (HPB) são condições tumorais prostáticas não relacionadas, de alta prevalência e que afetam homens com idade avançada. Suas etiologias não são bem compreendidas, havendo poucos fatores de risco reconhecidos, o que torna difícil a identificação dos indivíduos suscetíveis a estas doenças. A condição imunológica é um fator determinante no desenvolvimento e progressão tumoral. O antígeno leucocitário humano G (HLA-G) é uma molécula imunomodulatória relacionada a mecanismos de tolerância imunológica. Inúmeras evidências vêm apoiando o papel do HLA-G como um mecanismo de escape das células tumorais da imunidade antitumoral, sugerindo que a expressão desta molécula em pacientes com câncer possa ser prejudicial. Em condições patológicas e fisiológicas da próstata, pouco se conhece sobre o papel do HLA-G. No presente trabalho, investigamos a influência do HLA-G em câncer de próstata e hiperplasia prostática benigna. Participaram do estudo homens do sul do Brasil com CaP, HPB e indivíduos saudáveis predominantemente euro-descendentes. Inicialmente, oito polimorfismos da região 3’ não traduzida (UTR) do gene HLA-G foram analisados em 468 indivíduos, incluindo o polimorfismo de inserção/deleção de 14 pares de bases (rs371194629), e os polimorfismos de nucleotídeo único (SNP) na posição +3003T/C (rs1707), +3010C/G (rs1710), +3027A/C (rs17179101), +3035C/T (rs17179108), +3142G/C (rs1063320), +3187A/G (rs9380142) e +3196C/G (rs1610696) do gene. Também foi caracterizado o perfil de expressão da proteína HLA-G em 53 tecidos cancerosos/hiperplásicos (CaP/HPB) e em porções avaliadas como normais de próstatas provenientes de pacientes com CaP e HPB. Por fim, o perfil imunológico sistêmico foi investigado em cada condição estudada através da caracterização do perfil de citocinas séricas Th1/Th2/Th17 nos três grupos (n=89). Para tanto, foram dosadas as citocinas interleucina (IL)-2, IL-4, IL-6, IL-10, fator de necrose tumoral alfa (TNF-α), interferon gama (IFN-γ) e IL-17A. Nesse estudo, encontramos evidência de uma influência significativa de polimorfismos da 3’UTR do gene HLA-G na suscetibilidade ao CaP e nossos dados apoiam o uso da variante +3003 como um tag SNP para o risco de câncer de próstata. Além disso, foi possível diferenciar tecidos de próstata com câncer de tecidos com hiperplasia e tecidos normais pelo nível de expressão da proteína HLA-G. Uma expressão elevada foi observada predominantemente nos tecidos com CaP, não sendo comum níveis elevados de expressão de HLA-G em tecidos normais e com HPB. Tais resultados fornecem evidência de considerável influência da expressão proteica de HLA-G no desenvolvimento de CaP. Como um potencial mecanismo de escape das células cancerosas da próstata da imunidade antitumoral, o HLA-G representa um potencial alvo terapêutico para CaP. Quanto ao perfil imunológico sistêmico nas condições avaliadas, observamos nível elevado de IL-10 e TNF-α diferenciando homens com CaP dos saudáveis, enquanto níveis elevados de IFN-γ e IL-17A diferenciaram pacientes com HPB dos com CaP. Concluindo, nossos dados apontam um perfil relacionado à tolerância imunológica em câncer de próstata, influenciado pelo HLA-G e capaz de favorecer o desenvolvimento deste câncer. Já em HPB, que apesar de ser uma condição tumoral, é benigna, indícios de maior responsividade do sistema imune foram encontrados. / Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are unrelated prostatic tumoral conditions, highly prevalent and affecting aged men. Their etiologies are not well understood, with few risk factors recognized, which make the identification of susceptible individuals to the disease difficult. The immunological condition is determinant in tumor development and progression. The human leukocyte antigen G (HLA-G) is an immunomodulatory molecule related to mechanisms of immunological tolerance. Several pieces of evidence have supported the role of HLA-G as an escape mechanism of tumor cells from antitumor immunity, suggesting that the expression of this molecule in cancer patients can be harmful. In pathological and phisiological prostate conditions, little is known about the role of HLA-G. In the present work, we investigated the influence of HLA-G in prostate cancer and benign prostatic hyperplasia. Men from South Brazil with PCa, BPH and healthy individuals predominantly euro-descendant were included in the study. Firstly, eight HLA-G 3′ untranslated region (UTR) polymorphisms were analyzed in 468 individuals, including the 14 bp insertion/deletion polymorphism (rs371194629) and the single nucleotide polymorphisms (SNP) at gene position +3003T/C (rs1707), +3010C/G (rs1710), +3027A/C (rs17179101), +3035C/T (rs17179108), +3142G/C (rs1063320), +3187A/G (rs9380142) and +3196C/G (rs1610696). The profile of HLA-G protein expression was characterized in 53 cancerous/hyperplastic (PCa/BPH) and in areas evaluated as normal prostate tissues provenient from patients with PCa and BPH. Finally, the systemic immunological profile was investigated in each studied condition through the characterization of the Th1/Th2/Th17 serum cytokine profile in the three groups (n=89). For that, the concentration of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and IL-17A cytokines was measured. In this study, we found evidence of a significant influence of HLA-G 3′UTR polymorphisms in PCa susceptibility and our data support the use of the +3003 variant as a tag SNP for PCa risk. In addition, it was possible diferentiate prostate tissues with cancer from with hyperplasia and normal tissues through the level of HLA-G protein expression. An elevated expression was observed predominantly in PCa tissues, but elevated level of HLA-G expression was not common in normal and BPH prostate tissues. These results provide evidence of a considerable influence of HLA-G protein expression in PCa development. As a possible escape mechanism of prostate tumor cells from antitumor immunity, HLA-G represents a potential therapy target in PCa. About the systemic immunological profile in the evaluated conditions, high IL-10 and TNF-α level differentiated PCa patients from healthy men, while high IFN-γ and IL-17A level differentiated BPH from PCa patients. In conclusion, our data point out to a profile related to immunological tolerance in PCa, influenced by HLA-G and able to favour the cancer development. In BPH, a benign tumor condition, the data point out to a higher responsiveness of the immune system.

Anemia falciforme (AF) é considerada a doença monogênica mais prevalente no Brasil, e resulta de uma mutação pontual no gene da beta-globina que leva à produção de uma molécula de hemoglobina anormal (HbS). A HbS polimeriza quando submetida a baixas tensões de oxigênio, precipitando e causando a deformação dos eritrócitos pois torna rígida sua membrana plasmática que apresentará uma série de alterações e danos. Além disso, a falcemização dos eritrócitos ocasiona anemia severa, lesão de isquemia/reperfusão, superprodução de espécies reativas de oxigênio, inflamação e vaso-oclusão (VO). A VO manifesta-se clinicamente como crises de dor, ou crises vaso-oclusivas (CVOs) que pode levar ao bloqueio de vasos e capilares e ao comprometimento de órgãos. Estes pacientes possuem alta susceptibilidade a infecções, principalmente na infância. Os mecanismos envolvidos no desenvolvimento da VO, no entanto, não estão totalmente elucidados. Estudos acerca deste tema têm sugerido que a VO seria o resultado da interação entre eritrócitos falcêmicos, leucócitos, plaquetas, células endoteliais e substâncias presentes no plasma dos indivíduos afetados. Tais observações como que AF seria o resultado de uma resposta inflamatória exacerbada que estes indivíduos desenvolvem, levaram à formulação da hipótese de que a anemia falciforme se comporta como uma condição inflamatória crônica e sugerindo que uma modulação do sistema imune poderá ser útil para a regulação da sintomatologia clínica. Salientando a carência de dados na literatura referentes à correlação de polimorfismos descritos para genes do sistema imune e a patofisiologia da AF, nosso estudo tem como objetivo principal: Analisar a correlação para polimorfismos descritos para genes do sistema imune e correlacionar-lho com a patofisiologia da AF. Sabemos que o HLA-G é uma molécula HLA não-clássica, que mostrou ser expressa em sítios de inflamação e nas doenças inflamatórias. Na região promotora além de ser altamente polimórfica, encontramos a região UTR 3' que parece desempenhar um papel importante na regulação da expressão do HLA-G. Então, dentre dos polimorfismos avaliados específicamente temos os dos genes HLA-G (14pb) e MiRNA (+3142). Nossos resultados indicam que os polimorfismos do HLA-G de 14pb e +3142 em 93 pacientes com AF, 21 pacientes apresentaram uma infecção pelo VHC e 16 pacientes com AF (22,2%) eram homozigotos para o genótipo +3142C e nenhum deles era positivo para HCV. Nenhum dos resultados obtidos indicou qualquer tipo de imunodeficiência mas pelo contrário, sugerem a existência de uma tendência inflamatória crônica na clínica da AF. Assim fica evidente a importância do polimorfismo +3142 sobre a susceptibilidade a infecções entre os pacientes com SCD.

Mit dieser Arbeit sollte untersucht werden, ob es eine Beeinträchtigung der körperlichen Leistungsfähigkeit bei Patienten bis zum 20. Lebensjahr mit inaktiver juveniler idiopathischer Arthritis bzw. einer Arthritis in Remission im Vergleich zu gesunden Gleichaltrigen gibt und wenn ja, ob ein Zusammenhang zu dem Eiweißkörper HLA B27 besteht. / 1 Introduction 1.1 Definition 1.2 Goal of this Thesis 2 Material and Methods 2.1 Description of the Test Persons 2.1.1 HLA B27-positive Patients 2.1.2 HLA B27-negative Patients with Arthritis 2.1.3 Healthy Controlls 2.2 Order of Study 2.2.1 Information and physical Examination 2.2.2 Wingate Test 2.2.3 Questionaire and VAS 2.2.4 Stresstest for measuring the Aerob Capacity 2.3 Analysis 3 Results 3.1 Wingate Test 3.1.1 Entire Test Persons 3.1.2 Triplets 3.2 Questionaire and VAS 3.2.1 Entire Test Persons 3.2.2 Triplets 3.3 Stresstest 3.3.1 Entire Test Persons 3.3.2 Triplets 4 Discussion 4.1 Test Persons 4.2 Wingate Tes 4.3 Questionaire and VAS 4.4 Stresstest 4.5 Summary 5 Conclusion Abbreviations Attachments Literature

Zusammenfassung Einleitung HLA-G, ein nicht-klassisches HLA bzw. MHC Klasse Ib Molekül, kann sowohl als membrangebundenes als auch als lösliches Molekül verschiedenste Immunzellpopulationen effektiv inhibieren. Unter physiologischen Bedingungen wird HLA-G vor allem in der Plazenta exprimiert, wo es dazu beiträgt den semiallogenen Embryo vor einer Abstoßung durch das mütterliche Immunsystem zu beschützen. Außerdem wird HLA-G in einer Vielzahl von Tumoren wie zum Beispiel in Ovarialkarzinomen überexprimiert. Ziel dieser Arbeit war es besonders die Rolle von löslichem HLA-G im Ovarialkarzinom und die Expression von HLA-G in verschiedenen Subtypen des Ovarialkarzinoms genauer zu untersuchen. Ergebnisse Anhand eines Tissue Microarrays wurde bestätigt dass HLA-G unter physiologischen Bedingungen nur in sehr wenigen Geweben wie Plazenta oder Testes exprimiert wird. Außerdem wurden erstmals auch im Nebennierenmark hohe Expressionslevel detektiert. Im Gegensatz zur physiologischen Expression wurde HLA-G in serösen, muzinösen, endometrioiden und Klarzellkarzinomen und somit in Tumoren aller untersuchten Subtypen des Ovarialkarzinoms detektiert. Am häufigsten war HLA-G in hochgradigen serösen Karzinomen überexprimiert. Hier konnte gezeigt werden dass auf Genexpressionslevel in Ovarialkarzinomen die Expression des immunsuppressiven HLA-G mit der Expression von klassischen MHC Molekülen wie HLA-A, -B oder -C hochsignifikant korreliert. Außerdem konnte in Aszitesproben von Patientinnen mit Ovarialkarzinomen hohe Konzentrationen von löslichem HLA-G nachgewiesen werden. Auch auf metastasierten Tumorzellen in regionalen Lymphknoten war HLA-G nachweisbar. Überraschenderweise wurde aber besonders viel HLA-G auf Dendritischen Zellen in Lymphknoten detektiert. Da in Monozyten und Dendritischen Zellen von gesunden Spendern durch IL-4 oder IL-10 im Gegensatz zu Literatur keine Expression von HLA-G induzierbar war, untersuchten wir ob Dendritische Zellen lösliches HLA-G binden. Es konnte gezeigt werden, dass besonders Dendritische Zellen die in Gegenwart von IL-4, IL-10 und GM-CSF aus Monozyten generiert wurden (DC-10) effektiv lösliches HLA-G über ILT Rezeptoren binden. In Abhängigkeit von ihrer Beladung mit HLA-G hemmen auch fixierte DC-10 Zellen noch die Proliferation von zytotoxischen CD8+ T Zellen. Zudem wurden regulatorische T Zellen induziert. Schlussfolgerungen Besonders in den am häufigsten diagnostizierten hochgradigen serösen Ovarialkarzinomen ist HLA-G in den meisten Fällen überexprimiert. Durch die Expression immunsuppressiver MHC Klasse Ib Moleküle wie HLA-G können wahrscheinlich auch Tumore wachsen, die noch klassische MHC Moleküle exprimieren und aufgrund ihrer Mutationslast eigentlich vom Immunsystem erkannt und eliminiert werden müssten. Lösliches HLA-G könnte zudem lokal Immunantworten gegen Tumorantigene unterdrücken indem es an Dendritische Zellen in regionalen Lymphknoten bindet. Diese Zellen präsentieren nomalerweise zytotoxischen T Zellen Tumorantigene und spielen daher eine entscheidende Rolle in der Entstehung von protektiven Immunantworten. Mit löslichem HLA-G beladene Dendritische Zellen hemmen jedoch die Proliferation von CD8+ T Zellen und induzieren regulatorische T Zellen. Dadurch könnten Ovarialkarzinome “aus der Ferne” auch in metastasenfreien Lymphknoten die Entstehung von gegen den Tumor gerichteten Immunantworten unterdrücken. Dieser erstmals beschriebene Mechanismus könnte auch in anderen malignen Erkrankungen eine Rolle spielen, da lösliches HLA-G in einer Vielzahl von Tumorindikationen nachgewiesen wurde. / Abstract Background HLA-G is a non-classical MHC class I molecule which exerts strong immunosuppressive effects on various immune cells. Several membrane-bound and soluble isoforms are known. Physiologically, HLA-G is predominantly expressed in the placenta, where it contributes to protecting the semi-allogeneic embryo from rejection by the maternal immune system. However, HLA-G is also often upregulated during tumourigenesis, such as in ovarian cancer. The aim of this thesis is to investigate how soluble HLA-G may contribute to local immunosuppression in ovarian carcinomas, and to characterize HLA-G expression in different ovarian carcinoma subtypes and metastases. Results As reported by others, physiological HLA-G expression is restricted to few tissues, such as placenta and testes. Here, HLA-G was also detected in the medulla of the adrenal gland. In contrast, HLA-G expression was frequently detected in tumours of all assessed subtypes of ovarian carcinomas (serous, mucinous, endometrioid and clear cell). Highest expression levels were detected in high-grade serous carcinomas. In primary tumours, expression of HLA-G correlated with expression of classical MHC class I molecules HLA-A, -B and -C. Surprisingly, high levels of HLA-G were also detected on dendritic cells in local lymph nodes. As no expression of HLA-G was inducible in monocytes or dendritic cells from healthy donors in response to IL-10 or IL-4, we speculated that tumour-derived soluble HLA-G might be transferred to dendritic cells via the lymphatic system. Accordingly, high levels of tumour-derived soluble HLA-G were detected in ovarian cancer ascites samples. In vitro, dendritic cells expanded in the presence of IL-4, IL-10 and GM-CSF (DC-10) were particularly prone to binding high amounts of soluble HLA-G via ILT receptors. Furthermore, HLA-G loaded DC-10 cells inhibited the proliferation of CD8 effector cells and induced regulatory T cells, even when the DC-10 cells had been fixed with paraformaldehyde. Conclusion The immunosuppressive molecule HLA-G is overexpressed in high-grade serous ovarian carcinomas, which account for the majority of ovarian cancers. In particular tumours with a high mutational burden and intact expression of classical, immunogenic MHC class Ia molecules may use HLA-G to escape from immunosurveillance. Additionally, tumour-derived soluble HLA-G may inhibit adaptive immune responses by binding to dendritic cells in local lymph nodes. Dendritic cells usually play a decisive role in the initiation of adaptive anti-tumour immune responses by presenting tumour antigens to cytotoxic T cells. In contrast, dendritic cells loaded with soluble HLA-G inhibit the proliferation of effector T cells and promote the induction of regulatory T cells. Thus, soluble HLA-G that is transferred to dendritic cells via lymphatic vessels may enable ovarian carcinomas to remotely suppress anti-tumour immune responses in local lymph nodes. This novel immune-escape mechanism may also exist in other solid tumours that express HLA-G.

Selection of HIV-1 variants resistant to antiretroviral therapy is well documented. However, the selection in vivo of HIV-1 mutant species that can escape host immune system HLA class I restricted cytotoxic T-lymphocyte responses has, to date, only been documented in a few individuals and its clinical importance is not well understood. This thesis analyses the observed diversity of the HIV-1 reverse transcriptase protein in a well characterised, stable, HLA-diverse cohort of HIV-1 infected patients with over two thousand patient-years of observation. The results show that HIV-1 polymorphism is selected within functional constraints and is associated with specific HLA class I alleles. Furthermore, these associations significantly cluster along the sequence and tend to occur within known corresponding HLA-restricted epitopes. Absence of polymorphism is also HLA-specific and more often seen with common HLA alleles. Knowledge of HLA specific viral polymorphisms can be used to model an individual’s viral load from their HLA type and viral sequence. These results suggest that cytotoxic T-lymphocyte escape mutation in HIV-1 is critical to the host at an individual and population level as well as to short and long term viral evolution. This work provides new insights into viral-host interactions and has clinical implications for individualisation of HIV-1 therapy and vaccine design.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation eventually leading to the destruction of cartilage and bone. The aetiopathogenesis is not completely understood, but previous studies have shown that the disease is multifactorial with genetic, environmental and hormonal factors involved. Immune cells, e.g., T- and B-cells, and macrophages, migrate into the joints, with increased expression of numerous soluble factors such as cytokines, chemokines and adhesion molecules functionally active both locally and systemically. Analyses of blood samples from the Medical Biobank in Umeå from individuals before the onset of symptoms of joint disease showed that anti-citrullinated protein/peptide antibodies (ACPA) preceded the development of disease by years and this finding has been confirmed by other studies.                                         The aim of this thesis was to identify signs of activation of the immune system analysed as up-regulation of pro- and anti-inflammatory cytokines, sero-positivity for autoantibodies, and genetic factors identified as relevant for the development and disease progression of RA. The concentrations of 30 cytokines and chemokines were measured in blood samples from individuals before the onset of symptoms, and when diagnosed with RA, together with population-based matched controls using a multiplex system. The predictive value of different isotypes (IgG, IgA, and IgM) of ACPA and rheumatoid factor (RF) before onset of symptoms and different types of ACPA (e.g., mutated citrullinated vimentin, MCV) were analysed for disease development and progression in patients with early RA and controls from Northern Sweden. These factors were related to the genetic markers, HLA- shared epitope (SE) alleles and the 1858C/T polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene.                            In paper I, it was shown that in individuals who later developed RA (i.e., pre-patients) the levels of several cytokines and related factors that represent the adaptive immune system (Th1, Th2, and T regulatory cell related factors) were significantly elevated compared with controls, whereas, after the onset of disease the involvement of the immune system was more general and widespread. In paper II, the presence of different isotypes (IgM, IgA and IgG) of ACPA in pre-patients, patients and controls was evaluated showing that both the IgG and IgA isotype predicted the onset of RA by years with the IgG isotype having the highest predictive value. In paper III, the association of the 1858T variant of PTPN22 with RA was confirmed. Furthermore, the association was restricted to autoantibody positive disease and this variant was correlated with an earlier age for disease onset. In paper IV, anti-MCV antibodies were identified as being associated with a more severe disease course of RA, measured by disease activity score, erythrocyte sedimentation rate, and swollen joint count over time compared with anti-CCP2, anti-CCP3, and anti-CCP3.1 antibodies.                                                                                                In conclusion, individuals who later developed RA had increased concentrations of inflammatory markers reflecting an activation of the immune system years before the clinical symptoms of the disease developed. Also, the presence of ACPA of IgG and IgA isotype prior to disease onset predicted the development of RA. The PTPN22 1858T variant was associated with sero-positive RA and anti-MCV antibodies were associated with a higher inflammatory activity compared with anti-CCP2, -CCP3 and -CCP3.1 antibodies. These findings together present a possibility to better predict the development and progression of RA.

The aim of the project was to develop a PCR method to detect HLA-B27 at the Immunology Department of St. James hospital in Dublin. The HLA-B27 gene is common among patients with ankylosing spondylitis (AS). Ninety percent of patients with AS have the HLA-B27 gene and it is therefore counted as a risk factor and could be used as part of the diagnosis. Twenty-two frozen blood samples from patients with AS or suspected AS were donated from the rheumatology department at St. James hospital. PCR is a well known and common technique, many hospital laboratories have a PCR machine and therefore PCR is a good choice for detection of the HLA-B27 gene. A multiplex PCR was developed where a PCR control, primers to the β-globin gene, was used in the same tube as the HLA-B27 primers, to secure that the PCR worked in every tube. Finally a blind test was performed to test the specificity of the PCR. The result shows that the specificity was 100%. Of all patient samples, sixteen was HLA-B27 positive and six were HLA-B27 negative. In addition, optimal conditions for the PCR and the way to extract DNA from frozen blood were successfully established. For future diagnosis, the described PCR can be used to detect the HLA-B27 gene in patients and it can be considered as a start for further development of a real-time PCR for detection of the HLA-B27.

<p><i>Successful integration of simulations within the Network-Based Defence (NBD), specifically use of simulations within Command and Control (C2) environments, enforces a number of requirements. Simulations must be reliable and be able to respond in a timely manner. Otherwise the commander will have no confidence in using simulation as a tool. An important aspect of these requirements is the provision of fault-tolerant simulations in which failures are detected and resolved in a consistent manner. Given the distributed nature of many military simulations systems, services for fault-tolerance in distributed simulations are desirable. The main architecture for distributed simulations within the military domain, the High Level Architecture (HLA), does not provide support for development of fault-tolerant simulations.</i></p><p><i>A common approach for fault-tolerance in distributed systems is check-pointing. In this approach, states of the system are persistently stored through-out its operation. In case a failure occurs, the system is restored using a previously saved state. Given the abovementioned shortcomings of the HLA standard this thesis explores development of fault-tolerant mechanisms in the context of the HLA. More specifically, the design, implementation and evaluation of fault-tolerance mechanisms, based on check-pointing, are described and discussed.</i></p>