Plattenepithelkarzinome im Kopf-Hals-Bereich, insbesondere HPV-assoziierte HNSCC, sind in den vergangenen Jahren zunehmend in den Fokus onkologischer Forschung gerückt. Während sich die Untersuchungen zunächst überwiegend auf die Identifizierung und Bestätigung lebensstilassoziierter Risikofaktoren wie Tabak- und Alkoholkonsum konzentrierten, die inzwischen als gesicherte und dominierende Risikofaktoren gelten, erlangten im Verlauf die huma-nen Papillomviren zunehmende Aufmerksamkeit. Auch der kausale Zusammenhang zwischen HPV und HNSCC (vor allem OPSCC) wird inzwischen nicht mehr in Frage gestellt. Im Jahr 2017 erhielt HPV mit dem Surrogatmarker p16 sogar Einzug in die aktuelle (8.) Edition der TNM-Klassifikation; p16-positive und -negative OPSCC werden seitdem verschieden klassifiziert. Obwohl Leitlinien dem entgegenstehen, wird eine De-Eskalation der Therapie trotz fehlender Evidenz aus randomisiert-kontrollierten klinischen Studien auch außerhalb von Studien diskutiert. Allerdings führen weder Tabak- und Alkoholkonsum noch HPV-Infektionen bei allen exponierten Menschen zur Entstehung maligner Erkrankungen inklusive HNSCC. Daher scheint neben diesen Risikofaktoren auch der genetische Hintergrund eine entscheidende Rolle bei der Entstehung von Malignomen zu spielen. Eine genetische Prädisposition für eine unwirksame Auseinandersetzung mit neu aufgetretenen neoplastisch transformierten Zellen sollte bei gleicher Histologie weitestgehend unabhängig von der Sub-Lokalisation des Gewebes sein, sofern die biologische Basis identisch ist. Onkogene HPV-Subtypen haben das Potential, eine Vielzahl epithelialer Gewebe zu infizieren und neoplastisch zu transformieren, die zugrundeliegenden molekularbiologischen Mechanismen sind seit längerem bekannt. Offen blieb bisher, warum die meisten HPV-Infektionen zu einer transienten neoplastischen Transformation führen, welche nur bei wenigen Patienten in einem manifesten Malignom resultiert. Aufgrund der zentralen Bedeutung von humanen Leukozyten-Antigenen für die Abwehr viraler Infektionen und für die Tumorentstehung lag ein Zusammenhang zwischen HLA-Merkmalen und HPV-getriebenen HNSCC nahe. Dass verschiedene HLA-Antigene und -Haplotypen Einfluss auf die Entstehung und das Outcome von HNSCC-Patienten haben, hatten Untersuchungen unserer Arbeitsgruppe bereits gezeigt. Diese Ergebnisse galt es in einer unabhängigen Kohorte zu überprüfen und weitere Erkenntnisse zur Rolle von HLA-Merkmalen in der Entstehung und Prognose sowie für mögliche individuelle Therapieentscheidungen zu gewinnen und außerdem die Frage der Bedeutung von HLA im Kontext HPV-getriebener HNSCC zu beantworten. Hierfür konnte ich innerhalb der Tumordatenbank und Biobank der HNO-Universitätsklinik 94 Patienten mit HPV-getriebenen HNSCC (p16-positiv und Nachweis von HPV-DNA und/oder RNA) inklusive Tumore verschiedener Lokalisationen außerhalb des Oropharynx identifizieren, die zwischen 2012 und 2018 am Universitätsklinikum Leipzig behandelt wurden. Zunächst galt es, nach Identifikation von Patienten mit HPV-getriebenen Tumoren verschiedener Lokalisationen und der HLA-Typisierung herauszufinden, ob sich korrelierend mit der Lokalisation Subgruppen von Patienten bezüglich klinischer, epidemiologischer und genetischer Merkmale identifizieren lassen. Bei 57 unserer 94 Patienten befand sich der Primärtumor im Oropharynx und bei 37 Patienten lag dieser außerhalb. Während einige Charakteristika wie Geschlecht, Alter bei Diagnose oder klassische Risikofaktoren für HNSCC wie Tabak- und Alkoholkonsum sich zwischen diesen beiden Gruppen nicht signifikant unterschieden, ließen sich in den folgenden klinischen Kategorien statistisch signifikante Unterschiede finden: T-Kategorie der 8. Edition, N-Kategorie der 7. und 8. Edition, UICC-Stadium der 8. Edition, kapselüberschreitendes Wachstum und Therapie inklusive Bestrahlung. Bei Untersuchungen bezüglich des Einflusses dieser klinischen und epidemiologischen Risikofaktoren auf das progressionsfreie Überleben der Patienten ergaben sich signifikante Unterschiede abhängig von Nikotinkonsum, Alkoholkonsum, der T-Kategorie und der N-Kategorie. Interessanterweise konnte für andere bekannte Risikofaktoren wie Geschlecht, Lokalisation des Primärtumors, kapselüberschreitendes Wachstum und Art der Therapie in unserer Kohorte kein signifikanter Einfluss auf das PFS gezeigt werden. Obwohl bei Diagnosestellung eine ähnliche Verteilung der T-Kategorien zwischen den beiden Lokalisationsgruppen beobachtet wurde, wiesen Fälle mit Primum im Oropharynx häufiger lokoregionäre Metastasen und eine höhere Anzahl positiver Halslymphknoten auf. Dies ist unabhängig vom HPV-Status und konnte zuvor auch in einer anderen Kohorte gezeigt werden. Bei lokoregionärer Metastasierung unterschied sich die Verteilung ENE-positiver bzw. -negativer Lymphknoten kaum zwischen OPSCC und anderen HNSCC, sie war in beiden Gruppen mit einem schlechteren Outcome verbunden und das kapselüberschreitende Wachstum stellt im Gegensatz zur N-Kategorie einen unabhängigen Prädiktor für das PFS dar. Die signifikanten Unterschiede in den T- und N-Kategorien nach der 8. TNM-Edition scheinen allerdings mehr mit der abweichenden Kategorisierung p16-positiver OPSCC als mit einer differenten Biologie der Tumore zusammenzuhängen. In diesem Kollektiv erfolgten die HLA-Typisierungen mittels SSO-Typisierungskits. Bei der Betrachtung der HLA-Antigene fanden wir nur bei A11 eine signifikant abweichende Häufigkeit zwischen den Lokalisations-Gruppen. Wir konnten auch die Anreicherung von B51-Trägern im Vergleich zu gesunden Blutspendern, die von einer anderen Forschungsgruppe bereits beschrieben wurde, bestätigen. Im Vergleich zu gesunden deutschen Blutspendern fanden sich jedoch in unserer Kohorte einige HLA-Haplotypen signifikant vermindert. Dies waren vor allem diejenigen, die nach Müller et al. die höchste Prävalenz bei den gesunden Blutspendern aufweisen. Neben abweichenden Haplotyp-Frequenzen stellten wir bei diesen Haplotypen auch ein geringeres Kopplungsungleichgewicht fest, was wahrscheinlich durch die zuvor durch unsere Arbeitsgruppe beschriebene Disruption der Haplotypen und Rekombination von Antigenen in anderen, seltenen Haplotypen zu erklären ist. Darüber hinaus wiesen Träger der bei gesunden Erwachsenen am häufigsten vorkommenden Haplotypen ein niedrigeres relatives Risiko für HPV-getriebene HNSCC auf. Im Gegensatz dazu fanden wir bei HPV-getriebenen HNSCC erhöhte Frequenzen von A3-B18, A3-B8, A1-B51 und A3-B51, welche alle zu den bei gesunden Blutspendern niederfrequenten Haplotypen gehören. Dies spiegelt sich in einem erhöhten relativen Risiko für die Träger dieser Haplotypen und insbesondere der A3-Haplotypen A3-B18 und A3-B8 wider, die wir in unserer Kohorte häufig in ungewöhnlichen DR-Anti-genkombinationen fanden. Die erhöhte Prävalenz von A24-B8-DR3 bei 8 Patienten ist ein Beispiel für solche ungewöhnlichen Haplotypen. A3-Triplotypen, die bei gesunden Blutspendern selten vorkommen, wiesen in unserer Kohorte eine signifikant höhere Frequenz auf. Bis auf das Antigen A11 und den Haplotyp A3-B18 als einzige Ausnahmen wurden allerdings keine signifikanten Unterschiede in den Haplotyp-Frequenzen in Abhängigkeit von der Lokalisation des Primärtumors beobachtet. Der höherfrequent gefundene Haplotyp A3-B18 erwies sich als unabhängiger Prädiktor für schlechteres PFS. Wir konnten außerdem 13 unabhängige Prädiktoren für das PFS unserer Patienten identifizieren. Zu diesen zählen neben dem bereits genannten kapselüberschreitenden Wachstum (ENE) auch Alkoholkonsum, Lokalisation, T-Kategorie und Therapie sowie 8 HLA-Merkmale (A1C, B12C, A11, B51, DR15, DQB1 homozygot, A3-B18, A1-B8). Weiterhin konnten wir unter Verwendung der natürlichen Logarithmen der Hazard Ratios der von uns identifizierten unabhängigen Prädiktoren des PFS ein Modell entwickeln, das zwischen vier Risikogruppen von HPV-getriebenen HNSCC differenzieren kann. Dieses Modell besteht aus den oben genannten 5 klinischen und 8 genetischen Kovariaten. Die 4 Risikogruppen unterscheiden sich auch bezüglich des Rezidiv-freien Überlebens bzw. der lokalen, nodalen und loko-regionären Kontrolle, des fernmetastasierungsfreien Überlebens, des HNSCC-spezifischen und des Gesamtüberlebens. Unsere Ergebnisse können nicht nur Grundlage für zukünftige Präventions- und Therapiestrategien (in Abhängigkeit vom HPV-Status und genetischen Besonderheiten) sein, sondern liefern ebenso Hinweise darauf, dass genetischen Faktoren auch bei der Festlegung der Prognose und Planung der Nachsorge von HNSCC-Patienten größere Aufmerksamkeit geschenkt werden sollte. Unterschiede in der HLA-Merkmalsausstattung können einen wichtigen Beitrag auf dem Weg zur individualisierten Diagnostik und Therapie leisten, welcher aktuell in allen Bereichen der Medizin beschritten wird. Um unsere Ergebnisse zu bestätigen und den Weg in die klinische Praxis zu ermöglichen, sind selbstverständlich weitere Untersuchungen notwendig, für die wir mit bereits erfolgten Sequenzierungen mittels NGS in einer weiteren Kohorte gute Voraussetzungen geschaffen haben. Als zentrales Ergebnis meines Promotionsprojektes kann der von uns erbrachte Nachweis gelten, dass HLA-Antigene und Haplotypen Risikofaktoren für HPV-getriebene Plattenepithelkarzinome der Kopf-Hals-Region sind und den Behandlungserfolg beeinflussen.:1. Einleitung 1.1 Einteilung maligner Tumore im Kopf-Hals-Bereich 1.2 Epidemiologie 1.3 Risikofaktoren 1.4 HNSCC und HPV 1.5 Die Humanen Leukozyten-Antigene (HLA) 1.5.1 HLA-Genkomplex und Kopplungsungleichgewicht 1.5.2 HLA/MHC Klasse I 1.5.3 HLA/MHC Klasse II 1.5.4 Cross Reactive Epitope Groups (CREGs) 1.5.5 Tumormilieu und Immun-Escape-Mechanismen 1.6 Präventionsstrategien 1.7 Therapieoptionen 1.8 Prognose 1.9 Fragestellung und Zielsetzung 2. Publikationen 2.1 Vortrag: International Symposium on HPV-infection in Head and Neck Cancer vom 10.-11.11.2022 in Amsterdam 2.2 Vortrag: Meeting on Experimental and Translational Research in Head and Neck Cancer vom 20.-21.01.2023 in Ulm 2.3 Abstract und Vortrag: HNO-Kongress und 94. Jahresversammlung der DGHNO-KHC vom 17.-20.05.2023 in Leipzig 2.4 Publikation in Frontiers in Oncology 3. Zusammenfassung 4. Verzeichnisse 4.1 Abkürzungsverzeichnis 4.2 Abbildungsverzeichnis 4.3 Literaturverzeichnis 5. Erklärungen 5.1 Eigenständigkeitserklärung 5.2 Erklärung zum Eigenanteil an der Publikation 5.3 Bestätigung der Koautoren zur Erklärung des Eigenanteils 6. Lebenslauf 7. Danksagung 8. Anhang

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

MHCII molecules expose a weave of antigens, which send survival or activation signals to T lymphocytes. The ongoing process of peptide binding to the MHC class II groove implicates three accessory molecules: the invariant chain, DM and DO. The invariant chain folds and directs the MHCII molecules to the endosomal pathway. Then, DM exchanges the CLIP peptide, which is a remnant of the degraded invariant chain, for peptides of better affinity. Expressed in highly specialized antigen presenting cells, DO competes with MHCII molecules for DM binding and favors the presentation of receptor-internalized antigens. Altogether, these molecules exhibit potential immunomodulatory properties that can be exploited to increase the potency of peptide vaccines. DO requires DM for maturation and to exit the ER. Interestingly, it is possible to monitor this interaction through a conformation change on DOβ that is recognized by the Mags.DO5 monoclonal antibody. Using Mags.DO5, we showed that DM stabilizes the interactions between the DO α1 and β1 chains and that DM influences DO folding in the ER. Thus, the Mags.DO5+ conformation correlates with DO egress from the ER. To further evaluate this conformation change, directed evolution was applied to DO. Of the 41 unique mutants obtained, 25% were localized at the DM-DO binding interface and 12% are at the solvent-exposed β1 domain, which is thought to be the Mags.DO5 epitope. In addition, I used the library to test the ability of HLA-DO to inhibit HLA-DM and sorted for the amount of CLIP. Interestingly, most of the mutants showed a decrease inhibitory effect, supporting the notion that the intrinsic instability of DO is a required for its function. Finally, these results support the model in which DO competes against classical MHCII molecules by sequestering DM chaperone’s function. MHCII molecules are also characterized by their ability to present superantigens, a group of bacterial or viral toxins that coerces MHCII-TCR binding in a less promiscuous fashion than what is observed in a canonical setting. While the mechanism of how bacterial superantigens form trimeric complexes with TCR and MHCII is well understood, the mouse mammary tumor virus superantigens (vSAG) are poorly defined. In the absence of a crystal structure, I chose a functional approach to examine the relation between vSAG, MHCII and TCR with the goal of uncovering the overall trimolecular architecture. I showed that TCR concomitantly binds both the MHCII α chain and the vSAG and that TCR-MHCII docking is almost canonical when coerced by vSAGs. Because many peptides may be tolerated in the MHCII groove, the pressure exerted by vSAG seems to tweak conventional TCR-MHCII interactions. Furthermore, my results demonstrate that vSAG binding to MHCII molecules is conformation-dependent and abrogated by the CLIP amino-terminal residues extending outside the peptide-binding groove. In addition, they also suggest that vSAGs cross-link adjacent MHCIIs and activate T cells via a TGXY motif. / Les molécules du CMH présentent une panoplie d'antigènes qui, lorsque reconnus par un lymphocyte T spécifique, indique à ce dernier de survivre ou de s'activer. Le processus menant à la liaison d'un peptide à la poche du CMH de classe II, implique trois molécules accessoires, soit la chaine invariante, DM et DO. La chaine invariante replie et dirige les molécules du CMHII jusqu'à la voie endosomale. Ensuite, DM échange CLIP, peptide découlant de la dégradation de la chaine invariante, pour d'autres ayant une meilleure affinité. Exprimé seulement chez certaines cellules présentatrices, DO compétitionne avec les molécules du CMHII pour la liaison à DM et ainsi favorise la présentation d'antigènes internalisés par des récepteurs membranaires. Ensemble, ces protéines ont un potentiel immunomodulatoire pouvant être exploité afin d'augmenter l'efficacité de vaccin peptidique. DO requiert DM pour arriver à maturité et sortir du RE. Cette interaction, qui induit un changement de conformation dans la chaine β de DO, peut être sondée à l'aide de l'anticorps monoclonal Mags.DO5. En utilisant cet anticorps, nous avons montré que DM stabilise l'interaction entre les domaines α1 et β1 de DO et influence son repliement dans le RE. Donc, la conformation qui révèle l’épitope Mags.DO5 corrèle avec la migration de DO hors du RE. Afin d'étudier plus précisément ce changement de conformation, DO fut contraint à une ronde d’évolution dirigée. Des 41 mutants obtenus, 25% se retrouvent à l'interface DO- DM et 12% se retrouvent à la surface exposée au solvant du domain β1, région hypothétique de l'épitope Mags.DO5. De plus, la bibliothèque de mutants a été testée pour son habileté à inhiber l'activité de DM. La plupart des mutants montre une activité inhibitrice diminuée, ce qui supporte le model où DO compétionne les molécules du CMHII en séquestrant le rôle chaperon de DM. Les molécules du CMHII ont l'unique habileté de présenter les superantigènes, une famille de toxines virales et bactériennes qui force l'interaction CMHII-TCR de façon beaucoup moins spécifique qu'en contexte canonique. Alors que la façon dont les superantigènes bactériens s'assemblent avec le CMHII et le TCR soit bien comprise, la nature du complexe trimoléculaire découlant des superantigènes du virus de la tumeur mammaire de la souris (vSAG) reste mal définie. En l'absence d'une structure cristalline, une approche fonctionnelle a été choisie pour examiner la relation des vSAGs avec le CMHII et le TCR avec le but de dévoiler l'architecture de ce multi-complexe protéique. Je montre que le TCR lie parallèlement la chaine α du CMHII et vSAG, ce qui résulte en une interaction presque canonique. Puisque différents peptides peuvent être tolérés lors de cet ancrage, il semble que vSAG ajuste les interactions CMHII-TCR conventionnelles. En outre, mes résultats montrent que vSAG reconnait un épitope conformationnel et que cette liaison peut être abrogée par l'extension amino-terminale du peptide CLIP, laquelle s'étend en deçà de la niche. Finalement, mes résultats suggèrent que vSAG peut réticuler plusieurs CMHII adjacents et active les cellules T via son motif TGXY.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Kidney transplantation is the best treatment for patients with end-stage renal failure. The main problem of kidney transplantation is however the development of a cellular and antibody-mediated (humoral) rejection. During the last decade, thanks to the advanced immunosuppression, prognosis of survival and function of transplanted organs has significantly improved. Nevertheless, humoral rejection remains very serious obstacle in high-risk patients, because it can permanently damage the graft. Therefore, before transplantation it is necessary to stratify patients into high and low risk groups for development of antibody-mediated rejection. Current immunogenetic tests performed before transplantation include, in addition to HLA typing, detection of panel-reactive antibodies. However, this test does not provide information about B cells which participate in the humoral response of the kidney recipient. Therefore, in the presented thesis we studied B cell reactivity and its regulation in transplanted patients. In this retrospective analysis we measured levels of the B cell activating factor, a cytokine regulating the function of B lymphocytes (BAFF). Current reports suggest that BAFF could serve as a marker of humoral rejection. Furthermore, we focused on B lymphocytes and their capacity to produce...

O objetivo geral da pesquisa foi associar os polimorfismos do gene HLA-G (região 3\' NT) com a coinfecção HIV/HCV e com os grupos (HIV, HCV e controles saudáveis). Trata-se de um estudo transversal, comparativo, descritivo. Participaram do estudo, 560 indivíduos, sendo 156 controles saudáveis, 102 coinfetados HIV/HCV, 186 infectados pelo HIV e 116 por HCV. Para a identificação dos polimorfismos, o DNA genômico foi extraído do sangue total e a genotipagem feita por PCR e visualizada em gel de poliacrilamida a 7%, no qual o polimorfismo de 14pb foi identificado, e por sequenciamento os outros sete SNPs. Os resultados sociodemográficos apontam que a amostra na sua grande maioria foi composta por indivíduos adultos e do sexo masculino. No que diz respeito à cor da pele, na comparação entre os grupos HCV e HIV/HCV, observou-se um maior número de coinfectados apresentando a cor preta e parda do que nos monoinfectados (P=0,0001). Com relação à categoria de exposição para aquisição do HIV, na comparação entre os grupos HIV e HIV/HCV, observou-se diferença significante na transmissão por via heterossexual, sendo sua frequência maior no grupo HIV (P=0,0000). No caso da comparação entre os grupos HCV e HIV/HCV, observou-se também diferença na transmissão heterossexual, sendo sua frequência significantemente maior no grupo HIV/HCV (P=0,0001). Quanto aos achados relacionados ao genótipo do HCV, na comparação entre os grupos HCV e HIV/HCV, o genótipo 1a apresentou frequência maior nos coinfectados (P=0,0001). No que diz respeito à carga viral do HIV, na comparação entre os grupos HIV e HIV/HCV, o grupo da monoinfecção apresentou maior carga viral do que o grupo da coinfecção (P=0,0350). Com relação ao grau de fibrose hepática, na comparação entre os grupos HCV e HIV/HCV, o grupo da coinfecção tem mais fibrose leve do que o grupo da monoinfecção (P=0,0009). Quanto aos polimorfismos genéticos da região 3\' NT do HLA-G, foi encontrado que o genótipo de heterozigose Del/Ins de 14 pb apresentou diferença significante nos indivíduos coinfectados pelo HIV/HCV (P=0,0216) quando comparados com o grupo controle. Em relação ao SNP +3003, a comparação dos grupos HCV e controle saudável mostrou que alelo +3003T apresentou uma frequência significantemente maior no grupo HCV (P=0,0147); o genótipo +3003C/T apresentou uma frequência maior no grupo controle (P=0,0095); o genótipo +3003T/T estava maior no grupo HCV (P=0,0095). A comparação entre os grupos HIV e HCV mostrou que a frequência do alelo +3003C estava maior no grupo HIV (P=0,0463); e o genótipo +3003T/T apresentou uma frequência maior no grupo HCV (P=0,0494). A frequência do genótipo +3187A/A estava maior no grupo HIV/HCV em comparação ao HIV (P=0,0193); e do +3187A/G estava maior no grupo HIV (P=0,0187). O genótipo +3196C/G apresentou frequência significamente maior no grupo HIV do que no controle saudável (P=0,0213). A UTR-10, na comparação entre os grupos HIV e controle, mostrou frequência maior no grupo HIV (P=0,0044); quando comparados os grupos HIV/HCV e HIV, frequência foi maior no grupo HIV (P=0,0300) e na comparação entre os grupos HIV e HCV, sua frequência também foi maior no grupo HIV (P=0,0140). A UTR-4, na comparação dos grupos HCV e controle saudável, revelou uma frequência maior no grupo controle (P=0,0147). A UTR-9, na comparação dos grupos HIV/HCV e HIV, mostrou frequência maior no grupo HIV/HCV (P=0,0460). Em relação aos dados clínicos, a presença do alelo T na posição +3035 foi significantemente associada à maior carga viral do HCV, acima de 400.000 cópias/mL (P=0,0244). Em relação aos tipos de genótipos do HCV, a presença do alelo +3027C foi associada ao subtipo 1a do HCV (P=0,0109). Adicionalmente, a presença do genótipo C/C na posição +3027 também foi significantemente associada com o subtipo 1a do HCV (P=0,0015). Ainda, o alelo A do SNP +3187 foi significantemente associado com os outros genótipos do HCV, excluindo o 1a (P=0,0369). Embora não esteja totalmente esclarecida a função do gene HLA-G, estudos têm sido desenvolvidos para melhor elucidar sua função nos contextos fisiológicos, como gestação, e patológicos, como tumores, transplantes, doenças inflamatórias e infecciosas. Tais estudos procuram ampliar o conhecimento sobre o sistema imunológico e contribuem para o desenvolvimento de novas estratégias diagnósticas e terapêuticas. Os resultados do presente estudo contribuem para a ampliação do conhecimento sobre os polimorfismos da região 3\' NT do gene HLA-G, na coinfecção HIV/HCV. Como também, na melhoria da assistência de enfermagem que deve buscar reduzir a morbimortalidade pela referida patologia. Porém, ainda há um longo percurso a ser percorrido na compreensão dos fatores imunogenéticos envolvidos na coinfecção pelo HIV/HCV / The general objective of the research was to associate the polymorphism of the gene HLA-G (region 3\' NT) with the co-infection HIV/HCV and with the groups (HIV, HCV and healthy control). It is a cross-sectional, comparative, descriptive study. 560 individuals participated of the study, being 156 healthy control individuals, 102 co- infected HIV/HCV, 186 infected by HIV and 116 by HCV. For identifying the polymorphisms, the genomic DNA was extracted from the total blood and the genotyping was made by PCR and visualized in gel of polyacrylamide at 7%, in which the polymorphism of 14pb was identified, and by sequencing the other seven SNPs. The social demographic results point that the most of the sample was composed by male adult individuals. Regarding the color of the skin, in the comparison between the groups HCV and HIV/HCV, a bigger number of co-infected with black skin and brown-skinned was observed than in the mono infected (P=0,0001). Regarding to the category of exposition for acquisition of the HIV, in the comparison between the groups HIV and HIV/HCV, a significant difference was observed in the transmission through heterosexual exposition, being its frequency bigger in the group HIV (P=0,0000). In the case of the comparison between the groups HCV and HIV/HCV, the difference in the heterosexual transmission was also observed, being its frequency significantly higher in the group HIV/HCV (P=0,0001). About the finding related to the genotype of the HCV, in the comparison between the groups HCV and HIV/HCV, the genotype 1a presented higher frequency in the co- infected (P=0,0001). Regarding to the viral load of the HIV, in the comparison between the groups HIV and HIV/HCV, the group of the mono infection presented bigger viral load that the group of the co-infection (P=0,0350). Regarding to the level of hepatic fibrosis, in the comparison between the groups HCV and HIV/HCV, the group of co-infection has a lighter fibrosis that the group of the mono infection (P=0,0009). Regarding to the genetic polymorphisms of the region 3\' NT of the HLA-G, it was found that the genotype of heterozygosis Del/Ins of 14 pb, presented significant difference in the individuals co-infected by the HIV/HCV (P=0,0216) when compared with the control group. About the SNP +3003, the comparison of the groups HCV and healthy control, it was showed that the allele +3003T presented a significant higher frequency in the group HCV (P=0,0147); the genotype +3003C/T presented a higher frequency in the control group (P=0,0095); the genotype +3003T/T was bigger in the group HCV (P=0,0095). The comparison between the groups HIV and HCV showed that the frequency of the allele +3003C was bigger in the group HIV (P=0,0463); and the genotype +3003T/T presented a bigger frequency in the group (P=0,0494). The frequency of the genotype +3187A/A was bigger in the group HIV/HCV in comparison to the HIV (P=0,0193); and of the +3187A/G was bigger in the group HIV (P=0,0187). The genotype +3196C/G presented frequency significantly bigger in the group HIV than in the healthy control (P=0,0213). The UTR-10, in comparison between the groups HIV and control, showed bigger frequency in the group HIV (P=0,0044); when compared the groups HIV/HCV and HIV, frequency was bigger in the group HIV (P=0,0300) and in the comparison between the groups HIV and HCV, its frequency was also bigger in the group (P=0,0140). The UTR-4, in the comparison of the groups HCV and healthy control, revealed a bigger frequency in the control group (P=0,0147). The UTR-9, in comparison of the groups HIV/HCV and HIV, showed bigger frequency in the group HIV/HCV (P=0,0460). Regarding to the clinical data, the presence of the allele T in the position +3035, was significantly associated to bigger viral load of the HCV, above 400.000 copies /mL (P=0,0244). About the types of genotypes of the HCV, the presence of the allele +3027C was associated with the subtype 1a of the HCV (P=0,0109). Additionally, the presence of the genotype C/C in the position +3027 was also significantly associated with the subtype 1a of the HCV (P=0,0015). Still, the allele A of the SNP +3187 was significantly associated with the other genotypes of the HCV, excluding the 1a (P=0,0369). Although the function of the gene HLA-G, is not totally clarified, studies have been developed for better elucidate its function in the physiological contexts, like gestation, and pathological, such as tumours, transplants, infectious and inflammatory diseases. These studies aim to extend the knowledge about the immunological system and contribute for the development of new diagnostic and therapeutic strategies. The results of this study contribute for enhancement of the knowledge about the polymorphisms of the region 3\' NT of the gene HLA-G, in the co-infection HIV/HCV. As well as, in the improvement of the assistance of nursing that must seek reducing the morbid mortality by the pathology referred. However, there is still a long path to be followed in the comprehension of the immunogenic factors involved in the co-infection by the HIV/HCV

A infecção por papilomavirus é a principal causa de desenvolvimento de neoplasias intraepiteliais cervicais (NIC) e câncer do colo do útero (CCU). Estudos epidemiológicos têm demonstrado que a persistência do genoma viral encontra-se associado a variantes moleculares específicas de papilomavirus humano (HPV) de alto risco. As moléculas HLA de classe II têm um importante papel na resposta imune. Associações entre HLA e CCU ou infecção por HPV tem sido demonstrado em diferentes populações. O nosso objetivo foi verificar se a variabilidade de HLA-DRB1 e DQB1 estavam associada ao CCU e NIC III em mulheres de Belém, uma população formada pelos 3 principais grupos étnicos humanos e uma área de alto risco para o CCU no Norte do Brasil. Foi investigada a existência de diferenças na distribuição de alelos HLA entre mulheres com CCU e NIC III portadoras de diferentes variantes de HPV-16 e mulheres citologicamente normais. Os genes HLA DQB1 e DRB1 foram tipados pelo método de PCR-SSO em 95 casos e 287 controles de mulheres com citologia normal atendidas em um centro de prevenção do colo do útero na mesma cidade. As variantes de HPV-16 foram tipadas por sequenciamento de um fragmento da região controladora do genoma viral (LCR). O polimorfismo na posição 350 do gene E6 foi tipado baseado em um protocolo de hibridização em pontos, para identificar a alteração na posição 350T→G. A magnitude das associações foi estimada por odds ratio (OR) e os respectivos intervalos de confiança (IC), ajustados para potenciais fatores de confusão. Uma associação positiva foi observada entre CCU e os haplótipos DRB1* 150 l-DQB1*0602, DRB1*04-DQB1*0301 e DRB1*1602-DQB1*0301. Ao contrário, DRB1*01-DQB1*0501 mostrou um efeito protetor. Os alelos DRB1*0804, DQB1*0402 apresentaram efeito protetor contra positividade por HPV. O alelo DQB1*0502 e o grupo DRB1*15 foram positivamente associados. Os nossos resultados mostram que as associações positivas de DRB1*1501 e DRB1*1602 podem ser atribuídas a variantes asiático-americanas quando comparado a variantes européias. O risco conferido a DRB1*1501 foi encontrado associado tanto a variantes E6350G quanto a variantes E6350T, entretanto, o maior efeito foi devido às variantes E6250T. A associação positiva de DRB1*1602 foi significativa somente no grupo de mulheres positivas para E6350G. Estes resultados estão de acordo com a composição étnica da população estudada bem como um maior potencial oncogênico de certas variantes. Nossos dados sugerem que a contribuição dos alelos HLA na susceptibilidade genética ao CCU difere de acordo com a distribuição das variantes de HPV em uma dada região geográfica ou grupo étnico. / Papillomavirus infection is the major cause for the development of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) in humano Epidemiological studies have demonstrated that persistence of the viral genome and development of cervical cancer are associated with specific molecular variants of high-risk human Papillomavirus (HPV). Human leukocyte antigen (HLA) molecules play a role in immune response and associations between HLA and IeC or HPV infections have been reported in several populations. We aimed to verify if HLA-DRB1 and DQB1 variability is related to ICC and CIN III in women from Belem, a population formed by the three main human ethnic groups and a high-risk area for this disease in Northem Brazil. We also investigated if there are differences in the HLA class II alleles distribution between women with ICC and CIN III that harbor different HPV-16 variants and women without cancer. HLA DRB1 and DQB1 were typed by PCR-SSO based methods in 95 ICC cases and 287 controls consisting of normal cytology from women attending cervical cancer screening programs in the same city. HPV-16 variants were typed by sequencing a PCR-amplified fragment of long control region (LCR) of the viral genome. The E6350 polymorphism was typed on the basis of a dot blot protocol targeting a specific nucleotide alteration in the position 350T→G of the E6 gene. The magnitude of associations was estimated by odds ratio (OR) and the respective 95% confidence interval (CI), adjusted for potential confounder factors. A positive association was found between ICC cases and DRB1*150 l-DQB1*0602, DRB1*04-DQB1*0301 and DRB1*1602-DQB1*0301 haplotypes. Conversely, DRB1*01-DQB1*05 showed a protective effect. DRB1*0804, DQB1*0402 showed negative association against HPV infection. DQB1*0502 and DRB1*15 were positively associated with HPV infection. Our study showed that positive association of DRB1*1501 and DRB1*1602 alleles may be attributed to AsianAmerican then European variants. Furthermore, the DRB1*1501 was found associated with both women carrying E6350G or E350T, however, a higher effect was observed for E6350T variants carriers. The positive association of DRB1*1602 was significant for women harboring E6350G then E6350T variants. These data are in agreement with ethnical component of the studied population as well as a higher oncogenic potential of certain HPV variants. Our results also suggest that the contribution of HLA class II alieles to the genetic susceptibility to ICC differs depending on the HPV-16 variants distribution in a given geographic and ethnic group.

INTRODUÇÃO: A Estomatite Aftóide Recorrente (EAR) é uma doença oral com incidência em 20% da população mundial, caracterizada por úlceras mucosas de caráter recidivante. O seu diagnóstico baseia-se principalmente na história clínica do paciente. Hereditariedade pode ser um fator de risco para a doença. Entretanto, os estudos disponíveis não são conclusivos quanto aos resultados obtidos, variando segundo a população estudada. OBJETIVOS: Neste trabalho tipificamos moléculas HLA de classe I e de classe II e avaliamos a freqüência destas moléculas em 31 pacientes, da cidade de São Paulo, portadores de Estomatite Aftóide Recorrente bem como em seus subgrupos (minor, major e herpetiforme), comparando com grupo controle, composto de 961 pacientes saudáveis. CASUÍSTICA E MÉTODOS: Prospectivamente, 58 pacientes com suspeita diagnóstica de Estomatite Aftóide Recorrente no período de fevereiro de 2004 a maio de 2006, foram estudados. Estes pacientes foram submetidos a protocolo de exames e, daqueles que obedeceram os critérios de inclusão, foi extraído o Ácido Desoxi Ribonucléico (DNA), por meio de amostra de 10 ml de sangue total periférico, com o fim de proceder a tipificação HLA por Reação de Polimerização em Cadeia. RESULTADO: Nos pacientes portadores de Estomatite Aftóide Recorrente do tipo minor encontramos as freqüências HLA A33 e B35, estatisticamente significantes quando comparadas com o grupo controle. CONCLUSÃO: As freqüências HLA-A33 e HLA-B35 podem estar associadas à Estomatite Aftóide Recorrente minor na população brasileira. / INTRODUCTION: Recurent aphthous stomatits (RAS) is a common oral mucosa disorders and affects 20% of the world\'s population, this disease is characterized by recurring painful ulcers of the mouth. The diagnosis is base on the clinic and the lesion\'s history. Hereditary may be involved in the morbidity of Recurent aphthous stomatits. Despite extensive investigations of etiology\'s Recurent aphthous stomatits, they are not conclusive and variety by different ethnic backgrounds. OBJECTIVES: In this work HLA class I and class II was typing from 31 subjects affected by Recurent aphthous stomatits, and compared with 961 healthy controls. CASUISTIC and METHOD: Fifty eight patients with diagnostic hypothesis of Recurent aphthous stomatits were treated, in the period of February of 2004 to May of 2006. A protocol of exams were obtained from those that obeyed the inclusion criteria. To obtain the DNA, 10 ml venous blood sample was collected to type HLA using Polymerase Chain Reaction - Sequence Specific Oligonucleotide (PCR-SSO). RESULTS: The investigation revealed that HLA-A33 and HLA-B35 were more frequent in minor Recurent aphthous stomatits patient, when compared with control group. CONCLUSION: HLA-A33 and HLA-B35 may be associated with Recurent aphthous stomatits type minor in the Brazilian\'s population.

Made available in DSpace on 2016-08-10T10:39:10Z (GMT). No. of bitstreams: 1 THYAGO PEDROSA MAGALHAES.pdf: 2261259 bytes, checksum: 42a1ba331ec79afdea4d0071a56fca2c (MD5) Previous issue date: 2015-03-10 / Multiple sclerosis is a chronic inflammatory disease of the central nervous system, characterized by demyelination and neuronal degeneration, a major cause of disability in young. Subjects an autoimmune response against neuronal auto antigen, with destruction of the myelin sheath, is observed in individuals with genetic predisposition exposed to certain environmental factors. Among the genetic factors, studies suggest a complex interaction between the alleles of the human leukocyte antigen (HLA-DRB1*1501 and DQB1*0602) and the risk of developing the disease. The allele DRB1 * 1501 was associated with the disease in Caucasian patients in North America and Northern Europe, and the DQB1*0602 allele in Caucasians of Northern Europe and African descendant. The aim of this study was to conduct a systematic review and a meta-analysis of studies that investigated the contribution of DRB1*1501 and DQB1*0602 in the risk of developing MS in different populations of the world. A systematic review and meta-analysis were performed on the main publications in the electronic database of PubMed, by using the terms "HLA" "MULTIPLE SCLEROSIS", "DR" "DQ" and "HLA" "MULTIPLE SCLEROSIS" and "BRAZIL "resulting in a total of 181 articles. After reading the articles, 18 met the inclusion criteria and were selected for review. Based on the results it was possible to conclude that the two alleles are more common in MS patients than in controls, with the allele DRB1*1501 more frequent in European and Caucasian populations and the DQB1 * 0602 with a distribution more heterogeneous in populations. The results obtained by the meta-analysis showed a statistically significant association between DRB1 * 1501 and DQB1 * 0602 allele and the risk of developing multiple sclerosis (OR combined DRB1 * 1501 = 2.934, 95% CI: 2.154 to 3.998, p <0, 0001 and DQB1 * 0602 combined OR = 2.906, 95% CI: 2.167 to 3.896, p <0.0001). / A Esclerose Múltipla é uma doença inflamatória crônica do sistema nervoso central, caracterizada por desmielinização e degeneração neuronal, sendo uma importante causa de incapacidade em jovens. Decorre de uma resposta autoimune contra autoantígenos neuronais, com destruição da bainha de mielina em indivíduos com predisposição genética, expostos a determinados fatores ambientais. Dentre os fatores genéticos, estudos apontam para uma interação complexa de alelos do Antígeno Leucocitário Humano (HLA-DRB1*1501 e DQB1*0602), e o risco de desenvolver a doença. O alelo DRB1*1501 foi associado à doença em pacientes caucasianos na América do Norte e Norte da Europa e o alelo DQB1*0602 em caucasianos da Norte da Europa e afrodescentes. O objetivo do estudo foi realizar uma revisão sistemática e meta-análise sobre os estudos que investigaram a contribuição dos alelos DRB1*1501 e DQB1*0602 no risco de desenvolver EM nas diversas populações do mundo. Uma revisão sistemática e uma meta-análise foram realizadas acerca das principais publicações encontradas na base de dados eletrônicas do PUBMED utilizando os termos HLA MULTIPLE SCLEROSIS , DR DQ e HLA MULTIPLE SCLEROSIS e BRAZIL , resultando em um total de 181 artigos. Após a leitura dos artigos, 18 preencheram os critérios de inclusão e foram selecionados para a revisão. Com base nos resultados obtidos dos artigos foi possível concluir que os dois alelos avaliados são mais frequentes em pacientes com Esclerose Múltipla do que em controles, sendo o alelo DRB1*1501 mais comum nas populações europeias e caucasianas e o alelo DQB1*0602 com uma distribuição mais heterogênea nas populações. Os resultados obtidos pela meta-análise demonstraram associações estatisticamente significativas entre os alelos DRB1*1501 e DQB1*0602 e o risco de desenvolver Esclerose Múltipla, (OR combinada DRB1*1501= 2,934, 95% IC: 2,154 3,998, p < 0,0001 e OR combinada DQB1*0602 = 2,906, 95% IC: 2,167 3,896, p < 0,0001).

A infecção crônica pelo vírus da hepatite C (HCV) é importante fator de risco para o desenvolvimento de cirrose hepática e de carcinoma hepatocelular. A evolução para formas mais graves está relacionada a fatores ligados ao vírus, ao hospedeiro e à resposta imune. O objetivo deste estudo foi avaliar a associação entre os polimorfismos do gene HLA-E, a expressão da molécula HLA-E e a gravidade da doença hepática pelo HCV. Foram incluídos 112 pacientes com hepatite C crônica e avaliados parâmetros clínicos, bioquímicos e histológicos (esteatose, atividade inflamatória e fibrose hepática). A variabilidade do gene HLA-E foi avaliada por sequenciamento de Sanger, e a expressão hepática da molécula, por imunoistoquímica. Para comparação da expressão hepática da molécula HLA-E e da variabilidade do gene HLA-E, foram usados dois grupos controles de indivíduos sem hepatopatia da mesma região geográfica. A imunoistoquímica para HLA-E identificou expressão da molécula nos hepatócitos e nas células de Kupffer. A expressão de HLAE em hepatócitos e células de Kupffer foi encontrada em 56,3% e 43,8% dos pacientes com HCV e em 20% e 10% nos controles (P = 0,008 e 0,02), respectivamente. Foi identificado que o percentual de pacientes do sexo masculino, com expressão moderada de HLA-E em células de Kupffer, foi maior em relação aos pacientes do sexo feminino (22,8% x 7,3%; P = 0,03). As amostras de fígado classificadas como esteatose, atividade necroinflamatória e fibrose graves apresentaram maior grau de expressão de HLA-E em células de Kupffer e hepatócitos, com associação linear significativa. Na análise multivariada, as variáveis que influenciaram significativamente a gravidade da doença foram a expressão da molécula HLA-E nos hepatócitos, a idade avançada e o índice de massa corporal maior que 25. Foram identificados 14 haplótipos diferentes do gene HLA-E, quatro deles ainda não descritos na literatura. A frequência do alelo HLA-E*01:01:01:03 foi menor no grupo de pacientes, quando comparada ao controle (P = 0,0001). O alelo HLA-E*01:03:05 associou-se a maior probabilidade (OR = 4,69) de expressão da molécula HLA-E, na célula de Kupffer (P = 0,046). O genótipo TT do polimorfismo +424 T/C (rs1059510) associou-se a menor probabilidade (OR = 0,06) de expressão da molécula HLA-E, na célula de Kupffer, em relação à ausência de expressão (P=0,009), a menor probabilidade (OR = 0,22) de atividade inflamatória moderada/grave em relação à leve (P = 0,047) e esteve associado a menor probabilidade (OR = 0,17) de fibrose hepática moderada/grave em relação à fibrose leve (P = 0,049). Os resultados do presente estudo sugerem que a pesquisa de fatores imunogenéticos, como a expressão hepática da molécula HLA-E e a identificação da variabilidade genética do HLA-E, pode ter aplicabilidade no manejo clínico dos pacientes, uma vez que auxilia na discriminação daqueles com maior risco de atingir formas avançadas da hepatite C crônica. / Chronic hepatitis C is an important risk factor for the development of cirrhosis and hepatocellular carcinoma. The severity of liver disease can be influenced by factors related to the virus, the host and the immune response. The aim of this study was to evaluate the association between HLA-E gene polymorphisms, HLA-E molecule expression and HCV liver disease severity. We included 112 patients with chronic hepatitis C and evaluated clinical, biochemical and histological parameters (steatosis, inflammatory activity and liver fibrosis). The variability of the HLA-E gene was assessed by Sanger sequencing and liver HLA-E expression by immunohistochemistry. Two control groups of individuals without hepatopathy from the same geographical region were used to compare the HLA-E expression and the gene variability. Immunohistochemistry for HLA-E showed positivity in hepatocyte and Kupffer cell. HLA-E positivity in hepatocytes and Kupffer cells were found in 56.3% and 43.8% of HCV patients and in 20% and 10% in the controls (P = 0.008 and 0.02), respectively. We found that the percentage of male patients with moderate HLA-E expression in Kupffer cells was higher than in females (22.8% vs. 7.3%, P = 0.03). The liver samples classified as severe fibrosis, necroinflammatory activity and steatosis presented greater expression of HLA-E on Kupffer cells and hepatocytes. There was a positive linear association between HLA-E expression and severity of liver damage (P<0.05). In the multivariate analysis, the variables that significantly influenced the severity of the disease were HLA-E molecule expression in hepatocytes, advanced age and body mass index greater than 25. Fourteen different HLA-E haplotypes were identified, four of them not yet described in the literature. The frequency of the HLA-E * 01: 01: 01: 03 allele was lower in the group of patients than in the control group (P = 0.0001). The HLA-E * 01: 03: 05 allele was associated with increased likelihood (OR = 4.69) of HLA-E expression in the Kupffer cell (P = 0.046). The TT genotype of the +424 T / C polymorphism (rs1059510) was associated with a lower probability (OR = 0.06) of HLA-E expression in the Kupffer cell in relation to the absence of its expression (P = 0.009), was associated with a lower probability (OR=0,22) of moderate/severe necroinflammatory activity in relation to the mild inflammatory activity (P=0,047) and was associated with a lower probability (OR = 0.17) of moderate / severe hepatic fibrosis in relation to mild fibrosis (P = 0.049). The results of the present study suggest that the study for immunogenic factors such as HLA-E liver expression and the identification of certain polymorphisms and alleles of the HLA-E gene may have applicability in the clinical management of patients since it aids in discrimination of those at greatest risk of reaching advanced forms of chronic hepatitis C.