Les cellules Natural Killer (NK) sont des lymphocytes capables de tuer les cellules tumorales avec une expression altérée des molécules du Complexe Majeur d’Histocompatibilité (CMH) de classe I . Chez l’homme, cette reconnaissance de “l’absence du soi” est relayée par l’absence d’engagement des antigènes des leucocytes humains (HLA) par les récepteurs inhibiteurs des cellules NK. Chez l’homme, ces récepteurs inhibiteurs incluent les récepteurs KIR qui ont comme analogues fonctionnels chez les rongeurs les lectines Ly49. Certaines tumeurs échappent à la surveillance des NK en augmentant leur expression des molécules HLA. Donc, bloquer les interactions entre les molécules KIR et HLA est une stratégie anti-tumorale intéressante qu’INNATE PHARMA a décidé d’explorer en développant l’anticorps thérapeutique anti-KIR 1-7F9. Mais, ces interactions sont nécessaires pour l’acquisition des fonctions des NK, i.e leur éducation. De plus, elles sont impliquées dans la tolérance aux cellules du soi par les cellules NK. Anticiper la toxicité de 1-7F9, évaluer ses propriétés anti-tumorales et son impact sur l’éducation des NK étaient les objectifs de notre travail. Deux modèles murins ont été développées à ces fins. Le premier, est basé sur les souris B6 : les récepteurs inhibiteurs Ly49 C/I ont pour ligand les molécules H-2b. Le second modèle est basé sur des souris transgéniques pour un unique récepteur KIR et son ligand, en l’absence des molécules endogènes de CMH de classe I murines. Ces deux modèles ont montré que le blocage des récepteurs inhibiteurs n’altère pas la tolérance au soi des NK et est une stratégie anti-tumorale efficace qui n’altère pas la fonctionnalité des NK. / Natural Killer cells (NK cells) are lymphocytes able to kill tumors with aletered expression of Major Histocompatibility Complex (MHC) class I molecules. This “missing self” recognition is mediated by the lack of engagement of Human Leukocytes Antigens (HLA) with NK inhibitory receptors that include Killer Immunoglobulin like Receptors (or KIR) in humans. In rodents, the functional analogues of KIR are Ly49 lectins. Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. So, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy that INNATE PHARMA decided to explore, developing the anti-KIR monoclonal antibody 1-7F9. Nevertheless, these interactions are necessary for the acquisition of NK functional properties, i.e for their “education”. They are also involved in self-tolerance on educated NK cells. For the pre-clinical development of 1-7F9, it was necessary to anticipate anti-KIR mAb safety and toxicity, evaluate its anti-cancer potential and its impact on NK education. A first part of our work was performed in a surrogate B6 mouse model:Ly49 C/I inhibitory receptors have H-2b molécules as endogenous ligand. Their interactions were blocked with anti-Ly49 C/I monoclonal antibody. In a second part, we report the generation of transgenic mice expressing a single inhibitory KIR in the context of its HLA ligand and in absence of endogenous mouse MHC class I molecules. Both models showed that the blockade of NK inhibitory receptors interactions with their endogenous ligands did not break self tolerance, had a strong anti-tumor effect and did not abrogate NK functionality.

Es ist schon lange bekannt, dass das Immunsystem eine wichtige Rolle in der Immunabwehr von malignen Tumoren spielt. In der vorliegenden Arbeit wurde das Verhalten der unten genannten Zellen, Rezeptoren und löslichem Protein MICA im peripheren Blut von Patienten mit soliden und hämatologischen Tumorerkrankungen sowie einer Kontrollgruppe mittels Durchflußzytometrie und ELISA-Verfahren untersucht. NK-Zellen ( u.a. CD 56+CD16-NK-Zellen, CD 56-CD16+ Nk-Zellen, CD56+CD16+NK-Zellen), NKT-Zellen und verschiedene T-Lymphozyten ( u.a. αβ-Lymphozyten, δγ-Lymphozyten, Vγ9Vδ1-Lymphozyetn, Vγ9Vδ2-Lymphozyten) sind auf direkten oder indirekten Weg über aktivierende Rezeptoren ( u.a. NKG2D, NKp44, NKp46, NKp33), inhibierende Rezeptoren ( u.a. p 58.1, p58.2, p70, NKG2A) und Chemokinrezeptoren ( u.a. CXCR1) an der Lyse / Apoptose von Tumorzellen über HLA-abhängige (MICA) und HLA-unabhängig Moleküle beteiligt. Es ließ sich zeigen, dass MICA genauso im Serum von hämatologischen Patienten wie auch bei soliden Tumorerkrankungen vorkommt, wobei vor allem Patienten mit fortgeschrittenen Stadien und Metastasierung hohe Werte aufwiesen. Es wurde eine Verminderung der Gesamtzahl an Lymphozyten und αβ-T-Lymphozyten bei hämatologischen und bei soliden Tumoren im peripheren Blut festgestellt. Weiterhin zeigte sich, wie bereits in der Literatur berichtet, bei hämatologischen Neoplasien ein signifikant erhöhter Wert an NK-Zellen und CD56-16+NK-Zellen sowie eine verminderte Expression von NKG2D auf NK-Zellen, auf αβ-T-Lymphozyten und auf γδ-T-Lymphozyten. Es ließ sich auch eine verminderte Expression von p58.1 und NKG2A auf γδ-Lymphozyten im Vergleich zur gesunden Kontrollgruppe nachweisen. Ein signifikanter Unterschied im Vergleich zu soliden Tumoren fand sich nicht, bis auf den oben genannten Lymphozytenanteil. Der Nachweis, dass erhöhte lösliche Serum MICA-Werte die Parameter wie KIRs und KARs sowie Chemokinrezetoren oder die Apopotose der Immunzellen bei hämatologischen oder bei soliden Tumorerkrankungen beeinflusst, konnte in dieser Arbeit nicht geführt werden. Die vorliegende Arbeit trägt dazu bei, die Immunabwehr gegenüber soliden und hämatologischen Tumorerkrankungen besser zu verstehen. / Natural killer cells (NK), NKT cells, αβ-T cells and δγ-T cells play an important role in tumor defence. These cells eliminate tumor cells through killer inhibitory receptors (KIR) and stimulatory killer activating receptors (KAR), which can lyse target cells by binding to the major histocompatibility complex class I-related chain A (MICA) protein. The aim of this study is to analyze the reduction of these immune cells, the association between the levels of soluble MICA with KIR- and KAR-expressing on αβ cells, γδ cells and NK cells and their interaction with chemokine receptor CXCR1in the peripheral blood of patients with haematological and epithelial malignancies. ELISA and flow cytometric analysis were used in comparison to controls. Reduced numbers of αβ-T- cells and the presence of soluble MICA could be demonstrated in the serum of patients with both haematological and epithelial malignancies. Higher levels of soluble MICA were associated with advanced stages of disease and metastasation. Patient samples showed also lower numbers of NK cells and CD56-16+NK cells as well as a reduced expression of NKG2D on NK, αβ and γδ T cells. Confirming existing evidence, there was also a reduced expression of p58.1 and NKG2A on γδ cells in comparison to controls. No significant differences between solid and haematological malignancies were found, except for this type of lymphocytes. The study did not provide evidence that increased levels of soluble MICA influence KIRs and KARs, the chemokine receptors or the apoptosis of immune cells.

Les cellules tueuses naturelles (NK) sont régulées par des récepteurs activateurs et inhibiteurs. La plupart des récepteurs inhibiteurs reconnaisse des molécules du complexe majeur d'histocompatibilité (CMH) de classe I, et protège les cellules saines des phénomènes d'auto-immunité médiés par les cellules NK. Cependant, certains récepteurs activateurs, incluant le récepteur killer cell Ig-like receptor (KIR) 2DS1, reconnaissent aussi des ligands CMH de classe I. Cela pose la question de savoir comment les cellules NK qui expriment des récepteurs activateurs deviennent tolérantes au soi. Nous avons cherché à déterminer si la présence de HLA-C2, le ligand du récepteurs 2DS1, peut induire les cellules NK qui expriment le 2DS1 à développer un état de tolérance au soi. Indépendamment de la présence ou de l'absence du ligand HLA-C2 dans le donneur, une activité anti-HLA-C2 a été identifiée in vitro dans certains clones NK 2DS1-positifs. La fréquence des clones NK avec réactivité anti-HLA-C2 était élevée parmi les donneurs homozygotes pour HLA-C1. De façon étonnante, nous n'avons pas constaté de différence statistiquement significative dans la fréquence de cytotoxicité anti-HLA-C2 entre les donneurs HLA-C2 hétérozygotes et les donneurs sans ligand HLA-C2. Par contre, les donneurs HLA-C2 homozygotes montrent une fréquence réduite de clones NK avec réactivité anti-HLA-C2 par rapport aux autres donneurs. Clones 2DS1-positifs qui co-expriment des KIR inhibiteurs spécifiques des molécules HLA de classe I du soi n’étaient pas communément cytotoxiques, et la cytotoxicité anti-HLA-C2 était limité presque exclusivement à des clones positifs seulement pour 2DS1 (« single positive » 2DS1 clones). Nous avons aussi identifié des clones 2DS1 « single positive » avec réactivité anti-HLA-C2 dans des patients recevant une greffe de cellules souches hématopoïétiques à partir de donneurs 2DS1. Ces résultats montrent que plusieurs cellules NK avec réactivité anti-HLA-C2 sont présentes dans des donneurs 2DS1 soit hétérozygotes soit homozygotes pour HLA-C1. En revanche, les clones 2DS1-positifs obtenus par des donneurs homozygotes pour HLA-C2 sont fréquemment tolérants aux antigènes HLA-C2. / NK cells are regulated by inhibiting and activating cell surface receptors. Most inhibitory receptors recognize MHC-class I antigens, and protect healthy cells from NK cell-mediated auto-aggression. However, certain activating receptors, including the human killer cell Ig-like receptor (KIR) 2DS1, also recognize MHC-class I. This raises the question of how NK cells expressing such activating receptors are tolerized to host tissues. We investigated whether the presence of HLA-C2, the cognate ligand for 2DS1, induces tolerance in 2DS1-expressing NK cells. Anti-HLA-C2 activity could be detected in vitro in some 2DS1 positive NK clones irrespective of presence or absence of HLA-C2 ligand in the donor. The frequency of anti-HLA-C2 reactivity was high in donors homozygous for HLA-C1. Surprisingly, there was no significant difference in frequency of anti-HLA-C2 cytotoxicity in donors heterozygous for HLA-C2 and donors without HLA-C2 ligand. However, donors homozygous for HLA-C2 had significantly reduced frequency of anti-HLA-C2 reactive clones as compared to all other donors. 2DS1 positive clones that express inhibitory KIR for self-HLA class I were commonly non-cytotoxic, and anti-HLA-C2 cytotoxicity was nearly exclusively restricted to 2DS1 single positive clones lacking inhibitory KIR. 2DS1 single positive NK clones with anti-HLA-C2 reactivity were also present post-transplantation in HLA-C2 positive recipients of hematopoietic stem cell transplants from 2DS1 positive donors. These results demonstrate that many NK cells with anti-HLA-C2 reactivity are present in HLA-C1 homozygous and heterozygous donors with 2DS1. In contrast, 2DS1 positive clones from HLA-C2 homozygous donors are frequently tolerant to HLA-C2.

We performed a retrospective study in 185 patients with myelogenous leukemias who had received hematopoietic cells from unrelated donors. The aim of this study was to answer the question wether the benefit of KIR ligand incompatibility seen in haploidentical tranplantations can also be seen using unrelated donors. We could not detect a significant difference in survival between patients with a KIR ligand incompatibility and those with either fully matched or partially mismatched unrelated donors in this patient cohort. / In der Therapie von Leukämien ist die Knochenmark- bzw. Stammzelltransplantation eine tragende Säule. Für den Transplantationserfolg ist eine Übereinstimmung der Haupthistokompatibilitätsantige (HLA-Antigene der Klassen I und II) zwischen Spender und Empfänger von zentraler Bedeutung. Diese Notwendigkeit ergibt sich aus der sogenannten MHC-Restriktion in der T-Zellrezeptorerkennung. Ob auch NK-Zellrezeptoren und deren Liganden in der Spenderauswahl berücksichtigt werden sollten, ist bisher unzureichend untersucht. Insbesondere trifft das für die KIR-Rezeptoren zu, die wie die T-Zellrezeptoren ebenfalls HLA-Antigene als Liganden besitzen. Velardi et al. haben 2002 erstmalig gezeigt, daß in der Therapie myeloischer Leukämien die Transplantation von Blutstammzellen verwandter Spender mit KIR-Liganden-Inkompatibilität von klinischem Vorteil ist. Ob KIR-Liganden-Inkompatibilität auch bei Knochenmark-/ Stammzelltransplantationen Unverwandter Bedeutung erlangen könnte, war zu Studienbeginn offen und blieb auch infolge diskrepanter Untersuchungsergebnisse von verschiedenen Arbeitsgruppen im Verlauf der Studie widersprüchlich. Im Rahmen dieser Arbeit wurde diese Fragestellung, die auch Teil einer internationalen Studie war, an 185 Spender-Empfänger-Paaren retrospektiv untersucht. Dabei wurde bei den Paaren einerseits die KIR-Liganden-Kompatibilität auf der Grundlage der HLA-C-Supertypen erschlossen (nach Velardi et al.). Andererseits konnte sie im internationalen Studienprogramm direkt aus dem KIR-Genotyp des Spenders und dem HLA-C-Supertyp des Empfängers ermittelt werden. Die Untersuchungen ergaben folgende Resultate: bei Vorliegen von KIR-Liganden-Inkompatibilität hat die Verwendung von ATG als Bestandteil der GvHD-Prophylaxe keinen Einfluß auf das klinische Ergebnis. Die Vermutungen von Giebel et al. wurden damit nicht gestützt. Die Bestimmung des KIR-Liganden-Status mit Hilfe der Rückschlußmethode allein aus dem HLA-Typ ist unzuverlässig. Für eine exakte Differenzierung ist die gleichzeitige KIR-Genotypisierung erforderlich. KIR-Liganden-Inkompatibilität ist bei unverwandten Knochenmark-/ Stammzelltransplantationen nicht von klinischem Vorteil. Auch ein gezieltes Aussuchen HLA-C-inkompatibler Spender auf der Grundlage einer KIR-Genotypisierung stellt derzeit keine therapeutische Option dar.

Introdução: A Doença de Gaucher (DG) é causada pela atividade reduzida da enzima lisossomal glucocerebrosidase, o que leva ao acúmulo de glicocerebrosídeo nas células e a uma estimulação crônica do sistema imune. Células natural killer (NK) possuem um papel importante na resposta imune e sua atividade é alterada na DG. Os receptores KIR (Killer Immunoglobulin-like Receptors) regulam a atividade das células NK através da interação com moléculas de HLA (Human Leukocyte Antigen) de classe I das célulasalvo. Objetivo: Analisar a variabilidade dos genes KIR em uma coorte de pacientes com DG do Sul do Brasil, compará-las a um grupo controle e buscar associações com manifestações clínicas. Metodologia: Trinta e um pacientes com DG tipo I (24 com forma leve, 4 com forma moderada e 3 com forma grave) foram analisados e comparados a 250 controles saudáveis quanto a frequência dos genes HLA e KIR. Resultados/Discussão: Não houve diferença significativa nas frequências de variantes dos genes KIR entre os grupos. O alelo HLA B37 é mais frequente nos pacientes com DG do que no grupo controle (p=0,011). A idade de início dos sintomas mostrou associação com a combinação das variantes KIR2DL2 e KIR2DS2 com seu ligante HLA-C1 (p=0,038). Pacientes que apresentam a variante HLA-C2 parecem apresentar maior susceptibilidade a desenvolver bandas mono ou policlonais na eletroforese de proteínas (p=0,007, OR=21,3). Foi encontrada associação entre os alelos DR11 (p=0.008) e DR13 (p=0.011) e gravidade da doença. O alelo DR11 parece estar associado a comprometimento neurológico, enquanto o alelo DR13 ao desenvolvimento de osteonecrose. Conclusão: Nossos dados sugerem uma possível associação entre variantes dos genes KIR e HLA e a DG. Devem ser estudadas em outras coortes de pacientes já que parecem ser um fator modificador de fenótipo. / Background: Gaucher disease (GD) is caused by the reduced activity of a lysosomal enzyme glucocerebrosidase, which leads to the accumulation of glucocerebroside in the cells and a chronic stimulation of the immune system. Natural Killer (NK) cells play an important role in the immune response, and their activity is impaired in GD. Killer immunoglobulin-like receptors (KIR) regulate the activity of NK cells through an interaction with specific human leukocyte antigen (HLA) class I molecules on target cells. Objectives: To analyze the variability of KIR genes in a Southern Brazilian sample of GD patients, to compare it with controls, and to look for associations with clinical manifestations. Methodology: Thirty one GD type I patients (24 mild, 4 moderate, and 3 severe) were analyzed and compared to 250 healthy controls regarding the frequency of HLA and KIR genes. Results/Discussion: There was no significative difference in the frequencies of KIR gene variants between the groups. The HLA B37 allele is more frequent in patients with GD than in control group (p=0.011). The age of onset of symptoms was associated with KIR2DL2 and KIR2DS2 combination with the ligand HLA-C1 (p=0.038). Patients who have the HLA-C2 variant appear to have more mono/polyclonal bands in protein electrophoresis (p=0.007, OR=21.3). An association between the DR11 (p=0.008) and DR13 (p=0.011) alleles and disease severity was found. The DR11 allele appears to be associated with neurological impairment, while the DR13 allele to the development of osteonecrosis. Conclusion: Our data suggest a possible association between KIR genes and HLA genes and GD. They should be studied in other cohorts of GD patients as they seem to be a phenotype modifying factor.

Made available in DSpace on 2016-04-04T12:45:12Z (GMT). No. of bitstreams: 2 nathalia_sa_ioc_mest_2015.pdf: 3537847 bytes, checksum: 61867e9568232ee7e431513765bfe8c5 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Atualmente, a tuberculose (TB) e a síndrome da imunodeficiência humana (HIV) são as duas principais doenças infecciosas que levam à óbito no mundo. A infecção pelo HIV aumenta o risco de adoecimento por TB, sendo essa uma das mais frequentes doenças oportunistas. Em certos pacientes com tuberculose e infectados pelo HIV-1 que recebem tratamento para os dois agravos, uma profunda reação patológica inflamatória pode surgir, causando um efeito contrário ao esperado. Esse quadro patológico paradoxal é denominado IRIS (Síndrome Inflamatória da Reconstituição Imune). Os fatores associados ao risco da IRIS ainda não estão completamente compreendidos. Estudos sobre a patogênese desta síndrome relatam que tanto a combinação da carga antigênica quanto a susceptibilidade genética do hospedeiro podem influenciar o aparecimento da síndrome. No presente estudo, avaliamos a distribuição e o impacto dos genótipos HLA-B, HLA-DRB1 e KIR em indivíduos com tuberculose infectados pelo HIV-1, além do papel desses genes na ocorrência da IRIS. O estudo é retrospectivo, e incluiu 61 pacientes acompanhados no período de 2006 a 2012 no âmbito do projeto \2018\2019Síndrome de reconstituição imune: avaliação da resposta imune em pacientes com tuberculose em uso de HAART\2019\2019, conduzido em colaboração com o Instituto Nacional de Infectologia (INI/FIOCRUZ) Os dados das frequências gênicas dos pacientes foram comparados com dados disponíveis para a população brasileira. Os alelos HLA-B mais frequentes foram: B*15; B*44; B*35 e B*07, enquanto que os alelos HLA-DRB1 mais frequentes no estudo foram: DRB1*07, DRB1*11, DRB1*04 e DRB1*15. Esses resultados corroboram com estudos prévios da população Brasileira e, apesar de terem sido observadas algumas diferenças nas frequências alélicas entre os grupos com IRIS e sem IRIS, estas não atingiram significância estatística. Uma tendência à significância envolvendo o alelo HLA-B*42 foi observada entre os grupos IRIS x não IRIS (p= 0,064). Com relação às frequências dos genes KIR, estas foram semelhantes às descritas para a população Brasileira, porém não houve diferenças estatisticamente significativas relativas à distribuição das frequências dos diferentes genótipos KIR e seus haplótipos quando se comparou o grupo de pacientes com IRIS versus sem IRIS. Portanto, com base nestes achados, não foi possível inferir associações entre estes marcadores genéticos e a ocorrência de IRIS. Contudo, esse trabalho foi pioneiro na descrição da distribuição dos alelos HLA-B, HLA-DRB1 e KIR em indivíduos com tuberculose infectados pelo HIV-1 que, no seu conjunto, visam contribuir para a discussão sobre o impacto de genes do hospedeiro no contexto dos dois agravos estudados e na ocorrência da IRIS / Currently, tuberculosis (TB) and human immunodeficiency syndrome (HIV) are the two major infectious diseases that lead to death in the world. HIV infection increases the risk of TB illness, being one of the most frequent opportunistic diseases. In some patients with tuberculosis and HIV - 1 that received treatment for the two diseases, a deep pathological inflammatory reaction can ar ise, causing an effect contrary to the expected. This paradoxical pathological condition is called IRIS ( inflammatory syndrome of reconstitution immunity ) . The factors associated with the risk of IRIS are not yet completely understood. Studies on the patho genesis of this syndrome report that both the combination of antigenic load and the genetic susceptibility of the host can influence the appearance of the syndrome. In the present study, we evaluated the distribution and the impact of HLA - B, HLA - DRB1 and K IR genotypes in individuals with tuberculosis infected with HIV - 1, as well as the role of these genes in the occurrence of IRIS. This study is retrospective and included 61 patients followed up between 2006 and 2012 in the context of the project ''Immune r econstitution syndrome: evaluation of immune response in patients with tuberculosis in use of HAART’', held in collaboration with the National Infectology Institute ( INI/FIOCRUZ). The gene frequency data of patients were compared with data from the Brazili an population. HLA - B alleles more frequent were B*15; B*44; B*35 and B*07, while HLA - DRB1 alleles frequent in the study were DRB1*07, DRB1*11, DRB1*04 and DRB1*15. These results corroborate previous studies in the Brazilian population and, although some di fferences in the allele frequencies could be observed between the groups with and without IRIS, none of these was statistically significant. A trend to significance involving the allele HLA - B*42 was observed between IRIS x non - IRIS groups (p =0.064). Conce rning KIR genes frequencies , they were similar to those described for the Brazilian population, but no statistically significant difference in the distribution of KIR genotypes and haplotypes was observed in the comparison of IRIS versus non - IRIS pa tients. Therefore, based on our findings it was not possible to infer any association between these genetic markers and the occurrence of IRIS. However, this study was pioneer in describing the distribution of HLA - B, HLA - DRB1 and KIR alleles among individu als with tuberculosis infected with HIV - 1. These results contribute to the discussion of the impact of host genes in the context of the two diseases studied and in the occurrence of IRIS.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento / Made available in DSpace on 2013-03-04T20:42:44Z (GMT). No. of bitstreams: 1 313825.pdf: 2234847 bytes, checksum: 0b50f1734f61ae413cbbdcea1df4feda (MD5) / A artrite reumatoide (AR) é uma doença autoimune caracterizada por inflamação crônica que causa erosões e deformidades nas articulações e manifestações inflamatórias sistêmicas como acometimentos pulmonar e cardíaco. As células natural killer (NK) são células efetoras que participam tanto da resposta imune inata quanto adaptativa através de produção de citocinas e ação citolítica. As NK são encontradas com frequência nas articulações afetadas pela AR. Dentre os receptores das células NK, destaca-se o receptor das células killer semelhante à imunoglobulina (KIR) que possui ação ativadora e/ou inibidora das NK. O objetivo deste trabalho foi investigar a presença/ausência dos genes KIR na população de Santa Catarina e a associação da presença dos genes KIR ativadores com pacientes de AR e com dados clínicos. Neste estudo foram avaliados 147 pacientes com AR (casos) e 164 indivíduos sem histórico de doença autoimune (controle) para a presença/ausência de 16 genes KIR, através da técnica de PCR por primer de sequência específica (PCR-SSP). No entanto, apenas os genes KIR ativadores (KIR2DS1, KIR2SD2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1) foram investigados para o estudo de associação caso-controle. As frequências gênicas foram estimadas e o odds ratio (OR) foi calculado para avaliar associação dos genes KIR ativadores com AR (p<0,05 foi considerado significativo). As amostras de pacientes e controles tiveram predominância de mulheres (87%) com média de 53(±12) anos e com ascendência europeia (86%). As frequências de presença/ausência dos 16 genes KIR em pacientes com AR assemelharam-se aos estudos com AR e outras doenças autoimunes no Brasil, bem como as frequências destes genes no grupo controle aproximaram-se aos demais estudos brasileiros para populações eurodescendentes. Não foi encontrada associação do polimorfismo de ausência e presença dos genes ativadores com AR. No entanto, a presença do gene KIR2DS2 foi mais frequente em pacientes com AR que apresentaram manifestação articular (MA) em relação ao grupo controle (OR=2,312; p=0,010). Além do mais, não houve associação do tagabismo com a AR, apenas com fator reumatoide (FR) em pacientes com AR (OR=2,947; p=0,02). Os genes KIR não foram associados ao desenvolvimento de AR neste estudo. Entretanto, o gene KIR2DS2 foi mais frequente em pacientes com a presença de MA, enquanto que o hábito tabagista foi relacionado com FR.

Introdução: A Doença de Gaucher (DG) é causada pela atividade reduzida da enzima lisossomal glucocerebrosidase, o que leva ao acúmulo de glicocerebrosídeo nas células e a uma estimulação crônica do sistema imune. Células natural killer (NK) possuem um papel importante na resposta imune e sua atividade é alterada na DG. Os receptores KIR (Killer Immunoglobulin-like Receptors) regulam a atividade das células NK através da interação com moléculas de HLA (Human Leukocyte Antigen) de classe I das célulasalvo. Objetivo: Analisar a variabilidade dos genes KIR em uma coorte de pacientes com DG do Sul do Brasil, compará-las a um grupo controle e buscar associações com manifestações clínicas. Metodologia: Trinta e um pacientes com DG tipo I (24 com forma leve, 4 com forma moderada e 3 com forma grave) foram analisados e comparados a 250 controles saudáveis quanto a frequência dos genes HLA e KIR. Resultados/Discussão: Não houve diferença significativa nas frequências de variantes dos genes KIR entre os grupos. O alelo HLA B37 é mais frequente nos pacientes com DG do que no grupo controle (p=0,011). A idade de início dos sintomas mostrou associação com a combinação das variantes KIR2DL2 e KIR2DS2 com seu ligante HLA-C1 (p=0,038). Pacientes que apresentam a variante HLA-C2 parecem apresentar maior susceptibilidade a desenvolver bandas mono ou policlonais na eletroforese de proteínas (p=0,007, OR=21,3). Foi encontrada associação entre os alelos DR11 (p=0.008) e DR13 (p=0.011) e gravidade da doença. O alelo DR11 parece estar associado a comprometimento neurológico, enquanto o alelo DR13 ao desenvolvimento de osteonecrose. Conclusão: Nossos dados sugerem uma possível associação entre variantes dos genes KIR e HLA e a DG. Devem ser estudadas em outras coortes de pacientes já que parecem ser um fator modificador de fenótipo. / Background: Gaucher disease (GD) is caused by the reduced activity of a lysosomal enzyme glucocerebrosidase, which leads to the accumulation of glucocerebroside in the cells and a chronic stimulation of the immune system. Natural Killer (NK) cells play an important role in the immune response, and their activity is impaired in GD. Killer immunoglobulin-like receptors (KIR) regulate the activity of NK cells through an interaction with specific human leukocyte antigen (HLA) class I molecules on target cells. Objectives: To analyze the variability of KIR genes in a Southern Brazilian sample of GD patients, to compare it with controls, and to look for associations with clinical manifestations. Methodology: Thirty one GD type I patients (24 mild, 4 moderate, and 3 severe) were analyzed and compared to 250 healthy controls regarding the frequency of HLA and KIR genes. Results/Discussion: There was no significative difference in the frequencies of KIR gene variants between the groups. The HLA B37 allele is more frequent in patients with GD than in control group (p=0.011). The age of onset of symptoms was associated with KIR2DL2 and KIR2DS2 combination with the ligand HLA-C1 (p=0.038). Patients who have the HLA-C2 variant appear to have more mono/polyclonal bands in protein electrophoresis (p=0.007, OR=21.3). An association between the DR11 (p=0.008) and DR13 (p=0.011) alleles and disease severity was found. The DR11 allele appears to be associated with neurological impairment, while the DR13 allele to the development of osteonecrosis. Conclusion: Our data suggest a possible association between KIR genes and HLA genes and GD. They should be studied in other cohorts of GD patients as they seem to be a phenotype modifying factor.

Introdução: A Doença de Gaucher (DG) é causada pela atividade reduzida da enzima lisossomal glucocerebrosidase, o que leva ao acúmulo de glicocerebrosídeo nas células e a uma estimulação crônica do sistema imune. Células natural killer (NK) possuem um papel importante na resposta imune e sua atividade é alterada na DG. Os receptores KIR (Killer Immunoglobulin-like Receptors) regulam a atividade das células NK através da interação com moléculas de HLA (Human Leukocyte Antigen) de classe I das célulasalvo. Objetivo: Analisar a variabilidade dos genes KIR em uma coorte de pacientes com DG do Sul do Brasil, compará-las a um grupo controle e buscar associações com manifestações clínicas. Metodologia: Trinta e um pacientes com DG tipo I (24 com forma leve, 4 com forma moderada e 3 com forma grave) foram analisados e comparados a 250 controles saudáveis quanto a frequência dos genes HLA e KIR. Resultados/Discussão: Não houve diferença significativa nas frequências de variantes dos genes KIR entre os grupos. O alelo HLA B37 é mais frequente nos pacientes com DG do que no grupo controle (p=0,011). A idade de início dos sintomas mostrou associação com a combinação das variantes KIR2DL2 e KIR2DS2 com seu ligante HLA-C1 (p=0,038). Pacientes que apresentam a variante HLA-C2 parecem apresentar maior susceptibilidade a desenvolver bandas mono ou policlonais na eletroforese de proteínas (p=0,007, OR=21,3). Foi encontrada associação entre os alelos DR11 (p=0.008) e DR13 (p=0.011) e gravidade da doença. O alelo DR11 parece estar associado a comprometimento neurológico, enquanto o alelo DR13 ao desenvolvimento de osteonecrose. Conclusão: Nossos dados sugerem uma possível associação entre variantes dos genes KIR e HLA e a DG. Devem ser estudadas em outras coortes de pacientes já que parecem ser um fator modificador de fenótipo. / Background: Gaucher disease (GD) is caused by the reduced activity of a lysosomal enzyme glucocerebrosidase, which leads to the accumulation of glucocerebroside in the cells and a chronic stimulation of the immune system. Natural Killer (NK) cells play an important role in the immune response, and their activity is impaired in GD. Killer immunoglobulin-like receptors (KIR) regulate the activity of NK cells through an interaction with specific human leukocyte antigen (HLA) class I molecules on target cells. Objectives: To analyze the variability of KIR genes in a Southern Brazilian sample of GD patients, to compare it with controls, and to look for associations with clinical manifestations. Methodology: Thirty one GD type I patients (24 mild, 4 moderate, and 3 severe) were analyzed and compared to 250 healthy controls regarding the frequency of HLA and KIR genes. Results/Discussion: There was no significative difference in the frequencies of KIR gene variants between the groups. The HLA B37 allele is more frequent in patients with GD than in control group (p=0.011). The age of onset of symptoms was associated with KIR2DL2 and KIR2DS2 combination with the ligand HLA-C1 (p=0.038). Patients who have the HLA-C2 variant appear to have more mono/polyclonal bands in protein electrophoresis (p=0.007, OR=21.3). An association between the DR11 (p=0.008) and DR13 (p=0.011) alleles and disease severity was found. The DR11 allele appears to be associated with neurological impairment, while the DR13 allele to the development of osteonecrosis. Conclusion: Our data suggest a possible association between KIR genes and HLA genes and GD. They should be studied in other cohorts of GD patients as they seem to be a phenotype modifying factor.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22371号 / 医博第4612号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 松田 文彦, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM