Interleukin-21 (IL-21), a recently discovered member of the IL-2 cytokine family, has been shown to have diverse regulatory effects on B cells including the induction of antibody secretion, differentiation, or apoptosis depending on the cell milieu and activation status. However, the effects of IL-21 on B cell neoplasms such as diffuse large B cell lymphoma (DLBCL) are largely unknown. Our research uncovered the widespread expression of the IL-21 receptor (IL-21R) in B cell lymphomas including DLBCL and that IL-21 stimulation resulted in potent phosphorylation of STAT1 and 3 and weak activation of STAT5. However, our findings also showed that treatment of DLBCL cell lines with IL-21 induced cell cycle arrest and apoptosis. The cell death was caspase-dependent and evident in a majority of DLBCL cell lines. To further examine the potential therapeutic applicability of IL-21, we assessed the effects of IL-21 on primary DLBCL tumors and in vivo DLBCL xenografts in mice. In primary tumors, IL-21 induced apoptosis in five of five DLBCLs compared to two of three follicular lymphomas and two of seven chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B lymphocytes. In mice bearing DLBCL xenograft tumors, in situ IL-21 injections induced tumor regression and dramatically extended the overall survival of mice (P<0.001). To elucidate the mechanism of IL-21-induced cell death we analyzed the expression of apoptosis-regulating proteins and observed a strong downregulation of anti-apoptotic Bcl-2 and Bcl-XL and an upregulation of pro-apoptotic Bax post IL-21 stimulation. Subsequent experiments showed that ectopic expression of Bcl-2 or Bcl-XL was able to partially reduce cell death induced by IL-21 while Bax knockdown with siRNA blocked apoptosis completely. To gain insight into the signaling pathways shifting the expression of these proteins toward cell death we performed microarray analysis on sensitive and resistant DLBCL cell lines. The most striking difference in gene expression was observed in C-MYC which was only induced in cell lines exhibiting apoptosis upon IL-21 treatment. Previous reports have shown that c-Myc, which has been studied extensively for its oncogenic properties, can induce apoptosis via downregulation of its transcriptional targets Bcl-2 and Bcl-XL. We then showed that IL-21-induced cell death is dependent on c-Myc by utilizing specific siRNA and shRNA to block the upregulation of this transcription factor and prevent apoptosis. Since c-Myc is a bona-fide target of STAT3 we also showed that siRNA-mediated knockdown of STAT3 abrogated apoptosis by preventing c-Myc upregulation and its subsequent effects on apoptosis-regulating proteins. Our results delineate a novel IL-21 pro-apoptotic signaling pathway and one of the first examples in which the STAT3 - c-Myc pathway, which usually promotes B cell survival and oncogenesis, can be exploited for treatment of cancer. Furthermore, our findings demonstrate that IL-21 is a highly potent anti-DLBCL agent in vitro and in animal models and should be examined in clinical studies of DLBCL.

In summary, this study will be focused on Il-21 and its implications in the antibody response in influenza. The isotype classes primarily involved in this process will also be examined. This will be accomplished by looking at the serum of mice and analyzing the present influenza specific antibodies using ELISA. Another goal was to optimize the ELISA in order to make it sensitive enough to catch small differences in the results. This topic is important due to its implications for improving influenza vaccinations and preventions as current vaccines are not 100% effective. Influenza contributes to significant disease and death around the world every year and each piece of this puzzle is significant in order for the scientific community to be able to eventually make strides to improve the burden of this disease.

Indiana University-Purdue University Indianapolis (IUPUI) / The immune system protects the body from foreign organisms. T cells and B cells are integral components of the ability of the immune system to generate focused immune responses. The development of specialized subsets of T helper cells is governed by transcription factors. Previous work demonstrated a requirement for the transcription factor PU.1 in the development of IL-9-secreting Th9 cells. Work in this dissertation demonstrates that the Th9 subset is not stable in vitro, and that PU.1 expression decreases during long-term culture. To examine a role for PU.1 in Th9-independent immunity we examined a model of multiple sclerosis termed experimental autoimmune encephalomyelitis (EAE). Mice that lack PU.1 expression in T cells (Sfpi1lck-/- mice) demonstrated more severe disease with attenuated recovery compared to control mice, and this was accompanied by an increase of T cells in the central nervous system. We also observed that following multiple routes of immunization Sfpi1lck-/- mice had increased numbers of T follicular helper (Tfh) cells and increased germinal center responses. This correlated with increased expression of the cytokine IL-21 and the surface protein CD40L in T cells that lacked PU.1 expression and resulted in increased numbers of germinal center B cells and antigen-specific antibody titers compared to control mice. The increased germinal center B cells and antibody titers were attenuated with blocking CD40L antibody but not with neutralizing IL-21 antibody. These results suggest that PU.1 limits the expression of CD40L on Tfh cells to regulate the humoral immune response. Together, the data in this dissertation demonstrate Th9-independent functions of PU.1. Moreover, this work shows that transcription factors promoting the development of one subset of T helper cells can simultaneously have negative effects on distinct T cell lineages.

A large number of cytokines are expressed in the feet and hands joints of patients with Rheumatoid arthritis. It is necessary to study the new cytokines which may help in prognosis and diagnosis of this autoimmune disease. Omentin1 and Interleukin 20 are the new cytokines and their expressions may have a role in the expression of proinflammatory cytokines; IL-1, IL-6 and TNF, in different cell tissues such as synovial fibroblasts, chondrocytes, peripheral blood mononuclear cells and, sera. It is conceivable that these biomarkers may be used in biological therapies.

IL-6 is an inflammatory cytokine that contributes to the pathogenesis of many immunological diseases including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, allergic asthma, as well as the protection against infections caused by various pathogens. These are linked to its role in regulating CD4 T cell differentiation and effector function. Most of these functions are dependent on the IL-6-mediated signaling through the transcription factor Stat3. In this thesis, we identify a novel molecular mechanism by which IL-6 regulates CD4 T cell effector function. We show that IL-6-dependent signal raises the levels of mitochondrial Ca2+ late during activation of CD4 T cells. This is further used to prolong the expression of effector cytokines IL-4 and IL-21. The modulation of mitochondrial Ca2+ is mediated by the regulation of mitochondrial Stat3 and the formation of respiratory supercomplexes. Thus, in addition to the canonical signaling of IL-6 through Stat3 as a transcription factor, IL-6 also modulates mitochondrial Stat3 to regulate mitochondrial function in CD4 T cells. This could be an alternative pathway by which IL-6 regulates effector function of CD4 T cells and it could contribute to the pathogenesis of inflammatory disease. Little is known about the effects of IL-6 on CD8 T cells. In this thesis, we reveal a paradigm-shifting mechanism by which IL-6 regulates antibody production by converting CD8 T cells into B cell helpers through IL-21. Briefly, IL-6 promotes the differentiation of a subset of naïve CD8 T cells into a unique population of effector CD8 T cells characterized by the production of high levels of IL-21. IL-21-producing CD8 T cells provide help to B cells to induce isotype switching and protective antibody production during infection. In summary, this thesis provides new insights into both mechanistic and functional aspects of IL-6 in regulating T cell function. These findings may shed light on the development of new therapeutic approaches in treating autoimmune disorders and preventing infectious diseases.

The world is grappling with an escalating risk of viral outbreaks of pandemic proportion, with zoonotic RNA viruses such as chikungunya virus (CHIKV) and SARS-CoV-2 posing significant threats to global health. Several environmental and evolutionary factors have fueled the emergence and spread of infection, creating a constant arms race against emerging pathogens. Current prevention and mitigation strategies are inadequate, necessitating tools to prevent and control viral infections; innovative strategies are needed in the pipeline to address significant challenges. CHIKV is a mosquito-borne virus that has caused millions of disease cases worldwide and is a reemerging threat with increasing potential to become endemic in the US. Currently, there are no licensed treatments available to protect against CHIK disease, making the development of a vaccine crucial. Live-attenuated vaccines (LAVs) have traditionally been a promising strategy due to their high immunogenicity and cost-effectiveness. However, concerns regarding adverse side effects and the potential for viral replication leading to pathogenic reversions or transmission into mosquitoes have limited their use. To that end, we have developed a new generation of safer vaccines by modifying the standard LAV platform through innovative attenuating strategies. Our dual-attenuated platform utilizes a previously developed chimera of CHIKV and the closely related Semliki Forest virus (SFV) as a vaccine backbone which expresses antiviral mouse cytokines IFN-γ or IL-21, as an additional mechanism to control infection. In several mouse models, both cytokine-expressing candidates showed reduced footpad swelling and minimal to no systemic replication or dissemination capacity compared to the parental vaccine post-vaccination. Importantly, these candidates conferred full protection from wildtype CHIK disease. Our IFNγ-expressing vaccine showed the most significant attenuation of viral replication. To understand the underlying mechanism, we identified three IFNγ-regulated antiviral genes (Gbp1/2 and Ido1) that were highly upregulated in 3T3 mouse fibroblasts post-infection with the IFN-γ-expressing candidate but not the parental backbone. To further investigate the role of these genes in restricting viral replication and enhance the clinical relevance of our vaccine platform, we redesigned our vaccine to express human IFNγ (hIFNγ) and performed viral growth kinetics in MRC5 human lung fibroblasts. Our vaccine showed reduced viral replication compared to controls and high expression of human GBP1/2/3 was observed post-infection. Overexpression of these genes demonstrated a direct impact on viral replication against wildtype CHIKV. These findings shed light on the mechanism of action of our vaccine and highlight the potential of targeting IFNγ-regulated antiviral genes for developing effective vaccines against CHIKV. Our results provided a foundation for investigating the broad-use application of IFN-γ against other alphaviruses for vaccine or therapeutic design. We evaluated the effects of increasing levels of exogenous hIFNγ on Mayaro virus (MAYV), Ross River virus (RRV), and Venezuelan Equine Encephalitis virus (VEEV). We observed a positive dose-dependent relationship between hIFNγ and decreasing viral titers for all three viruses. Interestingly, we also observed similar patterns of GBP upregulation with MAYV and RRV, both Old World alphaviruses, but not with VEEV, a New World alphavirus. This finding may indicate an alternative IFNγ-stimulated pathway responsible for controlling different alphaviruses. Overall, these studies establish a fundamental role of IFNγ in controlling viral infection and highlight its potential use in both vaccine and therapeutic intervention. While LAVs are a gold standard for developing immunity against a virus, the urgency of responding to an active and deadly pandemic has promoted the use of faster strategies such as mRNA vaccines. Once the viral sequence was known, these vaccines were comparatively quick to produce for SARS-CoV-2 and prevented millions of disease cases at the height of their introduction. However, the emergence of variants of concerns bypassing previous immunization efforts has demonstrated the need for complementary treatments such as antivirals to control disease. To that end, we evaluated several rhodium organometallic complexes as potential antivirals against SARS-CoV-2. We show that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(ICy)Cl2 and Cp*Rh(dpvm)Cl are non-toxic in Vero E6 and Calu3 cells and reduce SARS-CoV-2 plaque formation up to 99%. These complexes have previously demonstrated high antimicrobial activity against multiple antibiotic-resistance bacteria and with our results, support their potential application as pharmaceuticals, warranting further investigation into their activity. / Doctor of Philosophy / The global response to the COVID-19 pandemic, and its far-reaching impact, revealed significant shortcomings in public health preparedness for emerging viruses. Despite efforts to develop vaccines and antivirals to prevent and treat disease, current mitigation strategies have proven insufficient to eradicate the pathogen. The emergence of viral outbreaks caused by viruses such as chikungunya (CHIKV) and SARS-CoV-2 underscores the ongoing threat posed by emerging infectious diseases. Improved countermeasures are urgently needed to address gaps in vaccine and antiviral development. CHIKV is a mosquito-borne virus that has caused millions of infections across hundreds of countries with the emergent potential to become endemic in the US. Currently, there are no vaccines available to the public; therefore, it is important to generate and administer an effective vaccine before further spread of the virus. To this end, we developed innovative live-attenuated vaccines (LAVs) against CHIKV using a weakened chimeric backbone of CHIKV and its close relative, Semliki Forest virus (SFV), along with vaccine-driven expression of antiviral cytokines to control viral replication. Vaccination of highly susceptible mice with these cytokine-expressing vaccines produced significantly decreased side-effects compared to the parental virus not expressing the cytokines. Additionally, these viruses had significantly restricted viral replication capabilities while robustly protecting mice from a semi-lethal CHIKV infection. Our interferon-gamma (IFNγ) expressing vaccine had the greatest impact on viral replication, and we investigated the mechanism leading to this attenuation. To assess the clinical relevance of our vaccine platform, we redesigned the virus to express human IFNγ and identified a specific pattern of IFNγ-stimulated genes that are potentially responsible for limiting CHIKV replication. Furthermore, we demonstrated the broad therapeutic use of IFNγ against other medically relevant alphaviruses. Overall, these studies establish an improved mechanism to create safer vaccines without compromising efficacy and highlight the therapeutic potential of IFNγ against alphaviruses. Lastly, in a collaborative effort to respond to the COVID-19 pandemic, we also explored and characterized the use of a new class of antiviral drugs. With the advent of increasing drug resistance, it is essential to develop novel and resilient therapeutics. We demonstrated the first antiviral potential of rhodium organometallics, which was previously shown to be effective against multiple antibiotic-resistant bacteria. Two complexes demonstrated high virucidal activity against SARS-CoV-2 and low toxicity in mammalian cell lines. Moreover, these complexes can be further derivatized to improve efficacy, making them a promising new antiviral strategy.

O Diabetes Mellitus tipo 1 (DM1A) é uma doença autoimune caracterizada pela infiltração pancreática de linfócitos T e B, macrófagos e células dendríticas, levando à perda progressiva da capacidade de secreção de insulina pelas células beta pancreáticas. A homeostase das células T, ou seja, o desenvolvimento e manutenção apropriados dos números e funções das células T são essenciais para a integridade do sistema imune. Classicamente acreditava-se que as células T CD4+ poderiam se subdividir em duas populações efetoras distintas, T helper 1 e T helper 2. Recentemente, foram descritas duas novas vias de ativação de linfócitos T CD4+: a via Th17, que tem papel fundamental na autoimunidade; a via T regulatória, onde células T CD4+CD25+ high são essenciais na tolerância periférica e proteção contra autoimunidade. As Interleucinas (IL) de cadeia gama comum agem em várias etapas desta diferenciação linfocítica. A IL-21 é o membro mais recente desta família de citocinas, que inclui também: IL-2, IL-4, IL-7 , IL-9 e IL-15. A IL-21 atua através da interação com seu receptor, o IL-21R, apresentando ações pleiotrópicas e, como regra, pró-inflamatórias. Em estudos com modelos animais de diabetes autoimune verificou-se que a IL-21 e seu receptor são essenciais para o desenvolvimento da doença, porém ainda não há estudos sobre a ação desta interleucina no DM1 em humanos. O objetivo de nosso estudo foi avaliar o papel dos receptores das interleucinas de cadeia gama comum na patogênese do DM1A através da determinação da expressão da proteína de superfície e do RNA mensageiro destes receptores em pacientes com DM1A de início recente, em comparação com indivíduos controles normais, e da correlação destes valores com títulos de autoanticorpos pancreáticos. Estudamos a expressão da proteína de superfície do IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) e IL-7R (CD127) em linfócitos T periféricos de 35 pacientes com DM1 e 25 controles sadios utilizando citometria de fluxo. O tempo médio de diagnóstico do DM1 foi de 3 meses, e todos os pacientes estavam em uso de insulina no momento da coleta de sangue. Auto-anticorpos pancreáticos (anti-GAD65 e anti-IA2) foram dosados através de radioimunoensaio. A expressão do RNAm de IL-21R, IL-2R e IL-2R foi quantificada por PCR em tempo real em 23 dos pacientes portadores de DM1A. Detectamos, pela primeira vez, diminuição significativa na expressão proporcional de IL-21R, CD25 e CD122 em linfócitos TCD3+ e TCD4+, além de diminuição na expressão de CD124 em linfócitos T CD4+ e CD127 em linfócitos T CD3+. Verificamos também redução significativa na quantidade de células TCD4+CD25+high (T regulatórias) nos pacientes DM1A. Não houve correlação entre expressão dos receptores de superfície das interleucinas de cadeia gama comum e títulos de autoanticorpos pancreáticos. Realizamos o PCR em tempo real para quantificar a expressão do RNA mensageiro (RNAm) dos receptores de interleucinas de cadeia gama comum, e avaliar se esta correspondia à expressão das proteínas de superfície obtida através de citometria de fluxo. Comparamos a expressão do RNAm de IL-21R, IL-2R e IL-2R nos pacientes DM1A dividindo-os em tercis de acordo com os valores de expressão de proteína de superfície obtidos por citometria de fluxo em linfócitos T CD3+, e verificamos que não houve diferença entre os 3 grupos na expressão relativa dos genes estudados. Portanto, em nossa casuística a redução da expressão da proteína de superfície dos receptores de interleucinas de cadeia gama comum possivelmente decorreu de alterações posteriores à transcrição do RNA mensageiro / Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by pancreatic infiltration of T and B lymphocytes, macrophages and dendritic cells, leading to a progressive destruction of the insulin-producing -cells. Homeostasis of T cells can be defined as the ability of the immune system to maintain normal T-cell counts and to restore T-cell numbers following T-cell depletion or expansion. It was classically believed that the CD4+ T cells could be activated into two distinct effector populations, T helper1 and T helper2. It was recently described two new pathways of CD4+ T lymphocytes activation: the Th17 pathway, that plays a fundamental role in autoimmunity and the regulatory pathway (Treg), where CD4+CD25+high T cells are essential to maintain peripheral tolerance and therefore protect against autoimmunity. The common gamma chain cytokines interfere with several steps of the CD4+ T lymphocytes differentiation. Interleukin-21 (IL-21) is the most recent member of this family, that also includes IL-2, IL-4, IL-7, IL-9 and IL-15, and has pleiotropic effects on the immune system. Interleukin-21 acts through interaction with its receptor, the IL-21R, which is expressed in a great variety of immune cells. Various studies with animal models of autoimmune diabetes demonstrated that IL-21 and its receptor are essential for the development of the disease, but there are no studies evaluating the role of this interleukin and its receptor in T1DM in humans. The aim of our study was to assess the role of common gamma chain-dependent cytokine receptors in the pathogenesis of T1D, by determining the expression of the surface protein and mRNA of these receptors in recent-onset T1D patients and correlating these values with titles of pancreatic autoantibodies. We studied the surface protein expression of IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) and IL-7R (CD127) in peripheral T lymphocytes of 35 patients with T1D and 25 healthy controls using flow cytometry. Mean T1D duration was 3 months and all patients were using insulin at the time of blood withdraw. Pancreatic autoantibodies (anti-GAD65 and anti-IA2) were assessed by radioimmunoassay. The mRNA expression of IL-21R, IL-2R and IL-2R was quantified by real time PCR in 23 of the T1D patients. We detected for the first time a statistically significant decrease in the proportional expression of IL-21R, CD25 and CD122 on CD3+ and CD4+ T lymphocytes, a decrease in the expression of CD124 on CD4+ T cells and CD127 on CD3+ T lymphocytes. We also observed a significant reduction in the amount of CD4+ CD25+high (T regulatory cells) in T1D patients. There was no correlation between the expression of the surface receptors of common gamma chain cytokines and titles of pancreatic autoantibodies. We performed real-time PCR to quantify RNA expression of common gamma-chain interleukin receptors, and evaluate if these values corresponded to those of surface proteins obtained using flow cytometry. We compared the mRNA expression of IL-21R, IL-2R and IL-2R in T1D patients by dividing them into tertiles according to the expression values of surface protein obtained by flow cytometry in CD3+T lymphocytes. We observed that there was no difference in the relative expression of mRNA among the 3 groups of patients. Therefore, in our study, the reduction of surface protein expression of common gamma chain cytokines receptors was possibly due to alterations that occurred after the transcription of mRNA

Diabete Melito tipo 1A (DM1A) é uma doença autoimune resultante da interação de fatores ambientais, alterações imunológicas e predisposição genética. Decorre da perda da tolerância aos antígenos das células beta pancreáticas e ativação do sistema imunológico, notadamente dos linfócitos T e B. A via linfocitária T helper 17 está fortemente associada ao processo inflamatório denominado insulite, que resulta na destruição das células beta pancreáticas, levando à perda gradativa da produção de insulina e à instalação do DM1A. A via T helper 17 é regulada pelas interleucinas IL-17, IL-21, IL-23 e IL-27, que também atuam na agressão autoimune. Neste estudo nós avaliamos a importância dos receptores da IL-21 (IL-21RA) e da IL-17 (IL-17RA) na susceptibilidade ao DM1A e nas suas manifestações autoimunes (autoanticorpos) em 631 pacientes portadores de DM1A (24.6 ± 13.0 anos) comparados com 652 controles saudáveis (28.5 ± 11.4 anos). Não há dados prévios na literatura. As variantes selecionadas dos genes do IL-17RA (n=4), do IL-21R (n=5) e da cadeia gama comum (n=1) foram genotipadas através da metodologia Vera Code, Golden Gate (Illumina, EUA). Os autoanticorpos circulantes anti-descarboxilase do ácido glutâmico (anti-GAD65), anti-tirosina fosfatase (anti-IA2), anti-peroxidase (anti-TPO) e anti-tireoglobulina (anti-TG) foram dosados por radioimunoensaio, o antitransportador de zinco 8 (anti-ZnT8) por ELISA, o anti-célula parietal por imunofluorescência direta e o anti-receptor de TSH (TRAb) por ensaio radiorreceptor, marcado com iodo radioativo. As frequências dos genótipos das variantes foram testadas para o Equilíbrio de Hardy-Weinberg e as associações genotípicas, pelos testes do qui-quadrado e exato de Fisher. Estes foram ajustados para as covariáveis (idade, sexo, cor autorreferida e duração do diabete) na análise de regressão logística binária. Obtivemos os seguintes resultados para o gene IL17RA: a variante rs2241049 (genótipos AG/GG) foi associada à susceptibilidade para o DM1A (OR=1,42; IC95%=1,11-1,81; p=0,005), enquanto a variante rs879577 à proteção ao DM1A (genótipo AA: OR=0,61; IC95%= 0,4 - 0,93; p=0.021) e à menor frequência do anticorpo anti-IA2 (AA/AG; OR=0,67, IC95%= 0,45-0,99; p=0.043). O genótipo GG de rs5748863 foi relacionado à menor frequência dos autoanticorpos anti-IA2 (OR=0,52, IC95%= 0,32- 0,86; p=0.010). Em relação ao gene IL21R, os genótipos GC/CC de rs7199138 foram relacionados à susceptibilidade ao DM1A (OR= 1,33; IC95%= 1,05-1,68; p= 0,018). As variantes rs2214537(CG/GG) e rs2285452 (AG/AA) foram associadas à menor frequência dos autoanticorpos anti-célula parietal (OR=0,24; IC95%= 0,09-0,59; p < 0.001) e antiendomísio (OR=0,17; IC95%= 0,04-0,8; p=0.025), respectivamente. As variantes rs3093315 (TG/TT) e rs2285452 (AA) condicionaram maior frequência de TRAb (OR=5,89; IC95%=1,26-27,61; p=0.024) e anti-TPO (OR=2,38; IC95%= 1,1-5,13; p=0.028), respectivamente. Nossos resultados sugerem que variantes dos genes IL17RA e IL21R estão associadas à fisiopatologia do DM1A e à expressão de autoanticorpos pancreáticos e extrapancreáticos / Type 1 diabetes mellitus (T1D) is an autoimmune disease resulting from the interaction of environmental factors, immunological changes and genetic predisposition. It results from the loss of tolerance to pancreatic beta cell antigens and to the activation of the immune system, notably T and B lymphocytes. The T helper-17 pathway is strongly associated with the inflammatory process called insulitis, which results in pancreatic beta cells destruction, leading to the gradual loss of insulin production and to the manifestation of T1D. The T helper 17 pathway is regulated by the interleukins IL-17, IL-21, IL-23 and IL-27, which also act in the autoimmune aggression. In this study we evaluated the importance of the receptors of IL-21 (IL-21RA) and IL-17 (IL-17RA) genes in the susceptibility to T1D and its autoimmune manifestations (autoantibodies) in 631 patients with DM1A (24.6 ± 13.0 years) , compared with 652 health controls (28.5 ± 11.4 years).There is no previous data in the literature. The selected variants of the IL-17RA (n = 4), IL-21R (n = 5) and of the common gamma chain (n = 1) genes were genotyped using the Vera Code methodology, Golden Gate (Illumina, USA). The autoantibodies anti-decarboxylase of glutamic acid (anti-GAD65), anti-tyrosine phosphatase (anti-IA2), anti-peroxidase (anti-TPO) and anti-thyroglobulin (anti-TG) were measured by radioimmunoassay, anti zinc transporter 8 (Anti-ZnT8) by ELISA, anti-parietal cell by direct immunofluorescence and anti-TSH receptor (TRAb) by radiolabeled radioiodine receptor assay. The frequencies of the genotypes of the variants were tested by Hardy-Weinberg Equilibrium and the genotypic associations were performed by the chi-square and Fisher\'s exact tests. These were adjusted for the covariates (age, gender, self-reported color and diabetes duration) in binary logistic regression analysis.We obtained the following results for IL-17RA : the rs2241049 variant (AG/GG genotypes) was associated with susceptibility to T1D (OR = 1,42; CI65%= 1,11-1,81; p = 0,005), while the rs879577 variant to T1D protection (genotype AA: OR = 0,61; CI95%= 0,4 - 0,93; p = 0,021) and to lower anti-IA2 frequency (AA/AG; OR = 0,067; CI95%=0,45-0,99; p=0,043). Further, the GG genotype of rs5748863 variant was related to lower frequency of the autoantibody anti-IA2 (OR= 0,52; CI95%= 0,32- 0,86; p= 0,010). Regarding IL21R, the GC / CC genotypes of rs7199138 were related to susceptibility to T1D (OR = 1,33; CI95%= 1,05-1,68; p = 0,018). The variants rs2214537 (CG/GG) and rs2285452 (AG/AA) were associated with lower frequency of parietal cell (OR=0,24; CI95%= 0,09-0,59; p < 0,001) and antiendomysial autoantibodies (OR=0,17; CI95%= 0,04-0,8; p=0,025), respectively. The rs3093315 (TG/TT) and rs2285452 (AA) variants were related to higher frequency of TRAb (OR=5,89; CI95%= 1,26-27,61; p=0,024) and anti-TPO (OR=2,38; CI95%= 1,1-5,13; p=0,028), respectively. Our results suggest that variants of the IL17RA and IL21R genes are associated with the pathophysiology of T1D and the expression of pancreatic and extra-pancreatic autoantibodies

As citocinas têm papel importante como mediadores através das respostas imunológicas. A Interleucina-21, importante regulador dos linfócitos T e B, é produzida por linfócitos CD4 ativados, e está implicada na patogênese do diabetes autoimune em modelo animal, o NOD. A região promotora da IL-21, que contempla sítios de controle da expressão gênica em camundongos, o NFATc2, T-bet e c-MAF, foi estudada pela primeira vez em humanos portadores de diabetes tipo 1A, neste trabalho. Foi analisado também a freqüência do polimorfismo C1858T do gene PTPN22, que tem sido associado em estudos recentes como fator de risco importante para diabetes tipo1A e outras doenças autoimunes. Associou-se ainda, autoanticorpos pancreáticos e não-pancreáticos em diabéticos e grupo controle normal, e estes resultados foram analisados com ambos os genes. Foram estudados 612 DM1A e 792 indivíduos do grupo controle. Após extração de DNA genômico, a região 5proximal da região promotora do gene da Il-21, -448+83pb, foi seqüenciada em 309 brasileiros diabéticos tipo 1A e 189 indivíduos do grupo controle. A genotipagem do polimorfismo C1858T do gene PTPN22, por RFLP, foi realizada em 434 diabéticos e 689 controles, bem como os alelos HLA-DRB1. Foi encontrada uma variação alélica, em heterozigoze, na posição g.-241 T>A, em apenas uma paciente, que apresentou idade de diagnóstico aos 30 anos de idade. Esta variante alélica não foi encontrada nos 497 indivíduos (308 DM1A e 189 grupo controle). A freqüência dos alelos polimórficos (CT/TT) foi maior nos diabéticos (18.7%) que no grupo controle (10.6%), OR 1,94 e p<0,001. O polimorfismo C1858T do gene PTPN22 associou-se à maior freqüência dos autoanticorpos pancreático anti-GAD65 (p=0,002) e não-pancreático anti-TG (p=0,001), quando avaliados os dois grupos juntos, DM1A e grupo controle. Os diabéticos apresentaram maior freüência dos autoanticorpos como segue: autoanticorpos pancreáticos: anti-GAD65: 225 /482 (46.7%) vs 13/786 (1.7%), p<0.001; anti-IA2 : 204/469 (43.5%) vs 15/786 (1.9%), p<0.001. Autoanticorpos não-pancreáticos: FAN: 60/234 (25.6%) vs 13/239 (5.4%), p<0.001; anti-TPO: 64/279 (22.9%) vs 34/495 (6.9%), p<0.001; anti-TG : 65/278 (23.4%) vs 44/489 (9%), p<0.001; TRAb: 14/187 (7.5%) vs 1/327 (0.3%), p<0.001; anti-21-OH : 8/154 (5,2%) vs 1/160 (0,6%), p< 0,001. Os autoanticorpos a seguir foram realizados apenas nos pacientes diabéticos: anti-tTG 5/73% (6.8%), anti-Endom 10/176 (5.7%). Com exceção do anti-GAD65 e anti-TG, nenhum outro autoanticorpo associou-se ao polimorfismo do gene PTPN22. Os alelos HLA-DR3/DR$ predominaram nos diabéticos (p<0,001). Concluimos então que o polimorfismo C1858T do gene PTPN22 e os alelos HLA-DR3/DR4 estão associados ao risco de DM1A. Variantes alélicas na região 5 proximal do gene da IL-21 parece não ser predisponente à suscetibilidade ao DM1A e outras doenças autoimunes. Autoanticorpos órgão-específicos são mais freqüentes em diabéticos, principalmente nas glândulas adrenal e tireóide. O polimorfismo C1858T do gene PTPN22 está associado à maior freqüência dos autoanticorpos anti-GAD65 e antitireoglobulina / Objective: Cytokines are central mediators of inflammation through innate and adaptive immune responses. IL-21, a critical regulator of T and B cell function, is produced by various subsets of CD4+ T cells, and it has been implicated in the pathogenesis of non obese diabetes mouse. The proximal promoter of IL-21, which controls its Th-cell-subset-specific expression through the action of NFATc2, T-bet and c-MAF in animal models, was evaluated in type 1A diabetes (T1AD) patients for the first time. This study also analyzed the 1858T PTPN22 polymorphism, which has recently emerged as an important risk factor for T1AD and other autoimmune diseases. Moreover, islet and other organ-specific autoantibodies were quantified in T1AD patients and healthy controls and the results were correlated with both genes. Research design and methods: The case series comprised 612 T1AD patients and 792 healthy control (HC) individuals. Genomic DNA extraction was performed by salting-out in purified blood leukocytes. The region encompassing -448+83 bp of IL-21 gene was amplified and sequenced using genomic DNA from 309 Brazilian T1AD patients and 189 control individuals. RFLP genotyping of C1858T PTPN22 was performed in 689 controls and 434 T1D patients. HLA DR3/DR4 alleles were also evaluated. Results: A heterozygous allelic variant (g.-241 T>A) was found in only one patient, who was 30 years old at the onset of disease. This allelic variant was not found in 497 individuals (308 T1AD patients and 189 healthy controls). The PTPN22 1858T allele frequency was greater in patients (18.7%) than in controls (10.6%): odds ratio of 1.94; p<0.001. An association was found between C1858T polymorphism and higher frequency of GAD65 Ab (p=0.002) and TG Ab (p=0.011), among both T1AD and HC. Type 1 diabetes patients presented higher frequency of the following autoantibodies, compared with HC (p<0.001): GAD65 Ab (46.7% vs 1.7%); IA2 Ab (43.5% vs 1.9%); ANA (25.6% vs 5.4%); TPO Ab (22.9% vs 6.9%); TG Ab (23.4% vs 9.0%); TRAb (7.5% vs 0.3%); 21-OH Ab (5,2% vs 0,6%). The following antibodies were evaluated only in T1AD: tTG Ab (6.8%) anti-Endom (5.7%). Except by GAD65 Ab and TG Ab, no association was found between C1858T polymorphism and these autoantibodies.HLA-DR3/DR4 alleles predominated in T1D patients (p<0.001) Conclusions: C1858T PTPN22 polymorphism and the HLA-DR3 and/or DR4 alleles were associated with proneness to T1AD. Allelic variants at the 5\' proximal region of the IL-21 gene do not seem to predispose to susceptibility to T1AD and other autoimmune endocrine diseases. Autoantibodies specific to other organs and tissues are frequent in T1AD carriers, mainly to the thyroid glands. The 1858T PTPN22 polymorphism was associated with higher frequency of GAD65A and TGA.

As citocinas têm papel importante como mediadores através das respostas imunológicas. A Interleucina-21, importante regulador dos linfócitos T e B, é produzida por linfócitos CD4 ativados, e está implicada na patogênese do diabetes autoimune em modelo animal, o NOD. A região promotora da IL-21, que contempla sítios de controle da expressão gênica em camundongos, o NFATc2, T-bet e c-MAF, foi estudada pela primeira vez em humanos portadores de diabetes tipo 1A, neste trabalho. Foi analisado também a freqüência do polimorfismo C1858T do gene PTPN22, que tem sido associado em estudos recentes como fator de risco importante para diabetes tipo1A e outras doenças autoimunes. Associou-se ainda, autoanticorpos pancreáticos e não-pancreáticos em diabéticos e grupo controle normal, e estes resultados foram analisados com ambos os genes. Foram estudados 612 DM1A e 792 indivíduos do grupo controle. Após extração de DNA genômico, a região 5proximal da região promotora do gene da Il-21, -448+83pb, foi seqüenciada em 309 brasileiros diabéticos tipo 1A e 189 indivíduos do grupo controle. A genotipagem do polimorfismo C1858T do gene PTPN22, por RFLP, foi realizada em 434 diabéticos e 689 controles, bem como os alelos HLA-DRB1. Foi encontrada uma variação alélica, em heterozigoze, na posição g.-241 T>A, em apenas uma paciente, que apresentou idade de diagnóstico aos 30 anos de idade. Esta variante alélica não foi encontrada nos 497 indivíduos (308 DM1A e 189 grupo controle). A freqüência dos alelos polimórficos (CT/TT) foi maior nos diabéticos (18.7%) que no grupo controle (10.6%), OR 1,94 e p<0,001. O polimorfismo C1858T do gene PTPN22 associou-se à maior freqüência dos autoanticorpos pancreático anti-GAD65 (p=0,002) e não-pancreático anti-TG (p=0,001), quando avaliados os dois grupos juntos, DM1A e grupo controle. Os diabéticos apresentaram maior freüência dos autoanticorpos como segue: autoanticorpos pancreáticos: anti-GAD65: 225 /482 (46.7%) vs 13/786 (1.7%), p<0.001; anti-IA2 : 204/469 (43.5%) vs 15/786 (1.9%), p<0.001. Autoanticorpos não-pancreáticos: FAN: 60/234 (25.6%) vs 13/239 (5.4%), p<0.001; anti-TPO: 64/279 (22.9%) vs 34/495 (6.9%), p<0.001; anti-TG : 65/278 (23.4%) vs 44/489 (9%), p<0.001; TRAb: 14/187 (7.5%) vs 1/327 (0.3%), p<0.001; anti-21-OH : 8/154 (5,2%) vs 1/160 (0,6%), p< 0,001. Os autoanticorpos a seguir foram realizados apenas nos pacientes diabéticos: anti-tTG 5/73% (6.8%), anti-Endom 10/176 (5.7%). Com exceção do anti-GAD65 e anti-TG, nenhum outro autoanticorpo associou-se ao polimorfismo do gene PTPN22. Os alelos HLA-DR3/DR$ predominaram nos diabéticos (p<0,001). Concluimos então que o polimorfismo C1858T do gene PTPN22 e os alelos HLA-DR3/DR4 estão associados ao risco de DM1A. Variantes alélicas na região 5 proximal do gene da IL-21 parece não ser predisponente à suscetibilidade ao DM1A e outras doenças autoimunes. Autoanticorpos órgão-específicos são mais freqüentes em diabéticos, principalmente nas glândulas adrenal e tireóide. O polimorfismo C1858T do gene PTPN22 está associado à maior freqüência dos autoanticorpos anti-GAD65 e antitireoglobulina / Objective: Cytokines are central mediators of inflammation through innate and adaptive immune responses. IL-21, a critical regulator of T and B cell function, is produced by various subsets of CD4+ T cells, and it has been implicated in the pathogenesis of non obese diabetes mouse. The proximal promoter of IL-21, which controls its Th-cell-subset-specific expression through the action of NFATc2, T-bet and c-MAF in animal models, was evaluated in type 1A diabetes (T1AD) patients for the first time. This study also analyzed the 1858T PTPN22 polymorphism, which has recently emerged as an important risk factor for T1AD and other autoimmune diseases. Moreover, islet and other organ-specific autoantibodies were quantified in T1AD patients and healthy controls and the results were correlated with both genes. Research design and methods: The case series comprised 612 T1AD patients and 792 healthy control (HC) individuals. Genomic DNA extraction was performed by salting-out in purified blood leukocytes. The region encompassing -448+83 bp of IL-21 gene was amplified and sequenced using genomic DNA from 309 Brazilian T1AD patients and 189 control individuals. RFLP genotyping of C1858T PTPN22 was performed in 689 controls and 434 T1D patients. HLA DR3/DR4 alleles were also evaluated. Results: A heterozygous allelic variant (g.-241 T>A) was found in only one patient, who was 30 years old at the onset of disease. This allelic variant was not found in 497 individuals (308 T1AD patients and 189 healthy controls). The PTPN22 1858T allele frequency was greater in patients (18.7%) than in controls (10.6%): odds ratio of 1.94; p<0.001. An association was found between C1858T polymorphism and higher frequency of GAD65 Ab (p=0.002) and TG Ab (p=0.011), among both T1AD and HC. Type 1 diabetes patients presented higher frequency of the following autoantibodies, compared with HC (p<0.001): GAD65 Ab (46.7% vs 1.7%); IA2 Ab (43.5% vs 1.9%); ANA (25.6% vs 5.4%); TPO Ab (22.9% vs 6.9%); TG Ab (23.4% vs 9.0%); TRAb (7.5% vs 0.3%); 21-OH Ab (5,2% vs 0,6%). The following antibodies were evaluated only in T1AD: tTG Ab (6.8%) anti-Endom (5.7%). Except by GAD65 Ab and TG Ab, no association was found between C1858T polymorphism and these autoantibodies.HLA-DR3/DR4 alleles predominated in T1D patients (p<0.001) Conclusions: C1858T PTPN22 polymorphism and the HLA-DR3 and/or DR4 alleles were associated with proneness to T1AD. Allelic variants at the 5\' proximal region of the IL-21 gene do not seem to predispose to susceptibility to T1AD and other autoimmune endocrine diseases. Autoantibodies specific to other organs and tissues are frequent in T1AD carriers, mainly to the thyroid glands. The 1858T PTPN22 polymorphism was associated with higher frequency of GAD65A and TGA.