Type 1 diabetes (T1D) results from a loss of functional insulin-producing pancreatic beta cells. The etiology of T1D is poorly understood, but the detection of infiltrating inflammatory cells in the pancreas and circulating autoantibodies has led to the common notion that an autoimmune process plays a central role in the pathogenesis of the disease. The aim of this doctoral thesis was to assess various aspects of the immunopathology of type 1 diabetes. To this purpose, studies have been conducted on pancreatic material from the Network for Pancreatic Organ Donors with Diabetes (nPOD) collection, the Nordic Network for Islet Transplantation, and the Diabetes Virus Detection (DiViD) study. Paper I is a study on pancreatic tissue from organ donors with varying duration of T1D as well as non-diabetic donors and subjects with other types of diabetes, in which persistent expression of glucose transporters was shown on the beta cell membrane despite several years of T1D. Glucose transporter 1 was also confirmed as the predominant glucose transporter on human pancreatic islets. In paper II, we report on signs of inflammation in the exocrine but not in the endocrine pancreas in non-diabetic organ donors with diabetes-related autoantibodies, suggesting that diabetes-associated autoantibodies can occur in response to unspecific pancreatic lesions. Paper III aimed to characterize the T cell-infiltration of pancreatic islets in material from recent-onset T1D patients. Insulitis was shown in all subjects, but with distinct differences in expression analysis of T- and B cell activation to cell-mediated allorejected kidney transplant. Also Paper IV was conducted on material from recent-onset cases and showed increased islet glucagon content, in combination with a reduced number of islets but sustained mean islet size. Together, these results provide expansion of our knowledge of the immunopathology in T1D, and will hopefully assist in bringing us towards a deeper understanding of T1D aetiology and eventually an effective cure.

N-methyl-D-aspartate receptor (NMDAR) antibody encephalitis is a recently described autoimmune encephalopathy defined by the presence of serum antibodies that bind NMDARs (NMDAR-Abs). NMDAR-Ab encephalitis is a severe, but treatmentresponsive encephalitis with subacute onset. It can be associated with tumours and affects mainly young adults. Patients present with cognitive dysfunction, seizures, psychiatric and sleep disorders and most develop dyskinesias, autonomic instability and reduced consciousness. To explore further the NMDAR-Abs and their potential pathogenicity, a series of in vitro investigations were performed and preliminary attempts at passive transfer of disease. Human embryonic kidney (HEK) cells transfected with the NR1 and NR2B subunits, and live cultured neurons, were used first to detect NMDAR-Ab binding. Immunocytochemistry and ow cytometry demonstrated that binding to transfected HEK cells could be improved when NMDAR were presented in clusters by cotransfection with the postsynaptic density protein PSD-95. The NR1 subunit was identified as the target of NMDAR-Abs, and a novel quantitative assay based on immunoprecipitation of NR1 tagged by fusion with green uorescent protein was developed. Measurement of NMDAR-Ab levels showed that antibody levels corresponded to the clinical disease score within individual patients. Although the purification of full length NR1 was not successful, a secreted N-terminal construct was created and expressed in HEK cells. The binding of NMDAR-Abs was confirmed and this construct will be used for active immunisation in future. To explore pathogenic mechanisms in vitro, the main antibody subclasses were shown to be IgG1 and IgG3. Moreover the patients' autoantibodies, but not healthy control antibodies, were able to activate the complement cascade in vitro in cell lines and primary cultures. Finally, the NMDAR-Abs were shown to bind to primary microglial cultures and to cause morphological changes corresponding to early activation processes after prolonged exposure. The research has developed new assays that could be used for diagnosis and serial studies and revealed new potential mechanisms in NMDAR-Ab encephalitis.

A surdez neurossensorial imunomediada (SNIM) é uma das formas reversíveis de surdez neurossensorial, justificando a necessidade de identificação de marcadores mais específicos que ajudem na sua abordagem. O Western blot com antígenos de tecidos bovinos detecta um anticorpo contra a proteína de 68kD (hsp-70) em SNIM. Entretanto, anticorpos antihsp- 70 são comumente encontrados em indivíduos sadios. O objetivo do estudo foi determinar a reatividade de soros de pacientes com doenças otológicas freqüentemente relacionadas à etiologia auto-imune contra antígeno celular de linhagem humana (HeLa) através do Western blot, comparando com outros marcadores sorológicos de auto-imunidade. Soros de 81 pacientes com surdez neurossensorial (25 surdez súbita, 35 rapidamente progressiva e 21 doença Ménière) foram testados por Western blot utilizando extrato total de célula HeLa como antígeno alvo. Os pacientes com surdez foram comparados com indivíduos com audição normal e sem queixas otológicas ou história de doença sistêmica auto-imune (n=48). Observou-se reatividade contra célula HeLa principalmente nas regiões de 42, 48 and 62kD no grupo com surdez. O padrão de reatividade foi diferente entre os diferentes subgrupos de surdez. A reatividade contra as bandas de 48 and 62kD foi observada em surdez súbita e surdez rapidamente progressiva. A proteína de 48kD proveniente da surdez rapidamente progressiva parece ser diferente daquela encontrada em surdez súbita devido a sua resistência à tripsina. Este estudo demonstrou a existência de autorreatividade contra células HeLa na surdez súbita, surdez rapidamente progressiva e doença de Ménière, sugerindo a presença de novos autoanticorpos. O seu papel é ainda desconhecido. Estudos detalhados são necessários para avaliar a real relevância clínica desta autorreatividade na patologia de orelha interna ou como marcador prognóstico ou diagnóstico / Immune-mediated sensorineural hearing loss (SNHL) is one of few forms of reversible SNHL, justifying the need to define more specific markers to help clinical approach. Western blot with bovine tissues detect an autoantibody against the 68kD protein (hsp70) in immune-mediated SNHL. However, antihsp70 antibodies are also common in healthy individuals. The objective of this study was to determinate the reactivity of serum from patients with otologic entities often related to autoimmune etiology against human cell line antigen (HeLa) by Western blot, comparing to other serological markers. Sera of 81 patients with SNHL (25 sudden SNHL, 35 rapidly progressive SNHL and 21 Ménière\'s disease patients) were tested by Western blot using HeLa cell total extract as target. Western blot outcome was compared with detection of other current autoimmune markers. Experimental group data were compared to normal-hearing subjects (n=48) without otologic or systemic autoimmune disease. Reactivity to HeLa cells was observed mostly at 42, 48 and 62kD region which pattern was different among different groups. Binding to 48 and 62kD HeLa cell antigen was observed in sudden SNHL and rapidly progressive SNHL. The 48kD protein from rapidly progressive SNHL seems to be different from sudden SNHL\'s according to resistance to trypsin. This study detected autoreactivity to HeLa cells in sudden SNHL, rapidly progressive SNHL and Ménière\'s disease, suggesting the presence of new autoantibodies. Their role is still unknown and further studies should evaluate their relevance on pathology in the inner ear or as diagnostic or prognostic marker

INTRODUÇÃO: A urticária crônica é caracterizada pelo aparecimento de placas eritêmato-edematosas, pruriginosas, que perduram por mais de seis semanas. A etiologia é desconhecida na maioria dos pacientes sendo definida como idiopática (UCI). A desregulação imunológica na UCI pode ser devido a distúrbios na produção de citocinas e quimiocinas. OBJETIVOS: Avaliar o perfil citocinas e quimiocinas em pacientes submetidos ao teste de soro autólogo (ASST) avaliando os soros, a expressão de RNAm e a expressão intracelular de células mononucleares do sangue periférico (CMN) induzidas por estímulos policlonais. METODOLOGIA: Pacientes com UCI (n=37) foram selecionados do Ambulatório de Dermatologia do HC-FMUSP e submetidos ao ASST. O grupo controle foi constituído por indivíduos saudáveis (n=33). Os níveis séricos de citocinas e quimiocinas foram determinados por citometria de fluxo ou por ELISA e a expressão de RNAm de citocinas foi determinada por Real-Time PCR. RESULTADOS: Uma elevação dos níveis séricos de TNF-, IL-6, IL-1, IL-12p70 e IL-10 foi detectada nos pacientes com UCI comparados ao grupo controle, independente da resposta ao ASST. A secreção in vitro de citocinas por CMN estimuladas por fitohemaglutinina (PHA) mostrou aumento da produção de IL-2 nos pacientes com UCI e de IL-17A e IL-10 no grupo ASST positivo em relação ao grupo controle. A expressão de RNAm para IL-10 em CMN, foi diminuída no grupo ASST negativo comparado ao grupo controle. Além disto, um aumento da capacidade linfoproliferativa ao mitógeno Pokeweed foi observado nos pacientes ASST positivo em relação aos indivíduos controles. Os níveis séricos de CXCL8, CCL2, CXCL10 e CXCL9 foram encontrados elevados nos pacientes com UCI em relação aos controles. A secreção de quimiocinas in vitro, foi observado aumento dos níveis basais de CCL2 pelas CMN dos pacientes em relação aos controles, que se elevaram em resposta a enterotoxina A de Staphylococcus aureus (SEA). Já o estímulo com PHA promoveu aumento na produção de CXCL8 e CCL5 pelas CMN dos pacientes. A expressão intracelular de CXCL8 foi detectada principalmente nas células CD14+. A intensidade média de fluorescência (MFI) e a porcentagem da expressão de CXCL8 em CD14+ nos níveis basais e estimulados com SEA encontram-se diminuídos nos pacientes com UCI comparado ao grupo controle. A expressão intracelular de CCL2 em células CD14+ mostrou uma queda na porcentagem dos níveis basais somente nos pacientes ASST negativo em relação ao grupo controle. Além disto, em condições basais de cultura houve um aumento na porcentagem da expressão de CCR5 em células T CD8+ de pacientes com UCI, em função do aumento no grupo ASST positivo. CONCLUSÕES: Os resultados enfatizam o conceito de desequilíbrio imunológico na UCI, independente da resposta ao ASST, evidenciado pelo aumento na secreção de quimiocinas e citocinas pró-inflamatórias. Estes dados sugerem que na UCI, os linfócitos e monócitos estão ativados, os quais podem contribuir para a imunopatogênese da doença / INTRODUCTION: Chronic urticaria is skin disorder characterized by recurrent and transitory itchy weals occurring regularly for more than 6 weeks. The aetiology is not identified in most patients being considered as idiopathic (CIU). The immunological dysregulation in CIU could be due to a disturbed cytokines and chemokines production. OBJECTIVES: To evaluate the pattern of cytokine and chemokine in CIU patients who undergone autologous serum skin test (ASST), assessing sera, mRNA expression and intracellular expression of peripheral blood mononuclear cells (PBMC) through the secretion upon induced by policlonal stimuli. METHODS: CIU patients (n=37) were selected from the Dermatological Outpatient Clinic of the Hospital das Clínicas de São Paulo (HC-FMUSP) and submitted to the ASST. The control group consisted of healthy subjects (n=33). Cytokine and chemokine levels were assessed by flow cytometer and ELISA and mRNA expression was analyzed by Real-Time PCR. RESULTS: Elevated levels of TNF-, IL-6, IL-1, IL-12p70 and IL-10 were observed in sera from CIU patients compared to healthy control group. CIU patients also showed increased IL-2 production by PBMC stimulated with phytohemagglutinin (PHA). Moreover, it was observed higher IL-17A and IL-10 levels in the ASST+ group compared to control group. The IL-10 mRNA expression was diminished in the ASST- group compared to control group. Furthermore, an increased lymphoproliferative response to Pokeweed mitogen was observed in the ASST+ patients compared to healthy subjects. Seric levels of CXCL8, CCL2, CXCL10 and CXCL9 were higher in CIU patients. Regarding the in vitro chemokines secretion, it was detected higher basal levels of CCL2 in CIU patients, which was increased by Staphylococcus aureus enterotoxin A (SEA). Stimulation with PHA increased the CXCL8 and CCL5 production by CIU mononuclear cells. The main source of CXCL8 was the CD14+ cells. CIU CD14+ cells showed decreased mean fluorescence intensity and percentage of CXCL8 expression with and without SEA stimuli. The percentage of CD14+ producing CCL2 was lower in ASST- patients compared to healthy control subjects. Furthermore, in the absence of stimuli the percentage of CCR5-expressing CD8+ T cells was higher in CIU patients, mainly due to an increased expression by the ASST+ group. CONCLUSIONS: These results indicate an immunological dysregulation in CIU, without association to ASST response, which was evidenced by the increased production of pro-inflammatory cytokines and chemokines. The data suggest a higher activation of monocytes and lymphocytes in CIU, which may contribute to its immunopathogenesis

Objetivo: este estudo objetivou avaliar a presença do anticorpo anti-alfaenolase (AAAE) IgM na doença de Behçet (DB) e suas possíveis associações com as manifestações clínicas e atividade da doença. Métodos: noventa e sete pacientes com DB foram comparados a 36 pacientes com enteroartrite (EA) [24 com doença de Crohn (DC) e 12 com retocolite ulcerativa (RCU)], além de 87 controles saudáveis. Os testes para detecção do AAAE IgM foram realizados por Immunoblotting. A atividade de doença foi avaliada por índices padronizados, como o Formulário de Atividade Atual da Doença de Behçet (BR-BDCAF) para os pacientes com DB e o Índice de Harvey-Bradshaw (HBI) para os pacientes com DC e RCU. Uma segunda avaliação foi realizada somente nos pacientes com DB (n=56) para a detecção do AAAE IgM, avaliação de atividade de doença e dosagem de proteína-C-reativa (PCR). Resultados: maior prevalência do AAAE IgM foi encontrada na DB (17,7%) comparativamente à EA (2,8%) e aos controles saudáveis (2,3%), p < 0,001. A frequência do AAAE IgM foi maior na DB ativa quando comparada à DB inativa (30,2% vs. 7,4%, p=0,006). Este achado foi confirmado em uma segunda avaliação de 56 pacientes do grupo com DB (45,5% vs. 13,3%, p=0,02). A média do BR-BDCAF foi maior no grupo com AAAE IgM positivo, em ambas avaliações (9,1 ± 5,4 vs. 4,9 ± 4,9, p=0,002; 5,0 ± 4,9 vs. 2,2 ± 2,9, p=0,01, respectivamente). Os pacientes com DB em atividade mucocutânea e articular apresentaram maior incidência do AAAE IgM, tanto na primeira quanto na segunda avaliação (64,7% vs. 27,5%, p=0,005; 36,4% vs. 7,1%, p=0,039, respectivamente). Conclusões: os presentes dados corroboram que a alfa-enolase é um antígeno alvo na DB, particularmente associada à atividade mucocutânea e articular da doença. Além disso, o AAAE IgM pode distinguir a DB da EA, especialmente em pacientes com alta atividade de doença / Objective: this study aimed to assess IgM AAEA in systemic Behçet\'s disease (BD) and its possible association with clinical manifestations and disease activity. Methods: ninety-seven consecutively selected BD patients were compared to 36 enteropathic spondyloarthritis (ESpA) [24 Crohn\'s disease (CD) and 12 ulcerative colitis (UC)] patients and 87 healthy controls. IgM AAEA was detected by Immunoblotting. Disease activity was assessed by standardized indexes, Brazilian BD Current Activity Form (BR-BDCAF) for BD and Harvey-Bradshaw Index (HBI) for CD and UC patients. A second evaluation was performed in BD patients (n=56), regarding IgM AAEA presence, disease activity scores and C-reactive protein (CRP). Results: higher IgM AAEA prevalence was found in BD (17.7%) compared to ESpA (2.8%) and healthy controls (2.3%), p < 0.001. IgM AAEA frequency was higher in active BD compared to inactive BD (30.2% vs. 7.4%, p=0.006), a finding confirmed in the second cross-sectional evaluation of 56 of these BD patients (45.5% vs. 13.3%, p=0.02). Mean BR-BDCAF scores were higher in IgM AAEA positive group on both evaluations (9.1 ± 5.4 vs. 4.9 ± 4.9, p=0.002; 5.0 ± 4.9 vs. 2.2 ± 2.9, p=0.01, respectively). BD patients with mucocutaneous and articular symptoms presented higher IgM AAEA positivity in the first and second evaluations (64.7% vs. 27.5%, p=0.005; 36.4% vs. 7.1%, p=0.039 respectively). Conclusions: these data support the notion that alpha-enolase is a target antigen in BD, particularly associated with disease activity, mucocutaneous and articular involvement. In addition, IgM AAEA may distinguish BD from ESpA, especially in patients with high disease activity

A surdez neurossensorial imunomediada (SNIM) é uma das formas reversíveis de surdez neurossensorial, justificando a necessidade de identificação de marcadores mais específicos que ajudem na sua abordagem. O Western blot com antígenos de tecidos bovinos detecta um anticorpo contra a proteína de 68kD (hsp-70) em SNIM. Entretanto, anticorpos antihsp- 70 são comumente encontrados em indivíduos sadios. O objetivo do estudo foi determinar a reatividade de soros de pacientes com doenças otológicas freqüentemente relacionadas à etiologia auto-imune contra antígeno celular de linhagem humana (HeLa) através do Western blot, comparando com outros marcadores sorológicos de auto-imunidade. Soros de 81 pacientes com surdez neurossensorial (25 surdez súbita, 35 rapidamente progressiva e 21 doença Ménière) foram testados por Western blot utilizando extrato total de célula HeLa como antígeno alvo. Os pacientes com surdez foram comparados com indivíduos com audição normal e sem queixas otológicas ou história de doença sistêmica auto-imune (n=48). Observou-se reatividade contra célula HeLa principalmente nas regiões de 42, 48 and 62kD no grupo com surdez. O padrão de reatividade foi diferente entre os diferentes subgrupos de surdez. A reatividade contra as bandas de 48 and 62kD foi observada em surdez súbita e surdez rapidamente progressiva. A proteína de 48kD proveniente da surdez rapidamente progressiva parece ser diferente daquela encontrada em surdez súbita devido a sua resistência à tripsina. Este estudo demonstrou a existência de autorreatividade contra células HeLa na surdez súbita, surdez rapidamente progressiva e doença de Ménière, sugerindo a presença de novos autoanticorpos. O seu papel é ainda desconhecido. Estudos detalhados são necessários para avaliar a real relevância clínica desta autorreatividade na patologia de orelha interna ou como marcador prognóstico ou diagnóstico / Immune-mediated sensorineural hearing loss (SNHL) is one of few forms of reversible SNHL, justifying the need to define more specific markers to help clinical approach. Western blot with bovine tissues detect an autoantibody against the 68kD protein (hsp70) in immune-mediated SNHL. However, antihsp70 antibodies are also common in healthy individuals. The objective of this study was to determinate the reactivity of serum from patients with otologic entities often related to autoimmune etiology against human cell line antigen (HeLa) by Western blot, comparing to other serological markers. Sera of 81 patients with SNHL (25 sudden SNHL, 35 rapidly progressive SNHL and 21 Ménière\'s disease patients) were tested by Western blot using HeLa cell total extract as target. Western blot outcome was compared with detection of other current autoimmune markers. Experimental group data were compared to normal-hearing subjects (n=48) without otologic or systemic autoimmune disease. Reactivity to HeLa cells was observed mostly at 42, 48 and 62kD region which pattern was different among different groups. Binding to 48 and 62kD HeLa cell antigen was observed in sudden SNHL and rapidly progressive SNHL. The 48kD protein from rapidly progressive SNHL seems to be different from sudden SNHL\'s according to resistance to trypsin. This study detected autoreactivity to HeLa cells in sudden SNHL, rapidly progressive SNHL and Ménière\'s disease, suggesting the presence of new autoantibodies. Their role is still unknown and further studies should evaluate their relevance on pathology in the inner ear or as diagnostic or prognostic marker

Objetivo: o objetivo deste estudo foi avaliar a prevalência de autoanticorpos e a dosagem da concentração de células Natural Killer (NK) no sangue periférico em pacientes com endometriose. Métodos: Entre dezembro de 2004 e dezembro de 2007 foram avaliadas 155 pacientes submetidas a videolaparoscopia, divididas em um grupo sem endometriose(n=55) e outro com endometriose (n=100). Foi coletada amostra de sangue periférico de todas as pacientes no momento da laparoscopia e nessa amostra foi realizada a quantificação do percentual de células NK em relação aos linfócitos periféricos (por citometria de fluxo), e a determinação dos seguintes auto-anticorpos: anticorpos antinucleares (ANA, por imunofluorescência indireta), anticorpos antitireoglobulina e antiperoxidase (anti-TG e anti-TPO, por eletroquimioluminescência), anticorpos anticardilipina e antifosfatidilserina (aCL e aPS IgG, IgM e IgA, todos por ensaio imunoenzimático). Além da presença de endometriose, essas pacientes também foram avaliadas quanto ao estadiamento, os locais de doença, relações com a fase do ciclo, e a classificação histológica dessa doença. Resultados: as pacientes com endometriose apresentaram percentual de células NK (média DP de 15,3 9,8%) superiores àquelas sem a doença (média DP de 10,6 5,8%), p<0,001. Quanto aos autoanticorpos, as portadoras de endometriose também apresentaram positividade para ANA mais frequentemente (33%) que as pacientes do grupo controle (12,7%), p=0,006. Quanto aos anti-TG, anti-TPO, anti-CL (IgG, IgM e IgA) e aPS ( IgG, IgM e IgA), não houve diferenças estatísticas quanto à sua positividade. As células NK também mostraram-se mais elevadas nas protadoras de endometriose em estádios avançados e naquelas com comprometimento de retossigmóide, grupo no qual encontramos o maior percentual de células NK com concentração média de 19,8 10,3%. Concentrações de células NK 12,5% podem ser usadas como marcadores de endometriose em retossigmóide, com sensibilidade de 73% e especificidade de 65%. Utilizando-se de um modelo estatístico de probabilidades, demonstramos que associação desse marcador (NK 12,5%) com a presença de sintomas como dor e/ou sangramento intestinal durante a menstruação nos possibilitou estimar uma probabilidade de comprometimento de retossigmóide de 60,4%. Conclusões: pacientes com endometriose apresentam maior concentração de células NK periféricas, além de maior prevalência de ANA positivo em relação àquelas sem endometriose. As células NK aumentam nas pacientes com endometriose predominantemente nos estádios avançados, com comprometimento de retossigmóide. Nesse sentido poderiam ser utilizadas como marcadores diagnósticos desse tipo de comprometimento da doença, principalmente se forem avaliadas em conjunto com os sintomas das pacientes / Objectives: The objective of this study was to evaluate the prevalence of autoantibodies and the percentage of natural killer (NK) cells in the peripheral blood of patients with endometriosis. Methods: Between December 2004 and December 2007, 155 patients submitted to videolaparoscopy were evaluated. Patients were divided into two groups: one group of women without endometriosis (n = 55) and another in which all the women had endometriosis (n = 100). Samples of peripheral blood were collected from all the patients at the time of laparoscopy and flow cytometry was used to determine the percentage of NK cells in relation to peripheral blood lymphocytes in these samples. In addition, the following autoantibodies were measured: antinuclear antibodies (ANA) by indirect immunofluorescence, anti-thyroglobulin and anti-thyroid peroxidase antibodies (anti-TG and anti-TPO) by electrochemiluminescence, and anticardiolipin and anti-phosphatidylserine antibodies (aCL and aPS IgG, IgM and IgA), all performed using immunoenzymatic assay. In addition to the presence of endometriosis, these patients were also evaluated with respect to staging, to the sites of the disease, any association with the phase of the menstrual cycle and the histological classification of the disease. Results: The patients with endometriosis had a higher percentage of NK cells (15.3 ± 9.8%; mean ± SD) compared to those without the disease (10.6 ± 5.8%; mean ± SD), (p<0.001). Evaluation of the autoantibodies showed that positivity for ANA was more common in the group of patients with endometriosis (33%) compared to the patients in the control group (12.7%), (p = 0.006). With respect to anti-TG, anti-TPO, aCL (IgG, IgM and IgA) and aPS (IgG, IgM and IgA), no statistically significant differences were found between the groups of patients with or without endometriosis. NK cell concentrations were also found to be higher in patients with advanced stages of endometriosis and in those in whom the rectosigmoid was affected by the disease, this being the group in which the highest percentage of NK cells was found, with mean concentrations of 19.8 ± 10.3%. NK cell concentrations 12.5% may be used as markers of endometriosis of the rectosigmoid, with sensitivity of 73% and specificity of 65%. Using a statistical model of probability, these findings showed that the association of this marker (NK 12.5%) with the presence of symptoms such as pain and/or intestinal bleeding during menstruation permitted an estimation to be made of a likelihood of 60.4% of rectosigmoid endometriosis. Conclusions: Patients with endometriosis have higher percentages of peripheral NK cells, as well as a greater prevalence of positive ANA compared to those without endometriosis. The concentration of peripheral NK cells increases in patients with endometriosis, predominantly in patients with advanced stages of the disease and those in whom the rectosigmoid is affected. Therefore, the concentration of NK cells in peripheral blood could be used as a diagnostic marker of this type of endometriosis, particularly when evaluated together with patients symptoms

Objetivo: o objetivo deste estudo foi avaliar a prevalência de autoanticorpos e a dosagem da concentração de células Natural Killer (NK) no sangue periférico em pacientes com endometriose. Métodos: Entre dezembro de 2004 e dezembro de 2007 foram avaliadas 155 pacientes submetidas a videolaparoscopia, divididas em um grupo sem endometriose(n=55) e outro com endometriose (n=100). Foi coletada amostra de sangue periférico de todas as pacientes no momento da laparoscopia e nessa amostra foi realizada a quantificação do percentual de células NK em relação aos linfócitos periféricos (por citometria de fluxo), e a determinação dos seguintes auto-anticorpos: anticorpos antinucleares (ANA, por imunofluorescência indireta), anticorpos antitireoglobulina e antiperoxidase (anti-TG e anti-TPO, por eletroquimioluminescência), anticorpos anticardilipina e antifosfatidilserina (aCL e aPS IgG, IgM e IgA, todos por ensaio imunoenzimático). Além da presença de endometriose, essas pacientes também foram avaliadas quanto ao estadiamento, os locais de doença, relações com a fase do ciclo, e a classificação histológica dessa doença. Resultados: as pacientes com endometriose apresentaram percentual de células NK (média DP de 15,3 9,8%) superiores àquelas sem a doença (média DP de 10,6 5,8%), p<0,001. Quanto aos autoanticorpos, as portadoras de endometriose também apresentaram positividade para ANA mais frequentemente (33%) que as pacientes do grupo controle (12,7%), p=0,006. Quanto aos anti-TG, anti-TPO, anti-CL (IgG, IgM e IgA) e aPS ( IgG, IgM e IgA), não houve diferenças estatísticas quanto à sua positividade. As células NK também mostraram-se mais elevadas nas protadoras de endometriose em estádios avançados e naquelas com comprometimento de retossigmóide, grupo no qual encontramos o maior percentual de células NK com concentração média de 19,8 10,3%. Concentrações de células NK 12,5% podem ser usadas como marcadores de endometriose em retossigmóide, com sensibilidade de 73% e especificidade de 65%. Utilizando-se de um modelo estatístico de probabilidades, demonstramos que associação desse marcador (NK 12,5%) com a presença de sintomas como dor e/ou sangramento intestinal durante a menstruação nos possibilitou estimar uma probabilidade de comprometimento de retossigmóide de 60,4%. Conclusões: pacientes com endometriose apresentam maior concentração de células NK periféricas, além de maior prevalência de ANA positivo em relação àquelas sem endometriose. As células NK aumentam nas pacientes com endometriose predominantemente nos estádios avançados, com comprometimento de retossigmóide. Nesse sentido poderiam ser utilizadas como marcadores diagnósticos desse tipo de comprometimento da doença, principalmente se forem avaliadas em conjunto com os sintomas das pacientes / Objectives: The objective of this study was to evaluate the prevalence of autoantibodies and the percentage of natural killer (NK) cells in the peripheral blood of patients with endometriosis. Methods: Between December 2004 and December 2007, 155 patients submitted to videolaparoscopy were evaluated. Patients were divided into two groups: one group of women without endometriosis (n = 55) and another in which all the women had endometriosis (n = 100). Samples of peripheral blood were collected from all the patients at the time of laparoscopy and flow cytometry was used to determine the percentage of NK cells in relation to peripheral blood lymphocytes in these samples. In addition, the following autoantibodies were measured: antinuclear antibodies (ANA) by indirect immunofluorescence, anti-thyroglobulin and anti-thyroid peroxidase antibodies (anti-TG and anti-TPO) by electrochemiluminescence, and anticardiolipin and anti-phosphatidylserine antibodies (aCL and aPS IgG, IgM and IgA), all performed using immunoenzymatic assay. In addition to the presence of endometriosis, these patients were also evaluated with respect to staging, to the sites of the disease, any association with the phase of the menstrual cycle and the histological classification of the disease. Results: The patients with endometriosis had a higher percentage of NK cells (15.3 ± 9.8%; mean ± SD) compared to those without the disease (10.6 ± 5.8%; mean ± SD), (p<0.001). Evaluation of the autoantibodies showed that positivity for ANA was more common in the group of patients with endometriosis (33%) compared to the patients in the control group (12.7%), (p = 0.006). With respect to anti-TG, anti-TPO, aCL (IgG, IgM and IgA) and aPS (IgG, IgM and IgA), no statistically significant differences were found between the groups of patients with or without endometriosis. NK cell concentrations were also found to be higher in patients with advanced stages of endometriosis and in those in whom the rectosigmoid was affected by the disease, this being the group in which the highest percentage of NK cells was found, with mean concentrations of 19.8 ± 10.3%. NK cell concentrations 12.5% may be used as markers of endometriosis of the rectosigmoid, with sensitivity of 73% and specificity of 65%. Using a statistical model of probability, these findings showed that the association of this marker (NK 12.5%) with the presence of symptoms such as pain and/or intestinal bleeding during menstruation permitted an estimation to be made of a likelihood of 60.4% of rectosigmoid endometriosis. Conclusions: Patients with endometriosis have higher percentages of peripheral NK cells, as well as a greater prevalence of positive ANA compared to those without endometriosis. The concentration of peripheral NK cells increases in patients with endometriosis, predominantly in patients with advanced stages of the disease and those in whom the rectosigmoid is affected. Therefore, the concentration of NK cells in peripheral blood could be used as a diagnostic marker of this type of endometriosis, particularly when evaluated together with patients symptoms

Le cancer du sein est un problème de santé public majeur. Avec 500 000 décès dans le monde chaque année, il concentre de nombreux efforts dans la recherche de nouvelles thérapies et de biomarqueurs aussi bien diagnostiques, pronostiques que de suivi thérapeutique. D'un point de vue diagnostique, la mammographie est actuellement la méthode de dépistage de référence. Cependant, elle présente de nombreuses limites, notamment chez les femmes avec une densité mammaire élevée. L'identification de marqueurs permettant de mettre en évidence de petites tumeurs ou pronostiquer l'évolution de ces tumeurs serait une aide précieuse dans la prise en charge de la maladie. D'un point de vue thérapeutique, la radiothérapie est l'un des traitements principaux contre le cancer du sein. Cependant, certaines patientes peuvent développer d'importants effets secondaires tardifs sévères. La prédiction de cette radiotoxicité est un enjeu majeur car elle permettrait d'identifier les patients à risque, de leur proposer un traitement personnalisé et au final d'optimiser leur prise en charge thérapeutique. Malgré de nombreux efforts, il n'existe à l'heure actuelle aucun marqueur prédictif de la radiotoxicité tardive utilisé en routine clinique.Ce travail de thèse a pour objectif d'identifier des marqueurs protéiques, d'une part pour le diagnostic et le pronostic des formes précoces du cancer du sein, et d'autre part pour la prédiction de la réponse à la radiothérapie. Grâce à différentes approches de protéomique, j'ai pu identifier trois signatures protéiques : (1) une signature d'autoanticorps sériques (GAL3, RACK1, PAK2, PHB2 et RUVBL1) pour le diagnostic des carcinomes canalaires in situ (CCIS) et des cancers précoces, (2) une signature d'autoanticorps sériques (CIRBP, ECHDC1, HMGN1, PSRC1 et RBP-Jκ) pronostic de la progression de CCIS en cancer invasif et (3) cinq protéines lymphocytaires (AK2, ANX1, APEX1, HSPA8 et IDH2) prédictives d'une toxicité radio-induite tardive. Au delà de leur intérêt diagnostic, pronostic ou prédictif, ces signatures ont également permis d'identifier de nouveaux partenaires moléculaires susceptibles d'être impliqués dans les mécanismes de carcinogénèse mammaire et de radiotoxicité tardive. / Breast cancer is a major public health problem. With 500,000 deaths every year around the world, it focuses on many efforts in the search for new therapies and for therapeutic monitoring, diagnosis or prognosis biomarkers. From a diagnostic point of view, mammography remains gold standard for breast cancer detection. However, it has many limitations, especially in women with significant breast density. The identification of biomarkers for early diagnosis or for prognosis of in situ tumors in this population at risk would be a valuable improvement in the breast cancer management. Considering treatment strategies, radiotherapy has become a major treatment against breast cancer. However, some patients can develop severe radiation-induced late side effects. Their prediction is a main challenge as clinicians will be able to identify patients at risk and develop individualized treatment. Despite all efforts, no biomarkers are validated and used in clinical routine for normal tissues outcomes after irradiation. The aim of my PhD work was to identify protein markers for the diagnosis and prognosis of early-stage breast cancers, and also for the prediction of radiation-induced severe late effects. With different proteomic approaches, I was able to identify three protein signatures: (1) a serum autoantibody signature for the diagnosis of DCIS and node negative early-stage breast cancers (GAL3, RACK1, PAK2, and PHB2 RUVBL1), (2) a serum autoantibody signature of ductal carcinoma in situ progression to invasive breast cancer (CIRBP, ECHDC1, HMGN1, PSRC1 and RBP-Jκ) and (3) five lymphocyte proteins (AK2, ANX1, APEX1, HSPA8 and IDH2 ) that could predict late radiation-induced toxicity. In addition, these signatures allowed to identify new molecular partners likely to be involved in the mechanisms of mammary carcinogenesis and in the late radio-induced toxicity.

Made available in DSpace on 2014-07-29T15:25:24Z (GMT). No. of bitstreams: 1 tese Jozelia rego.pdf: 289983 bytes, checksum: 81f90ad780122cbecb664ab66f6306c0 (MD5) Previous issue date: 2009-12-17 / Autoimmune diseases are a clinical syndrome caused by the activation of T and/or B cells. They are multifactorial in nature and characterized by the presence of autoantibodies directed against cellular components. These autoantibodies can act as diagnostic markers or as predictors for these diseases. The ANA test is a very useful tool in the investigation of autoimmune diseases. OBJECTIVES: a) establishing a correlation between clinical diagnoses and fluorescence patterns in ANA tests on HEp-2 cells; b) determining the frequency of fluorescence patterns; c) establishing a correlation between clinical diagnosis and fluorescence titers; d) establishing possible correlations of changes in fluorescence patterns. CASES AND METHODS: All the ANA requests sent to the Immunorheumatology Laboratory of the Teaching Hospital of the Federal University of Goias, from January / 2000 to December / 2007 were analyzed and those with positive results were selected. For the ANA research, the investigator used the IFI technique and HEp-2 cells as substrate. To classify the fluorescence patterns decision trees proposed by the Brazilian Consensus for Standardization of ANA in HEp-2 cells were used. RESULTS: Among the 8,631 ANA requests, 1,167 presented positive results (13,52%). These positive tests were divided into two groups: Group I (tests requested in one occasion) and Group II (tests requested in more than one occasion). In Group I, nuclear patterns were more prevalent (89,41%). Speckled nuclear patterns were seen more frequently (78,81%), with special notice to fine speckled nuclear patterns (32,74%), coarse speckled nuclear patterns (29,86%) and fine dense speckled nuclear patterns (9,79%). Among the clinical diagnoses, rheumatic autoimmune diseases were the most prevalent (59,87%) and they correlated mostly with speckled nuclear patterns. A positive ANA was noted in 216 cases (34,67%) of non-immune conditions and in 22 cases (3,53%) of undetermined diagnosis. Cases with moderate (1:160) and high (1:640 and > 1:640) titers presented a high association with autoimmune diseases (54,25%; 73,23%; 83,91%, respectively). In Group II, the analytic clinical diagnosis and fluorescence titer factors showed a significant association with the change in the fluorescence pattern. CONCLUSIONS: 1) ANA was found to be positive in autoimmune (61,80%) and in non-autoimmune diseases (34,67%). 2) The most frequently found positive ANA correlation was seen with a diagnosis of lupus erythematosus (38,04%), mainly with coarse speckled nuclear pattern (32,91%), fine speckled nuclear pattern (25,73%), homogeneous nuclear pattern (19,40%) and fine dense speckled nuclear pattern (10,12%). 3) Nuclear patterns were more frequently found (89,41%), and among them, speckled patterns were prevalent (78,81%). 4) Low titers can be found in rheumatic autoimmune diseases and, therefore, can not be interpreted as an exclusion criteria for autoimmune disease, as long as there are clinical indications. 5) High titers can be found in non-autoimmune diseases and, therefore, can not be interpreted as specific to autoimmune diseases. 6) When the ANA test was requested in more than one occasion for the same patient, the clinical diagnosis (especially SLE) and the fluorescence titer (1:40 and 1:160) showed an association with the change of the fluorescence pattern. 7) A correct valuation of the ANA test should associate information from positive results to the clinical history and the physical examination of the patient when they are suggestive of an autoimmune disease, most notably, of rheumatic autoimmune diseases / As doenças autoimunes são síndromes clínicas causadas pela ativação de células T e/ou B, de origem multifatorial, caracterizadas pela presença de autoanticorpos dirigidos contra componentes celulares. Os autoanticorpos podem atuar como marcadores diagnósticos ou como marcadores preditores destas doenças. O teste de FAN é um exame útil na investigação de doenças autoimunes. OBJETIVOS: a) verificar a correlação entre os diagnósticos clínicos e os padrões de fluorescência na pesquisa de FAN em células HEp-2; b) determinar a freqüência dos padrões de fluorescência; c) verificar a correlação entre os diagnósticos clínicos e os títulos de fluorescência; d) verificar as possíveis correlações da mudança dos padrões de fluorescência. CASUÍSTICA E MÉTODOS: Foram analisadas todas as solicitações de FAN encaminhadas ao Laboratório de Imuno-Reumatologia do Hospital das Clínicas da Universidade Federal de Goiás, durante o período de jan./ 2000 a dez./ 2007, e selecionadas as solicitações com resultados positivos. A pesquisa do FAN foi realizada pela técnica de IFI, utilizando-se como substrato células HEp-2. Para a classificação dos padrões de fluorescência utilizou-se as árvores de classificação definidas pelo I Consenso Nacional para padronização dos laudos de FAN em células HEp-2. RESULTADOS: Das 8631 solicitações de FAN, 1167 apresentaram resultados positivos (13,52%). Os testes positivos foram divididos em dois grupos: Grupo I (exames solicitados em uma ocasião) e Grupo II (exames solicitados em mais de uma ocasião). No Grupo I, os padrões encontrados em maior freqüência foram os nucleares (89,41%). Os padrões nucleares pontilhados foram observados em maior freqüência (78,81%), destacando-se os padrões nuclear pontilhado fino (32,74%), nuclear pontilhado grosso (29,86%) e nuclear pontilhado fino denso (9,79%). Dentre os diagnósticos clínicos descritos, as doenças autoimunes reumáticas foram observadas em maior freqüência (59,87%) e correlacionaram-se, principalmente, com os padrões nucleares pontilhados. FAN positivo foi observado em 216 casos (34,67%) de situações não-autoimunes e em 22 casos (3,53%) de diagnóstico não definido. Os casos com títulos moderados (1:160) e elevados (1:640 e > 1:640) apresentaram maior associação com enfermidades autoimunes (54,25%; 73,23%; 83,91%, respectivamente). No Grupo II, os fatores analíticos diagnóstico clínico e título de fluorescência mostraram associação significativa com a mudança do padrão de fluorescência. CONCLUSÕES: 1) A positividade do teste de FAN foi observada em doenças autoimunes (61,80%) e em doenças não-autoimunes (34,67%). 2) A correlação mais freqüente do FAN positivo foi observada com o diagnóstico de lupus eritematoso (38,04%), principalmente com os padrões nuclear pontilhado grosso (32,91%), nuclear pontilhado fino (25,73%), nuclear homogêneo (19,40%) e nuclear pontilhado fino denso (10,12%). 3) Os padrões nucleares foram observados em maior freqüência (89,41%), sendo os pontilhados os mais comuns (78,81%). 4) Títulos baixos podem ser encontrados em doenças autoimunes reumáticas e, portanto, não devem ser interpretados como critério para exclusão de doença autoimune, desde que exista suspeita clínica. 5) Títulos altos podem ser encontrados em doenças não-autoimunes e, portanto, não devem ser interpretados como específicos de doença autoimune. 6) Quando o teste de FAN foi solicitado em mais de uma ocasião, para o mesmo paciente, o diagnóstico clínico (principalmente de LES) e o título de fluorescência (1:40 e 1:160) mostraram associação com a mudança do padrão de fluorescência. 7) A valorização correta do teste de FAN deve associar as informações fornecidas pelo resultado positivo à história clínica e exame físico do paciente, quando sugestivos de doença autoimune, principalmente de doenças autoimunes reumáticas.