Dissertations / Theses on the topic 'IgA'

Orientador: Claudio Lucio Rossi
Tese (doutorado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas
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Resumo: A neurocisticercose (NC) e uma importante causa de doença neurológica em muitos paises em desenvolvimento, incluindo o Brasil. O diagnostico clinico da NC e dificultado pelo polimorfismo e pela não especificidade dos sintomas. As tecnicas de neuroimagem e pesquisa de anticorpos específicos tem contribuído para o diagnostico da NC e uma melhor compreensão dos processos fisiopatológicos dessa infecção. O presente trabalho teve como objetivo avaliar, por meio de técnicas imunoenzimaticas (ELISA), a resposta imune humoral na NC, utilizando como preparações antigênicas o liquido vesicular (LV) e uma fração glicoproteica obtida do extrato bruto de cisticercos de Taenia solium (T. solium) com afinidade por lentil-lectina (fração Gp). Cinquenta e seis amostras de liquido cefalorraquidiano (LCR), 22 de pacientes com NC e 34 de pacientes com outros problemas neurológicos, foram utilizadas para a pesquisa de IgG e suas subclasses, com os seguintes resultados: IgG-LV: 100% de sensibilidade e especificidade; IgG1 -LV: 72,73% de sensibilidade e 100% de especificidade; IgG2-LV: 81,81% de sensibilidade e 100% de especificidade; IgG3-LV: 59,09% de sensibilidade e 97,06% de especificidade; IgG4-LV: 90,91% de sensibilidade e 97,06% de especificidade; IgG-fração Gp: 90,91% de sensibilidade e 97,06% de especificidade; IgG1-fração Gp: 59,09% de sensibilidade e 91,18% de especificidade; IgG2-fração Gp: 68,18% de sensibilidade e 94,12% de especificidade; IgG3-fração Gp: 36,36% de sensibilidade e 100% de especificidade; IgG4-fração Gp: 86,36% de sensibilidade e 100% de especificidade. Quarenta e sete amostras de LCR, 16 de pacientes com NC e 31 de pacientes com outros problemas neurológicos foram utilizadas para a pesquisa de IgE, com os seguintes resultados: IgE-LV e IgE-fração Gp: 93,75% de sensibilidade e 100% de especificidade. Cinquenta e sete amostras de soros, 22 de pacientes com NC, 18 de pacientes com outras infecções e 17 de pessoas presumivelmente sadias, foram utilizadas para a pesquisa da IgG e suas subclasses, IgE, IgA e IgM, com os seguintes resultados: IgG-LV: 100% de sensibilidade e especificidade; IgG1-LV: 86,36% de sensibilidade e 94,28% de especificidade; IgG2-LV: 90,91% de sensibilidade e 97,14% de especificidade; IgG3-LV: 86,36% de sensibilidade e 97,14% de especificidade; IgG4-LV: 100% de sensibilidade e de especificidade; IgG-fração Gp: 95,45% de sensibilidade e 100% de especificidade; IgG1-fração Gp: 63,64% de sensibilidade e 94,28% de especificidade; IgG2-fração Gp: 68,18% de sensibilidade e 97,14% de especificidade; IgG3-fração Gp: 54,54% de sensibilidade e 88,57% de especificidade; IgG4-fração Gp: 90,91% de sensibilidade e 100% de especificidade; IgELV: 90,91% de sensibilidade e 97,14% de especificidade; IgE-fração Gp: 86,36% de sensibilidade e 100% de especificidade; IgA-LV: 54,54% de sensibilidade e 94,28% de especificidade; IgA-fração Gp: 13,63% de sensibilidade e 100% de especificidade. Anticorpos IgM não foram detectados com as preparações de LV e fração Gp. Nossos resultados mostraram que, com ambas as preparações antigênicas, tanto em amostras de LCR quanto em amostras de soros, a maior positividade foi obtida na detecção de anticorpos das classes IgG e IgE, seguida da positividade da IgA. Anticorpos IgM não foram detectados em amostras de soros com reações de ELISA realizadas com LV e fração Gp. Com relação as subclasses da IgG, a IgG4 apresentou, tanto em amostras de LCR como em amostras de soros, valores de positividade e concentração iguais ou superiores as outras subclasses. As reações ELISA realizadas com LV mostraram sensibilidades iguais ou superiores aquelas obtidas com a fração Gp. Considerando a complexidade e o custo final da obtenção da fração Gp, o LV pode ser considerado mais adequado para a pesquisa de anticorpos em amostras de LCR e soros de pacientes com NC.
Abstract: Neurocysticercosis (NC) is an important cause of neurological disease in many developing countries, including Brazil. The clinical diagnosis of NC is hindered by the polymorphism and non-specificity of the symptoms. Neuroimaging techniques and detection of specific antibodies have contributed to the diagnosis of NC and a better understanding of the physiopathological processes of this infection. The purpose of this study was to evaluate the humoral immune response in NC by using immunoenzymatic techniques (ELISA) in which vesicular fluid (VF) and a glycoprotein fraction purified from a crude extract of Taenia solium cysticerci with affinity for lentil-lectin (fraction Gp) were used as antigenic preparations. Fifty-six cerebrospinal fluid (CSF) samples, 22 from patients with NC and 34 from patients with other neurological disorders, were assayed for IgG and IgG subclasses, with the following results: IgG-VF: 100% sensitivity and specificity, IgG1 - VF: 72.73% sensitivity and 100% specificity, IgG2 -VF: 81.81% sensitivity and 100% specificity, IgG3 -VF: 59.09% sensitivity and 97.06% specificity, IgG4 -VF: 90.91% sensitivity and 97.06% specificity, IgG-fraction Gp: 90.91% sensitivity and 97.06% specificity, IgG1- fraction Gp: 59.09% sensitivity and 91.18% specificity, IgG2-fraction Gp: 68.18% sensitivity and 94.12% specificity, IgG3 -fraction Gp: 36.36% sensitivity and 100% specificity, IgG4 - fraction Gp: 86.36% sensitivity and 100% specificity. Forty-seven CSF samples, 16 from patients with NC and 31 from patients with other neurological disorders, were assayed for IgE, with the following results: IgE-VF and IgE-fraction Gp: 93.75% sensitivity and 100% specificity. Fifty-seven serum samples, 22 from patients with NC, 18 from patients with other infections and 17 from presumably healthy individuals, were assayed for IgG, IgG subclasses, IgE, IgA and IgM, with the following results: IgG-VF: 100% sensitivity and specificity, IgG1-VF: 86.36% sensitivity and 94.28% specificity, IgG2 -VF: 90.91% sensitivity and 97.14% specificity, IgG3 -VF: 86.36% sensitivity and 97.14% specificity, IgG4 -VF:100% sensitivity and specificity, IgG-fraction Gp: 95.45% sensitivity and 100% specificity, IgG1- fraction Gp: 63.64% sensitivity and 94.28% specificity, IgG2 -fraction Gp: 68.18% sensitivity and 97.14% specificity, IgG3 -fraction Gp: 54.54% sensitivity and 88.57% specificity, IgG4 - fraction Gp: 90.91% sensitivity and 100% specificity, IgE-VF: 90.91% sensitivity and 97.14% specificity, IgE-fraction Gp: 86.36% sensitivity and 100% specificity, IgA-VF: 54.54% sensitivity and 94.28% specificity, IgA-fraction Gp: 13.63% sensitivity and 100% specificity. No specific IgM antibodies were detected with VF and fraction Gp antigenic preparations. These results show that with the two antigenic preparations the highest positivity in CSF and serum samples was obtained for IgG and IgE antibodies, followed by positivity for IgA. No IgM antibodies were detected in serum samples assayed with VF and fraction Gp. With regard to IgG subclasses, IgG4 positivity and concentration in CSF and serum samples were higher than or equal to the other subclasses. ELISA reactions done with VF showed equal or higher sensitivities than those obtained with fraction Gp. Considering the complexity and high cost of obtaining fraction Gp, VF could be more suitable for detecting specific antibodies in CSF and serum samples from patients with NC.
Doutorado
Ciencias Biomedicas
Doutor em Ciências Médicas

IgA in IgA Nephropathy Lorna Layward IgA nephropathy is a common glomerulonephritis of unknown pathogenesis, characterised by the presence of IgA within the glomerular mesangium. The predominant subclass of deposited IgA is known to be of the subclass IgA1 and is, at least in part, polymeric. IgA subclass measurements showed that raised serum IgA levels were restricted to the IgAl subclass, and that increased IgA levels were only observed in the systemic (not mucosal) compartment of the IgA immune system. In vitro production of IgA by peripheral blood lymphocytes was increased, with a concomitant decrease in IgG production. The response to systemic challenge with tetanus toxoid demonstrated a bias towards serum IgA1 antibody production; an enhanced circulating antigen-specific B cell response; and a positive saliva response where none was observed in controls. Serum polymeric IgA antibody production in response to systemic antigen challenge was significantly elevated in IgA nephropathy. Patients with IgA nephropathy were more likely to have an IgG subclass antibody deficiency than controls, showing dysregulation of other isotypes besides IgA. The affinity of serum IgA antibodies produced was lower than controls, while IgG affinity was normal. The production of low affinity IgA antibodies may result in the formation of nephritogenic immune complexes and explain the predominance of IgA within the glomerular mesangium. Gut permeability was normal in IgA nephropathy, and the response to mucosal antigen challenge did not differ from controls except for a higher antigen-specific in vitro B cell response. These results suggest an enhanced overlap of circulating IgA immunocompetent IgA B cells between the two sites in IgA nephropathy, regardless of the route of antigen administration. These data show abnormalities of mainly systemic IgA lending support to the hypothesis that the source of IgA overproduction is the systemic rather than the mucosal compartment of the IgA immune system.

IgA nephropathy (IgAN) is a common glomerulonephritis characterised by deposition of IgA1 in the glomerular mesangium The underlying abnormality lies within the IgA system rather than the kidney, and modest irregularities of IgA biology have been described, but the mechanisms involved in IgA1 deposition and glomerular injury remain elusive. A few reports have suggested an abnormality of the carbohydrate component of IgA1 in IgAN. These studies sought to confirm and further characterise the putative glycosylation defect and to identify its biochemical basis. Lectin binding assays were developed and used to analyse the N- and O-linked glycans of IgA1 in IgAN and controls. No gross abnormality of N-glycosylation was detected in IgAN, though these studies were subject to technical limitations. IgA1 in IgAN displayed significantly increased binding to lectins with affinity for O-linked N-acetylgalactosamine (Ga1NAc) as compared to controls. One explanation for this finding is reduced terminal galactosylation of the O-linked sugars of IgA1. A novel assay was developed to measure the functional activity of alpha1,3 galactosyltransferase (alpha1,3GT), the enzyme responsible for O-galactosylation, in cell lysates. In IgAN, peripheral blood B cells appeared to show significantly lower alpha1,3GT activity than controls, and this was inversely proportional to Ga1NAc expression of serum IgA1 as measured by lectin binding. These studies confirm an abnormality of O-linked glycosylation of serum IgA1 in IgAN, which may be attributed to low B cell alpha1,3GT activity. Altered O-glycosylation of IgA1 may be relevant to the pathogenesis of IgAN.

L’immunoglobuline A (IgA) est l’immunoglobuline la plus abondamment synthétisée chez les mammifères. Ses propriétés ambivalentes l’impliquent non seulement dans des fonctions de protection contre les agents pathogènes mais aussi dans des phénomènes de tolérance immunitaire vis-à-vis des germes commensaux du microbiote. Toutefois, les IgA peuvent développer des propriétés pathogènes. Dans la première partie de mon travail de thèse, nous avons étudié les effets pathogènes de l’IgA. Les dépôts d’IgA sur le mésangium sont la caractéristique de l’IgAN. La physiopathologie de cette maladie est mal connue. L’hypothèse d’un défaut de glycosylation de l’IgA est souvent retenue ; ce défaut peut être la cause de sa polymérisation et de son antigénicité, il peut aussi favoriser le clivage du récepteur CD89. Nous avons analysé l’effet du défaut d’affinité de la région variable des IgA, de la substitution de la chaîne légère ainsi que de l’association des IgA à leur récepteur, le CD89 sur l’induction des lésions et le dysfonctionnement rénal chez quatre modèles murins différents générés au laboratoire et suivis pendant 12 mois. Nous avons également étudié les propriétés physico-chimiques des IgA de 28 patients ayant une dysglobulinémie et de 28 IgA produites par des hybridomes ; la relation entre ces propriétés et la capacité des IgA à se déposer a été observée. Dans une seconde partie, nous avons étudié l’aspect immunomodulateur et les propriétés antiinflammatoires conférées par l’IgA humaine surexprimée chez un modèle murin de lupus systémique (souris MRL/lpr). Dans la dernière partie du travail, nous avons contribué à la caractérisation d’un modèle de souris transgénique exprimant l’IgA de classe 2 et à l’étude de l’effet de signalisation médiée par cette IgA2 sur le développement des populations lymphocytaires. L’ensemble de ces travaux a montré l’effet pathogène des IgA naturelles ayant une faible affinité sur le développement de la néphropathie à IgA ; ainsi les analyses des IgA des patients et des hybridomes montrent que c’est la stabilité moléculaire de préférence au profil de glycosylation qui joue un rôle crucial dans leur capacité de dépôt. L’expression des IgA humaines chez les souris lupiques a considérablement prolongé leur durée de vie et a ralenti la survenue de l’auto-immunité et de l’atteinte rénale ce qui témoigne du rôle anti-inflammatoire des IgA. L’étude du modèle murin exprimant l’IgA2 humaine a montré que la signalisation via l’IgA2 joue un rôle inhibiteur sur le développement précoce de certaines sous-populations de cellules B. L’ensemble de ces résultats montrent la multitude d’effets de l’IgA lui permettant d’intervenir d’une part dans la pathogenèse d’une maladie complexe (l’IgAN) et d’autre part dans la protection de l’auto-immunité, témoignant de la complexité des interactions mises en jeu et du caractère régulateur de cette immunoglobuline
Immunoglobulin A (IgA) is the most synthetized immunoglobulin in mammals. IgA has ambivalent properties: it is implicated in the mechanisms of defense against pathogens but also in the immune tolerance of commensal microbiota. However, IgA can develop pathogenic properties. In the first part of my thesis, we studied the pathogenic effects of IgA. IgA deposits are the main characteristic of IgA nephropathy (IgAN). IgAN physiopathology is not yet clearly understood. The hypothesis of a glycosylation defect is strongly adapted. This defect can be due to IgA polymerization or antigenicity. It can also induce shedding of CD89 (IgA Fc receptor) or other factors. We studied the effect of variable region altered affinity, the light chain substitution and the association of IgA with CD89 on the development of kidney lesions and impairment of kidney function in four mouse models followed up during 12 months. In addition, we studied the physico-chemical properties of 28 IgA purified from patients with dysglobulinaemia and 28 chimeric IgA produced by hybridomas. The effect of these properties on the propensity of IgA for mesangial deposition was explored. In the second part, we studied the immunomodulatory and anti-inflammatory properties conferred by the overexpression of human IgA in a mouse model with systemic lupus (MRL/lpr model). In the last part, we contributed to the characterization of a transgenic mouse model producing IgA class 2 and to the study of the effect of IgA2-mediated signaling on B lymphocyte development. Altogether, obtained results show the pathogenic effect of low affinity-IgA on the development of IgA nephropathy. In addition, different analyses showed that molecular stability but not glycosylation profile is the determining factor for IgA deposition. On the other hand, IgA expression in lupus-prone mice extended their survival, delayed the onset of auto-immunity and ameliorated kidney functions in these animals which supports IgA anti-inflammatory properties. The study of IgA2-mediated signaling in the transgenic model showed the inhibitory effect of IgA2 on the early development of several B cell sub-populations. All of these results show the multiple effects of IgA which contribute on one hand to the pathogenesis of a complex disease (IgAN) and on the other hand to protection from autoimmunity, demonstrating the complexity of interactions and the regulatory character of this immunoglobulin

Orientador: Claudio Lucio Rossi
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A toxoplasmose, uma zoonose com ampla distribuição mundial, causada pelo parasita intracelular obrigatório Toxoplasma gondii, é geralmente adquirida por meio da ingestão de cistos ou oocistos viáveis do parasita, presentes, respectivamente, em carne crua ou mal cozida e no solo, alimento ou água contaminados com fezes de gatos infectados. A toxoplasmose pode ser altamente debilitante, e ocasionalmente fatal, em crianças infectadas no útero e em receptores de transplante. O diagnóstico de infecção aguda primária em mulheres grávidas é geralmente baseado em testes sorológicos, visto que, na grande maioria dos casos, a toxoplasmose não é reconhecida clinicamente. A longa persistência dos anticorpos IgM em algumas pessoas e a dificuldade para demonstrar soroconversão ou aumento significativo da concentração de anticorpos específicos, têm complicado a interpretação dos testes sorológicos, quando se suspeita de infecção aguda. Com relação à infecção toxoplámica em pacientes transplantados, em muitos casos o status sorológico do doador não é conhecido e a pesquisa periódica de anticorpos anti-T. gondii no receptor raramente é realizada. O objetivo do primeiro estudo foi determinar o valor da demonstração dos anticorpos IgA anti-T.gondii para o diagnóstico da fase aguda da infecção toxoplásmica. Nossos resultados mostraram que os anticorpos IgA são detectados com alta freqüência em amostras de soros obtidas de mulheres com evidência clínica e/ou sorológica de infecção toxoplásmica aguda. Entretanto, em 19% das mulheres apresentando persistência de anticorpos IgM e alto índice de avidez dos anticorpos IgG, anticorpos IgA anti-T. gondii foram detectados em amostras de soros coletadas mais de 9 meses após o início da infecção, indicando que esses anticorpos não podem ser considerados marcadores confiáveis de infecção aguda primária. No segundo estudo, nós relatamos o diagnóstico de infecção toxoplásmica primária em um paciente com mieloma múltiplo submetido a transplante alogênico não-mieloablativo de células hematopoiéticas, provenientes de doador com sorologia negativa para toxoplasmose. A resposta primária contra o T. gondii foi baseada na soroconversão dos anticorpos IgM, IgG e IgA. O paciente foi prontamente tratado e nenhuma complicação relacionada à toxoplasmose foi observada nos meses subseqüentes. Esse caso ressalta a necessidade da detecção dos anticorpos anti-T. gondii no doador e no receptor antes do transplante e a importância do monitoramento sorológico do receptor durante o seguimento pós-transplante
Abstract: Toxoplasmosis, a cosmopolitan zoonotic disease caused by the intracellular parasite Toxoplasma gondii, is usually acquired through the ingestion of viable parasite cysts or oocysts, present, respectively, in raw or undercooked meat and in soil, food or water contaminated with feces of infected cats. Toxoplasmosis can be highly debilitating and occasionally fatal in children infected in utero and in transplant recipients. The diagnosis of acute primary infection in pregnant women is usually based on serology, because in the great majority of cases primary infection is not recognized clinically. The sustained persistence, in some persons, of specific IgM antibodies and the difficulty in demonstrating seroconversion or a significant increase in specific antibody concentrations, have complicated the interpretation of serological tests when acute infection is suspected. With regard to toxoplasmic infection in transplant patients, in many cases the serological status of the donor is not known and the periodic research of anti-T. gondii antibodies in the receptor is rarely performed. In the first study, we investigated the usefulness of detecting anti-T. gondii IgA for the diagnosis of an acute acquired Toxoplasma infection. Our results showed that anti-T. gondii IgA antibodies are detected with a high frequency in serum samples obtained from women with clinical and/or serologic evidence of acute acquired Toxoplasma infection. However, in 19% of the women presenting a sustained IgM antibody response and a high IgG avidity index, anti-T. gondii IgA antibodies were detected in serum samples collected more than nine months after the beginning of infection, indicating that IgA cannot be considered a dependable marker for acute primary infection. In the second study, we report the diagnosis of a primary toxoplasmic infection in a patient with multiple myeloma following a non-myeloablative allogeneic transplant with hematopoietic stem cells from a donor with negative serology for toxoplasmosis. The primary response to T. gondii was supported by IgM, IgG and IgA seroconversion. The patient was promptly treated and there were no complications related to toxoplasmosis in the subsequent months. This case stresses the importance of detecting anti-T. gondii antibodies in the donor and in the recipient before transplantation, and of serologically monitoring the recipient during long-term follow-u
Mestrado
Ciencias Biomedicas
Mestre em Ciências Médicas

Existing immunoglobulin (Ig) tests only give a limited picture of the immunological response to food antigens. Furthermore, existing tests require large volumes of sample, over a limited number of foods, are not amenable to a high sample through-put system and the results are limited to normally just one immunoglobulin class. In order to investigate the global immune response towards food products we have developed the "all diet" microarray concept. The "all-diet food protein microarray contains extracts of over 400 food ingredients that cover most of the food products found in the UK. Using this system we have retrospectively determined food specific IgE, IgA, IgG and IgM from 17 well characterized sera. The results were analyzed by multivariate techniques and parametric methods. The proof-of-concept of the ''all diet microarray to investigate the relationships between food antigen specificity and multiple Ig type was demonstrated here. The novelity of this protein microarray is the use of arrayed food samples sequentially extracted with detergent and chaotropic agents. The array system possesses many advantages over traditional systems such as requirement of low sample volume, high sensitivity and a global view of the immune response. Notwithstanding these potential advantages to clinical practices, these benefits remain yet to be demonstrated. The development of the technique will allow further expansion into areas of research such as conjugation of the microarray with sensitized human basophils and also immunoglobulin binding to extracts of parasites.

El objetivo de esta Tesis es realizar un estudio retrospectivo de características clínicas histológicas de la nefropatía idiopática en una serie amplia de pacientes, aportar acerca de la historia de enfermedad y conocer qué factores pueden relacionarse con el de la enfermedad.

En ella se analiza la historia clínica y la biopsia renal de 100 pacientes, 77 adultos y 23 niños, diagnosticados de nefropatía entre septiembre de 1975 y diciembre de 1986 en el Servicio de Nefrología del Hospital Clínico de Barcelona. El diagnóstico se fundamenta en el hallazgo de depósitos aislados o predominantes de inmunoglobulina A en el mesangio glomerular. Se excluyen los pacientes con evidencia clínica o serológica de lupus eritematoso sistémico, enfermedad hepática crónica o púrpura de Schönlein-Henoch. Durante este período se efectúan un total de 1754 biopsias renales, 639 de las cuales son diagnosticadas de enfermedad glomerular primitiva (39,2%). Entre estas últimas biopsias, 112 reúnen los criterios de nefropatía IgA, lo que representa un 17,5 %, y se descartan 12 pacientes que no reúnen las condiciones requeridas en el presente estudio. El material histológico se obtiene por biopsia renal quirúrgica o percutánea y es procesado para microscopía óptica (MO) e inmunofluorescencia en todos los casos. Además se procesa para microscopia electrónica (ME) en 20 casos. Las biopsias se clasifican según el sistema propuesto por la OMS para las enfermedades glomerulares y que distingue 5 tipos histológicos: (I) Riñón normal, (II) Cambios menores, (III) GN segmentaria y focal, (IV) GN proliferativa difusa, y (V) GN esclerosante. Los datos se expresan utilizando media aritmética y la desviación estándar. En el análisis estadístico se emplea la "t" de Student, la prueba del X(2) y en ocasiones un método paramétrico. El deterioro de la función renal se valora mediante el coeficiente de regresión de los recíprocos de creatinina sérica (l/Cr) versus tiempo de evolución. La supervivencia renal se calcula con el método de las tablas de vida de Cutler y Ederer.

La serie de pacientes adultos consta de 77 casos, 57 varones y 20 mujeres (2,8/1). La edad de inicio 26 +/- 10 años (entre 15 y 50) y la edad al efectuar la biopsia 31 +/- 10 (entre 15 y 56). Existe un antecedente familiar de nefropatía en el 14,5% de los casos y 2 pacientes son hermanos. Las formas de presentación son: hematuria macroscópica en 37 pacientes (48%), proteinuria y/o microhematuria en 21 (27,3%), hipertensión arterial en 15 (19,5%), insuficiencia renal en 2 (2,6%), síndrome nefrítico agudo en 1 (1,3%) y síndrome nefrótico en 1 (1,3%). No se observan diferencias relacionadas con el sexo. En el momento de biopsia muestran proteinuria 70 pacientes (91%) y hematuria microscópica 62 de 69 (90%). Un total de 39 pacientes se hallan hipertensos (50,6%) y 11 presentan HTA maligna. Asimismo, 25 pacientes muestran una creatinina sérica (Cr) superior a 1,4 mg/dl y el aclaramiento de Cr es inferior a 70 ml/min en 29 de 62 pacientes. El nivel sérico de IgA se determina en 35 pacientes, siendo en 11 de ellos superior a 400 mg/dl (31,4%). Los niveles de C3 y C4 y la actividad CH(50) son normales y el antígeno HBsAb en todos los casos estudiados.

En la biopsia renal de pacientes adultos, las lesiones más constantes en MO son la proliferación (83%) y la expansión mesangial (100%). Las lesiones sobreañadidas (semilunas, adherencias y esclerosis) son frecuentes pero en general afectan un bajo porcentaje de glomérulos. Existe una asociación muy estrecha entre fibrosis intersticial, atrofia tubular y esclerosis glomerular (p=0,0005). La BR clasificada de tipo I: 0 casos (0%), tipo II: 13 casos (16,7%), tipo III: 35 (45,5%), tipo IV: 27 casos (35%) y tipo V: 2 (2,6%). En el examen por IF la IgA es constante y es la única inmunoglobulina presente en 35 casos (45,6%). La IgA tiene una localización mesangial en 55 casos y mesangial y capilar en los 22 restantes. Se detecta C3 en los glomérulos de 74 biopsias (96%) y fibrinógeno en 22 (28,6%). El examen por ME muestra depósitos en todos casos y depósitos subendoteliales en 5 de 13 casos.

La evolución es conocida en 67 pacientes adultos. La duración media del seguimiento ha sido de 3,7 +/- 3 años. Al final, 4 pacientes se hallan en remisión clínica (5,9%), 24 permanecen estables con función renal normal (35,8%), 4 desarrollan HTA no preexistente (5,9%) y los 35 restantes muestran una función renal deficiente, con Cr superior a 1,4 mg/dl (52,2%). En 18 se ha valorado la velocidad de insuficiencia renal calculando la pendiente de la línea de regresión de l/Cr sérica. En 5 es nula (inferior a 1 x 10(-3), en 6 muestra un lento deterioro (entre 1 y 8,3 x 10(-3) y en los 7 restante el deterioro es rápido (superior a 8,3 x 10(-3). Entre 44 pacientes con función renal normal en el momento de BR, 32 evolucionan de modo favorable y 12 de modo desfavorable. No se observan diferencias relacionadas con la edad o el sexo, pero son factores de mal pronóstico la ausencia de una historia recurrente y el hallazgo de proteinuria superior a 1 gr./24 h. Al comparar las lesiones histológicas, sólo es significativa una mayor incidencia de proliferación y de expansión mesiangial en los pacientes con evolución desfavorable. La probabilidad de supervivencia renal es del 93% a los 5 años, del 75% a los 10 años, del 70% a los 15 años y del 64% a los 20 años del inicio de los síntomas. La supervivencia renal es mejor en los pacientes con historia de hematuria recurrente, proteinuria inferior a 1g/24 h, hipertensión, con Cr sérica inferior a 1,4 mg/dl en el momento de la BR o con tipos histológicos II y III.

Asimismo se estudian 23 niños con nefropatía IgA (13 varones y 10 hembras) con una edad media de 10 +/- 3 años. La presentación común ha sido la hematuria macroscópica en 18 casos (78,3%), la cual tiene un carácter recurrente en 13 (56,7%). La nefropatía IgA muestra en la infancia un perfil clínico más benigno que en el adulto y caracterizado por la ausencia de HTA y de IR en el momento del diagnóstico. El patrón histológico es también menos severo, que se objetiva un tipo I en 4 casos (17,4%), un tipo Il en 7 (30,4%), un tipo lII en 9 (29,1%) y un tipo IV en sólo 3 (13%). Al final del seguimiento (media 4,2 +/-3 años) 6 niños se hallan en remisión (31.6%), 11 permanecen estables (57,9%) y sólo 2 muestran un discreto deterioro de la función renal (10,5 %).

Un total de 26 adultos con nefropatia IgA (33,8%) cursa con hematuria A recurrente (HR). La edad de inicio (21,3 +/- 2 años) y en el momento de la BR (28,4 +/- 8 años) es inferior la del resto de pacientes. El porcentaje de casos con proteinuria superior a 1 gr/ 24 h. (42%) y con Cr sérica superior1.4 mg/dl es menor. BR muestra en el 80% un tipo II o III y sólo en el 20% un tipo IV. Al final del seguimiento, la función renal es deficiente en el 27,3% de los casos con HR y sólo 3 pacientes se hallan en hemodiálisis. Sin embargo, cuando los pacientes con o sin HR se comparan para una edad media de 32 años, no hemos observado ninguna diferencia significativa en los niveles de Cr sérica, porcentaje de casos con Cr superior a 1,4 mg/dl o incidencia de hipertensión arterial.

Entre un total de 77 pacientes adultos, 11 muestran HTA maligna secundaria en el momento del diagnóstico (14,3%). Diez son varones y la edad media al efectuar la BR es 35,6 +/- 3 años. Ocho tienen una TA distólica igual o superior a 140 mm Hg. Cinco muestran muestran edema de papila y el resto retinopatía grado IIl. En 5 existe un antecedente de hematuria macroscópica. La proteinuria es superior a 1 g/24 h en todos los casos (media 4 +/-3g/24 h) y la Cr sérica varía entre 2,3 y 12 mg/dl (media 4,6 +/-3 mg/dl). La BR muestra una nefropatía IgA tipo III en 3 casos y tipo IV en los 8 restantes. Cuatro presentan necrosis fibrinoide y tres endoarteritis proliferativa. Todos reciben tratamiento hipotensor y sólo tres continúan hipertensos. Al final del seguimiento (media 18 +/- 12 meses), 7 se hallan en hemodiálisis. Estos 11 pacientes se comparan con 10 pacientes diagnosticados de HTA maligna "esencial" en el mismo período de tiempo y con una BR. Los hallazgos clínicos o analíticos no permiten el diagnóstico diferencial entre ambos grupos y se concluye que la BR es imprescindible para descartar la nefropatía IgA en los sujetos con HTA maligna.

Finalmente se destaca que la nefropatía IgA puede tener carácter familiar y que dos casos cursan con IRA reversible durante un brote de hematuria macroscópica.
A retrospective study of 100 patients with IgA nephropathy was undertaken and the histological patterns correlated with the clinical features at the time of biopsy and the prognosis of the disease. The study included 77 adul (57 males and 20 females). The mean age at apparent onset was 26 +/-10 years and the mean age biopsy was 31 +/-10 years. The common clinical presentations in adults macroscopic hematuria (48 %), asymptomatic microhematuria and proteinuria (27,3%) and hypertension (19,5%). At time of biopsy, proteinuria of more than 1 g/day as found in 54 cases (70,2%), hypertension in 39 cases (50,6%) and impaired renal function, with serum creatinine of more than 1,4 mg/dl in 25 cases (32,5%). Malignant HTA was observed in 11 cases. The WHO morphological criteria used in classifying IgA neoprhaty showed zero patients (0%) with Class I disease (normal kidney),13 patients (16,1%) with Class II (minor changes), 35 patients (45,5%) with Class III (focal and segmental glomerulonephritis), 27 patients (35%) with Class IV (diffuse proliferative glomerulonephritis) and 2 patients (2,6%) with Class V (sclerosing glomerulonephritis). At the final examination, after a follow-up period of 3,7 +/- 3 years, 35 patients (52,2%) had impaired renal function. 14 of these patients went into terminal failure. Among patients with normal renal function at time of biopsy, progression to renal failure was more common in patients with 1) no history of recurrent gross hematuria, 2) proteinuria of than 1 g/day and 3) significant degree of enlargement and proliferation. The actuarial renal survival rate at year 10 after the onset was 75%, and at year 20 was 64%. Patients recurrent gross hematuria were younger, showed a low frequency of hypertension and impaired renal function at time of biopsy and had a more benign course. Malignant HTA in IgA nephropathy is commoner than previously reported and it presents in a fashion similar to that of malignant essential HTA. Therefore, given a case of malignant HTA, it is difficult to rule out IgA nephropathy if a renal biopsy is not performed. A comparative study of 23 children (mean age at biopsy 10 +/-3 years) was made. Children exhibited less severe histological picture and had a better prognosis than adults.

IgA nephropathy (IgAN) is the commonest primary glomerulonephritis worldwide, with 20-40% of patients developing progressive kidney disease. The most accurate predictors of prognosis are the presence of proteinuria and tubulointerstitial fibrosis, while the degree of mesangial IgA deposition is not a prognostic factor. These findings imply that tubular-specific factors play a key role in progressive IgAN. The aim of this thesis was to explore whether filtered IgA has a direct effect on proximal tubular epithelial cell (PTEC) activation and generation of pro-inflammatory and pro-fibrotic cytokines. The interaction between IgA and PTEC was initially investigated in vivo in Munich Wistar Frömter rats by multiphoton microscopy. These studies demonstrated that IgA, that crossed the glomerular filtration barrier, interacted with PTEC and underwent endocytosis via their apical surface. This process was greatly upregulated in a model of podocyte injury, resulting in increased amounts of filtered IgA. In vitro, human IgA1, and especially galactose-deficient polymeric IgA1, stimulated release of pro-inflammatory and pro-fibrotic cytokines from cultured human HK-2 PTEC. A mouse model of IgAN was optimised that developed both glomerular and tubulointerstitial inflammatory cell infiltration. Although glomerular deposition of complement component C3 was increased in the model, mice genetically deficient in key initiators of the lectin pathway, Collectin-11 (CL-11) or Mannose-binding lectin-associated serine protease-2 (MASP-2), were not protected from interstitial macrophage infiltration, while reductions in glomerular cell number and T cell infiltration were observed. These studies provide evidence for the first time that filtered IgA is able to interact with the proximal tubule and undergo endocytosis. IgA1, and especially galactosedeficient polymeric IgA1, stimulated a pro-inflammatory and pro-fibrotic response from PTEC that may contribute towards progressive IgAN. Understanding this interaction further may reveal novel targets for therapy in this condition. Deficiencies in CL-11 and MASP-2 did not protect against tubulointerstitial inflammation in a mouse model of IgAN, and further studies should concentrate on whether the alternative pathway is activated in this model.

To improve diagnosis of Toxoplasma gondii in difficult patient groups, IgA- and IgE-immunosorbent agglutination assays (ISAGA), IgG avidity and Western blotting were developed and assessed. In the ISAGA, rabbit anti-human IgA or anti-human IgE (for the IgA-ISAGA and IgE-ISAGA respectively) was adsorbed onto microtitre plates; formaldehyde fixed tachyzoites were used to identify specific antibody. Both ISAGAs were specific; only 1/482 (0.2%) and 1/513 (0.19%) false positive results were recorded for the IgA and IgE-ISAGA respectively. Both were produced early in infection and were detected for up to 11 (IgA) and 10 (IgA) months. In the avidity ELISA, the performance of excretory/secretory, surface, cytoplasmic and mixed antigen was similar when tested with sera from patients with known duration of infection. Thirteen pregnant women were tested. Specific IgA was detected in all and so was not useful to time infection but specific IgE was absent in 5 women and may therefore have been useful to exclude recent infection. Specific IgE however, was not detected in one woman who seroconverted; detection of IgE may indicate severity of infection. High IgG avidity was measured in 4 patients and could have been useful in conjunction with IgE to exclude recent infection. In immunocompromised patients, IgA and IgE were detected with similar frequency to IgM, but their presence in some IgM negative patients makes them a useful addition to the repertoire of testing. High avidity in immunocompromised patients was not of use to improve diagnosis. Using Western blotting it was possible to differentiate between acute (< 4 months) and recent (4-10 months) infection. This test was less useful for timing infection of infection in pregnancy.

La néphropathie à IgA (NIgA), une cause majeure d'insuffisance rénale dans le monde, est caractérisée par une pathogenèse complexe, incluant des facteurs inconnus favorisant la formation des complexes d'IgAl dans la circulation et des dépôts de ces complexes dans le mésangium. Des anomalies des récepteurs aux IgA sont impliquées: de complexes circulants d'IgAl avec la forme soluble (s) du récepteur myéloïde CD89 et la surexpression du récepteur mesangial aux IgAl le TfRl (récepteur de la transferrine 1). Dans la première partie de cette thèse, en utilisant un nouveau model murin de NIgA exprimant à la fois PIgAl et le CD89 humains, nous avons démontré que les complexe IgAl-CD89s déclenchent une procédure pathogénique incluant la surexpression de TfRl et de l'enzyme transglutaminase2 (TGase2), permettant des dépôts mésangiaux de complexes d'IgAl et l'activation cellulaire. Des antigènes alimentaires, notamment le gluten, ont été associés avec la NIgA sans que le mécanisme soit connu. Avec u modèle murin innovant: des souris sensibilisées au gluten, développé dans la deuxième partie de cette étude, démontrant le rôle des IgA, TGase2, TfRl et de microbiotes intestinaux dans la maladie cœliaque induits par gluten, j'ai conclu cette thèse par une troisième partie axée sur l'effet du gluten dans la NIgA. J'ai montré que le gluten associé au CD89, est impliqué dans la formation des complexes IgAl-CD89s et dans la réponse mucosale IgAl exacerbée, induisant le développement de la NIgA. Des marqueurs cruciaux: CD89, TfRl, TGase2 et gluten, émergent de cette thèse, corrèlant la NIgA avec la maladie cœliaque et offrant de nouvelles cibles thérapeutiques aux deux pathologies
IgA nephropathy (IgAN), a major cause of renal failure Worldwide, is characterized by a complex pathogenesis, which consists of unknown factors favoring formation of macromolecular IgAl complexes in the circulation and complex deposition in the mesangium. IgA receptor abnormalities are implicated, including circulating complexes of IgAl with the soluble (s) form of the myeloid receptor CD89 and over-expression of the mesangial IgAl receptor TfRl (transferrin receptor 1). In the first part of this thesis, using a new IgAN mouse model expressing both human IgAl and CD89, w demonstrated that IgAl-sCD89 complexes initiated a process of auto-amplification involving over-expression of TfR and the cross-linking enzyme transglutaminase2 (TGase2), allowing increased mesangial deposition of pathogenic IgA complexes and chronic mesangial cell activation. Food antigens, notably gluten, are also associated with IgAN onset, bi the mechanism is still unknown. Inspired by a novel model of gluten-sensitized mice developed in the second part of this study showing the role of IgA, TGase2, TfRl and the intestinal microbiota in the induction of immune responses an coeliac-like disease induced by gluten, I concluded this thesis with a third part focused on the effect of gluten in IgA1 using the same sensitization method. I showed that gluten in association with CD89, was implicated in IgAl-sCD8 complex formation and exacerbating IgAl mucosal response, resulting in a breakdown of oral tolerance and IgA1 development. Crucial players: CD89, TfRl, TGase2 and gluten, emerge from this thesis, affecting both IgAN and coeliac disease and proposing new therapeutic targets for both pathologies

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Essa dissertação é composta de duas pesquisas complementares, realizadas no período de primeiro de janeiro de 2004 a 30 de setembro de 2005. No primeiro estudo, realizou-se a sorologia para IgG e IgM anti-toxoplasma no sangue do cordão de 1514 RN pela técnica MEIA e a comparação entre os resultados dos anticorpos IgG e IgM no sangue de cordão e periférico de 167 RN (86 suspeitos e 81 normais). A sorologia por MEIA permitiu que fossem selecionados 86 RN suspeitos de toxoplasmose congênita, cujas amostras foram testadas para detecção de IgM pela técnica ELFA. A comparação entre a sorologia para IgG e IgM por MEIA mostrou não haver diferença significativa entre os resultados obtidos no sangue de cordão e periférico. A triagem sorológica dos 1514 RN pela técnica MEIA revelou que: 0,59% (09/1514) apresentavam IgG e IgM reagentes; 64,60 (978/1514) apresentavam IgG reagente e IgM não reagente; 0,46% (7/1514) apresentavam IgG indeterminada e IgM não reagente e 34,35% (520/1514) apresentaram IgG e IgM não reagentes. A incidência da toxoplasmose diagnosticada pela presença de IgM pelas técnicas MEIA e ELFA foi de 6,6/1000 nascimentos, contudo essa incidência não reflete o número de RN infectados, pois muitos RN não produzem anticorpos de classe IgM ao nascer, sendo necessário que os 76 RN suspeitos e que tiveram IgM não reagentes sejam acompanhados até dois anos de idade. O segundo estudo foi realizado nas crianças suspeitas de toxoplasmose congênita acompanhadas no Ambulatório de Infecções Congênitas do HC. Das 86 encaminhadas para acompanhamento, apenas 56 retornaram para consulta. As amostras dessas crianças foram testadas para IgM anti T.gondii pelas técnicas MEIA, ELFA e IFI e para IgA por ELISA captura. O diagnóstico da infecção congênita foi concluído em 44 RN, sendo que 28 estavam infectados e 16 não estavam. Dos 28 infectados, 42,9% (12/28) apresentaram IgM reagente pelas técnicas usadas. A sensibilidade, 84 especificidade, acurácia, valores preditivos positivo e negativo das técnicas MEIA e ELFA foram iguais, respectivamente de 36,7%, 100%, 100%, 47,1% e 59,1%; da IFI 28,6%, 87,5%, 80,0%, 44,4% e 50% e da IgA de 25,0%, 100%, 100%, 43,2% e 52,3%. A IgM foi reagente em 81,8% (9/11) das crianças sintomáticas, demonstrando sua relação com a gravidade da transmissão vertical, com maiores concentrações em crianças mais afetadas pelo processo infeccioso intra-uterino. Por outro lado, não foi detectada em 57,1% (16/28) dos infectados, provavelmente em conseqüência do tratamento da mãe. A sensibilidade da IgM anti T.gondii, associando três técnicas (MEIA, ELFA e IFI) foi de 42,9% (12/28) e da IgA foi de 25% (7/28), mostrando que a suspeita de toxoplasmose congênita não pode ser afastada apenas pela ausência desses anticorpos.

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Introdução: O diagnóstico rápido, simples e preciso para confirmar a infecção pelo vírus dengue (DENV) é uma necessidade real, uma vez que a doença pode se manifestar com um amplo espectro de sinais e sintomas, similares a outros quadros febris agudos. Durante a infecção por dengue se verifica também a produção da imunoglobulina A (IgA) específica, que aumenta ao mesmo tempo que a imunoglobulina M (IgM), permanece positiva por um período de tempo mais curto e se apresenta em níveis mais elevados na infecção secundária. Objetivo: O presente estudo teve como objetivo investigar a presença de IgA no soro durante a infecção primária e secundária (sequencial) pelo DENV. Metodologia: Foram avaliadas amostras de soro por meio do teste imunoenzimático de captura da IgA (AAC-ELISA) in house. Resultados: Avaliou-se um total de 445 amostras de soro, sendo 171 caracterizados como infecção primária e 194 secundária; 40 amostras de indivíduos saudáveis negativos para dengue e 40 de vacinados contra febre amarela. As amostras foram distribuídas em 13 grupos. A positividade da IgA foi de 42,2% (154/365), sendo 27,5% (47/171) na infecção primária e 55,2% (107/194) na secundária. Na infecção secundária, a IgA foi detectada do 2º ao 4º dias de sintomas (grupo 1), antes mesmo da IgM, assim como no grupo 11 no qual a IgM não havia sido detectada (infecção secundária). Na infecção primária o maior valor da sensibilidade foi de 60,0 (36,4 - 80,0) no grupo com 30-35 dias de sintomas e na secundária foi de 87,5% (60,4 – 97,8), grupo com 8 dias de sintomas. A especificidade foi de 100% nas duas infecções (94,3 – 100). Ao aplicar o teste em paralelo para ambas técnicas observou-se um aumento global de 6,6% na sensibilidade do diagnóstico; sendo 2,7% para a infecção primária e de 15,2% para a secundária. A IgA não foi detectada nas amostras dos indivíduos saudáveis, nem nas amostras dos indivíduos recentemente vacinados contra febre amarela. Conclusões: A detecção da IgA demonstrou ser útil como forma de diagnóstico sorológico e em conjunto com a detecção da IgM poderá auxiliar na confirmação de casos agudos de dengue e na interpretação dos resultados de casos inconclusivos, permitindo a adoção de medidas preventivas para evitar a ocorrência de epidemias e ocorrência de casos graves e óbitos.

Com o objetivo de avaliar as imunidades humoral e celular de pacientes com endometriose, analisamos as concentrações das Imunoglobulinas ( A, G e M ) e das Interleucinas (1β, 10 e IFγ) no soro e no líquido peritonial. Também analisamos a sua associação com a presença de infertilidade, o nível de dor apresentado e a extensão da endometriose. Foram coletadas amostras de soro e líquido peritonial de 43 pacientes submetidas a laparoscopia, que foram divididas em 4 grupos : 20 pacientes inférteis com endometriose, 9 pacientes inférteis sem endometriose, 4 pacientes férteis com endometriose e 10 pacientes férteis sem endometriose. As concentrações de IL-1β, IL-10 e IFγ foram determinadas pela método de ELISA e as concentrações de IgA, IgG e IgM foram feitas pela técnica de imunodifusão radial. Nas pacientes com endometriose, as concentrações de IgA no soro e do líquido peritonial foram maiores nas pacientes inférteis do que nas férteis. A IgG estava mais elevada nas pacientes sem endometriose do que nas pacientes com endometriose, todas inférteis. Os níveis de interleucina 10 no líquido peritonial de pacientes com endometriose foram mais elevados nas pacientes inférteis do que nas férteis e Interferon γ apresentou concentrações mais baixas no soro e no líquido peritonial de pacientes inférteis sem endometriose. Não houve diferenças significativas entre os grupos para as concentrações de IgM e IL-1β, bem como nas correlações entre os níveis de citocinas e imunoglobulinas entre os diferentes grupos com os graus de endometriose e os níveis de dor nessas pacientes.

La nephropathie a iga est la glomerulonephrite la plus frequente dans tous les pays sauf l'afrique. Les signes d'atteinte renale sont etroitement lies a des episodes d'infections orl a repetition (angines, rhinopharyngites). Les iga sont par ailleurs l'isotype predominant produit par les tissus lymphoides associes aux muqueuses (malt). L'auteur a recherche les alterations possibles du malt chez les patients atteints de nephropathie a iga. Cette these rassemble les travaux de plusieurs annees sur cette thematique et comporte deux parties. Dans la premiere, les contributions personnelles sont replacees dans le contexte d'une large revue de la litterature internationale sur la nephropathie a iga. La seconde partie rassemble les textes des publications internationales nationales et locales de l'auteur sur ce sujet. La conclusion principale de ce travail est qu'il existe effectivement des anomalies du malt, au niveau des amygdales et d'autres territoires muqueux dans la nephropathie a iga. Ces alterations peuvent contribuer au developpement et a l'evolution de la maladie

Background. Antiphospholipid (aPL) antibodies are a heterogeneous group of autoantibodies directed against plasma protein-phospholipid complexes or single plasma proteins. Their presence in patients with thrombosis and/or pregnancy morbidity defines antiphospholipid syndrome (APS). APS is considered as primary (PAPS) if present alone, or secondary if associated with other systemic autoimmune diseases, particularly with systemic lupus erythematosus (SLE). Laboratory criteria for APS classification include lupus anticoagulant (LA) and/or medium-high levels of IgG/IgM anticardiolipin (aCL) and/or medium-high levels of IgG/IgM anti-Beta2glycoprotein I (anti-Beta2GPI) antibodies, all confirmed no earlier than 12 weeks later. Currently, ELISA assay for detection of aCL and anti-Beta2GPI antibodies, despite several attempts, is not a standardized technique. Recently, few studies have compared the performance of ELISA with that of other technologies also fully automated including the fluorescence enzyme immunoassay (FEIA) and the chemiluminescence immunoassays (CLIA), but they have produced debatable results. The search for new markers of APS through the use of laboratory techniques alternatives to ELISA, such as FEIA and CLIA methods is currently under interest. IgA aCL and IgA anti-Beta2GPI antibodies are not considered one of the recommended laboratory criteria for APS classification, and their clinical relevance is as yet controversial. Moreover, aPL are not only considered as a tool for APS classification, but they could be useful parameters to stratify the risk for developing clinical manifestations of the disease. In particular, anti-anti-Beta2GPI antibodies directed against the Domain I (anti-DI) of the molecule, were reported to be associated to thrombotic risk in antiphospholipid syndrome (APS), so correlating them with a more severe clinical picture. Objectives. The aim of the study was to compare the performance of a home-made ELISA with that of FEIA and CLIA assays in detecting aCL and anti-Beta2GPI antibodies in a large, homogeneous cohort of PAPS patients and in a group of subjects with clinical criteria for APS classification but ELISA negative for laboratory criteria. The results were compared with those obtained in a control group including healthy blood donors and patients with autoimmune diseases different from APS. Subsequently, the clinical relevance of IgA aCL, IgA anti- Beta2GPI antibodies and of IgG anti-DI antibodies was evaluated in a large homogeneous cohort of PAPS patients. Their diagnostic sensitivity was investigated in a group of seronegative patients for conventional aPL but with clinical manifestations of APS. Moreover, prognostic value of these antibodies in APS patients was studied. Methods. IgG/IgM/IgA aCL and IgG/IgM/IgA anti-Beta2GPI antibodies were determined using FEIA, (EliA TM, Phadia AB, Sweden). aCL/anti-Beta2GPI of IgG isotype and IgG anti-DI antibodies were assayed using CLIA (HemosIL AcuStar®, Inova, USA). The manufacturer's recommendations were followed carefully for both techniques. A home-made ELISA performed following the European Forum on aPL recommendations was used for the comparison between methods. All sera were also tested for LA following updated guidelines using diluted Russell viper venom time and diluted activated partial thromboplastin time as screening tests. Results and Conclusions. (1) Comparison between an ELISA home made and FEIA technique. The sensitivities of the ELISA and FEIA tecniques were similar with the exception of IgM aCL which was found to be significantly higher in the PAPS patients using the ELISA method. The two assays had a comparable specificity, a high significant agreement and a significant correlation between the antibody levels. FEIA testing uncovered no significant prevalence of any antiphospholipid antibody in the ELISA negative patients. In conclusion, our results suggest that FEIA tecnique is comparable to a home-made ELISA. If confirmed by large scale studies on PAPS patients, these results could support FEIA's routine use in detecting aCL and anti-Beta2GPI antibodies. Results and Conclusions. (2) Comparison between an ELISA home made and CLIA technique. When compared with the ELISA technique, it came to light that the CLIA method had a significantly lower sensitivity for IgM aCL and IgG/IgM anti-Beta2GPI antibodies; while, its specificity was higher with respect to ELISA for IgM aCL and IgM anti-Beta2GPI antibodies. The two techniques showed a high, significant agreement and a significant antibody titer correlation. CLIA also detected IgG/IgM aCL and IgG anti-Beta2GPI antibodies in the seronegative patients using ELISA method. There was a significantly higher prevalence of IgG aCL and IgG anti- Beta2GPI antibodies in those patients with respect to that in the control population. In conclusion, despite a lower sensitivity, CLIA showed a higher specificity for some aPL and a good level of agreement and correlation with a home made ELISA. CLIA also detected some aCL and anti-Beta2GPI antibodies in the seronegative patients not usually identified by home made ELISA. If confirmed by further studies, CLIA could be considered a valuable method to assess patients with clinical manifestations of APS but testing negative for aPL using a home made ELISA. Results and Conclusions. (3) ACL and anti-Beta2GPI antibodies of IgA isotype. Present respectively in 19% and 50% of the PAPS patients studied, IgA aCL and IgA anti- Beta2GPI antibody frequencies were both statistically significant .The mean titers of both IgA aCL and IgA anti-Beta2GPI antibodies were higher in the thrombotic patients, but only the latter were significantly associated with thrombosis. When analyzed, the patients FEIA negative for conventional IgG/IgM aPL, but with the clinical features of APS, in 10.6% of cases were tested positive for anti-Beta2GPI IgA, this data was found to be significant. In conclusion, positivity to IgA anti-Beta2GPI antibody detected using FEIA was found to be clinically relevant in PAPS patients. Moreover, the prevalence of isolated IgA anti- Beta2GPI antibody positivity was significant in the seronegative patients. These results suggest that patients with clinical signs/symptoms of APS but who do not meet conventional antiphospholipid antibody laboratory criteria could undergo at least of IgA anti-Beta2GPI antibody testing using FEIA technique. Results and Conclusions. (4) IgG anti-DI antibodies. The sensitivity and specificity of IgG anti-DI antibodies were comparable to those of IgG aCL and IgG anti-Beta2GPI antibodies. There was a significant agreement, association and antibody titre correlation between IgG anti-DI and IgG aCL as well as IgG anti-Beta2GPI antibodies. IgG anti-DI antibody showed lesser prevalence and mean titres in the pregnancy morbidity than in thrombotic and PAPS patients with both involvements. Regarding the conventional aPL antibody profiles, the triple positivity group had higher prevalence and mean titres than single and double positivity ones. In conclusion, as regards the anti-DI antibodies this study provides further evidence that these antibodies detected by CLIA, can be considered a promising biomarker for risk assessment particularly in patients having vascular thrombosis and triple conventional aPL positivity, which is considered an antibody profile associated to the most severe features of APS. Thus, anti-DI antibodies might constitute an additional useful tool in clinical and therapeutic decisions.
Introduzione. Gli anticorpi antifosfolipidi (aPL) sono un gruppo eterogeneo di autoanticorpi specifici per complessi fosfolipide-proteina o proteine leganti i fosfolipidi. La loro presenza in pazienti con trombosi e/o complicanze ostetriche definisce la sindrome da anticorpi antifosfolipidi (APS). L'APS viene considerata primaria (PAPS) se presente in forma isolata, altrimenti secondaria se associata ad altra malattia autoimmune sistemica che solitamente e' il lupus eritematosus sistemico (LES). I criteri di laboratorio per la classificazione di APS includono la presenza di tre aPL ed in particolare del lupus anticoagulant (LA) e/o di livelli medio-alti di anticorpi anticardiolipina (aCL) IgG/IgM e/o di livelli medio-alti di anticorpi anti-Beta2glycoproteina I (anti-Beta2GPI) IgG/IgM, tutti confermati non prima di 12 settimane. Attualmente, le metodiche ELISA per la determinazione degli aCL e anti-Beta2GPI di classe IgG/IgM, nonostante svariati tentativi, non sono ancora standardizzate. Di recente, alcuni studi hanno confrontato le performance dei test ELISA con quelle di altre tecnologie anche completamente automatizzate tra le quali rientrano sia il fluorescence enzyme immunoassay (FEIA) che il chemiluminescence immunoassay (CLIA). Questi lavori, tuttavia, hanno prodotto risultati non concordanti. Gli anticorpi aCL e anti-Beta2GPI di classe IgA non sono ancora considerati criterio di laboratorio per la classificazione dell'APS e la loro rilevanza clinica e' al momento oggetto di dibattito. Inoltre gli aPL non sono considerati soltanto strumenti di classificazione dell'APS, ma anche parametri per la stratificazione del rischio di sviluppare le manifestazioni cliniche della malattia. In particolare, gli anticorpi anti-Beta2GPI specifici per un preciso epitopo situato nel Dominio I della molecola sembrano essere associati maggiormente al rischio di trombosi piuttosto che all'impegno ostetrico e di conseguenza sarebbero correlati a un quadro clinico piu' severo dell'APS. Obiettivi. Lo scopo della tesi e' stato di confrontare la performance di un ELISA home made con quella delle tecniche FEIA e CLIA nel rilevamento degli anticorpi aCL IgG/IgM e anti-Beta2GPI IgG/IgM in un'ampia e omogenea coorte di pazienti affetti da sindrome da anticorpi antifosfolipidi primaria (PAPS) e in un gruppo di soggetti con i criteri clinici per la classificazione di APS ma ELISA negativi per i criteri di laboratorio. I risultati sono stati confrontati con quelli ottenuti in un gruppo di controllo comprendente donatori sani e pazienti con malattie autoimmuni diverse dall'APS. Successivamente e' stata valutata la rilevanza clinica degli aCL e anti-Beta2GPI di classe IgA e degli anticorpi anti-Dominio I (anti-DI) di isotipo IgG in un'ampia ed omogenea coorte di pazienti affetti esclusivamente da PAPS. Inoltre, la sensibilita'  diagnostica di questi anticorpi e' stata valutata in un gruppo di pazienti sieronegativi per gli aPL convenzionali, ma con manifestazioni cliniche di APS. Di entrambi gli anticorpi e' stato anche indagato il valore prognostico nell'ambito dell'APS. Metodi. Gli aCL e gli anti-Beta2GPI di classe IgG/IgM/IgA sono stati determinati usando il metodo FEIA (EliA TM, Phadia AB, Sweden). Inoltre gli aCL e anti-Beta2GPI di classe IgG/IgM sono stati anche analizzati assieme gli anticorpi anti-DI IgG utilizzando il metodo CLIA (HemosIL AcuStar®). Le raccomandazioni del produttore sono state seguite scrupolosamente per entrambe le tecniche. Per il confronto dei risultati ottenuti con le diverse metodiche e' stato usato un test ELISA home made, eseguito seguendo le raccomandazioni del Forum europeo sugli aPL. Tutti i sieri sono stati testati anche per LA seguendo le linee guida aggiornate utilizzando il tempo di veleno di vipera Russell ed il tempo di protrombina parziale attivata, entrambi con fosfolipidi diluiti, come tests di screening. Risultati e Conclusioni. (1) Confronto ELISA home made con FEIA. Le sensibilita'  delle tecniche ELISA home made e FEIA sono risultate essere simili ad eccezione degli aCL di classe IgM che sono risultati significativamente piu' frequenti nei pazienti PAPS con il metodo ELISA. I due metodi avevano una specificita' simile, un'elevata concordanza e una correlazione significativa tra i livelli anticorpali. Inoltre il metodo FEIA non ha rilevato alcuna significativa prevalenza degli anticorpi antifosfolipidi nei pazienti ELISA negativi, ma con manifestazioni cliniche di APS. In conclusione, questi risultati suggeriscono che il metodo FEIA e' paragonabile al test ELISA home made. Se confermato da altri studi su ampie casistiche di pazienti affetti da PAPS, questi risultati potrebbero supportare l'uso del FEIA nella determinazione degli aCL e anti-Beta2GPI nell'analisi di routine. Risultati e Conclusioni. (2) Confronto ELISA home made con CLIA. Quando e' stata confrontata la tecnica ELISA home made con la tecnica CLIA, e' emerso che il metodo CLIA aveva una sensibilita' significativamente piu' bassa per gli aCL IgM e gli anti-Beta2GPI IgG/IgM rispetto a quella dell'ELISA; invece la sua specificita' e' risultata significativamente piu' alta per gli anticorpi aCL IgM e anti-Beta2GPI IgM. Le due tecniche hanno mostrato un'alta e significativa concordanza e una significativa correlazione dei titoli anticorpali. Inoltre il CLIA ha rilevato gli anticorpi aCL IgG/IgM e anti-Beta2GPI IgG nei pazienti sieronegativi in ELISA. Vi era infatti una prevalenza di aCL IgG e di anti-Beta2GPI IgG significativamente maggiore nei pazienti sieronegativi con i criteri clinici di APS che nella popolazione sana di controllo. In conclusione, il metodo CLIA, nonostante una minore sensibilita', ha mostrato una specificita' piu' alta per alcuni aPL e un buon livello di concordanza e di correlazione con la metodica ELISA home made. Il CLIA, inoltre, e' stato in grado di rilevare gli aCL IgG e gli anti-Beta2GPI IgG nei pazienti sieronegativi non identificati dall'ELISA. Se confermato da ulteriori studi, il CLIA potrebbe essere considerato un metodo valido per la valutazione di pazienti con manifestazioni cliniche di APS, ma con gli aPL negativi al test ELISA home made. Risultati e Conclusioni. (3) Gli aCl e gli anti-Beta2GPI di classe IgA: Gli aCL e gli anti-Beta2GPI di classe IgA sono stati testati con il metodo FEIA e sono risultati sgnificativamente presenti rispettivamente nel 19% e nel 50% dei pazienti affetti da PAPS. I loro titoli medi erano piu' elevati nei pazienti con impegno trombotico rispetto alle pazienti con impegno ostetrico. Tuttavia solo gli anti-Beta2GPI IgA erano significativamente associati alla trombosi. Quando sono stati analizzati i pazienti FEIA negativi per aCL IgG/IgM e per anti-Beta2GPI IgG/IgM ma con le caratteristiche cliniche di APS, nel 10,6% di essi sono stati trovati gli anticorpi anti-Beta2GPI IgA. Questo dato e' risultato essere significativo nel confronto con la popolazione sana di controllo. In conclusione, la positivita' per gli anticorpi anti-Beta2GPI IgA definita con il metodo FEIA e' risultata clinicamente rilevante nei pazienti PAPS. Inoltre la presenza di anticorpi anti-Beta2GPI IgA era significativa nei pazienti sieronegativi per gli isotipi IgG e IgM. Questi risultati suggeriscono che nei pazienti con segni/sintomi clinici di APS, ma che non soddisfano i criteri di laboratorio per gli anticorpi antifosfolipidi convenzionali si potrebbero determinare gli anticorpi anti-Beta2GPI di classe IgA al fine di incrementare la sensibilita'  diagnostica per APS. Risultati e Conclusioni. (4) Gli anti-DI IgG: La sensibilita' e la specificita' degli anticorpi anti-DI IgG rilevati con il metodo CLIA erano paragonabili a quelle degli anticorpi aCL IgG e anti-Beta2GPI IgG. Si e' riscontrata una significativa concordanza, un'associazione e una correlazione dei titoli anticorpali degli anti-DI IgG con gli aCL IgG e gli anti-Beta2GPI IgG. Inoltre gli anticorpi anti-DI IgG hanno mostrato una minore prevalenza e titoli medi piu' bassi nelle complicanze ostetriche rispetto ai pazienti con trombosi e ai pazienti con entrambi i coinvolgimenti clinici. Per quanto riguarda i profili anticorpali degli aPL convenzionali, il gruppo con la triplice positivita' antifosfolipidica ha mostrato una maggiore prevalenza e maggior titoli medi degli anticorpi anti-DI, rispetto ai gruppi con singola e duplice positivita'. In conclusione, gli anti-DI, rilevati con la tecnica CLIA, possono essere considerati dei promettenti biomarkers per la valutazione del rischio clinico di trombosi vascolare e di triplice positivita' per gli aPL convenzionali, solitamente associata ai quadri clinici piu' severi di APS. Pertanto, essi possono costituire uno strumento aggiuntivo utile per eventuali decisioni cliniche e terapeutiche.

Candida albicans is an opportunistic yeast pathogen and can cause life-threatening systemic candidiasis. The cell surface of C. albicans is enriched with mannan that is resistant to complement activation in the absence of antimannan antibodies. To better appreciate antimannan antibody functions in human immunity, our laboratory constructed the human recombinant antimannan Fc-free fragment M1 Fab. M1 Fab was subsequently converted to full-length human recombinant antimannan antibodies: M1g1 (IgG1), M1a1 (IgA1), M1a2 (IgA2). Each retains the identical M1 Fab binding region but differ in the isotype. Previously, our laboratory has established that M1 Fab can increase C3b deposition to C. albicans via the alternative pathway and that M1g1 activates the alternative and classical pathways of complement and increases phagocytosis of C. albicans by murine macrophages. The purpose of this study was to assess the influence of M1a1 and M1a2 on M1g1 mediated complement activation and phagocytosis of C. albicans. M1a1 or M1a2 was found unable to promote C3b-deposition to C. albicans as determined by flow cytometry and immunofluorescence microscopy. The formation of the alternative pathway convertase on C. albicans was promoted by M1 Fab but not by M1-Fab contained within M1a1 or M1a2. Additionally, M1g1 mediated C3b deposition was inhibited by M1a1 or M1a2 in a dose-dependent manner. Finally, M1a1 or M1a2 each significantly increased phagocytosis of C. albicans (P <0.001) by human neutrophils independent of serum. The presence of M1a1 or M1a2 did not inhibit M1g1-mediated phagocytosis, indicating a redundant function of IgG1 and IgA antibodies in opsonophagocytosis. Thus, human antimannan IgA subclass variants hinder complement activation while increasing neutrophil phagocytosis of C. albicans. These results contribute to a more complete understanding of the role of serum IgA in host immunity.

In the present studies we have explored the link between food hypersensitivity and IgA nephropathy (IgAN) and evaluated treatment options in primary and recurrent disease. Approximately one third of our IgAN patients had a rectal mucosal sensitivity to gluten, as demonstrated by increased local mucosal nitric oxide production and/or myeloperoxidase release after gluten challenge. The gluten sensitivity seemed to be an innate immune reaction unrelated to the pathogenesis of celiac disease. Approximately half of the patients had a rectal mucosal sensitivity to soy or cow’s milk (CM). The levels of IgG antibodies to alfa-lactalbumin, beta-lactoglobulin and casein were significantly higher in CM sensitive as compared with non-sensitive IgAN patients, indicating that an adaptive immune response might be involved in addition to the innate immune reaction observed. With the knowledge of gastrointestinal reactivity enteric treatment was considered as a potential new treatment approach of IgAN. A 6-month prospective trial demonstrated proof-of-concept for the use of enteric budesonide targeted to the ileocaecal region of IgAN patients. We observed a modest, but significant reduction in urine albumin, a minor reduction of serum creatinine and a modest increase of eGFR calculated by the MDRD equation. eGFR calculated from the Cockcroft-Gault formula and cystatin C was not changed. In a retrospective study recurrence of IgAN and graft loss was evaluated in Norwegian and Swedish patients having received a primary renal transplant due to IgAN. Adjusting for relevant covariates, a multiple Cox-regression analysis on time to IgAN recurrence showed that use of statins was associated with reduced risk of recurrence and reduced risk of graft loss. The time lag from diagnosis to first transplantation and female gender were also associated with lower risk of recurrence. Improved graft survival was associated with related donor, low donor age and no or low number of acute rejection episodes.

Transport of polymeric immunoglobulin A (plgA) in rat salivary glands has been investigated by combined morphological and biochemical techniques in vivo and in vitro. The distribution of IgA and its cellular receptor secretory component (SC) was observed by immunoperoxidase staining of cryosections from parotid and submaxillary gland, showing serous acinar cells are the site of IgA transport into saliva. Binding of horse radish peroxidase specific IgA to parotid serous acinar cells in vitro, observed by electron microscopy, shows that only the basolateral domain of acinar cells possesses exposed SC. A combination of new cell fractionation methods and standard western blotting techniques shows that SC present on basolateral plasma membrane of parotid acinar cells has a molecular weight (mwt) >100,000 and shows a high affinity for plgA in vitro. The existence of a 73,000 mwt SC occurring with plgA in cellular fractions of parotid gland suggest cleavage of SC occurs prior to secretion. The kinetics of plgA trancytosis was studied using isolated parotid acini. Bound plgA was secreted into the incubation medium as slgA, within thirty minutes of incubation at 37°C. Secretion of plgA was initially rapid but slowed over a 2hr period of incubation at 37°C. In addition to facilitating plgA transport serous acinar cells also synthesise and secrete a diverse range of other salivary proteins which are packaged into secretion granules and secreted directly through the apical plasma membrane. It is improbable that one complex secretory pathway facilitates both bulk secretion of salivary protein and transport of plgA. Therefore secreted proteins must be selectively segregated during secretion into saliva. Secretion of proteins from acinar cells in vitro shows proteins are released at two distinct rates.

Les entérobactéries Salmonella sont divisées en plusieurs sérovars dont les quatre principaux Typhimurium, Enteritidis, Typhi et Paratyphi sont responsables soit de gastroentérites soit de fièvres typhoïdes, à raison de plus de 90 millions de cas et 400 000 décès par an. L’apparition de souches multi-résistantes nécessite la mise en place d’une vaccination prophylactique muqueuse. L’environnement intestinal est caractérisé par une balance entre tolérance immunitaire et réaction inflammatoire régulée par les immunoglobulines (Ig) A sécrétoires. Les IgA des sécrétions muqueuses sont dimériques, les IgA sériques sont monomérique et deux isotypes ont été décrits chez l’Homme: IgA1 et IgA2. Nous avons tout d’abord exploré les fonctions des différents isotypes et isoformes des IgA humaines. Nous avons pu noter un rôle anti-inflammatoire des IgA1 à l’inverse d’un rôle pro-inflammatoire des IgA2 et nous avons souligné un processus de régulation de l’expression des récepteurs aux IgA par les IgA elles-mêmes ainsi qu’un axe IgA/lymphocytes T CD8 cytotoxiques. Nous avons ensuite mis en place un vaccin multivalent composé des antigènes SseB et OmpC de Salmonella liés à des Ig sécrétoires. Cette étude a mis en évidence une solide réponse immunitaire humorale et cellulaire spécifique aux antigènes couplés à des IgA ou IgM après vaccination intra-nasale au niveau systémique et muqueux. Par ailleurs, de plus fortes réponses humorales et systémiques spécifiques ont été observées en couplant à la fois OmpC et SseB sur l’IgA. Ce travail de thèse ouvre de nouvelles perspectives pour la mise en place de vaccins muqueux multivalents et pourrait apporter des réponses quant au rôle des IgA
The enterobacteria Salmonella species are divided into several serovars such as Typhimurium, Enteritidis, Typhi and Paratyphi which are the major causative agents of either gastroenteritis or typhoid fever. They are responsible for more than 90 million cases and 400 000 deaths each year. The increase in multi-drug resistant strains requires the implementation of prophylactic mucosal vaccines. Besides, the intestinal environment is characterized by a balance between immune tolerance and inflammatory response tightly regulated by secretory immunoglobulins (Ig) A. Mucosal IgA are mainly dimeric, serum IgA monomeric and two IgA isotypes have been described in humans: IgA1 and IgA2. We firstly explored the functions of the different isotypes and isoforms of human IgA. We pointed out a pro-inflammatory role of IgA2 whereas IgA1 rather oriented the immunity towards an anti-inflammatory response. We have also highlighted both the regulation of IgA receptors expression by IgA and an IgA/CD8 cytotoxic T cells axis. We also designed a multivalent vaccine against Salmonella by coupling two antigens – SseB and OmpC – to secretory Ig. We pointed out solid specific humoral and cellular responses against both these antigens coupled to either IgA or IgM after intra-nasal immunization in mucosal but also systemic compartments. We have also demonstrated the possibility to preserve and increase the antigen immunogenicity with a multivalent vaccine. This thesis thus paves the way for new secretory Ig-vectorized mucosal vaccines. In addition, the immune response could be modulated through the chosen isotype or isoform and the differences in immune activation generated by structural changes in IgA could shed some light on their role in mucosal homeostasis

In the beginning of this millennium the increasing level of work related illness was de-scribed, in the public debate, as one of the most serious and costly social problems of our times. An important question in the present study is whether or not the newspapers contributed to make their readers, the politicians and other social actors aware of this vast and growing problem.

Thus, the main purpose was to find out the extent of the news media coverage on occupational health/ill-health in Swedish newspapers in the end of the 1990s, and the ways in which the topic was framed. Furthermore the intent was to produce a better and deeper understanding of the factors influencing the coverage.

Theoretically the study draws on framing theory. Framing here refers to the process through which complex issues are reduced to journalistically manageable dimensions in the construction of news stories, resulting in a text, a news story that presents and high-lights some aspects and perspectives of the perceived reality but not others.

A combination of research methods was used - A content and frame analysis of six months of occupational health coverage in seven newspapers; an interview study with journalists and their scientific sources about the news production; a one week’s news-room study aimed at observing the everyday production of news; and finally, a short email survey directed to the editorial staff at the examined news papers, with the purpose to get some indication on how the coverage of occupational health was organised and prioritized at the different newspapers.

In the empirical analysis the newspapers´ picturing of occupational health/ill-health was compared with picture emerging from official statistics on occupational sickness and injury. In many respects a deviation was found between the two. Furthermore, simi-larities and differences in content between different newspapers, between different news sections and between news stories written by journalists of different sex, were examined.

A key finding is that the Swedish newspapers did not draw their readers’ attention to the extensive and growing problem at the places of work. A majority of the stories related to occupational health/ill-health were episodic, and treated the issues as isolated and random events rather than predictable and preventable problems, although there were also more thematic articles written during special circumstances. The results indicate that a primary cause of the topics low priority in the newspapers was that the coverage of occupational health/ill-health had not been integrated into the journalistic routines.

Mestrado em Engenharia Mecânica
A few years ago drawings were made in the drawing boards and using pencils on vellum. There were no computers helping the designers in the parts modeling. After designing the object, the design was passed to the analysts. The designers and analysts were in constant communication. Nowadays, the designers used Computer Aided Design (CAD) tools in the parts modeling. For application the analysis at the geometries, initially a mesh to approximate the geometries is generated. After this, on the mesh the Finite Element Method (FEM) was applied. In complex engineering design, the generation and manipulation of meshes in FEA was estimated to take over 80% of the overall analysis time. The form to break down the barriers between engineering design and the analysis is with reconstruction the entire process, but at the same time maintaining compatibility with existing practices. Create only one geometric model is the focus of reconstruction process. This geometric model is used in the representation of the geometry, as well as in the analysis, and this concept is designated by Isogeometric Analysis (IGA). In this present work the development of the tools for generate the CAD and calculate the basis function for representation the object are proposed. Initially, the mathematical formulations for Bézier, B-Spline and NURBS, for curves and surfaces are presented. The algorithms developed to generate the curves and surfaces are demonstrated. The IGA and FEM formulation for tridimensional and bidimensional spaces are introduced. In this work, a development of a tools for application this method are proposed. The convergence of the results for FEM and IGA programs are studied and compared to the theoretical values and Abaqus comercial program. The results obtained with IGA formulation converge to the reference values.
Há alguns anos atrás, os objectos eram feitos pelos designers e a criação do desenho era feita com lápis e papel vegetal. Não existiam computadores nos gabinetes de desenho para ajudar na modelação dos objectos. Após o desenho estar concluído este era entregue aos analistas para calcularem a resistência do mesmos quando solicitados por cargas externas. Assim, o gabinete de design e o gabinete de análise estavam em constante comunicação. Nos tempos de hoje os designers utilizam as ferramentas de Computer-Aided Design (CAD) para gerar os objectos, representando assim a geometria original. Por outro lado, os analistas fazem a análise baseada no Método dos Elementos Finitos (MEF). Neste método, inicialmente, gera-se uma malha para fazer a aproximação do objecto e utiliza-se esta malha gerada na análise. A forma de combater esta barreira é a construção de um novo processo de análise, mas ao mesmo tempo manter a compatibilidade com a análise do Método de Elementos Finitos. Este novo método foca-se na geração de um modelo geométrico, sendo este modelo utilizado tanto para a representação da geometria como para a análise. A principal sustentação deste novo método é a utilização das funções de base da criação e representação dos objectos, posteriormente, utilizadas na análise dos mesmos. Este novo conceito é designado por Análise Isogeométrica. Neste trabalho é exposto o desenvolvimento de ferramentas para gerar curvas e superfícies utilizando as formulações de Bézier, B-spline e NURBS. Assim, desenvolveram-se sub-rotinas para calcular as funções de base. Inicialmente apresentaram-se as formulações matemáticas e posteriormente os algoritmos desenvolvidos para a representação das curvas e superfícies. O desenvolvimento de ferramentas de análise para problemas no espaço bidimensional e tridimensional utilizando o Método de Elementos Finitos e a Análise Isogeométrica também é abordado neste trabalho. Para ser mais fácil a sua aplicação, foi desenvolvida um interface. Por fim utilizaram-se problemas e estudaram-se as curvas de convergência dos resultados e compararando-os com as referência analíticas e com o programa Abaqus. Em termos de conclusão, os resultados obtidos com a Análise Isogeométrica convergem mais rapidamente para os valores de referência do que o Abaqus e o programa desenvolvido com base no método de elementos finitos.

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CNPQ; CAPES
O aumento na concentração de IgA Secretora em secreções externas é uma importante ferramenta de diagnóstico para infecções que acometem as mucosas. O uso de metodologia de imunoquimiluminescência para realizar tal diagnóstico permite uma maior sensibilidade que os métodos espectrofotométricos tradicionalmente utilizados. Neste trabalho, um ensaio Dotimunoquimiluminescente (Dot-CLIA) foi proposto para a determinação de IgA Secretora. Os anticorpos anti-IgAS e anti-IgG-peroxidase foram previamente conjugados com éster de acridina (AE). Dot-ELISA e Dot-CLIA foram então realizadas aplicando amostras de IgAS em discos de membranas de nitrocelulose (0,45 μm de diâmetro de poro) e foi medida a atividade da peroxidase e a quimiluminescência (expressa em unidade relativa de luz; URL), respectivamente. O complexo ternário formado por IgAS/anti- IgAS-AE/anti-IgG-peroxidase- AE e o controle (PBS em substituição a IgAS) forneceram valores de 302.255 ± 28.736 RLU e 8.247 ± 3.479 RLU, respectivamente. Dot-ELISA simultaneamente realizada forneceu cor marrom pelo complexo ternário e ausência de cor foi observada para o controle. A relação entre RLU versus quantidade de IgAS utilizando o método proposto mostrou uma curva hiperbólica. O leite materno sem lipídios e caseína purificado por HPLC apresentou três picos de proteína e os que correspondiam a IgAS e ao componente secretor livre apresentaram valores de cerca de 50.000 RLU e 30.000 RLU, respectivamente. Portanto, pode-se concluir que o ensaio Dot-CLIA é capaz de avaliar quantitativamente e especificamente IgAS em fluidos biológicos.

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Infections for intestinal parasites are one of the main morbidade causes in humans and, its relationships with socioeconomic levels and hygiene conditions in countries in development are already very established. Many works, even so, they are being accomplished to elucidate the complex interactions among nutrition, these infections and answer imunol?gica, because it is seen that malnutrition commits the immunity increasing the susceptibilidade for infectious diseases and these for its time can harm the state human nutricional. It is known that sponge helm?nticos they stimulate synthesis of IgE so much policlonal as specific for the same ones and that IgA secretora, main imunoglobulina of defense of the mucous ones, can act against protozoa as the Giardia lamblia and against helmintos as Trichuris tichiura and Strongyloides stercorales. Some studies show that the malnutrition energy prot?ica influences in the production of these answers, but some authors show results divergentes. In this work it was evaluated the levels of total IgE, IgA s?rica and secretora, contagem of sanguine eosin?filos, levels of proteins s?ricas and state nutricional, in 103 children of low socioeconomic level, to discover a correlation between those and infection for enteroparasitas. They participated in the study children of both sexes, with age of 3 to 6 years, visitors of the same creche and residents in a neighborhood with precarious hygiene conditions and basic saneamento, in the city of Christmas. The obtained results showed that the faulty environmental and socioeconomic conditions favored to a high infection frequency for enteroparasitas, mainly Trichuris trichiura and Ascaris lumbricoides between the helmintos and Endolimax sleep and Gi?rdia lamblia among the protozoa. Light malnutrition without deficit prot?ico was observed in 30% of the children, which didn't also present significant deficiencies of IgA s?rica and secretora. The sponged children
Infec??es por parasitas intestinais s?o uma das principais causas de morbidade em humanos e, suas rela??es com n?veis s?cio-econ?micos e condi??es de higiene em pa?ses em desenvolvimento j? s?o bem estabelecidas. Muitos trabalhos, por?m, est?o sendo realizados para elucidar as complexas intera??es entre nutri??o, estas infec??es e resposta imunol?gica, pois ? visto que desnutri??o compromete a imunidade aumentando a susceptibilidade para doen?as infecciosas e estas por sua vez podem prejudicar o estado nutricional humano. Sabe-se que parasitas helm?nticos estimulam s?ntese de IgE tanto policlonal como espec?fica para ant?geno dos mesmos e que IgA secretora, principal imunoglobulina de defesa das mucosas, pode atuar contra protozo?rios como a Giardia lamblia e contra helmintos como Trichuris tichiura e Strongyloides stercorales. Alguns estudos mostram que a desnutri??o energ?tico prot?ica influencia na produ??o destas respostas, mas outros autores mostram resultados divergentes. Neste trabalho avaliou-se os n?veis de IgE total, IgA s?rica e secretora, contagem de eosin?filos sangu?neos, n?veis de prote?nas s?ricas e estado nutricional, em 103 crian?as de baixo n?vel s?cio-econ?mico, para se averiguar uma correla??o entre esses e infec??o por enteroparasitas. Participaram do estudo crian?as de ambos os sexos, com idade de 3 a 6 anos, freq?entadoras da mesma creche e residentes em um bairro com prec?rias condi??es de higiene e saneamento b?sico, na cidade do Natal. Os resultados obtidos mostraram que as deficientes condi??es ambientais e s?cio-econ?micas favoreceram a uma alta freq??ncia de infec??o por enteroparasitas, principalmente Trichuris trichiura e Ascaris lumbricoides entre os helmintos e Endolimax nana e Gi?rdia lamblia entre os protozo?rios. Desnutri??o leve sem d?ficit prot?ico foi observada em 30% das crian?as, as quais tamb?m n?o apresentaram defici?ncias significativas de IgA s?rica e secretora. As crian?as parasitadas apresentaram eosinofilia sangu?nea e n?veis s?ricos de IgE total elevados confirmando a importante participa??o das mesmas na resposta imune contra helmintos. Pode-se, portanto, sugerir que as crian?as apesar de poliparasitadas n?o estavam com sua resposta imune de mucosa contra parasitas, prejudicada, provavelmente por ainda n?o estarem intensamente infectados, como observado na contagem de ovos por grama de fezes e tamb?m por n?o terem seu estado nutricional gravemente comprometido

Los niveles urinarios de moléculas relacionadas con la activación del complemento y con el entorno inflamatorio local que inducen lesión renal han sido ampliamente estudiados como potenciales marcadores predictivos de severidad y pronóstico en la nefropatía IgA (NIgA). Sin embargo, el papel de estas moléculas como potenciales biomarcadores permanece poco definido por varias razones. Por un lado, la asociación entre los niveles de excreción urinaria de algunas de estas moléculas y sus respectivos depósitos mesangiales no ha sido analizada adecuadamente, y se desconoce la contribución de las moléculas implicadas en la vía de las lectinas a la activación del complemento en las células tubulares. Tampoco hay estudios que comparen la capacidad predictiva de la fibrosis intersticial entre varios marcadores urinarios de progresión de la lesión renal. Además, los datos disponibles se basan en medidas obtenidas en muestras de orina puntual, y no se ha analizado la exactitud de dichas medidas para estimar la excreción de orina de 24h. Por último, no se han estimado las fuentes de variación sujetas a dichas medidas, necesarias para interpretar correctamente los resultados y definir las especificaciones metodológicas. Esta tesis proporciona nuevos datos a la literatura existente sobre estas moléculas, a través de cuatro estudios observacionales realizados en cohortes de pacientes con NIgA. Los estudios se centraron en las medidas de lectina de unión a manosa (MBL), C4d, C5b-9, properdina, interleucina 6 (IL-6), factor de crecimiento epidérmico (EGF), proteína quimioatractante de monocitos 1 (MCP-1) y factor de crecimiento transformante β 1 (TGFβ - 1). Se evaluó la capacidad de los niveles urinarios de proteínas relacionadas con el complemento para identificar las vías locales del complemento activadas en las biopsias renales, así como la capacidad de los niveles urinarios de citoquinas y factores de crecimiento para predecir la extensión de fibrosis intersticial. También se analizó la exactitud de las medidas de EGF en orina puntual para predecir la excreción diaria de EGF. Finalmente, se estimaron las fuentes de variación sujetas a las medidas de EGF urinario. Esta tesis concluye que la MBL urinaria contribuye a la activación del complemento por la vía de las lectinas en las células tubulares, y se asocia a la gravedad de la lesión tubulointersticial, por lo que podría considerarse como un potencial biomarcador de la severidad de la lesión renal en pacientes con NIgA. Además, los niveles urinarios de MBL y C4d podrían aplicarse en la práctica clínica como biomarcadores sensibles y específicos de la presencia de sus respectivos depósitos mesangiales. Los niveles urinarios de EGF, MCP-1 e IL-6 mejoran la capacidad predictiva de modelos que incluyen la el filtrado glomerular (FG) y la edad para estimar la fibrosis intersticial. La combinación de los niveles de MCP-1 y EGF, o de los niveles de MCP-1 e IL-6, proporcionan un poder predictivo similar y parecen ser los más apropiados para ser aplicados a la práctica clínica. Además, la exactitud de la ratio EGF/creatinina para predecir la excreción de 24 h depende del FG y del grado de fibrosis intersticial resultante. La excreción diaria de EGF se correlaciona más fuertemente con la fibrosis que la ratio EGF/creatinina y su determinación debería emplearse en estudios dirigidos a evaluar la excreción de EGF como un biomarcador pronóstico precoz en individuos con FG elevados, o en estudios diseñados para estimar la masa tubular en pacientes con enfermedad renal crónica avanzada. Por último, los resultados indican que en las medidas de la ratio EGF/creatinina debe aplicarse el valor de referencia del cambio (VRC) para evaluar un cambio clínico significativo en los pacientes. Además, deben emplearse distintos VRCs en función del grado de fibrosis intersticial y/o GFR.
Urinary levels of molecules related to the activation of complement and to the local inflammatory environment inducing renal injury have been widely studied as potential predictive markers of disease severity and prognosis in IgA nephropathy (IgAN). However, the role of these molecules as potential biomarkers remains poorly defined for several reasons. On one hand, the association between the urinary levels of some of these molecules and their respective mesangial deposits has not been adequately analyzed, and the contribution of molecules involved in the lectin pathway to complement activation in tubular cells is unknown. There are also no studies comparing the predictive ability of interstitial fibrosis among several urinary markers of renal injury progression. In addition, the data available are based on measurements obtained in spot-urine samples, and the accuracy of such measurements to estimate the 24h urine excretion has not been analyzed. Finally, the sources of variation subject to these measures have not been estimated, which are necessary to correctly interpret the results and define the methodological specifications. This thesis provides new data to the existing literature on these molecules through four observational studies performed in cohorts of patients with IgAN. The studies were focused on measurements of mannose binding lectin (MBL), C4d, C5b-9, properdin, interleukin 6 (IL-6), epidermal growth factor (EGF), monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor β1 (TGFβ-1). The ability of urinary levels of complement related proteins to identify the local complement pathways activated in kidney biopsies was assessed, as well as the ability of urinary levels of cytokines and growth factors to predict the amount of interstitial fibrosis. The accuracy of spot-urine EGF measurements to predict the daily EGF excretion was also analyzed. Finally, sources of variation subject to urinary EGF measurements were estimated. This thesis concludes that urinary MBL contributes to the activation of complement by the lectin pathway in tubular cells and is associated with the severity of tubulointerstitial injury, and therefore could be considered as a potential marker of the severity of kidney injury in patients with IgAN. Urinary levels of MBL and C4d might be used in clinical practice as sensitive and specific biomarkers of the presence of their respective mesangial deposits. Results also show that urinary levels of EGF, MCP-1 and IL-6 improve the predictive ability of models including glomerular filtration rate (GFR) and age to estimate kidney interstitial fibrosis. The combination of MCP-1 and EGF levels, or MCP-1 and IL-6 levels, provide similar predictive power and appear to be the most appropriate to be applied in clinical practice. In addition, the spot EGF/creatinine ratio do not provide an adequate prediction of 24h excretion of EGF. In addition, the accuracy of the EGF/creatinine ratio to predict the 24h excretion depends on GFR and on the ensuing degree of interstitial fibrosis. Daily EGF excretion correlates more strongly with fibrosis than EGF/creatinine ratio, and its use is preferred both in studies that aim to analyze the value of EGF excretion as an early prognostic biomarker in individuals with high GFR values, as well as in those designed to estimate the tubular mass from urinary levels of EGF in patients with advanced chronic renal disease. Finally, the results show that in the EGF/creatinine ratio measurements the reference change value (RCV) should be applied to assess a clinically significant change in patients. In addition, different RCVs should be used depending on the degree of interstitial fibrosis and/or GFR.

La NIgA primaire est la plus fréquente des glomérulonéphrites. Elle représente, selon l’origine géographique et ethnique des populations, 10 à 40% des glomérulonéphrites primitives. Le diagnostic de NIgA est fait nécessairement sur la ponction biopsie rénale (PBR) en immunofluorescence. La grande difficulté dans cette maladie se trouve dans la prédiction au moment du diagnostic par les facteurs pronostiques de l’évolution vers l'insuffisance rénale chronique terminale à 10 et 20 ans après le début de la maladie. L’hypertension artérielle, la protéinurie ≥l g/24 h et la sévérité des lésions histologiques sur la biopsie rénale sont les facteurs de risque majeurs qui permettent la prédiction initiale. Objectifs de l’étude : confirmer et valider dans notre cohorte de NIgA les 3 facteurs de risque (FdR) comme facteurs significatifs et indépendants prédictifs d’une évolution ultérieure vers la Dialyse ou le Décès (avant dialyse) et utiliser ces 3 FdR pour développer un modèle simple de Risque Rénal Absolu (RRA) dont le score évalué au moment du diagnostic permettrait une prédiction du devenir à long terme (10 et 20 ans). Notre cohorte de patients (332) avec NIgA a été recrutée par le Service de Néphrologie, Dialyse, et Transplantation Rénale du CHU de Saint Etienne à l’Hôpital Nord (IGAN-STET-CO). La cohorte est prospective et inclut tous les patients avec diagnostic de Néphropathie à IgA primaire dont la biopsie rénale diagnostique a été réalisée entre le 1er janvier 1990 et le 31 décembre 1999. Le diagnostic histologique de NIgA est défini par la présence de dépôts mésangiaux d’IgA, d’intensité au moins 1+ comme immunoglobuline dominante ou codominante en immunofluorescence. L’intervalle de temps entre le début la maladie et la PBR diagnostique a été de : moyenne (déviation standard, DS) = 5.7 (8.5) ans et médiane (extrêmes) = 2.5 (0.1-46.9) ans. La durée totale d’exposition au risque principal entre le début de NIgA et le dernier recul ou le l’évènement principal était: moyenne (DS) = 12.9 (9.5) ans et médiane (extrêmes) = 11.3 (0.l-56.0) ans; chez 44 patients cette durée d’exposition était supérieure à 20 ans (l3.3%). La progression de la NIgA a été basée sur l’apparition de deux évènements : l’évènement principal (primary end-point) était la dialyse (Di; correspondant à la mort rénale avec un DFG de stade V autour de 8 ml/mn/1.73m2S) ou le décès du patient (De; s’il survenait avant le début de la dialyse), et représenté par Di/De ou plus simplement par D/D; l’évènement secondaire (secondary end-point) était la survenue d’un DFGe<60 ml/mn/1.73m2S marquant le début de l’IRC et correspondant à l’entrée dans le stade III de la maladie rénale chronique (MRC-3+). Les facteurs de risque majeurs étudiés dans cette étude sont l’hypertension artérielle (supérieure à 140/90 mmHg), la protéinurie (≥1g/24 h), et le score optique global (GOS ≥8). Le risque rénal absolu (RRA) de dialyse/décès (D/D) a été calculé à partir de ces trois facteurs simplifiés et dichotomiques après avoir confirmé leur caractère indépendant (les uns des autres) et que leur poids spécifique dans cette prédiction était quantitativement très similaire. 0 pour aucun de ces FdR, 3 pour leur présence simultanée et un score intermédiaire de 1 ou de 2 pour la présence de 1 ou 2 parmi ces 3 facteurs. Ce RRA a été utilisé comme une variable qualitative avec 4 catégories. Nous avons utilisé les courbes de survie sans l’évènement étudié selon Kaplan-Meier (KM) et la méthode de régression de Cox qui permet d’étudier l’influence d’une variable sur la survie sans l’évènement et permet ainsi d’isoler des facteurs pronostiques. Le DFG estimé au moment du diagnostic est de: moyenne(DS) = 74.7 (28.3) et médiane = 80 ml/mn/1.73m2S contre au dernier recul: moyenne (DS) = 68.0 (3 l .3) et médiane = 72.5 avec un P très significatif. La répartition des patients selon le RRA était la suivante: 151 patients (45,5%) avec un score de 0, 69 (20,8%) avec un score de 1,65 (19,6%) avec un score de 2, et 47 patients (14,2%) avec un score de 3. Le taux d'incidence cumulative de D/D, respectivement à 10 ans et 20 ans, a été de 2% (81 à risque) et 4% (15 à risque) pour RRA = 0,2% (38 à risque) et 9% (12 à risque) pour RRA = 1,7% (42 à risque) et 18% (9 à risque) pour RRA = 2, et 29% (27 à risque) et 64% (8 à risque) pour RRA = 3 (test du Logrank global; Chi2 = 62,1; P <0,0001). Les courbes de survie des patients sans D/D, selon Kaplan-Meier ont montré une meilleure survie avec un contrôle effectif de l’HTA. Le taux d'incidence cumulée pour dialyse/décès à 10 et à 20 ans était respectivement, pour le groupe non hypertendu de 4% (84 patients à risque) et 5% (17 patients à risque); pour le groupe des patients avec HTA contrôlée de 1% (54 patients à risque) et 19% (14 patients à risque), et pour le groupe des hypertendus non- contrôlés de 19% (50 patients à risque) et 42% (13 patients à risque) avec une différence significative entre les groupes des non hypertendus et des hypertendus contrôlés avec le groupe des hypertendus non contrôlés (P <0,0001). Pour la protéinurie, les courbes de survie des patients sans D/D, selon Kaplan-Meier, ont montré une amélioration significative (p<0,0001) en cas de réduction effective de la protéinurie. Ainsi, le taux d'incidence cumulée pour dialyse/décès à 10 et 20 ans était de 3% (118 à risque) et 5% (25 à risque) pour le groupe avec protéinurie faible/absente, 2% (40 à risque) et 2% (10 à risque) pour le groupe avec « protéinurie réduite», et 29% (30 à risque) et 67% (9 à risque) pour le groupe avec protéinurie non-réduite. Le contrôle des lésions histologiques graves est difficile à estimer à partir de notre cohorte en l'absence de biopsies répétées. Notre score de Risque Rénal Absolu est basé sur trois facteurs de risque majeurs, indépendants et simplifiés prédictifs de la progression vers la MRC-3+, puis vers la dialyse/décès. Ces FdR avaient finalement un poids identique dans cette prédiction à long terme, comme en témoignent : - une valeur de β/SE comprise entre 4 et 5 dans l’analyse monofactorielle et entre 2 et 3 dans l’analyse multifactorielle de Cox, - ainsi que des paramètres d’exactitude quasi similaires: même valeur prédictive négative autour de 0,95 et même valeur prédictive positive autour de 0,30; de même la probabilité de survenue de dialyse/décès était d'environ 31% en leur présence, mais seulement de 3 à 6% en leur absence. Au moment du diagnostic, 47 patients présentaient les 3 FdR ensemble (RRA = 3) et 28 (60%) sont arrivés en dialyse ou sont décédés. La probabilité de l'évolution à long terme vers la dialyse/décès était de 60% chez les patients avec RRA = 3 contre seulement 6% chez les patients avec RRA =0. Nous sommes les premiers à publier sur le Risque Rénale Absolu de dialyse e/o de décès et de proposer un score global avec une application clinique importante. L'utilisation de ce score RRA, nous permet de prédire à 10 et 20 ans après le début de la maladie, le taux d’incidence cumulative de dialyse/décès en retenant les chiffres arrondis à 20ans de 5% si RRA=0, de 10% si RRA=1, de 20% si RRA= 2, et de 60% si RRA=3, et cela dans une population activement traitée. Notre RRA est très simple à calculer par une simple addition des FdR présents au diagnostic, ne nécessite pas de calcul et s’affranchit des autres facteurs de risque (cliniques, biologiques, pathologiques et génétiques) décrits chez les patients avec NIgA comme le sexe ou l’âge du patient au moment de la biopsie ou l’indice de masse corporelle, IMC . Le point important est que l'utilisation du RRA, permettrait de bien sélectionner les patients à inclure dans des essais randomisés et contrôlés L’autre avantage sera dans les études génétiques de démontrer l'impact d'un génotype / allèle spécifique lié à la progression de la maladie en montrant une augmentation pas à pas de la fréquence du génotype/allèle dans les sous- groupes de RRA= 0 à RRA=3
For the individual patient with primary IgA nephropathy (IgAN), it remains a challenge to predict accurately the long term outcome at 10 and 20 years after disease onset. We studied it in a prospective cohort of 332 (237 males, 71.4%) biopsy-proven IgAN patients, the IGAN-STET-CO, aged at diagnosis of mean = 41.4 (SD=15.1) years, with a total exposure time of mean = 12.9 (9.5) y. Using three consensual risk factors (RF) simplified as dichotomous covariates : occurrence of hypertension, proteinuria ≥1 g/d, and severe pathological lesions (global optical score ≥8), we calculated an absolute renal risk (ARR) of dialysis/death (D/D), in analogy to the absolute cardiovascular risk. The ultimate prediction according to the number of RF present at diagnosis (ARR score: 0, 1, 2, or 3) was done by the Cox regression and the Kaplan-Meier survival methods. Overall, this ARR scoring permitted significant (P<0.0001) stratification of the risk. The cumulative incidence rate of D/D events (N=45), respectively at 10 and 20y, was 2 and 4% for ARR=0 (45.5% of all cases), 2 and 9% for ARR=1 (20.8%), 7 and 18% for ARR=2 (19.6%), and 29 and 64% for ARR=3 (N=47; 14.1%) in adequately treated patients. Effective control of hypertension and reduction of proteinuria improved survival without D/D when achieved. This absolute renal risk score evaluated at diagnosis, allowed accurate prediction of ultimate dialysis/death risk and was also validated in another cohort. This is a significant progress in the management of the individual patient with lgA nephropathy