Abstract There is evidence that the process leading to type 1 diabetes may start in early infancy or even in utero, with a prodrome of variable duration preceding clinical manifestation. The purpose of the present work was to learn more about the occurrence and significance of humoral beta-cell autoimmunity during pregnancy and in infancy, to search for possible signs of prenatal or early postnatal induction of beta-cell autoimmunity and to explore the role of enterovirus infections as potential triggers of such autoimmunity. The population comprised mothers and their newborn infants from families with type 1 diabetes who had entered the first (n=20) or the second pilot study (n=208) of the Trial to Reduce IDDM in the Genetically at Risk (TRIGR). Almost 40% of the mothers with type 1 diabetes had antibodies to islet cells (ICA), 55% to glutamic acid decarboxylase (GADA) and 54% to the IA-2 protein (IA-2A) in the two samples taken during pregnancy, where the frequencies for the unaffected mothers were 5%, 5% and 3%, respectively. All autoantibody specificities were detected in the cord blood largely at the same frequencies as in the maternal circulation. In addition, ICA was found in 2.7%, GADA in 0.6%, IA-2A in 0.3% and insulin autoantibodies (IAA) in 0.1% out of a series of 1002 cord blood samples from infants representing the normal population. None of the infants of the autoantibody-negative mothers in these series had autoantibodies detectable in their cord blood. The rate of decline of transplacentally transferred autoantibodies during the first months of life was observed to be similar to that reported for the disappearance of maternally acquired IgG antibodies, the estimated mean elimination time ranging from 3.1-4.5 months. The higher the initial autoantibody level, the longer was the elimination time, and transplacentally transferred autoantibodies were occasionally detected up to the age of 9-12 months, and even at 15 months in a very few cases. The peak incidence of enterovirus RNA in serum was observed at the age of 6-12 months, while that of infections, based on changes in antibody titres, was seen at the age of 18 months. The frequency of enterovirus infections in the autoantibody-positive infants during the 6 months before the appearance of the first autoantibodies was almost three times higher than in age-matched infants testing negative for autoantibodies. These observations suggest that pregnancy does not have any strong modulating effect on the prevalence and titres of diabetes-associated autoantibodies. If such autoantibodies are present in the mother, most of them are transferred to the foetal circulation and are detectable in the cord blood. No signs of foetal induction of beta-cell autoimmunity were observed, indicating that such a phenomenon is extremely rare. Most of the transplacentally transferred autoantibodies disappear within the first 3-6 months of postnatal life, but they may persist even up to the age of 15 months in exceptional cases, suggesting that the optimal age for the initiation of large-scale screening in the general population is 18-24 months. The temporal association between enterovirus infections and the first signs of beta-cell autoimmunity supports the hypothesis that enteroviruses may induce a primary beta-cell insult.

Abstract The purpose of this research was to assess the role of antibodies to glutamic acid decarboxylase (GAD) in children with newly diagnosed type 1 diabetes in relation to other disease-associated autoantibodies and HLA-defined genetic disease susceptibility, to evaluate the role of GAD antibodies (GADA) in relation to clinical characteristics at the diagnosis of type 1 diabetes and to compare the frequency and levels of GADA between adult and childhood onset type 1 diabetes.The study population comprised altogether 999 children and adolescents with type 1 diabetes, 100 affected adult subjects and more than 370 non-diabetic controls. GADA were measured with a liquid radioligand assay, and a similar assay was used for the analysis of antibodies to the islet antigen 2 (IA-2) molecule. Islet cell antibodies (ICA) were determined with conventional immunofluorescence and insulin autoantibodies (IAA) with a liquid phase radioimmunoassay either in a tube or a plate format (microassay). GADA were detected at diagnosis in 68 to 73% of the children and adolescents with type 1 diabetes. GADA were more frequent in girls and in those older than 10 years of age at clinical disease manifestation. Subjects testing positive for GADA had higher levels of ICA and IAA than those negative for GADA. Multiple antibodies ( 2) were observed more often in girls and in children under the age of 5 years. Children with the HLA DR3/non-DR4 phenotype had the highest GADA levels, significantly higher than those seen in children with the DR4/non-DR3 combination. The highest prevalence of multiple autoantibodies was seen in subjects heterozygous for DR3/4. When studying HLA DQB1 genotypes those with the DQB1*02/y (y = other than *0302) genotype had the highest GADA levels as expected since DQB1*02 and DR3 are in strong linkage disease equilibrium. The same group had the lowest frequency of multiple antibodies among the children younger than 10 years of age.Patients diagnosed with type 1 diabetes before the age of 20 had a higher frequency of all four autoantibodies analysed than those presenting with clinical disease after the age of 20. The proportion of subjects testing negative for all four antibodies was substantially higher among adults than in those under the age of 20. The smallest age-related difference in antibody frequencies was observed for GADA, and the GADA-positive adult patients had on an average about three times higher antibody levels than the GADA-positive children. No association was observed between positivity for GADA and the degree of metabolic decompensation at the clinical presentation of type 1 diabetes. No significant differences were either seen between the subjects who tested positive for GADA at diagnosis and those who were negative in serum C-peptide concentrations, metabolic control or exogenous insulin requirement over the first 2 years of observation. The proportion of children in clinical remission was, however, lower among GADA-positive subjects than in GADA-negative patients at 18 months after the clinical manifestation. Positivity for multiple antibodies was associated with accelerated beta-cell destruction and increased exogenous insulin requirements over the 2-year observation period. The observations that GADA are related to female gender, older age and the HLA-DR3/ DQB1*02 haplotype suggest that a strong humoral immune response to GAD may reflect a propensity to general autoimmunity rather than specific beta-cell destruction.

Abstract The purpose of this work was to assess the value of humoral and genetic risk markers in the prediction of type 1 diabetes in siblings of children with type 1 diabetes, to characterise preclinical course of beta-cell autoimmunity in siblings, and to investigate the frequency of autoantibodies and their relations to genetic markers, beta-cell function and progression to type 1 diabetes in a schoolchild population. The prevalence and predictive value of autoantibodies was studied in 755 initially unaffected siblings, and the combination of genetic markers and autoantibodies in 701 of these siblings. Islet cell autoantibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase antibodies (GADA) and IA-2 antibodies (IA-2A) were all shown to be of value in the prediction of type 1 diabetes in siblings initially tested at or close to the diagnosis of type 1 diabetes in the index case in the family. The risk of progression to type 1 diabetes was related to the number of autoantibodies detected, and the PPV of multiple autoantibodies was 55% over a period of 8 years. Autoantibodies were closely associated with HLA risk markers. A combination of the genetic markers and autoantibodies increased the PPVs of all autoantibodies substantially but also markedly reduced the sensitivity. The preclinical course of type 1 diabetes was investigated in 39 initially unaffected siblings who progressed to clinical disease during the follow-up. These individuals were characterised by the high-risk genetic markers, decreased beta-cell function and humoral autoimmunity against multiple beta-cell targets. However, all measures implied a remarkable individual variation in the rate of the disease process and the pattern of humoral beta-cell autoimmunity. Furthermore, the autoimmune process resulting in clinical presentation of type 1 diabetes could not be unambiguously distinguished from autoimmunity not leading to clinical disease within almost 10 years of follow-up. The frequencies of ICA, IA-2A, GADA and IAA in 3652 healthy Finnish schoolchildren were 2.8%, 0.6%, 0.5% and 0.9%, respectively, and multiple antibodies were detected in 0.6% of these children. GADA and multiple antibodies were related to the DQB1*0302 allele and the DQB1*02/0302 genotype. A reduced first-phase insulin reponse (FPIR) was associated with IA-2A, GADA, IAA and multiple antibodies, but not with ICA or any specific DQB1 allele or genotype. Four subjects progressed to type 1 diabetes, all of them having multiple autoantibodies and those two who underwent an intravenous glucose tolerance test had also a reduced FPIR. None of the progressors carried the high risk DQB1*0302 allele and two of them even carried the protective DQB1*0602 or *0603 allele. In conclusion, autoantibodies alone are recommended as first-line screening in siblings, whereas subsequent determination of HLA-DQB1 markers and their combination with autoantibodies provides a valuable tool for more precise risk assessment. Wide heterogeneity in the course of preclinical type 1 diabetes complicates an accurate estimation of the individual risk of progression to type 1 diabetes among siblings of children with type 1 diabetes. Combined screening for autoantibodies is recommended for the assessment of the risk of progression to type 1 diabetes in schoolchild populations, whereas the present observations challenge the value of current genetic risk markers in predictive strategies targeting schoolchildren.

No description available.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of immune tolerance and the production of auto-antibodies which target various nuclear components. There is a 9:1 women to men ratio among lupus patients, indicating differing mechanisms of lupus pathogenesis between the sexes. Although lupus patients may develop many different manifestations, lupus nephritis (LN) remains to be one of the most devastating manifestations and an indicator of poor prognosis. Although both sexes develop LN, the nephritis in males often develops more rapidly and is more severe. Necrotic cell death is a characteristic of lupus nephritis and contributes to the exacerbation of the inflammatory immune response within the glomeruli. Previously our laboratory found that absence or pharmacological inhibition of Poly [ADP-ribose] polymerase 1 (PARP-1), an enzyme involved in necrotic cell death, results in milder nephritis, reduced necrotic lesions, and higher survival rates only among males. Although RIPK3-mediated necrosis was a likely candidate for inducing necrosis during female LN, murine models of glomerulonephritis revealed that the development of LN occurs independently of RIPK3. In addition, during LN, there is no crosstalk between the RIPK3- and PARP-1 mediated pathways to induce necrotic cell death. The sex bias in SLE indicates sex hormones may play a role in pathogenesis. Interestingly, estrogen receptor alpha (ERα) in the renal tissue is highly expressed and the renal specific estrogen-induced gene activation is second only to that of reproductive organs. The absence of estrogen receptor alpha protects female mice from developing nephritis, despite the presence of immune complexes in the kidneys and production of pro-inflammatory cytokines. Analysis of gene expression changes during LN progression indicate the protection seen in ERKO females may be due to alterations in metabolic pathways, including PPAR and retinol metabolism. These results demonstrate the complexity of lupus nephritis. Despite the presence of necrosis in LN, this manifestation occurs in a RIPK3-independent manner, which leaves the pathway responsible for necrosis in female kidneys to still be investigated. In addition, lupus nephritis occurs in an ER-dependent manner in females, demonstrating the significant impacts sex-hormone environments play in the pathogenesis of immune-mediated nephropathies. / Biomedical Sciences

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

O diabetes mellitus tipo 1A (DM1A), doença autoimune órgão-específica, resulta da destruição seletiva das células pancreáticas produtoras de insulina pela infiltração progressiva de células inflamatórias, particularmente linfócitos T auto-reativos. O DM1A tem etiologia complexa, resultante da interação de fatores ambientais e vários genes, particularmente os do sistema HLA (alelos -DR3 e -DR4). Paralelamente, genes que codificam outros componentes da resposta imune, como as citocinas, também são fortes candidatos à predisposição à autoimunidade. Sabe-se que a intensidade da resposta imunológica tem relação com a ativação e recrutamento de linfócitos T e B, produção de citocinas e autoanticorpos, associados às respostas imunológicas TH1 e TH2. Recentemente, uma nova sub-população de células T, a TH17, de intenso poder inflamatório, tem sido implicada em doenças autoimunes. O desenvolvimento das células TH17 sofre influência da citocina heterodimérica IL-27, composta pelas subunidades p28 e EBI3 (proteína do gene 3 induzida pelo vírus Epstein Barr), expressa predominantemente em macrófagos ou células dendríticas. A IL-27 tem sido associada à doença de Crohn, encefalomielite experimental autoimune e diabetes autoimune em camundongos. O seu bloqueio retarda o aparecimento do diabetes nestes animais. A implicação da IL-27 no DM1A em humanos é pouco conhecida. O presente estudo tem como objetivo pesquisar mutações ou polimorfismos na região 5 proximal e regiões codificadoras do gene da IL-27p28 e sua possível associação com predisposição ao DM1A. Estas regiões foram amplificadas pelo método de Reação em Cadeia da Polimerase (PCR) e submetido ao seqüenciamento automático e PCR-RFLP (Restriction Fragments Lenght Polymorphisms) utilizando DNA genômico obtido de leucócitos de sangue periférico. A casuística envolveu 614 indivíduos, sendo 318 pacientes portadores de DM1A (idade 19,6 ± 11,2 anos, 129M/189F), caracterizados por apresentarem hiperglicemia e necessidade precoce de insulinoterapia e um grupo controle, com 296 indivíduos saudáveis (idade 30,3±13,2 anos, 131M/165F), com glicemia de jejum e HbA1c normais. Na análise dos resultados do sequenciamento, observamos oito variantes alélicas, seis delas já descritas na literatura. As duas novas variantes alélicas causaram a substituição de uma citosina por uma timina na posição c.-347 C>T na região 5 proximal e a substituição de uma guanina por uma citosina no exon 5, na posição c.498 G>C, levando à mudança do aminoácido, de ácido glutâmico para ácido aspártico no resíduo 166 da seqüência da proteína. A distribuição dos genótipos na população estudada foi consistente com o equilíbrio de Hardy-Weinberg. As freqüências genotípicas e alélicas destas variantes não diferiram entre portadores de DM1 A e controles Não encontramos associação destas variantes com sexo e grupo étnico nos dois grupos e, com idade de diagnóstico do diabetes, presença de auto- anticorpos pancreáticos e extra-pancreáticos no grupo com DM1A. Não houve diferença na freqüência dos haplótipos estimados entre os pacientes DM1A e grupo controle. Sendo assim, nossos resultados sugerem que variantes alélicas no gene da IL-27p28 não estão implicadas na susceptibilidade ao DM1A na nossa população / Type 1A diabetes mellitus (T1D), an organ-specific autoimmune disease, results from the selective destruction of insulin-producing pancreatic cells by progressive infiltration of inflammatory cells, particularly autoreactive T lymphocytes. The complex etiology of T1D includes the interaction of environmental factors and multiple genes, particularly those of the HLA system (DR3 and DR4 alleles). In parallel, genes encoding other components of the immune response, such as cytokines, are also strong candidates for predisposition to autoimmunity. It is known that the intensity of immune response depends on the activation and recruitment of T and B lymphocytes, production of cytokines and autoantibodies, related to TH1 and TH2 immune responses. Recently, a new subpopulation of T cells exhibiting intense inflammatory activity, the TH17 subset, has been implicated in autoimmune diseases. The development of TH17 cells is influenced by IL-27, a heterodimeric cytokine composed of p28 and EBI3 (Epstein-Barr Virus- induced gene 3 protein) subunits, expressed predominantly in macrophages or dendritic cells. IL-27 has been associated with Crohn\'s disease, experimental autoimmune encephalomyelitis and autoimmune diabetes in mice. IL-27 blockage delays the onset of diabetes in these animals, but the role and involvement of IL-27 in T1D in humans has not yet been reported. The aim of this study was identify mutations or polymorphisms in the coding regions and boundary intron sequences of IL-27p28, including the 5 proximal region, and their possible association with the disease. Those regions were amplified by Polymerase Chain Reaction (PCR) and the polymorphisms determined by automatic sequencing and PCR-RFLP (Restriction Fragment length polymorphisms) The cohort involved 614 individuals - 318 patients with T1D ( 19.6 ± 11.2 years of age, 129M/189F) and 296 control subjects (30.3 ± 13.2 years of age, 131M/165F) with normal fasting glucose. We identified eight allelic variants in the 5 proximal and coding regions of IL- 27p28, six of them already described in database repositories. The two new allelic variants were: the substitution of a cytosine by a thymine at position c.- 347 C>T of the 5 proximal region and the substitution of a guanine by a cytosine in exon 5, at position c.498 G>C, determining a change in the aminoacid sequence of the protein at residue 166 from glutamic acid to aspartic acid. The genotypic frequencies of these variants were in Hardy- Weinberg equilibrium in both groups. The frequency of the alleles and genotypes did not differ between T1D patients and controls. There was no association between IL-27p28 variants with gender or ethnical origin in the population analyzed . Also, no association was found between these variants with age at diagnosis of diabetes nor with the presence of pancreatic and extrapancreatic autoantibodies in T1D patients The frequency of the estimated haplotypes were similar between groups. Our results suggest that allelic variants in the IL-27p28 gene are not involved in susceptibility to T1D in our population

O diabetes mellitus tipo 1A (DM1A), doença autoimune órgão-específica, resulta da destruição seletiva das células pancreáticas produtoras de insulina pela infiltração progressiva de células inflamatórias, particularmente linfócitos T auto-reativos. O DM1A tem etiologia complexa, resultante da interação de fatores ambientais e vários genes, particularmente os do sistema HLA (alelos -DR3 e -DR4). Paralelamente, genes que codificam outros componentes da resposta imune, como as citocinas, também são fortes candidatos à predisposição à autoimunidade. Sabe-se que a intensidade da resposta imunológica tem relação com a ativação e recrutamento de linfócitos T e B, produção de citocinas e autoanticorpos, associados às respostas imunológicas TH1 e TH2. Recentemente, uma nova sub-população de células T, a TH17, de intenso poder inflamatório, tem sido implicada em doenças autoimunes. O desenvolvimento das células TH17 sofre influência da citocina heterodimérica IL-27, composta pelas subunidades p28 e EBI3 (proteína do gene 3 induzida pelo vírus Epstein Barr), expressa predominantemente em macrófagos ou células dendríticas. A IL-27 tem sido associada à doença de Crohn, encefalomielite experimental autoimune e diabetes autoimune em camundongos. O seu bloqueio retarda o aparecimento do diabetes nestes animais. A implicação da IL-27 no DM1A em humanos é pouco conhecida. O presente estudo tem como objetivo pesquisar mutações ou polimorfismos na região 5 proximal e regiões codificadoras do gene da IL-27p28 e sua possível associação com predisposição ao DM1A. Estas regiões foram amplificadas pelo método de Reação em Cadeia da Polimerase (PCR) e submetido ao seqüenciamento automático e PCR-RFLP (Restriction Fragments Lenght Polymorphisms) utilizando DNA genômico obtido de leucócitos de sangue periférico. A casuística envolveu 614 indivíduos, sendo 318 pacientes portadores de DM1A (idade 19,6 ± 11,2 anos, 129M/189F), caracterizados por apresentarem hiperglicemia e necessidade precoce de insulinoterapia e um grupo controle, com 296 indivíduos saudáveis (idade 30,3±13,2 anos, 131M/165F), com glicemia de jejum e HbA1c normais. Na análise dos resultados do sequenciamento, observamos oito variantes alélicas, seis delas já descritas na literatura. As duas novas variantes alélicas causaram a substituição de uma citosina por uma timina na posição c.-347 C>T na região 5 proximal e a substituição de uma guanina por uma citosina no exon 5, na posição c.498 G>C, levando à mudança do aminoácido, de ácido glutâmico para ácido aspártico no resíduo 166 da seqüência da proteína. A distribuição dos genótipos na população estudada foi consistente com o equilíbrio de Hardy-Weinberg. As freqüências genotípicas e alélicas destas variantes não diferiram entre portadores de DM1 A e controles Não encontramos associação destas variantes com sexo e grupo étnico nos dois grupos e, com idade de diagnóstico do diabetes, presença de auto- anticorpos pancreáticos e extra-pancreáticos no grupo com DM1A. Não houve diferença na freqüência dos haplótipos estimados entre os pacientes DM1A e grupo controle. Sendo assim, nossos resultados sugerem que variantes alélicas no gene da IL-27p28 não estão implicadas na susceptibilidade ao DM1A na nossa população / Type 1A diabetes mellitus (T1D), an organ-specific autoimmune disease, results from the selective destruction of insulin-producing pancreatic cells by progressive infiltration of inflammatory cells, particularly autoreactive T lymphocytes. The complex etiology of T1D includes the interaction of environmental factors and multiple genes, particularly those of the HLA system (DR3 and DR4 alleles). In parallel, genes encoding other components of the immune response, such as cytokines, are also strong candidates for predisposition to autoimmunity. It is known that the intensity of immune response depends on the activation and recruitment of T and B lymphocytes, production of cytokines and autoantibodies, related to TH1 and TH2 immune responses. Recently, a new subpopulation of T cells exhibiting intense inflammatory activity, the TH17 subset, has been implicated in autoimmune diseases. The development of TH17 cells is influenced by IL-27, a heterodimeric cytokine composed of p28 and EBI3 (Epstein-Barr Virus- induced gene 3 protein) subunits, expressed predominantly in macrophages or dendritic cells. IL-27 has been associated with Crohn\'s disease, experimental autoimmune encephalomyelitis and autoimmune diabetes in mice. IL-27 blockage delays the onset of diabetes in these animals, but the role and involvement of IL-27 in T1D in humans has not yet been reported. The aim of this study was identify mutations or polymorphisms in the coding regions and boundary intron sequences of IL-27p28, including the 5 proximal region, and their possible association with the disease. Those regions were amplified by Polymerase Chain Reaction (PCR) and the polymorphisms determined by automatic sequencing and PCR-RFLP (Restriction Fragment length polymorphisms) The cohort involved 614 individuals - 318 patients with T1D ( 19.6 ± 11.2 years of age, 129M/189F) and 296 control subjects (30.3 ± 13.2 years of age, 131M/165F) with normal fasting glucose. We identified eight allelic variants in the 5 proximal and coding regions of IL- 27p28, six of them already described in database repositories. The two new allelic variants were: the substitution of a cytosine by a thymine at position c.- 347 C>T of the 5 proximal region and the substitution of a guanine by a cytosine in exon 5, at position c.498 G>C, determining a change in the aminoacid sequence of the protein at residue 166 from glutamic acid to aspartic acid. The genotypic frequencies of these variants were in Hardy- Weinberg equilibrium in both groups. The frequency of the alleles and genotypes did not differ between T1D patients and controls. There was no association between IL-27p28 variants with gender or ethnical origin in the population analyzed . Also, no association was found between these variants with age at diagnosis of diabetes nor with the presence of pancreatic and extrapancreatic autoantibodies in T1D patients The frequency of the estimated haplotypes were similar between groups. Our results suggest that allelic variants in the IL-27p28 gene are not involved in susceptibility to T1D in our population

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Because tumours are comprised of "self" cells and antigens, they escape recognition by the immune system, which discriminates between "self" and "non-self". One such antigen is cathepsin B, a lysosomal cysteine proteinase, that has been implicated as one of the proteolytic enzymes involved in tumour invasion and metastasis. Cathepsin B autoantibodies could open possibilities which may be useful in cancer immunotherapy. In this study generation of cathepsin B autoantibodies was attempted by manipulating the immune system into recognising and responding to cathepsin B in complex with a "foreign" protein, bovine serum albumin (BSA). Cathepsin B was isolated from rabbit liver using the three phase partitioning (TPP) method, modified by adding t-butanol in the homogenisation buffer. Isolation of cathepsin Band cathepsin L, using this novel method, minimised the formation of artefacts such as a covalent cathepsin L-stefin B complex and produced higher yields of enzyme. Pure rabbit liver cathepsin B was conjugated to BSA, using glutaraldehyde as coupling agent, and administered intramuscularly into rabbits. Another three inoculation protocols, which functioned as controls were: i) free cathepsin B administered intramuscularly, ii) complexed cathepsin B administered intravenously, and iii) free cathepsin B administered intravenously. IgGs isolated from inoculated rabbits' serum were assayed by a three layer ELISA system, immunoinhibition assays and dot blots. The anti-complex (intramuscular) antibodies showed the highest recognition for cathepsin B and were the only antibodies that were immunoinhibitory. This suggests that the immune system was, to some extend, successfully manipulated into recognising the complexed "self" cathepsin B. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.