RESUMO
Adenine and adenosine metabolism has been studied in intact human erythrocytes in vitro using high performance liquid chromatography, isotopic labeling and electrophoresis. Their metabolism to nucleotides was controlled by phosphoribose diphosphate synthesis which was phosphate dependent. Adenosine formed hypoxanthine or IMP depending upon Pi concentration, but adenosine kinase and deaminase activities were not affected by P levels. Free [14C]adenine and [14C]hypoxanthine were found in cellular extracts. Rapid interconversions occurred to give a distribution for ATP : ADP : AMP of 10 : 1 : 0.1. Marked decomposition of ATP to ADP and AMP occurred during incubations in plasma and Earle's media in air on nitrogen, but ATP levels remained stable in phosphate buffers and in the presence of oxygen. At physiological Pi (1 mM) adenosine kinase activity grossly exceeded adenine phosphoribosyltransferase activity. The latter was approximately 7 fold that of hypoxanthine phosphoribosyltransferase activity. These differences decreased with increasing Pi levels. No significant increase in corresponding nucleotides was obtained by incubation with high levels (0.5 mM) of adenine, guanine or guanosine at physiological Ii, ATP increased by 10% independently of the substrate employed and significant amounts of IMP and GTP were formed adenosine and guanosine, respectively. The existence of a bound intracellular pool of ATP is suggested.
Assuntos
Adenina/sangue , Adenosina/sangue , Eritrócitos/metabolismo , Difosfato de Adenosina/sangue , Trifosfato de Adenosina/sangue , Glicemia/metabolismo , Cromatografia Líquida de Alta Pressão , Glicólise , Humanos , Nucleotídeos de Inosina/sangue , Cinética , Lactatos/sangue , Purinas/sangueRESUMO
The ability of recombinant interferon-gamma (rIFN-gamma) to induce major histocompatibility complex (MHC) antigen expression in the rat insulinoma cell line RINm5F was investigated. The cells were stained with monoclonal antibodies specific for rat class I and class II MHC antigens. RINm5F cells endogenously expressed class I antigens; this was enhanced by rIFN-gamma. Class II antigens could not be detected on RINm5F cells, but both I-A and I-E were induced by rIFN-gamma.
Assuntos
Adenoma de Células das Ilhotas Pancreáticas/imunologia , Antígenos HLA/imunologia , Insulinoma/imunologia , Interferon gama/imunologia , Neoplasias Pancreáticas/imunologia , Animais , Anticorpos Monoclonais , Linhagem Celular , Antígenos HLA/classificação , Insulinoma/patologia , Neoplasias Pancreáticas/patologia , Proteínas RecombinantesRESUMO
The second international workshop on insulin autoantibodies (IAAs) demonstrated improved concordance among laboratories with both radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) when measurements were based on signals displaceable by preincubation with excess insulin. This feature has been incorporated into our original ELISA for IAAs, and the assay was used to compare IAAs in diabetes-related and healthy populations. The serum from a healthy islet cell antibody (ICA)+ and IAA+ subject was used to construct standard curves with and without preincubation with 1 U/ml human insulin. Reporting results in arbitrary displacement (delta +/- IAA) units derived from the standard curve improved precision and increased the specificity of the assay. Frequency analysis of the results from 200 control adults did not show a normal distribution, and cumulative frequency analysis demonstrated two populations: 10 of 200 (5%) control adults, 8 of 241 (3.3%) healthy schoolchildren, and 18 of 229 (7.9%) non-insulin-dependent diabetes mellitus (NIDDM) patients had IAAs greater than 12 delta +/- IAA units. On the same basis, we tested samples from 89 individuals in the prospective Bart's Windsor Family Study for insulin-dependent diabetes mellitus (IDDM), 31 of whom had ICAs greater than 5 Juvenile Diabetes Foundation (JDF) units. Of those with ICAs greater than 5 JDF units, 12 developed IDDM and 1 developed NIDDM in 10 yr of study. IAAs greater than 12 delta +/- IAA units were found in 6 of 12 (50%) who became diabetic and in 4 of 18 (22%) who remain healthy.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Diabetes Mellitus/imunologia , Anticorpos Anti-Insulina/análise , Adulto , Autoanticorpos/análise , Biomarcadores/sangue , Criança , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Ilhotas Pancreáticas/imunologia , Valores de ReferênciaRESUMO
A detailed study of 133 subjects with insulin-dependent (type I) diabetes with severe microvascular disease has failed to substantiate the hypothesis that HLA factors influence the predisposition to this type of complication. A significant association between proliferative retinopathy and raised levels of circulating immune complexes was found. The distribution of insulin-binding levels in serum was similar to that in patients without complications. There was no correlation between insulin binding and the presence of immune complexes and no evidence was found that these complexes contained anti-insulin, anti-nuclear, or organ-specific antibodies. The distribution of insulin-binding levels in these subjects with diabetes of long duration was similar to that observed in 270 subjects with juvenile-onset short-duration type I diabetes. When the data were combined, significant associations between HLA-B8 and low/absent insulin binding levels were observed. HLA-BW62 was not associated with either high or low insulin-binding capacity. It is concluded that HLA genetic factors, insulin-binding capacity, and autoimmunity are unrelated to the pathogenesis of microvascular disease. Raised levels of circulating immune complexes may well be secondary to widespread tissue damage in diabetes of long duration.
Assuntos
Angiopatias Diabéticas/imunologia , Antígenos HLA/genética , Insulina/farmacologia , Adulto , Complexo Antígeno-Anticorpo/análise , Complicações do Diabetes , Diabetes Mellitus/tratamento farmacológico , Retinopatia Diabética/imunologia , Feminino , Antígeno HLA-B7 , Antígeno HLA-B8 , Humanos , Insulina/metabolismo , Masculino , Pessoa de Meia-IdadeAssuntos
Eritrócitos/metabolismo , Purinas/sangue , Adenina/sangue , Adulto , Alopurinol/sangue , Animais , Glicemia/análise , Radioisótopos de Carbono , Difosfatos/sangue , Guanina/sangue , Hemólise , Humanos , Hipoxantinas/sangue , Inosina/sangue , Ribose/sangue , Especificidade da Espécie , Suínos , Fatores de TempoAssuntos
Gota/metabolismo , Hipoxantinas/metabolismo , Pirimidinas/metabolismo , Ácido Úrico/metabolismo , Xantinas/metabolismo , Adulto , Consumo de Bebidas Alcoólicas , Alopurinol/metabolismo , Animais , Radioisótopos de Carbono , Duodeno/metabolismo , Humanos , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , Obesidade/metabolismo , Pirazóis/metabolismo , Especificidade da Espécie , Baço/metabolismo , Compostos de Sulfidrila/metabolismo , SuínosAssuntos
Diabetes Mellitus Tipo 1/etiologia , Modelos Biológicos , Animais , Anticorpos Anti-Idiotípicos/imunologia , Autoimunidade , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Meio Ambiente , Antígenos HLA-DR/genética , Humanos , Idiótipos de Imunoglobulinas/imunologia , MutaçãoRESUMO
1. The metabolism of [1-(14)C]glyoxylate to carbon dioxide, glycine, oxalate, serine, formate and glycollate was investigated in hyperoxaluric and control subjects' kidney and liver tissue in vitro. 2. Only glycine and carbon dioxide became significantly labelled with (14)C, and this was less in the hyperoxaluric patients' kidney tissue than in the control tissue. 3. Liver did not show this difference. 4. The metabolism of [1-(14)C]glycollate was also studied in the liver tissue; glyoxylate formation was demonstrated and the formation of (14)CO(2) from this substrate was likewise unimpaired in the hyperoxaluric patients' liver tissue in these experiments. 5. Glycine was not metabolized by human kidney, liver or blood cells under the conditions used. 6. These observations show that glyoxylate metabolism by the kidney is impaired in primary hyperoxaluria.
Assuntos
Glicina/metabolismo , Glicolatos/metabolismo , Glioxilatos/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Fígado/metabolismo , Adulto , Dióxido de Carbono/metabolismo , Isótopos de Carbono , Criança , Pré-Escolar , Cromatografia por Troca Iônica , Feminino , Humanos , Masculino , Oxalatos/urina , Fatores de TempoRESUMO
Indirect immunofluorescence techniques employing sheep monospecific antisera to human IgG subclasses on unfixed cryostat sections have revealed the IgG subclass distribution in autoantibodies to pancreatic islets (ICA), thyroid epithelial (TMA), gastric parietal (PCA) and adrenal fasciculata (AdA) cells. Whereas antibodies were detected in all four subclasses in 21 of 27 TMA positive sera (mean IgG titre 29 +/- 5 . 2), 13 of 15 PCA (mean IgG titre 41 +/- 3 . 8) and eight of 14 AdA sera (mean IgG titre 10 +/- 3 . 1), only four of 35 ICA positive sera (mean IgG titre 45 +/- 3 . 6) reacted in all four subclasses. Approximately 50% of ICA positive sera showed a restricted polyclonal response to the 'common' pancreatic antigen and 12% of these sera reacted only with IgG2 subclass. The restriction rarely applied to co-existent thyrogastric antibodies in these sera and was independent of the ability of ICA to fix complement. Lesser subclass restrictions were observed in antibody responses to the 'common' antigen of the adrenal cortex.
Assuntos
Autoanticorpos/análise , Imunoglobulina G/classificação , Glândulas Suprarrenais/imunologia , Adulto , Especificidade de Anticorpos , Testes de Fixação de Complemento , Feminino , Humanos , Ilhotas Pancreáticas/imunologia , Masculino , Pessoa de Meia-Idade , Estômago/imunologia , Glândula Tireoide/imunologiaRESUMO
Biochemical changes in the blood following induction of renal failure by glycerol or mercuric chloride have been studied in 16 rats. Plasma creatinine, urea and Pi levels indicated that renal impairment followed the same time course in both renal failure models, with the severest effects on day 3 and returning to normal by day 7. Erythrocyte ATP and guanine triphosphate (GTP) levels were significantly elevated above contorl values on day 1 and remained elevated in both models. ATP/ADP and GTP/GDP ratios also increased in both models. In renal failure the increased purine 'salvage' in the erythrocyte may be attributed to accumulation of purine metabolites in the serum associated with increased P-ribose-PP levels due to elevated cellular Pi. Nucleotide changes in both these models are analogous to those found in chronic renal failure in man.
Assuntos
Injúria Renal Aguda/sangue , Eritrócitos/metabolismo , Glicerol/farmacologia , Mercúrio/farmacologia , Nucleotídeos/sangue , Injúria Renal Aguda/induzido quimicamente , Difosfato de Adenosina/sangue , Monofosfato de Adenosina/sangue , Trifosfato de Adenosina/sangue , Animais , Cloretos/farmacologia , Creatinina/sangue , Modelos Animais de Doenças , Feminino , Guanosina Difosfato/sangue , Guanosina Trifosfato/sangue , Fosfatos/sangue , Ratos , Ureia/sangueRESUMO
1 The metabolites of thiopurinol have been investigated in intact human and pig erythrocytes using [6-(14)C]-thiopurinol, high voltage electrophoresis and automated cation exchange chromatography. 2 Pre-incubation of human erythrocytes in vitro with thiopurinol increased the formation of hypoxanthine from [8-(14)C]-inosine and simultaneously reduced IMP synthesis. 3 Reduced inosine formation, following one week of thiopurinol therapy, has also been observed in vitro using the intact red cells from three patients suffering from gout. 4 Binding of thiopurinol, and to lesser extents of 6-mercaptopurine, uric acid, oxipurinol and allopurinol, has been demonstrated electrophoretically to human and pig serum proteins. 5 Thiopurinol is shown to have a greater binding capacity for purified human serum albumin than uric acid or 6-mercaptopurine at varying substrate and albumin concentrations covering the physiological range. Under these conditions the binding of uric acid is reduced in the presence of thiopurinol. 6 A comparison of the distribution of thiopurinol, 6-mercaptopurine, allopurinol and oxipurinol in whole blood has revealed that allopurinol and oxipurinol are not irreversibly bound to cellular proteins; whereas 30% and 13% of the total cellular uptake was irreversibly bound in the case of thiopurinol and 6-mercaptopurine respectively at substrate levels of 1 nmol per µl blood.
RESUMO
The prevalences of autoimmune endocrine disease and relevant organ-specific autoantibodies were determined in 141 patients with type I (insulin-dependent) diabetes and their families. All available members of the families were genotyped for HLA. Islet-cell antibody was found in 10 (4%) out of 248 unaffected siblings, all of whom were genetically potential cases of diabetes. One developed classical symptoms six months later. In contrast, thyroid and gastric parietal-cell antibodies occurred independent of the HLA-linked susceptibility to diabetes. These results suggest that different genes control the production of these autoantibodies and the susceptibility to type I diabetes.
Assuntos
Autoanticorpos/análise , Diabetes Mellitus/imunologia , Genes MHC da Classe II , Estômago/imunologia , Glândula Tireoide/imunologia , Adolescente , Adulto , Doenças Autoimunes/genética , Criança , Pré-Escolar , Diabetes Mellitus/genética , Feminino , Antígenos HLA/análise , Humanos , Lactente , Ilhotas Pancreáticas/imunologia , MasculinoRESUMO
Evidence is presented for the existence of a separate species of islet-cell antibodies which fix complement. Investigations in type I diabetics, non-diabetic polyendocrine patients, and unaffected first-degree relatives of type I diabetic probands show that the complement-fixing islet-cell antibodies are more closely related to the onset of clinical disease than the conventional islet-cell antibody, and they tend to disappear more rapidly. The complement-fixing antibodies may reflect damage of pancreatic beta cells more selectively and may be preferable to the conventional antibody as a serological marker for studying the natural history of type I diabetes.
Assuntos
Autoanticorpos/análise , Proteínas do Sistema Complemento/imunologia , Diabetes Mellitus/imunologia , Ilhotas Pancreáticas/imunologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Complemento C1/imunologia , Complemento C3/imunologia , Complemento C4/imunologia , Testes de Fixação de Complemento , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/genética , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Children with newly diagnosed insulin-dependent diabetes mellitus (IDDM) had increased numbers of CD25 positive lymphocytes in peripheral blood and peripheral blood mononuclear cells responded to insulin antigens by proliferation. The CD25 positivity and insulin proliferation were associated to the duration of symptoms before the diagnosis of IDDM. Thus increased numbers of CD25 positive cells were found in 89% and insulin induced proliferation in 100% of patients with symptoms of diabetes of less than 1 week's duration before diagnosis, while CD25 positivity and insulin-induced proliferation were observed in 36% and 29% of children who had had symptoms for 4 weeks or more before diagnosis. Children with IDDM also had increased numbers of CD4 positive T cells in peripheral blood. The frequency of HLA-DR4 and HLA-DR3/4 in diabetic children was higher and that of HLA-DR2 lower than in the normal population. Insulin, islet cell, gastric-parietal cell, thyroid and antinuclear antibodies did not correlate to the duration of symptoms before diagnosis.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Insulina/imunologia , Linfócitos T/classificação , Adolescente , Autoanticorpos/análise , Divisão Celular , Criança , Pré-Escolar , Feminino , Antígenos HLA/análise , Antígenos HLA-B , Antígenos HLA-DR/análise , Humanos , Leucócitos Mononucleares/patologia , Ativação Linfocitária , MasculinoRESUMO
Use of monoclonal antibodies specific for rat lymphocyte subsets and an anti-insulin marker has allowed us to document the following sequence of events leading to the development of clinical diabetes in this animal model. The first change observed in the pancreas is increased expression of MHC class II molecules on vascular endothelium and this precedes lymphocytic infiltration. Next, T cells of the T helper phenotype infiltrate the pancreas around blood vessels. Many of the infiltrating T cells show class II expression indicating that they are activated. A few cytotoxic and suppressor cells and B lymphocytes are also present and their numbers increase proportionately with rat age. Some macrophages are also seen. Finally, at a late stage class II MHC molecules can be detected in partially destroyed islets on beta cells which are still actively synthesising insulin. We have never observed expression of class II molecules on glucagon or somatostatin secreting cells which are invariably well preserved.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Antígenos de Histocompatibilidade Classe II/análise , Ilhotas Pancreáticas/imunologia , Linfócitos/imunologia , Ratos Mutantes/imunologia , Animais , Anticorpos Monoclonais , Linfócitos/classificação , Complexo Principal de Histocompatibilidade , Pâncreas/imunologia , RatosRESUMO
A cohort of BB/E rats derived from litters with a high and low incidence of IDDM was studied prospectively to examine the relationship between circulating autoantibodies, islet insulin secretion, pancreatic infiltration, and islet cell replication during the pre-diabetic period. Although a higher incidence of islet cell surface (ICSA) and insulin autoantibodies (IAA) was detected in the diabetes-prone than in the low diabetic-incidence BB/E rats there was no correlation between the two antibodies in individual animals. Moreover, ICSA, but not IAA, were associated with loss of first phase islet insulin release. Between 75 and 105 days of age the number of diabetes-prone rats with ICSA and impaired islet insulin secretory function increased. Over the same period, there was a concomitant increase in the proportion of diabetes-prone animals with pancreatic infiltration, and increased islet endocrine cell proliferation. All these interrelated phenomena were observed in diabetes-prone BB/E rats at a time when the animals were normoglycaemic.
Assuntos
Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/patologia , Animais , Autoanticorpos/análise , Autorradiografia , Divisão Celular , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Insulina/metabolismo , Anticorpos Anti-Insulina/análise , Secreção de Insulina , Ilhotas Pancreáticas/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos BBRESUMO
Complement-fixing islet-cell antibodies (CF-ICA) were found in 20 out of 685 unaffected first-degree relatives of children with type 1 diabetes. During a 5-year follow-up, 7 of the 20 became diabetic, 1 continued to show the antibodies without any abnormality of glucose tolerance, and 12 subjects lost them without the disease developing. Although CF-ICA are useful as a marker of active insulitis they should not at present be used to define subjects who might benefit from preventive immunosuppression.
Assuntos
Anticorpos/análise , Doenças Autoimunes/genética , Diabetes Mellitus Tipo 1/genética , Ilhotas Pancreáticas/imunologia , Testes de Fixação de Complemento , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Suscetibilidade a Doenças , Meio Ambiente , Seguimentos , Genótipo , Teste de Tolerância a Glucose , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Terapia de Imunossupressão , Ilhotas Pancreáticas/microbiologia , Estudos ProspectivosRESUMO
The observations emerged from the pancreatic transplant experiments in identical twins indicate that Type I diabetic patients maintain memory cytotoxic T cells for several years and these can be "re-awakened" when Class I identical beta cells are re-introduced into the diabetic melieu. In addition, these data have shown that these lymphocytes can (in a matter of a few weeks) cause rapid irreversible decompensation of beta cell function. From this, an important lesson is learnt: cytotoxic T cells, when generated in sufficient number against beta cells do not leave much "breathing space" for these cells. This has important implications for explaining the long latency period preceding the acute onset of Type I diabetes. ICA, when produced, seem unable to cause gross damage to beta cells. They persist for several years in the blood, but beta cell function remains apparently unaltered. It is only when cytotoxic T cells, with fine specificity for beta cells are generated that the 'killing cycle' is completed. Whether these cells are present all the time, and kept under tight control by active suppressor mechanisms or whether they appear only after an environmental trigger (e.g., retroviruses) is unknown. If the former is the case, this would give strong support to the suggested important role of suppressor T cells in the pathogenetic circuit. Some evidence for this has been produced (rev. in [116]) but, obviously, it requires confirmation (see debate which followed [116]). If, on the other hand, the latter is experimentally confirmed, one can return to the theory that cytotoxic T cells acquire the characteristics of autoreactivity by expressing receptors on their surface in a configuration which enables combination with self-autoantigens (rev. in [45]). In summary, the study of the etiopathogenesis of Type I diabetes and human autoimmunity in general has attracted a great deal of interest among immunologists. It is only by further dissecting the various limbs of the undesirable immune response against beta cells and, by trying to formulate novel hypotheses, sometimes against accepted dogmas [32], that the complete picture will be finally disclosed. At this stage it will be possible to design effective therapy trials, so that Type I diabetes and other related autoimmune disorders ultimately may be prevented.