RESUMO
In coeliac disease (CD), anti-tissue transglutaminase 2 immunoglobulin (Ig)A antibodies (anti-TG2) are produced and deposited in the intestine. PreventCD (www.preventcd.com) is a European multi-centre study, which investigates the influence of infant nutrition and that of genetic, immunological and other environmental factors on the risk of developing CD. The aim of the current study was to evaluate the appearance of intestinal anti-TG2 deposits in very early intestinal biopsies from at-risk infants and their predictive value for villous atrophy. Sixty-five small bowel biopsies, performed in 62 children, were investigated for the presence of intestinal anti-TG2 extracellular IgA deposits by using double immunofluorescence. The biopsies were performed in the presence of elevated serum levels of CD-associated antibodies and/or symptoms suggesting disease. Deposits of anti-TG2 IgA were present in 53 of 53 CD patients and three of three potential CD patients. In potential CD patients, mucosal deposits showed a patchy distribution characterized by some areas completely negative, whereas active CD patients had uniformly present and evident mucosal deposits. Only one of six patients without CD (negative for serum anti-TG2 and with normal mucosa) had intestinal deposits with a patchy distribution and a weak staining. Two of the 53 CD patients received a definitive diagnosis of CD after a second or third biopsy; mucosal deposits of anti-TG2 IgA were evaluated in all samples. Before developing villous atrophy, both patients had anti-TG2 deposits in normal mucosal architecture, antibodies in one patient being absent in serum. We demonstrated that in CD the intestinal deposits of anti-TG2 are a constant presence and appear very early in the natural history of disease.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Autoanticorpos/metabolismo , Doença Celíaca/imunologia , Proteínas de Ligação ao GTP/imunologia , Imunoglobulina A/metabolismo , Mucosa Intestinal/imunologia , Transglutaminases/imunologia , Atrofia , Biópsia , Doença Celíaca/diagnóstico , Criança , Pré-Escolar , Progressão da Doença , Europa (Continente) , Feminino , Humanos , Lactente , Mucosa Intestinal/patologia , Masculino , Prognóstico , Proteína 2 Glutamina gama-Glutamiltransferase , Fatores de RiscoRESUMO
It has always been known that anti-tissue transglutaminase 2 (anti-TG2) antibodies are produced in the small intestine. Their serum titres correlate with mucosal damage degree and decrease on a gluten-free diet (GFD). We aimed to correlate intestinal anti-TG2 antibodies levels with degree of mucosal damage and GFD duration. Thirty-four active, 71 potential and 24 CD patients on GFD for at least 2 years were enrolled. Anti-TG2 deposits were detected in intestinal biopsies by double immunofluorescence. Biopsies were cultured for 24 h with medium, and with gliadin peptic tryptic digest (PTG) or A-gliadin peptide 31-43 (P31-43). Anti-TG2 antibodies secreted into supernatants were measured by enzyme-linked immunosorbent assay (ELISA). All active CD patients secreted high titres of anti-TG2 antibodies into culture medium that increased with the worsening of mucosal injury (Spearman's r = 0·71; P < 0·0001). Seventy of 71 potential CD patients and 15 of 24 treated CD patients secreted low titres of anti-TG2 antibodies into supernatants, eight of nine negative treated patients being on GFD for more than 10 years. An inverse correlation between antibody titres and duration of GFD was found, (Spearman's r = -0·52; P < 0·01). All active, 53 of 71 potential and six of 24 treated, CD patients showed anti-TG2 mucosal deposits. Five of six positive treated CD patients had been on GFD for fewer than 6 years and were also positive for secreted anti-TG2. In treated patients, PTG/P31-43 was not able to induce secretion of anti-TG2 antibodies into culture medium. Measurement of anti-TG2 antibodies in biopsy supernatants proved to be more sensitive than detection by immunofluorescence to reveal their intestinal production. Intestinal antiTG2 antibodies titres correlated positively with the degree of mucosal damage and inversely with the duration of GFD.
Assuntos
Autoanticorpos/imunologia , Doença Celíaca/imunologia , Dieta Livre de Glúten , Proteínas de Ligação ao GTP/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Transglutaminases/imunologia , Adolescente , Adulto , Autoanticorpos/sangue , Biomarcadores/sangue , Biomarcadores/metabolismo , Biópsia , Doença Celíaca/sangue , Doença Celíaca/metabolismo , Criança , Pré-Escolar , Humanos , Imunoglobulina A Secretora/imunologia , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Adulto JovemRESUMO
Anti-tissue transglutaminase 2 (anti-TG2) antibodies are present in the serum of the great majority of untreated coeliac disease (CD) patients. They are produced and deposited in the small intestinal mucosa. Potential CD patients present serum anti-TG2 antibodies higher than cut-off, but a normal duodenal mucosa where mucosal deposits of anti-TG2 are not always detectable. The aim of our work was to investigate the presence of anti-TG2 intestinal antibodies in patients with potential CD, and identify the most sensitive test to detect them. Twelve active CD patients, 28 potential CD patients and 39 non-CD controls were enrolled. Biopsy fragments from all patients were analysed by double immunofluorescence to detect mucosal deposits of anti-TG2 antibodies. Fragments from the same subjects were also cultured for 24 h with medium in the presence or absence of gliadin peptides. Anti-TG2 autoantibodies secreted into supernatants were measured by enzyme-linked immunosorbent assay. All active CD, 68% of potential CD patients and 20% of non-CD controls showed mucosal deposits of immunoglobulin (Ig)A anti-TG2; at the same time 100, 96 and 8% of active CD, potential CD and non-CD control patients secreted these antibodies in culture supernatants, respectively. Our data showed that, to detect intestinal anti-TG2 antibodies, the measurement of antibodies secreted into culture supernatants has higher sensitivity and specificity (97·5 and 92·3%, respectively) than the detection of mucosal deposits (77·5 and 80·0%, respectively). The measurement of intestinal anti-TG2 antibodies may prove useful in clinical practice to predict evolution towards mucosal atrophy in potential coeliac patients and identify patients with gluten sensitivity.
Assuntos
Autoanticorpos/análise , Doença Celíaca/diagnóstico , Proteínas de Ligação ao GTP/imunologia , Intestino Delgado/imunologia , Transglutaminases/imunologia , Adolescente , Autoanticorpos/imunologia , Biópsia , Doença Celíaca/imunologia , Doença Celíaca/patologia , Células Cultivadas , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Gliadina/imunologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Masculino , Proteína 2 Glutamina gama-Glutamiltransferase , Sensibilidade e EspecificidadeRESUMO
The diagnosis of coeliac disease (CD) represents a special challenge in selective immunoglobulin (Ig)A deficiency (IgAD). A high density of T cell receptor (TCR)gammadelta(+) intraepithelial lymphocytes (IELs) and intestinal IgA anti-tissue transglutaminase 2 (anti-TG2) antibody deposits are suggestive of CD. We analysed the density of TCRgammadelta(+) IELs and the deposition of IgM anti-TG2 antibodies in the jejunal mucosa of IgAD patients with and without CD. Immunohistochemical analyses for the number of CD3+ and TCRgammadelta(+) IELs and double immunofluorescence assay for IgM anti-TG2 antibody deposits were performed in biopsies from 25 children with IgAD (nine untreated CD, seven potential CD and nine without CD). Sixteen immunologically intact children without CD represented the controls. IgAD without CD had a higher number of CD3+ and TCRgammadelta(+) IELs than controls (P < 0.05), but lower than IgAD with CD (P < 0.01). No significant differences were noted between IgAD subjects without CD and those with potential CD. Furthermore, IgAD patients without CD showed a higher TCRgammadelta(+)/CD3+ ratio than the control group (P < 0.05), while the ratio was similar to subjects with CD and potential CD. Intestinal IgM anti-TG2 antibody deposits were present in six of seven of the IgAD patients with untreated CD, one of seven with potential CD and none of those without CD. Most of the patients with IgAD show immune activation in the jejunal mucosa. IgM anti-TG2 antibody deposits are present only in CD. Intestinal IgM anti-TG2 and immunohistochemical markers do not discriminate between IgAD and potential CD with IgAD. Therefore, the serum IgG CD-associated autoantibodies remains very important for the diagnosis of CD in IgAD.
Assuntos
Autoanticorpos/análise , Autoantígenos/imunologia , Doença Celíaca/imunologia , Deficiência de IgA/imunologia , Imunoglobulina M/análise , Jejuno/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/patologia , Transglutaminases/imunologia , Autoanticorpos/imunologia , Biópsia , Doença Celíaca/diagnóstico , Doença Celíaca/etiologia , Doença Celíaca/patologia , Criança , Pré-Escolar , Epitélio/imunologia , Epitélio/patologia , Feminino , Proteínas de Ligação ao GTP , Antígenos HLA-DR/análise , Humanos , Deficiência de IgA/complicações , Deficiência de IgA/patologia , Imunoglobulina M/imunologia , Lactente , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Jejuno/patologia , Masculino , Proteína 2 Glutamina gama-Glutamiltransferase , Subpopulações de Linfócitos T/imunologia , Adulto JovemRESUMO
By GWAS studies on celiac disease, gene expression was studied at the level of the whole intestinal mucosa, composed by two different compartments: epithelium and lamina propria. Our aim is to analyse the gene-expression and DNA methylation of candidate genes in each of these compartments. Epithelium was separated from lamina propria in biopsies of CeD patients and CTRs using magnetic beads. Gene-expression was analysed by RT-PC; methylation analysis required bisulfite conversion and NGS. Reverse modulation of gene-expression and methylation in the same cellular compartment was observed for the IL21 and SH2B3 genes in CeD patients relative to CTRs. Bioinformatics analysis highlighted the regulatory elements in the genomic region of SH2B3 that altered methylation levels. The cREL and TNFAIP3 genes showed methylation patterns that were significantly different between CeD patients and CTRs. In CeD, the genes linked to inflammatory processes are up-regulated, whereas the genes involved in the cell adhesion/integrity of the intestinal barrier are down-regulated. These findings suggest a correlation between gene-expression and methylation profile for the IL21 and SH2B3 genes. We identified a "gene-expression phenotype" of CeD and showed that the abnormal response to dietary antigens in CeD might be related not to abnormalities of gene structure but to the regulation of molecular pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Doença Celíaca/patologia , Metilação de DNA , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Interleucinas/genética , Adolescente , Biópsia , Doença Celíaca/genética , Criança , Pré-Escolar , Duodeno/química , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Mucosa Intestinal/química , Masculino , Proteínas Proto-Oncogênicas c-ret/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genéticaRESUMO
Celiac disease (CeD) is a multifactorial disease influenced by both genetic and environmental risk factors. CeD genetic components are mainly due to HLA class II genes, which account for approximately 40% of the disease heritability. The environmental factor is linked to gliadin ingestion. Despite genetic and epigenetic studies, the pathological molecular mechanism remains unclarified. The strong genetic component does not explain more than half of the hereditability; we identified several epigenetic features that contribute to the understanding of the missing hereditability. The lipid profile of infants has been proposed as a potential biomarker of CeD metabolism that can be measured before they exhibit developmental disorders and clinical symptoms. We suggest that the state of the host is a main factor for the abnormal immune response to gluten. Long before any exposure to the offending agent or any production of specific antibodies, several molecular mechanisms are differentially expressed in infants who will develop CeD compared to their peers matched for the same genetic profile. The present study explored the serum phospholipid profile of a group of infants at risk for celiac disease, followed up to 8 years to monitor the onset of CeD. We compared 30 patients who developed the disease with 20 age- and sex-matched peers with similar genetic profiles who did not develop the disease within 8 years. Serum phospholipids were analysed at 4 months, before exposure to gluten, and at 12 months of age, when none showed any marker of disease. In the 30 CeD patients, we also analysed the serum at the time of diagnosis (>24 months). The serum phospholipid profile was fairly constant across 4 and 12 months of age and, in CeD, up to 24-36 months. The phospholipid signature was dramatically different in infants who developed CeD when compared to that of control NY-CeD (Not Yet developing Celiac Disease) peers. We identified a specific serum phospholipid signature that predicts the onset of celiac disease in HLA at-risk infants years before the appearance of antibodies specific for CeD in the serum and before any clinical symptoms, even before gluten introduction into the diet at 4 months. Specifically, lysophosphatidylcholine, phosphatidylcholine, alkylacyl-phosphatidylcholine, phosphoethanolamines, phosphatidylserines, phosphatidylglycerol and phosphatidylinositol were found to be differentially represented in CeD versus NY-CeD. A set constituted by a limited number of alkylacyl-phosphatidylcholine and lyso-phosphatidylcholine, together with the duration of breast-feeding, allows the discrimination of infants who develop celiac disease before 8 years of age from those at a similar genetic risk who do not develop the disease. In addition to recent discovery, our paper unveiled a specifc phopholipid profile, able to discriminate infants who eventually develop celiac disease years before antibodies or clinical symptoms ensue.
Assuntos
Doença Celíaca/diagnóstico , Testes Diagnósticos de Rotina , Fosfolipídeos/sangue , Biomarcadores/sangue , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Glutens/imunologia , Humanos , Lactente , Lipidômica , Masculino , Fatores de RiscoRESUMO
BACKGROUND: A gluten-free diet is currently the only reliable therapeutic strategy that is approved for coeliac disease (CD). For many patients, however, compliance remains inadequate. AIM: To investigate the immunogenicity of wheat flour that was pre-treated with selected lactobacilli and fungal proteases (hydrolysed wheat gluten) in coeliac patients. METHODS: The immunogenicity of hydrolysed wheat gluten was evaluated both in vitro in intestinal T cell lines (TCLs) and in vivo in treated CD patients after a short-term gluten challenge. Twenty treated CD patients were enrolled and equally randomised into two groups. The patients ate bread that was prepared with hydrolysed wheat flour or natural wheat flour (10 g of gluten/d for 3 days). The interferon (INF)-γ responses to natural gliadin and a 33-mer peptide were assessed by the enzyme-linked immunospot (ELISPOT) assay on peripheral blood mononuclear cells (PBMCs) both before and 6 days after the start of the challenge. RESULTS: Hydrolysed wheat was not able to activate the TCLs from the coeliac intestinal mucosa. Consistent with the in vitro results, no significant increase in INF-γ secretion was observed in patients who consumed hydrolysed wheat flour. Conversely, the consumption of natural wheat gluten mobilised INF-γ secreting cells in the blood (P<.05). CONCLUSIONS: We confirm that fermentation of wheat flour with sourdough lactobacilli and fungal proteases is capable of abolishing the T cell immunogenicity of gluten in coeliac patients. Our data also validate the short-term oral challenge as a useful tool for testing the efficacy of novel therapeutic approaches.
Assuntos
Doença Celíaca/dietoterapia , Farinha , Glutens/administração & dosagem , Triticum , Adolescente , Pão , Criança , Dieta Livre de Glúten , Feminino , Fermentação , Fungos/metabolismo , Gliadina/metabolismo , Humanos , Interferon gama/imunologia , Mucosa Intestinal/metabolismo , Lactobacillus , Leucócitos Mononucleares/metabolismo , Masculino , Linfócitos T/metabolismoRESUMO
BACKGROUND: Small bowel biopsy is the gold standard for Celiac Disease (CD) diagnosis, nevertheless serum assays are the first step in ascertaining a diagnosis of CD. New ESPGHAN Criteria 2012 (European Society of Pediatric Gastroenterology Hepatology and Nutrition) suggest using exclusively anti-tissue Transglutaminase IgA antibodies (anti-tTGA) as initial approach to symptomatic subjects. The aim of our study was to evaluate the diagnostic accuracy of anti-tTGA as initial screening assay for CD in a large cohort of pediatric patients. METHODS: We selected 730 subjects aged between 6 months and 4 years ("Group A") and 348 subjects younger than 2 years (which are part of the 730 subjects) ("Group B"). We performed anti-Deamidated Gliadin Peptides IgA and IgG antibodies (a-DGP IgA/IgG) and anti-tTGA assays by ELISA test. We evaluated the agreement between anti-tTGA and a-DGP IgA/IgG assays and compared the diagnostic accuracy of a-DGP IgA/IgG with that of anti-tTGA in both groups of patients. RESULTS: There was a substantial agreement between anti-tTGA and a-DGP IgA in "Group A" and an almost perfect agreement in "Group B"; the strength of agreement between anti-tTGA and a-DGP IgG was moderate in "Group A" and substantial in "Group B". anti-tTGA were more sensitive and specific than a-DGP IgA/IgG in both groups. CONCLUSIONS: anti-tTGA could be used as initial screening assay for CD in all subjects from 6 months of age according to ESPGHAN Criteria 2012.
Assuntos
Autoanticorpos/sangue , Doença Celíaca/diagnóstico , Proteínas de Ligação ao GTP/sangue , Imunoglobulina A/química , Imunoglobulina G/química , Transglutaminases/sangue , Doença Celíaca/sangue , Doença Celíaca/imunologia , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/imunologia , Gliadina/química , Gliadina/imunologia , Humanos , Lactente , Recém-Nascido , Masculino , Triagem Neonatal , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Proteína 2 Glutamina gama-Glutamiltransferase , Sensibilidade e Especificidade , Transglutaminases/antagonistas & inibidores , Transglutaminases/imunologiaRESUMO
BACKGROUND: New evidence emerged on early feeding practices and the risk of coeliac disease. AIM: To systematically update evidence on these practices to find out whether there is a need to revise current recommendations. METHODS: MEDLINE, EMBASE and the Cochrane Library were searched from July 2012 (end of last search) to February 2015 for studies of any design that assessed the effect of gluten consumption and breastfeeding on the development of coeliac disease and/or coeliac disease-related autoimmunity. RESULTS: We identified 21 publications, including two, new, large, randomised controlled trials performed in high-risk infants. Exclusive or any breastfeeding, as well as breastfeeding at the time of gluten introduction, did not reduce the risk of developing coeliac disease during childhood. For infants at high risk of developing coeliac disease, gluten introduction at 4 months of age in very small amounts, or at 6 or 12 months of age, resulted in similar rates of coeliac disease diagnosis in early childhood. Later gluten introduction was associated with later development of coeliac specific autoimmunity and coeliac disease during childhood, but not total risk reduction. Observational studies indicate that consumption of a higher amount of gluten at weaning may increase the risk for coeliac disease development. CONCLUSIONS: Infant feeding practices (breastfeeding, time of gluten introduction) have no effect on the risk of developing coeliac disease during childhood (at least at specific timeframes evaluated in the included studies), necessitating an update of current European recommendations.
Assuntos
Aleitamento Materno , Doença Celíaca/epidemiologia , Comportamento Alimentar/fisiologia , Doença Celíaca/etiologia , Glutens/administração & dosagem , Glutens/efeitos adversos , Humanos , Lactente , Fatores de Tempo , DesmameRESUMO
A case of superior vena cava obstruction due to carcinoma of unknown origin is reported. A superior vena cava bypass with polytetrafluoroethylene was performed by suturing the prosthesis to the left innominate vein and the right atrium, respectively. Long-term satisfactory results were achieved.
RESUMO
BACKGROUND: PREVENTCD, Prevent Coeliac Disease, is an international project investigating the hypothesis of possible induction of tolerance to gluten in genetically predisposed children through introducing small quantities of gluten during the period of breastfeeding. AIM: To summarise current knowledge on the possible relationship between early feeding practices and the risk of coeliac disease (CD). METHODS: The Cochrane Library, MEDLINE, and EMBASE databases were searched in May 2011, and the search was updated in January 2012, and again in July 2012. RESULTS: Breastfeeding (BF) and CD: some studies show a protective effect of BF, while others show no effect. No studies have shown a long-term preventive effect. BF at the time of gluten introduction and CD: Results from a meta-analysis of five observational case-control studies suggest that BF at gluten introduction is associated with a lower risk of CD compared with formula feeding. It is unclear whether BF provides a permanent protection or only delays the onset of CD. Timing of gluten introduction: The data suggest that both early (≤4 months) and late (≥7 months) introduction of gluten may increase the risk of CD. Amount of gluten at weaning (and later) and CD: One incident case-referent study documented that the introduction of gluten in large amounts compared with small or medium amounts increased the risk of CD. CONCLUSIONS: In the absence of clear evidence, in order to decrease the risk of later coeliac disease, it is reasonable to avoid both early (<4 months) and late (≥7 months) introduction of gluten, and to introduce gluten while the infant is still being breastfed. Future studies may clarify the remaining uncertainties.
Assuntos
Aleitamento Materno/métodos , Doença Celíaca/prevenção & controle , Estudos de Casos e Controles , Doença Celíaca/etiologia , Doença Celíaca/fisiopatologia , Predisposição Genética para Doença , Glutens/administração & dosagem , Glutens/efeitos adversos , Humanos , Lactente , Fatores de Risco , Fatores de Tempo , DesmameRESUMO
BACKGROUND: An association between coeliac disease (CD) and functional gastrointestinal disorders (FGIDs) has at present only been demonstrated in adults. AIMS: To assess the prevalence of FGIDs at 1 year and the role of psychological aspects on the development of FGIDs in CD children. METHODS: One-hundred consecutive CD children (36M and 64F) were followed up for 1 year. Fifty-six children (25M and 31F) represented the control group. All children and/or their parents completed validated questionnaires for GI symptoms, depression, and anxiety. GI symptoms at diagnosis and after 1 year of gluten-free diet (GFD) were compared. RESULTS: Twenty-three/82 (28%) CD patients followed up prospectively, on GFD from at least 1 year, fulfilled the Rome III criteria for FGIDs compared with 5/56 (8.9%) controls (P = 0.008; χ² = 6.8; OR: 3.97; 95% CI: 1.40-11.21). Children complaining with GI symptoms alone [21/52 (40.3%)] more likely fulfilled Rome III criteria for FGIDs after 1 year of GFD than children with extra-intestinal symptoms (P = 0.045). CD children with FGDIs presented significantly higher anxiety and depression compared to CD children without FGIDs and controls (P = 0.02). CONCLUSIONS: This study demonstrates that children with CD on a GFD for a year have a much higher prevalence of functional GI symptoms than do controls. Whether the risk is due to the residua of a chronic inflammatory process, and/or due to psychological factors remains to be further tested.
Assuntos
Doença Celíaca/complicações , Dieta Livre de Glúten , Gastroenteropatias/etiologia , Dor Abdominal , Adolescente , Fatores Etários , Estudos de Casos e Controles , Doença Celíaca/dietoterapia , Doença Celíaca/fisiopatologia , Criança , Pré-Escolar , Feminino , Gastroenteropatias/dietoterapia , Gastroenteropatias/fisiopatologia , Humanos , Itália , Modelos Logísticos , Masculino , Prontuários Médicos , Estudos ProspectivosRESUMO
Subfraction 2R of fraction 9 from a peptic-tryptic-pancreatic digest of wheat gliadin is known to be toxic in vivo to celiac patients. We have found that fractions 9 and 2R inhibit the in vitro development of fetal rat intestine and the increase of enterocyte height occurring in organ culture of atrophic celiac mucosa (0.1-0.5 mg/ml medium). Other peptide fractions of the gliadin digest are devoid of such in vitro effects. Subfraction 2R, after incubation with morphologically normal small intestinal mucosa of celiacs in remission and ultrafiltration, was still very active in both culture systems at low concentration (0.1 mg/ml); on the contrary, subfraction 2R was inactivated after incubation with normal mucosa. These results are compatible with the hypothesis that there is a mucosal defect in handling gliadin peptides in celiac disease, and suggest that there is either a primary (or secondary) enzyme deficiency or some other mechanism operating in the intestinal mucosa of celiac patients in remission.
Assuntos
Doença Celíaca/patologia , Gliadina/farmacologia , Intestino Delgado/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Animais , Atrofia , Feto , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Jejuno/patologia , Técnicas de Cultura de Órgãos , Ratos , Ratos EndogâmicosRESUMO
Insulin increased protein kinase C (PKC) activity by 2-fold in both membrane preparations and insulin receptor (IR) antibody precipitates from NIH-3T3 cells expressing human IRs (3T3hIR). PKC-alpha, -delta, and -zeta were barely detectable in IR antibody precipitates of unstimulated cells, while increasing by 7-, 3.5-, and 3-fold, respectively, after insulin addition. Preexposure of 3T3hIR cells to staurosporine reduced insulin-induced receptor coprecipitation with PKC-alpha, -delta, and -zeta by 3-, 4-, and 10-fold, respectively, accompanied by a 1.5-fold decrease in insulin degradation and a similar increase in insulin retroendocytosis. Selective depletion of cellular PKC-alpha and -delta, by 24 h of 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure, reduced insulin degradation by 3-fold and similarly increased insulin retroendocytosis, with no change in PKC-zeta. In lysates of NIH-3T3 cells expressing the R1152Q/K1153A IRs (3T3Mut), insulin-induced coprecipitation of PKC-alpha, -delta, and -zeta with the IR was reduced by 10-, 7-, and 3-fold, respectively. Similar to the 3T3hIR cells chronically exposed to TPA, untreated 3T3Mut featured a 3-fold decrease in insulin degradation, with a 3-fold increase in intact insulin retroendocytosis. Thus, in NIH-3T3 cells, insulin elicits receptor interaction with multiple PKC isoforms. Interaction of PKC-alpha and/or -delta with the IR appears to control its intracellular routing.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Receptor de Insulina/metabolismo , Células 3T3 , Animais , Transporte Biológico , Regulação para Baixo , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Humanos , Camundongos , Testes de Precipitina , Proteína Quinase C/antagonistas & inibidores , Receptor de Insulina/química , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Eosinophils may be involved in the pathogenesis of inflammation in inflammatory bowel disease. The purpose of this study was to verify whether concentrations of eosinophilic cationic protein in gut lavage fluid from children with inflammatory bowel disease correlate with clinical and laboratory indexes of disease activity. METHODS: Twenty-three children with Crohn's disease, 14 with ulcerative colitis, and 22 age-matched control subjects entered the study. Radioimmunoassay and sandwich enzyme-linked immunosorbent assay techniques were used to measure eosinophilic cationic protein, total immunoglobulin G and interleukin-1beta, respectively. RESULTS: Gut lavage eosinophilic cationic protein levels were significantly (p < 0.005) higher in patients with Crohn's disease and ulcerative colitis than in control subjects. Intestinal eosinophilic cationic protein levels decreased in three of four children with Crohn's disease who were fed an elemental diet. There was a significant (p < 0.001) correlation between eosinophilic cationic protein concentrations and immunoglobulin G and interleukin-1beta levels in gut lavage fluid. CONCLUSIONS: Elevated intestinal eosinophilic cationic protein levels in inflammatory bowel disease suggest that eosinophils are involved in the gastrointestinal inflammation in this disease. Intestinal eosinophilic cationic protein concentration is another marker with which to discriminate between active and inactive inflammatory bowel disease.
Assuntos
Proteínas Sanguíneas/metabolismo , Mediadores da Inflamação/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Ribonucleases , Irrigação Terapêutica , Adolescente , Criança , Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Proteínas Granulares de Eosinófilos , Feminino , Humanos , MasculinoRESUMO
OBJECTIVE: Tissue transglutaminase (tTG) is the main autoantigen recognized by endomysial antibodies. The aim of this study was to assess sensitivity, specificity, and predictive value of IgA and IgG antibodies to tTG in the diagnosis of celiac disease compared with endomysial antibodies. STUDY DESIGN: We established enzyme-linked immunosorbent assay procedures to measure IgA and IgG antibodies to tTG in sera from 48 untreated and 33 treated patients with celiac disease and from 63 patients with gastrointestinal disease who were in a control group. Sera from 10 patients with celiac disease were examined at various times after gluten was reintroduced into the patients' diet. RESULTS: Both IgA and IgG to tTG were significantly (P <.001) higher in serum of untreated patients with celiac disease versus those in the control group; IgA but not IgG was significantly (P <.001) higher in untreated versus treated patients with celiac disease. IgA and IgG antitissue tTG had a diagnostic sensitivity, specificity, and positive predictive value of 92% and 21%, 98% and 97%, and 98% and 83%, respectively. The concordance rate of IgA anti-tTG with IgA antiendomysial antibodies was 95%. In 5 of the 10 patients undergoing gluten challenge, IgA antiendomysium antibodies were detected earlier than IgA anti-tTG antibodies. CONCLUSIONS: tTG-based enzyme-linked immunosorbent assay is an effective diagnostic test, although immunofluorescent-based assays are more sensitive, particularly during gluten challenge.
Assuntos
Doença Celíaca/sangue , Doença Celíaca/diagnóstico , GTP Fosfo-Hidrolases/imunologia , Proteínas de Ligação ao GTP , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Transglutaminases/imunologia , Adolescente , Adulto , Biomarcadores/sangue , Doença Celíaca/enzimologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , GTP Fosfo-Hidrolases/metabolismo , Humanos , Lactente , Masculino , Proteína 2 Glutamina gama-Glutamiltransferase , Sensibilidade e Especificidade , Transglutaminases/metabolismoRESUMO
L6 myotubes expressing the constitutively active Arg1152-->Gln insulin receptor (L6(1152)) featured a 31% increased glucose consumption as compared with L6 cells expressing wild-type receptors (L6(WT)). However, insulin treatment decreased glucose consumption of the mutant cells by 20% while increasing that of the L6(WT) by 30%. In the L6(WT), insulin elicited a significant increase in glucose transport and GLUT1 and GLUT4 plasma membrane expression, while in the L6(1152), all of these functions were constitutively activated and not further stimulated by insulin. Similarly, glycogen content and glycogen synthase activity were increased by 80 and 125%, respectively, in the L6(1152 )versus the L6(WT) and unaffected by insulin (while a 2-fold increase was measured in insulin-exposed L6(WT)). Glucose oxidation and pyruvate dehydrogenase activity were also 25% higher in the mutant compared with the L6(WT). However, in the L6(1152), both functions decreased by 35% in response to insulin (while increasing by 60 and 80%, respectively, in the L6(WT)). Similarly as in the L6(1152), in vivo, forearm glucose uptake in IR1152 patients was 2-fold higher than in control subjects. This difference was not accounted for by higher plasma glucose levels. We conclude that, in skeletal muscle, glucose storage and oxidation are differentially impaired by the expression of IR1152, suggesting that their regulation by insulin involves divergent signaling pathways. Muscle expression of IR1152 may contribute to impairing glucose tolerance in IR1152 individuals.
Assuntos
Glucose/metabolismo , Músculo Esquelético/metabolismo , Receptor de Insulina/metabolismo , Linhagem Celular , Humanos , Oxirredução , Mutação Puntual , Receptor de Insulina/genética , Transdução de SinaisRESUMO
In L6 muscle cells expressing the Arg1152 --> Gln insulin receptor (Mut), basal tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was increased by 35% compared with wild-type cells (WT). Upon exposure to insulin, IRS-1 phosphorylation increased by 12-fold in both the Mut and WT cells. IRS-2 was constitutively phosphorylated in Mut cells and not further phosphorylated by insulin. The maximal phosphorylation of IRS-2 in basal Mut cells was paralleled by a 4-fold increased binding of the kinase regulatory loop binding domain of IRS-2 to the Arg1152 --> Gln receptor. Grb2 and phosphatidylinositol 3-kinase association to IRS-1 and IRS-2 reflected the phosphorylation levels of the two IRSs. Mitogen-activated protein kinase activation and [3H]thymidine incorporation closely correlated with IRS-1 phosphorylation in Mut and WT cells, while glycogen synthesis and synthase activity correlated with IRS-2 phosphorylation. The Arg1152 --> Gln mutant did not signal Shc phosphorylation or Shc-Grb2 association in intact L6 cells, while binding Shc in a yeast two-hybrid system and phosphorylating Shc in vitro. Thus, IRS-2 appears to mediate insulin regulation of glucose storage in Mut cells, while insulin-stimulated mitogenesis correlates with the activation of the IRS-1/mitogen-activated protein kinase pathway in these cells. IRS-1 and Shc-mediated mitogenesis may be redundant in muscle cells.