Elena Bazzigaluppi

Increased intestinal permeability precedes clinical onset of type 1 diabetes

Aims/hypothesisRecent observations have shown subclinical intestinal abnormalities in human type 1 diabetes. Whether these are related to the pathogenetic process or secondary to the diabetes remains to be clarified. The aim of this study was to investigate this issue by examining intestinal permeability to sugars in subjects at different stages of type 1 diabetes: preclinical, new-onset and long-term established disease.MethodsEighty-one subjects with islet autoimmunity (18 preclinical, 28 new-onset and 35 long-term type 1 diabetes) and 40 healthy control subjects were investigated by a lactulose-mannitol test, consisting of oral administration of the two sugars and measurement of their urinary excretion.ResultsAll groups of subjects with islet autoimmunity showed an increase in intestinal permeability (p ≤ 0.009 vs controls) to the disaccharide lactulose, indicative of a damaged barrier, but a similar permeability to the monosaccharide mannitol (NS vs controls), indicative of an integral surface mucosa; consequently there was an increase in the lactulose:mannitol excretion ratio (p ≤ 0.025 vs controls).Conclusions/interpretationThese findings indicate the presence of a subclinical enteropathy associated with type 1 diabetes that is already detectable before clinical onset of the disease, and suggest that the small intestine is an organ participating in the pathogenetic process of type 1 diabetes.

Islet autoantibody markers in IDDM : risk assessment strategies yielding high sensitivity

SummaryIdentification of islet autoantigens offers the possibility that antibody tests other than islet cell antibodies may be used for assessing risk of insulin-dependent diabetes mellitus (IDDM). The aim of this study was to determine the combination of islet autoantibody markers that could identify most future cases of IDDM. Islet cell antibodies, antibodies to glutamic acid decarboxylase (GAD)65, 37,000/ 40,000 Mr islet tryptic fragments, carboxypeptidase-H, and islet cell autoantigen (ICA)69 were measured in sera from 100 newly-diagnosed IDDM patients, 27 individuals prior to onset of IDDM, and 83 control subjects. Islet cell antibodies were detected in 88 % of IDDM patients and 81 % with pre-IDDM, GAD65 antibodies in 70 % of IDDM patients and 89 % with pre-IDDM, and antibodies to 37,000/40,000 Mr islet tryptic fragments in 54 % of IDDM patients and in 48 % with pre-IDDM. The latter were found only in conjunction with islet cell antibodies and were more frequent in young onset cases. All 20 IDDM patients and the 3 pre-IDDM subjects who had islet cell antibodies without GAD65 antibodies had antibodies to 37,000/40,000 Mr islet tryptic fragments, and all but one had disease onset before age 15 years. No sera strongly immunoprecipitated in vitro translated ICA69 or carboxypeptidase-H; 4 % of patients had anti-ICA69 and 11 % anti-carboxypeptidase-H levels above those of the control subjects. The findings suggest that none of the single antibody specificities are as sensitive as islet cell antibodies, but that a combination of GAD65 antibodies and antibodies to 37,000/40,000 Mr islet tryptic fragments has the potential to identify more than 90 % of future cases of IDDM. Such a strategy could eventually replace islet cell antibodies in population screening for IDDM risk assessment.

Association of IA-2 autoantibodies with HLA DR4 phenotypes in IDDM

SummaryInsulin, glutamate decarboxylase (GAD) and the protein tyrosine phosphatase-like molecule IA-2 are major targets of humoral autoimmunity in insulin-dependent diabetes mellitus (IDDM). These autoantibodies are heterogeneous with respect to age and patient human leukocyte antigen (HLA) phenotype. We have previously demonstrated that GAD and IA-2 antibodies potentially identify different subsets of IDDM patients. The aim of this study was to determine whether GAD and IA-2 autoantibodies were associated with different HLA DR phenotypes. We studied 160 patients with IDDM onset before age 16 years. At disease onset serum was tested for GAD and IA-2 antibodies by immunoprecipitation of in vitro-translated35S-methionine labelled recombinant proteins. IA-2 antibodies were significantly associated with HLA DR4: 67 (86 %) of 78 patients with HLA DR4 vs 31 (38 %) of 82 non-DR4 patients had IA-2 antibodies (pc<0.0001) and IA-2 antibody levels were higher in patients with HLA DR4 (pc<0.0001). In contrast, GAD antibodies were more prevalent (pc<0.05) and antibody levels highest (pc<0.01) in patients with HLA DR3 phenotypes. These data provide further evidence that, in IDDM, production and titre of major autoantibody specificities are associated with HLA class II alleles.

Antibodies to tissue transglutaminase C in Type I diabetes

Aims/hypothesis. Silent coeliac disease is a gluten driven autoimmune disease which is relatively frequent in patients with Type I (insulin-dependent) diabetes mellitus. To determine the extent of gluten associated autoimmunity in Type I diabetes, autoantibodies to tissue transglutaminase C, a major autoantigen in coeliac disease, were measured in patients with new-onset Type I diabetes. Methods. We measured IgG and IgA tissue transglutaminase C autoantibodies using human recombinant antigen and radio-binding assays in a cohort of 287 patients with new-onset Type I diabetes, 119 with Type II (non-insulin-dependent) diabetes mellitus and in 213 control subjects. Results. We found IgA and IgG tissue transglutaminase C antibodies in 24 (8 %) patients with Type I diabetes; 97 (33 %) patients had IgG antibodies only and 1 IgA antibodies only. Antibody concentrations were highest in those with both IgA and IgG antibodies. Only 2 (2 %) patients with Type II diabetes and 2 (1 %) control subjects had either IgG or IgA tissue transglutaminase C antibodies. Patients with HLA DRB1*04 alleles had the highest prevalence of IgG tissue transglutaminase C antibodies. Conclusion/Interpretation. These data show that almost 10 % of patients have autoimmunity typical of coeliac disease and that another 30 % have low level tissue transglutaminase C antibody binding. This high prevalence suggests either involvement of the gut in the pathogenesis of Type I diabetes or that transglutaminase is a secondary autoantigen resulting from beta-cell destruction. [Diabetologia (1999) 42: 1195–1198]

Parameters associated with residual insulin secretion during the first year of disease in children and adolescents with Type 1 diabetes mellitus.

Factors associated with residual insulin secretion and spontaneous remission in Type 1 diabetic patients are important in the evaluation of treatment aimed at modifying the natural history of Type 1 DM. We investigated the effect of parameters at onset on residual beta cell function in 215 Type 1 DM children and adolescents. Blood gas analysis, HLA, GAD and IA‐2 antibodies before the start of insulin treatment were recorded for each patient. Residual C‐peptide secretion was assessed by the glucagon test, and parameters of metabolic control (HbA1c and insulin dose U kg−1 day−1) were examined at disease onset and after 3, 6, and 12 months. Residual C‐peptide secretion throughout the first year of disease was significantly reduced in patients with disease onset before age 5. Multiple regression analysis showed that low pH at onset showed a significant and independent association with reduced C‐peptide at 3 months (p = 0.02) and that the detection of GAD antibodies had a significant independent association with decreased C‐peptide secretion at 6 months of follow‐up (p = 0.02). Insulin requirement was higher in the youngest patients group and in patients with GAD antibodies. Spontaneous insulin remission (HbA1c <6 % and insulin <0.3 U kg−1 day−1) occurred in 22/192 (11 %) patients at 3 months of follow‐up, in 15/190 (8 %) patients at 6 months and in 8/169 (5 %) patient at 12 months. Remission was more prevalent in older patients (p = 0.01) and in patients without detectable GAD antibodies: (14/64 vs 8/128, p = 0.001). Sex, IA‐2 antibodies and HLA DR were not independently associated with C‐peptide secretion, insulin requirement or remission in the first year of Type 1 DM. This study confirms the association of young age, severe acidosis at disease onset, and GAD antibodies with decreased residual beta‐cell function and spontaneous remission during the first year of insulin treatment. These factors should be considered in trials evaluating therapies to retain beta‐cell function and induce remission at and after disease onset.

Fulminant autoimmune Type 1 diabetes during interferon-alpha therapy: a case of Th1-mediated disease?

Aim  A case of autoimmune Type 1 diabetes with some unique characteristics developing in a 29‐year‐old male during treatment with interferon‐alpha (IFN‐α) for chronic hepatitis C virus (HCV) hepatitis is reported.

Two-Step Islet Autoantibody Screening for Risk Assessment of Type 1 Diabetes in Relatives

OBJECTIVE To examine the performance of islet cell antibodies (ICAs) and antibodies to glutamate decarboxylase (GADA), IA-2 (IA-2 antibody [IA-2A]), and insulin (insulin autoantibody [IAA]), alone and in combination, in assessing type 1 diabetes risk within type 1 diabetic families to identify a practical and effective screening strategy for predicting type 1 diabetes in relatives. RESEARCH DESIGN AND METHODS ICA, GADA, IA-2A, and IAA were determined in 806 first-degree relatives participating in a prospective type 1 diabetes family study (median follow-up 6.17 years, range 0.6–8.3). The conferred risk of developing type 1 diabetes within 6 years was evaluated by Kaplan-Meier for each antibody marker, used alone or in combination. RESULTS ICAs were detected in 3%, GADA in 5.1%, IA-2A in 2.5%, and IAA in 3.7% of relatives; ≥1 antibody markers were detected in 10.7% of relatives and ≥2 were detected in 1.9% of relatives. The risk of type 1 diabetes at 6 years was 1.5% in relatives with only 1 marker and 24.8% in relatives with ≥2 markers. As a practical and effective strategy for type 1 diabetes risk assessment in relatives, this study indicates a first-step screening based on GADA and IA-2A measurement—which identified 6.5% of relatives, including all who developed the disease, with a 6-year type 1 diabetes risk of 9.0%—followed by a second step based on ICA and IAA measurement in relatives with either GADA or IA-2A, which identified a total of 1.9% of all relatives as having ≥2 markers, and a 6-year risk of 24.8%, including 6 of 7 who developed type 1 diabetes. CONCLUSIONS A two-step antibody screening, based first on GADA and IA-2A and then on ICA and IAA measurements in identified individuals, is likely to be a practical, sensitive, and effective strategy for predicting type 1 diabetes in first-degree relatives.

Development of an immunoassay for all human isoferritins, and its application to serum ferritin evaluation

Calibrated mixtures of anti-H and anti-L ferritin subunit monoclonal antibodies were used in a sandwich enzyme-immunoassay for the evaluation of all isoferritins. The assay was designed to have overlapping calibration plots for human liver (95% L-chain) and recombinant human H-chain ferritin (100% H-chain). It appeared to recognize all the heart isoferritins including the ones in the middle of the isoferritin spectrum. By direct comparison it was shown that these isoferritins are under-evaluated by the assays for H- and L-subunit-rich ferritins, based on the two separated antibodies. The three assays (for total, H-rich and L-rich ferritins) provide an index of the presence of the isoferritins with intermediate H/L composition. Sera from 30 tumor and non-tumor patients were analyzed. Intermediate isoferritins were found in 2 non-tumor subjects and in none of the 14 patients with Hodgkins disease or mammary carcinoma. It is concluded that the evaluation of the total and intermediate isoferritins is possible, but does not have an evident clinical significance for tumor monitoring.

Autoantibodies to Harmonin and Villin Are Diagnostic Markers in Children with IPEX Syndrome

Autoantibodies to enterocyte antigens harmonin (75 kDa USH1C protein) and villin (actin-binding 95 kDa protein) are associated with the Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. In this study we evaluated the diagnostic value of harmonin and villin autoantibodies in IPEX and IPEX-like syndromes. Harmonin and villin autoantibodies were measured by a novel Luminescent-Immuno-Precipitation-System (LIPS) quantitative assay, in patients with IPEX, IPEX-like syndrome, Primary Immunodeficiencies (PID) with enteropathy, all diagnosed by sequencing of the FOXP3 gene, and in type 1 diabetes (T1D), celiac disease and healthy blood donors as control groups. Harmonin and villin autoantibodies were detected in 12 (92%) and 6 (46%) of 13 IPEX patients, and in none of the IPEX-like, PID, T1D, celiac patients, respectively. All IPEX patients, including one case with late and atypical clinical presentation, had either harmonin and/or villin autoantibodies and tested positive for enterocyte antibodies by indirect immunofluorescence. When measured in IPEX patients in remission after immunosuppressive therapy or hematopoietic stem cell transplantation, harmonin and villin autoantibodies became undetectable or persisted at low titers in all cases but one in whom harmonin autoantibodies remained constantly high. In one patient, a peak of harmonin antibodies paralleled a relapse phase of enteropathy. Our study demonstrates that harmonin and villin autoantibodies, measured by LIPS, are sensitive and specific markers of IPEX, differentiate IPEX, including atypical cases, from other early childhood disorders associated with enteropathy, and are useful for screening and clinical monitoring of affected children.

Antineutrophil cytoplasmic antibody positivity in IgG4-related disease: A case report and review of the literature.

Background:IgG4-related disease (IgG4-RD) is a fibroinflammatory condition characterized by serum IgG4 elevation and tissue infiltration of IgG4-positive plasma cells. Substantial overlap between IgG4-RD and antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) exists in terms of organ involvement and histopathological features. A positive ANCA assay is regarded as a highly specific finding in favor of an AAV, and generally influences away from a diagnosis of IgG4-RD. Recent reports, however, have raised the possibility that some patients with IgG4-RD are ANCA positive, thus suggesting reconsideration of the role of ANCA in the diagnostic workup. In the present work, we describe the first case of concomitant biopsy-proven IgG4-RD and granulomatosis with polyangiitis (GPA), demonstrating antiproteinase 3 (PR3) ANCA of the IgG4 subclass in the patients serum. We also review the literature in order to provide clinicians with tools for interpreting ANCA positivity in IgG4-RD patients. Case summary:A 51-year-old woman was referred for left exopthalmos due to lacrimal gland enlargement and increased serum IgG4 concentration. IgG4-RD was suspected and further imaging studies disclosed multiple pulmonary masses in the right lung. Histological analysis of the left lacrimal gland was diagnostic for IgG4-RD, but lung biopsy showed typical features of GPA. ANCA assay was positive for anti-PR3 antibodies. Further immunofluorescence studies demonstrated anti-PR3 antibodies of IgG1 and IgG4 subclass. Treatment with rituximab induced swift remission of both IgG4-RD and GPA manifestations. We identified 9 other reports of patients with IgG4-RD and positive ANCA in the English literature, 5 cases with biopsy-proven IgG4-RD and 4 cases in whom IgG4-RD was diagnosed presumptively. Four patients had also histological evidence of concomitant AAV. Conclusion:The present work demonstrates that ANCA positivity in patients with biopsy-proven IgG4-RD should prompt the exclusion of a concomitant vasculitic process; a positive ANCA does not exclude the diagnosis of IgG4-RD; confirmation through immunoenzymatic assays of the ANCA specificity, clinical-pathological correlation, and histopathological evaluation remain crucial steps for the differential diagnosis between AAV and IgG4-RD.