Vito Lampasona

Similar low frequency of anti-MOG IgG and IgM in MS patients and healthy subjects.

The authors used a liquid-phase radiobinding assay to measure serum anti-myelin oligodendrocyte protein (MOG) immunoglobulin (Ig) G in 87 patients with multiple sclerosis (MS), in 12 patients with encephalomyelitis, and in 47 healthy subjects. Anti-MOG IgM was determined in samples obtained at onset from 40 of 87 patients with MS and in control subjects. The frequency of positive samples with low titers of anti-MOG IgG (≤5.7%) and IgM (≤8.3%) was similar in all the groups and subgroups. Binding competition experiments showed that these antibodies had low affinity. Anti-MOG antibodies are not disease specific.

Zinc Transporter 8 Antibodies Complement GAD and IA-2 Antibodies in the Identification and Characterization of Adult-Onset Autoimmune Diabetes: Non Insulin Requiring Autoimmune Diabetes (NIRAD) 4

OBJECTIVE Zinc transporter 8 (ZnT8) is an islet β-cell secretory granule membrane protein recently identified as an autoantibody antigen in type 1 diabetes. The aim of this study was to determine the prevalence and role of antibodies to ZnT8 (ZnT8As) in adult-onset diabetes. RESEARCH DESIGN AND METHODS ZnT8As were measured by a radioimmunoprecipitation assay using recombinant ZnT8 COOH-terminal or NH2-terminal proteins in 193 patients with adult-onset autoimmune diabetes having antibodies to either GAD (GADAs) or IA-2 (IA-2As) and in 1,056 antibody-negative patients with type 2 diabetes from the Non Insulin Requiring Autoimmune Diabetes (NIRAD) study. RESULTS ZnT8As-COOH were detected in 18.6% patients with autoimmune diabetes and 1.4% with type 2 diabetes. ZnT8As-NH2 were rare. ZnT8As were associated with younger age and a high GADA titer. The use of GADAs, IA-2As, and ZnT8As in combination allowed a stratification of clinical phenotype, with younger age of onset of diabetes and characteristics of more severe insulin deficiency (higher fasting glucose and A1C, lower BMI, total cholesterol, and triglycerides) in patients with all three markers, with progressive attenuation in patients with two, one, and no antibodies (all Ptrend < 0.001). Autoantibody titers, association with high-risk HLA genotypes, and prevalence of thyroid peroxidase antibodies followed the same trend (all P < 0.001). CONCLUSIONS ZnT8As are detectable in a proportion of patients with adult-onset autoimmune diabetes and seem to be a valuable marker to differentiate clinical phenotypes.

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.

Diabetes Antibody Standardization Program: First Proficiency Evaluation of Assays for Autoantibodies to Zinc Transporter 8

BACKGROUND Zinc transporter 8 (ZnT8) is a recently identified major autoantigen in type 1 diabetes, and autoantibodies to ZnT8 (ZnT8A) are new markers for disease prediction and diagnosis. Here we report the results of the first international proficiency evaluation of ZnT8A assays by the Diabetes Antibody Standardization Program (DASP). METHODS After a pilot workshop in 2007, an expanded ZnT8A workshop was held in 2009, with 26 participating laboratories from 13 countries submitting results of 63 different assays. ZnT8A levels were measured in coded sera from 50 patients with newly diagnosed type 1 diabetes and 100 blood donor controls. Results were analyzed comparing area under the ROC curve (ROC-AUC), sensitivity adjusted to 95% specificity (AS95), concordance of sample ZnT8A positive or negative designation, and autoantibody levels. RESULTS ZnT8A radio binding assays (RBAs) based on combined immunoprecipitation of the 2 most frequent ZnT8 COOH-terminal domain polymorphic variants showed a median ROC-AUC of 0.848 [interquartile range (IQR) 0.796-0.878] and a median AS95 of 70% (IQR 60%-72%). These RBAs were more sensitive than assays using as antigen either 1 ZnT8 variant only or chimeric constructs joining NH(2)- and COOH-terminal domains, assays based on immunoprecipitation and bioluminescent detection, or assays based on immunofluorescent staining of cells transfected with full-length antigen. CONCLUSIONS The DASP workshop identified immunoprecipitation-based ZnT8A assays and antigen constructs that achieved both a high degree of sensitivity and specificity and were suitable for more widespread clinical application.

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]

Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients

Background: Antibodies against aquaporin-4 (AQP4), a water channel particularly expressed on perivascular astrocytic podocytes, are proposed as a marker for the diagnosis of neuromyelitis optica (NMO). However, a consensus on seroprevalence and optimal detection method has not yet been reached. Objectives: To investigate the performance of different assays to detect anti-AQP4 antibodies. Methods: We set up five different assays. Two of them were capable to detect perivascular IgG reactivity on brain tissue by immunofluorescence (NMO-IgG). Other three assays have been set to detect anti-AQP4 antibodies: immunofluorescence and flow cytometry on AQP4-expressing cells, and a radioimmunoprecipitation assay. We assessed sensitivity and specificity of these assays by interrogating sera of 33 NMO patients, 13 patients at high risk to develop NMO (hrNMO), 6 patients affected by acute partial transverse myelitis (APTM), 20 patients with multiple sclerosis (MS), and 67 age- and sex-matched healthy controls. Results: We found that the presence of serum NMO-IgG and anti-AQP4 reactivity is almost exclusively restricted to patients with NMO and hrNMO. Seroprevalence and sensitivity ranged from 30 to 47%, depending on the assay. Specificity ranged from 95 to 100%. Comparing results obtained in the five assays, we noticed lack of concordance in some samples. Conclusions: Detection of NMO-IgG or anti-AQP4 antibodies may represent a valuable tool to assist neurologists in the differential diagnosis between patients with NMO, hrNMO, APTM, or MS. The current lack of a gold standard to detect anti-AQP4 antibodies implies the necessity to standardize the detection of these antibodies.

Insulin gene mutations as cause of diabetes in children negative for five type 1 diabetes autoantibodies

OBJECTIVE—Heterozygous, gain-of-function mutations of the insulin gene can cause permanent diabetes with onset ranging from the neonatal period through adulthood. The aim of our study was to screen for the insulin gene in patients who had been clinically classified as type 1 diabetic but who tested negative for type 1 diabetes autoantibodies. RESEARCH DESIGN AND METHODS—We reviewed the clinical records of 326 patients with the diagnosis of type 1 diabetes and identified seven probands who had diabetes in isolation and were negative for five type 1 diabetes autoantibodies. We sequenced the INS gene in these seven patients. RESULTS—In two patients whose diabetes onset had been at 2 years 10 months of age and at 6 years 8 months of age, respectively, we identified the mutation GB8S and a novel mutation in the preproinsulin signal peptide (ASignal23S). CONCLUSIONS—Insulin gene mutations are rare in absolute terms in patients classified as type 1 diabetic (0.6%) but can be identified after a thorough screening of type 1 diabetes autoantibodies.

Alloantibody and Autoantibody Monitoring Predicts Islet Transplantation Outcome in Human Type 1 Diabetes

Long-term clinical outcome of islet transplantation is hampered by the rejection and recurrence of autoimmunity. Accurate monitoring may allow for early detection and treatment of these potentially compromising immune events. Islet transplant outcome was analyzed in 59 consecutive pancreatic islet recipients in whom baseline and de novo posttransplant autoantibodies (GAD antibody, insulinoma-associated protein 2 antigen, zinc transporter type 8 antigen) and donor-specific alloantibodies (DSA) were quantified. Thirty-nine recipients (66%) showed DSA or autoantibody increases (de novo expression or titer increase) after islet transplantation. Recipients who had a posttransplant antibody increase showed similar initial performance but significantly lower graft survival than patients without an increase (islet autoantibodies P < 0.001, DSA P < 0.001). Posttransplant DSA or autoantibody increases were associated with HLA-DR mismatches (P = 0.008), induction with antithymocyte globulin (P = 0.0001), and pretransplant panel reactive alloantibody >15% in either class I or class II (P = 0.024) as independent risk factors and with rapamycin as protective (P = 0.006) against antibody increases. DSA or autoantibody increases after islet transplantation are important prognostic markers, and their identification could potentially lead to improved islet cell transplant outcomes.

BRAFV600E-mutation is invariably present and associated to oncogene-induced senescence in Erdheim-Chester disease

Objectives Erdheim-Chester disease (ECD) is a rare form of histiocytosis characterised by uncontrolled chronic inflammation. The oncogenic BRAFV600E mutation has been reported in biopsies in 19 out of 37 patients with ECD from the largest published cohort, but never found in the patients’ peripheral blood. Also, the role of the mutation in the pathogenesis of the disease has not been elucidated yet. BRAFV600E has been associated with oncogene-induced senescence (OIS), a protective mechanism against oncogenic events, characterised by the induction of proinflammatory pathways. Methods We verified the BRAF status in biopsies and peripheral blood from 18 patients with ECD from our cohort and matched controls by means of immunohistochemistry and of an ultrasensitive assay, based on the combination of a locked nucleic acid PCR and pyrosequencing. Droplet digital PCR was used to confirm the findings. We also evaluated the presence of senescence markers in ECD histiocytes. Results BRAFV600E mutation was present in all the biopsy and peripheral blood samples from patients with ECD and in none of the controls. ECD histiocytes and a fraction of circulating monocytes from patients with ECD showed signs of a constitutive activation of the MAPK pathway. Moreover, BRAF-mutated histiocytes expressed markers of OIS. Conclusions The oncogenic BRAFV600E mutation is present in biopsies and in the peripheral blood from all patients with ECD who were evaluated and is associated with OIS. These findings have significant implications for the pathogenesis, diagnosis and treatment of ECD.

Contribution of Antibodies Against IA-2β and Zinc Transporter 8 to Classification of Diabetes Diagnosed Under 40 Years of Age

OBJECTIVE We investigated whether measuring autoantibodies against zinc transporter 8 (ZnT8A) and IA-2β (IA-2βA) may improve classification of new-onset type 1 diabetic patients based on detection of autoantibodies against insulin (IAA), GAD (GADA), and IA-2 (IA-2A). In addition, we studied the correlation of IA-2βA and ZnT8A with other biological and demographic variables. RESEARCH DESIGN AND METHODS Circulating autoantibodies were determined by liquid-phase radiobinding assays from 761 healthy control subjects and 655 new-onset (<1 week insulin) diabetic patients (aged 0–39 years) with clinical type 1 diabetes phenotype consecutively recruited by the Belgian Diabetes Registry. RESULTS At diagnosis, IA-2βA and ZnT8A prevalences were 41 and 58%, respectively. In IAA-negative, GADA-negative, and IA-2A–negative patients, one IA-2βA–positive and eleven ZnT8A-positive individuals were identified at the expense of eight and seven additional positive control subjects (1%), respectively, for each test. ZnT8A or IA-2βA screening increased (P < 0.001; McNemar) the number of patients with ≥2 antibodies both under (from 78 to 87% for ZnT8A and 82% for IA-2βA) and above age 15 (from 51 to 63% for ZnT8A and 56% for IA-2βA) versus 0% in control subjects. IA-2βA and ZnT8A were preferentially associated with IA-2A, and with younger age at diagnosis. Unlike ZnT8A, IA-2βA levels were positively correlated with HLA-DQ8 and negatively with HLA-DQ2. ZnT8A could replace IAA for classification of patients above age 10 without loss of sensitivity or specificity. CONCLUSIONS ZnT8A, and to a lesser degree IA-2βA, may usefully complement GADA, IA-2A, and IAA for classifying insulin-treated diabetes under age 40 years.