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Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare recessive disorder resulting from mutations in the autoimmune regulator (AIRE) gene. There is no information on AIRE mutations in Indians. In a cross-sectional study, nine patients (eight families), from four referral hospitals in India, were studied for AIRE mutations by direct sequencing. We screened for new mutations in 150 controls by allele-specific PCR. The patients had 1-7 known components of APECED. Three patients had unusual manifestations: presentation with type 1 diabetes; chronic sinusitis and otitis media; and facial dysmorphism. All patients carried homozygous, probably recessive, AIRE mutations. Two unrelated patients from a small in-bred community (Vanika Vaisya) in south India carried an unreported missense mutation, p.V80G, in the N-terminal caspase recruitment domain. Another unique mutation, p.C302X, resulting in a truncated protein with deletion of both zinc-finger domains, was detected in a patient from Gujarat. Neither mutation was detected in controls. Other mutations, previously described in Caucasians, were: 13 base pair deletion (p.C322fsX372) in 4 (38%), and Finn-major (p.R257X) and p.R139X (Sardinian) mutation in one subject each. In conclusion, in this first series of APECED in Indians, we detected AIRE mutations previously reported in Caucasians, as well as unique mutations. Of these, p.V80G is possibly an ancestral mutation in an in-bred community.

We characterized insulin receptors on a human lymphoblastoid cell line (IM-9) and studied their regulation using anti-receptor antibodies and fluorescence flow cytometry. The fluorescence intensity distribution of insulin receptors on cells was determined by incubating the cells with one of three different anti-receptor antisera (human serum B-9 containing polyclonal autoantibodies, serum from a rabbit with polyclonal antibodies, and a monoclonal antibody to the receptor produced in mouse hybridomas), followed by incubation with an appropriate fluorescein isothiocyanate-labeled second antibody and analysis on an Epics-V flow cytometer. however, the human anti-receptor antisera B-2 and B-9 inhibited the binding of the monoclonal anti-receptor antibody by about 50%, suggesting that these antisera contained autoantibodies directed at the monoclonal antibody binding site. These data indicate that insulin receptors can be regulated by both insulin and anti-receptor antibody and demonstrate the utility of immunofluorescence and flow cytometry as a tool for the study of the insulin receptor.The first step in the action of insulin is binding to a specific glycoprotein receptor on the surface of cells. The primary approach for characterization of the insulin receptor has been through the study of its interaction with 125I-labeled insulin (125I-insulin) (1,2). Antibodies to the insulin receptor also have provided experimental probes of receptor structure and function (3). Anti-insulin-receptor antibodies were initially detected in the sera of patients with a rare form of insulin-resistant diabetes (4), although subsequently it has been possible to raise such antibodies in rabbits and mice by immunization with partially purified receptor (5) and even to produce monoclonal antibodies to the insulin receptor (6, 7). In addition to binding to the receptor, many of these antibodies block insulin binding (8) and mimic many of insulin's biological effects (9).The use of fluorescence probes to study insulin receptors or action has been limited. Murphy et al. studied the binding and internalization of a fluorescein derivative of insulin to Swiss 3T3 cells by flow cytometry (10, 11). Fluorescein has been coupled to insulin directly or via ovalbumin; using these ligands, Schlessinger et al. have shown that insulin receptors undergo patch-and-cap formation at 370C (12). Subsequently, it was shown that fluorescently-labeled anti-receptor antibody cocapped with the rhodamine-coupled insulin (12) and that the rhodamine-insulin-receptor complex was internalized and degraded (13).In the present study, we used fluorescence flow cytometry for identification of insulin receptors and for studying their turnover. Using this technique, we have been able to study (i) the expression of receptors and their regulation by insulin, (ii) compare the effects of acute and chronic exposure of target cells to antibodies directed against insulin receptors, (iii) demonstrate shared antigenic determinants between polyclonal human autoantibodies to th...

In a prospective screening program for type I diabetes mellitus, we identified a unique family in which several members (mother and three siblings) expressed an unusual set of HLA-DR alleles (DR2+, DR3/4-) and were in different phases of immunologically mediated islet beta cell dysfunction. Immunologic and/or clinical manifestations of type I diabetes were absent in all siblings not sharing both HLA haplotypes in common with the proband. This article illustrates: the clinical utility of prospective family screening for predictive markers, such as islet cell antibodies, progressive autoimmune beta cell destruction can occur in the absence of the "high-risk" alleles HLA-DR3 and DR4, and HLA identity with the proband, rather than specific HLA alleles, i.e., presence of DR3, DR4 and absence of DR2, is an essential factor.