Role of coxsakie virus B4 in the pathogenesis of type 1 diabetes mellitus

Aetiopathogenesis of Type 1 Diabetes Mellitus (T1DM) includes genetic, immunologic and environmental factors. An autoimmune origin had been suggested based on the findings of auto-antibodies (ICA,GAD,IA2). Among enviromental factors interesting findings on the role played by Enteroviruses, such as Coxsackie B4, have been reported. However much more data are necessary to establish a clear association between viruses and T1DM in humans. To identify pathogenetically relevant autoantigens targets in patients with T1DM and to evaluate the possible involvement infection agents in the pathogenesis of T1DM we used a peptide library approch. We enrolled 58 patients (35 M, 23 F) aged 0.8-18.7 years with newly-diagnosed T1DM and 30 healthy age-and sex matched individuals as controls. We screened a random peptide library with a pool of immunoglobulins G derived from sera of patients and controls. A set of peptides, obtained from the last biopanning round, was synthetized and used in an ELISA assay to test individual patients’ sera as well as control IgG immunoglobulins. We identified an immunodominant peptide of 12 aa (that we call peptide T1DM) that was recognised by 43/58 (74%) of patients' sera but by none of the controls’sera. The peptide was compared with know microbial sequences using BLAST network service. We found hight degree of homology with viral VP1, a protein expressed on Coxsackie virus capside (the omologus peptide was called Coxsa peptide). IgG antibodies against the T1DM peptide were affinity purified from patients' sera and were able to recognise the viral protein VP1 and the Coxsa peptide. We then compare the peptide T1DM sequence with know human sequence using BLAST network service and found hight degree of homology with three human proteins in beta cells: phogrin (a protein localized in secretory granules of beta cells), 6-phosphofructo-1-kinase (6PKF an enzyme that catalyzes an important step of glycolysis) and L-type calcium channel that in beta cell is of great importance in insulin secretion. Anti T1DM and anti Coxa peptides antibodies recognized phogrin, 6PKF and L-type calcium channel by immunoprecipitetion of beta cell lysates and bind phogrin and 6PKF (demonstred using FACS analysis) in beta cells. Finally we found that antipeptides antibodies were able to induce apoptosis of pancreatic beta cells. Therefore we suggest that in genetically predisposed subjets Coxsackievirus virus B4 infection may induce an antiviral response able to trigger destruction of beta cells through a molecular mimicry mechanism between VP1 protein of coxsackie virus B4 and human autoantigens (phogrin, 6PKF and L-type calcium channel) leading to the onset of type 1 diabetes mellitus.