No abstract

Celiac disease in humans is activated by the dietary ingestion of wheat, rye, triticale, barley, and possibly oats. Gliadins in wheat and similar proteins in the other grains are known to activate disease in susceptible individuals. There is a striking association between celiac disease and HLA-B8, -DR3 and/or -DR7, and -DC3. Nonetheless, less than 0.2% of individuals with those serologic HLA specificities develop celiac disease and disease is not always concordant among monozygotic twins. We propose that additional environmental factors may be important in the pathogenesis of celiac disease. To investigate that possibility, we examined a data bank of protein sequences for other proteins that might share amino acid sequence homologies with A-gliadin, an alpha-gliadin component known to activate celiac disease and whose complete primary amino acid sequence is known. These studies demonstrate that A-gliadin shares a region of amino acid sequence homology with the 54-kD E1b protein of human adenovirus type 12 (Ad12), an adenovirus usually isolated from the intestinal tract. The region spans 12 amino acid residues, includes 8 residue identities and an identical pentapeptide, and is hydrophilic in both proteins. Antibody reactive with the 54-kD Ad12 E1b protein cross-reacts with A-gliadin, a 119 amino acid cyanogen bromide peptide of A-gliadin that spans the region of homology and a synthetic heptapeptide of A-gliadin from within the region of homology. We suggest that an encounter of the immune system with antigenic determinants produced during intestinal viral infection may be important in the pathogenesis of celiac disease.

Celiac disease has one of the strongest associations with HLA (human leukocyte antigen) class H markers of the known HLA-linked diseases. This association is primarily with the class II serologic specificities HLA-DR3 and -DQw2. We previously described a restriction fragment length polymorphism (RFLP) characterized by the presence of a 4.0-kilobase Rsa I fragment derived from an HLA class II

Celiac disease is activated in genetically susceptible individuals by the ingestion of dietary gluten and similar proteins in other grains (reviewed in 1). The HLA class II specificity DQw2 (previously known as DC3 or MB2) is present, as assessed serologically, in 80-90% of individuals with clinically diagnosed celiac disease (2-4). However, ~25% of the normal population also has this serologic marker and ingests dietary gluten without the development of disease (3). This observation suggests that the HLA antigen associated with increased susceptibility to celiac disease may be a subspecificity of HLA-DQw2, or may be encoded by another HLA-D region locus that is in linkage disequilibrium with the DQ locus. To identify such a celiac disease-associated HLA-D region specificity, we examined the structure of class II HLA genes in celiac disease patients and control subjects using restriction fragment length polymorphism (RFLP) analysis (5). We now report a polymorphic 4.0 kb Rsa I fragment detected using a DQ/3 chain cDNA probe. This 4.0 kb fragment provides a more accurate means of identifying individuals at risk for the development of celiac disease than the serologic marker, HLA-DQw2. Materials and Methods Patient and Control Cell Lines.Patients for these studies were 20 unrelated Caucasians with clinically diagnosed celiac disease. Diagnosis was based on clinical evidence of malabsorption, a small bowel biopsy compatible with celiac disease, clinical and/or biopsy improvement on a gluten-free diet, and clinical and/or biopsy abnormalities upon rechallenge with a gluten-containing diet. Controls were 11 unrelated individuals without clinical evidence of celiac disease. Patients and controls in this study were HLA typed by a standard complement-dependent microcytotoxicity assay, and were all positive for at least one allele of DQw2. Lymphoblastoid B cell lines were initiated from donors by culturing 5 X 10 6 peripheral blood leukocytes with supernatant from the Epstein-Barr virusproducing cell line, 1437, in the presence of 2 ttg/ml cyclosporin A. These B cell lines were maintained in RPMI 1640 culture medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 #g/ml streptomycin and 10% FCS.DNA Purification. Nuclei from B cell lines were prepared by lysis of l0 s cells in 20 ml of 10 mM Tris HCI (ph 8.0), 150 mM sodium chloride, 1 mM magnesium chloride, and 0.5% NP-40. After centrifugation at 750 g, the nuclear pellet was resuspended in 75 mM sodium chloride, 25 mM EDTA with gentle pipetting. SDS and proteinase K were added This work was supported by grant AM35108 from the National Institutes of Health, Bethesda, MD.J. ExP. MEn.

Ribavirin, 1-,-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole; Viratek, Inc., Covina, Calif.), has a broad spectrum of antiviral activity. However, the study of the absorption, metabolism, and excretion of this compoupd has been limited by the lack of an appropriate assay for ribavirin and its metabolites. Since ribavirin has definite potential for therapeutic use, we developed a radioimmunoassay to measure ribavirin levels in clinical specimens. To prepare an effective immunogen, ribavirin was monosuccinylated and coupled to ovalbumin. The competitive binding radioimmunoassay, in which tritium-labeled ribavirin and rabbit antiribavirin serum were used, was quantitative for ribavirin at concentrations of 1 pmol/100 ,ul in urine or plasma samples. The rabbit antibody cross-reacted with the major metabolite of ribavirin, 1,2,4-triazole-3-carboxamide, at a low level (2 to 5%) which did not interfere with ribavirin binding until concentrations of 1,2,4-triazole-3-carboxamide 10-to 100-fold higher than ribavirin were present in mock samples, a condition not present in biological specimens. We used the ribavirin radioimmunoassay to determine the ribavirin concentration in mouse plasma after intraperitoneal administration, in the sera of adults fromn Sierra Leone after oral or intravenous administration for treatment of suspected Lassa fever, and in the sera of children in the United States after smallparticle aerosol administration. Our experience with the radioimmunoassay indicates that it is sensitive, accurate, and reproducible. The assay will permit studies leading to a better understanding of the pharmacology and pharmacokinetics of this potentially useful antiviral drug.The synthetic nucleoside-like compound ribavirin (1-,-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; Virazole; Virjtek, Inc., Covina, Cal-