Insulin autoantibodies before and after carbimazole therapy in Asian Indian patients with Graves' disease.

Carbimazole therapy can induce insulin autoantibodies (IAA) in Japanese Graves' disease patients, a phenomenon possibly linked to their immunogenetic profile. This phenomenon is not observed in Caucasians. We assessed IAA levels in 114 North Indian Graves' disease patients before and after carbimazole therapy (mean duration 6.2 +/- 3.9 months). The functional significance of IAA was assessed in 46 of them by first phase (sum of +1 and +3 min) insulin response to intravenous glucose (IVGTT) and an oral glucose tolerance test (OGTT) undertaken before commencement of the carbimazole therapy. IAA were measured using a radiobinding assay and expressed as the assay precision unit, S.D. scores (S.D.S), over healthy controls. Before treatment 22 of 114 (19.3%) patients were IAA positive (mean +/- S.D., 5.9 +/- 3.2 S.D.S). After carbimazole therapy a further 11 (9.6%) showed positive for IAA (mean +/- S.D., 3.5 +/- 1 S.D.S). Of the 22 patients who were IAA positive before treatment, 12 became negative after carbimazole therapy. The fasting insulin and first phase insulin responses were similar in IAA positive and IAA negative Graves' disease patients (mean +/- S.D., 61.7 +/- 35.9 versus 88.3 +/- 46.6 pmol/l, P = 0.123 and 1127 +/- 696 versus 1033 +/- 430 pmol/l, P = 0.716, respectively). The OGTT results were comparable in the IAA positive and the IAA negative groups. Thus, North Indian Graves' disease patients, who resemble Caucasians in their HLA haplotypes, behave like Japanese in their tendency to become IAA positive with carbimazole therapy. A subset of the patients who were IAA positive before treatment also demonstrated negative IAA (12/22) after carbimazole therapy.

A monoclonal antibody against the X protein of hepatitis B virus: fine mapping of its epitope and application in a quantitative ELISA of the X protein in sera of hepatitis B patients.

A HBx-specific mouse monoclonal antibody was developed and its epitope mapped to a hydrophilic segment 94HKRTLGL100 using the multipin peptide synthesis technique. A sensitive ELISA with a threshold of 5 to 10 ng was developed to identify the HBx-positive hepatitis B cases and measure the levels of HBx in sera. The same patient sera were also analyzed for the presence of anti-HBx using the purified recombinant antigen. HBx was present in 23% of the cases (15/65) whereas only 14% of the cases (9/65) were positive for anti-HBx. The mean value of HBx in acute hepatitis sera was higher (522 ng/ml) than in cirrhosis cases (48 ng/ml). PCR amplification of the S gene showed that all 15 HBx-positive cases were also positive for the viral DNA.

A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function.

The hepatitis B virus X protein (HBx) sequence (154 aa) has been divided into six regions (A-F) based on its sequence homology with X proteins of other mammalian hepadnaviruses. Regions A, C, and E are more conserved and include all the four conserved cysteines (C7, C61, C69, and C137). To localize the regions of HBx important for transactivation, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was constructed by site-directed mutagenesis. A HBx-specific monoclonal antibody was developed and used to confirm the expression of mutants by Western blot. Transactivation property of the HBx mutants was studied on Rous sarcoma virus-long terminal repeat (RSV-LTR) in transient transfection assays. We observed that deletion of the most conserved region A or substitution of the N-terminal cysteine (C7) had no effect on transactivation. Deletion of the nonconserved regions B or F also had no deleterious effects. Deletions of regions C and D resulted in a significant loss of function. Substitution of both C61 and C69 present in region C, caused almost 90% loss of activity that could be partially overcome by transfecting more expression plasmid. The fully conserved 9 amino acid segment (residues 132 to 140) within region E including C137 appeared to be crucial for its activity. Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able to stimulate the RSV-LTR quite efficiently, suggesting a crucial role played by this domain in transactivation function.

Assignment of disulphide bonds in the X protein (HBx) of hepatitis B virus.

We have established the disulphide arrangement of cysteines in E. coli expressed HBx by chromatographic analysis of enzymatically cleaved protein and sequence analysis of cysteine containing fragments. Eight of the nine cysteines are disulphide linked in an interesting pattern. Each cysteine is linked to the fourth cysteine in a sequential manner and the last cysteine is free; the disulphide linkages are between Cys7 and Cys78, Cys17 and Cys115, Cys61 and Cys137, Cys69 and Cys143 while Cys148 is free.

Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification.

The presence of hepatitis B virus DNA in the sera of individuals is the most definitive marker of an active viral infection. We have used polymerase chain reaction detection of hepatitis B virus DNA directly on whole blood dried as a spot on filter paper. The method is rapid, specific, and sensitive and has the ability to detect as little as 10 virus particles by ethidium bromide staining of the polymerase chain reaction-amplified products. The method is cost-effective, and the stability of the spots makes the collection and transportation of potentially infectious blood safe.

Studies on engraftment following fetal liver infusion.

Studies to find engraftment following fetal liver infusion (FLI) in aplastic anaemia (AA) and acute myeloid leukaemia (AML) were carried out in 24 patients (17 AA and 7 AML patients) out of the 56 who received FLI. HLA studies done in 13 patients (3 AA and 5 AML), repeatedly after FLI, showed no significant change in HLA antigen pattern before and after FLI. Red cell antigen studies were done in five (1 AA and 4 AML) patients, 3 weeks to 7 months after FLI. One patient with AML who was Rh negative prior to reinduction chemotherapy became Rh positive two months after FLI; six months later he was Rh negative again. In the remaining patients there was no change in red cell antigen pattern after FLI. Radio-immuno-assay to detect alpha-fetoprotein levels, carried out in 10 (8 AA and 2 AML) patients repeatedly after FLI, demonstrated no increase. In 13 patients (8 AA and 5 AML) in whom there was a sex difference between donor and recipient, bone marrow cultures for sex chromosomes revealed mixture of XX and XY cells in 3 male patients with aplastic anaemia. One male patient with AML demonstrated complete engraftment after induction chemotherapy and FLI: all the mitoses studied were of XX pattern. Engraftment was however temporary as repeated studies revealed reversion to XY pattern. The present work suggests that infusion of fetal liver cells may sometimes induce temporary chimerism or engraftment in an adult host; in the majority of cases, however, engraftment could not be established.