A mouse model for cystinuria type I.

Cystinuria, one of the most common inborn errors of metabolism in humans, accounts for 1-2% of all cases of renal lithiasis. It is caused by defects in the heterodimeric transporter system rBAT/b0,+AT, which lead to reduced reabsorption of cystine and dibasic amino acids through the epithelial cells of the renal tubules and the intestine. In an N-ethyl-N-nitrosourea mutagenesis screen for recessive mutations we identified a mutant mouse with elevated concentrations of lysine, arginine and ornithine in urine, displaying the clinical syndrome of urolithiasis and its complications. Positional cloning of the causative mutation identified a missense mutation in the solute carrier family 3 member 1 gene (Slc3a1) leading to an amino acid exchange D140G in the extracellular domain of the rBAT protein. The mouse model mimics the aetiology and clinical manifestations of human cystinuria type I, and is suitable for the study of its pathophysiology as well as the evaluation of therapeutic and metaphylactic approaches.

Correlation of interferon treatment response with GBV-C/HGV genomic RNA and anti-envelope 2 protein antibody.

The clinical significance of GB virus C/hepatitis G virus (GBV-C/HGV) co-infection was studied retrospectively in 100 consecutive patients with hepatitis C virus (HCV) infection. All 100 patients had been treated with interferon-alpha (IFN-alpha). Co-infection with GBV-C/HGV and HCV was detected in 10 of the 100 patients (10%) and anti-envelope 2 region (anti-E2) antibody was detected in 25 patients. None of the patients with GBV-C/HGV RNA had anti-E2 antibody. Co-infected patients were younger (P < .005) and their serum transaminase levels were lower than HCV-only infected patients (P< .01). In 7 of the 10 co-infected patients, HCV RNA was eradicated from serum after IFN-alpha treatment and normal alanine transaminase (ALT) levels continued in 6 of these 7 patients. In one patient who was negative for HCV RNA but positive for GBV-C/HGV RNA, the ALT level relapsed transiently. The rate of clearance of HCV and normalization of the ALT level was significantly higher in co-infected patients than in HCV-only infected patients (P < .05). GBV-C/HGV RNA disappeared from 6 of the 10 co-infected patients (60%) upon cessation of IFN-alpha treatment. However, continuous clearance of GBV-C/HGV was observed in only two patients and anti-E2 antibody could not be detected in the serum of these patients. These results indicate that co-infected patients tend to be younger and more sensitive to IFN-alpha treatment. However, long-term clearance of GBV-C/HGV after IFN-alpha treatment may be difficult. Moreover, anti-E2 antibody may act to neutralize GBV-C/ HGV.

Humoral immune response to the E2 protein of hepatitis G virus is associated with long-term recovery from infection and reveals a high frequency of hepatitis G virus exposure among healthy blood donors.

The second envelope protein (E2) of the hepatitis G virus (HGV) was expressed in Chinese hamster ovary (CHO) cells and showed a molecular weight of approximately 60 to 70 kd, with 15 to 25 kd of the size contributed by N-linked glycosylation. An enzyme-linked immunosorbent assay (ELISA) using HGV-E2 was developed to test for antibodies to this protein (anti-E2) in human sera. High sensitivity was achieved by developing monoclonal antibodies (mAbs) to HGV-E2, which were used as capture antibodies in the ELISA. Our studies revealed that 16% of healthy Spanish blood donors were exposed to HGV, indicating that additional routes of viral transmission besides parenteral exposure might exist. An even higher prevalence of exposure to HGV (52%-73%) was found in several groups at risk of parenteral exposure to infectious agents, i.e., intravenous drug users, transfusion history, hemophiliacs, and hepatitis C virus (HCV)-positive patients. Most anti-E2-positive patients were HGV-RNA-negative and vice versa, indicating an inverse correlation of these two viral markers. A panel of 16 posttransfusion patients followed for up to 16 years revealed that patients who develop an anti-E2 response become HGV-RNA-negative, while patients who do not develop anti-E2 are persistently infected. Immunity to HGV seems to be long-lasting, because circulating antibody to E2 could still be detected 14 years after seroconversion. Sequence comparisons showed that E2 is highly conserved among isolates collected worldwide, indicating that immune escape variants are not common in HGV infections. This reflects on a molecular level why HGV infections usually are cleared spontaneously by the host. However, possible mechanisms of HGV persistence, as found in some patients, remain to be elucidated.

Detection of antibodies to a putative hepatitis G virus envelope protein.

BACKGROUND: A flavivirus designated hepatitis G virus (HGV) has been isolated from the serum of patients with non-A-E hepatitis. Hitherto, the presence of HGV RNA in serum has been detected with the reverse transcription-polymerase chain reaction (RT-PCR) amplification method. We have now developed an immunoassay for antibodies against an HGV protein. METHODS: Recombinant HGV envelope protein E2 was used as antigen in an ELISA. 80 blood donors, 99 intravenous-drug users, and 11 patients with acute post-transfusion hepatitis were tested for antibodies to E2. The HGV-RNA status was assessed by RT-PCR. FINDINGS: Anti-E2 seroprevalence was 9% among the blood donors and 41% among the drug users; HGV-RNA prevalence was 2.5% and 38%, respectively. Whereas anti-E2 prevalence increased with the duration of drug use, HGV-RNA prevalence declined in parallel. In each group, the presence of anti-E2 and HGV RNA was almost mutually exclusive: none of the blood donors and only 4% of the drug users were positive for both markers at the same time. Of the 11 post-transfusion patients--who were all HGV-RNA positive and anti-E2 negative at the onset of disease--four developed antibodies to E2 during the following year, and two of the four subsequently became HGV-RNA negative. INTERPRETATION: We conclude that a humoral immune response to E2 is associated with loss of detectable HGV viraemia. Thus, E2-specific antibodies might serve as a useful marker for diagnosing recovery from HGV infections. The immunoassay we describe should facilitate investigation of suspected infections and may be helpful in the elucidation of the clinical significance of HGV.

Reverse transcription-PCR detection of hepatitis G virus.

Hepatitis G virus (HGV) was recently identified as a new member of the Flaviviridae, but its clinical significance is still unclear. Since no immunoassay for the diagnosis of HGV is available, we developed a sensitive reverse transcription-PCR (RT-PCR) assay to facilitate the detection of the viral genome by mass screening in the clinical laboratory. Sequences within the 5'-noncoding region and within the putative NS5a region are independently amplified in the presence of digoxigenin-11-dUTP and are detected by hybridization with biotinylated capture probes binding to a streptavidin-coated matrix. Semiquantitative Enzymun-Test DNA detection via chemiluminescence can be performed either in a microtiter plate format or on fully automated ES 300 machines. We were able to detect at least 8 x 10(2) genome equivalents per ml of serum using both primer pairs. HGV was shown to be present in 43 of 130 (33%) serum samples from intravenous drug abusers with a high risk of parenteral exposure. However, only two of the patients were positive when the NS5a primers only were used, and only one patient was positive when only the 5'-noncoding region primers were used, demonstrating the increased sensitivity of HGV detection with two sets of primers. Among these patients, there was no obvious correlation with other viral infections like hepatitis B virus, hepatitis C virus, or human immunodeficiency virus. Within a blood donor panel, 3 of 92 (3%) samples were found to be HGV positive, suggesting that donated blood may need to be screened for HGV.