Suramin blocks hepatitis C binding to human hepatoma cells in vitro.

It was demonstrated recently that the binding of dengue virus to its target cell receptor could be effectively blocked by both heparin and by the polysulphonate pharmaceutical, Suramin [Chen et al. (1997) Nature Medicine 3:866-871]. Because both dengue and hepatitis C virus (HCV) belong to the Flaviviridae and because the HCV envelope is predicted to possess a heparin-binding motif, we tested heparin, Suramin, and a number of other polyanionic compounds for their ability to block HCV binding in vitro. The compounds, at concentrations ranging from 0.5 to 5,000 microg/ml, were tested using the human hepatoma cell line HepG2 cultured under conditions designed to enhance hepatocyte differentiation. Cells were harvested at 2 weeks postinoculation and HCV-RNA was quantified by means of a chemiluminescent reverse transcription polymerase chain reaction (PCR) assay. Suramin was found to be capable of blocking HCV binding in this system at a concentration similar to that reported to be effective against dengue virus. Removal of the viral envelope by treatment with chloroform also prevented HCV infection. Neither chondroitin sulphate nor the Suramin analogue CPD14 were able to block HCV under these conditions.

Combination therapy with interferon alpha and ribavirin for chronic hepatitis C virus infection in thalassaemic patients.

Hepatitis C virus (HCV) infection is common in multi-transfused thalassaemic patients, and, in combination with transfusional iron overload, can result in progressive liver disease. Therapy with interferon-alpha causes a sustained loss of HCV in only 15-25% of patients, and there is as yet no established effective therapy for those who fail to respond. We have conducted a pilot study of combination anti-viral therapy for patients who failed to respond, or relapsed after an initial response to single-agent interferon-alpha. Patients were treated for 6 months with interferon-alpha 2b, given subcutaneously three mega units thrice weekly, together with ribavirin, orally 1 g daily. 11 patients were enrolled, their median age was 24.9 years. 8/10 evaluable patients had cirrhosis on biopsy, five were infected with HCV type 1 and all but one had initial HCV RNA titres > 10(6) genomes/ml. Five patients (45.5%) had a sustained virological response with loss of serum HCV RNA for > 6 months after finishing therapy. There was no clear association between response to therapy and age, histology, HCV genotype, or HCV RNA titre. Transfusion requirements were significantly increased during the treatment phase, probably due to ribavirin-induced haemolysis, and this necessitated intensification of iron chelation therapy. Serum ferritin levels decreased significantly in those who responded. These results suggest that combination therapy is potent in clearing HCV infection, and may provide effective second-line therapy for thalassaemic patients who have failed to respond to interferon-alpha monotherapy.

Virological, biochemical and histological effects of human lymphoblastoid interferon in Swedish patients with chronic hepatitis C.

Thirty-eight Swedish patients with chronic hepatitis C were randomly assigned to receive either 3 million units (MU) or 5 MU of human lymphoblastoid interferon-alpha-n1 (Wellferon) three times per week for either 6 or 12 months. The patients were monitored biochemically, histologically and by quantitative polymerase chain reaction for circulating HCV RNA, during therapy and for the following year. Overall, 22 (58%) of the patients lost detectable hepatitis C virus (HCV) viraemia during therapy but eight of these patients relapsed during follow-up, leaving 14 (37%) sustained responders. Patients infected with HCV non-type 1 genotypes were significantly more likely to achieve a sustained response than were those infected with HCV type 1 (63% vs 10.5%, P = 0.001). Sustained virological responses were also associated with lower pretreatment viraemia level, younger age, absence of cirrhosis and the higher interferon dosage regimens but these associations failed to reach statistical significance. In 97% of patients there was concordance between virological and biochemical responses, and a statistically significant (P = 0.005) improvement in the Knodell histological activity index was observed in the virological sustained responders.

Lack of susceptibility of the cottontop tamarin to hepatitis C infection.

The only species apart from man that is known to be susceptible to HCV infection is the chimpanzee but the availability of this primate for research is strictly limited. In an attempt to find an alternative and more practical model for HCV studies three cottontop tamarins were inoculated intravenously with HCV-containing serum from patients with chronic HCV infection. The tamarins were monitored regularly for biochemical indications of hepatic inflammation and serum samples were assayed at weekly intervals for the presence of HCV-RNA and HCV antibodies. HCV-RNA was detectable at 10 minutes postinoculation in all three animals but not at any later time point over a 6 month period. No evidence of an active humoral immune response to the inoculated HCV was obtained although passively transferred anti-HCV was detectable in one animal until 1 week postinoculation. Biochemical findings did not indicate hepatic inflammation and liver histology remained normal. It is concluded on the basis of these negative findings that the cottontop tamarin is not susceptible to HCV infection.

A single tube two compartment reverse transcription polymerase chain reaction system for ultrasensitive quantitative detection of hepatitis C virus RNA.

European legislation requiring polymerase chain reaction (PCR) screening of certain plasma pools to exclude contamination with hepatitis C virus (HCV) will be introduced shortly. The concentration of HCV-RNA in contaminated pools is likely to be much lower than that typically found in single donations, so the sensitivity of the PCR assays used for the screening will be critical. We describe here the development of a quantitative PCR assay for HCV-RNA with a detection limit of approximately 40 genomes/ml, which represents a sensitivity increase of 10-100 fold over that achieved by currently available assays. This approach to quantitative reverse transcription PCR (RT-PCR) may have wide applications.

Acute hepatitis C viral infection during pregnancy: failure of mother to infant transmission.

A pregnant woman developed an acute hepatitis C virus (HCV) type 3a infection during the second trimester of pregnancy. The clinical virological features are presented, including HCV RNA quantification of maternal serum samples collected during pregnancy. These findings are discussed in light of the child's remaining uninfected after 5 years of follow-up.

Analysis of clinical and virological factors associated with response to alpha interferon therapy in chronic hepatitis C.

Interferon alpha (IFN-alpha) therapy is currently the treatment of choice for chronic hepatitis C (HCV) infection, but it fails to achieve a sustained response in approximately 75% of those treated. The factors which determine whether or not an individual will respond to IFN-alpha are uncertain, although a number of potentially predictive factors have been proposed. In this study a wide range of clinical, demographic, and virological parameters were evaluated in relation to therapeutic outcome in a group of 30 Italian patients with chronic hepatitis C. All patients received 3 MU leukocyte-derived IFN-alpha three times a week for 6 months and were then followed prospectively for at least 12 months. 53% of patients responded initially, but a sustained response was observed in only 17%. Responders were found to be significantly younger than nonresponders (45.6 +/- 3.1 vs. 55.4 +/- 2.7), and less frequently cirrhotic (2/16 vs. 7/14). Sustained responders had a mean pretreatment HCV-RNA titer approximately tenfold lower than that of those who did not have a sustained response, but the difference was not statistically significant. HCV genotype was found to be significantly associated with both initial and sustained response. Patients infected with HCV-2a were more likely to respond (89%) than those who were infected with HCV-1b (37%), and they were also more likely to sustain that response (33% vs. 6%).(ABSTRACT TRUNCATED AT 250 WORDS)

Optimisation and evaluation of a quantitative chemiluminescent polymerase chain reaction assay for hepatitis C virus RNA.

A quantitative non-isotopic assay for measuring hepatitis C virus (HCV) RNA has been developed and evaluated. Viral RNA extracted from serum is reverse transcribed and amplified by the polymerase chain reaction (PCR) using biotinylated 5' non-coding region primers. PCR products are captured on streptavidin coated microtitre plates, denatured with sodium hydroxide and hybridised with an alkaline phosphatase-labelled oligonucleotide probe. Quantification is achieved by measuring the intensity of light emitted by a dioxetane-based chemiluminescent substrate. The chief advantages of the assay are: (i) extreme sensitivity with the ability to detect single molecules of HCV cDNA, (ii) a 5 log10 dynamic range sufficient to cover the 10(3)-10(8) genomes/ml viraemia levels typically seen in patient samples, (iii) specificity and reproducibility suitable for application in a clinical context, and (iv) a rapid non-nested assay format with the ability to handle large throughputs and with a potential for automation. The feasibility of using the assay to monitor viraemia level changes in patients undergoing interferon therapy for chronic HCV infection has been demonstrated.

A pilot study of combination therapy with ribavirin plus interferon alfa for interferon alfa-resistant chronic hepatitis C.

BACKGROUND/AIMS: In chronic hepatitis C, interferon alfa (IFN-alpha) therapy fails to achieve a sustained response in approximately 75% of patients. Similarly, ribavirin induces only a transient response. The aim of this study was to evaluate whether ribavirin and IFN-alpha in combination could be effective in IFN-alpha-resistant chronic hepatitis C. METHODS: Twenty patients with chronic hepatitis C resistant to a previous course of IFN-alpha were randomly assigned to receive either ribavirin combined with IFN-alpha or IFN-alpha alone for 6 months. RESULTS: Serum alanine aminotransferase levels decreased significantly during therapy in both treatment groups, but after therapy, the levels remained significantly decreased only in the combination therapy group. Nine months after treatment, sustained normalization of aminotransferase levels, associated with sustained loss of serum hepatitis C virus RNA, was observed in 40% of the patients in the combination therapy group but in none of the patients treated with IFN-alpha alone (P < 0.05). The sustained response was accompanied by reduced hepatic necroinflammatory activity on biopsy. CONCLUSIONS: These findings suggest that ribavirin plus IFN-alpha combination therapy is able to induce a sustained biochemical and virological response in a significant proportion of patients with IFN-alpha-resistant chronic hepatitis C.

Developmental effects of combined exposure to ethanol and vitamin A.

The potential for ethanol (EtOH) to influence the developmental toxicity of vitamin A was investigated. 11 groups of approximately 31 FDA-bred Osborne-Mendel rats received either a control or isocaloric 6.4% EtOH liquid diet (containing 4000 IU vitamin A/litre) ad lib. The vehicle control, EtOH and pair-fed (pair-fed against the EtOH group) groups received corn oil (the vehicle) by gavage. Vitamin A was administered by gavage without EtOH at 40,000, 80,000, 120,000 or 160,000 IU/kg daily. Vitamin A was administered by gavage at 10,000, 20,000, 40,000 or 80,000 IU/kg with EtOH ad lib., daily throughout the study. Combined EtOH and vitamin A resulted in significant reductions in maternal diet consumption and body weight when doses of vitamin A were as low as 10,000 IU/kg. The most severe effects on overall (days 0-20) maternal body weight gain were observed in the groups receiving 120,000 or 160,000 IU vitamin A/kg alone or EtOH in combination with 80,000 IU vitamin A/kg. The overall diet consumption (days 0-20) paralleled the overall weight gain. In general, pups exposed to ethanol and vitamin A had a tendency to weigh less than those exposed to vitamin A alone, but to weigh more than those exposed to EtOH alone. EtOH combined with vitamin A at 80,000 IU/kg resulted in an increased incidence of cleft palate relative to the vehicle control or either treatment alone. The incidence of exencephaly and protruding tongue was significantly greater in the group given vitamin A at 160,000 IU/kg, compared with the vehicle control group. The most consistent statistically significant skeletal finding in the groups receiving combined treatment was a treatment-related increased incidence of supernumerary ribs [14th rib (C7), 14th rib bud (L1) and 15 ribs]. In addition, the incidence of misshapen zygomatic arch was also significantly increased in the group exposed to EtOH and vitamin A at 80,000 IU/kg. The incidence of moderately enlarged renal pelvis and severely enlarged ureter proximal to the kidney was increased in the group exposed to EtOH and vitamin A at 80,000 IU/kg relative to the vehicle control, or either treatment alone. Therefore, for some of the endpoints examined in this investigation, it would appear that ethanol potentiates the developmental effects of vitamin A.

Effect of ethanol and vitamin A excess on vitamin A status in the liver, plasma and foetuses of pregnant rats.

The effect of maternal consumption of dietary ethanol and high doses of vitamin A by gavage was investigated by evaluating plasma, liver and foetal vitamin A in Osborne-Mendel pregnant rats with a view to assessing whether ethanol modulated the potential toxicity of excess vitamin A. All groups received 4000 IU vitamin A/litre in a liquid diet. Ethanol-exposed groups also received 6.4% (v/v) ethanol in the liquid diet. Vitamin A was administered by gavage once per day in corn oil in doses ranging from 10,000 to 160,000 IU/kg body weight. Plasma vitamin A levels in ethanol-exposed groups were similar to levels in a pair-fed group. Plasma vitamin A levels were similar in the group given ethanol plus 40,000 IU vitamin A/kg and the group given 40,000 IU vitamin A/kg only, but were higher in the group receiving ethanol plus 80,000 IU vitamin A/kg than in the group given 80,000 IU vitamin A/kg only. Retinyl esters were present in the plasma of animals receiving 160,000 IU vitamin A/kg only, indicating possible saturation of the liver with vitamin A. Retinyl palmitate levels in female foetuses of the group administered ethanol plus 80,000 IU vitamin A/kg were significantly higher than those of the group administered 80,000 IU vitamin A/kg only; no significant differences in levels of retinyl palmitate in male foetuses were observed between these two groups. This observation suggests a possible sex difference in the modulation of vitamin A toxicity by ethanol in the foetus.

Teratogenic potential of triphenyl phosphate in Sprague-Dawley (Spartan) rats.

Male and female Sprague-Dawley (Spartan) rats were fed dietary levels of 0, 0.25, 0.50, 0.75 or 1.00% triphenyl phosphate (TPP) from 4 weeks post weaning for 91 days, through mating and gestation. At these dietary levels, the daily intake of TPP during pregnancy was 0, 166, 341, 516 and 690 mg/kg body weight, respectively. TPP exposure had no toxic effects on mothers or offspring at these dosages. The types of developmental anomalies were similar in both treated and control animals. No significant increases in the incidence of anomalies were seen in the treated groups as compared to control values. TPP was not teratogenic in Sprague-Dawley rats at the levels tested.

Potential reversibility of skeletal effects in rats exposed in utero to caffeine.

Groups of Osborne-Mendel rats, killed at three time intervals following mating, were studied to determine whether prenatal skeletal ossification delays observed following low-level caffeine administration represent transient or persistent ossification problems. Group A litters were killed on gestation day 20; group B neonates were killed on post-natal day 0; and group C pups were killed on post-natal day 6. Within each group, dose levels of 0, 0.018, 0.036 or 0.07% caffeine in distilled water were available ad lib. to groups of 30-60 dams from gestation day 0 to day 20. Average daily caffeine consumption was 24.7-29.0 mg/kg body weight for the 0.018% group, 42.7-48.8 mg/kg body weight for the 0.036% group and 70.6-75.1 mg/kg body weight for the 0.07% group. In group A litters, the mean number of viable foetuses was significantly less in the mid-dose and high-dose animals than in the controls. In the same group, the average number of foetuses per litter with at least one sternebral ossification delay was increased significantly in all treated groups and the average number of foetuses per litter with at least two sternebral variations was significantly increased in the mid- and high-dose groups. The percentages of litters containing foetuses with at least two and at least three sternebral variations and the average number of foetuses per litter with at least three sternebral variations were significantly increased only in the high-dose group. Foetuses from the mid- and high-dose groups also had significant increases in certain skeletal defects, namely missing centra and reduced ossification of the dorsal arch. Foetuses from the high-dose group also had significant increases in bipartite supraoccipital, and reduced ossification of the hyoid, metacarpals and metatarsals. In group B, day 0 neonates from all treated groups showed a significantly increased incidence of delayed sternebral ossification (average number of foetuses per litter with one or more missing, incomplete or bipartite sternebrae). The percentages of litters containing neonates with delayed sternebral ossification were also increased significantly in all treated groups. Neonates from the 0.07% level in group B also exhibited a significant increase in the incidence of supernumerary rib bud, and in reduced ossification of the metacarpals, metatarsals and calcaneus bones. Significant increases were also seen, in group B, in the low- and mid-dose animals, respectively, in supernumery rib bud and in reduced ossification of the metatarsals.(ABSTRACT TRUNCATED AT 400 WORDS)

Teratological research using in vitro systems. III. Embryonic organs in culture.

A search of literature published through the spring of 1986 yielded approximately 95 citations for the following embryonic organs in culture: kidney, pancreas, skin, palate, craniofacial tissue, tooth, lens, bones, digits, and liver. However, only the in vitro organ culture of the palate and tooth are reviewed in this paper. The other organ culture systems were not reported as teratogenic screens. Although some organs may have the potential for such use, many are currently used for evaluation of the pathogenesis associated with congenital abnormalities, cancer, or transplacental carcinogenesis, or in studies of the structure and function of differentiating organs, and will not be included in this review.

Reproduction study of caffeine administration to male Osborne-Mendel rats.

The potential for caffeine, administered twice daily by gavage in a total dose of 40 or 80 mg/kg/day, to adversely affect the reproductive performance of male rats was investigated. Treatment was continued through 3 wk of serial mating; mating and dosing were terminated concurrently, after which the sires were autopsied and their testes weighed. Controls were treated similarly with distilled water. Significant dose-related differences were detected for sire body weight, but not for testes weight. The majority of the significant effects on the offspring were for those born to the low-dose sires. Statistically significant differences were sporadically detected for the number of pups born and their body weights and survival; however, these differences were not consistently detected in either a dose- or temporal-related fashion. Thus, caffeine appears to have little potential to produce adverse reproductive effects when administered by gavage to male rats at the levels tested in this study.

Nonideal collection characteristics of single stage and cascade impactors.

Experimental studies of single stage and cascade impactors showed that bounce was severe for dry particles on dry solid surfaces. Oil drops on dry surfaces or dry particles on oil coated surfaces were collected as predicted by theory. While glass fiber media collection surfaces reduced particle bounce, the impactor collection efficiency characteristics were changed.

Precision aerosol divider.

A precision aerosol divider with a 17 to 1 flow split has been designed and tested. The divider, primarily a mass monitor calibration device, collects 94.4% of the aerosol on a filter to serve as a standard and delivers 5.6% of the aerosol to the mass monitor to be calibrated.