In vitro hepatitis B virus infection of human bone marrow cells.

Infection of humans with hepatitis B virus (HBV) frequently results in suppression of hematopoiesis; in some cases this may lead to severe bone marrow failure. The mechanism whereby HBV infection affects hematopoiesis is unknown. In vitro exposure of human bone marrow to HBV results in a dose-dependent inhibition of erythroid (erythroid burst forming units, BFU-E; erythroid colony-forming units CFU-E), myeloid (colony-forming units-granulocyte macrophage CFU-GM), and lymphoid (CFU-[T-lymphocytic]-TL) hematopoietic stem cells. Inactivation or immunoabsorption of HBV from sera resulted in loss of HBV-induced inhibition of hematopoietic stem cells. De novo gamma interferon was not detectable in the supernatants of cultures of bone marrow cells with HBV. Antibodies to gamma interferon did not affect the suppression of hematopoietic stem cells by HBV. Hepatitis B surface antigen (HBsAg) was detected by immune electron microscopy in nuclei of greater than 70% of immature hematopoietic cells including myeloblasts, normoblasts, and lymphoblasts; granulocytes had mostly cytoplasmic HBsAg. Hepatitis B virus core antigen (HBcAg) was also detected in about 5% of HBV infected bone marrow cells by immunoperoxidase staining. These data indicate that HBV can infect hematopoietic cells and their progenitors, thus suggesting a wider range of tropism for HBV than previously reported. These results may provide a basis to study HBV infection in vitro, and the effects of HBV on hematopoiesis.

Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.

Serum components inhibit DNA polymerase, thereby obviating direct detection of serum viral DNA sequences by the polymerase chain reaction (PCR). This has necessitated extraction of nucleic acid from sera before performing PCR and has resulted in loss of sensitivity. By adsorbing virus to a solid surface (microcentrifuge tubes or antibody coated microparticles) followed by proteinase K digestion, as little as three viruses per 200 microliters serum may be directly detected by PCR without nucleic acid extraction. The sensitivity is dependent on the surface area of the adsorptive surface and is increased by having antibodies on the adsorptive surface. The nucleic acid sequence of the amplified DNA fragments may be directly determined by the dideoxy method. Of 24 plasma samples from HBsAg+ volunteer blood donors, HBV DNA was detected in 7 by dot blot assay, 7 by liquid hybridization, and 9 by PCR. PCR detected DNA in every sample that was positive by another assay. Analysis of serial samples of two patients with acute self-limited hepatitis B found detectable HBsAg and pre-S2 antigenemia before HBV DNA by the PCR method. These results suggest that surface antigenemia may precede viremia during acute hepatitis.

A randomized, double-blind, placebo-controlled trial of lenalidomide in the treatment of moderately severe active Crohn's disease.

BACKGROUND: Therapy targeted at tumour necrosis factor-alpha has an established role in Crohn's disease. Lenalidomide, an analogue of thalidomide, is an oral immunomodulatory agent with powerful antitumour necrosis factor-alpha properties. It is licensed for myeloma and myelodysplastic syndrome. Based upon reports of thalidomide efficacy, lenalidomide was evaluated in Crohn's disease. AIM: To evaluate the efficacy and safety of lenalidomide in subjects with moderately severe active Crohn's disease. METHODS: In a multicentre, double-blind, placebo-controlled parallel group study 89 subjects were randomized to lenalidomide 25 mg daily, 5 mg daily or placebo. Subjects were treated for 12 weeks. The primary end point was a 70-point reduction in Crohn's Disease Activity Index. RESULTS: The overall clinical response rate was not significantly different between the three groups: lenalidomide 25 mg 26%, lenalidomide 5 mg 48% and placebo 39%. Lenalidomide was generally well tolerated with only one serious adverse event, a deep vein thrombosis, being attributed to treatment. CONCLUSION: Lenalidomide, an oral agent with antitumour necrosis factor-alpha properties, was not effective in active Crohn's disease in contrast to reports of benefit from thalidomide. The reasons for this lack of efficacy are speculative, other physiological activities may offset its action on inflammatory cytokines, or its antitumour necrosis factor-alpha action without apoptosis may be insufficient for activity in Crohn's disease.

Thalidomide and its analogues inhibit lipopolysaccharide-mediated Iinduction of cyclooxygenase-2.

We investigated the effect of thalidomide, a compound with immunomodulatory and antiangiogenic properties, on lipopolysaccharide (LPS)-mediated induction of cyclooxygenase-2 (Cox-2) and prostaglandin (PG) biosynthesis in murine macrophages. Thalidomide caused a dose-dependent inhibition of LPS-mediated induction of PGE(2) synthesis in RAW 264.7 cells. The induction of Cox-2 protein and mRNA by LPS was also suppressed by thalidomide. Based on the results of nuclear run-off assays and transient transfections, treatment with LPS stimulated Cox-2 transcription, an effect that was unaffected by thalidomide. Thalidomide decreased the stability of Cox-2 mRNA. A series of structural analogues of thalidomide also inhibited LPS-mediated induction of Cox-2 and PGE(2) synthesis. Taken together, these data provide new insights into the antineoplastic and anti-inflammatory properties of thalidomide.

Aplastic anemia and non-A, non-B hepatitis.

Severe aplastic anemia is a rare but important complication of hepatitis. The agent(s) responsible for the hepatitis in these cases have not been well defined. Sixteen patient with hepatitis-associated aplastic anemia were studied for evidence of recent infection with hepatitis A virus, hepatitis B virus, cytomegalovirus, Epstein-Barr virus, and Toxoplasma. Results were compared with data from 10 randomly selected patients with aplastic anemia unassociated with hepatitis. Of the 16 patients, recent acute hepatitis A infection could be excluded in at least 14 patients. Hepatitis B surface antigen (HBsAg) was present in only one patient. A diagnosis of recent hepatitis B infection could not be excluded with confidence in two others. Tests for cytomegalovirus, Epstein-Barr virus, and Toxoplasma gave negative results. No patient with aplasia unassociated with hepatitis had evidence of recent hepatitis A infection, and the frequency of hepatitis B antibodies in this group was indistinguishable from that in patients with hepatitis. These data indicate that most cases of hepatitis that preceded aplastic anemia were not caused by hepatitis A virus or hepatitis B virus; non-A, non-B agents were probably involved in at least 13 of the 16 cases studied.

Hepatitis in an adult with rubella.

Rubella accompanied by serum aminotransferase elevations occurred in a 24-year-old woman. Although not generally recognized, hepatic involvement in adult rubella was the probable cause of her liver function test abnormalities. Sporadic hepatitis labeled as non-A, non-B may result from infection by common viruses such as rubella.

Serologic and DNA follow-up data from HBsAg-positive patients treated with orthotopic liver transplantation.

Fifteen hepatitis B surface antigen (HBsAg) positive patients treated with orthotopic liver transplantation were studied to determine whether any clinical, serologic, or histologic data were predictive for recurrent hepatitis B infection leading to graft failure. Six patients died early, one due to primary graft nonfunction and the remaining five due to septic complications. There were nine patients surviving longer than two months, eight of whom are alive at a mean follow-up of 556 days. HBsAg and hepatitis B core antibody (anti-HBc) reappeared in the sera of all survivors after a variable transient period of clearance. One patient died 3 months posttransplant of fungal sepsis and was found to have histologic evidence for recurrent hepatitis and positive immunoperoxidase staining postmortem. The remaining eight survivors are home and clinically well, with no histologic evidence of hepatitis. Seven of these eight patients have hepatitis B viral DNA in their sera. We conclude that while there is a high early mortality, usually from sepsis, none of the serologic, histologic, or DNA data analyzed can be used to predict graft loss from recurrent hepatitis. No grafts have been lost due to recurrent hepatitis B in this series, and therefore we believe that HBsAg positive patients should not be excluded from transplantation.

Hepatitis B disease in dialysis and transplant patients. Further epidemiologic and serologic studies.

As hepatitis B virus (HBV) infection in renal transplant recipients is associated with a high incidence of progressive liver disease it may be inadvisable to transplant hemodialysis patients with hepatitis B antigenemia. To determine the natural history of HBV disease in hemodialysis patients, all 49 patients on hemodialysis treatment for at least 1 year, at 3 centers, who developed circulating hepatitis B surface antigen (HBsAG), were studied. A subgroup of these patients (n = 31) aged less than or equal to 50 years, followed for 55 +/- 6 months after detection of HBsAg was compared with 22 previously studied HBsAg-positive transplant patients followed for 81 +/- 9 months. Significantly more transplant patients developed chronic hepatitis defined biochemically (P less than .001) and none of the transplant patients became HBsAg-negative compared with 19% of the hemodialysis group. Taking difference in follow-up into account, mortality was significantly higher in the transplant recipients (P less than .005) following development of HBsAg antigenemia, and the mortality difference was attributable to deaths from liver disease. A total of 36 serum samples from 14 of the 22 HBsAg-positive renal transplant recipients was analyzed for hepatitis B e antigen (HBeAg), antibody to hepatitis D virus (anti-HD), and hepatitis B virus deoxyribonucleic acid (HBVDNA) concentration. No serum sample was anti-HD-positive. Twelve of the 14 patients were HBeAg-positive. Five patients became HBeAg-negative, 3 of whom developed aggressive liver disease. One HBeAg-negative anti-HBe-positive patient had progression of liver disease from asymptomatic carrier status to chronic active hepatitis (CAH). Of 14 patients, 9 developed progressive CAH. HBVDNA concentration was not diagnostic of disease activity on liver biopsy. However only 1 sample of 10 measured in 5 patients with nonprogressive disease had a level greater than 100 pg/L, compared with 9 of 17 in the group who progressed to CAH. During the interval when the liver histology progressed from asymptomatic carriage or chronic persistent hepatitis (CPH) to CAH, the HBVDNA concentration increased by greater than 10 times baseline in 4 of 5 patients who had serial samples, whereas this did not occur in 4 patients with nonprogressive disease. We conclude that the long-term outcome of hepatitis B infection in transplant recipients is significantly worse than in hemodialysis patients. Therefore it may be inadvisable to transplant HBsAg-positive hemodialysis patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Variation in antibody reactivity to the hepatitis C virus by comparative immunoscreening and enzyme immunoassay.

The detection of antibody to the hepatitis C virus C100-3 antigen from the nonstructural region (NS3/NS4) of the viral genome was the first useful marker developed to detect past or potentially active infection with the hepatitis C virus. A systematic epitope survey of the nonstructural region has uncovered other immunogenic antigens. In order to assess the possible diagnostic utility of these antigens, their reactivity against a limited panel of sera from patients with chronic liver disease due to hepatitis C virus and other etiologies was tested. Antibody assays were performed using an immunoblot plaque assay and an enzyme-linked immunosorbent assay (ELISA). In a study of 16 C100-3-reactive individuals, all 16 patients were reactive using the plaque assay for the NS3 3' (409-1-1) and NS3 5' (C33u). In this same group of patients, antibodies by ELISA were reactive to NS3 3' in 12 of 16 patients (75%), NS3 5' in 15 of 16 patients (93%), and a capsid antigen (NC450) in 14 of 16 patients. In a group of five patients who were diagnosed with cryptogenic liver disease (C100-3 negative), 4 of 5 patients were reactive for antibody to all of the above epitopes. In a survey of 23 patients with other forms of chronic liver disease (nonviral liver disease, hepatitis B, alcoholic liver disease, cholestatic liver disease, and autoimmune hepatitis), only 1 of 23 patients was reactive for antibody to the C100-3 and 4 of 23 patients were reactive for antibodies to structural and nonstructural regions of the virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Fulminant hepatic failure following bone marrow transplantation for hepatitis-associated aplastic anemia.

A case of aplastic anemia associated with non-A, non-B hepatitis was initially successfully treated by bone marrow transplantation. The patient subsequently developed fulminant hepatic failure. Fulminant hepatic failure is rare in bone marrow transplantation and only occurs in association with aplastic anemia associated with viral hepatitis. This case helps to highlight the relationships between the immune system, hepatitis, and bone marrow failure.

Correlation of HBV DNA and monoclonal reactivity to HBsAg in serum of patients with HBV infection.

Hepatitis B virus (HBV) DNA hybridization assay, a monoclonal radioimmunoassay (M-RIA) for hepatitis B surface antigen (HBsAg) and conventional polyclonal immunoassays for HBV associated antigens were used to study sera from patients on dialysis and with acute hepatitis B. HBV DNA was detectable in hepatitis B e antigen (HBeAg) negative patients with acute hepatitis but not in HBsAg+ HBeAg- dialysis patients. In acute hepatitis, HBsAg immunoreactivity by M-RIA could still be detected even though a commercial immunoassay for HBsAg, the AUSRIA II, and the HBV DNA assay were no longer positive. Unlike in acute HBV infection, serum HBV DNA was detectable in dialysis patients who were AUSTRIA II negative but M-RIA positive. Serial determination of HBsAg by M-RIA and HBV DNA revealed episodes of HBV DNA positivity months after both the HBsAg was no longer positive by polyclonal immunoassay. Thus, the M-RIA for HBsAg and the molecular hybridization technique for HBV DNA are sensitive and specific assays for the identification of potentially infectious individuals who would not have been characterized as such based on the results of conventional polyclonal immunoassays.

S.T.E.P.S.: a comprehensive program for controlling and monitoring access to thalidomide.

In July 1998, the US Food and Drug Administration approved the marketing of thalidomide for the treatment of cutaneous manifestations of erythema nodosum leprosum. To ensure that fetal exposure to this teratogenic agent does not occur, the manufacturer has instituted a comprehensive program to control prescribing, dispensing, and use of the drug. This program, known as the System for Thalidomide Education and Prescribing Safety (S.T.E.P.S. [Celgene Corporation, Warren, New Jersey]), is based in part on experience gained with other drugs--specifically isotretinoin and clozapine--that offer important clinical benefits but carry the potential for serious harm. To achieve its goal of the lowest possible incidence of drug-associated teratogenicity, the S.T.E.P.S. program uses a three-pronged approach: (1) controlling access to the drug; (2) educating prescribers, pharmacists, and patients; and (3) monitoring compliance. Clinicians who wish to prescribe thalidomide must be registered in the S.T.E.P.S. Prescriber Registry and agree to prescribe the drug in accordance with S.T.E.P.S. patient eligibility criteria and monitoring procedures. Pharmacies must also register and agree to comply with patient identification and monitoring criteria. Finally, patients receive visual aids, including a videotape, written material, and verbal counseling about the benefits and risks of thalidomide therapy, the importance of not becoming pregnant during therapy, and the types of contraception required (including emergency contraception) and their availability. Women of childbearing potential must agree to undergo pregnancy testing before starting therapy and on a regular schedule during therapy. All patients must agree to complete a confidential survey about their compliance with contraception, testing, and drug therapy. The manufacturer is monitoring survey results and outcome data and is prepared to make whatever modifications to the S.T.E.P.S. program are necessary to ensure its effectiveness. In addition to minimizing the potential risk for fetal harm associated with thalidomide therapy, the S.T.E.P.S. program may provide a model for future cases in which a drug offers compelling benefits but poses profound risks unless its distribution is carefully controlled.

Gastrointestinal illness in managed care: healthcare utilization and costs.

Identification of inefficiencies is a first step to improving the quality of gastrointestinal (GI) care at the most reasonable cost. This analysis used administrative data to examine the healthcare utilization and associated costs of the management of GI illnesses in a 2.5 million-member private managed care plan containing many benefit designs. An overall incidence of 10% was found for GI conditions, with a preponderance in adults (patients older than 40 years) and women. The most frequently occurring conditions were abdominal pain, nonulcer peptic diseases, lower GI tract diseases, and other GI tract problems. These conditions, along with gallbladder/biliary tract disease, were also the most costly. Claims submitted for care during GI episodes averaged $17 per member per month. Increasing severity of condition was associated with substantial increases in utilization and costs (except for medication use). For most GI conditions, approximately 40% of charges were for professional services (procedures, tests, and visits) and 40% of charges were for facility admissions. The prescription utilization analysis indicated areas where utilization patterns may not match accepted guidelines, such as the low use of anti-Helicobacter pylori therapy, the possible concomitant use of nonsteroidal anti-inflammatory drugs in patients with upper GI diseases, and the use of narcotics in treating patients with lower GI disease and abdominal pain. Also, there was no clear relationship between medication utilization and disease severity. Thus, this analysis indicated that GI disease is a significant economic burden to managed care, and identified usage patterns that potentially could be modified to improve quality of care.

Comparative study of the immunogenicity and safety of two doses of recombinant hepatitis B vaccine in healthy adolescents.

PURPOSE: A prospective, two-armed, open-label, randomized trial was performed to compare the geometric mean titers (GMT), seroprotection (SP) and seroconversion (SC) rates found after administration of two doses of recombinant hepatitis B vaccine. METHODS: Recombinant hepatitis B vaccine 10 or 20 micrograms was administered IM at 0, 1, and 6 months in healthy adolescents. RESULTS: Volunteers who received either dose of the vaccine had similarly high seroconversion and seroprotection rates at all visits. At Month 8, both doses of the vaccine were highly immunogenic with GMTs of 1989 mIU/mL (10 micrograms dose) and 7672 mIU/mL (20 micrograms dose) and nearly equivalent SP rates (97% and 99% in the 10 and 20 micrograms dose groups, respectively). The geometric mean titers of seroconverters at Months 3, 6 and 8 were significantly greater in the 20 micrograms group as compared to the 10 micrograms group (p < or = 0.003). Both doses were well-tolerated, with injection site pain the most common reported adverse event. Injection site pain was reported significantly (p = 0.004) more by volunteers who received the 20 micrograms dose (10.7%) compared with volunteers who received the 10 micrograms dose (3.8%). CONCLUSION: Vaccination with 10 micrograms of recombinant hepatitis B vaccine may provide a clinically effective and economical alternative to the use of the 20 micrograms dose in healthy adolescents.

Cationic lipid enhances in vitro receptor-mediated transfection.

The efficiency of cell-specific transfection by receptor-mediated uptake is improved by the use of cationic lipids. Asialoglycoprotein (AP) was conjugated to poly-L-lysine (PL) and complexed with the plasmid pCMVL that contains a luciferase reporter gene. The asialoglycoprotein-poly-L-lysine:pCMVL (AP-PL:pCMVL) complexes then were mixed with the cationic lipid dioctadecylamidoglycylspermine (DOGS). This complex was taken up by the hepatocyte-like cell line, Hep G2, via the asialoglycoprotein receptor. The expression of luciferase in cells transfected with the DOGS/AP-PL: pCMVL complexes were significantly increased compared with AP-PL:pCMVL complexes without DOGS. The ratio of AP-PL to DOGS is an important determinant for both transfection efficiency and for maintaining receptor specificity. Therefore, cationic lipids significantly increased the efficiency of asialoglycoprotein receptor mediated transfection in the hepatoblastoma cell line, Hep G2. The use of cationic lipids with receptor-mediated gene delivery systems could potentially increase transfection efficiency yet maintain cell-target specificity.

Hepatitis C in liver transplant recipients.

HCV infection is commonly found in patients with chronic liver disease undergoing liver transplantation. However, the presence of antibody to HCV does not appear to be associated with the development of hepatitis posttransplant. No other risk factors were identified that appear to predispose patients to development of hepatitis in the posttransplant period, including amount of blood product exposure. The role of immunosuppression in the acquisition and expression of liver disease caused by HCV remains to be determined.

Molecular biology of viral hepatitis and hepatocellular carcinoma.

Five viruses are the major causes of hepatitis. These viruses are totally unrelated to each other in structure and mode of replication despite the similarity in the acute syndrome produced by each virus. HAV is a single-stranded RNA virus that has a very stable capsid and whose proteins are derived from a single polyprotein. HBV is a DNA virus that replicates through an RNA intermediate. HCV is a labile single-stranded RNA virus whose proteins are derived from a polyprotein. HDV is a defective RNA virus related to viroids that encodes a capsid antigen, delta antigen, and requires the envelope protein of HBV (HBsAg) for its propagation. HEV is a labile RNA virus that is unrelated to other known viruses. Hepatitis B, C, and D can cause chronic hepatitis. Both chronic hepatitis B and C virus infections are associated with primary hepatocellular carcinoma. The most likely mechanism for hepatitis B and C promotion of primary hepatocellular carcinoma in that these viruses cause chronic inflammation and increased mitotic activity of the pluripotent oval cells of the liver. Most likely, primary hepatocellular carcinoma arises out of synergy between chronic viral infection and some other carcinogenic stimulus such as exposure to a hepatotoxin.