A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.

Bacterial infection of the mammalian bloodstream can lead to overwhelming sepsis, a potentially fatal syndrome of irreversible cardiovascular collapse (shock) and critical organ failure. Cachectin, also known as tumour necrosis factor, is a macrophage-derived peptide hormone released in response to bacterial lipopolysaccharide, and it has been implicated as a principal mediator of endotoxic shock, although its function in bacterial sepsis is not known. Anaesthetized baboons were passively immunized against endogenous cachectin and subsequently infused with an LD100 dose of live Escherichia coli. Control animals (not immunized against cachectin) developed hypotension followed by lethal renal and pulmonary failure. Neutralizing monoclonal anti-cachectin antibody fragments (F(ab')2) administered to baboons only one hour before bacterial challenge protected against shock, but did not prevent critical organ failure. Complete protection against shock, vital organ dysfunction, persistent stress hormone release and death was conferred by administration of antibodies 2 h before bacterial infusion. These results indicate that cachectin is a mediator of fatal bacteraemic shock, and suggest that antibodies against cachectin offer a potential therapy of life-threatening infection.

No abstract

A specific assay has been developed for a blood-borne non-A, non-B hepatitis (NANBH) virus in which a polypeptide synthesized in recombinant yeast clones of the hepatitis C virus (HCV) is used to capture circulating viral antibodies. HCV antibodies were detected in six of seven human sera that were shown previously to transmit NANBH to chimpanzees. Assays of ten blood transfusions in the United States that resulted in chronic NANBH revealed that there was at least one positive blood donor in nine of these cases and that all ten recipients seroconverted during their illnesses. About 80 percent of chronic, post-transfusion NANBH (PT-NANBH) patients from Italy and Japan had circulating HCV antibody; a much lower frequency (15 percent) was observed in acute, resolving infections. In addition, 58 percent of NANBH patients from the United States with no identifiable source of parenteral exposure to the virus were also positive for HCV antibody. These data indicate that HCV is a major cause of NANBH throughout the world.

We measured antibody (anti-HCV) to hepatitis C virus, which causes non-A, non-B hepatitis, by radioimmunoassay in prospectively followed transfusion recipients and their donors. Of 15 patients with chronic non-A, non-B hepatitis documented by liver biopsy, all seroconverted for the antibody; of 5 with acute resolving non-A, non-B hepatitis, 3 (60 percent) seroconverted. The development of anti-HCV was delayed (mean delay, 21.9 weeks after transfusion, or 15 weeks after the onset of clinical hepatitis) and took approximately one year in one patient. Antibody has persisted in 14 of the 15 patients with chronic disease (mean follow-up, greater than or equal to 6.9 years; maximum, greater than or equal to 12), but has disappeared in the 3 with acute resolving disease after a mean of 4.1 years. Anti-HCV was detected in samples of donor serum given to 14 (88 percent) of the 16 anti-HCV-positive patients for whom all donor samples were available. Only 33 percent of the anti-HCV-positive donors tested had an elevated serum concentration of alanine aminotransferase; 54 percent were positive for antibody to the hepatitis B core antigen (anti-HBc). We conclude that hepatitis C virus is the predominant agent of transfusion-associated non-A, non-B hepatitis and that screening of donors for anti-HCV could prevent the majority of cases of the disease. "Surrogate" assays for anti-HBc and alanine aminotransferase would have detected approximately half the anti-HCV-positive donors involved in the transmission of hepatitis that we identified.

No abstract

A high incidence of community-acquired hepatitis C virus infection that can lead to the progressive development of chronic active hepatitis, liver cirrhosis, and primary hepatoceflular carcinoma occurs throughout the world. A vaccine to control the spread of this agent that represents a major cause of chronic liver disease is therefore needed. Seven chimpanzees (Pan troglodytes) have been immunized with both putative envelope glycoproteins [El (gp33) (4) and leads to the development of chronic hepatitis and liver cirrhosis in "50%o and 10% of cases, respectively (5). A significant proportion of patients with liver cirrhosis will also develop primary hepatocellular carcinoma (6). The prevalence of HCV infection around the world is generally between 0.4 and 2% (7-10), although a much higher level has been reported in Egypt (14%; see ref. 11). Therefore, HCV constitutes a major cause of chronic liver disease throughout the world. With the recent development of recombinant-based diagnostic assays for the detection of circulating HCV antibodies (2, 3), the risk of being infected with HCV after transfusion of blood or cellular components has been substantially reduced (12,13). However, community-acquired infection is much more common and occurs at various frequencies in high-risk groups such as i.v. drug users, health-care workers, and sexual and household contacts ofhepatitis patients, although -40%o of cases in the United States appear to have no known risk factor for acquisition of infection (14). Thus, the development of an HCV vaccine to prevent transmission within the community is highly desirable.HCV is distantly related genetically to both the pestiviruses and flaviviruses and, like these relatives, appears to process virion structural proteins from the N-terminal region of the polyprotein precursor encoded by the positivestranded RNA genome (15). The host signal peptidase mediates the cleavage of a basic, presumed nucleocapsid protein (C; -20 kDa) from the N terminus of the polyprotein precursor followed by two glycoproteins (El, glycoproteins (gp33 and gp72) under nondenaturing conditions from the endoplasmic reticulum. A fraction of the purified material was shown to exist in the form of a large E1/E2 oligomeric complex (22). We now report on the efficacy of this purified preparation in vaccinating chimpanzees against experimental infection with HCV-1. MATERIALS AND METHODSVaccine Preparation. A Stu I-Bgl II cDNA restriction fragment ofthe HCV-1 genome (nt 63 to +2901; aa 1-967; ref. 15) encoding the complete C (20 kDa), El (gp33 kDa), and E2 (gp72 kDa) proteins along with a C-terminally truncated NS2 product was cloned into the Sma I site of plasmid SC59 downstream of a hybrid early/late vaccinia promoter (S. Chakrabarti and B.M., unpublished work). BSC40 cells preinfected with wild-type WR vaccinia were transfected with the SC59 recombinant and thymidine kinase-negative recombinants, selected, and purified through three rounds ofplaque purification (23). Spinner cultures of HeLa cells (109 c...

Severe weight loss and debilitative wasting of lean body mass frequently complicate the treatment of patients suffering from malignancy or chronic infection. Termed cachexia, this syndrome of anorexia, anemia, and weakness further increases cancer mortality; some data indicate that as many as 30% of cancer patients die from cachexia, rather than tumor burden (1-3). The severity of cachexia may be unrelated to tumor size or parasite load, and profound wasting has been observed in patients with tumor burdens of only 0.01-5.0% body mass (4). If not reversed, cachexia-associated derangements of homeostasis lead to immunological deficiencies, organ failure, and multiple metabolic abnormalities . While it is clear that a variety of mechanisms participate in the pathogenesis of cachexia, and that cachexia adversely affects prognosis, the etiology of this syndrome is not known.For a number of years we have been searching for endogenous, humoral mediators of cachexia, beginning with the characterization of metabolic changes in trypanosome-infected rabbits that develop profound cachexia and lose up to 50% of lean body mass within weeks. In later stages of disease a paradoxical increase in circulating triglycerides occurs, attributable to systemic suppression of lipoprotein lipase (LPL)t (5). A bacterial LPS-inducible serum factor which suppresses LPL in mice, and several other key lipogenic enzymes in the adipocyte cell line 3T3-L1, was isolated and named cachectin (6, 7). Cachectin evokes a state of cellular cachexia by suppressing the expression of several mRNAs encoding essential lipogenic enzymes (8, 9). Myocytes also show changes in cellular metabolism after exposure to cachectin in vitro, including a prompt decrease in resting transmembrane potential difference and depletion of intracellular glycogen stores with increased lactate efflux, and a later increase in hexose transporters (10,11). It has been suggested that cachectin may play a