HCV Infection Alters Salivary Gland Histology and Saliva Composition.

Hepatitis C virus (HCV) infection is the most common blood-borne chronic infection in the United States. Chronic lymphocytic sialadenitis and sicca syndrome have been reported in chronic HCV infection. Up to 55% of these patients may have xerostomia; the mechanisms of the xerostomia and salivary gland (SG) hypofunction remain controversial. The objectives of this project are to establish if xerostomia associates with SG and HCV infection and to characterize the structural changes in SG and saliva composition. Eighteen HCV-infected patients with xerostomia were evaluated for SG dysfunction; 6 of these patients (patients 1-6) were further evaluated for SG histopathological changes and changes in saliva composition. The techniques used include clinical and laboratory assessment, SG ultrasonography, histological evaluation, sialochemical and proteomics analysis, and RNA in situ hybridization. All the HCV patients had low saliva flow, chronic sialadenitis, and SG fibrosis and lacked Sjögren syndrome (SS) characteristic autoantibodies. Further evaluation of a subgroup of 6 HCV patients (patients 1-6) demonstrated diffuse lymphocytic infiltrates that are predominantly CD8+ T cells with a significant increase in the number of inflammatory cells. Alcian Blue/periodic acid-Schiff staining showed significant changes in the ratio and intensity of the acinar secretory units of the HCV patients' minor SG. The submandibular glands showed significant ultrasonographic abnormalities in the parenchyma relative to the parotid glands. Significant changes were also observed in the concentration of sodium and mucin 5b. Although no significant correlation was observed between the lymphocytic infiltrates and the years of HCV chronic infection, a positive correlation was observed between HCV RNA-positive epithelial cells and the years of HCV infection. Consistent with the low saliva flow and xerostomia, patients showed changes in several markers of SG acinar and ductal function. Changes in the composition of the saliva suggest that HCV infection can cause xerostomia by mechanisms distinct from SS.

Non-A, non-B hepatitis: ultrastructural evidence for two agents in experimentally infected chimpanzees.

Two different ultrastructural alterations were observed in liver cells of chimpanzees inoculated with plasma derived from two different patients with non-A, non-B hepatitis. During the acute phase of illness in one group of four chimpanzees, peculiar tubular structures, composed of two unit membranes with electron-opaque material in between, were observed in the cytoplasm of hepatocytes. In contrast, these structures were never detected in the liver cells of the second group of five chimpanzees that received the second inoculum, However, nuclear changes, usually associated with aggregates of 20- to 27-nanometer particles, were found in hepatocytes of the latter animals. Although these particles resembled viruses, they were not as uniform as small virus particles often appear. In five other chimpanzees inoculated with non-A, non-B hepatitis material not known to be related to the first two inocula, cytoplasmic structures were found in four, and nuclear structures were found in the remaining one. Thus, all 14 chimpanzees inoculated with transmissible non-A, non-B hepatitis agents could be classified as having either nuclear or cytoplasmic changes. These observations add support to epidemiologic data suggesting that there may be more than one agent of non-A, non-B hepatitis.

An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis.

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.

Lack of protective immunity against reinfection with hepatitis C virus.

Some individuals infected with hepatitis C virus (HCV) experience multiple episodes of acute hepatitis. It is unclear whether these episodes are due to reinfection with HCV or to reactivation of the original virus infection. Markers of viral replication and host immunity were studied in five chimpanzees sequentially inoculated over a period of 3 years with different HCV strains of proven infectivity. Each rechallenge of a convalescent chimpanzee with the same or a different HCV strain resulted in the reappearance of viremia, which was due to infection with the subsequent challenge virus. The evidence indicates that HCV infection does not elicit protective immunity against reinfection with homologous or heterologous strains, which raises concerns for the development of effective vaccines against HCV.

Transmission of HTLV-III infection from human plasma to chimpanzees: an animal model for AIDS.

Two of three chimpanzees given plasma from patients with acquired immune deficiency syndrome (AIDS) or pre-AIDS showed serum antibodies to type III human T-cell leukemia virus (HTLV-III) 10 to 12 weeks after transfusion. One animal also developed lymphadenopathy, transient depression of the ratio of T4 to T8 lymphocytes, and impaired blastogenic responses. No opportunistic infections occurred. Adenopathy persisted for 32 weeks, and antibody to HTLV-III persisted for at least 48 weeks. This transmission of HTLV-III by lymphocyte-poor plasma confirms the potential risk of such plasma or plasma derivatives to recipients. The susceptibility of the chimpanzee to HTLV-III infection and the ability to simulate the human lymphadenopathy syndrome in this animal makes it a valuable model for further study of AIDS.

The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.

The mechanisms by which hepatitis C virus (HCV) induces chronic infection in the vast majority of infected individuals are unknown. Sequences within the HCV E1 and E2 envelope genes were analyzed during the acute phase of hepatitis C in 12 patients with different clinical outcomes. Acute resolving hepatitis was associated with relative evolutionary stasis of the heterogeneous viral population (quasispecies), whereas progressing hepatitis correlated with genetic evolution of HCV. Consistent with the hypothesis of selective pressure by the host immune system, the sequence changes occurred almost exclusively within the hypervariable region 1 of the E2 gene and were temporally correlated with antibody seroconversion. These data indicate that the evolutionary dynamics of the HCV quasispecies during the acute phase of hepatitis C predict whether the infection will resolve or become chronic.

Identification of a murine CD4+ T-lymphocyte response site in hepatitis C virus core protein.

The T cell response to a recombinant HCV truncated core protein (cp1-10) was measured in a proliferation assay. Based on a 10-fold greater response to this truncated core protein than to its shorter form (cp1-8), a predominant epitope was mapped to the carboxyl quarter of this sequence. This epitope was further mapped to a synthetic peptide corresponding to amino acids 121-140 of the core protein. The peptide was antigenic for T cells of all three H-2 types tested, H-2 r, b and d, and the proliferating T cells were CD4+. Besides inducing specific proliferation in vitro, peptide aa121-140 can prime helper T cells in vivo. When boosted with core protein, mice primed with peptide produced 64-fold higher antibody titer than without priming in 1 week. The identification of a broadly immunogenic T cell helper epitope on core protein may be important for vaccine design against HCV.

Lack of isolate-specific neutralizing activity is correlated with an increased viral burden in rapidly progressing HIV-1-infected patients.

OBJECTIVE: To delineate the interaction between in vivo HIV replication and host antiviral immunity during disease progression in order to elucidate the pathogenesis of AIDS. DESIGN: In a cohort of HIV-seropositive patients, the serum concentration of viral particles, the blood concentration of mononuclear cells harbouring infectious virus and the serum titre of isolate-specific neutralizing antibodies were correlated with the rates of CD4+ T-cell depletion and disease progression. METHODS: Using a quantitative reverse-transcriptase linked polymerase chain reaction assay, the concentration of viral particles was measured in blood samples from 103 initially symptom-free subjects who were followed up for > or = 24 months. The concentration of infectious virus and the neutralizing antibodies to autologous HIV isolates were assessed in 37 out of the 103 subjects. The rate of decrease in CD4 cells over the 24 months was calculated for each subject. RESULTS: Rapidly progressing patients (rate of decrease in CD4 cells > or = 60%) had a high concentration of viral particles and a high concentration of infectious virus associated with an undetectable serum titre of isolate-specific neutralizing antibodies. Stable patients (rate of decrease in CD4 cells < 30%) had a low concentration of infectious virus and either a low concentration of viral particles with the absence of isolate-specific neutralizing antibodies or a high concentration of viral particles with the presence of isolate-specific neutralizing antibodies. Slowly progressing patients (rate of decrease in CD4 cells > or = 30 and < 60%) showed an intermediate profile. CONCLUSIONS: Progression to AIDS is associated with a shift in the balance between viral replication and host immunity that increases the concentration of infected cells and destroys the CD4+ T-lymphocyte population.

Comparison of four different antigen-capture assays for the detection of human immunodeficiency virus antigen (HIV-Ag).

Three enzyme immunoassays (EIA), two polyclonal and one monoclonal, and one polyclonal radioimmunoassay (RIA) for the detection of human immunodeficiency virus antigen (HIV-Ag) have been evaluated and compared. The three EIAs had similar sensitivity in detecting HIV viral lysate and were more sensitive than the RIA, which was the only assay with a one-step probing-detection format. However, the EIA that used a monoclonal anti-p24 as the capturing reagent was unable to detect any of the serial supernatant samples of a positive viral culture from an HIV-infected patient. Only the two polyclonal, non-p24-restricted assays were able to detect an unusual expression of HIV-Ag in the serum of an acute HIV-infected patient. Overall, the sensitivity of HIV-Ag capture assays was enhanced when (a) the capture antibody was polyclonal rather than monoclonal, (b) the polyclonal antibody was broad rather than p24-restricted, and (c) the probing-detecting procedure was in a two-step format rather than one-step format. In addition, the use of neutralizing assays to confirm the results was absolutely necessary.

Expression and characterization of HIV type 1 envelope protein associated with a broadly reactive neutralizing antibody response.

We have studied envelope protein from a donor with nonprogressive HIV-1 infection whose serum contains broadly cross-reactive, primary virus NA. DNA was extracted from lymphocytes, which had been collected approximately 6 and 12 months prior to the time of collection of the cross-reactive serum, and env genes were synthesized, cloned, expressed on pseudoviruses, and phenotyped in NA assays. Two clones from each time point had identical V3 region nucleotide sequences, utilized CCR5 but not CXCR4 for cell entry, and had similar reactivities with reference sera. Analysis of the full nucleotide sequence of one clone (R2) demonstrated it to be subtype B and have normal predicted glycosylation. R2 pseudovirus was compared with others expressing env genes of various clades for neutralization by sera from U.S. donors (presumed or known subtype B infections), and from individuals infected with subtypes A, C, D, E, and F viruses. Neutralization by the U.S. sera of R2 and other clade B pseudoviruses was low to moderate, although R2 was uniquely neutralized by all. R2 was neutralized by 3/3, 3/3, 2/5, 5/8, and 3/4 clade A, C, D, E, and F sera, respectively. R2 and a clade E pseudovirus were neutralized by largely complementary groups of sera, potentially defining two antigenic subgroups of HIV-1. The results suggest that the epitope(s) that induced the cross-clade reactive NA in donor 2 may be expressed on the R2 envelope.

Stages in the progression of HIV infection in chimpanzees.

Circulating HIV antigens and HIV specific antibodies in sera of experimentally infected chimpanzees were monitored by ELISA immunoassay, Western blot, and radioimmunoprecipitation procedures. Three of three chimpanzees given plasma from patients with AIDS or ARC tested positive for HIV antigens beginning six to ten weeks after transfusion. Antigen production rose sharply but was of short duration. Despite their proven infectivity and the presence of anti-HIV antibody, all donors to these chimpanzees tested negative for the HIV antigen. Of the three animals that developed HIV antigen one animal did not produce any HIV antibodies or evidence of disease. A second produced antibodies to only the p24 and p18 antigens and remained clinically well. The third produced antibodies beginning with anti-p24, to all the major HIV proteins except gp120, and then developed marked lymphadenopathy which persisted for 32 weeks. Antibody persistence after the disappearance of clinical disease was variable and was greatest for gp41 and least for p24. These data may be of value in the interpretation of human serological testing for HIV and in further studies of the sequence of events leading to the pathological effects of HIV infection. A significant value of the chimpanzee model is the capacity of this animal to respond in a variety of ways to HIV infection, suggesting the existence of successive or alternate states of early HIV infection, and may have implications in the design of early interventions.

Non-A, non-B viral hepatitis.

Non-A, non-B hepatitis is a newly recognized disease entity. Although initially described as a transfusion related viral infection, the disease can occur in sporadic, endemic, and epidemic settings. There are no confirmed, reproducible serologic tests for associated antigens or antibodies, but electron microscopy has revealed virus-like particles of different sizes. Nonspecific laboratory tests of hepatic dysfunction, especially alanine aminotransferase, are currently utilized to diagnose non-A, non-B hepatitis in patients and may be used to implicate blood donor carriers of this virus. The existence of an infectious non-A, non-B hepatitis agent and proof of a chronic carrier state in humans have been documented by transmission studies in chimpanzees. Cross challenge studies in chimpanzees, as well as some epidemiologic data, suggest that more than one agent causes non-A, non-B hepatitis.

Seroprevalence of hepatitis C virus in the general population of northwest Tanzania.

Sera from 516 participants enrolled in a population-based cross-sectional study in northwest Tanzania were tested for antibodies to hepatitis C virus (HCV). The mean age of study subjects was 29 years (range = 16-49 years); 43% were men, 6% reported a history of blood transfusion, and 4% were infected with human immunodeficiency virus-1 (HIV-1). Although 53 of 516 sera (10.3%, 95% confidence interval [CI] = 7.8-13.2%) were repeatedly reactive by a third-generation enzyme immunoassay (EIA-3), only 6 of the 53 were positive when tested with a third-generation recombinant immunoblot assay (confirmed HCV seroprevalence = 1.2%, 95% CI = 0.4-2.5%). The positive predictive value of the HCV EIA-3 in this population was 18.8% (95% CI = 7.0-36.4%). False positivity was not correlated with EIA-3 optical density values, age, sex, infection with HIV-1, or a history of blood transfusion, but it was marginally associated with increased serum IgG levels. We conclude that the prevalence of HCV is low in this region and that the HCV EIA-3 has a higher false-positivity rate in this population than has been reported among U.S. blood donors.

Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical, in situ hybridization and ultrastructural study.

Monoclonal antibodies (Mabs) were developed against antigens from a pure culture of Mycoplasma incognitus grown in modified SP-4 medium. All the Mabs obtained were shown to react only with M. incognitus, and not with other species of human mycoplasma. The Mabs identified M. incognitus immunohistologically in thymus, liver, spleen, lymph node, or brain from 22 patients with AIDS, as well as in 2 placentas delivered by patients with AIDS. Using an 35S-labeled DNA probe specific for M. incognitus and in situ hybridization technique, we also identified M. incognitus-specific genetic material in these tissues. Furthermore, ultrastructural studies of the specific areas of tissues which were highly positive for M. incognitus antigens revealed characteristic structures of mycoplasma organisms. These mycoplasma-like particles could be identified intracellularly and extracellularly. Histopathology of the tissues infected by M. incognitus varied from no pathological changes to fulminant necrosis with or without an associated inflammatory reaction. M. incognitus, a novel pathogenic mycoplasma, was cytopathic and cytocidal.

The clinical significance of hepatitis B virus antigens and antibodies.

There are two well-characterized antigen-antibody systems which relate specificially to viral hepatitis B. Tests for HBsAg and anti-HBs are readily available and of great benefit to the diagnosis, prevention and understanding of hepatitis B. Tests for HBcAg and anti-HBc are still research techniques which requires further development before they can be used at the level of everyday medical practice. HBsAg in an individual indicates that he harbors the virus of hepatitis B; it may be present in the absence of liver disease or be found in association with both acute and chronic type B hepatitis. The presence of HBsAg also suggests that HBV may be causally related to some cases of periarteritis nodosa, chronic glomerulonephritis, and hepatoma. Although HBV is readily transmitted in blood, the major portion of post-transfusion hepatitis now appears to be serologically unrelated to either the hepatitis B virus ("serum") or the hepatitis A virus ("infectious"); the etiology of these cases is currently undetermined. There is increasing evidence that HBV may be transmitted by modes other than blood, but the exact mechanisms of such transmission is not established. The combined transmission of HBV by blood and other routes has resulted in a large number of persistent carriers of HBsAg in the world. There is no current method to alter this carrier state. The hepatitis risk of such persistent carriers to their personal and professional contacts is under investigation.

A study of street heroin lots for the presence of the hepatitis B surface antigen.

Hepatitis B surface antigen (HBsAg) of subtype ay predominates among narcotic addicts infected with hepatitis B virus (HBV) in Europe, Australia and the United States. However, the ad subtype predominates among the non-addict carriers of HBsAg. We investigated the possibility that heroin lots were contaminated with HBV at a source of opium production, the Middle East, a geographical region where HBsAg/ay predominates in the general population. One hundred and nine lots of street heroin were assayed for HBsAg by radioimmunoassay. None of the lots tested was reproduceably HBsAg positive. These results suggest that the heroin itself is not responsible for the high incidence of HBV infection or for the predominance of HBsAg/ay in the addict population. The predominance of HBsAg/ay among addicts in Europe and Australia as well as the United States might be due to extensive needle sharing among a mobile population of drug abusers, although such worldwide dissemination of one subtype by these means is unlikely.

Health status disparities among public and private emergency department patients.

OBJECTIVE: To explore whether patients in a public ED had poorer health than patients in a private ED, the authors compared the physical and mental health statuses of patients seeking emergency care. METHODS: A cross-sectional observational study of all adult patients, regardless of acuity, seen during two 24-hour periods in spring 1997 in an urban county trauma center (68,000 annual visits) and a private community ED (35,000 annual visits). Scores on the Physical Component Summary (PCS) and the Mental Component Summary (MCS) scales of the Medical Outcomes Study 12-Item Short-Form Health Survey (SF-12) were compared between sites, with published national norms, and with hospital admission. RESULTS: Of 571 eligible patients, 392 (69%) completed the SF-12. Patients in the public ED had a mean PCS score of 40.1, compared with 43.7 for patients in the private ED, for a difference of 3.6 points (p < 0.01; 95% CI = 0.9 to 6.1). After controlling for age, sex, ethnicity, triage acuity, ambulance arrival, and insurance status, this difference increased to 3.9 points (p = 0.02; 95% CI = 0.7 to 7.0). The mean MCS score among public ED patients was 44.1, compared with 46.5 in the private ED population, for a difference of 2.4 (p = 0.08; 95% CI = -0.3 to 5.0); after adjustment this difference increased to 2.5 (p = 0.15; 95% CI = -0.9 to 5.8), but remained statistically not significant. While all scores were significantly lower than national norms (mean PCS 50.1, mean MCS 50.0), patients in the public ED scored consistently lowest. PCS score was significantly inversely correlated with admission, with each point decrease in PCS score increasing the odds of admission by 0.05 (95% CI = 0.01 to 0.08), and conferring an odds ratio of 5.1 (95% CI = 1.2 to 21.1) for admission among the 25th percentile for PCS scores. CONCLUSIONS: Patients seeking care in the public ED had lower adjusted physical health status scores than comparable patients obtaining care in a private ED. The SF-12 is sufficiently responsive to detect hypothesized differences between ED populations, and correlates well with admission decisions.

The emerging pattern of post-transfusion hepatitis.

The exclusion of commercial and hepatitis B surface antigen (HBs Ag)-positive donors, as measured by counterelectrophoresis, has markedly reduced the frequency of post-transfusion hepatitis (PTH). A further, significant reduction in type-B PTH can be achieved by prescreening donors for HBs Ag by solid-phase radioimmunoassay (RIA); When a voluntary donor population, pretreated by RIA, is used, approximately 90 per cent of residual hepatitis is serologically unrelated to either the type-A or type-B hepatitis viruses. Similarly, cytomegalovirus and the Epstein-Barr virus are not serologically implicated in "non-A, non-B" hepatitis. Additional human hepatitis virus(es) may exist.