Hepatitis B virus infection in cultured human lymphoblastoid cells.

Since it has been postulated that liver hepatocytes may become infected by hepatitis B virus (HBV) in vivo through direct contact with infected macrophages, the possibility that a circulating cell of hematopoietic origin might be susceptible to infection with HBV was investigated. Cells positive for HBV surface antigen were identified in aspirates of bone marrow cells from people infected with HBV. These cells were used to prepare a lymphoblastoid suspension culture that contains HBV-infected cells.

New human T-lymphotropic retrovirus related to simian T-lymphotropic virus type III (STLV-IIIAGM).

This report describes serologic evidence for a virus similar to that known as simian T-lymphotropic virus type III of African Green monkeys (STLV-IIIAGM) infecting apparently healthy people in Senegal, West Africa, and the isolation of virus from these individuals. Serum samples from selected healthy West African people showed unusual serologic profiles when tested with antigens of HTLV-III/LAV, the etiologic agent of AIDS, and of STLV-IIIAGM. The samples reacted strongly with all of the major viral antigens of STLV-IIIAGM but showed variable or no reactivity with the major viral antigens of HTLV-III/LAV by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A new human T-lymphotropic virus (HTLV-IV) isolated from these people was grown in vitro and shown to have retroviral type particles, growth characteristics, and major viral proteins similar to those of the STLV-III and HTLV-III/LAV group of retroviruses. The gp120/160, gp32, p64, p55, p53, p24, and p15 proteins precipitated were the same size as and reactive with STLV-IIIAGM proteins. The serologic data suggest that this virus shares more common epitopes with STLV-IIIAGM than with the prototype HTLV-III/LAV that infects people in the United States and Europe. Further study of this virus and of the origin of the HTLV-III/LAV group of viruses may expand our understanding of the human AIDS virus.

Generation of phagocytic MAK and MAC-DC for therapeutic use: characterization and in vitro functional properties.

Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures. Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC). A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low-intermediate phagocytosis capacity by MAK. Both CD14+ FCgammaR+ (FcgammaRI/CD64, FcgammaRII/CD32, FcgammaRIII/CD16) MAK and CD1a+/CD86+, CD14- MAC-DC were able to phagocytose whole tumor cells. However, only MAK phagocytosis was enhanced by FcgammaR engagement. MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcgammaRI. Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.

Clinical, hematologic, and immunologic cross-sectional evaluation of individuals exposed to human immunodeficiency virus type-2 (HIV-2).

We studied the clinical status and certain hematologic and immunologic parameters in healthy prostitutes from Dakar, Senegal who were seropositive for antibodies to human immunodeficiency virus type-2 (HIV-2). Generalized lymphadenopathy and clinical signs or symptoms similar to those which are seen with human immunodeficiency virus type-1 (HIV-1) infection were not present. Comparison to seronegative prostitutes and minor surgery control patients were made and significant elevations were seen in T8 lymphocytes (p = .03), IgG (p = .0001), and beta 2-microglobulin (p = .03). The mean T4 lymphocyte count in seropositive prostitutes was lower than in seronegative prostitutes (757 vs. 1179, p = .15), but this difference was not statistically significant and appeared to be correlated with age. No significant differences were noted between the seronegative and seropositive prostitutes in lymphocyte stimulation studies to certain mitogens. Antilymphocyte antibodies above background were not present in either population. We conclude that HIV-2 is a sexually transmitted agent that produces immunologic alterations consistent with a persistent viral infection. HIV-2 seropositive prostitutes studied to date do not show clinical signs of immune suppression, as has been described with HIV-1 infection. The pathogenic potential of HIV-2 appears to differ from that of HIV-1, the etiologic agent of the AIDS pandemic.

Immune therapy with macrophages: present status and critical requirements for implementation.

Adoptive transfer of activated macrophages has been validated in animal experimental tumor models; clinical trials are ongoing (70 patients up to now). The mechanisms involved are reviewed as well as improved and standardized procedures for macrophage differentiation and activation. New developments including specific Ag presentation and gene therapy are discussed.

[Adoptive immunotherapy: evaluation and perspectives in the treatment of certain cancers]. / Immunothérapie adoptive: bilan et perspectives dans le traitement de certains cancers.

INTRODUCTION: Adoptive immunotherapy was first introduced in the 1980s. This new anticancer therapeutic approach has already demonstrated promising results in both animal models and humans affected by various tumors. CURRENT KNOWLEDGE AND KEY POINTS: This review summarizes the requirements of such therapies involving either activated lymphocytes, tumor-infiltrating lymphocytes or activated macrophages. It focuses more particularly on the promising approaches that represent antigen presenting cells such as macrophages and antigen-loaded dendritic cells in the development of safe and effective cancer vaccines. FUTURE PROSPECTS AND PROJECTS: Standardized procedures for macrophages and dendritic cell generation, as well as preliminary results of clinical applications in patients with either prostate cancer or melanoma, are also discussed.

Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA.

Closed-circular HBV DNA was introduced into cells of the established human hepatoma culture HepG2. The culture medium of one of 40 single-cell clones contained HBV surface antigen (HBsAg), core-related antigens (HBc/eAg), and HBV DNA sequences. HBV DNA and DNA polymerase activity were detected in particles resembling both nucleocapsids and complete virions (Dane particles). Intracellular integrated and extrachromosomal HBV DNA sequences were detected. Relaxed-circular and single-stranded forms of viral DNA were identified as likely replicative intermediates of the HBV genome. In conclusion, in vitro production of Dane-like particles by transformed human hepatocytes has been achieved. This model should be valuable as a cell culture system for studying virus replication and virus-host cell interactions.

Monocyte-derived dendritic cells: development of a cellular processor for clinical applications.

Since dendritic cells (DCs) are the most professional antigen-presenting cells, (Schuler et al., 1997), increasing interest in their use in clinical approaches has been observed. (Nestle et al., 1998; Murphy G. et al., 1996). We have developed an ex vivo standardized process for the generation of dendritic-like cells (MAC-DCs) from human blood circulating monocytes. Human monocytes can differentiate into very different functional cells according to the conditions of culture, media and cytokines used. In the present study, we demonstrate that both pure monocytes and mononuclear cells differentiate into DCs when they are grown in defined medium AIM-V in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IL13 and in approved biocompatible non-adherent bags. Quality and functional controls of the immature DCs obtained rely on bacterial sterility, viability, morphology and recovery. The MAC-DCs also present an immature DC phenotype with a low expression of CD14 and CD64, and high expression of MHC-I, MHC-II and CD40. They also express B7 costimulatory molecules (CD80, CD86), CD83, and CD1a molecules. They induce strong allogenic T-cell proliferation (mixed lymphocyte reaction as well as proliferation of autologous memory T lymphocytes when incubated in the presence of recall antigens (tuberculosis, Candida albicans, and tetanus toxoid). They also show an increase in phagocytic uptake of yeast, tumour cells and debris. The global closed system which, under reproducible good medical practice (GMP) conditions, enables the production of dendritic cells of clinical quality, has been optimized ("Vac Cell Processor"). It contains all bags, connections, media, reagents, washing solutions, control antibodies, standard operating procedures, data management, traceability and help in the form of dedicated software.

Bispecific antibodies and targeted cellular cytotoxicity.

The Second International Conference on Bispecific Antibodies (BsAbs) and Targeted Cellular Cytotoxicity considered how targeted cytotoxicity can be used (1) to increase understanding of the general mechanisms of cellular cytotoxicity and (2) clinically in the treatment of cancer and infectious disease. BsAbs, the main mediators of targeted cellular cytotoxicity, can be made by chemical crosslinking, by fusing hybridoma cells and by molecular genetic approaches. BsAbs bind to target cells via one V region and trigger molecules such as T-cell receptors (TCRs) or FcR for IgG (Fc gamma R) on cytotoxic cells via their other V region. This linking of triggering structures to target cells induces target cell lysis and provides important clues to the signals used to elicit the lytic process.

Bispecific anti-human red blood Rhesus-D antigen x anti Fc gamma RI targeted antibody-dependent cell-mediated cytotoxicity and phagocytosis by mononuclear leucocytes.

The Fc receptor mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis induced by bispecific antibody (BsAb) to the high-affinity Fc receptor for IgG (Fc gamma RI) and to human red blood group antigen RhD were studied in vitro, using human mononuclear leucocytes as effector cells. The results were compared with those obtained by using a human monoclonal IgG1 anti-RhD used alone and a reference human polyclonal anti-RhD antibody. The effect of non-specific human IgG on FcR-mediated functions by mononuclear leucocytes was checked. The results demonstrate that BsAb presents a high resistance of Fc-mediated function to blockade by non-specific human IgG compared with that of both polyclonal and monoclonal anti-RhD antibodies. These results further encourage possible clinical application of bispecific antibody in passive immunotherapy.

Fatal Selenomonas sputigena septicemia probably originating from lung abscess.

A case of fatal septicemia with Selenomonas sputigena in an immunocompromised patient is reported. The patient had a lung abscess from which the septicemia is believed to have originated. In contrast to the only other case reported in the literature, the isolate from our patient was characterized by very slow and difficult growth.

[Hepatitis B virus and hepatoma: evidence for a cytoplasmic tumor antigen in transformed hepatocytes]. / Virus de l'hépatite B et cancer primitif du foie: mise en évidence d'un antigène tumoral cytoplasmique dans l'hépatocyte transformé.

A tumoural antigen (HBV/T Ag) has been found in the cytoplasm of the human hepatoma cell line PLC/PRF/5 by indirect immunofluorescence and immunoperoxidase assays using anti-sera from Senegalese patients with primary hepatocellular carcinoma. The same antigen was detected in PHC tissues. According to the data obtained with serum and tissue controls the HBV/T Ag-anti-HBV/T system seems associated with transformation of hepatocytes by hepatitis B virus.

Further studies on production and characterization of HBsAg derived from a human hepatoma cell line (PLC/PRF/5).

Four aspects for the use of PLC/PRF/5 cell line as an alternative source of HBsAg for hepatitis B vaccine production were assayed in this study: improvement in HBsAg production with chemical inducers, tests for the presence of hepatitis B virions, antigenic topology of HBsAg polypeptides and immunogenicity of HBsAg in guinea pigs. 10(-6) M dexamethasone and/or 10(-4) M sodium butyrate treatments enhanced HBsAg production by a factor of approximately two when compared with untreated cells. Particles of 35 nm diameter produced by PLC/PRF/5 cells were observed at a CsCl density of 1.22-1.24, associated with typical HBsAg 22 nm spheres. These particles did not contain HBV DNA as proved by negative results of hybridization using cloned HBV/DNA as a probe. Western blot analysis revealed that only polypeptides P 22000 (P1) and P 26000 (P2) were specifically recognized by a human anti-HBs serum, irrespective of the origin of HBsAg, i.e. human serum or PLC/PRF/5 hepatoma cell line. HBsAg purified from PLC/PRF/5 cell line was immunogenic in guinea pigs. However, the humoral response was delayed and lower in terms of anti-HBs titers when compared to the response obtained after immunization with a licensed hepatitis B vaccine (HEVAC B, Institut Pasteur Production).

Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers.

Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (10(7) - 2 x 10(7) cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.

Hepatitis B virus core antigen (HBc Ag) accumulation in an HBV nonproducer clone of HepG2-transfected cells is associated with cytopathic effect.

Two clones of the hepatoblastoma HepG2 cell line transfected with complete hepatitis B virus deoxyribonucleic acid (HBV DNA) were studied. The kinetics and cytopathic effect of HBV Ag production in these two clones (one of which was an HBV producer) were compared to those of the parent HepG2 cell line. The presence of hepatitis B surface antigen (HBs Ag) and hepatitis B e antigen (HBe Ag) was determined by commercial enzyme-linked immunosorbent assay (ELISA). A hepatitis B core antigen (HBc Ag)-specific ELISA assay was developed, using monoclonal anti-HBc to detect HBc Ag. Amounts of HBs, HBe, and HBC Ags were partially quantified in both intracellular and extracellular compartments. The HBV producer clone excreted high levels of HBc, HBe, and HBs Ags from the beginning of the growth phase, and no cytopathic effect was observed. The HBV nonproducer clone produced high levels of HBs and HBe Ags, but there was no detectable HBc Ag in the supernatant; instead, HBc Ag accumulated in the intracellular compartment. In this clone, significant cell death was observed 4 days after cell confluency, corresponding with notable HBc Ag release into the supernatant. These results suggest a cytopathic effect associated with HBc Ag accumulation in the HBV nonproducer clone, but no cytopathic effect in the HBV producer clone. This suggests that virological factors as well as the host's immune response may be considered in explaining liver injury occurring in hepatitis B.

A molecularly cloned hepatitis B virus produced in vitro is infectious in a chimpanzee.

The infectivity of hepatitis B virus (HBV) produced in vitro by HepG2 cells transfected with HBV DNA (HepG2T14) has been assayed in a chimpanzee. Following inoculation, the chimpanzee underwent a typical course of type B hepatitis infection, characterized by elevation of serum aminotransferases and by histological identification of hepatic damage. Hepatitis B surface antigen and core-related antigen appeared in the serum at weeks 5 and 7, respectively, after infection. HBV DNA was detected in serum samples, and replicative forms of the HBV genome were identified in liver biopsies. Subtype identification of hepatitis B surface antigen and restriction enzyme analysis of HBV DNA in both the inoculum and the serum of the infected chimpanzee confirmed that the hepatitis B infection observed in this animal was caused by viral particles produced by HepG2T14 cells. These findings indicate that, although HepG2 cells do not seem to be susceptible to infection by HBV in vitro, they can produce biologically active infectious virions after transfection with cloned HBV DNA.

Evidence of extrachromosomal forms of hepatitis B viral DNA in a bone marrow culture obtained from a patient recently infected with hepatitis B virus.

A cell culture that produces Dane-like particles was initiated from a bone marrow aspirate of an acute hepatitis B patient. By using Southern blot analysis and a recombinant hepatitis B virus (HBV) DNA plasmid probe, extrachromosomal forms of HBV DNA were detected. The two forms of HBV DNA migrate as a closed circular 2.2-kb form and an open circular 3.9-kb form. There was no evidence of HBV DNA integration into the host genome.

Structural and functional characterization of Factor VIII-delta II, a new recombinant Factor VIII lacking most of the B-domain.

A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.

Immunocytochemical and electron microscopic study of hepatitis B virus antigen and complete particle production in hepatitis B virus DNA transfected HepG2 cells.

The relationship between the presence of hepatitis B virus antigens, their localization and hepatitis B virus replication was studied in different clones of cultured HepG2 hepatoblastoma cells transfected with cloned hepatitis B virus DNA. Intracellular hepatitis B virus antigens were detected by immunofluorescence. The production of these antigens was evaluated in the culture media by enzyme-linked immunoassay. Hepatitis B virus DNA was detected using dot-blot hybridization. Three types of HBcAg staining were observed in transfected HepG2 cells: (a) cells with nuclear HBcAg, (b) cells with cytoplasmic HBcAg and (c) cells with both nuclear and cytoplasmic HBcAg. Cell types b and c also expressed hepatitis B virus DNA in their culture media. Our results suggest that cytoplasmic HBcAg may be more involved than nuclear HBcAg in hepatitis B virus replication. The site of hepatitis B virus formation in hepatocytes was studied by electron microscopic examination of a specific hepatitis B virus producer clone, thereby allowing detection of intracellular Dane particles more easily than liver biopsy samples from infected patients. Dane particles and HBsAg filaments were found in large, dilated structures probably related to the endoplasmic reticulum. Budding of core particles into cisternae of endoplasmic reticulum-related structures appears to be a possible mechanism for hepatitis B virus formation; our results suggest that the exocytosis of cisternae to extracellular spaces may be a mechanism for release of hepatitis B virus particles.