CYP1A1 alleles in women with focal nodular hyperplasia of the liver (FNH).

OBJECTIVE: Disorders of steroid hormone metabolism might be related to the etiology of focal nodular hyperplasia of the liver (FNH), a benign tumor, especially prevalent in women. The cytochrome P450 1A1 (CYP1A1) enzyme is implicated in the bioactivation of multiple precarcinogens as well as in the metabolism of steroids. Genetic polymorphisms of CYP1A1 have been associated with altered catalytic activity in the hydroxylation of sex hormones and this may account for interindividual variability in exposure to hormone-mediated cell proliferation signals and reactive steroid metabolites. In the study at hand, we aimed to evaluate a possible association between CYP1A1*1, *2A, *2B, and *4 alleles and FNH. METHOD: Genotyping of 26 affected female patients of Caucasian origin was carried out using PCR/RFLP. RESULTS: Allele frequencies for the CYP1A1 variants *2A, *2B and *4 in 26 female patients with FNH were 0.058, 0.019 and 0.058, respectively. Crude odds ratios for the individual alleles were 0.75 (95% CI 0.23-2.44), 0.72 (95% CI 0.10-5.34) and 1.96 (95% CI 0.59-6.50), respectively. There were no significant differences between these values and corresponding allele frequencies obtained in a large German sample of unaffected Caucasian women. CONCLUSION: The present data do not suggest a relevant association between CYP1A1 polymorphisms and focal nodular hyperplasia of the liver in female Caucasians.

Cationic polyhexylcyanoacrylate nanoparticles as carriers for antisense oligonucleotides.

After antisense oligodeoxynucleotides (ODNs) were suggested for therapeutic use in 1978, major advances were made in developing modified oligonucleotides with increased nuclease resistance and improved cellular uptake. In the present report, positively charged nanoparticles prepared from diethylaminoethyl (DEAE)-dextran and polyhexylcyanoacrylate (PHCA) were evaluated as carriers for ODNs. The oligonucleotides were analyzed by anion-exchange HPLC. The nanoparticles exhibited a high loading capacity, with approximately 35 mumol ODNs adsorbed per gram of polymeric material. The adsorption efficacy was found to be dependent on the pH, on the ionic strength of the medium, and on the amount of DEAE-dextran. Highest loading for ODNs was achieved at pH 5.5, using a 10 mM phosphate buffer. Oligonucleotides adsorbed to the surface of the nanoparticles were nearly completely protected against degradation by the endonuclease DNase I and under in vitro cell culture conditions, whereas unprotected ODNs were totally digested under these conditions. Nanoparticles led to a 20-fold increase in cellular uptake of FITC-oligonucleotides. The internalized oligonucleotides were frequently localized as vesicular structures in the cytoplasmatic compartment. Because of their temperature-dependent uptake, we propose an active uptake mechanism, such as endocytosis, for the internalization of the ODN-nanoparticle formulations.

[Zinc sulfate and heparin for local therapy of herpes. Antiherpetic drugs, not leading to selection of HSV variants]. / Zinksulfat und Heparin zur Lokaltherapie des Herpes. Antiherpetische Wirkstoffe, die nicht zur Selektion resistenter HSV-Varianten führen.

The massive widespread use of a substance to treat HSV could lead to the spread of resistant HSV strains and hence to a failure of treatment in a vital indication. In Germany, three defined substances suitable for treating HSV are available over the counter: Zinc sulfate, heparin and acyclovir. Experiments show that heparin retains its activity even after 20 passages whereas acyclovir produces a completely resistant virus after only a single passage. The inactivation of free HSV by zinc sulfate is reduced after serial passages. However, this partial resistance disappears again spontaneously and may be considered clinically irrelevant. With respect to drug efficacy and resistance, therefore, a combination of zinc sulfate and heparin would appear superior to zinc sulfate alone. The use of the genotoxic DNA polymerase inhibitor acyclovir should be restricted to severe cases of HSV.

Interferon-gamma-induced assembly block in the replication cycle of adenovirus 2: augmentation by tumour necrosis factor-alpha.

Replication of adenovirus 2 (Ad-2) is inhibited in A 549 cells pretreated with interferon-gamma (IFN-gamma). The antiviral effect is synergistically enhanced by the simultaneous presence of tumor necrosis factor-alpha (TNF-alpha) before infection. Under conditions of strong inhibition of virus progeny formation, viral DNA synthesis and [35S]methionine incorporation into most late viral proteins are only marginally impaired. Pulse chase experiments indicate a partial inhibition of processing of viral proteins. Viral proteins are not degraded and capsomeres accumulate in the inhibitor-treated cells. Capsid formation, on the other hand, is strongly inhibited in the cytokine-treated cells. The inhibition of Ad-2 replication in A 549 cells by IFN-gamma and TNF-alpha is caused, therefore, by a block in the maturation of Ad-2.

[Therapy of banal HSV lesions: molecular mechanisms of the antiviral activity of zinc sulfate]. / Zur Therapie banaler HSV-Läsionen: der molekulare Mechanismus der antiviralen Aktivität von Zinksulfat.

Preparations of heparin in combination with the physiologically harmless and even beneficial zinc sulphate are available for the topical treatment of superficial mucocutaneous lesions caused by HSV (herpes simplex virus). We study the molecular mechanism of the antiviral effects of zinc ions. A concentration of 100 mumol/l Zn2+ in the culture medium reduces the virus yield in a HSV infected AGMK cell line to less than 1/1000 of the control. At this concentration zinc sulphate does not exert any major cytotoxic effects, nor does it block the synthesis of viral or cellular DNA. Free virus, however, is inactivated by 8 orders of magnitude by 15 mmol/l zinc sulphate within a few hours. The inactivated virus is limited in the glycoprotein-dependent functions adsorption and penetration. Electron micrographs show massive deposition of zinc onto virion components. The dramatic antiviral effect in vivo is therefore explained by an inhibition of virion glycoprotein functions after accumulation of zinc in the virion, presumably by binding to sulphhydryl groups of glycoprotein B.

The mechanism of the antiherpetic activity of zinc sulphate.

The molecular mechanism of the effects of zinc ions against herpes simplex virus (HSV) infection was investigated. Zinc sulphate (100 microM) in the culture medium of an HSV-infected African green monkey kidney cell line did not block viral DNA synthesis and, at this concentration, only moderate cytotoxic effects were observed in uninfected cells. Nevertheless, virus yields were reduced to less than 1% of the control. Thus the long standing hypothesis that zinc might block multiplication of HSV by selective intranuclear inhibition of the viral DNA polymerase apparently has lost its validity. Inhibition of virus growth in the absence of severe cytotoxicity must therefore result from other effects of ZnSO4. Free virus is inactivated by 15 mM-ZnSO4 within a few hours of its addition. The inactivated virus is defective in the glycoprotein-dependent functions of penetration and, to some extent, adsorption. Electron micrographs show massive deposition of zinc onto virion components. In a virion, transmembrane transport of zinc ions is not expected and the established antiviral effect is therefore explained by an inhibition of virion glycoprotein function after non-specific accumulation of zinc into many virion membrane components.

Immunoglobulin and T cell receptor gene rearrangements in lymphoproliferative disorders.

Thirty-one samples representing Hodgkin's and non-Hodgkin's lymphomas, angioimmunoblastic lymphadenopathy (AILD), and benign follicular hyperplasia in HIV infections were examined for rearrangements of the immunoglobulin (Ig) and T cell receptor (TcR) beta-chain gene loci. In 11 of 12 non-Hodgkin's lymphomas (classified as Burkitt lymphoma (2), centrocytic lymphoma (1), centrocytic-centroblastic lymphoma (5), centroblastic lymphoma (3], only rearranged Ig genes could be detected. The exceptional case was an unclassified high-grade lymphoma, which represented a rearrangement of the TcR beta-chain. We also examined DNA from lymphoid neoplasms in which the lineage of the malignant cell was still controversial. Rearrangement of the TcR could exclusively be demonstrated in all 3 cases of AILD. One Ig gene rearrangement and 4 TcR beta-chain rearrangements were found in 13 samples of Hodgkin's lymphomas (11 lymph nodes, 1 pleura effusion and 1 bone biopsy with proven infiltration). Examination of 3 cases of benign follicular hyperplasia in HIV infection represented one Ig rearrangement.

Neurovirulence and latency in inbred mice of two HSV-1 intrastrain variants of divergent pathogenicity.

The pathogenicity pattern of the HSV-1 strain ANG which is nonencephalitogenic in mice is compared with that of a selected neurovirulent variant of this strain in DBA-2 mice. After i.p. inoculation both variants replicate to high titers in the mouse peritoneum and build up a virus reservoir in the spleen. Both viruses have no effect on visceral mouse organs other than the spleen; both viruses lead to an inefficient and masked viraemia and both replicate efficiently in CNS tissue after direct intracranial injection. Only the pathogenic variant, however, spreads to the CNS and leads to lethal encephalitis upon intraperitoneal infection. The assumption that infection of the CNS would be mediated by hematogenous transport is not supported by the data obtained from transfer and cocultivation experiments with lymphocytes or experiments involving artificial viraemia. In a model to analyse the capacity of the viruses to invade nerve axons and to induce a latent infection both viruses were found to be latency positive in dorsal root ganglia. It is clear that non-neurovirulent HSV-1 strains are subjected to a postganglionic block of virus spread from the periphery to the CNS. The experiments led to the hypothesis that axonal transport even beyond the dorsal root ganglia to the CNS proceeds unrestricted, whereas lethal CNS invasion is prevented by a restriction of viral replication of HSV-1 ANG in the CNS by a virus-induced host defence mechanism.

Specificity of human natural killer cells in limiting dilution culture for determinants of herpes simplex virus type 1 glycoproteins.

The frequency and specificity of human cells with natural killer (NK) cytotoxic activity for herpes simplex virus type 1 (HSV-1)-infected targets was measured by limiting dilution culture. The frequency of NK cell precursors (NK-p) reactive with HSV-1-infected cells was 2- to 11-fold higher than that of NK-p reactive with mock-infected cells. The frequency of NK-p reactive with infected target cells lacking viral glycoprotein C or presenting an antigenically altered glycoprotein B was approximately twofold lower than that with wild-type virus-infected cells. Specificity analysis demonstrated that NK cells with a high statistical probability of being monoclonal were reactive with either glycoprotein B or C. These results provide the first evidence that cells with human NK activity possess clonal specificity for HSV-1-infected target cells.

Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity.

Virus-neutralizing monoclonal antibodies specific for 13 different genetically defined epitopes of glycoproteins gC, gB, and gD of herpes simplex virus type 1, strain KOS-321, were compared for their ability to provide passive immunity to DBA-2 mice challenged intracranially. Protection was highly specific, since individual monoclonal antibodies failed to protect against infection with monoclonal antibody-resistant (mar) mutants altered in the single epitope recognized by the injected antibody. The dose-response kinetics of passive immunity paralleled the in vitro neutralization titers for each antibody. No correlation was observed between immune protection and antibody isotype or complement-dependent in vitro neutralization titers. This suggests that virus neutralization was not the protective mechanism. In general, antibodies reactive with epitopes of gC were protective at the lowest antibody doses, antibodies specific for gB were less efficient in providing immunity, and antibodies against gD were the least effective. mar mutants with single epitope changes in gC and multiple epitope changes in gB showed highly reduced pathogenicity, requiring up to 5 X 10(6) PFU to kill 50% of infected animals. These findings indicated that antigenic variation affects virus growth and spread in the central nervous system. Thus, mutations which affect antigenic structure also can alter virus pathogenicity. The alteration of these epitopes does not, however, appreciably reduce the development of resistance to infection. Infection of mice with these mutants or inoculation of mice with UV-inactivated, mutant-infected cells before challenge rendered the animals resistant to infection with wild-type herpes simplex virus type 1.

Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes.

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.

Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity.

The relative antigenicity of the individual herpes simplex virus type 1 (KOS) glycoproteins gC and gB was analyzed in BALB/c mice by using KOS mutants altered in their ability to present these antigens on cell surface membranes during infection. The mutants employed were as follows: syn LD70 , a non-temperature-sensitive mutant defective in the synthesis of cell surface membrane gC; tsF13 , a temperature-sensitive mutant defective in the processing of the precursor form of gB to the mature cell surface form at 39 degrees C; and ts606 , an immediate early temperature-sensitive mutant defective in the production of all early and late proteins including the glycoproteins. By comparing the relative susceptibility to immunolysis of mouse 3T3 cells infected at 39 degrees C with wild-type virus, presenting the full complement of the glycoprotein antigens, gC, gB, and gD, with target cells infected with mutants presenting only subsets of these antigens, we determined that a major portion of cytolytic antibody contained in hyperimmune anti-herpes simplex virus type 1 (KOS) mouse antiserum was directed against glycoproteins gC and gB. The relative immunogenicity of wild-type and mutant virus-infected cells also was compared in BALB/c mice. Immunogen lacking the mature form of gB induced a cytolytic antibody titer comparable to that of the wild-type virus, whereas that lacking the mature form of gC showed a 70% reduction in titer. The absence of the mature cell surface forms of gB and gC in immunogen preparations resulted in a 4- to 15-fold reduction in in virus neutralizing titer. Animals immunized with ts606 -infected cells (39 degrees C) induced relatively little virus-specific cytolytic and neutralizing antibody. Analysis of the glycoprotein specificities of these antisera by radioimmunoprecipitation showed that the antigens immunoprecipitated reflected the viral plasma membrane glycoprotein profiles of the immunogens. The absence of the mature forms of gC or gB in the immunizing preparation did not appreciably affect the immunoprecipitating antibody response to other antigens. Mice immunized with wild-type and mutant virus-infected cells were tested for their resistance to intracranial and intraperitoneal challenge with the highly virulent WAL strain of herpes simplex virus type 1. Despite the observed alterations in serum virus-specific antibody induced with the individual immunogens, all animals survived an intraperitoneal challenge of 10 50% lethal doses. However, differences in the survival of animals were obtained upon intracranial challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropathogenicity of herpes simplex virus in mice: protection against lethal encephalitis by co-infection with a non-encephalitogenic strain.

Intraperitoneal infection of susceptible mice with an apathogenic herpes simplex virus type 1 (HSV-1) strain prevented the lethal outcome of a challenge infection with a pathogenic strain, even if the challenge preceded the protective infection. It was found that the protective inoculation blocks the initial replication of the challenge virus. In addition, intraperitoneal infection with the protective HSV-1 strain led to the induction of a refractory state in the central nervous system, resulting in resistance to direct intracranial infection with HSV-1. This state is also inducible locally by intracerebral inoculation of a non-replicating mutant virus. The results indicate that HSV-1 strains differing in neurovirulence may differ in the induction or the sensitivity to this protective effect. Experiments with non-replicating HSV-1 temperature-sensitive strains demonstrated that protection against lethal infection does not depend on replication or expression of late genes of the protective strain. Inoculation of animals with detergent-soluble extracts of infected cells or infected and u.v.-irradiated syngeneic cells protected the animals against co-infection with encephalitogenic challenge virus. The experiments define this protective effect as an antigen-induced-immediate host defence mechanism active within 24 h post-infection.

Genetic variability of herpes simplex virus: development of a pathogenic variant during passaging of a nonpathogenic herpes simplex virus type 1 virus strain in mouse brain.

Herpes simplex virus type 1 ANG (HSV-1 ANG) is originally nonpathogenic for inbred mice upon intraperitoneal intravenous, or intravaginal inoculation. In contrast, mice died of encephalitis within 4 to 5 days after intracerebral inoculation with this strain. HSV-1 ANG was serially passaged in mouse brains. In two independent series, peripherally pathogenic virus variants had developed and accumulated in the virus progeny after 12 to 15 intracerebral passages. In mixed infections both nonpathogenic and pathogenic viruses replicated at the primary site of infection and spread to various organs. However, only the pathogenic phenotype could be recovered from the spinal cord and the brain. Comparison of the restriction enzyme cleavage patterns of pathogenic ANG and nonpathogenic ANG virus DNAs revealed distinct alterations in the S-segment (US) sequences bounded by coordinates 0.953 and 0.958 in the prototype orientation and by coordinates 0.862 to 0.867 in the IS orientation of the viral genome. However, it is not known whether these alterations are physiologically relevant to the observed changes in pathogenicity. When coinjected intraperitoneally at 50 to 100-fold excess, the nonpathogenic HSV-1 ANG protected mice against its own pathogenic variant as well as against other pathogenic HSV-1 strains. Pathogenic HSV-1 ANG proved to be genetically and phenotypically stable for at least 25 serial passages in tissue culture at either high or low multiplicity of infection.