Decreased efficacy of twice-weekly intravaginal dehydroepiandrosterone on vulvovaginal atrophy.

OBJECTIVE: While daily intravaginal administration of 0.50% (6.5 mg) dehydroepiandrosterone (DHEA, prasterone) for 12 weeks has shown clinically and statistically significant effects on moderate to severe (MS) dyspareunia as the most bothersome symptom (MBS), the present study analyzes the effect of a reduced dosing regimen on MBS vaginal dryness. METHOD: Daily intravaginal 0.50% prasterone for 2 weeks followed by twice weekly for 10 weeks versus placebo. RESULTS: Maximal beneficial changes in vaginal parabasal and superficial cells and pH were observed at 2 weeks as observed for intravaginal 10 µg estradiol (E2). This was followed by a decrease or lack of efficacy improvement after switching to twice-weekly dosing. The decrease in percentage of parabasal cells, increase in percentage of superficial cells and decrease in vaginal pH were all highly significant (p < 0.0001 to 0.0002 over placebo) at 12 weeks. In parallel, the statistical significance over placebo (p value) on MBS vaginal dryness at 6 weeks was 0.09 followed by an increase to 0.198 at 12 weeks. For MBS dyspareunia, the p value of 0.008 at 6 weeks was followed by a p value of 0.077 at 12 weeks, thus illustrating a decrease of efficacy at the lower dosing regimen. The improvements of vaginal secretions, color, epithelial integrity and epithelial surface thickness were observed at a p value < 0.01 or 0.05 over placebo at 2 weeks, with a similar or loss of statistical difference compared to placebo at later time intervals. No significant adverse event was observed. Vaginal discharge related to the melting of Witepsol was reported in 1.8% of subjects. CONCLUSION: The present data show that daily dosing with 0.50% DHEA for 2 weeks followed by twice-weekly dosing is a suboptimal treatment of the symptoms/signs of vulvovaginal atrophy resulting from a substantial loss of the efficacy achieved at daily dosing.

Binding of nuclear factors to functional domains of the duck hepatitis B virus enhancer.

We have analyzed the structures, relative organization, and activities of binding sites for nuclear factors in the duck hepatitis B virus (duck HBV) enhancer. DNase I footprinting analysis and mobility shift assays demonstrate that this enhancer of 192 bp contains at least three binding sites for transcription factors: one for hepatocyte-adipocyte C/EBP, a second for the liver-specific transactivator hepatocyte nuclear factor 1 HNF-1, and a third for a factor, called F3, which binds to a DNA sequence bearing some resemblance to that for the ubiquitous factor EF-C. Analysis of transcriptional activity reveals that oligonucleotides corresponding to the individual binding sites, inserted upstream from a heterologous promoter, display very weak enhancer activity, whereas the enhancer encompassing these three sites displays very high activity. Analysis of duck HBV enhancer mutants indicates that the deletion of any of these sites leads to a modification of transcriptional enhancer activity. The hepatocyte nuclear factor 1 binding site is crucial, since an internal deletion of 14 bp abolishes the activity. The C/EBP site can act as repressor, and the F3 site is required for full activity. Comparative analysis reveals that the nuclear factors are similar to those bound to the human HBV enhancer but that the organization of their binding sites in the duck HBV enhancer is different.

Characterization of the hepatitis B virus preS/S region encoded transcriptional transactivator.

A transactivating function generated by carboxy-terminal truncation of the HBV envelope proteins has been recently described. To characterize the preS/S protein domains responsible for transactivation, preS1/S2/S and preS2/S 3' deletion mutants under the control of the adenoviral major late promoter were tested for their transactivating potential in cotransfection experiments using the c-myc and c-fos regulatory sequences as targets. Deletion of the carboxyterminal hydrophobic domain of the S protein and the presence of the endoplasmic reticulum insertion signal I (ER signal I) are required for the generation of the preS/S transactivating function. Multiple transcription factors binding sites (i.e., TRE, SRE, and NFkB sites) mediated the truncated preS/S-induced activation of the target regulatory sequences. The transactivation phenomenon is linked, at least in part, to the protein kinase C signaling pathway.

Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction.

The specificity of first-generation enzyme-linked immunosorbent assays (ELIAs) for antibody detection in individuals with hepatitis C virus (HCV) infection has been questioned in some pathological situations. We observed a surprisingly high prevalence of anti-HCV antibodies in alcoholic patients, and thus, false-positive reactions in anti-HCV tests were strongly suspected. The introduction of new epitopes, particularly a core protein, C22 (second-generation tests), seems to increase the sensitivity of anti-HCV detection. In order to study the specificity of the second-generation tests, 60 serum samples from alcoholic patients found to be positive by the first-generation anti-HCV ELISA (Ortho) were reexamined by a second-generation anti-HCV enzyme immunoassay (Abbott) and a recombinant immunoblot assay (RIBA II; Chiron). Fifteen serum samples gave contradictory results when they were tested by the two assays. We performed nested polymerase chain reactions (PCRs) to confirm that the discrepancies that we observed could be due to the presence of low levels of anti-HCV antibodies, which were detected by a more sensitive test, or to unspecific positive reactions. Nested PCR revealed the presence of HCV RNA sequences in all anti-HCV-positive sera or sera that were weakly positive by ELISA. Anti-HCV positive by RIBA II was always correlated with the presence of viral RNA in serum, but HCV RNA was detected in RIBA II-negative sera. These results indicate that the specificity of the second-generation tests is an important improvement but that an HCV infection can still persist without detectable antibodies. PCR remains the reference assay to clear up controversial serology results and to detect HCV infection in patients with no anti-HCV-detectable immune response.

The hepatitis B virus X protein transactivation of c-fos and c-myc proto-oncogenes is mediated by multiple transcription factors.

We have constructed two expression vectors in order to study the action of the HBV 17 Kd X protein on the c-fos and c-myc promoters. The results show that the promoters contain multiple elements that respond to X protein, suggesting involvement of multiple transcription factors. The exact mechanism of the interaction remains elusive, but our data allow speculation about the factors that may be influenced.

Truncated pre-S/S proteins transactivate multiple target sequences.

In order to investigate the transactivational function of HBV truncated preS/S proteins we have constructed two sets of plasmids and have tested their transactivational potential on the c-myc regulatory sequences and the TPA-responsive element. We found that preS/S proteins only become transactivationally active when truncated at the carboxy terminal end. Furthermore, using immunofluorescence microscopy we determined that the proteins are located exclusively in the cytoplasm, apparently ruling out DNA binding and activation of factors in the nucleus.

Identification of a strong enhancer element upstream from the pregenomic RNA start site of the duck hepatitis B virus genome.

The genome of the duck hepatitis B virus (DHBV) contains an enhancer element. This sequence, of 192 bp, is located in the 3'-terminal coding region of the DNA polymerase gene (nucleotides 2159 to 2351), upstream from the pregenomic RNA start site. This enhancer potentiates a marked increased activity from the heterologous thymidine kinase promoter in an orientation-independent manner and at a proximal, as well as a distal, location. The DHBV enhancer activates transcription in a relatively cell-type-independent manner. Sequence homologies with the nuclear factor EF-C binding site are located in the DHBV enhancer. By using the HepG2 nuclear extracts and the DHBV enhancer as probes, a complex was observed in mobility shift assays.

Hepatitis B virus X protein transactivates the long terminal repeats of human immunodeficiency virus types 1 and 2.

The X gene product of the hepatitis B virus (HBV) has been expressed transiently in HepG2 cells, and the 17-kilodalton protein has been detected by Western (immuno-) blot analysis. Cotransfection of the X gene with the long terminal repeat of human immunodeficiency virus type 1 or 2 results in a stimulation of long terminal repeat-directed expression that is higher than the X-induced stimulation of the HBV enhancer linked to either autologous promoter or to the heterologous simian virus 40 promoter. A frameshift mutation abolished this transactivation. In vitro nuclear transcription assays revealed that HBV X acts at the transcriptional level. The carboxy terminus of the HBV X protein does not seem to be necessary for its transactivating activity, as demonstrated by using HBV X protein deletion mutants.

Cytomegalovirus infection and trans-activation of HIV-1 and HIV-2 LTRs in human astrocytoma cells.

Susceptibility of a human astrocytoma cell line to human cytomegalovirus (HCMV) infection was investigated. Infection of U-373MG astrocytoma cells with two strains of HCMV resulted in both production of extracellular, infectious virus and expression of immediate early and early antigens within 18 hours and late antigens after 72 hours of infection. The kinetics of infection in U-373MG cells were the same as in human diploid fibroblasts (MRC-5). Since HCMV and human immunodeficiency virus (HIV) have reportedly been found in astrocytic cells in vivo, we studied the possible interaction between HCMV and HIV long terminal repeat (LTR) elements in this cellular environment. HCMV infection transactivated the LTR of HIV-1 and HIV-2 to similar levels. Interestingly, transfection of these cells with infectious HIV-1 provirus did not result in expression of gag, env, or F proteins detectable by immunofluorescence. However, provirus gene expression was not completely silent, since it transactivated HIV-1 LTR. The level of this transactivation was similar to that seen following cotransfection with a tat expression vector. These results suggest that opportunistic infection with HCMV may reactivate latent HIV genomes in glial cells.

Broad specificity of the hepatitis B enhancer function.

The genome of hepatitis B virus (HBV) contains an enhancer element located in the coding region of the DNA polymerase open reading frame between the 3' end of the S gene and the X gene. To determine whether HBV enhancer is species and/or tissue specific, several cell lines were transfected with CAT plasmids containing different subgenomic fragments of the HBV genome. The activity of the HBV transcriptional enhancer sequence was shown not to be strictly hepatotropic, since it was found in hematopoietic tissues. This activity was not species specific either, since it was found in feline and mouse cells.

Detection of hepatitis B virus X product using an open reading frame Escherichia coli expression vector.

The genome of the hepatitis B virus (HBV) contains a sequence, designated X, capable of encoding a protein of 154 amino acids. To determine whether the putative protein synthesized from this region is antigenic, we examined the sera of HBV-infected patients for the ability to react with a hybrid protein that contained 133 amino acids encoded by the X region and portions of the bacterial ompF and beta-galactosidase genes. Some HBV-positive sera tested contained antibodies that specifically recognized the hybrid protein. All sera were from patients diagnosed as suffering from chronic active hepatitis. We conclude that the X region of HBV encodes a protein and that this protein is antigenic in some patients.

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.

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.

[Characterization of viral RNA in Friend tumor cells having lost their capacity to produce viral particles]. / Caractérisation de l'ARN viral dans les cellules tumorales de Friend ayant perdu leur capacité de produire des particules virales.

Cytoplasmic RNA from Friend tumoral cells which had lost their ability to produce viral particles was analyzed for its viralRNA content. A major 32S RNA corresponding to the genome of the defective SFFV virus was detected by hybridization with a synthetic DNA complementary to the Rauscher virus genome. Very low amounts of a 34S species were also found. No 38S RNA with the size of the helper virus genome was present in the cells. It was concluded that the cessation of virus production resulted from a disappearance of the helper provirus from these cells or from a block in the transcriptional process of this provirus.