Rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal Caco-2 cell monolayers.

We provide here new insights into rotavirus (RRV) pathogenicity by showing that RRV infection promotes structural and functional injuries localized at the tight junctions (TJ) in the cell-cell junctional complex of cultured polarized human intestinal Caco-2 cells forming monolayers. RRV infection resulted in a progressive increase in the paracellular permeability to [(3)H]mannitol as a function of the time postinfection. We observed a disorganization of the TJ-associated protein occludin as a function of the time postinfection, whereas distribution of the zonula adherens associated E-cadherin was not affected. These structural and functional RRV-induced TJ injuries were not accompanied by alteration in cell and monolayer integrity, as assessed by the lack of change in transepithelial membrane resistance and lactate dehydrogenase release. Finally, using the stabilizer of actin filaments Jasplakinolide, we demonstrated that the RRV-induced structural and functional alterations in TJ are independent of the RRV-induced apical F-actin rearrangements.

Synthesis, in vitro antiviral evaluation, and stability studies of bis(S-acyl-2-thioethyl) ester derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) as potential PMEA prodrugs with improved oral bioavailability.

A new series of hitherto unknown 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) phosphonodiester derivatives incorporating carboxyesterase-labile S-acyl-2-thioethyl (SATE) moieties as transient phosphonate-protecting groups was prepared in an attempt to increase the oral bioavailability of the antiviral agent PMEA. We report here a direct comparison of the in vitro anti-HIV and anti-HSV activities as well as the in vitro stability between the bis(SATE) derivatives and the already known PMEA prodrugs, namely, bis[(pivaloyloxy)methyl (POM)]- and bis[dithiodiethyl (DTE)]PMEA. All of the compounds tested showed an enhanced in vitro antiviral activity compared to the parent PMEA. The bis(POM)- and bis(tBu-SATE)PMEA derivatives were the most effective. However, striking differences between these two compounds were found during the stability studies. In particular the bis(tBu-SATE)PMEA was found to be more stable than bis(POM)PMEA in human gastric juice and human serum, suggesting it could be considered as a promising candidate for further in vivo development.

Sodium valproate, an anticonvulsant drug, stimulates human immunodeficiency virus type 1 replication independently of glutathione levels.

Since modulation of the glutathione (GSH) level has been implicated in the regulation of human immunodeficiency virus (HIV) transcription and expression, we have undertaken an analysis of the effect of sodium valproate (VPA) on HIV-1 replication. VPA, which is an anti-epileptic drug in widespread use in clinical medicine, has been shown to depress the activity of GSH reductase, an enzyme required for maintaining high cellular levels of reduced GSH. The effect of this drug on HIV-1 replication has been studied in primary infected cells, i.e. peripheral blood mononuclear cells (PBMC) and monocyte/macrophages, in the CEM-SS cell line, and in chronically infected stimulated and non-stimulated U1 cells. We have shown that VPA markedly enhanced viral replication in all infected cells tested. Virus production was induced in U1 cells by VPA treatment and the stimulatory effects of tumour necrosis factor-alpha, interleukin-6 and granulocyte/macrophage colony-stimulating factor were augmented. The LTR-driven gene expression in Jurkat T cells was increased. However, the elevated viral production did not correlate with the effect of VPA on the intracellular GSH level. Thus, VPA stimulated in vitro HIV-1 replication in acutely and chronically infected cells and enhanced LTR-driven gene expression. These effects were observed for concentrations that are reached in the plasma of VPA-treated patients. Therefore, although the clinical significance of these data remains to be demonstrated, these results should be considered in the choice of an anticonvulsant drug in HIV-infected individuals.

Longitudinal study of human herpesvirus 6 infection in organ transplant recipients.

Human herpesvirus 6 (HHV-6) has been frequently isolated from immunocompromised patients. To determine if a routine survey of HHV-6 infection is needed after organ transplantation, as is the case for human cytomegalovirus infection, we observed patients who had received kidney, liver, and kidney-liver transplants; these patients were followed up for the first 3 months after transplantation. HHV-6 infection was diagnosed by isolation of the virus and by the results of serological tests. Antibodies to HHV-6 were detected in 28 (87.5%) of the 32 recipients, before the transplant, whereas only 4 (12.5%) of the 32 recipients were seronegative for HHV-6. After engraftment, HHV-6 infection occurred in 10 (31%) of the 32 recipients; infection was diagnosed by isolation of the virus (6 of 32 recipients) or by the results of serological tests (4 of 32 recipients). Regardless of whether they had HHV-6 primary infection or reactivation, severe clinical manifestations were observed only in patients who had concomitant cytomegalovirus infection, and no correlation could be found between graft rejection and HHV-6 infection. These results suggest that HHV-6 infection occurs frequently in organ transplant recipients and that it is usually not associated with severe clinical manifestations unless accompanied by a concomitant CMV infection.

Sodium valproate, an anticonvulsant drug, stimulates human cytomegalovirus replication.

Valproic acid (VPA), a simple branched-chain fatty acid having anticonvulsant activity and used in the treatment of many forms of epilepsy, markedly stimulated human cytomegalovirus (HCMV) replication in human fibroblasts (MRC-5 cells). The maximum level of stimulation was reached when cells were treated for 24 h before infection. The enhancement of virus replication correlated with an increase in the number of immediate early (IE) and early (E) antigen-positive cells. VPA also induced expression of IE antigens after transfection of fibroblasts with a plasmid containing the entire IE1-2 region. Moreover, VPA stimulated the HCMV IE1-2 promoter/enhancer-mediated expression of beta-galactosidase in a stably transfected Jurkat T cell line. Recently, VPA was shown to inhibit glutathione reductase in human red blood cells, but an action through the glutathione metabolic pathway can be eliminated in this case, since VPA decreased the intracellular level of glutathione in Jurkat T cells but not in MRC-5 cells. The ability of VPA to stimulate HCMV replication provides an attractive model for studying the molecular mechanism of the regulation of HCMV IE1-2 gene expression.

Seric and local antibodies against a synthetic peptide of HPV16.

An ELISA method was used to detect IgG and IgA directed against a synthetic peptide derived from the E2 ORF of the human papillomavirus (HPV) 16 in sera and in cervico-vaginal secretions from 20 women without evidence of HPV infection and from 41 women with histological diagnosis of HPV infection. The proportion of IgA positive sera (63.4% in the case-group vs. 20.0% in the control-group) and secretions (48.8% in the case-group vs. 15.0% in the control-group) was significantly higher in women with HPV infection and seemed to increase with the severity of the cervical lesion. Such a difference was not found for specific IgG. Comparing, for each patient, the antibody level in the serum and in the secretions, we found that the amount of IgA was at mean 2.4 times higher in the sera than in the secretions.

Valproic acid reduces the intracellular level of glutathione and stimulates human immunodeficiency virus.

Modifications of the glutathione (GSH) intracellular level have been implicated in the regulation of human immunodeficiency virus (HIV) transcription and expression. In regard to this hypothesis, we have investigated the effects of valproic acid (VPA) on HIV replication. Indeed, it has been recently reported that VPA inhibits the human red blood cell glutathione reductase. In the supernatant of a CEM-SS T-lymphocytic cell line infected with the LAI strain of HIV-1, we observed an increase, in a dose-dependent fashion, of the reverse transcriptase activity after treatment of cells with VPA. VPA also induced HIV expression in the chronically infected monocytic U1 cell line which constitutively expresses low levels of virus, enhanced the HIV-long terminal repeat (LTR)-directed expression of beta-galactosidase in transiently transfected Jurkat T-cells, and potentiated the PMA effect on the LTR transactivation. GSH assays showed that VPA treatment led to a decrease in the intracellular level of this thiol compound in U937 (U1 parent-cell line) and in Jurkat T-cells. Work to understand the molecular mechanism of VPA-induced HIV transcription and expression are now in progress. VPA seems to be an adequate molecule to study the implications of a GSH decrease in the stimulation of HIV replication. However, a modification of the intracellular balance between reduced and oxidized glutathione, rather than a simple reduction of the intracellular glutathione level, could be of importance in the regulation of HIV replication and we are now testing this hypothesis. Finally, these findings already suggest that VPA, which is an anticonvulsive drug frequently prescribed for the management of various seizure disorders, should not be recommended for treatment of epilepsy or other related illnesses in HIV-positive individuals.

Effects of glutathione precursors on human immunodeficiency virus replication.

Asymptomatic human immunodeficiency virus (HIV)-seropositive individuals have reduced glutathione (GSH) levels. This has led to the suggestion that elevated intracellular thiols levels may inhibit HIV replication and progression of the disease. We confirmed that N-acetyl-L-cysteine (NAC), a cysteine prodrug which maintains intracellular GSH levels during oxidative stress, inhibits in the chronically infected U1 cells, the stimulation of HIV replication induced by phorbol 12-myristate 13-acetate (PMA), interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF). However, we found no significant inhibition of PMA-mediated long terminal repeat (LTR)-directed beta-galactosidase expression in transiently transfected Jurkat T-cells. We have compared NAC effects with the effects of other GSH precursors on HIV expression. Treatment of the U1 cell line by L-2-oxo-4-thiazolidine carboxylic acid (OTC), which is converted to cysteine by 5-oxoprolinase, or by homocysteine (HC), a natural cysteine precursor, reduced the PMA-induced HIV expression, but surprisingly, markedly stimulated the expression mediated by IL-6 and GM-CSF. Several experiments to investigate the effect of OTC on LTR transactivation were carried out, but beta-galactosidase activity was never modified in a significant fashion in PMA-induced Jurkat T-cells after OTC treatment. Furthermore, HC stimulated the PMA-mediated HIV-LTR transactivation in Jurkat T-cells. GSH assays showed that treatment of U937 and Jurkat T-cells with NAC and OTC moderately increased the GSH level, while HC led to a significantly higher increase of the thiol level. In conclusion, it appeared that an increase of the GSH intracellular level did not lead solely to an inhibition of HIV replication but could also lead to an activation of viral expression. This seemed the case when HIV replication was stimulated by compounds which act mainly at a post-transcriptional level.

Environmental modulation of the autonomy of cytotoxic T lymphocytes.

The extent to which one compartment of the immune system depends on another for efficient function is important to establish to fully comprehend disease phenotypes arising from selective immunodeficiency. Just how much the major histocompatibility complex class I-restricted cytotoxic T cell responses depend on class II-restricted T cell help has been controversial. Using the influenza A virus system, we show that mice unable to make class II-restricted T cell responses due to an engineered defect in class II molecule expression are able to mount virtually normal cytotoxic responses when bred under specific-pathogen-free conditions. However, when exposed to the more diverse environmental challenges of a conventional breeding facility, a situation that more closely parallels immunodeficient states in man, they show impaired cytotoxic responses.

Enterovirus infections in neonates. A retrospective study of 21 cases.

OBJECTIVES: The aim of this study was to define the major features of enterovirus infections in the neonatal period based on our own experience. METHODS: Epidemiology, clinical manifestations and laboratory investigations concerning 21 neonates having experienced a Coxsackie B or an Echovirus infection between 1987 and 1991, were retrospectively reviewed. Aetiological diagnosis was made by classical viral isolation and/or by evidencing Coxsackie B-specific IgM antibodies with an immunocapture enzyme immunoassay. RESULTS: In 13 neonates the infection occurred between June and September. The onset of clinical signs ranged from day 1 to day 25 after birth with two separate periods: before 7 days of age, suggesting a perinatal transmission of the virus, or beyond this date, more likely connected with a postnatal transmission. Clinical manifestations included hyperthermia, gastroenteritis, meningitis, encephalitis, pneumonia and myocarditis, with a diphasic pattern in 6 cases. Most of the neonates improved gradually and developed normally. The Coxsackie B-specific IgM assay was the most rapid method whereas viral isolation, even though it took more time, was the most sensitive technique to establish the aetiological diagnosis in neonates. CONCLUSIONS: Enterovirus infections in neonates are difficult to diagnose and to differentiate from bacterial infections. A viral-like illness in the environment of the neonate allows the clinician to anticipate the clinical signs and a possibly fatal disease. Identification of the causal virus should be performed by both viral isolation and search for specific IgM antibodies. Treatment and prophylaxis are so far disappointing.

[Role of herpes virus simplex and cytomegalovirus as cofactors of papillomavirus in dysplastic and cancerous lesions of the uterine cervix]. / Rôle des virus herpes simplex et du cytomegalovirus en tant que co-facteurs des papillomavirus dans l'apparition des lésions dysplasiques et cancéreuses du col de l'utérus.

Human papillomaviruses (HPV) play an important role in cervical carcinogenesis but are probably not the only factors. To assess the role of HPV 6 and 11, of herpes simplex virus (HSV) and of cytomegalovirus (CMV) as cofactors for HPV 16 and HPV 18, we used polymerase chain reaction (PCR). The genomes of these six viruses was searched for in cervical biopsies, inbred in paraffin, from 22 normal cervices, 21 low grade cervical intraepithelial neoplasia (CIN), 21 high grade CIN and 20 squamous cancers. HPV 16 DNA had the highest prevalence in cervical lesions. Its frequency reached 24% in low grade CIN, 57% in high grade CIN, 50% in cancers and 9% in normal cervices. The simultaneous presence of HPV 16 and/or HPV 18 DNA and CMV and/or HSV DNA was significantly more frequent in high grade CIN and cancers than in normal cervices. On the other hand the presence of HPV 16 and/or HPV 18 DNA in samples lacking the other viral genomes did not significantly differ between the groups. Consequently the disease-viral infection relation was not apparent when these two "potentially oncogenic" HPV are isolated without coinfection by HSV or CMV. Our results are in agreement with the hypothesis of the role of herpesviruses as cofactors in cervical carcinogenesis. These viruses may disturb the control mechanisms of cellular growth and differentiation in HPV infected cells.

Time-resolved fluoroimmunoassay compared with virus isolation for rapid detection of respiratory syncytial virus in nasopharyngeal aspirates.

Two monoclonal antibodies against two distinct conserved epitopes of the respiratory syncytial virus (RSV) nucleocapsid protein were used in a direct time-resolved fluoroimmunoassay (TR-FIA) for the detection of RSV antigens in nasopharyngeal aspirates. The capture antibody was adsorbed to the solid phase of microdilution strip wells, and the indicator antibody was labeled with a europium chelate. Specimens and label were incubated simultaneously for 1 h at 37 degrees C in the coated wells. After the test samples were washed, fluorescence enhancement solution was added, strips were shaken, and the time-resolved fluorescence was measured. The test procedure took only 75 min, and the total time for 20 specimens, with pretreatment by sonication, was 2 to 3 h. We prospectively evaluated the detection of RSV in nasopharyngeal aspirates of pediatric patients by TR-FIA and by virus isolation in human diploid fibroblasts. TR-FIA detected 40 of 42 isolation-positive specimens. Nine additional isolation-negative specimens were positive by TR-FIA; all proved to be true positives by a blocking-type confirmatory assay. The sensitivity, specificity, positive predictive value, and negative predictive value for TR-FIA were 95, 96, 82, and 99%, respectively, of the values obtained by virus isolation and 96, 100, 100, and 99%, respectively, of the values obtained by virus isolation and the confirmatory assay.

An enzyme-linked immunosorbent assay using monoclonal antibodies for the detection of respiratory syncytial virus in clinical specimens.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of respiratory syncytial virus in nasopharyngeal secretions. This assay employed as immunoreagents two monoclonal antibodies directed against two distinct epitopes of the viral nucleocapsid. One of them (RSV 4) was used for antigen capture and the other (NC 4) was labelled with N-hydroxy-succinimide-epsilon-caproil biotin and used for antigen detection. Streptavidin biotin-peroxidase complexes were employed as amplification mode. The immunoassay was performed in 6 hours and was able to detect as little as 1 ng/ml of purified nucleocapsid. When 87 nasopharyngeal secretions were analyzed by an indirect immunofluorescence assay using commercial reagents and by the newly developed ELISA, the sensitivity and the specificity of the two assays were found to be very similar.

Etiology of pituitary apoplexy: review and case presentation.

Pituitary apoplexy is an acute neurologic emergency typically occurring in patients who suffer rapid and fatal expansion of a pre-existing adenoma. We report an example of pituitary apoplexy secondary to necrotizing hypophysitis due to a mycobacterial or tuberculous infection with limited systemic infection.

Use of egg-yolk antibody for detection of respiratory syncytial virus in nasal secretions by ELISA.

Egg-yolk immunoglobulins extracted from the eggs of hens immunized with respiratory syncytial virus (RSV) have been used as a reagent in double sandwich ELISA for detecting RSV in nasal secretions. The sensitivity of virus detection was the same in indirect ELISA, using rabbit anti chicken globulin conjugate, as when biotinylated yolk globulin and labeled avidin were used for detection. The specificity of ELISA for detecting RSV using yolk antibody was similar to that achieved by indirect immunofluorescence using commercial reagents of mammalian origin. Purified immunoglobulin was easily extracted from egg yolk; the amount of globulin present in a single preparation obtained from a batch of ten eggs was sufficient to carry out 10(6) ELISA tests for RSV detection.

Sodium dodecyl sulfate-mediated transfer of electrophoretically separated DNA-binding proteins.

A modified procedure for the transfer of electrophoretically-separated proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels onto nitrocellulose filters has been developed. During the diffusion mediated transfer, the SDS-protein complexes were maintained and SDS was added to the buffer. This increases the number of polypeptide species bound to the filter thereby giving an accurate replica of the original gel pattern. The immobilization in the gel of certain polypeptides characterized as DNA-binding proteins, which is observed when SDS is eliminated prior to blotting is avoided. The molecules blotted in the presence of SDS remain immunoreactive and able to bind DNA.

[Isolation of 503 strains of virus in meningeal syndromes. Virologic and epidemiologic study]. / Isolement de cinq cent trois souches de virus au cours de syndromes méningés. Etude virologique et épidémiologique.

An epidemiological and virological study on meningitis in Alsace during the 13-year period 1968-1981 is reported. 503 viruses isolated were associated with the diagnosis of meningitis. The Echovirus type 30 accounted for 48 p. 100 of the cases, the mumps virus of 12 p. 100. The remarkable high incidence of Echo 30 virus-associated cases is related to three outbreaks which occurred in Alsace during the summers of 1968, 1975 and 1980. Children below 12 years of age are the most sensitive population and particularly boys who made up 2/3 of the number of cases.

Comparison of immunosorbent electron microscopy, enzyme immunoassay and counterimmunoelectrophoresis for detection of human rotavirus in stools.

The detection of human rotaviruses by routine electron microscopy examination of stool specimens has been compared with the sensitivity of detection obtainable by three different immunoassays. These assays are: 1) immunosorbent electron microscopy (ISEM), which consists of the serological trapping of viruses on electron microscopy grids coated with protein A and specific viral antiserum; 2) an enzyme-linked immunosorbent assay (ELISA), in which the primary antibody is rabbit anti-rotavirus immunoglobulin, the secondary antibody is chicken anti-rotavirus immunoglobulin extracted from egg yolk of immunized hens, and the indicator antibody is alkaline phosphatase-conjugated rabbit anti-chicken immunoglobulin; 3) counterimmunoelectrophoresis (CIE). A total of 63 stool specimens from infants with gastroenteritis were examined. Of these, 23 and 24 specimens were found to contain rotavirus by electron microscopy and CIE, respectively. When scored by ELISA and ISEM, 37 and 39 were found to be positive, respectively. Confirmatory inhibition assays were necessary to eliminate some false positive reactions in ELISA. Detection of human rotaviruses in stools by ISEM is as sensitive as by ELISA, but in weakly positive specimens, ISEM offers the additional advantage of a direct visual demonstration of the presence of the aetiological agent.