Deciphering the mode of action of the synthetic antimicrobial peptide Bac8c.

Bac8c (RIWVIWRR-NH(2)) is an 8-amino-acid peptide derived from Bac2A (RLARIVVIRVAR-NH(2)), a C3A/C11A variant of the naturally occurring bovine peptide, bactenecin (also known as bovine dodecapeptide), the smallest peptide with activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. The effects of Bac8c on Escherichia coli were examined by studying its bacteriostatic and bactericidal properties, demonstrating its effects on proton motive force generation, and visually analyzing (via transmission electron microscopy) its effects on cells at different concentrations, in order to probe the complexities of the mechanism of action of Bac8c. Results were consistent with a two-stage model for the Bac8c mode of action. At sublethal concentrations (3 µg/ml), Bac8c addition resulted in transient membrane destabilization and metabolic imbalances, which appeared to be linked to inhibition of respiratory function. Although sublethal concentrations resulted in deleterious downstream events, such as methylglyoxal formation and free radical generation, native E. coli defense systems were sufficient for full recovery within 2 h. In contrast, at the minimal bactericidal concentration (6 µg/ml), Bac8c substantially but incompletely depolarized the cytoplasmic membrane within 5 min and disrupted electron transport, which in turn resulted in partial membrane permeabilization and cell death.

[Prophylactic antiretroviral therapy after sexual exposure to HIV: 93 cases]. / Traitement antirétroviral prophylactique après exposition sexuelle au VIH: 93 cas.

PATIENTS AND METHODS: We studied prospectively the feasibility of post exposure prophylaxis against HIV in 93 subjects consulting after sexual exposure at STD Center of Hopital Saint-Louis. Among the 93 subjects, 76 were men (45 homosexual) and 17 women. RESULTS: Delay to consultation was 38 h. Among sexual exposure 90 p. 100 were anal or vaginal intercourse and 10 p. 100 oral intercourse. Fifty percent were unprotected. Seventy-five percent of source subject HIV status was unknown, but controlled negative in 14 p. 100 of cases. Three subjects were infected initially. Seventy-two subjects were treated, with triple regimen, for 30 days without severe adverse event. Twenty-five percent were lost to follow up before the end of treatment, only 54 controlled their serology after the end of treatment (after 1 month: 70 p. 100, after 2 months: 51 p. 100 and after 4-6 months: 13 p. 100). DISCUSSION: This study underlines the difficulty in obtaining clinical and serological control after post exposure prophylaxis, even in a STD Department involved in prevention and counseling.

Seroprevalence of herpes simplex virus type 2 antibodies in an STD clinic in Paris.

Our objective was to evaluate the seroprevalence of herpes simplex virus (HSV)-2 and HSV-1 in a population of men and women attending the STD clinic of Hôpital St-Louis (Paris, France). Four hundred and eighty-seven patients (264 men and 223 women) were tested for HSV-2 and HSV-1 antibodies by specific enzyme immunoassay (EIA) (Smithkline-Beecham Biologicals). Univariate and multivariate analyses were carried out for correlations with clinical, socio-epidemiological and behavioural data. HSV-2 seroprevalence was 55% (44.7% in men, 67.3% in women). HSV-1 seroprevalence was 93% (94.7% in men, 91% in women). The predictive factors of HSV-2 seropositivity being female (OR: 3.37), age (OR: 1.04), country of origin (Central Africa OR: 3.52, North Africa OR: 1.36), history of genital herpes (OR: 10.97), hepatitis B virus (HBV) markers (OR: 1.92) and hepatitis C virus (HCV) markers (OR: 3.96). The only protective factor was HSV-1 seropositivity (OR: 0.25). The predictive factors of HSV-1 seropositivity were only the country of origin (Central Africa OR: 2.95, North Africa OR: 1.83) and the absence of genital herpes (OR: 11.01). Only 23 (8.6%) HSV-2 seropositive patients had a history of genital herpes. This study underlines the very high HSV-2 seroprevalence of patients with STDs, only a few of whom have a history of genital herpes. Detection and counselling is urgently needed for these patients.

A prospective study of the influence of HIV status on the seroreversion of serological tests for syphilis.

UNLABELLED: The evolution of serological tests for syphilis (STSs) after therapy in HIV+ patients is a major point of controversy, with possible seroreactivation and illicit seroreversion in these patients. The aim of our study was to evaluate the long-term outcome of STSs in a cohort of HIV+ male homosexuals with a history of treated syphilis as compared with HIV- controls. PATIENTS AND METHODS: Sixty-nine HIV+ male homosexuals with a documented history of treated syphilis and positive baseline treponemal tests were prospectively studied between 1986 and 1993. A medical examination, HIV staging, CD4+ cell count, VDRL, FTA-Abs tests and TPHA were performed every 6 months. Controls consisted of 49 HIV- patients with similar inclusion criteria over the same period. Comparisons between subgroups were based on chi(2) and Kruskal-Wallis tests. Analysis of negativation of the STS used the failure data methods (Kaplan-Meier, log-rank and Cox's model). RESULTS: Patients had a mean age of 38 years, a baseline CD4+ cell count of 578/mm(3), elapsed time since last syphilis of 7.5 years and a median follow-up of 4.3 years. Controls had a mean age of 42 years, elapsed time since last syphilis of 5.3 years and a median follow-up of 4.7 years. Time to seroreversion was shorter in HIV+ patients for TPHA (p = 0.009, log-rank test) and FTA-Abs test (p = 0.001, log-rank test), even after adjustment for stage of syphilis, age and time since the last episode of syphilis. The decrease in VDRL titres was not different between the 2 groups (p = 0.053, log-rank test). Seroreversion of the TPHA, FTA-Abs test and VDRL test was not significantly related to stage of syphilis, time elapsed since the last episode of syphilis, age or history of STDs in both groups. Seroreversion of the TPHA and VDRL test was not related to baseline CD4+ cell count. However, seroreversion of the FTA-Abs test was related to a low baseline CD4+ cell count (p = 0. 003). In HIV+ patients, a significant decrease in titres was noticed for TPHA, FTA-Abs test and VDRL test over time, but this time effect remained only for TPHA titres after adjustment for the CD4+ cell count. CONCLUSION: TPHA may serorevert in HIV+ patients. Thus, a non-reactive TPHA does not exclude a past syphilis infection in such patients. Evolution of the VDRL test after therapy is regular in HIV+ patients. The VDRL test remains adequate for controlling the efficacy of treatment in these patients.

Daily multidrug therapy for leprosy; results of a fourteen-year experience.

Between 1980 and 1994, 67 new or relapsing leprosy patients were treated by daily administered multidrug regimens. Tuberculoid patients (23 TT/BT) received either bitherapy [rifampin + dapsone or clofazimine (RMP + DDS or CLO)] or tritherapy [RMP + DDS and/or CLO and/or ethionamide (ETH)] until clinical cure. Lepromatous patients (44 BB/BL/LL) received tritherapy (RMP + DDS and/or CLO and/or ETH) at least until bacteriological negativity. Of the 23 tuberculoid patients only one patient (5%) was cured at 6 months and about 70% needed between 6 and 24 months of treatment to obtain clinical cure (mean 19.5 months). In the 44 lepromatous patients, the achievement of bacteriological negativity was significantly linked to the initial bacterial index (BI), and it occurred after 2 to 7 years (mean 66.5 months) of multidrug therapy (MDT). The average BI decrease per year was 1.1+ during the first year, 0.9+ the second year, and then < 0.5+ per year. Reactional states significantly (p < 0.01) influenced the BI course: reversal reactions (RR) accelerated while erythema nodosum leprosum (ENL) delayed the BI decrease. Three of the 23 (13%) tuberculoid and 19 of the 44 (43%) lepromatous patients (p < 0.02) exhibited a RR and 18 of 44 (41%) lepromatous patients had ENL during MDT. A late RR (LRR) was observed in 1 (5%) and 6 (17%) of our tuberculoid and lepromatous patients, respectively, and 3 (8%) of our lepromatous patients suffered post-MDT ENL. No confirmed relapse has been observed within a follow-up period of 6 months to 7 years and 3 months [59 person-years at risk (PYR)] for TT/BT patients and of 4 months to 5 years and 10 months (100 PYR) for BB/BL/LL patients. When compared to the recommended WHO/MDT, it appears that daily MDT does not increase the clinical or the bacteriological cure rates either at 6 months in paucibacillary tuberculoid patients or at 2d years in multibacillary lepromatous patients. Moreover, as does the WHO/MDT, our regimens show a high frequency of reactional states both during and after treatment. This fact constitutes the main new problem of the actual treatment of leprosy.

Membranes and polymer structures--biocompatibility aspects with respect to production limits.

Plasmapheresis can be performed by centrifugation and by use of membrane technology. With the latter technique we receive a plasma which is absolutely free from platelets. This is why membranes are gaining market shares in this particular field of medical application. Today plasmapheresis membranes are mostly fabricated from synthetic polymers, such as polypropylene (e.g. PLASMAPHAN), polysulfone, polyacrylonitrile, polymethylmethacrylate, polyvinylalcohol and others, the only exception being cellulose acetate. Parameters determining the biocompatibility of plasmapheresis membranes are generation of complement C3a or C5a, hemolysis and possible thrombus formation. These parameters depend on various properties of the membrane polymer: e.g. the nature of the molecular end/side-groups, the distribution of electrical charges on the polymer surface and the different chemical structures and conformation of the polymer. In addition, membrane properties like pore distribution and geometry or the flow characteristics of a particular device-design may trigger cell activation or influence biocompatibility through the adsorption of various plasmacomponents. Most of the polymers which are used today for manufacturing plasmapheresis membranes have not been developed for this purpose. They were originally selected to be used as textile fibers. Further, no present membrane polymer has been specifically developed to achieve high biocompatibility. The membrane profile was designed in such a way that pheresis properties were met rather than optimizing biochemical blood/polymer interactions. One reason for this decision may be that the market volume of plasmapheresis technology is too small in order to justify specific and high-cost developments of polymers for this purpose. Polymer selection to achieve excellent biocompatibility profiles is determined by polymer-availability, costs, membrane-forming processes and environmental aspects related to possible pollution during the manufacturing process. The production of PLASMAPHAN by the unique Accurel-process combines several of these parameters. The main membrane production processes and especially the Accurel-process are described here. The influence of polymer-surface properties, membrane structure and module-design on the biocompatibility of plasmapheresis treatments are discussed and explained by appropriate examples.

The role of stress in interpreting the dexamethasone suppression test.

Stress has been implicated as a major confounding factor in the interpretation of Dexamethasone Suppression Test (DST) results. This study was designed to examine the effects of stress on DST results. Fifty patients with high levels of acute, chronic, and environmental stress participated in the study. Each patient was given a comprehensive psychiatric and psychological assessment, a routine administration of dexamethasone, and blood tests of cortisol values. The results indicate that the three measures of stress do not appear to affect levels of cortisol suppression, however, all three measures of stress predicted depression. As expected, DST cortisol levels were related to depression. Results are discussed in terms of their implications for understanding the associations among stress, depression and DST results.

[Acute uveitis in reversal reactions]. / Uvéites aiguës au cours des réactions de réversion.

Two cases of acute uveitis have been reported in reversal reactions in lepromatous patients treated with anti-hansenian multidrug therapy with daily rifampicin. This type of eye damage has seldom been reported in reversal reactions.

Imaging of dialysis-related amyloid (AB-amyloid) deposits with 131I-beta 2-microglobulin.

The diagnosis of dialysis-related amyloid (AB-amyloid) has been based usually on clinical and radiological criteria. Following the discovery that beta 2-microglobulin was the major protein of this amyloid, we isolated and radiolabelled uremic plasma beta 2-microglobulin. After intravenous injection, gamma-camera images of selected joint areas were obtained from 42 patients who were on regular hemodialysis therapy. Positive scans involving the shoulder, hip, knee and carpal regions were found in 13 of 14 patients treated for more than 10 years and 10 of 16 patients treated for 5 to 10 years. Patients treated for less time had negative scans. Specificity was indicated by negative scans in non-amyloid inflammatory lesions in control hemodialysis patients. Up to 48-fold tracer enrichment was detected in excised AB-amyloid containing tissue as compared to amyloid-free tissue. These findings suggest that circulating radiolabelled beta 2-microglobulin is taken up by the amyloid deposits. This method may non-invasively detect tissue infiltrates of amyloid. It may also permit prospective evaluation of the efficacy of prophylactic dialysis strategies which are designed to prevent or delay the onset of this complication of long-term dialysis.

Prosomes discriminate between mRNA of adenovirus-infected and uninfected HeLa cells.

Prosomes are small cytoplasmic RNP complexes associated with repressed mRNA. In in vitro translation, they discriminate between the mRNA of adenovirus-infected HeLa cells and those of uninfected cells grown under normal conditions. Prosomes as well as their RNA constituents interact much more strongly with poly(A)+ mRNA of infected cells and inhibit their translation in vitro preferentially. A possible role of prosomes in the differential regulation of translation is discussed.

Specific imaging of dialysis-related amyloid deposits using 131I-beta-2-microglobulin.

Dialysis-related amyloidosis (DRA), characterized by its association with beta 2-microglobulin (beta 2m), has become a major concern in long-term hemodialysis patients. Hitherto the diagnosis was based on histological examinations of tissue obtained by biopsy or during surgery. In this preliminary study a new noninvasive diagnostic method was developed using the affinity of beta 2m for its derived fibrils. 3 patients on long-term hemodialysis for 10-16 years with biopsy-proven DRA and 1 patient on chronic hemodialysis for only 6 months were examined after intravenous injection of 131I-labelled beta 2m. Specific local accumulation of radioactivity was noted in the DRA patients after 48 h, persisting for further 96 h and corresponding to clinically or radiologically evident sites of amyloid deposition and to several other hitherto unsuspected sites. Examination of an excised amyloid tumor subsequent to in vivo labelling confirmed a highly specific accumulation of radioactivity in the amyloid tissue but not in control tissue. In the patient on chronic hemodialysis for only 6 months, no specific local accumulation was detected even after 1 week. These findings provide in vivo evidence in man that a specific uptake of circulating amyloid precursor molecules into deposits occurs and that this uptake may be used to radiolabel even small tissue infiltrates of amyloid. This method therefore may not only allow an objective, noninvasive detection of DRA but may also be used to obtain new pathophysiologic insights into amyloid formation in man, as well as permitting the evaluation of preventive therapeutic strategies in prospective studies on new patients.

Monoclonal antibodies produced by in vitro immunization with sera of human-xenotransplant-bearing mice.

In vitro immunization procedures, using sera of athymic mice bearing human WOC ovarian tumors or CM III mammary tumors as immunizing antigen, induced a highly efficient formation of mABs (44% of antibody-producing clones) reacting with human ovarian and/or mammary tumor cells. More than half of these mABs showed cross-reactivity with mouse cell lines. Immunogenicity of normal mouse components in the sera from tumor bearers can be excluded since control immunization with sera of normal athymic mice yielded no mABs reacting with mouse or human cell lines. Furthermore, immunization with sera from tumor bearers did not induce mABs only reacting with mouse cells since 20% of the antibody-producing clones showed an exclusive specificity for the human tumor cells. On the basis of these results we concluded that the human-mouse cross-reacting mABs were induced by circulating human TAA with epitopes shared by mouse cellular components.

Monoclonal antibody-defined circulating human tumor-associated antigen with epitope shared by cytokeratins.

Sera of human colonic carcinoma xenografted rnu/nu rats were used to immunize rnu/+rats in order to obtain an immune response against circulating human tumor-associated components. After fusion of rat spleen cells with mouse myeloma cells monoclonal antibody MAB 108 could be established which reacted with two 40 and 45 kD cytokeratins as well as with vimentin, with a soluble 37 kD protein apparently derived from the 45 kD protein and with a 37 kD protein released by tumor cells. The MAB 108-specific epitope was also detected in tissue polypeptide antigen (TPA), a human tumor-associated antigen originally described by Björklund et al. (22).

R-loop mapping of calf-thymus RNA polymerase II. Transcripts in vitro on restriction fragments of adenovirus 2 DNA.

Nascent RNA, synthesized by calf thymus ENA polymerase II on the restriction endonuclease EcoRI fragment A and BamHI fragment B of adenovirus 2 DNA, was rehybridized to the template strand under conditions allowing transcription R-loop formation. Hybrids, visualized by electron microscopy, were found in looped and in branched configurations, the former being abundant, with an average loop number of roughly four per EcoRI fragment A and three per BamHI fragment B. Frequency distributions of transcription R-loops, synthesized on identical restriction fragments for different periods of time, showed similar patterns, thus pointing to a nonrandom type of transcription. A comparison of the frequency distribution of R-loops seen on the two overlapping restriction fragments reveals reasonable coincidence in the loop pattern in one out of the four possible fragment orientations, permitting correlation of the transcription R-loop pattern with the physical map of the adenovirus 2 DNA. Comparison of start sites tentatively mapped in vitro with the known promoters for RNA polymerase II in vivo suggests that a major transcription site in vitro is located in close proximity to, or identical with, two promoters in vivo at 16.1 and 16.5 map units.

Molecular cloning and preliminary characterization of a Drosophila melanogaster gene from a region adjacent to the centromeric beta-heterochromatin.

Using recombinant DNA technology we have isolated a 4.4 kb DNA fragment from Drosophila melanogaster which can be localized by in situ hybridization to the region 80C on the left arm of chromosome III. This DNA fragment codes for a 1.4 kb long poly(A)-containing RNA which comprises about 0.6% of the mass of cytoplasmic poly(A) RNA in Kc cells and Oregon R Embryos. This RNA codes for a 26,000 MW protein of still unknown function.

Comparison of proteins bound to the different functional classes of messenger RNA.

Proteins from nuclear ribonucleoproteins, informosomes, polysomal messenger ribonucleoproteins and cytoplasmic "binding factor" are characterized. 1. Nuclear ribonucleoproteins are purified from nuclei disrupted by ultrasonication. Possible contamination by nucleoplasm, histones or remaining cytoplasmic structures is controlled. 2. Informosomal proteins are obtained by mild RNAase degradation. This method gives informosomal proteins without appreciable contamination. 3. Polysomal messenger ribonucleoproteins are obtained from cells where the initiation of protein synthesis is arrested in order to release the messenger ribonucleoproteins from the polysomes. Their proteins are obtained like the informosomal proteins by mild RNAase digestion. No contamination by informosomes could be detected by sodium dodecyl sulfate gel electrophoresis. 4. Cytoplasmic "binding factor" proteins are purified by affinity chromatography. 5. The four sets of proteins are analysed by sodium dodecylsulfate acrylamide gel electrophoresis. In spite of the fact that some proteins from one or another kind of messenger ribonucleoprotein, have apparently the same molecular weight, the majority of proteins differ.