Véronique Fontaine

Evaluation of Combined General Primer-Mediated PCR Sequencing and Type-Specific PCR Strategies for Determination of Human Papillomavirus Genotypes in Cervical Cell Specimens

ABSTRACT A strategy combining human papillomavirus general primer (mainly the PGMY primers)-directed PCR sequencing and type-specific PCR is presented. DNA samples were first tested in general primer-mediated PCR. The amplified fragments of positive samples after ethidium bromide-stained DNA gel analysis were further sequenced, and corresponding DNA samples were further analyzed by PCR using type-specific primers for human papillomavirus (HPV) types 16, 18, 31, and 52. The comparison of the results of 157 samples analyzed by this strategy in parallel with the Hybrid Capture 2 tests and with the HPV INNO-LiPA (Innogenetics line probe assay) shows that this method is suitable for HPV detection and genotyping in cervical cell samples. Although the PCR sequencing method is as sensitive as the HPV INNO-LiPA for HPV detection, our method allows the identification of a broader range of HPV types. In contrast, the HPV INNO-LiPA was less time-consuming and better identified coinfections.

Effects of HPV-16 E5, E6 and E7 proteins on survival, adhesion, migration and invasion of trophoblastic cells

Among high-risk human papillomaviruses (HPV), HPV-16 infection is the most prevalent causative factor for cervical cancer. Beside other mucosal targets, HPV-16 was reported to infect the placenta and to replicate in trophoblastic cells. Since these cells share invasive properties of tumoral cells, they represent an ideal model to investigate several oncogenic processes. In the present work, we analyzed the impacts of HPV-16 E5, E6 and E7 oncoproteins on the trophoblastic model. Our results showed that E5 impaired the viability of trophoblastic and cervical cell lines but E6 and E7, favoring cell growth, neutralized the E5 cytotoxic effect. In addition, E5 decreased the adhesiveness of trophoblastic cells to the tissue culture plastic and to endometrial cells similarly as described previously for E6 and E7. E5 and E6 plus E7 increased also their migration and their invasive properties. Cells expressing HPV-16 early proteins under the control of the long control region endogenous promoter displayed growth advantage and were also more motile and invasive compared with control cells. Interestingly, the E-cadherin was downregulated in trophoblastic cells expressing E5, E6 and E7. Nuclear factor-kappaB and activator protein-1 activities were also enhanced. In conclusion, HPV-16 early proteins enhanced trophoblastic growth and intensify the malignant phenotype by impairing cell adhesion leading to increased cellular motile and invasive properties. HPV-16 E5 participated, with E6 and E7, in these changes by impairing E-cadherin expression, a hallmark of malignant progression.

Evidence of Human Papillomavirus in the Placenta

Human papillomavirus (HPV) is an epitheliotropic virus typically infecting keratinocytes but also possibly epithelial trophoblastic placental cells. In the present study, we set out to investigate whether HPV can be recovered from transabdominally obtained placental cells to avoid any confounding contamination by HPV-infected cervical cells. Thirty-five placental samples from women undergoing transabdominal chorionic villous sampling were analyzed, and we detected HPV-16 and HPV-62 in 2 placentas. This study suggests that HPV infection of the placenta can occur early in pregnancy. The overall clinical implication of these results remains to be elucidated.

Internal deletions of amino acids 29-42 of human interleukin-6 (IL-6) differentially affect bioactivity and folding.

Internal deletions of the human interleukin‐6 (IL‐6) cDNA have been generated in the region encoding residues 29 to 42. Mutant proteins were produced by in vitro transcription—translation or in Escherichia coli and tested for their biological activity using the hybridoma growth factor (HGF) assay or a transcriptional activation assay on human hepatoma cells. The folding of the mutants was also checked by immunoprecipitation with conformation‐specific monoclonal antibodies. The results show that only residues 29 to 34 are crucial for IL‐6 activity and that the first two amino acids are probably involved in the definition of the IL‐6 active site.

Detection of HPV and the role of p16INK4A overexpression as a surrogate marker for the presence of functional HPV oncoprotein E7 in colorectal cancer.

BackgroundBased on the well-recognized etiological role of human papillomavirus (HPV) in cervical, anogenital and oropharyngeal carcinogenesis, a potential role of HPV in colorectal carcinogenesis has been suggested. For that reason, the aim of the present study was to investigate the presence of HPV DNA in colorectal carcinomas (CRC) and to study overexpression of p16INK4A as a marker for the presence of an active HPV oncoprotein E7. These findings were correlated with clinical and pathological prognostic factors of CRC.MethodsThe presence of HPV was assessed using a multiplex PCR system of 10 non-biotinylated primers. The amplified fragments of HPV positive samples were further analyzed by a highly sensitive, broad spectrum SPF10 PCR and subsequently genotyped using reverse hybridization in a line probe assay.P16INK4A protein expression was investigated in a subset of 90 (30 HPV positive and 60 HPV negative) CRC samples by immunohistochemistry.ResultsHPV DNA was found in 14.2% of the CRC samples with HPV16 as the most prevalent type. No significant differences in clinical and pathological variables were found between HPV positive and negative CRCs, except for age. HPV positive patients were significantly younger (p = 0.05). There was no significant correlation between the presence of HPV and overexpression of p16INK4A (p = 0.325).ConclusionsIn conclusion, the presence of oncogenic HPV DNA in a small cohort of CRC samples may suggest that HPV may be involved in the carcinogenesis of some CRC. However, contrary to what has been observed in head and neck squamous cell cancer and cancer of the uterine cervix, p16INK4A does not seem to be a surrogate marker for an active HPV infection in CRC. Therefore, further functional analyses are necessary to elucidate the role of HPV in CRC.

Efficiency of an inexpensive liquid-based cytology performed by cytocentrifugations: a comparative study using the histology as reference standard

Background Although liquid-based cytology (LBC) is now recommended for cervical cancer screening, it requires expensive automated devices and materials. To evaluate the efficiency of inexpensive LBC methods relying on an inexpensive fixative liquid, Easyfix®, we compared the results obtained by the liquid-based cytology (LBC) diagnoses performed by cytocentrifugations (Papspin® and Turbitec®) with those obtained by histology. Furthermore, we evaluated the efficiency of the fixative liquid, Easyfix®, to preserve HPV DNA in the collected samples. Method 266 LBC were compared with 174 colposcopies and 91 Loop Electrosurgical Excision Procedure (LEEP). Among the LBC, 51 were performed using the Papspin® system and 215 were performed using the Turbitec® system. To control the quality of the preservation liquid, Easyfix®, we correlated the results of HCII assays with those of HPV PCR. Results For Papspin® and Turbitec® systems, the sensitivities were respectively 82.6% (95% CI: 61.2–95.0%, p < 0.001) and 75.0% (95% CI: 64.4–89.8%, p < 0.001) and the specificities were 92.6% (95%CI: 76.5–99.1%, p < 0.001) and 96.2% (95% CI: 91.3–98.7%, p < 0.001). We find no statistical difference between the results of the both systems (p = ns). The sensitivity of the HCII was 86.4% (95% IC: 77.4–92.8%, p < 0.001) and the specificity was 39.4% (95% CI: 31.2–48.1%, p < 0.001). The comparison between HCII and HPV-PCR shows a good correlation: the kappa was 0.89. Conclusion LBC performed by cytocentrifugations are inexpensive, reduce inadequate smears, show excellent efficiency and allow HPV detection by molecular biology.

Internal deletions in human interleukin-6: structure-function analysis

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAbs): mAbs that show preference towards denatured hIL-6, or conformation-specific mAbs. The binding pattern of these two series of mAbs indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAbs. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative evaluation of 3 selective media for primary isolation of Helicobacter pylori from gastric biopsies under routine conditions

This study evaluates 3 selective media (Pylori agar [bioMérieux, France], BD Helicobacter agar, modified [Becton Dickinson, USA], and an in-house medium) designed for Helicobacter pylori isolation. Ninety-eight strains were isolated from 400 gastric biopsies. The media were equally efficient for Helicobacter pyloris growth. However, contaminations were only observed using commercial media.

Increased Vancomycin Susceptibility in Mycobacteria: a New Approach To Identify Synergistic Activity against Multidrug-Resistant Mycobacteria

ABSTRACT Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing Mycobacterium bovis BCG and M. tuberculosis mutants that lack phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids with wild-type strains, we observed that glycopeptides strongly inhibited PDIM-deprived mycobacteria. Vancomycin together with a drug targeting lipid synthesis inhibited multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential antituberculosis (TB) agents and might provide a new antimycobacterial drug-screening strategy.

Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria

ABSTRACT Tuberculosis is still a cause of major concern, partly due to the emergence of multidrug-resistant strains. New drugs are therefore needed. Vancomycin can target mycobacteria with cell envelope deficiency. In this study, we used a vancomycin susceptibility assay to detect drugs hampering lipid synthesis in Mycobacterium bovis BCG and in Mycobacterium tuberculosis. We tested three drugs already used to treat human obesity: tetrahydrolipstatin (THL), simvastatin, and fenofibrate. Only vancomycin and THL were able to synergize on M. bovis BCG and on M. tuberculosis, although mycobacteria could also be inhibited by simvastatin alone. Lipid analysis allowed us to identify several lipid modifications in M. tuberculosis H37Rv treated with those drugs. THL treatment mainly reduced the phthiocerol dimycocerosate (PDIM) content in the mycobacterial cell wall, providing an explanation for the synergy, since PDIM deficiency has been related to vancomycin susceptibility. Proteomic analysis suggested that bacteria treated with THL, in contrast to bacteria treated with simvastatin, tried to recover, inducing, among other reactions, lipid synthesis. The combination of THL and vancomycin should be considered a promising solution in developing new strategies to treat multidrug-resistant tuberculosis.