Simona Marchetti

RNA (E6 and E7) Assays versus DNA (E6 and E7) Assays for Risk Evaluation for Women Infected with Human Papillomavirus

ABSTRACT In the majority of cases, high-risk human papillomavirus (HR HPV) infections regress spontaneously, with only a small percentage progressing to high-grade lesions. Current screening methods are based on DNA detection. An alternative would be to monitor expression of the E6 and E7 viral oncogenes continuously expressed by malignant phenotypes. In the work reported in this paper, we compared the two methods for a group of women with high-risk HPV infections. Cervical specimens from 400 women, previously found to be HPV DNA positive, were analyzed for HPV DNA by a liquid hybridization assay and typed by multiplex PCR (for types 16, 18, 31, and 33). Identification of HR HPV E6 and E7 RNA transcripts was performed using real-time reverse transcription-PCR and nucleic acid sequence-based amplification assays. Results were compared with concurrent cytological data. HR HPVs were found in 61.2% of patients. The most common genotype was HPV type 16 (HPV-16) (47.1%), followed by HPV-18, HPV-31, and HPV-33. Nine percent of cases involved other genotypes. Among 223 HPV DNA-positive samples, only 118 were positive in the RNA test. Among HPV DNA-positive patients with normal cytology, we detected E6 and E7 RNA transcripts in two cases (18.2%). The rate of detection increased gradually with the grade of the observed lesions, rising from 20% for patients with atypical squamous cells of undetermined significance to 48.1% for women with low-grade squamous intraepithelial lesions and 86.3% for those with high-grade squamous intraepithelial lesions. These results suggest that testing for HPV E6 and E7 transcripts could be a useful tool for screening and patient management, providing more accurate predictions of risk than those obtained by DNA testing.

Porphyromonas gingivalis and the pathogenesis of rheumatoid arthritis: analysis of various compartments including the synovial tissue

IntroductionWe evaluated the presence of Porphyromonas gingivalis (Pg) DNA in the synovial tissue through synovial biopsy and in other compartments of rheumatoid arthritis (RA) patients in comparison with patients affected by other arthritides. Possible links with clinical, immunologic and genetic features were assessed.MethodsPeripheral blood (PB), sub-gingival dental plaque, synovial fluid (SF) and synovial tissue samples were collected from 69 patients with active knee arthritis (32 with RA and 37 with other arthritides, of which 14 had undifferentiated peripheral inflammatory arthritis - UPIA). Demographic, clinical, laboratory and immunological data were recorded. The presence of Pg DNA was evaluated through PCR. The HLA-DR haplotype was assessed for 45 patients with RA and UPIA.ResultsNo differences arose in the positivity for Pg DNA in the sub-gingival plaque, PB and SF samples between RA and the cohort of other arthritides. Full PB samples showed a higher positivity for Pg DNA than plasma samples (11.8% vs. 1.5%, P = 0.04). Patients with RA showed a higher positivity for Pg DNA in the synovial tissue compared to controls (33.3% vs. 5.9%, P <0.01). UPIA and RA patients carrying the HLA DRB1*04 allele showed a higher positivity for Pg DNA in the synovial tissue compared to patients negative for the allele (57.1% vs. 16.7%, P = 0.04). RA patients positive for Pg DNA in the sub-gingival plaque had a lower disease duration and a higher peripheral blood leucocyte and neutrophil count. The presence of Pg DNA did not influence disease activity, disease disability or positivity for autoantibodies.ConclusionsThe presence of Pg DNA in the synovial tissue of RA patients suggests a pathogenic role of the bacterium. The higher positivity of Pg DNA in full peripheral blood and synovial tissue samples compared to plasma and synovial fluid suggests a possible intracellular localization of Pg, in particular in patients positive for HLA-DR4.

Virological suppression reduces clinical progression in patients with multiclass-resistant HIV type 1

The virological and immunological outcomes in patients carrying multiclass-resistant HIV-1, their predictors, and their impact on disease progression were investigated. Antiretroviral-experienced patients carrying at least one primary resistance mutation (IAS-USA 2006) to two to three classes of antiretroviral drugs were analyzed for achieving an HIV-1 RNA <50 copies/ml, a CD4 count increase of >200 cells/microl from baseline, and progression to an AIDS-defining event or death. Survival analysis was performed using the Kaplan-Meier estimates and predictors of different outcomes were analyzed using Coxs regression models. A total of 236 patients were identified. Of these 73% reached HIV-1 RNA <50 copies/ml. Higher genotypic sensitivity score of the salvage regimen, lower viral load, and more recent calendar year at genotyping were independently associated with virological response. Immunological response (58%) was predicted by a more recent calendar year, the achievement of an undetectable viral load, and higher CD4 counts at genotyping. Thirty-three patients showed clinical progression: achieving HIV-1 RNA <50 copies/ml predicted AIDS-free survival, independently from other significant cofactors. In individuals with multiclass-resistant HIV-1, virological suppression and immunological recovery are becoming more easily accessible with more recent therapies. The achievement of virological suppression is a strong predictor of reduced clinical progression.

Bilirubin: An Endogenous Molecule with Antiviral Activity in vitro.

Bilirubin-IX-alpha (BR) is the final product of heme metabolism through the heme oxygenase/biliverdin reductase (HO/BVR) system. Previous papers reported on the microbicidal effects of the HO by-products biliverdin-IX-alpha, carbon monoxide and iron, through either direct or indirect mechanisms. In this paper the evidence of a virucidal effect of BR against human herpes simplex virus type 1 (HSV-1) and the enterovirus EV71 was provided. Bilirubin-IX-alpha, at concentrations 1–10 μM, close to those found in blood and tissues, significantly reduced HSV-1 and EV71 replication in Hep-2 and Vero cell lines, respectively. Bilirubin-IX-alpha inhibited viral infection of Hep-2 and Vero cells when given 2 h before, concomitantly and 2 h after viral infection. Furthermore, BR retained its antiviral activity even complexed with a saturating concentration of human serum-albumin. Moreover, 10 μM BR increased the formation of nitric oxide and the phosphorylation of c-Jun N-terminal kinase in Vero and Hep-2 cell lines, respectively, thus implying a role of these two pathways in the mechanism of antiviral activity of the bile pigment. In conclusion, these results support the antiviral effect of BR against HSV-1 and enterovirus in vitro, and put the basis for further basic and clinical studies to understand the real role of BR as an endogenous antiviral molecule.

Prevalence, incidence and predictors of anal high-risk HPV infections and cytological abnormalities in HIV-infected individuals

OBJECTIVES Our purpose was to investigate prevalence, incidence and risk factors of anal high risk-HPV infections and cytological abnormalities in HIV-positive individuals. METHODS A cohort of consecutively enrolled HIV-positive patients underwent, at baseline visit, a sexual behaviors questionnaire, anoscopy, HPV testing and cytological examination. Hybridization and multiplex-PCR were used for DNA detection and typing; HPV E6-E7 mRNA expression was analyzed in HR-HPV+ patients. Logistic regression was used to assess predictors of HR-HPV infection and anal dysplasia. RESULTS 233 HIV-infected patients were enrolled (81% males, median age 44 years). HR-HPV was detected in 144 anal swabs and showed a positive association with CDC stage C and a negative association with a higher CD4 count and the use of a NNRTI-based antiretroviral regimen. HR-HPV DNA detection and anal warts at baseline were associated to cytological abnormalities; a detectable HIV-RNA independently predicted new onset anal dysplasia at follow-up (incidence 15.4 per 100 patients-year). Incidence of new HR-HPV infection was 44.2 per 100 patients-year. CONCLUSIONS The relevance of screening for anal dysplasia in HIV+ patients is emphasized, especially in those with detectable plasma HIV-RNA, anal HR-HPV infection or compromised immunological status.