RNA-Seq Analysis of Differentiated Keratinocytes Reveals a Massive Response to Late Events during Human Papillomavirus 16 Infection, Including Loss of Epithelial Barrier Function.

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalized keratinocytes (NIKS) and NIKS stably transfected with HPV16 episomal genomes (NIKS16) were compared using next-generation sequencing (RNA-Seq). HPV16 infection altered expression of 2,862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNA-Seq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log2 = 1.8, 3.5-fold change), 670 genes were downregulated and 296 genes were upregulated. HPV downregulated many genes involved in epithelial barrier function, which involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant CCL20, and proinflammatory cytokines interleukin 1α (IL-1α) and IL-1ß. However, the type I interferon regulator IRF1, kappa interferon (IFN-κ), and viral restriction factors (IFIT1, -2, -3, and -5, OASL, CD74, and RTP4) were upregulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions, and cell adhesion. Quantitative PCR (qRT-PCR) and Western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.IMPORTANCE HPV genome amplification and capsid formation take place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNA-Seq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3,000 genes were differentially expressed in keratinocytes due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into the virus-host interaction that is crucial for the production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle.

[POLYHEXAMETHYLENE BIGUANID HYDROCHLORIDE (BIGUANELLE) THERAPY OF BACTERIAL VAGINOSIS].

UNLABELLED: The aim of our study was to determine the efficacy and tolerability of local therapy with polyhexamethylene biguanid hydrochloride (BIGUANELLE) in women with anaerobic vaginal infection. MATERIAL AND METHODS: We include in our study 23 women (18-50) with established by AMSEL criteria bacterial vaginosis. In all of the women at the beginning and at the end of the survey was performed gynecological examination and microbiological research (AMSEL). The therapeutic scheme at all women is with a single vaginal application of gynecological solution BIGUANELLE. Effectiveness of the treatment was evaluated according to clinical complaints and microbiological research (Amsel criteria: Ph 4.5 >; KOH (+); "clue cells"; specific vaginal fluorine). Tolerability of patients to treatment was assessed by questionnaire. RESULTS: Clinical complaints of patients after the therapy decreased as follows: vaginal fluorine with 73.9%; odor--75%; pruritus--50%; discomfort--90%. Microbiological research and their evaluation by AMSEL, showed the therapeutic efficacy of the therapy in 16 (69.6%) of all (n-23) patients. At 7 (30.4%) women, the treatment remained without effect. At questionnaire answers, 73.9% patients were satisfied with the application of BIGUANELLE, 95.6% of them have implemented it easily, 95.6% of women believe that BIGUANELLE is more convenient to use in comparison with similar products which have a daily application, and none of the patients (100%) have any complaints in applying this gynecological solution. CONCLUSIONS: BIGUANELLE showed good clinical efficacy in the treatment of bacterial vaginosis. It is easily applied and well tolerated by the patients.

Can oral anaerobic bacteria cause adverse pregnancy outcomes?

AIM: Maternal periodontal infection has been recognized as a risk factor for premature and low birthweight infants. It is suspected that pathogens causing periodontal disease may translocate to the amniotic cavity and so contribute to triggering an adverse pregnancy outcome. The aim of this study was to evaluate whether the presence of specific periodontal pathogens may influence the incidence of preterm labor and premature birth. MATERIAL AND METHODS: This study was designed as a hospital-based case-control study. A total of 70 pregnant women, aged 18-40 with single live pregnancy were recruited from the Departement of Gynecolgy and Obstetrics at a General hospital in Sibenik, Croatia, between March 2013 to March 2014. The case group: 30 pregnant women who were hospitalised with signs of premature labor. CONTROL GROUP: 40 patients with normal pregnancy post-delivery up to 48 hrs, who had given birth at term, and the baby had a weight of more than 2500 gr. These women had undergone microbiological examination at the time of recruitment, microbial samples, paper point subgingival swabs were obtained in both groups and processed by anaerobic culturing. Standard procedures were used for culture and identification of bacteria. Information was collected on demographics, health behaviors, and obstetric and systemic diseases that may have influence the premature delivery. RESULTS: The levels of periodontal pathogens tended to be higher in the premature (case group) labor compared to the term deliveries (control group). Levels of Porphyromonas gingivalis, Fuscobacterium nucleatum, Actinomyces actinomycetecomitans were statistically significantly higher in premature births as compared to term deliveries, adjusting for baseline levels. The joint effects of red and orange microbial clusters were significantly higher in the premature group compared to the term group. CONCLUSIONS: The study shows a significant association betwen periodontal anaerobic infection and adverse pregnancy outcome. High levels of periodontal pathogens during pregnancy are associated with an increased risk for preterm delivery. Further studies elucidating the role of the microbial load and maternal immune response as related to pregnancy outcome seem merited.

Combination of two anti-stress procedures: our original 4-corner deltoid-like vaginal suspension and suburethral duplication sec. Lazarevski in patients with genital prolapse.

AIM: The effectiveness of the combination of two anti-stress procedures: 1) our original 4-corner deltoid-like vaginal suspension; and 2) suburethral duplication sec. Lazarevski are analysed. STUDY DESIGN: 144 women, undergoing vaginal hysterectomy, combined with our original 4-corner deltoid-like vaginal suspension and suburethral duplication sec. Lazarevski were divided into: SUIGP group (n = 48) with stress urinary incontinence (SUI) and genital prolapse (GP); and GP group (n = 96) with GP. All operations were performed by the first author of this study. RESULTS: At the last follow-up (mean 18.3 months for GP, and 18.7 months for SUIGP), point C was significantly higher in both groups and total vaginal length (tvl) longer in GP vs. preoperative positions (p < 0.05; p < 0.05; p < 0.05 respectively). There were no postoperative significant differences between groups. Regarding the severity of prolapse: stage I for anterior segment and stage 0 for most severe segment predominated in SUIGP (p < 0.05; p < 0.05 respectively). The recurrent prolapse was: 1) in anterior segment: stage I 6.25% in GP and 18.7% in SUIGP; stage II 6.25% in GP and 2.1% in SUIGP; 2) in posterior segment: stage I 18.7% in GP and 18.7% in SUIGP. There was no recurrent prolapse of the apical segment, and only one patient with stage III anterior segment. The recurrent SUI was 8.3% in SUIGP, and de novo SUI 1% in GP. CONCLUSION: This proposed combination seems to be safe and effective (Tab. 8, Fig. 2, Ref. 24). Full Text (Free, PDF) www.bmj.sk.