The E6 gene polymorphism of Human papillomavirus 16 in relation to the risk of cervical cancer in Tunisian women.

Human papillomavirus type 16 (HPV-16) is the most prevalent HPV type worldwide and in Tunisia and the major carcinogenic HPV type found in cervical precancers and cancers. Previous studies have reported that genetic diversity of HPV16-E6 oncoprotein might be associated with cervical intraepithelial neoplasia progression. In this study we aimed to investigate the prevalence of HPV-16 E6 variants in precancerous lesions in Tunisian population to assess potential correlation with disease severity. Positive HPV cervical samples were obtained from the Laboratory of Anatomy Pathology of Pasteur Institute of Tunis. Cytological study was performed to identify cervical precancerous lesions. HPVs were typed using Reverse Line Hybridization. Only samples with HPV-16 single infection were selected for HP16-E6 genetic diversity investigation. HPV-16 E6 gene amplification was performed by PCR using specific primers and sequenced by Sanger Sequencing. The multiple alignment of generated sequences was performed using MEGAX software. Phylogenetic tree was constructed using Maximum Likehood method. The ternary complex of E6, E6AP and p53 core domain was used to perform in silico point mutations and thermodynamic calculations to assess stability and binding affinity. Genetic analysis of Tunisian E6-HPV16 sequences showed the presence of three lineages: European (A), African (C) and Asian American (D). Interestingly, the EUR variants were identified as the dominant lineage of HPV-16 and HPV-16 E6 350 G (L83V) was the most detected mutation in precancerous lesions. Modelling data showed that African variants induced the largest destabilizing effect on E6 structure and decreasing thereby in the affinity toward E6AP. Therefore, women infected with European variants are associated with low and high intraepithelial lesions. The findings give useful information for personalized decision algorithms of intra-epithelial cervical neoplasia in Tunisian women.

The Prevalence, Genotype Distribution and Risk Factors of Human Papillomavirus in Tunisia: A National-Based Study.

There are limited national population-based studies on HPV genotypes distribution in Tunisia, thus making difficult an assessment of the burden of vaccine-preventable cervical cancer. In this context, we conducted a national survey to determine the HPV prevalence and genotypes distribution and the risk factors for HPV infections in Tunisian women. This is a cross-sectional study performed between December 2012 and December 2014. A liquid-based Pap smear sample was obtained from all women and samples' DNAs were extracted. Only women with betaglobin-positive PCR were further analysed for HPV detection and typing by a nested-PCR of the L1 region followed by next-generation sequencing. A multiple logistic regression model was used for the analysis of associations between the variables. A total of 1517 women were enrolled in this study, and 1229 out of the 1517 cervical samples were positive for the betaglobin control PCR and tested for HPV. Overall HPV infection prevalence was measured to be 7.8% (96/1229), with significant differences between the grand regions, ranging from 2% in the North to 13.1% in Grand Tunis. High-risk HPV genotypes accounted for 5% of the infections. The most prevalent genotypes were HPV 31 (1%), 16 (0.9%), 59 (0.7%). HPV18 was detected only in four cases of the study population. Potential risk factors were living in Grand Tunis region (OR: 7.94 [2.74-22.99]), married status (OR: 2.74 [1.23-6.13]), smoking habit (OR: 2.73 [1.35-5.51]), occupation (OR: 1.81 [1.09-3.01]) and women with multiple sexual partners (OR: 1.91 [1.07-3.39]). These findings underscore the need to evaluate the cost effectiveness of HPV vaccine implementation, contribute to the evidence on the burden of HPV infections, the critical role of sexual behaviour and socioeconomic status, and call for increased support to the preventive program of cervical cancer in Tunisia.

TLR9 and Glioma: Friends or Foes?

Toll-like receptor 9 (TLR9) is an intracellular innate immunity receptor that plays a vital role in chronic inflammation and in recognizing pathogenic and self-DNA in immune complexes. This activation of intracellular signaling leads to the transcription of either immune-related or malignancy genes through specific transcription factors. Thus, it has been hypothesized that TLR9 may cause glioma. This article reviews the roles of TLR9 in the pathogenesis of glioma and its related signaling molecules in either defending or promoting glioma. TLR9 mediates the invasion-induced hypoxia of brain cancer cells by the activation of matrix metalloproteinases (2, 9, and 13) in brain tissues. In contrast, the combination of the TLR9 agonist CpG ODN to radiotherapy boosts the role of T cells in antitumor effects. The TLR9 agonist CpG ODN 107 also enhances the radiosensitivity of human glioma U87 cells by blocking tumor angiogenesis. CpG enhances apoptosis in vitro and in vivo. Furthermore, it can enhance the antigen-presenting capacity of microglia, switch immune response toward CD8 T cells, and reduce the number of CD4CD25 Treg cells. CpG ODN shows promise as a potent immunotherapeutic drug against cancer, but specific cautions should be taken when activating TLR9, especially in the case of glioblastoma.

Distribution of human papillomavirus in precancerous and cancerous cervical neoplasia in Tunisian women.

BACKGROUND: High-risk human papillomavirus (HR-HPV) are responsible for cervical cancer (CC) which represents the second most prevalent gynecological cancer among Tunisian women. Preventive strategies against CC are based on prophylactic vaccines that have not yet been implemented into the national vaccination program of Tunisia. Therefore, the present study aimed to investigate the HPV genotypes distribution in cervical neoplasia in Tunisian women in order to predict the impact of using current HPV vaccines on cancer prevention in Tunisia. METHODS: A total of 200 formalin-fixed paraffin embedded biopsies were collected in our study. DNA was extracted using Qiagen Mini prep kit. DNA quality was controlled by Beta Globin PCR. Only positive samples for Beta Globin test were used. HPV detection was performed by a nested PCR using PYGMY and GP5+/6+ primers. Genotyping was performed by Reverse Line hybridization using 31 probes. RESULTS: The mean age of participants was 38.97 years and 75% were over 30 years. Cervical neoplasia distribution according to age showed that CINII/CINIII was observed among women over 30 years old. All samples were positive for Beta Globin PCR. Overall HPV prevalence in cervical lesions was 83% (166/200). HPV was present in 65% of CINI, 82% of CINII/CINIII and 85% of CC. HR-HPV was statistically significantly associated with cervical intraepithelial neoplasia (p < 10-3). HR-HPV distribution according to lesion grade and cervical cancer showed that HPV16 and HPV18 were present in all lesions. For CINII/CINIII, HPV 35 (37.5%) was the most detected type, followed by HPV18 (33.3%) HPV 45 (28.5%) and HPV 16 (18.9%). HPV 45(57.5%), HPV 18 (53.3%) were the most detected in CC. HPV58, 59, 68 were only detected in CC and associated with HPV45, 18 and HPV16. HPV39, 31, 33, 52, 56 and HPV70 was associated only with CINI. CONCLUSIONS: Our findings can give useful information for vaccine implementation by helping the health policymakers to choose the most appropriate vaccine type in Tunisia.

A naturally occurring variant of HPV-16 E7 exerts increased transforming activity through acquisition of an additional phospho-acceptor site.

Human Papillomavirus E6 and E7 play critical roles in cancer development, although not all isolates of the viral oncoproteins are identical. A common E7 variant encodes an amino acid change at N29S. We show that this change increases the levels of phosphorylation by CKII by creating an additional phospho-acceptor site at S29. This confers increased phospho-dependent interaction with a number of cellular targets, including TATA Box Binding Protein (TBP) and pRb. A further consequence is an increased ability to target pRb and p130 for degradation. Biologically, these biochemical differences are reflected in an increased ability of the N29S variant to transform primary rodent cells. This is the first study to demonstrate an important biochemical change in E7 function caused by a naturally occurring variation, and we suggest that the N29S variant merits further assessment to determine whether it has an increased association with the development of HPV-associated malignancies.

Expression of Toll-like receptor 9 increases with progression of cervical neoplasia in Tunisian women--a comparative analysis of condyloma, cervical intraepithelial neoplasia and invasive carcinoma.

Toll-like receptors (TLRs) are expressed in immune and tumor cells and recognize pathogen-associated molecular patterns. Cervical cancer (CC) is directly linked to a persistent infection with high risk human papillomaviruses (HR-HPVs) and could be associated with alteration of TLRs expression. TLR9 plays a key role in the recognition of DNA viruses and better understanding of this signaling pathway in CC could lead to the development of novel immunotherapeutic approaches. The present study was undertaken to determine the level of TLR9 expression in cervical neoplasias from Tunisian women with 53 formalin-fixed and paraffin-embedded specimens, including 22 samples of invasive cervical carcinoma (ICC), 18 of cervical intraepithelial neoplasia (CIN), 7 of condyloma and 6 normal cervical tissues as control cases. Quantification of TLR9 expression was based on scoring four degrees of extent and intensity of immunostaining in squamous epithelial cells. TLR9 expression gradually increased from CIN1 (80% weak intensity) to CIN2 (83.3% moderate), CIN3 (57.1% strong) and ICC (100% very strong). It was absent in normal cervical tissue and weak in 71.4% of condyloma. The mean scores of TLR9 expression were compared using the Kruskall-Wallis test and there was a statistical significance between normal tissue and condyloma as well as between condyloma, CINs and ICC. These results suggest that TLR9 may play a role in progression of cervical neoplasia in Tunisian patients and could represent a useful biomarker for malignant transformation of cervical squamous cells.

Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells.

BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.