Cross-sectional associations between cutaneous viral infections and regulatory T lymphocytes in circulation.

BACKGROUND: Cutaneous viral infections and immune suppression are risk factors for some forms of nonmelanoma skin cancer; however, their interrelationship is poorly understood. OBJECTIVES: To examine cross-sectional associations between cutaneous viral infections and circulating forkhead-box P3 (FOXP3)-expressing T-regulatory (Treg) cells, suppressive cells that dampen effective antitumour immunity. MATERIALS AND METHODS: Blood, eyebrow hair (EBH) and skin swab (SSW) samples were collected from 352 patients 60 years and older undergoing skin screening, without prevalent skin cancer, while participating in an ongoing prospective cohort study of cutaneous viral infections and skin cancer. DNA corresponding to 98 cutaneous human papillomavirus (HPV) types and five human polyomaviruses (HPyV) was assessed in EBH and SSW. Distinct classes of circulating Treg-cell subpopulations were defined by flow cytometry including cutaneous lymphocyte antigen (CLA) and CCR4high Treg cells, both previously associated with cutaneous diseases. Age- and sex-adjusted associations between circulating T-cell populations and infection were estimated using logistic regression. RESULTS: Total Treg-cell proportion in peripheral blood was not associated with ß HPV or HPyV infection. However, the proportion of circulating CLA+ Treg cells was inversely associated with γ HPV EBH infection [odds ratio (OR) 0·54, 95% confidence interval (CI) 0·35-0·84]. Interestingly, circulating Treg cells expressing markers indicative of antigen activation (CD27- CD45RA- FOXP3+ CD4+ ) were also inversely associated with γ HPV infection in SSW (OR 0·55, 95% CI 0·30-0·99) and EBH (OR 0·56, 95% CI 0·36-0·86). CONCLUSIONS: Inverse associations between circulating Treg cells and γ HPV infection suggest that localized viral infection may promote immunosuppressive cell migration into skin.

Predictors of oropharyngeal cancer survival in Europe.

OBJECTIVES: HPV16-positive oropharyngeal cancer (OPC) patients experience better outcomes compared to HPV16-negative patients. Currently, strategies for treatment de-escalation are based on HPV status, smoking history and disease stage. However, the appropriate cut-point for smoking and the role of other non-clinical factors in OPC survival remains uncertain. MATERIALS AND METHODS: We examined factors associated with OPC outcome in 321 patients recruited in a large European multi-center study. Seropositivity for HPV16 E6 was used as a marker of HPV16 positive cancer. Hazard ratios (HR) and confidence intervals (CI) were estimated using Cox proportional models adjusted for potential confounders. RESULTS: Overall 5-year survival following OPC diagnosis was 50%. HPV16-positive OPC cases were at significantly lower risk of death (aHR = 0.51, 95% CI: 0.32-0.80). A significant effect on OPC survival was apparent for female sex (aHR 0.50: 95% CI: 0.29-0.85) and being underweight at diagnosis (aHR: 2.41, 95% CI: 1.38-4.21). A 10 pack year smoking history was not associated with overall survival. Higher stage at diagnosis appeared as the only factor significantly associated with OPC recurrence (aHR: 4.88, 95% CI: 2.12-11.21). CONCLUSION: This study confirms that HPV16 status is an independent prognostic factor for OPC survival while female sex lowers risk of death and being underweight at diagnosis increases the risk of death. Smoking was not an independent predictor of OPC survival.

Mechanism of translation control of the alternative Drosophila melanogaster Voltage Dependent Anion-selective Channel 1 mRNAs.

The eukaryotic porin, also called the Voltage Dependent Anion-selective Channel (VDAC), is the main pore-forming protein of the outer mitochondrial membrane. In Drosophila melanogaster, a cluster of genes evolutionarily linked to VDAC is present on chromosome 2L. The main VDAC isoform, called VDAC1 (Porin1), is expressed from the first gene of the cluster. The porin1 gene produces two splice variants, 1A-VDAC and 1B-VDAC, with the same coding sequence but different 5' untranslated regions (UTRs). Here, we studied the influence of the two 5' UTRs, 1A-5' UTR and 1B-5' UTR, on transcription and translation of VDAC1 mRNAs. In porin-less yeast cells, transformation with a construct carrying 1A-VDAC results in the expression of the corresponding protein and in complementation of a defective cell phenotype, whereas the 1B-VDAC sequence actively represses VDAC expression. Identical results were obtained using constructs containing the two 5' UTRs upstream of the GFP reporter. A short region of 15 nucleotides in the 1B-5' UTR should be able to pair with an exposed helix of 18S ribosomal RNA (rRNA), and this interaction could be involved in the translational repression. Our data suggest that contacts between the 5' UTR and 18S rRNA sequences could modulate the translation of Drosophila 1B-VDAC mRNA. The evolutionary significance of this finding is discussed.

The pathogenesis of cutaneous squamous cell carcinoma in organ transplant recipients.

The pathogenesis of keratinocyte carcinoma following organ transplantation is multifactorial, and recent evidence suggests a complex and often synergistic interplay between the carcinogenic effects of ultraviolet radiation, compromised immune surveillance, direct pro- and anticarcinogenic effects of drugs, oncogenic viruses (in particular, beta-genus human papillomaviruses) and host genetic susceptibility factors. We present an overview of those factors for which there is currently the most convincing evidence and highlight important gaps in our knowledge. In particular, a clear understanding of the interdependence and relative contributions of these co-factors is currently lacking, yet has important implications for rational development of clinically relevant biomarkers and targeted strategies for treatment and prevention of post-transplant keratinocyte cancers.

Detection of oncogenic viruses in water environments by a Luminex-based multiplex platform for high throughput screening of infectious agents.

Recent studies documented the detection of viruses strongly associated with human cancer in urban sewages and other water environments worldwide. The aim of this study was to estimate the occurrence of human oncogenic viruses in environmental samples (sewage, river, marine, and pool/spa water) using highly sensitive and specific multiplex bead-based assays (Luminex technology). A total of 33 samples were analysed for 140 oncogenic viral agents, including mucosal and cutaneous human papillomaviruses (HPVs), human polyomaviruses (HPyV), human herpesviruses (HHV) and mouse mammary tumour virus (MMTV). Eighty-eight percent of the samples tested positive for at least one viral pathogen and the simultaneous presence of more than one virus was frequent (mean number of positivities/sample = 3.03). A total of 30 different Alpha, Beta and Gamma HPVs were detected, including mucosal and cutaneous types. The high-risk type HPV16 was the most frequently detected virus, identified in 73% of the samples. Of the 12 HPyVs tested, only two (BKPyV and MCPyV) were detected. At least one of these two was present in 48% of the samples. MMTV was detected in 21% of the samples, while herpesviruses - HHV-6 and HHV-1 - were detected in two samples (6%). The present study is the first to provide a comprehensive picture of the occurrence of oncogenic viruses belonging to different families and species in diverse water environments, and the first to successfully use, in environmental samples, a Luminex-based multiplex platform for high throughput screening of infectious agents. Our findings, showing that oncogenic viruses are ubiquitous in water environments, pave the way for future studies on the fate of these pathogens in water environments as well as on their potential for transmission via the waterborne route.

TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16(INK4a) protein expression.

Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16(INK4a) was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16(INK4a) post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis.

Fusobacterium nucleatum associates with stages of colorectal neoplasia development, colorectal cancer and disease outcome.

Commensal bacteria in the colon may play a role in colorectal cancer (CRC) development. Recent studies from North America showed that Fusobacterium nucleatum (Fn) infection is over-represented in disease tissue versus matched normal tissue in CRC patients. Using quantitative real-time polymerase chain reaction (qPCR) of DNA extracted from colorectal tissue biopsies and surgical resections of three European cohorts totalling 122 CRC patients, we found an over-abundance of Fn in cancerous compared to matched normal tissue (p < 0.0001). To determine whether Fn infection is an early event in CRC development, we assayed Fn in colorectal adenoma (CRA) tissue from 52 Irish patients. While for all CRAs the Fn level was not statistically significantly higher in disease versus normal tissue (p = 0.06), it was significantly higher for high-grade dysplasia (p = 0.015). As a secondary objective, we determined that CRC patients with low Fn levels had a significantly longer overall survival time than patients with moderate and high levels of the bacterium (p = 0.008). The investigation of Fn as a potential non-invasive biomarker for CRC screening showed that, while Fn was more abundant in stool samples from CRC patients compared to adenomas or controls, the levels in stool did not correlate with cancer or adenoma tissue levels from the same individuals. This is the first study examining Fn in the colonic tissue and stool of European CRC and CRA patients, and suggests Fn as a novel risk factor for disease progression from adenoma to cancer, possibly affecting patient survival outcomes. Our results highlight the potential of Fn detection as a diagnostic and prognostic determinant in CRC patients.

Human papillomavirus infection among human immunodeficiency virus-infected women in Maharashtra, India.

INTRODUCTION: Frequency and distribution of HPV types in HIV-infected women with and without cervical neoplasia and their determinants have not been widely studied in India. We report and discuss HPV prevalence and type distribution in HIV-infected women. METHODS: HPV genotyping was done using cervical samples from 1109 HIV-infected women in a cross-sectional study. RESULTS: Any HPV was detected in 44.8% and high-risk ones in 41.0% women. Frequency of single and multiple high-risk infections were 26.7% and 14.3%, respectively. Frequencies of high-risk HPV infections in women with and without cervical neoplasia were 73.5% and 37.6%, respectively. HPV16 was the most common genotype, present in 11.5%, and 58.5% of women with cervical intraepithelial neoplasia (CIN) 2 and 3. Other most common high-risk HPV types in CIN 2-3 lesions were HPV 31 (22.6%); 56 (13.2%); 18 and 68a (11.3%) and 33, 35 and 51 (9.4%); and 70 (7.5%). Women under 30 or over 44 years, no abortions, and women with diagnosis of HIV infection within the last 5 years were at high risk of multiple oncogenic HPV infection. CONCLUSION: We observed a very high frequency of high-risk HPV and multiple infections in HIV-infected women.

Prevalence of human Papillomavirus DNA in eyebrow hairs plucked from patients with psoriasis treated with TNF inhibitors.

BACKGROUND: Tumour necrosis factor alpha (TNF-α) inhibitors are associated with an increased risk of infections and with a still debatable risk of skin cancer. Furthermore, cutaneous human papillomavirus (HPV) infection may be involved in skin cancer. OBJECTIVES: Our primary objective was to assess the HPV DNA prevalence in psoriasis patients treated with TNF inhibitors and the secondary objective was to assess the same parameter before and during treatment. METHODS: Plucked eyebrow hairs were collected from 151 consecutive patients with moderate to severe chronic plaque psoriasis, including 48 patients treated with anti-TNF-α agents, 21 patients treated with methotrexate (MTX) and 82 patients with no previous systemic treatment. Among them, 38 patients were subsequently treated with either MTX or anti-TNF-α agents. HPV genotyping was performed using the HPV type-specific E7 PCR bead-based multiplex allowing the detection of 27 genus-α types, 25 genus-ß types, 16 genus-γ types and one single genus-µ type. Follow-up provided a total of 972.7 person-months of overall exposure for patients treated with TNF inhibitors and 326.9 person-months for patients treated with MTX. RESULTS: Our data confirm the high prevalence of ß-HPV infection in healthy skin of psoriasis patients (68.9%), with no significant difference between untreated patients (64.6%), patients treated with MTX (76.2%) and patients treated with anti-TNF-α agents (72.9%). The mean number of different HPV types and the distribution of HPV types were similar in different groups of patients. Moreover, in prospectively treated patients, we did not observe any change in the HPV DNA prevalence in the distribution of HPV types and the number of HPV types after a mean duration of treatment of 332 ± 39.8 days. CONCLUSION: Despite the small number of patients in our cohort, our results are quite encouraging in view of the increased use of anti-TNF-α agents in different auto inflammatory immune diseases.

Human beta papillomavirus DNA study in primary cutaneous squamous cell carcinomas and their corresponding metastases.

The association between beta human papillomavirus (HPV) types and cutaneous squamous cell carcinomas (cSCCs) is controversial. Several studies have found such an association, especially at early stages of carcinogenesis, but the presence of beta HPV types in aggressive cSCCs has only been reported in three patients previously. We aimed to search for beta HPV DNA in primary cSCCs and their corresponding lymph node metastases in a series of patients. The presence of DNA from 25 beta HPV types was determined using a multiplex PCR protocol in 35 primary cSCCs from 35 patients and their corresponding lymph node metastases. DNA from beta HPV types was detected in 9 % of primary cSCCs and in 13 % of metastases. No primary cutaneous SCC or lymphatic metastases were found to share the same HPV DNA. These data suggest that beta HPV types do not play an etiopathogenic role in advanced stages of squamous cell carcinogenesis.

HPV16 genetic variation and the development of cervical cancer worldwide.

BACKGROUND: Factors that favour a small proportion of HPV16 infections to progress to cancer are still poorly understood, but several studies have implicated a role of HPV16 genetic variation. METHODS: To evaluate the association between HPV16 genetic variants and cervical cancer risk, we designed a multicentre case-control study based on HPV16-positive cervical samples (1121 cervical cancer cases and 400 controls) from the International Agency for Research on Cancer biobank. By sequencing the E6 gene, HPV16 isolates were classified into variant lineages and the European (EUR)-lineage isolates were subclassified by the common polymorphism T350G. RESULTS: Incidence of variant lineages differed between cases and controls in Europe/Central Asia (P=0.006, driven by an underrepresentation of African lineages in cases), and South/Central America (P=0.056, driven by an overrepresentation of Asian American/North American lineages in cases). EUR-350G isolates were significantly underrepresented in cervical cancer in East Asia (odds ratio (OR)=0.02 vs EUR-350T; 95% confidence interval (CI)=0.00-0.37) and Europe/Central Asia (OR=0.42; 95% CI=0.27-0.64), whereas the opposite was true in South/Central America (OR=4.69; 95% CI=2.07-10.66). CONCLUSION: We observed that the distribution of HPV16 variants worldwide, and their relative risks for cervical cancer appear to be population-dependent.

Assessment of the efficacy of a new formulation for plantar wart mummification: new experimental design and human papillomavirus identification.

Cutaneous warts are caused by infection of the epidermis with human papillomavirus (HPV). Cryotherapy using liquid nitrogen is one of the most common local treatments. In this study, we used a novel ex vivo approach to compare the efficacy of a new product with conventional liquid-nitrogen cryotherapy by studying epidermal histology and assessing the presence of HPV types 1 and 2 DNA in plantar warts. The studied formulation, which acts by tissues mummification, is a combination of nitric acid, organic acids and metallic salts. We found that, similar to liquid nitrogen, the studied product induced alterations in the wart structure. In addition, unlike liquid nitrogen, this product also reduced the amount of HPV DNA. The results suggest that there is a poor correlation between the histological response and the antiviral efficacy of standard wart treatment.

Differential presence of Papillomavirus variants in cervical cancer: an analysis for HPV33, HPV45 and HPV58.

BACKGROUND: Certain human papillomaviruses (HPVs) are the causative agents of cervical carcinomas in humans. The identification of the link between infection and cancer has resulted in the successful establishment of clinical strategies such as screening or vaccination programs, aiming to prevent this pathology. More than 150 different HPVs have been described and classified and the large majority of them are not related to cancer. The genus Alphapapillomavirus encompasses many PVs, some of which are identified in humans as oncogenic, according to the epidemiological connection between infection and cervical cancer. Variants of some of these "high-risk" HPVs may have an increased involvement in cervical cancer, although definitive data are still wanting. The aim of the present work was to analyze the presence of HPV33, HPV45 and HPV58 variants in cases of cervical cancer. METHODS: Samples from cervical lesions in the context of different cervical cancer surveys were analyzed for presence of HPV DNA. Samples positive for HPV33, HPV45 or HPV58 DNA were selected and the E6/E7 genes were amplified and sequenced. The phylogenetic relationships of these sequences were inferred using an evolutionary placement algorithm and accordingly classified at the variant level. RESULTS: All viral E6/E7 sequences were successfully placed in the classification schemes of the corresponding viruses. For HPV33 (n=23), 45 (n=61) or 58 (n=29), the distribution of variants found in cases of cervical cancer is not a random sample of the corresponding diversity. In all three HPVs, the respective A variants were more prevalent in the viral DNA-positive cases of cervical cancer analyzed. This is the first study trying to discern the phylogenetic connection between variants of the oncogenic HPV33, 45 and 58, and squamous cell carcinoma of the cervix.

Urinary human polyomavirus and papillomavirus infection and bladder cancer risk.

BACKGROUND: The association of transitional cell carcinomas of the bladder (TCB) with Schistosoma haematobium suggested a possible role of infections in the aetiology of TCB. METHODS: In all, 114 TCB cases and 140 hospital controls from Pordenone Province were enrolled within an Italian multi-centric case-control study. Urine samples were screened for DNA from five human polyomaviruses (HPyV) (JCV, BKV, MCV, WUV, and KIV); SV40; and 22 mucosal human papillomaviruses (HPV) using highly sensitive PCR assays. Odds ratios (ORs) and corresponding confidence intervals (CIs) were computed for risk of TCB by HPyV- or HPV-positivity using unconditional logistic regression. RESULTS: Human polyomavirus prevalence was similar in TCB cases (71.7%) and controls (77.7%) (OR for TCB=0.85; 95% CI: 0.45-1.61). JCV was the most frequently detected HPyV type. No individual HPyV showed a significant association. Among cases, HPyV-positivity was not associated with tumour characteristics, but it was significantly lower in women than men and among current and former smokers than never smokers. Human papillomavirus was detected in seven cases and five controls (OR=1.52; 95% CI: 0.42-5.45). CONCLUSION: The present small study does not support an involvement of HPyV or HPV infection in TCB aetiology in immunocompetent individuals. Differences in HPyV-positivity by sex and smoking may derive from differences in either acquisition or persistence of the infection.

Assessment of transformed properties in vitro and of tumorigenicity in vivo in primary keratinocytes cultured for epidermal sheet transplantation.

Epidermal keratinocytes are used as a cell source for autologous and allogenic cell transplant therapy for skin burns. The question addressed here is to determine whether the culture process may induce cellular, molecular, or genetic alterations that might increase the risk of cellular transformation. Keratinocytes from four different human donors were investigated for molecular and cellular parameters indicative of transformation status, including (i) karyotype, (ii) telomere length, (iii) proliferation rate, (iv) epithelial-mesenchymal transition, (v) anchorage-independent growth potential, and (vi) tumorigenicity in nude mice. Results show that, despite increased cell survival in one keratinocyte strain, none of the cultures displayed characteristics of cell transformations, implying that the culture protocol does not generate artefacts leading to the selection of transformed cells. We conclude that the current protocol does not result in an increased risk of tumorigenicity of transplanted cells.