Poly(I:C) Treatment Prevents Skin Tumor Formation in the Preclinical HPV8 Transgenic Mouse Model.

Actinic keratoses and cutaneous squamous cell carcinomas are associated with infections with human papillomavirus of genus beta (betaHPV) in immunosuppressed patients. To date, targeted therapy against betaHPV-associated skin cancer does not exist because of the large number of betaHPV without defined high-risk types. In this study, we hypothesized that the activation of innate antiviral immunity in the skin, asymptomatically infected with betaHPV, induces an antitumor response by in situ autovaccination and prevents the formation of betaHPV-associated skin cancer. To test this, we used the preclinical keratin-14-HPV8 transgenic mouse model, which develops skin tumors after mechanical wounding. Remarkably, treatment with the antiviral immune response activating polyinosinic-polycytidylic acid (poly[I:C]) completely prevented cutaneous tumor growth. The induction of the IFN-induced genes Cxcl10 and Ifit1 by poly(I:C) depended on MDA5 activation. Increased numbers of total and activated CD4 and CD8 T cells were detected in poly(I:C)-treated skin. T cells were found in the skin of poly(I:C)-treated mice but not in the skin tumors of untreated mice. T-cell depletion showed a predominant role of CD4 T cells in poly(I:C)-mediated tumor prevention. Our findings identify the MDA5 ligand poly(I:C) as a promising candidate for in situ autovaccination approaches, which might serve as a treatment strategy against betaHPV-related skin diseases.

Natural History of Cutaneous Human Polyomavirus Infection in Healthy Individuals.

Several human polyomaviruses (HPyVs) were recently discovered. Merkel cell polyomavirus (MCPyV) induces Merkel cell carcinoma. HPyV6, HPyV7, and TSPyV have been associated with rare skin lesions in immunosuppressed patients. HPyV9, HPyV10, and Saint Louis Polyomavirus (STLPyV) have not been convincingly associated with any disease. The aim of this prospective study was to evaluate the cutaneous prevalence, persistence and viral load of HPyVs in healthy individuals. Eight hundred seventy forehead and hand swabs were collected from 109 volunteers 4-6 weeks apart (collection period-1). Fifty-nine participants were available for follow-up a decade later (collection period-2). HPyV-DNA prevalence and viral loads of MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, HPyV10, and STLPyV were determined by virus-specific real-time PCRs. Risk factors for HPyV prevalence, short- and long-term persistence were explored by logistic regression analyses. Baseline prevalence rates were similar for forehead and hand: MCPyV 67.9/67.0%, HPyV6 31.2/25.7%, HPyV7 13.8/11.0%, HPyV10 11.9/15.6%, STLPyV 7.3/8.3%, TSPyV 0.9/0.9%, and HPyV9 0.9/0.9%. Short-term persistence in period-1 was found in 59.6% (MCPyV), 23.9% (HPyV6), 10.1% (HPyV7), 6.4% (HPyV10), 5.5% (STLPyV), and 0% (TSPyV and HPyV9) on the forehead, with similar values for the hand. Long-term persistence for 9-12 years occurred only for MCPyV (forehead/hand 39.0%/44.1% of volunteers), HPyV6 (16.9%/11.9%), and HPyV7 (3.4%/5.1%). Individuals with short-term persistence had significantly higher viral loads at baseline compared to those with transient DNA-positivity (p < 0.001 for MCPyV, HPyV6, HPyV7, and HPyV10, respectively). This was also true for median viral loads in period-1 of MCPyV, HPyV6, and HPyV7 of volunteers with long-term persistence. Multiplicity (two or more different HPyVs) was a risk factor for prevalence and persistence for most HPyVs. Further risk factors were older age for HPyV6 and male sex for MCPyV on the forehead. Smoking was not a risk factor. In contrast to MCPyV, HPyV6, HPyV7, and rarely STLPyV, polyomaviruses TSPyV, HPyV9, and HPyV10 do not seem to be long-term constituents of the human skin virome of healthy individuals. Furthermore, this study showed that higher viral loads are associated with both short- and long-term persistence of HPyVs on the skin. HPyV multiplicity is a risk factor for prevalence, short-term and/or long-term persistence of MCPyV, HPyV6, HPyV7, and HPyV10.

German evidence and consensus-based (S3) guideline: Vaccination recommendations for the prevention of HPV-associated lesions.

Anogenital and oropharyngeal infections with human papilloma viruses (HPV) are common. Clinically manifest disease may significantly impact quality of life; the treatment of HPV-associated lesions is associated with a high rate of recurrence and invasive neoplasms, such as cervical, anal, vulvar, penile, and oropharyngeal cancers, which are characterized by significant morbidity and mortality. Vaccination against HPV is an effective and safe measure for the primary prevention of HPV-associated lesions, but immunization rates are still low in Germany. The present publication is an abridged version of the German evidence and consensus-based guideline "Vaccination recommendations for the prevention of HPV-associated lesions", which is available on the website of the German Association of the Scientific Medical Societies (AWMF). On the basis of a systematic review with meta-analyses, a representative panel developed and agreed upon recommendations for the vaccination of different populations against HPV. In addition, consensus-based recommendations were developed for specific issues relevant to everyday practice. Based on current evidence and a representative expert consensus, these recommendations are intended to provide guidance in a field in which there is often uncertainty and in which both patients and health care providers are sometimes confronted with controversial and emotionally charged points of view.

Human papillomavirus and posttransplantation cutaneous squamous cell carcinoma: A multicenter, prospective cohort study.

Organ transplant recipients (OTRs) have a 100-fold increased risk of cutaneous squamous cell carcinoma (cSCC). We prospectively evaluated the association between ß genus human papillomaviruses (ßPV) and keratinocyte carcinoma in OTRs. Two OTR cohorts without cSCC were assembled: cohort 1 was transplanted in 2003-2006 (n = 274) and cohort 2 was transplanted in 1986-2002 (n = 352). Participants were followed until death or cessation of follow-up in 2016. ßPV infection was assessed in eyebrow hair by using polymerase chain reaction-based methods. ßPV IgG seroresponses were determined with multiplex serology. A competing risk model with delayed entry was used to estimate cumulative incidence of histologically proven cSCC and the effect of ßPV by using a multivariable Cox regression model. Results are reported as adjusted hazard ratios (HRs). OTRs with 5 or more different ßPV types in eyebrow hair had 1.7 times the risk of cSCC vs OTRs with 0 to 4 different types (HR 1.7, 95% confidence interval 1.1-2.6). A similar risk was seen with high ßPV loads (HR 1.8, 95% confidence interval 1.2-2.8). No significant associations were seen between serum antibodies and cSCC or between ßPV and basal cell carcinoma. The diversity and load of ßPV types in eyebrow hair are associated with cSCC risk in OTRs, providing evidence that ßPV is associated with cSCC carcinogenesis and may present a target for future preventive strategies.

Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis.

Epidemiological evidence is accumulating that beta-human papillomaviruses (HPV) synergize with UV-light in the development of precancerous actinic keratosis, and cutaneous squamous cell carcinomas (cSCC), one of the most common cancers in the Caucasian population. We previously demonstrated the tumorigenic activity of beta-HPV type 8 (HPV8) in the skin of transgenic mice and its cooperation with UV-light. Analysis of underlying mechanisms now showed that in keratinocytes expressing the HPV8E6 protein a transient increase of tyrosine phosphorylated epidermal growth factor receptor (EGFR) in response to UV-irradiation occurred, while EGFR tyrosine phosphorylation, i.e., receptor tyrosine kinase (RTK)-activity was hardly affected in empty vector control cells. FACS and immunofluorescences revealed that the EGFR was internalized into early endosomes in response to UV-exposure in both, HPV8E6 positive and in control cells, yet with a higher rate in the presence of HPV8E6. Moreover, only in HPV8E6 expressing keratinocytes the EGFR was further sorted into CD63+ intraluminal vesicles, indicative for trafficking to late endosomes. The latter requires the ubiquitination of the EGFR, and in correlation, we could show that only in HPV8E6 positive keratinocytes the EGFR was ubiquitinated upon UV-exposure. HPV8E6 and tyrosine phosphorylated EGFR directly interacted which was enhanced by UV-irradiation. The treatment of K14-HPV8E6 transgenic mice with Canertinib, an inhibitor of the RTK-activity of the EGFR, suppressed skin papilloma growth in response to UV-irradiation. This confirms the crucial role of the RTK-activity of the EGFR in HPV8E6 and UV-mediated papillomatosis in transgenic mice. Taken together, our results demonstrate that HPV8E6 alters the signaling of the UV-activated EGFR and this is a critical step in papilloma formation in response to UV-light in transgenic mice. Our results provide a molecular basis how a beta-HPV type may support early steps of skin tumor formation in cooperation with UV-light.

HPV8 Field Cancerization in a Transgenic Mouse Model Is due to Lrig1+ Keratinocyte Stem Cell Expansion.

ß-Human papillomaviruses (HPVs) cause near ubiquitous latent skin infection within long-lived hair follicle (HF) keratinocyte stem cells. In patients with epidermodysplasia verruciformis, ß-HPV viral replication is associated with skin keratosis and cutaneous squamous cell carcinoma. To determine the role of HF keratinocyte stem cells in ß-HPV-induced skin carcinogenesis, we utilized a transgenic mouse model in which the keratin 14 promoter drives expression of the entire HPV8 early region (HPV8tg). HPV8tg mice developed thicker skin in comparison with wild-type littermates consistent with a hyperproliferative epidermis. HF keratinocyte proliferation was evident within the Lrig1+ keratinocyte stem cell population (69 vs. 55%, P < 0.01, n = 7), and not in the CD34+, LGR5+, and LGR6+ keratinocyte stem cell populations. This was associated with a 2.8-fold expansion in Lrig1+ keratinocytes and 3.8-fold increased colony-forming efficiency. Consistent with this, we observed nuclear p63 expression throughout this population and the HF infundibulum and adjoining interfollicular epidermis, associated with a switch from p63 transcriptional activation isoforms to ΔNp63 isoforms in HPV8tg skin. Epidermodysplasia verruciformis keratosis and in some cases actinic keratoses demonstrated similar histology associated with ß-HPV reactivation and nuclear p63 expression within the HF infundibulum and perifollicular epidermis. These findings would suggest that ß-HPV field cancerization arises from the HF junctional zone and predispose to squamous cell carcinoma.

Dynamics of BKPyV reactivation and risk of hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation.

OBJECTIVES: Aim of this retrospective study was to analyze the dynamics of BKPyV reactivation in allogeneic hematopoietic stem cell transplant recipients in order to identify patients with higher risk to develop BKPyV-associated hemorrhagic cystitis (BKPyV-associated HC). METHODS: The study included 58 allo-HSCT recipients from the University Hospital of Cologne detected BKPyV positive by real-time PCR between 2009 and 2015. For correlative analysis, the first detected BKPyV-DNA load in urine and in plasma as well as the onset and severity of HC following the first day of conditioning regimen was considered. Phylogenetic analysis of BKPyV isolates was performed. RESULTS: In 25 of 58 patients, BKPyV-DNA was detected in urine only (group U), whereas 33 patients developed additional viremia (group P). A chronologic sequence viruria-viremia-HC was identified. Viral load of >106 copies/mL at first viruria and evidence of viremia after 45 days from the start of conditioning represented risk factors for the onset of HC. Molecular characterization revealed a non-stereotypic distribution of viral subtypes across groups U and P. CONCLUSIONS: Monitoring of BKPyV-DNA by real-time PCR after initiation of conditioning, regularly performed in clinical practice, can be a crucial tool for the early identification of patients with higher risk of BKPyV-associated HC.

Genome Sequence of Novel Human Parechovirus Type 17.

Human parechoviruses (HPeV) circulate worldwide, causing a broad variety of symptoms, preferentially in early childhood. We report here the nearly complete genome sequence of a novel HPeV type, consisting of 7,062 nucleotides and encoding 2,179 amino acids. M36/CI/2014 was taxonomically classified as HPeV-17 by the picornavirus study group.

Anal chromoendoscopy using gastroenterological video-endoscopes: A new method to perform high resolution anoscopy for diagnosing intraepithelial neoplasia and anal carcinoma in HIV-infected patients.

Introduction Anal carcinoma represents an increasing problem in HIV-infected patients. Anal intraepithelial neoplasia (AIN), the precursor lesion, is currently diagnosed by high-resolution anoscopy (HRA) using optical magnification derived from gynecological colposcopy. This prospective study evaluates anal chromoendoscopy (ACE) using standard gastroenterological video-endoscopes in diagnosing AIN. Methods After clinical examination, proctoscopy and surface staining with acetic acid followed by Lugol's solution, ACE was performed with a mucosectomy cap on the tip of the endoscope. Biopsy specimens were collected from areas with a pathological staining pattern and from areas with normal appearance; combined results were considered as reference. Results Two hundred eleven HIV-positive patients seen between 2007 and 2013 were evaluated. Of these, 95.7 % were males, and the median age was 45 years. In 86.7 %, the mode of HIV transmission was sex among males. Combination antiretroviral treatment was applied in 75.8 %. The sensitivity of ACE in diagnosing AIN was 0.85, the specificity was 0.55, the positive predictive value was 0.50, and the negative predictive value (NPV) was 0.87. Diagnostic performance increased in individuals with high-grade lesions (NPV: 0.99) and in the second study period from 2011 to 2013. Side effects were rare and of minor clinical relevance. Conclusions Anal chromoendoscopy is safe and effective in diagnosing AIN in a population of HIV-infected patients. It is particularly useful for the exclusion of high-grade lesions that have the strongest risk of progression to anal carcinoma. Therefore, ACE may become a valuable new tool to manage AIN and to prevent anal malignancy in HIV-positive patients.

Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.

The response of the skin to harmful environmental agents is shaped decisively by the status of the immune system. Keratinocytes constitutively express and secrete the chemokine-like mediator, macrophage migration inhibitory factor (MIF), more strongly than dermal fibroblasts, thereby creating a MIF gradient in skin. By using global and epidermis-restricted Mif-knockout (Mif-/- and K14-Cre+/tg; Miffl/fl) mice, we found that MIF both recruits and maintains antigen-presenting cells in the dermis/epidermis. The reduced presence of antigen-presenting cells in the absence of MIF was associated with accelerated and increased formation of nonmelanoma skin tumors during chemical carcinogenesis. Our results demonstrate that MIF is essential for maintaining innate immunity in skin. Loss of keratinocyte-derived MIF leads to a loss of control of epithelial skin tumor formation in chemical skin carcinogenesis, which highlights an unexpected tumor-suppressive activity of MIF in murine skin.-Brocks, T., Fedorchenko, O., Schliermann, N., Stein, A., Moll, U. M., Seegobin, S., Dewor, M., Hallek, M., Marquardt, Y., Fietkau, K., Heise, R., Huth, S., Pfister, H., Bernhagen, J., Bucala, R., Baron, J. M., Fingerle-Rowson, G. Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.

High prevalence of human papillomaviruses in Ghanaian pregnant women.

Data about the prevalence of human papillomaviruses (HPV) in African women with normal and abnormal cervical cytology are still scarce. Current HPV vaccines contain HPV types, which mainly represent the HPV epidemiology of industrial countries. As further developments of HPV vaccines are going on, it is necessary to regard regional differences in HPV type prevalence to ensure optimal protection by the vaccine. Vaginal swabs of Ghanaian pregnant women, routinely collected before delivery to rule out bacterial infections causing early onset sepsis, were screened for 12 high-risk (HR), 13 probably/possibly (pHR), and 18 low-risk (LR) HPV types. Most pregnant women come for delivery to the hospital. This was considered as appropriate possibility to have an unselected group of women. HPV DNA were detected in 55/165 women (33.3, 95 % CI 26.3-41.1 %). Thirty-four out of fifty-five (61.8, 95 % CI 47.7-74.3 %) of HPV-positive women were infected with HR and/or pHR HPV types. The five most prevalent HR or pHR HPV types were HPV-52 and HPV-67 (7 women each, 4.2, 95 % CI 1.9-8.9 %), HPV-53 (six women, 3.6, 95 % CI 1.5-8.1 %), HPV-45 (five women, 3.0, 95 % CI 1.1-7.3 %), and HPV-18 (four women, 2.4, 95 % CI 0.8-6.5 %), respectively. HPV-16 was found in two women only (1.2, 95 % CI 0.2-4.8 %). Future HPV vaccine research may devote special interest to HPV-67 and HPV-53 provided further studies confirm their high prevalence in the general population of Sub-Saharan African countries. The true carcinogenic potential of HPV-67, which is a member of species alpha9 including HPV-16, and so far categorized as pHR, should be clarified.

Deletion of epidermal Rac1 inhibits HPV-8 induced skin papilloma formation and facilitates HPV-8- and UV-light induced skin carcinogenesis.

Overexpression and increased activity of the small Rho GTPase Rac1 has been linked to squamous cell carcinoma of the epidermis and mucosa in humans. Targeted deletion of Rac1 or inhibition of Rac1 activity in epidermal keratinocytes reduced papilloma formation in a chemical skin carcinogenesis mouse model. However, a potential role of Rac1 in HPV- and UV-light induced skin carcinogenesis has not been investigated so far, solar UV radiation being an important carcinogen to the skin.To investigate this, we deleted Rac1 or modulated its activity in mice with transgenic expression of Human papilloma virus type-8 (HPV-8) in epidermal keratinocytes. Our data show that inhibition or deletion of Rac1 results in reduced papilloma formation upon UV-irradiation with a single dose, whereas constitutive activation of Rac1 strongly increases papilloma frequency in these mice. Surprisingly, we observed that, upon chronic UV-irradiation, the majority of mice with transgenic expression of HPV-8 and epidermis specific Rac1 deletion developed squamous cell carcinomas. Taken together, our data show that Rac1 exerts a dual role in skin carcinogenesis: its activation is, on one hand, required for HPV-8- and UV-light induced papilloma formation but, on the other, suppresses the development of squamous cell carcinomas.

High-grade Dysplasia in Anogenital Warts of HIV-Positive Men.

Importance: Human papillomavirus (HPV)-induced anogenital lesions are very frequent in men who have sex with men (MSM) who are HIV-positive (HIV+). Anogenital warts (AGWs) are considered benign lesions caused by low-risk HPV-types, whereas anogenital dysplasias are potential cancer precursors associated with high-risk HPV-types. Both types of lesions can usually be distinguished clinically. Objective: To describe a case series of HIV+ MSM with typical AGW that harbored different grades of dysplasia. Design, Setting, and Participants: For this retrospective virological analysis, we recruited 25 HIV+ MSM with AGWs (n = 38) harboring areas of dysplasia and 22 patients who were HIV-negative (HIV-) with AGWs seen between February 2013 and March 2015 at a tertiary dermatological referral center for anal cancer screening. Dysplasia-containing AGW tissue of HIV+ MSM were compared with randomly selected AGWs of patients who were HIV-. Main Outcomes and Measures: Histopathological analysis, immunohistochemical staining for p16INK4a and Ki67, HPV-typing, and viral load determination in AGWs of HIV+ compared with patients who were HIV-. Results: Overall, 25 HIV+ MSM with AGWs (mean [SD] age, 47.3 [11.1] years) harboring areas of dysplasia and 22 patients who were HIV- (5 women, 17 men; mean [SD] age, 35.5 [12.8] years) with AGWs were included in this study. The 38 dysplasia-containing AGWs of HIV+ MSM harbored low-grade dysplasia in 6 cases (16%), high-grade dysplasia in 31 cases (81%), and areas of invasive anal carcinoma in 1 (3%) case. With the exception of 1 biopsy, all low-grade lesions were p16INK4a-negative, whereas 25 of 31 (81%) AGWs with high-grade lesions or an anal carcinoma were p16INK4a-positive. Only low-risk HPV-types were present in 11 samples (29%; 2 low-grade lesions and 9 high-grade lesions), low-risk and high-risk types were found in 19 samples (50%; 1 low-grade lesion and 18 high-grade lesions), and only high-risk HPV-types were present in 8 samples (21%; 3 low-grade lesions, 4 high-grade lesion, and 1 cancer-containing lesion). High low-risk HPV DNA loads were found in low-grade and high-grade lesions, while high high-risk HPV DNA loads were only found in AGWs harboring high-grade lesions. The 22 AGWs of patients who were HIV- showed no signs of dysplasia, and p16INK4a-staining was always negative. All of these samples carried low-risk HPV types, and in 2 cases high-risk HPV-types were detected additionally. Conclusions and Relevance: In contrast to immunocompetent patients, AGWs of HIV+ MSM may harbor high-grade dysplasia or even invasive squamous cell carcinoma. A substantial proportion of theses lesions may only contain low-risk HPV-types. Anogenital warts in patients who are HIV+ should be evaluated histopathologically to exclude cancer precursors.

Persistent CMV infection after allogeneic hematopoietic stem cell transplantation in a CMV-seronegative donor-to-positive recipient constellation: Development of multidrug resistance in the absence of anti-viral cellular immunity.

We describe a case of persistent cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with discordant and high-risk (D-/R+) constellation of CMV serostatus. Despite the use of different and innovative antiviral strategies, viral replication could not be suppressed successfully promoting a protracted CMV colitis associated with severe gastrointestinal graft-versus-host disease (GI GVHD). We illustrate that the development of multidrug viral resistance, the failure to mount a CMV-specific cellular immune response, as confirmed by QuantiFERON(®)-CMV (Qiagen) assay, and the refractory GVHD requiring prolonged immunosuppression were the main factors contributing to persistent viral replication and the fulminant unfavorable course.

The levels of epithelial anchor proteins ß-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

Infection with viruses of the genus Betapapillomavirus, ß-human papillomaviruses (ß-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins ß-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that ß-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of ß-catenin was elevated, no signs of ß-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of ß-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that ß-catenin and ZO-1 are direct targets of E7 of the oncogenic ß-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin.

Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis.

BACKGROUND: The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC). High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality. In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (ß-PV) play an important role in accelerating the early stages of skin tumorigenesis. METHODS: We investigated the effects of UV induced DNA damage in murine models of ß-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain. Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting. Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry. RESULTS: Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase. Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the C-terminal part of the protein for prevention of DNA damage repair and p300 binding. Whereas all K14-HPV8-E6wt animals develop skin tumors after UV expression of the HPV8-E6-K136N mutant significantly blocked skin tumor development after UV treatment. The persistence of CPDs in hyperproliferative epidermis K14-HPV8-E6wt skin resulted in the accumulation of γH2AX foci. DNA damage sensing was impaired in E6 positive cells grown as monolayer culture and in organotypic cultures, due to lack of phosphorylation of ATM, ATR and Chk1. CONCLUSION: We showed that cells expressing E6 fail to sense and mount an effective response to repair UV-induced DNA lesions and demonstrated a physiological relevance of E6-mediated inhibition of DNA damage repair for tumor initiation. These are the first mechanistical in vivo data on the tumorigenicity of HPV8 and demonstrate that the impairment of DNA damage repair pathways by the viral E6 protein is a critical factor in HPV-driven skin carcinogenesis.

Detection and characterization of enteroviruses and parechoviruses in healthy people living in the South of Côte d'Ivoire.

BACKGROUND: Human enteroviruses (EVs) and parechoviruses (HPeVs) belong to the family Picornaviridae. Although most EV and HPeV infections remain asymptomatic, both pathogens can cause a wide spectrum of clinical manifestations ranging from respiratory or gastrointestinal symptoms to myocarditis, neonatal sepsis, and infections of the central nervous system. OBJECTIVES: Aim of the present study was to investigate the spectrum of EVs and HPeVs in apparently healthy adults and children living in the South of Côte d'Ivoire. STUDY DESIGN: The study included 105 stool samples obtained from healthy individuals aged 0-53 years between June 2013 and December 2014 in the Sud-Como region of Côte d'Ivoire. After collection and shipment to Germany, the samples were analyzed by real-time PCR for the presence of EVs and HPeVs RNA. Molecular typing and virus isolation of all samples were performed.''é RESULTS: Out of 105 samples, 24 (22.8%) were EV positive and six (5.2%) were HPeV positive. Twenty-one EV positive samples could be characterized with serotypes belonging to EV group A-C, while three could not be further specified. Interestingly, several rarely described serotypes were identified, e.g., EV-C99, EV-B93, EV-C116, and EV-A119. Typing of HPeV positive samples resulted in HPeV-1 and -5 detections, while one isolate could not be assigned to the known HPeV types. CONCLUSIONS: This study showed a large variety of EV strains in healthy people in the South of Côte d'Ivoire and provided the first available data about HPeV infections in a sub-Saharan African country.

Application of Luminex Gastrointestinal Pathogen Panel to human stool samples from Côte d'Ivoire.

INTRODUCTION: Gastrointestinal infections caused by viruses, bacteria, and parasites are endemic in most developing countries due to inadequate provision of safe water supplies, sanitation, and hygiene. To investigate the enteric pathogens infecting people living in Côte d'Ivoire, the Luminex Gastrointestinal Pathogen Panel (xTAG GPP) assay was used to analyze 34 human fecal samples. This study represents the first application of this technology to samples from a sub-Saharan African country. METHODOLOGY: Thirty-four stool samples from asymptomatic and symptomatic patients, 1-15 years of age, were analyzed by xTAG GPP. The Luminex assay represents a qualitative bead-based multiplexed molecular diagnostic test able to identify concurrently 15 enteric pathogens, including bacteria, viruses, and parasites. RESULTS: Overall, 22 out of 34 (64.7%) fecal specimens were detected to be positive by xTAG GPP. Sixteen were from asymptomatic subjects, and 10 patients (45.4%) showed co-infections. G. duodenalis was detected in 15 patients, in both mono- and co-infections, representing the most frequent pathogen, followed by enterotoxigenic Escherichia coli (ETEC) LT/ST. Four norovirus isolates were also detected and assigned to genogroups I and II. CONCLUSIONS: Considering the burden of enteric infections in developing countries, particularly among children, and the high rate of co-infections in asymptomatic subjects, this study shows the need for diagnostic tools such as xTAG GPP to improve diagnosis and treatment of these infections in endemic areas.