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PDF file - 45K, Associations between cumulative HPV load and SCC, stratified by skin type and time since transplantation (for OTR). In each case the reference category is HPV negative combined with the lowest load tertile.

Background: Beta-human papillomavirus (betaPV) may play a role in the development of cutaneous squamous cell carcinoma (SCC). However betaPV is highly prevalent, and it may only be people with a higher viral load who have increased risk of SCCs. We therefore examined the association between betaPV load and SCCs.Methods: We recruited 448 immunocompetent cases with SCCs and 464 controls from Italy and Australia and 497 immunosuppressed organ transplant recipients (OTR; 179 cases and 318 controls) from Europe. We used reverse hybridization to genotype 25 betaPV types in eyebrow hair follicles and determined the viral load for eight selected types using quantitative PCR. We used logistic regression to assess associations between type-specific and cumulative viral load and SCCs.Results: Australian and OTR participants in the highest cumulative load tertile were at significantly higher risk of SCCs than those in the lowest tertile. Those with more than four betaPV types in the high load tertile were at approximately three-fold increased risk of SCCs. In Australia, HPV23 and 36 loads were significantly associated with SCCs, with borderline associations for HPV5 and 38. In OTR, HPV8 and 38 loads were significantly associated and HPV20 and 36 were borderline. We found little evidence for an association between load and SCCs in Italy.Conclusions: High viral load may be associated with risk of cutaneous SCCs, with total load seemingly more important than the load of any specific type.Impact: Our findings lend weight to the hypothesis that HPV plays a role in skin carcinogenesis. Cancer Epidemiol Biomarkers Prev; 22(4); 719–27. ©2013 AACR.

Supplementary Table 1 from Cutaneous Human Papillomaviruses Down-regulate AKT1, whereas AKT2 Up-regulation and Activation Associates with Tumors

Supplementary Figure 2 from Cutaneous Human Papillomaviruses Down-regulate AKT1, whereas AKT2 Up-regulation and Activation Associates with Tumors

Supplementary Figure S3. FACS sorted keratinocytes.

Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous β HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous HPV8 early genes down-regulate epidermal AKT activity by down-regulating AKT1 isoform levels. This down-regulation occurs before papilloma formation or tumorigenesis and leads to cutaneous differentiation changes that may weaken the epidermal squame for viral release. We find that, in viral warts (papillomas) and HPV gene-induced epidermal tumors, AKT activity can be activated focally by up-regulation and phosphorylation of the AKT2 isoform. In squamous cell carcinomas (SCC), AKT1 down-regulation is also common, consistent with a viral influence, whereas AKT2 up-regulation is widespread. Activation of up-regulated AKT2 by serine phosphorylation associates with high-grade tumors. Our data suggest that AKT2 up-regulation is characteristic of SCC and that coincident AKT2 activation through serine phosphorylation correlates with malignancy. These findings highlight differences between the effects of anogenital and cutaneous HPV on epithelial AKT activity and furthermore show that AKT isoforms can behave differently during epidermal tumorigenesis. These findings also suggest AKT2 as a possible therapeutic tumor target in SCC. [Cancer Res 2007;67(17):8207–15]

Supplementary Figure 1 from Cutaneous Human Papillomaviruses Down-regulate AKT1, whereas AKT2 Up-regulation and Activation Associates with Tumors

Supplementary Figure Legends. Legends for Supplementary Figures S1-S3.

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 due to the large number of betaHPV without defined high-risk types. Here, we hypothesized that the activation of innate antiviral immunity in skin, asymptomatically infected with betaHPV, induces an anti-tumor response by in situ autovaccination and prevents the formation of betaHPV-associated skin cancer. To test this, we used the preclinical K14-HPV8 transgenic mouse model, which develops skin tumors after mechanical wounding. Remarkably, treatment with antiviral immune response activating poly(I:C) completely prevented cutaneous tumor growth. Induction of the interferon-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 skin tumors of untreated mice. T cell depletion demonstrated 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 novel treatment strategy against betaHPV-related skin diseases.

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.

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.

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.

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.

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.

// Jayesh Deshmukh 1 , Ruth Pofahl 1 , Herbert Pfister 2 , Ingo Haase 1 1 Department of Dermatology, University of Cologne, Cologne, 50937, Germany 2 Institute of Virology, University of Cologne, Cologne, 50935, Germany Correspondence to: Jayesh Deshmukh, email: jayesh.deshmukh@uk-koeln.de Keywords: HPV-8, Rac1, skin cancer, SCC, UV (Ultraviolet) light Received: April 01, 2016 Accepted: July 19, 2016 Published: August 05, 2016 ABSTRACT 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.

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.

Organ transplant recipients (OTRs) have a 100‐fold increased risk of cutaneous squamous cell carcinoma (cSCC). We prospectively evaluated the association between β genus human papillomaviruses (βPV) 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. βPV infection was assessed in eyebrow hair by using polymerase chain reaction–based methods. βPV 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 βPV by using a multivariable Cox regression model. Results are reported as adjusted hazard ratios (HRs). OTRs with 5 or more different βPV 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 βPV loads (HR 1.8, 95% confidence interval 1.2‐2.8). No significant associations were seen between serum antibodies and cSCC or between βPV and basal cell carcinoma. The diversity and load of βPV types in eyebrow hair are associated with cSCC risk in OTRs, providing evidence that βPV is associated with cSCC carcinogenesis and may present a target for future preventive strategies., In two cohorts of organ transplant recipients, those with five and more different beta‐genus human papillomavirus types and high virus loads in eyebrow hairs subsequently develop significantly more cutaneous squamous cell carcinomas than others, suggesting that these virus types may increase the risk of cutaneous squamous cell carcinoma in these patients.

Concrete calculations of (linear) dragging effects on test masses were performed by Einstein in the period 1912–1913 by introducing the model of an infinitely thin mass shell. Einstein’s impact on the subsequent work of Thirring and Lense from 1918 on the induction of gravitational dragging forces by accelerated (rotating) masses is revealed. Generalizations to strong fields were performed not earlier than in 1966 by Brill and Cohen. Extensions to higher orders of the angular velocity ω of rotation by Pfister and Braun (1985–1989) led to a solution of the “centrifugal force problem” and to a “quasiglobal principle of equivalence.” The present experimental status of dragging phenomena is shortly reviewed, and cosmological and Machian aspects of gravitomagnetism are discussed.

HIV-positive men who have sex with men (MSM) have an increased risk for anal human papillomavirus (HPV) infection, anal high-grade intraepithelial lesions (HSIL), and anal cancer. Smoking is associated with abnormal anal cytology and with an increased risk for anal cancer. We collected 3736 intraanal swabs from 803 HIV-positive MSM who participated in an anal cancer screening program between October 2003 and August 2014. HPV prevalence, anal cytology and HPV DNA load of high-risk (HR) HPV-types 16, 18, 31 and 33 of non-smokers and smokers were compared. HPV-typing was performed by alpha-HPV genus-specific PCR and hybridization with 38 type-specific probes using a multiplex genotyping assay. In samples positive for HPV16, 18, 31, or 33, HPV DNA loads were determined by type-specific real-time PCRs and expressed as HPV DNA copies per betaglobin gene copy. At baseline, HR-HPV DNA (80.5 vs. 89.0%, p=0.001), HPV16 DNA (41.6 vs. 52.3%, p=0.003), HPV18 DNA (15.5 vs. 26.0%, p

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.

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.

Recently, several novel human polyomaviruses (HPyVs) have been detected. HPyV6, 7, 9 and 10 are not associated with any disease so far. Trichodysplasia spinulosa (TS)-associated polyomavirus (TSPyV) can cause the rare skin disease TS. We have evaluated cutaneous DNA prevalence and viral loads of five HPyVs in HIV-infected men compared to healthy male controls. 449 forehead swabs were analysed by HPyV-specific real-time PCR. HPyV6, HPyV7, TSPyV and HPyV10 were found significantly more frequently on the skin of 210 HIV-infected compared to 239 HIV-negative men (HPyV6, 39.0 vs 27.6 %; HPyV7, 21.0 vs 13.4 %; TSPyV, 3.8 vs 0.8 %; HPyV10, 9.3 vs 3.4 %; P

Activation-induced cytidine deaminase (AID) not only promotes immune diversity by initiating somatic hypermutation and class switch recombination in immunoglobulin genes but also provokes genomic instability by introducing translocations and mutations into non-immunoglobulin genes. To test whether AID is essential for virus-induced tumor development, we used two transgenic tumor models: mice expressing hepatitis C virus (HCV) core proteins (HCV-Tg), driven by the hepatitis B virus promoter, and mice expressing human papillomavirus type 8 proteins (HPV8-Tg), driven by the Keratin 14 promoter. Both strains were analyzed in the absence and presence of AID by crossing each with AID−/− mice. There was no difference in the liver tumor frequency between the HCV-Tg/AID+/+ and HCV-Tg/AID−/− mice at 20 months of age although the AID+/+ mice showed more severe histological findings and increased cytokine expression. Furthermore, a low level of AID transcript was detected in the HCV-Tg/AID+/+ liver tissue that was not derived from hepatocytes themselves but from intra-hepatic immune cells. Although AID may not be the direct cause of HCV-induced oncogenesis, AID expressed in B cells, not in hepatocytes, may prolong steatosis and cause increased lymphocyte infiltration into HCV core protein-induced liver lesions. Similarly, there was no difference in the time course of skin tumor development between the HPV8-Tg/AID−/− and HPV8-Tg/AID+/+ groups. In conclusion, AID does not appear to be required for tumor development in the two virus-induced tumor mouse models tested although AID expressed in infiltrating B cells may promote inflammatory reactions in HCV core protein-induced liver pathogenesis.

Human papillomavirus (HPV) XS2 was isolated from warts on an immunosuppressed patient. After HPV vaccination, the warts resolved. HPVXS2 was also found in warts and normal skin of HIV-positive patients and rarely in HIV-negative controls. Further studies should elucidate the mechanisms that lead to wart clearance.

Ludwig Lange (1863–1936) published in the years 1885–1886 (at the age of 22– 23 years!) three papers on the law of inertia which have to be judged as a stroke of genius because, nearly 200 years after Newton’s Principia, he succeeded in eliminating the concepts of absolute space and absolute time. A particular merit of Lange’s papers is that he clearly analyzed which parts of Newton’s first law are useful definitions and which parts are non-trivial experimental facts, if not wonders of nature, a distinction which is even missing in most modern textbooks on mechanics. (And it should be clear from the outset that any basic law of physics has to contain both definitions – here inertial system, in other cases electric field, temperature, entropy, quantum state, . . . – and new non-trivial facts about nature.) The papers [1] and [2] are quite extended works, containing, besides Lange’s innovative contributions, also historical and philosophical overviews of the law of inertia, the booklet [2] also containing Lange’s PhD-thesis of the year 1886 at the university of Leipzig. The shorter paper [3], translated here, concentrates on Lange’s own contributions and their mathematical foundations. In the year 1902 Lange published again a quite extended analysis [4] of the law of inertia, together with reactions of other authors on his work from 1885–1886; see below. Lange’s main new findings and his results of lasting significance are contained in part I of the paper [3]. In this part he also gives a clear motivation for his work by saying: “Newton’s absolute space is a phantom that should never be made the basis of an exact science”. “To find a fully valid substitute for it is the goal of the following”. The absolute highlight of Lange’s work are his Definitions I and II and his Theorems I and II, and these are also the parts which are later highly praised and often literally quoted by other authors. Lange’s papers obviously are the first place where the nowadays standard terms ‘inertial system’ and ‘inertial timescale’ are introduced into the literature. As Lange freely admits, his Definition II and Law II concerning the inertial timescale owe much to somewhat earlier work by Neumann [5] and Thomson, Tait [6]. Neumann’s work particularly was a decisive stimulus for Lange, as is clear from the quotation: “The question now arises whether it is possible to eliminate also absolute space by a similar procedure [as Neumann’s one]. Indeed this is possible”. Concerning Definition I we like to point to the fact that originally, in [1], Lange tried to base an inertial system on only two points left to themselves. After an objection

In this issue, Genders et al. (2015) demonstrate in a cohort of organ transplant recipients that betapapillomavirus seropositivity around transplantation significantly increases the risk of developing keratinocyte carcinomas. These results further substantiate an etiologic role of betapapillomaviruses in skin cancer and raise prospects of anti-viral immunotherapy.

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a “stem cell-like” state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44 high EpCAM high cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.

Cutaneous human papillomaviruses (HPV) have been reported in cutaneous squamous cell carcinoma (SCC). We conducted a clinic-based case-control study to investigate the association between genus-beta HPV DNA in eyebrow hairs (EBH) and SCC. EBH from 168 SCC cases and 290 controls were genotyped for genus-beta HPV DNA. SCC tumors from a subset of cases (n = 142) were also genotyped. Viral load was determined in a subset of specimens positive for a single HPV type. Associations with SCC were estimated by odds ratios (OR) and 95% confidence intervals (CI) adjusted for age and sex using logistic regression. Statistical tests were two-sided. EBH DNA prevalence was greater in cases (87%) than controls (73%) (p < 0.05), and the association with SCC increased with the number of HPV types present, (≥ 4 types vs. HPV-negative: OR = 2.02, 95% CI = 1.07-3.80; p(trend) = 0.02). Type-specific associations were observed between SCC and DNA in EBH for HPV23 (OR = 1.90, 95% CI = 1.10-3.30) and HPV38 (OR = 1.84, 95% CI = 1.04-3.24). Additionally, when compared with the controls, the DNA prevalence in EBH was significantly higher among cases for 11 of the 25 genus-beta types tested, when accounting for DNA for the same HPV type in the tumor (ORs = 3.44-76.50). Compared to controls, the mean viral DNA load in EBH among the selected cases was greater for HPV5, HPV8 and HPV24, but lower for HPV38. SCC cases were more likely than controls to have HPV DNA+ EBH for single and multiple HPV types, providing additional support for the potential role of genus-beta HPV infections in SCC development.

In Europe and beyond, several advisory bodies have been monitoring the developments in the field of Synthetic Biology. Reports have been sent to national governments for information on the developments and possible regulatory and risk assessment questions raised by this field. To put the issues in a broader perspective, four national biosafety advisory bodies (the French High Council for Biotechnology, the German Central Committee on Biological Safety, the Netherlands Commission on Genetic Modification and the Belgian Scientific Institute of Public Health (Biosafety and Biotechnology Unit)) decided to join forces and organize an international scientific workshop to review some of the latest scientific insights and look into possible challenges in the risk assessment of Synthetic Biology. The SynBio Workshop (Paris 2012) – Risk assessment challenges of Synthetic Biology took place on the 12th of December 2012 and gathered scientists from biosafety advisory bodies from fifteen European countries, from the European Food Safety Authority as well as representatives of the European Commission, together with research scientists selected for their excellence in the field. The workshop was divided into two sessions: the first session gave an overview of four major fields in Synthetic Biology. The second session was set up for discussion with a scientific panel and the audience to identify and address relevant questions for risk assessment raised by recent and future developments of Synthetic Biology. An overview of the workshop and the discussion points put forward during the day are discussed in this document.

The evolution of hepatitis-B virus (HBV) drug resistance is characterized by the emergence of resistance conferring mutations and compensatory mutations. Therefore HBV drug-resistant isolates often harbor multiple mutations in the reverse transcriptase (RT) and corresponding mutations in the hepatitis B surface antigen (HBsAg). In this study mutational patterns of 60 HBV isolates harboring drug resistance mutations rtM204V or rtM204I were retrospectively analyzed. Both mutations, especially mutation rtM204V, were most often accompanied by compensatory mutations rtV173L and rtL180M but also by mutations conferring entecavir (n = 5) or adefovir resistance (n = 4). In addition, 22 (36.7%) drug resistant HBV isolates carried mutations related to immune escape in the HBsAg. In seven cases premature stop codons in HBsAg were detected resulting in the expression of truncated HBsAg. Clonal analysis of these seven quasispecies even disclosed the presence of HBV isolates carrying further stop codons and in one case the occurrence of resistance mutation rtA181T without the stop codon mutation sW172*. Interestingly, only one HBV clone carried the resistance mutations rtM204V and rtA181T. HBV drug resistant isolates frequently harbored HBsAg mutations associated with immune escape or disease progression pointing to a complex interaction of both proteins. HBV genotypic resistance tests based on population sequencing methods seemed to be inappropriate for determining the clinical relevance of stop codons in the HBsAg.

Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Although the ultraviolet irradiation is a key contributor to the establishment of this keratinocyte malignancy, the infection by some types of human papillomavirus (HPV) has also been implicated in NMSC development. Cancers occur as a result of a complex series of interactions between the cancer cell and its surrounding matrix. The matrix metalloproteinases (MMPs) play a role in degrading the extracellular matrix. MMP9 is an important gelatinase involved in processes such as cell migration, invasion and metastasis. In this report, we demonstrated by EMSA experiments that the MMP9 promoter contains a binding site for the transcriptional regulator E2 of HPV8. Transient reporter gene assays showed that HPV8-E2 activated the MMP9 promoter in a dose-dependent manner in human epidermal keratinocytes. An E2 transactivation-defective mutant (I73L) as well as a DNA-binding deficient mutant (R433K) demonstrated no activation of the MMP9 promoter, suggesting that both an intact transactivation and DNA-binding domain are required for E2 activation of the MMP9-promoter. The functional role of the E2 binding site within the MMP9 promoter was also confirmed by mutating the E2 binding site. In organotypic cultures of human skin, an overexpression of MMP9 was observed in suprabasal layers of the HPV8 E2-expressing epidermis thus confirming the results of the monolayer cultures. These results demonstrate that the early gene E2 of HPV8 is able to increase the expression of MMP9 by direct activation of the MMP9-promoter.

The E6 proteins from high-risk alpha human papillomavirus (HPV) types (e.g., HPV16) are characterized by the presence of a PDZ-binding motif through which they interact with a number of cellular PDZ domain-containing substrates and cooperate in their degradation. The ability of these E6 proteins to bind to PDZ domain proteins correlates with the oncogenic potential of the virus. The E6 proteins of oncogenic HPV from the genus Betapapillomavirus (betaPV, e.g., HPV8) do not encode a PDZ-binding motif. We found that the PDZ domain protein syntenin-2 is transcriptionally downregulated in primary human epidermal keratinocytes (PHEK) by HPV8 E6. The mRNA levels of the known HPV16 E6 PDZ protein targets Dlg, Scribble, Magi-1, Magi-3, PSD95, and Mupp1 were not changed by HPV8 E6. Decreased protein levels of syntenin-2 were observed in cell extracts from PHEK expressing HPV5, -8, -16, -20, and -38 E6 but not in HPV1 and -4 E6-positive keratinocytes. Surprisingly, HPV16 E6 also repressed transcription of syntenin-2 but with a much lower efficiency than HPV8 E6. In healthy human skin, syntenin-2 expression is localized in suprabasal epidermal layers. In organotypic skin cultures, the differentiation-dependent expression of syntenin-2 was absent in HPV8 E6- and E6E7-expressing cells. In basal cell carcinomas of the skin, syntenin-2 was not detectable, whereas in squamous cell carcinomas, expression was located in differentiated areas. Short hairpin RNA-mediated knockdown of syntenin-2 led to an inhibition of differentiation and an increase in the proliferation capacity in PHEK. These results identified syntenin-2 as the first PDZ domain protein controlled by HPV8 and HPV16 at the mRNA level.

Background Anal human papillomavirus (HPV) infection and anal dysplasia are frequent in HIV-positive men who have sex with men (HIV + MSM), and progression of low-grade (LSIL) to high-grade squamous intraepithelial lesions (HSIL) or anal cancer (AC) occurs faster than in HIV-negative individuals. High-risk (HR)-HPV-E6/E7 oncogene mRNA testing has a higher specificity and a higher positive predictive value (PPV) than HR-HPV-DNA testing for detecting high-grade cervical lesions. Objective To evaluate the diagnostic accuracy of the NucliSENS-EasyQ HPV1.1 E6/E7-mRNA-assay for the detection of anal dysplasia in HIV + MSM. Study design 289 intraanal swabs from HIV + MSM participating in a screening program that included anal cytology, high-resolution anoscopy and histology were analyzed. HR-HPV-DNA detection was performed by PCR and hybridization using a bead-based multiplex genotyping assay. E6/E7-mRNA detection of HR-HPV-types 16, 18, 31, 33 and 45 was performed using the NucliSENS-EasyQ assay. Results 269 swabs had valid results in both test formats (111 normal, 10 ASCUS, 105 LSIL, 42 HSIL, 1 AC). For the detection of LSIL + (LSIL + HSIL + cancer) sensitivity, specificity, negative predictive value (NPV) and PPV were 80.4%, 26.4%, 52.5%, and 57.2% for HR-HPV-DNA testing, respectively, compared to 75.7%, 57.9%, 66.0% and 68.7% for E6/E7-mRNA testing. The respective values for the detection of HSIL/cancer were 95.3%, 26.1%, 96.7%, 19.7% for HR-HPV-DNA and 95.3%, 46.0%, 98.1%, 25.2% for E6/E7-mRNA detection. Conclusion Compared to HR-HPV-DNA detection, E6/E7-mRNA testing has an increased specificity (approximately two-fold), similar sensitivity and higher NPV and PPV for the detection of low- and high-grade anal dysplasia in HIV + MSM.

The balance between analytical (low) and clinical (high) sensitivity is crucial for the specificity of a routine HPV test as limited specificity will be harmful due to unnecessary treatment of healthy women. Up to now HPV diagnostics is mainly based on DNA detection for which target and signal amplification methods are available. PCR techniques can be divided into type-specific and consensus PCRs. Due to its high clinical sensitivity and its relatively high specificity the HC2 test is still regarded as the gold standard in routine HPV testing. It hybridizes 13 (near) full-length stabilized RNA probes of high-risk HPV types to denatured target DNA followed by detection via antibodies and chemiluminescence. To avoid costly validation studies for each new HPV test standards for evaluation have been defined. Recently several new HPV detection assays have been commercialized. They all show promising data in first published studies but still await full validation according to the criteria mentioned above. Among them only the cobas HPV test has already been fully validated for use in triage and as adjunct to cytology. HPV 16 and 18 confer a much higher risk for development of a CIN 2+ compared to the other HPV high-risk types. It is therefore appropriate to test for these HPV types independently. Apart from that testing for individual HPV types is of very limited clinical value up to now. HPV RNA testing is a promising option with potentially higher specificity. As a first system, the APTIMA test has now received an FDA approval.

Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-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 β-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 β-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 β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-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 β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-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.

Prostate cancer is one of the most prevalent types of cancer in men. Controversial data exist concerning the role of BKPyV and the xenotropic murine leukaemia virus-related gammaretrovirus (XMRV) in prostate cancer development. We therefore assessed the association between prostate cancer and viral infections. We could detect BKPyV in only 1 out of 85 prostate cancer samples, whereas none of the tissue samples showed evidence for XMRV positivity. Lack of detection of BKPyV and XMRV in prostate cancer tissues suggests that these viruses do not play a role in the pathogenesis of this type of cancer.

Merkel cell polyomavirus (MCPyV or MCV) is the first polyomavirus to be clearly implicated as a causal agent underlying a human cancer, Merkel cell carcinoma (MCC). Infection with MCPyV is common in the general population, and a majority of adults shed MCPyV from the surface of their skin. In this study, we quantitated MCPyV DNA in skin swab specimens from healthy volunteers sampled at different anatomical sites over time periods ranging from 3 months to 4 years. The volunteers were also tested using a serological assay that detects antibodies specific for the MCPyV virion. There was a positive correlation between MCPyV virion-specific antibody titers and viral load at all anatomical sites tested (dorsal portion of the hands, forehead, and buttocks) (Spearman’s r 0.644, P