A New Cellular Factor Recognizes E2 Binding Sites of Papillomaviruses Which Mediate Transcriptional Repression by E2

Virology. Feb, 2002  |  Pubmed ID: 11853404

Repression of transcription by the full-length E2 protein of papillomaviruses (PV) seems to occur when the E2 binding sites and those of positively acting cellular factors overlap. Previously, we showed that RUNX1 (formerly called CBF) binds to the repression-mediating E2 binding site P2 of human PV type 8 (HPV8). By a yeast one-hybrid system we could identify an unknown protein binding also to P2, tentatively called PBF (papillomavirus binding factor). PBF recognizes the sequence CCGG, which represents the 3' half of the E2 binding site just adjacent to the RUNX1 motif. PBF also binds to the repression-mediating E2 BS-1 in BPV1, which is conserved to P2 of HPV8. Point mutations destroying PBF binding to HPV8 P2 and BPV-1 E2 BS-1 in vitro reduce promoter activity in corresponding reporter constructs. Our results suggest that PBF might play a role in transcription of PV genes and in E2-mediated repression.

Expression of P16 Protein Identifies a Distinct Entity of Tonsillar Carcinomas Associated with Human Papillomavirus

The American Journal of Pathology. Mar, 2003  |  Pubmed ID: 12598309

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.

Detection of a Novel 1905C-->T Mutation Within the Dihydropyrimidine Dehydrogenase Gene and Potential for Misclassification with the Exon 14-skipping Mutation

Clinical Chemistry. Apr, 2003  |  Pubmed ID: 12651840

Chapter 8: Human Papillomavirus and Skin Cancer

Journal of the National Cancer Institute. Monographs. 2003  |  Pubmed ID: 12807946

A high prevalence of human papillomavirus (HPV) DNA, particularly in squamous cell skin carcinoma of immunosuppressed but also of immunocompetent patients, has renewed great interest in a possible etiologic role of HPV in nonmelanoma skin cancer. It is difficult, however, to interpret these findings against a background of low-level infections with multiple HPV types from supergroup B (HPV4-related and epidermodysplasia verruciformis [EV] HPV), probably acquired by everyone early in and throughout life. Thus far, no high-risk HPV types have been identified. Because of the low copy numbers of HPV DNA in skin cancers, probably not every tumor cell contains a viral genome, which is compatible with cutaneous HPV being possibly important for tumor initiation and progression, but not for maintenance of the malignant phenotype. The question with regard to high-risk types should, therefore, be readdressed in case-control studies on the basis of serology, which can reveal viral activities over years. The viruses lingering in all people are apparently activated by sunlight (UV) exposure, by immunosuppression, and by hyperproliferation of the epithelium (psoriasis) and/or in the specific genetic background of the host (EV). It is intriguing that most of these factors are established risk factors in skin carcinogenesis. The weak transforming activity of cutaneous HPV in vitro compared with the transforming activity of genital HPV may explain the need for activators and synergistic factors. The antiapoptotic activities of E6 proteins of cutaneous HPV could be relevant to oncogenesis in the interplay with UV exposure. Prospective studies should determine the kinetics of HPV activation relative to tumor development.

Evidence for the Association of Human Papillomavirus Infection and Cutaneous Squamous Cell Carcinoma in Immunocompetent Individuals

Archives of Dermatology. Jul, 2003  |  Pubmed ID: 12873884

The aim of our study was to evaluate human papillomavirus (HPV) infection as a risk factor for cutaneous squamous cell carcinoma (SCC) in immunocompetent individuals.

Human Papillomavirus-positive Tonsillar Carcinomas: a Different Tumor Entity?

Medical Microbiology and Immunology. Aug, 2003  |  Pubmed ID: 12920586

Human papillomavirus (HPV) infections are thought to be one of the causal factors in the development of head and neck squamous cell carcinomas (HNSCC), particularly in tumors arising from the Waldeyer's tonsillar ring. We screened 98 carefully stratified HNSCC and different control tissues for the presence of HPV DNA by nested polymerase chain reaction (PCR) specific for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs and by HPV16-specific single step PCR. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. On average HNSCC showed rather low HPV DNA prevalences; 18% of the oral cavity cancers, 8% of nasopharyngeal cancers, 25% of hypopharyngeal cancers and 7% of laryngeal cancers were HPV DNA positive. In contrast, HPV sequences could be detected in 45% of the oropharyngeal cancers, particularly tonsillar carcinomas (58%). Tonsillar carcinomas were significantly more likely to be HPV positive than tumors from any other site ( P<0.001). All tonsillar cancers contained oncogenic HPV types, predominantly HPV16 (13 of 14; 93%). Unaffected tonsils were available from two of these patients, but both tested negative for HPV DNA. Furthermore, no HPV DNA could be found in tonsillar biopsy specimens from control groups. Localization and load of HPV DNA was determined in HPV16-positive tonsillar carcinomas, their metastases and in unaffected mucosa using laser-assisted microdissection and subsequent real time fluorescence PCR. We demonstrated that the HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Quantification of HPV16 DNA in samples of seven patients yielded viral loads from 6 to 153 HPV DNA copies per beta-globin gene copy and the load values in both locations were roughly comparable. These loads are comparable with data shown for other HPV-associated lesions. Statistical evaluation of data related to clinicopathological parameters showed a significant correlation of the HPV positivity of tonsillar carcinomas with tumor grading ( P=0.008) and alcohol consumption ( P=0.029). Taken together our findings show a preferential association of HPV DNA with tonsillar carcinomas. Furthermore our results argue for HPV-positive tonsillar carcinomas representing a separate tumor entity, which is less dependent on conventional HNSCC risk factors.

High Prevalence of Epidermodysplasia Verruciformis-associated Human Papillomavirus DNA in Actinic Keratoses of the Immunocompetent Population

Archives of Dermatological Research. Dec, 2003  |  Pubmed ID: 14618345

Skin cancers in both immunosuppressed and immunocompetent populations are associated with epidermodysplasia verruciformis human papillomavirus (EV-HPV) DNA. However, little is known about the prevalence of EV-HPVs in actinic keratoses in immunocompetent individuals. Actinic keratoses from 114 patients were classified as low-grade ( n=76) or high-grade ( n=38) according to the extent of histological atypia. HPV DNA was amplified from 54 frozen and 60 paraffin-embedded biopsy specimens by nested polymerase chain reaction (PCR) with several consensus and type-specific primers. PCR products were sequenced for typing. These results were compared with HPV detection in skin cancers ( n=20) and Bowen's disease ( n=18). A broad spectrum of EV-HPV types including oncogenic HPV5 and HPV8 and partially characterized sequences were detected in actinic keratoses and cutaneous cancers. In actinic keratoses a higher prevalence of EV-HPV DNA was found in frozen tissues than in formalin-fixed tissues (85% vs 67%). There was no difference between the low- and high-grade actinic keratoses either in terms of EV-HPV DNA prevalence or the results of serological study using HPV8 virus-like particles. The detection rate of EV-HPVs was lower in skin cancers and Bowen's disease. This would suggest involvement of EV-HPVs in the early stages of cutaneous oncogenesis.

The Papillomavirus E2 Protein Binds to and Synergizes with C/EBP Factors Involved in Keratinocyte Differentiation

Journal of Virology. May, 2003  |  Pubmed ID: 12692227

The papillomavirus life cycle is closely linked to the differentiation program of the host keratinocyte. Thus, late gene expression and viral maturation are restricted to terminally differentiated keratinocytes. A variety of cellular transcription factors including those of the C/EBP family are involved in the regulation of keratinocyte differentiation. In this study we show that the papillomavirus transcription factor E2 cooperates with C/EBPalpha and -beta in transcriptional activation. This synergism was independent of an E2 binding site. E2 and C/EBP factors synergistically transactivated a synthetic promoter construct containing classical C/EBPbeta sites and the C/EBPalpha-responsive proximal promoter of the involucrin gene, which is naturally expressed in differentiating keratinocytes. C/EBPalpha or -beta coprecipitated with E2 proteins derived from human papillomavirus type 8 (HPV8), HPV16, HPV18, and bovine papillomavirus type 1 in vitro and in vivo, indicating complex formation by the cellular and viral factors. The interaction domains could be mapped to the C terminus of E2 and amino acids 261 to 302 located within the bZIP motif of C/EBPbeta. Our data suggest that E2, via its interaction with C/EBP factors, may contribute to enhancing keratinocyte differentiation, which is suppressed by the viral oncoproteins E6 and E7 in HPV-induced lesions.

Dual Role of Tumor Suppressor P53 in Regulation of DNA Replication and Oncogene E6-promoter Activity of Epidermodysplasia Verruciformis-associated Human Papillomavirus Type 8

Virology. Apr, 2003  |  Pubmed ID: 12706078

Human papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3' part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs "CCAAC" (nt 57-73) and M29 (nt 84-112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P(175). In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8.

[Comment. HPV7-induced Skin Warts in Butchers: an Occupational Disease?]

Journal Der Deutschen Dermatologischen Gesellschaft = Journal of the German Society of Dermatology : JDDG. May, 2003  |  Pubmed ID: 16285309

Natural Cell-mediated Cytotoxicity of Peripheral Blood Lymphocytes Against Target Cells Transfected with Epidermodysplasia Verruciformis-specific Human Papillomavirus Type 8 L1 DNA Sequences

International Journal of Molecular Medicine. Jan, 2004  |  Pubmed ID: 14654993

The aim of the present study was to characterise natural cell-mediated cytotoxicity against COS-7 cells transfected with potentially oncogenic HPV-8 L1 DNA sequences cloned in sense and antisense orientation and to evaluate their lysis by peripheral blood lymphocytes (PBL) from patients with epidermodysplasia verruciformis (EV), a rare disease associated with life-long infection by specific HPV types. COS-7 cells were transfected with HPV-8 Hinc II restriction fragment (nucleotide positions 5434-7654) cloned in sense (COS-L1S) and antisense (COS-L1A) orientation into pCB6 expression vector. Cytotoxic activity of isolated PBL against COS cell lines as well as K562 erythroleukaemic cells was evaluated by 51Cr-release assay. We found that lymphocytes responsible for natural lysis of COS and K562 cells are CD3-negative CD56-positive natural killer (NK) cells. Analysis of NK cell cytotoxic activity against different COS cell lines has revealed that lymphocytes from healthy subjects killed COS-L1S cells significantly more efficiently than wild COS-7 and COS-L1A cells. Significantly more efficient lysis of COS-L1S cells was also observed in EV patients. Thus, expression of HPV L1 renders target cells more susceptible to NK-mediated cytotoxicity that may enable more effective elimination of transformed cells.

Increased Prevalence of Human Papillomavirus in Hairs Plucked from Patients with Psoriasis Treated with Psoralen-UV-A

Archives of Dermatology. Mar, 2004  |  Pubmed ID: 15023775

Patients with psoriasis treated with psoralen-UV-A (PUVA) are at increased risk of skin cancer; however, the exact causes of this increased incidence are not well understood. It has been suggested that PUVA may increase expression of the tumorigenic agent human papillomavirus (HPV) in skin by directly stimulating virus replication, immune suppression, or both, thereby leading to skin cancer formation.

The Human Papillomavirus Type 8 E2 Protein Suppresses Beta4-integrin Expression in Primary Human Keratinocytes

Journal of Virology. Oct, 2004  |  Pubmed ID: 15367640

Human papillomaviruses (HPVs) infect keratinocytes of skin and mucosa. Homeostasis of these constantly renewing, stratified epithelia is maintained by balanced keratinocyte proliferation and terminal differentiation. Instructions from the extracellular matrix engaging integrins strongly regulate these keratinocyte functions. The papillomavirus life cycle parallels the differentiation program of stratified epithelia, and viral progeny is produced only in terminally differentiating keratinocytes. Whereas papillomavirus oncoproteins can inhibit keratinocyte differentiation, the viral transcription factor E2 seems to counterbalance the impact of oncoproteins. In this study we show that high expression of HPV type 8 (HPV8) E2 in cultured primary keratinocytes leads to strong down-regulation of beta4-integrin expression levels, partial reduction of beta1-integrin, and detachment of transfected keratinocytes from underlying structures. Unlike HPV18 E2-expressing keratinocytes, HPV8 E2 transfectants did not primarily undergo apoptosis. HPV8 E2 partially suppressed beta4-integrin promoter activity by binding to a specific E2 binding site leading to displacement of at least one cellular DNA binding factor. To our knowledge, we show for the first time that specific E2 binding contributes to regulation of a cellular promoter. In vivo, decreased beta4-integrin expression is associated with detachment of keratinocytes from the underlying basement membrane and their egress from the basal to suprabasal layers. In papillomavirus disease, beta4-integrin down-regulation in keratinocytes with higher E2 expression may push virally infected cells into the transit-amplifying compartment and ensure their commitment to the differentiation process required for virus replication.

Circulating Soluble Tumour Necrosis Factor Receptors in Patients with Epidermodysplasia Verruciformis As Compared to Patients with Cutaneous Tumours in the General Population

Oncology Reports. Jan, 2005  |  Pubmed ID: 15583817

Soluble tumour necrosis factor receptors type I and II (sTNF-RI and II) were evaluated in sera from patients with epidermodysplasia verruciformis and patients with cutaneous warts, actinic keratoses, squamous cell carcinomas or basal cell carcinomas by specific enzyme-linked immunobiological assays. In patients with widespread epidermodysplasia verruciformis lesions, the levels of both sTNF-Rs were in normal range. Both types of sTNF-Rs were significantly increased in patients with warts. The levels of sTNF-RI were significantly increased in patients with multiple actinic keratoses, squamous cell carcinoma and basal cell carcinoma. Increased levels of circulating sTNF-Rs may facilitate development of cutaneous tumours. Normal levels of sTNF-Rs in patients with epidermodysplasia verruciformis might, at least partially, contribute to a slow growth and low metastatic potential of cancers in these patients.

Expression of P16 Protein is Associated with Human Papillomavirus Status in Tonsillar Carcinomas and Has Implications on Survival

Advances in Oto-rhino-laryngology. 2005  |  Pubmed ID: 15608419

Our recent analysis of papillomavirus (HPV) DNA in different malignant head and neck tumors revealed that HPV infections occurred most frequently in tonsillar carcinomas (58%) and that 84% of positive cases contained the highly oncogenic HPV type 16. We could also present data in favor of the hypothesis that in view of their clinical behavior and the involved risk factors HPV-positive and HPV-negative tonsillar carcinomas may represent two separate tumor entities. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection/typing we analyzed p16 protein expression in 34 tonsillar carcinomas for their correlation with HPV status. p16 is an inhibitor of cyclin-dependent kinases 4 and 6 which activate the negative cell cycle regulator protein pRB which in turn downregulates p16 expression. It could be shown that in neoplastic cells of the cervix uteri E7 protein of the high-risk HPVs can interfere with this regulatory circuit by its virtue to inactivate pRB and thus lead to the overexpession of p16. We found 53% of the tested tonsillar carcinomas to be HPV positive. 56% of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only 1 of the HPV-negative carcinomas showed focal p16 staining (p < 0.001). Clinical outcome analysis revealed a significant correlation of p16 expression with increased disease-free survival (p = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV DNA detection.

Presence of Epstein-Barr Virus in Esophageal Cancer is Restricted to Tumor Infiltrating Lymphocytes

Medical Microbiology and Immunology. Aug, 2005  |  Pubmed ID: 15692828

As representatives of low and high incidence countries respectively, 72 esophageal squamous cell carcinomas and 40 adenocarcinomas from Germany, and 43 esophageal squamous cell carcinomas from Russia were tested for the presence of Epstein-Barr virus (EBV) DNA by PCR and in situ hybridization. Thirty-four percent of the squamous cell carcinomas (SCC) and 26% of the adenocarcinomas (AC) contained EBV DNA as detected by nested PCR. Quantitative analysis using real time PCR revealed one copy of the EBV genome per every 27-200,000 cells. EBER RNA in situ hybridization showed no EBV-specific transcripts in the nuclei of the tumor cells. However, EBER transcripts were expressed in the nuclei of tumor infiltrating lymphocytes in 7 SCC and 1 AC of 24 EBV DNA positive cases. The present data provide no evidence for the persistence of EBV in the tumor cells of esophageal cancer. In contrast to a previous report from Taiwan, EBV is unlikely to play a role in esophageal carcinogenesis.

Development of Skin Tumors in Mice Transgenic for Early Genes of Human Papillomavirus Type 8

Cancer Research. Feb, 2005  |  Pubmed ID: 15735026

The cutaneous human papillomavirus (HPV) 8 is clearly involved in skin cancer development in epidermodysplasia verruciformis patients and its early genes E2, E6, and E7 have been implicated in cell transformation in vitro. To examine the functions of these genes in vivo we integrated the complete early region of HPV8 into the genome of DBA/Bl6 mice. To target their expression to the basal layer of the squamous epithelia the transgenes were put under the control of the keratin-14 promoter. Transgenic mice were back-crossed for up to six generations into both FVB/N and Bl6 mouse strains. Whereas none of the HPV8 transgene-negative littermates developed lesions in the skin or any other organ, 91% of HPV8-transgenic mice developed single or multifocal benign tumors, characterized by papillomatosis, acanthosis, hyperkeratosis, and varying degrees of epidermal dysplasia. Squamous cell carcinomas developed in 6% of the transgenic FVB/N mice. Real-time reverse transcription-PCR showed highest expression levels for HPV8-E2, followed by E7 and E6. There was no consistent difference in relative viral RNA levels between healthy or dysplastic skin and malignant skin tumors. Whereas UV-induced mutations in the tumor suppressor gene p53 are frequently detected in human skin carcinomas, mutations in p53 were not observed either in the benign or malignant mouse tumors. Nonmelanoma skin cancer developed in HPV8-transgenic mice without any treatment with physical or chemical carcinogens. This is the first experimental proof of the carcinogenic potential of an epidermodysplasia verruciformis-associated HPV-type in vivo.

The E7 Protein of Cutaneous Human Papillomavirus Type 8 Causes Invasion of Human Keratinocytes into the Dermis in Organotypic Cultures of Skin

Cancer Research. Mar, 2005  |  Pubmed ID: 15781634

Human papillomaviruses (HPV) have been implicated in the development of nonmelanoma skin cancer (NMSC). The molecular mechanisms by which these viruses contribute towards NMSC are poorly understood. We have used an in vitro skin-equivalent model generated by transducing primary adult human epidermal keratinocytes with retroviruses expressing HPV genes to investigate the mechanisms of viral transformation. In this model, keratinocytes expressing HPV genes are seeded onto a mesenchyme composed of deepidermalized human dermis that had been repopulated with primary dermal fibroblasts. Expression of the HPV8 E7 gene caused both an enhancement of terminal differentiation and hyperproliferation, but most strikingly, the acquisition of the ability to migrate and invade through the underlying dermis. The basement membrane integrity was disrupted in a time-dependent manner in areas of invading keratinocytes, as evidenced by immunostaining of its protein components collagen types VII, IV, and laminin 5. This was accompanied by the overexpression of extracellular matrix metalloproteinases MMP-1, MMP-8, and MT-1-MMP. These results suggest that the cutaneous HPV type 8 that is frequently found in NMSC of epidermodysplasia verruciformis patients may actively promote an invasive keratinocyte phenotype. These findings also highlight the importance of epithelial-extracellular matrix-mesenchymal interactions that are required to support cell invasion.

Clinical Spectrum and Virologic Characteristics of Anal Intraepithelial Neoplasia in HIV Infection

Journal of the American Academy of Dermatology. Apr, 2005  |  Pubmed ID: 15793509

Anal intraepithelial neoplasia (AIN) represents a precursor lesion of invasive squamous cell carcinoma with a clear association to high-risk human papillomavirus (HPV) types. HIV infection is strongly associated with a higher prevalence of genital HPV infection, a higher incidence of AIN, and, consecutively, an increased risk for anal cancer.

Expression of Membrane Type 1 Matrix Metalloproteinase in Papillomavirus-positive Cells: Role of the Human Papillomavirus (HPV) 16 and HPV8 E7 Gene Products

The Journal of General Virology. May, 2005  |  Pubmed ID: 15831939

Matrix metalloproteinases (MMPs) degrade extracellular matrix. They are involved in cellular proliferation, migration, angiogenesis, invasion and metastasis. MT-1 MMP, a membrane-bound MMP, is expressed in carcinomas of the uterine cervix in vivo. This type of cancer is associated with human papillomavirus (HPV) infection. Here it was shown that keratinocytes transformed with HPV16 or HPV18 in vitro, and HPV-positive cervical carcinoma cell lines, constitutively expressed MT-1 MMP. Expression of the E7 protein from the mucosal and cutaneous high-risk types HPV16 and HPV8, but not from the cutaneous low-risk type HPV1, was sufficient to induce MT-1 MMP expression in primary human keratinocytes and HaCaT cells. As a consequence, MMP-2 was activated. MT-1 MMP expression might play a role in the HPV life cycle by promoting proliferation of host cells and might contribute to their invasive phenotype during malignant progression.

Human Papillomavirus-DNA Loads in Actinic Keratoses Exceed Those in Non-melanoma Skin Cancers

The Journal of Investigative Dermatology. Jul, 2005  |  Pubmed ID: 15982308

Recent studies suggest a role of cutaneous human papillomaviruses (HPV) in non-melanoma skin cancer (NMSC) development. In this study viral DNA loads of six frequent HPV types were determined by quantitative, type-specific real-time-PCR (Q-PCR) in actinic keratoses (AK, n=26), NMSC (n=31), perilesional tissue (n=22), and metastases of squamous cell carcinomas (SCC) (n=8) which were previously shown to be positive for HPV5, 8, 15, 20, 24, or 36. HPV-DNA loads in AK, (partially microdissected) NMSC, and perilesional skin ranged between one HPV-DNA copy per 0.02 and 14,200 cell equivalents (median: 1 HPV-DNA copy per 344 cell equivalents; n=48). In 32 of the 79 HPV-positive skin biopsies and in seven of the eight metastases viral loads were even below the detection limit of Q-PCR. Low viral loads in NMSC were confirmed by in situ-hybridization showing only a few HPV-DNA-positive nuclei per section. Viral loads in SCC, basal cell carcinomas, and perilesional tissue were similar. But, viral loads found in AK were significantly higher than in SCC (p=0.035). Our data suggest that persistence of HPV is not necessary for the maintenance of the malignant phenotype of individual NMSC cells. Although a passenger state cannot be excluded, the data are compatible with a carcinogenic role of HPV in early steps of tumor development.

Use of Interferon-alpha in Recurrent Respiratory Papillomatosis: 20-year Follow-up

The Annals of Otology, Rhinology, and Laryngology. Jun, 2005  |  Pubmed ID: 16042104

The aim of this study was analysis of the results of use of interferon-alpha (IFN-alpha) in patients with recurrent respiratory papillomatosis (RRP) and correlation of the results with human papillomavirus (HPV) type.

Modifications of Keratinocyte Functions by Human Papillomaviruses

Experimental Dermatology. Oct, 2005  |  Pubmed ID: 16176295

Evolution of HIV Resistance During Treatment Interruption in Experienced Patients and After Restarting a New Therapy

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. Dec, 2005  |  Pubmed ID: 16191482

To analyse the evolution of resistance patterns in patients undergoing treatment interruption (TI) and re-initiating highly active anti-retroviral therapy (HAART).

Human Papillomavirus Type 26-associated Periungual Squamous Cell Carcinoma in Situ in a HIV-infected Patient with Concomitant Penile and Anal Intraepithelial Neoplasia

Journal of the American Academy of Dermatology. Oct, 2005  |  Pubmed ID: 16198810

5% Imiquimod Suppositories Decrease the DNA Load of Intra-anal HPV Types 6 and 11 in HIV-infected Men After Surgical Ablation of Condylomata Acuminata

Archives of Dermatology. Feb, 2006  |  Pubmed ID: 16490857

Imiquimod Treatment of Anal Intraepithelial Neoplasia in HIV-positive Men

Archives of Dermatology. Nov, 2006  |  Pubmed ID: 17116834

To evaluate the treatment of anal intraepithelial neoplasia (AIN) with the local immune response modifier imiquimod in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM).

Compensatory Mutations at the HIV Cleavage Sites P7/p1 and P1/p6-gag in Therapy-naive and Therapy-experienced Patients

Antiviral Therapy. 2006  |  Pubmed ID: 17302250

Mutations in the genome of HIV conferring drug resistance are a major reason for the failure of antiretroviral therapy, but they often compromise viral fitness. Protease (PR) cleavage site (CS) mutations could compensate for impaired replication capacity of drug-resistant viruses.

Expression of Matrix Metalloproteinase (MMP)-2, MMP-9, MMP-13, and MT1-MMP in Skin Tumors of Human Papillomavirus Type 8 Transgenic Mice

Experimental Dermatology. Jan, 2006  |  Pubmed ID: 16364029

Human papillomaviruses (HPV) are small DNA viruses that induce a wide variety of hyperproliferative lesions in cutaneous and mucosal epithelia. It is proposed that HPV is involved in non-melanoma skin cancer development. We have previously shown that HPV8 transgenic mice spontaneously develop papillomatous skin tumors. Histology revealed epidermal hyperplasia, acanthosis and hypergranulosis and in some cases squamous cell carcinomas (SCC). Zymographic and immunoblot analysis of normal skin extracts identified increased amounts of matrix metalloproteinase (MMP)-9, MMP-13 and MT1-MMP in HPV8-positive mice compared with HPV8-negative animals. In situ gelatin zymography of tumor specimens displayed a strong proteolytic activity in papillomas, and SCC putatively attributed to the increased amounts of activated MMP-9 found in tissue extracts. In addition, immunoblot analysis revealed increased amounts of active MMP-13 and MT1-MMP in tumor extracts as compared with control extracts. Immunohistochemical stainings of SCC specimens depicted MMP-13 to be specifically expressed in stromal fibroblasts neighboring the tumor islands, whereas MT1-MMP was detected both in tumor cells and in stromal cells. Taken together, these results implicate a role for MMPs in the development of HPV8-induced cutaneous tumors.

Primary HIV Drug Resistance and Efficacy of First-line Antiretroviral Therapy Guided by Resistance Testing

Journal of Acquired Immune Deficiency Syndromes (1999). Apr, 2006  |  Pubmed ID: 16652031

Primary HIV drug resistance has been associated with poor treatment outcome of first-line highly active antiretroviral therapy (HAART) in several trials. The aim of the study was to assess the efficacy of first-line HAART guided by resistance testing.

Children and Partners of Patients with Recurrent Respiratory Papillomatosis Have No Evidence of the Disease During Long-term Observation

International Journal of Pediatric Otorhinolaryngology. Dec, 2006  |  Pubmed ID: 16945430

Recurrent respiratory papillomatosis (RRP) is the most common benign neoplasm affecting the larynx and upper respiratory tract. The aim of our study was to investigate whether children and partners of patients with RRP develop the same disease and to determine whether there is an impact of pregnancy on the course of RRP.

Reactivation of an Occult Hepatitis B Virus Escape Mutant in an Anti-HBs Positive, Anti-HBc Negative Lymphoma Patient

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. Jan, 2007  |  Pubmed ID: 17134939

Hepatitis B virus (HBV) often persists after resolution, but its replication is suppressed by antiviral T cells. Immunosuppressive treatment may lead to viral reactivation and severe hepatitis. Early antiviral therapy prevents reactivation but some occult HBV infections are not easily detectable.

Combined Analysis of HPV-DNA, P16 and EGFR Expression to Predict Prognosis in Oropharyngeal Cancer

International Journal of Cancer. Journal International Du Cancer. Apr, 2007  |  Pubmed ID: 17236202

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV-DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV-DNA and expression of p16 and EGFR were analyzed. The 5-year disease-free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty-eight percent of the cases contained oncogenic HPV-DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV-DNA (p < 0.001). Univariate analysis of the 5-year DFS revealed a significantly better outcome for patients with p16-positive tumors (84% vs. 49%, p = 0.009). EGFR-negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5-year DFS of 93% for p16+/EGFR- tumors vs. 39% for p16-/EGFR+ tumors (p = 0.003) and to a 5-year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5-fold increased risk for relapse in patients with p16-negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV-positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

HPV8 Early Genes Modulate Differentiation and Cell Cycle of Primary Human Adult Keratinocytes

Experimental Dermatology. Jul, 2007  |  Pubmed ID: 17576239

Human papillomaviruses (HPV) have been associated with the development of non-melanoma skin cancer (NMSC) but the molecular mechanisms of the role of the virus in NMSC development are not clearly understood. Abnormal epithelial differentiation seen in malignant transformation of keratinocytes is associated with changes in keratin expression. The purpose of this study was to investigate the phenotype of primary human adult keratinocytes expressing early genes of HPV8 with specific reference to their differentiation and cell cycle profile to determine whether early genes of HPV8 lead to changes that are consistent with transformation. The expression of HPV8 early genes either individually or simultaneously caused distinct changes in the keratinocyte morphology and induced an abnormal keratin expression pattern, that included simple epithelial (K8, K18, K19), hyperproliferation-specific (K6, K16), basal-specific (K14, K15) and differentiation-specific (K1, K10) keratins. Our results indicate that expression of HPV8 early genes disrupts the normal keratin expression pattern in vitro. Expression of HPV8-E7 alone caused polyploidy that was associated with decreased expression of p21 and pRb. Expression of individual genes or in combination differentially influenced cell morphology and cell cycle distribution which might be important in HPV8-induced keratinocyte transformation.

Cutaneous Human Papillomaviruses Down-regulate AKT1, Whereas AKT2 Up-regulation and Activation Associates with Tumors

Cancer Research. Sep, 2007  |  Pubmed ID: 17804734

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 beta 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.

Trends of Prevalence of Primary HIV Drug Resistance in Germany

The Journal of Antimicrobial Chemotherapy. Oct, 2007  |  Pubmed ID: 17715124

Primary HIV drug resistance (PDR) is associated with poor treatment outcome of first-line highly active antiretroviral therapy (HAART). The aim of the study was to observe the trend of prevalence of PDR between 2001 and 2005.

Human Papilloma Virus (HPV)-associated Gynecological Alteration in Mothers of Children with Recurrent Respiratory Papillomatosis During Long-term Observation

Cancer Detection and Prevention. 2007  |  Pubmed ID: 17935912

Human papilloma virus (HPV) is one of the most frequently observed sexually transmitted infections. The study' purpose was to investigate the relation between a mother's gynecological history and the local status of her child with recurrent respiratory papillomatosis (RRP).

[Condylomata Acuminata and Other HPV Associated Disease Pictures of the Genitals, Anus and Urethra]

Journal Der Deutschen Dermatologischen Gesellschaft = Journal of the German Society of Dermatology : JDDG. Feb, 2008  |  Pubmed ID: 18261086

Imiquimod Leads to a Decrease of Human Papillomavirus DNA and to a Sustained Clearance of Anal Intraepithelial Neoplasia in HIV-infected Men

The Journal of Investigative Dermatology. Aug, 2008  |  Pubmed ID: 18273049

Anal intraepithelial neoplasia (AIN), a human papillomavirus (HPV)-associated precursor lesion of anal carcinoma, is highly prevalent in HIV-infected men having sex with men (MSM). This prospective follow-up study evaluated the long-term results of imiquimod treatment of AIN in 19 HIV-infected MSM. Standardized follow-up examinations included high-resolution anoscopy, anal cytology/histology, HPV typing, and DNA load determination for HPV types 16, 18, 31, and 33. Mean follow-up time was 30.3 months. A total of 74% (14/19) of the patients remained free of AIN at the previously treated site. Five patients (26%) had recurrent high-grade AIN after a mean time of 24.6 months. At the end of follow-up, the numbers of HPV types as well as high-risk HPV-DNA loads were significantly lower than before therapy. During follow-up, 58% of all patients (11/19) developed new anal cytological abnormalities in previously normal, untreated anal regions. 55% of these new AIN lesions were high-grade lesions and most of them were located intra-anally and associated with high-risk HPV types not detectable before therapy. These results demonstrate that imiquimod leads to a high rate of long-term clearance of AIN in HIV-positive men together with a prolonged decrease of high-risk HPV-DNA load. However, new AIN lesions associated with previously undetected HPV types frequently occur in untreated areas.

A Human Papillomavirus-associated Disease with Disseminated Warts, Depressed Cell-mediated Immunity, Primary Lymphedema, and Anogenital Dysplasia: WILD Syndrome

Archives of Dermatology. Mar, 2008  |  Pubmed ID: 18347293

Epidermodysplasia verruciformis (EV) is a rare genodermatosis associated with infections with specific human papillomaviruses (HPVs) belonging to the beta genus of HPV. Patients with EV usually have a selective defect in cell-mediated immunity. Although skin cancer frequently develops in the sun-exposed cutaneous lesions of patients with EV, the anogenital area is usually not affected by squamous cell carcinomas related to mucosal HPV types.

Penile Intraepithelial Neoplasia is Frequent in HIV-positive Men with Anal Dysplasia

The Journal of Investigative Dermatology. Sep, 2008  |  Pubmed ID: 18385760

Anogenital human papillomavirus (HPV)-infection is common in HIV-infected men who have sex with men (HIV+MSM). These patients have a strongly increased risk of HPV-induced anal cancer and its precursor lesion, anal intraepithelial neoplasia (AIN), and a moderately increased risk for penile cancer. Only limited data exist on penile intraepithelial neoplasia (PIN) in HIV+MSM. We determined the prevalence and evaluated the virologic characteristics of PIN and AIN in 263 HIV+MSM. In case of histologically confirmed PIN (and AIN), HPV-typing, HPV-DNA load determination, and immunohistochemical staining for p16(INK4a) were performed. PIN was detected in 11 (4.2%) and AIN in 156 (59.3%) patients. Ten PIN patients also had AIN within the observation period. Four clinical types of PINs could be distinguished. High-risk-alpha-HPV-DNA was found in 10 PIN lesions, with HPV16 being the most frequent type. Infections with multiple HPV-types were common. All high-grade lesions had high-risk-HPV-DNA-loads > or = 1 HPV-copy/beta-globin-gene-copy. Cutaneous beta-HPVs were found in PIN and AIN, but beta-HPV-DNA loads were very low, irrespective of the histological grade. p16(INK4a) Expression was detectable in all PIN lesions and correlated both with the histological grade and with high-risk HPV-DNA loads. In view of the PIN prevalence found in our study, all HIV+MSM should be screened for PIN in addition to AIN screening.

The Human Papillomavirus Type 8 E2 Protein Induces Skin Tumors in Transgenic Mice

The Journal of Investigative Dermatology. Sep, 2008  |  Pubmed ID: 18401427

Transgenic mice expressing early genes of the cutaneous human papillomavirus 8 (HPV8) spontaneously develop skin papillomas, epidermal dysplasia, and squamous cell carcinoma (6%). As the HPV8 protein E2 revealed transforming capacity in vitro, we generated three epidermal specific HPV8-E2-transgenic FVB/N mouse lines to dissect its role in tumor development. The rate of tumor formation in the three lines correlated with the different E2-mRNA levels. More than 60% of heterozygous line 2 mice, but none of the HPV8-negative littermates, spontaneously developed ulcerous lesions of the skin over an observation period of up to 144 weeks, beginning on average 74+/-22 weeks after birth. Most lesions presented infundibular hyperplasia and acanthosis combined with low-grade dysplasia. Severe dysplasia of the epidermis occurred in 6%. Two carcinomas revealed a sharply demarcated spindle-cell component. Only 3 weeks after a single UV irradiation, 87% of heterozygous line 2 and 36% of line 35 mice developed skin tumors. A rapidly growing invasive tumor composed of spindle cells arose 10 weeks after irradiation of a line-35 animal. The histology of skin cancers in HPV8-E2 mice is reminiscent of a subset of highly aggressive squamous cell carcinoma in immunosuppressed transplant recipients with a massive spindle-cell component.

High Beta-HPV DNA Loads and Strong Seroreactivity Are Present in Epidermodysplasia Verruciformis

The Journal of Investigative Dermatology. Apr, 2009  |  Pubmed ID: 18923444

Epidermodysplasia verruciformis (EV) is a rare disease, characterized by cutaneous warts and associated with a strong predisposition to beta-genus human papillomavirus (HPV). Earlier studies reported high copy numbers of HPV-DNA in nearly all skin tumors from EV patients, but neither HPV replication status in non-lesional skin nor anti-HPV seroreactivity in these patients have been reported yet. We therefore performed a comprehensive viral load analysis for the more common beta-HPV types on skin samples and plucked eyebrow hairs from four EV patients treated at our dermatology department. The results clearly demonstrate that they carry a multiplicity (up to eighteen types) of beta-HPV genotypes in both skin sites. Worthy of note, a high intrapatient concordance for specific types between hair bulbs and skin biopsies was observed and the same beta-PV profile was maintained over time. Viral load analysis revealed a load range between less than one HPV-DNA copy per 100 cells to more than 400 HPV-DNA copies per cell in both eyebrow hairs and skin proliferative lesions. Evaluation of seroreactivity to beta-HPV types in the four EV patients revealed that antibodies against the 16 beta-HPV were significantly more prevalent and showed higher titers than in the controls.

Distribution and Density of CD1a+ and CD83+ Dendritic Cells in HPV-associated Laryngeal Papillomas

International Journal of Pediatric Otorhinolaryngology. Feb, 2009  |  Pubmed ID: 19062106

Respiratory papillomatosis associated with human papilloma virus (HPV) infection is the most common benign laryngeal neoplasm. The age of patients at disease onset, HPV type, number of surgeries are well known prognostic factors of the disease course. The correlation between dendritic cell (DC) density in tumor tissue and clinical prognosis was established.

Prevalence of C-terminal Gag Cleavage Site Mutations in HIV from Therapy-naïve Patients

The Journal of Infection. Jan, 2009  |  Pubmed ID: 19110315

HIV protease - as well as gag cleavage site (CS) - mutations occur in HIV with PI resistance but little is known about the prevalence of CS mutations in drug-naïve patients.

Prevalence and Associated Factors of Betapapillomavirus Infections in Individuals Without Cutaneous Squamous Cell Carcinoma

The Journal of General Virology. Jul, 2009  |  Pubmed ID: 19321753

Betapapillomavirus (betaPV) infections are often associated with squamous-cell carcinoma (SCC) and the prevalence of betaPV infections in (immunosuppressed) SCC patients is known to be high. The distribution and possible associated factors of betaPV infections in the general population, however, are largely unknown. To address this issue, betaPV infection was studied in 1405 SCC-free immunocompetent (n=845) and immunosuppressed (n=560) individuals from six countries of different latitudes. A standard study protocol was used to obtain information about age, sex, UV-irradiation and skin type, and from all participants eyebrow hairs were collected for detection and genotyping of 25 established betaPV types using the PM-PCR reverse hybridization assay (RHA) method. The frequency of betaPV-positive participants ranged from 84 to 91% in the immunocompetent population with HPV23 as the most prevalent type, and from 81 to 98% in the immunosuppressed population with HPV23 as the most or the second most prevalent type. The median number of infecting betaPV types ranged from four to six in the immunocompetent and from three to six in the immunosuppressed population. Increasing age in the immunocompetent participants and (duration of) immunosuppression in the immunosuppressed patients were associated with betaPV infection. In both groups, sex, skin phototype, sunburns and sun-exposure were not consistently associated with betaPV infection. This study demonstrates that betaPV infections are also highly prevalent in SCC-free individuals, with similar HPV types prevailing in both immunocompetent and immunosuppressed persons. Age and (duration of) immunosuppression were identified as betaPV infection-associated factors, whereas characteristics related to sun exposure and skin type were not.

Prevalence of Minor Variants of HIV Strains at Reverse Transcriptase Position 103 in Therapy-naïve Patients and Their Impact on the Virological Failure

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. May, 2009  |  Pubmed ID: 19375978

Minority HIV-1 populations with resistance mutations might result in therapy failure. The prevalence of transmitted minorities in therapy-naïve patients and their influence on the virological outcome of the first-line-therapy need clarification.

Oncogenic Viruses

Cancer Treatment and Research. 2009  |  Pubmed ID: 19415199

Evolution of Raltegravir Resistance During Therapy

The Journal of Antimicrobial Chemotherapy. Jul, 2009  |  Pubmed ID: 19447792

We investigated the prevalence of raltegravir resistance-associated mutations at baseline and their evolution during raltegravir therapy in patients infected with different HIV-1 subtypes.

Spontaneous Tumour Development in Human Papillomavirus Type 8 E6 Transgenic Mice and Rapid Induction by UV-light Exposure and Wounding

The Journal of General Virology. Dec, 2009  |  Pubmed ID: 19692543

Cutaneous human papillomavirus type 8 (HPV8) is carcinogenic in patients with epidermodysplasia verruciformis. Transgenic mice with the complete early region (CER) of HPV8 spontaneously developed papillomas, dysplasia and squamous cell carcinomas of the skin. To characterize the role of individual early genes in carcinogenesis, the E6 and E6/E7 genes were expressed separately in transgenic mice. Nearly all HPV8-E6-positive mice spontaneously developed multifocal tumours, characterized by papillomatosis, hyperkeratosis and varying degrees of epidermal dysplasia. In 6 % of the cases, the tumours became malignant, comparable with HPV8-CER mice. Thus, in the murine epidermis, E6 is the major oncogene necessary and sufficient to induce spontaneous tumour development up to the level of squamous cell carcinoma. To evaluate the synergistic effects of UV light and wound healing, the skin of HPV8 mice was irradiated with UVA/UVB light or wounded with punch biopsies. These treatments induced papillomatosis in HPV8-CER and -E6 mice within 3 weeks. Irradiation with UVA alone did not induce papillomatosis and UVB alone had a weaker effect than UVA/UVB, indicating a synergistic role of UVA in UVB-induced papillomatosis. An HPV8 infection persisting over decades in interaction with sun burns and wound healing processes may be a relevant cause of skin cancer in humans.

Merkel Cell Polyomavirus DNA in Persons Without Merkel Cell Carcinoma

Emerging Infectious Diseases. Sep, 2009  |  Pubmed ID: 19788824

Merkel cell polyomavirus (MCPyV) DNA was detected in 88% of Merkel cell carcinomas in contrast to 16% of other skin tumors. MCPyV was also found in anogenital and oral samples (31%) and eyebrow hairs (50%) of HIV-positive men and in forehead swabs (62%) of healthy controls. MCPyV thus appears to be widespread.

Seasonal Influenza Virus Species in Patient Swab Samples Analyzed for the Presence of the Pandemic (H1N1) 2009 Influenza Virus

Journal of Clinical Microbiology. Dec, 2009  |  Pubmed ID: 19794039

The Adenoviral E1A Oncoprotein Activates the Smad7 Promoter: Requirement of a Functional E-box

International Journal of Oncology. Dec, 2009  |  Pubmed ID: 19885573

DNA tumorviruses like adenoviruses (AdV) or human papillomaviruses (HPV) have adopted various strategies to interfere with antiproliferative transforming growth factor-beta (TGF-beta) signalling. Here we report that the AdV E1A oncoprotein is sufficient to induce Smad7 expression, an inhibitor of TGF-beta signalling. E1A but not HPV oncoproteins activated the Smad7 promoter. A promoter proximal E-box was crucial for E1A-mediated transcriptional activity. E1A but not HPV oncoproteins induced specific binding activity at this E-box, which was identified as upstream stimulatory factor. In conclusion, these results unravel a novel mechanism of how the AdV E1A oncoprotein induces a cellular inhibitor of TGF-beta signalling.

Detection of Adenoviruses and Rotaviruses in Drinking Water Sources Used in Rural Areas of Benin, West Africa

Applied and Environmental Microbiology. May, 2009  |  Pubmed ID: 19270143

Diseases associated with viruses also found in environmental samples cause major health problems in developing countries. Little is known about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. We established a method to analyze 10 liters of water from drinking water sources in a rural area of Benin for the presence of adenoviruses and rotaviruses. Overall, 541 samples from 287 drinking water sources were tested. A total of 12.9% of the sources were positive for adenoviruses and 2.1% of the sources were positive for rotaviruses at least once. Due to the temporary nature of viral contamination in drinking water sources, the probability of virus detection increased with the number of samples taken at one test site over time. No seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Overall, 3 of 15 surface water samples (20%) and 35 of 247 wells (14.2%) but also 2 of 25 pumps (8%) tested positive for adenoviruses or rotaviruses. The presence of latrines within a radius of 50 m in the vicinity of pumps or wells was identified as being a risk factor for virus detection. In summary, viral contamination was correlated with the presence of latrines in the vicinity of drinking water sources, indicating the importance of appropriate decision support systems in these socioeconomic prospering regions.

Human Papillomavirus 5 and 8 E6 Downregulate Interleukin-8 Secretion in Primary Human Keratinocytes

The Journal of General Virology. Apr, 2010  |  Pubmed ID: 20007354

Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early stages of skin cancer development, since both the prevalence and viral load are higher in precancerous actinic keratoses than in skin cancers. Interleukin-8 (IL-8) is an inflammatory cytokine that serves to alert the surrounding tissue after UV-induced damage. We examined the effects of the E2, E6 and E7 proteins of HPV8 and the E6 proteins of various HPV genotypes on IL-8 secretion from primary keratinocytes. HPV5 and HPV8 E6 showed the highest downregulation of basal IL-8 secretion. HPV8 E6 also negatively modulated IL-8 mRNA expression and protein secretion upon UVB irradiation. The downregulation of IL-8 in actinic keratoses may weaken the response to UV-induced damage and thus favour the accumulation of UVB-induced mutations.

Human Papillomavirus Type 8 E2 Protein Unravels JunB/Fra-1 As an Activator of the Beta4-integrin Gene in Human Keratinocytes

Journal of Virology. Feb, 2010  |  Pubmed ID: 19923172

The papillomavirus life cycle parallels keratinocyte differentiation in stratifying epithelia. We have previously shown that the human papillomavirus type 8 (HPV8) E2 protein downregulates beta4-integrin expression in normal human keratinocytes, which may trigger subsequent differentiation steps. Here, we demonstrate that the DNA binding domain of HPV8 E2 is sufficient to displace a cellular factor from the beta4-integrin promoter. We identified the E2-displaceable factor as activator protein 1 (AP-1), a heteromeric transcription factor with differentiation-specific expression in the epithelium. beta4-Integrin-positive epithelial cells displayed strong AP-1 binding activity. Both AP-1 binding activity and beta4-integrin expression were coregulated during keratinocyte differentiation suggesting the involvement of AP-1 in beta4-integrin expression. In normal human keratinocytes the AP-1 complex was composed of JunB and Fra-1 subunits. Chromatin immunoprecipitation assays confirmed that JunB/Fra-1 proteins interact in vivo with the beta4-integrin promoter and that JunB/Fra-1 promoter occupancy is reduced during keratinocyte differentiation as well as in HPV8 E2 positive keratinocytes. Ectopic expression of the tethered JunB/Fra-1 heterodimer in normal human keratinocytes activated the beta4-integrin promoter, while coexpression of HPV8 E2 reverted the JunB/Fra-1 effect. In summary, we identified a novel mechanism of human beta4-integrin regulation that is specifically targeted by the HPV8 E2 protein mimicking transcriptional conditions of differentiation. This may explain the early steps of how HPV8 commits its host cells to the differentiation process required for the viral life cycle.

Seroreactivity of 38 Human Papillomavirus Types in Epidermodysplasia Verruciformis Patients, Relatives, and Controls

The Journal of Investigative Dermatology. Mar, 2010  |  Pubmed ID: 19924140

Epidermodysplasia verruciformis (EV) is a rare recessive genodermatosis characterized by high susceptibility to infections with human papillomaviruses (HPVs) of genus beta. Knowledge about seroreactivity against HPV in these patients and their first-degree relatives is scarce. Using multiplex serology, we analyzed antibodies to 38 HPV types from five genera in 32 EV patients, 22 first-degree relatives, and 64 and 44 age- and sex-matched, non-related, healthy controls, respectively. EV patients showed higher seroprevalences than non-related controls with statistically significant odds ratios (ORs) for 5 of 10 investigated alpha (OR range 6.9-21.3), all 16 beta (OR range 12.3-61.3), 3 of 9 gamma (OR range 6.4-11.7), and 1 of 2 micro HPVs (OR 5.8). In comparison to their relatives, antibodies in EV patients were significantly more prevalent for 4 of 16 beta HPVs (OR range 12.5-25.6), but for none of the other genera. A significantly increased seroprevalence in relatives compared with their controls was only seen for HPV 5 (OR 22.1). The considerably elevated HPV seroprevalence in EV patients, especially for beta papillomaviruses (PVs), reflects the high viral load described for these individuals. Whether the observed differences between relatives and healthy controls depend on heterozygosity for EV-associated alleles requires further investigation.

High Prevalence of Bevirimat Resistance Mutations in Protease Inhibitor-resistant HIV Isolates

AIDS (London, England). Mar, 2010  |  Pubmed ID: 19926962

Bevirimat is the first drug of a new class of antivirals that hamper the maturation of HIV. The objective of this study was to evaluate the sequence variability of the gag region targeted by bevirimat in HIV subtype-B isolates.

Expression of Proliferative Biomarkers in Anal Intraepithelial Neoplasia of HIV-positive Men

Journal of the American Academy of Dermatology. Sep, 2010  |  Pubmed ID: 20006407

Anal intraepithelial neoplasia (AIN), a human papillomavirus (HPV)-associated precursor lesion of anal carcinoma, is highly prevalent among HIV-infected individuals, especially in men having sex with men (MSM). Early diagnosis and treatment of AIN might prevent development of anal cancer.

The Evolution of Protease Mutation 76V is Associated with Protease Mutation 46I and Gag Mutation 431V

AIDS (London, England). Mar, 2010  |  Pubmed ID: 20139751

Recently, first-line lopinavir failure was observed due to protease mutation 76V. In the present study, we found 76V associated with protease mutation 46I and gag cleavage-site mutation 431V. Longitudinal analysis of patients failing protease inhibitor therapies demonstrated that 76V strictly occurs either together with 46I and/or 431V or in HIV isolates already harbouring one of both mutations. Therefore, all three mutations seem to cooperate in terms of protease inhibitor resistance.

Evolution of Protease Inhibitor Resistance in the Gag and Pol Genes of HIV Subtype G Isolates

The Journal of Antimicrobial Chemotherapy. Jul, 2010  |  Pubmed ID: 20430786

To analyse HIV Gag cleavage site (CS) and non-CS mutations in HIV non-B isolates from patients failing antiretroviral therapy.

No Evidence That Human Papillomavirus is Responsible for the Aggressive Nature of Recessive Dystrophic Epidermolysis Bullosa-associated Squamous Cell Carcinoma

The Journal of Investigative Dermatology. Dec, 2010  |  Pubmed ID: 20739945

Multicenter Study of the Association Between Betapapillomavirus Infection and Cutaneous Squamous Cell Carcinoma

Cancer Research. Dec, 2010  |  Pubmed ID: 21098702

Human papillomaviruses (betaPV) from the beta genus cannot be classified according to their oncogenicity due to a paucity of information. This study evaluates the association between betaPV infection and cutaneous squamous cell carcinoma in conjunction with measures of UV exposure and susceptibility. We performed case-control studies in the Netherlands, Italy, and Australia, countries with profoundly different UV exposures. The presence of 25 betaPV types in eyebrow hair follicles was determined using a highly sensitive HPV DNA genotyping assay, and antibodies for the 15 most prevalent betaPV types in a total of 689 squamous cell carcinoma cases and 845 controls were detected using multiplex serology. Multivariate logistic regression models were used for case-control comparisons and interaction analyses. BetaPV DNA was detected in eyebrow hairs of more than 90% of all participants. The presence of betaPV DNA was associated with an increased risk of squamous cell carcinoma in the Netherlands (OR = 2.8; 95% CI 1.3-5.8) and Italy (OR = 1.7; 95% CI 0.79-3.6), but not in Australia (OR = 0.91; 95% CI 0.53-1.6). Seropositivity for betaPV in controls ranged between 52% and 67%. A positive antibody response against 4 or more betaPV types was associated with squamous cell carcinoma in Australia (OR = 2.2; 95% CI 1.4-3.3), the Netherlands (OR = 2.0; 95% CI 1.2-3.4) and fair-skinned Italians (OR = 1.6, 95% CI 0.94- 2.7). The association between UV susceptibility and squamous cell carcinoma was stronger in betaPV-seropositive people. These combined data support the hypothesis that betaPV may play a role in the development of cutaneous squamous cell carcinoma.

Keratinocyte-specific Stat3 Heterozygosity Impairs Development of Skin Tumors in Human Papillomavirus 8 Transgenic Mice

Cancer Research. Oct, 2010  |  Pubmed ID: 20876801

Human papillomaviruses (HPV) of the genus β are thought to play a role in human skin cancers, but this has been difficult to establish using epidemiologic approaches. To gain insight into the transforming activities of β-HPV, transgenic mouse models have been generated that develop skin tumors. Recent evidence suggests a central role of signal transducer and activator of transcription 3 (Stat3) as a transcriptional node for cancer cell-autonomous initiation of a tumor-promoting gene signature associated with cell proliferation, cell survival, and angiogenesis. Moreover, high levels of phospho-Stat3 have been detected in tumors arising in HPV8-CER transgenic mice. In this study, we investigate the in vivo role of Stat3 in HPV8-induced skin carcinogenesis by combining our established experimental model of HPV8-induced skin cancer with epidermis-restricted Stat3 ablation. Stat3 heterozygous epidermis was less prone to tumorigenesis than wild-type epidermis. Three of the 23 (13%) Stat3(+/-):HPV8 animals developed tumors within 12 weeks of life, whereas 54.3% of Stat3(+/+):HPV8 mice already exhibited tumors in the same observation period (median age for tumor appearance, 10 weeks). The few tumors that arose in the Stat3(+/-):HPV8 mice were benign and never progressed to a more malignant phenotype. Collectively, these results offer direct evidence of a critical role for Stat3 in HPV8-driven epithelial carcinogenesis. Our findings imply that targeting Stat3 activity in keratinocytes may be a viable strategy to prevent and treat HPV-induced skin cancer.

Quantification of Beta-human Papillomavirus DNA by Real-time PCR

Nature Protocols. 2010  |  Pubmed ID: 20010722

Quantitative PCR with hybridization probes allows the reliable quantification of viral DNA sequences in clinical samples with a dynamic range and sensitivity that cannot be achieved with other methods. The technical background for the establishment of protocols is described and established protocols are presented to estimate the viral load per cell of frequently occurring betapapillomaviruses (HPV5, -8, -15, -20, -23, -24, -36 and -38) in skin tumors, healthy skin and hair bulbs. This approach accurately adjusts dilution series of reference DNA of different viral types relative to pUC18, which is crucial for comparative analyses and for interlaboratory standardization. The type-specific determination of beta-HPV DNA loads is an important research tool toward discrimination between low-level persistence and activated possibly pathologically relevant infections. The analysis of 24 samples, starting with DNA extraction and followed by HPV typing and quantification of-on average-three of the described HPV types takes about 2 d.

Short Communication: Selection of Thymidine Analogue Resistance Mutational Patterns in Children Infected from a Common HIV Type 1 Subtype G Source

AIDS Research and Human Retroviruses. Mar, 2010  |  Pubmed ID: 20334563

In HIV-1, thymidine analogue mutations (TAMs) cluster in one of two groups (215Y, 41L, 210W, or 215F, 219E/Q), representing two independent mutational patterns (T215Y and T215F cluster, respectively). The mechanisms by which these pathways are selected are not fully understood. To investigate possible factors driving the selection of the TAMs, we analyzed the TAM patterns with regard to the respective treatment, viral load, and HLA in 18 children all infected from a common source of HIV-1 clade G virus and initially treated with zidovudine. The HIV reverse transcriptase sequences of 14/18 children carried at least one TAM. At first sampling date, the T215Y-linked pattern was observed in five cases and the T215F cluster was seen in nine. During the follow-up period, three patients changed their patterns. Children treated with identical NRTI combinations at the first sampling date developed different pathways. Under AZT/d4T therapies, an association was found between the HLA B*13 (in combination with HLA DRB1*0701) and the mutation T215Y. The mutation T215Y reverted in three out of four patients who discontinued AZT/d4T treatment. We speculate that in the context of these subtype G viruses, the development of the T215Y mutation may be strongly disfavored whereas the presence of HLA B*13 may counteract this effect and permit its development.

Prevalence and Characteristics of Hepatitis B and C Virus Infections in Treatment-naïve HIV-infected Patients

Medical Microbiology and Immunology. Feb, 2011  |  Pubmed ID: 20853118

In HIV-infected treatment-naïve patients, we analyzed risk factors for either chronic hepatitis B (HBV) infection, occult HBV infection (OHBV) or a positive hepatitis C (HCV) serostatus. A total of 918 patients of the RESINA-cohort in Germany were included in this study. Before initiating antiretroviral therapy, clinical parameters were collected and blood samples were analyzed for antibodies against HIV, HBV and HCV, HBs antigen and viral nucleic acids for HIV and HBV. Present or past HBV infection (i.e. HBsAg and/or anti-HBc) was found in 43.4% of patients. HBsAg was detected in 4.5% (41/918) and HBV DNA in 6.1% (34/554), resulting in OHBV infection in 2.9% (16/554) of patients. OHBV infection could not be ruled out by the presence of anti-HBs (50.1%) or the absence of all HBV seromarkers (25%). A HCV-positive serostatus was associated with the IVDU transmission route, non-African ethnicity, elevated liver parameters (ASL or GGT) and low HIV viral load. Replicative HBV infection and HCV-positive serostatus both correlated with HIV resistance mutations (P = 0.001 and P = 0.028). HBV and HCV infection are frequent co-infections in HIV treatment-naive patients. These co-infections influence viral evolution, clinical parameters and serological markers. Consequently, HIV patients should routinely be tested for HBV and HCV infection before initiating HIV treatment. OHBV infection constituted almost half of all HBV infections with detectable HBV DNA. Due to a lack of risk factors indicating OHBV infection, HBV diagnosis should not only include serological markers but also the detection of HBV DNA.

The E2 Protein of Human Papillomavirus Type 8 Increases the Expression of Matrix Metalloproteinase-9 in Human Keratinocytes and Organotypic Skin Cultures

Medical Microbiology and Immunology. May, 2011  |  Pubmed ID: 21274725

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.

Skin Tumor Formation in Human Papillomavirus 8 Transgenic Mice is Associated with a Deregulation of Oncogenic MiRNAs and Their Tumor Suppressive Targets

Journal of Dermatological Science. Oct, 2011  |  Pubmed ID: 21763111

Dysregulation of microRNA (miRNA) expression is regularly found in various types of cancer and contributes to tumorigenic processes. However, little is known about miRNA expression in non-melanoma skin cancer in which a pathogenic role of beta human papillomaviruses (HPV) is discussed. A carcinogenic potential of beta HPV8 could be demonstrated in a transgenic mouse model, expressing all early genes of HPV8 (HPV8-CER). A single UVA/B-dose induced oncogene expression and led to papilloma growth within three weeks.

No Evidence for a Role of Xenotropic Murine Leukaemia Virus-related Virus and BK Virus in Prostate Cancer of German Patients

Medical Microbiology and Immunology. Sep, 2011  |  Pubmed ID: 21898167

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.

Beta-papillomavirus DNA Loads in Hair Follicles of Immunocompetent People and Organ Transplant Recipients

Medical Microbiology and Immunology. Jul, 2011  |  Pubmed ID: 21792749

There is increasing evidence of an association between human papillomaviruses (HPV) of the beta-genus (beta-PV) and the development of cutaneous squamous cell carcinoma (SCC). The viral DNA load may be an important determinant of pathogenicity, but there are currently no baseline epidemiological data relating to load in people without SCC. We investigated DNA-loads of eight beta-PV types previously associated with risk of SCC. We collected eyebrow hairs from immunocompetent people (ICP) and organ transplant recipients (OTR), determined load by quantitative PCR and obtained demographic, phenotypic, and sun exposure information. Viral loads for ICP from Australia (n = 241) and Italy (n = 223) and OTR from across Europe (n = 318) spanned seven orders of magnitude. The median loads for all types were below one viral DNA copy per 60 cells and were highest for HPV5, HPV8 and HPV20. None of the populations had consistently higher viral loads for all 8 types. However, a higher proportion of OTR were in the top deciles of viral load distributions for six of the eight beta-PV types examined. In a nested analysis of Italian OTR and ICP, this finding was significant for six beta-PV types and cumulative load. Increasing age was significantly associated with higher viral loads in Australia, and there was a weak trend for higher loads with the time elapsed since transplantation in the OTR. We observed a wide distribution of beta-PV loads with OTR significantly more likely to have the highest viral loads. Thus, viral loads may be an important contributor to the higher risk of SCC in OTR.

Mutational Patterns in the Frameshift-regulating Site of HIV-1 Selected by Protease Inhibitors

Medical Microbiology and Immunology. Dec, 2011  |  Pubmed ID: 22200908

Sustained suppression of viral replication in HIV-1 infected patients is especially hampered by the emergence of HIV-1 drug resistance. The mechanisms of drug resistance mainly involve mutations directly altering the interaction of viral enzymes and inhibitors. However, protease inhibitors do not only select for mutations in the protease but also for mutations in the precursor Gag and Pol proteins. In this study, we analysed the frameshift-regulating site of HIV-1 subtype B isolates, which also encodes for Gag and Pol proteins, classified as either treatment-naïve (TN) or protease inhibitor resistant (PI-R). HIV-1 Gag cleavage site mutations (G435E, K436N, I437V, L449F/V) especially correlated with protease inhibitor resistance mutations, but also Pol cleavage site mutations (D05G, D05S) could be assigned to specific protease resistance profiles. Additionally, two Gag non-cleavage site mutations (S440F, H441P) were observed more often in HIV-1 isolates carrying protease resistance mutations. However, in dual luciferase assays, the frameshift efficiencies of specific clones did not reveal any effect from these mutations. Nevertheless, two patterns of mutations modestly increased the frameshift rates in vitro, but were not specifically accumulating in PI-resistant HIV-1 isolates. In summary, HIV-1 Gag cleavage site mutations were dominantly selected in PI-resistant HIV-1 isolates but also Pol cleavage site mutations influenced resistance profiles in the protease. Additionally, Gag non-cleavage site mutations accumulated in PI-resistant HIV-1 isolates, but were not related to an increased frameshift efficiency.

Upregulation of Lipocalin-2 in Human Papillomavirus-positive Keratinocytes and Cutaneous Squamous Cell Carcinomas

The Journal of General Virology. Feb, 2011  |  Pubmed ID: 21048034

It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) type 8 in keratinocytes induces cell invasion and accelerated differentiation. Looking for cellular genes deregulated by HPV-8 E7, lipocalin-2 was identified as being upregulated in these cells by cDNA microarray analysis. Lipocalin-2 is known to be overexpressed in many human cancers and is implicated in the regulation of cell proliferation, differentiation and apoptosis. In this study, increased levels of lipocalin-2 were observed in extracts from HPV-8 E7-positive keratinocytes and from keratinocytes expressing E7 of HPV-1, -4, -5, -15, -20 and -38, but not of HPV-16. Similar results were obtained when measuring secreted lipocalin-2 in the supernatants of the cell cultures. Lipocalin-2 expression was associated with cell differentiation in keratinocyte monolayers and in organotypic skin cultures. It was found in the uppermost layers of HPV-5, -8, -15, -16, -20 and 38 E7-expressing but not low-risk HPV-1 and -4 E7-expressing keratinocytes. Immunohistochemical staining of HPV-positive and -negative human skin squamous cell carcinomas (SCCs) revealed lipocalin-2 expression mostly in differentiated, filaggrin-positive areas of 15 out of 17 HPV-positive and three out of nine HPV-negative SCCs. These data indicate that lipocalin-2 expression correlates with HPV positivity of cutaneous SCCs.

Enhanced StefinA and Sprr2 Expression During Papilloma Formation in HPV8 Transgenic Mice

Journal of Dermatological Science. May, 2011  |  Pubmed ID: 21458245

The human papillomavirus type 8 (HPV8) is associated with the development of non-melanoma skin cancer. Transgenic mice expressing the complete early gene region of HPV8 (E6/E7/E1/E2/E4=CER) or E6 separately under the control of the keratin14 promoter spontaneously developed papillomas characterized by varying degrees of epidermal dysplasia. Papilloma growth could be synchronized by a single UVA/B irradiation of the skin, which led to the development of papillomas within three weeks.

HIV Prevalence and Route of Transmission in Turkish Immigrants Living in North-Rhine Westphalia, Germany

Medical Microbiology and Immunology. Nov, 2011  |  Pubmed ID: 21461764

The high number of Turkish immigrants in the German state North-Rhine Westphalia (NRW) compelled us to look for HIV-infected patients with Turkish nationality. In the AREVIR database, we found 127 (107 men, 20 women) Turkish HIV patients living in NRW. In order to investigate transmission clusters and their correlation to gender, nationality and self-reported transmission mode, a phylogenetic analysis including pol gene sequences was performed. Subtype distribution and the number of HIV drug resistance mutations in the Turkish patient group were found to be similar to the proportion in the non-Turkish patients. Great differences were observed in self-reported mode of transmission in the heterosexual Turkish male subgroup. Neighbour-joining tree of pol gene sequences gave indication that 59% of these reported heterosexual transmissions cluster with those of men having sex with men in the database. This is the first study analysing HIV type distribution, drug resistance mutations and transmission mode in a Turkish immigrant population.

Merkel Cell Polyomavirus Infection in HIV-positive Men

Archives of Dermatology. Apr, 2011  |  Pubmed ID: 21482890

To evaluate Merkel cell polyomavirus (MCPyV) DNA prevalence and load among men with human immunodeficiency virus (HIV) (hereafter referred to as HIV-positive men) and among healthy male control subjects.

Positive Correlation Between Merkel Cell Polyomavirus Viral Load and Capsid-specific Antibody Titer

Medical Microbiology and Immunology. Feb, 2012  |  Pubmed ID: 21614514

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 < 0.0001). The study results are consistent with previous findings suggesting that the skin is primary site of chronic MCPyV infection in healthy adults and suggest that the magnitude of an individual's seroresponsiveness against the MCPyV virion generally reflects the overall MCPyV DNA load across wide areas of the skin. In light of previous reports indicating a correlation between MCC and strong MCPyV-specific seroresponsiveness, this model suggests that poorly controlled chronic MCPyV infection might be a risk factor in the development of MCC.

Characterization of Beta Papillomavirus E4 Expression in Tumours from Epidermodysplasia Verruciformis Patients and in Experimental Models

Virology. Feb, 2012  |  Pubmed ID: 22217391

This study provides a first characterisation of β-HPV life-cycle events in tumours abscised from EV patients (the human model of β-HPV-induced skin cancer), and shows how changes in E4 expression patterns relate to disease severity. β-HPV life-cycle has also been reconstructed in organotypic raft cultures created using EV-derived keratinocytes. In EV lesions and raft cultures, abundant cytoplasmic E4 expression was detectable in differentiating cells along with viral genome amplification as reported for other HPV types. E4 expression was also seen in PCNA-positive basal cells in some EV skin cancers as well as in tumours from HPV8CER (Complete Early Region) transgenic mice. In these lesions, E4 staining extended throughout the full thickness of the epithelium and was apparent in the markedly atypical cells. The loss of such staining at the tumour border suggests a distinct type of E4 dysregulation that may be exploited as a marker of viral expression during β-HPV-associated skin cancer progression.

Human Papillomavirus Oncogene MRNA Testing for the Detection of Anal Dysplasia in HIV-positive Men Who Have Sex with Men

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. Jan, 2012  |  Pubmed ID: 22261122

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.

Epidemiology of Transmitted Drug Resistance in Chronically HIV-Infected Patients in Germany: The RESINA Study 2001-2009

Intervirology. 2012  |  Pubmed ID: 22286886

Objectives: Transmitted HIV drug resistance may impair treatment efficacy of combination antiretroviral therapy (ART). This study describes the epidemiology of transmitted resistance in chronically infected patients. Methods: In a prospective multicenter trial in Nordrhein-Westfalen, Germany, transmitted drug resistance was determined by genotypic resistance testing in patients on initiation of first-line ART. Results: From 2001 to 2009, 2,078 patients were enrolled in the study. 79.9% were male, 81.2% were Caucasians, and a homosexual transmission mode was found in 51.3%. Of these patients, 41.5% were at the stage of AIDS, median CD4 cell count was 230/μl, and median viral load was 64.466 copies/ml. Transmitted drug resistance mutations were seen in 9.2% (95% CI, 7.9-10.4). Resistance in the nucleoside reverse transcriptase inhibitor class was found in 5.8% (4.8-6.8), in the nonnucleoside reverse transcriptase inhibitor class in 2.8% (2.1-3.6), and in the protease inhibitor class in 2.7% (2.0-3.4). After a continuous increase to a level above 10% in the years 2006 and 2007, a decline of drug resistance prevalence followed in 2008 and 2009. Conclusions: Transmitted HIV drug resistance was found in around 10% of chronically infected patients in Germany who started their ART. We showed a moderate decline of the prevalence of mutant virus strains in recent years. Further surveillance of this phenomenon is mandatory.

The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

PloS One. 2012  |  Pubmed ID: 22292024

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+) CD57(+) CD7(-) phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+) T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.