P. Di Bonito

Aberrant expression of VEGF-C is related to grade of cervical intraepithelial neoplasia (CIN) and high risk HPV, but does not predict virus clearance after treatment of CIN or prognosis of cervical cancer

Aims: Increased angiogenesis leads to invasion in cervical cancer. Vascular endothelial growth factors (VEGFs) are involved in angiogenesis, but molecular links to the most important aetiological agent, human papillomavirus (HPV), need clarifying. Material/Methods: Archival samples—150 squamous cell carcinomas (SCCs) and 152 cervical intraepithelial neoplasia (CIN) lesions—were examined immunohistochemically for anti-VEGF-C antibody and for HPV by polymerase chain reaction (PCR). Follow up data were available for all SCC cases, and 67 CIN lesions were monitored with serial PCR to assess HPV clearance/persistence after treatment. Results: High risk (HR) HPV types were closely associated with CIN (odds ratio, 19.12; 95% confidence interval, 2.31 to 157.81) and SCC (27.25; 3.28 to 226.09). There was a linear increase of VEGF-C expression—weak in CIN1 and intense in CIN3 and SCC (20.49; 8.69 to 48.26). VEGF-C upregulation was a sensitive (93.5%; 95% CI, 90.1% to 96.9%) marker of HR-HPV type (4.70; 2.17 to 10.21), but lost its significance in multivariate regression—p16INK4a and survivin were equally strong independent predictors of HR-HPV. Aberrant expression of VEGF-C did not predict clearance/persistence of HR-HPV after treatment of CIN. In cervical cancer, VEGF-C had no prognostic value in univariate or multivariate survival analysis. After adjustment for HR-HPV, FIGO stage, age, and tumour grade, only FIGO stage and age remained independent prognostic predictors. Conclusions: VEGF-C is an early marker of cervical carcinogenesis, with linearly increasing expression starting from low grade CIN. VEGF-C expression is closely related to HR-HPV in cervical lesions, probably because of its p53 independent upregulation by the E6 oncoprotein of HR-HPV.

Immunomodulatory activity of a plant extract containing human papillomavirus 16-E7 protein in human monocyte-derived dendritic cells

This study reports the immunomodulatory activity on human Monocyte Derived Dendritic Cells (MDDCs) of a vaccine preparation shown to be effective against an HPV16-related tumour in an animal model. The vaccine is composed of extract from Nicotiana benthamiana leaves containing HPV16 E7 protein expressed by a potato virus X-derived vector (NbPVX-E7). The effect of the extract was evaluated on MDDC differentiation and maturation by monitoring the phenotypic expression of specific markers. The results show that NbPVX-E7 does not induce monocyte differentiation to dendritic cells, but does induce MDDC maturation. Plant extract does not influence MDDC-uptake of E7-FITC while it significantly improves the Ovalbumin-FITC uptake, considered as a model antigen. Importantly, NbPVX-E7-pulsed MDDCs/PBMCs are able to prime human blood-derived lymphocytes from healthy individuals to induce HPV16 E7-specific cytotoxic activity. This is a propaedeutic study for a possible use of E7-containing plant extract in human immunotherapy of HPV-related lesions.

Immunological characterization of Toscana virus proteins

Summary. The genome of Toscana virus (Bunyaviridae family, Phlebovirus genus) consists of three single stranded RNA segments (L, M, S), with negative polarity. The L and M segments contain a single ORF in viral complementary sense and the S segment contains two ORFs in “ambisense” orientation. The M segment codes for three proteins in 3′–5′ genomic orientation: a 30 kDa non structural protein and two 65 kDa glycoproteins, GN, and GC. In this paper we report the expression in E. coli of the S segment ORFs and of three regions of the L ORF. The expressed proteins were used to produce monospecific polyclonal antibodies in mice. By using these antibodies the N and the NSs proteins were unequivocally assigned to the S viral-complementary and viral-sense ORFs, respectively, and the L protein to the L ORF. We have found that like N and L proteins, NSs protein is associated with the viral nucleocapsids in mature virions, suggesting its possible involvement in early events of viral replication. NSs protein was also found associated with cellular polysomes. In virus-infected cells the anti-L antibodies recognized proteins shorter than the full-length L protein, possibly products of L subgenomic segments. Interestingly these defective products were not found in mature virions, suggesting specific mechanisms in virion assembly.

Down-regulated nucleoside diphosphate kinase nm23-H1 expression is unrelated to high-risk human papillomavirus but associated with progression of cervical intraepithelial neoplasia and unfavourable prognosis in cervical cancer

Objective: One of the factors leading to an invasive phenotype is the nm23 family of metastases-associated genes. Of the six known members, nm23-H1 is the most frequently studied potential anti-metastatic gene in cervical cancer. However, the possible molecular links to oncogenic human papillomavirus (HPV) are completely unexplored as yet. Materials and methods: As a part of the HPV-Pathogen Istituto Superiore di Sanità study, a series of 150 squamous cell carcinomas (SCCs) and 152 cervical intraepithelial neoplasia (CIN) lesions were examined by immunohistochemical staining for nm23-H1, and tested for HPV by polymerase chain reaction (PCR) with three sets of primers (MY09/11, GP5+/GP6+ and short PCR fragment). Follow-up data were available on all patients with SCC, and 67 CIN lesions were monitored by serial PCR for clearance or persistence of HPV after cone treatment. Results: A linear decrease (p = 0.001) was observed in nm23-H1 expression, starting from CIN1 (85% with normal expression), with the most dramatic down regulation on transition from CIN2 (70% normal) to CIN3 (39%) and further to SCC (25%). Reduced expression was associated with CIN3 or cancer at an odds ratio 8.72 (95% confidence interval 4.13 to 18.41). Nm23-H1 was of no use as a marker of the high-risk human papillomavirus (HR-HPV) type, and it did not predict clearance or persistence of HR-HPV after treatment of CIN. Importantly, nm23-H1 expression was a significant prognostic factor in cervical cancer, reduced expression being associated with lower survival (p = 0.022) in univariate analysis. In the multivariate (Cox) regression model, however, only the International Federation of Gynecology and Obstetrics stage (p = 0.001) and age (p = 0.011) remained independent prognostic predictors. Conclusions: Down-regulated nm23-H1 expression is markedly associated with progression from CIN2 to CIN3, and predicts poor prognosis in cervical cancer. Nm23-H1 down regulation is probably orchestrated by mechanisms independent of HR-HPV oncoproteins and is possibly related to the emergence of a proteolytic phenotype.

Oncogenic Papillomavirus and Polyomavirus in Water Environments: Is There a Potential for Waterborne Transmission?

Waterborne exposure to human viruses through contact with sewage-contaminated water environments can result in infections associated with a wide range of illnesses. Gastrointestinal symptoms are the most commonly encountered manifestations of waterborne viral illness. Respiratory diseases, neurological diseases and paralysis can also occur. Whether viral infections resulting in health outcomes like cancer might also be transmitted by the waterborne route is unknown. Recently, viruses belonging to two oncogenic groups—Human Papillomaviruses (HPVs) and Human Polyomaviruses (HPyVs)—have been detected in urban sewages worldwide. The latter have also been identified in other water environments. HPVs are epitheliotropic viruses responsible for several diseases of skin and mucosae, from common warts to squamous intraepithelial lesions that can either heal or progress to invasive carcinoma of the cervix, vulva, vagina, penis, anus or oropharynx. Human PyVs infect different tissues and organs, causing infections that are usually subclinical in immunocompetent individuals but can be serious in immunocompromised hosts. These pathogens belong to a family of DNA tumour viruses. Merkel cell polyomavirus, a HPyV identified in recent years, has attracted much attention due to its link with a rare and aggressive form of human cancer. Merkel cell carcinoma, the incidence of which has tripled over the past two decades. JC polyomavirus and BK polyomavirus are also potentially oncogenic. The observed abundance and wide dissemination of HPVs and HPyVs in water environments strongly suggest the need to shed light on the fate of these viruses in water environments and to elucidate their potential for waterborne transmission. Such information is essential for the improvement of wastewater management programs in terms of both sewage treatment and water quality surveillance.

A large spectrum of alpha and beta papillomaviruses are detected in human stool samples.

Human papillomaviruses (HPVs) have been detected in urban wastewaters, demonstrating that epitheliotropic viruses can find their way into sewage through the washing of skin and mucous membranes. Papillomavirus shedding through faeces is still an unexplored issue. The objective of the present study was to investigate the presence of HPVs in stool samples. We analysed 103 faecal specimens collected from hospitalized patients with diarrhoea using validated primers able to detect α, β and γ HPVs. PCR products underwent sequencing analysis and sequences were aligned to reference genomes from the Papillomavirus Episteme database. A total of 15 sequences were characterized from the faecal samples. Thirteen samples (12.6 %) were positive for nine genotypes belonging to the α and β genera: HPV32 (LR, α1), HPV39 (HR, α7), HPV44 (LR, α10), HPV8 (β1), HPV9, HPV23, HPV37, HPV38 and HPV120 (β2). Two putative novel genotypes of the β genus, species 1 and 2, were also detected. The tissue(s) of origin is unknown, since faeces can collect HPVs originating from or passing through the entire digestive system. To our knowledge, this is the first investigation on the occurrence and diversity of HPVs in faecal samples. Results from this study demonstrate that HPVs can find their way into sewage as a consequence of shedding in the faeces. This highlights the need for further studies aimed at understanding the prevalence of HPV in different water environments and the potential for waterborne transmission.

First detection of papillomaviruses and polyomaviruses in swimming pool waters: unrecognized recreational water-related pathogens?

Viral outbreaks associated with swimming pools have been described worldwide. The objective of this study was to examine the extent of viral contamination in indoor and outdoor swimming pools.