High-risk HPV types in Tunisia. A pilot study reveals an unexpectedly high prevalence of types 58 and 82 and lack of HPV 18 among female prostitutes.

"High-risk" HPVs (HR-HPV) have sharply different prevalences and there is evidence to suggest this may vary according to regions. Accurate description of HR-HPV circulation is a key feature for the rational design of prevention and screening campaigns. To gain insight into HR-HPV circulation in Tunisia, a pilot study was carried out on 64 healthy prostitutes working in the Tunis area. HPV detection and typing were carried out by MY09/MY11 PCR and restriction fragment length polymorphism (RFLP). A selected set of samples was also assayed by Gp5+/Gp6+ PCR and typed by direct sequencing. Out of 64 women, 28 were HPV positive. HPV-16 and HPV-58, both members of the genus Alpha-Papillomavirus, species 9, accounted for 10 and 7 cases with a prevalence rate of 38% and 27%, respectively. HPV-82, a member of species 5, ranked third with four cases (approximately 15%). Types 31, 33, 35; all members of species 9 were each detected once (approximately 4%) while neither HPV-18 nor related members of species 7 were detected. Type 72 and 83, both members of species 3, were the only low-risk types, each detected only once (4% each). Two samples could not be typed. The prevalence of HPV types appeared sharply different from that of neighboring areas. Should the existence of epidemiological pockets be confirmed by larger, more detailed studies, screening and vaccine campaigns will have to be designed carefully taking into account the actual epidemiological context of the target population.

Ectopic deposition of melanin pigments as detoxifying mechanism: a paradigm for basal nuclei pigmentation.

Melanins are UV shielding pigments found in skin and other light exposed tissues. However, a kind of melanin, named neuromelanin (NM), is found in those deep brain loci that degenerate in Parkinson's disease (PD), where no such a function may be imagined. The NM synthetic pathway, different from the one of eumelanin based on tyrosinase, is still obscure as well as its physiological function. Here we show that under conditions of excess of toxic quinone concentration, nonmelanocytic cell strains (i.e., primary keratinocytes) may accumulate a dark cytoplasmatic pigment that proved to be a melanin. The ability of pigment deposition, possibly driven by peroxidases, is restricted to diploid cells and increases cell survival acting as a sink for potentially hazardous quinones. We suggest that in the basal nuclei, exposed to high level of catecholaminergic neurotransmitters, NM deposition is a relevant antioxidant mechanism by trapping quinones and semiquinones, thus protecting neurons from accumulating damage over many years. In this perspective, just as a hypothesis, we may imagine that PD neuron degeneration is the consequence of a reduced/abrogated ability to produce neuromelanin.

Presence of bovine papillomavirus type 2 DNA and expression of the viral oncoprotein E5 in naturally occurring urinary bladder tumours in cows.

Samples of neoplastic and normal urothelium were obtained from cows originating from areas of southern Italy, a region in which chronic enzootic haematuria is endemic and bracken fern infestation is widespread. Specimens were analysed for bovine papillomavirus type 2 (BPV-2) DNA, BPV-2 E5 expression and telomerase activity. A total of 46 of 60 tumours and 17 of 34 normal bladder mucosa samples harboured BPV-2 DNA. Analysis of a subset of samples showed E5 protein expression and telomerase activity in tumour tissue only. No normal samples positive for BPV DNA showed E5 protein expression or telomerase activity, suggesting the presence of DNA in a latent state. Taken together, these data on naturally occurring bovine bladder tumours corroborate the hypothesis of their virus origin.

DNA vaccines against HPV-16 E7-expressing tumour cells.

BACKGROUND: Genetic immunisation induces the endogenous production of the encoded antigens, which favours their presentation by MHC class I molecules. The E7 protein from "high risk" Human Papillomavirus (HPV) is constitutively expressed in cervical cancer and represents a target for immunotherapy. MATERIALS AND METHODS: Several E7-encoding DNA vaccines were constructed including unmodified E7 and E7 fused to ubiquitin or to the Invariant chain in order to increase the presentation of E7-derived peptides by MHC class I or II molecules, respectively. These vaccines were administered i.m. to C57BL/6 mice that were subsequently challenged with E7-positive tumour cell lines expressing different levels of MHC class I molecules. RESULTS: The E7-Ii fusion sequence protected a number of animals from tumour challenging. No differences were associated with the MHC class I status of the challenging cell lines. CONCLUSION: Engineering the intracellular pathway for antigen presentation is able to produce a valid therapeutic response even against tumours with down-regulated MHC class I.

Tyrosinase protects human melanocytes from ROS-generating compounds.

The effects of two tetrahydroisoquinolines (TIQs), tetrahydropapaveroline (THP) and salsolinol (SAL), on human primary melanocytes were studied. These compounds are naturally occurring alkaloids deriving from the condensation of dopamine with aldehydes. The effects on the viability were studied by treating the cells with variable concentration of THP or SAL; both TIQs were well tolerated up to roughly 30 micro M. At higher concentrations, THP became overtly toxic while SAL showed no cytotoxic effect up to 100 micro M. TIQs treatment determined an impairment of intracellular activity of antioxidant enzymes, like SOD, DT-diaphorase, and glutathione peroxidase. A decrease of alpha-ketoglutarate dehydrogenase activity was also evidenced following TIQs treatment; a very strong diminution was found in THP-treated cells, whose viability was highly decreased. Both TIQs increased tyrosinase-specific mRNA transcription followed, in the case of SAL, by an activation of tyrosinase. In the presence of tyrosinase inhibitors TIQs treatment resulted in a sharp cytotoxic effect even at concentrations normally well tolerated. Taken together these data suggest that tyrosinase represents an outstanding protective mechanism against ROS-generating compounds, once primary detoxifying mechanisms are impaired or not available.

Cytotoxicity of dopamine-derived tetrahydroisoquinolines on melanoma cells.

Tetrahydroisoquinolines (TIQs) are endogenous alkaloid compounds detected in urine, central nervous system and some peripheral tissues in Mammalia. No data are at present available on TIQ levels in skin, although in vitro biochemical evidences indicate that they may undergo auto-oxidation with production of reactive oxygen species or may be enzymatically converted into melanin pigments. The effect of two catechol-bearing TIQs, salsolinol (SAL) and tetrahydropapaveroline (THP), on the viability of melanotic or amelanotic melanoma cell lines was investigated. Both SAL and THP were well tolerated up to roughly 30 microM and became overtly toxic at higher concentrations, with SAL being better tolerated than THP. Intracellular activity of some antioxidant enzymes, tyrosinase and alpha-ketoglutarate dehydrogenase was also evaluated to assess the cell response to oxidative and metabolic challenge of TIQs treatment. Catalase and superoxide dismutase pre-treatment only partially prevented TIQs toxicity while a complete protection was obtained with N-acetylcysteine and GSH. TIQs were able to provoke upregulation of the scavenging enzymes catalase and DT-diaphorase and to determine a decrease of the alpha-ketoglutarate dehydrogenase activity. SAL and THP enhanced tyrosinase activity and melanin production, suggesting that they were indeed tyrosinase substrates leading to melanin formation. The results support the evidence that TIQs were toxic toward melanoma cells, leading to their death by necrosis. TIQs toxicity was likely due to increased oxidative stress by generation of reactive oxygen species and oxidative metabolites. Our study represents an intent to furnish an additional contribution for the comprehension of catechol cytotoxicity.

HPV16 and HPV18 in genital tumors: Significantly different levels of viral integration and correlation to tumor invasiveness.

The integration of the high-risk HPV16 and HPV18 types into the cell genome is considered an important step in malignant transformation. The relationship between the physical status of the virus and clinical/pathological parameters was studied by type-specific and multiplex PCR for E6, E2, and E1 sequences in 86 genital tumors from different sites, consisting of 69 invasive carcinomas (including 5 microinvasive carcinomas), 9 carcinomas in situ, 6 severe dysplasias, and 2 moderate dysplasias. Forty tumors contained HPV16 (46.6%), 7 HPV18 (8.1%), and 39 both viruses (45.3%). HPV16 DNA was found either as pure integrant (35.4%), or pure episome (36.7%), or a mixture of both (27.8%). Conversely, all 46 lesions containing HPV18 showed pure integrated forms. The physical status of both types was not related to the tumor site, the tumor/node/metastasis stage, or the histological differentiation grade of the invasive carcinomas. HPV16 integration was significantly associated with invasiveness. Interestingly, in double infections when HPV16 coexisted with HPV18, its genome was found more frequently in episomal form than in single infections where, conversely, it was mostly integrated (P < 0.0001), suggesting a sort of competition for cell integration sites. The complete HPV18 integration, even in pre-neoplastic lesions, indicates a different behavior in genital transformation compared with HPV16 and may reflect a major aggressiveness of this viral type. In conclusion, virus typing in conjunction with the evaluation of the integration status may provide a better prognostic evaluation together with an improved diagnosis.