We found that recircularized high-risk (type 16 and 18) and low-risk mucosal (type 6b and 11) and cutaneous (type 5 and 8) human papillomavirus (HPV) genomes replicate readily when delivered into U2OS cells by electroporation. The replication efficiency is dependent on the amount of input HPV DNA and can be followed for more than 3 weeks in proliferating cell culture without selection. Cotransfection of recircularized HPV genomes with a linear G418 resistance marker plasmid has allowed subcloning of cell lines, which, in a majority of cases, carry multicopy episomal HPV DNA. Analysis of the HPV DNA status in these established cell lines showed that HPV genomes exist in these cells as stable extrachromosomal oligomers. When the cell lines were cultivated as confluent cultures, a 3-to 10-fold amplification of the HPV genomes per cell was induced. Two-dimensional (2D) agarose gel electrophoresis confirmed amplification of mono-and oligomeric HPV genomes in these confluent cell cultures. Amplification occurred as a result of the initiation of semiconservative two-dimensional replication from one active origin in the HPV oligomer. Our data suggest that the system described here might be a valuable, cost-effective, and efficient tool for use in HPV DNA replication studies, as well as for the design of cell-based assays to identify potential inhibitors of all stages of HPV genome replication.Due to their association with distinctive human cancers, human papillomaviruses (HPVs) are widely studied. Papillomaviruses have been phylogenetically grouped into genera, species, types, subtypes, and variants (11), and more than 100 HPV types have been identified thus far. The best-characterized ␣-genus HPVs are associated with infections of the mucosal epithelia that lead to the induction of benign tumors. These viruses are divided into high-risk types (e.g., high-risk HPV type 16 [HR-HPV-16] and -18), which have the capability of inducing anogenital malignancies, and low-risk types (e.g., LR-HPV-6 and -11), which induce hyperproliferative mucosal lesions and are rarely associated with malignancy. Mucosal HPV infections tend to clear on their own, but in some cases, latent infection could be established and may persist for years. Cutaneous ␤-genus HPV infections are highly prevalent in the general population and tend to persist (14). Vaccines based on virus-like particles made up of the capsid protein L1 have been developed against and Gardasil/Silgard [Merck Research Laboratories]). Because these virus types are responsible for only a portion of all HPV-induced malignant and benign tumors, a clear need exists for vaccines or antivirals against a broader spectrum of pathogenic HPV types.Despite the differences in viral pathogenesis, progeny virion production invariably depends on cell differentiation and occurs only in terminally differentiated keratinocytes. HPVs require the host's replication machinery to reproduce their genomes, and these viruses have developed a unique replication strategy that is adapted to keratinocyte di...

BackgroundThe skin proteome contains valuable information on skin condition, but also on how skin may evolve in time and may respond to treatments. Despite the potential of measuring regulatory-, effector- and structural proteins in the skin for biomarker applications in clinical dermatology and skin care, convenient diagnostic tools are lacking. The aim of the present study was to develop a highly versatile and non-invasive diagnostic tool for multiplex measurements of protein biomarkers from the surface of skin.ResultsThe Transdermal Analyses Patch (TAP) is a novel molecular diagnostic tool that has been developed to capture biomarkers directly from skin, which are quantitatively analyzed in spot-ELISA assays. Optimisation of protocols for TAP production and biomarker analyses makes TAP measurements highly specific and reproducible. In measurements of interleukin-1α (IL-1α), IL-1 receptor antagonist (IL-1RA) and human β-defensin (hBD-1) from healthy skin, TAP appears far more sensitive than skin lavage-based methods using ELISA. No side-effects were observed using TAP on human skin.ConclusionTAP is a practical and valuable new skin diagnostic tool for measuring protein-based biomarkers from skin, which is convenient to use for operators, with minimal burden for patients.

TAP measurements, especially IL-1α, IL-1RA, and hBD-1, from the skin surface were sensitive enough for monitoring dynamic changes in the skin in the two models of skin perturbation. We conclude that TAP holds promise for non-invasively unraveling the dynamics of processes related to skin perturbation and repair.

There is a need for non-invasive diagnostic tools that can objectively measure psoriasis activity and that can be used to monitor therapeutic effects of psoriasis treatment. This study aimed to determine whether non-invasive measurements of proteins from psoriasis lesional skin can be used to assess disease severity and to measure treatment efficacy.Using FibroTx TAP technology for protein-measurements directly from the surface of skin, clear differences in levels of IL-1α, IL-1RA and CXCL-1/2 were found between psoriasis lesional skin and non-lesional skin. No clear correlations were found between FibroTx TAP measurements and PASI scoring, with the exception of a mild correlation between CXCL-1/2 and scaling. Similarly, no clear correlations were found between FibroTx TAP measurements and ultrasound measurements of skin, with the exception of a weak correlation between IL-1RA and SLEB thickness. Monitoring IL-1RA and CXCL-1/2 on skin lesions undergoing narrow-band UVB phototherapy clearly reflected normalisation of skin.Skin-surface measurements of IL-1RA and CXCL-1/2 have potential for assessing severity of psoriasis and for monitoring treatment efficacy. Measurements of IL-1RA and CXCL-1/2 displayed a disease profile distinct from PASI or sonography, thus confirming that measuring the ‘molecular root’ of inflammation has value for scoring disease severity in its own right.

Background: There is a need for non-invasive diagnostic tools that can objectively measure psoriasis activity and that can be used to monitor therapeutic effects of psoriasis treatment. This study aimed to determine whether non-invasive measurements of proteins from psoriasis lesional skin can be used to assess disease severity and to measure treatment efficacy. Results: Using FibroTx TAP technology for protein-measurements directly from the surface of skin, clear differences in levels of IL-1a, IL-1RA and CXCL-1/2 were found between psoriasis lesional skin and non-lesional skin. No clear correlations were found between FibroTx TAP measurements and PASI scoring, with the exception of a mild correlation between CXCL-1/2 and scaling. Similarly, no clear correlations were found between FibroTx TAP measurements and ultrasound measurements of skin, with the exception of a weak correlation between IL-1RA and SLEB thickness. Monitoring IL-1RA and CXCL-1/2 on skin lesions undergoing narrow-band UVB phototherapy clearly reflected normalisation of skin. Conclusions: Skin-surface measurements of IL-1RA and CXCL-1/2 have potential for assessing severity of psoriasis and for monitoring treatment efficacy. Measurements of IL-1RA and CXCL-1/2 displayed a disease profile distinct from PASI or sonography, thus confirming that measuring the ‘molecular root’ of inflammation has value for scoring disease severity in its own right.