Genomic characterization of ten novel cutaneous human papillomaviruses from keratotic lesions of immunosuppressed patients.

Viral warts from immunosuppressed organ transplant recipients (OTR) persist over years and may progress into non-melanoma skin cancer. The types of human papillomaviruses (HPV) in such lesions are different from that seen in the general population. A subset of these lesions is not infected with the classical wart-associated HPV types. In order to gain a better understanding of the HPV types in those lesions, we isolated ten novel HPVs from persisting keratotic lesions of immunosuppressed OTRs by rolling circle amplification and subsequent long-template PCR. Additionally, we sequenced and characterized the whole genome of the ten novel HPV types. Phylogenetic analyses revealed that nine HPV types belonged to the genus Gammapapillomavirus (γ-PV) and one to the genus Betapapillomavirus. In a phylogenetic analysis using L1 fragments of human and non-human PV types, primate papillomaviruses and our novel HPV types nested within the genus γ-PV in a highly polyphyletic pattern. This study significantly broadens the knowledge concerning the diversity and evolution of the poorly known γ-PV types.

Co-expression of insulin-like growth factor-1 and interleukin-4 in an in vitro inflammatory model.

The ailment osteoarthritis (OA) has two aspects - inflammation and cartilage degradation - where combined transgene expression may offer an effective gene therapy. Our present study focuses on the co-expression of interleukin-4 (IL-4) and insulin-like-growth factor-1 (IGF-1), which specifically target inflammation and cartilage repair, respectively. In this study, we analyze the expression of IGF-1 and IL-4 from a single plasmid vector, where each gene is expressed through an independent promoter and enhancer sequence. Regenerative and anti-inflammatory effects of IGF-1 alone and of both IGF-1 and IL-4 were analyzed in an in vitro chondrocyte inflammatory model. Co-expression of both transgenes in primary chondrocytes was ascertained by immunoassays. Following stimulation with IL-1beta and TNFalpha, pro-inflammatory mediators as well as IGF-binding proteins were down-regulated more effectively in the presence of both genes to levels comparable to the non-stimulated control. Further, cartilage regeneration proteins type II collagen and proteoglycans were up-regulated in stimulated cells transfected with IGF-1 alone and in combination with IL-4. The co-expression of IGF-1 and IL-4 shows that both transgenes complement each other by effectively triggering cartilage regeneration and reducing inflammation. Use of combinatorial transgene expression offers a promising avenue in the area of gene therapy in OA.

Expression of canine interleukin-4 in canine chondrocytes inhibits inflammatory cascade through STAT6.

Interleukin-4 (IL-4) is a pleiotropic cytokine with broad spectrum of biological effects on target cells. This study deals with the mammalian expression of canine IL-4 (cIL-4) in canine articular chondrocytes (CAC) and its ability to down-regulate pro-inflammatory cytokines, enzyme mediators and their catabolites. We transfected cIL-4 in CAC which were then stimulated with canine recombinant IL-1beta and TNFalpha or left as untreated control CAC. The cIL-4 protein was detected by Western blot analysis and quantified by sandwich ELISA utilizing monoclonal and polyclonal antibodies raised against recombinant cIL-4. Pro-inflammatory cytokines and enzyme mediators were quantified by quantitative real-time PCR, and nitrite production was measured by a calorimetric assay. Our results show that cIL-4 is expressed in CAC as a 17kD protein, which inhibits various cytokines and inflammatory mediators when stimulated by IL-1beta and TNFalpha. Thus, cIL-4 expressed in CAC is biologically active and suppresses inflammatory mediators in vitro. Since STAT6 (signal transducer and activator of transcription 6) was solely expressed in transfected CAC and not at all detectable in control cells, it is likely that IL-4 exerts its anti-inflammatory effects through STAT6 signaling. These studies allowed us to identify the canine form of STAT6 which we have partially sequenced.

Quantification of cytokines and inflammatory mediators in a three-dimensional model of inflammatory arthritis.

Human recombinant IL-1beta and TNFalpha have been previously used to induce a cytokine response in canine chondrocytes. In order to establish this functional relation in a homologous system in vitro, we have developed both 2D and 3D models of inflammatory arthritis using canine recombinant cytokines in canine articular chondrocytes. IL-1beta and TNFalpha were cloned and subsequently expressed in Escherichia coli. The purified recombinant canine cytokines were used to simulate inflammation in vitro and the expression of typical inflammation markers such as proinflammatory cytokines (IL-1beta, IL-6, IL-8, GM-CSF and TNFalpha), enzyme mediators (MMP-3 MMP-13, iNOS, COX-2) and their catabolites (NO, PGE(2)) was measured. High expression of proinflammatory cytokines, enzyme mediators and their catabolites was only observed in IL-1beta/TNFalpha stimulated cells. We conclude that the canine IL-1beta and TNFalpha generated in this study are biologically active and equally effective in the canine cell culture systems. Inducing an inflammatory pathway by canine exogenous cytokines in canine chondrocytes provides a useful tool for the study of canine inflammatory arthritis.

Identification of dysregulated genes in cutaneous squamous cell carcinoma.

Carcinogenesis is a multi-step process resulting from the accumulation of genetic mutations and subsequently leading to dysregulated genes, but the number and identity of differentially expressed genes in cutaneous squamous cell carcinoma (SCC) is unknown at present. In order to identify dysregulated genes, we examined the relative mRNA expression present in cutaneous SCC and its precursor lesion actinic keratosis (AK) by comparison to normal skin. Snap frozen biopsies from 20 specimens of normal skin, 10 AK, and 10 cutaneous SCC were examined. Total-RNA was extracted, reversely transcribed, and 14 genes were investigated using gene-specific intron-flanking primers and quantitative real-time reverse transcription PCR. Specificity was confirmed by sequencing of the PCR amplicons. Ten of 14 genes were significantly dysregulated in AK and/or cutaneous SCC by comparison to normal skin. The genes CNN2, COX4I1, COX5B, COX7C, CRLF3, CTSC, NDRG1, and LMNA showed increased expression in skin cancer (p < 0.02), while RPL15 and LGTN were down-regulated (p < 0.03). The genes differentially expressed during skin carcinogenesis may prove useful in order to understand the origin and progression of cutaneous SCC and for diagnostic approaches.

Molecular and phenotypic modulations of primary and immortalized canine chondrocytes in different culture systems.

This study was conducted to determine physiological and functional features of primary and immortalized canine chondrocytes. Chondrocytes were immortalized by introducing the catalytic component of human telomerase namely human telomerase reverse transcriptase (hTERT). Primary chondrocytes lost their characteristic phenotype and growth properties whereas the immortalized cells remained polygonal with rapid growth rate. The expression of chondrocyte-specific markers decreased many-fold whereas that of chondrocyte-non-specific gene increased in primary chondrocytes. The immortalized cells did not express chondrocyte-specific genes in monolayers. Both primary and immortalized cells were encapsulated in alginate microspheres to construct three-dimensional (3D) culture system. As the primary chondrocytes, also the telomerase-transfected cells adopted a chondrocyte-specific gene expression pattern in alginate culture. Thus, the expression of telomerase represents possibility to expand chondrocytes without limitation while maintaining the chondrocyte-specific phenotype in 3D cultures. Use of such cells provides a standardized tool for testing different tissue engineering applications in canine model.