Age and synovitis affect the results of the treatment of knee osteoarthritis with Microfragmented Autologous Fat Tissue.

PURPOSE: This study aims to assess the effectiveness of Microfragmented Autologous Fat Tissue (MFAT) treatment for knee osteoarthritis and to investigate whether patients' pre-treatment clinical condition, such as synovitis, correlates with clinical outcomes, to identify potential predicting factors for the success or failure of the treatment. METHODS: In this prospective Cohort Study Level II multicentric trial, consecutive patients with a diagnosis of early/mild osteoarthritis and failure of previous conservative measures were enrolled to undergo diagnostic arthroscopy and a single MFAT injection. Patients were assessed with repeated scoring systems at baseline, 6 months, and 12 months after surgery. The demographic features, the arthroscopic findings, the immunophenotype of injected tissue and the histologic examination of synovia of failed patients were analyzed. RESULTS: Data from 91 patients showed a significant improvement in Lysholm, WOMAC scores at 1-year follow-up (p < 0.001). A significant decrease in VAS score was observed, while a significant improvement of measured flexion angle was registered at 1 year (p < 0.001). No major complications were reported. Age and synovitis were identified as significant factors influencing the clinical outcome (p < 0.05). Body mass index, previous or concomitant procedures, and specific cartilage defects had no influence. The mean number of injected adipose tissue-derived mesenchymal stem cells seem not to correlate with the clinical outcome. CONCLUSION: MFAT is effective in reducing pain when used with a single dose injection in early/mild OA of the knee, without major complications. Age over 60 and synovitis may be predictive for persistent pain at one year and should be considered before indications.

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role.

BACKGROUND: Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super-antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T-lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders. OBJECTIVES: To investigate the presence of HHV7 DNA on CTCL and healthy skin donors (HD). METHODS: We used quantitative real time polymerase chain reaction to evaluate the potential pathogenic role of HHV7. RESULTS: Twenty-seven of 84 (32.1%) HD were positive for HHV7 DNA. Twenty-one of 148 (14.2%) patients with CTCLs were positive for HHV7 DNA: nine of 39 (23.1%) SS, six of 14 (42.9%) CD30+ CTCLs and six of 24 (25.0%) LyP, and HHV7 DNA was negative in all 71 patients with MF. CONCLUSIONS: These results seem to exclude a pathogenic role of HHV7 in CTCLs, suggesting the possibility of skin as a latency site.

The role of quality control in a skin bank: tissue viability determination.

New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider linear range, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4 degrees C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative, whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation.

Mutant p53 subverts p63 control over KLF4 expression in keratinocytes.

Genetic experiments established that p63 is crucial for the development and maintenance of pluri-stratified epithelia and KLF4 for the barrier function of the skin. KLF4 is one of the factors that reprogram differentiated cells to iPS. We investigated the relationship between p63 and KLF4 using RNA interference, overexpression, chromatin immunoprecipitation and transient transfections with reporter constructs. We find that p63 directly represses KLF4 in normal keratinocytes (KCs) by binding to upstream promoter sites. Unlike p63, KLF4 levels are high in the upper layers of human skin and increase upon differentiation of KCs in vitro. In HaCaT KCs, which harbor two mutant alleles of p53, inactivation of p63 and of mutant p53 leads to KLF4 repression. p63 and p53 mutants are bound to sites in the KLF4 core promoter. Importantly, expression of the H179Y and R282Q p53 mutants in primary KCs is sufficient to activate endogenous KLF4. Finally, immunohistochemical analysis of tissue arrays confirms increased coexpression of KLF4 and mutant p53 in squamous cell carcinomas. Our data indicate that suppression of KLF4 is part of the growth-promoting strategy of p63 in the lower layers of normal epidermis, and that tumor-predisposing p53 mutations hijack p63 to a different location on the promoter, turning it into an activator of this reprogramming factor.

p63 regulates the caspase-8-FLIP apoptotic pathway in epidermis.

The transcription factor p63, member of the p53 family, is crucial for epithelial development. An RNAi screening identified the apoptotic gene Procaspase-8 as a target activated by p63. The caspase-8 inhibitor FLIP is also under p63 control. We analysed and detailed the direct transactivation through the use of RNAi, reporter assays, ChIPs, western blots, confocal studies in HaCat, as well as in primary human keratinocytes. The direct DeltaNp63 regulation of these targets was confirmed in vivo using transgenic DeltaNp63 mice under the K5 promoter, as compared with p63 knockout mice, and in vitro in normal human primary keratinocytes following UV irradiation. Lowering the steady state of p63 protein levels changes the relative ratio of FLIP isoforms, causing the activation of the expressed, inactive Procaspase-8, into the active isoform thus triggering the proapoptotic cascade. Therefore, p63 fine-tunes the Procaspase-8-FLIP pro- and antiapoptotic pathway in keratinocytes.