Development and evaluation of RADA-PDGF2 self-assembling peptide hydrogel for enhanced skin wound healing.

Background: Wound healing complications affect numerous patients each year, creating significant economic and medical challenges. Currently, available methods are not fully effective in the treatment of chronic or complicated wounds; thus, new methods are constantly sought. Our previous studies showed that a peptide designated as PDGF2 derived from PDGF-BB could be a promising drug candidate for wound treatment and that RADA16-I can serve as a release system for bioactive peptides in wound healing. Based on that, in this work, we designed a new self-assembling hydrogel RADA-PDGF2, connecting both peptides by a sequence specific for neutrophil elastase, and evaluated its activity in wound healing. Methods: The physicochemical properties of the designed scaffold were analyzed using transmission electron microscopy, atomic force microscopy, cryoSEM microscopies, and circular dichroism spectroscopy. The enzymatic cleavage was performed using human neutrophil elastase and monitored using high-performance liquid chromatography and MS spectroscopic techniques. The aforementioned techniques (HPLC and MS) were also used to assess the stability of the peptide in water and human plasma. The biological activity was analyzed on human skin cells using a colorimetric XTT test, collagen synthesis evaluation, and a migration assay. The biocompatibility was analyzed with LDH cytotoxicity assay and flow cytometric analysis of activation of immune cells. Finally, RADA-PDGF2 activity in wound healing was checked in a mouse dorsal skin injury model. Results: The analysis showed that RADA-PDGF2 can self-assemble, form a hydrogel, and release a bioactive sequence when incubated with human elastase. It shows pro-proliferative and pro-migratory properties and accelerates wound closure in the mouse model compared to RADA16-I. In addition, it is not cytotoxic to human cells and does not show immunogenicity. RADA-PDGF2 seems to be a promising drug candidate for wound management.

Associations of Selected Cytokines Levels in Organ Transplant Recipients Without and With Malignant Skin Neoplasms.

INTRODUCTION: Skin malignancies are the most prevalent neoplasms seen in organ transplant recipients (OTRs). Immunosuppressive treatment has been attributed to play a causative role in malignancy development. The aim of the study was to assess cytokine concentrations involved in cytotoxic and regulatory responses in patients after organ transplantation (Tx). We compared two OTR subgroups: those with malignant skin tumors and those without any known changes developed after Tx. MATERIALS AND METHODS: We enrolled 102 patients, 63: 3-360 (median: min-max) months after Tx, aged 54.3 ± 9.9 (mean ± SD) years (38.2% females). Seventeen patients were diagnosed with malignant skin neoplasms. The most frequent treatment schemes were cyclosporine A - mycophenolate mofetil - glucocorticosteroids (GS) (37.4%), mycophenolate mofetil-tacrolimus - GS (15.2%), and azathioprine-cyclosporine A-GS (14.1%). A 5-mL sample of venous blood was obtained from participants of two subgroups: those with malignant skin tumors and those without any known changes. The blood was tested for interleukin 2 (IL-2), interferon gamma, IL-10, and transforming growth factor beta concentrations (Multicytokine Flex Set, ELISA). The Kruskal-Wallis test was used to compare variables; P < .05 was considered valid. RESULTS: Age, gender distribution, and time from transplantation did not differ across the two subgroups. We found significantly lower blood concentrations of IL-2 and IL-10 in patients with post-transplantation skin cancers versus patients without any known skin changes (0 pgmL(-1) vs. 21.22 pgmL(-1), and 4.93 pgmL(-1) vs. 7.36 pgmL(-1), respectively). The differences between interferon gamma and transforming growth factor beta levels were insignificant across studied groups. CONCLUSIONS: Our findings suggest that immunosuppressive response assessed by cytokine IL-2 and IL-10 levels may be used in the risk stratification for the development of skin cancer in organ recipient patients.

Corticotropin-releasing hormone triggers differentiation in HaCaT keratinocytes.

BACKGROUND: Corticotropin-releasing hormone (CRH) is proposed to be involved in the regulation of the proliferative capacity of keratinocytes, based on its significant actions in the skin. These are mediated by CRH-R1alpha and represented by adenylate cyclase activation, Ca2+ influx, inhibition of cell proliferation and modifications in intracellular signal transduction by NF-kappaB. OBJECTIVES: To define CRH action in the cell cycle we investigated its effects on the differentiation programme using the HaCaT keratinocytes model. METHODS: HaCaT keratinocytes were incubated with CRH in Dulbecco's modified Eagles's medium (containing 1.8 mmol L(-1) calcium) or EpiLife (containing 0.06 mmol L(-1) calcium) medium. Cell proliferation was assessed with the MTT assay. Flow cytometry was used for the measurement of DNA content, cell size and granularity and the expression of cytokeratin 14, cytokeratin 1 and involucrin. The electrophoretic mobility shift assay was used to determine DNA binding activity by AP-1 transcription factor. Expression of cytokeratin 1 was also assessed with immunofluorescence microscopy. RESULTS: CRH did produce inhibition of proliferation, which was dose-dependent; the shape of the inhibition curve was determined by the media calcium concentration. CRH action was pinpointed at inhibition of the G0/1 to the S phase transition of the cell cycle. CRH also increased AP-1 binding activity, cell granularity, cytokeratin 1 and involucrin expression, and inhibited cytokeratin 14 expression. CONCLUSIONS: These results are consistent with CRH induction of the keratinocyte differentiation programme. Thus, the overall CRH cutaneous actions connote protective functions for the epidermis, that appear to include the triggering or acceleration of the differentiation programme.