Innate and adaptive immunity gene expression of human keratinocytes cultured of severe burn injury

PURPOSE:Evaluate the expression profile of genes related to Innate and Adaptive Immune System (IAIS) of human Primary Epidermal keratinocytes (hPEKP) of patients with severe burns.METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific IAIS PCR Arrays plates (SA Biosciences).RESULTS: After the analysis of gene expression we found that 63% of these genes were differentially expressed, of which 77% were repressed and 23% were hyper-regulated. Among these, the following genes (fold increase or decrease): IL8 (41), IL6 (32), TNF (-92), HLA-E (-86), LYS (-74), CCR6 (- 73), CD86 (-41) and HLA-A (-35).CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UNIFESP-EPM Department of SurgeryUNIFESP-EPMUNIFESPUniversidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Department of SurgeryUNIFESP, EPM, Department of SurgeryUNIFESP-EPMUNIFESP, EPM, Department of SurgeryFAPESP: 2011/12945-4FAPESP: 2013/10.905-0SciEL

Innate and adaptive immunity gene expression of human keratinocytes cultured of severe burn injury

PURPOSE:Evaluate the expression profile of genes related to Innate and Adaptive Immune System (IAIS) of human Primary Epidermal keratinocytes (hPEKP) of patients with severe burns.METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific IAIS PCR Arrays plates (SA Biosciences).RESULTS: After the analysis of gene expression we found that 63% of these genes were differentially expressed, of which 77% were repressed and 23% were hyper-regulated. Among these, the following genes (fold increase or decrease): IL8 (41), IL6 (32), TNF (-92), HLA-E (-86), LYS (-74), CCR6 (- 73), CD86 (-41) and HLA-A (-35).CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UNIFESP-EPM Department of SurgeryUNIFESP-EPMUNIFESPUniversidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Department of SurgeryUNIFESP, EPM, Department of SurgeryUNIFESP-EPMUNIFESP, EPM, Department of SurgeryFAPESP: 2011/12945-4FAPESP: 2013/10.905-0SciEL

Human beta defensin-4 and keratinocyte growth factor gene expression in cultured keratinocyte and fibroblasts of burned patients

PURPOSE: To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples.METHODS: Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR.RESULTS:In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn.CONCLUSIONS:KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UNIFESP-EPM Department of SurgeryUNIFESP-EPMUniversidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Department of SurgeryUNIFESP, EPM, Department of SurgeryUNIFESP-EPMUNIFESP, EPM, Department of SurgeryFAPESP: 2011/12.945-4FAPESP: 2013/10.905-0SciEL