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Human beta-defensin and keratinocyte growth factor in burned patients' keratinocytes and fibloblasts culture

Grant number: 11/24021-1
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): May 01, 2012
Effective date (End): April 30, 2015
Field of knowledge:Health Sciences - Medicine - Surgery
Principal Investigator:Alfredo Gragnani Filho
Grantee:Larissa Elias Lanziani
Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

The high infection incidence among burned patients is a major concern regarding their clinical evolution, being directly related to the morbidity and mortality rates of the group. Antimicrobial peptides - innate immune response components - protect the organism by directly intoxicating the microorganism or activating inflammatory response cells, therefore, raising the expression of these peptides by the epithelial cells to improve the host's resistance against microbial infections. Among the antimicrobial peptides involved in the inflammatory response we emphasize here the defensins family: they destabilize membranes rich in negative curvature lipids by creating pores that allow ions and essential composts to pathogens' survival efflux. Lots of studies regarding KGF (keratinocyte growth factor) actions in skin show qualitative relations between KGF and immune response peptides levels. As an example, KGF supplementation in an experimental burn model in a keratinocyte over dermal matrix culture and the posterior Pseudomonas aeruginosa's inoculation show that KGF decreases its proliferation. We can infer that KGF acts indirectly as a modulator of the immune response - since the substance alone does not have any effect on bacterial proliferation. The present study hypotheses that the KGF is a necessary trigger to some antimicrobial peptides synthesis by keratinocytes, such as human beta-defensins. Objective: evaluate KGF levels produced by cultivated fibroblasts and defensins production by cultivated keratinocytes in burned patients' skin samples. Method: 10 patients will be evaluated, 5 in the experimental group and 5 in the control group, all of them hospitalized at Burn Care Unity of the Plastics Surgery Discipline of the Federal University of São Paulo (UTD-DCP-HSP-UNIFESP). The experimental group will be composed of patients with second-degree deep partial-thickness or third-degree extending between 25% and 50% of total body surface area, while the control group will be composed by patients with second-degree deep partial-thickness or third-degree extending less than 5% of total body surface area, both groups with surgical procedures needs. Normal human keratinocytes derived from burned patients' skin fragments will be isolated and cultivated according to the standard method. The isolation and culture of human dermal fibroblasts, which would be disposed of in surgical procedures realized at UTD-DCP-HSP-UNIFESP, will be realized by the enzymatic dissociation technique. In the in vitro phase, beta-defensin-4 and KGF, analyzed of, respectively, epidermal keratinocytes and dermal primary fibroblasts previously cultivated, will be chosen for validation of the gene expression by RT-PCR. Three reaction replications of each sample will be used to guarantee statistical significance. Expected results: the beta-defensin-4 and KGF gene expression by the epidermal keratinocytes and dermal fibroblasts, respectively, should be more significant in samples derived from major burned patients than in the control group. (AU)

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