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Wednesday, September 28, 2005
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8588

Pseudomonas aeruginosa Proliferation in Experimental Burn Model Using Colagen Matrix, Human Cultured Keratinocytes and Keratinocyte Growth Factor KGF

Christiane Steponavicius Sobral, MD, PhD, Ian Kaplan, grad, student, Xudong Cao, PhD, Jeffrey Morgan, PhD, Alfredo Gragnani, MD, PhD, and Lydia Masako Ferreira, MD, PhD.

Objective : the aim of this original study was verify if KGF on the culture media decreases Pseudomonas aeruginosa proliferation Background Data: One of the most important functions of skin is the organism protection. The loss of epidermal protection leads to important changes, causing serious immunological damage. When keratinocyte lesion occurs, as in major burns, it turns down the organism protection mechanism, as physical as immunological. Besides many treatment innovations, these patients are vulnerable to bacterial infection, specially Pseudomona aeruginosa. These patients need special care and some times, cultured ketatinocytes auto-graft. The keratinocyte growth factor (KGF), a growth factor produced by fibroblasts, has receptors on keratinocytes. It is caractherized by its proliferative effect and has been used in many experimental models.As there were no previous studies relating Pseudomonas aeruginosa, KGF , experimental burn models and human cultured keratinocyte culture. Methods: Keratinocytes were cultured from neonate discharged foreskin using standard procedure. Once the primary keratinocyte culture was done the cells were seeded onto two different feeder layers. The keratinocytes seeded on genetically modified feeder layer produced keratinocytes which were KGF producers. The keratinocytes seeded on usual feeder layer gave origin to normal keratinocytes, incapable to produce KGF. That group , the media was supplemented with different concentrations of KGF, 0, 1 ng/ml, 5 ng/ml, 10 ng/ml and 40 ng/ml on the culture media. The supplementation was done 24 hours before the bacterial inoculation After genetical modification, both groups were seeded on a collagen matrix to develop an epithelia. The epidermis was cultivated on air/liquid interface for 7 days. After that the epidermis was burned and infected with two Pseudompnas aeruginosa strains transfected by Xenorhabdus luminescens lux CDABE plasmid, with the pennicilyn gene resistance. The bacterial number could be calculated by light emission. Bacterial proliferation was measured for 12 hours using a luminometer. Genetically modified cells KGF producers, and normal groups were supplemented on media with KGF different concentrations in different time points. Results: The presence of KGF on the culture media decreased bacterial proliferation on the genetically modified group. The supplemented group the most effective KGF concentration was 40 ng/ml of KGF on the supplemented media group. Conclusions: This result suggests that in this experimental model, KGF added to the culture media was as effective as KGF produced by genetically modified keratinocytes.


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