Quimiotaxia de polimorfonucleares em crianças gravemente enfermas

AUTOR(ES)
DATA DE PUBLICAÇÃO

1996

RESUMO

This study examined the in vitro chemotaxis of polymorphonuclear leukocytes (PMNs) obtained from critical care children admitted to a pediatric intensive care unit. The investigation was carried out in three stages. In the first, cells from eight patients and eight healthy volunteers were used to standardize the conditions for the chemotaxis assay. For this experiment, only non-activated sera from both the patients and the controls were used. In the second stage (Protocol A), the migration of PMNs from 17 patients and ten healthy individuals in response to E. coli lipopolysaccharide (100?g/mL) was studied, and in the third and final stage (Protocol B), the presence of chemotaxis inhibitors in the sera of seven patients was determined. For this, PMNs from 6 selected donors were incubated with sera from patients and healthy individuals in the presence of a chemo-attractant (E. coli lipopolisaccharide, 10p.g/mL, 30?g/mL and 100?g/mL). Blood sampling for the determination of white blood cell counts and for the quantification of Cortisol, C3c and C4 complement fraction levels were obtained from most patients and healthy individuals. In all cases, the blood samples were collected at the same time of day. PMNs were isolated using a double discontinuous Ficoll-Histopaque gradient and then washed twice in 5 mL of a balanced salt solution containing Eagles minimum essential medium, HEPES (0.85g%) and albumin (0.1 g%), pH 7.2 (adjusted with NaOH). The cells were centrifuged at 400 g for 10 min at room temperature between washes and then re-suspended to a concentration of 4 x 106/ml_ prior to use. The viability of these cells was >90% as determined by vital staining with Turk s solution. Chemotaxis was studied using a 48-well micro-chemotaxis chamber. The bottom chamber of each well contained 25 ?L of the desired chemo-attractant or serum while the upper chamber contained 50 ?L of the appropriate PMN suspension. PMN migration was allowed to proceed for 1 hr at 37°C in humidified air containing 5% C02. At the end of this period, the chambers were disassembled and the filter removed, stained, and the number of migrating cells counted. There was no significant difference in the migration of neutrophils obtained from patients and normal individuals in Protocol A (14.0±2.1 vs 11.2±1.0, respectively, counted in ten high power fields). Protocol B showed that the sera of most patients contained a factor(s) capable of significantly (p<0.05) inhibiting the migration of PMNs from healthy donors. The effect of this inhibitor sera factor(s) was definitely significant (p<0.05), being observed in all the concentrations of LPS mentioned above. The Cortisol levels in the patients (20.6±17 ?g /dL), including the three stages, were significantly (p<0.05) greater than in healthy donors (13.16±1.7 ?g/dL) but did not correlate with the ability of inhibit migration. Likewise, whole leukocyte counts were also higher in patients (9270±2470) than in donors (4400±253). The C3c and C4 complement levels were generally normal in most of the patients, but 20%(6/31) had C3c levels under normality. No significant differences were observed between patients and donors C3c levels. On the other side, although both groups had C4 normal levels, the patients levels (23.37±1.9mg/dL) were significantly (p<0.05) lower than in donors (32.34±3.5mg/dL). Based on the above results, we conclude that the ability of PMN from critical care children to migrate following stimulation is unimpaired but that sera from these individuals contain a factor(s) capable of inhibiting the migration of PMN from normal donors. Hormonal levels, white blood cell counts and complement fraction levels failed to correlate with PMN migration

ASSUNTO(S)

quimiotaxia crianças - doença - tratamento leucocitos

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