Abstract 1602 Poster Session II, Sunday, 5/2 (poster 101)

GBS is the major bacterial cause of neonatal sepsis. Cells of the monocyte lineage are the first cells to encounter the pathogen in the lung, and play an important role in host defense. Deficiencies in monocyte function may contribute to the enhanced susceptibility of neonates to GBS disease. Capsular sialic acid is known to be a virulence factor; a desialylated mutant (COH1-11) is much less virulent in animal models. This study was designed to evaluate phagocytosis of GBS by monocytes; our hypotheses were that cord blood monocytes have reduced phagocytosis of GBS and that phagocytosis of COH1-11 by both adult and cord blood cells is enhanced relative to the parent strain (COH1). Mononuclear cells were isolated by centrifugation over Ficoll Hypaque. Suspensions containing 106 monocytes plus 108 CFU/ml of lucifer yellow labelled GBS were incubated at 37°C for 15, 30, 60, 120 or 240min. Cells were washed to remove unassociated GBS and monocytes labelled with phycoerythrin conjugated anti-CD14. Two-color fluorescence flow cytometry was performed; the percentage of doubly labelled CD14+ cells (red anti-CD14 + yellow GBS) was calculated and is shown below (mean±SEM, n=4-12). (Table) As compared by ANOVA, the adult monocytes showed significantly increased association (P<.05] above baseline at 60 min; this did not occur until 120 min for cord blood. For the parent strain (COH1), association of monocytes and GBS peaked at 120 min for adult and 240 min for cord blood. Despite these differences in the temporal trend, there were no differences when adult and cord blood results were compared to one another. The desialylated strain (COH1-11) was rapidly associated with both adult and cord blood monocytes with no variation across time. To determine whether the demonstrated association was phagocytosis vs. nonspecific adherence, cytochalasin B was added to prevent uptake. At 120 min, the results for adult monocytes + COH1 (n=3) were 71±10; with cytochalasin B it was 16±6, p=.01. However, for COH1-11 (n=3), the values were 88±10 without and 86±22 with cytochalasin B. We concluded that although there is a different pattern of phagocytosis exhibited by cord blood and adult monocytes, the same number of cells ingest organisms. Thus, the enhanced susceptibility of neonates to GBS disease is not due to differences in phagocytosis by monocytes. The desialylated GBS binds nonspecifically to the monocyte; sialic acid may enhance virulence by preventing nonspecific binding and subsequent triggering of host defense mechanisms.

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