Signal transduction in human macrophages by gp83 ligand of Trypanosoma cruzi: trypomastigote gp83 ligand up-regulates trypanosome entry through protein kinase C activation.

We found that Trypanosoma cruzi trypomastigote cloned surface ligand (gp83 trans-sialidase) signals macrophages to up-regulate parasite entry by activating protein kinase C (PKC). Incubation of r-gp83 ligand with macrophages activates PKC and this activation is abolished when r-gp83 is depleted by immunoprecipitation with anti-r-gp83 antibodies, which recognize the secreted gp83 of trypomastigotes by immunoblotting. This activation is seen as early as 15 min with maximal activity at 60 min and correlates with the concentration of macrophage cell cytosol. Bisindolylmaleimide I, a PKC inhibitor, abolished the activation of PKC induced by r-gp83 ligand. Incubation of macrophages with r-gp83 ligand significantly enhanced the number of trypanosomes per cell. Bisindolylmaleimide I also inhibited the enhancement of trypomastigote uptake by macrophages induced by the r-ligand. These results demonstrate that T. cruzi uses a novel mechanism to signal cells in the process of trypanosome entry, via a secreted trypanosome ligand which signals macrophages through activation of PKC.

The cysteine-cysteine family of chemokines RANTES, MIP-1alpha, and MIP-1beta induce trypanocidal activity in human macrophages via nitric oxide.

This paper describes a new role for the cysteine-cysteine (CC) chemokines RANTES, MIP-1alpha, and MIP-1beta on human macrophage function, which is the induction of nitric oxide (NO)-mediated trypanocidal activity. In a previous report, we showed that RANTES, MIP-1alpha and MIP-1beta enhance Trypanosoma cruzi uptake and promote parasite killing by human macrophages (M. F. Lima, Y. Zhang, and F. Villalta, Cell. Mol. Biol. 43:1067-1076, 1997). Here we study the mechanism by which RANTES, MIP-1alpha, and MIP-1beta activate human macrophages obtained from healthy individuals to kill T. cruzi. Treatment of human macrophages with different concentrations of RANTES, MIP-1alpha, and MIP-1beta enhances T. cruzi trypomastigote phagocytosis in a dose peak response. The optimal response induced by the three CC chemokines is attained at 500 ng/ml. The macrophage trypanocidal activity induced by CC chemokines can be completely inhibited by L-N-monomethyl arginine (L-NMMA), a specific inhibitor of the L-arginine:NO pathway, but not by its D-enantiomer. Culture supernatants of chemokine-treated human macrophages contain increased NO2- levels, and NO2- production is also specifically inhibited by L-NMMA. The amount of NO2- induced by these chemokines in human macrophages is comparable to the amount of NO2- induced by gamma interferon. The killing of trypomastigotes by NO in cell-free medium is blocked by an NO antagonist or a NO scavenger. This data supports the hypothesis that the CC chemokines RANTES, MIP-1alpha, and MIP-1beta activate human macrophages to kill T. cruzi via NO, which is an effective trypanocidal mechanism.

Signal transduction in human macrophages by gp83 ligand of Trypanosoma cruzi: trypomastigote gp83 ligand up-regulates trypanosome entry through the MAP kinase pathway.

We found that Trypanosoma cruzi trypomastigote cloned surface ligand (gp83 trans-sialidase) signals human macrophages to up-regulate parasite entry by inducing tyrosine phosphorylation of MAP kinase. Preincubation of human macrophages with r-gp83 transsialidase significantly enhanced both the percentage of phagocytosed trypanosomes and the number of trypanosomes per cell in a concentration dependent fashion. Incubation of r.gp83 with macrophages induced tyrosine phosphorylation of several macrophage proteins. This enhancement was inhibited by genistein, a tyrosine kinase inhibitor. The r-trypanosome ligand enhanced tyrosine phosphorylation of ERK1 and this enhancement was specifically inhibited by the inhibitor of MAP kinase phosphorylation, PD 98059, or by genistein. PD 98050 or genistein also inhibited the enhancement of trypomastigote uptake by macrophages induced by the r-ligand. These results indicate that T. cruzi uses a novel mechanism to signal cells in the process of trypanosome entry, via a secreted trypanosome ligand which signals macrophages through the MAP kinase pathway.

Drug screening in newborns and mothers using meconium samples, paired urine samples, and interviews.

We evaluated the prevalence of illicit substance abuse by comparing drug screening results derived from meconium, urine pairs, and maternal interview. Mother/infant pairs (580) were entered into this blinded, prospective study. Prevalence of illicit substance abuse was 3.4%. The lack of prenatal care correlated with the use of cocaine (p < 0.001). Neonates born to cocaine-using mothers were more likely to be premature, to have a lower birth weight, decreased length, and smaller head circumference using unpaired t test (overall p < 0.05 using Bonferroni method for simultaneous inference). For mother/infant pairs who had positive drug screening for cocaine, the interview, maternal urine sample, and meconium sample showed equal sensitivity, although the newborn urine showed poor correlation. We suggest that the newborn urine sample could be deleted from newborn drug screening, and lack of perinatal care may serve as a marker of substance abuse.

Neonatal immune neutropenia following the administration of intravenous immune globulin.

BACKGROUND: In adults--but not neonates--neutropenia has been reported to complicate treatment with intravenous immunoglobulin, but the mechanism is unknown. PURPOSE: To describe for the first time the case of a newborn infant who, after intravenous immunoglobulin, demonstrated serum antineutrophil antibodies and neutropenia. PATIENTS AND METHODS: The 1,425-g, 36-week-gestation boy was healthy except for intrauterine growth retardation. Intravenous immunoglobulin (1g/dose x 3) was administered to treat alloimmune thrombocytopenia. Neutrophil-specific antibodies were detected by a granulocyte immunofluorescence assay. RESULTS: After the intravenous immunoglobulin, the platelet count normalized but the neutrophil count declined to 450/mm3. Neutrophil-specific antibodies were detected in the serum of the infant but not in the maternal serum. Furthermore, cross-matching revealed that the maternal serum did not react with the infant's granulocytes. Two of three random lots of intravenous immunoglobulin contained detectible anti-neutrophil antibodies. CONCLUSIONS: After intravenous immunoglobulin, the infant's serum contained one or more anti-neutrophil antibodies that were not maternal in origin. We speculate that the neutropenia resulted from the administration of intravenous immunoglobulin containing antineutrophil antibodies.