Judith E. Layton

The zebrafish is a useful model organism for developmental and genetic studies. The morphology and function of zebrafish myeloid cells were characterized. Adult zebrafish contain 2 distinct granulocytes, a heterophil and a rarer eosinophil, both of which circulate and are generated in the kidney, the adult hematopoietic organ. Heterophils show strong histochemical myeloperoxidasic activity, although weaker peroxidase activity was observed under some conditions in eosinophils and erythrocytes. Embryonic zebrafish have circulating immature heterophils by 48 hours after fertilization (hpf). A zebrafish myeloperoxidase homologue (myeloid-specific peroxidase; mpx) was isolated. Phylogenetic analysis suggested it represented a gene ancestral to the mammalian myeloperoxidase gene family. It was expressed in adult granulocytes and in embryos from 18 hpf, first diffusely in the axial intermediate cell mass and then discretely in a dispersed cell population. Comparison of hemoglobinized cell distribution, mpx gene expression, and myeloperoxidase histochemistry in wild-type and mutant embryos confirmed that the latter reliably identified a population of myeloid cells. Studies in embryos after tail transection demonstrated that mpx- and peroxidase-expressing cells were mobile and localized to a site of inflammation, indicating functional capability of these embryonic granulocytes. Embryonic macrophages removed carbon particles from the circulation by phagocytosis. Collectively, these observations have demonstrated the early onset of zebrafish granulopoiesis, have proved that granulocytes circulate by 48 hpf, and have demonstrated the functional activity of embryonic granulocytes and macrophages. These observations will facilitate the application of this genetically tractable organism to the study of myelopoiesis.

By three criteria, we have demonstrated that B cell stimulatory factor (BSF-1) and B cell differentiation factor (BCDF-gamma) are the same lymphokine. Highly purified preparations of high performance liquid chromatography-purified or affinity-purified BSF-1 had BCDF-gamma activity but not BCDF-mu activity. A monoclonal anti-BSF-1 antibody coupled to Sepharose depleted both BSF-1 and BCDF-gamma activity but not BCDF-mu activity from two different T cell supernatants. Soluble monoclonal anti-BSF-1 blocked the BSF-1 and BCDF-gamma but not the BCDF-mu responses. These results suggest that BSF-1 acts on both resting and activated B cells to induce different effects.

OBJECTIVE: To assess the effect of plasmafiltration (PF) on biochemical markers of inflammation, cytokines, organ dysfunction, and 14-day mortality in human sepsis. DESIGN: Multicenter, prospective, randomized, controlled clinical trial. SETTING: Seven university-affiliated intensive care units. PATIENTS: Thirty patients (22 adults, eight children) with new (<24 hrs) clinical evidence of infection and sepsis syndrome were enrolled. Fourteen of 30 (nine adults, five children) were randomized to PF. INTERVENTIONS: All patients received protocol-driven supportive intensive care, and those randomized to PF received continuous plasma exchange for 34 hrs using a hollow-fiber plasma filter. MEASUREMENTS AND MAIN RESULTS: Illness severity and risk of death were calculated with the Pediatric Risk of Mortality (children) and the Acute Physiology and Chronic Health Evaluation II (adults) scales. Plasma samples (0, 6, 24, and 48 hrs) were assayed for acute-phase proteins (albumin, globulin, C-reactive protein, alpha1-antitrypsin, haptoglobin), inflammatory mediators (complement fragment C3, thromboxane B2), and cytokines (interleukin-6, granulocyte colony-stimulating factor, leukemia inhibitory factor). Sieving coefficients were estimated from filtrate concentrations at 3 hrs. The two groups were matched for incidence of septic shock (13 of 14 vs. 11 of 16), refractory shock (three of 14 vs. six of 16), bacteremia (six of 14 vs. five of 16), severity of illness, and calculated risk of death (0.68 vs. 0.64). There was no difference in mortality. Eight of 14 PF patients (57%) and eight of 16 controls (50%) survived for 14 days (p = .73, Fisher's exact test). Multiple logistic regression revealed age (odds ratio, 16.4:1; 95% confidence interval, 2.12-infinity) and shock (10.6:1; 1.32-infinity) as significant predictors of death; plasmafiltration was associated with a nonsignificant reduction in the risk of death (odds ratio, 1.78:1; 95% confidence interval, 0.20-18.1). The mean (SD) number of organs failing in the first 7 days in the PF group was 2.57 (0.94) vs. 2.94 (0.85) in controls (p = .37, Mann-Whitney U test). Both groups had similarly elevated plasma concentrations of all inflammatory mediators except complement fragment C3 at study entry. Leukemia inhibitory factor was detectable in four patients only. PF did not influence mean concentrations of interleukin-6, granulocyte colony-stimulating factor, thromboxane B2, total white cell count, neutrophil count, or platelet count, but it was associated with significant reductions of alpha1-antitrypsin, haptoglobin, C-reactive protein, and complement fragment C3 in the first 6 hrs (p < .05). The sieving coefficients for all inflammatory mediators approached unity. CONCLUSIONS: PF caused a significant attenuation of the acute-phase response in sepsis. There was no significant difference in mortality, but there was a trend toward fewer organs failing in the PF group that suggests that this procedure might be beneficial.

Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipitation of JAK1 with the G-CSF receptor suggested a physical association which existed prior to G-CSF stimulation.