Pluripotent hemopoietic progenitors (CFU-GEMM) in polycythemia vera: analysis of erythropoietin requirement and proliferative activity

Pluripotent hemopoietic progenitors (CFU-GEMM) give rise to multilineage hemopoietic colonies in culture. We have examined the erythropoietin requirements of CFU-GEMM-derived erythroid progeny in patients with polycythemia vera (PV) and studied their proliferative activity by short-term exposure to 3HTdR. Mixed colonies with erythroid components were observed in all bone marrow and peripheral blood samples from patients with PV that were cultured without addition of exogenous erythropoietin. This response is consistent with previously reported growth patterns for CFU-E and BFU-E. The frequency of mixed colonies increased regularly when erythropoietin was added to the cultures. Short-term exposure of peripheral blood specimens to 3HTdR prior to plating yielded a reduction of the plating efficiency by 20%- 70% when compared to cells that were not exposed to 3HTdR. The observation of cycling CFU-GEMM in PV contrasts with the usually quiescent behavior of CFU-GEMM in peripheral blood of normal individuals under steady-state conditions. These results support the view that the increased proliferative rate observed for CFU-GEMM may be responsible for the increased formation of blood cells in PV.     

Cryopreservation of enucleated human neutrophils (PMN cytoplasts)

Previously, we have shown that enucleated human neutrophils (PMN cytoplasts), when activated by particulate or fluid stimuli, generate superoxide and hydrogen peroxide at rates comparable (per unit area of plasma membrane) to those observed with intact neutrophils. Moreover, PMN cytoplasts also ingest and, to a certain extent, kill bacteria. We now report that PMN cytoplasts can be cryopreserved with maintenance of their functional activity. The PMN cytoplasts were frozen in a medium with 10% (v/v) fetal calf serum and 10% (v/v) dimethyl sulfoxide, and stored at -70 degrees C. After thawing and washing, the recovery was 75%. The content of alkaline phosphatase and lactate dehydrogenase, the consumption of oxygen and generation of hydrogen peroxide, and the rate of phagocytosis of Staphylococcus aureus bacteria was the same for fresh and cryopreserved PMN cytoplasts. Identical values were obtained after preservation in liquid nitrogen. These results open possibilities to store neutrophil material, allowing longitudinal follow-up of patients, comparative studies between different patients, exchange of material between laboratories, and storage of reference material for experiments in series.     

Lymphokine(s) from isolated T lymphocyte subpopulations support multilineage hematopoietic colony and megakaryocytic colony formation

Conditioned medium derived from peripheral mononuclear low-density cells stimulated with phytohemagglutinin (PHA) supports the growth of noncommitted hematopoietic progenitors from marrow and peripheral blood cells. These immature progenitors (CFU-GEMM) can be identified in culture as multilineage hematopoietic colonies containing erythroblasts, eosinophilic, basophilic and neutrophilic granulocytes, megakaryocytes, macrophages, and T and B lymphocytes. In this report, we describe the effect of lymphokines released from purified T lymphocyte preparations of helper (T4) and suppressor/cytotoxic (T8) phenotype derived from peripheral blood on the growth of multilineage hematopoietic colonies and megakaryocytic colonies. It was found that PHA-stimulated lymphocytes of T4 phenotype and, to a lesser extent, of T8 phenotype elaborate lymphokine(s) that support the growth and development of multilineage colonies (CFU-GEMM), granulopoietic colonies (CFU-C), erythroid bursts (BFU-E) and megakaryocytic colonies (CFU-M) by nonadherent and T cell-depleted bone marrow cells.     

Chloramphenicol-induced bone marrow injury: possible role of bacterial metabolites of chloramphenicol

To explore the potential role of some bacterial metabolites of chloramphenicol (CAP) in CAP-induced hematotoxicity, we examined their cytotoxic effects on bone marrow cells in vitro using a number of cytotoxicity parameters. Among the metabolites tested, dehydro-CAP (DHCAP) and p-nitrophenyl-2-amino-3 hydroxypropanone-HCI (NPAP) were more toxic than CAP. DHCAP was at least as toxic as nitroso-CAP. At concentrations of less than or equal to 10(-4) mol/L, DHCAP caused total irreversible inhibition of myeloid colony (CFU-GM) growth and 80% inhibition of DNA synthesis in human bone marrow. Incubation of human bone marrow cells with 10(-4) mol/L nitroso-CAP or DHCAP for 24 hours resulted in 75% and 65% cell death respectively. Although DHCAP was 10- to 20-fold more cytotoxic than CAP, it was only one third as effective in inhibiting mitochondrial protein synthesis, indicating that DHCAP exerts its toxic effect by alternate mechanisms. The cytotoxicity of DHCAP and its relative stability, compared to the unstable nitroso CAP, suggest that this bacterial metabolite of CAP, and possibly others, may play a significant role in CAP-induced hematotoxicity.     

Release of interleukin-12 in experimental Escherichia coli septic shock in baboons: relation to plasma levels of interleukin-10 and interferon- gamma

Interleukin (IL)-12 is thought to be a key factor for the induction of interferon gamma (IFN-gamma), a cytokine essential for the lethal effects of endotoxin. We report here on the release of the nonfunctional subunit of IL-12, p40, as well as biologically active heterodimeric IL-12, p70, after administration of a lethal (n = 5) or sublethal (n = 8) dose of live Escherichia coli to baboons. Remarkably, on lethal challenge, peak levels of p40 were observed at 3 hours that were about twofold lower than those elicited after sublethal challenge (2,813 +/- 515 pg/mL v 4,972 +/- 732 pg/mL, P < .05). This disparity was also observed, although to a lesser extent, for IL-12 p70 antigen, of which maximum levels of 91 +/- 47 pg/mL and 151 +/- 41 pg/mL were measured 6 hours after a lethal or sublethal dose of E coli, respectively. Circulating p70 antigen correlated with IL-12 biologic activity (r = 0.869; P < .001). When comparing lethal to sublethal conditions, lower peak levels of IL-12 on lethal E coli sharply contrasted with higher levels of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8 observed in these animals. Lower IL-12 concentrations in the lethal group may have resulted in part from the enhanced production of IL-10, a known inhibitor of IL-12 synthesis in vitro, as peak levels of this cytokine 3 hours postchallenge inversely correlated with peak levels of IL-12, in particular p40 (r = -0.802; P < .01). Contrary to what might be expected if IFN-gamma were solely induced by IL-12, lethally challenged baboons generated threefold more IFN-gamma at 6 hours than those receiving a sublethal dose (P < .05). Moreover, higher levels of IFN- gamma were associated with lower p40/p70 ratios, suggesting that, in agreement with observations in vitro, IFN-gamma may have preferentially upregulated the release of p70 over p40. These data show that IL-12 is released in experimental septic shock in nonhuman primates and suggest that IL-10 and IFN-gamma are involved in the regulation of this release. Furthermore, this study indicates that the systemic release of IL-12 might be essential, but is not likely sufficient, to promote lethal production of IFN-gamma in sepsis.