Summary.Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor-a (TNF-a) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single-cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor-enriched bone marrow CD34 þ compartment from MDS patients (P ¼ 0 . 039), which are absent in both CD34 ¹ MDS cells (P ¼ 0 . 53) and also CD34 þ cells from normal subjects (P ¼ 0 . 55). MDS CD34 þ blood cells also showed oxidized pyrimidine nucleotides compared with CD34 ¹ cells (P ¼ 0 . 029). Within normal subjects no differences were seen between CD34 þ and CD34 ¹ bone marrow cell compartments. CD34 þ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF-a and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF-a, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.

No abstract

Phagocytosis in the solitary ascidian Ciona intestinalis was investigated using mixed and separated populations of blood cells in vitro. Only the vacuolar and granular amoebocytes were seen to ingest bacteria, and when the serine protease inhibitors, STI, or benzamidine were added to monolayers of mixed cell types, uptake was significantly reduced. Analyses carried out with isolated cells revealed that phagocytosis was enhanced by incubation of the bacteria in blood cell lysate supernatants (CLS) that had been pre-treated with LPS. By contrast, pre-incubation of the bacteria in CLS preparations that were inhibited by benzamidine produced lower levels of phagocytosis. Treatment of the bacteria with plasma also failed to promote uptake, and there was no detectable agglutination of the bacteria by CLS. As lysate supernatants made from morula cells, but not other cell types, were effective in promoting phagocytosis, we propose that opsonins are derived from the morula cells, and that phagocytosis involves cooperation between different cell populations. Moreover, as the morula cells are the principal repositories of prophenoloxidase and an associated serine protease (factors which are activated by LPS but blocked by STI or benzamidine), prophenoloxidase or the protease may be involved in the opsonic phenomenon in a similar way to that previously reported for arthropods.

No abstract

Blood cell-mediated cytotoxic activity against mammalian target cells by the hemocytes of the solitary ascidian Ciona intestinalis was investigated in vitro by fluorochromasia. Salt-conditioned target cells were labeled with carboxyfluorescein diacetate and challenged with mixed and separated hemocytes. The assay provided optimal conditions for the functioning of the effector hemocytes while maintaining low background leakage from the target cells. Comparison of different hemocyte populations, separated by density gradient centrifugation, revealed that only cell bands containing the phagocytic and nonphagocytic amoebocytes exhibited cytotoxicity. Experiments to characterize cytolysis demonstrated that activity increased with the effector to target cell ratio, occurred within 15 min, and was maximal at an incubation temperature of 20 degrees C. Both human (K562) and mouse [YAC-1, P815, WEHI (3B) and L929] target cell lines were killed by the ascidian effector hemocytes. This paper demonstrates a population of nonspecific cytotoxic effector cells in the blood of C. intestinalis that are able to spontaneously kill a range of mammalian targets in vitro.