S _nnuay A panel of human colonic adenocarcinoma cell lines was examined both for expression of mRNAs of the nitric oxide synthase (NOS) gene family and for evidence of enzymic activity based on citrulline and nitrite (NO,-) formation. Reverse transcription-polymerase chain reaction (RT-PCR). revealed that all lines (SW480. SW620. DLD-1 and WiDr) expressed mRNA for the Ca-+-dependent endothelial (e)NOS. while SW480 cells also expressed the Ca2'-dependent neuronal (n)NOS. The mRNA for the Ca'+-independent inducible (i)NOS was expressed both by cytokine-stimulated and by unstimulated SW480. SW620 and DLD-1 cells, but none was seen at any time in the WiDr cells. There was, however, little correlation between mRNA expression and enzynic activity based on citruUline and NO,-formation. Thus none of the cell lines exhibited measurable Ca'-dependent NOS activity, while Ca2'-independent NOS activity was seen in all but the WiDr cells. Furthermore. DLD-1 cells generated citrulline with resultant NO.-formation only after stimulation with lipopolysacchanrde (LPS) and or cytokines. while SW480 and SW620 did so constitutively. Thus RT-PCR studies indicate that tumour cells of similar epithelial origin display a diverse pattern of NOS gene family expression. and parallel biochemical studies clearly indicate that such expression does not always result in measurable enzymic activity leading to the generation of NO.

BALB/c mice are highly susceptible to Leishmania major infection. They develop a progressive fatal disseminating disease even with a minimum infecting dose. However, these mice are able to contain the disease if they are exposed to sublethal gamma-irradiation shortly before infection. Earlier studies demonstrated that CD4+ T cells from mice which had recovered from infection (Tr) can adoptively transfer resistance. In contrast, CD4+ cells from mice with progressive disease (Ts) not only failed to protect, but can reverse the protective effect of the Tr cells. Spleen cells from BALB/c mice which had recovered from L. major infection or which had progressive disease were cultured with leishmanial antigens in vitro. The culture supernatant from spleen cells of recovered mice (TrSN) contains high levels of macrophage-activating factor (MAF) activity which can activate peritoneal macrophages to kill 51Cr-labeled P815 cells and to eliminate intracellular parasites as measured by the reduction in [3H]thymidine uptake by residual parasites released from macrophages following sodium dodecyl sulfate treatment. The MAF activity of TrSN parallels that of recombinant interferon-gamma (IFN-gamma). In contrast, culture supernatant of spleen cells from mice with progressive disease (TsSN) contains no detectable MAF but it is able to neutralize the MAF activity of TrSN. The MAF-inhibiting function of TsSN appears to be mediated by interleukin (IL)3 and IL4, since the MAF activity of TrSN and rIFN-gamma also can be inhibited by the addition of rIL3 and rIL4 but not by rIL1 or rIL2. Furthermore, the MAF-inhibiting activity of TsSN can be partially reversed by the addition of specific anti-IL3 or anti-IL4, but completely reversed by the combination of the two antibodies in vitro. These findings provide a mechanism for the immune regulation in leishmaniasis and a means by which the two subsets of CD4+ T cells influence each other through their modulation of macrophage function.

Increased platelet reactivity has been implicated in various vascular diseases. Since the protein content of platelets is determined mainly by the megakaryocyte, alterations in megakaryocyte mRNA expression may influence platelet protein content and therefore activity. In order to determine whether DNA content (ploidy) of a megakaryocyte influences its mRNA expression, we have developed a method to investigate the relationship between megakaryocyte ploidy and gene expression. By measuring the ploidy and mRNA expression in individual cells, we have shown that in the physiological state there is an increase in mRNA expression for beta-actin, glycoprotein IIb and neuropeptide Y with increase in ploidy and that this increase levels off at high ploidy values. This may have relevance in the understanding of platelet reactivity in pathological events.

A rapid and simple method has been developed for the separation of pure populations of intact human megakaryocytes from whole bone marrow. Megakaryocytes were specifically recognized using monoclonal antibodies coupled to magnetizable articles. Cells labelled with the magnetizable probe were separated from unlabelled cells by introduction of a magnetic field. The technique yields megakaryocyte suspensions with a purity of greater than 98%. Electron microscope examination showed that the ultrastructure of the isolated megakaryocytes was well preserved. Using this method of cell purification, we have investigated expression of the gene for platelet-derived growth factor (PDGF). RNA was isolated from cells harvested from either ribs or posterior iliac crest. The RNA was spotted onto nitrocellulose, and then hybridized using a c-sis riboprobe specific for PDGF B chain mRNA. We demonstrate that mRNA for PDGF B-chain is identifiable in samples of 50,000 cells. We conclude that PDGF is synthesized by the megakaryocyte.

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Spleen cells from BALB/c mice infected with 2 x 107 L. major promastigotes and developing progressive disease produced significantly lower levels of interleukin-2 (IL-2) in response to concanavalin A stimulation than did spleen cells from uninfected mice. In contrast, spleen cells from sublethally irradiated and infected mice, which were able to contain lesion development, produced significantly higher levels of IL-2. The increase in IL-2 production closely paralleled lesion regression. Mice protectively immunized by four intravenous injections with lethally irradiated promastigotes also produced enhanced levels of IL-2, which were sustained after challenge infection. In contrast, spleen cells from BALB/c mice given four s.c. injections of irradiated promastigotes produced high levels of IL-2 before but not after infection. These mice eventually produced levels of IL-2 indistinguishable from those of unimmunized mice with progressive disease. There is thus an inverse relation between disease progression and the ability of spleen cells to produce IL-2. Spleen cells from mice with uncontrolled disease not only produced lower levels of IL-2 but also impaired IL-2 production by normal spleen cells. The ability to inhibit IL-2 was abrogated by (i) passing the cells through a Sephadex G-10 column, (ii) removal of plastic adherent cells, and (iii) removal of carbonyl iron-ingesting cells. Furthermore, Sephadex G-10 column-treated and plastic adherent, nonspecific esterase-positive spleen cells from mice with progressive disease were able to suppress IL-2 production by normal splenic T cells. The suppressive activity of the adherent cells was not affected by treatment with anti-Thy-1.2 antibody and complement. In contrast, adherent spleen cells from uninfected mice were devoid of such suppressor activity. The depressed IL-2 production by spleen cells from progressively infected mice could be restored to that of normal spleen cells by the addition of indomethacin to the culture. There was however, no correlation between IL-2 production and IL-1 activity in infected or immunized BALB/c mice. Thus, it appears that the suppression of IL-2 production is mediated by prostaglandins elaborated by macrophages from chronically infected mice.

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