The specificity of the syngeneic mixed leukocyte response, a primary anti-I region T cell proliferative response, is determined intrathymically

In previous studies, the syngeneic MLR of peripheral T cells was shown to be predominantly an I region-restricted function. In this report we show that adult thymocytes are also capable of responding to syngeneic irradiated stimulator cells in a syngeneic MLR, provided that TCGF is added to the culture system. Using this assay, it was possible for the first time to examine the pattern of I region restriction within the thymus itself. Analysis of the thymocyte syngeneic MLR in thymuses from radiation-induced bone marrow chimeras demonstrated that the MHC preference seen in the peripheral T cell population also existed in cells resident within the thymus. Experiments utilizing congenitally athymic mice transplanted with allogeneic thymic grafts demonstrated that both peripheral T cells and thymocytes from such animals displayed a strong preferential proliferation toward stimulator cells bearing thymic-type MHC determinants. The results in the nude model thus demonstrate that the thymus by itself is sufficient to impart such restriction specificity on a developing T cell repertoire. These results are consistent with the notion that the thymus exerts selective pressure on maturing T cell populations that results in a skewing of the T cell repertoire toward the recognition of thymic-type I region products, and that this MHC preference exists before expansion of T cells in the periphery.     

Effect of different physico-chemical conditions on malolactic fermentation of fourLactobacillus plantarum wild strains isolated from wines of northwestern Spain

Summary Four strains ofLactobacillus plantarum, were tested for malolactic fermentation under conditions of variations in temperature, pH and SO2, L-malate and ethanol levels. When the pH value was below 3.5, malolactic fermentation was lower and was more sensitive to temperature changes. Malolactic fermentation decreased when the SO2 and ethanol levels were increased. The effects of L-malate levels were not significant.     

Influence of the curing of the killer phenotype inSaccharomyces cerevisiae wine strains on their fermentative behaviour

Fermentative behaviour and cell growth have been studied in grape juice inoculated either with two killerSaccharomyces cerevisiae wild strains or with their Acridine Orange-cured isogenic counterparts. The number of viable cells/ml at the beginning of the fermentation, as well as during exponential growth, were higher in grape juices inoculated with the cured strains. The CO₂ production, fermentative rate and ethanol and acetic acid production were also higher in the cured strains, particularly during the stage of active fermentation. These differences, however, were minimal at the end of the fermentations.     

Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells

A murine monoclonal antibody, termed HeFi-1, was produced after immunization with the L428 Hodgkin's disease tissue culture cell line. HeFi-1 selectively stained only the Reed-Sternberg or Hodgkin's cells in 18 of 18 cases of Hodgkin's disease, including the nodular sclerosis, mixed cellularity, and lymphocyte-depleted histologic subtypes. HeFi-1 did not stain any cells in normal lung, brain, salivary gland, thyroid, gall bladder, pancreas, liver, testis, breast, endometrium, or kidney. Rare large cells at the edge of the lymphoid follicles were stained in normal tonsil, colon, and hyperplastic thymus. There was no staining of any cells in 14 cases of B cell non-Hodgkin's lymphoma; however, the malignant cells in three of 11 cases of non-Hodgkin's lymphoma which appeared to express T cell markers were also stained with HeFi-1. Tissue culture cell lines including the T cell acute lymphocytic leukemia lines MOLT4 and CEM, the histiocytic cell line U-937, and the amniotic cell line WISH were not stained. Seven Epstein Barr virus (EBV)-positive lymphoblastoid cell lines were stained with HeFi-1, but there was no staining of three EBV+ African Burkitt's lymphoma cell lines or three EBV- American Burkitt's cell lines. HeFi-1 did not block the ability of the L428 cells to stimulate a mixed lymphocyte reaction or function as accessory cells for mitogen-induced human T cell proliferative responses. Modulation of the HeFi-1 cell surface antigen on the L428 cells was not observed. HeFi-1 specifically immunoprecipitated a cell surface protein of approximately 120,000 daltons from both the L428 and EBV+ lymphoblastoid cell lines. HeFi-1 monoclonal antibody should prove useful not only in the diagnosis, staging, and potential therapy of Hodgkin's disease, but also for determining the cell of origin of the Reed-Sternberg cell.     

Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow

A variety of biologic response modifiers (BRM) can potently augment NK activity in nonlymphoid organs. By using the liver as a model organ, we have shown that this augmentation of organ-associated NK activity is coincident with a 10- to 15-fold increase in the number of large granular lymphocytes (LGL) which can be isolated. The present study was designed to investigate the mechanism by which BRM induce this increase in liver-associated LGL and the coincident increase in hepatic NK activity. Initial studies confirm that a single dose of the pyran copolymer, maleic anhydride divinyl ether (MVE-2), augmented hepatic NK activity and increased the number of liver-associated LGL from 3 x 10(4)/liver to 5 x 10(5)/liver (a 17-fold increase). Multiple injections of MVE-2 further augmented total liver-associated NK activity and LGL number (to 13 x 10(5)/liver). As expected, both the NK activity and detectable LGL were eliminated by treatment of the mice with antiasialo GM1 (asGM1) serum. Three possible mechanisms for the BRM-induced increase in liver-associated LGL have been investigated, including 1) the rapid proliferation of resident hepatic LGL, 2) the redistribution of mature LGL from peripheral sites such as the spleen, or by 3) a rapid output and subsequent hepatic localization of LGL or their precursors recently derived from the bone marrow (BM). Our results demonstrated that the contribution of in situ proliferation to the BRM-induced increase in liver-LGL was relatively small, since the number of cells expressing NK-associated markers (i.e., asGM1, Thy-1.2, and NK1.1) and in G2/M phase (as assessed by propidium iodide uptake) was only 4 to 8%. Further experiments demonstrated that splenectomy before the administration of MVE-2 did not inhibit the augmentation of liver-associated NK activity. This result argued against a recruitment of mature LGL from the spleen. In contrast, selective depletion of the BM following administration of 89Sr decreased the ability of MVE-2 to augment liver-associated NK activity by greater than 80%. This procedure also significantly decreased the ability of Propionibacterium acnes (85%) and multiple doses of IL-2 (49%) to augment liver-associated NK activity. These results demonstrate that the rapid augmentation of liver-associated NK activity by BRM is largely due to localization and accumulation in the liver of LGL recently derived from the BM.     

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin.

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.     

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 M benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.     

Antitumor effects of human recombinant interleukin-1α and etoposide against human tumor cells: mechanism for synergismin vitro and activityin vivo

Recombinant human interleukin 1α (rh IL-1α) and etoposide (VP-16) synergize for direct growth inhibition of several human tumor cell linesin vitro. Our previous studies demonstrated that VP-16 increased the number of membrane-associated IL-1 receptors (IL-1Rs) and also enhanced the internalization of receptor-bound rh IL-1α. The purposes of this study were to test our hypothess that these events were critical to the synergy between rhIL-1α and VP-16, to determine whether rhIL-1α and VP-16 synergize to increase superoxide (SO) anion radical productionin vitro since SO anion has been implicated in the toxic effects of IL-1, and to investigate the antitumor efficacy of the combinaton against tumors in vivo. A375/C6 melanoma cells and OVCAR-3 ovarian carcinoma cells were tested with IL-1 receptor antagonist (IL-1ra) before exposure to rhIL-1α, VP-16 and rhIL-1α plus VP-16. The synergistic or antagonistic effects were assessed by MTT assay. SO production was measured by reduction of cytochrome C. Athymic female mice bearing the A375/C6 melanoma were treated by rhIL-1α, VP-16, and rhIL-1α+VP-16. The antitumor effects were evaluated by quantitating tumor growth and survival time. Pretreatment with the IL-1ra abrogated the synergistic effects of rhIL-1α and VP-16. The production of SO radical by A375/C6 cells was increased 2.5 fold by the combination of rhIL-1α and VP-16, and the addition of exogenous SOD blocked the synergy between rhIL-1α and VP-16. However, when A375/S0D15 cells which over-expressed manganese superoxide dismutase (MnSOD) after MnSOD cDNA transfecton were exposed to rhIL-1α and VP-16, in vitro antagonism was observed. In vivo studies demonstrated that the combination of rhIL-1α and VP-16 delayed tumor growth better than either agent alone, although long-term survival was not improved because of substantial toxicity. Our results suggest that the synergistic antitumor effects of IL-1α and VP-16 may be due to IL-1R modulation and increased internalization of IL-1-IL-1R complex by VP-16 treatment, as well as to a subsequent increase in SO anion radical production from the tumor cells exposed to both drugs. Thus, the combnation of IL-1α and VP-16 might prove useful for the treatment of malignant diseasein vivo, if the increased toxicity can be reduced or managed. The US Government’s right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged.     

The effect of an interleukin receptor antagonist (IL-1ra) on colonocyte eicosanoid release

We investigated whether an interleukin 1 receptor antagonist (IL-1ra) altered cellular release of prostanoids and leukotrienes in a transformed colonic cell line (CACO-2) in the presence of proinflammatory stimuli. Cellular inflammation was induced by treatment with lipopolysaccharide (LPS) or the cytokine, interleukin 1 beta (IL-1(beta)). In a separate set of experiments, cells were pretreated with IL-1ra prior to exposure to LPS or IL-1(beta). Prostaglandin E(2) and leukotriene B(4) (LTB(4)) levels were quantified by ELISA assays. Both LPS and IL-1(beta) exposure were noted to stimulate cellular PGE(2) release, a response which was significantly inhibited by IL-1ra treatment. Either stimulant when administered alone failed to stimulate release of LTB(4). When administered after IL-1ra pretreatment however, both stimuli caused a significant increase in LTB(4) release. These results suggest that a cytokine receptor antagonist can selectively influence eicosanoid production in this cell line. Furthermore, this study suggests that a IL-1ra may have a future clinical role in the treatment of inflammatory disorders of the colon which are intimately linked to enhanced eicosanoid synthesis.