The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone has immunomodulatory activity.

Diverse gram-negative bacterial cells communicate with each other by using diffusible N-acyl homoserine lactone (AHL) signal molecules to coordinate gene expression with cell population density. Accumulation of AHLs above a threshold concentration renders the population "quorate," and the appropriate target gene is activated. In pathogenic bacteria, such as Pseudomonas aeruginosa, AHL-mediated quorum sensing is involved in the regulation of multiple virulence determinants. We therefore sought to determine whether the immune system is capable of responding to these bacterial signal molecules. Consequently the immunomodulatory properties of the AHLs N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) were evaluated in murine and human leukocyte immunoassays in vitro. OdDHL, but not OHHL, inhibited lymphocyte proliferation and tumor necrosis factor alpha production by lipopolysaccharide-stimulated macrophages. Furthermore, OdDHL simultaneously and potently down-regulated the production of IL-12, a Th-1-supportive cytokine. At high concentrations (>7 x 10(-5) M) OdDHL inhibited antibody production by keyhole limpet hemocyanin-stimulated spleen cells, but at lower concentrations (<7 x 10(-5) M), antibody production was stimulated, apparently by increasing the proportion of the immunoglobulin G1 (IgG1) isotype. OdDHL also promoted IgE production by interleukin-4-stimulated human peripheral blood mononuclear cells. These data indicate that OdDHL may influence the Th-1-Th-2 balance in the infected host and suggest that, in addition to regulating the expression of virulence determinants, OdDHL may contribute to the pathogenesis of P. aeruginosa infections by functioning as a virulence determinant per se.

An investigation of the temporal induction of cytokine mRNAs in LPS-challenged thioglycollate-elicited murine peritoneal macrophages using the reverse transcription polymerase chain reaction.

OBJECTIVE AND DESIGN: A comprehensive study to standardise interleukin (IL)-1 alpha, -1 beta, -6, -10, -12 and tumour necrosis factor alpha (TNF alpha) mRNA detection in murine peritoneal macrophages, using the reverse transcription polymerase chain reaction (RT-PCR) was carried out. SUBJECTS: Thioglycollate-elicited peritoneal exudate cells were harvested from female BALB/c mice and the adherent macrophage fraction isolated for use. TREATMENT: Peritoneal macrophages (1 x 10(6)) were incubated in the presence or absence of lipopolysaccharide (LPS; at a final concentration of 1 microgram/ml) for 0, 1, 2, 3, 4, 5 and 24 h. METHODS: Culture supernatants and cells were harvested at each time point, the secreted cytokine protein levels quantified by sandwich immunoassays and the cytokine mRNA levels assessed by RT-PCR. RESULTS: The IL-6 mRNA was not expressed in detectable amounts in the macrophages, unless challenged with LPS. TNF alpha, IL-1 alpha, IL-1 beta, IL-10 and IL-12 mRNAs were expressed in both stimulated and unstimulated macrophages. The levels of the PCR products and thus mRNAs of all the cytokines increased with LPS stimulation, maximal levels being achieved 3 to 5 h post stimulation. CONCLUSIONS: RT-PCR produced consistent results, indicating that this technique could be used to investigate the effect of biological mediators and novel pharmacological agents on cytokine mRNA levels in macrophages.

Effect of pharmacological agents on the productions of interleukin-10 by the murine D10.G4.1 TH2 cell line in vitro.

It is becoming increasingly clear that the clinical courses of a variety of autoimmune and infectious diseases are influenced by the balance of TH1 and TH2 cell subsets that are generated during the immune response. IL-10 is one of several cytokines which influences the differentiation of TH cell subsets and represents a target for therapeutic intervention. We have evaluated a variety of pharmacological agents for their ability to modulate IL-10 release by the murine D10.G4.1 TH2 cell line when stimulated with concanacavalin-A in the presence of IL-1 alpha. Several were inhibitory, and the concentrations which caused a 50% reduction in IL-10 production were 0.38 microM cyclosporin-A, 0.0073 microM dexamethasone, 0.045 microM prednisolone and 0.31 microM cycloheximide. Methotrexate and pentoxifyline caused a weak but statistically significant reduction in IL-10 production at a concentration of 10 microM (P < or = 0.05), whereas amrinone and azathioprine had no clear effect. The pharmacological agents tested are known to exert multiple effects and were evaluated with a view to their use as reference standards in an ongoing screening programme to identify novel compounds which specifically modulate Il-10 production.

BTS 71 412: in vitro profile of a novel pyrazolinone immunosuppressant.

The effect of BTS 71 412, 4-acetyl-1-(4-chlorophenyl)-3-(2-methylthiophenyl)- 3-pyrazolin-5-one, has been determined on a variety of immune reactions in vitro in order to gain a further insight into the mechanisms whereby this novel immunosuppressive drug suppresses cell and antibody mediated immune responses in vivo. BTS 71 412 markedly inhibited [3H]thymidine incorporation by mouse splenocytes activated with concanavalin-A (IC50 = 20.1 microM), phytohaemagglutinin (IC50 = 4.6 microM), lipopolysaccharide (LPS) (IC50 = 3.2 microM), anti-IgM (mu-chain specific) (IC50 = 2.6 microM), or mixed lymphocyte culture (IC50 = 8.4 microM). Activity of BTS 71 412 was not associated with a reduction in cell viability. BTS 71 412 also prevented [3H]thymidine incorporation by the murine HT-2 helper T-cell clone when cultured with IL-2 (IC50 = 7.6 microM) or IL-4 (IC50 = 7.3 microM), enriched Thy-1+ T-lymphocytes stimulated with phorbol 12-myristate 13-acetate plus ionomycin (IC50 = 3.6 microM), and enriched B220- B-lymphocytes stimulated with LPS (IC50 = 3.0 microM). Splenocytes cultured with BTS 71 412 produced lower levels of interleukin (IL)-2, IL-10 and interferon-gamma when stimulated with concanavalin-A (IC50 values 42 microM, 22 microM and 60 microM, respectively). The compound suppressed in vitro antibody responses to keyhole limpet haemocyanin (IC50 = 2.3 microM), but did not reproducibly inhibit IL-6 or tumour necrosis factor alpha production by adherent peritoneal macrophages stimulated with LPS in vitro. These data indicate that BTS 71 412 specifically inhibits both B- and T-lymphocyte activation and proliferation but does not affect macrophage activation in vitro.

Seminal plasma superoxide dismutase activity following radiotherapy: Protection by the exogenous enzyme and effects of intracellular inhibition.

For referred subfertile, radiation-exposed and known fertile males seminal plasma superoxide dismutase activity did not correlate with sperm density, % sperm motility, sperm velocity or penetration in the sperm penetration assay (SPA), but was found to correlate positively with seminal plasma zinc. A study of seminal plasma superoxide dismutase in relation to post-irradiation clinical treatment time provided tentative evidence that following a radiation challenge, superoxide dismutase might be subject to induction. When sperm intracellular superoxide dismutase was inhibited by incubation with diethyldithiocarbamate (DDC) in defined culture medium, motility ceased within 30 min but there was no associated rise in lipid peroxidation. Sperm pretreatment with DDC at concentrations as low as 10 mum could abolish penetration in the SPA provided sperm were prepared by passive incubation. Cells electropermeabilized in the presence of calcium could overcome the DDC block. Exogenous addition of superoxide dismutase and catalase could provide effective protection to sperm against the effects of ultraviolet treatment.

Interactions of interferons in the induction of histocompatibility antigens in mouse fibroblasts and glial cells.

The interactive effects of interferons (IFN) in the induction of class II major histocompatibility complex (MHC) antigens in a murine fibroblast line and murine glial cells (primary astrocytes and an oligodendroglioma line) were examined. It was found that IFN-alpha and -beta were additive with IFN-gamma in the induction of class I antigens but that both IFN-alpha and -beta down-regulated the induction of class II antigens by IFN-gamma. This was most clear cut when the IFN-alpha or -beta was present in large excess in terms of anti-viral activity. Recombinant IFN-alpha 2 was found weakly to induce class II antigens, unlike natural IFN-alpha. The results imply that IFN-alpha and -beta may be important in the control of class II antigen expression in vivo although not by themselves inducing class II antigens.

The rapid induction of the acrosome reaction of human spermatozoa by electropermeabilization.

Human spermatozoa are usually prepared for in vitro fertilization (IVF) by centrifugal washing and incubation in albumin based media. This is an inefficient technique contributing to poor IVF prospects, false-negative scores in the sperm penetration assay (SPA), and inadequate quantification in the sperm chromosome assay (SCA). High yields of fusiogenically functional sperm were rapidly and efficiently prepared by exposing sperm suspensions to an electrical pulse of 750 to 1500 V cm-1 for 2.5 msec. This process was calcium-dependent, and sperm incorporation into zona-free hamster eggs exhibited a linear relationship with Ca2+. At voltages greater than 1500 V cm-1, penetration declined. Sperm from subfertile men demonstrated significantly increased penetration after electropermeabilization. Sperm development was not hindered and in the SCA, yields of sperm metaphases approaching 100% were achievable.

Assessment of heterospecific zona-free ovum penetration under fully defined conditions.

The prognostic value of the sperm penetration assay (SPA) using zona-free hamster ova has not been universally accepted. This is partly due to technical problems with the assay, resulting in 5-17% false negative results. The advantage of replacing albumin with defined synthetic polymers in the SPA culture media, increasing the calcium gradient and incorporating potential active tract agents and antioxidants, has been investigated. These studies have resulted in the development of a procedure that has produced no negative results among known fertile donors (n = 55). Application of this technique to a non-specific group of patients with suspected sub-fertility produced a different pattern of sperm penetration compared with the 'standard' SPA in 27% of cases. Improved sperm velocity, motile survival, enhanced hyaluronidase release and rate of acrosome induction were noted using the synthetic media.

Role of interferon-gamma in T-cell responses to Semliki Forest virus-infected murine brain cells.

Primary brain cell cultures prepared from newborn C3H mice were infected with Semliki Forest virus (SFV) or treated with a beta-propiolactone-inactivated preparation of SFV (BPL-SFV). The effects of recombinant interferon-gamma (IFN-gamma) treatment on SFV replication, SFV antigen display, major histocompatibility complex (MHC) class I and class II antigen expression, susceptibility to lysis by SFV-specific cytotoxic T lymphocytes (CTL) and the ability to stimulate SFV-specific T lymphocytes to release IFN-gamma were determined. The IFN-gamma treatment prevented replication of SFV, as determined by incorporation of [3H]uridine into SFV-RNA, and reduced expression of SFV antigens on the cell surface, as determined by lysis with antibody and complement or indirect immunofluorescence. BPL-SFV-treated brain cells expressed no SFV antigen detectable by lysis with antibody and complement or indirect immunofluorescence. IFN-gamma increased expression of MHC class I and class II antigens, measured by indirect immunofluorescence, susceptibility to killing by alloreactive T-cell lines and ability to stimulate an allogeneic mixed lymphocyte reaction (MLR). Brain cells infected with SFV or treated with BPL-SFV were susceptible to killing by the CTL. The killing was MHC restricted and neither uninfected nor untreated cells were killed. IFN-gamma treatment prior to SFV infection or BPL-SFV treatment resulted in an augmentation of lysis by the CTL, indicating that even where SFV antigen expression is reduced or present at very low levels, in the context of enhanced MHC class I expression cells remain susceptible to CTL killing. Brain cells treated with BPL-SFV stimulated SFV-specific T cells to release IFN-gamma. Pretreatment of brain cells with IFN-alpha beta or IFN-gamma prior to BPL-SFV treatment markedly increased the ability of the cells to stimulate the SFV-specific T cells to release IFN-gamma. Release of IFN-gamma was MHC restricted and brain cells untreated with BPL-SFV did not stimulate IFN-gamma release. IFN-gamma released by T cells stimulated with BPL-SFV-treated brain cells increased class II MHC expression by brain cells as assessed by indirect immunofluorescence.

Infection of cultured murine brain cells by Semliki Forest virus: effects of interferon-alpha beta on viral replication, viral antigen display, major histocompatibility complex antigen display and lysis by cytotoxic T lymphocytes.

Primary brain cell cultures prepared from newborn mice were infected with Semliki Forest virus (SFV). The effects of interferon (IFN-alpha beta) treatment on SFV replication, SFV and major histocompatibility complex (MHC) class I antigen expression, and susceptibility to lysis by SFV-specific cytotoxic T lymphocytes (CTL) were determined. The IFN-alpha beta treatment prevented replication of SFV as determined by incorporation of [3H]uridine into SFV RNA and very markedly reduced the expression of SFV antigens on the cell surface as determined by lysis with antibody and complement or indirect immunofluorescence. However, IFN-alpha beta increased expression of MHC class I antigens, measured by indirect immunofluorescence and as assessed indirectly by susceptibility to killing by alloreactive T cell lines. SFV infection had no effect on MHC class I expression in either IFN-alpha beta-treated or -untreated cells. The infected IFN-alpha beta-untreated brain cells were susceptible to killing by the CTL at effector/target ratios in the range 3 to 30. The killing was MHC antigen-restricted, and uninfected cells were not killed. A target cell (YAC) highly susceptible to natural killer cell cytotoxicity was not killed by the CTL. IFN-alpha beta treatment prior to SFV infection resulted in an augmentation of lysis by the CTL, indicating that even where SFV antigen expression is reduced, in the context of enhanced MHC class I expression brain cells remain susceptible to CTL killing.

A fluorocarbon-coated diuresis cage for rodents.

A simple and inexpensive rat diuresis cage is described. It may be constructed from thin aluminium sheeting in combination with a standard laboratory holding cage. Application of a low resistance PTFE coating reduces urine losses to a level better than a leading commercial design. Some physiological strain comparison data obtained using the cage are given.