m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Interferon-gamma enhances cytotoxic T lymphocyte recognition of endogenous peptide in keratinocytes without lowering the requirement for surface peptide

Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven by the K14 promoter, form a murine model of HPV-mediated epithelial cancers in humans. Our previous studies have shown that K14E7 transgenic skin grafts onto syngeneic mice are not susceptible to immune destruction despite the demonstrated presence of a strong, systemic CTL response directed against the E7 protein. Consistent with this finding, we now show that cultured, E7 transgenic keratinocytes (KC) express comparable endogenous levels of E7 protein to a range of CTL-sensitive E7-expressing cell lines but are not susceptible to CTL-mediated lysis in vitro . E7 transgenic and non-transgenic KC are susceptible to conventional mechanisms of CTL-mediated lysis, including perforin and Fas/FasL interaction when an excess of exogenous peptide is provided. The concentration of exogenous peptide required to render a cell susceptible to lysis was similar between KC and other conventional CTL targets (e.g. EL-4), despite large differences in H-2D(b) expression at the cell surface. Furthermore, exposure of KC to IFN-gamma increased H-2D(b) expression, but did not substantially alter the exogenous peptide concentration required to sensitize cells for half maximal lysis. In contrast, the lytic sensitivity of transgenic KC expressing endogenous E7 is modestly improved by exposure to IFN-gamma. Thus, failure of CTL to eliminate KC expressing endogenous E7, and by inference squamous tumours expressing E7, may reflect the need for a sustained, local inflammatory environment during the immune effector phase.

A clonal culture system demonstrates that IL-4 induces a subpopulation of noncytolytic T cells with low CD8, perforin, and granzyme expression

Immune deviation of cytolytic T cell function, induced by type 2 cytokines like IL-4, is an attractive concept to explain failure of the immune system in some diseases. However, this concept is challenged by previous conflicting results on whether type 2 cytokine-producing CD8(+) T cells are cytolytic. Therefore, we have analyzed the relationship between cytolytic activity and cytokine production among large numbers of primary CD8(+) T cell clones. Single murine CD8(+) T cells of naive phenotype were activated at high efficiency with immobilized Abs to CD3, CD8, and CD11a in the presence of IL-2 (neutral conditions) or IL-2, IL-4, and anti-IFN-gamma Ab (type 2-polarizing conditions) for 8-9 days. Under neutral conditions, most clones produced IFN-gamma without IL-4 and were cytolytic. Under type 2-polarizing conditions, most clones produced IFN-gamma and IL-4 but displayed variable cytolytic activity and CD8 expression. Separation on the basis of surface CD8 levels revealed that, compared with CD8(high) cells from the same cultures, CD8(low) cells were poorly cytolytic and expressed low levels of perforin mRNA and protein and granzyme A, B, and C mRNA. A similar, smaller population of noncytolytic CD8(low) cells was identified among CD8(low) T cells activated in mixed lymphocyte reaction with IL-4. Variable efficiency of generation of the noncytolytic cells may account for the differing results of earlier studies. We conclude that IL-4 promotes the development of a noncytolytic CD8(low) T cell phenotype that might be important in tumor- or pathogen-induced immune deviation.

The relationship of natural killer cell counts, perforin mRNA and CD2 expression to post-exercise natural killer cell activity in humans

The aim of this study was to further investigate the mechanism of suppression of natural killer (NK) cell cytotoxic activity In peripheral blood following strenuous exercise. Blood was collected for analysis of NK cell concentration, cytotoxic activity, CD2 surface expression and perforin gene expression from runners (RUN, n = 6) and resting controls (CONTROL, n = 4) pre-exercise, 0, 1.5, 5, and 24 h following a 60-min treadmill run at 80% of VO2 peak. Natural killer cytotoxic activity, measured using a whole blood chromium release assay, fluctuated minimally in the CONTROL group and increased by 63% and decreased by 43% 0 and 1.5 h post-exercise, respectively, in the RUN group (group x time, P < 0.001). Lytic index (cytotoxic activity per cell) did not change. Perforin mRNA, measured using quantitative real-time polymerase chain reaction (ORT-PCR) decreased from pre- to post-exercise and remained decreased through 24 h, The decrease from pre- to 0 In post-exercise was seen predominately in the RUN group and was inversely correlated r = - 0.95) to pre-exercise perform mRNA. The NK cell surface expression of CD2 (lymphocyte function-associated antigen-2) was determined using fluorescent antibodies and flow cytometry, There was no change in the proportion of NK cells expressing CD2 or CD2 density, We conclude that (1) numerical redistribution accounted for most of the change in NK cytotoxic activity following a strenuous run, (2) decrease in perforin gene expression during the run was inversely related to pre-exercise levels but did not parallel changes in cytotoxic activity, and (3) CD2 surface expression was not affected by exercise.

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG(4) monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 mu g 10(6) cells(-1) mL(-1) at a PEI nitrogen: DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from similar to 13 to similar to 39 mg L-1. Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended activated hypothermic synthesis.

Correlation of habitual physical activity levels with flow mediated dilation of the brachial artery in 5-10 year old children

Endothelial dysfunction is an early key event of atherogenesis. Both fitness level and exercise intervention have been shown to positively influence endothelial function. In a cross-sectional study of 47 children, the relationship between habitual physical activity and flow-mediated dilation (FMD) of the brachial artery was explored. Habitual physical activity levels (PALs) were assessed using a validated stable isotope technique, and FMD of the brachial artery was measured via high-resolution ultrasound. The results showed that habitual physical activity significantly correlated with FMD (r=0.39, P=0.007), and remained the most influential variable on dilation in multivariate analysis. Although both fitness level and exercise intervention have previously been shown to positively influence FMD, this is the first time that a relationship with normal PALs has been investigated, especially, at such a young age. These data support the concept that physical activity exerts its protective effect on cardiovascular health via the endothelium and add further emphasis to the importance of physical activity in childhood.

Superconductivity mediated by charge fluctuations in layered molecular crystals

We consider the competition between superconducting, charge ordered, and metallic phases in layered molecular crystals with the theta and beta" structures. Applying slave-boson theory to the relevant extended Hubbard model, we show that the superconductivity is mediated by charge fluctuations and the Cooper pairs have d(xy) symmetry. This is in contrast to the kappa-(BEDT-TTF)(2)X family, for which theoretical calculations give superconductivity mediated by spin fluctuations and with d(x)2(-y)2 symmetry. We predict several materials that should become superconducting under pressure.

Acid-mediated conversion of methylene-interrupted bisepoxides to tetrahydrofurans: A biomimetic transformation

The acid-mediated transformation of syn and anti methylene interrupted cis,cis and cis,trans bisepoxides to tetrahydrofurans is high yielding, and demonstrates both regioselectivity and stereoselectivity. Trans,trans methylene interrupted bisepoxides do not yield tetrahydrofurans under the same conditions.

From sabotage to camouflage: viral evasion of cytotoxic T lymphocyte and natural killer cell-mediated immunity

The outcome of a virus infection is strongly influenced by interactions between host immune defences and virus 'anti-defence' mechanisms. For many viruses, their continued survival depends on, the speed of their attach: their capacity to replicate and transmit to uninfected hosts prior to their elimination by an effective immune response. In contrast, the success of persistent viruses lies in their capacity for immunological subterfuge: the evasion of host defence mechanisms by either mutation (covered elsewhere in this issue, by Gould and Bangham, pp. 321-328) or interference with the action of host cellular proteins that are important components of the immune response. This review will focus on the strategies employed by persistent viruses against two formidable host defences against virus infection: the CD8+ cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses.

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-H-3]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin I. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin I using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an cc-helical secondary structure. (C) 1998 Elsevier Science B.V, All rights reserved.

Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha 1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [H-3]strychnine binding assays. In the wild-type (WT) GlyR, 1 mu M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1-M2 and M2-M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2-M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.

Self-efficacy for refusal mediated by outcome expectancies in the prediction of alcohol-dependence amongst young adults

The present study examined the relative importance of outcome expectancies and self-efficacy [1] in the prediction of alcohol dependence [2] and alcohol consumption in a sample of young adult drinkers drawn from a milieu previously reported as supportive of risky drinking. In predicting alcohol dependence, outcome expectancies were found to mediate self-efficacy and the same pattern was found for both males and females. This suggests that male and female drinkers may become more similar as they progress along the drinking continuum from risky drinking to dependent drinking. However, in women, in comparison to men, a greater array of expectancies and self-efficacy scales were found to predict heavy drinking, as measured by quantity and frequency. These results suggest that heavy drinking women are particularly at risk of developing drinking related complications and that preventative education needs to take into account gender differences.

Complement-mediated regulation of Trichomonas vaginalis infection in mice

Trichomonas vaginalis is a flagellated protozoan which causes trichomoniasis, a sexually transmitted disease of the human genitourinary tract, The importance of the alternative complement pathway in host defence against T. vaginalis was investigated in vitro. Kinetic studies utilising immunofixation following electrophoresis showed that both a strongly and weakly virulent strain of T, vaginalis activated murine serum C3. In vivo studies with congenic-resistant, C5-deficient, B10.D2/OSn- and C5-sufficient, B10.D2/nSn mice showed that the presence of C5 is a significant factor in the innate host resistance to primary infection with a strongly virulent, but not a weakly virulent trichomonad strain.

p75 neurotrophin receptor-mediated neuronal death is promoted by Bcl-2 and prevented by Bcl-x(L)

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. We microinjected p75NTR expression plasmids into sensory neurons in the presence of growth factors and assessed the effect of the expressed proteins on cell survival. We show that, unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the death domain and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promoted, and was required for, p75NTR killing, whereas killing was inhibited by its homologue BcI-x(L). These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus.