Adenosine modulates LPS-induced cytokine production in porcine monocytes

Adenosine plays an important role during inflammation, particularly through modulation of monocyte function. The objective of the present study was to evaluate the effect of synthetic adenosine analogs on cytokine production by porcine monocytes. The LPS-stimulated cytokine production was measured by flow cytometry and quantitative real-time PCR. Adenosine receptor expression was measured by quantitative real-time PCR. The present study demonstrates that adenosine analog N-ethylcarboxyamidoadenosine (NECA) down-regulates TNF-α production and up-regulates IL-8 production by LPS-stimulated porcine monocytes. The effect was more pronounced in CD163^? subset of monocytes compared to the CD163⁺ subset. Although both monocyte subsets express mRNA for A1, A2A, A2B and A3 adenosine receptors, the treatment of monocytes with various adenosine receptor agonists and antagonists proved that the effect of adenosine is mediated preferentially via A2A adenosine receptor. Moreover, the study suggests that the effect of NECA on porcine monocytes alters the levels of the cytokines which could play a role in the differentiation of naive T cells into Th17 cells. The results suggest that adenosine plays an important role in modulation of cytokine production by porcine monocytes.     

Histamine modulates the cellular stress response in yeast

The cellular stress response is a universal protective reaction to adverse environmental or microenvironmental conditions, such as heat and drugs, associated in part with the highly conserved heat shock proteins (HSPs). Histamine is a key inflammatory mediator derived from l-histidine that governs vital cellular processes beyond inflammation, while recent evidence implies additional actions in both prokaryotes and eukaryotes. This study explored the possible role of histamine in the heat shock response in yeast, an established experimental model for the pharmacological investigation of the cellular stress response. The response was evaluated by determining growth and viability of post-logarithmic phase grown yeast cultures after heat shock at 53°C for 30 min. Thermal preconditioning at 37°C for 2 h served as a positive control. The effect of histamine was investigated following long-term administration through the post-logarithmic phase of growth or short-term administration for 2 h prior to heat shock. Short-term treatment with 1 mM histamine resulted in de novo protein synthesis-dependent acquisition of thermotolerance, while lower doses or long-term administration of histamine failed to induce the heat-resistant phenotype. Preliminary investigation of HSP104, HSP70 and HSP60 expression by western blotting showed an increase of these proteins after thermal preconditioning. However, a differential HSP and tubulin expression appeared to underlie the response of yeast cells to histamine. In conclusion, histamine was capable of inducing the adaptive phenotype, while the contribution of HSPs and tubulin and the potential implications remain largely elusive.     

Curosurf modulates cAMP accumulation in human monocytes through a membrane-controlled mechanism

The cellular mechanisms by which pulmonary surfactant exerts its effects, including anti-inflammatory or proinflammatory effects, have remained elusive. To address the issue of whether plasma membrane modifications represent a target for these mechanisms, we designed an experimental protocol involving the determination of changes in cAMP levels under membrane-dependent or -independent stimulatory pathways. The effects of a modified natural porcine surfactant, Curosurf, and the major surfactant protein A were evaluated on resting and stimulated cAMP levels of human monocytes. We found that agents that elevate intracellular cAMP exhibit different susceptibilities toward a preexposure to Curosurf. The rise in cAMP induced by membrane-active agents such as cholera toxin or the diterpene forskolin was significantly inhibited by monocyte preexposure to Curosurf. In contrast, the rise in cAMP induced by the membrane-permeant phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine or by the Bordetella pertussis toxin adenylate cyclase-hemolysin was unaffected by Curosurf. Surfactant protein A did not affect either cAMP levels or the inhibitory capacity of Curosurf. We suggest that a plasma membrane-associated event affecting the mechanism underlying the effects of cholera toxin or forskolin is involved in the inhibition of cAMP accumulation caused by Curosurf.     

Protein phosphatase Z modulates oxidative stress response in fungi

The genome of the filamentous fungus Aspergillus nidulans harbors the gene ppzA that codes for the catalytic subunit of protein phosphatase Z (PPZ), and the closely related opportunistic pathogen Aspergillus fumigatus encompasses a highly similar PPZ gene (phzA). When PpzA and PhzA were expressed in Saccharomyces cerevisiae or Schizosaccharomyces pombe they partially complemented the deleted phosphatases in the ppz1 or the pzh1 mutants, and they also mimicked the effect of Ppz1 overexpression in slt2 MAP kinase deficient S. cerevisiae cells. Although ppzA acted as the functional equivalent of the known PPZ enzymes its disruption in A. nidulans did not result in the expected phenotypes since it failed to affect salt tolerance or cell wall integrity. However, the inactivation of ppzA resulted in increased sensitivity to oxidizing agents like tert-butylhydroperoxide, menadione, and diamide. To demonstrate the general validity of our observations we showed that the deletion of the orthologous PPZ genes in other model organisms, such as S. cerevisiae (PPZ1) or Candida albicans (CaPPZ1) also caused oxidative stress sensitivity. Thus, our work reveals a novel function of the PPZ enzyme in A. nidulans that is conserved in very distantly related fungi.     

Environmental and biological context modulates the physiological stress response of bats to human disturbance

Oecologia - Environmental and biological context play significant roles in modulating physiological stress responses of individuals in wildlife populations yet are often overlooked when evaluating...     

Transcriptional profiling of stress response in cultured porcine islets

Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7 mM), inflammatory cytokine addition (IL-1β, TNF-α, and IFN-γ), or both, for 48 h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and β cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.     

Adherence of human monocytes and NK cells to human TNF-alpha-stimulated porcine endothelial cells

Discordant xenograft models undergoing delayed rejection response are characterized by xenograft infiltration with host monocytes and NK cells, associated with the release of large quantities of pro-inflammatory cytokines, such as TNF-alpha. In the present study, human monocytes (PBMo)/NK cells (PBNK) isolated from peripheral blood and cultured porcine aortic endothelial cells (PAEC) treated with recombinant human TNF-alpha (rhTNF-alpha) were used to investigate their adhesive interactions and mAbs against porcine E-selectin, human CD11a and CD49d were used to test their relative contributions to such intercellular adhesions. The PBMo exhibited significantly greater adherence to resting (unstimulated) PAEC than PBNK. The rhTNF-alpha upregulated E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression on PAEC and augmented the adhesiveness of PAEC for PBMo and PBNK in a time- and dose-dependent manner. In mAb blocking assays, anti-E-selectin, anti-CD11a and anti-CD49d mAbs did not inhibit PBMo adherence to rhTNF-alpha-stimulated PAEC when used singly, but resulted in a maximal inhibitory effect when used in combination. Regarding PBNK, anti-E-selectin mAb had no marked influence on PBNK adherence. The combined use of anti-CD11a and anti-CD49d mAbs produced additive reduction in the PBNK binding to rhTNF-alpha-stimulated PAEC, even to far below baseline (unstimulated) levels. Therefore, it is concluded that human TNF-alpha promotes the adhesiveness of PAEC for human monocytes and NK cells and that the mechanism underlying the increased adherence differs for PBMo and PBNK.     

Arabidopsis EIN2 modulates stress response through abscisic acid response pathway

The nuclear protein ETHYLENE INSENSITIVE2 (EIN2) is a central component of the ethylene signal transduction pathway in plants, and plays an important role in mediating cross-links between several hormone response pathways, including abscisic acid (ABA). ABA mediates stress responses in plants, but there is no report on the role of EIN2 on plant response to salt and osmotic stresses. Here, we show that EIN2 gene regulates plant response to osmotic and salt stress through an ABA-dependent pathway in Arabidopsis. The expression of the EIN2 gene is down-regulated by salt and osmotic stress. An Arabidopsis EIN2 null mutant was supersensitive to both salt and osmotic stress conditions. Disruption of EIN2 specifically altered the expression pattern of stress marker gene RD29B in response to the stresses, but not the stress- or ABA-responsive genes RD29A and RD22, suggesting EIN2 modulates plant stress responses through the RD29B branch of the ABA response. Furthermore, disruption of EIN2 caused substantial increase in ABA. Lastly, our data showed that mutations of other key genes in ethylene pathway also had altered sensitivity to abiotic stresses, indicating that the intact ethylene may involve in the stress response. Taken together, the results identified EIN2 as a cross-link node in ethylene, ABA and stress signaling pathways, and EIN2 is necessary to induce developmental arrest during seed germination, and seedling establishment, as well as subsequent vegetative growth, thereby allowing the survival and growth of plants under the adverse environmental conditions. Youning Wang and Chuang Liu contributed equally to this work.     

Hypermethylation of tobacco heterochromatic loci in response to osmotic stress

 Plants have to cope with a number of envi-ronmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methylation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension culture, and the stress was elicited with NaCl or D-mannitol. Using the restriction enzymes MspI/HpaII and MboI/Sau3AI in combination with Southern hydridization we observed a reversible hypermethylation of the external cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucleotides within the CCGG motifs (HpaII sites) appeared to be fully methylated in tobacco DNA repetitive sequences under normal physiological conditions. The data suggest epigenetic changes in the plant genome based on de novo methylation of DNA in response to environmental stress. Received: 26 November 1996/Accepted: 20 December 1996     

Heterogeneity of human monocytes: differences in response to IFN-gamma

Human peripheral blood monocytes can be separated into two subpopulations which differ in the efficiency of their adherence to glass after 16 hours of incubation. The adherent subpopulation was found to be about twice as effective in binding mannose-resistant E. coli 0-124, mannose-sensitive E. coli 0-128 and opsonised E. coli than the nonadherent one. In addition, reduction of cytochrome C in response to E. coli binding or 12-myristate 13-acetate (PMA) stimulation was two fold higher in adherent cells. The binding of E. coli O-124 and the superoxide generation stimulated by E. coli were inhibited by the addition of mannose only in the adherent monocytes, indicating the presence of mannose receptors on the cell surface in the adherent subpopulation. The treatment of the nonadherent cells with 0.1-1000 U/ml of Interferon (IFN-gamma) for 24 hours resulted in a dose dependent increase in superoxide generation. After 72 hours of incubation with IFN-gamma (1000 U/ml) the amount of superoxide generated by the nonadherent cells was elevated to 20.5 +/- 1.4 nmoles/10(6) cells/15 min, similar to that of the adherent cells (24.5 +/- 1.2 nmoles/10(6) cells/15 min untreated adherent monocytes). The generation of superoxide in the IFN-gamma treated nonadherent monocytes stimulated by E. coli 0-128 was significantly reduced by addition of mannose.     

Hfq modulates the sigmaE-mediated envelope stress response and the sigma32-mediated cytoplasmic stress response in Escherichia coli

Hfq, a chaperone for small noncoding RNAs, regulates many processes in Escherichia coli, including the sigma(S)-mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters. Many of these genes are involved in amino acid uptake and biosynthesis, sugar uptake and metabolism, and cell energetics. In addition, we find altered regulation of the sigma(E)- and sigma(32)-mediated stress responses, which we analyze further. We show that cells lacking Hfq induce the sigma(E)-mediated envelope stress response and are defective in sigma(E)-mediated repression of outer membrane proteins. We also show that the sigma(32)-mediated cytoplasmic stress response is repressed in cells lacking Hfq due to increased expression of DnaK. Furthermore, we show that cells lacking Hfq are defective in the "long-term adaptation" of sigma(32) to chronic chaperone overexpression. Together, our results indicate that Hfq may play a general role in stress response regulation in E. coli.     

Putrescine modulates antioxidant defense response in wheat under high temperature stress

Effects of putrescine (Put) on responses of wheat (Triticum aestivum) seedlings or detached tillers at mid-milky stage to high temperature (HT) stress were investigated. The heat tolerant cv. PBW 343 exhibited higher content of antioxidants and activities of antioxidative enzymes, while lower content of lipid peroxides as compared to the heat-sensitive cv. HD 2329. HT elevated peroxidase (POX) and superoxide dismutase (SOD) activities, while diamine oxidase (DAO) and polyamine oxidase (PAO) activities were reduced in roots, shoots and developing grains. Application of Put under HT further enhanced POX and SOD activities along with increased content of ascorbate and tocophereol in grains. Invariably POX and SOD revealed higher activities in roots while CAT, DAO and PAO activities were higher in shoots. The content of lipid peroxides was increased in roots and shoots of HT stressed seedlings but less in Put-treated cv. PBW 343.     

Neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells

Defensins, a family of small, cationic, antimicrobial peptides, are found in mammals, insects and plants. alpha-defensins are stored in granules of neutrophils and released upon activation by exocytosis. It was shown here that human neutrophil peptide (HNP), at concentrations of 10(-8) -10(-9) M, up-regulated the expression of TNF-alpha and IL-1 beta in monocytes activated with Staphylococcus aureus or PMA, while expression of IL-10 mRNA was down-regulated and production of IL-8 was not affected. HNP alone was unable to induce TNF-alpha or IL-1 beta expression in resting monocytes. At concentrations of 10(-4) -10(-5)M, HNP was cytotoxic for monocytes in serum-free medium. The cytotoxicity was abrogated in the presence of serum, while a cytokine-modulating effect of HNP was observed in the presence of serum and in whole blood, suggesting that this mechanism may function in vivo. Similarly, serum did not abrogate bactericidal activity of HNP. It was also demonstrated herein that HNP at 10 (-8) -10(-9) M, attenuated the inhibitory action of dexamethasone on TNF-alpha production. In parallel to monocyte studies, we have showed that HNP at concentrations ranging from 10(-9)M to 10(-6)M caused about 5-fold suppression of VCAM-1 expression in TNF-alpha-activated human umbilical vein endothelial cells, while the ICAM-1 expression was not affected. Our findings suggest that neutrophil defensins have the potential to modulate the inflammatory responses through regulation of cytokine production and adhesion molecule expression.     

RGD-mediated adhesion of porcine granulosa cells modulates their differentiation response to FSH in vitro

Porcine granulosa cells cultured in serum-free medium undergo metabolic and morphologic changes after follicle stimulating hormone (FSH) stimulation. Under these conditions, granulosa cells differentiate and tend to round up and their links with the plastic support are reduced. Coating of culture substratum with PepTite-2000, an integrin-binding synthetic peptide containing RGD (Arg-Gly-Asp) sequences enhanced the plating of granulosa cells. Whether the peptide be present or not, cells cultivated in basal synthetic medium (without FSH) were flattened and attached to the substratum by stress fibers at focal contacts where integrin β1, extracellular fibronectin, and urokinase plasminogen activator colocalized. After FSH stimulation, part of the cells rounded up and F-actin took a more uniform, cortical localization. Correlatively, extracellular fibronectin aggregated in a clump, while integrin β1 and urokinase plasminogen activator spread over rounded cells. These morphological changes elicited by FSH were little affected by the presence of PepTite-2000, yet a larger number of cells remained flattened. However, concerning steroidogenesis, increasing concentrations of peptide seemed to favor progesterone rather than estrogen production, and to restrain luteinizing hormone (LH) receptor expression, suggesting a premature committment of cells towards luteinization rather than completion of follicular preovulatory differentiation.     

Neonatal porcine Sertoli cells inhibit human natural antibody-mediated lysis

Sertoli cells protect cotransplanted cells from allogeneic and xenogeneic rejection. Additionally, neonatal porcine Sertoli cells (NPSCs) survive long-term as xenografts in nonimmunosuppressed rodents. This has led to the hypothesis that NPSCs could be used to prevent cellular rejection in clinical transplantation, thereby eliminating the need for chronic immunosuppression. Prior to transplantation of NPSCs in humans it is necessary to determine whether they are also protected from humoral-mediated xenograft rejection. The presence of Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (alphaGal epitope) as well as binding of human immunoglobulin G (IgG) and IgM to NPSCs was examined by immunocytochemical and fluorescence-activated cell sorter analysis. alphaGal was detected on 88.5% +/- 3.0% of NPSCs. Consistent with this, 71.7% +/- 1.0% and 65.4% +/- 5.2% of NPSCs were bound by IgG and IgM, respectively. When cultured NPSCs underwent an in vitro cytotoxicity assay by incubation with human AB serum plus complement, no increase in cellular lysis was observed, while controls--porcine aorta endothelial cells--were shown to contain > 60% dead cells. Finally, activation of the complement cascade was examined by immunohistochemistry. C3 and C4 were deposited on the surface of the NPSC membrane, indicating activation of complement. Although the complement cascade was activated, the membrane attack complex (MAC) was not formed. These data demonstrate that despite expression of alphaGal, binding of xenoreactive antibodies, and the activation of complement, NPSCs survive human antibody and complement-mediated lysis by preventing MAC formation. This suggests that NPSCs may be able to survive humoral-mediated rejection in a clinical situation.     

Lysosomal enzyme release from human monocytes in response to particulate stimuli

Lysosomal enzyme release from human monocytes was evaluated in response to opsonized zymosan, opsonized sheep erythrocytes, and latex beads. Monocytes were found to release lysosomal enzymes immediately upon challenge with all three phagocytosable particles. Cytochalasin B enhanced beta-glucosaminidase release from mononuclear cells challenged with opsonized zymosan or opsonized red blood cells, but inhibited the response to latex particles. Lysosomal enzyme release was found to be independent of protein synthesis, and in the absence of cytochalasin B required the stimulus to be presented either as a phagocytosable particle or immobilized on a surface. The kinetics of enzyme release and phagocytosis were also examined and found to be different, lending support to the hypothesis that lysosomal enzyme release may be a physiologic response to a biologic stimulus in vivo and not simply an "accidental" consequence of an ongoing phagocytic event.     

Type VI secretion modulates quorum sensing and stress response in Vibrio anguillarum

Type VI protein secretion systems (T6SS) are essential for virulence of several Gram‐negative bacteria. In this study, we identified a T6SS in Vibrio anguillarum, a marine bacterium that causes a hemorrhagic septicemia in fish. A partial operon vtsA‐H (v ibrio t ype s ix secretion) was sequenced and shown to encode eight proteins. VtsE‐H are signature proteins found in other T6SSs, while VtsA‐D are not associated with T6SS studied so far. In‐frame deletions were made in each gene. Secretion of a haemolysin‐co‐regulated‐like protein (Hcp), a protein secreted by all studied T6SSs, was decreased in VtsE‐H. Unexpectedly, VtsA, VtsC and VtsD activated while VtsB and VtsE‐H repressed hcp expression. The T6SS proteins also regulated expression of two extracellular proteases, EmpA and PrtV, but inversely to Hcp expression. This regulation was indirect as T6S positively regulated expression of the stress‐response regulator RpoS and the quorum‐sensing regulator VanT, which positively regulate protease expression. Moreover, VtsA‐H proteins were not needed for virulence but did play a role in various stress responses. Thus, these data characterize a new role for T6S in the ecology of bacteria and we hypothesize this role to be a signal sensing mechanism that modulates the expression of regulators of the general stress response.