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Biomedical subjects

E Bohn

Publications and source records attributed to E Bohn.

At least 19 recordsLinked to original sources
PubMed2006

The Yersinia enterocolitica invasin protein promotes major histocompatibility complex class I- and class II-restricted T-cell responses.

Yersinia enterocolitica invasin (Inv) protein confers internalization into and expression of proinflammatory cytokines by host cells. Both events require binding of Inv to beta1 integrins, which initiates signaling cascades including activation of focal adhesion complexes, Rac1, mitogen-activated protein kinase, and NF-kappaB. Here we tested whether Inv might be suitable as a delivery molecule and adjuvant if used as a component of a vaccine. For this purpose, hybrid proteins composed of Inv and ovalbumin (OVA) were prepared, applied as a coating to microparticles, and used for vaccination. Fusion of OVA to Inv did not significantly disturb the ability of Inv to promote host cell binding, internalization, and interleukin-8 (IL-8) secretion when applied as a coating to microparticles. The microparticles were used for vaccination of mice adoptively transferred with OVA-specific T cells from OT-1 or DO11.10 mice. Administration of OVA-Inv-coated microparticles induced OVA-specific T-cell responses. OVA-specific CD4 T cells produced both gamma interferon (IFN-gamma) and IL-4 as determined by enzyme-linked immunosorbent assay. Likewise, pronounced OVA-specific CD8 T-cell responses associated with IFN-gamma production were observed. Together, these results suggest that Inv might be an attractive tool in vaccination as it confers both host cell uptake and adjuvant activity by engagement of beta1 integrins of host cells, which leads to CD4 as well as CD8 T-cell responses.

Adhesins, Bacterial↗
PubMed2004

Gene expression patterns of epithelial cells modulated by pathogenicity factors of Yersinia enterocolitica.

Epithelial cells express genes whose products signal the presence of pathogenic microorganisms to the immune system. Pathogenicity factors of enteric bacteria modulate host cell gene expression. Using microarray technology we have profiled epithelial cell gene expression upon interaction with Yersinia enterocolitica. Yersinia enterocolitica wild-type and isogenic mutant strains were used to identify host genes modulated by invasin protein (Inv), which is involved in enteroinvasion, and Yersinia outer protein P (YopP) which inhibits innate immune responses. Among 22 283 probesets (14,239 unique genes), we found 193 probesets (165 genes) to be regulated by Yersinia infection. The majority of these genes were induced by Inv, whose recognition leads to expression of NF-kappa B-regulated factors such as cytokines and adhesion molecules. Yersinia virulence plasmid (pYV)-encoded factors counter regulated Inv-induced gene expression. Thus, YopP repressed Inv-induced NF-kappa B regulated genes at 2 h post infection whereas other pYV-encoded factors repressed host cell genes at 4 and 8 h post infection. Chromosomally encoded factors of Yersinia, other than Inv, induced expression of genes known to be induced by TGF-beta receptor signalling. These genes were also repressed by pYV-encoded factors. Only a few host genes were exclusively induced by pYV-encoded factors. We hypothesize that some of these genes may contribute to pYV-mediated silencing of host cells. In conclusion, the data demonstrates that epithelial cells express a limited number of genes upon interaction with enteric Yersinia. Both Inv and YopP appear to modulate gene expression in order to subvert epithelial cell functions involved in innate immunity.

Adhesins, Bacterial↗
PubMed2001

Biologic consequences of Stat1-independent IFN signaling.

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNgamma functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNgamma and IFNalpha/beta regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNgamma exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNgamma receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences.

Animals↗
PubMed1999

DNA immunization confers systemic, but not mucosal, protection against enteroinvasive bacteria.

Naked plasmid DNA (pRc/Y-hsp60) with a cytomegalovirus promoter and a sequence encoding Yersinia enterocolitica 60-kDa heat shock protein (Y-HSP60) was used for vaccination. After intramuscular injection of pRc/Y-hsp60, Y-hsp60 mRNA could be detected by reverse transcription-PCR in muscle, liver and spleen. A single immunization with pRc/Y-hsp60 induced significant Y-HSP60-specific T cell responses after 1 week. IFN-gamma production by spleen cells upon stimulation with Y-HSP60 was strictly dependent on the presence of CD4+ T cells, indicating the generation of a Th1 response upon DNA immunization. DNA immunization in addition induced strong Y-HSP60-specific IgG2a, weak IgG1, but not IgA antibodies. Immunization of BALB/c and C57BL/6 mice with pRc/Y-hsp60 conferred protection against disseminated Y. enterocolitica infection in spleen, but not at the site of mucosal entry, the Peyer's patches. Furthermore, pRc/Y-hsp60 vaccination did not induce cross-protection against related pathogens. Vaccination of beta2-microglobulin- and H2-I-Abeta-deficient mice was not protective, suggesting that both CD4+ and CD8+ T cells are required for protective immunity induced by DNA vaccination.

Animals↗
PubMed1998

IL-18 (IFN-gamma-inducing factor) regulates early cytokine production in, and promotes resolution of, bacterial infection in mice.

IL-12-induced IFN-gamma production is essential for clearance of Yersinia enterocolitica infection. Similar to IL-12, the recently described cytokine IL-18 (IFN-gamma-inducing factor) is produced by macrophages and induces IFN-gamma production in spleen cells. Therefore, we have investigated the role of IL-18 in Yersinia infection of mice. Heat-killed yersinia-triggered IL-18-promoted IFN-gamma production of splenocytes was predominantly dependent on endogenous IL-12 production, whereas IL-12-promoted IFN-gamma production was not IL-18 dependent. IL-18-induced IFN-gamma production was to a higher degree dependent on IFN-gammaR-mediated mechanisms and in synergism with IL-2 resulted in at least fivefold higher IFN-gamma levels as compared with the combination of IL-12 plus IL-2. Analysis of the effect of IL-18 on IL-12 production of LPS-stimulated peritoneal macrophages revealed that IL-18 decreased LPS-induced IL-12 production, indicating that IL-18 might be involved in negative regulation of IL-12 production. In vivo studies revealed that Yersinia-resistant C57BL/6 mice expressed fourfold higher IL-18 mRNA levels than did susceptible BALB/c mice. Administration of anti-IL-18 Abs caused a 100- to 1000-fold increase in bacterial counts in the spleen of infected mice but did not change IFN-gamma production levels. Taken together, our data demonstrate that IL-18 is involved in regulation of cytokine production during the early phase of bacterial infections as well as in clearance of Yersinia infection.

Animals↗
PubMed1998

In vivo tracking and protective properties of Yersinia-specific intestinal T cells.

After invasion via M cells enteropathogenic Yersinia enterocolitica subsequently establish an infection at three different sites: (i) Peyer's patches (PP), (ii) mesenteric lymph nodes (MLN), and after systemic dissemination in (iii) spleen, liver and lung. In order to characterize protective properties of intestinal T cells at the different sites of Y. enterocolitica infection, PP and MLN T cells were isolated from Y. enterocolitica-infected C57B1/6 mice and Yersinia-specific T cell lines were generated. These T cells exhibited the phenotype of CD4 Th1 cells. The adoptive transfer of Yersinia-specific Th1 cells from PP and MLN conferred protection against a lethal orogastric inoculum with Y. enterocolitica as revealed by survival post-infection. However, determination of bacterial counts in infected organs revealed that the transfer of PP T cells conferred protection in spleen but not in MLN and PP, whereas the transfer of T cells from MLN reduced bacterial counts in both spleen and MLN but not in PP. To elucidate the different protection pattern we wanted to track the transferred cells in vivo. For this purpose the cells were labelled with the stable green fluorescent cell linker PKH2-GL prior to the adoptive transfer. In vivo tracking of these cells revealed that the distribution pattern of transferred T cells in spleen, MLN and PP correlated closely with the protection pattern observed after Yersinia infection. Thus, most cells were recovered from the spleen, while only few cells were recovered from MLN and PP. In keeping with these results a rapid and significant increase in interferon-gamma (IFN-gamma) production in the spleen of mice after adoptive transfer of T cell lines was observed. Taken together, the present results demonstrate that intestinal CD4 Th1 cells from PP and MLN may be involved in the defence against Y. enterocolitica at different sites of the infection, and that PKH2-GL labelling is a suitable tool to characterize T cell functions in vivo.

Adoptive Transfer↗
PubMed1998

Ambiguous role of interleukin-12 in Yersinia enterocolitica infection in susceptible and resistant mouse strains.

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-gamma) production in NK and CD4+ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-gamma-receptor-deficient (IFN-gamma R-/-) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55-/-) mice. IFN-gamma R-/- mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-gamma R+/+ mice. Administration of IL-12 was protective in IFN-gamma R+/+ mice but not in IFN-gamma R-/- mice, suggesting that IFN-gamma R-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor beta (TGF-beta). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-beta. While administration of IL-12 alone increased TNF-alpha levels, administration of TGF-beta or TGF-beta plus IL-12 decreased both TNF-alpha and IFN-gamma levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55-/- mice, suggesting that TNF-alpha accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.

Animals↗
PubMed1997

On the mechanism of lysophospholipase activity of secretory phospholipase A2 (EC 3.1.1.4): deacylation of monoacylphosphoglycerides by intrinsic sn-1 specificity and pH-dependent acyl migration in combination with sn-2 specificity.

We show for the first time that secreted low-molecular weight phospholipase A2 (EC 3.1.1.4) catalyzes the deacylation of monoacylphosphoglycerides directly from the sn-1 position, although at a very low rate: purified phospholipase A2 enzymes from bee venom, crotalus atrox venom, and porcine pancreas hydrolyze the sn-1 ester bond in 1-palmitoyl-2-O-methyl-sn-glycero-3-phosphorylcholine. Hydrolytic rates with the corresponding isomer, 1-O-methyl-2-palmitoyl-sn-glycero-3-phosphorylcholine, are about 3-4 orders of magnitude higher. The similarities in Ca2+ requirement and inactivation profiles suggest that deacylation, albeit with different rates, from both sn-1 and sn-2 positions is catalyzed by the same catalytic site of phospholipase A2. Furthermore, evidence is provided that phospholipase A2-catalyzed 1-acyl lysophospholipid deacylation is mediated by sn-1-directed action, but above pH 7 acyl migration with subsequent enzyme-catalyzed hydrolytic cleavage from the sn-2 position contributes to the overall deacylation of monoacylphosphoglycerides, acyl migration becoming eventually the rate-limiting factor.

Acylation↗
PubMed1997

Cytokine mRNA expression in intestinal tissue of interleukin-2 deficient mice with bowel inflammation.

BACKGROUND: Mice deficient in interleukin-2 (IL-2) develop inflammatory bowel disease resembling ulcerative colitis in humans. Recent studies provided evidence that alpha beta T cells, particularly CD4 T cells, rather than B cells, are involved in the pathogenesis of bowel inflammation of IL-2 deficient mice. AIM: To analyse the pattern of expression of cytokine mRNA in intestinal tissue of normal and IL-2 deficient mice. METHODS: Expression of beta-actin, IL-1 alpha, IL-1 beta, IL-6, IL-10, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and transforming growth factor beta 1 (TGF-beta 1) mRNA was analysed in colon and small intestinal tissue of both IL-2 deficient (IL-2-/-) mice and normal (wild type) litter mates (IL-2+/+) at different ages by using qualitative, as well as semiquantitative, competitive reverse transcription polymerase chain reaction (RT-PCR). Results were correlated with the phase of progression of the disease, as determined by histology. RESULTS: IL-2-/- mice had expressed low levels of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, and IFN-gamma mRNA in the colon by 1.5 weeks of age. In advance of the development of histologically and clinically detectable bowel inflammation, expression of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IFN-gamma, and IL-10, but not TGF-beta 1, mRNA increased in the colon of IL-2 deficient mice. In contrast, IL-2+/+ mice expressed TGF-beta 1 mRNA in colon tissue at 13 and 23 weeks of age, but not IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IL-10, or IFN-gamma mRNA. Levels of expression of cytokine mRNA in tissue from the small intestine were comparable in IL-2-/- and IL-2+/+ mice. CONCLUSIONS: Bowel inflammation in IL-2 deficient mice is preceded by an increase in IL-1 alpha, IL-1 beta, TNF-alpha, and IFN-gamma mRNA expression in colon tissue. Low levels of TGF-beta 1, but high levels of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IFN-gamma, and IL-10 mRNA expression correlate with the manifestation of severe colitis, and suggest that T cells and macrophages are involved in bowel inflammation of IL-2 deficient mice.

Actins↗
PubMed1996

Subcellular distribution of glycosylphosphatidylinositol-specific phospholipase D in rat liver.

Glycosylphosphatidylinositol (GPI)-hydrolysing enzymes have been described in many mammalian tissues and body fluids; however, their site(s) of action and in vivo functions have remained unclear. In order to identify a possible intracellular site of GPI hydrolysis, we studied the subcellular distribution of GPI-hydrolysing activity in rat liver. We found that purified fractions from rat liver hydrolysed the GPI moieties of two GPI-anchored proteins with the specificity of a phospholipase D. This GPI-specific phospholipase D (GPI-PLD) activity was found to be highly enriched in a lysosomal fraction and showed a similar intracellular distribution to that of typical lysosomal enzymes. Our results indicate that lysosomes may represent a possible intracellular site of GPI-PLD action.

Animals↗
PubMed1996

IL-12 is essential for resistance against Yersinia enterocolitica by triggering IFN-gamma production in NK cells and CD4+ T cells.

Although Yersinia enterocolitica is extracellularly located in infected tissues, a specific T cell response is required to overcome infection. Recent work implicated that in contrast to Yersinia-susceptible BALB/c mice, C57BL/6 mice are Yersinia resistant due to the rapid development of a Yersinia-specific Th1 T cell response. This study focused on the role of IL-12 in Y. enterocolitica infections in both mouse strains. We found that C57BL/6 and BALB/c mice produced comparable quantities of IL-12 mRNA after Y. enterocolitica infection. Likewise, Yersinia-infected bone marrow macrophages from both mouse strains produced equal quantities of IL-12. Administration of neutralizing anti-IL-12 Abs abrogated resistance against yersiniae in either strain. In addition, administration of rIL-12 rendered BALB/c mice resistant to yersiniae, while this treatment was toxic to C57BL/6 mice. IL-12-mediated protection was partially dependent on IFN-gamma. Spleen cells from both strains of mice produced Yersinia-triggered IFN-gamma in an IL-12-dependent manner, although those from BALB/c mice produced 10-fold lesser quantities. Administration of rIL-12 in vivo increased Yersinia-induced IFN-gamma production by BALB/c spleen cells in vitro, but decreased IFN-gamma production by spleen cells from C57BL/6 mice. IL-10 was antagonistic to IL-12 only in BALB/c mice and inhibited Yersinia-triggered IFN-gamma production. In vivo depletion experiments revealed that IL-12 accounts for Yersinia-induced IFN-gamma production by both NK cells and CD4+ T cells, the latter of which are an essential source of IFN-gamma r while NK cell-derived IFN-gamma production can be compensated by other cells. In contrast to that in the spleen, IL-12 plays a minor role in protection against yersiniae in Peyer's patches after orogastric infection. In summary, our data suggest that IL-12 is rapidly induced by Y. enterocolitica infection and required for IFN-gamma production by NK cells as well by CD4+ T cells. Although BALB/c and C57BL/6 mice produced comparable quantities of IL-12, IFN-gamma production, and thus resistance to yersiniae, can be increased by exogenous IL-12 only in BALB/c, not in C57BL/6, mice.

Animals↗
PubMed1995

Deferoxamine B but not deferoxamine G1 inhibits cytokine production in murine bone marrow macrophages.

The iron chelator deferoxamine (DFO) B enhances virulence of Yersinia enterocolitica and modulates cellular immune responses. Since cytokines mediate effector mechanisms in resolution of yersiniae from infected tissues, the impact of DFO B and DFO G1 on cytokine production by murine bone marrow macrophages (BMM) was investigated. BMM were stimulated with lipopolysaccharide (LPS) of Salmonella typhimurium or infected with Y. enterocolitica. DFO B inhibited interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-alpha mRNA production 4-fold (shown by semiquantitative reverse transcription polymerase chain reaction). TNF-alpha and IL-6 protein production was reduced 50% by DFO B. In contrast, DFO G1 had no effect on cytokine production. Moreover, cytokine production by Yersinia-infected BMM was decreased by plasmid-encoded Yersinia proteins. Thus, plasmid-cured strains induced higher cytokine responses in BMM than did the wild type strain. These results suggest that DFO B acts in a bimodal fashion in yersiniosis: iron supply to the pathogen and immunosuppression of the host.

Actins↗
PubMed1994

Immune responses to Yersinia enterocolitica in susceptible BALB/c and resistant C57BL/6 mice: an essential role for gamma interferon.

Susceptibility of mice to infection with Yersinia enterocolitica has been shown to be related to neither the Ity locus encoding for resistance to Salmonella typhimurium and other pathogens nor the H-2 locus. Recent studies in our laboratory have demonstrated that T-cell-mediated immune responses are required for overcoming primary Yersinia infection. In the present study, we investigated the course of infection with Y. enterocolitica and the resulting immune responses in Yersinia-susceptible BALB/c and Yersinia-resistant C57BL/6 mice. In the early phase of infection, the clearance of the pathogen was comparable in both strains of mice, suggesting similar mechanisms of innate resistance. Splenic T cells from Yersinia-infected C57BL/6 mice exhibited marked proliferative responses and produced gamma interferon (IFN-gamma) upon exposure to heat-killed yersiniae. By contrast, the Yersinia-specific T-cell response in BALB/c mice was weak, and IFN-gamma production could not be detected before day 21 postinfection. T cells isolated from C57BL/6 mice 7 days after infection mediated immunity to Y. enterocolitica but those from BALB/c mice did not, while at 21 days postinfection T cells from both strains mediated protection. Neutralization of IFN-gamma abrogated resistance to yersiniae in C57BL/6 mice but to a far smaller extent in BALB/c mice. Administration of recombinant IFN-gamma or anti-interleukin-4 antibodies rendered BALB/c mice resistant to yersiniae, whereas this treatment did not significantly affect the course of the infection in C57BL/6 mice. These results indicate that the cellular immune response, in particular the production of IFN-gamma by Yersinia-specific T cells, is associated with resistance of mice to Y. enterocolitica.

Animals↗
PubMed1994

Early gamma interferon mRNA expression is associated with resistance of mice against Yersinia enterocolitica.

T cells in cooperation with macrophages play an important role in resolution of primary Yersinia enterocolitica infection in mice. Previous work from this laboratory demonstrated that gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) are essential mediators of these processes. In an attempt to elucidate early mechanisms of resistance, we investigated cytokine mRNA production, including that for interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, IL-10, TNF-alpha, and IFN-gamma, after primary as well as secondary Y. enterocolitica infection in Yersinia-susceptible BALB/c mice and Yersinia-resistant C57BL/6 mice. In both strains of mice, proinflammatory cytokines such as IL-1 beta, IL-6, and TNF-alpha were expressed rapidly and to comparable degrees, while IFN-gamma expression was enhanced two- to eightfold in C57BL/6 mice, as revealed by semiquantitative reverse transcription PCR. Similar results were found in both mouse strains after secondary Y. enterocolitica infection. IL-2 mRNA was detected only during secondary infection and disappeared rapidly in BALB/c mice. IL-4 mRNA expression was detectable in C57BL/6 but not BALB/c mice. The levels of cytokine mRNA expression correlated closely with the number of injected bacteria. The findings reported here support the hypothesis that early and enhanced production of IFN-gamma may be associated with a state of heightened resistance against Y. enterocolitica infection.

Animals↗
PubMed1992

Annexin II inhibits calcium-dependent phospholipase A1 and lysophospholipase but not triacyl glycerol lipase activities of rat liver hepatic lipase.

A member of the annexin family (the heterotetrameric annexin II2p11(2) complex purified from porcine intestinal epithelium) was tested for its ability to affect different calcium-dependent intrinsic lipolytic activities of rat liver hepatic lipase (HL). Whereas annexin II in the presence of calcium failed to interfere with HL triacyl glycerol lipase (EC 3.1.1.3) activity, it inhibited HL phospholipase A1 (EC 3.1.1.32) and lysophospholipase (EC 3.1.1.5) activities. Inhibition could be overcome by increasing the substrate concentration. Under phospholipase A1 assay conditions, annexin II did not bind to the purified HL enzyme. These results therefore suggest that only inhibitor/substrate interactions lead to inhibition of HL phospholipase A1 and lysophospholipase activities, an obviously general mechanism of phospholipase inhibition by annexins. Possible implications of HL inhibition in vivo by annexins are discussed.

Animals↗
PubMed1988

Inhibitors of liver lysosomal acid phospholipase A1.

Lysosomal acid phospholipase A1, as well as other lysosomal enzymes, may be released under pathophysiological conditions into extralysosomal compartments. As shown here, several unspecific mechanisms exist which inhibit the hydrolysis of membrane diacylphospholipids by lysosomal acid phospholipase A1 and hence prevent an uncontrolled membrane destruction. These findings were obtained by employing partially purified rat liver lysosomal acid phospholipase A1 and sonicated radioactively labeled phosphatidylethanolamine or phosphatidylcholine as substrate. The inhibitory principles found include (1) pH, (2) inorganic cations, and (3) various proteins. Inorganic cations and proteins, however, inhibited lysosomal acid phospholipase A1 activity only below pH 6.0, and inhibition never exceeded 96%. Of the inorganic cations studied, the divalent species, as compared to the monovalent one, impaired lysosomal acid phospholipase A1 activity at significantly lower concentrations. Virtually all of the intracellular and extracellular proteins studied inhibited the enzyme activity, but the inhibitory potencies of the different proteins varied considerably. In general, basic and hydrophobic proteins were the most potent inhibitors, whereas glycoproteins appeared to be less inhibitory. The degree of inhibition of the enzyme activity in both proteins and inorganic cations depended on the substrate concentration and not on that of the enzyme. Binding studies provided evidence for inhibitor-substrate and against inhibitor-enzyme interactions.

Animals↗