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Derek M McKay

Publications and source records attributed to Derek M McKay.

33 records · Page 2Linked to original sources
PubMed2003

Helicobacter pylori infection interferes with epithelial Stat6-mediated interleukin-4 signal transduction independent of cagA, cagE, or VacA.

Helicobacter pylori is a bacterial pathogen evolved to chronically colonize the gastric epithelium, evade immune clearance by the host, and cause gastritis, peptic ulcers, and even gastric malignancies in some infected humans. In view of the known ability of this bacterium to manipulate gastric epithelial cell signal transduction cascades, we determined the effects of H. pylori infection on epithelial IL-4-Stat6 signal transduction. HEp-2 and MKN45 epithelial cells were infected with H. pylori strains LC11 or 8823 (type 1; cagA(+)/cagE(+)/VacA(+)), LC20 (type 2; cagA(-), cagE(-), VacA(-)), and cagA, cagE, and vacA isogenic mutants of strain 8823, with some cells receiving subsequent treatment with the Th2 cytokine IL-4, a known Stat6 activator. Immunofluorescence showed a disruption of Stat6-induced nuclear translocation by IL-4 in LC11-infected HEp-2 cells. IL-4-inducible Stat6 DNA binding in HEp-2 and MKN45 cells was abrogated by infection, but MKN45 cell viability was unaffected. A decrease in IL-4-mediated Stat6 tyrosine phosphorylation in nuclear and whole cell lysates was also observed following infection with strains LC11 and LC20, while neither strain altered IL-4 receptor chain alpha or Janus kinase 1 protein expression. Furthermore, parental strain 8823 and its isogenic cagA, cagE, and vacA mutants also suppressed IL-4-induced Stat6 tyrosine phosphorylation to comparable degrees. Thus, H. pylori did not directly activate Stat6, but blocked the IL-4-induced activation of epithelial Stat6. This may represent an evolutionarily conserved strategy to disrupt a Th2 response and evade the host immune system, allowing for successful chronic infection.

Active Transport, Cell Nucleus↗
PubMed2003

Colonic bacterial superantigens evoke an inflammatory response and exaggerate disease in mice recovering from colitis.

BACKGROUND & AIMS: There is renewed interest in commensal bacteria as triggers of idiopathic disease, a concept that is prominent in inflammatory bowel disease (IBD). Here the effect of intracolonic instillation of Staphylococcus aureus enterotoxin B (SEB), a model superantigen (SAgs: potent T-cell stimuli), into mice was examined. METHODS: Mice (Balb/c, severe combined immunodeficient [SCID], V beta 8(+) ovalbumin transgenic [OVA-Tg], interleukin 10 [IL-10] knockout [KO]) received a single intrarectal (IR) dose of SAg and colonic form (histology, myeloperoxidase [MPO] activity) and function (ion transport) were assessed 12-72 hours later. In subsequent studies the potential for SEB to reactivate disease in mice recovering from dextran sodium sulfate (DSS)-induced colitis (5 days at 4% [wt/vol] followed by 14 days normal water) was examined. RESULTS: SEB-treated Balb/c mice displayed a time- and dose-dependent colonic inflammation (increased MPO, histologic damage score, and macrophage number). Similar events occurred in response to other SAgs, namely S. aureus enterotoxin A (SEA) and Yersinia pseudotuberculosis mitogen. Ion transport, the driving force for water movement, was unaffected by SEB treatment. SCID mice developed no inflammation after IR SEB delivery, whereas OVA Tg mice displayed enhanced responsiveness. Although SEB treatment of IL-10 KO mice did elicit a response, the inflammation was transitory and did not hasten the spontaneous colitis seen in these mice. Finally, mice recovering from DSS-induced colitis showed a worsening of the disease when challenged with SEB; IR SEB evoked significant increases in MPO, macrophage infiltration, T-cell activation (i.e., CD25 expression), and perturbed epithelial ion transport. CONCLUSIONS: Lumen-derived bacterial SAgs can elicit a local inflammation and aggravate enteric inflammatory disorders in which they were not the causative agent.

Animals↗
PubMed2003

Adenoviral transfer of the murine oncostatin M gene suppresses dextran-sodium sulfate-induced colitis.

The use of biologics has promising potential in the treatment of inflammation. Studies with cultured cells and mouse models of disease have ascribed proinflammatory and anti-inflammatory functions to oncostatin M (OSM) and the related cytokine, interleukin-6 (IL-6). Here, we examined the effect of systemic administration of adenoviral (Ad) vectors encoding either murine OSM (AdMuOSM) or murine IL-6 (AdMuIL-6) in a mouse model of colitis. BALB/c mice were treated with a 5-day course of 4% dextran-sodium sulfate (DSS) water with or without administration of adenoviral vectors (i.p. or i.m. at 10(7) plaque-forming units [pfu]) given as a cotreatment or therapy. The deletion variant of the adenovirus served as a control for adenoviral infection. Colitis was assessed by (1) morphology (damage score, macrophage infiltration, apoptosis) and (2) function (myeloperoxidase activity and Ussing chamber analysis of epithelial ion transport). Infection with adenovirus alone did not affect colonic form or function. AdMuOSM (either i.p. or i.m.) significantly reduced the severity of the DSS-induced colitis. There was less damage, reduced macrophage infiltration, fewer apoptotic bodies, and a significant improvement in stimulated ion transport in colonic tissues from the treated mice. No benefit of AdMuIL-6 treatment was observed in this model system. Thus, systemic administration of AdMuOSM given as a cotreatment and to a lesser extent as a therapy was found to be of benefit in DSS-induced colitis, a murine model of inflammatory bowel disease (IBD).

Acute-Phase Proteins↗
PubMed2003

Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-kappaB-independent pathway.

Recognition of bacterial products by the innate immune system is dependent on pattern-recognition receptors: toll-like receptor 9 (TLR-9) in the case of bacterial DNA. We hypothesized that bacterial DNA can directly affect enteric epithelial cells. RT-PCR revealed constitutive TLR-9 mRNA expression in three human colonic epithelial cell lines (T84, HT-29, Caco-2) and THP-1 monocytes. Epithelial cells, in six-well culture plates or on filter supports, were exposed to E. coli DNA (1-50 microg/ml), synthetic CpG-rich oligonucleotides, or calf thymus DNA for 6-48 h. Exposure to E. coli DNA resulted in an increase in IL-8 mRNA, and a time- and dose-dependent increase in IL-8 secretion. Also, CpG oligonucleotides induced epithelial IL-8 production, whereas calf thymus DNA did not. Exposure to E. coli DNA resulted in phosphorylation of ERK 1/2 MAPK and inhibitors of ERK activity (PD98059, UO126) significantly reduced the evoked IL-8 production. In contrast, inhibitors of NFkappaB activity (PDTC, SN50) did not block E. coli DNA-induced IL-8 production. Electrophoretic mobility shift assays revealed that E. coli DNA stimulated epithelial AP-1 but not NFkappaB activation. The barrier (i.e., transepithelial resistance) and ion transport parameters of epithelial monolayers (assessed in Ussing chambers) were unaltered following E. coli DNA exposure. Thus model gut epithelia express TLR-9 mRNA and, while maintaining their barrier function, can respond to E. coli DNA by increased IL-8 production.

Caco-2 Cells↗
PubMed2003

Enterohemorrhagic Escherichia coli O157:H7 disrupts Stat1-mediated gamma interferon signal transduction in epithelial cells.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a clinically important bacterial enteropathogen that manipulates a variety of host cell signal transduction cascades to establish infection. However, the effect of EHEC O157:H7 on Jak/Stat signaling is unknown. To define the effect of EHEC infection on epithelial gamma interferon (IFN-gamma)-Stat1 signaling, human T84 and HEp-2 epithelial cells were infected with EHEC O157:H7 and then stimulated with recombinant human IFN-gamma. Cells were also infected with different EHEC strains, heat-killed EHEC, enteropathogenic E. coli (EPEC) O127:H6, and the commensal strain E. coli HB101. Nuclear and whole-cell protein extracts were prepared and were assayed by an electrophoretic mobility shift assay (EMSA) and by Western blotting, respectively. Cells were also processed for immunofluorescence to detect the subcellular localization of Stat1. The EMSA revealed inducible, but not constitutive, Stat1 activation upon IFN-gamma treatment of both cell lines. The EMSA also showed that 6 h of EHEC O157:H7 infection, but not 30 min of EHEC O157:H7 infection, prevented subsequent Stat1 DNA binding induced by IFN-gamma, whereas infection with EPEC did not. Immunoblotting showed that infection with EHEC, but not infection with EPEC, eliminated IFN-gamma-induced Stat1 tyrosine phosphorylation in both dose- and time-dependent fashions and disrupted inducible protein expression of the Stat1-dependent gene interferon regulatory factor 1. Immunofluorescence revealed that EHEC infection did not prevent nuclear accumulation of Stat1 after IFN-gamma treatment. Also, Stat1 tyrosine phosphorylation was suppressed by different EHEC isolates, including intimin-, type III secretion- and plasmid-deficient strains, but not by HB101 and heat-killed EHEC. These findings indicate the novel disruption of host cell signaling caused by EHEC infection but not by EPEC infection.

Cell Line↗
PubMed2003

Canadian Association of Gastroenterology - Canadian Institutes of Health Research - pharmaceutical partner postdoctoral operating fellowship programme: an outstanding success that continues to excel!

The Canadian Association of Gastroenterology (CAG) postdoctoral fellowship programme was initiated in 1992 with the goal of promoting excellence in Canadian gastroenterological research. With backing from multiple pharmaceutical partners and the Canadian Institutes of Health Research, 87 fellows were funded over the next ten years for a total investment of $8,730,101. Between 1992 and 2000, fellows authored 247 articles; 176 being original research articles, 31 (17.5%) of which appeared in journals with impact factors of greater than 10. As testament to the program's success in developing young scientists, 31 former fellows (36%) have progressed to faculty positions. The fellowship programme continues to be an outstanding success and the flagship of CAG research activities.

Fellowships and Scholarships↗
PubMed2003

STAT-6 is an absolute requirement for murine rejection of Hymenolepis diminuta.

Infection with helminth parasites typically evokes a T helper 2-cell response in the mammalian host. Interleukin (IL)-4 and IL-13 are important in the rapid expulsion of parasitic nematodes, with the absence of either cytokine being compensated for by the other. However, the IL-4 and IL-13 common signaling molecule, signal transducer and activator of transcription 6 (STAT-6) appears to be mandatory for the spontaneous expulsion of enteric helminths. Mice genetically deficient in IL-4, IL-13, or STAT-6 were infected with the cestode Hymenolepis diminuta and worm infectivity and jejunal goblet cell responses assessed 12-18 days postinfection (PI). Only the STAT-6 knockout (KO) animals harbored adult worms; neither gravid adult nor stunted H. diminuta was obtained from the infected IL-4 KO or IL-13 KO mice > or = 12 days PI. Also, the establishment of worms in the intestine of STAT-6 KO animals was associated with a reduced goblet cell response. These findings support the hypothesis that increased mucin production is an important part of the host response to tapeworm infection and that functional STAT-6 signaling may be an absolute requirement for the rejection of intestinal cestodes and thus, helminth parasites in general.

Animals↗
PubMed2002

TGF-beta effects on epithelial ion transport and barrier: reduced Cl- secretion blocked by a p38 MAPK inhibitor.

Growth factors affect a variety of epithelial functions. We examined the ability of TGF-beta to modulate epithelial ion transport and permeability. Filter-grown monolayers of human colonic epithelia, T84 and HT-29 cells, were treated with TGF-beta (0.1-100 ng/ml, 15 min-72 h) or infected with an adenoviral vector encoding TGF-beta (Ad-TGF beta) for 144 h. Ion transport (i.e., short-circuit current, I(sc)) and transepithelial resistance (TER) were assessed in Ussing chambers. Neither recombinant TGF-beta nor Ad-TGF beta infection affected baseline I(sc); however, exposure to > or = 1 ng/ml TGF-beta led to a significant (30-50%) reduction in the I(sc) responses to forskolin, vasoactive intestinal peptide, and cholera toxin (agents that evoke Cl(-) secretion via cAMP mobilization) and to the cell-permeant dibutyryl cAMP. Pharmacological analysis of signaling pathways revealed that the inhibition of cAMP-driven epithelial Cl(-) secretion by TGF-beta was blocked by pretreatment with SB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors of JNK, ERK1/2 MAPK, or phosphatidylinositol 3'-kinase. TGF-beta enhanced the barrier function of the treated monolayers by up to threefold as assessed by TER; however, this event was temporally displaced from the altered I(sc) response, being statistically significant only at 72 h posttreatment. Thus, in addition to TGF-beta promotion of epithelial barrier function, we show that this growth factor also reduces responsiveness to cAMP-dependent secretagogues in a chronic manner and speculate that this serves as a braking mechanism to limit secretory enteropathies.

Biological Transport↗
PubMed2002

Dextran sodium sulphate-induced colitis perturbs muscarinic cholinergic control of colonic epithelial ion transport.

1. Neuronal cholinergic input is an important regulator of epithelial electrolyte transport and hence water movement in the gut. 2. In this study, colitis was induced by treating mice with 4% (w v(-1)) dextran sodium-sulphate (DSS)-water for 5 days followed by 3 days of normal water. Mid-colonic segments were mounted in Ussing chambers and short-circuit current (Isc, indicates net ion movement) responses to the cholinergic agonist, carbachol (CCh; 10(-4) M)+/-tetrodotoxin, atropine (ATR), hexamethonium (HEX), naloxone or phenoxybenzamine were assessed. 3. Tissues from mice with DSS-induced colitis displayed a drop in Isc in response to CCh (-11.3+/-3.3 microA/cm(2)), while those from control mice showed a transient increase in Isc (76.3+/-13.0 microA/cm(2)). The DeltaIsc in colon from DSS-treated mice was tetrodotoxin-sensitive, atropine-insensitive and was reversed by hexamethonium (HEX+CCh=16.7+/-7.8 microA/cm(2)), indicating involvement of a nicotinic receptor. 4. CCh induced a drop in Isc in tissues from controls only when they were pretreated with the cholinergic muscarinic receptor blocker, atropine: ATR+CCh=-21.3+/-7.0 microA/cm(2). Nicotine elicited a drop in Isc in Ussing-chambered colon from both control and DSS-treated mice that was TTX-sensitive. 5. The drop in Isc evoked by CCh challenge of colonic tissue from DSS-treated mice or ATR+CCh challenge of control tissue was not significantly affected by blockade of opiate or alpha-adrenergic receptors by naloxone or phenoxybenzamine, respectively. 6. The data indicate that DSS-colitis reveals a nicotinic receptor that becomes important in cholinergic regulation of ion transport.

Animals↗
PubMed2002

Nitric oxide reduces bacterial superantigen-immune cell activation and consequent epithelial abnormalities.

Inhibition of the inducible form of nitric oxide (NO) synthase prolonged the murine enteropathy evoked by the bacterial superantigen, Staphylococcus aureus enterotoxin B (SEB). We examined the ability of NO to alleviate SEB-induced epithelial dysfunction and immune cell activation. Human peripheral blood mononuclear cells (PBMC) were activated by SEB for 24 h +/- the NO donors, S-nitroso-N-acetylpenicillamine and spermine-NONOate. The conditioned medium (CM) was collected and applied to T84 epithelial monolayers, and permeability [i.e., transepithelial resistance (TER)] and stimulated ion transport (i.e., short-circuit current responses to carbachol and forskolin) were assessed 24 h later. Exposure to CM led to an approximately 40% drop in TER and hyporesponsiveness to both secretagogues. CM made in the presence of NO donors (10(-4) M) had no significant effect on epithelial barrier or ion transport parameters. NO donors alone had no effect on naive epithelia, and addition of the NO donors to previously made CM did not affect the ability of this CM to alter epithelial function. Moreover, the NO donors dose-dependently reduced SEB-evoked PBMC proliferation and cytokine production (i.e., interferon-gamma, tumor necrosis factor alpha) but did not affect viability. These findings suggest a beneficial role for NO in inflammation by reducing immune cell activation and thus ameliorating consequent physiological abnormalities, in this instance, perturbed epithelial permeability and active ion transport.

Antigens, Bacterial↗