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ABSTRACT: Salmonella infection is a common public health problem that can become chronic and increase the risk of cancer. Live, mutated Salmonella is used to target cancer cells. However, few studies have addressed chronic Salmonella infection in vivo. AvrA is a Salmonella type-three secretion effector that is multi-functional, inhibiting intestinal inflammation and enhancing proliferation. β-catenin is a key player in intestinal renewal, inflammation, and tumorigenesis. We hypothesize that in Salmonella-infected intestine, AvrA chronically activates the β-catenin pathway and increases cell proliferation, thus deregulating the intestinal responses to bacterial infection. We followed mice with Salmonella infection for 27 weeks and investigated the physiological effects and role of AvrA on β-catenin in chronically infected intestine. We found that AvrA persistently regulated β-catenin posttranslational modifications, including phosphorylation and acetylation. Moreover, the upstream regulator Akt, transcription factors T cell factors, nuclear β-catenin, and β-catenin target genes were enhanced in mice infected with Salmonella expressing AvrA. AvrA has a chronic functional role in promoting intestinal renewal. In summary, we have uncovered an essential role of Salmonella AvrA in chronically activating β-catenin, and impacting intestinal renewal in small intestine and colon. Our study emphasizes the importance of AvrA in chronic bacterial infection.
AJP Gastrointestinal and Liver Physiology 09/2012; · 3.43 Impact Factor
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ABSTRACT: OBJECTIVE:: Critical illness results in changes to the microbiology of the gastrointestinal tract, leading to a loss of commensal flora and an overgrowth of potentially pathogenic bacteria. Administering certain strains of live bacteria (probiotics) to critically ill patients may restore balance to the microbiota and have positive effects on immune function and gastrointestinal structure and function. The purpose of this systematic review was to evaluate the effect of probiotics in critically ill patients on clinical outcomes. DESIGN:: Systematic review INTERVENTIONS:: None. MEASUREMENTS AND MAIN RESULTS:: We searched computerized databases, reference lists of pertinent articles, and personal files from 1980 to 2011. We included randomized controlled trials enrolling critically ill adults, which evaluated probiotics compared to a placebo and reported clinically important outcomes (infections, mortality, and length of stay). A total of 23 randomized controlled trials met inclusion criteria. Probiotics were associated with reduced infectious complications as documented in 11 trials (risk ratio 0.82; 95% confidence interval 0.69-0.99; p = .03; test for heterogeneity p = .05; I 44%). When data from the seven trials reporting ventilator-associated pneumonia were pooled, ventilator-associated pneumonia rates were also significantly reduced with probiotics (risk ratio 0.75; 95% confidence interval 0.59-0.97; p = .03; test for heterogeneity p = .16; I 35%). Probiotics were associated with a trend toward reduced intensive care unit mortality (risk ratio 0.80; 95% confidence interval 0.59-1.09; p = .16; test for heterogeneity p = .89; I 0%) but did not influence hospital mortality. Probiotics had no effect on intensive care unit or hospital length of stay. Compared to trials of higher methodological quality, greater treatment effects were observed in trials of a lower methodological quality. CONCLUSIONS:: Probiotics appear to reduce infectious complications including ventilator-associated pneumonia and may influence intensive care unit mortality. However, clinical and statistical heterogeneity and imprecise estimates preclude strong clinical recommendations. Further research on probiotics in the critically ill is warranted.
Critical care medicine 09/2012; · 6.37 Impact Factor
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ABSTRACT: Axin1 and its homolog Axin2 are scaffold proteins essential for regulating Wnt signaling. Axin-dependent regulation of Wnt is important for various developmental processes and human diseases. However, the involvement of Axin1 and Axin2 in host defense and inflammation remains to be determined.
Here, we report that Axin1, but not Axin2, plays an essential role in host-pathogen interaction mediated by the Wnt pathway. Pathogenic Salmonella colonization greatly reduces the level of Axin1 in intestinal epithelial cells. This reduction is regulated at the posttranslational level in early onset of the bacterial infection. Further analysis reveals that the DIX domain and Ser614 of Axin1 are necessary for the Salmonella-mediated modulation through ubiquitination and SUMOylation.
Axin1 apparently has a preventive effect on bacterial invasiveness and inflammatory response during the early stages of infection. The results suggest a distinct biological function of Axin1 and Axin2 in infectious disease and intestinal inflammation while they are functionally equivalent in developmental settings.
PLoS ONE 01/2012; 7(4):e34942. · 4.09 Impact Factor
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ABSTRACT: The vitamin D metabolite, 1,25-(OH)₂D₃, binds the vitamin D receptor (VDR) to exert its regulatory effects at the transcription level. VDR is expressed in professional antigen-presenting cells (pAPCs), such as macrophages (Mø) and dendritic cells (DCs). We show for the first time that the 24-hydroxylase enzyme is activated in bone marrow-derived dendritic cell (BMDC), due to 1,25(OH)₂D₃ stimulation which resulted in the induction of its gene, CYP24A1. Furthermore, we provide evidence that the influence of 1,25-(OH)₂D₃ on TLR-4-L-induced activation of pAPC is dependent on the order of VDR and TLR-4 engagement. Thus, pre-treatment of pAPC with 1,25-(OH)₂D₃ partially inhibited LPS-induced nitric oxide (NO) production. However, these inhibitory effects were not observed when LPS and 1,25-(OH)₂D₃ were added simultaneously or when LPS preceded 1,25-(OH)₂D₃. Moreover, we found that 1,25-(OH)₂D₃ pre-treatment of pAPCs did not cause general suppression since it interfered with NO levels but not with the cytokines IL-6 or TNF-α. Consequently, engagement of VDR by 1,25-(OH)₂D₃ can partially interfere with TLR-4-L-induced activation of pAPCs only when it occurs before TLR-4 stimulation.
Immunobiology 04/2011; 216(9):988-96. · 3.20 Impact Factor
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ABSTRACT: Preterm infants face many challenges in transitioning from the in utero to extrauterine environment while still immature. Failure of the preterm gut to successfully mature to accommodate bacteria and food substrate leads to significant morbidity such as neonatal necrotizing enterocolitis. The intestinal epithelial barrier plays a critical role in gut protection. Heat shock protein 70 (Hsp70) is an inducible cytoprotective molecule shown to protect the intestinal epithelium in adult models. To investigate the hypothesis that Hsp70 may be important for early protection of the immature intestine, Hsp70 expression was evaluated in intestine of immature rat pups. Data demonstrate that Hsp70 is induced by exposure to mother's milk. Hsp70 is found in mother's milk, and increased Hsp70 transcription is induced by mother's milk. This Hsp70 colocalizes with the tight junction protein ZO-1. Mother's milk-induced Hsp70 may contribute to maintenance of barrier function in the face of oxidant stress. Further understanding of the means by which mother's milk increases Hsp70 in the ileum will allow potential means of strengthening the intestinal barrier in at-risk preterm infants.
Pediatric Research 01/2011; 69(5 Pt 1):395-400. · 2.70 Impact Factor
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ABSTRACT: The impermeant nature of the intestinal barrier is maintained by tight junctions (TJs) formed between adjacent intestinal epithelial cells. Disruption of TJs and loss of barrier function are associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal diseases in preterm infants. Human milk is protective against NEC, and the human milk factor erythropoietin (Epo) has been shown to protect endothelial cell-cell and blood-brain barriers. We hypothesized that Epo may also protect intestinal epithelial barriers, thereby lowering the incidence of NEC. Our data demonstrate that Epo protects enterocyte barrier function by supporting expression of the TJ protein ZO-1. As immaturity is a key factor in NEC, Epo regulation of ZO-1 in the human fetal immature H4 intestinal epithelial cell line was examined and demonstrated Epo-stimulated ZO-1 expression in a dose-dependent manner through the PI3K/Akt pathway. In a rat NEC model, oral administration of Epo lowered the incidence of NEC from 45 to 23% with statistical significance. In addition, Epo treatment protected intestinal barrier function and prevented loss of ZO-1 at the TJs in vivo. These effects were associated with elevated Akt phosphorylation in the intestine. This study reveals a novel role of Epo in the regulation of intestinal epithelial TJs and barrier function and suggests the possible use of enteral Epo as a therapeutic agent for gut diseases.
Journal of Biological Chemistry 01/2011; 286(14):12123-32. · 4.77 Impact Factor
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ABSTRACT: The ability of Salmonella typhimurium to enter intestinal epithelial cells constitutes a crucial step in pathogenesis. Salmonella invasion of the intestinal epithelium requires bacterial type three secretion system. Type three secretion system is a transport device that injects virulence proteins, called effectors, to paralyze or reprogram the eukaryotic cells. Avirulence factor for Salmonella (AvrA) is a Salmonella effector that inhibits the host's inflammatory responses. The mechanism by which AvrA modulates host cell signaling is not entirely clear. p53 is situated at the crossroads of a network of signaling pathways that are essential for genotoxic and nongenotoxic stress responses. We hypothesized that Salmonella infection activates the p53 pathway. We demonstrated that Salmonella infection increased p53 acetylation. Cells infected with AvrA-sufficient Salmonella have increased p53 acetylation, whereas cells infected with AvrA-deficient Salmonella have less p53 acetylation. In a cell-free system, AvrA possessed acetyltransferase activity and used p53 as a substrate. AvrA expression increased p53 transcriptional activity and induced cell cycle arrest. HCT116 p53-/- cells had less inflammatory responses. In a mouse model of Salmonella infection, intestinal epithelial p53 acetylation was increased by AvrA expression. Our studies provide novel mechanistic evidence that Salmonella modulates the p53 pathway during intestinal inflammation and infection.
AJP Gastrointestinal and Liver Physiology 03/2010; 298(5):G784-94. · 3.43 Impact Factor
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ABSTRACT: The initiation of T-cell immune responses requires professional antigen-presenting cells. Emerging data point towards an important role for macrophages (Mphi) in the priming of naïve T cells. In this study we analyzed the efficiency and the mechanisms by which Mphi derived from spleen (Sp-Mphi) or bone marrow (BM-Mphi) present Lymphocytic choriomeningitis virus (LCMV) antigens to epitope-specific T cells. We demonstrate that because of phagosomal maturation, Sp-Mphi downregulate their ability to cross-present cell-associated, but not soluble, antigens, as they are further differentiated in culture without altering their capacity to directly present virus antigens after infection. We propose that Sp-Mphi are extremely efficient at direct and cross-presentation. However, if these cells undergo further M-CSF-dependent maturation, they will adapt to be more scavenger and phagocytic and concurrently reduce their cross-presenting capacity. Accordingly, Sp-Mphi can have an important role in regulating T-cell responses through cross-presentation depending on their differentiation state.
Immunology and Cell Biology 11/2009; 88(1):3-12. · 3.66 Impact Factor
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Elaine O Petrof
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ABSTRACT: Our intestinal microbiota serve many roles vital to the normal daily function of the human gastrointestinal tract. Many probiotics are derived from our intestinal bacteria, and have been shown to provide clinical benefit in a variety of gastrointestinal conditions. Current evidence indicates that probiotic effects are strain-specific, they do not act through the same mechanisms, and nor are all probiotics indicated for the same health conditions. However, they do share several common features in that they exert anti-inflammatory effects, they employ different strategies to antagonize competing microorganisms, and they induce cytoprotective changes in the host either through enhancement of barrier function, or through the upregulation of cytoprotective host proteins. In this review we focus on a few selected probiotics - a bacterial mixture (VSL#3), a Gram-negative probiotic (E. coli Nissle 1917), two Gram-positive probiotic bacteria (LGG, L. reuteri), and a yeast probiotic (S. boulardii) - for which sound clinical and mechanistic data is available. Safety of probiotic formulations is also discussed.
Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry (Formerly Cu rrent Medicinal Chemistry - Anti-Inflammatory and Anti-Allergy Agents) 09/2009; 8(3):260-269.
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ABSTRACT: Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria have an important function; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns that may be critical to the development of this disease. Fecal samples from 20 preterm infants, 10 with NEC and 10 matched controls (including 4 twin pairs) were obtained from patients in a single site level III neonatal intensive care unit. Bacterial DNA from individual fecal samples was PCR-amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity that may accentuate the impact of single dominant microorganisms favored by empiric and widespread use of antibiotics.
The ISME Journal 05/2009; 3(8):944-54. · 7.38 Impact Factor
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ABSTRACT: Bacteria play a role in inflammatory bowel disease and other forms of intestinal inflammation. Although much attention has focused on the search for a pathogen or inciting inflammatory bacteria, another possibility is a lack of beneficial bacteria that normally confer anti-inflammatory properties in the gut. The purpose of this study was to determine whether normal commensal bacteria could inhibit inflammatory pathways important in intestinal inflammation.
Conditioned media from Lactobacillus plantarum (Lp-CM) and other gut bacteria was used to treat intestinal epithelial cell (YAMC) and macrophage (RAW 264.7) or primary culture murine dendritic cells. NF-kappaB was activated through TNF-Receptor, MyD88-dependent and -independent pathways and effects of Lp-CM on the NF-kappaB pathway were assessed. NF-kappaB binding activity was measured using ELISA and EMSA. 1kappaB expression was assessed by Western blot analysis, and proteasome activity determined using fluorescence-based proteasome assays. MCP-1 release was determined by ELISA.
Lp-CM inhibited NF-kappaB binding activity, degradation of IkappaBalpha and the chymotrypsin-like activity of the proteasome. Moreover, Lp-CM directly inhibited the activity of purified mouse proteasomes. This effect was specific, since conditioned media from other bacteria had no inhibitory effect. Unlike other proteasome inhibitors, Lp-CM was not toxic in cell death assays. Lp-CM inhibited MCP-1 release in all cell types tested.
These studies confirm, and provide a mechanism for, the anti-inflammatory effects of the probiotic and commensal bacterium Lactobacillus plantarum. The use of bacteria-free Lp-CM provides a novel strategy for treatment of intestinal inflammation which would eliminate the risk of bacteremia reported with conventional probiotics.
Inflammatory Bowel Diseases 05/2009; 15(10):1537-47. · 4.86 Impact Factor
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ABSTRACT: Platelet-activating factor (PAF) is a phospholipid inter- and intracellular mediator implicated in intestinal injury primarily via induction of an inflammatory cascade. We find that PAF also has direct pathological effects on intestinal epithelial cells (IEC). PAF induces Cl(-) channel activation, which is associated with intracellular acidosis and apoptosis. Using the rat small IEC line IEC-6, electrophysiological experiments demonstrated that PAF induces Cl(-) channel activation. This PAF-activated Cl(-) current was inhibited by Ca(2+) chelation and a calcium calmodulin kinase II inhibitor, suggesting PAF activation of a Ca(2+)-activated Cl(-) channel. To determine the pathological consequences of Cl(-) channel activation, microfluorimetry experiments were performed, which revealed PAF-induced intracellular acidosis, which is also inhibited by the Cl(-) channel inhibitor 4,4'diisothiocyanostilbene-2,2'disulfonic acid and Ca(2+) chelation. PAF-induced intracellular acidosis is associated with caspase 3 activation and DNA fragmentation. PAF-induced caspase activation was abolished in cells transfected with a pH compensatory Na/H exchanger construct to enhance H(+) extruding ability and prevent intracellular acidosis. As ClC-3 is a known intestinal Cl(-) channel dependent on both Ca(2+) and calcium calmodulin kinase II phosphorylation, we generated ClC-3 knockdown cells using short hairpin RNA. PAF induced Cl(-) current; acidosis and apoptosis were all significantly decreased in ClC-3 knockdown cells. Our data suggest a novel mechanism of PAF-induced injury by which PAF induces intracellular acidosis via activation of the Ca(2+)-dependent Cl(-) channel ClC-3, resulting in apoptosis of IEC.
AJP Gastrointestinal and Liver Physiology 06/2008; 294(5):G1191-200. · 3.43 Impact Factor
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ABSTRACT: Flagellin is a bacterial protein responsible for activation of Toll-like receptor 5 (TLR5), which we hypothesize is involved in Salmonella's induction of cytoprotective heat shock proteins in intestinal epithelial cells. Flagellin induces the cytoprotective heat shock protein Hsp25 in different intestinal epithelial cell lines and in mouse intestine. Flagellin induces Hsp25 expression in a time-dependent manner in vitro. This effect is transcriptional, as confirmed by luciferase reporter assays and actinomycin D treatment. In addition, Hsp25 induction requires p38 MAPK activation and is only observed when flagellin is added to the basolateral side of polarized intestinal epithelial cells, consistent with the known location of TLR5. Flagellin-mediated Hsp25 induction is associated with increased protective effects against oxidant stress, an effect that is at least partially mediated by p38 MAPK. Use of small interfering RNA against Hsp25 demonstrates that flagellin-mediated protection against oxidant stress is to some degree mediated through Hsp25 induction. This suggests that, by protecting against oxidant injury, the induction of Hsp25 expression by flagellin may contribute to intestinal homeostasis. In a coculture cell model and in a mouse model of Salmonella enterica Serovar Typhimurium infection, not only does infection with wild-type and a flagellin-deletion mutant strain of Salmonella show that flagellin induces Hsp25 in vivo, but it also demonstrates that in the case of live Salmonella infection, flagellin serves as a major stimulus for the induction of Hsp25 expression. These data provide evidence that flagellin is required for Salmonella-mediated induction of Hsp25 expression in intestinal epithelium.
AJP Gastrointestinal and Liver Physiology 04/2008; 294(3):G808-18. · 3.43 Impact Factor
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ABSTRACT: Salmonella Typhimurium is a major cause of human gastroenteritis. The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junction (TJ) structure and function in intestinal epithelial cells. AvrA is a newly described bacterial effector found in Salmonella. We report here that AvrA expression stabilizes cell permeability and tight junctions in intestinal epithelial cells. Cells colonized with an AvrA-deficient bacterial strain (AvrA-) displayed decreased cell permeability, disruption of TJs, and an increased inflammatory response. Western blot data showed that TJ proteins, such as ZO-1, claudin-1, decreased after AvrA- colonization for only 1 hour. In contrast, cells colonized with AvrA-sufficient bacteria maintained cell permeability with stabilized TJ structure. This difference was confirmed in vivo. Fluorescent tracer studies showed increased fluorescence in the blood of mice infected with AvrA- compared to those infected with the AvrA-sufficient strains. AvrA- disrupted TJ structure and function and increased inflammation in vivo, compared to the AvrA- sufficient strain. Additionally, AvrA overexpression increased TJ protein expression when transfected into colonic epithelial cells. An intriguing aspect of this study is that AvrA stabilized TJs, even though the other TTSS proteins, SopB, SopE, and SopE2, are known to disrupt TJs. AvrA may play a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. Our findings indicate an important role for the bacterial effector AvrA in regulation of intestinal epithelial cell TJs during inflammation. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampen the inflammatory response.
PLoS ONE 01/2008; 3(6):e2369. · 4.09 Impact Factor
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ABSTRACT: AvrA is a newly described bacterial effector existing in Salmonella. Here, we test the hypothesis that AvrA is a deubiquitinase that removes ubiquitin from two inhibitors of the nuclear factor-kappaB (NF-kappaB) pathway, IkappaBalpha and beta-catenin, thereby inhibiting the inflammatory responses of the host. The role of AvrA was assessed in intestinal epithelial cell models and in mouse models infected with AvrA-deficient and -sufficient Salmonella strains. We also purified AvrA and AvrA mutant proteins and characterized their deubiquitinase activity in a cell-free system. We investigated target gene and inflammatory cytokine expression, as well as effects on epithelial cell proliferation and apoptosis induced by AvrA-deficient and -sufficient bacterial strains in vivo. Our results show that AvrA blocks degradation of IkappaBalpha and beta-catenin in epithelial cells. AvrA deubiquitinates IkappaBalpha, which blocks its degradation and leads to the inhibition of NF-kappaB activation. Target genes of the NF-kappaB pathway, such as interleukin-6, were correspondingly down-regulated during bacterial infection with Salmonella expressing AvrA. AvrA also deubiquitinates and thus blocks degradation of beta-catenin. Target genes of the beta-catenin pathway, such as c-myc and cyclinD1, were correspondingly up-regulated with AvrA expression. Increased beta-catenin further negatively regulates the NF-kappaB pathway. Our findings suggest an important role for AvrA in regulating host inflammatory responses through NF-kappaB and beta-catenin pathways.
American Journal Of Pathology 10/2007; 171(3):882-92. · 4.89 Impact Factor
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ABSTRACT: Premature infants are susceptible to many conditions that are inflammatory in nature. For this patient population, which is expecting the intrauterine environment, pathways necessary for fetal life and development may not have completed the transitions necessary for extrauterine life. In this study, responses to tumor necrosis factor-alpha were compared in human fetal and adult intestinal epithelial cell lines along with preweaned and postweaned mouse intestinal sections to identify a potential developmental difference that may explain the heightened inflammatory response of preterm infants. The nuclear factor-kappaB (NF-kappaB) pathway regulates a wide variety of genes involved in immune and inflammatory processes. We report that, compared with adult intestinal epithelial cells, immature intestinal epithelial cells have increased NF-kappaB activity associated with increased NF-kappaB-DNA binding and transcriptional activity. This increased activity appears due to inadequate inhibition of signaling leading to NF-kappaB activation since there is also increased phosphorylation, ubiquitination, and degradation of the inhibitor of NF-kappaB in conjunction with decreased baseline expression and delayed resynthesis of this inhibitor. Thus we demonstrate a potential mechanism for the heightened inflammatory response of immature intestinal epithelial cells.
AJP Gastrointestinal and Liver Physiology 06/2007; 292(5):G1411-9. · 3.43 Impact Factor
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Olga Zaborina,
Francois Lepine,
Gaoping Xiao,
Vesta Valuckaite,
Yimei Chen,
Terry Li,
Mae Ciancio,
Alex Zaborin, Elaine O Petrof,
Elaine Petroff,
Jerrold R Turner,
Laurence G Rahme,
Eugene Chang,
John C Alverdy
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ABSTRACT: There is now substantial evidence that compounds released during host stress directly activate the virulence of certain opportunistic pathogens. Here, we considered that endogenous opioids might function as such compounds, given that they are among the first signals to be released at multiple tissue sites during host stress. We tested the ability of various opioid compounds to enhance the virulence of Pseudomonas aeruginosa using pyocyanin production as a biological readout, and demonstrated enhanced virulence when P. aeruginosa was exposed to synthetic (U-50,488) and endogenous (dynorphin) kappa-agonists. Using various mutants and reporter strains of P. aeruginosa, we identified involvement of key elements of the quorum sensing circuitry such as the global transcriptional regulator MvfR and the quorum sensing-related quinolone signaling molecules PQS, HHQ, and HQNO that respond to kappa-opioids. The in vivo significance of kappa-opioid signaling of P. aeruginosa was demonstrated in mice by showing that dynorphin is released from the intestinal mucosa following ischemia/reperfusion injury, activates quinolone signaling in P. aeruginosa, and enhances the virulence of P. aeruginosa against Lactobacillus spp. and Caenorhabditis elegans. Taken together, these data demonstrate that P. aeruginosa can intercept opioid compounds released during host stress and integrate them into core elements of quorum sensing circuitry leading to enhanced virulence.
PLoS Pathogens 04/2007; 3(3):e35. · 9.13 Impact Factor
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ABSTRACT: Conditioned media from the probiotic Lactobacillus GG (LGG-CM) induce heat shock protein (Hsp) expression in intestinal epithelial cells. LGG-CM induces both Hsp25 and Hsp72 in a time- and concentration-dependent manner. These effects are mediated by a low-molecular-weight peptide that is acid and heat stable. DNA microarray experiments demonstrate that Hsp72 is one of the most highly upregulated genes in response to LGG-CM treatment. Real-time PCR and electrophoretic mobility shift assay confirm that regulation of Hsp induction is at least in part transcriptional in nature, involving heat shock factor-1. Although Hsps are not induced for hours after exposure, transient exposure to LGG-CM is sufficient to initiate the signal for Hsp induction, suggesting that signal transduction pathways may be involved. Experiments confirm that LGG-CM modulates the activity of certain signaling pathways in intestinal epithelial cells by activating MAP kinases. Inhibitors of p38 and JNK block the expression of Hsp72 normally induced by LGG-CM. Functional studies indicate that LGG-CM treatment of gut epithelial cells protects them from oxidant stress, perhaps by preserving cytoskeletal integrity. By inducing the expression of cytoprotective Hsps in gut epithelial cells, and by activating signal transduction pathways, the peptide product(s) secreted by LGG may contribute to the beneficial clinical effects attributed to this probiotic.
AJP Cell Physiology 05/2006; 290(4):C1018-30. · 3.54 Impact Factor
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ABSTRACT: The extent and severity of mucosal injury in inflammatory bowel diseases are determined by the disequilibrium between 2 opposing processes: reparative and cytoprotective mechanisms vs. inflammation-induced injury. Probiotics may provide clinical benefit by ameliorating colitis; however, their mechanisms of action remain largely unknown. Our objective was to investigate microbial-epithelial interactions that could explain the beneficial therapeutic effects of probiotics.
The effect of VSL#3-conditioned media on the nuclear factor-kappaB pathway in young adult mouse colonic epithelial cells was assessed by using monocyte chemoattractant protein-1 enzyme-linked immunosorbent assays; IkappaBalpha, IkappaBbeta, and p105 immunoblot analysis; and nuclear factor-kappaB luciferase reporter gene and proteasome assays. Effects on heat shock proteins were determined by electrophoretic mobility shift assay and immunoblot for heat shock proteins 25 and 72 in young adult mouse colonic cells. Cytoprotection against oxidant injury was determined by chromium 51 release and filamentous and globular actin assays.
VSL#3 produces soluble factors that inhibit the chymotrypsin-like activity of the proteasome in gut epithelial cells. Proteasome inhibition is an early event that begins almost immediately after exposure of the epithelial cells to the probiotic-conditioned media. In addition, these bacteria inhibit the proinflammatory nuclear factor-kappaB pathway through a mechanism different from the type III secretory mechanisms described for other nonpathogenic enteric flora. They also induce the expression of cytoprotective heat shock proteins in intestinal epithelial cells.
The resulting inhibition of nuclear factor-kappaB and increased expression of heat shock proteins may account for the anti-inflammatory and cytoprotective effects reported for probiotics and may be a novel mechanism of microbial-epithelial interaction. These effects seem to be mediated through the common unifying mechanism of proteasome inhibition.
Gastroenterology 12/2004; 127(5):1474-87. · 11.68 Impact Factor
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ABSTRACT: While the pathological events evoked by infection are commonly described, effective host responses to bacteria and their products should primarily be protective. Heat shock protein (Hsp) expression is upregulated by many stimuli and serves to maintain intracellular protein integrity. The ability of the prototypic superantigen, Staphylococcus aureus enterotoxin B (SEB) to induce Hsps was investigated with BALB/c mice and by in vitro addition to the murine small intestinal epithelial cell line MSIE. SEB-treated (5 or 100 microg intraperitoneally) mice revealed increased Hsp25 and Hsp72, but not Hsc73, in jejunal lymphocytes and epithelial cells. A similar Hsp response to SEB occurred in MSIE cells and was preceded by activation of the ERK1/2 and p38 mitogen-activated protein kinases but not the SAPK/JNK pathway; pharmacological inhibition of ERK1/2, but not p38, significantly reduced SEB-induced Hsps. Moreover, SEB-treated MSIE cells were protected against oxidant-induced cytotoxicity (measured by 51Cr release) and F-actin depolymerization. Thus, SEB exposure results in a rapid induction of the Hsp25 and Hsp72 in intestinal epithelial cells, both directly and through lymphocyte activation, and we suggest that this event is important in protecting the gut from damage by Staphylococcus infection or in the reparatory process and may be a generalized response to lumen-derived bacterial toxins.
Infection and Immunity 07/2004; 72(6):3187-94. · 4.16 Impact Factor
