Plant-Derived Antimicrobials Reduce E. coli O157:H7 Virulence Factors Critical for Colonization in Cattle Gastrointestinal Tract In Vitro

This study investigated the effect of subinhibitory concentrations (SIC) of five plant-derived antimicrobials (PDAs), namely, trans cinnamaldehyde, eugenol, carvacrol, thymol, and β-resorcylic acid, on E. coli O157:H7 (EHEC) attachment and invasion of cultured bovine colonic (CO) and rectoanal junction (RAJ) epithelial cells. In addition, PDAs’ effect on EHEC genes critical for colonization of cattle gastrointestinal tract (CGIT) was determined in bovine rumen fluid (RF) and intestinal contents (BICs). Primary bovine CO and RAJ epithelial cells were established and were separately inoculated with three EHEC strains with or without (control) SIC of each PDA. Following incubation, EHEC that attached and invaded the cells were determined. Furthermore, the expression of EHEC genes critical for colonization in cattle was investigated using real-time, quantitative polymerase chain reaction in RF and BICs. All the PDAs decreased EHEC invasion of CO and RAJ epithelial cells (P<0.05). The PDAs also downregulated (P<0.05) the expression of EHEC genes critical for colonization in CGIT. Results suggest that the PDAs could potentially be used to control EHEC colonization in cattle; however follow-up in vivo studies in cattle are warranted

Plant-Derived Antimicrobials Reduce E. coli

This study investigated the effect of subinhibitory concentrations (SIC) of five plant-derived antimicrobials (PDAs), namely, trans cinnamaldehyde, eugenol, carvacrol, thymol, and β-resorcylic acid, on E. coli O157:H7 (EHEC) attachment and invasion of cultured bovine colonic (CO) and rectoanal junction (RAJ) epithelial cells. In addition, PDAs’ effect on EHEC genes critical for colonization of cattle gastrointestinal tract (CGIT) was determined in bovine rumen fluid (RF) and intestinal contents (BICs). Primary bovine CO and RAJ epithelial cells were established and were separately inoculated with three EHEC strains with or without (control) SIC of each PDA. Following incubation, EHEC that attached and invaded the cells were determined. Furthermore, the expression of EHEC genes critical for colonization in cattle was investigated using real-time, quantitative polymerase chain reaction in RF and BICs. All the PDAs decreased EHEC invasion of CO and RAJ epithelial cells (P<0.05). The PDAs also downregulated (P<0.05) the expression of EHEC genes critical for colonization in CGIT. Results suggest that the PDAs could potentially be used to control EHEC colonization in cattle; however follow-up in vivo studies in cattle are warranted

Long and Short Duration Exposures to the Selective Serotonin Reuptake Inhibitors (SSRIs) Fluoxetine, Paroxetine and Sertraline at Environmentally Relevant Concentrations Lead to Adverse Effects on Zebrafish Behaviour and Reproduction

Selective serotonin reuptake inhibitors (SSRIs) are currently the most prescribed class of psychotropic medications. Their increased global manufacture and use have become growing concerns for aquatic toxicologists and environmental biologists, who assess both the direct and indirect effects of substances on the environment and on human health. In order to assess the potential impact of environmentally relevant levels of SSRIs on fish development, behaviour and reproduction, we exposed juvenile and adult zebrafish to a select group of SSRIs using two separate exposure paradigms. In the first paradigm, juvenile zebrafish were exposed to Fluoxetine (Prozac), Paroxetine (Paxil), Sertraline (Zoloft) or a mixture of the three beginning at environmentally relevant levels (10 µg/L) for 135 days (long-term exposure) beginning at 5 days post fertilization (dpf). In the second paradigm, adult zebrafish were exposed to matching concentrations of the same SSRIs for 35 days (short-term exposure). The long-term exposure paradigm proved to have little to no overt effect on growth or development at sub-lethal concentrations (10 and 100 µg/L). However, both the stress/anxiety response (novel tank tests) and reproduction (fecundity and fertility) were dramatically reduced. Importantly, the short-term exposure of reproductively mature fish led to similar adverse effects on both the stress response and reproduction. Following both the short and long duration exposure paradigms, a 2-week washout period led to a small reduction in the adverse effects. These findings highlight the potential for SSRIs to negatively impact population dynamics in zebrafish and may be of particular value should they be found in other fish species in the environment

Mitochondrial Damage-Associated Molecular Patterns (MTDs) Are Released during Hepatic Ischemia Reperfusion and Induce Inflammatory Responses.

Ischemia / reperfusion injury (IRI) during the course of liver transplantation enhances the immunogenicity of allografts and thus impacts overall graft outcome. This sterile inflammatory insult is known to activate innate immunity and propagate organ damage through the recognition of damage-associate molecular pattern (DAMP) molecules. The purpose of the present study was to investigate the role of mitochondrial DAMPs (MTDs) in the pathogenesis of hepatic IRI. Using in vitro models we observed that levels of MTDs were significantly higher in both transplantation-associated and warm IR, and that co-culture of MTDs with human and rat hepatocytes significantly increased cell death. MTDs were also released in an in vivo rat model of hepatic IRI and associated with increased secretion of inflammatory cytokines (TNF-α, IL-6, and IL-10) and increased liver injury compared to the sham group. Our results suggest that hepatic IR results in a significant increase of MTDs both in vitro and in vivo suggesting that MTDs may serve as a novel marker in hepatic IRI. Co-culture of MTDs with hepatocytes showed a decrease in cell viability in a concentration dependent manner, which indicates that MTDs is a toxic mediator participating in the pathogenesis of liver IR injury

Long and Short Duration Exposures to the Selective Serotonin Reuptake Inhibitors (SSRIs) Fluoxetine, Paroxetine and Sertraline at Environmentally Relevant Concentrations Lead to Adverse Effects on Zebrafish Behaviour and Reproduction

Selective serotonin reuptake inhibitors (SSRIs) are currently the most prescribed class of psychotropic medications. Their increased global manufacture and use have become growing concerns for aquatic toxicologists and environmental biologists, who assess both the direct and indirect effects of substances on the environment and on human health. In order to assess the potential impact of environmentally relevant levels of SSRIs on fish development, behaviour and reproduction, we exposed juvenile and adult zebrafish to a select group of SSRIs using two separate exposure paradigms. In the first paradigm, juvenile zebrafish were exposed to Fluoxetine (Prozac), Paroxetine (Paxil), Sertraline (Zoloft) or a mixture of the three beginning at environmentally relevant levels (10 &micro;g/L) for 135 days (long-term exposure) beginning at 5 days post fertilization (dpf). In the second paradigm, adult zebrafish were exposed to matching concentrations of the same SSRIs for 35 days (short-term exposure). The long-term exposure paradigm proved to have little to no overt effect on growth or development at sub-lethal concentrations (10 and 100 &micro;g/L). However, both the stress/anxiety response (novel tank tests) and reproduction (fecundity and fertility) were dramatically reduced. Importantly, the short-term exposure of reproductively mature fish led to similar adverse effects on both the stress response and reproduction. Following both the short and long duration exposure paradigms, a 2-week washout period led to a small reduction in the adverse effects. These findings highlight the potential for SSRIs to negatively impact population dynamics in zebrafish and may be of particular value should they be found in other fish species in the environment

Fold changes in <i>TLR2</i>, <i>TLR4</i>, <i>TLR9</i>, <i>MyD88</i> and <i>NF-κB</i> mRNA levels.

<p>MTDs induce <i>MyD88</i> and <i>NFκB</i> expression and up-regulate expression of <i>TLR2</i>, <i>TLR4</i> and <i>TLR9</i> similar to warm hepatic IR. (A)-(E), The expression level of <i>TLR2</i>, <i>TLR4</i>, <i>TLR9</i>, <i>MyD88</i> and <i>NF-κB</i> significantly increased with MTDs co-culture, warm IR, and cold IR treatment compared to control; MTDs, co-culture MTDs with McA RH7777 hepatocyte cell line for 24 hours; W.IR, McA RH7777 hepatocyte cell line underwent warm ischemia for 1 hour followed by reperfusion for 24 hours <i>in vitro</i>; C.IR, McA RH7777 hepatocyte cell line underwent cold ischemia for 6 hours followed by reperfusion for 24 hours <i>in vitro</i>. *, P = 0.01, **, P = 0.001, ***, P = 0.0001 (n = 3/group).</p

Fold changes in <i>TNFα</i> mRNA levels.

<p>MTDs-induced hepatocytes up-regulate expression of TNFα similar to warm IR treatment. MTDs, co-culture 400ug/ml MTDs with McA RH7777 hepatocyte cell line for 24 hours; W.IR, McA RH7777 hepatocyte cell line underwent warm ischemia for 1 hour and reperfusion for 24 hours <i>in vitro</i>. **, P = 0.001, ***, P = 0.0001 (n = 3/group).</p

Warm ischemia and warm reperfusion <i>in vitro</i> elevates mtDNA levels in both rat and human hepatocyte cell lines.

<p>(A) One hour of warm ischemia and two hours of warm IR significantly increased the mtDNA levels in the rat hepatocyte cell line, McA RH7777. (B) One hour warm ischemia and two hours warm IR significantly increased the mtDNA levels in the human hepatocyte cell line, HepG2. Control, cells were cultured in normal cell culture incubator for two hours; 1h Isc, one hour warm ischemia incubation; 1+1 IR, one hour warm ischemia incubation followed by one hour warm reperfusion treatment, CytB, cytochrome B; COXIII, cytochrome C oxidase subunit III; NADH, NADH dehydrogenase. *, P = 0.01, **, P = 0.001, ***, P = 0.0001.</p