First Report of bla CTX-M-15-Type ESBL-Producing Klebsiella pneumoniae in Wild Migratory Birds in Pakistan

EcoHealth - We investigated wild migratory birds faecal swabs for extended-spectrum β-lactamases-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) from wetland habitats in Pakistan. ESBL-K....     

Trauma-hemorrhage inhibits splenic dendritic cell proinflammatory cytokine production via a mitogen-activated protein kinase process

Although splenic dendritic cell (DC) functions are markedly altered following trauma-hemorrhage, the mechanism(s) responsible for the altered DC functions remains unknown. We hypothesized that trauma-hemorrhage inhibits DC function via suppressing toll-like receptor 4 (TLR4) expression and mitogen-activated protein kinases (MAPKs). To examine this, male C3H/HeN (6-8 wk) mice were randomly assigned to sham operation or trauma-hemorrhage. Trauma-hemorrhage was induced by midline laparotomy and approximately 90 min of hypotension [blood pressure (BP) 35 mmHg], followed by fluid resuscitation (4x the shed blood volume in the form of Ringer lactate). Two hours later, mice were euthanized, splenic DCs were isolated, and the changes in their MAPK activation, TLR4-MD-2 expression, and ability to produce cytokines were measured. The results indicate that trauma-hemorrhage downregulated the lipopolysaccharide (LPS)-induced MAPK activation in splenic DCs. In addition to the decrease in MAPK activation, surface expression of TLR4-MD-2 was suppressed following trauma-hemorrhage. Furthermore, LPS-induced cytokine production from splenic DCs was also suppressed following trauma-hemorrhage. These findings thus suggest that the decrease in TLR4-MD-2 and MAPK activation may contribute to the LPS hyporesponsiveness of splenic DCs following trauma-hemorrhage. Hyporesponsiveness of splenic DCs was also found after stimulation with the TLR2 agonist zymosan. Our results may thus explain the profound immunosuppression that is known to occur under those conditions.     

Mechanism of estrogen-mediated intestinal protection following trauma-hemorrhage: p38 MAPK-dependent upregulation of HO-1

p38 MAPK has been reported to regulate the inflammatory response in various cell types via extracellular stimuli. p38 MAPK activation also results in the induction of heme oxygenase (HO)-1, which exerts potent anti-inflammatory effects. Although studies have shown that 17beta-estradiol (E(2)) prevented organ dysfunction following trauma-hemorrhage, it remains unknown whether p38 MAPK/HO-1 plays any role in E(2)-mediated attenuation of intestinal injury under those conditions. To study this, male rats underwent trauma-hemorrhage (mean blood pressure approximately 40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E(2) (1 mg/kg body wt), the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt) or E(2) plus SB-203580. Two hours thereafter, intestinal myeloperoxidase (MPO) activity and lactate, TNF-alpha, IL-6, ICAM-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and macrophage inflammatory protein (MIP)-2 levels were measured. Intestinal p38 MAPK and HO-1 protein levels were also determined. Trauma-hemorrhage led to an increase in intestinal MPO activity and lactate, TNF-alpha, IL-6, ICAM-1, CINC-1, and MIP-2 levels. This was accompanied with a decrease in intestinal p38 MAPK activity and increase in HO-1 expression. Administration of E(2) normalized all the above parameters except HO-1, which was further increased following trauma-hemorrhage. Administration of SB-203580 with E(2) abolished the E(2)-mediated restoration of the above parameters as well as the increase in intestinal HO-1 expression following trauma-hemorrhage. These results suggest that the p38 MAPK/HO-1 pathway plays a critical role in mediating the salutary effects of E(2) on shock-induced intestinal injury.     

Estrogen prevents intestinal inflammation after trauma-hemorrhage via downregulation of angiotensin II and angiotensin II subtype I receptor

Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17beta-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-alpha levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation.     

Role of p38 mitogen-activated protein kinase pathway in estrogen-mediated cardioprotection following trauma-hemorrhage

p38 mitogen-activated protein kinase (MAPK) activates a number of heat shock proteins (HSPs), including HSP27 and alpha(B)-crystallin, in response to stress. Activation of HSP27 or alpha(B)-crystallin is known to protect organs/cells by increasing the stability of actin microfilaments. Although our previous studies showed that 17beta-estradiol (E(2)) improves cardiovascular function after trauma-hemorrhage, whether the salutary effects of E(2) under those conditions are mediated via p38 MAPK remains unknown. Male rats (275-325 g body wt) were subjected to soft tissue trauma and hemorrhage (35-40 mmHg mean blood pressure for approximately 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were injected intravenously with vehicle, E(2) (1 mg/kg body wt), E(2) + the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt), or SB-203580 alone, and various parameters were measured 2 h thereafter. Cardiac functions that were depressed after trauma-hemorrhage were returned to normal levels by E(2) administration, and phosphorylation of cardiac p38 MAPK, HSP27, and alpha(B)-crystallin was increased. The E(2)-mediated improvement of cardiac function and increase in p38 MAPK, HSP27, and alpha(B)-crystallin phosphorylation were abolished with coadministration of SB-203580. These results suggest that the salutary effect of E(2) on cardiac function after trauma-hemorrhage is in part mediated via upregulation of p38 MAPK and subsequent phosphorylation of HSP27 and alpha(B)-crystallin.     

Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1β and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury.     

Tissue compartment-specific role of estrogen receptor subtypes in immune cell cytokine production following trauma-hemorrhage.

Although 17beta-estradiol administration following trauma-hemorrhage attenuates plasma cytokines and alteration in immune cell cytokine production, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. Accordingly, we examined which ER subtype predominantly mediates the salutary effects of 17beta-estradiol on systemic inflammatory response/immune cell cytokine production in various tissues following trauma-hemorrhage. Male rats underwent trauma-hemorrhage (mean blood pressure: 40 mmHg for 90 min) and fluid resuscitation. The ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), the ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), 17beta-estradiol (50 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation, and various measurements were made 24 h thereafter. 17beta-Estradiol or PPT administration following trauma-hemorrhage prevented the increase in plasma IL-6 and IL-10 levels that were observed in vehicle-treated animals. IL-6 and TNF-alpha production by Kupffer cells increased; however, splenic macrophages (SMPhi), alveolar macrophages (AMPhi), and peripheral blood mononuclear cells (PBMC) had decreased release of these cytokines after trauma-hemorrhage. IL-10 production, however, increased in all macrophage populations. Administration of 17beta-estradiol following trauma-hemorrhage prevented all of these alterations. PPT had the same effects as 17beta-estradiol on IL-6 and TNF-alpha production by Kupffer cells and SMPhi, and DPN had the same effects on AMPhi and PBMC. The same effects as 17beta-estradiol on IL-10 production were observed by PPT on Kupffer cells and DPN on PBMC. Both agonists were equally effective on SMPhi and AMPhi. Thus ER subtypes have tissue compartment-specific roles in mediating the effects of 17beta-estradiol on immune cell functions following trauma-hemorrhage.     

Gut-associated lymphoid T cell suppression enhances bacterial translocation in alcohol and burn injury

The mechanism of alcohol-mediated increased infection in burn patients remains unknown. With the use of a rat model of acute alcohol and burn injury, the present study ascertained whether acute alcohol exposure before thermal injury enhances gut bacterial translocation. On day 2 postinjury, we found a severalfold increase in gut bacterial translocation in rats receiving both alcohol and burn injury compared with the animals receiving either injury alone. Whereas there were no demonstrable changes in intestinal morphology in any group of animals, a significant increase in intestinal permeability was observed in ethanol- and burn-injured rats compared with the rats receiving either injury alone. We further examined the role of intestinal immune defense by determining the gut-associated lymphoid (Peyer's patches and mesenteric lymph nodes) T cell effector responses 2 days after alcohol and burn injury. Although there was a decrease in the proliferation and interferon-gamma by gut lymphoid T cells after burn injury alone; the suppression was maximum in the group of rats receiving both alcohol and burn injuries. Furthermore, the depletion of CD3(+) cells in healthy rats resulted in bacterial accumulation in mesenteric lymph nodes; such CD3(+) cell depletion in alcohol- and burn-injured rats furthered the spread of bacteria to spleen and circulation. In conclusion, our data suggest that the increased intestinal permeability and a suppression of intestinal immune defense in rats receiving alcohol and burn injury may cause an increase in bacterial translocation and their spread to extraintestinal sites.     

Salutary effects of estrogen receptor-beta agonist on lung injury after trauma-hemorrhage

Although 17beta-estradiol (E2) administration after trauma-hemorrhage attenuates lung injury in male rodents, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. We hypothesized that the salutary effects of E2 lung are mediated via ER-beta. Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). E2 (50 microg/kg), ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropiolnitrile (DPN; 5 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. At 24 h after trauma-hemorrhage or sham operation, bronchoalveolar fluid (BALF) was collected for protein concentration, LDH activity, and nitrate/nitrite and IL-6 levels. Moreover, lung tissue was used for inducible nitric oxide synthase (iNOS) mRNA/protein expression, nitrate/nitrite and IL-6 levels, and wet/dry weight ratio (n = 6 rats/group). One-way ANOVA and Tukey's test were used for statistical analysis. The results indicated that E2 downregulated lung iNOS expression after trauma-hemorrhage. Protein concentration, LDH activity, and nitrate/nitrite and IL-6 levels in BALF and nitrate/nitrite and IL-6 levels in the lung increased significantly after trauma-hemorrhage; however, administration of DPN but not PPT significantly improved all parameters. Moreover, DPN treatment attenuated trauma-hemorrhage-mediated increase in iNOS mRNA/protein expression in the lung. In contrast, no significant change in the above parameters was observed with PPT. Thus the salutary effects of E2 on attenuation of lung injury are mediated via ER-beta, and ER-beta-induced downregulation of iNOS likely plays a significant role in the DPN-mediated lung protection after trauma-hemorrhage.     

Maintenance of lung myeloperoxidase activity in proestrus females after trauma-hemorrhage: upregulation of heme oxygenase-1

Previous studies showed that females in the proestrus stage of the reproductive cycle maintain organ functions after trauma-hemorrhage. However, it remains unknown whether the female reproductive cycle is an important variable in the regulation of lung injury after trauma-hemorrhage and, if so, whether the effect is mediated via upregulation of heme oxygenase (HO)-1. To examine this, female Sprague-Dawley rats during diestrus, proestrus, estrus, and metestrus phases of the reproductive cycle or 14 days after ovariectomy underwent soft tissue trauma and then hemorrhage (mean blood pressure 40 mmHg for 90 min followed by fluid resuscitation). At 2 h after trauma-hemorrhage or sham operation, lung myeloperoxidase (MPO) activity and intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and HO-1 protein levels were measured. Plasma 17beta-estradiol concentration was also determined. The results indicated that trauma-hemorrhage increased lung MPO activity and ICAM-1, CINC-1, and CINC-3 levels in ovariectomized females. These parameters were found to be similar to sham-operated animals in proestrus female rats subjected to trauma-hemorrhage. Lung HO-1 protein level in proestrus females was increased significantly compared with female rats subjected to trauma-hemorrhage during diestrus, estrus, and metestrus phases of the reproductive cycle and ovariectomized rats. Furthermore, plasma 17beta-estradiol level was highest in proestrus females. Administration of the HO inhibitor chromium mesoporphyrin prevented the attenuation of shock-induced lung damage in proestrus females. Thus these findings suggest that the female reproductive cycle is an important variable in the regulation of lung injury following trauma-hemorrhage and that the protective effect in proestrus females is likely mediated via upregulation of HO-1.