Eleonora Feketeova

Hypertonic saline resuscitation limits neutrophil activation after trauma-hemorrhagic shock

There is evidence suggesting that the ischemic gut is a major source of factors that lead to neutrophil activation, and that neutrophil activation can be reduced by hypertonic saline resuscitation. Thus, we tested whether trauma-hemorrhagic shock-induced neutrophil activation can be reduced by hypertonic saline resuscitation, as well as whether hypertonic saline reduces the ability of mesenteric lymph from shocked animals to activate neutrophils. Male Sprague-Dawley rats subjected to trauma (laparotomy), plus 90 min of shock [mean arterial pressure (MAP) MAP = 30 mmHg] or sham shock were resuscitated with Ringers lactate or 7.5% hypertonic saline at an equivalent sodium load. Whole blood samples were collected before shock as well as at 1 and 2 h after the end of the shock period for neutrophil CD11b and CD18 expression measurements. In a second set of experiments, mesenteric lymph samples collected from rats subjected to trauma plus hemorrhagic shock (T/HS) or trauma plus sham-shock (T/SS) and resuscitated with Ringers lactate or hypertonic saline were tested for their ability to modulate PMN CD11b, CD18, or L-selectin expression, as well as prime PMN for an augmented respiratory burst. To avoid confounding results due to interspecies differences, while at the same time looking at potential human responses, both naive rat and human PMN were tested. Both CD11b and CD18 expression were increased in PMN harvested from rats subjected to T/HS and resuscitated with Ringers lactate solution, but not in T/HS rats resuscitated with hypertonic saline. These results indicate that PMN activation is increased to a greater extent in Ringers lactate-resuscitated than hypertonic saline-resuscitated animals. Likewise, mesenteric lymph from the T/HS rats resuscitated with Ringers lactate increased naive rat and human PMN CD11b and CD18 expression to a greater extent than did T/HS lymph from the hypertonic saline-treated rats. Additionally, T/HS lymph from the Ringers lactate-but not the hypertonic saline-treated rats induced PMN L-selectin shedding. Lastly, T/HS lymph from the Ringers lactate-treated rats induced the greatest PMN respiratory burst. These results indicate that resuscitation from T/HS with hypertonic saline is associated with less PMN activation than resuscitation with Ringers lactate, and that factors produced or released by the postischemic intestine and carried in the mesenteric lymph contribute to neutrophil activation after an episode of T/HS.

Serine proteases are involved in the pathogenesis of trauma-hemorrhagic shock-induced gut and lung injury.

The objective of this work was to test the hypothesis that intraluminal serine proteases are involved in trauma-hemorrhagic shock (T/HS)-induced intestinal and lung injury. Male Sprague-Dawley rats were administrated the serine protease inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, Nafamostat) either intraluminally into the gut or intravenously after a laparotomy (trauma) and then subjected to 90 min of hemorrhagic shock (T/HS) or sham shock (T/SS). Intestinal and lung injury was assessed at 3 h after resuscitation with Ringers lactate solution. In a second set of experiments, mesenteric lymph was collected from the groups of rats subjected to T/HS or T/SS and its ability to activate normal neutrophils was tested. Lung permeability, pulmonary myeloperoxidase levels, and the bronchoalveolar lavage fluid protein to plasma protein ratio were increased after T/HS but were significantly decreased in the T/HS rats receiving intraluminal (P < 0.05), but not intravenous, nafamostat. Likewise, T/HS-induced intestinal villus injury was less in the nafamostat-treated shock rats (P < 0.05). Last, the ability of T/HS mesenteric lymph to increase PMN CD11b expression or prime neutrophils for an augmented respiratory burst was significantly reduced by the intraluminal administration of nafamostat. Because intraluminal nafamostat reduced T/HS-induced gut and lung injury as well as the neutrophil activating ability of intestinal T/HS lymph, the presence of serine proteases in the ischemic gut may play an important role in T/HS-induced gut and hence lung injury.

Targeting immune response induction with cochleate and liposome-based vaccines

The immune response generated by infection with a pathogenic organism, or by vaccination with a live attenuated or whole killed pathogen, often does not stimulate optimal protection against that organism. Lipid matrix-based subunit vaccines can be used to produce custom-designed vaccines, that elicit desired immune responses targeted to specific parts of the pathogen that are relevant to protection. Harmful or competitive responses can be minimized or avoided. Earlier work with liposomes has allowed the development of a new class of subunit vaccines called cochleate delivery vehicles, whose structure and properties are very different from liposomes. Protein and DNA cochleates are highly effective vaccines when given via mucosal or parenteral routes, including oral, intranasal, intramuscular, or subcutaneous. Strong, long-lasting, mucosal and circulating, antibody and cell-mediated responses are generated. Protection from challenge with live viruses following oral or intramuscular administration has been achieved.

Attenuation of Shock-induced Acute Lung Injury by Sphingosine Kinase Inhibition

BACKGROUND Prolonged elevations of cytosolic calcium concentrations ([Ca2+]i) are required for optimal neutrophil (PMN) activation responses to G-Protein coupled chemoattractants. We recently showed that the coupling of endosomal Ca2+ store depletion to more prolonged entry of external Ca2+ depends on cellular conversion of sphingosine to sphingosine 1-phosphate (S1P) by sphingosine kinase (SK). We therefore hypothesized that inhibition of SK might inhibit PMN activation and thus ameliorate lung injury after trauma and hemorrhagic shock (T/HS). METHODS Chemotaxis (CTX) of human PMN was studied using modified Boyden chambers in the presence or absence of the selective SK inhibitor, SKI-2. After determining the concentration of SKI-2 that inhibited human PMN CTX by 50% (IC50) we subjected rats to T/HS (laparotomy, hemorrhage to 30-40 mm Hg x 90 minutes, 3 hours resuscitation). We then studied rat PMN CD11b expression using flow cytometry and lung injury using the Evans Blue dye technique in the presence of IC50 doses of SKI-2 or vehicle given in pretreatment at laparotomy. RESULTS Human PMN CTX was suppressed slightly more than 50% by 40 micromol/L SKI-2 (233 +/- 20 vs 103 +/- 12 x 10(3) cells/well, p < 0.001). Rat PMN expression of CD11b after T/HS was decreased from 352 +/- 30 to 232 +/- 7 MFU (p < 0.001) in the presence 30 micromol/L SKI-2. Lung permeability to Evans Blue was decreased from 9.5 +/- 2 to 4.1 +/- 0.7% (p = 0.036.). SKI-2 did not cause hemodynamic instability or alter resuscitation requirements. CONCLUSION Modulation of PMN Ca entry via SK inhibition inhibits PMN CTX in vitro, and inhibits CD11b expression in vivo without major effects on hemodynamics. These cellular changes were associated with amelioration of lung injury in vivo in a rat model of T/HS. These findings suggest that SK inhibition allows modulation of inflammation via control of [Ca2+]i without the cardiovascular compromise expected with Ca2+ channel blockade. SK inhibition therefore appears to be an important novel candidate therapy for inflammatory organ injury after shock.

Factors in intestinal lymph after shock increase neutrophil adhesion molecule expression and pulmonary leukosequestration.

BACKGROUND Because the ischemic gut may produce factors that initiate systemic inflammation, we tested the hypothesis that factors released from the gut into the mesenteric lymphatics increase neutrophil (PMN) adhesion molecule expression after trauma and shock. METHODS At 1 and 4 hours after hemorrhagic shock (30 mm Hg x 90 minutes) plus trauma (laparotomy) (T/HS) or sham-shock (T/SS), with or without mesenteric lymph duct ligation, PMN CD11b and CD18 expression was assessed in male rats. In additional rats, mesenteric lymph samples were tested for their ability to increase PMN CD11b expression in vitro. Lastly, at 4 hours after T/SS or T/HS with or without lymph duct ligation, pulmonary PMN sequestration was measured. RESULTS Compared with T/SS rats, T/HS was associated with up-regulation of PMN CD11b and CD18 expression, which was largely prevented by ligation of the mesenteric lymph duct (p < 0.01). Lymph duct ligation also prevented T/HS-induced pulmonary leukocyte sequestration (p < 0.01). In addition, mesenteric lymph from rats subjected to T/HS but not T/SS increased CD11b expression (p < 0.01). CONCLUSION Factors produced or released by the postischemic intestine and carried in the mesenteric lymph appear responsible for PMN activation and pulmonary PMN sequestration after an episode of T/HS.

Mesenteric lymph from burned rats induces endothelial cell injury and activates neutrophils

ObjectiveOur previous studies indicated that mesenteric lymph duct ligation prevented burn-induced lung injury. Thus, the goal of the present study was to begin to investigate potential mechanisms of this protective effect. DesignProspective animal study with concurrent control. SettingSmall animal laboratory. SubjectsAdult male Sprague-Dawley rats. InterventionsMesenteric lymph and portal vein plasma were collected from male rats subjected to a 40% third-degree scald burn or sham burn. The biological effects of these lymph and plasma samples were tested for their ability to kill human umbilical vein endothelial cells (HUVECs), increase HUVEC monolayer permeability, and activate polymorphonuclear leukocytes (PMNs), as reflected in CD11b adhesion molecule expression and superoxide production. Additionally, ileal specimens were harvested at the end of the experiment (6 hrs postburn) for histologic analysis. Measurements and Main ResultsPostburn mesenteric lymph produced during the first 2 hrs after burn injury and tested at a 5% concentration, but not sham-burn lymph or portal plasma from burned rats, was toxic for HUVECs resulting in cell death after an 18-hr incubation period. Similarly, only postburn lymph increased HUVEC monolayer permeability. Postburn lymph activated both rat and human PMNs as reflected in increased CD11b expression and augmentation of the phorbol myristate acetate-induced superoxide response. Neither sham-burn lymph nor postburn portal vein plasma activated PMNs. Both the burn and sham-burn lymph samples were sterile, indicating that the effects of burn lymph on the HUVECs or PMNs were not due to translocating bacteria. Last, an association was found between burn-induced gut injury and the production of toxic burn lymph. ConclusionsBurn-induced gut injury results in the production of biologically active factors that are carried in the mesenteric lymph, but not the portal plasma, which injure endothelial cells and activate PMNs and thus could contribute to distant organ injury.

Store-operated calcium channel inhibition attenuates neutrophil function and postshock acute lung injury

Background:A wide variety of neutrophil (PMN) functions are regulated by cytosolic calcium concentration. Calcium channel blockade might therefore decrease postshock inflammation but could also limit important cardiovascular compensations. PMN Ca2+ entry occurs, however, through store-operated calci

Resistance of the female, as opposed to the male, intestine to I/R-mediated injury is associated with increased resistance to gut-induced distant organ injury.

We tested the hypothesis that the female intestine is more resistant to gut I/R injury than the male intestine by comparing the effects of the isolated pure gut I/R superior mesenteric artery occlusion (SMAO) model on gut morphology and whether SMAO-induced distant organ injury (lung, bone marrow [BM], neutrophils, and red blood cells [RBCs]) would differ between male and proestrus female rats. At 6 or 24 h after SMAO or sham SMAO, gut injury, lung permeability, pulmonary neutrophil sequestration, RBC deformability, and BM RBC and white blood cell progenitor growth were measured, as was the ability of the plasma from these rats to activate naive rat neutrophils. At both 6 and 24 h after SMAO, the female rats had significantly less intestinal injury and reduced gut-induced lung injury, BM suppression, RBC dysfunction, and neutrophil activation than male rats subjected to SMAO. These results indicate that the resistance of proestrus female rats to gut injury and gut-induced distant organ injury is greater than that observed in male rats.

LOSS OF THE INTESTINAL MUCUS LAYER IN THE NORMAL RAT CAUSES GUT INJURY, BUT NOT TOXIC MESENTERIC LYMPH NOR LUNG INJURY

There is substantial evidence that gut barrier failure is associated with distant organ injury and systemic inflammation. After major trauma or stress, the factors and mechanisms involved in gut injury are unknown. Our primary hypothesis is that loss of the intestinal mucus layer will result in injury of the normal gut that is exacerbated by the presence of luminal pancreatic proteases. Our secondary hypothesis is that the injury produced in the gut will result in the production of biologically active mesenteric lymph and consequently distant organ (i.e., lung) injury. To test this hypothesis, five groups of rats were studied: 1) uninstrumented naive rats; 2) control rats in which a ligated segment of distal ileum was filled with saline; 3) rats with pancreatic proteases placed in their distal ileal segments; 4) rats with the mucolytic N-acetylcysteine (NAC) placed in their distal ileal segments; and 5) rats exposed to NAC and pancreatic proteases in their ileal segments. The potential systemic consequences of gut injury induced by NAC and proteases were assessed by measuring the biological activity of mesenteric lymph as well as gut-induced lung injury. Exposure of the normal intestine to NAC, but not saline or proteases, led to increased gut permeability, loss of mucus hydrophobicity, a decrease in the mucus layer, as well as morphological evidence of villous injury. Although proteases themselves did not cause gut injury, the combination of pancreatic proteases with NAC caused more severe injury than NAC alone, suggesting that once the mucus barrier is impaired, luminal proteases can injure the now vulnerable gut. Because comparable levels of gut injury caused by systemic insults are associated with gut-induced lung injury, which is mediated by biologically active factors in mesenteric lymph, we next tested whether this local model of gut injury would produce active mesenteric lymph or lead to lung injury. It did not, suggesting that gut injury by itself may not be sufficient to induce distant organ dysfunction. Therefore, loss of the intestinal mucus layer, especially in the presence of intraluminal pancreatic proteases, is sufficient to lead to injury and barrier dysfunction of the otherwise normal intestine but not to produce gut-induced distant organ dysfunction.

Intestinal bacterial overgrowth induces the production of biologically active intestinal lymph.

OBJECTIVE We have previously documented that gut-derived lymph from rats subjected to trauma plus hemorrhagic shock (T/HS) is injurious to vascular endothelial cells and activates neutrophils (PMNs), two key events in postshock organ injury. Because T/HS leads to gut injury, intestinal bacterial overgrowth, and the loss of gut barrier function, the relative role of gut injury as opposed to intestinal bacterial overgrowth per se in the pathogenesis of biologically active intestinal lymph is unclear. We therefore studied whether mesenteric lymph can injure endothelial cells and/or active PMNs in an intestinal bacterial overgrowth model where there is no gut injury (monoassociation). METHODS Bacterial overgrowth was established in male rats by treating the animals with 4 days of oral antibiotics followed by administration of a nonpathogenic, streptomycin-resistant strain of Escherichia coli C25. Mesenteric lymph was then collected from rats with normal flora and from E. coli C25 monoassociated rats. Its effects were tested on human umbilical vein endothelial cells (HUVECs) and human PMNs. As an additional control, lymph was collected from antibiotic-decontaminated rats that received antibiotics but were not colonized with E. coli C25. RESULTS As compared with medium, normal flora intestinal lymph, antibiotic-decontaminated lymph, or portal plasma from the monoassociated rats, mesenteric lymph from the monoassociated rats killed HUVECs and increased the permeability of a HUVEC monolayer. In contrast to the effects on HUVECs, lymph from the monoassociated rats did not increase PMN CD11b expression or prime PMNs for an augmented respiratory burst, as compared with lymph from the rats with normal flora or from antibiotic-decontaminated rats. The effects of lymph from the monoassociated rats was not caused by bacteria, because these lymph samples were sterile. CONCLUSION These results indicate that disruption of the normal intestinal microflora resulting in bacterial overgrowth with enteric bacilli may participate in the production of mesenteric lymph that is injurious to endothelial cells in shock, but this mechanism does not appear to be significantly involved in the activation of PMNs.