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The bast fibres, a subgroup of natural fibre family, have emerged as a strong competitor of widely used man-made glass fibre for use as fillers or reinforcing materials in certain types of composite materials, which do not require very high mechanical resistance. This paper investigates the manufacturing of multi-layered jute fabric-reinforced thermoplastic composite and its mechanical performance. Hessian jute fabrics in two, four and six layers without any pre-treatment were sandwiched in 0° orientation into seven layers of high-density polyethylene sheets and pressed at high temperature and pressure to form composite laminates having three different structural designs. The laminates with two, four and six layers contain approximately 6.70 wt%, 12.90 wt% and 18.50 wt% of jute fibres, respectively. Mechanical performance of the composite laminates having four and six layers of jute fabric was found to have improved significantly when compared to the pure high-density polyethylene laminates. Within a given sample thickness of 6.5 mm, the laminate with six layers of jute fabric exhibited the best mechanical performance. Optical microscopic analysis revealed that the yarn orientation of the fabrics within the composites remained stable, and there was no visible void in the laminate structure. Fracture morphology of the composite investigated by a scanning electron microscope showed good adhesion of the jute fabrics with the high-density polyethylene matrix. [ABSTRACT FROM AUTHOR]

Sepsis-induced systemic inflammation results in coagulation abnormalities that may be different in gram-positive and gram-negative infections. We used ciprofloxacin to induce a predominantly gram-positive Enterococcus faecalis polymicrobial sepsis in rats. Ciprofloxacin-untreated rats exhibited a predominantly gram-negative polymicrobial sepsis. Rats were subjected to 30% body surface area burn (B), cecal ligation puncture (CLP) with a 22-gauge needle, and B + CLP. Ciprofloxacin-treated B + CLP rats showed a significant decrease in plasma thrombin activatable fibrinolysis inhibitor (TAFI) levels compared with sham rats. However, plasma tissue factor pathway inhibitor (TFPI) levels decreased significantly in B, CLP, and B + CLP groups compared with sham rats. The ciprofloxacin-untreated group showed a significant decrease in plasma TAFI levels in CLP and B + CLP and plasma TFPI levels decreased in all 3 groups compared with sham rats. Histological changes in the liver and kidney included vascular congestion and parenchyma bleed following B + CLP in ciprofloxacin-untreated rats. These results suggest that plasma TAFI and TFPI levels differ depending on the type of bacteria involved in the septic process.

Infection remains the major cause of morbidity and mortality in burn patients. Furthermore, the use of antibiotics in such patients has led to the prevalence of antibiotic-resistant microbial infections; one such infection in intensive care unit turns out to be caused by the enterococcal organisms. Our laboratory studies have used a rat model of bum injury and Enterococcus faecalis infection. Sprague-Dawley male rats ( approximately 250 g) were initially given an intragastric gavage of the antibiotic ciprofloxacin for 3 days. This procedure allowed for decontamination of intestine of gram-negative and some gram-positive organisms. The remainders of the gram-positive organisms were, to a large extent, Enterococci. After the decontamination procedure, rats were intra-abdominally inoculated with E. faecalis; inoculation involved preparation of sterilized rat fecal pellets impregnated with E. faecalis (10 colony-forming units) and their implants through a midline abdominal wall incision. Some of the rats that were implanted with the fecal pellets were subject to full-thickness skin bums ( approximately 30% total body surface area; 95 degrees C water for 7 s). Sham abdominal infection rats received a sterile pellet only; sham bum procedure consisted of exposing the skin to room temperature water. All sham and bum and/or E. faecalis infection procedures were carried out on rats under pentobarbital anesthesia. Inflammation and innate host defense-related responses were assessed via measurements of neutrophil effector responses, i.e., oxygen anion free radical (O2)/eIastase production, CD11b/CD18 expression, apoptosis, and tissue infiltration. Determining epithelial lactulose permeability, microvascular albumin leakage, and epithelial tight junction integrity assessed the status of intestinal function/structural derangements. The animals' metabolic and cardiovascular integrity was evaluated determining blood pH, p02, pC02, heart rate, respiratory rate, blood pressure, and cardiac output. Whereas the aforementioned measurements were carried out at 24 to 48 h postbum injury with and without the Enterococcal infection, animal mortality was determined for up to 5 days after the experimental injuries. The results of the studies indicated that whereas bum or E. faecalis infection alone did not produce significant mortality, the dual insult with bum and E. faecalis infection resulted in significant animal death accompanied by relatively more profound metabolic and cardiovascular derangements. Inappropriately heightened neutrophil effector responses were present with bum alone as well as with the dual bum and infection complications. These studies suggest that animal models of bum injury with Enterococcal infection complications simulate the adverse outcomes bum patients infected with Enterococcal organisms.

We evaluated MAPK (Erk 1/2 and p38) signaling mechanisms of altered T-cell-mediated immune responses in thermal injury condition. Rats were subjected to 30% body surface scald burn, and their mesenteric lymph node (MLN) and Peyer's patch (PP) T cells were purified using nylon wool method. Activation of MAPKs, Erk 1/2 and p38 was assessed in T cells by determining its phosphorylation using immunoblot analysis, intracellular immunostaining and confocal microscopy. The results showed a down-regulation of Erk 1/2 and p38 activation in anti-CD3-stimulated T cells from thermally injured animals, compared to Erk 1/2 and p38 in sham rat T cells. The down-regulation of MAPKs in T cells was reversed by treatment of T cells with calcium agonist, ionomycin. These data indicate that attenuated MAPKs (Erk 1/2, p38) activation in thermally injured animals' T cells could result from derangement of Ca(2+) mobilization. This finding suggests that T cell signaling derangements with thermal injury involve an altered cross-talk between Ca(2+) mobilization and MAPK signaling mechanisms.

Neutrophil apoptosis is delayed under trauma and/or sepsis injury conditions. The molecular mechanism for the delay in apoptosis has not been well defined. We investigated whether activation of phosphatidyl inositol 3-kinase (PI3-kinase)/PKB signaling pathway contributes to the delay in neutrophil apoptosis with thermal injury. Rats were subjected to burns (30% total body surface area, 98 degrees C for 10 s), and euthanized 24 h later. Blood neutrophils were isolated with the use of Ficoll gradient centrifugation and cultured for the indicated time periods. Apoptosis was determined using annexin V and PI labeling and flow cytometry. NF-kappaB activation was examined using gel mobility shift assay and confocal microscopy. Expression levels of inhibitory apoptosis proteins (IAPs), including cellular IAP1 (cIAP1), cIAP2, X-linked IAP (XIAP), and survivin, and Bcl-2 family members such as Bcl-xl and Bad, were determined by Western blot analysis and/or RT-PCR, real-time PCR. The results showed that in culture, the decrease in apoptosis of neutrophils from thermally injured rats was prevented in the presence of PI3-kinase inhibitors wortmannin and LY-294002. There was upregulation of PKB and Bad phosphorylation and NF-kappaB activation in N-formyl-l-methionyl-l-leucyl-l-phenylalanine-stimulated neutrophils from thermally injured rats compared with the sham injured group. Increased Bad phosphorylation and NF-kappaB activation were also attenuated by wortmannin. Bcl-xl expression in neutrophils was upregulated with thermal injury and inhibited in the presence of wortmannin. However, the expression of IAP family members was neither affected by thermal injury nor inhibited by wortmannin. These data suggest that the delay in neutrophil apoptosis with thermal injury is partly caused by activation of PI3-kinase/PKB signaling and NF-kappaB, which appeared to be related to the increased Bcl-xl expression and phosphorylation of Bad, but not IAP expression.

Previous studies from our laboratory have shown that acute alcohol (EtOH) ingestion prior to burn injury enhances intestinal bacterial translocation. This study tested if increased intestinal bacterial translocation in alcohol and burn injured rats is due to an overgrowth in intestinal bacteria. We determined if the translocation was accompanied with alterations in intestinal permeability and immune cell population. Rats (225-250 g) were gavaged with alcohol to achieve a blood EtOH level in the range of 100 mg/dl prior to burn or sham injury (25% total body surface area). Two days after injury, we found that acute alcohol ingestions prior to burn injury results in a significant increase in bacterial counts in small intestine. The increase in intestinal bacterial counts accompanied a significant increase in intestinal permeability. Finally, immunohistochemical analysis revealed a substantial (p<0.05) loss of both T cell and dendritic cells in intestine of alcohol and burn injured rats compared with intestine of rats receiving either burn or sham injury. Altogether, results presented in this manuscript suggest that increase in intestinal bacterial growth along with alterations in intestinal permeability and immune status contribute to the increase in bacterial translocation observed in alcohol and burn injured rats.

Objective: To determine alterations in intestinal epithelial permeability to solutes in burn injured rats with and without Enterococcus faecalis infection and the role of neutrophils in the intestinal permeability changes., Design: Prospective sham-controlled animal study., Setting: University research laboratory., Subjects: Male Sprague-Dawley rats., Interventions: Rats were subjected to 30% total body surface burn (B group), E. faecalis infection (EF group) induced via intra-abdominal implantation of bacterial pellet, or combination of burn injury and E. faecalis infection (B+EF group)., Measurements and Main Results: In vivo measurements of intestinal permeability were carried out after intraluminal injection of H lactulose and C mannitol in the ileum of sham, B, EF, and B+EF groups of rats, 1 and 2 days after injury. Lactulose permeability was increased in the injured rat groups (B, EF, B+EF) on day 1 postinjury compared with sham. The combined injury group (B+EF) had the highest level of lactulose permeability. Although a significant change in lactulose permeability from day 1 to day 2 postinjury could not be demonstrated in the B and EF groups, lactulose permeability in the B+EF group on day 2 postinjury markedly decreased from day 1 but was still significantly higher than that in the sham group. Mannitol permeability was increased in all injured rat groups on day 1 postinjury; on day 2 it remained elevated post-B, decreased post-EF, and further increased after B+EF. Ex vivo measurements of lactulose movements across intestinal epithelial monolayers (IEC-18) were carried out in the presence of blood neutrophils from sham, B, EF, or B+EF rats. We also measured ex vivo transepithelial migration of neutrophils from sham, B, EF, or B+EF rat groups. Neither the transepithelial lactulose movement in the presence of neutrophils from, nor neutrophil migration in, the B or EF rats was significantly different from sham. However, a significant increase in transepithelial lactulose movement and neutrophil migration occurred in the B+EF group. Immunoblot analyses and in situ histochemical localizations of intestinal tight junction proteins, occludin and claudin-3, showed decreases in the distribution of occludin but not claudin-3 in the B, EF, and B+EF groups., Conclusions: Alterations in intestinal solute permeability and disruption of tight junction integrity after a two-hit injury with burn and E. faecalis infection, but not after individual injuries of burn or E. faecalis infection, are likely associated with heightened neutrophil flux across the intestinal epithelium.

Burn and septic injuries induce profound changes in coagulation status. This study examined the changes in plasma tissue factor pathway inhibitor (TFPI) and thrombin activatable fibrinolytic inhibitor (TAFI) levels in a rat model of burn and septic injuries. Rats underwent 30% TBSA cutaneous scald burn injury and septic insult was induced by caecal ligation and puncture (CLP). CLP was superimposed on burn injury to mimic the clinical model of sepsis complicating burn injury. Rats were pretreated with Cprofloxacin orally to colonize their gut with Enterococcus faecalis. TFPI and TAFI plasma levels were measured using functional activity assay kit with a chromogenic method at 24 and 72 hours following the injuries. TFPI levels decreased significantly at 24 hours in burn, CLP, and burn+CLP groups, followed by incomplete rebound recovery at 72 hours in all three groups. On the other hand, TAFI levels increased significantly at 24- and 72-hour time points in all three groups. These results suggest that burn, septic, and their combined injuries perturb coagulation cascade and thrombotic process toward the procoagulant pathway by impairing fibrinolysis.

Recent studies from our laboratory have suggested that acute alcohol ingestion prior to burn injury enhances gut bacterial translocation by suppressing T cell-mediated intestinal immune defense. To determine the mechanism responsible for suppressed T cell function, we examined the activation of mitogen-activated protein kinases (MAPK) members p-38 and ERK-1/2. Both p-38 and ERK-1/2 are known to play a significant role in the T cell proliferation and their cytokine production. Rats were gavaged with ethanol to achieve a blood alcohol level of approximately 100 mg/dl, before they were subjected to a 25% total body surface area burn injury. Two days after injury, rats were sacrificed and mesenteric lymph node (MLN) T cells were isolated and their ability to proliferate in response to anti-CD3 was determined. For p-38 and ERK-1/2 determination, T cells were divided into two groups. Cells in one group were stimulated with anti-CD3 for 3 min and lysed. The cells in the second group were cultured for approximately 18 h in the presence of anti-CD3 and lysed. MAPK status in 18-h cultured cells allowed us to determine whether or not the changes in p-38 and ERK-1/2 are transient or persist in the proliferating cells. Two days after injury, anti-CD3-mediated MLN T cell proliferation was more suppressed in rats gavaged with alcohol prior to burn injury compared to rats receiving either burn injury alone or sham-injured rats regardless of their exposure. Western blot analyses showed significant inhibition of ERK-1/2 phosphorylation in both freshly isolated and 18-h cultured T cells from alcohol and burn-injured rats compared to the sham rat T cells. The inhibition of p-38 phosphorylation in T cells derived from alcohol and burn-injured rats was found to be transient as no significant difference in p-38 phosphorylation was noted between the 18 h incubated MLN T cells of sham and alcohol and burn-injured rats. Taken together, our findings suggest that low levels of ERK-1/2 activation is likely to play a significant role in MLN T cell proliferative suppression in alcohol and burn-injured rats.

Objective: Our group has previously shown that the intestinal epithelium exhibits increased postburn barrier permeability and bacterial translocation associated with deranged neutrophil activity. The purpose of this investigation is to explore possible underlying intestinal structural mechanisms, leading to those functional changes with emphasis on (1) neutrophil influx and extravasation in the intestinal lamina propria 1-3 days after burn and (2) enterocyte proliferation, migration, apoptosis, and E-cadherin junctional epithelium levels 3 days after burn., Design: Freshly isolated ileum specimens were quick frozen, then cut by a cryostat into 30-micron-thick sections. Sections from day 1 postburn rats were immunostained with (1) anti-granulocyte or anti-elastase antibodies to assess neutrophil influx or (2) combined anti-granulocyte and anti-von Willebrand factor double immunolabeling to compare levels of neutrophil extravasation. Sections from day 3 postburn rats were immunostained with (1) bromodeoxyuridine immunohistochemistry 1, 3, 6, or 18 hrs after bromodeoxyuridine injection to assess enterocyte proliferation and migration, (2) cytokeratin-18 M30-immunohistochemistry to compare levels of enterocyte apoptosis, and (3) E-cadherin immunohistochemistry to compare junctional E-cadherin integrity. Ileal myeloperoxidase activity and bacterial translocation of Enterococcus faecalis were assessed biochemically and by E. faecalis-specific bacterial cultures, respectively, in day 3 postburn rats., Setting: : Research laboratories in a medical center and an academic institution., Subjects: Male Sprague-Dawley rats given sham treatment or treatment as a burn model with full-thickness skin scald over 30% total body surface area., Conclusions: We report (1) increased levels of neutrophil influx and extravasation in villi lamina propriae, including elastase-positive cells (postburn day 1), (2) heightened levels of intestinal myeloperoxidase activity (postburn day 3), (3) decreased levels of epithelial cell proliferation, migration, and E-cadherin (postburn day 3), and (4) increased enterocyte apoptosis and E. faecalis bacterial translocation (postburn day 3). Based on these structural and functional abnormalities, we propose a mechanism for burn injury-related intestinal barrier dysfunction that includes increased trans- and para-cellular leakage caused by impaired enterocyte renewal and decreased junctional E-cadherin levels subsequent to increased neutrophil influx and extravasation within the villus lamina propria microenvironment.

Alcohol intoxication is being recognized increasingly as the major factor in pathogenesis after burn injury. Findings from multiple studies support the suggestion that, in comparison with burn-injured patients who sustained injury in the absence of alcohol intoxication, burn-injured patients who sustained injury under the influence of alcohol exhibit higher rates of infection and are more likely to die. Thus, infection becomes the primary cause of death in burn-injured patients. Because the intestine is considered to be a major source of bacteria, studies in experimental animals have been designed to examine whether alcohol intoxication before burn injury enhances bacterial translocation from the intestine. Results of these studies have shown a several-fold increase in bacterial translocation from the intestine in the group of animals receiving combined insult of alcohol intoxication and burn injury compared with findings for the groups receiving either insult alone. Alcohol intoxication and burn injury independent of each other have also been shown to cause an increase in bacterial translocation. The gastrointestinal tract normally maintains a physical mucosal and immunologic barrier that provides an effective defense in keeping bacteria within the intestinal lumen. However, in injury conditions these defense mechanisms are impaired. Intestinal bacteria consequently gain access to extraintestinal sites. Intestine-derived bacteria are implicated in causing systemic infection and in subsequent multiple organ dysfunction in both immunocompromised patients and patients with injury, such as burn and trauma. In this article, we discuss three potential mechanisms that are likely to contribute to the increase in bacterial translocation in alcohol intoxication and burn injury: (1) increase in bacterial growth in the intestine, (2) physical disruption of mucosal barrier of the intestine, and (3) suppression of the immune defense in the intestine.

Neutrophil apoptosis is delayed under trauma and/or sepsis conditions. The mechanism for the delay has remained unclear. We hypothesize that modulation of the mitochondrial pathway of apoptosis contributes to the delay in neutrophil apoptosis with burn injury. Rats were subjected to burn injury (30% of total body surface area, 98 degrees C for 10 s) and euthanatized 24 h postinjury. Blood neutrophils from sham and burn-injured rats were isolated by Ficoll gradient centrifugation and cultured for 2 or 8 h. Neutrophil apoptosis was determined by annexin V and propidium iodide (PI) labeling and flow cytometry. Neutrophil mitochondrial morphology was assessed via histochemical staining (MitoTracker GreenFM) and confocal microscopy. Neutrophils from rats with burn injury showed a decreased level of apoptosis compared with sham rat neutrophils at both 2 and 8 h of incubation. In incubated sham rat neutrophils, mitochondria showed a change from normal "tubular" to an "aggregated" morphology. In contrast, cultured neutrophils from burn rats did not exhibit this mitochondrial morphological transition until 8 h of incubation. Compared with sham rat neutrophils, neutrophils from burn rats showed decreased levels of active caspase-9 and -3. Whereas an upregulation of Bcl-xL and a downregulation of Bax seemed to contribute to decreased apoptosis in burn rat neutrophils at 2 h of incubation, the decreased apoptosis at 8 h appeared to be associated with a decrease in Bax and increased phosphorylated Bad. These data suggest that suppression of the mitochondrial pathway plays an essential role in the delay of polymorphonuclear neutrophil apoptosis with burn injury.

Cutaneous burn injury-induced T lymphocyte suppression is a well-known phenomenon. In this study, we evaluated the effect of treatment of burn rats with pentoxifylline (PTX) on the burn-induced suppression of T lymphocytes. Anesthetized rats were subjected to 30% total body surface area burn by exposing skin to 95 degrees C water for 10 s. T lymphocytes were isolated from sham and burn rats with or without PTX treatment (120 mg/kg, ip). T cell proliferation and interleukin (IL)-2 production in response to T cell mitogen concanavalin A was measured using 3 H-thymidine uptake and enzyme-linked immunosorbent assay, respectively. P59 fyn autophosphorylation and its kinase activity was determined using in vitro kinase assay. In addition, T lymphocyte Ca2+ signaling was assessed using Ca2+ imaging technique. Two days after injury, there was a significant decrease in mesenteric lymph node T cell proliferation and IL-2 production in burn injured rats compared with those obtained from sham-injured rats. This decrease in T cell proliferation and IL-2 production in burn-injured rats was accompanied by a significant suppression in both P59 autophophorylation and kinase activity as well as Ca2+ signaling. Treatment of burn-injured rats with PTX produced a near complete recovery of T cell proliferation and IL-2 production. Furthermore, PTX treatment also prevented the burn-mediated suppression in P59fyn and kinase activity as well as restored Ca2+ signaling similar to those observed in sham injured rats. These findings altogether suggested that PTX treatment attenuate T cell suppression in burn-injured rats and that the effects of PTX are mediated via modulating P59 fyn and Ca2+ signaling.

Objective: To determine the effects of an immune-enhancing diet supplemented with glutamine, arginine, fish oil, and dietary nucleotides on mesenteric lymph node T-cell functional disturbances encountered after burn injury in rats., Design: A prospective animal study., Setting: University medical center research laboratory., Subjects: Adult male Sprague-Dawley rats., Interventions: Rats received a 30%, total body surface, full-thickness burn. Burn-injury rats received the IMPACT diet supplemented with glutamine, arginine, fish oil, and nucleotides or arginine, fish oil, and nucleotides, or an isocaloric/isonitrogenous diet without supplementation with glutamine, arginine, fish oil, or nucleotides., Measurements and Main Results: Two days after injury, we found a significant decrease in the proliferation and interleukin-2 production by mesenteric lymph node T cells derived from rats fed on conventional chow compared with sham rats. The burn-related suppression of mesenteric lymph node T-cell proliferation and interleukin-2 production was prevented when the rats were fed on a high-protein diet rich in glutamine, arginine, fish oil, and nucleotides. We found that the immunostimulatory effects of the enriched diet are dependent on the presence of glutamine, arginine, fish oil, and nucleotides as feeding of rats on the isocaloric/isonitrogenous diet deficient in glutamine, arginine, fish oil, and nucleotides did not prevent the burn-related suppression of mesenteric lymph node T-cell dysfunction. Finally, our studies suggested that immunostimulatory effects of the diet are mediated by prostaglandin E(2) regulation of T-cell activation signaling molecule P59fyn., Conclusion: These results suggest that a diet rich in arginine, fish oil, and nucleotides, with and without glutamine, can effectively prevent T-cell dysfunction encountered after burn injury.

Both experimental and clinical evidence suggest a suppression of T-cell function in burn and sepsis. The objective of the present study was to evaluate splenocyte and purified T-cell proliferative response and IL-2 production in septic neonatal rats. We also examined if alterations in T-cell proliferation and IL-2 production in neonatal sepsis is due to elevation in PGE2. PGE2 is known to play a significant role in T-cell suppression during sepsis in adults. Sepsis was induced in 15-day-old neonatal Sprague-Dawley rats by implanting 0.1 cm3 of fecal pellet impregnated with Escherichia coli (50 CFU) and Bacteroides fragilis (10(3) CFU). Animals receiving fecal pellets without the bacteria were designated as sterile. A group of septic and sterile rats were treated with PGE2 synthesis inhibitors, NS398 and resveratrol. These treatments of animals allowed us to evaluate the role of PGE2 in T-cell suppression during neonatal sepsis. Splenocytes as well as purified T cells were prepared and then proliferative response and IL-2 productive capacities were measured. A significant suppression of splenocyte proliferation and IL-2 production was noticed in both sterile and septic animals compared to the T cells from unoperated control rats. In contrast, the proliferation and IL-2 production by nylon wool purified T cells in sterile rats was not significantly different from control rats, whereas, a significant suppression in Con A-mediated T-cell proliferation and IL-2 production noticed in septic rat T cells compared to the sterile and control rat T cells. Such decrease in T-cell proliferation and IL-2 production was accompanied with 20-25% deaths in neonates implanted with septic pellets. No mortality was noted in sterile-implanted neonates. Treatment of animals with COX-1 inhibitor had no effect on T-cell proliferation response in both septic and sterile groups, whereas COX-2 inhibitor abrogated the decrease in T-cell proliferative response in the septic group. The treatment of animals with COX-2 inhibitor also significantly prevented the sepsis-associated mortality in neonates. In conclusion, the present study demonstrated T-cell suppression during neonatal sepsis is accompanied by a decrease in IL-2 production. Such suppressions were ameliorated with COX-2 inhibitor suggesting a role for PGE2 in the suppressed T-cell-mediated immune function in neonatal sepsis., (Copyright 2003 S. Karger AG, Basel)

Pathophysiology of burn injury with complications of gram-positive infections is not well characterized. We have developed an in vivo rat model to study the effects of burn injury along with intra-abdominal inoculation of Enterococcus faecalis. We hypothesized that although burn injury or E. faecalis inoculation by itself may not induce significant pathophysiological responses, the combination of the two can lead to adverse pathophysiological consequences. Sprague-Dawley rats were divided into 4 groups: group 1(C), controls; group 2(B), burn injury on 30% total body surface area; group 3(EF), intra-abdominal implantation of bacterial pellet impregnated with E. faecalis; group 4(B+EF), burn injury plus bacterial pellet implantation. The mortality was 25% and 60% on day 1 and 2 in Group 4(B+EF), respectively; no significant mortality was observed in other groups. In group 4(B+EF), metabolic acidosis, respiratory alkalosis, and a hyperdynamic state developed on day 1, and metabolic and respiratory acidosis and a hypodynamic state on day 2. There were no significant alterations in metabolic or hemodynamic measurements in other groups. Intestinal microvascular permeability to albumin on day 1 and 2 was increased in group 4(B+EF). In group 2(B), microvascular permeability was not increased significantly. Although the permeability was increased on day 1 in group 3(EF), it declined on day 2. The metabolic and hemodynamic alterations were correlated with increased intestinal microvascular permeability to albumin. E. faecalis appeared to be involved in initiating a vicious cycle of burn injury-mediated disruption of intestinal integrity along with metabolic and hemodynamic derangements.

We evaluated the dependency of neutrophil O production on PTK-Lyn and MAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn was assessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessed by Western blot analysis. O production was measured by isoluminol-enhanced luminometry. Imaging technique was employed to measure neutrophil [Ca2+](i) in individual cells. Thermal injury caused marked upregulation of Lyn and ERK1/2 accompanying enhanced neutrophil O production. Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478) before burn injury caused complete inhibition of the respective kinase activation. Both AG556 and AG1478 produced an ~66% inhibition in O production. Treatment with diltiazem (DZ) produced an ~37% inhibition of O production without affecting Lyn or ERK1/2 activation with burn injury. Ca2+ mobilization was upregulated with burn injury but not affected by treatment of burn rats with AG556. Unlike the partial inhibition of burn-induced O production by AG556, AG1478, or DZ, platelet-activating factor antagonist (PAFa) treatment of burn rats produced near complete inhibition of O production. PAFa treatment also blocked activation of Lyn. The findings suggest that the near complete inhibition of O production by PAFa was a result of blockade of PTK as well as Ca2+ signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result of potentiation of Ca2+ -linked and -independent signaling triggered by inflammatory agents such as PAF.

PGE2 is known to suppress T cell proliferation and IL-2 production in many inflammatory conditions. Previous studies from our laboratory have shown that such suppression of T cell proliferation in burn and sepsis could result from alteration in T cell activation signaling molecule p59fyn. In this study, we examined the role of downstream signaling molecules NFAT and AP-1 in PGE2-mediated suppression of T cell in burn injury. These studies were carried out utilizing splenic T cells from sham and burn rats 3 days after injury. The data presented in this manuscript suggest a significant suppression of IL-2 production by T cells from burn injured rats compared with the T cells from sham rats. The suppression in T cell IL-2 production was accompanied by a decrease in the activation of NFAT and AP-1 as well as a decrease in T cell p59fyn kinase activity. The treatments of burn-injured animals with PGE2 synthesis blocker indomethacin prevented both the decrease in NFAT and AP-1 binding to IL-2 sequences. In vitro incubation of control rat T cells with PGE2 suppressed the activation of NFAT and AP-1. These results suggested that the suppression of T cell IL-2 production could result from PGE2-mediated alterations in the T cell signaling molecule p59fyn and NFAT/AP-1.

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.

Objective: To evaluate the effect of burn injury with and without an Escherichia coliseptic complication on T-cell proliferation, interleukin-2 production, and Ca(2+) signaling responses in intestinal Peyer's patch and splenic T cells., Design: Prospective, randomized, sham-controlled animal study., Setting: University medical center research laboratory., Subjects: Adult male Sprague-Dawley rats., Interventions: Rats were subjected to a 30% total body surface area, full skin thickness burn. Infection in rats was induced via intraperitoneal inoculation of E. coli, 10(9) colony forming units/kg, with or without a prior burn., Measurements and Main Results: Rat Peyer's patch and splenic T lymphocytes were isolated by using a nylon wool cell purification protocol. T-cell proliferation, interleukin-2 production, and Ca(2+) signaling responses were measured after stimulation of cells with the mitogen, concanavalin A. T-cell proliferation was determined by measuring incorporation of (3)H-thymidine into T-cell cultures. Interleukin-2 production by T-cell cultures was measured by using enzyme-linked immunosorbent assay. Intracellular T-cell Ca2(+ )concentration, [Ca(2+)](i), was measured by the use of Ca(2+)-specific fluorescent label, fura-2, and its fluorometric quantification. [Ca(2+)](i) was also evaluated by the use of digital video imaging of fura-2 loaded individual T cells. T-cell proliferation and interleukin-2 production were suppressed substantially in both Peyer's patch and splenic T cells 3 days after either the initial burn alone or burn followed by the E. coli inoculation at 24 hrs after the initial burn. There seemed to be no demonstrable additive effects of E. coli infection on the effects produced by burn injury alone. The T-cell proliferation and interleukin-2 production suppressions with burn or burn-plus-infection insults were correlated with attenuated Ca(2+) signaling. E. coli infection alone suppressed T-cell proliferation in Peyer's patch but not in splenic T cells at 2 days postbacterial inoculation; E. coli infection had no effect on Peyer's patch or splenic T cells at 1 day postinjury. On the other hand, burn injury alone caused a substantial T-cell proliferative suppression at 2 days postburn in both Peyer's patch and splenic cells and a significant suppression in T-cell proliferation on day 1 postburn in Peyer's patch but not in the spleen., Conclusion: An initial burn injury suppressed T-cell proliferation at a level that it would not be further affected by a subsequent infection even if the infection by itself has the potential of suppressing T-cell proliferation. An earlier onset of T-cell suppression in Peyer's patch cells than in the spleen with burn could be attributable to an initial hypoperfusion-related intestinal mucosal tissue injury. Overall, our study supports the concept that burn injury per se can significantly suppress T-cell mediated immunity and that the intestine is an early tissue site of such suppression.

The role of platelet-activating factor (PAF) in Ca(2+) signaling and Ca(2+)-related enhancement of reactive oxygen intermediate (ROI) generation in neutrophils of burn-injured rats was ascertained by evaluating the effect of treatment of the rats with a PAF receptor antagonist. The treatment of rats with the antagonist also allowed us to evaluate the role of PAF in the priming of neutrophil ROI response with burn in vivo. A full skin thickness burn injury was produced in anesthetized rats by exposing 30% of total body surface area to 98 degrees C water for 10 s. Sham and burn rats were killed 1 day later, and their blood was collected to obtain neutrophils. Fluorescence-activated cell sorter analysis was used to quantify ROI production by the neutrophils. Cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) imaging technique was employed to measure neutrophil [Ca(2+)](i) in individual cells and microfluorometry for the assessment of [Ca(2+)](i) responses in suspensions of neutrophils. There was an overt enhancement of ROI generation by burn rat neutrophils. ROI release was accompanied by a marked elevation of [Ca(2+)](i) signaling. The treatment of rats with PAF receptor antagonist before burn prevented the upregulation of both [Ca(2+)](i) and ROI generation in neutrophils. These studies indicate that enhanced ROI production in neutrophils in the early stages after burn injury results from a PAF-mediated priming of the [Ca(2+)](i) signaling pathways in vivo.

Increased gut bacterial translocation in burn and trauma patients has been demonstrated in a number of previous studies, however, the mechanism for such an increased gut bacterial translocation in injured patients remains poorly understood. Utilizing a rat model of burn injury, in the present study we examined the role of intestinal immune defense by analyzing the T cell functions. We investigated if intestinal T cells dysfunction contributes to bacterial translocation after burn injury. Also our study determined if burn-mediated alterations in intestinal T cell functions are related to enhanced release of PGE2. Finally, we examined whether or not burn-related alterations in intestinal T cell function are due to inappropriate activation of signaling molecule P59fyn, which is required for T cell activation and proliferation. The results presented here showed an increase in gut bacterial accumulation in mesenteric lymph nodes after thermal injury. This was accompanied by a decrease in the intestinal T cell proliferative responses. Furthermore, the treatments of burn-injured animals with PGE2 synthesis blocker (indomethacin or NS398) prevented both the decrease in intestinal T cell proliferation and enhanced bacterial translocation. Finally, our data suggested that the inhibition of intestinal T cell proliferation could result via PGE2-mediated down-regulation of the T cell activation-signaling molecule P59fyn. These findings support a role of T cell-mediated immune defense against bacterial translocation in burn injury.

TGF-beta is known to inhibit many of the immune cell functions including T cell proliferation and IL-2 production. The mechanism of such TGF-beta-mediated inhibition of T cell functions is poorly understood. The present study examined the effects of TGF-beta on the activation of protein tyrosine kinases (PTK) P56lck, P59fyn, and Zap-70, and protein tyrosine phosphatases (PTP) SHP-1 and SHP-2. A balance between the actions of PTK and PTP is critical for appropriate T cell activation. These studies were carried out using nylon wool-purified splenic T cells from healthy Sprague-Dawley rats. Results from these studies showed that incubation of T cells with TGF-beta inhibited the activation of P56lck, P59fyn and Zap-70. The decrease in these three protein tyrosine kinases was accompanied by an increase in the activation of the protein tyrosine phosphatase SHP-1. There was no change in the phosphorylation of SHP-2 with and without pretreatment of T cells with TGF-beta. The decrease in P56lck, P59fyn kinase activity, and Zap-70 phosphorylation was prevented when T cells were stimulated with anti-CD3 in the presence of pervanadate, an inhibitor of PTP. These results suggested that TGF-beta-mediated inhibition of P56lck, P59fyn, and Zap-70 is likely due to an up-regulation of protein tyrosine phosphatases such as SHP-1.

Sepsis-induced suppression in T-cell proliferation follows deranged Ca(2+) signaling in adult rats. In preliminary studies, we observed suppression in T-cell proliferation in septic neonatal rats as well. In this study, we assessed splenic T-cell cytosolic Ca(2+) concentration, [Ca(2+)](i), as its elevation plays an important role in T-cell proliferation. Also, we investigated the role of PGE(2) in sepsis-related changes in T-cell [Ca(2+)](i) in animals pretreated with cyclooxygenase-1 (COX-1) inhibitor (resveratrol) and cyclooxygenase-2 (COX-2) inhibitor (NS-398). Sepsis was induced in 15-day-old rat pups by intraperitoneal implantation of fecal pellets containing Escherichia coli and Bacteroides fragilis. The sham group consisted of pups implanted with sterile fecal pellets. Septic and sham pups were sacrificed 24 h after implantation and their spleens were removed. The spleens from sham and septic pups, along with spleens from unoperated control pups, were processed for single cell suspensions, and T cells were isolated using nylon wool columns. Fura-2 fluorophotometry was employed for the measurement of [Ca(2+)](i) (in nM units) in T cells stimulated with concanavalin A (ConA). Our results show that ConA-mediated T-cell [Ca(2+)](i) response is significantly suppressed in septic neonatal rats. Pretreatment of pups with COX-2, but not COX-1 inhibitor, prevented the decrease in the [Ca(2+)](i) response. These findings suggest that PGE(2) might induce the attenuation in T-cell Ca(2+) signaling during sepsis in neonatal rats., (Copyright 2001 S. Karger AG, Basel)

In this study, we have evaluated the role of cytokine-induced neutrophil chemoattractant (CINC), in the upregulation of neutrophil Ca(2+) signaling in neutrophils from thermally injured rats treated with anti-CINC antibody. Additionally, we have determined the effect of the treatment with CINC antibody on the accumulation of activated neutrophils in the intestinal wall, and the effect of such accumulation on gut bacterial translocation. Measurements of myeloperoxidase (MPO) activity and immunohistochemical localization of neutrophils determined neutrophil sequestration in the rat intestine. Agar culture analyses and a specific Escherichia coli beta-galactosidase gene polymerase chain reaction was carried out to detect gut indigenous bacterial invasion into intestinal wall and extraintestinal mesenteric lymph nodes (MLN). The results showed that pretreatment of rats with anti-CINC antibody attenuated the thermal injury-induced enhancement in [Ca(2+)](i) responses in neutrophils both in the basal and Formyl-Met-Leu-Phe stimulated conditions. Moreover, treatment with the CINC antibody decreased neutrophil infiltration into the gut and attenuated thermal injury-caused translocation of bacteria into the MLN.

Alterations in cellular responses in various organ systems contribute to trauma-, burn-, and sepsis-related multiple organ dysfunction syndrome. Such alterations in muscle contractile, hepatic metabolic, and neutrophil and T-cell inflammatory-immune responses have been shown to result from cell-signaling modulations and/or impairments in the respective cell types. Altered Ca(2+) signaling would seem to play an important role in the myocardial and vascular smooth muscle contractile dysfunction in the injury conditions; Ca(2+)-linked signaling derangement also plays a crucial role in sepsis-induced altered skeletal muscle protein catabolism and resistance to insulin-mediated glucose use. The injury-related increased hepatic gluconeogenesis and acute-phase protein response could also be caused by a pathophysiologic up-regulation of hepatocyte Ca(2+)-signal generation. The increased oxidant production by neutrophil, a potentially detrimental inflammatory response in early stages after burn or septic injuries, seems to result from an up-regulation of both the Ca(2+)-dependent as well as Ca(2+)-independent signaling pathways. The injury conditions would seem to cause an inappropriate up-regulation of Ca(2+)-signal generation in the skeletal myocyte, hepatocyte, and neutrophil, while they lead to a down-regulation of Ca(2+) signaling in T cells. The crucial signaling derangement that causes T-cell proliferation suppression seems to be a decrease in the activation of protein tyrosine kinases, which subsequently down-regulates Ca(2+) signaling. The delineation of cell-signaling derangements in trauma, burn, or sepsis conditions can lead to development of therapeutic interventions against the disturbed cellular responses in the vital organ systems.

The present study evaluated burn-induced vascular permeability alterations of rat small intestine in vivo and assessed the effect of neutrophil depletion in burn-injured rats on the altered intestinal microvascular permeability. 125I-labeled bovine serum albumin (125I-BSA) was injected intravenously, and its leakage from circulation into the intestinal tissue was determined by measuring tissue counts of 125I-BSA. Compared with sham, vascular albumin permeability increased 1.7-fold on day 1 post-burn and 3.0-fold on day 3 post-burn in ileum. In the jejunum, albumin permeability increased 1.8- and 2.5-fold on day 1 and day 3 post-burn, respectively. Intestinal tissue edema, determined as increases in tissue water contents, was noted in both intestinal segments on day 1 post-burn; no further increase in edema was found on day 3 post-burn. Neutrophil depletion before burn injury prevented the vascular leakage of albumin and edema in the ileum and jejunum on day 1 post-burn. On day 3 post-burn, the effect of prior neutrophil depletion on vascular permeability was less marked, and edema formation was not affected at all. These findings indicate that an absence of neutrophils prevents the loss of intestinal vascular barrier properties only in the initial periods after burns.

To understand the mechanism of suppressed immunity following alcohol consumption and thermal injury, we analyzed T cell functions in a mouse model of acute alcohol exposure and burn injury. Mice with blood alcohol levels at approximately 100 mg/dl were given a 15% scald or sham injury. Mice were sacrificed 48 h after injury. Our data demonstrated a 20-25% decrease in Con A-mediated splenic T cell proliferation (p<0.01) and 45-50% decrease in interleukin-2 (IL-2) production (p<0.01) following burn injury compared to the T cells from sham animals. A further decrease in the proliferation (25-30%) and IL-2 production (40-45%) was detected in T cells derived from burned animals receiving alcohol as compared to burn alone. No significant change in the proliferation and IL-2 production was observed in splenic T cells derived from sham-injured mice regardless of alcohol exposure. Additionally, there was no demonstrable difference in splenocyte apoptosis in any treatment group. These results suggest that alcohol consumption prior to burn injury causes a greater decrease in T cell proliferation and IL-2 production compared to either burn or alcohol injury alone that may further attenuate the cell-mediated immunity and thus enhance susceptibility to infection.

Under inflammatory conditions after burn/trauma injuries, circulating neutrophils are frequently hyperactive, contributing to excessive superoxide production and related tissue damage. Although normal neutrophil activation is cooperatively controlled by Ca(2+)-independent and Ca(2+)-linked signaling pathways, exuberant Ca(2+)-linked signaling appears to cause neutrophil hyperactivation in the injury conditions.

Cutaneous thermal injury increases intestinal mucosal permeability. The mechanisms of this functional disturbance are not fully understood. We investigated whether accumulation of neutrophils in the intestine contributes to the increase in mucosal permeability. Labeled and unlabeled lactulose and mannitol were infused into a segment of rat ileum or jejunum. Blood concentrations of [(3)H]lactulose and [(14)C]mannitol were measured after 30, 60, and 90 min. On day 1 postburn, lactulose permeability increased fourfold in the ileum and twofold in the jejunum compared with sham-burned rats; mannitol permeability increased twofold in the ileum and 1. 5-fold in the jejunum. A greater increase in permeability occurred on day 3 postburn in the ileum, but not in the jejunum. The depletion of neutrophils in burned rats prevented the increase in permeability in both segments on day 1 postburn. Histological studies of intestines from burned, with or without neutrophil depletion, and sham-burned rats showed similar morphology. However, numerous neutrophils were found in the extravascular compartment in day 1 postburn, but not in neutrophil-depleted and sham-burned rats. These findings support the concept that the burn-induced increase in mucosal permeability is produced during the accumulation of neutrophils in the intestine and can be abrogated by the depletion of neutrophils.

Objective: To determine whether neutrophil depletion could eradicate intestinal bacterial translocation in bum-injured rats., Design: Prospective, randomized, controlled study., Setting: University research laboratory., Subjects: Adult male Sprague-Dawley rats., Interventions: The rats were intravenously administered a rabbit anti-rat neutrophil antibody causing profound neutropenia and subjected to a 30% total body surface area scald burn., Measurements and Main Results: The depletion of neutrophils from the intestine was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates. In addition, the presence of activated/extravasated neutrophils in intact intestines was determined via immunohistochemical localization of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component protein p47phox. Bacterial translocation was measured using agar cultures and by determining Escherichia coli beta-galactosidase gene via polymerase chain reaction/Southern blot analyses of mesenteric lymph node and spleen, liver, lung, and blood. MPO measurements demonstrated a six-fold increase above the control value in the intestinal tissue in rats on day 1 postburn. The presence of activated neutrophils (expression of p47phox protein) was also markedly increased in the intestines of these rats. The increased MPO activity and p47phox expression accompanied a translocation of indigenous E. coli into the mesenteric lymph node without a spread to other organs. The administration of anti-neutrophil antibody to burn animals prevented an increase in MPO activity and bacterial translocation., Conclusion: These studies indicate that enhanced intestinal bacterial translocation caused by burn injury could be related to the increased infiltration of activated neutrophils into the intestinal tissue after bum. The release of neutrophil products such as superoxide anion may effect intestinal tissue damage leading to bacterial translocation of indigenous E. coli.

PGE2-mediated suppression of T cell proliferation during sepsis could result from altered Ca2+ signaling. The present study evaluated the effects of PGE2 on Ca2+ release from intracellular stores and its influx through the plasma membrane in splenic T cells from Sprague-Dawley rats. Intracellular Ca2+ concentration ([Ca2+]i) responses in individual T cells were assessed using the Ca2+ imaging technique, and the release of Ca2+ from intracellular stores and Ca2+ influx were spectrofluorometrically quantified in T cell suspensions. Under unstimulated conditions, nearly 85% of T cells exhibited [Ca2+]i