Thyyar Ravindranath

PGE2 suppresses intestinal T cell function in thermal injury : A cause of enhanced bacterial translocation

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.

Computed Tomography in Diagnosing Retropharyngeal Abscess in Children

tered the clinical presentation. 7,8 Complications associated with retropharyngeal abscess include meningitis,9 epiglottitis, pneumonia, empyema, spontaneous rupture of the abscess with aspiration, bronchial erosion,l° pyopneumothorax, and purulent pericarditis.l°-1~ The interpretation of the standard lateral neck radiographic film in children is fraught with difficulty, making it hard to differenti-

Effects of burn with and without Escherichia coli infection in rats on intestinal vs. splenic T-cell responses.

Objective To evaluate the effect of burn injury with and without an Escherichia coli septic complication on T-cell proliferation, interleukin-2 production, and Ca2+ 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, 109 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 Ca2+ signaling responses were measured after stimulation of cells with the mitogen, concanavalin A. T-cell proliferation was determined by measuring incorporation of 3H-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, [Ca2+]i, was measured by the use of Ca2+-specific fluorescent label, fura-2, and its fluorometric quantification. [Ca2+]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 Ca2+ 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.

Enteral nutritional supplementation prevents mesenteric lymph node T-cell suppression in burn injury

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.

TNFα Decreases Gluconeogenesis in Hepatocytes Isolated from 10-Day-Old Rats

Gluconeogenesis decreases during septic shock, but its mechanism is not well known. Tumor necrosis factor alpha (TNF-α), which is a key cytokine in septic shock, can increase GLUT1 gene expression and glucose uptake in muscles and fatty tissues. TNF-α does not alter the metabolism of hepatocytes in which GLUT2 is the predominant glucose transporter. However, GLUT1 is the predominant glucose transporter in hepatocytes of 10-d-old rats. Thus, we hypothesized that TNF-α might increase glucose uptake and glycolysis in those cells, and decrease gluconeogenesis. In the present study, hepatocytes isolated from 10-d-old rats were incubated with TNF-α at the concentrations of 0, 0.98, 9.8, 98, and 980 ng/mL to evaluate TNF-α effects on gluconeogenesis and glucose uptake. TNF-α increased glucose uptake (41.1 ± 8 to 114 ± 21.4 μmol/106 cells at the concentration of 980 ng/mL of TNF-α) in a dose-dependent manner, and decreased gluconeogenesis (98.2 ± 8.2 to 1.1 ± 3.2 μmol/106 cells at the concentration of 980 ng/mL of TNF-α) in a dose-dependent manner. The decrease of glucokinase mRNA and GLUT1 mRNA abundance correlated with glucose uptake (r = 0.988 and 0.997, respectively), and the decrease of phosphoenolpyruvate carboxykinase mRNA abundance correlated with the decrease of gluconeogenesis (r = 0.972). The decrease of gluconeogenesis by TNF-α correlated with the increase of glucose uptake (r = −0.988). We concluded that TNF-α reciprocally suppressed gluconeogenesis in hepatocytes isolated from 10-d-old rats.

Effects of pentoxyfylline on mesenteric lymph node T-cells in a rat model of thermal 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°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.

Burn injury exacerbates hemodynamic and metabolic responses in rats with polymicrobial sepsis.

The most common and life-threatening complication of severe burn injury is infection, which often results in multiple organ failure (MOF). However, the mechanism of development of MOF after burn injury associated with infection is not fully understood. Our previous studies showed that when polymorphonuclear neutrophils (PMNs) are depleted, burn injury-induced increase in microvascular permeability to albumin is markedly attenuated. Thus, we hypothesized that the combination of burn injury and polymicrobial infection exacerbates PMN activation, increases intestinal microvascular permeability to albumin, and alters hemodynamics and metabolism more than burn injury or infection alone. Sprague–Dawley rats (250–275 g) were divided into four groups. In the burn group, rats were subjected to a 30% TBSA burn injury. In the cecal–ligation puncture (CLP) group, CLP was performed using a 22-gauge needle with one puncture. In burn + CLP group, rats were subjected to CLP immediately after burn procedure. In sham group, rats were subjected to sham procedures. Transient polymicrobial bacteremia and persistent polymicrobial bacteremia were induced in the CLP group and burn + CLP group, respectively. Microvascular permeability, myeloperoxidase, and PMN production of elastase and reactive oxygen species increased in the burn group and CLP group and further increased in the burn + CLP group. Hemodunamic and metabolic alterations on day 1 and 3 after injury correlated with those alterations. Although there was only a low mortality in the burn group and CLP group, there was a high mortality in burn + CLP group (79%). The mechanism of MOF that leads to high mortality in burn injury complicated by infection may involve uncontrolled microvascular damage mediated by PMN activation.

Endotoxemia Following Enteral Refeeding in Children

Plasma endotoxin-like activity, tumor necrosis factor α (TNFα) concentrations, core body temperature, and liver functions were measured before and after enteral feeding in children who had been deprived of enteral feeding for 5 days because of their illness. Transient endotoxemia and elevations in plasma TNFα concentrations occurred. Core body temperature, aspartate aminotransferase, alamine aminotransferase, and bilirubin concentrations were normal in patients who had elevated plasma endotoxin-like activity. Transient endotoxemia following enteral feeding may be due to the translocation from the gastrointestinal (GI) tract as a result of increased mesenteric circulation and peristalsis. No clinical consequences were noted despite transient endotoxemia. The transient endotoxemia is not due to the immature GI tract; instead, it results from enteral feeding following the deprivation of enteral feeds.

Tissue Factor Pathway Inhibitor and Thrombin Activatable Fibrinolytic Inhibitor Plasma Levels following Burnand Septic Injuries in Rats

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.

T-Cell Proliferative Responses following Sepsis in Neonatal Rats

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 (103 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.