[Clinical study on bacterial translocation in severe multiple trauma patients].

OBJECTIVE: To investigate bacterial translocation in severe multiple trauma patients using polymerase chain reaction (PCR) to detect the presence of bacteria in the blood. METHODS: Sixteen severe multiple trauma patients [injury severity score (ISS)>20] in surgery intensive care unit (SICU) were selected. Blood samples were collected 2, 24 and 48 hours after trauma for bacterial culture and microbial DNA detection. Meanwhile, plasma levels of D-lactate and lipopolysaccharide (LPS) in systemic circulation were determined. PCR was performed after DNA extraction, with target beta-lactosidase gene of E. coli and 16SrRNA gene of most pathogenic bacteria. All patients were observed within 30 days for infectious complications. D-lactate and LPS levels were determined in 63 patients before selective operation. RESULTS: Microbial DNA could be detected in blood as early as 2 hours following severe trauma, and altogether positive results were found in 10 patients (62.50%). All PCR-positive patients manifested sepsis, but none of the PCR-negative patients did (P<0.01). Bacterial DNA was discovered in 100.00% of sepsis patients and none in non-sepsis patients (P<0.01). Seventy percent of PCR-positive patients developed infectious complications, while none of PCR-negative patients did (P<0.01). The blood culture was positive only in 3 patients (18.75%), all of them were PCR-positive. E.coli DNA was found in 70.00% of all the PCR positive blood specimens. Systemic plasma concentration of D-lactate and LPS of all patients was significantly higher than that in control group, which consisted of 63 inpatients waiting for elective operations. Systemic plasma level of D-lactate showed a positive correlation with that of LPS (r=0.94, P<0.01). CONCLUSION: Intestinal bacterial translocation (most commonly E. coli) might occur early (2 hours) after severe trauma. Infection and sepsis have a close relationship with bacterial translocation. Detection of blood microbial DNA using PCR could reflect bacteria translocation and forecast imminent infection and sepsis.

Tissue lipopolysaccharide-binding protein expression in rats after thermal injury: potential role of TNF-alpha.

Recent studies have suggested that levels of lipopolysaccharide-binding protein (LBP) might play a harmful role by up-regulating the host's sensitivity to endotoxin. Our previous studies demonstrated that local endotoxin could up-regulate LBP expression after acute insults, however, the definite molecular mechanisms downstream of endotoxin action remain unclear. This study investigates whether tumor necrosis factor (TNF-alpha) might be responsible for the LBP formation during endogenous endotoxemia postburn. Wistar rats were anesthetized, and a 35% TBSA full-thickness burn was created. Animals were randomly divided into normal control, thermal injury and anti-TNF-alpha mAb treatment group. A significant elevation of plasma endotoxin concentration was observed after acute insults. TNF-alpha levels in plasma also rapidly increased after thermal injury. Meanwhile, LBP mRNA expression markedly increased in liver, lungs, kidneys and intestine postburn. There was no detectable TNF-alpha in the plasma of anti-TNF-alpha mAb treated animals. Treatment with anti-TNF-alpha mAb also resulted in significantly lower concentrations of LBP mRNA in local tissues. Additionally, several organ function parameter levels in plasma significantly decreased in treatment group. These results demonstrated that an increase of plasma TNF-alpha levels caused by burns might be associated with a marked elevation of tissue LBP mRNA expression, which could contribute to the development of multiple organ damage.

[Early fracture fixation alleviates gut barrier function damage after multiple firearm injuries in pigs].

OBJECTIVE: To explore if early fracture fixation can alleviate gut barrier function damage caused by multiple firearm injuries in pigs. METHODS: Twelve healthy pigs were subjected to tangential fracture of parietal bone and comminuted fractures of bilateral femora (ISS >or= 16) due to 5.8 mm bullets shooting and these pigs were divided randomly into 2 groups. Control group (n = 6) were not treated at all. Fracture fixation Group (n = 6) were managed by immediate fracture fixation of bilateral femora with intramedullary nails. Plasma concentration of D-lactate, DAO and endotoxin (in portal vein) were detected at different intervals before and after trauma. The portal vein blood was cultured and the percentage of positive isolation was calculated. The concentration of DAO in small bowel was also detected 72 hours later after trauma. RESULTS: In control group, the plasma concentrations of D-lactate, DAO and endotoxin increased at early stage and kept high till 72 hours after trauma; the percentage of positive blood culture was 63.3%. In Group F, the levels of plasma D-lactate, DAO and endotoxin were also elevated at early stage (6 - 12 h), but declined significantly from 24 h or 48 h after trauma compared with control group (P < 0.05), and the percentage of positive blood culture was lower (30.0%, P < 0.05). The concentrations of DAO in small bowel decreased in both groups, but to a less extent in Group F. CONCLUSION: Bacterial and endotoxin translocation emerged with increasing gut permeability after multiple firearm injuries. The damage of gut barrier function could be alleviated and the chance of enterogenous infection could be by early fracture fixation after trauma.

The potential role of Staphylococcal enterotoxin B in rats with postburn Staphylococcus aureus sepsis.

Staphylococcal enterotoxin B (SEB) is an important member of the superantigen family, which exerts a number of pathological effects in the human, as well as susceptible animals. The present study was conducted to observe the time course and tissue distribution of SEB in postburn Staphylococcus aureus infection; meanwhile, the relationship between SEB and multiple organ dysfunction was also studied. Eighty-six male Wistar rats were randomly divided into four groups as follows: normal control group (n = 10); scald control group (n = 10); postburn sepsis group (n = 50) in which rats inflicted with 20% total body surface area (TBSA) III degrees scald followed by SEB-producing S. aureus challenge were further divided into 0.5-, 2-, 6-, 12-, and 24-h subgroups, with 10 rats in each subgroup; and SEB monoclonal antibody (MAb) treatment group (n = 16) in which a dose of 4 mg/kg SEB MAb was given intravenously just before S. aureus challenge, and the rats were further divided into 2- and 6-h subgroups. It was found that after thermal injury combined with S. aureus infection, SEB was widely distributed to the liver, kidneys, lungs, and heart, exacerbating the pathophysiology of multiple organ dysfunction induced by postburn sepsis. At the same time, the gene and protein expressions of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were also markedly upregulated in various tissues. Early treatment with SEB-specific MAb-MAb2D(1)-could markedly decrease SEB levels in plasma as well as in various tissues, and could significantly reduce the 6-h mortality rate (17.64% [3/17] vs. 55.6% [20/36], P = 0.02). These data suggested that neutralization of SEB is effective in ameliorating S. aureus sepsis and subsequent multiple organ damage, which might be attributed to its inhibitory effect on inflammatory mediator formation.

Significance of biopterin induction in rats with postburn Staphylococcus aureus sepsis.

It has been demonstrated that biopterin, an essential cofactor of nitric oxide synthase (NOS), plays an important role in the pathogenesis of endotoxin-induced shock, yet its biological significance in gram-positive sepsis remains unclear. In this study, we adopted a rat model of postburn Staphylococcus aureus sepsis to investigate the potential role of biopterin in the pathogenesis of gram-positive sepsis. Wistar rats were inflicted with a 20% total body surface area (TBSA) full-thickness scald injury followed by S. aureus challenge, and then guanosine triphosphate-cyclohydrolase I (GTP-CHI) mRNA expression and biopterin levels in liver, kidneys, lungs, and heart were determined. We found that after S. aureus challenge, GTP-CHI gene expressions and biopterin levels were markedly upregulated in various tissues. Meanwhile, multiple organ dysfunction was induced by S. aureus challenge. It was shown that cardiac GTP-CHI mRNA expression and renal BH(4) levels were positively correlated with MB isoenzyme of creatine kinase (CK-MB) and creatinine (r = 0.892, P = 0.0012 and r = 0.9423, P = 0.0015, respectively). These results suggested that thermal injury combined with S. aureus challenge could induce de novo biosynthesis of biopterin, which might play a role in the development of multiple organ dysfunction syndrome secondary to postburn sepsis.

The mRNA expression patterns of tumor necrosis factor-alpha and TNFR-I in some vital organs after thermal injury.

AIM: To investigate changes of tumor necrosis factor-alpha (TNF-alpha) and TNFR-I expression in vital organs and their significance in the pathogenesis of multiple organ damage associated with endogenous endotoxin following major burns. METHODS: Wistar rats subjected to a 35 % full-thickness scald injury were sacrificed at 12 h, 24 h, 48 h, and 72 h postburn, respectively. Meanwhile, eight rats were taken as normal controls. Tissue samples from liver, spleen, kidney, lung and intestine were collected to assay tissue endotoxin levels and measure TNF-alpha and TNFR-I expression. In addition, blood samples were obtained for the determination of organ function parameters. RESULTS: Endotoxin levels in liver, spleen and lung increased markedly after thermal injury, with the highest level in liver. The gene expression of TNF-alpha in liver, lung and kidney was up-regulated after thermal injury, while the TNFR-I mRNA expression in liver, lung, kidney and intestine was shown decreased throughout the observation period. Thus, the mRNA expression ratio of TNF-alpha to TNFR-I was significantly increased postburn, particularly in pulmonary tissue (67-fold). In addition, the significant correlations between the expression of TNFR-I or the expression ratio of TNF-alpha/TNFR mRNA in liver tissue and serum aspartate aminotransferase levels were noted (P<0.05-0.01). Similar results were also obtained between pulmonary TNF-alpha mRNA expression and myeloperoxidase activities (P<0.01), whereas there was a highly negative correlation between levels of renal TNFR-I mRNA expression and serum creatinine. CONCLUSION: Burn injury could result in the translocation of gut-derived endotoxin that was mainly distributed in the liver, spleen and lung. The translocated endotoxin then made the expression of TNF-alpha and TNFR-I mRNA up-regulated and down-regulated respectively in various organs, which might be involved in the pathogenesis of multiple organ damage following burns.

Lipopolysaccharide-binding protein and lipopolysaccharide receptor CD14 gene expression after thermal injury and its potential mechanism(s).

BACKGROUND: We hypothesized that lipopolysaccharide-binding protein (LBP) and lipopolysaccharide receptor CD14 would present a pair of key molecules in pathophysiologic alterations induced by low concentrations of endotoxin after trauma. The aim of this study was to investigate the relationship between endotoxin translocation and tissue LBP/CD14 messenger ribonucleic acid (mRNA) expression after burn injury, and to define the potential role of LBP/CD14 in mediating inflammatory mediator induction, as well as the pathogenesis of organ damage. METHODS: Wistar rats were subjected to a 35% full-thickness scald injury, and tissue samples from liver, kidneys, lungs, and intestine were collected to measure LBP/CD14 and tumor necrosis factor-alpha (TNF-alpha) mRNA expression. Peritoneal macrophages were harvested by peritoneal lavage to determine CD14 mRNA expression. RESULTS: It was found that endotoxin levels in liver, spleen, and lung increased markedly after thermal injury, with the highest level in liver. Both tissue LBP and CD14 mRNA expression increased markedly after burns, peaking at 12 hours, and then decreasing gradually. At 48 hours, LBP gene expression had a tendency to the baseline level, whereas CD14 mRNA expression increased again. Likewise, CD14 mRNA levels were up-regulated markedly in peritoneal macrophages. Conversely, gene expression of TNF-alpha in tissues elevated markedly after acute insults. There were positive correlations between lipopolysaccharide levels and LBP/CD14 mRNA as well as TNF-alpha mRNA expression in tissues. Similar results were also obtained between CD14, TNF-alpha mRNA expression in liver tissue and liver function parameters, and between pulmonary TNF-alpha mRNA and myeloperoxidase activities (p < 0.01). CONCLUSION: Thermal injury per se can markedly up-regulate both LBP and CD14 gene expression in various organs. Excessive LBP and CD14 mRNA expression might be associated with enhanced synthesis and release of TNF-alpha stimulated by endotoxin translocation after major burns.

Effect of 2,4-diamino-6-hydroxy-pyrimidine on postburn Staphylococcus aureus sepsis in rats.

OBJECTIVE: Guanosine triphosphate-cyclohydrolase I (GTP-CHI) is the first and rate-limiting enzyme for the de novo biosynthesis of biopterin. The objective of present study was to observe the effect of 2,4-diamino-6-hydroxy-pyrimidine (DAHP), an inhibitor of GTP-CHI, on the development of postburn Staphylococcus aureus sepsis. DESIGN: A prospective, controlled animal study. SETTING: A research laboratory in a hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: Fifty-six male Wistar rats were randomly divided into four groups as follows: normal control group (n = 10), scald control group (n = 10), postburn sepsis group (n = 20), and DAHP treatment group (n = 16). In the scald control group, rats were subjected to a 20% total body surface area third-degree scald injury and then were killed at 24 hrs. In the postburn sepsis group (n = 20), rats were inflicted with 20% total body surface area third-degree scald followed by Staphylococcus aureus challenge, and they were further divided into 2- and 6-hr groups. In the DAHP treatment group (n = 16), animals were intraperitoneally injected with a dose of 1 g/kg DAHP before Staphylococcus aureus challenge and then were further divided into 2- and 6-hr groups. Tissue samples from liver, kidneys, lungs, and heart were collected to determine GTP-CHI, inducible nitric oxide synthase, and tumor necrosis factor-alpha messenger RNA expression. Meanwhile, biopterin and nitric oxide concentrations in these tissues were also measured. MEASUREMENTS AND MAIN RESULTS: After the scald injury followed by Staphylococcus aureus challenge, GTP-CHI messenger RNA expression and biopterin concentrations were significantly elevated in various tissues such as liver, heart, kidneys, and lungs, as were the values of inducible nitric oxide synthase messenger RNA expression and nitric oxide formation (p <.01). Pretreatment with DAHP significantly reduced GTP-CHI/biopterin induction (p <.05-.01), and the up-regulation of inducible nitric oxide synthase/nitric oxide was also suppressed. Furthermore, DAHP administration inhibited the gene expression of tumor necrosis factor-alpha. Two hours after septic challenge, tumor necrosis factor-alpha messenger RNA expression in liver, kidneys, and lungs in the DAHP-treated group was 35.7%, 37.3%, and 33.0% of that in the postburn septic group, respectively. Additionally, in animals without DAHP treatment, the 6-hr mortality rate was 55.6% (20 of 36), whereas it was only 25.0% in DAHP-treated animals (4 of 16, p =.08). CONCLUSIONS: Early treatment with DAHP might be a potential strategy to prevent the development of postburn Staphylococcal sepsis, which appears to be associated with down-regulation of biopterin and nitric oxide formation by DAHP.

The significance of changes in high mobility group-1 protein mRNA expression in rats after thermal injury.

There has been a widespread impression that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mediate the toxicity of high doses of lipopolysaccharide (LPS, endotoxin) and are key factors in septic shock. However, the clinical efficacy of treatment with antagonists of TNF-alpha and IL-1beta is still controversial, suggesting that mediators other than TNF-alpha and IL-1beta might contribute causally to endotoxin-induced death. Recent studies implicated high mobility group-1 (HMG-1) protein as a late mediator of endotoxin lethality in mice. However, the role of HMG-1 in mediating multiple organ damage-associating trauma has not been studied. This study was designed to investigate changes in HMG-1 gene expression in vital organs, and its potential role in mediating multiple organ damage following major burns. Wistar rats were subjected to a 35 percent full-thickness thermal injury, and randomly divided into three groups as follows: normal controls (n = 7), thermal injury (n = 24), and recombinant bactericidal/permeability-increasing protein (rBPI21) treatment (n = 12). Tissue samples from liver and lungs were collected to measure tissue endotoxin levels and HMG-1 mRNA expression. In addition, blood samples were obtained for measurement of organ function parameters. Our data demonstrated a significant increase in HMG-1 gene expression in tissues at 24 h postburn, which remained markedly elevated up to 72 h after thermal injury (P< 0.05-0.01). Treatment with rBPI21 could significantly decrease tissue HMG-1 mRNA expression in the liver and lung (P < 0.01). In addition, there were high positive correlations between hepatic HMG-1 mRNA and serum aminoleucine transferase (ALT) and aspartate aminotransferase (AST) levels, and also between pulmonary HMG-1 mRNA and myeloperoxidase activities (P < 0.05-0.01). Taken together, these findings indicate that thermal injury per se can markedly enhance HMG-1 gene expression in various organs. Up-regulation of HMG-1 expression may be involved in the pathogenesis of endogenous endotoxin-mediated multiple organ damage secondary to major burns.

Traditional Chinese medicine Qing Yi Tang alleviates oxygen free radical injury in acute necrotizing pancreatits.

AIM:To observe the changes in oxygen free radical (OFR) and the curative effect of traditional Chinese medicine Qing Yi Tang in acute necrotizing pancreatitis (ANP).METHODS:After induction of ANP by injection of sodium taurocholate into pancreatic duct, 16 dogs were randomly divided into control group and Chinese medicine group.Serum amylase, SOD and MDA were determined on postoperative day 1, 2, 4 and 7. The animals were sacrificed on day 7. SOD and MDA in organs were determined, and pathological changes in pancreas were observed.RESULTS: As compared with control group, the serum level of amylase (734U/L vs 2783U/L) and MDA (7.8nmol/ml vs 14.8nmol/ml) in Chinese medicine group were decreased on day 7 (P < 0.05), while SOD increased significantly (281nU/ml vs 55nU/ml, P < 0.01), and similar changes occurred in MDA and SOD in organs, especially in the pancreas; the pathological changes in the pancreas were alleviated as well.CONCLUSION: Qing Yi Tang is effective in clearing OFRs and alleviating pathological changes in ANP.