Limulus Research Articles

Leptospiral lipopolysaccharide-induced expression of inflammatory cytokines and mononuclear-macrophage infiltration in viscera of mice with Leptospira interrogans infection

Objective To understand the role of leptospiral lipopolysaccharide (L-LPS) in inducing human monocytes or mouse mononuclear-macrophages to produce inflammatory cytokines and to analyze the infiltration of peripheral blood-derived mononuclear-macrophages in mice with Leptospira interrogans (L.interrogans) infection. Methods Phenol-water method was used to extract L-LPS from L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai. The endotoxic activity of L-LPS was determined by using limulus test. Cytokine antibody array was used to detect the cytokines produced by human THP-1 monocytes or mouse J774A.1 mononuclear-macrophages after treatment with L-LPS or infection with L.interrogans strain Lai. The infiltration of peripheral blood-derived mononuclear-macrophages in lung, liver and kidney tissues, and the cytokines in serum samples collected from L.interrogans strain Lai-infected C3H/HeJ mice were detected by immunohistochemical examination using HRP-labeled peripheral blood-derived mononuclear-macrophage-specific antibody against CD68 and cytokine antibody array, respectively. Results The activity of L-LPS was about 1/5 of that of Escherichia coli LPS (E-LPS) with respect to solidifying limulus amebocyte lysate. Treating THP-1 and J774A.1 cells with L-LPS significantly increased the production of 29 and 9 cytokines (P<0.05 or P<0.01), respectively, including the major proinflammatory cytokines IL-6 and TNF-α and multiple chemokines of mononuclear-macrophages. The results of cytokine detection in the supernatants of L.interrogans-infected cells and the serum samples collected from L.interrogans-infected mice were similar to those in L-LPS-treated cells. A great number of peripheral blood-derived mononuclear-macrophages were presented in the lung, liver and kidney tissues of the infected mice after infection with the L.interrogans strain Lai. Conclusion L-LPS played a critical role in inducing the expression of multiple inflammatory cytokines by mononuclear-macrophages and in promoting the expression of mononuclear-macrophage chemokines. Mononuclear-macrophages were the major inflammatory cells infiltrating the viscera of L.interrogans-infected mice. Key words: Leptospira interrogans; Lipopolysaccharide; Inflammatory cytokine; Mononuclear-macrophage; Infiltration

Effect of extracellular histones on intestinal mucosa barrier function in mice and role of extracellular histones in development of sepsis

Objective To investigate the effect of extracellular histones (EH) on intestinal mucosal barrier function in mice and the correlation of EH with the pathogenesis of sepsis. Methods Twenty male C57BL/6 mice were assigned into experiment group (n=10) and control group (n=10) according to the random number table. Same dose (50 mg/kg) of EH and saline were administered through the caudal vein of mice in experiment and control groups respectively. Blood and intestinal samples in each group were collected 3 h after the administration. Morphology of intestinal mucosal tissue was detected by light scope and transmission electron microscope. Expressions of tight junction related proteins (ZO-1, Occludin and Claudin-1) were detected by western blot. Plasma levels of diamine oxidase (DAO) and intestinal fatty acid binding protein (I-FABP) were determined by ELISA method. Plasma level of endotoxin (ET) was determined by limulus test. Results Under transmission electron microscope, experiment group showed disorganized microvilli of intestinal epithelial cells with partially twisted, broken and lost, unclear tight junctions, and widened cellular space. Under light scope, experiment group showed substantial inflammatory cell infiltration in the intestinal wall, disorganized intestinal villi, edema and hemorrhage of mucosa and submucosa, and edematous goblet cells. Experimental versus control group showed significant reduction in levels of Claudin-1 (0.587 7±0.060 6 vs. 0.677 2±0.038 3), Occludin (0.127 7±0.085 7 vs. 0.430 6±0.086 9) and ZO-1 (0.393 3±0.080 8 vs. 0.812 8±0.096 3) (P<0.05). Experimental versus control group showed significantly up-regulated plasma levels of DAO [(1.61±0.20)U/ml vs. (0.69±0.15)U/ml], I-FABP [(548.5±36.8)EU/ml vs. (178.8±26.9) EU/ml] and ET [(0.182±0.076) EU/ml vs. (0.091±0.029) EU/ml](P<0.05). Conclusion EH can obviously impair the integrity of intestinal mucosal barrier in mice and hence induce endotoxin translocation. Key words: Histones; Intestines; Sepsis

Clinical study on application of intermittent hemofiltration combined with hemoperfusion in the early stage of severe burn in the prevention and treatment of sepsis

To investigate the effects of application of intermittent hemofiltration combined with hemoperfusion (HP) in the early stage of severe burn in the prevention and treatment of sepsis. Forty severely burned patients, admitted to our burn ward from June 2011 to March 2013, conforming to the study criteria, were divided into conventional treatment group (CT, n=20) and blood purification group (BP, n=20) according to the random number table. Patients in group CT received CT according to the accepted principles of treatment for a severe burn. Patients in group BP received CT and intermittent hemofiltration combined with HP once respectively on post injury day (PID) 3, 5, and 7, spanning 6 to 8 hours for each treatment. On PID 3, 5, 7, 10, and 14, body temperature, heart rate, and respiratory rate were recorded; white blood cell count (WBC), neutrophil granulocytes, blood urea nitrogen (BUN), and creatinine were determined; levels of IL-1, IL-6, TNF-α, and high-mobility group box 1 (HMGB1) in serum were determined by ELISA; level of LPS in serum was determined with the chromogenic substrate limulus amebocyte lysate method; level of procalcitonin (PCT) in serum was determined by double antibody sandwich immune chemiluminescence method. The symptoms and signs of sepsis were observed during the treatment. Data were processed with Fisher's exact test, chi-square test, analysis of variance for repeated measurement, and LSD-t test. (1) Except for that on PID 5, the mean body temperature of patients in group BP was significantly lower than that of group CT at each of the rest time points (with t values from 1.87 to 2.97, P values below 0.05). The heart rate was significantly slower in patients of group BP than in group CT from PID 3 to 14 (with t values from 1.78 to 3.59, P values below 0.05). Except for that on PID 3, the respiratory rate of patients in group BP was significantly slower than that of group CT at each of the rest time points (with t values from 1.93 to 2.85, P values below 0.05). (2) The levels of WBC, neutrophil granulocytes, BUN, and creatinine of patients in group BP were significantly lower than those of group CT (with t values from 1.78 to 4.23, P values below 0.05). (3) Except for that on PID 3, the level of IL-1 of patients in group BP was significantly lower than that of group CT at each of the rest time points (with t values from 1.97 to 4.16, P values below 0.05). Except for that on PID 7, the level of IL-6 of patients in group BP was significantly lower than that of group CT at each of the rest time points (with t values from 2.11 to 6.34, P values below 0.05). The levels of TNF-α and HMGB1 of patients in group BP were significantly lower than those of group CT from PID 3 to 14 (with t values from 1.98 to 5.29, P values below 0.05). (4) On PID 3, 5, 7, 10, and 14, the levels of LPS and PCT of patients in group BP were respectively (0.23 ± 0.07), (0.27 ± 0.09), (0.22 ± 0.06), (0.20 ± 0.08), (0.15 ± 0.07) EU/mL, and (0.44 ± 0.12), (0.67 ± 0.13), (0.74 ± 0.13), (0.64 ± 0.12), (0.71 ± 0.10) ng/mL, and they were lower than those of group CT [(0.37 ± 0.08), (0.45 ± 0.09), (0.56 ± 0.09), (0.48 ± 0.08), (0.40 ± 0.08) EU/mL, and (0.74 ± 0.11), (1.16 ± 0.12), (1.40 ± 0.13), (1.55 ± 0.15), (1.49 ± 0.14) ng/mL, with t values from 1.88 to 3.43, P values below 0.05]. (5) The incidence of sepsis of patients in group BP was obviously lower than that of group CT (χ² = 6.94, P<0.01). Intermittent hemofiltration combined with HP can effectively improve blood biochemical indexes and vital signs and reduce the occurrence of burn sepsis by decreasing the levels of proinflammatory cytokines, LPS, and PCT.

Function of colonic mucosal barrier of patients with colonic slow transit constipation

Objective To explore the function of colonic mucosal barrier of patients with colonic slow transit constipation (STC).Methods From June 2008 to June 2012,a total of 136 patients with STC were enrolled.Among them,course of disease of 55 cases was between one and six years,of 43 cases was between six and 10 years,and of 38 cases was over 10 years.The colonic transit time of 66 cases was between three and five days,of 42 cases was between five and seven days,and of 28 cases was over seven days.Altogether,35 cases received subtotal resection of the colon.At same time,individuals who received partial resection of the colon because of single polypus were set as control group.Fasting blood and urine samples of all subjects were collected.The colonic specimens of STC patients who received surgery and control group were harvested.The urinary lactulose and mannitol ratio (L/M) was detected by high performance liquid chromatography (HPLC).The level of blood Dlactic acid (D-LAC) was tested by enzymatic spectrophotometric.The level of blood diamine oxidase (DAO) was determined by speetrophotometry.The level of endotoxin (ET) was detected by azo chromogenic substrate limulus test.The colonic epithelial cells membrane resistance (TER) and paracellular mannitol permeability (PMP) were measured with Ussing perfusion chamber.t-test was performed for comparison between groups.Results Urinary L/M of STC group and control group was 0.16±0.03 and 0.10±0.02,respectively.The level of blood D-LAC was (1.81±0.19) and (1.04±0.13) mmol/L.The level of blood DAO was (17.07±1.81) and (9.78±1.14) U/L.The level of blood ET was (64.20±6.85) and (51.30±5.90) EU/L.The TER of colonic epithelia cell was (61.23±7.76) and (75.87±9.65) Ω/cm2.The PMP of colonic epithelia cell was (3.17±0.35) ％ and (2.14 ±0.22)％.All the differences were statistically significant (t =3.185,3.378,3.863,3.201,3.125 and 3.543,all P＜0.05).Among patients with disease course between one and six years,six to 10 years and over 10 years,colonic transit time of STC between three and five days,five to seven days and over seven days,urinary L/M,blood D-LAC level and blood DAO level increased along with the disease course and colonic transit time and the differences were statistical significantly compared with control group (urinary L/M:t=1.993,2.311,2.356,2.204,2.347 and 3.673; blood D-LAC level:t=2.023,2.886,4.124,1.999,2.998 and 3.465; blood DAO level:t=1.994,2.995,4.423,2.203,3.673 and 5.211; all P＜0.05).Compared with control group,there were significant differences in blood ET level of course of STC between six and 10 years,over 10 years,colonic transit time of STC between five and seven days and over seven days (t=2.121,4.245,3.241 and 4.657,all P＜0.05).Conclusion The permeability of colonic mucosal barrier increased and which was more significant in longer colonic transit time and long course of disease. Key words: Constipation; Colonic diseases; Intestinal mucosa

The role of CD4+CD25+ regulatory T cells in peripheral blood and intestinal endotoxin in liver injury of rat severe acute pancreatitis

Objective To investigate the role of CD4+ CD25+ regulatory T cells(Treg) in peripheral blood and intestinal endotoxin (ET) in liver injury of rat severe acute pancreatitis (SAP).Methods Sixty male Wistar rats were randomly allocated into sham operation group(SO group) and severe acute pancreatitis group(SAP group).The rats in SAP group recevied the injection of Sodium Taurocholate(NaTc) into their far-end of bile-pancreatic duct and were sacrificed in 3-,6-and 12-hour,serum amylase(AMY) and alanine aminotransferase(ALT) determined by full-automatic biochemistry instrument,limulus reagent method is used to determine ET content in plasma,the proportion of Treg among peripheral lymphocytes was determined by Flow Cytometry(FCM),HE stain is used to observe pathological changes in liver and pancreas,the protein of Foxp3 activity was evaluated by immunohistochemistry staining,and the relationships between these indicators were analyzed using Pearson correlation analysis test.Results Histopathologic examination and the level of ALT revealed the occurrence of pancreatic inflammation and pathological changes of liver in SAP group.The percentage of Treg in SAP groups significantly increased at 3 h(2.26％ ±0.32％),6 h(2.36 ％ ±0.48％)and 12 h(2.80％ ±0.35％) comparing to the SO groups(P＜0.01) ; plasma ET levels compared with the SO group was statistically significant (P＜ 0,01 ),and 12 h (0.85 ± 0.11) compared to,3 h (0.74±0.11) and 6 h (0.78-±-0.07) was no significant difference (P＞0.05).The expression of Foxp3 protein on the livers were upregulated markedly.Pearson correlation analysis teat showed that quantities of Treg were positively proportional to the levels of ET(r=0.89,P＜0.01) after liver injury of SAP.Conclusions SAP may lead to liver injury and the high plasma levels of ET and Treg in peripheral blood may play an important role in liver injury of SAP. Key words: Pancreatitis,acute necrotizing; Hepatitis,toxic; Endotoxins

The serum lipopolysaccharide level in people with different glucose tolerances and its relationship with insulin resistance and β-cell secretory function

Objective To investigate serum lipopolysaccharide (LPS) level in people with different glucose tolerances and to explore the relationship between LPS and insulin resistance/β-cell secretory function.Methods Sixty-seven subjects were recruited,including 23 with newly diagnosed type 2 diabetes ( T2DM),21 impaired glucose tolerance ( IGT),and 23 normal glucose tolerance (NGT).Serum LPS was assayed by limulus amebocyte lysate test ;expression of LPS toll-like receptor 4 (TLR4) on surface of plasma monocytes was measured by flow cytometric assays,and the changes of LPS levels by 0.5 hours and 2 hours after a high-fat diet were detected.Insulin resistance was evaluated by homeostasis model assessment for insulin resistance (HOMA-IR); β-cell secretory function was evaluated by homeostasis model assessment for β3 cell function ( HOMA-β )/HOMA-IR,increment in insulin in the first 30 minutes/increment in glucose in the first 30 minutes ( AIns30/ΔG30)/HOMA-IR,AUCIns120min/HOMA-IR.Results 2 h LPS levels after a high-fat diet were significantly higher than fasting LPS levels [ NGT:0.96(0.33,0.99)vs 0.62 (0.22,0.64),IGT:1.08(0.53,1.22)vs 0.71 (0.39,0.82),T2DM:1.23 (0.62,1.43)vs 0.86( 0.45,0.94 ),EU/ml,all P＜0.01 ].Fasting,0.5 h,and 2 h LPS levels and fasting TLR4 levels of T2 DM group and IGT group were respectively higher than those of NGT group [ fasting LPS:0.86( 0.45,0.94 ),0.71 ( 0.39,0.82 ) vs 0.62(0.22,0.64),EU/ml;0.5 h LPS:1.10(0.55,1.18),0.84(0.50,1.07) vs 0.73(0.31,0.76),EU/ml;2 h LPS:1.23(0.62,1.43),1.08(0.53,1.22)vs 0.96(0.33,0.99),EU/ml; fasting TLR4:36.96( 17.22,55.19),30.34 ( 15.00,45.18 )vs 15.66 (6.09,9.76),MIF/105 cells,all P＜0.01 ].Fasting LPS,AUCLPS 120 min,and fasting TLR4 were positively correlated with insulin resistance index and negatively correlated with β-cell secretory function index ( P＜0.05 ).Multiple linear regression analysis showed that fasting LPS was an independent correlative factor of HOMA-IR and 0.5 h LPS was an independent correlative factor of (AIns30/AG30)/HOMA-IR and AUCIns Ins120min/HOMA-IR.Conclusion People with different glucose tolerances show differed LPS levels and its receptor TLR4 levcls,both of which are correlated with insulin resistance and β-cell secretory function,suggesting that LPS is associated with the pathogenesis of abnormal glucose regulation. Key words: Lipopolysaccharide ; Toll-like receptor 4 ; Insulin resistance ; β-cell secretory function

Effects of lymphatic drainage and omega-3 polyunsaturated fatty acids on intestinal ischemia-reperfusion injury in rats

To investigate the effects of lymphatic drainage and omega-3 polyunsaturated fatty acid (omega-3PUFA) on high mobility group box 1 (HMGB1), inflammatory cytokines and endotoxin in rats with intestinal ischemia-reperfusion (I/R) injury. A total of 72 SD rats were randomly divided into drainage-alone group, I/R group, ischemia-reperfusion plus drainage (I/R + D) group (n = 8 each) and 3 groups with 16 rats undergoing gastrostomy in each group: normal diet (N) group, enteral nutrition (EN) group and enteral nutrition & omega-3PUFA (PUFA) group. And they were further divided into 2 subgroups (n = 8). The rats in I/R and I/R + D groups were subjected to a 60-min ischemia follow by 120-min reperfusion injury of superior mesenteric artery. When the rats suffered I/R injury, intestinal lymph was drained for 180 min in the I/R + D group. The rats in the drainage-alone group received 180-min lymph drainage without I/R injury. After 5 days with different nutrition regimes, the models were established similarly. The rats in the I/R + D sub-groups were treated with intestinal lymph drainage for 180 min. The serum and lymph samples were collected post-operatively. Endotoxin was detected by a Limulus kit. The inflammatory cytokines and high mobility group box 1 (HMGB1) were analyzed by enzyme-linked immunosorbent assay (ELISA). Endotoxin, inflammatory cytokines and lymphatic HMGB1 of lymphatic in the I/R + D group were higher than those in the drainage-alone group [all P < 0.05, IL-6: (30 +/- 8) pg/ml vs (20 +/- 6) pg/ml, endotoxin: (0.029 +/- 0.011) U/ml vs (0.008 +/- 0.005) U/ml]. The serum levels of endotoxin and inflammatory cytokines in the I/R + D group were lower than those in the I/R group (P < 0.05). The lymphatic levels of TNF-alpha (tumor necrosis factor-alpha) and HMGB1 in the N and EN groups were higher than those in the PUFA group[ TNF-alpha: (46 +/- 17) pg/ml, (54 +/- 16) pg/ml vs (28 +/- 9) pg/ml, HMGB1: (4.8 +/- 1.6) ng/ml, (5.3 +/- 1.8) ng/ml, (3.0 +/- 1.0) ng/ml, all P < 0.05)]. The serum levels of endotoxin, inflammatory cytokines and HMGB1 in the PUFA (I/R) group were lower than those in the N (I/R) group (P < 0.05). The levels of TNF-alpha and HMGB1 were lower in the PUFA (I/R + D) group than those in the N (I/R + D) group (both P < 0.05). Lymphatic drainage may reduce the levels of endotoxin, inflammatory cytokines and HMGB1 so as to alleviate the intestinal I/R injury. The intervention of omega-3PUFA has some protective effect through relieving inflammation.

Intravenous glutamine support downmodulats the release of plasma LPS and the activities of PLA2 and neutrophil elastase in taurocholate-induced acute severe pancreatitis in pigs

Objective To observe the effects of intravenous glutamine (Gln) support on plasma lipopolysaccharide (LPS,endotoxin) concentration and acitvities of phospholipase A2(PLA2) and neutrophil elastase (NE) in taurocholate-induced acute severe pancreatitis (ASP) in pigs. Methods Twenty-one pigs weighed 16 kg-22 kg were randomly divided into four groups: including sham-controlled group (sham group, n=5), ASP-controlled group(ASP group, n=5), ASP+glycine controlled group(Gly group, n=5) and ASP+Gln supported group (Gln group, n=6). Anesthesized pigs were subjected to ASP induced by injecting 1 ml/kg of mixed solution of 5% sodium taurocholate and 8 000～10 000 BAEE units trypsin/ml into pancreas via pancreatic duct, which was replaced by 0.9% sodium chloride phosphate buffer solution to be taken as sham group. Blood samples from caval vein was collected for the determinations of Gln, LPS, NE and PLA2. Plasma glutamine were measured by high performance liquid chromatography. Systemic plasma endotoxin levels was quantified by the chromogenic limulus amebocyte lysate (LAL) technique. All pigs were intravenously sacrificed by injecting 20 ml of 10%KCl. Anything involved in the processes of the sampling, preserving, measuring and so on had to be depyrogenated and to be asepsis. Results ASP porcine induced by sodium taurocholate mixed solution was associated with awfully lowed glutamine concentration but higher LPS, PLA2 and neutrophil elastase (NE) activities in plasma. There existed highly significant difference compared with the normal values (all P＜0.01 ). Intravenous glutamine support could effectively prevent the decrease of systemic plasma glutamine level (P＜0.01), and reduced the higher levels of systemic plasma LPS and the activities of plasma NE and PLA2 followed by acute severe pancreatitis induced by the mixed solusion of 5% sodium taurocholate and trypsin in pigs (all P＜0.01), but all were still higher than the normal values except for NE activities at 72 h after the induction of ASP.Conclusion It is suggested that maintaining higher plasma glutamine level could downregulate the level of plasma LPS and the activities of plasma PLA2 and NE, which is beneficial to lessen the cascade inflammatory responses irritated by these proinflammatory mediators and the functional lesions of extro-pancreas organs in pigs with ASP. Key words: Acute pancreatitis; Glutamine; Lipopolysaccharide; Neutrophil elastase; Phospholipase A2

Endotoxin Retention Capabilities of Positively Charged Nylon and Positively Charged Polysulphone Membrane Intravenous Filters

We have studied the endotoxin retention capabilities of two filters claimed to be suitable for 96-h use. The filters were, a positively charged nylon membrane filter (Pall Biomedical Ltd) and a positively charged polysulphone membrane filter (Gelman Sciences Ltd). Simulated patterns of intravenous therapy that would be used over a 96-h period were set up. The administration sets were injected with either a 1-mL bolus of an Escherichia coli (108 cells suspension in 5% glucose or in a parenteral nutrition solution. After 96 h, 2-mL filter effluent samples were tested for endotoxin using the Limulus amoebocyte lysate test (sensitivity: 0.015 endotoxin units (EU) mL−1). In the E. coli in 5% glucose experiments, no endotoxin was found downstream of the positvely charged nylon membrane filters, but large amounts of endotoxin (5 min= > 10.0 EU mL−1, 72 h = 0.153 EU mL−1) were found downstream of the positively charged polysulphone membrane filters. After challenging the filters with endotoxin in parenteral nutrition solution, again no endotoxin was detected downstream with the positively charged nylon membrane filters, but one of the positively charged polysulphone membrane filters allowed endotoxin to pass through after 48 h (4.45 EU mL−1). These results indicate that only the positively charged nylon membrane can be safely used for up to 96 h without detecting the presence of endotoxin downstream to the filter.

The alterations of intestinal mechanical barrier in rat models of non-alcoholic fatty liver disease

Objective To explore the alterations of intestinal mechanical barrier during the nonalcoholic fatty liver disease (NAFLD) progression. Methods To establish NAFLD rats' model,ninety male SD rats were divided into three groups equally: normal-diet group, high-sucrose diet group and high-fat diet group. At the 4th, 8th and 12th week each time point, ten rats were sacrificed in each group. Another twenty SD rats were randomly divided into carbon tetrachloride (CCl4) group and control group. The liver injured model of CCl4 group was induced by CCl4 ; all the rats of these two groups were killed at the 4th week. The degree of liver steatosis was observed through HE staining of liver paraffin sections. The lipopolysaccharide (LPS) level of portal vein blood was measured by limulus test. The occludin expression in intestinal mucosal was detected by immunofluorescent assay,and the fluorescence intensity was scored. The morphology change of intestinal mucosa tight junction was observed through electron microscopy. Results The liver histopathology suggested that NAFLD and liver injured rats' model was successfully established. There was no statistical significance of LPS level between high-sucrose diet group and normal diet group at all the time point (P＞0.05). At the 4th and 12th week, there was no statistical significance of LPS level between high-fat diet group and normal diet group (P＞0.05), while it was significantly higher in high-fat diet group than normal diet group at the 8th week (P＜0. 05). There was no statistical significance of LPS level between CCl4 group and control group (P＞0. 05). At the 8th week, the expression of occludin in normal-diet group, high-sucrose diet group and high-fat diet group was 1.80±0. 42, 1. 50 ± 0. 53 and 1.30±0.67, respectively, the expression was a little bit lower in high-sucrose diet group and high-fat diet group than in normal diet group, the difference was statistical significance (P＜0. 05). At the 12th week, the expression of occludin was significantly lower in high-sucrose diet group and high-fat diet group than normal diet group. The expression of occludin was significantly lower in CCl4 group (0.60±0.16) than in control group (1.80±0. 42) (P＜0. 05). No obvious morphology changes of tight junction was found in high-sucrose diet group, high-fat diet group and CCl4 group at each time point.Conclusion The expression of occludin decreased gradually during the non-alcoholic fatty liver disease progression. So in the NAFLD treatment, besides improving insulin resistance and protecting liver function,the protection of intestinal mucosa barrier as early as possible may slow down the progression of liver injury. Key words: Fatty liver; Intestinal mucosa; Tight junctions; Membrane proteins; Lipopolysacchariides

Dynamic comparison between endotoxemia and transforming growth factors after partial hepatectomy in rats

Objective To compare and evaluate the dynamic changes between endotoxemia and transforming growth factors after partial hepatectomy(PH)in rats.Methods One-hundred and two adult male Wistar rats were randomized into three groups:A normal control (NC)group,a partial hepatectomized(PH)group and a sham-operated(SO)group.To study the dynamic changes of endotoxemia and transforming growth factors,the rats were sacrificed before and at different time points after partial hepatectomy(6,12,24,36,48,72,120 and 168 h).NC group was used as 0 h observation time point,namely 0 h group.For each time point indicated,six rats were used in parallel.Level of endotoxin(ET)was determined in serum by using Limulus Lysate test with chromogenie substrate.Levels of TNF-α and TGF-α were determined in serum by using RIA.Level of TGF-β1 was determined in serum by using ELISA.Results Serum ET levels significantly increased during 6-72 h after PH,there were two peak values at 12,48 h,and compared respectively with the ET levels of NC group (P＜0.05,P＜0.01).Serum TNF-α levels were transiently increased significantly compared with NC (P＜0.05)at 6 h after PH,then decreased rapidly to the lowest value at 72 h(P＜0.01).Serum TGF-α levels significantly increased during 6 h-72 h after PH.There was positive relationship be tween the dynamic changes of TGF-α and ET in serum(r=0.778,P＜0.05).Serum TGF-β1 levels were increased significantly compared with NC(P＜0.01)at 6 h after PH.There was positive rela tionship between the dynamic changes of TGF-β1 and TNF-α in serum(r=0.757,P＜0.01).Conclusion Endotoxemia and inflammatory mediators induced bv PH can regulate liver regeneration by influencing transforming growth factors which are related to hepatocyte proliferation. Key words: Hepatectomy; Endotoxemia; TGF-α; TGF-β1

Measuring of free endotoxin in alum-precipitated vaccine of haemorrhagic septicaemia by limulus amebocyte lysate test

Summary Haemorrhagic septicaemia (HS) vaccine which is prepared in Razi Institute is used in endemic areas of Iran. Aluminum-hydroxide gel was used as adjuvant for preparing this vaccine. Post-vaccinal shock reactions were the main complaint after use of this vaccine. In a previous study, we could improve the vaccine by alum-precipitation Pasteurella multocida cells and removing the liquid phase. In this study, the amount of free endotoxin in aluminum-hydroxide and alum-HS vaccines was determined. It was found that endotoxin level was considerably decreased from 0.22 EU/ml to 0.03 EU/ml after alum-precipitation.

EDUCATION: Limulus in the Limelight

EDUCATION: <i>Limulus</i> in the Limelight

Imprisonment in a death-row cell: the fates of microbes entrapped in the Limulus blood clot.

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Development of the lateral eye of juvenile Limulus.

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Endogenous Endotoxemia in Patients with Liver Cirrhosis

INTRODUCTION Since the limulus test, a method for the evaluation of endotoxin levels using extract from horsehoe crabs (limulus, spp), was developed in the year 1969 by Levin and Bang, there have been reports on endotoxin from portal blood without negative gram bacterial infection, a condition known as endogenous endotoxemia. Up to now, endogenous endotoxemia in patients with liver cirrhosis (LC) is still controversial. This can be seen from the great variation in the prevalence of endotoxemia in peripheral vein in patients with liver cirrhosis, ranging from 0 to 80%. The main cause for this difference is the methodological factor, reflecting a difference in examination technique. Most studies conducted without a specific method demonstrate endotoxemia in patients with liver cirrhosis. While studies using a sensitive method of evaluation for endotoxin found the opposite, detecting no increase in endotoxin in patients with liver cirrhosis.

γ-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

A γ-hydroxyphosphonate P 6 ( O 1 -methyl- O 2 -(1, 2, 2-trimethylpropyl)-2-hydroxy-5- nitrophenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P 6 to the carrier proteins BSA (bovine serum albumin) and LPH ( Limulus polyphenus hemocyanin). Mice were immunized with P 6 -LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P 6 -BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP 6 ) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number k cat = 198 min -1 and the rate enhancement k cat / k uncat = 122 419. When 0.16 mg mL -1 EP 6 was preincubated in vitro with 0.132 mmol L -1 (220 mg kg -1 = 1.1×LD 95 ) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP 6 could prolong the latent time of spasm and death when mice were passively immunized with EP 6 intravenously 15 min before 1×LD 95 of soman challenge. These results demonstrate that EP 6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.

Site selection by migratory shorebirds in Delaware Bay, and its relationship to beach characteristics and abundance of horseshoe crab (Limulus polyphemus) eggs

-The distribution of migratory shorebirds within Delaware Bay, New Jersey, was examined relative to prey abundance and the physical characteristics of the intertidal beaches. Red Knots (Calidris canutus), Sanderlings (C. alba), Ruddy Turnstones (Arenaria interpres), and peeps comprised nearly 100% of the shorebirds on seven study beaches from mid-May to early June in both 1990 and 1991. The most abundant food item on these beaches was horseshoe crab (Limulus polyphemus) eggs, and there were few other available macroinvertebrates. Beaches preferred by shorebirds had higher numbers of crab eggs; the density of eggs in some surface (0-5 cm) sediments exceeded 1 05 / M2. In general, both horseshoe crab eggs and shorebirds increased along the bay shore from Higbee's Beach (near Cape May Point) to Moore's Beach, 32 km up-bay. However, shorebirds were widely distributed within the bay, possibly because eggs were sufficiently abundant on most beaches to support foraging by at least four birds per meter of shoreline. Shorebirds aggregated near shoreline discontinuities, such as salt-marsh creeks and jetties, that acted as concentrating mechanisms for passively drifting eggs. Sediment grain size and heterogeneity were probably not a primary determinant of shorebird distribution within Delaware Bay. Intertidal sand flats were not extensively used by foraging shorebirds, but the potential importance of nearby salt marshes as foraging sites requires further investigation. Received 20 January 1993, accepted 27 March

Endotoxemia in burn patients: levels of circulating endotoxins are related to burn size.

With use of a quantitative limulus assay, the levels of circulating endotoxins were examined in a population of burn patients with injuries covering 1% to 88% of the total body surface area (TBSA). In cases in which the injury was less than 20% TBSA, the increases in endotoxins were only 35% as compared with those of normal controls. As the extent of injury increased, the levels of endotoxins also increased: burns between 21% and 40% TBSA showed average increases of over 350% and burns in excess of 40% showed increases of 500%. The relationship between burn size and total endotoxin burden was significant (p = less than 0.01). Time-course studies indicated that in most cases, peak endotoxin levels occurred 3 to 4 days after injury. The data also showed that there was no relationship between the age of the patient and the extent of the endotoxin increase.

Inhibition of [formula omitted] agglutinins with N-acetyl-neuraminic acid and derivatives

Inhibition of [formula omitted] agglutinins with N-acetyl-neuraminic acid and derivatives