Development and characterisation of anti-peptide antibodies to rat lipopolysaccharide binding protein.
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Acute-Phase Proteins↗
Biomedical subjects
M I Halliday
Publications and source records attributed to M I Halliday.
Lower limb ischaemia-reperfusion injury causes endotoxaemia and endogenous antiendotoxin antibody consumption but not bacterial translocation.
BACKGROUND: It has been suggested that reperfusion of the acutely ischaemic lower limb alters gut permeability. The effect of lower limb ischaemia-reperfusion on systemic endotoxin and antiendotoxin antibody concentrations and the incidence of bacterial translocation was investigated. METHODS: Systemic endotoxin and antiendotoxin antibody concentrations were measured in five groups of male Wistar rats: control, after 3 h of bilateral hind limb ischaemia alone, and after 3 h of bilateral hind limb ischaemia followed by 1, 2 or 3 h of reperfusion. A second experiment examined translocation of indigenous bacteria following 2 h of reperfusion in a similar model. RESULTS: Ischaemia followed by reperfusion for 1, 2 or 3 h caused a significant increase in plasma endotoxin concentration to mean(s.e.m.) 10.0(3.0), 44.8(19.2) and 20.2(6.2) pg/ml compared with that in control animals (2.58(0.91) pg/ml) or animals in the ischaemia alone group (1.2(0.9) pg/ml) (P < 0.05). This was associated with a significant reduction in endogenous antiendotoxin antibody (immunoglobulin (Ig) G and IgM) concentration. No significant bacterial translocation was detected in any of the groups studied. CONCLUSION: These results demonstrate that a remote and isolated ischaemia-reperfusion injury to the lower limb, in the absence of infection or bacterial translocation, causes endotoxaemia. Further studies are needed to evaluate the role of endogenous antiendotoxin antibodies in this situation.
Animals↗
Intramucosal acidosis and systemic host responses in abdominal aortic aneurysm surgery.
OBJECTIVES: To assess the specific host responses to systemic endotoxemia and tumor necrosis factor (TNF) activation after abdominal aortic aneurysm surgery by measuring antiendotoxin core antibodies (EndoCab) immunoglobulin (Ig)G and IgM, and soluble p55TNF receptor concentrations. The role of the gut in initiating these immune responses was evaluated by correlating the systemic markers to changes in the intramucosal pH of the sigmoid colon. DESIGN: Retrospective, reevaluation study. SETTING: Vascular unit of a university hospital. PATIENTS: Eleven patients who underwent abdominal aortic aneurysm repair surgery were selected from a larger patient cohort (n = 42) on the basis of their clinical outcome (four patients had fatal complications and seven patients had an uneventful recovery). INTERVENTIONS: After induction of anesthesia, intramucosal pH of the sigmoid colon was measured using tonometry. Blood samples were obtained from indwelling catheters or direct venipuncture preoperatively, during surgery, and daily until postoperative day 5. MEASUREMENTS AND MAIN RESULTS: Those patients who died developed intramucosal acidosis of the sigmoid colon intraoperatively. Significant consumption of both IgG and IgM EndoCab antibodies was found in all patients. By measuring the concentration of antibodies to a neutral antigen, i.e., tetanus toxoid, the consumption of IgG EndoCab antibody concentrations was shown to be a specific host response. In all patients, reciprocal changes in the serum concentrations of p55TNF receptor and interleukin (IL)-6 were observed. The percentage increase in p55TNF receptor and the concentration of IL-6 were significantly higher in the nonsurvivor group by 48 hrs. There were significant correlations between, intramucosal pH and EndoCab antibodies, intramucosal pH and p55 TNF receptor, and p55 TNF receptor and IL-6. CONCLUSIONS: The development of colonic ischemia is associated with a significant consumption of IgG EndoCab antibodies and a simultaneous increase in soluble p55TNF receptor. This study provides further support for the concept that gut-derived endotoxin and the generation of TNF may play a role in the pathogenesis of complications after abdominal aortic aneurysm surgery.
Acidosis↗
Soluble TNF receptors as prognostic factors for mortality.
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Biomarkers↗
An ELISA for measuring tumour necrosis factor alpha in rat plasma.
Tumour necrosis factor (TNF) is a cytokine with multiple biological activities which plays a pivotal role in the response of the body to infection. TNF is secreted in the the monomeric form and associates to yield a biologically active oligomeric molecule. Bioactive TNF can be measured in plasma using cytotoxic assays which employ the murine cell-lines L929 or WEHI 164 clone 13. However, it has become clear that inactive TNF also circulates in vivo, which is not detected by bioassay. These inactive forms may play an important role in the regulation of TNF activity. The rat is often used as a model for the study of acute infection and its systemic effects. Our aim was to develop an ELISA for rat TNF which would provide a convenient and cost effective method of assay. The assay employs two commercially available antibodies raised against recombinant murine TNF (rMuTNF) which exhibit cross-reactivity with rat TNF. The lower limit of detection for this assay was determined to be 39.0 pg/ml rMuTNF. The inter and intra-assay coefficients of variation were < 12.0%. Specificity of the assay was shown by the high degree of parallelism obtained between rMuTNF and commercially available rRatTNF. The assay described measures rat TNF in both plasma and tissue culture supernatant. The measurement of plasma concentrations of TNF using both the ELISA and bioassay may help elucidate more fully the biological importance of TNF.
Animals↗
Tumour necrosis factor and inflammatory bowel disease.
BACKGROUND: Tumour necrosis factor (TNF) is a pleiotropic cytokine produced largely by macrophages and T lymphocytes. It has been implicated in the pathogenesis of numerous immunoinflammatory processes. Recently, a number of studies have indicated that anti-TNF antibodies may be of value in the treatment of inflammatory bowel disease. METHODS: The literature is reviewed regarding the role of TNF in the pathogenesis of inflammatory bowel disease and the results of administering TNF inhibitors. RESULTS AND CONCLUSIONS: TNF may have a role in the pathogenesis of inflammatory bowel disease. The effects of TNF inhibitors are complex and incompletely understood. Anti-TNF antibody strategies may have a role in the treatment of acute exacerbations of the disease but are unlikely to be appropriate therapies for long-term management.
Antibodies↗
Lower limb ischaemia-reperfusion injury alters gastrointestinal structure and function.
BACKGROUND: It has been suggested that bowel permeability is altered following abdominal aortic aneurysm surgery. The effect of ischaemia-reperfusion injury to the lower limb on the morphological structure, neutrophil infiltration and permeability of the bowel was investigated. METHODS: Histological assessment of the bowel was undertaken in five groups of Wistar rats: control, 3 h of bilateral hind limb ischaemia and 3 h of bilateral hind limb ischaemia followed by 1, 2 or 3 h of reperfusion. Using an everted gut sac model and 14C-labelled polyethylene glycol, the effect of ischaemia-reperfusion on small bowel permeability was studied. RESULTS: The small bowel showed a significant decrease in mucosal thickness, villus height and crypt depth in animals subjected to ischaemia followed by 2-hr reperfusion (mean(s.e.m.) 420(15), 217(9) and 163(6) microns respectively) compared with controls (481(11), 245(6) and 195(6) microns) (P < 0.05). Neutrophil count within the lamina propria was similar in the different groups. A significant increase in mean(s.e.m.) 14C-labelled polyethylene glycol translocation was detected in animals subjected to ischaemia-reperfusion compared with controls (760(40) versus 560(27) c.p.m. per ml per h) (P < 0.05). CONCLUSION: These data suggest that reperfusion of acutely ischaemic extremities produces structural and functional changes in the small intestine, although these changes are not associated with increased neutrophil infiltration within the bowel wall.
Animals↗
Modulation of TNF alpha and IL-6 in a peritonitis model using pentoxifylline.
Sepsis leads to release of reactants that play an important role in the development of multiple organ failure. The kinetics of two early mediators of the response to sepsis, tumour necrosis factor (TNF alpha) and interleukin 6 (IL-6), and their modulation with pentoxifylline (PTF), were investigated. An established and clinically relevant animal model was employed, and sepsis was induced by cecal ligation and puncture (CLP) in Wistar rats. Six hours after the operation, there was an increase in IL-6 in all animals, which declined toward normal by 18 hours. This early phase of IL-6 production was not influenced by PTF. TNF alpha and IL-6 were significantly higher in the CLP group than in the animals treated with PTF at 24 hours. The blood pressure of the CLP group at 24 hours was significantly lower than that of the shams, and this decrease was not influenced by PTF. This decline in blood pressure may have been the stimulus to TNF production and the second phase of IL-6 production, which appeared to be inhibited by PTF. Pentoxifylline appears to attenuate systemic cytokine production in this model and may have a role in the management of clinical sepsis.
Animals↗
Cytokine profile in chronic cardiac failure.
Elevated tumour necrosis factor alpha (TNF-alpha) has been demonstrated in chronic cardiac failure (CCF) and may relate to severity of CCF and development of cachexia. We measured TNF receptor p55 in addition to TNF-alpha in an attempt to improve the detection rate of TNF-alpha activation, and simultaneously measured interleukin 6 (IL-6), interleukin 8 (IL-8) and C-reactive protein. Thirty-four patients with CCF and 24 control subjects were studied. Only TNF receptor p55 [6.95 (0.77-42.3) vs. 5.52 (1.50-13.36) ng mL-1 (median (range)] and IL-6 [0.335 (0-9.79) vs. 0(0-14.71) pg mL-1) were significantly elevated in patients compared with control subjects (both P < 0.05). All inflammatory markers were more frequently elevated in patients, but none correlated with any of the clinical parameters studied. Reasons for inflammatory marker elevation in CCF are uncertain, but future studies should measure the p55 TNF receptor and IL-6 in addition to TNF-alpha, to improve detection of cytokine activity.
Aged↗
Urinary excretion of TNF receptors.
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Humans↗
Role of the gut in the pathophysiology of extrahepatic biliary obstruction.
BACKGROUND: Gram negative septic events are the commonest source of morbidity and mortality as a result of surgery in jaundiced patients. The large intestine provides the major source of Gram negative bacteria in mammals and is implicated in the pathogenesis of systemic endotoxaemia in obstructive jaundice. Bile salts have an important part in maintaining indigenous microecological homeostasis through their emulsifying properties. AIMS: The aim was to investigate the effects of biliary obstruction and isolated external biliary diversion on gastro-intestinal structure and caecal bacterial flora in relation to bacterial translocation. METHOD: Six groups of adult male Wistar rats were studied (no operation, sham operated, and bile duct ligated (BDL) for one and three weeks and a choledocho-vesical fistula (CDVF) for one week). At the end of the study period plasma was assayed for evidence of endotoxaemia and the animals were tested for bacterial translocation to the mesenteric lymph node complex (MLNC), liver, lungs, and spleen. Quantitative and qualitative bacteriological studies were performed on the caecal contents and segments of colon and terminal ileum were washed and prepared for histological assessment. RESULTS: Bacterial translocation was significantly increased in the BDL1 (68.8%) and BDL3 (60%) groups compared with the sham1 (6.3%), sham3 (9.1%), No operation (0%), and CDVF1 (16.7%) groups. Although translocation was more pronounced in the BDL1 group, this was almost exclusively to the MLNC compared with the more widespread translocation to other organs in the BDL3 group. The BDL3 group was the only group with significantly raised concentrations of endotoxin and anticore glycolipid. The caecal Gram negative aerobic counts were significantly increased in the BDL1 and CDVF1 groups compared with all other groups. There was evidence of structural abnormalities in the terminal ileum of rats jaundiced for three weeks, but not in the other groups. CONCLUSIONS: Biliary obstruction for one and three weeks promotes bacterial translocation although the mechanisms may be different. Absence of intralumenal bile results in a significant but self limiting increase in the Gram negative aerobic population, which may account for translocation in the early stages of biliary obstruction. As the duration of biliary obstruction increases systemic endotoxaemia is a consistent feature which, combined with factors such as immunological depression and physical disruption of gut barrier function, may promote bacterial translocation perpetuating systemic sepsis.
Animals↗
Bacterial translocation and gut microflora in obstructive jaundice.
Bacterial translocation from the gut is implicated in the pathophysiology of complications associated with obstructive jaundice. Absence of intraluminal bile salts and their antiendotoxic effects may result in overgrowth of bacteria, promoting bacterial translocation. The large bowel is the largest source of gram negative bacteria but the small bowel is more permeable. This study investigated the effect of obstructive jaundice on bacterial translocation and on the indigenous luminal microflora at 3 sites in the gastrointestinal tract. Significant bacterial translocation was demonstrated following 7 d of bile duct ligation compared with control or sham operated groups. A qualitative disturbance of the caecal indigenous microflora was demonstrated in jaundiced rats. There was no 'site-to-site' variation in the indigenous microflora at the 3 intestinal sites studied. We conclude that experimental obstructive jaundice for 1 wk promotes bacterial translocation without significant quantitative disturbance of the intestinal microflora in the small intestinal or caecum.
Animals↗
Lactulose as an antiendotoxin in experimental colitis.
The efficacy of lactulose as an antiendotoxin was studied and the effect of lactulose or colistin on faecal flora was investigated in a hapten-induced rat model of colitis. Enteral administration of lactulose to rats with colitis was associated with a significant reduction in the systemic concentration of endotoxin (median (range) 5.4 (0-19.9) versus 23.7 (0-145.0) pg/ml in colitic rats treated with water; 4.6 (0-10.8) pg/ml in healthy animals). Enteral administration of colistin significantly reduced the faecal count of aerobic Gram-negative bacilli (median (range) 2.84 (1.40-8.43) versus 8.26 (4.50-10.40) log10 colony-forming units per g faeces after treatment with water) but not the faecal load of endotoxin. Patients with inflammatory bowel disease may benefit from enteral treatment with lactulose to prevent systemic endotoxaemia and/or with colistin to modify enteric bacteria.
Animals↗
Effect of low-dose dopamine on sigmoid colonic intramucosal pH in patients undergoing elective abdominal aortic aneurysm repair.
The effect of low-dose dopamine administration on intramucosal pH (pHi) of the sigmoid colon and on postoperative function of various organs in patients undergoing elective abdominal aortic aneurysm repair was examined. Nineteen patients were randomized to two groups; nine received dopamine at a rate of 3 micrograms per kg per min for 24 h from induction of anaesthesia and ten control patients received fluids without dopamine. pHi was measured with a silicone tonometer and daily samples of blood were taken for measurement of liver transaminase activity, arterial oxygen saturation and creatinine concentration. Mean(s.e.m.) pHi fell to a significantly lower minimum value in those receiving dopamine compared with control patients (6.86(0.10) versus 7.11(0.08), P < 0.05). Five of the nine patients given dopamine developed intramucosal acidosis compared with only one of the ten control patients (P = 0.06). After operation the mean(s.e.m.) aspartate transaminase concentration in patients given dopamine rose from 33(2) to 80(17) units/l (P < 0.01); in control patients it rose from 32(3) to 59(16) units/l (P = 0.054). No differences between the groups was observed in the postoperative ratio of arterial oxygen saturation to inspired oxygen fraction or creatinine concentrations. These results indicate that dopamine has no beneficial effect on bowel mucosal oxygenation and function of the various organs in patients undergoing aortic aneurysm repair.
Aged↗
Significance of systemic endotoxaemia in inflammatory bowel disease.
Quantitative and qualitative disturbances in faecal flora suggest a role for enteric bacteria and their products in the pathogenesis of inflammatory bowel disease (IBD). This study investigated the hypothesis that systemically circulating endotoxins are of pathogenic significance in IBD by measuring antibody, cytokine, and acute phase protein responses. Systemic endotoxaemia was found in 88% patients with ulcerative colitis (n = 25) and 94% with Crohn's disease (n = 31) during clinical relapse. Systemic endotoxaemia correlated positively with anatomic extent and clinical activity of ulcerative colitis. Circulating tumour necrosis factor (TNF) was detected in 40% of patients with ulcerative colitis and 45% with Crohn's disease. Plasma TNF concentrations correlated with clinical and laboratory measures of disease activity and were associated with a surgical outcome to the disease episode. Plasma soluble TNF receptor p55 concentration correlated positively with disease activity and endotoxin core antibody concentrations. Plasma IgG endotoxin core antibody concentrations were significantly increased in patients with Crohn's disease and correlated with systemic endotoxaemia. The presence of systemic endotoxaemia, its correlation with disease activity, disease extent, and endotoxin core antibody concentration and the detection of circulating TNF and soluble TNF receptors all support a pathogenic role for endotoxins in IBD.
Adolescent↗
Bowel ischaemia and organ impairment in elective abdominal aortic aneurysm repair.
In 30 patients undergoing elective repair of abdominal aortic aneurysm the intramucosal pH (pHi) of the sigmoid colon was measured. Blood for endotoxin assay was taken at intervals before, during and after surgery. Daily measurements were made of liver transaminase activity and of arterial partial pressure of oxygen (PaO2). The mean (s.e.m.) peak systemic endotoxin concentration in those who developed intramucosal acidosis (pHi below 7.00) was 90(14) pg/ml, compared with 42(5) pg/ml in those who did not (P < 0.01). In the 14 patients whose pHi fell below 7.00, the mean (s.e.m.) postoperative rise in aspartate transaminase activity was 346(74) per cent, compared with 181(20) per cent in those whose pHi remained above this level (P < 0.05). The mean (s.e.m.) postoperative ratio of PaO2 to the fraction of inspired oxygen was 177(11) mmHg in those with intramucosal acidosis, compared with 260(24) mmHg in those whose pHi remained above 7.00 (P < 0.01). These results demonstrate a relationship between bowel ischaemia, endotoxaemia and organ impairment following elective aneurysm repair.
Aged↗
Modulation of immune function and weight loss by L-arginine in obstructive jaundice in the rat.
Jaundiced surgical patients have a high incidence of postoperative complications. Many causative factors have been identified including cachexia and immune suppression. The amino acid L-arginine has anabolic and immunostimulatory properties. It was hypothesized that dietary supplementation with L-arginine would diminish the weight loss and immune suppression of obstructive jaundice. Sixteen male Wistar rats rendered jaundiced by bile duct ligation were allocated to two groups. The test group (n = 8) received drinking water supplemented with 1.8 percent L-arginine ad libitum and the control group (n = 8) received a solution of isonitrogenous glycine. Both groups had free access to standard chow. Body-weight, and fluid and food intake were recorded. After 21 days, delayed-type hypersensitivity to 2,4-dinitrofluorobenzene was assessed. Animals receiving L-arginine consumed more food than controls (mean(s.e.m.) 414(16) versus 360(13) g, P < 0.05) and lost less weight (mean(s.e.m.) proportion of initial body-weight lost 7.8(1.2) versus 14.8(1.4) percent, P < 0.05). The delayed-type hypersensitivity response was significantly greater in rats receiving L-arginine (mean(s.e.m.) increase in ear thickness 23.9(2.7) versus 9.4(2.1) percent, P < 0.05). In this animal model of obstructive jaundice dietary supplementation with L-arginine diminished both weight loss and immune suppression.
Animals↗
Investigating lipopolysaccharide-binding proteins in serum using biotin-labelled lipopolysaccharide.
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Acute-Phase Proteins↗