Polymorphonuclear leukocyte activation. An early marker of the postsurgical sepsis response.

It has been suggested that major surgery induces polymorphonuclear leukocyte (PMNL) dysfunction, which exposes patients to the development of sepsis. Conversely, the sepsis response and multisystem organ failure in patients after surgery is thought to be mediated by activated PMNLs. In a preliminary attempt to investigate this paradox, we studied functional (hydrogen peroxide production) and phenotypic (the adhesion/complement receptor CD11b) markers of PMNL activation in 28 patients undergoing elective major resectional surgery; 11 (39%) of these patients developed postoperative sepsis (the septic group). The mean (SEM) preoperative level of neutrophil CD11b expression (97.8 [6.2] mean channel fluorescence [MCF] and 101.42 [7.9] MCF; P = .74) and hydrogen peroxide production (109.51 [4.91] MCF and 104.53 [6.3] MCF; P = .5) were similar for the uncomplicated and septic groups, respectively. However, on the first postoperative day, both mean CD11b expression and hydrogen peroxide production were greater in those patients who subsequently developed postoperative sepsis (192.5 [38] MCF vs 128.6 [8.1] MCF for the septic group vs the uncomplicated group, respectively [P < .05], and 120.43 [2.56] MCF vs 109.61 [3.05] MCF for the septic group vs the uncomplicated group, respectively [P < .0001]). We suggest that an exaggerated PMNL activation response to surgery is an early event in those patients destined to develop postsurgical sepsis.

Surgery and the release of a neutrophil Fc gamma receptor.

BACKGROUND: The fact that the incidence and mortality from postsurgical sepsis have remained unchanged over the past 15 years raises the possibility that some patients possess an idiosyncratic predisposition to the development of a postoperative sepsis response. Genetic polymorphisms of the neutrophil receptor for immunoglobulin G, CD16, are known, and their inheritance is associated with functional differences in neutrophil phagocytosis. The present studies were designed to provide preliminary data on the effects of surgery on the level of expression of CD16 and its metabolism preparatory to detailed examination of the relationship of these polymorphisms to sepsis responses. PATIENTS AND METHODS: Neutrophil CD16 expression was measured by flow cytometry before and after operation in patients undergoing major resectional surgery of the digestive tract. Assays were performed on whole blood preparations as well as on isolated and activated neutrophil preparations from these patients. RESULTS: Neutrophil CD16 expression was constitutively higher both before and after surgery in patients who developed a postoperative sepsis response than in those who did not. Surgery had no effect on the level of surface neutrophil CD16 expression in either group. Surgery depleted intracellular CD16 stores despite the maintenance of a constant level of CD16 on the neutrophil surface, membrane-bound CD16 being more readily cleaved by physiological neutrophil activators after surgery than before surgery. CONCLUSIONS: The intrinsic level of expression and postsurgical metabolism of neutrophil CD16 may be an important component of the predisposition of some patients to develop infection or sepsis after injury. Further studies of the distribution of CD16 allotypes and neutrophil function among surgical patients are warranted.

The use of Fab' fragments in a screening method for the detection of human anti-tumor monoclonal antibodies.

The use of immunoperoxidase staining methods to detect human monoclonal antibodies that react with frozen tissue sections of human tumors is limited due to endogenous immunoglobulin present in the sections. The endogenous immunoglobulin produces a significant background staining which makes the detection of a specific reaction difficult and unreliable. A method of incubating the sections with papain fragments (Fab') of goat anti-human immunoglobulin was evaluated. It was demonstrated that Fab' fragments of goat anti-human polyvalent immunoglobulin can block endogenous immunoglobulin background staining after prolonged washing of tissue sections (20h) and thus facilitating the screening of monoclonal antibodies against frozen sections of tumor tissue.

Flood-related contamination in catchments affected by historical metal mining: an unexpected and emerging hazard of climate change.

Floods in catchments affected by historical metal mining result in the remobilisation of large quantities of contaminated sediment from floodplain soils and old mine workings. This poses a significant threat to agricultural production and is preventing many European river catchments achieving a 'good chemical and ecological status', as demanded by the Water Framework Directive. Analysis of overbank sediment following widespread flooding in west Wales in June 2012 showed that flood sediments were contaminated above guideline pollution thresholds, in some samples by a factor of 82. Most significantly, silage produced from flood affected fields was found to contain up to 1900 mg kg(-1) of sediment associated Pb, which caused cattle poisoning and mortality. As a consequence of climate related increases in flooding this problem is likely to continue and intensify. Management of contaminated catchments requires a geomorphological approach to understand the spatial and temporal cycling of metals through the fluvial system.

Novel flow cytometric method for quantifying nuclear binding of the transcription factor nuclear factor kappa B in unseparated human monocytes and polymorphonuclear cells.

The transcription factor nuclear factor kappa B (NF kappa B) regulates the production of a number of pro-inflammatory mediators involved in polymorphonuclear and mononuclear cell activation. Measurement of NF kappa B DNA binding is used as an estimate of cellular activation and is usually measured by the DNA mobility shift assay. Results from the mobility shift assay can be difficult to quantify and do not allow discrimination of activity in different populations of cells in whole blood without prior separation. This paper describes a new flow cytometric method which allows rapid detection and quantification of DNA-bound NF kappa B in white cell populations of whole blood. The technique is sensitive and allows discriminate analysis and quantification of bound NF kappa B in nuclei from polymorphonuclear and mononuclear cell populations in whole blood.

Transcription factor NF kappa B expression and postsurgical organ dysfunction.

OBJECTIVE: To examine the role of neutrophil NF kappa B activation in organ dysfunction after major surgery. SUMMARY BACKGROUND DATA: NF kappa B is a transcription factor involved in the signal transduction of many stimuli that may participate in the pathogenesis of sepsis and resultant multiple organ dysfunction syndrome (MODS). It may therefore be a potential target for modulation in the reduction of postsurgical MODS. METHODS: Twenty-five patients undergoing major vascular surgery (thoracoabdominal aortic aneurysm repair) were studied. Perioperative levels of neutrophil NF kappa B, CD11b, and glutathione were measured. In vitro inhibition experiments using NF kappa B inhibitors were also performed. RESULTS: No differences in clinical parameters were apparent before surgery between the patients who subsequently developed MODS and those who did not. However, there was a significant difference in preoperative levels of NF kappa B between the patients who developed postoperative organ dysfunction and those who did not. There was also a significant preoperative difference between patients who survived surgery and those who did not. Glutathione levels were reduced both in patients who developed MODS and those who did not at the onset of surgery. NF kappa B inhibitors suppressed patient plasma-stimulated NF kappa B activation in healthy neutrophils. CONCLUSIONS: Preoperative neutrophil NF kappa B status may be a marker of postoperative outcome after major surgery, and therapy aimed at attenuating neutrophil NF kappa B activation may reduce postoperative sepsis and organ dysfunction.

Changes in major histocompatibility complex class II expression in monocytes and T cells of patients developing infection after surgery.

Expression of class II major histocompatibility complex (MHC) on monocytes is a prerequisite for effective antigen presentation and processing, an important component of the immune response to infection. It has been reported that the level of monocyte class II expression may identify patients who go on to develop infective complications following trauma. In the present study, flow cytometry was used to measure MHC class II (human leucocyte antigen (HLA)-DR) expression on circulating monocytes and T cells in 36 patients undergoing elective major resectional surgery, of whom 12 developed septic complications. The percentage of HLA-DR positive monocytes fell significantly on the first day after operation in both groups (P < 0.001) but was significantly higher in those without than in those with sepsis on days 1, 3 and 5 (P < 0.05). In contrast, the level of T cell HLA-DR expression rose significantly on the first day after operation (P < 0.05) in patients without sepsis to a level higher than in those who developed infection (P < 0.05). These findings have important implications, as predictive biological elements and for biological response modification, in patients at risk of developing sepsis after surgery.

Glutathione S-transferase Mu phenotype in patients with familial adenomatous polyposis and in unaffected controls.

Patients with familial adenomatous polyposis (FAP) are at high risk for duodenal tumors, the distribution of which suggests that bile is important in their development. Studies of the bile of FAP patients suggest that it contains an excess of active carcinogens. Defective hepatic metabolism of carcinogens might account for these findings. The isozyme glutathione S-transferase mu (GST-mu) plays a major role in the hepatic metabolism of carcinogens. Peripheral blood GST-mu status reflects hepatic GST-mu status. The concentration of GST-mu was therefore measured by enzyme-linked immunosorbent assay of heparinized peripheral blood samples taken from 31 unrelated patients with FAP and from 38 unrelated control patients. FAP and control patients were matched for age, sex, diet, and smoking status. The median GST-mu concentration (micrograms per milliliter) was 0.5 (interquartile range, 0 to 11.6) in the FAP group and 8.85 (0.9 to 29.4, p = 0.0013) in the control group. Of the 31 FAP patients, ten had no detectable GST-mu activity compared with only one of the 38 controls (p = 0.002), while 71% of FAP patients had GST-mu concentrations less than a supplied positive control, compared with 50% of control patients (p = 0.064). Abnormal hepatic metabolism of carcinogens by GST-mu might contribute to the development of intestinal tumors in patients with FAP.

Swainsonine, a glycosylation inhibitor, enhances both lymphocyte efficacy and tumour susceptibility in LAK and NK cytotoxicity.

Swainsonine (SW) inhibits the formation of N-linked complex oligosaccharides and has previously been shown to inhibit experimental metastasis in nude mice models. The present studies with human effector cells have shown that SW enhanced both lymphokine activated killer cell (LAK) and natural killer (NK) cytotoxicity in standard 51Cr-release assays. SW also increased the susceptibility of human K562 and Colo 320 target cells to NK and LAK cytotoxicity. The peak response of both LAK effectors and targets to SW occurred at 1-2 micrograms/ml SW. A novel finding was that SW enhanced the interleukin 2 (IL-2) beta chain receptor subunit expression on both LAK and NK cells to a greater extent than its enhancement of the IL-2R alpha (CD25 or TAC) receptor expression on LAK effectors. In addition, increases in both these receptors occurred at the doses of SW which augmented LAK cytotoxicity. We conclude that the anti-metastatic effects of SW have an immunological component which is maximal at 1-2 micrograms/ml SW. This suggests that dosage may be an important consideration to obtain optimal potential of SW in any future human cancer therapy.

Neutrophil activation and hyperamylasaemia after endoscopic retrograde cholangiopancreatography: potential role for the leukocyte in the pathogenesis of acute pancreatitis.

BACKGROUND AND STUDY AIMS: Hyperamylasaemia occurs in up to 60% of patients following endoscopic retrograde cholangiopancreatography (ERCP), and in a small proportion of patients (1-5%) acute pancreatitis may develop. We evaluated the role of the neutrophil in post-ERCP hyperamylasaemia and acute pancreatitis by measuring circulating CD11b adhesion receptor expression--an indicator of leukocyte activation. PATIENTS AND METHODS: A total of 43 patients undergoing elective ERCP were studied. Peripheral blood measurements of amylase activity and neutrophil CD11b content (by flow cytometry) were made immediately before ERCP (baseline), and at 2 and 24 hours after the procedure. RESULTS: ERCP induced an increase in amylase level above baseline in 41 of 43 patients. The 2-hour and 24-hour post-ERCP amylase levels were directly related (R = 0.9, P < 0.01). Baseline CD11b receptor status was positively correlated with post-ERCP amylase activity (R = 0.4, P < 0.05), and this relationship was stronger when pancreatography had been performed (R = 0.67, P < 0.01). Three patients (7%) developed clinical acute pancreatitis, with post-ERCP amylase levels persistently elevated above 1000 IU/l. Multiple linear regression identified CD11b expression as the most significant explanatory variable for amylase level after ERCP (multiple R = 0.74, P < 0.01). CONCLUSIONS: The findings from this pilot study indicate an association between neutrophil activation and hyperamylasaemia following ERCP, and suggest a role for this leukocyte in the pathogenesis of pancreatitis. Further study of neutrophil characteristics may allow identification of individual susceptibility to ERCP-induced pancreatic injury.

Endotoxin related early neutrophil activation is associated with outcome after thoracoabdominal aortic aneurysm repair.

BACKGROUND: Thoracoabdominal aortic aneurysm (TAAA) repair is associated with high mortality rates, especially in patients who develop postoperative renal and pulmonary impairment. Organ damage during TAAA repair may be mediated by activated polymorphonuclear neutrophils (PMNs) during a systemic inflammatory response. The association between intraoperative neutrophil activation and postoperative outcome was studied after TAAA surgery. METHODS: Perioperative PMN activation (surface CD11b expression by flow cytometry), plasma endotoxin, plasma endotoxin core antibody, tumour necrosis factor (TNF), and interleukin 1 (IL-1) levels were measured in 21 patients undergoing TAAA repair. RESULTS: Intraoperative PMN CD11b expression was significantly greater in the 11 patients who developed pulmonary and renal complications than in those who made an uneventful recovery (P = 0.0009). In addition, CD11b expression during visceral reperfusion was preceded by a rise in plasma endotoxin level and a fall in antibody level (reflecting binding by absorbed endotoxin) which was significantly greater in patients who developed complications (P = 0.031 and P = 0.001 respectively). Plasma endotoxin level correlated with CD11b expression (r = 0.828, P = 0.001). There were no significant differences in intraoperative plasma levels of TNF and IL-1, or aortic cross-clamp times and blood transfusion volumes between the complicated and uncomplicated repairs. CONCLUSION: Intraoperative neutrophil activation is a marker for the development of postoperative complications after TAAA reconstruction and is associated with endotoxin absorption.

Visceral ischemia and neutrophil activation in sepsis and organ dysfunction.

BACKGROUND: It has previously been shown that a rise in intraoperative neutrophil CD11b expression during supracoeliac cross-clamping is a marker for subsequent development of postoperative organ dysfunction. Prolonged visceral ischemia and increased aneurysm extent are associated with higher risks of morbidity and mortality after TAAA repair. This study investigates the relationship between visceral ischemia and neutrophil activation in sepsis and organ dysfunction following visceral reperfusion. METHOD: Fifty-one patients undergoing supracoeliac cross-clamping, 5 patients undergoing suprarenal clamping, and 8 patients undergoing infrarenal clamping for repair of aortic aneurysms were studied. Perioperative neutrophil CD11b expression was measured by flow cytometry. RESULTS: There was significant correlation between visceral clamp time and intraoperative CD11b expression. More extensive aneurysms resulted in increased visceral clamp times and CD11b expression. There were no differences between bypass and non-bypass-assisted surgery with regard to neutrophil expression. There were increased clamp time in patients who developed severe sepsis and postoperative organ dysfunction. Differences in preoperative levels of CD11b expression were observed between groups and high levels of preoperative CD11b expression were observed in patients who died intraoperatively, in type II patients who went on to develop severe sepsis and organ failure, and in patients who developed multiple organ failure rather than single organ failure. CONCLUSION: Longer periods of visceral ischemia are associated with higher levels of intraoperative CD11b expression, severe sepsis, and organ failure. High preoperative levels of CD11b may identify an "at-risk" subset of patients.

Expression of a suppressive p15E-related epitope in colorectal and gastric cancer.

mRNA for the suppressive epitope of p15E was found to be present in 24 of 30 samples of human colorectal cancer and in all four specimens of gastric cancer. mRNA for p15E was seldom seen in nonmalignant colonic or gastric mucosa but, when present, was associated with inflammatory or pre-malignant conditions of the digestive tract. Synthetic peptides derived from the conserved p15E sequence were found to suppress some aspects of the immune response implicated in anti-tumour activity. These data suggest that a p15E-related material with immunomodulatory properties is elaborated within human tumours, either by the tumour itself or as a normal component of the endogenous anti-tumour reaction.