Absence of lipopolysaccharide-induced inhibition of neutrophil apoptosis in patients with diabetes.

BACKGROUND: Patients with diabetes mellitus are considered to be at increased risk for infections and often experience more serious and prolonged infections. Defects in neutrophil microbicidal function have been identified in diabetic patients and are believed to contribute to this problem. Neutrophil apoptosis or programmed cell death regulates functional longevity, and is an integral component of inflammation and its resolution. It remains unknown whether neutrophils from diabetic patients demonstrate defects in apoptosis. HYPOTHESIS: Neutrophils from diabetic patients have defects in spontaneous and/or endotoxin (lipopolysaccharide [LPS])-induced apoptosis. DESIGN: Peripheral venous blood samples were collected from healthy volunteers and insulin-dependent diabetic patients to harvest neutrophils for in vitro study. INTERVENTIONS AND MAIN OUTCOME MEASURES: Neutrophils were cultured for 0 and 24 hours in the absence and presence of LPS. Normal neutrophils were also cultured under conditions of normal or high glucose concentration. Percentage of apoptotic cells was determined by flow cytometry. RESULTS: Normal neutrophils undergo significant apoptosis after 24 hours, and the percentage of apoptotic cells is reduced in the presence of LPS. Diabetic neutrophils undergo normal spontaneous apoptosis, but do not demonstrate LPS-induced inhibition of apoptosis. The LPS-induced inhibition of apoptosis in normal neutrophils is prevented under high-glucose conditions. CONCLUSIONS: Neutrophils from diabetic patients demonstrated defects in LPS-induced apoptosis. High-glucose environment may mediate these findings. The inability of diabetic neutrophils to reduce apoptosis following LPS exposure resulted in relatively increased apoptosis. This would cause decreased functional longevity of neutrophils and increased neutrophil clearance from infectious sites, possibly contributing to the increased susceptibility and severity of infections in diabetic patients.

Helicobacter pylori products upregulate neutrophil superoxide anion production.

Helicobacter pylori-neutrophil interactions may play a pathogenic role in H. pylori-induced gastritis and peptic ulcer disease. To understand these interactions, we explored the effects of H. pylori-derived products on neutrophil chemotaxis and superoxide anion production. H. pylori bacteria were cultured and supernatants fractionated. Neutrophil chemotactic activity was confirmed in the crude supernatants and in one fractionated peak corresponding to a previously described neutrophil chemotactic factor. H. pylori-derived crude supernatant, sonicate and all chromatography-derived peaks failed to directly stimulate neutrophil superoxide anion production. However, after pretreatment with sonicate, neutrophils demonstrated increased superoxide anion production (priming) following subsequent exposure to the secretagogue fmet-leu-phe. These results suggest that H. pylori products may attract neutrophils to the gastric mucosa without initially stimulating superoxide anion production or tissue injury. Oxygen radical-mediated gastric mucosal injury may subsequently result when these primed neutrophils undergo additional stimulation by as yet unidentified factors.

A prospective randomized trial of an antibiotic- and antiseptic-coated central venous catheter in the prevention of catheter-related infections.

OBJECTIVE: To test the efficacy of the ARROWgard (Arrow International Inc, Reading, Pa) central venous catheter (CVC) coated with silver sulfadiazine and chlorhexidine (A-CVC) in the prevention of CVC-related infections. DESIGN: Prospective, randomized trial. SETTING: A tertiary care medical center. PATIENTS AND INTERVENTION: Two hundred eighty-two patients who required CVC placement were evaluated in this study. Patients were prospectively randomized to receive either a standard CVC (S-CVC) or the A-CVC. Only fresh-stick double- and triple-lumen catheters were studied. MAIN OUTCOME MEASURES: Patients were evaluated for catheter site inflammation, catheter site colonization, local catheter-related infection, and catheter-related septicemia. RESULTS: The 2 groups were matched for age, percentage in the intensive care unit, percentage receiving total parenteral nutrition, percentage with triple-lumen catheters, and duration of catheterization. Rates of catheter site inflammation in the 2 groups were similar (12% vs 10%, S-CVC group and A-CVC group, respectively). The A-CVC was associated with a significantly decreased catheter site colonization rate (49% vs 28%; 43% reduction; P<.001) and local catheter-related infection rate (22.4% vs 5.8%; 74% reduction; P<.001). Rates of catheter-related septicemia were reduced by 41% in the A-CVC group (6.4% vs 3.8%, S-CVC group and A-CVC group, respectively), but this was not statistically significant. CONCLUSIONS: Despite a marked decrease in catheter site colonization and catheter-related infection rates, the A-CVC did not significantly reduce the incidence of catheter-related septicemia. This may be due to a greater pathogenic dependence on catheter hub contamination rather than catheter site colonization or local catheter-related infection, or the relatively short (5.2 days) duration of catheterization in this study.

Endotoxin-induced, neutrophil-mediated endothelial cytotoxicity is enhanced by T-lymphocytes.

The role of T-lymphocytes (T cells) and interferon-gamma (IFN-gamma) in the pathogenesis of sepsis-induced microvascular endothelial injury remains unclear. We sought to determine whether the syngeneic coculture of human T cells in the presence of LPS promoted subsequent neutrophil (PMN)-mediated endothelial cytotoxicity. Syngeneic T cells were cocultured with 51Cr-loaded human adipose microvascular endothelial cell (HAMVEC) monolayers in the absence and presence of LPS. Subsequent PMN-mediated HAMVEC cytotoxicity (measured as percent specific 51Cr release) was absent in cultures that contained T cells but no LPS and was significantly increased when T cells were cocultured in the presence of LPS. This was true both following addition of unstimulated PMNs (-0.8 +/- 3.0% vs 4.9 +/- 4.7% for T cells alone vs T cells plus LPS, respectively) and PMNs stimulated with f-Met-Leu-Phe (-0.4 +/- 3.1% vs 10.7 +/- 3.0% for T cells alone vs T cells plus LPS, respectively). Increased cytotoxicity was associated with increased expression of the endothelial adhesion molecules ICAM-1 and VCAM-1. Control experiments failed to demonstrate cytotoxicity when HAMVEC were cultured in the presence of IFN-gamma alone, LPS alone, or T cells without LPS. It appears that there is a necessary requirement of both LPS and (presumably activated) T cells or their products (other than IFN-gamma) for enhanced PMN-mediated endothelial cytotoxicity. This phenomenon may also be mediated by increased expression of endothelial adhesion molecules that promote subsequent PMN adhesion.

A prospective, randomized study of open vs laparoscopic inguinal hernia repair. An assessment of postoperative pain.

OBJECTIVE: To compare postoperative pain after laparoscopic hernia repair and conventional open hernia repair. DESIGN: Prospective, randomized study. SETTING: Veterans Affairs Medical Center. PATIENTS: Sixty-two patients scheduled for elective inguinal hernia repair. INTERVENTIONS: Patients were randomized in the operating room to have a laparoscopic hernia repair (30 patients) or a conventional open hernia repair (32 patients). All operations were performed while the patient was under general anesthesia to avoid anesthesia as a confounding variable. MEASURES: Postoperative pain following laparoscopic hernia repair and open hernia repair were compared using the McGill Pain Score, the McGill Visual Analogue Pain Scale score, and the number of acetaminophen with 30-mg codeine sulfate (Tylenol 3) tablets needed for pain during the first and second 24-hour periods postoperatively. All of the patients were interviewed and the postoperative pain was evaluated by a special study nurse (P.M.L.) who was blinded to the repair technique. RESULTS: At 24 hours, the patients with laparoscopic hernia repair had 26% less pain by the McGill Pain Score (P = .02) and 31% less pain by the McGill Visual Analogue Scale (P = .006) than those who underwent an open hernia repair. At 48 hours the patients who underwent laparoscopic hernia repair had 28% less pain by the McGill Pain Score (P = .03), 42% less pain by the McGill Visual Analogue Scale (P = .002), and used 42% fewer analgesic tablets (P = .004). CONCLUSION: Patients with a laparoscopic hernia repair had significantly less pain postoperatively than those with standard open hernia repairs.

Endotoxin activates T cell interferon-gamma secretion in the presence of endothelium.

T lymphocytes (T cells) and their secreted lymphokine interferon-gamma (IFN-gamma) play important mediator roles in endotoxin-induced inflammation. We sought to explore the necessary conditions for and degree of LPS-induced T cell activation for IFN-gamma secretion in a human syngeneic microvascular endothelial-T cell coculture system. Human peripheral blood T cells, with or without monocytes, were cocultured in the presence or absence of a syngeneic human adipose microvascular endothelial cell (HAMVEC) monolayer. Cocultures were stimulated with LPS (1 microgram/ml) and 3-day coculture supernatants assayed for IFN-gamma and IL-2 by ELISA. In the absence of HAMVEC, LPS-induced T cell activation for IFN-gamma secretion was only minimally demonstrated in the presence of monocytes. However, in the presence of HAMVEC, LPS activated T cells for IFN-gamma secretion in the absence of monocytes and markedly augmented the response in the presence of monocytes. A subset of donor cocultures showed no IFN-gamma response to LPS. IL-2 was not secreted as part of the LPS-induced T cell activation response. Our data support a hypothesis that endothelium serves as an accessory cell for T cell IFN-gamma secretion in endotoxin-induced inflammation. T cell-endothelial interactions may play a crucial role in promoting T cell activation during LPS-induced inflammation.

Interferon-gamma primes neutrophil-mediated gastric surface cell cytotoxicity.

The T lymphocyte product interferon-gamma (IFN-gamma) upregulates or primes polymorphonuclear leukocyte (PMN) oxidative responses to the receptor-initiated stimulant N-formyl-methionyl-leucyl-phenylalanine (FMLP) but not to the transduction-mediated stimulant phorbol myristate acetate (PMA). We sought a functional correlation between IFN-gamma-induced oxidative priming of PMNs and PMN-mediated cytotoxicity, using an in vitro assay of 51Cr release from rabbit gastric surface cells. Compared with control PMNs, IFN-gamma-primed PMNs exhibited a significant increase in cytotoxicity when stimulated with FMLP, but not when stimulated with PMA. IFN-gamma-induced, FMLP-stimulated, PMN-mediated cytotoxicity was reduced by adding superoxide dismutase to scavenge superoxide anion or by adding catalase or glutathione peroxidase to scavenge hydrogen peroxide. Cytotoxicity was also reduced by inhibiting myeloperoxidase activity with azide or scavenging HOCl with alanine or methionine. Cytotoxicity was blocked by a monoclonal antibody against the CD11/CD18 integrin of PMNs. The results indicate that the immunoregulatory lymphokine IFN-gamma primes the FMLP-stimulated cytotoxic activity of PMNs via the increased generation of reactive oxygen metabolites and indicate that cytotoxicity may require effector-target cell adherence. Therefore, T lymphocyte-derived IFN-gamma may have a role in the pathogenesis of PMN-mediated injury to gastric and gastrointestinal tract mucosa.

Gangrenous cholecystitis in an urban VA hospital.

Gangrenous cholecystitis is an advanced form of acute cholecystitis associated with increased morbidity and mortality. We sought to determine the incidence of gangrenous cholecystitis in an urban VA hospital patient population and identify any distinguishing characteristics that may aid in its preoperative diagnosis. We retrospectively reviewed all urgent admissions that underwent cholecystectomy (n = 65) over the past 7 years at the Allen Park VAMC. Using histologic criteria, 17 (26%) of these patients had gangrenous cholecystitis. As a group compared to patients with nongangrenous cholecystitis, patients with gangrenous cholecystitis were statistically older (64 vs 54) and had an elevated WBC (15.4 vs 11.5) and increased serum glucose levels (203 vs 141). Preoperative imaging studies (ultrasound and cholescintigraphy) correctly identified only 31% of the gangrenous cholecystitis patients. We conclude that in an urban VA hospital patient population, the diagnosis of gangrenous cholecystitis cannot be accurately made or ruled out among urgent admissions with acute biliary disease. Considering the high incidence (26%) and difficulty confirming the diagnosis of gangrenous cholecystitis in this setting, we recommend early surgical intervention for this and similar patient populations.

Oxidative priming of neutrophils by interferon-gamma.

Priming of neutrophil (PMN) oxidative responses is an integral component of host defense and inflammation and may contribute to cell-mediated tissue injury. The characteristics and mechanisms of interferon-gamma (IFN-gamma)-induced oxidative priming of PMNs were explored in vitro. Following pretreatment of human PMNs with recombinant IFN-gamma, superoxide anion release was selectively primed toward the receptor-initiated stimulants f-Met-Leu-Phe (fMLP) and C5a but not toward the transduction-mediated stimulants phorbol myristate acetate and A23187, a calcium ionophore. IFN-gamma also induced priming toward the stimulant NaF, a direct activator of guanine nucleotide regulatory proteins. Priming was not associated with changes in fMLP surface receptor expression or degranulation. Priming was dependent on de novo mRNA and protein synthesis. The immuno-regulatory lymphokine IFN-gamma primes PMN oxidative responses, apparently via production of proteins that are involved in the early postreceptor transductional pathways of oxidative metabolism.

Complement activation and lung permeability during cardiopulmonary bypass.

Pulmonary dysfunction after cardiopulmonary bypass has been attributed to the damaging effects of complement activation on the lung. To further explore this phenomenon, we measured plasma levels of activated complement components (radioimmunoassay), assessed neutrophil n-formyl-methionyl-leucyl-phenylalanine (FMLP) receptor status (radioligand saturation binding assay), and quantified pulmonary epithelial permeability as radioaerosol lung clearance of technetium 99m-labeled diethylenetriamine pentaacetic acid in a series of 8 patients undergoing cardiopulmonary bypass. Significant elevations of plasma C3adesArg, C4adesArg, and C5adesArg levels were seen just after CPB, indicating activation of both the classic and alternate complement pathways. Neutrophil activation was evident as increased expression of neutrophil FMLP surface receptors after bypass. Despite the presence of complement and neutrophil activation, increased pulmonary epithelial permeability was not seen. These data support the hypothesis that complement and neutrophil activation during cardiopulmonary bypass is not associated with acute lung injury, at least not pulmonary epithelial injury. One can therefore infer that increased pulmonary epithelial permeability in patients at high risk for and experiencing sepsis-induced and trauma-induced adult respiratory distress syndrome may be due to factors other than complement and neutrophil activation.

Activation of neutrophils by cachectin/tumor necrosis factor: priming of N-formyl-methionyl-leucyl-phenylalanine-induced oxidative responsiveness via receptor mobilization without degranulation.

Human recombinant cachectin/tumor necrosis factor (TNF) was shown to prime neutrophils (PMNs), in a dose-dependent fashion, for subsequent oxidative responsiveness toward n-formyl-methionyl-leucyl-phenylalanine (FMLP). One basis for this phenomenon appeared to be TNF-mediated FMLP receptor mobilization. The maximal observed priming response was associated with a nearly twofold increase in the expression of PMN FMLP surface receptors, without changes in receptor affinity. Priming was not seen following stimulation with phorbol myristate acetate, possibly eliminating a role for the protein kinase C-dependent transductional components of FMLP-induced oxidative activity in the priming process. FMLP receptor mobilization occurred without significant degranulation as evident by an absence of increased granular enzyme release. These data support a potential role of macrophage-derived TNF in the augmentation of PMN host-defense during infectious and inflammatory challenge. TNF-mediated PMN oxidative priming may also promote oxidant tissue injury as seen in septic shock, adult respiratory distress syndrome, and multiple system organ failure.

Characterization of N-formyl-methionyl-leucyl-phenylalanine receptors on human neutrophils. Effects of isolation and temperature on receptor expression and functional activity.

The study of polymorphonuclear neutrophil (PMN) surface receptor expression provides a means for the assessment of PMN function and state of cellular activation. In this study, we characterized binding of the chemotactic peptide FMLP to whole PMN, with particular attention to those variables that may account for the wide variation reported in the literature. These included avoidance of oxidized FMLP as a radioligand contaminant, determination of the optimal cold ligand concentration necessary for achieving minimal nonspecific binding throughout the range of radioligand concentrations used in saturation experiments (greater than or equal to 5 x 10(-5) M), avoidance of radioligand concentrations that equal or exceed receptor saturation and are not suitable for Scatchard analysis (greater than or equal to 60 to 80 nM), and avoidance of inadvertent receptor mobilization due to room temperature PMN isolation techniques and cell warming. PMN isolated and maintained at 4 degrees C expressed a single, high affinity population of FMLP receptors (approximately 6000 receptors per cell) with a KD of 15.5 nM. These characteristics, and in particular the single-affinity nature of the expressed FMLP receptor site, were derived from saturation experiments and confirmed with agonist competition studies. PMN subjected to room temperature isolation or 37 degrees C warming exhibited a 2.5-fold increase in FMLP receptor expression (approximately 15,000 receptors per cell) without changes in receptor affinity. These latter PMN, in correlation with increased receptor expression, had increased initial, maximal rates of FMLP-induced superoxide generation (10.2 vs 6.3 nmol/min/10(6) PMN for cells isolated and maintained at 4 degrees C) as a manifestation of their functional activation. The avoidance of inadvertent cellular activation during PMN isolation is essential to studies of PMN function, activation and the role of FMLP receptor expression/mobilization in these processes.

Neutrophil activation in sepsis. The relationship between fmet-leu-phe receptor mobilization and oxidative activity.

To elucidate further the manifestations and mechanisms of neutrophil (PMN) activation, PMNs from control and septic subjects were studied at baseline and under conditions of graded, in vitro activation. At baseline (4 degrees C PMN isolation), septic-derived PMNs were activated, as manifested by twofold increases in fmet-leu-phe (FMLP)-induced oxidative activity and concomitant FMLP surface receptor expression, compared with controls. Following degranulationlike maximal activation (phorbol myristate acetate pretreatment), both PMN populations exhibited maximal FMLP-induced oxidative priming and receptor up-regulation. However, following exudation-like moderate activation (37 degrees C pretreatment), control PMNs underwent significant receptor mobilization and oxidative priming but septic-derived PMNs exhibited oxidative deactivation (decreased FMLP-induced oxidative activity) without changes in FMLP receptor expression. Our data support the theory that while circulating PMNs in sepsis may promote oxidant-related microvascular lung injury, their oxidative deactivation following transpulmonary exudation (simulated by 37 degrees C pretreatment) may underlie the increased incidence of pulmonary infections seen in sepsis-induced adult respiratory distress syndrome.

Surface receptor mobilization in complement-mediated neutrophil activation: characterization and effects of methylprednisolone.

Complement-mediated neutrophil activation (CMNA) is an important host defense mechanism that is essential for effective neutrophil (PMN) proinflammatory activity. It has also been implicated as a pathogenic mechanism contributing toward the development of adult respiratory distress syndrome. Utilizing zymosan-activated serum pretreatment as an in vitro model for CMNA, we characterized the effects of CMNA on PMN superoxide (SO) production and N-formyl-methionyl-leucyl-phenylalanine (FMLP) receptor status. CMNA was associated with a 132 +/- 38% increase in FMLP-induced SO generation and a 110 +/- 30% increase in FMLP receptor expression. Methylprednisolone pretreatment prevented both the FMLP receptor mobilization and the SO priming effects of CMNA. These data support a concept that FMLP receptor mobilization is an important element in the PMN activation process. In addition, blocking this phenomenon may have clinical significance in attempts to modulate the potential damaging effects of the increased PMN oxidative metabolism associated with CMNA.

Increased pulmonary alveolar-capillary permeability in patients at risk for adult respiratory distress syndrome.

Two methods for predicting adult respiratory distress syndrome (ARDS) were evaluated prospectively in a group of 81 multitrauma and sepsis patients considered at clinical high risk. A popular ARDS risk-scoring method, employing discriminant analysis equations (weighted risk criteria and oxygenation characteristics), yielded a predictive accuracy of 59% and a false-negative rate of 22%. Pulmonary alveolar-capillary permeability (PACP) was determined with a radioaerosol lung-scan technique in 23 of these 81 patients, representing a statistically similar subgroup. Lung scanning achieved a predictive accuracy of 71% (after excluding patients with unilateral pulmonary contusion) and gave no false-negatives. We propose a combination of clinical risk identification and functional determination of PACP to assess a patient's risk of developing ARDS.

Complement-mediated neutrophil activation in sepsis- and trauma-related adult respiratory distress syndrome. Clarification with radioaerosol lung scans.

Complement-mediated neutrophil activation (CMNA) has been proposed as an important pathogenic mechanism causing acute microvascular lung injury in the adult respiratory distress syndrome (ARDS). To clarify the relationship between CMNA and evolving lung injury, we studied 26 patients with multiple trauma and sepsis within 24 hours of risk establishment for ARDS. Pulmonary alveolar-capillary permeability (PACP) was quantified as the clearance rate of a particulate radioaerosol. Seventeen patients (65%) had increased PACP (six developed ARDS) while nine (35%) had normal PACP (none developed ARDS; clearance rates of 3.4%/min and 1.5%/min, respectively). These patients, regardless of evidence of early lung injury, had elevated plasma C3adesArg levels and neutrophil chemotactic desensitization to C5a/C5adesArg. Plasma C3adesArg levels correlated weakly, but significantly, with PACP. Thus, CMNA may be a necessary, but not a sufficient, pathogenic mechanism in the evolution of ARDS.

The effects of methylprednisolone on complement-mediated neutrophil activation during cardiopulmonary bypass.

Complement-mediated neutrophil activation (CMNA) has been implicated as an important pathophysiologic mechanism contributing to acute microvascular lung injury in the adult respiratory distress syndrome (ARDS). Using cardiopulmonary bypass (CPB) as a clinical model for complement-mediated microvascular injury, we studied the effects of methylprednisolone (MPSS) pretreatment on manifestations of CMNA in 28 pediatric patients undergoing CPB. Six patients not receiving MPSS served as controls. Results demonstrated that MPSS did not prevent complement activation as noted by 4.5- and 7.7-fold increases in plasma C3a des Arg levels during and immediately after CPB, respectively. However, detectable in vivo and in vitro manifestations of CMNA were altered. Neutropenia during CPB was attenuated to 65% of prebypass values compared with 47% in the control group. Neutrophil selective chemotactic desensitization toward C5a/C5a des Arg during the on bypass and postbypass periods was evident in the control group (0.41 and 0.76 cm specific migration, respectively) and prevented in the MPSS group (1.55 and 2.00 cm specific migration, respectively). We conclude that CMNA during CPB is ameliorated and/or prevented by MPSS pretreatment. These findings suggest that MPSS pretreatment may ameliorate complement-mediated microvascular (lung) injury in CPB and ARDS.

Catabolism of the apolipoproteins of HDL in control and nephrotic rats.

125I-labeled high density lipoprotein (HDL) from control rats, or from rats made nephrotic by puromycin aminonucleoside, was injected into control or nephrotic rats. At 5 and 20 h, the amount and distribution of label remaining in apolipoproteins HDL, A-I, E, A-IV and the C apolipoproteins was measured after ultracentrifugal isolation of HDL and SDS-polyacrylamide gel electrophoretic separation of each apolipoprotein. There were no significant differences in the removal rates of apolipoprotein HDL or of the individual apolipoproteins when the removal of HDL of controls was compared to HDL of nephrotics. HDL from nephrotic rats contains less than 10% of either the apolipoprotein A-IV or apolipoprotein E content of control HDL, indicating that neither apolipoprotein A-IV nor apolipoprotein E play a significant role in determining the catabolic fate of rat HDL. In severely nephrotic animals the apoliprotein C content of HDL was reduced to 50% of control values and the apolipoprotein A-I content of HDL rose to 87% of the total apolipoprotein. The individual apolipoproteins of HDL from either nephrotics or controls were catabolized at the same rates irrespective of the degrees of nephrosis or altered HDL apolipoprotein composition. The apparent fractional catabolic rates for apolipoprotein HDL and for each of the apolipoproteins, determined after 20 h, did not differ from one another, and all were reduced by half in the nephrotic rats compared to the normal controls. These results support the concept that HDL is catabolized as a particle mediated by apolipoprotein A-I recognition, and they reinforce earlier work indicating that increased synthesis is the dominant factor responsible for increased plasma HDL concentrations in experimental nephrosis.