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Biomedical subjects

O D Rotstein

Publications and source records attributed to O D Rotstein.

At least 91 records · Page 5Linked to original sources
PubMed1995

Posttranscriptional regulation of macrophage tissue factor expression by antioxidants.

Tissue factor (TF) expression by cells of monocyte/macrophage lineage represents an important mechanism underlying the initiation of fibrin deposition at sites of extravascular inflammation. Recent evidence suggests a role for oxidant stress in the signalling pathway of various cell types by virtue of its ability to induce DNA binding of various transcription factors, including nuclear factor kappa B and AP-1. The effect of antioxidant treatment on lipopolysaccharide (LPS)-induced TF expression was examined in murine peritoneal macrophages and human monocytes. Both pyrrolidine dithiocarbamate, an oxidant scavenger, and N-acetyl-cysteine, a precursor of the endogenous antioxidant glutathione, inhibited stimulation of macrophage procoagulant activity by LPS. Northern blot analysis showed that neither of these agents reduced LPS-stimulated TF mRNA accumulation, thereby suggesting a posttranscriptional mechanism for the effect. Immunofluorescence studies of human monocytes using polyclonal anti-TF antibody showed that N-acetyl-cysteine treatment prevented the characteristic plasmalemmal localization of TF antigen that occurs in response to LPS. Western blot analysis showed that N-acetyl-cysteine reduced the accumulation of the 47-kD mature glycoprotein in LPS-treated cells, a finding consistent with the results of the immunofluorescence studies. Furthermore, these conditions did not result in an accumulation of the less mature forms of TF. When considered together, these data suggest that antioxidants exert their effects by impairing translation and/or by causing degradation of newly translated protein. The effect of antioxidants on tumor necrosis factor appeared to be species specific, with no effect on LPS-induced tumor necrosis factor in murine cells, but with inhibition in human monocytes. The posttranscriptional effect of antioxidants on TF expression data suggests a novel mechanism whereby these agents might modulate monocyte/macrophage activation.

Acetylcysteine↗
PubMed1995

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance.

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.

Animals↗
PubMed1995

Mast cell modulation of macrophage procoagulant activity and TNF production.

The expression of surface procoagulants by macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. Mast cells by virtue of their perivascular location are in a potent position to influence the inflammatory process. The present studies investigated the role of the mast cell in the generation of macrophage procoagulant activity (PCA) and tumor necrosis factor (TNF) production. Mast cell lysates caused a marked induction of macrophage PCA (dose and time dependent) and TNF release while whole mast cells had little effect. This effect was prevented by the tyrosine kinase inhibitor herbimycin. At the molecular level, Northern blot analysis revealed marked induction of the murine macrophage tissue factor transcript in response to incubation with mast cell lysate compared to control. These studies thus suggest that mast cell-macrophage interactions promote macrophage-mediated fibrin deposition and TNF release and that this effect is in part mediated via induction of tyrosine phosphorylation. These observations suggest novel mechanisms of involvement of the mast cell in the inflammatory microenvironment and macrophage activation.

Animals↗
PubMed1995

Definition of the role of enterococcus in intraabdominal infection: analysis of a prospective randomized trial.

BACKGROUND: The role of enterococcus in intraabdominal infection is controversial. This study examines the contribution of enterococcus to adverse outcome in a large intraabdominal infection trial. METHODS: A randomized prospective double-blind trial was performed to compare two different antimicrobial regimens in combination with surgical or percutaneous drainage in the treatment of complicated intraabdominal infections. A total of 330 valid patients was enrolled from 22 centers in North America. RESULTS: In 330 valid patients, 71 had enterococcus isolated from the initial drainage of an intraabdominal focus of infection. This finding was associated with a significantly higher treatment failure rate than that of patients without enterococcus (28% versus 14%, p < 0.01). In addition, only Acute Physiology and Chronic Health Evaluation II score and presence of enterococcus were significant independent predictors of treatment failure when stepwise logistic regression was performed (p < 0.01 and < 0.03). Risk factors for the presence of enterococcus include age, Acute Physiology and Chronic Health Evaluation II, preinfection hospital length of stay, postoperative infections, and anatomic source of infection. There was no difference between the clinical trial treatment regimens with regard to overall failure, failure associated with enterococcus, or frequency of enterococcal isolation. CONCLUSIONS: This study is the first to report enterococcus as a predictor of treatment failure in complicated intraabdominal infections. This trial also identifies several significant risk factors for the presence of enterococcus in such infections.

Abscess↗
PubMed1995

Intestinal epithelial cells down-regulate macrophage tumor necrosis factor-alpha secretion: a mechanism for immune homeostasis in the gut-associated lymphoid tissue.

BACKGROUND: The gut lumen contains more than 10(6) organisms per gram of luminal contents. The mechanisms that limit the response of macrophages in the lamina propria to these microbial antigens are unknown, although an intrinsic defect in this mechanism may contribute to the development of inflammatory bowel disease. Intestinal epithelial cells (IEC) may play an important role in mediating tonic down-regulation of local immune cell activation. The purpose of this study was to discern whether IEC might modulate macrophage activation in response to a variety of microbial stimuli. METHODS: Thioglycollate-elicited murine peritoneal macrophages were activated by endotoxin, zymosan, Escherichia coli, and Candida albicans in the presence or absence of IEC from the rat intestinal epithelial cell line IEC-6. Macrophage tumor necrosis factor-alpha (TNF-alpha) secretion was determined by enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide or zymosan-activated macrophages in coculture with IEC secreted significantly less TNF-alpha than macrophages cultured alone. The inhibitory effect of the IEC was dependent on their activation by lipopolysaccharide. Interleukin-1 alpha production was not affected. IEC-mediated suppression of macrophage TNF-alpha secretion was reversed by indomethacin but not by neutralizing antibody to TGF-beta. CONCLUSIONS: Lipopolysaccharide-activated IEC down-regulate macrophage TNF-alpha secretion in response to microbial stimuli through a prostanoid-mediated mechanism. IEC may mediate tonic down-regulation of immune cell activation in the gut-associated lymphoid tissue and may thereby regulate local and systemic inflammatory responses.

Animals↗
PubMed1995

Effect of alterations in early signal transduction events on the induction of procoagulant activity by murine hepatitis virus strain 3 in vitro.

The induction of macrophage procoagulant activity (PCA) has been shown to correlate with the development of fulminant hepatic necrosis after infection with murine hepatitis virus strain 3 (MHV-3). However, comparatively little is known about the early events in cells after viral infection leading to PCA expression. Accordingly, we investigated the early cellular events in the induction of macrophage PCA by MHV-3. MHV-3 stimulation of macrophages did not result in a detectable increase in intracellular calcium levels nor did stimulation of macrophages by calcium ionophores result in induction of PCA, suggesting that calcium transients were neither necessary nor sufficient for induction of PCA by MHV-3. Treatment of cells with phorbol myristate acetate had no effect on PCA induction; however, inhibition of protein kinase C (PKC) by staurosporine or H7 resulted in attenuation of macrophage PCA following MHV-3 stimulation (P < 0.05 compared with untreated macrophages), suggesting that although activation of PKC alone is insufficient for PCA induction, PKC may be an integral component of PCA induction by MHV-3. We have previously demonstrated that dimethyl prostaglandin E2 inhibited induction of PCA by MHV-3. In this study, treatment of cells by agents that increase intracellular cAMP (forskolin, isobutylmethyl xanthine) significantly inhibited PCA induction (P < 0.02). These results demonstrate that induction of macrophage PCA by MHV-3 involves PKC, but proceeds independently of changes in intracellular calcium, and that PCA expression is down-regulated by increases in intracellular cAMP.

16,16-Dimethylprostaglandin E2↗
PubMed1995

Induction of macrophage procoagulant activity by murine hepatitis virus strain 3: role of tyrosine phosphorylation.

The induction of a unique macrophage procoagulant molecule by murine hepatitis virus strain 3 correlates with the severity of viral hepatitis. The role of tyrosine phosphorylation in the signalling pathway leading to procoagulant expression was studied. Murine hepatitis virus strain 3 initiated a rapid increase in phosphotyrosine accumulation. Tyrosine kinase inhibition precluded this increase and abrogated expression of the virus-induced procoagulant mouse fibrinogen-like protein (musfiblp) gene. These findings suggest that manipulation of this signalling pathway in vivo might represent a novel approach to treating this disease.

Animals↗
PubMed1995

Signaling events in monocytes and macrophages.

The cellular signaling events leading to the systemic inflammatory response syndrome and sepsis in monocytes/macrophages activated by lipopolysaccharide (LPS) are well understood. LPS is a glycolipid component of Gram-negative bacterial cell wall. It exerts its effect through the lipid A moiety. LPS binds to monocytes/macrophages via a membrane-bound receptor, CD14, an interaction which is optimized in the presence of plasma factors, LPS-binding protein, and septin. Although LPS is known to bind to other receptors, the roles of these receptors in transmembrane signaling and activation of monocytes/macrophages are not as well understood as is that of the CD14 receptor. Intracellular events in response to LPS stimulation are mediated by phospholipase (PL) C, protein kinases, PLA2, and PLD. Activation of PLC by LPS results in the release of diacylglycerol and inositol 1,4,5-trisphosphate. The former mediates the stimulation of protein kinase C, and the latter induces an increase in intracellular calcium concentration. LPS stimulation of monocytes/macrophages also results in the phosphorylation and activation of several protein kinases, including protein tyrosine kinases which mediate cytokine production, and mitogen-activated protein kinase which activates cytosolic PLA2 to release arachidonate. LPS also plays a role in cellular proliferation and differentiation. Upregulation of the secretory form of PLA2 has also been documented in response to LPS. PLD is stimulated by LPS to release phosphatidic acid (PA). PA can activate the respiratory burst by increasing diacylglycerol production and by modulating the effects of guanine nucleotide-binding proteins. Therapeutic strategies to decrease the clinical effects of sepsis would logically include agents which block at initial receptor-ligand interaction, as well as those which attenuate the intracellular events that follow LPS stimulation. Early in vivo studies are promising, but clearly much work remains to be done.

Animals↗
PubMed1995

Stoma closure and wound infection: an evaluation of risk factors.

OBJECTIVE: To assess the infection rate in the nonstoma wound in patients who undergo stoma closure. DESIGN: Chart review. SETTING: A tertiary-care hospital. PATIENTS: Ninety-five patients who underwent elective closure of an abdominal wall stoma requiring a separate abdominal incision. INTERVENTIONS: Elective general surgery procedures. MAIN OUTCOME MEASURE: Wound infection rate. RESULTS: The overall wound infection rate was 29%. Primary wound closure was associated with a markedly increased wound infection rate (41%) compared with delayed primary or secondary wound closure (15%). No other preoperative factor specifically predicted a high rate of postoperative nonstoma wound infection. CONCLUSIONS: The nonstoma wound during elective closure of an abdominal stoma is at high risk for infection postoperatively. Delayed primary or secondary closure may lessen this risk.

Anti-Bacterial Agents↗
PubMed1994

Antimicrobial prophylaxis in surgery. Committee on Antimicrobial Agents, Canadian Infectious Disease Society.

OBJECTIVE: To provide guidelines for antimicrobial prophylaxis on the basis of the type of surgical procedure. OPTIONS: Standard drug regimens for prophylaxis of infection in a variety of surgical procedures were considered, including a first-generation cephalosporin; an aminoglycoside in combination with metronidazole, clindamycin or erythromycin; a second-generation cephalosporin; and trimethoprim-sulfamethoxazole. OUTCOMES: In order of importance: efficacy, side effects and cost. EVIDENCE: A MEDLINE search of articles published between January 1980 and December 1991. For clinical trial data, greatest emphasis was placed on randomized, double-blind studies using appropriate controls. VALUES: The Committee on Antimicrobial Agents of the Canadian Infectious Disease Society (CIDS) and two recognized experts (T.K.W. and O.D.R.) recommended antimicrobial regimens suitable for prophylaxis of infections in surgery. Whenever possible, recommendations were based on data from randomized controlled trials. BENEFITS, HARMS AND COSTS: Implementation of the guidelines is expected to reduce the incidence of postoperative infections, the inappropriate use of antibiotics and costs to hospitals. RECOMMENDATIONS: Antibiotic prophylaxis is recommended for operations with a high risk of postoperative wound infection or with a low risk of infection but significant consequences if infection occurs. These operations include clean-contaminated procedures and certain clean procedures. Drugs should be administered intravenously immediately before the operation. In colorectal operations oral administration also appears to be effective. A single dose is sufficient for most procedures. The regimen chosen depends on the pathogens usually associated with wound infection in a given operation, the serum half-life of the drugs, the antimicrobial susceptibility patterns in the local hospital and the cost of the drugs. VALIDATION: The guidelines were compared with others in standard textbooks of surgery and peer-reviewed articles. The guidelines were prepared and revised by the Committee on Antimicrobial Agents of the CIDS. They were then reviewed and revised further by the Council of the CIDS. SPONSOR: The CIDS was solely responsible for developing, funding and endorsing these guidelines.

Anti-Bacterial Agents↗
PubMed1994

Protein kinase C activation accelerates proton extrusion by vacuolar-type H(+)-ATPases in murine peritoneal macrophages.

The role of protein kinase C in the regulation of vacuolar-type H(+)-ATPase (V-ATPase) activity was studied in thioglycolate-elicited mouse peritoneal macrophages. Acid-loaded macrophages suspended in a Na(+)- and HCO(3-)-free K(+)-medium containing Zn2+, a H(+)-conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 +/- 0.04 pH/min (n = 9). Pretreatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V-ATPase inhibitor, bafilomycin A1 (200 nM) indicating that the effect of the protein kinase C agonist was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4-phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V-ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V-ATPase-mediated proton extrusion by protein kinase C.

Animals↗
PubMed1994

Unilamellar liposomes modulate secretion of tumor necrosis factor by lipopolysaccharide-stimulated macrophages.

Liposomal encapsulation of antimicrobial agents has been used to improve drug delivery, particularly against intracellular pathogens. The effect of unilamellar liposomes on macrophage activation in response to Escherichia coli lipopolysaccharide was examined. Liposomes caused a dose- and time-dependent inhibition of tumor necrosis factor release by lipopolysaccharide-treated cells. The accumulation of tumor necrosis factor mRNA transcripts was unaffected, suggesting a posttranscriptional mechanism for this effect. However, induction of macrophage procoagulant activity was unaffected by liposomes, indicating a selective rather than a global inhibition. These data suggest that liposomes used for drug delivery may modulate the host response to infection.

Animals↗
PubMed1994

Endotoxin-stimulated alveolar macrophages impair lung epithelial Na+ transport by an L-Arg-dependent mechanism.

The Na+ transport function of alveolar epithelium represents an important mechanism for air space fluid clearance after acute lung injury. We studied the effect of endotoxin-stimulated rat alveolar macrophages on lung epithelial ion transport and permeability in vitro. Cultured rat distal lung (alveolar) epithelial monolayers incubated with both endotoxin and macrophages demonstrated a 75% decline in transepithelial resistance and a selective 60% reduction in amiloride-sensitive short-circuit current (Isc). Single-channel patch-clamp analysis demonstrated a 60% decrease in the density of 25-pS nonselective cation (NSC) channels on the apical membrane of epithelium exposed to both endotoxin and macrophages. A concurrent reduction in epithelial F-actin content suggested a role for actin depolymerization in mediating this effect. Incubation of cocultures with the methylated L-arginine (Arg) derivative NG-monomethyl-L-arginine prevented the reduction in epithelial Isc, as did substitution of L-Arg with D-Arg or incubation in L-Arg-free medium. Furthermore, the stable and products of Arg metabolism were found to have no effect on epithelial ion transport. These studies show that endotoxin-stimulated alveolar macrophages impair distal lung epithelial ion transport by an L-Arg-dependent mechanism by inactivating amiloride-sensitive 25-pS NSC channels. This may represent a novel mechanism whereby local inflammatory cells regulate lung epithelial ion transport. This could affect the ability of the lung to clear fluid from the air space.

Adenine↗
PubMed1994

Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations.

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

Animals↗
PubMed1994

In situ ischemia and hypoxia enhance alveolar macrophage tissue factor expression.

Alveolar and interstitial fibrin deposition is a prominent pathologic feature in many acute lung injury syndromes. Previous studies have suggested that ischemic lung preservation has a stimulatory effect on donor alveolar macrophages (Mphis) during transplantation. An animal model of lung preservation was developed to examine the hypothesis that ischemia enhances Mphi procoagulant activity (PCA) as a potential mechanism contributing to lung reperfusion injury. Histologic examination of ischemic lungs reperfused ex vivo revealed evidence of alveolar fibrin deposition. Mphis lavaged from lungs stored for at least 8 h at 21 degrees C exhibited increased PCA. The use of factor-deficient human plasma characterized this Mphi procoagulant as tissue factor (TF). Since increased PCA correlated with decreased airspace pO2 at the end of preservation, the effect of various O2 concentrations on PCA induction in vivo and in vitro was examined. Lung inflation during ischemia with decreasing O2 concentrations confirmed that hypoxia was associated with a rise in Mphi PCA in situ. However, in vitro exposure of Mphis to hypoxia did not increase Mphi PCA, suggesting that hypoxia alone was not responsible for induction of this procoagulant effect. Northern blot analysis demonstrated an increase in TF mRNA levels from in situ but not in vitro Mphis, thereby confirming transcriptional TF induction in this group. In addition, enhanced PCA was observed when Mphis were suspended in the bronchoalveolar lavage supernatant from the ischemic lungs stored at 21 degrees C. This suggests that in situ lung ischemia and hypoxia may produce soluble factors that either directly or indirectly stimulate Mphi TF expression. These factors may contribute to Mphi-mediated ischemic lung injury.

Animals↗
PubMed1994

Oxidant stress inhibits pH regulatory mechanisms in murine peritoneal macrophages.

BACKGROUND: Maintenance of cytoplasmic pH (pHi) close to the physiologic range is vital to normal cellular homeostasis. We have previously reported that a vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pHi regulatory mechanism. Since the inflammatory microenvironment is frequently characterized by the influx of cells known to release reactive oxygen metabolites, we performed studies to examine the effect of oxidant stress on pHi regulation in peritoneal macrophages. Specifically, the effect of hydrogen peroxide on V-ATPase-mediated proton extrusion from acid-loaded macrophages was investigated. METHODS: Thioglycollate-elicited murine peritoneal macrophages were exposed to varying concentrations of hydrogen peroxide and examined for their ability to recover from an acid-load. pHi was studied by preloading cells with the pH-sensitive fluorescent dye, bis-carboxyethyl-carboxyfluorescein, and monitoring changes in fluorescence under various conditions using a fluorescence spectrometer. RESULTS: Hydrogen peroxide caused a time- and dose-dependent decrease in proton pump-mediated pHi recovery in peritoneal macrophages. This effect occurred without cytotoxicity and was a specific effect as evidenced by the ability of catalase to reverse the inhibition. Since hydrogen peroxide is known to deplete intracellular ATP, a substrate for V-ATPase activity, we hypothesized that ATP depletion may underlie the effect. These studies showed that hydrogen peroxide-mediated ATP depletion was both necessary and sufficient for the effect. Finally, depletion of intracellular glutathione in vivo by using diethyl maleate increased the sensitivity of V-ATPase activity to oxidant stress. CONCLUSIONS: Oxidant stress within the inflammatory milieu impairs macrophage pHi regulation. This effect is magnified by depletion of intracellular antioxidants, as occurs during sepsis. This represents another mechanism whereby oxidants may contribute to cellular dysfunction associated with inflammatory states.

Adenosine Triphosphate↗
PubMed1994

Therapeutic options in peritonitis.

The fundamental principles of management of peritonitis include general supportive management, antibiotic therapy, and prompt surgical intervention. Several empiric antibiotic regimens have proven to be effective. All have in common activity against gram-negative bacilli and anaerobic bacteria. The choice of a particular regimen depends primarily on patient-specific factors. Surgical intervention includes source control, peritoneal toilet, and prevention of recurrent infection. Control of the source of contamination involves resecting, excluding, or patching the diseased viscus at laparotomy or, more recently, laparoscopic methods in selected cases. Attempts to prevent recurrent infection using intraoperative saline lavage and radical peritoneal débridement have shown no benefit in well-designed clinical trials. The roles for continuous postoperative peritoneal lavage, planned relaparotomy, and laparostomy in the current management of severe peritonitis remain to be established.

Anti-Bacterial Agents↗