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Biomedical subjects

C Nathan

Publications and source records attributed to C Nathan.

At least 19 recordsLinked to original sources
PubMed1997

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase.

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.

Animals↗
PubMed1997

A novel antioxidant gene from Mycobacterium tuberculosis.

Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.

Amino Acid Sequence↗
PubMed1997

Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide.

To explore regulation of potentially lethal responses to bacterial lipopolysaccharide (LPS), we used differential display under LPS-free conditions to compare macrophage cell lines from two strains of mice congenic for a locus affecting LPS sensitivity. LPS-hyporesponsive cells, primary macrophages, and polymorphonuclear leukocytes transcribed secretory leukocyte protease inhibitor (SLPI), a known epithelial cell-derived inhibitor of leukocyte serine proteases. Transfection of macrophages with SLPI suppressed LPS-induced activation of NF-kappa B and production of nitric oxide and TNF alpha. The ability of interferon-gamma (IFN gamma) to restore LPS responsiveness is a hallmark of the LPS-hyporesponsive phenotype. IFN gamma suppressed expression of SLPI and restored LPS responsiveness to SLPI-producing cells. Thus, SLPI is an LPS-induced IFN gamma-suppressible phagocyte product that serves to inhibit LPS responses.

Amino Acid Sequence↗
PubMed1997

The effect of epidermal growth factor on the septic complications of acute pancreatitis.

Bacterial translocation (BT) from the gastrointestinal tract to mesenteric lymph nodes (MLN) and other extraintestinal organs is an important source of infection in acute pancreatitis (AP). Epidermal growth factor (EGF), a peptide hormone with trophic effects on gut mucosa, has decreased intestinal mucosal injury in septic rats and decreased burn-induced BT in mice. The purpose of this study is to examine whether EGF could affect BT in acute necrotizing pancreatitis. Forty-eight male Sprague-Dawley rats (250-350 g) were studied. AP was induced in Group I and Group II by pressure injection of 3% taurocholate and trypsin into the biliopancreatic duct (1 ml/kg of body weight). Group III and Group IV underwent laparotomy without induction of acute pancreatitis. Group I rats received human recombinant EGF (100 micrograms/kg, subcutaneously twice daily) and Group II rats received a similar volume of 0.1% bovine serum albumin as a placebo postoperatively. Group III and Group IV received EGF and placebo, respectively. At 48 hr postoperatively, blood was drawn for culture and amylase determinations. Jejunum and ileum were obtained to measure mucosal protein content, mucosal thickness, villus height, and crypt depth. Specimens from MLN, spleen, liver, pancreas, and cecum were harvested for pathology and culture of gram positive (G+), gram negative (G-), and anaerobic bacteria. Ileal mucosal protein levels were increased significantly in Group I (1.96 +/- 0.14 mg/cm) compared to Group II (0.95 +/- 0.15 mg/cm intestinal segment) (P < 0.01). Jejunal and ileal mucosal thickness, villus height, and crypt depth in Group I were significantly increased when compared to Group II (P < 0.05). All 12 rats in Group II had BT to MLN compared to 58% (7 of 12 rats) in Group I (P < 0.05). Thirty-three percent (4 of 12 rats) had BT to distant sites such as pancreas, spleen, liver, and/or blood in Group I vs 83% (10 of 12 rats) in Group II (P < 0.05). EGF treatment minimizes intestinal damage, decreases BT to MLN and bacterial spread to distant sites, and may be beneficial in preventing septic complications in AP.

Acute Disease↗
PubMed1997

Recovery of nosocomial fecal flora from frozen stool specimens and rectal swabs: comparison of preservatives for epidemiological studies.

The recovery of antibiotic-susceptible and -resistant aerobic Gram-negative bacilli from stool specimens and from mock rectal swabs after freezing (-20 degrees C) for as long as 4 weeks was studied using three preservatives: Cary-Blair (CB) transport medium, buffered glycerol saline (BGS), and Para Pak C&S solution (CS). In addition, the recovery of enterococci from rectal swabs was investigated after storage of swabs in Stuart's transport media at 4 degrees C as long as 4 weeks. The log10 decreases in bacterial counts from seeded stool suspensions frozen in BGS were 0.64 (i.e., fourfold) and 1.16 after 1 and 4 weeks, respectively, which were significantly less (p < .05) than 1 and 4 week decreases following freezing in CB (1.57 and 2.85) or in CS (1.50 and 2.45). The recovery of Gram-negative bacilli from patients' rectal swabs preserved in BGS was consistent with the results of the experiments with seeded stool suspensions. There was no detectable decrease in recovery of enterococci from rectal swabs stored at 4 degrees C. BGS performed well as a preservative for freezing stool specimens or rectal swabs for later recovery of nosocomial Gram-negative bacilli; enterococci survived well in refrigerated rectal swab specimens.

Bacteriological Techniques↗
PubMed1997

Skin colonization with vancomycin-resistant enterococci among hospitalized patients with bacteremia.

To assess the prevalence of skin and rectal colonization by vancomycin-resistant enterococci (VRE) in hospitalized bacteremic patients and to determine the relation between colonization and bacteremia, we compared 14 case patients who had bacteremia due to VRE with 30 control patients who had bacteremia due to other pathogens. Rectal colonization and skin (inguinal area and/or antecubital fossa) colonization with VRE were common among both case patients (100% had rectal colonization, and 86% had skin colonization) and control patients (37% had rectal colonization and 23% had skin colonization). Among patients with rectal colonization, skin colonization was more common when diarrhea or fecal incontinence was present. The bloodstream cleared without appropriate antimicrobial therapy in nine of the 14 patients with bacteremia due to VRE. The high prevalence of skin colonization with VRE may increase the risk of catheter-related sepsis, cross-infection, or blood culture contamination (which may explain the frequent spontaneous resolution of bacteremia due to VRE).

Anti-Bacterial Agents↗
PubMed1997

Inducible nitric oxide synthase-deficient mice have enhanced leukocyte-endothelium interactions in endotoxemia.

Nitric oxide (NO) from constitutive NO synthase (NOS) has been postulated to be a homeostatic regulator of leukocyte-endothelial cell interactions. By contrast, the inducible NO synthase (iNOS) isoform has been invoked as a potential pathogenic enzyme in numerous inflammatory diseases. The objective of this study was to determine whether the iNOS isoform is also capable of functioning as a regulator of leukocyte recruitment. Mice received endotoxin (LPS, 30 microg/kg, i.v.); 2-4 h later, intravital microscopy was used to examine leukocyte rolling and adhesion in postcapillary venules of the cremaster muscle and the sinusoids and postsinusoidal venules of the hepatic microcirculation. Leukocyte recruitment into the lung was also examined. RT-PCR confirmed that this treatment induced iNOS mRNA expression in wild-type mice as early as 2 h after LPS treatment. Between 2 and 4 h after LPS administration, the number of rolling and adherent leukocytes in cremasteric postcapillary venules and of adherent cells in liver postsinusoidal venules of iNOS-deficient mice were significantly higher than in wild-type mice. Leukocyte accumulation in the lung (measured by myeloperoxidase assay) was also significantly elevated in iNOS-deficient animals. These effects could not be attributed to differences in systemic blood pressure, shear rates, circulating leukocyte numbers, or baseline levels of rolling and adhesion because these parameters were not different between the two groups. To establish whether the differences in leukocyte recruitment were related to the leukocytes per se, perfusion of iNOS+/+ or iNOS-/- septic blood over purified E-selectin (using parallel plate flow chambers) revealed much larger recruitment of iNOS-/- leukocytes. These results suggest that iNOS induced in response to LPS releases NO that is capable of reducing leukocyte accumulation by affecting leukocytes directly and raises the possibility that induction of iNOS is a homeostatic regulator for leukocyte recruitment.

Analysis of Variance↗
PubMed1997

Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay.

To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S-nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91-kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.

Animals↗
PubMed1997

Nitric oxide and macrophage function.

At the interface between the innate and adaptive immune systems lies the high-output isoform of nitric oxide synthase (NOS2 or iNOS). This remarkable molecular machine requires at least 17 binding reactions to assemble a functional dimer. Sustained catalysis results from the ability of NOS2 to attach calmodulin without dependence on elevated Ca2+. Expression of NOS2 in macrophages is controlled by cytokines and microbial products, primarily by transcriptional induction. NOS2 has been documented in macrophages from human, horse, cow, goat, sheep, rat, mouse, and chicken. Human NOS2 is most readily observed in monocytes or macrophages from patients with infectious or inflammatory diseases. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths, and tumor cells. The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as acid, glutathione, cysteine, hydrogen peroxide, or superoxide. Although the high-output NO pathway probably evolved to protect the host from infection, suppressive effects on lymphocyte proliferation and damage to other normal host cells confer upon NOS2 the same protective/destructive duality inherent in every other major component of the immune response.

Animals↗
PubMed1996

Epidemiology of colonisation of patients and environment with vancomycin-resistant enterococci.

BACKGROUND: Vancomycin-resistant enterococci (VRE) have emerged as nosocomial pathogens during the past 5 years, but little is known about the epidemiology of VRE. We investigated colonisation of patients and environmental contamination with VRE in an endemic setting to assess the importance of different sources of colonisation. METHODS: Between April 12, and May 29, 1995, cultures from body sites (rectum, groin, arm, oropharynx, trachea, and stomach) and from environmental surfaces (bedrails, drawsheet, blood-pressure cuff, urine containers, and enteral feed) were obtained daily from all newly admitted ventilated patients in our medical intensive-care unit (MICU). Rectal cultures were obtained from all non-ventilated patients in the MICU. Strain types of VRE were determined by pulsed-field gel electrophoresis. FINDINGS: There were 97 admissions of 92 patients, of whom 38 required mechanical ventilation. Colonisation with VRE on admission was more common in ventilated than in non-ventilated patients (nine [24%] vs three [6%], p < 0.05). Of the nine ventilated patients colonised with VRE on admission, one acquired a new strain of VRE in the MICU. Of the 29 ventilated patients who were not colonised with VRE on admission, 12 (41%) acquired VRE in the MICU. The median time to acquisition of VRE was 5 days (interquartile range 3-8). Of the 13 ventilated patients who acquired VRE, 11 (85%) were colonised with VRE by cross-colonisation. VRE were isolated from 157 (12%) of 1294 environmental cultures. The rooms of 13 patients were contaminated with VRE, but only three (23%) of these patients subsequently acquired colonisation with VRE. Pulsed-field gel electrophoresis of 262 isolates showed 20 unique strain types of VRE. INTERPRETATION: Frequent colonisation with VRE on MICU admission and subsequent cross-colonisation are important factors in the endemic spread of VRE. Persistent VRE colonisation in the gastrointestinal tract and on the skin, the presence of multiple-strain types of VRE, and environmental contamination may all contribute to the spread of VRE.

Adult↗
PubMed1996

Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer's disease.

In Alzheimer's disease (AD), affected neurons accumulate beta amyloid protein, components of which can induce mouse microglia to express the high-output isoform of nitric oxide synthase (NOS2) in vitro. Products of NOS2 can be neurotoxic. In mice, NOS2 is normally suppressed by transforming growth factor beta 1 (TGF-beta 1). Expression of TGF-beta 1 is decreased in brains from AD patients, a situation that might be permissive for accumulation of NOS2. Accordingly, we investigated the expression of NOS2 in patients with AD, using three monospecific antibodies: a previously described polyclonal and two new monoclonal antibodies. Neurofibrillary tangle-bearing neurons and neuropil threads contained NOS2 in brains from each of 11 AD patients ranging in age from 47 to 81 years. NOS2 was undetectable in brains from 6 control subjects aged 23-72 years, but was expressed in small amounts in 3 control subjects aged 77-87 years. Thus, human neurons can express NOS2 in vivo. The high-output pathway of NO production may contribute to pathogenesis in AD.

Adult↗
PubMed1996

A comparison of the effect of universal use of gloves and gowns with that of glove use alone on acquisition of vancomycin-resistant enterococci in a medical intensive care unit.

OBJECTIVE: To determine the efficacy of the use of gloves and gowns compared with that of the use of gloves alone for the prevention of nosocomial transmission of vancomycin-resistant enterococci. DESIGN: Epidemiologic study and controlled, nonrandomized clinical trial. SETTING: University-affiliated, 900-bed, urban teaching hospital in which vancomycin-resistant enterococci are endemic. PATIENTS: 181 consecutive patients admitted to the medical intensive care unit for 48 hours or more. INTERVENTION: It was determined that all hospital employees would always use gloves and gowns when attending 8 particular beds in the medical intensive care unit and would always use gloves alone when attending 8 others. Compliance with precautions was monitored weekly. Rectal surveillance cultures were taken from patients daily. Cultures of environmental surfaces, such as those of bed rails, bedside tables, and other frequently touched objects in patient rooms and common areas, were taken monthly. Pulsed-field gel electrophoresis was used for molecular epidemiologic typing of vancomycin-resistant enterococci. MEASUREMENTS: The number of patients becoming colonized by vancomycin-resistant enterococci; the number of days to acquisition of vancomycin-resistant enterococci; and other measurements, including nosocomial infections, length of hospital stay, and mortality rates. RESULTS: The 93 patients in glove-and-gown rooms and the 88 patients in glove-only rooms had similar demographic and clinical characteristics. Fifteen (16.1%) patients in the glove-and-gown group and 13 (14.8%) in the glove-only group had vancomycin-resistant enterococci on admission to the medical intensive care unit. Twenty-four (25.8%) patients in the glove-and-gown group and 21 (23.9%) in the glove-only group acquired vancomycin-resistant enterococci in the medical intensive care unit. The mean times to colonization among the patients who became colonized were 8.0 days in the glove-and-gown group and 7.1 days in the glove-only group. None of these comparisons were statistically significant. Risk factors for acquisition of vancomycin-resistant enterococci induced length of stay in the medical intensive care unit, use of enteral feeding, and use of sucralfate. Compliance with precautions was 79% in glove-and-gown rooms and 62% in glove-only rooms (P < 0.001). Only 25 of 397 (6.3%) environmental cultures were positive for vancomycin-resistant enterococci. Nineteen types of vancomycin-resistant enterococci were documented by pulsed-field gel electrophoresis during the study period. CONCLUSIONS: Universal use of gloves and gowns was no better than universal use of gloves only in preventing rectal colonization by vancomycin-resistant enterococci in a medical intensive care unit of a hospital in which vancomycin-resistant enterococci are endemic. Because the use of gowns and gloves together may be associated with better compliance and may help prevent transmission of other infectious agents, this finding may not be applicable to outbreaks caused by single strains or hospitals in which the prevalence of vancomycin-resistant enterococci is low.

Adult↗
PubMed1996

Inducible nitric oxide synthase requires both the canonical calmodulin-binding domain and additional sequences in order to bind calmodulin and produce nitric oxide in the absence of free Ca2+.

All three mammalian isoforms of nitric oxide synthase (NOS) must bind calmodulin (CaM) for enzymatic activity. Only NOS2 (the inducible isoform, iNOS) does so at the low levels of free Ca2+ in resting cells and when almost all Ca2+ is chelated in cell-free preparations. To test directly whether the predicted CaM-binding region of mouse NOS2 accounts for its Ca2+ independence, we prepared chimeric NOS's in which mouse NOS2 residues 503-532 were reciprocally exchanged with the corresponding residues 725-754 of rat NOS1 (neuronal NOS). Unlike either parent, both chimeras required an intermediate level of free Ca2+ to bind CaM and generate NO. In cell lysates, the concentration of Ca2+ necessary for half-maximal activity (EC50) was approximately 0 for NOS2, 200-300 n for NOS1, and 7-10 n for the chimeras. Results were similar when the region exchanged was enlarged by 7-8 residues toward the amino terminus. In contrast, when the carboxyl-terminal half of NOS2 (residues 454-1144) was replaced with that of NOS1 (residues 675-1429), the resulting chimera resembled NOS1 (EC50, 200-300 n free Ca2+). Truncation analysis suggested that NOS2 residues within the sequence 484-726 were required for Ca2+-independent CaM-binding. Thus, both the canonical CaM-binding domain and additional residues within the region 484-726 are necessary for NOS2's ability to bind CaM and produce NO when Ca2+ levels approach zero.

Amino Acid Sequence↗
PubMed1996

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.

Animals↗
PubMed1996

Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis.

The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.

Amino Acid Sequence↗
PubMed1996

Ceramide selectively inhibits early events in the response of human neutrophils to tumor necrosis factor.

Cell spreading and the respiratory burst of neutrophils responding to soluble, physiological agents and adherent to model biological surfaces are typically delayed in onset by 15 min or more. The lag period may be a physiologically important feature of the action of such agents on neutrophils in that it may allow for migration before secretion. However, the mechanism that interposes such a long delay between stimulus and response is unknown. Tumor necrosis factor alpha (TNF-alpha) mediates some of its actions by triggering sphingomyelinase to generate ceramide. In adherent human neutrophils, however, exogenous ceramide did not mimic TNF-alpha's ability to stimulate cell spreading, paxillin tyrosine phosphorylation, or the respiratory burst. On the contrary, ceramide suppressed each such response. Ceramide did so by extending the lag period in the cells' response to TNF-alpha. Ceramide extended the lag period whether it was added exogenously or generated endogenously by sphingomyelinase, and the effect was reversible. Remarkably, however, ceramide inhibited cell spreading or the respiratory burst only if added together with TNF-alpha or within the next few minutes. Neutrophils ignored ceramide if it was added later, even if the TNF-alpha-triggered respiratory burst had not yet commenced. These features suggest that an early, brief elevation of ceramide in response to TNF-alpha could mediate the lag period. By temporarily inhibiting tyrosine phosphorylation, cell spreading, and the respiratory burst, ceramide or a functionally similar mediator could serve as a phase coordinator of the neutrophil's response to soluble agonists.

Adult↗