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Biomedical subjects

L D Gray

Publications and source records attributed to L D Gray.

At least 19 recordsLinked to original sources
PubMed1996

Performance of Meridian ImmunoCard Mycoplasma test in a multicenter clinical trial.

Serology is the principal laboratory method used to diagnose Mycoplasma pneumoniae infection. Meridian Diagnostics has developed the ImmunoCard Mycoplasma kit, a 10-min card-based enzyme-linked immunosorbent assay (ELISA) designed to detect immunoglobulin M (IgM) antibodies to M. pneumoniae. We compared the ImmunoCard with two M. pneumoniae IgM-specific assays (immunofluorescence assay [IFA] and ELISA) and a standard complement fixation (CF) procedure using 896 specimens submitted to clinical laboratories for M. pneumoniae serology. Equivocal results obtained by CF, IFA, or ELISA were resolved by testing with an additional method or by reviewing patient chart information. The ImmunoCard had sensitivities ranging from 74% compared with the ELISA to 96% compared with CF results with IFA. ImmunoCard specificities ranged from 85% compared with the IgM-specific ELISA to 98% compared with IgM-specific IFA results resolved with clinical chart review. We also compared the ImmunoCard results with consensus results of 694 specimens tested on at least two non-ImmunoCard methods because of the lack of a "gold standard" for M. pneumoniae serology. Overall, the ImmunoCard Mycoplasma IgM assay had 90% sensitivity, 93% specificity, and 92% agreement with the consensus results. The ImmunoCard is technically less complex and requires less equipment that the three other assays. Our results indicate that the ImmunoCard Mycoplasma IgM assay is a valid and simple procedure which can reduce technologist time (and, thus, labor cost) and turnaround time for laboratories analyzing small numbers of specimens (< 10 per batch) submitted for IgM anti-M. pneumoniae testing.

Adult↗
PubMed1994

Interleukin-1-mediated release of interleukin-8 by asbestos-stimulated human pleural mesothelial cells.

The pleuropulmonary response to inhaled asbestos frequently involves inflammation and release of various cytokines from lung cells. Among these, interleukin-8 (IL-8) released from the mesothelium could augment inflammation of the pleura by attracting neutrophils to the pleural space. We used cultures of human pleural mesothelial cells (HPMC) to examine the mechanism of IL-8 production by asbestos and cytokines. Suspensions of amosite, chrysotile, or crocidolite asbestos in concentrations as low as 5 micrograms/ml enhanced release of IL-8 from HPMC during 6 h of incubation at 37 degrees C. Electron microscopy of asbestos-treated HPMC showed that the cells avidly engulfed each of the different types of asbestos fibers. Two proinflammatory cytokines, interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha, enhanced IL-8 release within 2 h and had an even greater effect after 6 h. Release of IL-8 was measured by an enzyme-linked immunosorbent assay, and functional activity of the cytokine was assessed by chemotaxis of human neutrophils. Identity of IL-8 in HPMC supernatants was established by absorption with an antibody to IL-8. Preincubation of HPMC with IL-1 receptor antagonist (IL-1ra) significantly decreased release of IL-8 after stimulation with amosite or crocidolite asbestos. We conclude that HPMC release IL-8 in response to asbestos stimulation and that the response is, in part, mediated by IL-1, mainly in the form of IL-1 alpha.

Antibodies, Monoclonal↗
PubMed1993

A bacterial protease perturbs the paracellular barrier function of transporting epithelial monolayers in culture.

Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the epithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas elastase (PE) on the barrier function of epithelia by using Madin-Darby canine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. Immunostaining of selected proteins of the ZO and zonula adherens was used to explore the effects of PE on junctional proteins. PE-treated monolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated that, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescence staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zonula adherens, was unaffected by the enzyme. Elastases from porcine pancreas and human leukocytes with similar enzymatic activity (6 U/ml) did not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithelial monolayers, in part, by changing the cell architecture and altering at least one protein of the ZO.

Animals↗
PubMed1992

Laboratory diagnosis of bacterial meningitis.

Bacterial meningitis is relatively common, can progress rapidly, and can result in death or permanent debilitation. This infection justifiably elicits strong emotional reactions and, hopefully, immediate medical intervention. This review is a brief presentation of the pathogenesis of bacterial meningitis and a review of current knowledge, literature, and recommendations on the subject of laboratory diagnosis of bacterial meningitis. Those who work in clinical microbiology laboratories should be familiar with the tests used in detecting bacteria and bacterial antigens in cerebrospinal fluid (CSF) and should always have the utmost appreciation for the fact that results of such tests must always be reported immediately. Academic and practical aspects of the laboratory diagnosis of bacterial meningitis presented in this review include the following: anatomy of the meninges; pathogenesis; changes in the composition of CSF; etiological agents; processing CSF; microscopic examination of CSF; culturing CSF; methods of detecting bacterial antigens and bacterial components in CSF (counter-immunoelectrophoresis, coagglutination, latex agglutination, enzyme-linked immunosorbent assay, Limulus amebocyte lysate assay, and gas-liquid chromatography); use of the polymerase chain reaction; and practical considerations for testing CSF for bacterial antigens.

Animals↗
PubMed1992

Aerosolized Pseudomonas elastase and lung fluid balance in anesthetized sheep.

The role of the lung epithelium in lung fluid balance was studied by ventilating anesthetized sheep with an aerosol of 20 mg of elastase from Pseudomonas aeruginosa (Ps. elastase) to increase lung epithelial permeability without affecting lung endothelial permeability or lung vascular pressures. Ps. elastase had no effect on the lung vascular pressures, the alveolar-arterial PO2 gradient (A-aPO2), the flow or protein concentration of the lung lymph, or the postmortem water volume of the lungs. The morphological alveolar flooding score in these sheep was 2.5 times the control level, but this was only marginally significant. Elevation of the left atrial pressure by 20 cmH2O alone increased the postmortem lung water volume but had no effect on A-aPO2, the alveolar flooding score, or the lung epithelial permeability assessed by the clearance of 99mTc-labeled human serum albumin. Addition of aerosolized Ps. elastase to these sheep had no effect on the total lung water volume, but it caused a redistribution of water into the air spaces, as evidenced by significant increases in the alveolar flooding score and A-aPO2 (P less than 0.01). Elevation of the left atrial pressure by 40 cmH2O without elastase caused the same response as elevation of the left atrial pressure by 20 cmH2O with elastase, except the higher pressure caused a greater increase in the total lung water volume. We conclude that alteration of the integrity of the lung epithelium with aerosolized Ps. elastase causes a redistribution of lung water into the alveoli without affecting the total lung water volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosols↗
PubMed1990

Neutral endopeptidase from nuchal ligament of fetal calves.

The nuchal ligament of unborn calves contains a neutral endopeptidase that is biochemically and immunologically similar to the neutral endopeptidase (NEP), or enkephalinase, from human kidney. Enzymatic activity was inhibited more than 90% by phosphoramidon (1 microM). The specific activity in membrane fractions, as determined by hydrolysis of the dansylated substrate, DAPGN, was similar in tissue from fetuses of gestational ages ranging from 100 to 280 days. NEP activity in adult ligament tissue, however, was less than 10% of that in fetal tissue. Fibroblasts dissociated from ligament tissue by collagenase displayed less NEP activity than did preparations of intact ligament, and activity was even lower in cultured cells. By contrast, fibroblasts cultured from fetal calf lungs had NEP activity comparable to that in the ligament tissue. When ligament fibroblasts were cultured on subcellular matrices derived from fetal lung fibroblasts the NEP activity increased relative to those cultured on plastic alone. These studies confirm the presence of neutral endopeptidase (NEP) in the nuchal ligament of the fetal calf. The consistent activity through a range of gestational ages and the influence of the subcellular matrix suggest that this enzyme might be involved in growth of the ligament during fetal life.

Animals↗
PubMed1990

Effects of Pseudomonas aeruginosa elastase on alveolar epithelial permeability in guinea pigs.

Elastase-deficient mutants of Pseudomonas aeruginosa are less virulent than the wild type and are easily cleared from the lungs of guinea pigs. The effect of P. aeruginosa elastase on lung epithelium, however, is not yet understood. We addressed the hypothesis that breach of the epithelial barrier by elastase from P. aeruginosa allows invading organisms and toxic substances to penetrate the interstitium. We measured the clearance of aerosolized technetium-labeled albumin (molecular weight, 69,000) from the lungs of anesthetized guinea pigs with the aid of a gamma camera and a dedicated computer. Aerosols of the elastase (0.1 to 5 micrograms) increased the rate of clearance of labeled albumin from the lungs in proportion to the elastase dose. Electron microscopic studies using horseradish peroxidase as a tracer revealed that elastase interrupts intercellular tight junctions of the epithelial lining, thereby increasing the permeability to macromolecules. The amounts of elastase used in this report did not cause interstitial or alveolar edema, as determined by both postmortem extravascular lung water volume measurement and morphological examination. The data indicate that the elastase is a potentially important virulence factor in acute lung infection.

Animals↗
PubMed1990

Liberation of a neutrophil enzyme-releasing peptide from the surface of human alveolar macrophages.

The human alveolar macrophage product, enzyme-releasing peptide (ERP), has a molecular mass of 8,000 Da, and releases azurophilic and specific granule constituents from neutrophils. A murine monoclonal anti-ERP antibody (12E10H), previously used to show a lack of antigenic identity between ERP and C5a, interleukin 1, tumor necrosis factor, and gamma-interferon, showed no cross-reactivity with interleukin 8. 12E10H and a fluorescein-labeled second antibody were used to visualize ERP on the macrophage surface. ERP was removed from alveolar macrophages by a 3-min incubation with 5 X 10(-7) M bovine pancreatic trypsin at 37 degrees C. The washed trypsinized cells could readhere to plastic and exclude trypan blue. Dilution of the trypsin-derived ERP released myeloperoxidase from cytochalasin-B-treated neutrophils dose dependently. The enzyme-releasing ability of the trypsin-derived material was removed by immunoprecipitation using antibody 12E10H bound to Staphylococcal protein A Sepharose 4B. The estimated molecular mass of the trypsin-derived ERP (by molecular sieve chromatography on HPLC) was approximately 8,500 Da. Other proteases (plasmin, thrombin, and cathepsin G) also released ERP from the cell surface, but the ERP was not an active secretagogue for neutrophils. However, macrophages cultured with protease inhibitors did not show decreased ERP accumulation in the medium. Our data indicate that ERP exists on the surface of human alveolar macrophages and can be released by proteases found within the lung environment in some disease states.

Antibodies, Monoclonal↗
PubMed1990

A modified bronchoalveolar lavage procedure that allows measurement of lung epithelial lining fluid volume.

We replaced the standard serial bronchoalveolar lavage technique with a new "rewash" lavage procedure to allow estimation of the volume and protein concentration of the epithelial lining fluid (ELF) in anesthetized sheep. A bronchoscope 6.0 mm in diameter wedged in an airway was used to lavage a segment of lung with four cycles of instillation and aspiration of the lavage solution containing a radioactive tracer (technetium pertechnetate, 99mTcO4-). Errors caused by the fall in concentration of the tracer during the lavage were minimized by extrapolating the tracer concentration back to time zero when the lavage solution had mixed with the ELF, but had not had time to be affected by loss of the tracer or influx of fluid from the interstitium. In control sheep, the ELF of these lavaged segments had a mean volume of 1.6 +/- 1.0 ml and a mean protein concentration that was 26 +/- 19% of the protein concentration measured in the plasma. Increasing the left atrial pressure 19 +/- 5 cm H2O to cause "cardiac lung edema" had no significant effect on the ELF volume, but it increased the mean protein concentration to 57 +/- 30% of the plasma value (p less than 0.01). Lung injury caused by intravenous oleic acid caused lung edema, increased the mean ELF volume to 6.8 +/- 2.2 ml, and increased the mean ELF protein concentration to 86 +/- 26% of the plasma value (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Detection of Vibrio vulnificus cytolysin in V. vulnificus-infected mice.

Enzyme-linked immunosorbent assays using polyclonal and monoclonal antibodies specific for V. vulnificus cytolysin detected the toxin in an extract of skin lesions and in serum from mice showing local and systemic V. vulnificus disease after subcutaneous injection of the bacterium. The cytolysin also was detected in skin lesions by an indirect immunofluorescence procedure using polyclonal and monoclonal antibodies. Our findings provide direct evidence that the cytolysin is produced in vivo during the development of the disease process, and this observation is consistent with the hypothesis that the toxin is involved in the pathogenesis of V. vulnificus disease.

Animals↗
PubMed1989

Wound infection caused by Branhamella catarrhalis.

Branhamella catarrhalis was isolated from sputum, tracheal secretions, and a nonhealing and infected thoracic surgical wound in a 59-year-old woman who had a history of a chronic, interstitial fibrosis and who had undergone an open lung biopsy procedure. The patient's upper respiratory tract was the likely source of the organism. To our knowledge, this is the first report of a wound infection caused by B. catarrhalis.

Biopsy↗
PubMed1988

Isolated levator myositis.

A 54-year-old woman presented with a 1-day history of ptosis of the left upper lid. On examination, the patient exhibited a moderate ptosis, poor levator function, lid lag on down-gaze, and no limitation of ocular motility. A computed tomography (CT) scan with contrast demonstrated enhancement of the levator muscle and levator aponeurosis on the involved side. Treatment consisted of systemic steroid administration and led to complete resolution of the ptosis in 2 weeks. The combined clinical and CT scan findings give a characteristic pattern of an isolated levator myositis.

Blepharoptosis↗
PubMed1988

Conservation of protective and nonprotective epitopes in M proteins of group A streptococci.

Carefully controlled hybridization experiments with probes from a cloned serotype 5 M protein (M5) gene (smp5) were performed with DNA isolated from heterologous M types of group A streptococci, and the homologies detected by hybridization were compared with the ability of anti-pepM5 serum to cross-opsonize heterologous M types. As previously reported (J.R. Scott, S.K. Hollingshead, and V.A. Fischetti, Infect. Immun. 52:609-612, 1986), extensive structural homologies exist among the 3' ends of heterologous M protein genes, but there appears to be an increase in sequence variation as one moves towards the 5' ends. However, a clear, predictive correlation between the hybridization patterns and cross-opsonization was not observed. Antibodies raised to a synthetic peptide corresponding to central, conserved sequences adjacent to the C-terminal sides of the pepsin cleavage sites in M5, serotype 6 M protein, and serotype 24 M protein cross-reacted with heterologous acid-extracted M antigens but were not protective and did not bind to intact streptococcal cells, indicating that these epitopes are inaccessible on the intact cell surface. Removal of the N-terminal half of M5, serotype 6 M protein, or serotype 24 M protein by pepsin exposed the conserved epitope on the cell surface. These results suggest that immunoaccessible protective epitopes are confined to the highly variable N-terminal halves of M proteins and that a single, broadly conserved protective M protein epitope does not exist.

Amino Acid Sequence↗
PubMed1988

Experience with the use of pronase to eliminate interference factors in the latex agglutination test for cryptococcal antigen.

Cryptococcal antigen titers of 70 cerebrospinal fluid and 57 serum specimens from patients suspected of having cryptococcosis were determined both before and after treatment with pronase. Median titers of cerebrospinal fluid specimens before and after treatment were 128 and 128, respectively; mean geometric titers of these specimens before and after treatment were 102 and 204, respectively. Median titers of the serum specimens before and after treatment were 16 and 512, respectively; mean geometric titers of these specimens before and after treatment were 19 and 631, respectively. The modified latex agglutination test did not detect antigen in any of 50 cerebrospinal fluid and 51 serum specimens from patients not suspected of having cryptococcosis. These results suggest that the pronase modification increases the sensitivity of the latex agglutination test and that the modification be routinely incorporated into it.

Agglutination Tests↗
PubMed1988

Asbestos-induced endothelial cell activation and injury. Demonstration of fiber phagocytosis and oxidant-dependent toxicity.

Vascular endothelial cell injury is important in the development of a variety of chronic interstitial lung disorders. However, the involvement of such injury in the inflammatory response associated with the inhalation of asbestos fibers is unclear and the mechanism of asbestos fiber cytotoxicity remains unknown. In the present study, human umbilical vein endothelial cells were challenged with amosite asbestos and several parameters of cellular function were examined. Electron microscopic examination revealed that endothelial cell exposure to asbestos resulted in active phagocytosis of these particulates. Biochemical evidence of dose-dependent asbestos-mediated endothelial cell activation was indicated by increased metabolism of arachidonic acid. For example, amosite asbestos (500 micrograms/ml) produced a ninefold increase in prostacyclin (PGI2) levels over those levels in non-exposed cells. Incubation of human endothelial cells with asbestos fibers induced specific 51Cr release in both a dose- and time-dependent fashion indicative of cellular injury. Injury induced by amosite asbestos was not significantly attenuated by treatment of the endothelial cell monolayer with either the iron chelator deferoxamine, which prevents hydroxyl radical (.OH) formation, or by the superoxide anion (O2-) scavenger, superoxide dismutase. However, significant dose-dependent protection was observed with the hydrogen peroxide (H2O2) scavenger, catalase. Chelation of elemental iron present within amosite asbestos fibers by deferoxamine produced a 33% reduction in asbestos cytotoxicity, suggesting a potential role for hydroxyl radical-mediated injury via the iron-catalyzed Haber-Weiss reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Local abnormalities of coagulation and fibrinolysis and alveolar fibrin deposition in sheep with oleic acid-induced lung injury.

Extravascular, primarily intra-alveolar, fibrin deposition is a histologic hallmark of acute lung injury in humans and experimental animals, but the mechanisms leading to this finding are poorly understood. To determine whether local abnormalities in the fibrinolytic-procoagulant balance contribute to alveolar fibrin deposition in acute lung injury, we studied bronchoalveolar lavage (BAL) fluids of anesthetized sheep that received intravenous oleic acid. Prominent alveolar fibrin deposition was observed within 2 h after oleic acid-induced lung injury. Procoagulant and fibrinolytic activities were determined in BAL samples of anesthetized, mechanically ventilated sheep before and 2 h after intravenous oleic acid or saline. BAL procoagulant activity was found to be due mainly to tissue factor associated with Factor VII. In baseline BAL samples, we found relatively low levels of procoagulant activity and relatively high levels of fibrinolytic activity. After induction of oleic acid-induced lung injury, the procoagulant activity of BAL was markedly increased, whereas fibrinolytic activity was either depressed or undetectable. Antiplasmin activity was detectable in BAL of sheep after oleic acid-induced lung injury, which contributed at least in part to the depressed fibrinolytic activity observed. These perturbations occurred with the appearance of extensive alveolar fibrin deposition. In control sheep, BAL fibrinolytic activity was decreased, and antiplasmin activity increased modestly after 2 h of mechanical ventilation, but procoagulant activity was unchanged and alveolar fibrin was not observed. Procoagulant activity in lung lymph and plasma after lung injury did not differ from baseline values, and fibrinolytic activity was undetectable in lymph or plasma samples. These data indicate that increased procoagulant activity and concurrent disruption of the balance of coagulation and fibrinolysis establish local conditions that promote acute fibrin deposition in the alveoli of mechanically ventilated, oleic acid-injured sheep.

Animals↗