Biomedical subjects

L E Martin

Publications and source records attributed to L E Martin.

At least 19 recordsLinked to original sources

PubMed1990

The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population.

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.

Amino Acid Sequence↗

PubMed1990

A 59 kiloDalton outer membrane protein of Salmonella typhimurium protects against oxidative intraleukocytic killing due to human neutrophils.

We have isolated a Salmonella typhimurium (ST) mutant, JKS400, deficient in the production of a surface-exposed outer membrane protein (Omp) and phenotypically hypersensitive to the oxidative antimicrobial mechanism of polymorphonuclear leukocytes (PMNs). This Omp migrated at approximately 59 kiloDaltons (kD) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). We found with P22 transduction that the capacities to produce the protein and to exert wild-type resistance to oxidative killing were tightly linked. Transduction of JKS400 with a P22(HT)int- bacteriophage grown on a Tn10 insertion library in LT2 yielded tetracycline-resistant isolates that had been returned to wild-type protein production. Further experiments showed that restoration of protein production was accompanied by restoration of the parental resistance phenotype to killing by PMNs and by restoration to wild-type resistance to H2O2. The map position of the Tn10 was determined to be at 96 minutes in the Salmonella chromosome. This protein appears to behave as a virulence factor, promoting the capacity of Salmonella typhimurium LT2 to survive oxygen-dependent killing mechanisms in neutrophils.

Acid Phosphatase↗

PubMed1990

CAP37, a human neutrophil-derived chemotactic factor with monocyte specific activity.

CAP37, an antimicrobial protein of human neutrophil granules, is a specific chemoattractant for monocytes. Purified to homogeneity by sequential chromatography over carboxymethyl Sephadex, G-75 Sephadex, and hydrophobic interaction HPLC, demonstratively endotoxin-free CAP37 was maximally chemotactic over a range of 1.3 X 10(-9)-10(-8) M. Thus it was active in the same molar concentrations as formyl-methionyl-leucyl-phenylalanine. CAP37 lacked chemotactic activity for neutrophils and lymphocytes. In checkerboard assays CAP37 had some chemokinetic activity as well. It was also chemotactic for rabbit mononuclear cells. Higher concentrations (2.7 X 10(-8) M) were required for activity with rabbit cells than with human. Sequence analysis of the first 42 NH2-terminal amino acid residues of CAP37 showed strong homologies with known serine proteases that mediate various functions in inflammation. However, a critical substitution of a serine for a histidine at position 41 suggested that CAP37 lacked serine protease action. This impression was supported by the failure of CAP37 to bind tritiated diisopropyl fluorophosphate. 89% of total CAP37 was released extracellularly from human neutrophils while they phagocytized Staphylococcus aureus. We propose that CAP37 released from neutrophils during phagocytosis and degranulation may mediate recruitment of monocytes in the second wave of inflammation.

Amino Acid Sequence↗

PubMed1989

Quantitation of a cationic antimicrobial granule protein of human polymorphonuclear leukocytes by ELISA.

The quantitation of CAP57, a highly hydrophobic, native cationic antigen of human polymorphonuclear leukocytes has been achieved using ELISA. An important feature determining the sensitivity and precision of the ELISA was the reduction of non-specific protein-protein binding, particularly in the inhibition assays, thus eliminating high backgrounds obtained with presently available methodology. Washing of the solid phase-bound antigen and blocking of the non-specific binding sites using a potassium phosphate buffer containing heparin largely contributed to this increased sensitivity. The inhibition assays were conducted using antigen concentrations over the range of 0.9-120 ng. The assay is highly specific and can be performed using monoclonal antibodies and polyclonal antibodies. Non-specific reactions were observed only when high concentrations of antigen (greater than 100 ng) were present in the inhibition mixture. The technique as described is extremely simple, highly reproducible and could be of value in the detection of cationic antimicrobial proteins in the clinical setting in the future.

Animals↗

PubMed1989

O antigen and lipid A phosphoryl groups in resistance of Salmonella typhimurium LT-2 to nonoxidative killing in human polymorphonuclear neutrophils.

We have compared the intraleukocytic survival of isogenic strains of Salmonella typhimurium, whose outer membrane lipopolysaccharide differed in O antigen and lipid A composition and whose susceptibility to nonoxidative antimicrobial granule proteins of human polymorphonuclear neutrophilis (PMN) could be established. We found that the order of resistance to the bactericidal activity of intact PMN of the three bacterial strains utilized closely resembled their ordered resistance to the purified human cationic antimicrobial 57,000-dalton protein (CAP57). LT-2, a smooth wild-type strain, was far more resistant than SH9178, its rough (Rb LPS) mutant. It was most significant that SH7426, a polymyxin B-resistant pmrA mutant of SH9178, not only was substantially more resistant to CAP57 and to intraphagocytic killing than SH9178 but also came close to being as resistant as LT-2. These experiments confirm earlier work that showed the importance of the glycosyl groups of O antigens of S. typhimurium for their resistance to O2-independent antimicrobial phagocytosis by PMN. The surprising result was that a rough strain, very susceptible to bactericide, became substantially more resistant when a mutation led to its lipid A phosphoryl groups being 100% substituted with amino pentoses. Yet unresolved is whether the protection is due to the loss of negative charges on the lipid A, the substitution of sugar molecules in vulnerable loci in the outer membrane, or both.

Antigens, Bacterial↗

PubMed1989

Transgenic mice as a model to test the immunogenicity of proteins altered by site-specific mutagenesis.

Second generation therapeutic proteins are now being produced by in vitro mutagenesis of the relevant genes. Of some concern, however, is the possibility that these altered proteins will be immunogenic and the antibodies raised will also recognize the endogenous protein with undesirable consequences. We have designed a biological system to test these possibilities. In this model, transgenic mice produce and secrete human tissue plasminogen activator (h.tPA) to which these mice are immunologically tolerant. However, when challenged with a form in which a single amino acid has been substituted these mice will produce antibodies capable of recognizing h.tPA. These results indicate that there are immunological consequences connected with the design and administration of second generation therapeutic proteins.

Animals↗

PubMed1988

Killing of Proteus mirabilis by polymorphonuclear leukocyte granule proteins: evidence for species specificity by antimicrobial proteins.

Low-molecular-weight (Mr, ca. 3,800) polypeptides containing human defensins HNP-1 and HNP-2 (T. Ganz, M. S. Selsted, D. Szlarek, S. L. Harwig, K. Daher, D. F. Bainton, and R. I. Lehrer, J. Clin. Invest. 76:1427-1434, 1985) prepared in our laboratory from acid extracts of human polymorphonuclear granulocyte granules and purified human defensins were found to exert potent bactericidal action against Proteus mirabilis. The antimicrobial action of the extracts of human polymorphonuclear leukocytes granules against P. mirabilis appears to be due to the presence of the defensins. Because P. mirabilis resists the antimicrobial action of other granule proteins, we interpret the present results to mean that the various antimicrobial proteins display species specificity in their microbicidal action.

Blood Bactericidal Activity↗

PubMed1987

A monoclonal antibody that inhibits the antimicrobial action of a 57 KD cationic protein of human polymorphonuclear leukocytes.

Two monoclonal antibodies (mAb) specific for epitopes of a 57,000 m.w., cationic antimicrobial protein (CAP57) purified from granules of human polymorphonuclear leukocytes (PMN) have been produced. Both were IgG1 mouse antibodies with typical heavy and light chain structure. The mAb reactive with CAP57 failed to react specifically with other heretofore defined PMN or serum proteins as shown by ELISA. Both mAb showed specific reactivity in Western blots with CAP57. One of these mAb (P1G8) inhibited the antimicrobial action of CAP57 by 50% at a ratio of 62.5 micrograms antibody per microgram CAP57. The other mAb, P2A5, had no inhibitory capacity for CAP57. Binding constants of the two mAb for the antigen were determined and were found to be virtually identical. Thus, the greater inhibitory capacity of P1G8 for bacterial killing by CAP57 was not directly related to binding strength of the mAb. Competition experiments showed that unlabeled P1G8 could compete as well against radiolabeled P2A5 as could unlabeled P2A5. In the reverse experiment, it was seen that P1G8 competed with radiolabeled P1G8 for CAP57 better than unlabeled P2A5. These findings could be due to two antibodies that recognize different but adjacent epitopes on CAP57, one of the epitopes (P1G8) being closer to structure(s) of the protein essential to its antimicrobial action. Immunocytochemical studies showed positive staining with both mAb. The reaction was restricted to the cytoplasm of peripheral blood PMN and was of a granular pattern. Other peripheral blood cells (which included red blood cells, eosinophils, monocytes, and lymphocytes) failed to bind either mAb.

Antibodies, Monoclonal↗

PubMed1987

Automated sample preparation on-line with thermospray high-performance liquid chromatography-mass spectrometry for the determination of drugs in plasma.

The combination of a solid-phase extraction module, the AASP, on-line with thermospray high-performance liquid chromatography-mass spectrometry for the automated determination of drugs in plasma is described. The technique was evaluated successfully using, as an example, the determination of labetalol in human plasma. [2H7]Labetalol was used as an internal standard to compensate for changes in ionization efficiencies between analyses. The chromatographic and mass spectrometric conditions were optimized for labetalol. The combined technique was demonstrated as being robust and reliable for the analysis of plasma samples from a clinical study.

Autoanalysis↗

PubMed1987

Effects of one year's treatment with ranitidine and of truncal vagotomy on gastric contents.

Fifteen patients with peptic ulcer underwent 24 hour studies of gastric contents: before and on completing six weeks' treatment with oral ranitidine 150 mg bd, twice on maintenance treatment for nine to 12 months and one month after stopping the drug. For comparison, 11 patients underwent identical 24 hour studies three to 38 months after truncal vagotomy for duodenal ulcer. During treatment with ranitidine median 24 hour intragastric pH, nitrate concentration, and counts of total and nitrate reducing bacteria increased significantly regardless of dietary nitrate content; there was no significant increase in the median day time concentration of N-nitroso compounds. Despite these changes, an acid tide at some point in each 24 hour study period prevented persistent bacterial colonisation of the stomach. There were no significant differences between the biochemical and microbiological changes recorded during one year of treatment with ranitidine, and the observations on patients after truncal vagotomy. One month after stopping one year's treatment with ranitidine all variables examined returned to pretreatment levels. Treatment with ranitidine or vagotomy was associated with significant positive correlations among pH, nitrate concentration and bacterial counts. Correlations between pH and N-nitroso compound concentration and between concentrations of nitrite and N-nitroso compounds were not significant.

Adult↗

PubMed1986

Late intraphagosomal hydrogen ion concentration favors the in vitro antimicrobial capacity of a 37-kilodalton cationic granule protein of human neutrophil granulocytes.

We described previously (W.M. Shafer, L.E. Martin, and J.K. Spitznagel, Infect. Immun. 45:29-35, 1984) the presence of a 37-kilodalton cationic antimicrobial protein (37K CAP) in extracts of granules prepared from human polymorphonuclear granulocytes (PMN). In this investigation, we prepared 37K CAP from PMN granule extracts by sequential ion-exchange and molecular-sieve chromatography and examined its antimicrobial activity against a number of gram-negative and gram-positive bacteria. At concentrations of 5 micrograms/ml or lower, 37K CAP exerted selective antimicrobial activity against gram-negative bacteria. These bacteria included Acinetobacter lwoffii, Escherichia coli, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Pseudomonas cepacia, Salmonella typhi, Salmonella typhimurium, and Shigella sonnei. However, at 5 micrograms of 37K CAP per ml, Proteus mirabilis, Proteus vulgaris, and Serratia marcescens resisted this antimicrobial activity. The bactericidal activity of 37K CAP was greatest in acidic (pH 5.5) as opposed to alkaline (pH 7.5) media. The level of S. typhimurium resistance to 37K CAP correlated with the presence of O antigen in the lipopolysaccharide. In the absence of O antigen repeat units, resistance was proportional to the length of the core oligosaccharide. These results suggest that 37K CAP may contribute significantly to the ability of PMN to kill gram-negative bacteria by nonoxidative means, particularly as the maturing phagolysosome becomes acidified.

Anti-Bacterial Agents↗

PubMed1986

Antigonococcal activity of human neutrophil cathepsin G.

We have shown that lysosomal cathepsin G, prepared from acid extracts of granules derived from human polymorphonuclear granulocytes, exhibits potent in vitro antimicrobial activity against Neisseria gonorrhoeae. An isolated isozyme of cathepsin G was found to exhibit antigonococcal activity by a nonenzymatic mechanism in a time-dependent manner. Moreover, we observed that the antigonococcal activity of cathepsin G was relatively independent of pH and evident over a pH range resembling that invoked for maturing phagolysosomes. Using a number of isogenic strains, we determined that certain mutations known to alter cell envelope structure rendered gonococci more susceptible to cathepsin G. This suggests that the susceptibility of gonococci to cathepsin G, and possibly other antimicrobial proteins derived from PMN granules, is genetically determined and possibly related to the structure of the gonococcal cell envelope.

Antimicrobial Cationic Peptides↗

PubMed1985

Determination of salbutamol in human plasma and urine by high-performance thin-layer chromatography.

A rapid, accurate and sensitive method for the determination of salbutamol in plasma and urine is described. Salbutamol is extracted using solid-phase techniques and converted to an indoaniline dye by reaction with dimethyl-p-phenylenediamine. The indoaniline is separated using high-performance thin-layer chromatography and quantified by absorption microdensitometry at 650 nm. The method is sensitive down to 20 ng/ml in urine and to 1 ng/ml in plasma and provides data in good agreement with that obtained by gas chromatography--mass spectrometry. The method can be used for analysis of pharmacokinetic studies.

Albuterol↗

PubMed1985

Qualitative and quantitative analysis of ranitidine and its metabolites by high-performance liquid chromatography-mass spectrometry.

Reversed-phase high-performance liquid chromatography systems for the separation of ranitidine and its metabolites ranitidine-N-oxide, ranitidine-S-oxide, and desmethylranitidine have been developed for use in high-performance liquid chromatography-mass spectrometry. A direct liquid introduction-high-performance liquid chromatography-mass spectrometry system to analyse qualitatively and quantitatively solutions containing ranitidine and its metabolites by reversed-phase chromatography is described. A sample of urine collected from a subject given an oral dose of 75 mg of ranitidine and 75 mg of tris-deuterated ranitidine was analysed by this system. Ranitidine and its metabolites were identified by the ion doublets in the mass spectra which were 3 a.m.u. apart.

Biotransformation↗

PubMed1984

Ranitidine disposition and systemic availability in hepatic cirrhosis.

Single-dose ranitidine kinetics were studied in 10 patients with cirrhosis as proved by liver biopsy. All were clinically stable. After an overnight fast, ranitidine was given in a randomized crossover order as a bolus intravenous injection (50 mg) or was taken by mouth (150 mg). Terminal t1/2 was 2.7 +/- 0.4 hr after oral dosing and 2.9 +/- 0.4 hr after intravenous injection. Total plasma clearance was 470 +/- 170 ml/min and the steady-state volume of distribution was 1.2 +/- 0.2 l/kg. There was considerable intersubject variability in the ranitidine serum concentration-time profile after oral dosing. Systemic availability as assessed by AUC analysis was 58% +/- 11%. Not all of the dose could be recovered in the urine as unchanged ranitidine and its known metabolites after intravenous injection. At 0.5 microgram/ml the serum protein binding of ranitidine was 4.6% +/- 1.3%. It is concluded that disposition of ranitidine in these 10 stable subjects with cirrhosis was not significantly altered. The minor changes observed in some were as likely to be the result of secondary perturbations in physiologic status as to effects of cirrhosis on drug metabolism.

Administration, Oral↗

PubMed1984

Automated extraction of drugs from biological fluids.

An automated continuous flow liquid-liquid extraction procedure is described for the separation of the H2-antagonist loxtidine from plasma samples containing two metabolites which interfere in the radioimmunoassay of the drug. The extraction of the bronchodilator salbutamol was studied using the DuPont Prep I automated liquid solid extraction apparatus, with a 12 cartridge capacity, and a vacuum extraction box designed in this laboratory to hold 30 Sep-pak C-18 (Waters Associates) cartridges. Twenty-four plasma samples per hour can be automatically processed with the Prep I. Although the vacuum box is not fully automated 45 plasma samples per hour can be processed. The Prep I can only be used with DuPont XAD, strong cation and anion exchange cartridges. Cartridges containing alumina, silica, florisil, cation and anion exchange resins and reverse phase packings can all be used with the vacuum extraction box. The latter costs only a fraction of the Prep I and therefore each analyst can have his own unit.

Acetates↗

PubMed1984

Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate.

Acid (0.2 M sodium acetate, pH 4.0) extracts of granules recovered from disrupted human polymorphonuclear granulocytes (PMNs) exhibited in vitro antimicrobial activity against Salmonella typhimurium. To minimize proteolytic destruction or modification of antimicrobial proteins derived from these granules, we pretreated the PMNs with the serine protease inhibitor diisopropyl fluorophosphate. Fractionation of such extracts by carboxymethyl Sephadex and Sephadex G-75 chromatography resulted in the recovery of at least two antimicrobial, cationic proteins. These proteins differed substantially in antimicrobial activity, amino acid composition, and molecular weight (Mr, 37,000 and 57,000). As we have shown before (Shafer et al., Infect. Immun. 43:834-858), with unfractionated proteins, these two proteins exhibited diminished activity against a polymyxin B-resistant (PBr) mutant of S. typhimurium compared with their activity against the isogenic parental polymyxin B-sensitive (PBs) strain. Expression of the relevant mutation (prmA) in the PBr mutant decreases the electronegativity of lipid A, owing to increased 4-amino-4-deoxy-L-arabinosylation at the 4' phosphate residue (Vaara et al., FEBS Lett. 129:145-149). The data suggest that at least two different cationic proteins account for the antimicrobial capacity of extracts from human PMN granules. Moreover, the availability of anionic charges in the outer membrane of S. typhimurium due to free lipid A phosphates apparently dictates phenotypic levels of resistance to both of the cationic proteins extracted from human PMN granules.

Amino Acids↗

PubMed1983

Differential distribution of distinct forms of myeloperoxidase in different azurophilic granule subpopulations from human neutrophils.

Myeloperoxidase (MPO), a characteristic enzyme of human polymorphonuclear neutrophils (PMN), is localized in specialized lysosomal or azurophilic granules, and can be resolved into three distinct forms (I, II, III) by ion-exchange chromatography. Granules were isolated from single donor PMN and fractionated with centrifugation into two different azurophilic subpopulations (high and low density) by banding in a continuous sucrose density gradient. Ion-exchange chromatography of granule extracts indicated that the lower density granules contained mainly MPO forms II and III while the higher density granules appeared to contain all three forms, but in much reduced amounts. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed that, the mobilities of the heavy subunits of MPO appeared to be inversely related to the density of the granule population from which they were extracted. These observations suggest that the different forms of MPO may have distinct functional roles and/or are a possible reflection of maturational differences among the granule subpopulations.

Adult↗