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Biomedical subjects

R T Coughlin

Publications and source records attributed to R T Coughlin.

35 records · Page 2Linked to original sources
PubMed1992

Saponin adjuvant induction of ovalbumin-specific CD8+ cytotoxic T lymphocyte responses.

The ability of a saponin adjuvant, QS-21, to induce OVA-specific, class I MHC Ag-restricted CTL was investigated using different forms of soluble OVA and OVA adsorbed onto alum as immunogens. C57BL/6 mice were immunized with soluble native or denatured OVA in formulations that contained increasing quantities of QS-21, and CTL responses were measured using EL4 and E.G7-OVA cells as targets and splenic mononuclear cells as effectors. Ag-specific CTL responses were produced but only if the QS-21 adjuvant was used. Similar responses were induced using alum-adsorbed OVA when mixed with the QS-21 adjuvant but not when used alone. The CTL were specific for an epitope present on the OVA258-276 synthetic peptide, which contains the dominant CTL epitope recognized by C57BL/6 mice. The CD8+ subpopulation of lymphocytes in immune mice was not increased in spleens but increased significantly in vitro after culture with soluble OVA. The CTL activity of splenic mononuclear cell preparations was totally destroyed by treatment with mAb specific to the CD8 Ag plus complement. The ability of the QS-21 adjuvant to induce class I MHC Ag-restricted CTL after immunization with soluble proteins is a characteristic unique to saponin adjuvants.

Adjuvants, Immunologic↗
PubMed1992

Potentiation of HIV envelope glycoprotein and other immunogens by endotoxin (ET) and its molecular fragments.

The structural requirements for the immunopotentiating (adjuvant) effect of endotoxin (ET) were investigated. Mild hydrolysis (0.2 N acetic acid at 95 degrees C) was applied to various ET preparations and the lipid rich (Lipid A) and polysaccharide-rich (PS) preparations obtained were tested as adjuvants on three immunogens: sheep red blood cells (SRBC), L-glutamine: L-lysine: L-alanine containing random synthetic polypeptide (GLA-40), and recombinant HIV viral envelope polypeptide (CBre3). It was found that not only the Lipid A precipitates, but under certain hydrolytic conditions the non-toxic PS preparations were also potent adjuvants. The exact conditions of hydrolysis which led to the isolation of immune adjuvant bacterial products were established. These materials were also tested for endotoxicity (Limulus lysate clotting, chick embryo lethality and local Shwartzman skin reactivity), as well as for TNF generating activities. It was found that TNF generation runs parallel with toxicity of the samples, but it does not follow the adjuvant activity of the isolates. Chemical analysis of the preparations indicated that they did not contain residual ET or Lipid A, however, they did not exclude that deacylated and dephosphorylated skeletal remains of ET are among those components in these preparations which have immunomodulatory activity.

Adjuvants, Immunologic↗
PubMed1992

Immunogenicity and toxicity testing of an experimental HIV-1 vaccine in nonhuman primates.

A highly purified saponin from Q. saponaria (QS-21) was tested in juvenile rhesus macaques for adjuvant activity and toxicity. The QS-21 was tested alone or as part of an experimental subunit HIV-1 vaccine containing a truncated recombinant HIV-1 envelope protein (gp160D) adsorbed to alum. Antibody responses were measured using ELISA and cell-mediated immunity was measured using cellular proliferation assays. Potential toxicity was monitored by standard clinical pathology testing using peripheral blood and urine samples. No toxic effects were observed, even after the administration of the experimental vaccines three times at monthly intervals. The QS-21 saponin adjuvant enhanced total antibody production levels by greater than 100-fold and broadened the specificity of the response so that additional epitopes were recognized, when compared with alum-adsorbed HIV-1 gp160D formulation. Low-level, antigen-specific proliferative responses to HIV-1 recombinant gp160 were induced by either vaccine formulation. Proliferative responses were induced by a sham challenge with soluble recombinant HIV-1 gp160 for all of the animals that had been vaccinated. However, those that received the HIV-complete vaccine formulation containing QS-21 responded significantly better. These data demonstrated that the QS-21 adjuvant augmented both antibody responses and cell-mediated immunity and established immunological memory. The potent adjuvant activity and lack of toxicity suggest that this adjuvant should be safe and effective for use in HIV-1 vaccines.

AIDS Vaccines↗
PubMed1992

Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody enzyme immunoassay for toxin A only and two cytotoxicity assays.

A total of 320 stool specimens obtained from 262 patients suspected of having Clostridium difficile-associated gastrointestinal disease were examined with two cytotoxicity assays (CTAs) and two commercially available enzyme immunoassays (EIAs). The CTAs were an in-house-developed procedure (University of Massachusetts Medical Center [UMMC], Worcester, Mass.) and a commercial test (Bartels CTA; Baxter Healthcare Corp., West Sacramento, Calif.). One EIA was a monoclonal antibody-based assay for C. difficile toxins A and B (Cambridge Biotech Corp. [CBC], Worcester, Mass.). The other EIA employed monoclonal antibodies directed against only toxin A (Meridian Diagnostics, Cincinnati, Ohio). True-positive and true-negative results were defined on the basis of the results of the four assays, clinical assessments of patients, and the results of other laboratory tests. The sensitivities of the four assays were as follows: Bartels CTA, 100%; UMMC CTA, 97.2%; CBC EIA, 84.5%; and Meridian EIA, 69.0%. The Bartels CTA demonstrated a specificity of 99.2%. The other three assays had a specificity of 100%.

Antibodies, Monoclonal↗
PubMed1991

A purified saponin acts as an adjuvant for a T-independent antigen.

Three strains of mice were injected with a T-independent antigen, Escherichia coli 055:B5 polysaccharide (PS) combined with purified saponin, QS-21, isolated from Quillaja saponaria bank. PS was prepared by hydrolysis of lipopolysaccharide (LPS). Nine week old mice were injected intradermally with 60 micrograms PS, as determined by an anthrone assay, with or without 15 micrograms QS-21 on days 0 and 14. On day 22 sera were assayed by EIA for PS specific antibodies. Titers were 11-fold higher in CD-1 mice with QS-21. C3H/HeJ (Ipsd) and C3H/HeSnJ (Ipsr) mice also showed an adjuvant associated increase in titer with saponin. Therefore, LPS responsiveness was not required for the adjuvant effect. PS vaccinated C3H and CD-1 mice with and without QS-21 had similar antibody isotype profiles. IgG2b titers accounted for more than half of the total Ig response. IgG2a was next highest followed by IgG3, IgM, IgG1, and IgA. In comparison, CD-1 mice injected with 0.1 microgram intact LPS had a different LPS specific isotype profile. IgG3 was the highest followed by IgG1, IgG2b, IgM, IgG2a, and IgA.

Adjuvants, Immunologic↗
PubMed1988

Thin-layer chromatography of endotoxins, their derivatives and contaminants.

Thin-layer chromatographic (TLC) separation techniques were used to analyze the heterogeneity of various preparations which included smooth and rough endotoxins (ET), Lipid A precipitates and synthetic Lipid A samples and a novel cytotoxic bacterial lipid. Furthermore, carbohydrate-rich split products (PS) of ET were also separated on commercial silica-coated plates. Satisfactory results were obtained by two-dimensional TLC or by the combination of chromatography followed by high-voltage electrophoresis in the separation of PS of ET cleaved by mild acetic hydrolysis. Several spray reagents were found which were eminently suitable to detect carbohydrate containing compounds. Less specific but generally useful spray reagents were also developed which gave strong color reactions with lipids, proteinaceous and carbohydrate containing split products of the ET preparations. Improved chromatographic resolution has also revealed substantial heterogeneity in both rough and smooth ET samples. Three biological activities of the separated components could be determined. These were antigenicity detected by reactivity with monoclonal antibodies on the TLC plates, endotoxicity, determined by the Limulus amoebocyte lysate (LAL) test and direct cytotoxicity of P815 cells in vitro. Considerable amounts of non-endotoxic and non-antigenic contaminants could be detected in all preparations tested. Significant amounts of free Lipid A were also found in smooth ETs. Thus a new level of complexity is recognized by TLC within these preparations.

Animals↗
PubMed1987

Immunoprotective murine monoclonal antibodies specific for the outer-core polysaccharide and for the O-antigen of Escherichia coli 0111:B4 lipopolysaccharide (LPS).

The antigen specificity of two immunoprotective monoclonal antibodies derived from mice immunized with Escherichia coli 0111:B4 bacteria and boosted with purified lipopolysaccharide (LPS) were investigated. One of the antibodies, B7, was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunostaining to bind to the O-antigen containing LPS species, whereas the other antibody, 5B10, reacted with both O-antigen containing homologs and the O-antigen-deficient LPS. 5B10 did not bind to LPS from E. coli J5, an Rc mutant of E. coli 0111:B4 that lacks both the O-antigen and outer core sugars. 5B10 did not cross-react with LPS from several other E. coli strains. Thus 5B10 appeared to recognize a type-specific epitope in the outer core of LPS exclusive of Rc determinants. The monoclonal antibody specific for the polymeric O-antigen is of the IgG3 subclass, and the monoclonal antibody 5B10 specific for the outer core of LPS is an IgG2a. Although B7 and 5B10 were equally able to protect mice from a lethal challenge of E. coli 0111:B4 organisms, the outer core-specific IgG2a antibody was much more efficient at mediating the binding of human complement C3 than the O-antigen-specific IgG3 monoclonal antibody.

Animals↗
PubMed1987

Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12.

Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies. Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype). Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000. After heating to 100 degrees C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000. The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydrate-specific silver stain, while the heated monomer band showed no staining. In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer. Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE. LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies. Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer. Thus, some but not all of the LPS could be released from trimer complexes by boiling in SDS. The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1987

Ligand effects on membrane lipids associated with sodium, potassium-activated adenosine triphosphatase: comparative spin probe studies with rat brain and heart enzyme preparations.

Physical properties of membrane lipids associated with rat or dog brain and heart Na+, K+-ATPase preparations were compared using an electron spin resonance probe, 5-doxyl stearate. The degree of acyl chain order of the membrane lipids was greater for brain enzyme than for heart enzyme preparations; membrane lipids in the rat heart enzyme preparations were the most disordered. In the absence of added ligands, membrane lipids did not appear to undergo a detectable temperature-dependent rearrangement or structural transition. An apparent transition was observed in the simultaneous presence of Na+, Mg2+, and ATP. These ligands increased lipid order at temperatures above the structural transition, but not below it. In the presence of the above ligands, K+ caused a marked decrease in the transition temperature in the rat brain enzyme preparations, but only a modest decrease in rat heart enzyme preparations. Arrhenius plots of rat brain and heart Na+, K+-ATPase activity revealed a break point roughly corresponding to respective membrane lipid transition temperatures observed in the presence of Na+, K+, Mg2+, and ATP. A low concentration of ouabain (1 microM) failed to affect either the lipid transition temperature estimated by the spin probe or the value of lipid order of the rat brain enzyme preparations observed in the presence of Na+, Mg2+, and ATP, but markedly reduced the effect of K+ to lower the transition temperature observed in the presence of the above ligands. A high concentration (100 microM) of ouabain which was needed to completely inhibit rat heart enzyme eliminated the thermally induced structural rearrangement observed in the presence of Na+, Mg2+, and ATP, apparently through a nonspecific lipid perturbation. These results indicate that differences in the physical properties of the membrane lipids per se are unlikely to account for the low affinity of rat heart Na+, K+-ATPase for ouabain and also suggest that the use of high concentrations of ouabain required to completely inhibit Na+, K+-ATPase activity may cause nonspecific changes in addition to inhibition of Na+, K+-ATPase or the sodium pump.

Animals↗
PubMed1985

A pH titration study on the ionic bridging within lipopolysaccharide aggregates.

The packing of lipopolysaccharide aggregates from rough strains of Escherichia coli was examined at different pH values. Lipopolysaccharide head-group motion, measured with an electron spin resonance probe, was found to be dependent on pH, and indicated the existence of multiple ionizable groups. Lipopolysaccharide from a rough (Ra) and a heptose-less (Re) mutant were more rigid at pH 5 than at pH 10.5. In addition, head-group mobility of the magnesium salt of Ra lipopolysaccharide was substantially less than that of the sodium salt at pH 7.0, whereas at high pH (pH 12) the two salts were equally fluid. Changes in head-group packing were also reflected in pH-dependent changes in the phase transition measured with differential scanning calorimetry. The enthalpy of the transition, delta Ht, for the sodium salt of Re lipopolysaccharide was greatest at pH 7.5 and approached zero in both the acidic and the basic pH ranges. We propose that fixed charges in the core and lipid A regions significantly influence lipopolysaccharide head-group motion and the lipopolysaccharide aggregation state. Furthermore, ionic bridging among phosphate groups dramatically rigidifies head group interactions in the neutral to acidic pH ranges.

Calorimetry, Differential Scanning↗
PubMed1983

Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli.

Inductively coupled plasma emission spectroscopy was used to quantitate the metal cations bound to outer and cytoplasmic membranes and to extracted lipopolysaccharide from several Escherichia coli K12 strains. The outer membrane was found to be enriched in both calcium and magnesium relative to the cytoplasmic membrane. Both membranes contained significant levels of iron, aluminum, and zinc. The multivalent cation content of the lipopolysaccharide resembled that of the intact outer membrane. Lipopolysaccharide extracted from wild-type k12 strains contained higher levels of Mg than Ca regardless of the growth medium, but the medium used for growth did affect the relative amounts of bound Mg as well as the levels of the minor cations iron, aluminum, and zinc. In contrast, lipopolysaccharide isolated from a deep rough mutant strain, D21f2, contained more Ca than Mg. Electrodialysis of lipopolysaccharide from wild-type k12 strains removed 1 mol of Mg per mol of lipopolysaccharide but did not significantly affect the level of other bound metal ions. Dialysis of lipopolysaccharide against sodium (ethylenedinitrilo)tetraacetate removed most of the Mg and Ca, resulting in a sodium salt. The equimolar replacement of divalent cations with sodium in the sodium salt resulted in a net loss of counterion change. The sodium salt was dialyzed against either tris(hydroxymethyl)aminomethane hydrochloride, CaCl2, MgCl2, or TbCl3, and the resulting lipopolysaccharide salts were analyzed for their ionic composition. It was shown that tris(hydroxymethyl)aminomethane and Ca can replace some but not all of the Na bound to the sodium salt, but all of the other multivalent cations tested replaced Na, resulting in uniform lipopolysaccharide salts. Lipopolysaccharide isolated from the deep rough mutant strain D21f2 was also converted into a sodium salt. Relative to the wild-type lipopolysaccharide, Na was able to neutralize the anionic charge to a greater extent in the mutant lipopolysaccharide. Our results suggest that the loss of specific groups in the core region of the lipopolysaccharide from the mutant strain results in a more open structure that allows the binding of larger cations and of more monovalent cations.

Cations↗
PubMed1983

Physical properties of defined lipopolysaccharide salts.

The electron spin resonance probes 5-doxylstearate and 4-(dodecyldimethylammonio)-1-oxy-2,2,6,6-tetramethylpiperidine bromide were used to characterize the fluidity of the acyl chain and head-group regions, respectively, of defined salts of lipopolysaccharide (LPS) from Escherichia coli K12. The removal of the weakly bound divalent cations from native LPS by electrodialysis and their replacement by sodium had little effect on the midpoint of the lipid-phase transition or on head-group mobility. In contrast, lipopolysaccharide acyl chain mobility increased following electrodialysis. The replacement of most of the remaining cations with sodium resulted in a further dramatic increase in mobility in both the polar and nonpolar regions of lipopolysaccharide. Head-group mobility of the sodium salt of LPS was shown to be reduced with the addition of divalent cations. Furthermore, evidence is presented which suggests that low magnesium concentrations may induce phase separations in the sodium salt. The magnesium salt of lipopolysaccharide closely resembled the native form in both head-group and acyl chain mobility although the cation charge to phosphorus ratio in the magnesium salt was greater than that detected in the native isolate. Analyses of other lipopolysaccharide salts support our hypothesis that many of the observed differences in the physical and pathological properties of lipopolysaccharide salts may simply be explained by the degree of charge neutralization.

Carbohydrate Conformation↗
PubMed1981

The modification of human erythrocyte membrane structure by membrane stabilizers: an electron spin resonance study.

Membrane structure in intact human erythrocytes was analyzed by electron-spin-resonance (ESR) spectroscopy. The spin probes 5-doxyl stearate and 5-doxyl stearate methyl ester revealed thermally-induced structural transitions in the membrane at 37 degree C and 15 degree C. The addition of propranolol, diazepam, chlorpromazine, or Pluronic F68 all caused a decrease in the temperature of the upper transition, but did not markedly alter the temperature of the lower transition. In addition, diazepam caused a significant decrease in the ordering or packing of the membrane-lipid acyl chains. It is proposed here that the protection from hypotonic hemolysis that has been reported in the presence of these drugs is mediated by a structural rearrangement in the erythrocyte membrane involving a change in protein-lipid interactions.

Chlorpromazine↗