Biomedical subjects

J L Ryan

Publications and source records attributed to J L Ryan.

At least 19 recordsLinked to original sources

PubMed1993

Anti-PRP antibody levels after a primary series of PRP-OMPC and persistence of antibody titres following primary and booster doses.

The Haemophilus influenzae capsular polysaccharide-outer membrane protein conjugate, PRP-OMPC (PedvaxHIB) elicits very good antibody responses in infants > or = 2 months of age after the first dose. Increasing age at time of first vaccination correlates with higher antibody responses. Anti-PRP responses are consistently high with the first injection among all population groups studied. Booster doses stimulate anamnestic antibody responses after one year of age. Among US children (excluding Navajo and Apache children) given a primary injection at 14-18 months of age, the geometric mean titre (GMT) after 2 to 3 years was > 1 micrograms/ml. US children (excluding Navajo and Apache children) given a primary series at 2 and 4 months of age and a booster at 18 months of age also had an anti-PRP GMT > 1 micrograms/ml 2.5 years later. Navajo and Apache children given a primary series at 2 and 4 months of age and a booster at 12-15 months had antibody levels of 1.50 micrograms/ml one year later. Antibody persistence data suggest there will be long-term protection against Haemophilus influenzae b disease following immunization with PRP-OMPC.

Antibodies, Bacterial↗

PubMed1991

The presence of sialidase-sensitive sialosylgangliotetraosyl ceramide (GM1b) in stimulated murine macrophages. Deficiency of GM1b in Escherichia coli-activated macrophages from the C3H/HeJ mouse.

The stimulated murine macrophage was found to contain 11 major gangliosides of which 8 were determined to be monosialylated. The thin-layer chromatographic patterns were complicated by the presence of both sialic acid and ceramide fatty acid heterogeneity. N-glycolyl and N-acetylneuraminic acid-containing species were present for each ganglioside characterized. Although C18 sphingosine was the only long chain base detected, ceramide fatty acid ranged from C16 to C24 carbon moieties. Based on gas-liquid chromatographic and antibody analyses, all major tetraosyl structure gangliosides were ganglio series types. Comprising 43 to 60% of thioglycollate-stimulated cells and 60 to 70% of Escherichia coli-activated cells, monosialosyl-gangliotetraosyl ceromides (Gm1 gangliosides) were the major monosialo species of which four were present: sialidase-resistant NeuGc-GM1a and NeuAc-GM1a and sialidase sensitive NeuGc-GM1b and NeuAc-GM1b. Analyses of thioglycollate-elicited murine peritoneal macrophage ganglioside patterns from four strains of mice, including the C3H/HeJ strain, indicated that, in the absence of any expression of a genetic defect, the pattern is conserved. However, when E. coli was used as the activating agent, the normal C3H/HeN macrophage contained little Gm1a with the sialidase-sensitive Gm1b predominant; the converse was true for the congenic endotoxin hyporesponsive C3H/HeJ strain. Therefore, C3H/HeJ mice are not defective in ganglioside metabolism per se but in the processing of an endotoxin stimulus such that one manifestation is an altered macrophage ganglioside pattern deficient in Gm1b.

Animals↗

PubMed1991

Murine peritoneal macrophage gangliosides inhibit lymphocyte proliferation.

Gangliosides have been shown to act as immunoregulatory agents by altering proliferative responses of lymphocytes to both antigens and mitogens. Most early studies have utilized brain gangliosides and have required high concentrations. The role of endogenous gangliosides from macrophages has remained unexplored. In this study, thioglycolate-elicited murine peritoneal macrophage gangliosides were purified and added to cultures of murine lymphocytes. Nanogram amounts caused a profound inhibition of LPS-induced DNA synthesis of splenocytes and of purified B lymphocytes, without demonstrable cellular toxicity. No effect was seen using asialo-GM1. This effect was present across a wide range of lipopolysaccharide (LPS) doses. Nanogram amounts of macrophage gangliosides also inhibited concanavalin A (ConA)-mediated lymphocyte proliferation. Inhibition of LPS-induced mitogenesis was present even if gangliosides were removed from the extracellular environment after 15-60 min of incubation prior to the addition of LPS. This inhibition was reversible with incubation of ganglioside pre-treated lymphocytes in medium containing serum. These inhibitory properties of macrophage gangliosides are distinct from those found in studies using brain gangliosides, and support a potential role for macrophage gangliosides as negative modulators of lymphocyte proliferation.

Animals↗

PubMed1991

Tumor necrosis factor-alpha/cachectin enhances human immunodeficiency virus type 1 replication in primary macrophages.

Macrophages are important target cells for human immunodeficiency virus type 1 (HIV-1). The ability of HIV-1 to productively infect macrophages may be influenced by endogenous cytokines that alter the activation state of these cells. In this study, the effect of tumor necrosis factor-alpha/cachectin (TNF alpha), a cytokine with macrophage-activating properties, on HIV-1 replication in primary blood monocyte-derived macrophages was examined. Treatment of macrophages with recombinant human TNF alpha (rTNF alpha), starting before or after HIV-1 infection, consistently enhanced viral production fivefold or greater above control (P less than .01). rTNF alpha was active at low concentrations (0.05-50 ng/ml) and increased the replication of both lymphocyte-tropic (human T lymphotropic virus type IIIB) and macrophage-tropic (human T lymphotropic virus type III BaL) strains of HIV-1. These findings provide additional evidence that TNF alpha may play a role in the pathogenesis of HIV-1 infection by upregulating viral expression in macrophages.

Cells, Cultured↗

PubMed1990

Altered B-lymphocyte membrane architecture indicated by ganglioside accessibility in C3H/HeJ mice.

We have analyzed both the total ganglioside composition and the surface accessibility of C3H/HeN B lymphocytes and C3H/HeJ B lymphocytes. Seventeen individual resorcinol-positive moieties were visualized by two-dimensional thin-layer chromatography of the purified gangliosides from both strains. Complete homology between strains was seen in the patterns of total gangliosides purified from the endotoxin-responsive and -hyporesponsive strains, with only minor differences in the relative concentrations of four gangliosides. In comparison, only 12 individual gangliosides were accessible to surface labeling following galactose oxidase treatment in these same strains, suggesting that some gangliosides are masked at the cell surface in both strains. However, labeling of the more polar components was greatly reduced in the endotoxin-hyporesponsive (C3H/HeJ) strain, suggesting that these gangliosides have decreased accessibility to galactose oxidase at the cell surface. Therefore, while the total ganglioside compositions of the two strains were nearly equivalent, there were dramatic differences in ganglioside surface accessibility. These findings indicate that an alteration in membrane structure that is associated with the endotoxin hyporesponsiveness observed in C3H/HeJ B lymphocytes exists.

Animals↗

PubMed1989

Factors mediating lipopolysaccharide-induced ganglioside expression in murine peritoneal macrophages.

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.0 micrograms/ml LPS for 6-96 hr, but showed no differences in membrane ganglioside patterns. Coincubation of macrophages with lymphocytes and treating with LPS again elicited no ganglioside changes. In contrast, interferon gamma (IFN-gamma)-primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN-gamma-primed, LPS-treated macrophages were coincubated with lymphocytes. Ganglioside expression induced in vitro still did not approach the complex changes seen in vivo. However, transplanting C3H/HeN macrophages intraperitoneally into C3H/HeJ mice, followed by administration of intraperitoneal LPS, did reveal striking changes in ganglioside expression that resembled the pattern seen in vivo. Thus, LPS alone does not provide the necessary direct signal to promote macrophage ganglioside change even though it alters macrophage function. IFN-gamma appears to be one important mediator; however, complex interactions involving other cytokines or migration of independent populations of mononuclear cells may be required for the full manifestation of LPS-induced ganglioside expression in macrophages.

Animals↗

PubMed1989

Antimicrobial use in hospital-acquired infections.

The appropriate use of antibiotics in nosocomial infections has been a goal of infectious disease practitioners and hospital epidemiologists since the emergence of resistant staphylococci more than 30 years ago. This challenge has assumed new proportions in the 1980s for many reasons. First, resistant gram-positive and gram-negative bacteria are increasingly common. Thus many traditional drugs or combinations of drugs lack efficacy. Second, there are so many new antibiotics available that it is difficult for any but the most knowledgeable to use each of them appropriately. Third, economic reality has forced hospitals and physicians to alter their activities and focus more on cost considerations than was ever necessary before. Last, infectious disease as a subspecialty in internal medicine is profoundly different in the 1980s; many infections must be controlled for life rather than cured because of underlying infection with human immunodeficiency virus, which suppresses immunity. The use of antibiotics is now rapidly changing and the state of the art in 1989 is likely to be outdated very soon as new pathogens emerge and new drugs are developed. The appropriate use of the quinolones is a great challenge that we now face. Can we preserve this valuable class of drugs, or will resistance develop quickly because of widespread, indiscriminate, and inappropriate use? To what extent can we phase out aminoglycoside use without compromising efficacy and without promoting the development of resistance? Do the monobactams have a real role to play in combination therapy? How should formularies approach the diverse cephalosporins that now inundate the market? These are only a few of the fundamental questions that must be addressed as this decade ends.

Anti-Bacterial Agents↗

PubMed1989

Treatment of experimental invasive aspergillosis with novel amphotericin B/cholesterol-sulfate complexes.

An immunosuppressed rabbit model of invasive aspergillosis was used to evaluate a novel micellar preparation of cholesterol sulfate complexed to amphotericin B. The acute LD50 of amphotericin B-deoxycholate was 5.1 mg/kg versus 20 mg/kg for the amphotericin/cholesterol-sulfate complexes. Amphotericin B-deoxycholate given iv at a dose of 1.5 mg/kg was more effective in sterilizing liver and kidney than the amphotericin/cholesterol-sulfate complexes given iv at 1.5-4.5 mg/kg, but infection persisted in the lungs of all rabbits treated with those doses. Infection persisted even when the rabbits were given a lethal dose of amphotericin B-deoxycholate (4.5 mg/kg), but a dose of 15 mg/kg of the amphotericin/cholesterol-sulfate complexes sterilized tissues and was associated with no acute lethality. Equivalent doses of the amphotericin/cholesterol-sulfate complexes were less effective than amphotericin B-deoxycholate, but a fourfold decrease in acute lethality improved the therapeutic index of amphotericin B. The amphotericin/cholesterol-sulfate complexes appear to be an improved means of amphotericin B delivery and may improve therapy for invasive aspergillosis.

Amphotericin B↗

PubMed1989

Incorporation of recombinant gamma interferon into liposomes enhances its ability to induce peritoneal macrophage antitoxoplasma activity.

In this study we compared the ability of free- and liposome-incorporated murine recombinant gamma interferon (rIFN-gamma) to enhance peritoneal macrophage H2O2 release and antitoxoplasma activity in vitro. rIFN-gamma was efficiently (37 to 47%) incorporated into multilamellar vesicles composed of phosphatidylglycerol/cholesterol in a 2:1 molar ratio. The amount of rIFN-gamma incorporated into multilamellar vesicles and added to macrophages (0.1 to 1,000 U/ml) was quantitated with [3H]rIFN-gamma. The concentration of liposomal rIFN-gamma required to enhance macrophage H2O2 release (1 U/ml) and maximally inhibit Toxoplasma gondii growth (10 U/ml) was one-tenth the concentration required for free rIFN-gamma (10 and 100 U/ml, respectively). This increase in potency was observed in both thioglycolate-elicited and resident peritoneal macrophages. Control liposomes containing encapsulated buffer had no effect on the potency of free rIFN-gamma. The duration of macrophage activation induced by 24 h of liposomal rIFN-gamma treatment was also considerably longer than that induced by free rIFN-gamma (2 days versus less than 1 day). These data indicate that liposomal rIFN-gamma is more active than free rIFN-gamma as an inducer of macrophage microbicidal properties in vitro. This enhanced activity, combined with the potential for selective delivery of liposomal rIFN-gamma to phagocytic cells in vivo, may improve the therapeutic efficacy of rIFN-gamma in infections characterized by parasitization of phagocytes.

Animals↗

PubMed1989

Altered in vivo activity of liposome-incorporated lipopolysaccharide and lipid A.

We compared the abilities of free and liposome-incorporated Salmonella minnesota wild-type lipopolysaccharide (LPS) and lipid A to activate peritoneal macrophages and induce lethal toxicity in mice. Incorporation of lipid A into multilamellar vesicles resulted in a 100-fold-decreased potency to prime macrophages for phorbol myristate acetate-triggered release of H2O2. In addition, liposome incorporation reduced the lethality of LPS and lipid A at least 10-fold in dactinomycin-sensitized mice. Similar results were obtained with multilamellar liposomes delivered intravenously and when small unilamellar vesicles were employed. The observed difference in toxicity was not dependent on dactinomycin treatment, since a similar decrease was obtained with large doses of liposomal LPS in unsensitized mice. Control liposomes, prepared without LPS and lipid A, did not reduce the activities of the free compounds. The administration of a sublethal amount of liposomal LPS induced within 20 days, but not during the first week, tolerance to a subsequently injected lethal dose of free endotoxin. The latter observation suggests that early-phase tolerance is not the mechanism responsible for the reduced toxicity of liposomal LPS. These data show that liposomal LPS and lipid A have reduced endotoxic activity in vivo and are consistent with our hypothesis that a direct interaction of lipid A with appropriate plasma membrane components is necessary to efficiently trigger biologic responses. This interaction, however, is prevented by the stable insertion of LPS into the liposomal membrane.

Animals↗

PubMed1988

A procedure for the efficient incorporation of wild-type lipopolysaccharide into liposomes for use in immunological studies.

Previous studies on the mechanism of action of lipopolysaccharides (LPS) on macrophages have used wild-type lipopolysaccharide (wt-LPS) containing liposomes. In these studies the endotoxin was incorporated into liposomes by suspending the wt-LPS in the buffer used to rehydrate the lipid. Using this approach (buffer method), we observed that less than 10% of Salmonella minnesota smooth LPS is incorporated into multilamellar vesicles (MLV). If the non-incorporated material is not effectively separated from the liposomal form, erroneous conclusions on the mechanism of action of LPS can be drawn. Prolonged sonication of the wt-LPS-MLV suspension resulted in almost complete incorporation of the LPS into the resulting small unilamellar vesicles (SUV). In order to prepare MLV, we briefly soniated the buffer preparation, dehydrated the resulting smaller vesicles and then rehydrated the mixture (dry method). This procedure resulted in almost complete incorporation of the wt-LPS into MLV. The ability of wt-LPS in MLV prepared by the dry method to activate macrophages or trigger gelation of Limulus amoebocyte lysate was reduced by 100-1000-fold compared to the non-incorporated wt-LPS. This indicates that at least 99% of the wt-LPS is incorporated in MLV made by the dry method.

Animals↗

PubMed1988

Incorporation of LPS in liposomes diminishes its ability to induce tumoricidal activity and tumor necrosis factor secretion in murine macrophages.

We investigated the effect of lipopolysaccharide (LPS) incorporated into phospholipid vesicles (liposomes) on the induction of macrophage-mediated tumor cytotoxicity and tumor necrosis factor (TNF) secretion. The incorporation of Salmonella minnesota rough (Re)-LPS into multilamellar or small unilamellar vesicles (liposomes) resulted in an 100- to 1,000-fold reduction in its potency to activate both the macrophage cell line RAW 264.7 and murine thioglycolate elicited peritoneal macrophages to become cytotoxic for L929 and P815 tumor cells. Liposomal LPS was also a 100- to 1,000-fold less potent inducer of TNF secretion from RAW 264.7 cells. Cytokines secreted by the activated macrophages contributed to the cytotoxic effect on the L929 cells but not the P815 cell line. Human recombinant TNF was not cytotoxic for either cell line but was cytostatic for the L929 cell line. Morphological examination of the cells after uptake of fluorescent, free, and liposomal LPS revealed that both forms were internalized by the endocytic pathway. This, together with the considerably reduced potency of liposomal LPS to induce tumor cytotoxicity and TNF secretion, suggests that the interaction of the hydrophobic part of the lipid A moiety of LPS with the macrophage plasma membrane is needed to optimally activate these cells. Incorporation of LPS into liposomes effectively abrogates this interaction.

Animals↗

PubMed1988

Comparison of thymocyte and T lymphocyte gangliosides from C3H/HeN and C3H/HeJ mice.

Gangliosides have been prepared from resting murine thymocytes and splenic T cells. Profoundly different two-dimensional thin layer chromatography (2D TLC) patterns were observed between these two cell types. Thymocytes contained 28-30 discrete gangliosides of which eight represented major gangliosides. Splenic T lymphocytes from both strains had much simpler patterns, with six to seven major gangliosides and 12-13 minor gangliosides. Computerized analysis of the thymocyte ganglioside patterns between LPS-responder C3H/HeN mice and lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice revealed no significant difference in the major gangliosides. However, with splenic T cell gangliosides, there is a striking difference in the relative proportion of three homologous gangliosides between the two strains. Consistent with previous observations on macrophage gangliosides, the ratio of N-acetylneuraminic acid-containing ganglioside to N-glycolylneuraminic acid-containing ganglioside was higher in both thymocytes and T-cells from the LPS-responder strain. These results show that sialic acid-containing glycolipids from thymocytes and T lymphocytes between endotoxin responder and hyporesponder strains manifest small but significant changes. These differences are present in unstimulated cell populations and may represent a manifestation of the Lps gene.

Animals↗

PubMed1988

Effect of immunosuppression and amphotericin B on Aspergillus antigenemia in an experimental model.

A modified enzyme-linked immunosorbent assay (ELISA) for crude carbohydrate antigen was used to evaluate the kinetics of aspergillus antigenemia and to determine the effect of therapy on circulating antigen levels in an experimental model. The ELISA was rapid, simple to perform, and able to detect less than 10 ng of antigen/mL of serum. The model was also used to evaluate the effect of temporary and persistent immunosuppression on experimental disease. Antigen levels rose progressively in untreated control rabbits; all 15 animals had significant antigenemia. Treated animals had markedly reduced antigen levels, but nine of 13 rabbits had detectable antigen after 72 h of therapy, a result that correlated with persistent disease. Therapy begun 24 h after challenge in temporarily immunosuppressed animals was more likely to sterilize tissues than was therapy begun 48 h after challenge. Therapy in persistently immunosuppressed rabbits was less effective and may require improved antifungal regimens to be successful.

Amphotericin B↗

PubMed1988

Acylation of monocyte and glomerular mesangial cell proteins. Myristyl acylation of the interleukin 1 precursors.

Acylation of cellular proteins with the fatty acids myristate or palmitate represents an important mechanism for the co- or posttranslational modification of proteins. Lipid A, the biologically active component of bacterial endotoxin, exerts a number of biochemical effects on responsive cell types. Evidence is presented that lipid A stimulates the synthesis and subsequent myristyl acylation of intracellular monocyte and glomerular mesangial cell proteins. Two of the myristylated monocyte proteins were identified by specific immunoprecipitation as the 33-kD IL 1 alpha and beta precursors; a similar myristylated protein was found in mesangial cells. The 17-kD secretory form of monocyte IL 1 beta did not contain covalently linked myristate. Myristyl acylation of the IL 1 precursor proteins may facilitate the processing or membrane localization of these proteins, which lack characteristic hydrophobic signal sequences. The acylated 33-kD IL 1 alpha may remain preferentially associated with the membrane in an active form, whereas limited proteolysis may convert the biologically inactive IL 1 beta precursor into the extracellular, nonacylated, active 17-kD protein.

Acylation↗

PubMed1988

Activation of glomerular mesangial cells by gram-negative bacterial cell wall components.

The cell walls of gram-negative bacteria contain several biologically active components, including lipopolysaccharide (LPS), lipoprotein, and protein 1. The effects of these individual components and a synthetic analog of lipoprotein, TPP, on several activation parameters of glomerular mesangial cells (MC) were examined. Prostaglandin secretion, synthesis of the autogrowth factor, mesangial interleukin-1 (IL-1), and new synthesis of cellular proteins were assessed as markers of MC activation. All bacterial cell wall components evaluated were active in varying degrees as stimulants of prostaglandin secretion. In general, PGE was the predominant product. TPP and protein 1 also induced substantial secretion of thromboxane. Each cell-wall component was effective in stimulating mesangial IL-1 secretion. The activation of MC was associated with the enhanced synthesis of many cellular proteins in addition to IL-1. Stimulation by these bacterial components was dependent on the state of the mesangial cell cycle, because nonproliferating cells did not respond to these factors. Activation of MC by gram-negative bacterial cell wall components, with release of vasoactive prostaglandins and peptide mitogens, may be responsible for some of the glomerular hemodynamic alterations and cellular proliferative events associated with sepsis or chronic bacterial infection.

Animals↗

PubMed1988

The clinical value of gas-liquid chromatography in the detection of intra-amniotic microbial invasion.

Short-chain organic acids are byproducts of bacterial metabolism, and their detection by gas-liquid chromatography (GLC) in amniotic fluid has been proposed as a rapid, sensitive, and specific method for the diagnosis of infection. This study was conducted to determine whether GLC analysis of amniotic fluid would be helpful in a population of women in whom the Gram stain examination was negative. Amniotic fluid was retrieved by transabdominal amniocentesis from three groups of patients. Group 1 (negative control) contained 14 women with term pregnancies without clinical and microbiologic evidence of infection of the amniotic cavity; group 2 (positive control) included 17 women with a positive Gram stain examination and culture of amniotic fluid; group 3 (study group) comprised 25 women at risk for intra-amniotic infection who had a negative Gram stain of amniotic fluid. None of the patients in group 1 and 16 of 17 patients (94.1%) in group 2 had pathologic short-chain organic acids in the amniotic fluid. Seven of the 25 patients in group 3 had a positive amniotic fluid culture and only two had an abnormal GLC pattern. Of the remaining 18 patients with negative amniotic fluid cultures, eight had abnormal GLC results. In group 3, the sensitivity of GLC was 28.5% and the specificity was 55.5%. Gas-liquid chromatographic analysis of amniotic fluid has limited clinical value in the prediction of amniotic fluid culture when the Gram stain examination is negative.

Amniocentesis↗

PubMed1987

Oral ciprofloxacin in resistant urinary tract infections.

Thirty-two patients (18 men and 14 women), who ranged in age from 28 to 91 years (mean, 71.2 years), with urinary tract infections caused by Pseudomonas species or other organisms resistant to trimethoprim-sulfamethoxazole were treated with 500 mg of orally administered ciprofloxacin every 12 hours. Thirty patients completed at least five days of therapy and were evaluated for efficacy. Of these, the treatment of 28 (93 percent) patients was considered successful, with urine cultures yielding negative results five to nine days after cessation of therapy. Three of these patients were found to be reinfected with their primary pathogens when culture specimens were obtained again three to four weeks later. The two patients who received treatment that was classified as having failed had urine cultures that persistently grew Pseudomonas aeruginosa. Superinfections occurred in eight patients, four with diabetes and four with underlying central nervous system disease. Adverse reactions required discontinuation of therapy in two patients. Although the rates of reinfection and superinfection were somewhat high, these patients had a high frequency of underlying diseases that predisposed them to recurrent or difficult-to-treat infections. Despite these shortcomings, ciprofloxacin is a welcome addition to the oral antibiotic regimen for the treatment of antibiotic-resistant urinary infections.

Administration, Oral↗