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Biomedical subjects

J L Ryan

Publications and source records attributed to J L Ryan.

At least 19 recordsLinked to original sources
PubMed2001

Targeted mutagenesis of Smad1 reveals an essential role in chorioallantoic fusion.

The Smad family of intracellular signaling intermediates transduce signals downstream from the transforming growth factor beta (TGF-beta) family of receptor serine threonine kinases. The original member of this family, Smad1, has been shown to mediate signals from receptors for the bone morphogenetic proteins (BMPs), a large group of ligands in the TGF-beta superfamily that mediate important developmental events. We have targeted the Smad1 gene in mice and created mutants null at this locus. Smad1 mutant mice die at approximately 9.5 days postcoitum due to defects in allantois formation. In Smad1 mutant mice, the allantois fails to fuse to the chorion, resulting in a lack of placenta and failure to establish a definitive embryonic circulation. Although vasculogenesis is initiated in the mutant allantois, the vessels formed are disorganized, and VCAM-1 protein, a marker for distal allantois development, is not expressed. Smad1 null fibroblasts are still able to respond to BMP2, however, suggesting that the defect observed in the developing extraembryonic tissue is caused by a very specific loss of transcriptional activity regulated by Smad1. Our data further demonstrate that although highly similar structurally, Smad proteins are not functionally homologous.

Allantoin↗
PubMed2001

Differences in splenic B-lymphocyte ganglioside expression and accessibility in normal and endotoxin-hyporesponsive mice.

Endotoxin-responsive (C3H/HeN) and -hyporesponsive (C3H/HeJ) murine B lymphocytes purified by adherence to anti-immunoglobulin ("antibody panning") possess identical gangliosides but different ganglioside surface accessibilities. We investigated the distribution and surface accessibility of gangliosides of B lymphocytes purified by adherence to plastic ("plastic panning") or by subtraction of non-B-lymphocyte components. As with antibody panning, there were no entirely new or absent gangliosides in plastic-panned or subtraction-purified B lymphocytes of each strain. However, striking changes in relative expression of five gangliosides were detected with each purification protocol. Moreover, five gangliosides of antibody-panned and plastic-panned B lymphocytes but only two gangliosides of subtraction-purified B lymphocytes were inaccessible to surface labeling. Unlike the situation for antibody-panned B lymphocytes, no interstrain (HeN vs. HeJ) surface accessibility differences existed in gangliosides of plastic-panned or subtraction-purified cells. Exposure of subtraction-purified B lymphocytes to anti-immunoglobulin failed to elicit changes in ganglioside expression. Murine B lymphocytes have distinct protocol-dependent differences in glycolipid phenotype which likely denote individual subpopulations.

Animals↗
PubMed2000

A comparative trial of imipenem versus ceftazidime in the release of endotoxin and cytokine generation in patients with gram-negative urosepsis. Urosepsis Study Group.

Evidence from in vitro experiments and animal and human studies indicate that antibiotic therapy may induce the release of endotoxin from the outer membrane of Gram-negative bacteria. Antibiotics that bind preferentially to penicillin-binding protein-2 (PBP-2)--such as imipenem--are associated with little release of endotoxin, while antibiotics that preferentially bind to PBP-3--such as ceftazidime--are associated with far greater release of endotoxin. We conducted a randomized, multicenter, double-blind study comparing imipenem to ceftazidime in patients with urinary tract infections caused by Gram-negative bacilli associated with signs and symptoms of systemic inflammation. A total of 33 patients were randomized to receive either imipenem (n = 14) or ceftazidime (n = 19) for initial treatment for urosepsis. No differences in plasma endotoxin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) or urine endotoxin, IL-6 or IL-8 levels were found between the two treatment groups within the first 8 h after antibiotic administration. We conclude that, if differences exist with respect to endotoxin release by these two antimicrobial agents, these differences are not readily demonstrable in this clinical study with carefully defined patients with Gram-negative urinary tract infections.

Adult↗
PubMed1997

Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production.

Interleukin-12 (IL-12) is a key regulator of cell-mediated immunity that has therapeutic potential in cancer and infectious disease. In a previous Phase 1 dose escalation study of a single test dose of recombinant human IL-12 (rhIL-12) followed 14 days later by cycles of five consecutive daily intravenous injections every 3 weeks, we showed that a dose level up to 500 ng/kg could be administered with acceptable levels of safety. Based on these results, a Phase 2 study was conducted. In the Phase 2 study, however, administration of rhIL-12 at this same dose level resulted in severe toxicities with some patients unable to tolerate more than two successive doses. Of the 17 patients receiving rhIL-12 in the Phase 2 study, 12 patients were hospitalized and two patients died. A thorough scientific investigation to determine the cause of this unexpected toxicity failed to identify any difference in the drug products used or the patient populations enrolled in the Phase 1 and Phase 2 studies that could have accounted for the profound difference in toxicity. The focus of the investigation therefore shifted to the schedule of rhIL-12 administration. We determined that a single injection of rhIL-12 2 weeks before consecutive dosing included in the Phase 1 study, but not in the schedule of administration in the Phase 2 study, has a profound abrogating effect on IL-12-induced interferon-gamma (IFN-gamma) production and toxicity. This observation of schedule-dependent toxicity of IL-12 has been verified in mice, as well as nonhuman primates. In this regard, a single injection of IL-12 before consecutive daily dosing protected mice and cynomolgus monkeys from acute toxicity including mortality and was associated with an attenuated IFN-gamma response. Because of this unique biologic response, careful attention to the schedule of administration is required to assure safe and effective clinical development of this highly promising cytokine.

Adult↗
PubMed1996

Activation of murine lymphocytes by lipopolysaccharide incorporated in fusogenic, reconstituted influenza virus envelopes (virosomes).

We have studied the in vitro activation of murine lymphocytes with LPS incorporated in the membranes of both phospholipid vesicles (liposomes) and vesicles composed of fusogenic, reconstituted influenza virus envelopes (virosomes). The incorporation of Salmonella minnesota rough-LPS in liposomes reduced the potency of LPS to stimulate splenocyte proliferation and cell surface kappa-light chain expression on 70 Z/3 pre-B cells by over 100-fold. Salmonella minnesota rough-LPS inserted into virosomes was at least 10-fold more potent than free LPS, both when prebound virosomes were allowed to be taken up by the cells at neutral pH and when the virosomes were fused into the plasma membrane by low pH treatment. Inactivation of the virosomes by low pH pretreatment reduced the potency of the virosomal LPS to the level of liposome-incorporated LPS. The association of the various LPS forms with the cells was quantitated using radio-iodinated LPS. Correcting for uptake, virosomal LPS remained 2- to 10-fold more potent than free LPS in stimulating B lymphocytes and at least 100-fold more active than liposomal LPS or fusion-inactivated virosomes. After low pH-induced fusion with the plasma membrane, the majority (80%) of the prebound virosomes had fused with the cells, compared with about 8% after neutral uptake. From these results we conclude that LPS inserted into the plasma or endosomal membranes efficiently activates murine lymphocytes. The fusion data suggest that the incorporation into endosomal membranes might be a more effective stimulus.

Animals↗
PubMed1994

Drowning prevention. A case study in EMS epidemiology.

A central theme for EMS systems is the reduction of death and disability from emergency illnesses and injuries, with most systems focusing exclusively on treatment to fulfill their missions. Unfortunately, this has often resulted in the neglect or complete disregard of prevention, which actually may be the more powerful intervention strategy. Yet hopefully, as EMS systems begin to learn and embrace the principles of continuous quality improvement (CQI) in their organizational cultures, the advantages of prevention vs. treatment will become more apparent and internally recognized.

Data Collection↗
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↗