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R M Locksley

Publications and source records attributed to R M Locksley.

106 records · Page 6Linked to original sources
PubMed1987

Loss of granule myeloperoxidase during in vitro culture of human monocytes correlates with decay in antiprotozoa activity.

Human monocytes maintained in culture lose microbicidal activity against intracellular protozoa which has been correlated with attenuation of the respiratory burst. The granule enzyme myeloperoxidase, which can markedly amplify hydrogen peroxide-dependent antimicrobial activity, is also lost in vitro. Adherent monocytes were examined immediately, 3 and 10-14 days following explantation, for the magnitude of the stimulated respiratory burst and for cellular myeloperoxidase. Fresh cells generated 254 +/- 38 nmol O2-/mg protein as compared to a peak of 782 +/- 45 nmol O2-/mg at 3 days and less than 100 nmol O2-/mg after 10-14 days. The myeloperoxidase content of the cells also decreased; over 85% was lost after 3 days. Fresh monocytes killed over 90% of ingested Toxoplasma gondii or Leishmania major. In contrast, 10-14 day explanted monocytes killed only 12% of ingested Toxoplasma and 33% of Leishmania, and surviving organisms replicated readily. The 3-day monocytes were significantly less able to kill protozoa than were fresh cells despite their nearly 3-fold greater generation of O2-. If peroxidase was reintroduced into 3-day monocytes by coating organisms with eosinophil peroxidase prior to phagocytosis, their antiprotozoa activity was nearly restored to that of freshly explanted cells.

Animals↗
PubMed1986

Monoclonal antibodies binding to the human neutrophil C3bi receptor have disparate functional effects.

Three monoclonal antibodies (MAb)--OKMI, 7C3, and 60.3--immunoprecipitated a common 170-kd neutrophil membrane antigen closely associated with, or identical to, the C3bi receptor (CR3). Despite binding to a common receptor, these antibodies displayed marked differences in their effects on C3bi-mediated neutrophil function as assessed by the binding and ingestion of opsonized zymosan and the subsequent triggering of the respiratory burst. Antibody 7C3 caused a time-dependent, irreversible inhibition of the neutrophil oxidative response to opsonized zymosan that correlated with capping of the bound antibody. In contrast, antibody 60.3 caused an immediate inhibition of the neutrophil oxidative response to opsonized zymosan that required the continuous presence of exogenous antibody to achieve the maximal inhibitory effect. Antibody OKMI demonstrated minimal inhibition of O2- release. Despite their functional differences, binding of either 7C3 or 60.3 led to up-regulation of new antigen, presumably from intracellular sites as previously described using OKMI. Crossed immunoprecipitations of radiolabeled neutrophil lysates indicated that each MAb bound to different antigens near or within the CR3 complex. Thus three MAb binding to the neutrophil CR3 receptor each caused receptor up-regulation but had markedly different functional effects on the cell.

Antibodies, Monoclonal↗
PubMed1986

Murine cutaneous leishmaniasis: resistance correlates with the capacity to generate interferon-gamma in response to Leishmania antigens in vitro.

The kinetics of cell-mediated immunity developed during the course of Leishmania major infection in resistant (C57BL/6) and susceptible (BALB/c) mice were determined by using in vitro bioassays. Cells isolated from the lymph nodes draining the infected footpads were assayed for their proliferative responses to leishmania antigens (promastigote and amastigote) or to concanavalin A (Con A). Although lymph node cells (LNC) from both mouse strains proliferated to mitogen and antigen early after infection, both C57BL/6 and BALB/c mice developed diminished in vitro proliferative reactivity within 3 to 5 wk after infection. LNC from both mouse strains recovered lymphoproliferative reactivity to Con A (week 6), but only C57BL/6 mice regained reactivity to leishmania antigens. BALB/c cells remained unresponsive to leishmania antigens throughout the subsequent course of the infection. Supernatants derived from cultures of LNC that had been stimulated with Con A or leishmania antigens were assayed for interferon-gamma (IFN-gamma) by analyzing three distinct activities associated with IFN-gamma. Culture supernatants derived from leishmania antigen stimulation of LNC from infected C57BL/6 mice, but not BALB/c mice, were able to induce surface Ia on murine P388D1 cells. Ia-inducing activity was detectable in supernatants from C57BL/6 cells as early as 3 wk, and peaked by 5 wk after infection. Although cells from infected BALB/c mice never produced detectable IFN-gamma in response to leishmania antigens, LNC from both mouse strains produced readily detectable IFN-gamma in response to Con A throughout the course of infection. Culture supernatants that induced Ia on P388D1 cells were also capable of activating resident peritoneal macrophages to display leishmanicidal activity and of inhibiting encephalomyocarditis virus replication in murine fibroblasts. Each of these activities could be removed by prior incubation of the supernatants with rabbit heterologous anti-murine IFN-gamma sera or monoclonal rat-anti-murine IFN-gamma. The correlation of healer status with the capacity to generate IFN-gamma in vitro in response to leishmania antigens was examined in BALB/c mice that had been exposed to sublethal irradiation (550 rad) before infection. These animals have been previously shown to effectively resist L. major infection. Consistent with observations in the genetically resistant C57BL/6 mice, LNC from these animals demonstrated the capacity to respond to in vitro leishmania antigen stimulation with lymphoproliferation, and more importantly, by producing IFN-gamma.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Alteration of leukotriene release by macrophages ingesting Toxoplasma gondii.

Mouse resident peritoneal macrophages incubated with ionophore A23187 or opsonized zymosan released leukotrienes (LT) B4 and C4 (LTB4 and LTC4) and LTC4 and LTD4, respectively. In contrast, incubation with Toxoplasma gondii, an obligate intracellular protozoan, led to the formation of 11-, 12-, and 15-hydroxyicosatetraenoic acids (HETEs), together with an unidentified compound, designated compound X. Each of these compounds incorporated [3H]arachidonic acid from the macrophage during phagocytosis of T. gondii. Compound X migrated immediately prior to 15-HETE by reverse-phase HPLC and was distinct from authentic monoHETE, monohydroperoxyicosatetraenoic acid (mono-HPETE), and dihydroxyicosatetraenoic acid (diHETE) standards. The generation of compound X by macrophages correlated with the extent of phagocytosis of T. gondii and with intracellular survival of the organisms. Prior antibody-coating of T. gondii or activation of macrophages, either of which inhibited survival and replication of ingested organisms, was associated with production of LTD4 but not compound X. Killed organisms also stimulated LTD4 release only. Although T. gondii concentrated arachidonic acid, they did not metabolize the compound to identifiable lipoxygenase products. Preincubation of macrophages with the relative lipoxygenase inhibitors nordihydroguaiaretic acid or 5,8,11,14-icosatetraynoic acid inhibited the formation of compound X. The absence of leukotriene production by macrophages ingesting T. gondii may explain the relative lack of a neutrophil inflammatory response in diseases due to obligate intracellular organisms. Alternatively, compound X may have functional activities that might mediate some of the host responses to cellular parasitism.

Animals↗
PubMed1985

Infection with varicella-zoster virus after marrow transplantation.

Infection with varicella-zoster virus (VZV) occurred in 231 (16.6%) of 1,394 patients undergoing marrow transplantation in Seattle, Washington, between 1969 and 1982. The probability of VZV infection was 30% by one year after transplant. Eighty percent of infections occurred within the first nine months after transplant, and of these cases 45% had cutaneous or visceral dissemination. Twenty-three deaths were associated with VZV infection, all within the initial nine months after transplant. Postherpetic neuralgia, scarring, and bacterial superinfection were also significantly more frequent among patients with VZV in the first nine months after transplant (32%) than among patients with later infection (19%; P less than .05). By multivariate analysis, allogeneic transplant, acute or chronic graft-vs.-host disease, patient age between 10 and 29 years, diagnosis other than chronic myelogenous leukemia, and posttransplant use of antithymocyte globulin were each risk factors for VZV infection. Among infected patients, the only significant risk factor for VZV dissemination or death was acute graft-vs.-host disease (P less than .03 and P less than .0002, respectively.

Adolescent↗
PubMed1984

Development of cellular immunity in cutaneous leishmaniasis due to Leishmania tropica.

A laboratory worker was inadvertently inoculated with infectious material while passaging amastigotes of Leishmania tropica. The subsequent development of a cutaneous lesion was correlated with results of sequential in vitro bioassays of cell-mediated immunity. Induction of lymphocyte proliferation and interleukin 2 production by soluble L. tropica antigens appeared within five weeks of infection and reached maximal levels coincident with ulceration of the skin lesion. Thereafter interleukin 2 production rapidly decreased, whereas the lymphocyte proliferative response declined more slowly. Biopsy of the lesion after 12 weeks, at a time when antigen-induced lymphocyte proliferation was still significantly elevated but interleukin 2 activity had returned to near baseline values, demonstrated no organisms either histologically or by culture. Healing was complete after 20 weeks. These studies provide data on the kinetics of the development of cell-mediated immunity in human cutaneous leishmaniasis and suggest the usefulness of in vitro bioassays of specific immune reactivity to parasites for evaluating effective cell-mediated immunity.

Adult↗
PubMed1984

Interaction of primate alveolar macrophages and Legionella pneumophila.

We studied the interaction between Legionella pneumophila, which is principally a pulmonary pathogen, with primate alveolar macrophages (AM), which are the primary pulmonary cellular defense mechanism. For these studies we used L. pneumophila, type I, which were grown in albumin-yeast extract broth, were greater than 80% viable, and were comparable in virulence for guinea pigs to organisms from guinea pig spleen homogenates. For comparison, avirulent agar-passed L. pneumophila, type I, and a strain of Escherichia coli were also used. In the absence of detectable antibody, AM phagocytosed similar numbers of virulent and avirulent Legionella and killed the majority of ingested Legionella in 15-30 min, as determined by two different assays. The virulent and avirulent Legionella appeared to be equally susceptible to the cidal systems of the AM and both were killed more readily than were E. coli under both assay conditions. Phagocytosis of Legionella by AM was associated with a localized respiratory burst, as indicated by nitroblue tetrazolium reduction around ingested organisms. Killing of AM-associated Legionella was inhibited by the hydroxyl radical (OH.) scavenger mannitol (but not by an equiosmolar concentration of sodium sulfate), and by a combination of superoxide dismutase and catalase (but not by either enzyme alone). These findings suggest a contribution by OH., one generated by the metal-catalyzed interaction of superoxide and hydrogen peroxide (Haber-Weiss reaction) in the anti-Legionella activity of AM. The virulent Legionella that survived intracellularly increased in number from 4 X 10(4) at 1 h to 6 X 10(6) at 96 h after infection. In contrast, avirulent Legionella replicated more slowly, increasing in number from 4 X 10(4) to 1 X 10(5) over the same period. Replication of virulent Legionella destroyed the AM monolayers by 120 h, whereas monolayers containing avirulent organisms remained intact. Thus, virulence of Legionella appears not to correlate with its ability to survive early killing by AM, but rather with the ability of the small fraction of surviving organisms to replicate within these cells.

Animals↗
PubMed1984

Reversal of dementia associated with Whipple's disease by trimethoprim-sulfamethoxazole, drugs that penetrate the blood-brain barrier.

A previously healthy 67-yr-old man presented with progressive dementia over an 11-mo period. Evaluation revealed evidence of malabsorption. Jejunal biopsy established the diagnosis of Whipple's disease. No other etiology for the patient's dementia was uncovered. Treatment with trimethoprim-sulfamethoxazole resulted in rapid elimination of Whipple's bacilli from the jejunum and complete reversal of the patient's dementia over a 6-mo period. Significant levels of trimethoprim and sulfamethoxazole were easily quantitated in the cerebrospinal fluid during therapy. There is increasing recognition of progressive neurologic disease in patients with Whipple's disease who were treated with tetracycline. The reversal of presumed central nervous system disease in this case suggests that drugs that penetrate the blood-brain barrier might be preferable for the initial treatment of Whipple's disease.

Aged↗
PubMed1983

Oxidative response of phagocytes to parasite invasion.

Phagocytes destroy intracellular pathogens and extracellular targets in part by the production of toxic oxygen metabolites--namely, superoxide, hydrogen peroxide, hydroxyl radicals and possibly singlet molecular oxygen. The toxicity of hydrogen peroxide is increased greatly by peroxidase and a halide. A peroxidase that can be used for this purpose is present in neutrophils and monocytes (myeloperoxidase), but is lost when the monocyte matures into a macrophage; a different peroxidase is present in eosinophils. The latter enzyme, because of its strong positive charge, binds to the surface of parasites; any phagocyte in the region, when appropriately stimulated, may provide the hydrogen peroxide required for completion of the peroxidase system. Further, peroxidase-coated organisms are more readily killed when ingested by macrophages than are uncoated organisms. Oxygen-dependent toxicity requires the production of toxic oxygen products by phagocytes in amounts sufficient to overcome the protective capacity of endogenous scavengers in the parasite. The latter include catalase and glutathione peroxidase, which degrade hydrogen peroxide, and superoxide dismutase which dissipates superoxide. The host defence against parasites appears to depend in part on this balance between toxic oxygen metabolites and scavengers.

Animals↗
PubMed1983

Oxygen-dependent microbicidal systems of phagocytes and host defense against intracellular protozoa.

The role of oxygen-dependent microbicidal systems of leukocytes in the host defense against the major nonerythrocytic intracellular protozoa which infect man--Toxoplasma gondii, Trypanosoma cruzi, and the Leishmania species--is reviewed. The hydrogen peroxide-halide-peroxidase microbicidal system is uniformly cidal to these organisms in vitro. Peroxidase-independent oxygen product(s) toxicity is more variable. Studies to data indicate that phagocytes which contain granule peroxidase and which have the capacity to generate a vigorous respiratory burst; eg, neutrophils and monocytes, possess substantial activity against these protozoa. The absence of granule peroxidase together with the markedly attenuated respiratory burst of resident macrophages leaves these cells with a severe microbicidal defect. These protozoa can enter resident macrophages in the absence of antibody and survive and replicate within the intracellular environment. Enhancement of the antiparasite activity of resident macrophages can be accomplished either by activation of these cells by exposure to sensitized T-cell products, or by the introduction of exogenous peroxidase into the vacuole. Other factors influencing the ability of protozoa to survive intracellularly include the capacity of these organisms to avoid effective triggering of the macrophage respiratory burst and the levels of endogenous scavengers of oxygen products within the parasite.

Animals↗
PubMed1983

Increased respiratory burst in myeloperoxidase-deficient monocytes.

Studies of the respiratory burst in myeloperoxidase (MPO) deficient monocytes were undertaken to assess the physiologic consequence of the absence of MPO in these cells. As previously demonstrated with neutrophils, MPO-deficient monocytes had a greater initial rate, duration, and total superoxide production in response to phagocytosis of zymosan than did normal monocytes. Introduction of purified eosinophil peroxidase (EPO) into the phagosome by binding the enzyme to the surface of the zymosan particles changed the hypermetabolic characteristics of superoxide production in MPO-deficient cells to more closely resemble normal cells, but had no effect on superoxide generation by the normal monocytes. Further, inactivation of the bound EPO before ingestion restored the supranormal respiratory burst by the MPO-deficient cells. Iodination by MPO-deficient monocytes was significantly depressed as compared to normal monocytes following the ingestion of zymosan (1.9 versus 10.1 nmole I-/10(7) monocytes/30 min; p less than 0.01). In contrast, iodination was markedly augmented in MPO-deficient cells compared to normal cells after ingestion of zymosan coated with EPO (208 versus 70 nmole I-/10(7) monocytes/30 min; p less than 0.005), presumably reflecting the greater amounts of hydrogen peroxide formed by MPO-deficient cells. There were no differences in the levels of endogenous scavengers of reactive oxygen products (catalase, superoxide dismutase, glutathione peroxidase and reductase, and total glutathione) in MPO-deficient and normal monocytes that would account for the enhanced respiratory burst of MPO-deficient cells. These findings support a role for peroxidase in the termination of the respiratory burst of monocytes.

Humans↗
PubMed1982

Role for endogenous and acquired peroxidase in the toxoplasmacidal activity of murine and human mononuclear phagocytes.

Oxygen products generated by the respiratory burst of mononuclear phagocytes are microbicidal to intracellular pathogens including Toxoplasma gondii. The toxicity of one of these products, H(2)O(2), is markedly amplified by the granule peroxidase of circulating phagocytes in the presence of a halide. Eosinophil peroxidase (EPO) binds firmly to the surface of T. gondii and such organisms remain viable as determined by vital staining, uptake of 2-deoxyglucose, and survival and replication in human fibroblasts. They are, however, rapidly killed by the addition of H(2)O(2) and iodide under conditions in which control organisms are unaffected. We have used EPO bound to T. gondii to explore the role of peroxidase in the toxoplasmacidal activity of mononuclear phagocytes. Resident mouse peritoneal macrophages lack a granule peroxidase and have a weak respiratory burst; toxoplasma survive and replicate within these cells. However, these cells acquire significant toxoplasmacidal activity, as assessed microscopically and by the inhibition of uracil uptake, when organisms are coated with EPO before ingestion, an effect which is decreased by the hemeprotein inhibitors, aminotriazole and azide. EPO on the surface of Toxoplasma does not increase their ingestion by macrophages or the associated respiratory burst. Monocytes from patients with hereditary myeloperoxidase deficiency have a significant toxoplasmacidal defect that is abolished when EPO-coated organisms are used. In contrast, the toxoplasmacidal defect of monocytes from chronic granulomatous disease patients is unaffected by surface-bound EPO. In these studies, replication of surviving intracellular organisms varied inversely with the magnitude of the respiratory burst: replication was greatest in fibroblasts, slightly less in resident macrophages, and least in monocytes; it was significantly greater in chronic granulomotous disease than in normal or myeloperoxidase-deficient monocytes. These studies support a role for oxygen products and endogenous peroxidase in the optimal killing of T. gondii by monocytes and demonstrate that peroxidase-negative phagocytes can utilize peroxidase on the surface of ingested organisms to augment microbicidal activity.

Animals↗
PubMed1982

Multiply antibiotic-resistant Staphylococcus aureus: introduction, transmission, and evolution of nosocomial infection.

A burn patient with a multiply antibiotic-resistant Staphylococcus aureus infection was transferred to Harborview Medical Center from a burn unit in another state. Despite standard wound precautions, transmission to 34 patients occurred during the subsequent 15 months. Twenty-seven of the patients were infected. Disease included pneumonia, empyema, bacteremia, endocarditis, osteomyelitis, and burn and wound infections. Seventeen of the 34 patients died. Phage typing and plasmid analysis showed the spread of multiply resistant S. aureus from the burn unit to the surgical intensive care unit where a study evaluating the use of chloramphenicol in cases of bowel sepsis was in progress. During this period the organism became resistant to chloramphenicol by acquiring either of two chloramphenicol R-plasmids. Using plasmid profiles and antibiograms, four epidemic strains were identified that assisted in identifying patient and personnel reservoirs. The outbreak was controlled only after rifampin was added to vancomycin treatment of infected patients, which correlated with eradication of the carrier state.

Adult↗
PubMedDate not supplied

Murine cutaneous leishmaniasis: susceptibility correlates with differential expansion of helper T-cell subsets.

BALB/c mice develop fatal illness following infection with Leishmania major despite expansion of helper L3T4+ T cells in the draining lymph nodes and spleen. Healer mice, either genetically resistant C57BL/6 or BALB/c that have been pretreated with monoclonal antibody GK 1.5, also develop expanded numbers of L3T4+ T cells at the time of healing. Lymph node cells from healer mice produce gamma-interferon in vitro and message for gamma-interferon can be recovered from the lymph nodes during healing in vivo. Conversely, cells harvested from non-healer mice during the course of infection produce minimal gamma-interferon in vitro and have little message for gamma-interferon detectable in vivo. When the same Northern blots are hybridized for IL-4, large amounts of IL-4 message are detected only in cells from non-healer mice. The data are consistent with the expansion of type 1 helper cells (Th1) during healing and type 2 helper cells (Th2) during progressive leishmania infection.

Animals↗