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C J Morrison

Publications and source records attributed to C J Morrison.

At least 37 records · Page 2Linked to original sources
PubMed1995

Role of the Leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis.

The Leishmania surface protease gp63 has been identified as a parasite virulence factor. To better define the role of gp63 in Leishmania infectivity, the interaction of recombinant gp63 with complement and complement receptors was examined. On Leishmania, gp63 was not necessary for complement fixation. Complement activation occurred on transfected organisms expressing varying amounts of gp63 and on organisms expressing a mutant form of gp63 devoid of proteolytic activity. However, organisms expressing wild-type gp63 on their surface fixed only small amounts of the terminal complement components and were dramatically more resistant to lysis by complement than were those lacking functional gp63. Organisms expressing wild-type gp63 more rapidly converted C3b on their surface to a form that exhibited the neoantigen of iC3b and interacted avidly with cells expressing Mac-1, the receptor for iC3b. Complement fixation by transfected mammalian cells expressing recombinant Leishmania gp63 on their surface was also examined. The presence of Leishmania gp63 on the surface of these cells converted them into efficient activators of complement. Cells expressing gp63 on their surface fixed complement and bound avidly to the human complement receptors. The proteolytic activity of this molecule was not necessary for complement activation or adhesion to complement receptors. Thus, gp63 may contribute to parasite virulence by exerting a novel type of control over complement fixation. Organisms expressing gp63 can exploit the opsonic properties of complement while avoiding its lytic effects.

Animals↗
PubMed1995

Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood.

We developed a microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species. Nucleotide sequences derived from the internal transcribed spacer (ITS) region of fungal rDNA were used to develop species-specific oligonucleotide probes for Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridization was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Saccharomyces cerevisiae DNA but with no other DNAs tested. Genomic DNA purified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated capture probe, and streptavidin-coated microtitration plates, amplified DNA from a few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay.

Animals↗
PubMed1995

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Complementary Therapies↗
PubMed1994

Mutational and functional analysis of the Leishmania surface metalloproteinase GP63: similarities to matrix metalloproteinases.

The major surface glycoprotein of Leishmania, referred to as GP63, is a zinc metalloproteinase of 63,000 M(r) present on promastigotes and amastigotes from diverse species of Leishmania. GP63 shares several characteristics with the members of the matrix metalloproteinase family including degradation of at least one component of the extracellular matrix, location at the cell surface, requirement for Zn2+ for proteinase activity and inhibition of the proteinase activity by chelating agents and alpha 2-macroglobulin. Site-directed mutagenesis of the cloned L. major GP63 genes was carried out to determine whether the proposed active site of Leishmania GP63 was homologous to those of other zinc metalloproteinases. The codon encoding the catalytic glutamic acid was modified to encode an aspartic acid and when expressed in COS-7 cells the resulting mutant GP63 had no demonstrable proteinase activity compared to wild type GP63. GP63 was predicted to be synthesized as a precursor protein containing a pro region at the NH2-terminus of GP63 implicated to be involved with the regulation of proteinase activity. As with many other proteinases, including matrix metalloproteinases, these enzymes are synthesized as latent proteinases that require activation for full proteinase activity. L. major recombinant GP63 (rGP63) has been produced in the baculovirus expression system where rGP63 was secreted as a latent proteinase. To study the activation of baculovirus rGP63, purified rGP63 was incubated with the mercurial compound, HgCl2, at concentrations previously shown to result in activation of other latent matrix degrading metalloproteinases and resulted in a significant enhancement of GP63 proteinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Purification and characterization of the extracellular aspartyl proteinase of Candida albicans: removal of extraneous proteins and cell wall mannoprotein and evidence for lack of glycosylation.

Aspartyl proteinase (AP) is an extracellular enzyme of Candida albicans implicated as a pathogenic factor. Previous reports on the purification and characterization of AP suggested that a single DEAE-Sephadex chromatographic step was sufficient for the removal of extraneous proteins and that the final product was glycosylated. We purified AP using a chromatographic series consisting of DEAE-Sephadex A25, Sephadex G75 and rechromatography on DEAE-Sephadex A25. Use of DEAE-Sephadex alone did not remove extraneous proteins and removed little contaminating mannoprotein (MP). The addition of a Sephadex G75 column to the purification scheme removed the majority of contaminating MP and proteins. The final DEAE-Sephadex A25 chromatographic step resulted in (a) removal of detectable extraneous proteins, (b) removal of immunologically detectable MP by dot blot and Western blot enzyme immunoassay, (c) loss of periodic acid-silver stain positivity, and (d) a high AP yield (1295 U l-1) and specific activity (1749 U mg-1). We conclude that a single DEAE-Sephadex A25 purification step is insufficient to remove extraneous proteins and MP, which could interfere with the production of AP-specific antibodies and the dissection of moieties responsible for immune reactivity. Reports of periodic acid-Schiff or anthrone positivity of AP preparations may reflect the presence of extraneous MP, which can be removed by the chromatographic series we describe.

Aspartic Acid Endopeptidases↗
PubMed1993

Nonculture methods for diagnosis of disseminated candidiasis.

Two of the nonculture approaches to the diagnosis of DC, enzymatic-fluorometric determination of serum D-arabinitol and detection of marker antigens in antigenemia (enolase and CWMP), have been commercialized and have shown promise in limited clinical trials. These approaches are not new but are the culmination of efforts made over 10 or more years. Clearly, further fine-tuning of both metabolite and antigen detection is needed to simplify the methods and to improve their sensitivity and specificity so that they will be valuable in guiding clinical treatment decisions. An alternative approach, detection of DC by DNA amplification methods such as PCR, is a special case of a compelling technology and one that is capable of standardization across microbial genera. The availability of simplified PCR diagnostic methods for DC remains a tantalizing prospect. Nevertheless, the development of methods to release DNA from very small numbers of Candida organisms in the blood in a form that is sufficiently free of inhibitors of PCR will require further intensive effort. The maturation of these converging laboratory approaches to nonculture diagnosis of DC leads to more optimism about the eventual use of these methods in clinical laboratories.

Antigens, Fungal↗
PubMed1993

Heterogeneity of the purified extracellular aspartyl proteinase from Candida albicans: characterization with monoclonal antibodies and N-terminal amino acid sequence analysis.

Three dominant proteins (41, 48, and 49 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in purified preparations of the extracellular aspartyl proteinase (AP) of Candida albicans. All three proteins bound to the specific carboxyl proteinase ligand, pepstatin A, and were associated with maximum AP activity. The N-terminal amino acid sequence for the 48- and 49-kDa proteins matched that reported by others for AP, whereas the sequence for the 41-kDa protein was unique and was not homologous to any known protein. Time course studies demonstrated the simultaneous presence of all three proteins, supporting evidence that the 41- and 48-kDa proteins were not breakdown products of AP. Previous studies did not detect carbohydrate in SDS-polyacrylamide gels of purified AP preparations stained with periodic acid and silver, making glycosylation an unlikely explanation for the observed differences in the molecular masses of the proteins. Some monoclonal antibodies directed against the 49-kDa protein reacted with the 41- and 48-kDa proteins, indicating cross-reactive epitopes. Other monoclonal antibodies, however, reacted only with the 49-kDa protein. We conclude that three pepstatin A-binding proteins occur in purified AP preparations: two have the same amino acid N terminus as that reported for AP, whereas the third has a unique sequence. All three proteins should be considered when undertaking studies to determine the role of AP in candidal pathogenesis or when preparing specific antibodies for antigen capture assays.

Amino Acid Sequence↗
PubMed1991

Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation.

A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.

Amino Acid Sequence↗
PubMed1991

Mechanisms of fungal pathogenicity: correlation of virulence in vivo, susceptibility to killing by polymorphonuclear neutrophils in vitro, and neutrophil superoxide anion induction among Blastomyces dermatitidis isolates.

Seven Blastomyces dermatitidis isolates varying in virulence for mice were compared for susceptibility to polymorphonuclear neutrophil (PMN) killing and the ability to induce superoxide anion (O2-) production by PMNs in vitro. In vitro killing of six B. dermatitidis isolates by murine peripheral blood PMNs or by PMNs elicited from the peritoneal cavity by a local immune reaction (B. dermatitidis-immune mice given killed B. dermatitidis intraperitoneally 24 h earlier) inversely correlated with in vivo virulence (most to least virulent) isolates: VV, V, V40, KL-1, A2, and GA-1). The capacity of isolates to induce O2- production by PMNs also inversely correlated with in vivo virulence. Isolate A, of intermediate in vivo virulence, was a good inducer of O2- production in vitro but was no more susceptible to in vitro killing by PMNs than isolate V, VV, or V40. Fungal intracellular superoxide dismutase or catalase content did not correlate with in vivo virulence or in vitro killing by PMNs. Isolate A, however, had two to four times the intracellular catalase activity as did other B. dermatitidis isolates, suggesting a possible mechanism for its enhanced resistance to in vitro killing by PMNs. Therefore, while in vitro killing by PMNs and the capacity to induce O2- production by PMNs inversely correlated with virulence for six B. dermatitidis isolates, isolate A was an exception: its resistance to killing by PMN-generated oxygen metabolites in vitro but its susceptibility to killing in vivo suggest that its in vivo killing occurs by other, perhaps nonoxidative, mechanisms.

Animals↗
PubMed1990

Comparative effects of cilofungin and amphotericin B on experimental murine candidiasis.

The effectiveness of cilofungin (LY121019, referred to hereafter as LY), a lipopeptide, was studied in a murine candidiasis model. CD-1 mice (5 weeks old) were injected intravenously with 3 x 10(5) Candida albicans yeast cells. Intraperitoneal LY or amphotericin B (AmB) therapy was begun 4 days after infection and was continued daily for 2 weeks. LY and AmB were compared at 62.5, 6.25, and 0.625 mg/kg per day, with the LY dose split into two treatments per day. Mice were observed for 30 days postinfection, and survivors were necropsied. AmB at 62.5 mg/kg per day was lethal in the absence of infection. Cumulative mortality for infected controls was 94% (17 of 18). Survival of mice treated with the control diluent for LY was the same as survival with no treatment. Survival after 0.625 mg of LY per kg per day was the same as that of the controls, and 6.25 or 62.5 mg of LY per kg per day was significantly superior. AmB treatment at 0.625 or 6.25 mg/kg per day was protective and superior to the same LY doses. Atrophied kidneys were common in AmB-treated mice, and mice treated with 6.25 mg of AmB per kg per day appeared ill during therapy. The number of CFU recovered from kidneys and spleens of surviving mice reflected the same relationships between drugs and doses as those described for mortality. C. albicans was not cleared from the kidneys of mice in any group, and only in the 6.25-mg/kg-per-day AmB treatment group was not detectable C. albicans found in the spleens. These data indicate that LY or AmB suppresses candida infection but neither is curative in this model.

Amphotericin B↗
PubMed1989

Enhanced killing of Blastomyces dermatitidis by gamma interferon-activated murine peripheral blood polymorphonuclear neutrophils.

Normal peripheral blood polymorphonuclear neutrophils (PB-PMNs), challenged in vitro with yeast form Blastomyces dermatitidis, reduced inoculum colony-forming units of a virulent strain by 37.5 +/- 9.5%. Pre-incubation of PB-PMNs with 10-100,000 U/ml of purified recombinant murine gamma-interferon (IFN) for 1 h prior to challenge with fungi resulted in significant enhancement of PB-PMN fungicidal activity. No direct fungicidal activity by IFN alone was observed. Pretreatment of selected concentrations of IFN shown to have PMN-enhancing activity (100 or 1000 U/ml) with rabbit hyperimmune anti-IFN antiserum for 1 h before addition to PB-PMNs abrogated the enhancement of fungicidal activity. Isolated peripheral blood mononuclear cells failed to kill B. dermatitidis, even when mononuclear cells were present at a concentration ten times greater than that normally used in killing assays, and failed to be activated by IFN. Treatment of unstimulated or IFN-activated PB-PMNs with complement and hybridoma-derived monoclonal antibody specific for PMNs eliminated PB-PMN fungicidal activity. Exogenously added lipopolysaccharide (0.0005-50,000 ng/ml) did not activate PB-PMNs, whether added alone or in conjunction with IFN. The PB-PMN activating capacity of IFN could be destroyed by heat treatment (100 degrees C, 15 min) or by acid treatment with HCl (pH 2). These results demonstrate that recombinant gamma-interferon can stimulate PB-PMNs to kill B. dermatitidis, that the PB-PMN activating moiety is IFN and that PB-PMNs are responsible for fungal killing in this assay system.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

In vivo activation of peripheral blood polymorphonuclear neutrophils by gamma interferon results in enhanced fungal killing.

The effect of in vivo administration of murine recombinant gamma interferon (IFN) on the fungicidal activity of murine peripheral blood polymorphonuclear neutrophils (PB-PMNs) was studied. Mice were injected intramuscularly with 250, 2,500, 25,000 or 250,000 U of IFN 5 h before collection of peripheral blood. Purified PB-PMNs were cocultured in vitro with Blastomyces dermatitidis yeast cells for 2 h. PB-PMNs from untreated mice killed 44.5 +/- 12.5% of the fungal inoculum, whereas PB-PMNs from mice treated with 25,000 or 250,000 U of IFN showed significantly enhanced in vitro killing (68.0 +/- 9.4% [P less than 0.005] and 72.3 +/- 1.1% [P less than 0.001], respectively). Treatment with 250 or 2,500 U of IFN or 25,000 U of heated (100 degrees C, 15 min) IFN had no effect. The IFN-induced activation of PB-PMNs was transitory. Significant enhancement of PB-PMN killing activity occurred 1, 2, or 5 h after in vivo IFN administration, but no enhancement was observed 16 or 24 h after IFN treatment. Enhanced fungicidal activity by PB-PMNs from mice treated for 5 h with 25,000 U of IFN correlated with an increased release of superoxide anion (O2-) in vitro after stimulation of PB-PMNs with phorbol ester; normal PB-PMNs and IFN-activated PB-PMNs, respectively, produced 2.2 +/- 2.5 and 23.5 +/- 4.8 nmol of O2- per 10(6) PB-PMNs per 30 min (P less than 0.005). The exogenous addition of compounds that antagonize or inhibit the formation of oxygen radicals (superoxide dismutase, catalase, dimethyl sulfoxide, or sodium azide) significantly inhibited fungal killing by both normal and IFN-activated PB-PMNs. In addition to the enhanced microbicidal activity and superoxide generation demonstrated in vitro with constant cell numbers, there was a transient leukocytosis (particularly neutrophilia) in peripheral blood at doses of IFN and at times after IFN administration where enhanced activity was also demonstrated. In summary, our results indicate that PB-PMNs can be activated in vivo for enhanced killing of a fungal target. The enhanced killing capacity of IFN-activated PB-PMNs is due at least in part to the enhancement of oxidative killing mechanisms.

Animals↗
PubMed1988

Enhanced oxidative mechanisms in immunologically activated versus elicited polymorphonuclear neutrophils: correlations with fungicidal activity.

Peritoneal polymorphonuclear neutrophils (PMN) from mice were tested for their ability to kill the yeast form of Blastomyces dermatitidis (Bd) in vitro and for their fungicidal mechanisms. PMN elicited from immune mice by the intraperitoneal injection of non-viable Bd (referred to as immunologically activated PMN or ActPMN) showed significantly enhanced fungicidal activity in comparison with PMN elicited with thioglycollate medium (ThioPMN) [means = 44.7% (SD 12.8%) and 16.4% (SD 9.2%) killed; n = 14; p less than 0.001]. Production of superoxide anion (O2-) by ActPMN after stimulation with phorbol myristate acetate was enhanced in comparison with production by ThioPMN. Superoxide dismutase, which removes O2-, inhibited ActPMN killing by 75% (p less than 0.001) when added to cultures immediately before challenge with Bd (optimal concentration: 6000 U/ml). Sodium azide, which inhibits myeloperoxidase and scavenges singlet oxygen (1O2), and catalase, which breaks down hydrogen peroxide (H2O2), inhibited ActPMN killing by 64% (p less than 0.001) and 52% (p less than 0.001), with optimal concentrations of 1 mM and 10,000 U/ml, respectively. Two agents that both scavenge 1O2 and antagonise hypochlorous acid (HOCl-), histidine and tryptophan, were also powerful inhibitors of ActPMN killing. Quenchers of hydroxyl radical (.OH), dimethylsulfoxide and sodium benzoate, had less effect, and required higher concentrations. These data suggest that the enhanced killing of Bd by ActPMN involves one or more oxidative mechanisms, and that there is a prominent role for O2-, either directly or as a precursor of other active oxygen species, a probable role for H2O2, and possible roles for 1O2, HOCl-, and .OH.

Animals↗
PubMed1987

Activation of murine polymorphonuclear neutrophils for fungicidal activity by recombinant gamma interferon.

Recombinant murine gamma-interferon (IFN) was tested for its ability to enhance murine polymorphonuclear neutrophil (PMN) fungicidal activity in vitro. PMNs, elicited by intraperitoneal injection of thioglycollate 4 hr prior to collection, were treated with 0.00003-300,000 units of IFN per milliliter for 1 hr prior to challenge with yeast form Blastomyces dermatitidis. These PMNs were not fungicidal for Blastomyces in the absence of IFN; significant enhancement of PMN fungicidal activity by IFN treatment occurred in a dose-dependent manner with maximal enhancement observed at 30,000 U/ml (21% killing). Pretreatment of IFN with rabbit anti-IFN antiserum before addition to PMNs eliminated the enhancement of fungicidal activity by effective doses of IFN. PMN fungicidal activity against phagocytizable Candida albicans was significantly (P less than .001) higher (71.3 +/- 17.4%) than against B. dermatitidis. Candidacidal activity was not significantly enhanced by IFN treatment of PMNs. Exogenously added lipopolysaccharide, at levels corresponding to those found in this preparation of IFN, did not activate PMNs for enhanced fungicidal capacity. These data indicate a stimulatory role for IFN in the killing of B. dermatitidis by PMNs, suggesting that IFN is an active component of the communication between T lymphocytes and PMNs with respect to antimicrobial resistance. They suggest a natural role for IFN in host defense against blastomycosis and other fungal infections, and a possible therapeutic use for exogenous IFN in fungal disease.

Animals↗
PubMed1987

Effect of a local immune reaction on peripheral blood polymorphonuclear neutrophil microbicidal function: studies with fungal targets.

Peripheral blood polymorphonuclear neutrophils (PMN) from mice immunized with Blastomyces dermatitidis and then stimulated locally (intraperitoneally, ip) with B. dermatitidis antigen had enhanced killing of B. dermatitidis in vitro (54.4 +/- 19.49 of inoculum) compared to nonimmune mice (32.7 +/- 8.7%; P less than 0.02), nonimmune mice given antigen ip (30.6 +/- 14.0%; P less than 0.05), or immune mice not given antigen ip (15.4 +/- 9.9%; P less than 0.01). Peripheral blood PMN from all four groups had marked killing ability against Candida albicans (91.8-99.3% of inoculum). That the killing of B. dermatitidis was due to PMNs was demonstrated by lack of killing by isolated peripheral blood mononuclear cells from all four groups. A local immune reaction can result in enhancement of PMN fungicidal activity, and this is reflected even in peripheral blood PMN. We hypothesize this is an important component of normal host defenses against fungal infection, and likely other microbial infections. Enhancement of PMN microbicidal function by the soluble mediators presumed to be responsible for the effects observed may be an approach to immunomodulating therapy or prophylaxis of infection.

Animals↗
PubMed1985

Recombinant and natural gamma-interferon activation of macrophages in vitro: different dose requirements for induction of killing activity against phagocytizable and nonphagocytizable fungi.

Recombinant murine gamma-interferon (IFN) and supernatants from concanavalin A (ConA)-stimulated spleen cells were tested for their ability to activate resident peritoneal macrophages (M phi) for fungicidal activity. M phi monolayers pulsed overnight with IFN exhibited significantly enhanced fungicidal activity against Candida albicans (44 +/- 12 versus 0.0%) and Blastomyces dermatitidis (34 +/- 1 versus 3 +/- 3%). The effect of IFN was dose dependent; however, less IFN (10 U/ml) was required to activate M phi to kill phagocytizable C. albicans than to kill nonphagocytizable B. dermatitidis (1,000 U/ml). ConA-stimulated spleen cell supernatants were also able to activate M phi for fungicidal activity against both fungi. The capacity of ConA-stimulated spleen cell supernatants to activate M phi for fungicidal activity was neutralized in the presence of antibody to murine IFN. ConA-treated monolayers acquired the ability to kill C. albicans, but not B. dermatitidis, which was shown to be associated with residual (10%) lymphocytes in the monolayers. Lipopolysaccharide (0.001 to 10 micrograms/ml) failed to consistently activate M phi for fungicidal activity. These data show that IFN can exert an immunoregulatory role on M phi defense against these fungal pathogens.

Animals↗
PubMed1984

Enhanced killing of Candida albicans by cultured peritoneal exudate cells treated with SM-1213, a synthetic immunomodulator.

The effect of SM-1213 [1,2-O-isopropylidene-3-O-3'-(N', N'-dimethylamino-n-propyl)-D-glucofuranose] on the candidastatic and candidacidal capacity of guinea pig peritoneal exudate cells was investigated. Phagocytes treated with SM-1213 demonstrated an enhanced ability to inhibit intracellular hyphal formation and elongation of Candida albicans cells as well as to reduce the percentage of viable cells remaining after phagocytosis. Significant drug-induced anticandidal effects were also observed when peritoneal exudate cells were preincubated with SM-1213 and washed before infection with C. albicans. SM-1213 had no direct anticandidal effect against the yeast or hyphal form of the fungus, suggesting that the observed candidastatic and candidacidal effect of this drug was due to the enhancement of antimicrobial functions of phagocytes.

Adjuvants, Immunologic↗