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S M Levitz

Publications and source records attributed to S M Levitz.

At least 19 recordsLinked to original sources
PubMed1999

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages.

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 microM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.

Calibration↗
PubMed1999

Interactions of Penicillium marneffei with human leukocytes in vitro.

Penicillium marneffei, a dimorphic fungus endemic in parts of Asia, causes disease in those with impaired cell-mediated immunity, especially persons with AIDS. The histopathology of penicilliosis marneffei features the intracellular infection of macrophages. We studied the interactions between human leukocytes and heat-killed yeast-phase P. marneffei. Monocyte-derived macrophages bound and internalized P. marneffei in the presence of complement-sufficient pooled human serum (PHS). Binding and phagocytosis were still seen if PHS was heat inactivated or omitted altogether. The binding of unopsonized P. marneffei to monocyte-derived macrophages occurred in the absence of divalent cations and was not affected by inhibitors of mannose and beta-glucan receptors or monoclonal antibodies directed against CD14 and CD11/CD18. Binding was profoundly inhibited by wheat germ agglutinin. A vigorous respiratory burst was seen in peripheral blood mononuclear cells (PBMC) stimulated with P. marneffei, regardless of whether the fungi were opsonized. However, tumor necrosis factor alpha (TNF-alpha) release from PBMC stimulated with P. marneffei occurred only if serum was present. These data demonstrate that (i) monocyte-derived macrophages bind and phagocytose P. marneffei even in the absence of opsonization, (ii) binding is divalent cation independent but is inhibited by wheat germ agglutinin, suggesting that the major receptor(s) recognizing P. marneffei is a glycoprotein with exposed N-acetyl-beta-D-glucosaminyl groups, (iii) P. marneffei stimulates the respiratory burst regardless of whether opsonins are present, and (iv) serum factors are required for P. marneffei to stimulate TNF-alpha release. The ability of unopsonized P. marneffei to parasitize mononuclear phagocytes without stimulating the production of TNF-alpha may be critical for the virulence of this intracellular parasite.

Adult↗
PubMed1997

Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation.

Infections due to Cryptococcus neoformans are common in AIDS patients. We investigated the effect of chloroquine, which raises the pH of phagolysosomes, on the anticryptococcal activity of mononuclear phagocytes. C. neoformans multiplied within monocyte-derived macrophages (MDM) in the absence of chloroquine but were killed with the addition of chloroquine. Ammonium chloride was also beneficial, suggesting that effects were mediated by alkalinizing the phagolysosome. Chloroquine inhibits growth of other intracellular pathogens by limiting iron availability. However, chloroquine-induced augmentation of MDM anticryptococcal activity was unaffected by iron nitriloacetate, demonstrating that chloroquine worked by a mechanism independent of iron deprivation. There was an inverse correlation between growth of C. neoformans in cell-free media and pH, suggesting that some of the effect of chloroquine on the anticryptococcal activity of MDM could be explained by relatively poor growth at higher pH. Chloroquine enhanced MDM anticryptococcal activity against all tested cryptococcal strains except for one large-capsule strain which was not phagocytosed. Positive effects of chloroquine were also seen in monocytes from both HIV-infected and -uninfected donors. Finally, chloroquine was therapeutic in experimental cryptococcosis in outbred and severe combined immunodeficient mice. Thus, chloroquine enhances the activity of mononuclear phagocytes against C. neoformans by iron-independent, pH-dependent mechanisms and is therapeutic in murine models of cryptococcosis. Chloroquine might have clinical utility for the prophylaxis and treatment of human cryptococcosis.

Ammonium Chloride↗
PubMed1997

Priming with IFN-gamma restores deficient IL-12 production by peripheral blood mononuclear cells from HIV-seropositive donors.

Production of IL-12 is deficient in PBMC from HIV-infected individuals. Because of recent studies demonstrating that IFN-gamma priming increases the production of IL-12 in normal PBMC, we examined the role of IFN-gamma in the production of IL-12 in PBMC from HIV-seropositive donors. In response to Staphylococcus aureus, production of IFN-gamma and IL-12 was reduced in PBMC from HIV-seropositive compared with that from HIV-seronegative donors. Priming with IFN-gamma, through increases in both IL-12 p40 and p35 mRNA levels, caused a significant increase in IL-12 release by PBMC from both HIV-seropositive and HIV-seronegative donors. However, the increase was greater for PBMC from HIV-seropositive donors, largely restoring the deficit in IL-12 production seen in unprimed cells. In response to Cryptococcus neoformans, Candida albicans, and Mycobacterium tuberculosis, three pathogens that frequently cause opportunistic infections in persons with AIDS, IFN-gamma production was also reduced in PBMC from HIV-seropositive compared with seronegative donors. When primed with IFN-gamma, PBMC from both HIV-seropositive and seronegative donors released substantial and similar quantities of IL-12 in response to these organisms. Taken together, these results demonstrate that IFN-gamma can restore the deficit in IL-12 production seen in HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Induction of human immunodeficiency virus type 1 expression in monocytic cells by Cryptococcus neoformans and Candida albicans.

Because candidiasis and cryptococcosis are common in human immunodeficiency virus (HIV)-infected persons, the effect of Cryptococcus neoformans and Candida albicans on HIV expression in monocytic cells was examined. Stimulation of the latently HIV-infected myelomonocytic cell line OM-10.1 with C. neoformans and C. albicans in the presence of pooled human serum caused a ratio-dependent increase in HIV production. Induction of HIV by C. neoformans was enhanced by anti-capsular antibody, while induction by both organisms was inhibited by anti-TNF-alpha antibody. In THP-1 cells transfected with HIV plasmid constructs, both organisms induced transcription from the HIV long terminal repeat that was dependent on intact NF-kappaB binding sequences. Thus, C. neoformans and C. albicans enhance HIV expression in monocytic cells through a TNF-alpha- and NF-kappaB-dependent mechanism. In HIV-infected patients, such enhancement may further impair host immunity and could accelerate the course of HIV disease.

Binding Sites↗
PubMed1997

Mechanisms of impaired anticryptococcal activity of monocytes from donors infected with human immunodeficiency virus.

The mechanisms by which monocytes from patients infected with human immunodeficiency virus (HIV) have reduced growth inhibitory activity against Cryptococcus neoformans was examined. Monocyte-enriched peripheral blood mononuclear cells from 12 HIV-seropositive donors with CD4 cell counts of 10-210 cells/mm3 (median, 85) and HIV-seronegative donors were compared in assays to determine the binding and phagocytosis of C. neoformans and the respiratory burst and degranulation in response to C. neoformans and zymosan. Monocytes from HIV-infected and uninfected persons bound and ingested C. neoformans equally well; however, generation of hydrogen peroxide and specific release of beta-glucuronidase in response to C. neoformans was significantly reduced in monocyte-enriched cells from the HIV-infected donors. The impaired anticryptococcal activity of monocytes from persons with HIV may be related to defects in both oxidative and nonoxidative effector pathways that occur after the binding and internalization of the organism.

Blood Donors↗
PubMed1997

Variables affecting production of monocyte chemotactic factor 1 from human leukocytes stimulated with Cryptococcus neoformans.

The chemokine monocyte chemoattractant protein 1 (MCP-1) is produced predominantly by mononuclear phagocytes and stimulates recruitment into infected tissues of blood monocytes and T cells. These cell types are thought to be critical to host defenses against infections due to Cryptococcus neoformans, a major cause of disease in persons with AIDS and other disorders of cell-mediated immunity. Accordingly, in the present study, we examined the conditions under which human monocytes and bronchoalveolar macrophages (BAM) are stimulated by C. neoformans to produce MCP-1. C. neoformans was a potent inducer of MCP-1 release from monocytes, with levels of chemokine secreted similar to that seen following stimulation with lipopolysaccharide (LPS). BAM, in contrast, were stimulated by LPS, but not by C. neoformans, to secrete MCP-1. A peak in MCP-1 mRNA was seen 8 h following cryptococcal stimulation of monocytes. Nine strains of C. neoformans stimulated monocytes to release MCP-1, and there was only modest variation between strains. However, when an individual strain was used, the capacity of C. neoformans to stimulate monocyte MCP-1 release did vary, depending upon the conditions used to grow the fungal stimuli. Finally, C. neoformans stimulated comparable quantities of MCP-1 release in monocytes from donors with and without human immunodeficiency virus infection. These data establish C. neoformans as a potent stimulator of MCP-1 in monocytes, but not in BAM. The failure of C. neoformans to stimulate MCP-1 in BAM, if occurring in vivo, could result in a diminished cell-mediated inflammatory response following inhalation of airborne fungi.

Chemokine CCL2↗
PubMed1997

Binding of Cryptococcus neoformans to heterologously expressed human complement receptors.

Previously, we demonstrated that monoclonal antibodies (MAb) directed against any of the three defined complement receptors (CR) for the third component of complement (CR1, CR3, and CR4) profoundly inhibited the binding of serum-opsonized Cryptococcus neoformans to monocyte-derived macrophages. These studies suggested either that a synergistic interaction between multiple CR was required for optimal binding of C. neoformans or that the MAb were exerting nonspecific effects (such as receptor coassociation). In the present studies, we took a novel approach to dissecting out the contributions of individual receptors to binding of a microbial pathogen. Chinese hamster ovary (CHO) cells stably transfected with human CR1, CR3, or CR4 were challenged with serum-opsonized C. neoformans. We found that CHO cells transfected with any of the three receptors bound C. neoformans, with the avidity of binding to CR3 being the greatest followed in decreasing order by CR1 and CR4. Following binding of C. neoformans to transfected CHO cells, most organisms remained surface attached only, although for each receptor a significant percentage (18.5 to 27.3%) of C. neoformans was internalized. Both C. neoformans and sheep erythrocytes that were selectively opsonized with the fragments of the third component of complement, C3b and iC3b, were bound preferentially by CHO cells transfected with CR1 and CR3, respectively. These data establish CR1, CR3, and CR4 as receptors independently capable of binding C. neoformans opsonized with fragments of C3. Moreover, our study demonstrates the usefulness of transfected cell lines as a powerful tool for identifying the contribution of individual receptors to the binding of a microbial pathogen.

Animals↗
PubMed1996

Role of IL-12 in peripheral blood mononuclear cell responses to fungi in persons with and without HIV infection.

Clinical trials of IL-12 in persons infected with HIV have been proposed based on recent evidence suggesting IL-12 plays a critical role in the development of protective immune responses, and the HIV infection is associated with a deficiency of IL-12. As fungal infections are among the most common opportunistic infections associated with AIDS, we examined whether IL-12 p40 gene expression and p70 release in response to Cryptococcus neoformans and Candida albicans were deficient in monocyte-enriched PBMC from HIV-seropositive donors and whether rIL-12 could augment the proliferation of PBMC from HIV-seropositive donors in response to these fungi and to Pneumocystis carinii. PBMC from HIV-seronegative donors expressed IL-12 p40 mRNA in response to C. neoformans, C. albicans, and the positive control Staphylococcus aureus Cowan strain 1 (SAC), although the induction of IL-12 p40 mRNA was later and more prolonged with C. neoformans as the stimulus. Expression of IL-12 p40 mRNA in response to the three stimuli was similar in cells from HIV-seropositive and HIV-seronegative donors. However, when stimulated with SAC, cells from HIV-seropositive donors released significantly less IL-12, suggesting HIV infection induces a post-transcriptional defect in IL-12 release in response to SAC. While PBMC from HIV-seropositive donors had impaired proliferative responses to the three fungi tested, addition of rIL-12 did not enhance proliferation. These studies do not lend further support for the therapeutic use of IL-12 to prevent or treat fungal infections in persons infected with HIV.

AIDS-Related Opportunistic Infections↗
PubMed1996

Virulence factors of medically important fungi.

Human fungal pathogens have become an increasingly important medical problem with the explosion in the number of immunocompromised patients as a result of cancer, steroid therapy, chemotherapy, and AIDS. Additionally, the globalization of travel and expansion of humankind into previously undisturbed habitats have led to the reemergence of old fungi and new exposure to previously undescribed fungi. Until recently, relatively little was known about virulence factors for the medically important fungi. With the advent of molecular genetics, rapid progress has now been made in understanding the basis of pathogenicity for organisms such as Aspergillus species and Cryptococcus neoformans. The twin technologies of genetic transformation and "knockout" deletion construction allowed for genetic tests of virulence factors in these organisms. Such knowledge will prove invaluable for the rational design of antifungal therapies. Putative virulence factors and attributes are reviewed for Aspergillus species, C. neoformans, the dimorphic fungal pathogens, and others, with a focus upon a molecular genetic approach. Candida species are excluded from coverage, having been the subject of numerous recent reviews. This growing body of knowledge about fungal pathogens and their virulence factors will significantly aid efforts to treat the serious diseases they cause.

Antigens, Fungal↗
PubMed1996

Monocyte chemoattractant protein 1 and interleukin-8 production in mononuclear cells stimulated by oral microorganisms.

Chemokines are a family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. Chemokine expression contributes to the presence of different leukocyte populations observed in normal and pathologic states. In the present studies, peripheral blood mononuclear cells (PBMC) were stimulated by microbes (Candida albicans, Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) selected based upon their importance as oral pathogens. IL-8 and MCP-1 gene expression and protein release were determined by Northern blot (RNA blot) analysis and enzyme-linked immunosorbent assay. C. albicans, P. gingivalis, and A. actinomycetemcomitans induced high levels of production of both MCP-1 and IL-8. S. mutans was a strong inducer of MCP-1, but it did not stimulate significant production of IL-8. C. albicans, S. mutans, and A. actinomycetemcomitans were 500 to 5,000 times more potent than P. gingivalis in terms of MCP-1 production. In general, the microbe-to-PBMC ratios required for maximum gene expression of MCP-1 were lower than those for IL-8. However, for actual protein release of MCP-1 versus IL-8, differences in the effects of various microbe concentrations were observed only for A. actinomycetemcomitans. These results demonstrate that different oral pathogens induce specific dose-dependent patterns of chemokine gene expression and release. Such patterns may help explain the immunopathology of oral infections, particularly with regard to inflammatory leukocyte recruitment.

Aggregatibacter actinomycetemcomitans↗
PubMed1996

Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide.

Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection. We compared the effects of the deactivating cytokine interleukin 10 (IL-10) on human peripheral blood mononuclear cell (PBMC) responses to lipopolysaccharide (LPS), Cryptococcus neoformans, and Candida albicans. IL-10 effected dose-dependent inhibition of tumor necrosis factor alpha (TNF-alpha) release in PBMC stimulated by LPS and C. neoformans, with significant inhibition seen with 0.1 U/ml and greater than 90% inhibition noted with 10 U/ml. In contrast, even at doses as high as 100 U/ml, IL-10 inhibited TNF-alpha release in response to C. albicans by only 50%. IL-10 profoundly inhibited release of IL-1beta from PBMC stimulated by all three stimuli. TNF-alpha mRNA and release was inhibited even if IL-10 was added up to 8 h after cryptococcal stimulation. In contrast, inhibition of IL-1 beta mRNA was of lesser magnitude and occurred only when IL-10 was added within 2 h of cryptococcal stimulation. IL-10 inhibited translocation of NF-kappaB in response to LPS but not the fungal stimuli. All three stimuli induced IL-10 production in PBMC, although over 10-fold less IL-10 was released in response to C. neoformans compared with LPS and C. albicans. Thus, while IL-10 has deactivating effects on PBMC responses to all three stimuli, disparate stimulus- and response-specific patterns of deactivation are seen. Inhibition by IL-10 of proinflammatory cytokine release appears to occur at the level of gene transcription for TNF-alpha and both transcriptionally and posttranscriptionally for IL-1beta.

Candida albicans↗
PubMed1996

gamma Interferon gene expression and release in human lymphocytes directly activated by Cryptococcus neoformans and Candida albicans.

Previous studies in our laboratory and others have demonstrated that T and/or NK cells can directly bind to and inhibit the growth of the medically important fungal pathogens Cryptococcus neoformans and Candida albicans by apparently non-major histocompatibility complex-restricted mechanisms. Here, we examined whether this direct interaction between lymphocytes and fungi also results in cytokine gene expression and release. Nonadherent lymphocytes (NAL), isolated from human peripheral blood mononuclear cells by depletion of cells adherent to plastic and nylon wool, released gamma interferon (IFN-gamma), but not interleukin-4 (IL-4) and IL-10, following stimulation with C. neoformans yeast cells and C. albicans yeast cells, hyphae, and supernatants. The fungal stimuli also induced IFN-gamma mRNA, with peak gene expression seen at or after 18 h. IFN-gamma release was still seen even when either NK cells or T lymphocytes were depleted by negative selection, suggesting that both cell types can be stimulated by fungi to produce IFN-gamma. Release of IFN-gamma from fungus-stimulated NAL occurred in the absence of an intact complement system and was not especially enhanced by culture with IL-2 or IL-12. These data expand the mechanisms by which the direct interaction of NAL with fungal targets can lead to immune activation. Moreover, to our knowledge, this is the first demonstration of direct stimulation of T-cell cytokine release by microbial pathogens.

Candida albicans↗
PubMed1996

Influence of opsonization conditions on C3 deposition and phagocyte binding of large- and small-capsule Cryptococcus neoformans cells.

Previous studies demonstrated that, following opsonization with normal human serum (NHS), phagocytes bind greater numbers of small-capsule Cryptococcus neoformans cells than yeast cells with large capsules. The present study tested the hypothesis that suboptimal deposition of opsonic C3 fragments contributes to this disparity. C neoformans was grown under conditions promoting large or small capsules and was incubated at various concentrations in NHS. At low concentrations of yeast cells (125 cells per microl of NHS), the deposition of C3 fragments per unit of capsule volume and the binding of yeast cells to cultured human monocytes were similar for yeast cells having large and small capsules. However, at higher cell concentrations, large-capsule cells exhibited suboptimal coating with C3 fragments and markedly diminished monocyte binding compared with small-capsule cells. Thus, the inverse correlation between capsule size and phagocyte binding can be overcome by conditions promoting optimal C3 deposition.

Complement C3↗
PubMed1995

Direct antimicrobial activity of T cells.

T cells are generally thought to contribute to antimicrobial activity either by releasing lymphokines, which recruit and activate other cell types, or by major histocompatibility complex (MHC)-restricted lysis of infected host cells. Recently, it has become apparent that T cells can also mediate antimicrobial activity by direct interaction with microbial targets. Such interactions, which can be either antigen specific or nonspecific, occur in the apparent absence of MHC restriction and do not require the presence of other host cells. Microbial targets recognized by T cells include fungi, parasites and bacteria. Here, Stuart Levitz, Herbert Mathews and Juneann Murphy discuss the direct antimicrobial activity of T cells and speculate on its in vivo relevance.

Animals↗
PubMed1995

The effect of infection with human immunodeficiency virus on the anticryptococcal activity of lymphocytes and monocytes.

The effect of infection with human immunodeficiency virus (HIV) on the capacity of human lymphocytes and monocytes to inhibit and kill Cryptococcus neoformans in an 18-h assay was examined. In vitro infection of the Jurkat human T cell line with each of 3 HIV strains caused significant loss of anticryptococcal activity, which peaked 3-4 weeks after HIV infection. Lymphocytes from HIV-seropositive and -seronegative persons had similar activity, even if highly enriched for CD4 cells. The activity of lymphocytes from both seropositive and seronegative donors was increased by culture with interleukin-2 and phytohemagglutinin, but only activated lymphocytes from seronegative donors caused a reduction in C. neoformans colony-forming units. Both peripheral blood mononuclear cells and monocytes from HIV-positive persons had significantly reduced antifungal activity compared with cells from seronegative donors. Thus, under defined conditions, infection with HIV can impair the anticryptococcal activity of both lymphocytes and monocytes. Such qualitative defects may contribute, together with CD4 lymphocytopenia, to the particular susceptibility of HIV patients to cryptococcosis.

CD4 Antigens↗
PubMed1995

A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanil ide (XTT).

Studies of antimycotic host defenses have been limited by the paucity of rapid, reproducible quantitative assays for fungal cell damage. Prior studies defined a colorimetric method that uses MTT, a tetrazolium dye, to quantify polymorphonuclear leukocyte (PMNL)-mediated damage to fungi. These relatively simple, rapid, and reproducible assays require cumbersome extraction of precipitated MTT-formazan and high cell densities to overcome relatively low sensitivity. In experiments that compared assays with MTT and another tetrazolium dye, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-+ ++carboxanilide (XTT), estimates of damage to Aspergillus fumigatus hyphae by human PMNL were similar with both dyes. However, XTT reduction was more rapid and sensitive, yielding accurate results with fewer organisms and PMNL. The water-soluble XTT-formazan product also simplified measurements by eliminating the need for solvent-extraction steps that are obligatory in MTT assays. Thus, XTT is advantageous for quantitative assessment of fungal cell damage, although MTT remains useful for assessing fungal cell viability by direct microscopic visualization of precipitated formazan.

Aspergillus fumigatus↗