Anticryptococcal Activity Research Articles

Human neutrophil-mediated nonoxidative antifungal activity against Cryptococcus neoformans.

It has long been appreciated that polymorphonuclear leukocytes (PMN) kill Cryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing of C. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation of C. neoformans with these fractions for 18 h resulted in percent inhibition of growth of 67.4 +/- 3.4, 84.6 +/- 4.4, and 29.2 +/- 10.5 (mean +/- standard error, n = 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C(4) column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules.

Human immunodeficiency virus type 1 infection of alveolar macrophages impairs their innate fungicidal activity.

Impaired adaptive immunity is the hallmark of AIDS, but the effects of human immunodeficiency virus type 1 (HIV-1) infection on innate immunity are less clear. Cryptococcus neoformans (CN) is a common AIDS-related fungal pathogen acquired by inhalation. Alveolar macrophages (AM) comprise the initial host defense in cryptococcosis and they may arrest infection before dissemination occurs. We hypothesized that HIV-1 infection of AM impairs their anti-cryptococcal activity. This was tested by infection of normal AM with the M-tropic strain HIV-1(Bal). Two weeks postinfection we measured fungistatic activity against CN by colony counting, binding, and internalization of CN by confocal microscopy and AM cell viability by Alamar Blue assay. Uninfected AM from most donors demonstrated innate fungicidal activity against CN. In HIV-1-infected AM, there was a significant reduction, and in most cases loss, of fungicidal activity compared with the uninfected AM. The reduced antifungal activity was not due to any cytotoxic effect of HIV-1, and HIV-1 infection did not impair binding or internalization of yeast by AM. Thus, the innate fungicidal activity of primary human AM is impaired after HIV-1 infection in vitro by a mechanism involving a defect of intracellular antimicrobial processing.

Conditional lethality of the diprotic weak bases chloroquine and quinacrine against Cryptococcus neoformans.

Chloroquine at 10 microM enhances the activity of macrophages against Cryptococcus neoformans but does not directly inhibit cryptococcal growth. The antifungal activity of higher chloroquine concentrations likely to be found within the acidic cryptococcal phagosome was tested. Concentrations of >/=30 microM inhibited cryptococcal growth, and there was fungal killing at concentrations of >/=100 microM. Activity was dependent on physiologic temperature and pH. Quinacrine was 50-fold more active than chloroquine, and concentrations as low as 100 nM enhanced macrophage anticryptococcal activity. Quinacrine was concentrated within a vacuolar system within the fungal cell and highly concentrated within intracellular C. neoformans. Ammonium chloride and bafilomycin A both inhibited cryptococcal growth, suggesting that the activity of chloroquine and quinacrine may in part be due to disruption of pH-dependent processes. These findings add to the known spectrum of activity of chloroquine and quinacrine. These, and related compounds, may have utility for the treatment of cryptococcosis.

T lymphocyte and monocyte interaction by CD40/CD40 ligand facilitates a lymphoproliferative response and killing of Cryptococcus neoformans in vitro.

This study explored the role of CD40 / CD40 ligand (CD40L) in the induction of a lymphoproliferative response and killing of Cryptococcus neoformans in vitro. In our experimental system, monocytes exposed to C. neoformans were used as antigen-presenting cells (APC) and co-cultured with autologous T cells. The results showed that CD40 / CD40L strongly regulated the blastogenic response of T cells to C. neoformans. The fungus up-regulated CD40 expression on APC. An acapsular strain appeared to be a better inducer than an encapsulated strain. Time course experiments showed optimal regulation of CD40 expression at 48 h of incubation. Blocking the interaction of CD40 on APC with CD40L on T cells using mAb to CD40L resulted in a significant inhibition of IFN-gamma production. The anti-cryptococcal activity of monocytes was greatly influenced by the CD40 / CD40L interaction, and a positive correlation was found between nitric oxide secretion and enhanced killing of C. neoformans. Finally, the CD40 / CD40L interaction was critical for induction of optimal secretion of pro-inflammatory cytokines such as TNF-alpha and IL-1beta. These results indicate an important role for CD40 / CD40L interaction in inducing activation of T cells. Such cell-to-cell contact promotes anti-cryptococcal activity as well as secretion of pro-inflammatory cytokines by monocytes.

Potent Antifungal Activity of Garlic (Allium Sativum) Against Experimental Murine Dissemenated Cryptococcosis

The adequate management of cryptococcosis is difficult with the available armamentarium of synthetic antifungals. We made an effort to evaluate fresh extracts of garlic ( Allium sativum L.) to assess potential therapeutic use of this herb as an anticryptococcal agent. The in vitro antifungal activity in broth showed greatest activity in the aqueous extract (MIC 125–250 µg/ml; MFC 250 to = 500 µg/ml) compared to the alcoholic, crude or commercial extracts. Treatment of Balb/c mice with garlic extracts in mice (orally) showed that alcoholic extracts (5 mg/kg, q.i.d.) provided 100% protection of mice against disseminated cryptococcosis. A concomitant increase in MST (> 35 days) was observed, along with a significant reduction in cfu (log 10 ) burden of cryptococci in visceral (liver, spleen, kidney, lung and heart) tissues, especially brain (> 4.5 log 10 ). The efficacy of extracts was also evident in the sera of mice. Thus, alcoholic extracts of fresh garlic elicit potential anticryptococcal activity against murine disseminated cryptococcosis.

Antimicrobial activity of polycationic peptides

The in vitro activity of six polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated against several clinical isolates of Gram-positive and Gram-negative aerobic bacteria, yeasts, Pneumocystis carinii and Cryptosporidium parvum, by using microbroth dilution methods. The peptides exhibited different antibacterial activities and rapid time-dependent killing. The Gram-negative organisms were more susceptible to buforin II and cecropin P1, whereas buforin II and ranalexin were the most active compounds against the Gram-positive strains. Similarly, ranalexin showed the highest activity against Candida spp., whereas magainin II exerted the highest anticryptococcal activity. Finally, the peptides showed high anti- Pneumocystis activity, whereas no compound had strong inhibitory effect on C. parvum.

In-vitro activity of polycationic peptides against Cryptosporidium parvum, Pneumocystis carinii and yeast clinical isolates.

The in-vitro activity of magainin II, indolicidin and ranalexin against 14 clinical isolates of eukaryotic microorganisms was evaluated. Antifungal susceptibility testing was performed by broth microdilution, and activity against Pneumocystis carinii and Cryptosporidium parvum was determined by inoculation on to cell monolayers. For yeasts, peptide MICs and MFCs ranged from 6.25 to > 50 mg/L. Ranalexin showed the highest activity against Candida spp., while magainin II demonstrated greatest anticryptococcal activity. The peptides suppressed the growth of P. carinii by > or = 50% and > or = 90% at 5 and 50 microM, respectively, with the exception of indolicidin. Ranalexin, the most effective compound against C. parvum, suppressed its growth by > or = 40% at 50 microM.

Role of TNF-α in the Induction of Fungicidal Activity of Mouse Peritoneal Exudate Cells againstCryptococcus neoformansby IL-12 and IL-18

We have recently demonstrated that two IFN-γ-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) againstCryptococcus neoformansthrough NK cell production of interferon (IFN)-γ and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-α in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-α mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-γ production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-α by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-γ mAb. The cell type producing TNF-α among PEC was mostly macrophage. TNF-α significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-γ, and furthermore anti-TNF-α mAb partially inhibited these responses. Considered together, our results indicated that TNF-α contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity againstC. neoformansthrough enhancement of IFN-γ-induced production of NO by macrophages, but not through increased production of IFN-γ by NK cells.

Probing the N-5 Region of the Indoloquinoline Alkaloid, Cryptolepine for Anticryptococcal Activity

N-5 Alkylated analogues of cryptolepine were synthesized and tested for anticryptococcal activity. Evidence provided in this study suggests that the active form of cryptolepine consists of the flat tetracyclic aromatic ring with the methyl group on the N-5 atom. It was also found that changes in the electronic density around the N-5 atom do not appear to affect activity. Steric hindrance of the N-5 substituents seems to decrease activity. Through systematic modification of the N-5 alkyl groups, ω-phenylpentyl group was shown to possess the highest potency thus far. ©

Differential effects of iron load on basal and interferon-gamma plus lipopolysaccharide enhance anticryptococcal activity by the murine microglial cell line BV-2

Here we evaluated the influence of intracellular iron levels on the constitutive and interferon (IFN)-γ plus lipopolysaccharide (LPS) enhanced anticryptococcal activity by the murine microglial cell line BV-2. We demonstrated that iron loading via ferric nitrilotriacetate (FeNTA) resulted in a significant increase in the constitutive levels of anticryptococcal activity, while the enhancing effects by IFN-γ plus LPS were prevented. Accordingly, a major increase was observed in the levels of thiobarbituric reactive substance (TBARS) produced upon iron loading under basal conditions, whereas IFN-γ plus LPS treatment, that per se did not affect TBARS production, prevented by about 50% the enhancement otherwise occurring in response to iron loading. The potential involvement of multiple effector system and their relation to intracellular iron will be discussed.

Granulocyte-macrophage colony-stimulating factor and fluconazole enhance anti-cryptococcal activity of monocytes from AIDS patients.

To investigate the effect of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and fluconazole on anti-cryptococcal activity of monocytes from AIDS patients and normal subjects. The effect of GM-CSF and fluconazole on fungistatic and fungicidal activity of monocytes was studied in an in vitro system. Monocytes were treated in vitro with hrGM-CSF and fluconazole or either agent alone for 24 or 48 h, and fungistatic and fungicidal activity was evaluated in a colony-forming unit inhibition assay. CD11b/CD18 expression in monocytes was measured by flow cytometry analysis. Superoxide anion generation by peripheral blood monocytes was measured in the presence of pre-opsonized zymosan. Defective antifungal capacity of monocytes from AIDS patients was observed. GM-CSF treatment of monocytes from AIDS patients increased fungistatic activity, and the combination of hrGM-CSF and fluconazole resulted in fungicidal activity. The mechanisms involved in the GM-CSF-mediated effect appeared to be mediated by (i) enhancement of phagocytic activity, (ii) increase of superoxide anion generation, and (iii) upregulation of CD11b/CD18 expression on the monocyte surface. Our data highlight the effect of GM-CSF on anti-cryptococcal activity of human monocytes and show a synergistic effect of GM-CSF with fluconazole, suggesting a new therapeutic strategy in the treatment of cryptococcosis.

Development of Macrophage Anticryptococcal Activityin VitroIs Dependent on Endogenous M-CSF

We previously showed that nonactivated resident murine peritoneal macrophages (PM) from five strains (e.g., BALB/c) have C′-dependent fungistasis forCryptococcus neoformansin 24-h coculture, but not CD-1 PM unless culture time was extended or M-CSF treatment was used. We studied effect of a rat IgG1monoclonal (m) antibody (Ab) to murine M-CSF receptor on this anticryptococal activity. Culture of BALB/c PM with mAb, diluted 1:10, prechallenge reduced fungistasis from 58 to 21% (P< 0.01), whereas further 10-fold dilutions did not. Moreover, M-CSF pretreatment (5000 U/ml) significantly enhanced fungistasis (to 85%), whereas adding mAb 1:10 or 1:100 reduced that (to 58 and 77%, respectively,P< 0.01). In 48-h culture CD-1 PM had 39% fungistasis, reduced to 0% by mAb, M-CSF treatment of CD-1 PM increased fungistasis to 72%, which was reduced to 13 or 58% (P< 0.001) by 1:10 or 1:100 mAb, respectively. Complete blocking by mAb of CD-1 PM activity was consistent with lack of measurable early endogenous CD-1 M-CSF production. Increasing exogenous M-CSF could overcome the inhibition by mAb (64% fungistasis BALB/c PM reduced to 11% with inhibition by mAb or increased to 94% with 5000 U/ml M-CSF; 37% with both mAb and M-CSF, 51% with mAb and 10,000 U/ml;P< 0.05, 5000 U/ml + mAb vs 10,000 U/ml + mAb). Moreover, rabbit Ab to M-CSF significantly reduced anticryptococcal activity of untreated BALB/c macrophages. In summary, development of PM fugistatic activity is dependent on endogenous M-CSF, since it is blocked by anti-receptor mAb (as is exogenous M-CSF stimulation) or anti-M-CSF Ab, and macrophages of the mouse strain with delayed activity had no measurable early M-CSF production.

Interleukin-15 induces antimicrobial activity after release by Cryptococcus neoformans-stimulated monocytes.

A newly described cytokine, interleukin (IL)-15, shares many activities with IL-2; however, little is known about the stimuli for release of IL-15, and its role in antimicrobial host defense has not previously been demonstrated. This study found that Cryptococcus neoformans is a potent stimulus for the release of biologically active IL-15 from monocytes. Both IL-15 and IL-2 made significant contributions to lymphocyte proliferation and lymphocyte-mediated anticryptococcal activity to encapsulated and acapsular C. neoformans. IL-15 restored lymphocyte proliferation and anticryptococcal activity that had been abrogated by blocking IL-2. IL-15 also enhanced the anticryptococcal activity of lymphocytes but did not enhance the activity of monocytes. This suggests that IL-15 and IL-2 cooperate for lymphocyte activation and proliferation in vitro and demonstrates that IL-15 can induce antimicrobial activity. Taken together, these data suggest that microbes, and in particular C. neoformans, are an important stimulus for IL-15 and that IL-15 may have an important role in induction of antimicrobial effector mechanisms.

Effect of cytokines on anticryptococcal activity of human microglial cells.

The effect of selected cytokines on the antifungal activity of human microglia was studied with encapsulated and acapsular strains of Cryptococcus neoformans. None of the cytokines tested increased the fungistatic activity of microglia, suggesting that killing of cryptococci within the central nervous system is dependent on other host defense mechanisms.

Antifungal Activity of D0870 against Murine Infections and Its Mechanism of Action

We evaluated the in vivo antifungal activity of D0870, a new triazole agent, in comparison with that of fluconazole in two murine infection models. The therapeutic mechanism of D0870 in these models was also investigated in vitro. In a pulmonary infection with Cryptococcus neoformans in immunosuppressed mice, D0870 at 10–100 mg/kg significantly reduced viable counts in lungs infected with C. neoformans to 1/10–1,000 of the control on day 14, whereas fluconazole, only at 100 mg/kg, showed a significant reduction in the viable counts and was less active than D0870 at 10 mg/kg. D0870 at 3–30 mg/kg also showed excellent therapeutic efficacy against murine vaginal candidiasis and completely eliminated viable yeasts from the vaginal cavity, whereas positive cultures were found in 20% of mice treated with 30 mg fluconazole/kg. At pH 7 and 37°C, D0870 was active against C. neoformans in synthetic amino acid medium, fungal or sabouraud dextrose broth. By reducing the pH of the medium, the in vitro anticryptococcal activity of D0870 was enhanced and found to be fungicidal under all culture conditions at pH 4–5. D0870 also showed a stronger fungistatic activity against Candida albicans at pH 4. These results suggest that D0870 may exhibit a potent activity against these two murine infections by exerting an excellent antifungal activity at the infection sites thought to be acidic environments.

Isolation of a human serum protein that inhibits the growth of Cryptococcus neoformans.

Human serum at 5 to 10% (v/v) in tissue culture medium RPMI-1640, inhibits the growth of Cryptococcus neoformans by 80 to 93%. Serum fractionated on molecular sieve columns (Sephadex G-200) yielded an active protein fraction. This fraction at 100 micrograms protein/ml inhibited the growth of C. neoformans by 54%. When an active G-200 fraction was applied to a dye affinity column (Affi-Gel Blue) the fraction with inhibitory activity was bound by the column and was eluted with 1.4 M NaCl in 0.1 M phosphate buffer (pH 7.4). The bound fraction at 62.5 micrograms protein/ml inhibited C. neoformans growth by 82%. On native polyacrylamide gel electrophoresis (Nu-PAGE) the bound fraction migrated as a major and a minor band. Under the reducing conditions of sodium dodecyl sulfate (SDS)-PAGE the bound fraction yielded 4 prominent bands with MW ranging from 175 kDa to 45 kDa. Purification of the active Sephadex G-200 peak was achieved using an anion exchange column (DEAE-Sephacel). Protein eluted with 0.1 M NaCl had strong anticryptococcal activity (12.5 micrograms/ml, 79% inhibition), which in SDS-PAGE migrated as a single band with an approximate MW of 85 kDA. This protein appears important in natural host resistance to C. neoformans and polymorphisms or deficiencies may have epidemiologic and diagnostic relevance.

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.

Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells.

We examined the ability of interleukin-12 (IL-12) and IL-18 to induce the production of gamma interferon (IFN-gamma) and nitric oxide (NO) by murine peritoneal exudate cells (PEC) and to stimulate the growth-inhibitory activity of these cells against Cryptococcus neoformans. PEC produced IFN-gamma and NO when stimulated with a combination of IL-12 and IL-18 but little or no IFN-gamma or NO when either cytokine was used alone. PEC anticryptococcal activity was mediated by IFN-gamma and NO production, since it was completely inhibited by a neutralizing anti-IFN-gamma monoclonal antibody (MAb) and N(G)-monomethyl-L-arginine, a competitive inhibitor of NO synthesis, respectively. To identify the IFN-gamma-producing cells among PEC stimulated with IL-12 and IL-18, we depleted NK cells, gammadelta T cells, or CD4+ T cells by treating PEC with specific Abs and complement. NK cell depletion strongly suppressed IFN-gamma production and almost completely inhibited NO production and anticryptococcal activity, while depletion of other cells had no such influence. Alternatively, purified NK cells by two cycles of glass adherence and magnetic separation with anti-CD3, -CD4, -CD8, and -B220 MAbs produced a greater amount of IFN-gamma by stimulation with IL-12 and IL-18 than unseparated non-glass-adherent PEC. Our results demonstrated that IL-12 and IL-18 synergistically induced NO-dependent anticryptococcal activity of PEC by stimulating NK cells to produce IFN-gamma.

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.

Enhanced resistance to Cryptococcus neoformans infection induced by chloroquine in a murine model of meningoencephalitis.

Although the pathogenesis of cerebral cryptococcosis is poorly understood, local immune cells, such as microglia and astrocytes, likely play a critical role in containing infection. Chloroquine (CQ) is a weak base that accumulates within acidic vacuoles and increases their pH. Consequently, proteolytic activity of lysosomal enzymes and intracellular iron release/availability are impaired, resulting in decreased availability of nutrients crucial to microorganism survival and growth in the host. We found that CQ enhances BV2 microglial-cell-mediated anticryptococcal activity in vitro. The phenomenon is (i) evident when both unopsonized and opsonized microorganisms are used and (ii) mimicked by NH4Cl, another weak base, and by bafilomycin A1, an inhibitor of vacuolar-type H+-ATPases. In vivo, intracerebral administration of CQ before lethal local challenge with Cryptococcus neoformans results in a significant augmentation of median survival time and a marked reduction of yeast growth in the brain and is associated with the enhancement of local interleukin 1beta (IL-1beta) and IL-6 mRNA transcripts. Overall, these results provide the first evidence that CQ enhances anticryptococcal host defenses.