Maria Simitsopoulou

Interferon-γ and Granulocyte-Macrophage Colony-Stimulating Factor Augment the Activity of Polymorphonuclear Leukocytes against Medically Important Zygomycetes

Zygomycetes cause serious invasive infections, predominantly in immunocompromised and diabetic patients with poor prognoses and limited therapeutic options. We compared the antifungal function of human polymorphonuclear leukocytes (PMNLs) against hyphae of Rhizopus oryzae and R. microsporus, the most frequently isolated zygomycetes, with that against the less frequently isolated Absidia corymbifera. We then evaluated the effects of interferon (IFN)- gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF), alone or combined, on PMNL antifungal function against these zygomycetes. Both PMNL oxidative burst in response to hyphae and PMNL-induced hyphal damage were significantly lower in response to Rhizopus species than in response to A. corymbifera. Incubation of PMNLs with IFN- gamma and GM-CSF alone or combined for 22 h increased the PMNL-induced hyphal damage of all 3 species. The treatment of PMNLs with the combination of IFN- gamma and GM-CSF significantly increased the release of tumor necrosis factor- alpha in response to R. microsporus and A. corymbifera hyphae. IFN- gamma significantly reduced interleukin-8 release in response to all zygomycetes. Although Rhizopus species demonstrate a decreased susceptibility to the antifungal activity of human PMNLs, in comparison with A. corymbifera, IFN- gamma and GM-CSF augment the hyphal damage of all 3 zygomycetes, suggesting a role for IFN- gamma and GM-CSF in the management of invasive zygomycosis.

Differential Activities of Newer Antifungal Agents against Candida albicans and Candida parapsilosis Biofilms

ABSTRACT The activities of voriconazole, posaconazole, caspofungin, and anidulafungin against Candida albicans and Candida parapsilosis biofilms were evaluated. In contrast to planktonic cells, the MICs for voriconazole and posaconazole for the biofilms of the two species were high (≥256 and >64 mg/liter, respectively) but relatively low for the echinocandins caspofungin and anidulafungin (≤1 and ≤2 mg/liter, respectively).

Interactions between Human Phagocytes and Candida albicans Biofilms Alone and in Combination with Antifungal Agents

BACKGROUND Biofilm formation is an important component of vascular catheter infections caused by Candida albicans. Little is known about the interactions between human phagocytes, antifungal agents, and Candida biofilms. METHODS The interactions between C. albicans biofilms and human phagocytes alone and in combination with anidulafungin or voriconazole were investigated and compared with their corresponding planktonic counterparts by means of an in vitro biofilm model with clinical intravascular and green fluorescent protein (GFP)-expressing strains. Phagocyte-mediated and antifungal agent-mediated damages were determined by 2,3-bis[ 2- methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide assay, and structural effects were visualized by confocal microscopy. Oxidative burst was evaluated by flow cytometric measurement of dihydrorhodamine 123 oxidation, and cytokine release was measured by enzyme-linked immunosorbent assay. RESULTS Phagocytes alone and in combination with antifungal agents induced less damage against biofilms compared with planktonic cells. However, additive effects occurred between phagocytes and anidulafungin against Candida biofilms. Confocal microscopy demonstrated the absence of phagocytosis within biofilms but marked destruction caused by anidulafungin and phagocytes. Anidulafungin but not voriconazole elicited tumor necrosis factor alpha release from phagocytes compared with that from untreated biofilms. CONCLUSIONS C. albicans within biofilms are more resistant to phagocytic host defenses but are susceptible to additive effects between phagocytes and an echinocandin.

Differential Expression of Cytokines and Chemokines in Human Monocytes Induced by Lipid Formulations of Amphotericin B

ABSTRACT The immunomodulatory effects of liposomal amphotericin B (LAMB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD) on mRNA and protein profiles of five cytokines and chemokines expressed by human monocyte-enriched mononuclear leukocytes (MNCs) were comprehensively evaluated by semiquantitative reverse transcription-PCR and enzyme-linked immunosorbent assays; they were compared to those of deoxycholate amphotericin B (DAMB). mRNAs of interleukin-1β (IL-1β), IL-1 receptor antagonist (IL-1ra), tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1β (MIP-1β) were assessed after treatment of MNCs with each drug for 0.5, 2, 6, and 22 h. The cytokine protein profiles were obtained after incubation of MNCs with the drugs for 2 h (TNF-α) or 6 h (all the others). In the mRNA studies, DAMB resulted in an early increase of inflammatory cytokines or chemokines IL-1β, TNF-α, MCP-1, and MIP-1β (2 to 6 h) and in a late increase of anti-inflammatory IL-1ra (22 h). ABCD showed a general similar trend of inflammatory gene up-regulation. LAMB and ABLC decreased or did not affect IL-1β and TNF-α, whereas ABLC additionally decreased MIP-1β. In protein measurement studies, DAMB and ABCD up-regulated production of IL-1β (P < 0.05), decreased the IL-1ra/IL-1β ratio, and up-regulated the production of MCP-1 and MIP-1β. In comparison, LAMB and ABLC down-regulated or did not affect the production of these cytokines/chemokines compared to untreated MNCs; furthermore, ABLC tended to increase the IL-1ra/IL-1β ratio. These studies demonstrate that amphotericin B formulations differentially affect gene expression and release of an array of proinflammatory and anti-inflammatory cytokines that potentially may explain the differences in infusion-related reactions and dose-dependent nephrotoxicity as well as modulation of the host immune response to invasive fungal infections.

Inteferon gamma and granulocyte /macrophage colony- stimulating factor augment the antifungal activity of human polymorphonuclear leukocytes against Scedosporium spp.: comparison with Aspergillus spp.

While Aspergillus spp. have been the most frequent filamentous fungi causing infections in immunocompromised patients, Scedosporium spp. are emerging as life-threatening pathogens. We studied the effects of interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or combined on the antifungal activities of human polymorphonuclear leukocytes (PMN) against Scedosporium apiospermum and Scedosporium prolificans. We paralleled these activities to those against Aspergillus fumigatus and Aspergillus flavus. Incubation of PMN with IFN-gamma and GM-CSF for 22 h enhanced PMN-induced hyphal damage of both Aspergillus spp. and S. prolificans (p < 0.05) but not of S. apiospermum. However, hyphae of S. apiospermum were damaged significantly more after incubation with PMN that had been treated with IFN-gamma and GM-CSF for 2 h. In addition, incubation of PMN with GM-CSF for 2 h enhanced PMN oxidative burst measured as superoxide anion (O2-) production in response to nonopsonized hyphae of A. flavus and Scedosporium spp. (p < 0.05). In contrast, after 2 h, IFN-gamma and GM-CSF alone did not enhance PMN O2- in response to opsonized hyphae of A. flavus and Scedosporium spp.; however, the combination of IFN-gamma and GM-CSF showed significant enhancement against these species. Thus, IFN-gamma and GM-CSF, particularly in combination, demonstrate a species- and time-dependent augmentation of PMN responses to Scedosporium spp.

Immunomodulatory Effects of Voriconazole on Monocytes Challenged with Aspergillus fumigatus: Differential Role of Toll-Like Receptors

ABSTRACT Voriconazole (VRC) has activity against Aspergillus fumigatus, the most frequent cause of invasive aspergillosis in immunocompromised patients. The combination of VRC and A. fumigatus hyphae induced a more pronounced profile of expression of genes encoding inflammatory molecules in human monocytes than Aspergillus alone did. Herein, we provide further evidence of the potential mechanism underlying this immunomodulatory effect of VRC on human monocytes in response to A. fumigatus hyphae. A significant additive antifungal effect was shown when VRC was combined with monocytes against A. fumigatus hyphae. Both A. fumigatus hyphae and VRC induced pronounced profiles of mRNA and protein expression of Toll-like receptor 2 (TLR2) as well as tumor necrosis factor alpha (TNF-α) in THP-1 monocytic cells compared to untreated cells. The VRC-induced increase was greater than that induced by hyphae. The combination of VRC and hyphae increased mRNA and protein expression of TLR2 and TNF-α to even higher levels than did either VRC or hyphae alone. In contrast, TLR4 expression, both at the mRNA and protein levels, was not increased by either VRC or hyphae or their combination. In addition, significantly more NF-κB was translocated to the nuclei of THP-1 cells treated with VRC than untreated cells. While VRC induced more NF-κB than hyphae did, treatment with the combination of the two factors induced the greatest NF-κB expression. The pronounced profile of TLR2 signaling, TNF-α expression, and NF-κB activation in the presence of VRC suggests an immunomodulatory effect leading to a more efficient response to A. fumigatus.

Species-specific and drug-specific differences in susceptibility of Candida biofilms to echinocandins: characterization of less common bloodstream isolates

ABSTRACT Candida species other than Candida albicans are increasingly recognized as causes of biofilm-associated infections. This is a comprehensive study that compared the in vitro activities of all three echinocandins against biofilms formed by different common and infrequently identified Candida isolates. We determined the activities of anidulafungin (ANID), caspofungin (CAS), and micafungin (MFG) against planktonic cells and biofilms of bloodstream isolates of C. albicans (15 strains), Candida parapsilosis (6 strains), Candida lusitaniae (16 strains), Candida guilliermondii (5 strains), and Candida krusei (12 strains) by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. Planktonic and biofilm MICs were defined as ≥50% fungal damage. Planktonic cells of all Candida species were susceptible to the three echinocandins, with MICs of ≤1 mg/liter. By comparison, differences in the MIC profiles of biofilms in response to echinocandins existed among the Candida species. Thus, C. lusitaniae and C. guilliermondii biofilms were highly recalcitrant to all echinocandins, with MICs of ≥32 mg/liter. In contrast, the MICs of all three echinocandins for C. albicans and C. krusei biofilms were relatively low (MICs ≤ 1 mg/liter). While echinocandins exhibited generally high MICs against C. parapsilosis biofilms, MFG exhibited the lowest MICs against these isolates (4 mg/liter). A paradoxical growth effect was observed with CAS concentrations ranging from 8 to 64 mg/liter against C. albicans and C. parapsilosis biofilms but not against C. krusei, C. lusitaniae, or C. guilliermondii. While non-albicans Candida planktonic cells were susceptible to all echinocandins, there were drug- and species-specific differences in susceptibility among biofilms of the various Candida species, with C. lusitaniae and C. guilliermondii exhibiting profiles of high MICs of the three echinocandins.

Expression of Immunomodulatory Genes in Human Monocytes Induced by Voriconazole in the Presence of Aspergillus fumigatus

ABSTRACT We assessed the effect of voriconazole (VRC) on the expression and release of selected cytokines and chemokines in the THP-1 human monocytic cell line in response to Aspergillus fumigatus hyphal fragments (HF) by cDNA microarray analysis, reverse transcriptase (RT) PCR, and enzyme-linked immunosorbent assay. Stimulation of THP-1 cells by HF alone caused a significant up-regulation of CCL4 (MIP1B) and CCL16, while CCL2 (MCP1) was down-regulated. By comparison, in the presence of VRC, a large number of genes such as CCL3 (MIP1A), CCL4 (MIP1B), CCL5 (RANTES), CCL7 (MCP3), CCL11 (EOTAXIN), CCL15 (MIP1Δ), CXCL6, and CXCL13 were strongly up-regulated in THP-1 cells challenged by HF, whereas CCL20 (MIP3A) and CCL21 (MIP2) were down-regulated. Among five genes differentially expressed in THP-1 cells, IL12A, IL12B, and IL-16 were down-regulated whereas IL-11 and TGFB1 were significantly up-regulated in the presence of VRC. The inflammation-related genes IFNγ, IL1R1, and TNFA were also up-regulated in THP-1 cells exposed to HF only in the presence of VRC. RT-PCR of four selected genes validated the results of microarrays. The release of interleukin 1β (IL-1β) and IL-12 was significantly increased from monocytes stimulated either by HF alone (P < 0.05) or in the presence of VRC (P < 0.01 and P < 0.05, respectively). In contrast, tumor necrosis factor alpha release from monocytes was enhanced only in the presence of VRC (P < 0.01). The chemokines monocyte chemoattractant protein 1 and macrophage inflammatory protein 1β were decreased under both conditions (P < 0.01). These results demonstrate that in the presence of VRC, HF induces a more pronounced profile of gene expression in THP-1 cells than HF alone, potentially leading to more-efficient host resistance to A. fumigatus.

Activities of Triazole-Echinocandin Combinations against Candida Species in Biofilms and as Planktonic Cells

ABSTRACT Biofilm formation complicates the treatment of various infections caused by Candida species. We investigated the effects of simultaneous or sequential combinations of two triazoles, voriconazole (VRC) and posaconazole (PSC), with two echinocandins, anidulafungin (AND) and caspofungin (CAS), against Candida albicans and Candida parapsilosis biofilms in comparison to their planktonic counterparts. Antifungal activity was assessed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide (XTT) metabolic assay. Antifungal-agent interactions were analyzed by the Bliss independence model in the simultaneous-treatment studies and by analysis of variance (ANOVA) in the sequential-treatment studies. Against C. albicans planktonic cells, the simultaneous combination of PSC (32 to 128 mg/liter) and CAS (0.008 to 0.25 mg/liter) was synergistic; the combinations of PSC (128 to 1,024 mg/liter) with AND (0.03 to 0.5 mg/liter) and VRC (32 to 512 mg/liter) with AND (0.008 to 0.03 mg/liter) were antagonistic. Against C. parapsilosis planktonic cells, the interaction between VRC (32 to 1,024 mg/liter) and CAS (1 to 16 mg/liter) was antagonistic. All simultaneous antifungal combinations demonstrated indifferent interactions against biofilms of both Candida species. Damage to biofilms of both species increased (P < 0.01) in the presence of subinhibitory concentrations of echinocandins (0.008 to 0.064 mg/liter), followed by the addition of PSC (512 mg/liter for C. albicans and 64 to 512 mg/liter for C. parapsilosis) or VRC (256 to 512 mg/liter for C. albicans and 512 mg/liter for C. parapsilosis). Triazole-echinocandin combinations do not appear to produce antagonistic effects against Candida sp. biofilms, while various significant interactions occur with their planktonic counterparts.

Antifungal Activities of Posaconazole and Granulocyte-Macrophage Colony-Stimulating Factor Ex Vivo and in Mice with Disseminated Infection Due to Scedosporium prolificans

ABSTRACT Invasive infection due to Scedosporium prolificans is characterized by drug resistance and a high rate of mortality. The effects of posaconazole (POS), an investigational antifungal triazole, murine granulocyte-macrophage colony-stimulating factor (GM-CSF), and their combination against S. prolificans were evaluated ex vivo and in a newly developed murine model of disseminated infection due to this organism. When POS was combined with polymorphonuclear leukocytes from untreated or GM-CSF-treated mice (P < 0.01) ex vivo, it had increased activity in terms of the percentage of hyphal damage. Immunocompetent BALB/c mice were infected with 4 × 104 conidia of S. prolificans via the lateral tail vein. At 24 h postinfection the mice were treated with GM-CSF (5 μg/kg of body weight/day subcutaneously), POS (50 mg/kg/day by gavage), both agents, or saline only. Half of the brain, lung, liver, and kidney from each animal were cultured; and the other half of each organ was processed for histopathology. The mean survival times were 7.0 ± 0.3 days for the controls, 7.4 ± 0.4 days for POS-treated mice, 8.0 ± 0.3 days for GM-CSF-treated mice (P = 0.08 compared with the results for the controls), and 7.3 ± 0.3 days for POS-GM-CSF-treated mice. Fungal burdens (determined as the numbers of CFU per gram of tissue) were found in descending orders of magnitude in the kidneys, brains, livers, and lungs. The burdens were significantly reduced in the brains of GM-CSF-treated mice (P < 0.05) and the livers of POS-treated mice (P < 0.05). The numbers of lesions in the organs closely corresponded to the fungal burdens. GM-CSF tended to prolong survival (P = 0.08 compared with the results for the controls). While the combination of POS and GM-CSF showed enhanced activity ex vivo, it did not increase the activities of the two agents against this highly refractory filamentous fungus in mice.