againstAspergillus Fumigatus Research Articles

PU.1-CD23 signaling mediates pulmonary innate immunity against Aspergillus fumigatus infection by driving inflammatory response

BackgroundAspergillosis is a common cause of morbidity and mortality in immunocompromised populations. PU.1 is critical for innate immunity against Aspergillus fumigatus (AF) in macrophages. However, the molecular mechanism underlying PU.1 mediating immunity against AF infection in human alveolar macrophages (AMs) is still unclear.MethodsIn this study, we detected the expressions of PU.1, CD23, p-ERK, CCL20 and IL-8 and key inflammatory markers IL-1β, IL-6, TNF-α and IL-12 in human THP-1-derived macrophages (HTMs) or PU.1/CD23-overexpressed immunodeficient mice with AF infection. Moreover, we examined these expressions in PU.1-overexpressed/interfered HTMs. Additionally, we detected the phagocytosis of macrophages against AF infection with altered PU.1 expression. Dual luciferase, ChIP and EMSAs were performed to detect the interaction of PU.1 and CD23. And we invested the histological changes in mouse lung tissues transfected with PU.1/CD23-expressing adenoviruses in AF infection.ResultsThe results showed that the expressions of PU.1, CD23, p-ERK, CCL20, IL-8, IL-1β, IL-6, TNF-α and IL-12 increased significantly with AF infection, and PU.1 regulated the later 8 gene expressions in HTMs. Moreover, CD23 was directly activated by PU.1, and overexpression of CD23 in PU.1-interfered HTMs upregulated IL-1β, IL-6, TNF-α and IL-12 levels which were downregulated by PU.1 interference. PU.1 overexpression strengthened the phagocytosis of the HTMs against AF. And injection of PU.1/CD23-expressing adenoviruses attenuated pathological defects in immunodeficient mouse lung tissues with AF infection. Adenovirus (Ad)-PU.1 increased the CD23, p-ERK, CCL20, IL-8 levels.ConclusionsOur study concluded that PU.1-CD23 signaling mediates innate immunity against AF in lungs through regulating inflammatory response. Therefore, PU.1-CD23 may be a new anti-aspergillosis therapeutic for the treatment of invasive aspergillosis with the deepening of gene therapy and its wide application in the clinic.

Evaluation of Rezafungin Provisional CLSI Clinical Breakpoints and Epidemiological Cutoff Values Tested against a Worldwide Collection of Contemporaneous Invasive Fungal Isolates (2019 to 2020).

Rezafungin is a new echinocandin under development for the treatment of candidemia and invasive candidiasis. CLSI recently approved provisional susceptible-only breakpoints and epidemiological cutoff values for Candida spp. and rezafungin. The activities of rezafungin and comparators against 2019 to 2020 invasive fungal isolates was evaluated by applying the new CLSI breakpoints. Rezafungin demonstrated potent activity against Candida albicans (MIC50/MIC90, 0.03/0.06 mg/L; 100.0% susceptible), Candida tropicalis (MIC50/MIC90, 0.03/0.06 mg/L; 100% susceptible), Candida glabrata (MIC50/MIC90, 0.06/0.06 mg/L; 98.3% susceptible), Candida krusei (MIC50/MIC90, 0.03/0.03 mg/L; 100% susceptible), and Candida dubliniensis (MIC50/MIC90, 0.06/0.12 mg/L; 100% susceptible) when tested by the CLSI broth microdilution method. Rezafungin inhibited 99.6% of Candida parapsilosis isolates (MIC50/MIC90, 1/2 mg/L) at the susceptible breakpoint of ≤2 mg/L. All C. albicans, C. tropicalis, and C. krusei isolates, as well as most C. glabrata (96.2% to 97.9%) and C. parapsilosis (86.2% to 100%) isolates, were susceptible to comparator echinocandins. Fluconazole resistance was detected among 0.5%, 4.5%, 10.5%, and 1.2% of C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis isolates, respectively. All echinocandins displayed limited activity against Cryptococcus neoformans. Rezafungin and other echinocandins were active against Aspergillus fumigatus (minimum effective concentration for 90% of isolates tested [MEC90] range, 0.015 to 0.06 mg/L) and Aspergillus section Flavi (MEC90 range, 0.015 to 0.03 mg/L). All but 16 (8.6%) A. fumigatus isolates were susceptible to voriconazole, and 100% of Aspergillus section Flavi isolates were WT to mold-active azoles. When applying the CLSI clinical breakpoints, rezafungin displayed high susceptibility rates (>98.0%) against Candida isolates from invasive fungal infections and showed potent activity against Aspergillus isolates.

ASP2397 Is a Novel Natural Compound That Exhibits Rapid and Potent Fungicidal Activity against Aspergillus Species through a Specific Transporter.

Current therapies against invasive pulmonary aspergillosis (IPA) have a limited cure rate. Given that a delay in treatment initiation may be fatal, a new drug with rapid-onset and potent fungicidal activity is needed. The novel cyclic hexapeptide ASP2397 (currently known as VL-2397) exhibited antifungal activity against Aspergillus fumigatus (including azole-sensitive and azole-resistant isolates), A. terreus, and A. flavus at an MIC range of 1 to 4 μg/ml in human serum. Time-kill curve experiments showed that ASP2397 reduced germinated conidia of A. fumigatus by more than 1 log10 CFU within 6 h. In addition, ASP2397 inhibited hyphal elongation from germinated conidia of A. fumigatus, A. terreus, and A. flavus more rapidly than voriconazole. Under conditions of delayed treatment initiation in an IPA mouse model, ASP2397 had efficacy superior to that of posaconazole, with 100% survival and over 1 log10 CFU/g reduction in lung fungal burden. Histopathological investigation of lungs also showed that ASP2397 markedly suppressed disease progression. To clarify its mechanism of action, we generated a UV-induced mutant of A. fumigatus with low susceptibility to ASP2397. The mutant had a point mutation in the siderophore transporter gene sit1, which is absent in mammalian cells. These findings suggest that ASP2397 may improve clinical treatment options for IPA.

Antifungal synergy of a topical triazole, PC945, with a systemic triazole against respiratory Aspergillus fumigatus infection

Invasive pulmonary Aspergillosis is a leading cause of morbidity and mortality in immunosuppressed patients and treatment outcomes using oral antifungal triazoles remain suboptimal. Here we show that combining topical treatment using PC945, a novel inhaled triazole, with systemic treatment using known triazoles demonstrated synergistic antifungal effects against Aspergillus fumigatus (AF) in an in vitro human alveolus bilayer model and in the lungs of neutropenic immunocompromised mice. Combination treatment with apical PC945 and either basolateral posaconazole or voriconazole resulted in a synergistic interaction with potency improved over either compound as a monotherapy against both azole-susceptible and resistant AF invasion in vitro. Surprisingly there was little, or no synergistic interaction observed when apical and basolateral posaconazole or voriconazole were combined. In addition, repeated prophylactic treatment with PC945, but not posaconazole or voriconazole, showed superior effects to single prophylactic dose, suggesting tissue retention and/or accumulation of PC945. Furthermore, in mice infected with AF intranasally, 83% of animals treated with a combination of intranasal PC945 and oral posaconazole survived until day 7, while little protective effects were observed by either compound alone. Thus, the combination of a highly optimised topical triazole with oral triazoles potentially induces synergistic effects against AF infection.

APX001 Pharmacokinetic/Pharmacodynamic Target Determination against Aspergillus fumigatus in an In Vivo Model of Invasive Pulmonary Aspergillosis.

APX001, the prodrug of APX001A, is a first-in-class antifungal agent that has a potent activity against Aspergillus fumigatus The goal of current study was to determine the pharmacodynamic (PD) index and target of APX001 in an immunocompromised murine model of invasive pulmonary aspergillosis against 6 A. fumigatus isolates. Minimum effective concentration (MEC) values ranged from 0.03 to 0.06 mg/liter. Dose fractionation was performed against isolate AF293 using total doses of APX001 ranging from 81 to 768 mg/kg of body weight/day fractionated into every 3-, 6-, and 8-h regimens over a 96-h treatment duration. Efficacy was assessed by A. fumigatus quantitative PCR (qPCR) of conidial equivalents from lung homogenates. Nonlinear regression analysis using the Hill equation demonstrated that the 24-h area under the concentration-time curve (AUC)/MEC ratio was the pharmacokinetic (PK)/PD index that best correlated with efficacy (coefficient of determination [R2] = 0.79). Treatment studies with the remaining strains utilized regimens of 40 to 1,536 mg/kg of APX001 administered every 3 h for a 96-h duration. Exposure-response relationships for all strains were similar, and the median free drug AUC/MEC PK/PD targets for stasis and 1-log-kill endpoints were 47.6 and 89.4, respectively. The present studies demonstrated in vitro and in vivo APX001A/APX001 potency against A. fumigatus These results have potential relevance for clinical dose selection and evaluation of susceptibility breakpoints.

Chemoenzymatic Synthesis, Nanotization, and Anti-Aspergillus Activity of Optically Enriched Fluconazole Analogues.

Despite recent advances in diagnostic and therapeutic methods in antifungal research, aspergillosis still remains a leading cause of morbidity and mortality. One strategy to address this problem is to enhance the activity spectrum of known antifungals, and we now report the first successful application of Candida antarctica lipase (CAL) for the preparation of optically enriched fluconazole analogues. Anti-Aspergillus activity was observed for an optically enriched derivative, (-)-S-2-(2',4'-difluorophenyl)-1-hexyl-amino-3-(1‴,2‴,4‴)triazol-1‴-yl-propan-2-ol, which exhibits MIC values of 15.6 μg/ml and 7.8 μg/disc in broth microdilution and disc diffusion assays, respectively. This compound is tolerated by mammalian erythrocytes and cell lines (A549 and U87) at concentrations of up to 1,000 μg/ml. When incorporated into dextran nanoparticles, the novel, optically enriched fluconazole analogue exhibited improved antifungal activity against Aspergillus fumigatus (MIC, 1.63 μg/ml). These results not only demonstrate the ability of biocatalytic approaches to yield novel, optically enriched fluconazole derivatives but also suggest that enantiomerically pure fluconazole derivatives, and their nanotized counterparts, exhibiting anti-Aspergillus activity may have reduced toxicity.

In vitro combination of isavuconazole with micafungin or amphotericin B deoxycholate against medically important molds.

Whether isavuconazole, an extended-spectrum triazole, possesses synergistic activity in combination therapy with echinocandins or amphotericin B for the treatment of invasive molds infections has not been studied. Our in vitro combination studies showed that isavuconazole and micafungin are synergistically active against Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Cunninghamella bertholletiae. These results suggest that isavuconazole, in combination with micafungin, may have a role in the treatment of invasive aspergillosis and warrants further investigation.

Ultrashort Peptide Bioconjugates Are Exclusively Antifungal Agents and Synergize with Cyclodextrin and Amphotericin B

Many natural broad-spectrum cationic antimicrobial peptides (AMPs) possess a general mode of action that is dependent on lipophilicity and charge. Modulating the lipophilicity of AMPs by the addition of a fatty acid has been an effective strategy to increase the lytic activity and can further broaden the spectrum of AMPs. However, lipophilic modifications that narrow the spectrum of activity and exclusively direct peptides to fungi are less common. Here, we show that short peptide sequences can be targeted to fungi with structured lipophilic biomolecules, such as vitamin E and cholesterol. The conjugates were active against Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans but not against bacteria and were observed to cause membrane perturbation by transmission electron microscopy and in membrane permeability studies. However, for C. albicans, selected compounds were effective without the perturbation of the cell membrane, and synergism was seen with a vitamin E conjugate and amphotericin B. Moreover, in combination with β-cyclodextrin, antibacterial activity emerged in selected compounds. Biocompatibility for selected active compounds was tested in vitro and in vivo using toxicity assays on erythrocytes, macrophages, and mice. In vitro cytotoxicity experiments led to selective toxicity ratios (50% lethal concentration/MIC) of up to 64 for highly active antifungal compounds, and no in vivo murine toxicity was seen. Taken together, these results highlight the importance of the conjugated lipophilic structure and suggest that the modulation of other biologically relevant peptides with hydrophobic moieties, such as cholesterol and vitamin E, generate compounds with unique bioactivity.

Anidulafungin in Combination with Amphotericin B against Aspergillus fumigatus

We investigated the effects of anidulafungin alone and in combination with amphotericin B against Aspergillus fumigatus. Indifference was the only type of interaction observed in vitro. Anidulafungin at 1 and 5 mg/kg of body weight/day, amphotericin B at 1 mg/kg/day, and combination therapy prolonged the survival of mice with invasive aspergillosis. Anidulafungin at 5 mg/kg/day, alone and in combination with amphotericin B, reduced the kidney fungal burden. Overall, the combination was not superior to the most active single drug.

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

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-alpha) 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-alpha 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-kappaB was translocated to the nuclei of THP-1 cells treated with VRC than untreated cells. While VRC induced more NF-kappaB than hyphae did, treatment with the combination of the two factors induced the greatest NF-kappaB expression. The pronounced profile of TLR2 signaling, TNF-alpha expression, and NF-kappaB activation in the presence of VRC suggests an immunomodulatory effect leading to a more efficient response to A. fumigatus.

Calcineurin Inhibition or Mutation Enhances Cell Wall Inhibitors against Aspergillus fumigatus

Calcineurin mutation or inhibition enhanced the antifungal morphological effect of cell wall inhibitors caspofungin or nikkomycin Z against Aspergillus fumigatus. Quantification of 1,3-beta-d-glucan revealed decreased amounts in the calcineurin A (DeltacnaA) mutant. Calcineurin can be an excellent adjunct therapeutic target in combination with other cell wall inhibitors against A. fumigatus.

Concentration-Dependent Synergy and Antagonism within a Triple Antifungal Drug Combination againstAspergillusSpecies: Analysis by a New Response Surface Model

Triple antifungal combinations are used against refractory invasive aspergillosis without an adequate understanding of their pharmacodynamic interactions. We initially studied the in vitro triple combination of voriconazole, amphotericin B, and caspofungin against Aspergillus fumigatus, A. flavus, and A. terreus by a spectrophotometric microdilution broth method after 48 h of incubation. We then analyzed these results with a recently described nonlinear mixture response surface E(max)-based model modified to assess pharmacodynamic interactions at various growth levels. The new model allows flexibility in all four parameters of the E(max) model and is able to describe complex pharmacodynamic interactions. Concentration-dependent pharmacodynamic interactions were found within the triple antifungal combination. At the 50% growth level, synergy (median interaction indices of 0.43 to 0.82) was observed at low concentrations of voriconazole (<0.03 mg/liter) and amphotericin B (</=0.20 mg/liter) and at intermediate concentrations of caspofungin (0.95 to 14.88 mg/liter), whereas antagonism (median interaction indices of 1.17 to 1.80) was found at higher concentrations of voriconazole and amphotericin B. Ternary plot and interaction surface analysis further revealed the complexity of these concentration-dependent interactions. With increasing concentrations of amphotericin B, the synergistic interactions of voriconazole-caspofungin double combination decreased while the antagonistic interactions increased. A similar effect was observed when voriconazole was added to the double combination of amphotericin B and caspofungin. In conclusion, the new nonlinear mixture-amount response surface modeling of the triple antifungal combination demonstrated a net antagonism or synergy against Aspergillus species depending upon drug concentrations and species.

Monoclonal Immunoglobulin G1 Directed againstAspergillus fumigatusCell Wall Glycoprotein Protects against Experimental Murine Aspergillosis

Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log(10) units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 x 10(5) CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed.

In vitro and in vivo activities of NS-718, a new lipid nanosphere incorporating amphotericin B, against Aspergillus fumigatus.

We evaluated the in vitro and in vivo potencies of a new lipid nanosphere that incorporates amphotericin B (AmB), NS-718, against Aspergillus fumigatus. The in vitro activity of NS-718 (the MIC at which 90% of strains are inhibited [MIC90], 0.25 microgram/ml) against 18 isolates of A. fumigatus was similar to that of deoxycholate AmB (D-AmB; Fungizone; MIC90, 0.25 microgram/ml), but NS-718 was more potent than liposomal AmB (L-AmB; AmBi-some; MIC90, 1.0 microgram/ml). The in vivo efficacy of NS-718 in a rat model of invasive pulmonary aspergillosis was compared with those of D-AmB and L-AmB. A low dose (1 mg/kg of body weight) of L-AmB was ineffective (survival rate, 0%), although equivalent doses of D-AmB and NS-718 were more effective (survival rate, 17%). However, a higher dose of NS-718 (3 mg/kg) was more effective (survival rate, 100%) than equivalent doses of D-AmB and L-AmB (survival rate, 0%). To explain these differences, pharmacokinetic studies showed higher concentrations of AmB in the plasma of rats treated with NS-718 than in the plasma of those treated with D-AmB. Our results suggest that NS-718, a new preparation of AmB, is a promising antifungal agent with activity against pulmonary aspergillosis.

In vitro antifungal activities of a series of dication-substituted carbazoles, furans, and benzimidazoles.

Aromatic dicationic compounds possess antimicrobial activity against a wide range of eucaryotic pathogens, and in the present study an examination of the structures-functions of a series of compounds against fungi was performed. Sixty-seven dicationic molecules were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. The MICs of a large number of compounds were comparable to those of the standard antifungal drugs amphotericin B and fluconazole. Unlike fluconazole, potent inhibitory compounds in this series were found to have excellent fungicidal activities. The MIC of one of the most potent compounds against C. albicans was 0.39 microg/ml, and it was the most potent compound against C. neoformans (MIC, </=0.09 microg/ml). Selected compounds were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. Since some of these compounds have been safely given to animals, these classes of molecules have the potential to be developed as antifungal agents.

Structure-in vitro activity relationships of pentamidine analogues and dication-substituted bis-benzimidazoles as new antifungal agents.

Twenty analogues of pentamidine, 7 primary metabolites of pentamidine, and 30 dicationic substituted bis-benzimidazoles were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. A majority of the compounds had MICs at which 80% of the strains were inhibited (MIC80s) comparable to those of amphotericin B and fluconazole. Unlike fluconazole, many of these compounds were found to have potent fungicidal activity. The most potent compound against C. albicans had an MIC80 of </=0.09 microg/ml, and the most potent compound against C. neoformans had an MIC80 of 0.19 microg/ml. Selected compounds were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. It is clear from the data presented here that further studies on the structure-activity relationships, mechanisms of action and toxicities, and in vivo efficacies of these compounds are warranted to determine their clinical potential.

Skin tests in a primate model of allergic bronchopulmonary aspergillosis.

We have previously reported producing a primate model of allergic bronchopulmonary aspergillosis in which monkey precipitating IgG and transfused human IgE antibodies against Aspergillus fumigatus (AF) combined with aerosolized AF to produce an inflammatory response in the lung. In this study we attempt to demonstrate similar changes in the skin. Immunized and unimmunized monkeys with and without IgG precipitating antibody to AF were injected at multiple sites intradermally with normal human serum or human serum rich in IgE against AF. 1 day later each site was injected with AF. Serial skin biopsies were taken at intervals of 15 min, 1, 2, 6, 12, 24, and 48 h and light microscopic, immunofluorescent, and electron microscopic studies were performed. The most profound changes were associated with simian antibody (IgG) and human antibody (IgE) directed against AF and consisted or perivascular and interstitial neutrophilic infiltration at 2 h, eosinophils at 6 h, and mononuclear cells at 24 h. Immunofluorescent staining for fibrin-fibrinogen, IgM, and C3 was present and diapedesis of cells to extra-vascular dermal areas was evident. The skin appear to mirror the inflammatory changes seen in the lung in this primate model of allergic bronchopulmonary aspergillosis and should afford a useful model for further studies.

In vitro studies with ambruticin, a new antifungal antibiotic.

The in vitro antifungal inhibitory activities of ambruticin and of various antifungal drugs of choice against 190 fungal pathogens representative of the major human mycoses were compared using a modification of the ICS agar dilution technique. Ambruticin compared favorably with amphotericin B and miconazole when tested against the dimorphic pathogens Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis and against Aspergillus fumigatus. Miconazole was the most active compound against Sporothrix schenckii, Allescheria (Petriellidium) boydii, and selected dematiaceous fungi, with ambruticin giving minimal inhibitory, concentrations from 3- to 74-fold higher. Ambruticin compared unfavorably with amphotericin B and 5-fluorocytosine when tested against Candida and Torulopsis species. Ambruticin was not as active in vitro as tolnaftate when tested against the three genera of dermatophytic fungi, but compared favorably with miconazole.