Interplay of Cytokines and Chemokines in Aspergillosis.

Aspergillosis is a fungal infection caused by various species of Aspergillus, most notably A. fumigatus. This fungus causes a spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma, chronic pulmonary aspergillosis, and invasive aspergillosis. The clinical manifestations and severity of aspergillosis can vary depending on individual immune status and the specific species of Aspergillus involved. The recognition of Aspergillus involves pathogen-associated molecular patterns (PAMPs) such as glucan, galactomannan, mannose, and conidial surface proteins. These are recognized by the pathogen recognition receptors present on immune cells such as Toll-like receptors (TLR-1,2,3,4, etc.) and C-type lectins (Dectin-1 and Dectin-2). We discuss the roles of cytokines and pathogen recognition in aspergillosis from both the perspective of human and experimental infection. Several cytokines and chemokines have been implicated in the immune response to Aspergillus infection, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), CCR4, CCR17, and other interleukins. For example, allergic bronchopulmonary aspergillosis (ABPA) is characterized by Th2 and Th9 cell-type immunity and involves interleukin (IL)-4, IL-5, IL-13, and IL-10. In contrast, it has been observed that invasive aspergillosis involves Th1 and Th17 cell-type immunity via IFN-γ, IL-1, IL-6, and IL-17. These cytokines activate various immune cells and stimulate the production of other immune molecules, such as antimicrobial peptides and reactive oxygen species, which aid in the clearance of the fungal pathogen. Moreover, they help to initiate and coordinate the immune response, recruit immune cells to the site of infection, and promote clearance of the fungus. Insight into the host response from both human and animal studies may aid in understanding the immune response in aspergillosis, possibly leading to harnessing the power of cytokines or cytokine (receptor) antagonists and transforming them into precise immunotherapeutic strategies. This could advance personalized medicine.

Protective role of CFTR during fungal infection of cystic fibrosis bronchial epithelial cells with Aspergillus fumigatus.

Lung infection with the fungus Aspergillus fumigatus (Af) is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function. We established a fungal epithelial co-culture model to examine the impact of Af infection on CF bronchial epithelial barrier function using Af strains 10AF and AF293-GFP, and the CFBE41o- cell line homozygous for the F508del mutation with (CF+CFTR) and without (CF) normal CFTR expression. Following exposure of the epithelial surface to Af conidia, formation of germlings (early stages of fungal growth) was detected after 9-12 hours and hyphae (mature fungal growth) after 12-24 hours. During fungal morphogenesis, bronchial epithelial cells showed signs of damage including rounding, and partial detachment after 24 hours. Fluorescently labeled conidia were internalized after 6 hours and more internalized conidia were observed in CF compared to CF+CFTR cells. Infection of the apical surface with 10AF conidia, germlings, or hyphae was performed to determine growth stage-specific effects on tight junction protein zona occludens protein 1 (ZO-1) expression and transepithelial electrical resistance (TER). In response to infection with conidia or germlings, epithelial barrier function degraded time-dependently (based on ZO-1 immunofluorescence and TER) with a delayed onset in CF+CFTR cell monolayers and required viable fungi and apical application. Infection with hyphae caused an earlier onset and faster rate of decline in TER compared to conidia and germlings. Gliotoxin, a major Af virulence factor, caused a rapid decline in TER and induced a transient chloride secretory response in CF+CFTR but not CF cells. Our findings suggest growth and internalization of Af result in deleterious effects on bronchial epithelial barrier function that occurred more rapidly in the absence of CFTR. Bronchial epithelial barrier breakdown was time-dependent and morphotype-specific and mimicked by acute administration of gliotoxin. Our study also suggests a protective role for CFTR by turning on CFTR-dependent chloride transport in response to gliotoxin, a mechanism that will support mucociliary clearance, and could delay the loss of epithelial integrity during fungal development in vivo.

The role of occupational Aspergillus exposure in the development of diseases.

Aspergillus spp. have a high nutritional versatility and good growth on a large variety of construction materials. They also colonize soil or food, but decaying vegetation is their primary ecological niche. Therefore, exposure to fungi may occur at home, during hospitalization, during specific leisure activities, or at the workplace. The development of Aspergillus infections depends on the interplay between host susceptibility and the organism. Environments with high counts of fungal elements (conidia, hyphal fragments and others), high levels of bioarerosols, and elevated concentrations of mycotoxins or other volatile organic compounds should be considered as potential hazards, since they may present a risk to the exposed person. Rural tasks as well as work related to wood and food industries, poultries, swineries, waste handling plants, and other occupational environments involving contaminated organic material are among the ones posing higher respiratory risks to the workers. This paper presents a review of several studies related to occupational and indoor exposure to Aspergillus, potential health effects related to that exposure, and associated exposure assessment procedures.

Aspergillus-Pseudomonas interaction, relevant to competition in airways.

In airways of immunocompromised patients and individuals with cystic fibrosis, Pseudomonas aeruginosa and Aspergillus fumigatus are the most common opportunistic bacterial and fungal pathogens. Both pathogens form biofilms and cause acute and chronic illnesses. Previous studies revealed that P. aeruginosa is able to inhibit A. fumigatus biofilms in vitro. While numerous P. aeruginosa molecules have been shown to affect A. fumigatus, there never has been a systematic approach to define the principal causative agent. We studied 24 P. aeruginosa mutants, with deletions in genes important for virulence, iron acquisition, or quorum sensing, for their ability to interfere with A. fumigatus biofilms. Cells, planktonic or biofilm culture filtrates of four P. aeruginosa mutants, pvdD-pchE-, pvdD-, lasR-rhlR-, and lasR-, inhibited A. fumigatus biofilm metabolism or planktonic A. fumigatus growth significantly less than P. aeruginosa wild type. The common defect of these four mutants was a lack in the production of the P. aeruginosa siderophore pyoverdine. Pure pyoverdine affected A. fumigatus biofilm metabolism, and restored inhibition by the above mutants. In lungs from cystic fibrosis patients, pyoverdine production and antifungal activity correlated. The key inhibitory mechanism for pyoverdine was iron-chelation and denial of iron to A. fumigatus. Further experiments revealed a counteracting, self-protective mechanism by A. fumigatus, based on A. fumigatus siderophore production.

Microhemorrhage-associated tissue iron enhances the risk for Aspergillus fumigatus invasion in a mouse model of airway transplantation.

Invasive pulmonary disease due to the mold Aspergillus fumigatus can be life-threatening in lung transplant recipients, but the risk factors remain poorly understood. To study this process, we used a tracheal allograft mouse model that recapitulates large airway changes observed in patients undergoing lung transplantation. We report that microhemorrhage-related iron content may be a major determinant of A. fumigatus invasion and, consequently, its virulence. Invasive growth was increased during progressive alloimmune-mediated graft rejection associated with high concentrations of ferric iron in the graft. The role of iron in A. fumigatus invasive growth was further confirmed by showing that this invasive phenotype was increased in tracheal transplants from donor mice lacking the hemochromatosis gene (Hfe-/- ). The invasive phenotype was also increased in mouse syngrafts treated with topical iron solution and in allograft recipients receiving deferoxamine, a chelator that increases iron bioavailability to the mold. The invasive growth of the iron-intolerant A. fumigatus double-knockout mutant (ΔsreA/ΔcccA) was lower than that of the wild-type mold. Alloimmune-mediated microvascular damage and iron overload did not appear to impair the host's immune response. In human lung transplant recipients, positive staining for iron in lung transplant tissue was more commonly seen in endobronchial biopsy sections from transplanted airways than in biopsies from the patients' own airways. Collectively, these data identify iron as a major determinant of A. fumigatus invasive growth and a potential target to treat or prevent A. fumigatus infections in lung transplant patients.

Studies of Pseudomonas aeruginosa Mutants Indicate Pyoverdine as the Central Factor in Inhibition of Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are common opportunistic bacterial and fungal pathogens, respectively. They often coexist in airways of immunocompromised patients and individuals with cystic fibrosis, where they form biofilms and cause acute and chronic illnesses. Hence, the interactions between them have long been of interest and it is known that P. aeruginosa can inhibit A. fumigatusin vitro We have approached the definition of the inhibitory P. aeruginosa molecules by studying 24 P. aeruginosa mutants with various virulence genes deleted for the ability to inhibit A. fumigatus biofilms. The ability of P. aeruginosa cells or their extracellular products produced during planktonic or biofilm growth to affect A. fumigatus biofilm metabolism or planktonic A. fumigatus growth was studied in agar and liquid assays using conidia or hyphae. Four mutants, the pvdD pchE, pvdD, lasR rhlR, and lasR mutants, were shown to be defective in various assays. This suggested the P. aeruginosa siderophore pyoverdine as the key inhibitory molecule, although additional quorum sensing-regulated factors likely contribute to the deficiency of the latter two mutants. Studies of pure pyoverdine substantiated these conclusions and included the restoration of inhibition by the pyoverdine deletion mutants. A correlation between the concentration of pyoverdine produced and antifungal activity was also observed in clinical P. aeruginosa isolates derived from lungs of cystic fibrosis patients. The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatusIMPORTANCE Interactions between human pathogens found in the same body locale are of vast interest. These interactions could result in exacerbation or amelioration of diseases. The bacterium Pseudomonas aeruginosa affects the growth of the fungus Aspergillus fumigatus Both pathogens form biofilms that are resistant to therapeutic drugs and host immunity. P. aeruginosa and A. fumigatus biofilms are found in vivo, e.g., in the lungs of cystic fibrosis patients. Studying 24 P. aeruginosa mutants, we identified pyoverdine as the major anti-A. fumigatus compound produced by P. aeruginosa Pyoverdine captures iron from the environment, thus depriving A. fumigatus of a nutrient essential for its growth and metabolism. We show how microbes of different kingdoms compete for essential resources. Iron deprivation could be a therapeutic approach to the control of pathogen growth.

Small Colony Variants of Pseudomonas aeruginosa Display Heterogeneity in Inhibiting Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are major microbes in cystic fibrosis (CF). We reported non-mucoid P. aeruginosa isolates more inhibitory to A. fumigatus than mucoid ones. Another CF P. aeruginosa phenotype, small colony variants (SCVs), is an unknown factor in intermicrobial competition with A. fumigatus. Clinical SCV isolates and reference CF non-mucoid isolate (Pa10, producing normal-sized colonies) were compared. Live cells of P. aeruginosa or filtrates from P. aeruginosa planktonic or biofilm cultures were co-incubated with A. fumigatus growing under conditions allowing biofilm formation or with preformed biofilm. Metabolic activity of A. fumigatus biofilm was then measured. When necessary, assays were done after adjustment for growth differences by adding fresh medium to the planktonic culture filtrate. Pyoverdine determinations were performed spectrophotometrically on the planktonic culture filtrates. In all experimental conditions (live cells and planktonic or biofilm culture filtrates of P. aeruginosa versus A. fumigatus biofilm formation or preformed biofilm), three SCV isolates were less inhibitory than Pa10, two equal or more inhibitory. Adjusting planktonic culture filtrates for growth differences showed SCV inhibition differences variably related to growth or deficient inhibitor production. Studies suggested the principal P. aeruginosa inhibitor to be pyoverdine. SCV isolates appear heterogeneous in their capacity to inhibit A. fumigatus biofilm. SCV isolates can be important in the CF microbiome, because they are capable of intermicrobial inhibition.

Effect of Anaerobiasis or Hypoxia on Pseudomonas aeruginosa Inhibition of Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are the major bacterial and fungal pathogens in the airways of cystic fibrosis (CF) patients. This is likely related to their ability to form biofilms. Both microbes have been associated with CF disease progression. The interplay between these two pathogens has been studied under aerobic conditions, though accumulating data indicates that much of the CF airway is hypoxic or anaerobic. We studied the microbial interaction in these latter environments. Pa is an aggressor against Af forming biofilm or as established Af biofilm, whether Pa is cultivated in aerobic, hypoxic, or anaerobic conditions, or tested in aerobic or hypoxic conditions. Pa cells are generally more effective than planktonic or biofilm culture filtrates. Pa growth is less in anaerobic conditions, and filtrates less effective after anaerobic or hypoxic growth, or against hypoxic Af. These, and other comparisons shown, indicate that Pa would be less effective in such environments, as would be the case in a CF mucus plug. These observations would explain why Pa becomes established in CF airways before Af, and why Af may persist during disease progression.

Are Cystic Fibrosis Aspergillus fumigatus Isolates Different? Intermicrobial Interactions with Pseudomonas.

Pseudomonas aeruginosa and Aspergillus fumigatus are the leading bacterial and fungal pathogens in cystic fibrosis (CF). We have shown that Af biofilms are susceptible to Pseudomonas, particularly CF phenotypes. Those studies were performed with a reference virulent non-CF Aspergillus. Pseudomonas resident in CF airways undergo profound genetic and phenotypic adaptations to the abnormal environment. Studies have also indicated Aspergillus from CF patients have unexpected profiles of antifungal susceptibility. This would suggest that Aspergillus isolates from CF patients may be different or altered from other clinical isolates. It is important to know whether Aspergillus may also be altered, as a result of that CF environment, in susceptibility to Pseudomonas. CF Aspergillus proved not different in that susceptibility.

Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

Biofilm Filtrates of Pseudomonas aeruginosa Strains Isolated from Cystic Fibrosis Patients Inhibit Preformed Aspergillus fumigatus Biofilms via Apoptosis.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation.

Effect of Media Modified To Mimic Cystic Fibrosis Sputum on the Susceptibility of Aspergillus fumigatus, and the Frequency of Resistance at One Center.

Studies of cystic fibrosis (CF) patient exacerbations attributed toPseudomonas aeruginosainfection have indicated a lack of correlation of outcome within vitrosusceptibility results. One explanation is that the media used for testing do not mimic the airway milieu, resulting in incorrect conclusions. Therefore, media have been devised to mimic CF sputum.Aspergillus fumigatusis the leading fungal pathogen in CF, and susceptibility testing is also used to decide therapeutic choices. We assessed whether media designed to mimic CF sputa would give different fungal susceptibility results than those of classical methods, assaying voriconazole, the most utilized anti-Aspergillusdrug in this setting, and 30 CFAspergillusisolates. The frequency of marked resistance (defined as an MIC of >4 µg/ml) in our CF unit by classical methods is 7%. Studies performed with classical methods and with digested sputum medium, synthetic sputum medium, and artificial sputum medium revealed prominent differences inAspergillussusceptibility results, as well as growth rate, with each medium. Clinical correlative studies are required to determine which results are most useful in predicting outcome. Comparison of MICs with non-CF isolates also indicated the CF isolates were generally more resistant.

Mycologic Endocrinology.

The interactions of fungi and chemical messenger molecules, hormones or pheromones, are addressed in this chapter. These interactions include mammalian fungal pathogens, also plant pathogens, or non-pathogenic fungi, which can result in functional responses in receptor- or non-receptor-mediated fashions. Endogenous ligands in the fungi have been demonstrated to be important for mating in a number of systems. Mammalian hormones have been demonstrated to have stimulatory or inhibitory effects on growth for organisms such as Candida albicans, Paracoccidioides brasiliensis, Saccharomyces cerevisiae, Rhizopus nigricans, Aspergillus fumigatus, Coccidioides, and dermatophytic fungi. A number of fungi have been shown to have specific binding proteins for corticosteroid, estrogen and progesterone that are stereo-specific and high affinity. In some instances, the interactions of a mammalian hormone with the organism, in vivo, affects pathogenesis. Genome expression profiles of C. albicans in the presence of estradiol or progesterone, and S. cerevisiae with progesterone, indicate major up-regulation of various drug resistance pumps, like CDR1, and CDR2, can affect antifungal susceptibility. Azole antifungal interactions occur with fungal hormone binding proteins. Azoles also can block mammalian steroidogenesis. The finding of interactions of mammalian hormones with fungi and subsequent functional responses by the fungi, suggest that hormonal interactions with fungal systems has been conserved throughout evolution and have an important role in fungal pathogenesis, as well as in the overall biology of the organisms.

The cryptococcal antigen lateral flow assay: A point-of-care diagnostic at an opportune time.

Cryptococcal meningitis is a devastating HIV-related opportunistic infection, affecting nearly 1 million individuals and causing over 500 000 deaths each year. The burden of disease is greatest in sub-Saharan Africa and Southeast Asia, where cryptococcal disease is the most common cause of meningitis. Rapid, accurate and affordable diagnosis of cryptococcal disease has been lacking in many of the most heavily affected areas. Here, we review a point-of-care assay for cryptococcal disease, the dipstick-formatted cryptococcal antigen lateral flow assay (LFA) (IMMY, Norman, OK). In comparison to culture, the assay is 99.5% sensitive and 98% specific. In comparison to other commercially available tests for cryptococcal antigen, the LFA has equal or superior sensitivity and specificity in CSF, plasma and serum samples. We discuss potential applications for the use of the assay in resource-limited settings, including what is likely to be an important role of the LFA in screening for early cryptococcal infection before clinical disease and in evaluating pre-emptive treatment.

Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm.

Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3 alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50 against planktonic A. fumigatus was 1,250 µM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 µM. By XTT testing, DFM concentrations of <1,250 µM had no effect, whereas DFP at 2,500 µM increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 µM inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 µM DFP plus any concentration of FeCl3 was lower than that in the controls (P < 0.05 to 0.001). FeCl3 at ≥625 µM reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 µM was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl3 at ≥625 µM overcame inhibition by 625 µM DFP (P < 0.001). FeCl3 alone at ≥156 µM stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 µM FeCl3 only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.

Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium.

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients.

BACKGROUND: Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. METHODS: Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), ß-tubulin, and calmodulin sequencing. RESULTS: Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. CONCLUSIONS: To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.

Vitamin D and experimental invasive aspergillosis.

Immune cells express the vitamin D receptor and vitamin D metabolizing enzymes. Favorable vitamin D effects have been indicated in tuberculosis. Vitamin D deficiency increases T helper (Th) 2 responses to Aspergillus, and it suppresses Th2 responses in cystic fibrosis-allergic bronchopulmonary aspergillosis. Can vitamin D modulate the proinflammatory effects of amphotericin B (AmB) therapy in aspergillosis? Groups of mice were infected intravenously (IV) with 3-8 × 10(6) Aspergillus fumigatus conidia. In six experiments, doses of 0.08, 2, or 4 µg/kg calcitriol (active form of vitamin D) were given intraperitoneally +/- AmB-deoxycholate (AmBd) at 0.4, 0.8, 1.2, 1.8, 3.3, or 4.5 mg/kg or 0.8 or 1.2 mg/kg IV. Calcitriol doses were selected to range from doses used in humans to those just below doses shown to decalcify murine bones. In most experiments, doses of calcitriol and AmBd (or control diluents) were given five times, on alternate days, to minimize drug-drug interactions. Calcitriol treatment began on the day of challenge, and survival assessed for 10 days. In no experiments did calcitriol alone significantly worsen or enhance survival or affect residual infection in survivors. Calcitriol also did not affect the efficacy of AmBd. In a representative experiment, AmBd at 0.8 or 1.2 mg/kg IV alone +/- calcitriol at 2 µg/kg enhanced survival (P ≤ 0.01). However, the AmBd regimens with calcitriol were not different than those without, and calcitriol alone was identical to controls. In disseminated invasive aspergillosis, calcitriol did not affect outcome nor influence antifungal efficacy.

Protein targets for broad-spectrum mycosis vaccines: quantitative proteomic analysis of Aspergillus and Coccidioides and comparisons with other fungal pathogens.

Aspergillus species are responsible for most cases of fatal mold infections in immunocompromised patients, particularly in those receiving hematopoietic stem cell transplants. Experimental vaccines in mouse models have demonstrated a promising avenue of approach for the prevention of aspergillosis, as well as infections caused by other fungal pathogens, such as Coccidioides, the etiological agent of valley fever (coccidioidomycosis). Here, we investigated the hyphal proteomes of Aspergillus fumigatus and Coccidioides posadasii via quantitative MS(E) mass spectrometry with the objective of developing a vaccine that cross-protects against these and other species of fungi. Several homologous proteins with highly conserved sequences were identified and quantified in A. fumigatus and C. posadasii. Many abundant proteins from the cell wall of A. fumigatus present themselves as possible cross-protective vaccine candidates, due to the high degree of sequence homology to other medically relevant fungal proteins and low homologies to human or murine proteins.