Development Of Invasive Aspergillosis Research Articles

Characterization of Pulmonary Fibroblast Response to Aspergillosis fumigatus Exposure and Clinical Implications on the Development of Invasive Aspergillosis

Aspergillosis fumigatus(AF) is a ubiquitous environmental mold that spreads by producing airborne conidia (spores). Playing a crucial role in the recycling of environmental carbon and nitrogen, A. fumigatus is inhaled abundantly by animals and humans alike. Unfortunately, it is also responsible for one of the worst life‐threatening pneumonias known as invasive aspergillosis with a mortality rate above 30%, even with treatment. Healthy individuals that are immunocompetent can quickly clear inhaled AF spores before their germination via immune cells. However, in immunocompromised individuals the inability to clear the fungus allows the spores to germinate releasing a plethora of proteolytic enzymes as they infiltrate the tissue resulting in severe damage to the lung. Recent studies have shown that the proper response of the immune system hinges upon complex cellular cross‐talk between resident interstitial cells and circulating immune cells. Tissue resident fibroblasts, which are responsible for the production and maintenance of the extracellular matrix (ECM), have been recently suggested to play a pivotal role in regulating immune response. We hypothesize that A. fumigatus‐induced damage to the lungs would result in activation of pulmonary fibroblasts (PFs) and aid in protective inflammatory response. Utilizing our recently developed in vivo cre‐loxP lineage tracing mouse models we have interrogated and manipulated all activated lung fibroblasts during AF infection, clearing, and reinfection. Data from our lab shows that PFs can be activated following acute inhalation of AF spores even when they are cleared from the lungs of immunocompetent mice before pathological tissue damage occurs. Furthermore, repeated AF infections which are pathologically asymptomatic result in the accumulation of activated PFs. Here we show for the first time that fibroblast activation occurs in both transient and chronic pulmonary infections revealing a role in the immunomodulatory response and subsequent development of pulmonary disease.

Retrospective analysis of the association of the expression and single nucleotide polymorphisms (SNPs) of the TLR4, PTX3 and Dectin-1 (CLEC/A) genes with development of invasive aspergillosis among haematopoietic stem cell transplant recipients with oncohaematological disorders.

Several studies described single nucleotide polymorphisms (SNPs) on pattern recognition receptor (PRR) such as toll-like receptors (TLRs), dendritic cell-associated C-type lectin-1 (Dectin-1/CLEC7A) genes of patients with invasive fungal infections (IFIs) caused by Candida and Aspergillus. We screened TLR4, Dectin-1 and PTX3 polymorphisms in a Turkish population with invasive aspergillosis (IA) underlying haematological malignancies. In this case-control study, a cohort of 59 patients with haematological malignancies were included. There were 26 IA patients assigned by the EORTC-MSG criteria and 33 patients with no evidence of fungal disease. DNA and RNA were isolated from frozen bone marrow and serum samples. RNA levels and polymorphisms of TLR4 (rs4986790, rs4986791), Dectin-1 (rs16910526, rs7309123) and PTX3 (rs2305619, rs3816527) were determined. The odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated by unconditional logistic regression analysis. TLR4, PTX3 and Dectin-1 genes were downregulated in aspergillosis cohort under similar haematological conditions. TLR4 expression was 0.0626±0.032 in controls when compared to IA patients as 0.0077±0.014, and the difference was significant (P=.026). There was a difference in also the PTX3 gene among IA (0.0043±0.004) and control (0.5265±0.0043) groups (P=.035). The Dectin-1 (CLEC/A) expression was downregulated in IA group (0.1887±0.072 & 0.0655±0.010) but not statistically significant (P>.05). Conditional logistic regression analyses indicated that the GT genotype of rs16910526 polymorphism in Dectin-1 gene was associated with lower risk of IA (odds ratio=3.635, 95% confidence interval=0.690-3.138, P=.04).

Characterisation of the cellular and proteomic response of Galleria mellonella larvae to the development of invasive aspergillosis

BackgroundGalleria mellonella larvae were infected with conidia of Aspergillus fumigatus and the cellular and humoral immune responses of larvae to the pathogen were characterized as invasive aspergillosis developed.ResultsAt 2 h post-infection there was an increase in hemocyte density to 7.43 ± 0.50 × 106/ml from 0.98 ± 0.08 × 106/ml at 0 h. Hemocytes from larvae immune primed for 6 h with heat killed A. fumigatus conidia displayed superior anti-fungal activity. Examination of the spread of the fungus by Cryo-imaging and fluorescent microscopy revealed dissemination of the fungus through the larvae by 6 h and the formation of distinct nodules in tissue. By 24 h a range of nodules were visible at the site of infection and at sites distant from that indicating invasion of tissue. Proteomic analysis of larvae infected with viable conidia for 6 h demonstrated an increase in the abundance of gustatory receptor candidate 25 (37 fold), gloverin-like protein (14 fold), cecropin-A (11 fold). At 24 h post-infection gustatory receptor candidate 25 (126 fold), moricin-like peptide D (33 fold) and muscle protein 20-like protein (12 fold) were increased in abundance. Proteins decreased in abundance included fibrohexamerin (13 fold) and dimeric dihydrodiol dehydrogenase (8 fold).ConclusionThe results presented here indicate that G. mellonella larvae may be a convenient model for studying the stages in the development of invasive aspergillosis and may offer an insight into this process in mammals.

An investigation on the relationship between the occurrence of CMV DNAemia and the development of invasive aspergillosis in the allogeneic stem cell transplantation setting

Cytomegalovirus (CMV) infection might increase the risk of fungal superinfection in allogeneic stem cell transplant patients. The potential association between the occurrence of CMV DNAemia and the development of invasive aspergillosis in this clinical setting was investigated. The current retrospective observational study included 167 patients undergoing T cell-replete allogeneic stem cell transplantation. Virological monitoring of active CMV infection was performed by the pp65 antigenemia assay and/or by a plasma real-time PCR assay. A total of 109 out of 167 patients developed CMV DNAemia. Twenty-three patients had proven (n = 4) or probable (n = 19) invasive aspergillosis. The occurrence of CMV DNAemia was not significantly associated with the subsequent development of invasive aspergillosis (P = 0.38). Overall, the duration of the episodes of active CMV infection and the peak level of CMV DNAemia within the episodes were comparable, irrespective of whether invasive aspergillosis developed subsequently or not (P = 0.99; P = 0.70, respectively). Peak CMV DNA load in patients with proven or probable invasive aspergillosis who died was higher (median, 5,461 copies/ml) than that in those who survived (median 1,179 copies/ml) (P = 0.41). The data argue against the existence of an association between the occurrence of CMV DNAemia and the development of invasive aspergillosis, however, CMV replication, particularly at high levels, might aggravate the prognosis of this disease.

The cell biology of the innate immune response to Aspergillus fumigatus

The development of invasive aspergillosis is a feared complication for immunocompromised patients. Despite the use of antifungal agents with excellent bioactivity, the morbidity and mortality rates for invasive aspergillosis remain unacceptably high. Defects within the innate immune response portend the highest risk for patients, but detailed knowledge of molecular pathways in neutrophils and macrophages in response to this fungal pathogen is lacking. Phagocytosis of fungal spores is a key step that places the pathogen into a phagosome, a membrane-delimited compartment that undergoes maturation and ultimately delivers antigenic material to the class II MHC pathway. We review the role of Toll-like receptor 9 (TLR9) in phagosome maturation of Aspergillus fumigates-containing phagosomes. Advanced imaging modalities and the development of fungal-like particles are promising tools that will aid in the dissection of the molecular mechanism to fungal immunity.

Importance of elastase production in development of invasive aspergillosis

To evaluate the relationship between elastase activity of different Aspergillus fumigatus (A.fumigatus) isolates and their pathogenicity in mice. The animals were intravenously (IV) infected with 1×10(6) conidia/mouse. The elastase activity in mice sera and survival time were determined. All animals were sacrified at the end of the fourth week, and autopsies were performed for histopathological examination. The tissue sections were stained with Periodic Acid Schiff (PAS) and Verhoeff's techniques. The highest elastase activity was associated with the sera of infected mice with elastase-producing A.fumigatus AIR78 as standard strain (0.18±0.02mm), followed by infected animals with elastase-producing isolates (0.16±0.02mm) and control mice (0.14±0.02mm). The elastase activity index (EAI) was determined in value of 0.92±0.27. The survival times in mice infected with elastase-producing isolate and in control group were 21.6 and 30 days, respectively. Histopathological studies showed that the heart and kidneys were more infected by Aspergillus hyphae than other tissues. It was concluded that there is a significant relationship between elastase activity and survival time in mice with aspergillosis.

Investigation of extracellular elastase, acid proteinase and phospholipase activities as putative virulence factors in clinical isolates of Aspergillus species

We investigated the production of extracellular elastase, acid proteinase and phospholipase enzyme activities in clinical isolates of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger and any possible correlation between the existence of these enzymes and development of invasive aspergillosis. A total of 73 strains (45 A. fumigatus, 23 A. flavus, 5 A. niger) isolated from patients with invasive aspergillosis (n = 55), superficial aspergillosis (n = 5), allergic bronchopulmonary aspergillosis (n = 1), and from those colonized with Aspergillus (n = 12) were included in the study. The enzymatic activities were tested on solid media supplemented with the corresponding substrates. Elastase activity was detected in 95.6, 82.6, and 0% of A. fumigatus, A. flavus, and A. niger isolates, respectively. Acid proteinase activity was detected only for A. fumigatus and in 20 of 45 isolates belonging to this species. Phospholipase activity was present in all strains of A. fumigatus and A. niger but in none of the isolates of A. flavus. No statistical correlation could be established between the existence of elastase or acid proteinase activity and development of invasive disease (p > 0.05). While high phospholipase production was found to be associated with development of invasive aspergillosis (p < 0.01), not all isolates that caused invasive diseases showed high phospholipase activities.

Disruption of a Nonribosomal Peptide Synthetase in Aspergillus fumigatus Eliminates Gliotoxin Production

The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in DeltagliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the DeltagliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the DeltagliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.

TLR1 and TLR6 Polymorphisms Are Associated with Susceptibility to Invasive Aspergillosis after Allogeneic Stem Cell Transplantation

Toll-like receptors (TLRs) transmit signals in response to Aspergillus fumigatus conidia and hyphae. In this preliminary study, we examined the association between single nucleotide polymorphisms (SNPs) in TLR1, TLR4, and TLR6 genes and development of invasive aspergillosis (IA) in 127 allogeneic hematopoietic stem cell transplant recipients consisting of 22 patients with IA and 105 unaffected control subjects. The following SNPs and their pairwise interactions were considered in the model: TLR1 (239G > C, 743A > G, 914A > T, 1805G > T), TLR4 (896A > G, 1196C > T), and TLR6 (359T > C, 745C > T, 764C > T). No association was found between donor SNP and the risk of IA. Analysis of recipient SNP data showed that the presence of TLR1 239G > C (Arg80 > Thr) or the presence of both TLR1 743A > G (Asn248 > Ser) and TLR6 745C > T (Ser249 > Pro) is associated with IA (odds ratio = 1.30, 95% confidence interval = 1.13 to 1.50; P < .001). Further analyses using a prospective cohort may enable us to identify TLR polymorphisms associated with the susceptibility to IA within a defined interval among immunocompromised patients.

Recent advances in the therapy against invasive aspergillosis.

AbstractThe members of the genus Aspergillus are opportunistic fungi that cause disease almost exclusively in patients with chronic lung pathology or depressed immune defenses. Aspergillus disease has a variety of clinical manifestations, ranging from allergic responses occurring in the absence of fungal growth or airway colonization to invasion and destruction of lung parenchyma with or without dissemination to other organs (Denning and Stevens, 1990; Greenberger, 2002; Marr et al., 2002; Muller et al., 2002). Over the past decades, the incidence of invasive aspergillosis has increased dramatically as a result of an expansion of the population of immunocompromised patients. At particular risk for invasive aspergillosis are those patients with persistent and profound neutropenia as a result of aggressive anticancer chemotherapy, hematopoietic stem cell transplant, or aplastic anemia (Gerson et al., 1984; Weinberger et al., 1992; Walsh et al., 1994; Perea and Patterson, 2002). In addition, congenital or acquired qualitative defects of the phagocytic cells have also been associated with the development of invasive aspergillosis: examples are patients with chronic granulomatous disease, Job syndrome, or those receiving prolonged courses of immunosuppressive agents such as corticosteroids. Aspergillosis is uncommon in patients with AIDS but when present it is associated with short survival after diagnosis (Shetty et al., 1997; Holding et al., 2000).KeywordsHematopoietic Stem Cell TransplantInvasive AspergillosisChronic Granulomatous DiseaseInvasive Fungal InfectionLiposomal AmphotericinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Aspergillus fumigatus causes in vitro electrophysiological and morphological modifications in human nasal epithelial cells.

The role of the airway epithelium in the development of invasive aspergillosis in immunocompromised hosts has rarely been studied although patients at risk for this infection frequently have epithelial damage. We developed an in vitro model of primary culture of human nasal epithelial cells (HNEC) in air-liquid interface, which allows epithelial cell differentiation and mimics in vivo airway epithelium. We subsequently tested 7-day and 24-hour Aspergillus fumigatus filtrates on the apical side of HNEC to know whether A. fumigatus, the main species responsible for invasive aspergillosis, produces specific damage to the epithelial cells. The results were compared with those obtained with non-pathogenic filamentous fungi. Seven-day culture filtrates of A. fumigatus and Penicillium chrysogenum induced electrophysiological modifications whatever the fungus tested. In contrast, only 24-hour A. fumigatus filtrates induced a specific decrease in transepithelial resistance, hyperpolarization of the epithelium, and cytoplasmic vacuolization of HNEC compared with both A. niger and Penicillium chrysogenum. The inhibition of the A. fumigatus effects with amiloride suggests that the 24-hour fungal filtrate acts through sodium channels of HNEC. These early modifications of the epithelial cells could facilitate colonization of the airways by A. fumigatus. To know whether the molecules involved are specific to A. fumigatus or simply produced more rapidly than by other filamentous fungi warrants further investigation. In this perspective, the primary culture of HNEC represents a suitable model to study the interactions between airway epithelial cells and A. fumigatus.

Adhesion of Aspergillus Species to Extracellular Matrix Proteins: Evidence for Involvement of Negatively Charged Carbohydrates on the Conidial Surface

Invasive lung disease caused by Aspergillus species is a potentially fatal infection in immunocompromised patients. The adhesion of Aspergillus fumigatus conidia to proteins in the basal lamina is thought to be an initial step in the development of invasive aspergillosis. The purpose of this study was to determine the mechanism of adhesion of A. fumigatus conidiospores to basal-lamina proteins and to determine whether conidia possess unique adhesins which allow them to colonize the host. We compared conidia from different Aspergillus species for the ability to bind to purified fibronectin and intact basal lamina. Adhesion assays using immobilized fibronectin or type II pneumocyte-derived basal lamina showed that A. fumigatus conidia bound significantly better than those of other Aspergillus species to both fibronectin and intact basal lamina. Neither desialylation nor complete deglycosylation of fibronectin decreased the binding of A. fumigatus conidia to fibronectin, suggesting that oligosaccharides on fibronectin were not involved in conidiospore binding. Further evidence for this hypothesis came from experiments using purified fragments of fibronectin; A. fumigatus conidia preferentially bound to the nonglycosylated 40-kDa fragment which contains the glycosaminoglycan (GAG) binding domain. Negatively charged carbohydrates, including dextran sulfate and heparin, as well as high-ionic-strength buffers, inhibited binding of A. fumigatus conidia to both fibronectin and intact basal lamina, suggesting that negatively charged carbohydrates on the surface of the conidium may bind to the GAG binding domain of fibronectin and other basal-lamina proteins. These data provide evidence for a novel mechanism of conidial attachment whereby adherence to fibronectin and other basal-lamina proteins is mediated via negatively charged carbohydrates on the conidial surface.

Predictive value of a positive nasal swab for Aspergillus SP. in the diagnosis of invasive aspergillosis in adult neutropenic cancer patients

To evaluate the value of a positive nasal swab for Aspergillus in the diagnosis of invasive aspergillosis, we prospectively evaluated nasal colonization in 173 episodes of neutropenia in 92 patients with hematological malignancies. Weekly nasal swabs were taken, and the patients were followed until death or resolution of neutropenia. The outcome variables were the development of invasive aspergillosis, empirical antifungal therapy and death. In 31 episodes of neutropenia (18%) there was at least one positive nasal swab for Aspergillus sp. Only two patients developed invasive aspergillosis, both with a positive nasal swab ( p = 0.03). The positive and negative predictive values of a nasal swab were 6.4% and 100%, respectively. There was no difference between patients with positive or negative swabs regarding antifungal therapy or death. In this population of patients, a nasal swab for Aspergillus sp. had a low positive predictive value and a high negative predictive value for invasive aspergillosis.