Monoclonal immunoglobulin G1 directed against Aspergillus fumigatus cell 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.     

Coevolution of TH1, TH2, and TH17 responses during repeated pulmonary exposure to Aspergillus fumigatus conidia

Aspergillus fumigatus, a ubiquitous airborne fungus, can cause invasive infection in immunocompromised individuals but also triggers allergic bronchopulmonary aspergillosis in a subset of otherwise healthy individuals repeatedly exposed to the organism. This study addresses a critical gap in our understanding of the immunoregulation in response to repeated exposure to A. fumigatus conidia. C57BL/6 mice were challenged intranasally with A. fumigatus conidia weekly, and leukocyte composition, activation, and cytokine production were examined after two, four, and eight challenges. Approximately 99% of A. fumigatus conidia were cleared within 24 h after inoculation, and repeated exposure to A. fumigatus conidia did not result in hyphal growth or accumulation of conidia with time. After 2 challenges, there was an early influx of neutrophils and regulatory T (T(reg)) cells into the lungs but minimal inflammation. Repeated exposure promoted sustained expansion of the draining lymph nodes, while the influx of eosinophils and other myeloid cells into the lungs peaked after four exposures and then decreased despite continued A. fumigatus challenges. Goblet cell metaplasia and low-level fibrosis were evident during the response. Repeated exposure to A. fumigatus conidia induced T cell activation in the lungs and the codevelopment by four exposures of T(H)1, T(H)2, and T(H)17 responses in the lungs, which were maintained through eight exposures. Changes in CD4 T cell polarization or T(reg) numbers did not account for the reduction in myeloid cell numbers later in the response, suggesting a non-T-cell regulatory pathway involved in dampening inflammation during repeated exposure to A. fumigatus conidia.     

Aspergillus fumigatus corneal infection is regulated by chitin synthases and by neutrophil–derived acidic mammalian chitinase

Aspergillus fumigatus is an important cause of pulmonary and systemic infections in immune compromised individuals, and of corneal ulcers and blindness in immune competent patients. To examine the role of chitin synthases in Aspergillus corneal infection, we analyzed Aspergillus mutants of chitin synthase family 1 and family 2, and found that compared with the parent strain, the quadruple mutants from both families were more readily killed by neutrophils in vitro, and that both also exhibited impaired hyphal growth in the cornea. Further, inhibition of chitin synthases using Nikkomycin Z enhanced neutrophil killing in vitro and in vivo in a murine model of A. fumigatus corneal infection. Acidic mammalian chitinase (AMCase) is mostly produced by macrophages in asthmatic lungs; however, we now demonstrate that neutrophils are a major source of AMCase, which inhibits hyphal growth. In A. fumigatus corneal infection, neutrophils are the major source of AMCase, and addition of AMCase inhibitors or adoptive transfer of neutrophils from AMCase^?/? mice resulted in impaired hyphal killing. Together, these findings identify chitin synthases as important fungal virulence factors and neutrophil‐derived AMCase as an essential mediator of host defense.     

Binding of pulmonary surfactant proteins A and D to Aspergillus fumigatus conidia enhances phagocytosis and killing by human neutrophils and alveolar macrophages.

To determine whether the lung surfactant proteins A (SP-A) and D (SP-D) are involved in the initial protective immunity against opportunistic pulmonary fungal infections caused by Aspergillus fumigatus, we performed a series of in vitro functional studies to see if SP-A and SP-D enhanced binding, phagocytosis, activation, and killing of A. fumigatus conidia by human alveolar macrophages and circulating neutrophils. Both SP-A and SP-D bound to carbohydrate structures on A. fumigatus conidia in a calcium-dependent manner. SP-A and SP-D were also chemoattractant and significantly enhanced agglutination and binding of conidia to alveolar macrophages and neutrophils. Furthermore, in the presence of SP-A and SP-D, the phagocytosis, oxidative burst, and killing of A. fumigatus conidia by neutrophils were significantly increased. These findings indicate that SP-A and SP-D may have an important immunological role in the early antifungal defense responses in the lung, through inhibiting infectivity of conidia by agglutination and by enhancing uptake and killing of A. fumigatus by phagocytic cells.     

Association of presence of Aspergillus antibodies with hemoptysis in patients with old tuberculosis or bronchiectasis but no radiologically visible mycetoma

Old tuberculosis and bronchiectasis are the two most important causes of chronic structural changes of lungs in our locality. In the absence of radiologically visible mycetoma, the cause of hemoptysis in these two groups of patients is largely unknown. A 17-month prospective study was carried out to compare the prevalence of Aspergillus fumigatus and Aspergillus flavus antibodies in hemoptysis patients with old tuberculosis or bronchiectasis but no radiologically visible mycetoma (cases, n = 38), hemoptysis patients with other diagnosis (control group 1, n = 29), and patients with old tuberculosis or bronchiectasis but no hemoptysis (control group 2, n = 47) by a recently developed sensitive and specific A. fumigatus and A. flavus antibody assay. There were a significantly larger number of patients with antibody against A. fumigatus or A. flavus among the cases than among the patients in control groups 1 and 2 (P < 0.05 in both comparisons). Molds were not recovered from any of the patients. Among the 10 cases with Aspergillus antibody, eight and two had antibody against A. flavus and A. fumigatus, respectively. We conclude that there was an association between the presence of Aspergillus antibodies and hemoptysis in patients with old tuberculosis or bronchiectasis, suggesting that these patients probably had occult infections caused by the corresponding fungi. Development of serological tests against other Aspergillus species as well as other causes of mycetoma will probably increase the detection of occult mold infections in patients with existing parenchymal lung diseases, and treatment of fungal microinvasion may help to alleviate hemoptysis in these patients with bronchiectasis or old tuberculosis who have Aspergillus antibodies.     

Immunoreactivity of a 38-kilodalton Penicillium marneffei antigen with human immunodeficiency virus-positive sera.

Penicillium marneffei produced and secreted a 38-kDa antigen that appeared to be specific for this dimorphic fungus. This component could not be detected in antigenic extracts of Histoplasma capsulatum, Cryptococcus neoformans, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Candida albicans, and two other species of Penicillium by immunoblot analysis against the sera from patients with culture-confirmed penicilliosis marneffei. Antibody reactive with this antigen was found in a large proportion of human immunodeficiency virus (HIV)-positive patients, indicating a presumptive diagnosis of P. marneffei infection. A small number of asymptomatic HIV-seropositive patients and HIV-seropositive patients with other fungal infections were also found to be positive by this analysis, suggesting that subclinical or mixed fungal infections involving P. marneffei are not uncommon.     

Neutrophils produce interleukin 17A (IL-17A) in a dectin-1- and IL-23-dependent manner during invasive fungal infection

We have previously reported that compromised interleukin 17A (IL-17A) production in the lungs increased susceptibility to infection with the invasive fungal pathogen Aspergillus fumigatus. Here we have shown that culturing lung cells from A. fumigatus-challenged mice ex vivo demonstrated Dectin-1-dependent IL-17A production. In this system, neutralization of IL-23 but not IL-6, IL-1β, or IL-18 resulted in attenuated IL-17A production. Il23 mRNA expression was found to be lower in lung cells from A. fumigatus-challenged Dectin-1-deficient mice, whereas bone marrow-derived dendritic cells from Dectin-1-deficient mice failed to produce IL-23 in response to A. fumigatus in vitro. Addition of recombinant IL-23 augmented IL-17A production by wild-type (WT) and Dectin-1-deficient lung cells, although the addition of IL-6 or IL-1β did not augment the effect of IL-23. Intracellular cytokine staining of lung cells revealed lower levels of CD11b(+) IL-17A(+) and Ly-6G(+) IL-17A(+) cells in A. fumigatus-challenged Dectin-1-deficient mice. Ly-6G(+) neutrophils purified from the lungs of A. fumigatus-challenged Dectin-1-deficient mice displayed lower Il17a mRNA expression but surprisingly had intact Rorc and Rora mRNA expression. We further demonstrated that Ly-6G(+) neutrophils required the presence of myeloid cells for IL-17A production. Finally, upon in vitro stimulation with A. fumigatus, thioglycolate-elicited peritoneal neutrophils were positive for intracellular IL-17A expression and produced IL-17A in a Dectin-1- and IL-23-dependent manner. In summary, Dectin-1-dependent IL-17A production in the lungs during invasive fungal infection is mediated in part by CD11b(+) Ly-6G(+) neutrophils in an IL-23-dependent manner.     

Murine model of invasive pulmonary Aspergillosis: Follow-up of tissue injury,fungal burden and mortality with distinct elastase production strains

To study invasive pulmonary Aspergillosis (IPA), we depleted neutrophils in mice using the monoclonal antibody anti-Gr-1/Ly-6G. Immunocompetent and neutropenic mice were infected via intratracheal with conidia of Aspergillus fumigatus clinical isolates, characterized as either higher or lower elastase producers. Neutropenic animals exhibited 100% mortality in 5 days, for both strains, and were observed survival curves overlapped, lungs with angioinvasion, rupture of bronchial and vascular walls, associated with exuberance of conidia filamentation. The immunocompetent animals infected with the lower elastase producer strain presented with upregulated inflammatory processes, and a lack of conidia filamentation in the tissue. The fungal burden in the lungs was not different in the immunocompetent and neutropenic groups. These findings confirm the protective role of neutrophils against A. fumigatus and suggest that the fungal elastinolytic activity is not a critical virulence factor but may be involved in tissue injury.     

Monoclonal antibodies against a 97-kilodalton antigen from Aspergillus flavus.

We prepared a panel of five monoclonal antibodies (MAbs) directed against Aspergillus flavus that all reacted against one 97-kDa antigen by western blot (immunoblot). Flow cytometry demonstrated that these antibodies bound (in increasing degrees) to all morphologic stages of A. flavus growth: conidia, swollen conidia, and hyphae. Cross-reactivity among species was examined by enzyme-linked immunosorbent assay of fungal culture filtrates. Four MAbs reacted with 10 of 11 A. flavus isolates, and the fifth one reacted with 9 of them. One MAb also reacted with A. fumigatus, two reacted with A. niger, A. wentii, and A. nidulans, and all five reacted with A. ochraceus. None reacted with A. terreus, A. glaucus, A. versicolor, or a Penicillium species. Each MAb bound to A. flavus hyphae in formalin-fixed paraffin sections of a muscle biopsy from a confirmed human case of invasive aspergillosis. In summary, these MAbs identified a 97-kDa antigen found on A. flavus that is both surface bound and an exoantigen. Either the same or a cross-reacting antigen is present in A. fumigatus and other Aspergillus species.     

Rat monoclonal antibodies against Aspergillus galactomannan.

Monoclonal antibodies (MAbs) against Aspergillus fumigatus galactomannan were produced in rats. Seven of them, EB-A1 through EB-A7, were characterized in more detail. They were all immunoglobulin M antibodies, reacting in an indirect enzyme-linked immunosorbent assay with purified A. fumigatus galactomannan, with avidity constants of between 2 x 10(9) and 5 x 10(9)/M. Enzyme-linked immunosorbent assay inhibition experiments with modified galactomannan and synthetic oligomers of beta (1----5)galactofuranose demonstrated that the MAbs bound to an epitope located on the beta(1----5)galactofuranose-containing side chains of the galactomannan molecule. An identical or similar epitope also seemed to be present in other fungi. Immunofluorescence and immunoelectron microscopy experiments with EB-A2 revealed the presence of the antigen in the fungal wall and inside the cell. Immunoblotting experiments demonstrated that the epitope recognized by the MAbs was a common oligosaccharide moiety of a wide range of intracellular and extracellular glycoproteins in A. fumigatus. The characteristics of the MAbs justify their use in the diagnosis of invasive aspergillosis by antigen detection.     

Fetuin A, a serum component, promotes growth and biofilm formation by Aspergillus fumigatus

Aspergillus fumigatus is an all-important pathogenic fungus and is known for its angiotropism. When it invades human organs, A. fumigatus makes direct contact with blood and its components by causing inflammation and invading vascular structures. To learn the effect of its contact with blood on the development of infection, we examined the effect of serum on A. fumigatus growth. In Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, hyphal tip growth was accelerated, forming a thickened and well-networked biofilm associated with extracellular matrix, and fetuin A was identified as the active component in the serum that accelerates fungal growth leading to formation of a community. These results suggest that fetuin A is a novel accelerator of the growth of A. fumigatus and that it participates in the formation of thick biofilm.     

Vaccinations with recombinant variants of Aspergillus fumigatus allergen Asp f 3 protect mice against invasive aspergillosis

A vaccine that effectively protects immunocompromised patients against invasive aspergillosis is a novel approach to a universally fatal disease. Here we present a rationale for selection and in vivo testing of potential protein vaccine candidates, based on the modification of an immunodominant fungal allergen for which we demonstrate immunoprotective properties. Pulmonary exposure to viable Aspergillus fumigatus conidia as well as vaccination with crude hyphal extracts protects corticosteroid-immunosuppressed mice against invasive aspergillosis (J. I. Ito and J. M. Lyons, J. Infect. Dis. 186:869-871, 2002). Sera from the latter animals contain antibodies with numerous and diverse antigen specificities, whereas sera from conidium-exposed mice contain antibodies predominantly against allergen Asp f 3 (and some against Asp f 1), as identified by mass spectrometry. Subcutaneous immunization with recombinant Asp f 3 (rAsp f 3) but not with Asp f 1 was protective. The lungs of Asp f 3-vaccinated survivors were free of hyphae and showed only a patchy low-density infiltrate of mononuclear cells. In contrast, the nonimmunized animals died with invasive hyphal elements and a compact peribronchial infiltrate of predominantly polymorphonuclear leukocytes. Three truncated versions of rAsp f 3, spanning amino acid residues 15 to 168 [rAsp f 3(15-168)], 1 to 142, and 15 to 142 and lacking the known bipartite sequence required for IgE binding, were also shown to be protective. Remarkably, vaccination with either rAsp f 3(1-142) or rAsp f 3(15-168) drastically diminished the production of antigen-specific antibodies compared to vaccination with the full-length rAsp f 3(1-168) or the double-truncated rAsp f 3(15-142) version. Our findings point to a possible mechanism in which Asp f 3 vaccination induces a cellular immune response that upon infection results in the activation of lymphocytes that in turn enhances and/or restores the function of corticosteroid-suppressed macrophages to clear fungal elements in the lungs.     

Investigation into the production of 2-Pentylfuran by Aspergillus fumigatus and other respiratory pathogens in vitro and human breath samples.

Our objective was to identify, for diagnostic purposes, potential volatile biomarkers of human microbial pathogens. We analysed the head space of cultures of medically important bacterial and fungal respiratory pathogens for 2-Pentylfuran (2PF) production through the use of Solid Phase Micro Extraction (SPME) and Gas Chromatography/Mass Spectroscopy (GC/MS). Our results confirm that 2PF is consistently produced by Aspergillus fumigatus, Fusarium spp., Aspergillus terreus, Aspergillus flavus and to a lesser extent by Aspergillus niger. 2-Pentylfuran was not detected from most of the bacterial strains except for Streptococcus pneumoniae. In human studies, four litre breath samples were collected from patients with cystic fibrosis (CF), with and without colonisation by A. fumigatus and other pathogens, as well as healthy volunteers. 2-Pentylfuran was detected in breath samples collected from 4/4 patients with CF and A. fumigatus colonization, 3/7 patients with CF and no microbiological evidence of A. fumigatus and 0/10 healthy control individuals. These results suggest that 2PF may be a biomarker for lung colonization/infection by fungal pathogens. To our knowledge, this is the first report describing the detection in breath samples of a volatile biomarker of a pathogen resident in the lungs. Breath analysis has the potential of being a non-invasive diagnostic method of detecting respiratory infection including invasive aspergillosis.     

Cytokine and chemokine responses following pulmonary challenge with Aspergillus fumigatus: obligatory role of TNF-alpha and GM-CSF in neutrophil recruitment.

Aspergillus fumigatus causes life-threatening invasive pulmonary aspergillosis in the immunocompromised patient. In this study we have used a murine model of intratracheal challenge with A. fumigatus to investigate the recruitment of inflammatory cells in the lung and the expression of proinflammatory cytokines and chemokines. Our results show that A. fumigatus causes an acute pulmonary inflammatory response which is dominated by neutrophils and to a lesser extent macrophages. During the peak of infection, proinflammatory cytokines (TNF-alpha, GM-CSF and IL-1beta) and chemokines (MIP-1alpha, MCP-1 and MIP-2), are induced within the lung. Furthermore, treatment of mice with neutralizing anti-TNF-alpha and anti-GM-CSF mAbs reduced the influx of neutrophils into the lung and delayed fungal clearance. Our observations show that Aspergillus conidia are effective inducers of host chemokine responses both in vitro and in vivo. Furthermore, TNF-alpha and GM-CSF play a central role in the recruitment of neutrophils into the lung in response to this clinically important pathogen.     

Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR

The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.     

The monoclonal antibody against the major diagnostic antigen of Paracoccidioides brasiliensis mediates immune protection in infected BALB/c mice challenged intratracheally with the fungus

The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.     

Human albumin promotes germination, hyphal growth and antifungal resistance by Aspergillus fumigatus.

Invasive aspergillosis is one of the most common deep-seated fungal infections among patients with an impaired immune system. Albumin is a serum protein commonly administered to critical patients. Our objective was to evaluate the in vitro effect of human albumin upon germination and hyphal growth of Aspergillus species, especially the most pathogenic, Aspergillus fumigatus, as well as its influence on antifungal drug activity. Human albumin induced, at normal serum concentrations (2-4%), a significant promotion of conidial germination by A. fumigatus, but not by Aspergillus flavus and Aspergillus niger. However, mycelium growth following germination was enhanced in all Aspergillus species. Minimal inhibitory concentrations (MIC) of all strains tested to amphotericin B and itraconazole increased in the presence of physiological concentrations of human albumin. Voriconazole activity was not, however, significantly affected by the presence of the protein. Conidial germination represents a crucial initial step in the progression to invasive disease, involving metabolic pathways that may differ considerably among Aspergillus species. Our results support the concept that human albumin may promote a faster onset and enhanced dissemination of invasive aspergillosis.     

Comparison between Aspergillus fumigatus conidia and hyphae susceptibilities to amphotericin B, itraconazole, and voriconazole by use of the mold rapid susceptibility assay.

Although hyphae are the morphological form observed in tissue during invasive Aspergillus fumigatus infections, antifungal susceptibility testing for A. fumigatus utilizes conidial inocula. Previous studies have yielded conflicting results as to whether conidia adequately reflect antifungal susceptibility of hyphae, but the ease of handling and quantification of conidia have prompted their use in such assays. The mold rapid susceptibility assay, which utilizes a conidial inoculum (cRSA), was adapted as a novel method to assess the utility of conidial versus hyphal inocula (hRSA) to further evaluate the susceptibility of A. fumigatus conidia and hyphae to amphotericin B (AMB), itraconazole (ITC), and voriconazole (VRC). Conidial inocula were prepared as previously described for the cRSA and minimum inhibitory values (MIC) were determined. For the hRSA, microtiter test wells lacking antifungal drug were inoculated with a standardized conidial inoculum and incubated for 12 h at 35-37 degrees C to allow formation of hyphae. Following addition of antifungal drug and 48 h incubation at 35-37 degrees C, hRSA antifungal minimum inhibitory concentration (MIC) values were determined by analysing the pattern of residual glucose levels in hRSA test wells. hRSA MIC values of each strain were influenced by hyphal inoculum size, with increasing hyphal inoculum size corresponding to increased AMB, ITC and VRC MIC values. Comparisons between the hRSA and cRSA MIC values demonstrated insignificant differences in conidial and hyphal susceptibility to drug, thus justifying the use of either fungal form in RSA-based susceptibility testing of A. fumigatus isolates. The RSA may be adapted for use of similar testing of other invasive molds that predominate as hyphal forms in tissue.     

Necessary and sufficient role for T helper cells to prevent fungal dissemination in allergic lung disease

Mucosal immune responses to fungal infection range from T helper type 2 (Th2) cell-directed allergic inflammation to Th1-predominant neutrophilic inflammation, but the mechanisms directing these divergent mucosal immune outcomes and the role of T cells in host defense against mucosal fungal infections are not known. Here we examined the mouse mucosal immune responses to 12 filamentous environmental fungal species over a broad range of exposure doses and determined the requirement of T cells for host defense. For all tested fungi, low-grade conidium exposures induced Th2- and eosinophil-predominant allergic lung disease, whereas higher exposures led to rapid conversion to neutrophil- and Th1 cell-predominant inflammation, a phenomenon we term immune phenotype switching. All fungal exposure doses were further linked to the secretion of interleukin-17A (IL-17A). Fungal infections with Curvularia lunata and Aspergillus fumigatus were typically confined to the airway during allergic inflammation but became locally invasive and disseminated to the brain at higher conidium challenge doses, in association with predominant Th1 responses. Fungal dissemination occurred at relatively low challenge doses with the conidia of Aspergillus fumigatus administered to recombinase activating gene 1 (Rag-1)-deficient mice, which lack B and T cells, but B cell-deficient μMT mice and T helper cell-reconstituted Rag-1-deficient mice were comparable to wild-type mice in preventing fungal dissemination. Our findings demonstrate that Th2 cell-predominant allergic responses followed by immune phenotype switching and fungal dissemination are highly predictable outcomes with progressive fungal infectious burdens and that T helper cell responses are protective against lethal fungal dissemination.     

Damage to hyphal forms of fungi by human leukocytes in vitro. A possible host defense mechanism in aspergillosis and mucormycosis.

Evidence suggests that neutrophils are important in host defenses against invasive aspergillosis and mucormycosis, although hyphae in these lesions are too large to be phagocytized. Interactions of neutrophils with hyphae of Aspergillus fumigatus and Rhizopus oryzae were studed in vitro. Light and electron microscopic observations indicated that neutrophils attached to and spread over the surfaces of hyphae, even in the absence of serum. This was followed by dramatic morphologic changes which suggested severe damage and probably death of hyphae. An assay of neutrophil-induced reduction of uptake of radioisotopes was used to quantitate damage to the fungi by neutrophils from normal subjects. Damage to hyphae was inhibited by a variety of compounds which are known to affect neutrophil surface functions, motility, and metabolism. Use of inhibitors of oxidative microbicidal mechanisms of neutrophils indicated the central importance of these mechanisms in damage to hyphae. Inhibitors of neutrophil cationic proteins altered damage only to Rhizopus. Damage to hyphae by lysozyme suggested that it may play a secondary role in the neutrophil, primarily against Aspergillus. This new nonphagocytic mechanism may play an important role in host defenses against these and other hyphal forms of fungi.