IL-33, IL-25 and TSLP contribute to development of fungal-associated protease-induced innate-type airway inflammation.

Certain proteases derived from house dust mites and plants are considered to trigger initiation of allergic airway inflammation by disrupting tight junctions between epithelial cells. It is known that inhalation of proteases such as house dust mite-derived Der p1 and/or papaya-derived papain caused airway eosinophilia in naïve mice and even in Rag-deficient mice that lack acquired immune cells such as T, B and NKT cells. In contrast, little is known regarding the possible involvement of proteases derived from Aspergillus species (fungal-associated proteases; FAP), which are ubiquitous saprophytic fungi in the environment, in the development of allergic airway eosinophilia. Here, we found that inhalation of FAP by naïve mice led to airway eosinophilia that was dependent on protease-activated receptor-2 (PAR2), but not TLR2 and TLR4. Those findings suggest that the protease activity of FAP, but not endotoxins in FAP, are important in the setting. In addition, development of that eosinophilia was mediated by innate immune cells (ILCs) such as innate lymphoid cells, but not by acquired immune cells such as T, B and NKT cells. Whereas IL-33, IL-25 and thymic stromal lymphopoietin (TSLP) are involved in induction of FAP-induced ILC-mediated airway eosinophilia, IL-33-rather than IL-25 and/or TSLP-was critical for the eosinophilia in our model. Our findings improve our understanding of the molecular mechanisms involved in induction of airway inflammation by FAP.

AST to ALT Ratio is elevated in disseminated histoplasmosis as compared to localized pulmonary disease and other endemic mycoses.

Severe pulmonary or disseminated histoplasmosis often necessitates presumptive antifungal treatment while awaiting definitive diagnosis. Histoplasma antigen assays have improved sensitivity but results may lag up to 7 days. In order to increase diagnostic certainty, "soft clues" may be looked for in laboratory and radiologic data, such as elevated alkaline phosphatase or ferritin levels and findings of mediastinal adenopathy or hepatosplenomegaly. To determine if elevated aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio is specific to histoplasmosis or a non-specific marker for disseminated fungal infection or sepsis in general, we retrospectively examined records of all patients diagnosed with an endemic fungal infection (EFI) at Rush University Medical Center from January of 1997 to October of 2012, and a cohort of septic patients with elevated liver enzymes. We identified 90 cases of EFIs during the study period that met all inclusion criteria (Histoplasma 21, Blastomyces 56, Coccidioides 12, Paracoccidioides 1). We also evaluated 10 control patients with bacterial sepsis. The mean ratio of AST to ALT in patients with disseminated histoplasmosis was 2.69 (95% CI:1.22, 4.16) while for other EFIs, the mean ratio ranged from 0.38 to 1.14 with disseminated coccidioidomycosis and blastomycosis respectively (P < 0.0001). The ratio in patients with bacterial sepsis was 0.84. We propose the use of the AST/ALT ratio as a clinical "soft clue" suggestive of disseminated histoplasmosis in the appropriate host, and to possibly distinguish cross reactivity of the Histoplasma antigen assay with other EFIs.

Coccidioidomycosis: adenosine deaminase levels, serologic parameters, culture results, and polymerase chain reaction testing in pleural fluid.

BACKGROUND: In a patient with positive serum serology for coccidioidomycosis, the differential diagnosis of concurrent pleural effusions can be challenging. We, therefore, sought to clarify the performance characteristics of biochemical, serologic, and nucleic-acid-based testing in an attempt to avoid invasive procedures. The utility of adenosine deaminase (ADA), coccidioidal serology, and polymerase chain reaction (PCR) in the evaluation of pleuropulmonary coccidioidomycosis has not been previously reported. METHODS: Forty consecutive patients evaluated for pleuropulmonary coccidioidomycosis were included. Demographic data, pleural fluid values, culture results, and clinical diagnoses were obtained from patient chart review. ADA testing was performed by ARUP Laboratories, coccidioidal serologic testing was performed by the University of California-Davis coccidioidomycosis serology laboratory, and PCR testing was performed by the Translational Genomics Research Institute using a previously published methodology. RESULTS: Fifteen patients were diagnosed with pleuropulmonary coccidioidomycosis by European Organization for the Research and Treatment of Cancer/Mycoses Study Group criteria. Pleural fluid ADA concentrations were < 40 IU/L in all patients (range, < 1.0-28.6 IU/L; median, 4.7). The sensitivity and specificity of coccidioidal serologic testing was 100% in this study. The specificity of PCR testing was high (100%), although the overall sensitivity remained low, and was comparable to the experience of others in the clinical use of PCR for coccidioidal diagnostics. CONCLUSION: Contrary to prior speculation, ADA levels in pleuropulmonary coccidioidomycosis were not elevated in this study. The sensitivity and specificity of coccidioidal serologic testing in nonserum samples remained high, but the clinical usefulness of PCR testing in pleural fluid was disappointing and was comparable to pleural fluid culture.

Immune regulation and tolerance to fungi in the lungs and skin.

The balance of pro- and anti-inflammatory signaling is a prerequisite for successful host/fungal interactions and requires the coordinate actions of both innate and adaptive immune systems. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases and limit protective antifungal immune responses. The newly described Th17 develop - mental pathway may play an inflammatory role previously attributed to uncontrolled Th1 responses and serve to accommodate the seemingly paradoxical association of chronic inflammatory responses with fungal persistence in the face of an ongoing inflammation. In this scenario, unrestricted fungal growth could result from the activation of not only pathogenic Th17 cells, but also Th2 cells whose activation is strictly dependent on fungal burden. The capacity of regulatory T cells (Tregs) to inhibit aspects of innate and adaptive antifungal immunity is required for protective tolerance to fungi. Indoleamine 2,3-dioxygenase (IDO) and tryptophan catabolites contribute to such a homeostatic condition by providing the host with immune defense mechanisms adequate for protection, without necessarily eliminating fungal pathogens - which would impair immune memory - or causing an unacceptable level of tissue damage. IDO and tryptophan metabolites may prove to be potent regulators capable of taming overzealous or heightened inflammatory host responses.

Pulmonary cryptococcosis induces chitinase in the rat.

BACKGROUND: We previously demonstrated that chronic pulmonary infection with Cryptococcus neoformans results in enhanced allergic inflammation and airway hyperreactivity in a rat model. Because the cell wall of C. neoformans consists of chitin, and since acidic mammalian chitinase (AMCase) has recently been implicated as a novel mediator of asthma, we sought to determine whether such infection induces chitinase activity and expression of AMCase in the rat. METHODS: We utilized a previously-established model of chronic C. neoformans pulmonary infection in the rat to analyze the activity, expression and localization of AMCase. RESULTS: Our studies indicate that intratracheal inoculation of C. neoformans induces chitinase activity within the lung and bronchoalveolar lavage fluid of infected rats. Chitinase activity is also elicited by pulmonary infection with other fungi (e.g. C. albicans), but not by the inoculation of dead organisms. Enhanced chitinase activity reflects increased AMCase expression by airway epithelial cells and alveolar macrophages. Systemic cryptococcosis is not associated with increased pulmonary chitinase activity or AMCase expression. CONCLUSION: Our findings indicate a possible link between respiratory fungal infections, including C. neoformans, and asthma through the induction of AMCase.

Urease produced by Coccidioides posadasii contributes to the virulence of this respiratory pathogen.

Urease activity during in vitro growth in the saprobic and parasitic phases of Coccidioides spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of Coccidioides posadasii lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of C. posadasii is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of C. posadasii resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a well-organized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of C. posadasii contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.

A recombinant aspartyl protease of Coccidioides posadasii induces protection against pulmonary coccidioidomycosis in mice.

Coccidioidomycosis is a respiratory disease of humans caused by the desert soil-borne fungal pathogens Coccidioides spp. Recurrent epidemics of this mycosis in the southwestern United States have contributed significantly to escalated health care costs. Clinical and experimental studies indicate that prior symptomatic coccidioidomycosis induces immunity against subsequent infection, and activation of T cells is essential for containment of the pathogen and its clearance from host tissue. Development of a human vaccine against coccidioidomycosis has focused on recombinant T-cell-reactive antigens which elicit a durable protective immune response against pulmonary infection in mice. In this study we fractionated a protective multicomponent parasitic cell wall extract in an attempt to identify T-cell antigens. Immunoblots of electrophoretic separations of this extract identified patient seroreactive proteins which were subsequently excised from two-dimensional polyacrylamide gel electrophoresis gels, trypsin digested, and sequenced by tandem mass spectrometry. The full-length gene which encodes a dominant protein in the immunoblot was identified using established methods of bioinformatics. The gene was cloned and expressed, and the recombinant protein was shown to stimulate immune T cells in vitro. The deduced protein was predicted to contain epitopes that bind to human major histocompatibility complex class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes induced gamma interferon production by immune T lymphocytes. The T-cell-reactive antigen, which is homologous to secreted fungal aspartyl proteases, protected mice against pulmonary infection with Coccidioides posadasii. We argue that this immunoproteomic/bioinformatic approach to the identification of candidate vaccines against coccidioidomycosis is both efficient and productive.

Changes in the elastase activity and colonization ability of Aspergillus fumigatus after successive inoculations in mice.

In a previous work we demonstrated a clear link between elastase activity and pathogenicity using what we have named the Elastase Activity Index (EAI). In the present study we have evaluated the possible variability of this index as a consequence of successive inoculations in mice. Two strains of Aspergillus fumigatus isolated from the environment without elastase activity were used. These strains were inoculated into successive batches of ten mice. Our results showed that with each inoculation there was an increase in the number of mice on each batch from which the strain could be isolated and an increase in the number of strains with an EAI>1. This study suggests that A. fumigatus could adapt to the environment in which it is developed, increasing its pathogenic capabilities from host to host.

The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence.

Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immunocompromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes l-ornithine N(5)-oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal l-ornithine N(5)-oxygenases. A stable DeltasidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N',N",N'''-triacetylfusarinine C (TAF) and ferricrocin. Growth of the DeltasidA strain was the same as that of the wild type in rich media; however, the DeltasidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the DeltasidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the DeltasidA strain was unable to remove iron from human transferrin. A rescued strain (DeltasidA + sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the DeltasidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.

Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin production and virulence.

Oxylipins comprise a family of oxygenated fatty acid-derived signaling molecules that initiate critical biological activities in animals, plants, and fungi. Mammalian oxylipins, including the prostaglandins (PGs), mediate many immune and inflammation responses in animals. PG production by pathogenic microbes is theorized to play a role in pathogenesis. We have genetically characterized three Aspergillus genes, ppoA, ppoB, and ppoC, encoding fatty acid oxygenases similar in sequence to specific mammalian prostaglandin synthases, the cyclooxygenases. Enzyme-linked immunosorbent assay analysis showed that production of PG species is decreased in both Aspergillus nidulans and A. fumigatus ppo mutants, implicating Ppo activity in generating PGs. The A. fumigatus triple-ppo-silenced mutant was hypervirulent in the invasive pulmonary aspergillosis murine model system and showed increased tolerance to H(2)O(2) stress relative to that of the wild type. We propose that Ppo products, PG, and/or other oxylipins may serve as activators of mammalian immune responses contributing to enhanced resistance to opportunistic fungi and as factors that modulate fungal development contributing to resistance to host defenses.

Urokinase-type plasminogen activator is required for the generation of a type 1 immune response to pulmonary Cryptococcus neoformans infection.

Urokinase-type plasminogen activator (uPA)(-/-) mice cannot mount protective host defenses during infection with the opportunistic yeast Cryptococcus neoformans (52D). Because effective host defense against C. neoformans requires specific immune responses and the generation of type 1 (T1) cytokines, we determined how the absence of uPA impacts these processes. Wild-type (WT) and uPA(-/-) mice were inoculated with C. neoformans. Macrophage antifungal activity was assessed histologically, T lymphocyte responses in vivo and proliferation in vitro were quantified, and cytokine concentrations were determined by ELISA. uPA(-/-) macrophages have impaired antimicrobial activity. Regional lymph nodes of infected uPA(-/-) mice contained fewer cells than WT, suggesting impaired T cell proliferation in response to the pathogen in vivo. In vitro, uPA(-/-) T lymphocytes had impaired proliferative responses to C. neoformans rechallenge compared with WT. Infected WT mice generated T1 cytokines in the lung, characterized by high levels of IFN-gamma and IL-12. uPA(-/-) mice had decreased levels of IFN-gamma and IL-12, and increased IL-5, a type 2 cytokine. In the absence of uPA, the cytokine profile of regional lymph nodes shifted from a T1 pattern characterized by IFN-gamma and IL-2 to a weak, nonpolarized response. We conclude that in the absence of uPA, lymphocyte proliferative responses are diminished, and mice fail to generate protective T1 cytokines, resulting in impaired antimicrobial activity. This study provides novel evidence that uPA is a critical modulator of immune responses and of immune cell effector functions in vivo.

Expression of inducible nitric oxide synthase and enzymes of arginine metabolism in Fusarium kyushuense-exposed mouse lung.

Expression of inducible nitric oxide (NO) synthase (iNOS) and related enzymes of arginine metabolism in the mouse lung exposed to filamentous fungus Fusarium kyushuense was studied by RNA blot, immunoblot, and histological analyses. When mice were exposed intranasally to the fungi only once, no induction of iNOS mRNA was observed. However, when the animals were infected again 6 days after the first exposure, iNOS mRNA was induced, reached a maximum 12-24 h after the exposure, and decreased to an undetectable level at 48 h. mRNAs for cationic amino acid transporter-2 (CAT2) and argininosuccinate synthetase were induced gradually, reached a maximum at 24 h, and decreased at 48 h. Arginase II mRNA increased at 24 h and decreased markedly at 48 h. On the other hand, arginase I mRNA started to increase at 24 h and reached to a much higher level at 48 h. Ornithine decarboxylase and ornithine aminotransferase mRNAs were also induced. Immunoblot analysis showed that iNOS, argininosuccinate synthetase, and arginase I and II proteins were induced with similar kinetics as those of their respective mRNAs. In histological examination, fungal elements were observed in the bronchoalveolar lumen at 3-6 h, decreased at 12 h, and almost disappeared at 48 h. Small granuloma appeared 3 h after the infection and their size increased with time. These results suggest that NO is produced in the mouse lung in response to F. kyushuense exposure and that the NO production is regulated by CAT2, the citrulline-NO cycle, and arginase isoforms. Enhanced synthesis of polyamines and proline (and thus collagen) is also suggested.

Expression of lung inducible nitric oxide synthase protein does not correlate with nitric oxide production in vivo in a pulmonary immune response against Cryptococcus neoformans.

Mice infected intratracheally with Cryptococcus neoformans (Cne) require CD4 and CD8 T cells, IFN-gamma, and M phi production of nitric oxide (NO) for effective resolution of the pulmonary infection. Differences exist among strains of mice in clearing the infection. C.B-17 mice reduced Cne lung burden at a significantly greater rate than C57BL/6 (B6) mice and resistance correlated with greater IFN-gamma production by C.B-17 lung-associated lymph node cells. We examined whether the differences observed in the ability of B6 vs C.B-17 mice to clear Cne was due to 1) numbers of inflammatory cells recruited to the lung, 2) the activation state of the recruited cells as measured by expression of inducible nitric oxide synthase (iNOS), and/or 3) the in vivo production of NO as measured by quantitating urine nitrates. The level of iNOS protein was identical in lungs from both strains of mice during Cne infection as determined by Western blot analysis of whole lung homogenates and immunocytochemistry of isolated lung macrophages. Surprisingly, in vivo studies of iNOS activity indicated that NO production in B6 mice was significantly less than that in C.B-17 mice. While single cell suspensions from lungs of either mouse strain produced identical amounts of NO, NO production by lung explants paralleled in vivo urinary nitrate excretion, suggesting that the maintenance of pulmonary architecture and cell-cell interaction was necessary for suppression of iNOS activity in B6 mice. These data strongly implicate the existence of mechanisms that regulate NO production at the level of enzyme activity during infections and have important implications for analyzing the role of iNOS during an immune response in in vivo models.

Expression of inducible nitric oxide synthase in rat pulmonary Cryptococcus neoformans granulomas.

Rats, like humans, have extremely effective immune mechanisms for controlling pulmonary Cryptococcus neoformans infection. The mechanism(s) responsible for efficient immunity in rat experimental infection is unknown. Recently, induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) have been implicated as an important microbicidal mechanism by which activated macrophages effect cytotoxicity against microbes. In this report, we investigated the expression of iNOS in rat pulmonary cryptococcosis. Localization and regulation of NO production was studied by immunohistochemistry for iNOS in conjunction with immunohistochemistry for cell markers, cytokines, and cryptococcal capsular polysaccharide. iNOS immunoreactivity was detected in macrophages, neutrophils, vascular endothelium, and respiratory epithelium. Double-immunolabeling studies revealed that the most prominent iNOS immunoreactivity was localized to epithelioid macrophages (CD11b/c+) within granulomas; CD4+ and CD8+ T cells were numerous around granulomas but did not express iNOS. iNOS immunoreactivity was detected in a selective population of epithelioid macrophages within some granulomas but not others. iNOS- granulomas were identical to iNOS+ granulomas with respect to morphology and immunohistochemical profiles. Macrophage iNOS immunoreactivity was detected 1 week after infection in one out of four rats and was strongly expressed in all rats at 2 weeks (in up to 50 percent of the granulomas) but declined considerably by 25 days. iNOS expression coincided with granuloma formation and preceded a decrease in lung fungal burden, suggesting an anticryptococcal role for NO. By double labeling, cytokines that have been shown to promote (interferon-gamma, granulocyte/macrophage colony-stimulating factor) and inhibit (transforming growth factor-beta) macrophage iNOS expression were detected around iNOS+ granuloma. iNOS immunoreactivity was expressed in selected neutrophils (1 and 2 weeks) and endothelial cells (1 and 2 weeks and 25 days) in the inflamed lung. Airway iNOS immunoreactivity was limited to the luminal border of rare bronchiolar epithelial cells. iNOS immunoreactivity was not detected in uninfected rats. The present study provides the first evidence for association of iNOS expression with protective cellular responses to cryptococcal infection in vivo.

[Invasive pulmonary aspergillosis in a patient with acute leukemia--the role of neutrophil elastase in in cavity formation].

A 47-year-old man complaining of common cold-like symptoms was admitted to our hospital. Acute myelogenous leukemia was diagnosed and the patient was treated with induction chemotherapy. During granulocytopenia caused by induction chemotherapy, a nodular lesion appeared in the right upper lobe. The nodular lesion changed to a cavitary lesion after the recovery of peripheral white blood cell counts. A transbronchial biopsy specimen obtained from the right B3b showed Aspergillus. Oral itraconozole, flucytosine, and intravenous amphotericin B were given. The cavitary lesion in the right upper lobe regressed after anti-fungal therapy was started. During granulocytopenia caused by consolidation chemotherapy, the nodular lesion enlarged again. Thereafter, as bone marrow recovered, it changed to a cavitary lesion with a lung ball inside. In both episodes, a nodular lesion appeared during granulocytopenia, and changed to a cavitary lesion after bone marrow recovery. In addition, the level of neutrophil elastase reached its maximum at the time of the bone marrow recovery. These findings suggest that white blood cells of the host as well as neutrophil elastase play an important role in cavitation in pulmonary aspergillosis.

Bronchoalveolar lavage fluid angiotensin-converting enzyme in interstitial lung diseases.

In this study we evaluated the disease specificity of bronchoalveolar lavage fluid angiotensin-converting enzyme (BALF-ACE), its correlation with cellular constituents of bronchoalveolar lavage fluid (BALF), and for sarcoidosis, with other proposed markers of disease activity. Furthermore, the question of the clinical value of BALF-ACE determinations in in interstitial lung diseases or any of its subgroups was addressed. The study population consisted of 222 patients, 69 with biopsy proven sarcoidosis, 3 with hypersensitivity pneumonitis, 4 with acute histoplasmosis, 27 with idiopathic pulmonary fibrosis (IPF), 4 with rheumatoid arthritis-related interstitial fibrosis, 9 with pulmonary drug toxicity, 16 with pulmonary malignancies, 26 with other parenchymal lung disease entities, and 30 in whom the final diagnosis remained indeterminate. Elevated BALF-ACE concentrations were seen in all diagnostic categories. In sarcoidosis BALF-ACE levels correlated well with lavage lymphocyte counts (r = 0.49; p less than 0.0001), in contrast to IPF where they correlated well with lavage neutrophil counts (r = 0.51; p less than 0.007). The correlation of BALF-ACE and serum-ACE was significant. In sarcoidosis the mean BALF-ACE level was lower for patients with Stage-I chest roentgenographic patterns (0.664 U/L), compared to those with Stage II (1.112 U/L) and Stage III (1.083 U/L). It was concluded that elevated BALF-ACE levels are not specific for sarcoidosis. The correlations of BALF-ACE levels with different cellular constituents of BALF suggest a different cellular origin of BALF-ACE. In sarcoidosis BALF-ACE levels correlate well with other proposed markers of disease activity and seem to reflect pulmonary activity better than serum ACE.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated serum angiotensin-converting enzyme (SACE) activity in acute pulmonary histoplasmosis.

Serum angiotensin-converting enzyme (SACE) levels were measured in 44 subjects six weeks after acute pulmonary histoplasmosis. All patients were infected in a common-source outbreak of histoplasmosis which occurred on one day. All patients had both strictly defined clinical and serologic evidence of infection. The SACE activity was elevated at six weeks compared to normal controls, and seven of the 44 had levels more than 2 SD above the normal mean. SACE levels were also measured at three and 24 weeks after acute infection in a smaller number of the same subjects. Serial observations demonstrated that all subjects (including those with normal and elevated SACE at six weeks) had a rise and fall in SACE activity following symptomatic acute pulmonary histoplasmosis. Our findings suggest that elevated SACE does not reliably separate sarcoidosis from histoplasmosis, although elevations in histoplasmosis are much less common and may occur only briefly following acute pulmonary histoplasmosis. More important, it seems that SACE activity rises acutely in all patients with symptomatic acute histoplasmosis and then falls gradually toward baseline over several months, coinciding temporally with the granulomatous response.