Exopolysaccharide from Paecilomyces lilacinus modulates macrophage activities through the TLR4/NF­κB/MAPK pathway.

Multiple exopolysaccharides (EPSs) have been isolated from various organisms in extreme environments and have yielded a variety of activities. The present study evaluated the immunomodulatory capabilities of an EPS (termed PH­EPS) derived from the fungus Paecilomyces lilacinus PH0016, which was isolated from a tropical and hyperhaline environment in southern China. The macrophage RAW 264.7 cell line was used to investigate the mechanism of PH­EPS­induced macrophage activation. The results indicated that RAW 264.7 macrophages were activated by PH­EPS, in an effect slightly inferior to lipopolysaccharide (LPS), as evidenced by secretion of interleukin (IL)­1ß, tumor necrosis factor (TNF)­α and nitric oxide (NO), and by significantly increased phagocytosis in the cells treated with PH­EPS. Nuclear factor (NF)­κB p65 was significantly translocated into the nucleus in the PH­EPS­treated cells. In addition, expression of inducible NO synthase (iNOS) and IκB­α degradation were enhanced in PH­EPS­treated cells. The phosphorylation levels of p38, JNK and ERK were also significantly increased in the PH­EPS­treated cells. Furthermore, IL­1ß and TNF­α production was markedly decreased in PH­EPS­treated cells when the mitogen­activated protein kinase (MAPK) pathways were blocked by the inhibitor Dectin­1 and by antibodies against Toll­like receptor 4 (TLR4). The present results indicated that PH­EPS from Paecilomyces lilacinus possessed the capability of activating RAW 264.7 cells via the TLR4/NF­κB/MAPKs signaling pathway.

Change in kidney damage biomarkers after 13 weeks of exposing rats to the complex of Paecilomyces sinclairii and its host Bombyx mori larvae.

Complex of Paecilomyces sinclairii and host larvae, Bombyx mori, is a well known health food; however, concerns about nephrotoxicity have been raised. Kidney toxicity was investigated after 13 weeks of administering the complex orally to rats with parameters including blood urea nitrogen (BUN), creatinine, and kidney damage biomarkers, beta-2-microglobulin (ß2m), glutathione S-transferase alpha (GST-α), kidney injury molecule 1 (KIM-1), tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), vascular endothelial growth factor (VEGF), calbindin, clusterin, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin. Dose-dependent kidney cell karyomegaly and tubular hypertrophy were observed, with higher severity in males. There was a dose-dependent increase in KIM-1 and TIMP-1 levels in kidney and urinary KIM-1, cystatin C, ß2m, and osteopontin levels. KIM-1 and TIMP-1 increased in male kidneys had not recovered by 2 weeks after stopping exposure. Cystatin C in kidney was significantly lowered in all treatment groups at 13 weeks of administration. All the changes were more noticeable in males. These data indicate that the complex damage renal tubule cells with histopathological lesions and changes in biomarker levels. Kidney and urinary KIM-1 and cystatin C were the most markedly affected and early increased indicators among biomarkers tested, whereas BUN and creatinine were not affected.

[Immunorehabilitation in patients with paecilomycosis-complicated echinococcosis and asthma].

Paecilomycosis is a new type of systemic mycosis caused by different species of fungi of the genus Paecilomyces. Paecilomycosis-complicated echinococcosis and asthma run a severe course. A complication of mycosis is accompanied by secondary immunodeficiency. A good result was obtained in the treatment of ill children by using the fungicide diflucan and the immunomodulator polyoxidonium. In the examinees with paecilomycosis-complicated echinococcosis, secondary immunodeficiency was characterized by a statistical significant reduction in the blood levels of the lymphoid cells CD3+, CD4+, CD8+, CD16+, CD21+, by phagocytosis, a decrease in its quantitative parameters, and an increase in the counts of immunoglobulins and circulating immunocomplexes. To normalize the immune status in patients with paecilomycosis-complicated echinococcosis, it is expedient to postsurgicallyuse fungicides, such as nizoral, diflucan, orungal, mycosyst, and the immunomodulators polyoxidonium and irillen.

Hypersensitivity pneumonitis in a hardwood processing plant related to heavy mold exposure.

Two workers employed in a hardwood floor plant presented symptoms suggestive of hypersensitivity pneumonitis (HP). At that plant, kiln-dried wood often shows moldy growth and is subsequently brought inside for processing. This study evaluated the environment in attempt to identify the causative antigen and verify whether other workers of this and similar plants had or were at risk of developing HP. Dust from dust-removing systems and molds on the surface of wood planks were collected and air samples taken from a sister plant. Blood samples, spirometry, and symptoms' questionnaires were obtained from 11 co-workers. Dense Paecilomyces growth was observed on the surface of the dried processed wood in the index plant. This fungal genus was not detected in the sister plant. An additional worker had symptoms suggestive of HP, and his bronchoalveolar lavage revealed a lymphocytic alveolitis. The 3 confirmed cases of HP and the other 10 workers had positive specific IgG antibodies to Paecilomyces. We report 3 cases of HP out of 13 workers and a 100% sensitization to molds in workers of a hardwood processing plant. This rate is much higher than what is commonly seen in other environments associated with HP. The drying process is suspected of being responsible for the massive Paecilomyces contamination likely responsible for the HP.

Glucan-binding activity of silkworm 30-kDa apolipoprotein and its involvement in defense against fungal infection.

The silkworm Bombyx mori 30-kDa lipoproteins (6G1 and 19G1), major components of the hemolymph, were shown to bind to glucans. 6G1 apolipoprotein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant 6G1 apolipoprotein specifically bound to beta-glucan, but not to chitin, mannan, peptidoglycan, or oligosaccharide chains on glycoproteins. The beta-glucan binding of the recombinant 6G1 was inhibited by laminaribiose and laminarin, a soluble glucan, but not by lipopolysaccharide or insect blood sugar, trehalose at physiological concentration. Furthermore, the recombinant 6G1 was shown to participate in the activation of prophenoloxidase cascade and to interfere with hyphal growth of the entomopathogenic fungus Paecilomyces tenuipes, injected into pupae of B. mori. These results suggest that 6G1 lipoprotein plays a role in the protection of B. mori against invading fungi.

The liquid culture filtrates of Paecilomyces tenuipes (Peck) Samson (=Isaria japonica Yasuda) and Paecilomyces cicadae (Miquel) Samson (=Isaria sinclairii (Berk.) Llond) regulate Th1 and Th2 cytokine response in murine Peyer's patch cells in vitro and ex vivo.

The effects of liquid culture filtrates of medicinal entomogenous fungi, Paecilomyces tenuipes (Peck) Samson (=Isaria japonica Yasuda or Isaria tenuipes) (PTCF) and Paecilomyces cicadae (Miquel) Samson (=Isaria sinclairii (Berk.) Llond) (PCCF), on cytokine productions in cultured Peyer's patches (PP) from C57BL/6J mice were investigated in vitro and ex vivo. In an in vitro experiment, PTCF (100 and 10 microg/ml) enhanced the production of T helper 1 (Th1) cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, in cultured PP cells stimulated with 5 microg/ml concanavalin A (Con A) but did not influence on the production of T helper 2 (Th2) cytokines, IL-4 and IL-5. PTCF also enhanced the production of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-10 in the cultured PP cells. While, PCCF enhanced the production of IFN-gamma but did not alter the level of IL-2 in the PP cells. In an ex vivo experiment using PP cells removed from the mice after oral treatment of PTCF (10 and 100 mg/kg daily for 7 consecutive days), the production of IL-2 and IFN-gamma were increased in response to Con A. On the other hand, orally treated PCCF (10 mg/kg/day) suppressed IL-2 production but did not change the levels of IFN-gamma and IL-10 in the isolated PP cells. The flow cytometric analysis revealed that the population of CD3(+) cells in the PP cells slightly but significantly increased after oral administration of PCCF. Orally administered PTCF did not change the population of T (CD3(+)), B (CD19(+)), T cell subset (CD4(+)and CD8(+)) and Th1 (IFN-gamma(+)) and Th2 (IL-4(+)). From PTCF, the fraction rich in proteoglycans was separated as active fraction that stimulates Th1 immune response. These results indicate that the mode of action of PTCF and PCCF on mucosal immune response is different and this is contributed to their metabolites. Taken together, there is a possibility of PTCF and PCCF being therapeutic or preventive agents for immune diseases such as cancer, allergy and parasitic disease through activation of mucosal immune response.

Galactomannans from the cell walls of species of Paecilomyces sect. Paecilomyces and their teleomorphs as immunotaxonomic markers.

An alkali-extractable and water-soluble fraction (F1S) was obtained from cell walls of Paecilomyces variotii and species of the related genera Talaromyces, Byssochlamys and Thermoascus. The structure of the main polysaccharide of these fractions was studied and found to consist of a core of (1 --> 6)-alpha-mannopyranose partially substituted at 0.2 by chains of galactofuranose and shorter chains of mannopyranose. The differences in the regularity of the branching points and the length of the galactofuranose side chains are useful to distinguish between species. These differences were detected by immunological methods, since highly specific polyclonal antibodies were raised against these polysaccharides. Mycelium of P. variotii CBS 990.73A was stained by indirect immunofluorescence. The polysaccharides studied in this work differ from the one described for species from section Isarioidea, and this is another indication of the heterogeneity of the genus Paecilomyces.

Characterization of monoclonal antibodies against cell wall epitopes of the insect pathogenic fungus, Nomuraea rileyi: differential binding to fungal surfaces and cross-reactivity with host hemocytes and basement membrane components.

Monoclonal antibodies (MAbs) were generated against epitopes on yeast-like hyphal bodies and hyphae of the entomopathogenic hyphomycete, Nomuraea rileyi. Two MAbs (4C10, 2H4) bind to epitopes common to both hyphal bodies and hyphae, whereas MAb 4E9 binds only to hyphal surfaces. 4C10 and 2H4 appear to be directed towards carbohydrate portions of cell surface mannoproteins, as evidenced by similarities in staining patterns between these MAbs and Concanavalin A on Western blots of N. rileyi cell wall extracts. These MAbs cross-react with antigens on blastospore and hyphal surfaces of two other entomopathogenic fungi, Beauveria bassiana and Paecilomyces farinosus in fluorescence microscopy assays, but do not cross-react with a non-entomopathogenic strain of Candida albicans or with Saccharomyces cerevisiae yeasts. MAb 4C10 also cross-reacts with immunocompetent granular hemocytes from Spodoptera exigua (beet armyworm) and Trichoplusia ni (cabbage looper) larvae and with S. exigua plasmatocytes. Electron microscopy revealed that this MAb binds to a component in cytoplasmic granules in the hemocytes, and that surface labeling may be due to the release of this MAb-positive component upon degranulation. MAb 2H4 does not cross-react with granular hemocytes, but does bind to plasmatocytes and hemocytes that tightly adhere to the substrate in monolayer assays. Additionally, MAb 4C10 specifically labels a basement membrane epitope on S. exigua fat body, suggesting that this antibody binds to mannose residues on extracellular matrix glycoproteins. Cross-reactivity of these N. rileyi MAbs with insect hemocyte and tissue components indicates that fungal surface epitopes can mimic host surface molecules, which could explain why N. rileyi hyphal bodies are not recognized by granulocytes and are able to circulate freely in the hemolymph without binding to basement membranes lining the hemocoel.

Immunohistologic identification of Aspergillus spp. and other hyaline fungi by using polyclonal fluorescent antibodies.

Isolation and identification of pathogenic Aspergillus and Fusarium spp. from clinical materials provide the most accurate means for establishing a diagnosis of infections by these molds. Such efforts, however, are not always successful. Histologic diagnosis also has its limitations. In vivo the hyphae of Aspergillus and Fusarium spp. are very similar and their in situ manifestations are not pathognomonic. To improve the histologic diagnosis of infections by Aspergillus and Fusarium species, we developed polyclonal fluorescent-antibody reagents to Aspergillus fumigatus and Fusarium solani and evaluated their diagnostic utilities. Our studies revealed that A. fumigatus and F. solani share epitopes not only with one another but also with other Aspergillus and Fusarium spp. as well as with Paecilomyces lilacinus and Pseudallescheria boydii. Adsorption of the A. fumigatus conjugate with cells of Fusarium proliferatum and F. solani and F. solani antiserum with cells of Aspergillus flavus resulted in reagents that distinguished Aspergillus spp. from Fusarium spp. but that still cross-stained P. lilacinus and P. boydii. Adjunctive use of a specific P. boydii conjugate enabled the identification of Aspergillus spp., Fusarium spp., P. lilacinus, and P. boydii in formalin-fixed tissue sections from 19 humans with culture-proven cases of mycotic infection.

Production of reactive oxygen metabolites by opsonized fungi and bacteria isolated from indoor air, and their interactions with soluble stimuli, fMLP or PMA.

Changes in the levels of free intracellular calcium ([Ca2+]i) and the production of reactive oxygen metabolites (ROM) induced by opsonized indoor air fungi and bacteria in human polymorphonuclear leukocytes (PMNL) were measured. Moreover, modification of a chemotactic peptide (fMLP)-and a tumor promoter (PMA)-induced production of ROM by opsonized fungi and bacteria were studied. The cells were exposed to graded doses of opsonized Candida sp., Aspergillus sp., Cladosporium sp., Stachybotrys sp., Penicillium sp., Paecilomyces sp., or A4 or A91 Streptomyces sp. alone, or together with fMLP or PMA. All the organisms were isolated from air samples of mold-problem buildings. None of the fungi or bacteria induced changes in [Ca2+]i or the production of ROM without opsonization with human serum. Of all opsonized fungi and bacteria, only Candida sp. elevated [Ca2+]i. All fungi and bacteria, except Paecilomyces sp. and Stachybotrys sp., markedly increased the production of ROM in PMNL. Furthermore, A91 Streptomyces sp. and Aspergillus sp. amplified fMLP-induced production of ROM. Only Candida sp. increased PMA-induced phenomen that normally occurs in the lung, was required for biological activity of the fungi and bacteria. Amplification by opsonization of fungi- or bacteria-induced leukocyte activation revealed remarkable changes between these biologically active particles. The present results suggest that many indoor air fungi and bacteria may activate leukocytes to produce oxidative stress, perhaps associated with harmful effects in exposed individuals.

Comparative analysis of phagocytosis of fungal cells by insect hemocytes versus horse neutrophils.

In this study, the phagocytic ability of Spodoptera exigua hemocytes was compared to horse neutrophils. In vitro assays showed that the insect granulocytes and horse neutrophils actively phagocytose FITC-labeled Paecilomyces farinosus blastospores opsonized with S. exigua hemolymph lectin or horse serum, respectively. Killing of fungal cells by the neutrophils and hemocytes was analyzed under in vitro conditions. Neutrophils reduced the growth of P. farinosus up to 65% whereas no fungicidal activity was observed with hemocyte monolayers. The production of oxygen metabolites by both phagocytic cells incubated with various elicitors (fungal cells, bacteria, phorbol myristate acetate) was examined using luminol-enhanced chemiluminescence. Phagocytosis of opsonized microbes by horse neutrophils resulted in marked increase of chemiluminescence activity whereas no chemiluminescence was detected in similarly challenged phagocytic insect hemocytes. Electron microscopy was used to examine phagocytic events and confirmed that insect phagocytes were unable to kill tested microbes.

Serum IgG antibodies to mold spores in two Norwegian sawmill populations: relationship to respiratory and other work-related symptoms.

Wood trimmers and planing operators from two separate sawmill populations (N = 303 and 170) were studied by serology assessment and a self-administered questionnaire. IgG antibodies to Rhizopus microsporus ssp. rhizopodiformis, Paecilomyces variotii, and Aspergillus fumigatus were measured by ELISA. The questionnaire included questions about general respiratory symptoms and symptoms after handling moldy timber. Personal exposure of wood trimmers to mold spores and wood dust was measured in one part of the sawmills. R. microsporus was the most prevalent mold assessed by serology. Antibody levels were higher and symptoms suggestive of mucous membrane irritation, chronic nonspecific lung disease, allergic alveolitis, and organic dust toxic syndrome were more frequently reported by wood trimmers than by planing operators. The mean level of IgG antibodies to R. microsporus in sawmill workers working in the same work area was the best predictor of symptoms in both populations. The consistent results indicate that exposure to spores of R. microsporus may cause several respiratory symptoms in wood trimmers.

Relationships between exposure to spores from Rhizopus microsporus and Paecilomyces variotii and serum IgG antibodies in wood trimmers.

A longitudinal study of wood trimmers exposed to mould spores was carried out from 1985 to 1988. Exposure to airborne spores from Rhizopus microsporus ssp. rhizopodiformis and Paecilomyces variotii was measured by scanning electron microscopy of filter samples. Specific IgG antibodies to these moulds were measured by ELISA in serum samples collected at half-year intervals. Antibody levels to R. microsporus and P. variotii were higher in wood trimmers than in other sawmill workers whose jobs had an assumed lower exposure to mould spores. Antibody levels were significantly elevated after periods with high exposure compared to antibody levels in the same wood trimmers after periods with low exposure. Antibodies were also found in newly employed wood trimmers. These wood trimmers were exposed to 1,300 x 10(3) R. microsporus spores/m3 and 130 x 10(3) P. variotii spores/m3 (arithmetic mean exposure). Antibody levels in the newly employed wood trimmers were similar to antibody levels in wood trimmers who had already worked in the trimming department before the start of the study. Antibody levels to R. microsporus and P. variotii in wood trimmers can be regarded as indicators of fairly recent exposure. There were, however, large differences between the individual antibody levels of similarly exposed wood trimmers.

[Infectious-allergic bronchopulmonary paecilomycosis]. / Infektsionno-allergicheskii bronkholegochnyi petsilomikoz.

Primary or secondary infection of the lungs with fungi of the Paecilomyces family (P. variotii and P. viridis) gives rise to the development of infectious allergic bronchopulmonary paecilomycosis characterized by the presence of chronic allergic interstitial pneumonia and obstructive bronchitis, bronchial asthma, total and pulmonary eosinophilia, the presence of the tissue parasitic form of the fungus in sputum, blood, pulmonary tissue, the presence of allergen-specific IgE and/or IgG antibodies in patients' sera, immediate or double (20 min and 6 h) reaction of the skin to administration of allergen of Paecilomyces, by not infrequent combination of lung damage and impairment of other organs as well as by chronic relapses.