Evaluation of blood cell viability rate, gene expression, and O-GlcNAcylation profiles as indicative signatures for fungal stimulation of salmonid cell models.

Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in primary head kidney and secondary embryonic cells from salmonid fish after stimulation with the inactivated fungi Mucor hiemalis and Fusarium aveneacium and with purified fungal molecular patterns. The transcript levels of most of the 45 selected genes were altered in head-kidney cells after 24 h of stimulation with fungal antigens. Stimulation with the inactivated fungus M. hiemalis induced the most pronounced transcriptional changes, including the pathogen receptor-encoding genes CLEC18A and TLR22, the cytokine-encoding genes IL6 and TNF, and the gene encoding the antimicrobial peptide LEAP2. In parallel, we analyzed the total GlcNAcylation status of embryonic salmonid cells with or without stimulation with inactivated fungi. O-GlcNAcylation modulates gene expression, intracellular protein, and signal activity, but we detected no significant differences after a 3-h stimulation. A pathway analysis tool identified the "apoptosis of leukocytes" based on the expression profile 24 h after fungal stimulation. Fluorescence microscopy combined with flow cytometry revealed apoptosis in 50 % of head-kidney leukocytes after 3 h stimulation with M. hiemalis, but this level decreased by > 5% after 24 h of stimulation. The number of apoptotic cells significantly increased in all blood cells after a 3-h stimulation with fungal molecular patterns compared to unstimulated controls. This in vitro approach identified transcript-based parameters that were strongly modulated by fungal infections of salmonid fish.

In Vivo and In Vitro Impairments in T Helper Cell and Neutrophil Responses against Mucor irregularis in Card9 Knockout Mice.

Mucor irregularis is a frequently found fungus in Asia, especially China, and it causes primary cutaneous mucormycosis with a high rate of disfigurement. Caspase recruitment domain-containing protein 9 (Card9) is an essential adaptor molecule downstream of C-type lectin receptors. It mediates the activation of nuclear factor kappa B (NF-κB), regulates T helper 1 (Th1) and Th17 differentiation, and plays an important role in fungal immune surveillance. CARD9 deficiency correlates with the increased susceptibility to many fungal infections, including cutaneous mucormycosis caused by M. irregularis However, the underlying immunological mechanisms were not elucidated. Our study established a murine model of subcutaneous M. irregularis infection, and we isolated immune cells, including bone marrow-derived macrophages, bone marrow-derived dendritic cells, naive T cells, and neutrophils, from wild-type (WT) and Card9 knockout (Card9-/- ) mice to examine the antifungal effect of Card9 on M. irregularis in vivo and in vitroCard9-/- mice exhibited increased susceptibility to M. irregularis infection. Impaired local cytokine and chemokine production, NF-κB (p65) activation, and Th1/17 cell differentiation and partially impaired neutrophil-dependent antifungal immunity were observed in Card9-/- mice. This work enriches our knowledge of the relationship between CARD9 deficiency and mucormycosis.

Human mucosal-associated invariant T cells respond to Mucorales species in a MR1-dependent manner.

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.

Invasive Mucormycosis in Children With Malignancies: Report From the Infection Working Group of the Hellenic Society of Pediatric Hematology-Oncology.

Mucormycosis is an invasive, life-threatening fungal infection that mainly affects immunocompromised hosts. We collected data of pediatric mucormycosis cases from all 7 Greek Hematology-Oncology Departments for the years 2008-2017. Six cases of invasive mucormycosis diagnosed during treatment for malignancies were included in the study. In 4 children (66%) mucormycosis occurred within the first 20 days after diagnosis of the underlying disease. Two cases were classified as proven mucormycosis and 4 as probable. The most frequently recorded species was Rhizopus arrhizus (2 patients), followed by Mucor spp (1), and Lichtheimia spp (1). All patients received liposomal amphotericin B. Combined antifungal treatment was used in 5 cases. Surgical excision was performed in 4 cases (66%). Two patients died at 6 and 12 months after the diagnosis, respectively, 1 (17%) because of mucormycosis. Our data suggest that mucormycosis may occur early after the initiation of intensive chemotherapy in children with malignancies.

Identification of Mucor circinelloides antigens recognized by sera from immunocompromised infected mice / Identificación de antígenos de Mucor circinelloides reconocidos por sueros de ratones inmunodeprimidos infectados

BACKGROUND: Mucor circinelloides is an opportunistic fungus capable of causing mucormycosis, a highly aggressive infection of quick spreading. Besides, it also has a high mortality rate due to late diagnosis and difficult treatment. AIMS: In this study we have identified the most immunoreactive proteins of the secretome and the total protein extract of M. circinelloides using sera from immunocompromised infected mice. METHODS: The proteins of the secretome and the total extract were analyzed by two-dimensional electrophoresis and the most immunoreactive antigens were detected by Western Blot, facing the sera of immunocompromised infected mice to the proteins obtained in both extracts of M. circinelloides. RESULTS: Seven antigens were detected in the secretome extract, and two in the total extract, all of them corresponding only to three proteins. The enzyme enolase was detected in both extracts, while triosephosphate isomerase was detected in the secretome, and heat shock protein HSS1 in the total extract. CONCLUSIONS: In this work the most immunoreactive antigens of the secretome and the total extract of M. circinelloides were identified. The identified proteins are well known fungal antigens and, therefore, these findings can be useful for future research into alternatives for the diagnosis and treatment of mucormycosis

ANTECEDENTES: Mucor circinelloides es un hongo oportunista causante de la mucormicosis, una infección altamente agresiva y de rápida expansión. Además, también presenta una alta mortalidad debido al diagnóstico tardío y el difícil tratamiento. OBJETIVOS: En este estudio se han identificado las proteínas más inmunorreactivas del secretoma y del extracto total de proteínas de M. circinelloides mediante el uso de sueros obtenidos de ratones inmunodeprimidos infectados. MÉTODOS: Las proteínas del secretoma y del extracto total se analizaron mediante electroforesis bidimensional y se detectaron los antígenos más inmunorreactivos mediante Western Blot, enfrentando el suero de los ratones inmunodeprimidos infectados a las proteínas obtenidas en ambos extractos de M. circinelloides. RESULTADOS: Se identificaron 7 antígenos en el secretoma y 2 en el extracto total, todos ellos correspondientes a 3 proteínas. La enolasa se detectó en ambos extractos, mientras que la triosafosfato isomerasa se detectó en el secretoma, y la proteína de choque térmico HSS1 en el extracto total. CONCLUSIONES: En este trabajo se identificaron los antígenos más inmunorreactivos del secretoma y del extracto total de M. circinelloides. Todas las proteínas identificadas son antígenos fúngicos muy conocidos y, por ello, estos resultados pueden ser de gran utilidad en futuras investigaciones relacionadas con la mejora del diagnóstico y el tratamiento de la mucormicosis

The pattern recognition receptors dectin-2, mincle, and FcRγ impact the dynamics of phagocytosis of Candida, Saccharomyces, Malassezia, and Mucor species.

Phagocytosis is a receptor-mediated process critical to innate immune clearance of pathogens. It proceeds in a regulated sequence of stages: (a) migration of phagocytes towards pathogens, (b) recognition of PAMPs and binding through PRRs, (c) engulfment and internalisation into phagosomes, (d) phagosome maturation, and (e) killing of pathogen or host cells. However, little is known about the role that individual receptors play in these discrete stages in the recognition of fungal cells. In a previous study, we found that dectin-2 deficiency impacted some but not all stages of macrophage-mediated phagocytosis of Candida glabrata. Because the C-type lectin receptor dectin-2 critically requires coupling to the FcRγ chain for signalling, we hypothesised that this coupling may be important for regulating phagocytosis of fungal cargo. We therefore examined how deficiency in FcRγ itself or two receptors to which it couples (dectin-2 and mincle) impacts phagocytosis of six fungal organisms representing three different fungal taxa. Our data show that deficiency in these proteins impairs murine bone marrow-derived macrophage migration, engulfment, and phagosome maturation, but not macrophage survival. Therefore, FcRγ engagement with selective C-type lectin receptors (CLRs) critically affects the spatio-temporal dynamics of fungal phagocytosis.

Natural killer cell-mediated damage of clinical isolates of mucormycetes.

Haematopoietic stem cell transplant (HSCT) recipients are at high risk for mucormycosis, which has a mortality of up to 90%. The adoptive transfer of Natural killer (NK) cells is a promising therapeutic option in order to improve the reconstitution of host immunity after HSCT and to directly combat the fungal pathogen. As a number of fungal pathogens have developed strategies to evade the innate immune system, we investigated the interaction of human NK cells with various clinical isolates of different species of mucormycetes. Our results show that human IL-2 prestimulated NK cells damaged all mucormycetes tested. The extent of the damage depended, at least in part, on the growth curve characteristics of the individual fungal isolate. All isolates decreased the secretion of interferon-γ by NK cells to a similar extent. Our data suggest that NK cells damage a wide spectrum of mucormycetes, but that the antifungal effect is higher if NK cells are administered at an early time point of infection.

Changes in PH in exhaled breath condensate after specific bronchial challenge test in patients with chronic hypersensitivity pneumonitis: a prospective study.

BACKGROUND: The aim of this study was to investigate the influence of the specific inhalation challenge (SIC) on changes of pH values in exhaled breath condensate (EBC) in patients with hypersensitivity pneumonitis (HP). METHODS: A prospective study of 85 patients with suspected HP, of whom 63 were diagnosed with HP due to exposure to avian or fungal antigens. In all cases, EBC samples were collected before and after completion of the SIC and pH values were determined. RESULTS: Taken as a whole, patients with HP did not present changes in EBC pH after SIC. However, considering only patients with exposure to molds, those diagnosed with HP had a significantly more acid pH post-SIC than those with another diagnosis (p = 0.011). This fact is not observed in patients exposed to bird's antigens. A ROC curve showed that a reduction in EBC pH of 0.3 units or more after SIC in patients diagnosed with HP due to exposure to molds had a sensitivity of 30 % (CI: 12.8 to 54.3 %) and a specificity of 100 % (CI: 65.5 to 100 %). CONCLUSION: EBC pH may be useful in interpreting SIC results in patients with HP, especially in those patients exposed to molds. Further studies are now required to test the validity of these proposals.

A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides.

Mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. Individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. These individuals often present with defects in phagocytic effector cell function. Research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence against mucormycosis. However, these model systems have not been satisfactory for direct analysis of the interaction between innate immune effector cells and infectious sporangiospores in vivo. Here, we report the first real-time in vivo analysis of the early innate immune response to mucormycete infection using a whole-animal zebrafish larval model system. We identified differential host susceptibility, dependent on the site of infection (hindbrain ventricle and swim bladder), as well as differential functions of the two major phagocyte effector cell types in response to viable and non-viable spores. Larval susceptibility to mucormycete spore infection was increased upon immunosuppressant treatment. We showed for the first time that macrophages and neutrophils were readily recruited in vivo to the site of infection in an intact host and that spore phagocytosis can be observed in real-time in vivo. While exploring innate immune effector recruitment dynamics, we discovered the formation of phagocyte clusters in response to fungal spores that potentially play a role in fungal spore dissemination. Spores failed to activate pro-inflammatory gene expression by 6 h post-infection in both infection models. After 24 h, induction of a pro-inflammatory response was observed only in hindbrain ventricle infections. Only a weak pro-inflammatory response was initiated after spore injection into the swim bladder during the same time frame. In the future, the zebrafish larva as a live whole-animal model system will contribute greatly to the study of molecular mechanisms involved in the interaction of the host innate immune system with fungal spores during mucormycosis.

Sialoglycoproteins in morphological distinct stages of Mucor polymorphosporus and their influence on phagocytosis by human blood phagocytes.

The possible role of sialic acids in host cells-fungi interaction and their association with glycoproteins were evaluated using a clinical isolate of the dimorphic fungus Mucor polymorphosporus. Lectin-binding assays with spores and yeast cells denoted the presence of surface sialoglycoconjugates containing 2,3- and 2,6-linked sialylglycosyl groups. Western blotting with peroxidase-labeled Limulus polyphemus agglutinin revealed the occurrence of different sialoglycoprotein types in both cell lysates and cell wall protein extracts of mycelia, spores, and yeasts of M. polymorphosporus. Sialic acids contributed to the surface negative charge of spores and yeast forms as evaluated by adherence to a cationic substrate. Sialidase-treated spores were less resistant to phagocytosis by human neutrophils and monocytes from healthy individuals than control (untreated) fungal suspensions. The results suggest that sialic acids are terminal units of various glycoproteins of M. polymorphosporus, contributing to negative charge of yeasts and spore cells and protecting infectious propagules from destruction by host cells.

Pulmonary mucormycosis.

Mucormycosis (formerly zygomycosis) is a life-threatening opportunistic mycosis that infects a broad range of hosts with qualitative or quantitative defects in innate immunity, including patients with severe neutropenia, recipients of corticosteroids or other immunosuppressive medications, poorly controlled diabetes mellitus, and those with iron overload states. Mucormycosis has recently emerged as breakthrough sinopulmonary infection in hematologic patients and recipients of transplantation being on antifungal prophylaxis with Aspergillus-active antifungals that lack activity against Mucorales. Unlike pulmonary aspergillosis, the prognosis and outcome of pulmonary mucormycosis have not improved significantly over the last decade, mainly because of difficulties in early diagnosis and the limited activity of current antifungal agents against Mucorales. Recent evidence suggests a critical role for iron metabolism and fungal-endothelial cell interactions in pathogenesis of mucormycosis, and holds promise for development of novel therapeutic strategies. Currently, prompt initiation of antifungal therapy with a lipid amphotericin B-based regimen, reversal of underlying host factors, and aggressive surgical approach offers the best chances for survival of patients infected with this devastating mycosis.

Spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides and their ability to activate human complement system in vitro.

Complement activation by spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides was studied using absorbed human serum in the presence or absence of chelators (EGTA or EDTA). We found that the spore caused full complement activation when incubated with EGTA-Mg2+ or without chelators, indicating that the alternative pathway is mainly responsible for this response. In order to compare activation profiles from each species, ELISAs for C3 and C4 fragments, mannan binding lectin (MBL), C-reactive protein (CRP) and IgG studies were carried out. All proteins were present on the species tested. Immunofluorescence tests demonstrated the presence of C3 fragments on the surface of all samples, which were confluent throughout fungal surfaces. The same profile of C3, C4, MBL, CRP and IgG deposition, observed in all species, suggests a similar activation behavior for these species.

[Hypersensitivity pneumonitis caused by Mucor species in a cork worker]. / Neumonitis por hipersensibilidad a Mucor sp. en un trabajador de la industria del corcho.

Suberosis is a type of hypersensitivity pneumonitis caused by exposure to cork proteins and molds such as Aspergillus fumigatus and Penicillium frequentans. We present the case of a man with suberosis caused by Mucor species, a mold that has been rarely reported to cause hypersensitivity pneumonitis. Furthermore, the patient had symptoms and lung function tests indicating bronchial obstruction-an atypical presentation of hypersensitivity pneumonitis. No imaging abnormalities were observed. A diagnosis was made on the basis of bronchoalveolar lavage and transbronchial biopsy findings. Mucor species was identified as the causative agent using a specific bronchial challenge test.

[Significance of MUC5B antibody in differential diagnosis between Aspergillus species and Mucorales of fungal sinusitis].

OBJECTIVE: To differentiate between Aspergillus species and Mucorales of fungal sinusitis by immunohistochemistry. METHODS: Formalin-fixed paraffin-embedded tissue blocks of 66 cases of fungal sinusitis were retrieved from the archival files of Department of Pathology of Beijing Tongren Hospital during the period from 2001 to 2006. The samples included 29 cases of fungal balls, 12 cases of allergic fungal sinusitis, 24 cases of chronic invasive fungal sinusitis and 1 case of acute invasive fungal sinusitis. The types of fungi were 44 Aspergillus species (31 cases of A. fumigatus, 7 cases of A. flavus and 6 cases of A. terreus) and 22 Mucorales (14 cases of Mucor species and 8 cases of Rhizopus species). Immunohistochemistry was performed with MUC2, MUC5AC and MUC5B antibodies. The results were compared with histochemical study for periodic acid-Schiff (PAS) and Grocott methenamine silver (GMS) stains. RESULTS: Immunohistochemical study for MUC5B showed that the positive rate of Aspergillus species was 90.9%, in contrast to 4.5% in Mucorales (P < 0.001). The expression of MUC2 and MUC5AC was completely negative, whereas PAS and GMS stains were positive in all cases. CONCLUSION: MUC5B antibody appears to be a useful immunohistochemical marker for identifying fungal types in tissue sections, especially in distinguishing between Aspergillus species and Mucorales in fungal sinusitis.

Effects of inhaled fine dust on lung tissue changes and antibody response induced by spores of opportunistic fungi in goats.

OBJECTIVE: To investigate the effects of sterile fine dust aerosol inhalation on antibody responses and lung tissue changes induced by Mucor ramosissimus or Trichoderma viride spores following intratracheal inoculation in goats. ANIMALS: 36 weanling Boer-Spanish goats. PROCEDURES: 6 goats were allocated to each of 2 M ramosissimus-inoculated groups, 2 T viride-inoculated groups, and 2 control (tent or pen) groups. One of each pair of sporetreated groups and the tent control group were exposed 7 times to sterilized fine feedyard dust (mean+/-SD particle diameter, <7.72+/-0.69 microm) for 4 hours in a specially constructed tent. Goats in the 4 fungal treatment groups were inoculated intratracheally 5 times with a fungal spore preparation (30 mL), whereas tent control goats were intratracheally inoculated with physiologic saline (0.9% NaCl) solution (30 mL). Pen control goats were not inoculated or exposed to dust. Goats received an IV challenge with equine RBCs to assess antibody responses to foreign antigens. Postmortem examinations were performed at study completion (day 68) to evaluate lung tissue lesions. RESULTS: 5 of 7 deaths occurred between days 18 and 45 and were attributed to fine dust exposures prior to fungal treatments. Fine dust inhalation induced similar lung lesions and precipitating antibodies among spore-treated goats. Following spore inoculations, dust-exposed goats had significantly more spores per gram of consolidated lung tissue than did their nonexposed counterparts. CONCLUSIONS AND CLINICAL RELEVANCE: Fine dust inhalation appeared to decrease the ability of goats to successfully clear fungal spores from the lungs following intratracheal inoculation.

Detection of fungi spectrum in industrial and home bakeries and determinated fungal allergy with skin prick test.

Airborne fungal pathogens such as Penicillium, Aspergillus, Cladosporium, Trichophyton, and Alternaria may cause health problems. In this research, the fungal flora at different bakeries and their potential allergenic effects on the workers were investigated. We investigated 148 workers at 17 industrial type bakeries and 62 workers at 17 home type bakeries in Afyon. Our study was performed in two different seasons and climates, between January 2004 and June 2004. Fungal flora was detected by using Petri-dish method. In the winter, Penicillium was the dominant genus, while Cladosporium was the dominant genus during the summer, in both types of bakeries. The allergenic properties of dominant culturable fungi on workers involved in the bakeries were determined with the skin-prick test. It was found that with workers in the industrial type bakeries, the most common skin test positivity was caused by Penicillium. In the other hand, the skin test positivity, performed on workers in the home type bakeries, was equally caused by Penicillium, Trichophyton and Aspergillus.

The effect of dietary administration of the fungus Mucor circinelloides on non-specific immune responses of gilthead seabream.

The immunostimulant potential of the whole fungus Mucor circinelloides administered in the diet to gilthead seabream (Sparus aurata L.) was studied. Three different lyophilised strains were used: the wild-type (R7B) and two mutant strains in the carotene-production pathway which are enriched in lycopene (MU224) or beta-carotene (T31). Fish were fed diets containing 0 (control) or 10 g of one of the whole M. circinelloides strains per kg feed. After 2, 4 or 6 weeks of treatment the growth rate as well as humoral (lysozyme activity) and cellular (phagocytosis and cytotoxicity) immune responses were determined. The specific growth rate increased slightly with all the M. circinelloides-supplemented diets. Serum lysozyme activity increased slightly (P>0.05) in fish fed the mutant strain-supplemented diets. Of the cellular responses, phagocytosis significantly increased after 6 weeks, in fish fed the wild-type strain-supplemented diets while cytotoxicity increased after 4 weeks in fish fed the beta-carotene-enriched strain-supplemented diet. The immune responses were increased to some degree by diets containing whole M. circinelloides strains. These results are discussed in the light of the on-going search for new microorganisms, wild or mutant-type, for use as immunostimulants in fish farming.

Haematological, serum biochemical and serological features of platypuses with and without mycotic granulomatous dermatitis.

OBJECTIVE: To determine whether there are haematological, serum biochemical and serological differences between platypuses (Ornithorhynchus anatinus) with and without granulomatous dermatitis due to Mucor amphibiorum infection. An additional objective was to establish reference haematological and serum biochemical ranges for the species in Tasmania. DESIGN: A clinicopathological and serological study. ANIMALS: A total of 37 free-living adult platypuses captured from streams and dams in Northern Tasmania were used in the clinicopathological study. Twenty-seven were clinically normal and 10 had mycotic granulomatous dermatitis. A total of 22 platypuses (20 adult and 2 juvenile) were used for the serosurvey. Eighteen were captured from streams in Northern Tasmania, and four were submitted for necropsy. RESULTS: Platypuses with mycotic ulcerative dermatitis had significantly smaller packed red cell volumes, haemoglobin concentrations, lymphocyte counts, serum cholesterol and calcium concentrations, and higher serum globulin and potassium concentrations than clinically normal animals. The lymphopenia and hyperkalaemia were thought to be clinically significant. Numbers of Trypanosoma binneyi in blood smears were similar between the two groups. Diseased platypuses had higher concentrations of serum antibody against Mucor amphibiorum as determined by ELISA compared to clinically normal platypuses. CONCLUSION: Platypuses affected by mycotic granulomatous dermatitis showed haematological and serum biochemical changes when compared to clinically normal animals from the same Tasmanian sites. A serological survey may be a useful method for detecting the prevalence of exposure to Mucor amphibiorum and humoral immunity in platypus populations both in Tasmania and the mainland of Australia.