Inhibition of extracellular traps by spores of Trichoderma stromaticum on neutrophils obtained from human peripheral blood.

Although the genus Trichoderma is widely used as a biocontrol agent in crops, little is known about its potential impact on the human immune system. In mice, our group has shown that exposition to T. asperelloides spores lead to reduced neutrophil counts in the peripheral blood and in the peritoneal cavity. In addition, T. stromaticum spores produced an inflammatory infiltrate on mice lungs, reducing the levels of IFN-γ and IL-10 cytokines, reactive oxygen species, and receptors of microbial patterns. Here we demonstrate that the interaction of human peripheral neutrophils with T. stromaticum spores also leads to a reduced release of neutrophil extracellular traps (NETs) after induction with the NET-inducer agent phorbol 12-myristate 13-acetate. This interaction also reduced the expression levels of multiple microRNAs, such as miR-221, miR-222, miR-223 and miR-27a, as well as genes related to NETs, such as ELANE, MPO and PADI4. Furthermore, T. stromaticum spores affected the expression of the genes SOCS3, TLR4, CSNK2A1, GSDMD, and NFFKBIA, related to the activation of inflammatory immune responses in neutrophils. Overall, our results suggest T. stromaticum as a potential NET inhibitor and as an immunomodulatory agent. Since this fungus is used as biocontrol in crops, our findings point to the importance of advancing our knowledge on the effects of this bioagent on the human immune system. Finally, the study of the active compounds produced by the fungus is also important for the prospection of new drugs that could be used to block the exacerbation of inflammatory immune responses present in several human diseases.

Immune-triggering effect of the foodborne parasite Kudoa septempunctata through the C-type lectin Mincle in HT29 cells.

Kudoa septempunctata is a myxozoan parasite that causes food poisoning in individuals consuming olive flounder. The present study aimed to investigate the currently insufficiently elucidated early molecular mechanisms of inflammatory responses in the intestine owing to parasite ingestion. After Kudoa spores were isolated from olive flounder, HT29 cells were exposed to spores identified to be alive using SYTO-9 and propidium iodide staining or to antigens of Kudoa spores (KsAg). IL-1ß, IL-8, TNF-α and NFKB1 expression and NF-κB activation were assessed using real-time PCR, cytokine array and western blotting. The immunofluorescence of FITC-conjugated lectins, results of ligand binding assays using Mincle-Fc and IgG-Fc, CLEC4E expressions in response to KsAg stimulation, and Mincle-dependent NF-κB activation were assessed to clarify the early immunetriggering mechanism. Inflammatory cytokines (IL-1α, GM-CSF and TNF-α), chemokines (IL-8, CCL2, CCL5 and CXCL1) and NF-κB activation (pNF-κB/NF-κB) in HT29 cells increased following stimulation by KsAg. The immunofluorescence results of spores and lectins (concanavalin A and wheat germ agglutinin) suggested the importance of Mincle in molecular recognition between Kudoa spores and intestinal cells. Practically, data for Mincle-Fc and KsAg binding affinity, CLEC4E mRNA expression, Mincle immunofluorescence staining and hMincledependent NF-κB activation demonstrated the involvement of Mincle in the early immune-triggering mechanism. The present study newly elucidated that the molecular recognition and immune-triggering mechanism of K. septempunctata are associated with Mincle on human intestinal epithelial cells. [BMB Reports 2020; 53(9): 478-483].

Upregulation of CD4+CD8+ memory cells in the piglet intestine following oral administration of Bacillus subtilis spores combined with PEDV whole inactivated virus.

Oral immunization is a commonly employed route for inducing local immunity. However, the application of oral immunization is limited by the short-term persistence of immunity, particularly for inactivated viruses. The ultimate goal for mucosal vaccination is to stimulate protective immunological memory. In the intestine, long-term persistence of immunity is related to CD4+CD8+ memory T-cells. In this study, piglets were orally immunized with Bacillus subtilis spores (B.s) plus whole inactivated porcine epidemic diarrhea virus (PEDV WIV), followed by booster oral immunization. Initially, the results showed that B.s plus PEDV WIV enhanced the anti-PEDV capability on mucosal surfaces, as evidenced by plaque reduction neutralization tests in serum and intestinal fluid. Elevated antigen-specific IgG titers in the serum and IgA titers in saliva, feces and nasal washing liquid were also observed. Meanwhile, B.s plus PEDV WIV increased the area of Peyer's patches and the number of intraepithelial lymphocytes in the ileum of piglets. Similarly, the percentage of CD4+CD8+ memory T-cells were upregulated and proliferation ability of antigen-specific memory T-cell was strengthened in intestinal mucosal-associated lymphocytes, which was accompanied with increased expression of CCR9 after oral immunization with B.s plus PEDV WIV. In addition, the activation of memory T-cells is correlated with the increased mRNA expression of Toll-like receptor 2 and 4, as well as interleukin-6 and induced by B.s. Collectively, the study provided further insight into the potential immunopotentiator ability of B.s to assist PEDV WIV in the potentiation of immunity by upregulating memory CD4+CD8+ T cells via oral immunization.

Environmental heat and airborne pollen concentration are associated with increased asthma severity in horses.

REASON FOR PERFORMING STUDY: Clinical exacerbations of severe equine asthma (formerly recurrent airway obstruction [RAO]) are more frequently reported during winter when horses are exposed to airborne dusts during stabling. However, we have also observed a worsening of clinical signs on days during a heatwave. OBJECTIVES: To investigate the association between environmental temperature and humidity and clinical signs of asthma in horses during clinical exacerbation of the disease. STUDY DESIGN: Retrospective longitudinal study. METHODS: Historical data on 14 severe asthmatic horses exposed to a dusty environment and evaluated using a previously validated clinical scoring system were analysed. Barn temperature and relative humidity values were obtained and air enthalpy (h) calculated. Correlation tests were used to study the relationship between mean daily clinical scores of horses and environmental variables. Lung function parameters recorded at 4 day intervals during hot (25°C) and warm (18°C) barn conditions were compared using a paired t test. RESULTS: Significant positive correlations were observed between the mean daily clinical score and temperature (r = 0.58, P = 0.01) and air enthalpy (r = 0.55, P = 0.02). Maximal daily temperature correlated with airborne pollen concentrations (r = 0.51, P = 0.0002). In the absence of changes in the management of horses, higher barn temperature and enthalpy were associated with increased transpulmonary pressure (P = 0.005), pulmonary resistance (P = 0.008) and elastance values (P = 0.005). CONCLUSIONS: Providing a cold environment may help attenuate the severity of airway obstruction in uncontrolled exacerbations of severe equine asthma. Furthermore, variations in environmental heat and associated pollen concentrations should also be taken into account when evaluating the response to therapy in clinical or research settings.

Comparison of pollen levels between 2 pollen traps in Salamanca, Spain

No disponible

A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model.

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes.

Comparative glycan profiling of Ceratopteris richardii 'C-Fern' gametophytes and sporophytes links cell-wall composition to functional specialization.

BACKGROUND AND AIMS: Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii 'C-Fern', a widely used model system for ferns. METHODS: Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of 'C-Fern' sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes. KEY RESULTS: While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. CONCLUSIONS: The differences and similarities in glycan cell-wall composition between 'C-Fern' gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte.

[About forest allergens]. / Jos metsään haluat mennä nyt.

In the summer forest there are allergens and irritating substances that cause respiratory or cutaneous symptoms. Birch and alder pollen allergy is common in Finland. Pollens of other trees cause sensitization only sporadically. Spores of molds and mushrooms cause allergic reactions, but the means to study spore allergy are inadequate. Even edible mushrooms may cause allergic abdominal discomforts, and trehalose intolerance is supposed to be present in a small percentage of the population. Lichen allergens may bring about IgE-mediated reactions, contact allergy and photoallergic reactions.

Associations between pollen counts, pollutants, and asthma-related hospital admissions in a high-density Indian metropolis.

BACKGROUND: The seasonal pattern of asthma-related hospitalization has often been correlated with ambient allergen/pollutant levels. OBJECTIVE: To examine the relationship between asthma-related hospital admissions (ARHA) and outdoor pollen, spore, and pollutant levels for adult patients in a densely populated Indian megacity Kolkata. METHODS: ARHA data were obtained from two major teaching hospitals of the city. Pollen and spores causing allergic sensitization were identified by skin prick tests (SPTs) among respiratory allergic subjects (N = 1353). Outdoor concentrations of aeroallergens were determined using a Burkard sampler for five consecutive years (2004-2009). Levels of NO(2), SO(2), suspended particulate matters (SPMs), and respirable particulate matters (RPMs) were made available by West Bengal Pollution Control Board (WBPCB, Government of West Bengal). Poisson multivariate Poisson regression (with adjustments for overdispersion) was used to model the data. Results. We found that ARHA in Kolkata increased with predictable regularity in March and September, while remaining low in January and July. SPT showed highly positive skin reactions with grass/weed and palm pollens in respiratory allergic patients, while Aspergilli spores also evoked good sensitivity. In our regression model, the airborne pollen types, Cheno-Amaranthaceae and Cyperaceae, and the inorganic pollutant, SO(2) and RPM, were significantly associated with ARHA (p < .05). CONCLUSION: ARHA in the megacity of Kolkata shows two seasonal peaks that can be correlated with outdoor grass/weed pollen and RPM concentrations. In contrast, the city's ambient fungal spore counts were not found to be significantly associated.

Correlation between Alt a 1 levels and clinical symptoms in Alternaria alternata-monosensitized patients.

BACKGROUND: Alternaria alternata is a risk factor for developing asthma.Alt a 1, which has been described as the major allergen in A alternata, shows a good correlation with A alternata spores only when they have germinated. OBJECTIVES: The objective of this study was to determine the correlation between spore counts and clinical symptoms in patients with allergic asthma and/or rhinitis monosensitized to A alternata. METHODS: Two types of samplers were used to determine exposure: a Burkard spore trap to collect A alternata spores and a high-volume air sampler to collect airborne particles. A total of 366 air filters were collected. Alt a 1 levels were measured by monoclonal antibody-based enzyme-linked immunosorbent assay. Eighteen monosensitized patients were asked to record their daily symptoms throughout the year. RESULTS: A alternata spores were detected throughout the year, whereas Alt a 1 was detected only between March and December. Symptoms showed positive and significant correlations with spore counts (r=0.459, P<.001), and Alt a 1 levels (r=0.294, P<.001). The correlation between spores and Alt a 1 was low. The negative binomial model proved that an increase of 10 pg/m3 in Alt a 1 levels increased the number of symptoms at a 3-day lag by 5%. CONCLUSIONS: In patients who are allergic to A alternata, Alt a 1 levels can be considered an important marker for predicting the risk of respiratory symptoms.

The effect of anti-spore antibody responses on the use of spores for vaccine delivery.

Among the different types of bacteria being exploited as live vaccines Bacillus endospores have a number of distinct advantages most importantly being their heat stability. Typically antigens are displayed on the spore surface or expressed in the germinating spore, i.e., the vegetative cell. How antigens are delivered by spores can significantly affect the nature of the resulting immune response. When antigens are expressed in the germinating spore, then, after the first dose of recombinant spores, ensuing humoral responses are abruptly arrested. We have investigated this phenomenon by first demonstrating that while immune responses against the encoded antigen are impaired this is not the case for anti-spore responses that progressively increase until hyperimmunity is reached. We reasoned that anti-spore IgG or sIgA could be responsible for inhibiting spore germination, thus preventing expression of the vegetatively expressed antigen. In this work we have demonstrated that antisera from both immunised and naïve animals can bind to spores non-specifically yet only IgG or sIgA from immunised animals can bind specifically. Both immune and naïve antibodies were found to inhibit germination in vitro and most probably non-specific binding could account for this. On the other hand only immune antibodies were capable of opsonising spores and enhancing their uptake by macrophages and we reason that specific antibody-spore binding is required. Opsonophagocytosis could be an important tool to reduce spore germination and subsequent outgrowth. With regard to use for heterologous antigen delivery though, this severely curtails the use of a repetitive dosing regime.

Immunostimulatory activity of Bacillus spores.

Bacillus species, typically Bacillus subtilis, are being used as probiotics and mounting evidence indicates that Bacillus species are important for development of a robust gut-associated lymphoid system (GALT). We used a number of gut isolates of Bacillus incorporating three species, B. subtilis, Bacillus licheniformis and Bacillus flexus to evaluate the nature of interaction between spores and the GALT. In mice orally administered with spores, evidence of cell proliferation was determined in the germinal centers of Peyer's patches. Stimulation of antigen-presenting cells and T lymphocytes was also markedly enhanced. Cytokines were shown to be induced in spleens and mesenteric lymph nodes of mice including the proinflammatory cytokines, tumour necrosis factor-alpha and IL-6. We also demonstrated that vegetative cells of B. subtilis can stimulate expression of the toll-like receptor (TLR) genes for TLR2 and TLR4. However, we were able to show that spores could not stimulate either and must, by default, interact with another TLR and by this mechanism help activate innate immunity.

Allergenic pollens and spores in the working environment of Japanese pear farmers.

Occupational allergies such as pollinosis are reported in several agricultural works in Japan. Many pollens and spores were observed in Japanese pear orchard during the artificial pollination season. By the study on daily symptoms in an allergic farmer, we confirmed that the pollinosis symptoms were most common and most severe during the artificial pollination. These results suggest that the exposure to allergenic pollens and spores induces allergic symptoms. Thus, caution should be paid for the avoidance of the exposure to these allergenic pollens and spores to prevent the allergy.

Comparison of enzyme immunoassay-based assays for environmental Alternaria alternata.

BACKGROUND: Alternaria alternata-derived allergenic materials are causes of human disease. Several immunoassays exist to quantify these materials. OBJECTIVE: To compare methods for evaluating Alternaria content. METHODS: Four methods, including 1 monoclonal antibody (MAb)-based assay specific for recombinant Alt a 1, 1 MAb-based assay for chromatographically purified Alt a 1, 1 polyclonal antibody (PAb)-based assay for chromatographically purified Alt a 1, and 1 PAb-based assay for whole Alternaria extract, were evaluated. Environmental samples collected as part of the National Survey of Lead and Allergens in Housing were examined. Alternaria spore counts were determined in dust by observation. RESULTS: The MAb-based assay for recombinant Alt a 1 detected Alternaria in few samples (25%); the PAb-based assay for whole Alternaria proteins detected antigen in 97% of the samples. The PAb- and MAb-based assays for purified Alt a 1 detected antigen in 100% of the samples. There was a significant positive correlation between the 2 assays directed against purified Alt a 1. There was a positive correlation between the PAb-based assay for whole Alternaria and the PAb-based assay for Alt a 1. Nearly all the dust samples contained Alternaria spores, and there was a strong positive correlation between counts and all assays. CONCLUSION: Because of the multifaceted nature of Alternaria, the disparities between methods for quantifying Alternaria, the cross-reactivity between fungal allergens, and the documented genetic promiscuity of this fungus, enzyme immunoassays using PAbs against a range of Alternaria proteins will probably produce the most reliable estimation of overall Alternaria exposure in house dust.

Occupational asthma caused by white mushroom.

The commercial growing of white mushroom (Agaricus bisporus) is a frequent activity in certain Spain regions as La Rioja. We report two cases of white mushroom workers suffering from asthma caused by hypersensitivity to basidiocarp and spores of white mushroom.

Hypersensitivity pneumonitis due to Bunashimeji mushrooms in the mushroom industry.

BACKGROUND: Detection of hypersensitivity pneumonitis (HP) in employees involved in the Bunashimeji mushroom industry is difficult. The level of precipitating antibody is not related with the prediction of progression and resolution of HP. The aims of this study were to examine the actual prevalence of HP in the Bunashimeji industry and the clinical differences among selected employees. METHODS: One hundred and fourteen employees worked in Bunashimeji enterprises. These subjects were divided into the following subgroups: office workers, pickers/packers with mask and pickers/packers without mask. We measured serum Krebs von der Lungen-6 (KL-6), surfactant protein (SP)-A and SP-D, and examined the stimulation index (SI) due to Bunashimeji spores. Chest high-resolution computed tomography (HRCT), pulmonary function tests and bronchoalveolar lavage (BAL) were performed for select employees who showed positive SI values (>200%) to examine the clinical differences. RESULTS: The proportion of respiratory symptoms was significantly higher in the pickers/packers than that in the office workers. The picker/packer group had high serum KL-6 concentrations and SI compared with the office worker group. Thirty select employees were divided into the following three subgroups: HP, select employees without HP, and SI <400% and KL-6 <500 U/ml , using high SI levels (>400%) and positive serum KL-6 concentration (>500 U/ml). Four exhibited ground glass opacities with centrilobular fine nodules on HRCT, and 8 had high numbers of lymphocytes in the BAL fluid. The BAL findings and serum KL-6 concentrations showed significant differences among the three groups. CONCLUSIONS: Four employees were evaluated as having HP. Serum KL-6 and SP-D may be related to the resolution of HP in addition to SI and chest HRCT.

Production of monoclonal antibodies against peripheral-vesicle proteins in zoospores of Phytophthora nicotianae.

A coimmunisation protocol using microsomal fractions from Phytophthora nicotianae cells has enhanced the production of monoclonal antibodies directed towards proteins produced during asexual sporulation. Over 40% of the antibodies targeted three categories of zoospore peripheral vesicles. Five antibodies label the contents of dorsal vesicles, with three of these reacting with two P. nicotianae polypeptides with a relative molecular mass of approximately 100 kDa. Two antibodies label the contents of large peripheral vesicles and react with two very high-molecular-weight polypeptides in extracts of P. nicotianae cells. These antibodies cross-react with the contents of large peripheral vesicles in P. cinnamomi zoospores. Ten antibodies label the contents of P. nicotianae zoospore ventral vesicles and react with a single polypeptide with a relative molecular mass of 230 kDa. A number of these antibodies against the contents of ventral vesicles in P. nicotianae zoospores cross-react with ventral-vesicle proteins in P. cinnamomi cells in immunofluorescence and immunoblot assays. The study illustrates the value of the coimmunisation protocol and has produced antibodies that could be instrumental in the cloning of genes encoding peripheral-vesicle proteins.

Correlation of environmental factors with asthma and rhinitis symptoms in Tulsa, OK.

BACKGROUND: Airborne allergens, pollutants, and climatic changes are known to influence the symptoms of asthma patients. OBJECTIVE: To correlate airborne fungal spore and pollen concentrations, meteorological data, and airborne pollutants with asthma and rhinitis symptoms to develop predictive models for asthma severity. METHODS: Patients from the Tulsa community participated in this study from September 1 to October 31, 2000, by filling out daily symptom diaries and measuring morning and evening peak expiratory flow rates. Air samples were collected using a volumetric spore trap. Meteorological variables and maximum and average pollutants were also included in the analysis. Linear regression analyses were performed for all environmental variables and symptom scores. Forward stepwise multiple regression analyses were performed to determine sets of variables that could be used to predict the conditions of increased symptom severity. RESULTS: Twenty-four patients participated in this study. The predominant spore types included Cladosporium, ascospores, and basidiospores. The predominant pollen type was Ambrosia. September was unusually hot and dry in Tulsa, but 161 mm of precipitation fell in October, primarily during the last 11 days. Two periods of peak symptoms occurred during the study, the first during the peak week of Ambrosia and the second after a 22 degrees C drop in temperature over 6 days. Numerous environmental variables showed significant correlations with symptom scores; however, there was no single predictive model for all symptoms. CONCLUSIONS: Ambrosia pollen and other environmental variables, including ozone levels, were significantly correlated with asthma and rhinitis symptoms.

Adaptive innate immunity? Responsive-mode prophylaxis in the mealworm beetle, Tenebrio molitor.

A primary infection by a parasite may indicate a higher risk of being reinfected in the near future (since infection may indicate that enemies are becoming more abundant). Acquired immunity does not exist in invertebrates despite the fact that they also face increased risks of reinfection following primary exposure. However, when subjected to immune insult, insects can produce immune responses that persist for long enough to provide prophylaxis. Because these immune responses are costly, persistence must be maintained through a selective advantage. We tested for the possibility that these long-lasting immune responses provided increased resistance to later infections by experimentally mimicking a primary immune insult (pre-challenge) in larvae of the mealworm beetle, Tenebrio molitor, with lipopolysaccharides (LPS) prior to early or late exposure to spores of the entomopathogenic fungus Metarhizium anisopliae. We found that pre-challenged larvae produced a long-lasting antimicrobial response, which provided a survival benefit when the larvae were exposed to fungal infection. These results suggest that the observed response is functionally "adaptive".

[Fungal allergy--part II]. / Alergia na grzyby--Czesc II.

Allergic reactions caused by fungi, may be present in the airways, skin, eyes, gastrointestinal tract, skin and to cause systemic reaction. Nasal symptoms react as allergic rhinitis. In a small group of patients the allergic fungal rhinosinusitis is present. It is important, because of the way of treatment, to differentiate the AFS from the other forms of chronic rhinosinusitis. The fungi may cause bronchial asthma, allergic bronchopulmonary aspergillosis and allergic alveolitis (hypersensitivity pneumonitis). Diagnostics of the allergic reactions caused by fungi includes routine allergological methods, mycologic culture and direct microscopic examination. The process of reaching the diagnosis of allergic fungal rhinosinusitis and allergic bronchopulmonary aspergillosis may require the CT and histopatologic examination. The treatment of fungal allergic reactions includes allergen avoidance, pharmacotherapy, antifungal treatment (id reactions) and surgical treatment (allergic fungal rhinosinusitis). The immunotherapy is a valuable, and accepted by the WHO therapy of the Cladosporium and Alternaria sensitive patients.