Transcription factor BZR2 activates chitinase Cht20.2 transcription to confer resistance to wheat stripe rust.

The brassinosteroid pathway promotes a variety of physiological processes in plants and the brassinosteroid insensitive1-ethylmethane sulfonate suppressor (BES)/brassinazole-resistant (BZR) functions as one of its key regulators. We previously showed that the BES/BZR-type transcription factor TaBZR2 mediates the drought stress response in wheat (Triticum aestivum) by directly upregulating the transcriptional activity of glutathione S-transferase 1. However, the function of TaBZR2 in plants under biotic stresses is unknown. In this study, we found that transcript levels of TaBZR2 were upregulated in response to inoculation with wheat stripe rust fungus (Puccinia striiformis f. sp. tritici, Pst) and treatment with flg22 or an elicitor-like protein of Pst, Pst322. Wheat lines overexpressing TaBZR2 conferred increased resistance, whereas TaBZR2-RNAi lines exhibited decreased resistance to multiple races of Pst. TaBZR2 targeted the promoter of the chitinase gene TaCht20.2, activating its transcription. Knockdown of TaCht20.2 in wheat resulted in enhanced susceptibility to Pst, indicating the positive role of TaCht20.2 in wheat resistance. Upon Pst infection in vivo, the overexpression of TaBZR2 increased total chitinase activity, whereas RNAi-mediated silencing of TaBZR2 reduced total chitinase activity. Taken together, our results suggest that TaBZR2 confers broad-spectrum resistance to the stripe rust fungus by increasing total chitinase activity in wheat.

Coronary Vasculitis Induced in Mice by Cell Wall Mannoprotein Fractions of Clinically Isolated Candida Species.

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances (CADS) such as the hot water extract of C. albicans and Candida water-soluble fractions (CAWS) induce coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the mannoprotein fractions (MN fractions) of clinically isolated Candida species induce vasculitis in mice. We prepared MN fractions from 26 strains of Candida species by conventional hot water extraction and compared vasculitis in DBA/2 mice. The results obtained revealed that the induction of vasculitis and resulting heart failure were significantly dependent on the species; namely, death rates on day 200 were as follows: Candida krusei (100%), Candida albicans (84%), Candida dubliniensis (47%), Candida parapsilosis (44%), Candida glabrata (32%), Candida guilliermondii (20%), and Candida tropicalis (20%). Even for C. albicans, some strains did not induce vasculitis. The present results suggest that MN-induced vasculitis is strongly dependent on the species and strains of Candida, and also that the MN fractions of some non-albicans Candida induce similar toxicity to those of C. albicans.

Self-reported adverse reactions associated with mycoprotein (Quorn-brand) containing foods.

BACKGROUND: Mycoprotein, which is produced by a mold and is the basis of Quorn-brand meat substitutes, is a novel cause of allergic and gastrointestinal reactions, but little information has been available on its associated symptomatology. OBJECTIVE: To describe the nature and frequency of adverse reactions to mycoprotein. METHODS: Self-reports of adverse reactions to mycoprotein were collected via a Web-based questionnaire (www.quorncomplaints.org) and then analyzed. RESULTS: Analysis of 1,752 adverse reactions found that Quorn products caused allergic and gastrointestinal symptoms, with some people experiencing both. Allergic reactions, including urticaria and anaphylaxis, occurred within 4 hours of consumption in 312 people. Of those reactions, 45.8%, 1 fatal, began within 1 hour of exposure. Of those 312 individuals, 188 (60.3%) reported repeated reactions after repeated consumption of Quorn, and 2 people experienced 8 reactions (13 people did not say whether they experienced more than 1 reaction). Quorn foods caused gastrointestinal symptoms, including emesis and diarrhea, within 8 hours of consumption in 1,692 people. Of the gastrointestinal symptoms, 66.6% occurred 46 to 180 minutes after consumption of the products. Symptoms ranged from mild nausea to emesis severe enough to warrant medical attention. CONCLUSION: Mycoprotein may be causing numerous and sometimes life-threatening allergic and gastrointestinal reactions. The acceptance in the food supply of this nonessential ingredient deserves reconsideration.

Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus.

The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and ΔsrbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Δerg25A accumulates more C4-methyl sterol intermediates than Δerg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Δerg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of ΔsrbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response.

Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study.

AIM: To assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to mitigate the immunogenic effects of gluten in celiac patients. METHODS: Patients with initial diagnosis of celiac disease as confirmed by positive serology with subtotal or total villous atrophy on duodenal biopsies who adhere to a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. In a randomised double-blind placebo-controlled pilot study, patients consumed toast (approximately 7 g/d gluten) with AN-PEP for 2 wk (safety phase). After a 2-wk washout period with adherence of the usual GFD, 14 patients were randomised to gluten intake with either AN-PEP or placebo for 2 wk (efficacy phase). Measurements at baseline included complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. Furthermore, serum and quality of life questionnaires were collected during and after the safety, washout and efficacy phase. Duodenal biopsies were collected after the safety phase and after the efficacy phase. A change in histological evaluation according to the modified Marsh classification was the primary endpoint. RESULTS: In total, 16 adults were enrolled in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The mean score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP did not significantly deteriorate and moreover no differences between the groups were observed. During the efficacy phase, neither the placebo nor the AN-PEP group developed significant antibody titers. The IgA-EM concentrations remained negative in both groups. Two patients were excluded from entering the efficacy phase as their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies, while 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, no significant deterioration was observed regarding immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, IgA-tTG deposit staining increased after 2 wk of gluten compared to baseline in four out of seven patients on placebo. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. CONCLUSION: AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP.

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA.

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0 mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4⁺ and CD8⁺ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8⁺ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs.

Relevance of sensitization to occupational allergy and asthma in the detergent industry.

There exists considerable historic experience of the relationship between exposure and both the induction of sensitization and the elicitation of respiratory symptoms from industrial enzymes of bacterial and fungal origin used in a wide variety of detergent products. The detergent industry in particular has substantial experience of how the control of exposure leads to limitation of sensitization with low risk of symptoms. However, the experience also shows that there are substantial gaps in knowledge, even when the potential occupational allergy problem is firmly under control, and also that the relationship between exposure and sensitization can be hard to establish. The latter aspect includes a poor appreciation of how peak exposures and low levels of exposure over time contribute to sensitization. Furthermore, while a minority of workers develop specific IgE, essentially none appear to have symptoms, a situation which appears to contradict the allergy dogma that, once sensitized, an individual will react to much lower levels of exposure. For enzymes, the expression of symptoms occurs at similar or higher levels than those that cause induction. In spite of some knowledge gaps, medical surveillance programs and constant air monitoring provide the tools for successful management of enzymes in the occupational setting. Ultimately, the knowledge gained from the occupational setting facilitates the completion of safety assessments for consumer exposure to detergent enzymes. Such assessments have been proven to be correct by the decades of safe use both occupationally and in consumer products.

The toxicology and immunology of detergent enzymes.

Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T(H))-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.

Characterization and preparation of Aspergillus niger naringinase for debittering citrus juice.

UNLABELLED: Naringinase from Aspergillus niger was prepared and characterized to evaluate its effectiveness in debittering citrus juice. The enzyme was purified to homogeneity by sulfate fractionation and chromatographies on Q-Sepharose, Sephacryl S-200, and S-100 HR columns, and estimated by gel filtration chromatography (GFC) to have a molecular weight (MW) of 131 kDa, of which its subunit was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be around 65.5 kDa. The enzyme showed active and stable pH ranges both within 4.5 to 5.0. Its optimal temperature was in the range of 45 to 55 °C. Freeze drying provided an estimated enzymatic recovery of 95.9%, greater than spray drying with the recovery at 55.6%. The freeze-drying powder could retain its enzymatic activity stably at 4 °C for 6 mo. Also, the enzyme in 0.220 U/mL citrus juice could sufficiently remove the naringin for the bitterness. Oral acute toxicity study revealed the maximum tolerated dose (MTD) of the naringinase powder was >10 g/kg in mice. The contents of arsenic (As), lead (Pb), mercury (Hg), the aerobic plate count, and coliform number in the enzyme powder all met the criteria for food use. These characteristics suggest that the naringinase from A. niger is efficient and suitable for debittering the citrus juice, and the process consisting of fermentation, salt precipitation, ion exchange, ultrafiltration, and freeze drying is a promising means to prepare the naringinase for food industry, setting up a strong base to enzymatically debitter citrus juice. PRACTICAL APPLICATION: This study focused on characterization, preparation, and validation of naringinase from A. niger, which provided useful information on how to prepare, store, and use the naringinase. In addition, this naringinase met the safety standards for food use and showed strong ability to remove the bitter taste from citrus juice, which provided useful information for interested readers, and the food industry.

Treatment of candidemia in adult patients without neutropenia--an inconvenient truth.

In 2009 the Infectious Diseases Society of America reviewed the guidelines on the treatment of candidemia in non-neutropenic patients. In this document the preferred treatment was either fluconazole or an echinocandin. Amphotericin-B formulations were considered an alternative. However, careful assessment of published data showed similar efficacy between these drugs.

Respiratory tract allergic disease and atopy: experimental evidence for a fungal infectious etiology.

Allergic asthma is an obstructive lung disease linked to environmental exposures that elicit allergic airway inflammation and characteristic antigen-specific immunoglobulin reactions termed atopy. Analyses of asthma pathogenesis using experimental models have shown that T helper cells, especially T helper type 2 (Th2) cells and Th2 cytokines such as interleukin 4 (IL-4) and IL-13, are critical mediators of airway obstruction following allergen challenge, but the environmental initiators of lung Th2 responses are less defined. Our studies demonstrate that fungal-derived proteinases that are commonly found in home environments are requisite immune adjuvants capable of eliciting robust Th2 responses and allergic lung disease in mice. We have further shown that common household fungi readily infect the mouse respiratory tract and induce both asthma-like disease and atopy to otherwise innocuous bystander antigens through the secretion of proteinases. These findings support the possibility that asthma and atopy represent a reaction to respiratory tract fungal infection, suggesting novel means for diagnosis and therapy of diverse allergic disorders.

Vaccine hypersensitivity--update and overview.

Concerns about possible reactions to vaccines or vaccinations are frequently raised. However, the rate of reported vaccine-induced adverse events is low and ranges between 4.8-83.0 per 100,000 doses of the most frequently used vaccines. The number of true allergic reactions to routine vaccines is not known; estimations range from 1 per 500,000 to 1 per 1,000,000 doses for most vaccines. When allergens such as gelatine or egg proteins are components of the formulation, the rate for serious allergic reactions may be higher. Nevertheless, anaphylactic, potentially life-threatening reactions to vaccines are still a rare event (approximately 1 per 1,500,000 doses). The variety of reported vaccine-related adverse events is broad. Most frequently, reactions to vaccines are limited to the injection site and result from a non specific activation of the inflammatory system by, for example, aluminium salts or the active microbial components. If allergy is suspected, an accurate examination followed by algorithms is the key for correct diagnosis, treatment and the decision regarding revaccination in patients with immediate-type reactions to vaccines.

Link between allergic asthma and airway mucosal infection suggested by proteinase-secreting household fungi.

Active fungal proteinases are powerful allergens that induce experimental allergic lung disease strongly resembling atopic asthma, but the precise relationship between proteinases and asthma remains unknown. Here, we analyzed dust collected from the homes of asthmatic children for the presence and sources of active proteinases to further explore the relationship between active proteinases, atopy, and asthma. Active proteinases were present in all houses and many were derived from fungi, especially Aspergillus niger. Proteinase-active dust extracts were alone insufficient to initiate asthma-like disease in mice, but conidia of A. niger readily established a contained airway mucosal infection, allergic lung disease, and atopy to an innocuous bystander antigen. Proteinase produced by A. niger enhanced fungal clearance from lung and was required for robust allergic disease. Interleukin 13 (IL-13) and IL-5 were required for optimal clearance of lung fungal infection and eosinophils showed potent anti-fungal activity in vitro. Thus, asthma and atopy may both represent a protective response against contained airway infection due to ubiquitous proteinase-producing fungi.

Treatment options for invasive fungal infections.

Clinicians face an increasing occurrence of invasive fungal infections. These are due not only to traditional yeast and mould species but also to rare pathogens that can be difficult to treat. The introduction of new agents has expanded the options for treating common and rare mycotic infections with antifungal efficacy at least equal, and safety far superior, to that of a once-limited choice of therapies. Patients with invasive mycoses frequently have concomitant disorders and require multidrug regimens. Clinicians must be aware of the potential for interactions among agents available for treating invasive mycoses in patients with serious underlying conditions.

Effects of consuming mycoprotein, tofu or chicken upon subsequent eating behaviour, hunger and safety.

This study tested if: (1) a preload of mycoprotein and tofu consumed before a lunch meal have a greater effect on satiety when compared to a chicken preload, (2) the mycoprotein and tofu preloads, compared to chicken, are not associated with compensation or eating more food at a subsequent dinner meal. These hypotheses were tested in a controlled laboratory study using universal eating monitors to measure food intake and visual analogue scales to monitor hunger and satiety. Forty-two overweight adult females consumed three meals in the laboratory on 3 test days. At lunch, isocaloric pasta preloads, containing mycoprotein, tofu, or chicken, varied across the days in a balanced order. The findings of the study supported the two hypotheses. Mycoprotein and tofu preloads, in comparison to the chicken preload, were associated with lower food intake shortly after consuming the preload at lunch. Food intake following consumption of mycoprotein and tofu did not differ, and participants did not compensate for lower food intake at lunch by consuming more food at dinner. The findings suggest that mycoprotein and tofu have satiating properties that persist for several hours after a meal. These findings have significant implications for the development of foods that are low in kilojoules, but are also filling.

Occupational protein contact dermatitis from shiitake mushroom and demonstration of shiitake-specific immunoglobulin E.

Shiitake are popular edible mushrooms all over the world, and eating raw shiitake may lead to relatively common 'shiitake dermatitis' or toxicodermia. Workers involved in shiitake cultivation and marketing have distinct occupational respiratory and skin diseases unrelated to 'shiitake dermatitis'. There are no previous reports of protein contact dermatitis (PCD) from shiitake, and there is only 1 report of shiitake-specific immunoglobulin (Ig) E. We report 2 shiitake growers who developed work-related eczematous eruption on their hands. Both of the patients had small prick test reactions to fresh shiitake, and specific IgE to shiitake was detected in their sera by immunospot. One of the patients had a large prick test reaction to dry shiitake and also a positive wheal reaction to fresh shiitake in an open application test. Neither of the patients had noticed any symptoms of contact urticaria at work. Both of the patients had immediate IgE-mediated allergy to shiitake, and the diagnosis of occupational PCD was made. There are no commercial in vitro tests for shiitake-specific IgE. Tests for immediate allergy are important when shiitake contact dermatitis is investigated.