Reactive Oxygen Species Localization Programs Inflammation to Clear Microbes of Different Size.

How the number of immune cells recruited to sites of infection is determined and adjusted to differences in the cellular stoichiometry between host and pathogen is unknown. Here, we have uncovered a role for reactive oxygen species (ROS) as sensors of microbe size. By sensing the differential localization of ROS generated in response to microbes of different size, neutrophils tuned their interleukin (IL)-1ß expression via the selective oxidation of NF-κB, in order to implement distinct inflammatory programs. Small microbes triggered ROS intracellularly, suppressing IL-1ß expression to limit neutrophil recruitment as each phagocyte eliminated numerous pathogens. In contrast, large microbes triggered ROS extracellularly, amplifying IL-1ß expression to recruit numerous neutrophils forming cooperative clusters. Defects in ROS-mediated microbe size sensing resulted in large neutrophil infiltrates and clusters in response to small microbes that contribute to inflammatory disease. These findings highlight the impact of ROS localization on signal transduction.

Role of microbial and chemical composition in toxicological properties of indoor and outdoor air particulate matter.

BACKGROUND: Ambient air particulate matter (PM) is increasingly considered to be a causal factor evoking severe adverse health effects. People spend the majority of their time indoors, which should be taken into account especially in future risk assessments, when the role of outdoor air particles transported into indoor air is considered. Therefore, there is an urgent need for characterization of possible sources seasonally for harmful health outcomes both indoors and outdoors. METHODS: In this study, we collected size-segregated (PM(10-2.5), PM(2.5-0.2)) particulate samples with a high volume cascade impactor (HVCI) simultaneously both indoors and outdoors of a new single family detached house at four different seasons. The chemical composition of the samples was analyzed as was the presence of microbes. Mouse macrophages were exposed to PM samples for 24 hours. Thereafter, the levels of the proinflammatory cytokines, NO-production, cytotoxicity and changes in the cell cycle were investigated. The putative sources of the most toxic groups of constituents were resolved by using the principal component analysis (PCA) and pairwise dependencies of the variables were detected with Spearman correlation. RESULTS: Source-related toxicological responses clearly varied according to season. The role of outdoor sources in indoor air quality was significant only in the warm seasons and the significance of outdoor microbes was also larger in the indoor air. During wintertime, the role of indoor sources of the particles was more significant, as was also the case for microbes. With respect to the outdoor sources, soil-derived particles during a road dust episode and local wood combustion in wintertime were the most important factors inducing toxicological responses. CONCLUSIONS: Even though there were clear seasonal differences in the abilities of indoor and outdoor air to induce inflammatory and cytotoxic responses, there were relatively small differences in the chemical composition of the particles responsible of those effects. Outdoor sources have only a limited effect on indoor air quality in a newly built house with a modern ventilation system at least in a low air pollution environment. The most important sources for adverse health related toxicological effects were related to soil-derived constituents, local combustion emissions and microbes.

Pneumonia and lung infections due to emerging and unusual fungal pathogens.

Invasive mold infections affecting the lungs are increasing in incidence and diversity. Severely immunocompromised patients are particularly vulnerable to infection from unusual, normally nonpathogenic fungi that are found naturally in the environment. Certain fungi such as Scedosporium and the dematiaceous fungi also cause lung disease in hosts without overt immune compromise. The impacts of these emerging pathogens range from airway colonization to locally invasive lung, and disseminated, disease. Diagnosis requires isolation and identification of the etiologic agent by either or both phenotypic and molecular biology methods. Evidence of tissue invasion on histopathology is often required to distinguish infection from colonization. Diagnostic imaging techniques are nonspecific, and there are no reliable serological biomarkers of infection. Many rare molds and yeasts demonstrate reduced in vitro susceptibility to antifungal agents. Although amphotericin B formulations remain clinically useful for many of these infections, voriconazole and posaconazole are more effective for some of these difficult-to-treat pathogens. Surgical resection of diseased tissue and support of the host immune system are often required to optimize outcomes.

Murine models of airway fungal exposure and allergic sensitization.

Inhalation of common indoor filamentous fungi has been associated with the induction or exacerbation of allergic respiratory disease. The understanding of fungal inhalation and allergic sensitization has significantly advanced with the use of small animal models, especially mouse models. Numerous studies have employed different animal exposure and sensitization techniques, each with inherent advantages and disadvantages that are addressed in this review. In addition, most studies involve exposure of animals to fungal spores or spore extracts while neglecting the influence of hyphal or subcellular fragment exposures. Recent literature examining the potential for hyphae and fungal fragments to induce or exacerbate allergy is discussed. Innate immune recognition of fungal elements and their contribution to lung allergic inflammation in animal models are also reviewed. Though physical properties of fungi play an important role following exposure, host immune development is also critical in airway inflammation and allergy. We discuss the importance of environmental factors that influence early immune development and subsequent susceptibility to allergy. Murine studies that examine the role of intestinal microflora and prenatal or early life environmental factors that promote allergic sensitization are also evaluated. Future studies will require animal models that accurately reflect natural fungal exposures and identify environmental factors that influence immune development and thus promote respiratory fungal allergy and disease.

A dose-dependent relationship between the severity of visible mold growth and IgE levels of pre-school-aged resident children in Taiwan.

UNLABELLED: To demonstrate a dose-dependent relationship between severity of indoor visible mold growth and serum total IgE levels of resident children. A total of 97 children (4-7 years old) identified from previously established birth-cohort, with information pertaining to indoor environmental conditions after child's birth, were successfully recruited while sera were concurrently collected for total IgE and specific IgE analysis during clinical visits. Severity of visible mold growth at homes was scaled into three levels accordingly. A statistically significant dose-dependent relationship was found between severity of indoor visible mold growth and total serum IgE levels. The trend sustains after the model was adjusted for resident child's age, gender, pet-keeping history, number of siblings, atopic history of parents, presence of incense burning, and environmental tobacco smoking (ETS) at home. Further analysis of specific IgE to commonly examined fungal allergens did not substantiate the correlation. Rather, resident child's specific IgE to mite allergens, although without statistical significance, seemed to better associate with the ranked severity of indoor mold growth in this study. An adjuvant role of fungal exposure to enhance sensitization in indoor environment is therefore suggested in Taiwanese population with high prevalence of building dampness. PRACTICAL IMPLICATIONS: The presence of indoor visible mold growth, potentially resulting in fungal exposure, was not associated directly with changing biomarker levels of allergic response in resident children, rather playing an adjuvant role to enhance sensitization. On the other hand, other allergens, such as mite allergen examined in this study, appeared to support a more plausible etiology for directly triggering the ultimate allergic symptoms and diseases of interest. Evidence as such may derive different priority-setting when designing preventive measures for managing indoor air quality.

[Diagnostics of mycotic sensitization in children with allergic respiratory diseases by the ImmunoCAP technique].

The need to verify sensitization to mycotic allergens in children with allergic bronchopulmonary diseases is due to the severity of their clinical course, the high frequency of complications, and the inadequate efficiency of conventional treatment regimens. The sensitivity of most techniques used in clinical practice to estimate the level of specific artibodies to mycotic antigens is inadequately high. Recently clinically introduced high-technology diagnostic methods allow one to attack the problem at a qualitatively new level. The application of one of these methods, namely the highly sensitive semiautomatic diagnostic technology ImmunoCAP, permits the determination of the rates of IgE- and IgG-associated specific immune reactions in children with allergic respiratory diseases.

Cytokinin response induces immunity and fungal pathogen resistance, and modulates trafficking of the PRR LeEIX2 in tomato.

Plant immunity is often defined by the immunity hormones: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). These hormones are well known for differentially regulating defence responses against pathogens. In recent years, the involvement of other plant growth hormones such as auxin, gibberellic acid, abscisic acid, and cytokinins (CKs) in biotic stresses has been recognized. Previous reports have indicated that endogenous and exogenous CK treatment can result in pathogen resistance. We show here that CK induces systemic immunity in tomato (Solanum lycopersicum), modulating cellular trafficking of the pattern recognition receptor (PRR) LeEIX2, which mediates immune responses to Xyn11 family xylanases, and promoting resistance to Botrytis cinerea and Oidium neolycopersici in an SA- and ET-dependent mechanism. CK perception within the host underlies its protective effect. Our results support the notion that CK promotes pathogen resistance by inducing immunity in the host.

Serine protease activity of Cur l 1 from Curvularia lunata augments Th2 response in mice.

RATIONALE: Studies with mite allergens demonstrated that proteolytic activity augments allergic airway inflammation. This knowledge is limited to few enzyme allergens. OBJECTIVE: The objective of this study is to investigate the effect of serine protease Cur l 1 from Curvularia lunata in airway inflammation/hyper-responsiveness. METHODS: Cur l 1 was purified and inactivated using a serine protease inhibitor. Balb/c mice were sensitized with enzymatically active Cur l 1 or C. lunata extract. Sensitized mice were given booster dose on day 14 with active or inactivated Cur l 1. Intranasal challenge was given on day 28, 29, and 30. Airway hyper-responsiveness was measured by plethysmography. Blood, bronchoalveolar lavage fluid (BALF), spleen, and lungs from mice were analyzed for cellular infiltration, immunoglobulins, and cytokine levels. RESULTS: Mice challenged with enzymatically active Cur l 1 demonstrated significantly higher airway inflammation than inactive Cur l 1 group mice (p < 0.01). There was a significant difference in serum IgE and IgG1 levels among mice immunized with active Cur l 1 and inactive Cur l 1 (p < 0.01). IL-4 and IL-5 were higher in BALF and splenocyte culture supernatant of active Cur l 1 than inactive Cur l 1 mice. Lung histology revealed increased eosinophil infiltration, goblet cell hyperplasia and mucus secretion in active group. CONCLUSION: Proteolytic activity of Cur l 1 plays an important role in airway inflammation and the inactivated Cur l 1 has potential to be explored for immunotherapy.

Monocyte-derived dendritic cells from patients with severe forms of chromoblastomycosis induce CD4+ T cell activation in vitro.

Dendritic cells (DCs) have been described as initiators and modulators of the immune response. Recently we have shown a predominant production of interleukin-10 cytokine, low levels of interferon-gamma and inefficient T cell proliferation in patients with severe forms of chromoblastomycosis. Chromoblastomycosis starts with subcutaneous inoculation of Fonsecaea pedrosoi into tissue where DCs are the first line of defence against this microorganism. In the present study, the interaction of F. pedrosoi and DCs obtained from patients with chromoblastomycosis was investigated. Our results showed that DCs from patients exhibited an increased expression of human leucocyte antigen D-related (HLA-DR) and co-stimulatory molecules. In the presence of conidia, the expression of HLA-DR and CD86 was up-regulated by DCs from patients and controls. Finally, we demonstrate the reversal of antigen-specific anergy and a T helper type 1 response mediated by DCs incubated with F. pedrosoi conidea.

Quantification and characterisation of IgG binding to mould spores by flow cytometry and scanning electron microscopy.

The concentration of mould-specific IgG antibodies in serum may objectively indicate mould exposure and can help identifying exposed individuals. Although inhaled spores probably are the most important source of mould exposure, the commonly used methods for detecting mould-specific IgG antibodies are based on extracts from all mould components, with only low contribution from spores. We have developed a flow cytometric method using surface antigens on mould spores for quantifying mould-specific IgG antibodies in serum. Flow cytometric results were evaluated by comparison with ImmunoCap and ELISA measurements. The flow cytometric assay showed a broad linear dose-dependency and correlated moderately to strongly (r=0.41-0.97) with ImmunoCap and ELISA measurements. The IgG antibody binding was studied in detail by immunolabelling in scanning electron microscopy (SEM), revealing that morphology and IgG antibody binding differed among spores, both within and between mould strains. Germination studies by flow cytometry and SEM showed that IgG antibody binding to mould spores was altered during germination due to loss of coat. The present spore based antibody assay are simple and suitable for quantification of mould-specific IgG antibodies in serum, and includes specificity to other and possibly more relevant antigens than existing methods.

The fungal biopesticide Metarhizium anisopliae has an adjuvant effect on the allergic response to ovalbumin in mice.

The parasitic fungus, Metarhizium anisopliae, is non-pathogenic to humans and licensed for indoor control of cockroach infestation. An important reason for the elimination of this vermin is that sensitisation to cockroaches is associated with asthma. Previously M. anisopliae has been shown to cause allergic- and asthma-like responses in mice and in the present study we have examined the adjuvant activity of M. anisopliae on the allergic response to the model allergen ovalbumin (OVA) in a mouse model. Levels of OVA-specific IgE, IgG1 and IgG2a in serum were measured and the weight and cell number of the excised popliteal lymph node were determined. Mice primed with mycelium+OVA and boosted with OVA had increased anti-OVA IgE and IgG1 levels compared with mice primed with OVA alone or mycelium. Priming with M. anisopliae (as mycelium or MACA) increased weight or cell number of the excised PLNs. These results suggest that M. anisopliae has the ability to increase an allergic response to an allergen and consequently, may worsen allergy in susceptible individuals.

Seasonal distribution of Alternaria, Aspergillus, Cladosporium and Penicillium species isolated in homes of fungal allergic patients.

BACKGROUND: Allergy to airborne fungi can cause rhinitis and severe asthma, hence the exposure to spores inside home is an important factor of sensitization. The aim of this study was to determine the distribution and prevalence of species of Alternaria, Aspergillus, Cladosporium and Penicillium inside and outside of homes of patients allergic to fungi and to evaluate seasonal variations. METHODS: Air samples were collected in 22 selected homes of patients with allergy to fungi using a volumetric method of impacting plates with culture media. The isolated species were identified and statistical analysis of the presence of the four fungi was carried out. RESULTS: A total of 431 indoor and 150 outdoor exposed plates were cultured, leading to isolation of 11,843 colonies of fungi (range 0- 1 666 colony-forming units per cubic meter (CFUs/m(3)). 85.5% of total colonies belonged to the four genera considered. The highest presence of Aspergillus, Cladosporium and Penicillium in indoor environment was registered in autumn. Alternaria was more frequent in summer. In the outdoor environment, Penicillium was more abundant in winter and Aspergillus in summer (P= .002). The largest numbers of isolations were of Cladosporium and Penicillium during all four seasons, indoors as well as outdoors. Alternaria was present in all the homes studied both in summer and in autumn. The most prevalent species were: Alternaria alternata, Cladosporium herbarum, Cladosporium cladosporioides, Aspergillus niger and Penicillium chrysogenum. CONCLUSIONS: The quantitative analysis of the four taxa related with respiratory allergies demonstrated considerable seasonal variability. Statistical differences between the indoor and outdoor prevalence were detected only in Alternaria. In summer and autumn, the greater level of exposure to the four studied taxas occurred inside homes.

Induced humoral immunity and vaccination against major human fungal pathogens.

Protection against fungal pathogens can theoretically be elicited by vaccines that stimulate humoral or cellular immunity, or both. There is conclusive evidence that humoral immunity can modify the course of infection against certain pathogenic fungi such as Candida albicans and Cryptococcus neoformans. However, for other fungi, such as Aspergillus fumigatus, the notion that humoral immunity contributes to host defence is unproven. Attempts to evaluate the potential efficacy of humoral immunity using immune sera are often inconclusive, whereas consistent results can be obtained with monoclonal antibodies. Protective monoclonal antibodies can be used to identify antigens that induce useful humoral responses.

Allergenic extracts from Metarhizium canisopliae: obtainment and characterization.

Metarhizium anisopliae is used as a biopesticide for insects that damage agricultural plantations like sugar cane and forage plants. In a previous study the sensitization to this fungus of asthmatic patients coming from sugar cane areas was showed. The aims of this work were: to compare crude extracts obtained with Tris-HCl and Coca liquid from several growth phases of M. anisopliae concerning the total content of proteins and their electrophoretic analysis profile; to evaluate in vivo allergic sensitization in Balb/c mice and allergic patients from a sugar cane area, and to characterize the allergenic fractions in the sera of patients positive for the prick test by means of Western-blotting. The extract obtained with Coca liquid on the 16th day was the one that presented the greatest number of proteic fractions, including all those present in the other extracts. Twelve fractions were verified in this extract with approximate molecular weights from 94 to 14 kDa. The allergenicity of the extract obtained on the 16th day was proven by the production of IgE antibodies in Balb/c mice, with titres of 200. Prick tests carried out with the extract of the 16th day in 79 atopic individuals (from sugar cane area), 35 atopic individuals (from urban area) and 11 non- atopic individuals showed respective positivity of 29%, 9% and 0%. The allergenic characterization in vitro was performed by means of Western blotting, and the fractions that reacted with the positive individuals' sera were those of approximate molecular weights of 67 kDa (95%); 20 kDa (55%); 94 kDa (36%); 34 and 36 kDa (23%); 43 and 48 kDa (14%); 16 kDa (9%) and 54kDa (5%). It was concluded that the crude allergenic extract, obtained with Coca liquid from the 16th day growth of Metarhizium anisopliae, contains allergenic fractions and can be used in diagnostic screening tests.

Immunohistochemical characterization of the cellular infiltrate in Jorge Lobo's disease.

Few studies have been conducted to evaluate the cellular composition of the granulomatous lesions induced by Lacazia loboi. Thus, the objective of the present study was to characterize the mononuclear cell population present in cutaneous lesions obtained from 15 patients with Jorge Lobo's disease. Histological sections were stained with hematoxylin-eosin and methenamine silver and the following mononuclear cells were identified by immunohistochemistry: T lymphocytes (CD3+), helper T lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), B lymphocytes (CD20+), plasma cells (CD79+), natural killer cells (CD57+) and histiocytes (CD68+). This study showed that the inflammatory infiltrate mainly consists of histiocytes and multinucleated giant cells, in addition to the presence of a large number of fungal cells. The identified inflammatory cells showed the following frequency: CD68+ histiocytes > CD3+ T lymphocytes > CD4+ T > CD8+ T lymphocytes > CD57+ natural killer cells > CD79+ plasma cells > CD20+ B lymphocytes. Based on the findings of a large number of fungal cells in the infected tissues and the disorganized cell arrangement in the granuloma, we hypothesize that patients with Jorge Lobo's disease present immunoregulatory disturbances, which are likely to be specific and perhaps responsible for the lack of containment of the pathogen.

Assessment of indoor air fungi in Western-Anatolia, Turkey.

This study was conducted to determine fungal spores in the indoor air of the houses in the city of Afyon, Western-Anatolia, Turkey. We investigated the seasonal properties of mould spores in 10 houses of Afyon over a period of one year. Viable moulds were recovered from all 10 houses. Twenty seven different moulds were isolated and identified from the indoor air of the houses. The most common genus was Cladosporium spp. (31.9%), followed by Aspergillus spp. (18.6%), Penicillium spp. (15.5%), Altemaria spp. (13.0%) and other species (21.0%). The mould concentration was higher in the kitchens than in other parts of the houses such as the living rooms and bedrooms (p < 0.05). The fungal flora of the air in the Afyon city region has a seasonal variation. All fungal species had their highest prevalence in summer and their lowest in winter, but only Aspergillus spp. had a significant seasonal variation (p = 0.012). Viable moulds are common in the houses of Afyon. Reducing these indoor fungi is necessary to improve the health of individuals with fungal-induced diseases like asthma.

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.

Purification and characterisation of a major allergen of Alternaria alternata.

A major allergen of Alternaria (Alt a Bd29K) was identified by SDS-PAGE and immunoblotting. It was purified by gel-permeation and ion-exchange HPLC. The apparent molecular mass was 29,000, with a cysteine linked sub-unit of 15,000 Mr. The apparent isoelectric point was 4.2. It was found to be a constituent of the Alt I allergen group.

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.

Activation of the alternative complement pathway by Fonsecaea pedrosoi.

Human sera were examined for evidence of complement activation by Fonsecaea pedrosoi. The serum that had been incubated with F. pedrosoi showed immunoelectrophoretic C3 conversion and generation of C5a, as measured by radioimmunoassay. C3 conversion and C5a generation did not occur in serum chelated with EDTA, or in serum heated to 56 degrees C for 30 min. Serum depleted of the heat (50 degrees C X 30 min)-labile factor B was also deficient in C3 conversion and C5a generation. Complement activation was not affected when the classical pathway was blocked by Mg++-EGTA. These studies indicate that F. pedrosoi can activate the complement system via the alternative pathway, with the resultant development of C5a.