Synthesis of oligosaccharides related to galactomannans from Aspergillus fumigatus and their NMR spectral data.

The synthesis of model oligosaccharides related to antigenic galactomannans of the dangerous fungal pathogen Aspergillus fumigatus has been performed employing pyranoside-into-furanoside (PIF) rearrangement and controlled O(5) → O(6) benzoyl migration as key synthetic methods. The prepared compounds along with some previously synthesized oligosaccharides were studied by NMR spectroscopy with the full assignment of 1H and 13C signals and the determination of 13C NMR glycosylation effects. The obtained NMR database on 13C NMR chemical shifts for oligosaccharides representing galactomannan fragments forms the basis for further structural analysis of galactomannan related polysaccharides by a non-destructive approach based on the calculation of the 13C NMR spectra of polysaccharides by additive schemes.

No effect of antifungal compounds on functional properties of human antifungal T-helper type 1 cells.

Despite the availability of new antifungal compounds, invasive fungal disease is associated with a high mortality in hematopoietic stem cell transplant (HSCT) recipients. A growing body of evidence suggests that T lymphocytes from the T-helper type 1 (TH 1) play an important role in the antifungal host defense, and preliminary data indicate a potential benefit of infusing donor-derived antifungal TH 1 cells to HSCT patients suffering from invasive fungal disease. Unfortunately, it is unclear to date whether the function of these cells is affected by concomitantly administered antifungal agents. We therefore analyzed the effects of various concentrations of commonly used antifungal compounds such as amphotericin B, caspofungin, fluconazole, voriconazole, and posaconazole on the functional properties of cultivated human antifungal TH 1 cells. None of the antifungal compounds tested significantly influenced the secretion of interferon-γ and tumor necrosis factor-α, and only posaconazole at high concentrations slightly decreased proliferation of antifungal TH 1 cells. Our data indicate that the antifungal agents tested do not significantly affect the functional properties of antifungal TH 1 cells and can therefore be concomitantly administered.

Susceptibility of mice to invasive aspergillosis correlates with delayed cell influx into the lungs.

Ubiquitous fungus Aspergillus fumigatus (A. fumigatus) is involved in invasive pulmonary aspergillosis (IPA), a frequent infection in immunocompromized patients. Genetic differences are likely to play a role predisposing to IPA. This study was aimed to compare six genetically different mouse strains in their susceptibility to IPA and to determine possible mechanisms involved in the pathogenesis of this infection. Immunosuppressed BALB/c and C57BL/6 mice infected with A. fumigatus conidia were more resistant to IPA than DBA/1, DBA/2, CBA, and A/Sn strains. Phagocytosis of A. fumigatus conidia by blood polymorphonuclear neutrophils (PMN) or bone marrow derived dendritic cells showed no difference between strains. All IPA susceptible strains demonstrated decreased PMN influx into the lungs during infection compared with resistant strains. Flow cytometry analysis of the composition of lung infiltrating cells showed that IPA susceptible mice had a decreased number of phagocytes before the infection. After infection the numbers of Gr-1(+)CD11b(+) PMN cells in the lungs of immunosuppressed mice increased from 10-20% to 50-60% while the percentage of CD11(+)F4/80(+) resident macrophages was unchanged. Among susceptible strains DBA/2 and A/Sn have a defect in C5 component of complement. Injection of normal serum into complement deficient but not into complement sufficient CBA or DBA/1 mice significantly improved their survival. We showed that complement replacement significantly increased PMN homing to the lungs of complement deficient mice. Thus, defect in complement system can predispose to IPA. Our results demonstrated that early influx of PMN into the lungs of mice is important for the resistance to IPA.

Clinical-scale generation of human anti-Aspergillus T cells for adoptive immunotherapy.

Invasive aspergillosis is a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic SCT. There is a growing body of evidence that T cells are important in the host defense against Aspergillus, and adoptively transferred anti-Aspergillus T-helper 1 (T(H)) 1 cells might reduce infectious mortality in hematopoietic transplant recipients. Here we present for the first time a simple and rapid method for the clinical-scale generation of functionally active anti-Aspergillus T cells according to good manufacturing practice conditions. A total of 1.1 x 10(9) WBCs derived from a leukapheresis product were incubated with Aspergillus antigens. Stimulated cells were selected by means of the IFN-gamma secretion assay and expanded. In three independent experiments, a median number of 2 x 10(7) CD3+CD4+cells (range, 0.9-3.2 x 10(7)) were obtained within 13 days. The cultured CD3+CD4+ cells exhibited almost exclusively a memory activated T-helper cell phenotype. Upon restimulation, the generated T cells produced IFN-gamma, but no IL-4 or IL-10, indicating a T(H)1-cell population. Additionally, the cells proliferated upon restimulation and showed reduced alloreactivity compared to unselected CD4+ cells. This method of generating is suitable for future prospective trials designed to evaluate the effect of adoptive immunotherapy in hematopoietic transplant recipients with invasive aspergillosis.

The role of the sakA (Hog1) and tcsB (sln1) genes in the oxidant adaptation of Aspergillus fumigatus.

The Hog1 MAP kinase pathway regulates stress adaptation in several fungi. To assess its role in stress adaptation in Aspergillus fumigatus, we constructed mutants in genes encoding the sensor histidine kinase (HK) tcsB as well as sakA, which are homologues of the Saccharomyces cerevisiae sln1 and Hog1, respectively. Compared to the wild type strain (Wt), growth of sakA (sakAtriangle up) mutant was reduced, and growth inhibition was increased when H(2)O(2), menadione, or SDS was added to the media. On the other hand, the tcsB mutant (tcsBtriangle up) was similar to the Wt strain in regard to growth and morphology, although a partial sensitivity to SDS was observed. Western blot analysis of Wt and the tcsBtriangle up strains indicated that when stressed with H(2)O(2), phosphorylation of Hog1p still occurs in the mutant. Since in Candida albicans, Hog1 regulates transcription of at least one histidine kinase, we performed RT-PCR of 6 histidine kinase genes as well as the ssk1 and skn7 response regulator genes of A. fumigatus. No significant differences in transcription were observed with the sakAtriangle up when compared to the Wt, indicating that the sakA does not regulate transcription of these genes. Our studies indicate that the A. fumigatus sakA is required for optimal growth of the organism with or without oxidant stress, while tcsB gene is dispensable.

Immunochemical comparison of the allergenic potency of spores and mycelium of cladosporium cladosporioides extracts by a nitrocellulose electroblotting technique / Comparación inmunoquímica de la potencia alergénica de los extractos de esporas y mecilio del cladosporium cladosporioides por una técnica de eletroblotting en nitrocelulosa

Background: The lack of well standardized or characterized extracts that contain the relevant allergens of the appropriate fungus is resulting in a high heterogeneity of the commercial preparation. Material and methods: Immunochemical detection of the allergens composition of spore and mycelium of C. cladosporioides was studied by electroblotting using sera from Cladosporium allergic patients and 125 I- anti- human IgE. A MW range of allergens between 16 to 88 KDa was identified. The most important with a MW of 16, 20,30, 39, 43, 50, 60 and 88 KDa. Results: The allergenic composition of spore and mycelium looked very similar. However, partial or total inhibition of the serum with a conidial or mycelial extract demonstrated that the total concentration of allergens (particulary 20 and 60 KDa molecules) was higher in the conidium than in the mycelium. Conclusions: These results indicated that conidium and mycelium contained the same allergenic determinants but at different concentration in the two propagule. Results with 50 % inhibited sera demonstrated also that the total concentration of allergens was higher in the conidium than in the mycelium

Antecedentes: La falta de extractos estandarizados o caracterizados que contienen alergenos relevantes de componentes fúngicos, da como resultado una elevada heterogeneidad de los preparados comerciales. Material and methods. La detección inmunoquímica de la composición alergénica de las esporas y del micelio de C. Cladosporioides ha sido estudiada por el método del electroblotting usando sueros procedentes de pacientes alérgicos a Cladosporium y anti-IgE humano marcado con Yodo 125. El peso molecular de los alergenos identificados fue situado entre 16 y 88 Kda. Los más importantes con un peso molecular de 16, 20, 30, 39, 43, 50, 60 y 88 Kda. Resultados: La composición alergénica detectada en las espora y el micelio ha sido idéntica. Sin embargo, la inhibición parcial o total del suero con extractos de esporas o micelios ha demostrado que la dosis de alergenos (especialmente de 20 y 60 KDa) ha sido más alta en las esporas que en el micelio. Conclusiones: Estos resultados indican que las esporas y los micelios contienen los mismos determinantes alergénicos pero a diferente concentración en ambos extractos. Con un 50% de inhibición sérica, la concentración de alergenos fue más elevada para las esporas que para los milecelios

Galactomannoproteins of Aspergillus fumigatus.

Galactofuranose-containing molecules have been repeatedly shown to be important antigens among human fungal pathogens, including Aspergillus fumigatus. Immunogenic galactofuran determinants have been poorly characterized chemically, however. We reported here the characterization of two glycoproteins of A. fumigatus with an N-glycan containing galactofuranose. These proteins are a phospholipase C and a phytase. Chemical characterization of the N-glycan indicates that it is a mixture of Hex(5-13)HexNAc(2) oligosaccharides, the major molecular species corresponding to Hex(6-8)HexNAc(2). The N-glycan contained one galactofuranose unit that was in a terminal nonreducing position attached to the 2 position of Man. This single terminal nonreducing galactofuranose is essential for the immunoreactivity of the N-glycans assessed either with a monoclonal antibody that recognizes a tetra-beta-1,5-galactofuran chain of galactomannan or with Aspergillus-infected patient sera.

Comparison of the allergenic potency of spores and mycelium of Cladosporium

The allergenic potency of spore and mycelium extracts of Cladosporium was estimated by RAST, RAST inhibition and PCA tests. Spores contained a concentration of allergens higher than mycelia. Results of PCA tests suggested that spores contained specific allergens. However, in a comparative study of extracts from different species of Cladosporium animal and human models gave different estimates of the allergenic potency of the different species. In spite of these variations it was shown that extracts from spores of Cladosporium contained the highest amount of Cladosporium allergens

El potencial alergénico de los extractos de las esporas y del micelio de Cladosporium ha sido valorado por los métodos de RAST, RAST inhibición y PCA. Las esporas contienen una dosis de alérgenos más elevada que el micelio. Los resultados del ensayo PCA sugieren que las esporas contienen alérgenos específicos. Sin embargo, en un estudio comparativo de los extractos procedentes de diferentes especies de Cladosporium, el modelo animal y humano han dado diferentes estimaciones del poder alérgenico entre las distintas especies. Aunque haya variaciones, se ha demostrado que los extractos de las esporas de Cladosporium contienen una cantidad más elevada de los alérgenos