ABSTRACT
DNA microarray technology was used to determine the gene expression profile of human monocyte-derived macrophages (hMDMs) after stimulation by Penicillium marneffei yeast. The expression levels of 175 macrophage genes were found to be altered by a minimum of two-fold in magnitude following 4 hours of P. marneffei exposure. Among those, 41 genes were upregulated in activated hMDMs while 134 genes were downregulated. Real-time PCR and RT-PCR were performed to further examine gene expression associated with the inflammatory response. Increased levels of TNF-alpha and IL-1 beta gene expression in both hMDMs and human monocyte-derived dendritic cells (hMoDCs) were observed after stimulation by P. marneffei yeast. Furthermore, the genes encoding T-bet, IL-6 and ICAM-1 were also upregulated in hMDMs. Functional analysis of the adhesion of P. marneffei to dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN, CD209) was performed in hMoDCs since the microarray data revealed an increased expression of DC-SIGN in activated hMDMs. We found that DC-SIGN-Fc bound preferentially to P. marneffei yeast rather than to conidia. Moreover, an anti-DC-SIGN monoclonal antibody inhibited the binding of P. marneffei yeast to hMoDCs, but did not inhibit endocytosis of P. marneffei yeast. The mannose receptor, on the other hand, was important in both adhesion and phagocytosis. These results suggest that P. marneffei may exploit DC-SIGN as a receptor to facilitate the systemic spread of infection. Taken together, our study demonstrates the usefulness of microarray technology in generating valuable expression data to permit conventional immunologic investigations of host-fungal interactions.
Subject(s)
Antigen-Presenting Cells/immunology , Cell Adhesion/genetics , Cell Adhesion Molecules/genetics , Cells, Cultured , Dendritic Cells/immunology , Gene Expression Profiling , Gene Expression Regulation/immunology , Host-Pathogen Interactions , Humans , Lectins, C-Type/genetics , Macrophages/immunology , Mycoses/immunology , Oligonucleotide Array Sequence Analysis , Penicillium/immunology , Phagocytosis/genetics , Receptors, Cell Surface/genetics , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Monoclonal antibody against P. mameffei yeast secreted antigen was produced in order to develop a serological test for penicilliosis marneffei. The yeast form of P. marneffei was cultured in brain heart infusion broth at 37 degrees C for 7 days. A secreted antigen was prepared, partially purified from culture supernatant and subsequently immunized in a BALB/c mouse. Mouse monoclonal antibody was produced from immune spleen cells by a standard hybridoma technique. Specificity of the obtained monoclonal antibody was assessed with yeast secreted antigens for P. mameffei, C. alblicans, C. neoformans, and H. capsulatum by an indirect ELISA. Three of 46 hybrid clones (1 F1, 2G5, and 3G4) reacted positively with P mameffei secreted antigen. 1 F1 and 3G4 were cloned by two rounds of limiting dilution. Partially purified monoclonal antibody and rabbit polyclonal antibody against P. marneffei yeast secreted antigen were used to develop a double antibody sandwich ELISA to detect P. marneffei antigen in plasma or serum samples of 7 patients with penicilliosis marneffei and 5 healthy controls. The sandwich ELISA developed using monoclonal antibody as a capture antibody and rabbit polyclonal antibody as a detector was able to detect P. marneffei antigen in all the plasma and serum samples of penicilliosis marneffei patients, while negative in all the healthy controls. Thus, the monoclonal antibody produced in the present study appeared to be highly specific for P. marneffei and the double antibody sandwich ELISA developed using monoclonal and polyclonal antibodies against the yeast secreted antigen of P. marneffei showed a strong potential for the diagnosis of penicilliosis marneffei.
Subject(s)
Animals , Antibodies, Monoclonal/biosynthesis , Antigens, Fungal/immunology , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Humans , Mice , Mice, Inbred BALB C , Mycoses/diagnosis , Penicillium/immunology , Rabbits , Sensitivity and Specificity , Serologic Tests , Thailand , Yeasts/growth & developmentABSTRACT
We report a case of hypersensitivity pneumonitis in a 30-yr-old female housewife caused by Penicillium species found in her home environment. The patient was diagnosed according to history, chest radiograph, spirometry, high-resolution chest CT, and transbronchial lung biopsy. To identify the causative agent, cultured aeromolds were collected by the open-plate method. From the main fungi cultured, fungal antigens were prepared, and immunoblot analysis with the patient's serum and each fungal antigen was performed. A fungal colonies were isolated from the patient's home. Immunoblotting analysis with the patient's sera demonstrated a IgG-binding fractions to Penicillium species extract, while binding was not noted with control subject. This study indicates that the patient had hypersensitivity pneumonitis on exposure to Penicillium species in her home environment.
Subject(s)
Adult , Female , Humans , Alveolitis, Extrinsic Allergic/etiology , Antibodies, Fungal/blood , Antigens, Fungal , Environmental Microbiology , Housing , Immunoglobulin G/blood , Korea , Penicillium/immunologyABSTRACT
El objetivo del estudio fue establecer la variación anual de la microflora en la ciudad de San Juan y en hogares de pacientes con patología respiratoria IgE dependiente. San Juan es una provincia del oeste de la República Argentina, con escasísimas precipitaciones pluviales anuales (menos de 10 mm), con un promedio de humedad relativa ambiente de 46 por ciento (min.: 36 por ciento y máx.: 55 por ciento) y una temperatura promedio de 17,6ºC (min.: 7,8ºC y máx.: 26,5ºC). Para poder conocer el desarrollo de hongos y definir mejor la correlación existente entre ellos y las enfermedades alérgicas respiratorias, estudiamos durante un período anual (julio 1994-junio 1995) la presencia de seis géneros de hongos: Alternaria, Cladosporium, Penicillium, Aspergillus, Mucor y Rhizopus, en el ambiente exterior de la ciudad de San Juan, y en el domicilio de nueve (9) niños con enfermedad respiratoria (rinitis y/o asma), con Prick Test positivo para todos los hongos en estudio. Se controló la reactividad sanguínea de estos pacientes a los mismos géneros de hongos por IgE RAST. La flora micológica fue estudiada por el método gravimétrico, exponiendo mensualmente cápsulas de Petri (diámetro: 10 cm) en medio ambiente externo y en los domicilios (4 cápsulas por domicilio), con medios de cultivo de Sabouraud y Czapek-Dox (Lab. Merck). Los Prick Test se realizaron con antígenos Allergon AB (Lab. Welt), y las IgE RAST (Allerex Labs. Inc - USA). El hongo que se desarrolló con más frecuencia en los domicilios estudiados fue el Penicillium (40,63 por ciento), seguido por Rhizopus (17,24 por ciento), Aspergillus (14,09 por ciento), Mucor (11,28 por ciento), Cladosporium (4,81 por ciento), Alternaria (4,75 por ciento), y otros (7,0 por ciento). Aunque en distinto porcentaje, la distribución de hongos en ambientes externos sigue una curva muy similar a las halladas en los domicilios: Penicillium (17,86 por ciento), Rhizopus (12,76 por ciento), Aspergillus (11,13 por ciento), Mucor (9,97 por ciento), Alternaria (8,12 por ciento), Cladosporium (6,03 por ciento) y otros (1,39 por ciento). La mayor concentración de hongos se alcanzó en octubre para domicilios y en noviembre-diciembre para ambiente exterior. La mejor correlación entre el Prick Test y el RAST se obtuvo para Alternaria y Aspergillus (87 por ciento), seguidos por Cladosporium (75 por ciento), Penicillium (62 por ciento), Rhizopus (50 por ciento), y Mucor (37 por ciento). No encontramos correlación directa entre la respuestas de los pacientes y la concentración de hongos en el período de estudio, confirmando lo esperado de acuerdo con las diferentes potencias alergénicas de cada género