Subject(s)
Cholecalciferol/therapeutic use , Immunoglobulin G/blood , Immunologic Deficiency Syndromes/drug therapy , Mononeuropathies/drug therapy , Sitagliptin Phosphate/therapeutic use , Cholecalciferol/administration & dosage , Humans , Immunoglobulin G/immunology , Immunologic Deficiency Syndromes/blood , Immunologic Deficiency Syndromes/immunology , Male , Middle Aged , Mononeuropathies/immunology , Sitagliptin Phosphate/administration & dosage , Time FactorsABSTRACT
Pentraxin 3 (PTX3) is a tumor necrosis factor and interleukin-1beta-stimulated gene that encodes a long PTX with proinflammatory activity. Here, we show that peritoneal macrophages derived from PTX3 transgenic (Tg) mice express higher levels of PTX3 mRNA than macrophages from wild-type (WT) mice, at basal level as well as upon stimulation with zymosan (Zy). Macrophages from Tg mice also showed improved opsonin-independent phagocytosis of Zy particles and the yeast form of the fungus Paracoccidioides brasiliensis. In the case of P. brasiliensis, an enhanced microbicidal activity accompanied by higher production of nitric oxide was also observed in macrophages from Tg mice. Using fluorescein-activated cell sorter analysis and reverse transcriptase-polymerase chain reaction, we demonstrated that basal level of Toll-like receptor-6 and Zy-induced dectin-1 expression was slightly but consistently higher in macrophages from Tg mice than in macrophages from WT mice. Recombinant (r)PTX3 protein binds to Zy particles as well as to yeast cells of P. brasiliensis and addition of rPTX3, to a culture of WT-derived macrophages containing Zy leads to an increase in the phagocytic index, which parallels that of Tg-derived macrophages, demonstrating the opsonin-like activity of PTX3. It is important that blockade of dectin-1 receptor inhibited the phagocytosis of Zy particles by WT and PTX3 Tg macrophages, pointing out the relevant role of dectin-1 as the main receptor involved in Zy uptake. Our results provide evidence for a role of PTX3 as an important component of the innate-immune response and as part of the host mechanisms that control fungal recognition and phagocytosis.
Subject(s)
C-Reactive Protein/genetics , Macrophages, Peritoneal/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Opsonin Proteins/genetics , Phagocytosis/genetics , Serum Amyloid P-Component/genetics , Zymosan/immunology , Animals , Binding Sites/drug effects , Binding Sites/genetics , C-Reactive Protein/metabolism , Female , Immunity, Innate/genetics , Lectins, C-Type , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Male , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/metabolism , Membrane Proteins/antagonists & inhibitors , Mice , Mice, Transgenic , Nerve Tissue Proteins/antagonists & inhibitors , Nitric Oxide/metabolism , Opsonin Proteins/metabolism , Paracoccidioides/immunology , Phagocytosis/drug effects , Phagocytosis/immunology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/metabolism , Serum Amyloid P-Component/metabolism , Toll-Like Receptor 6 , Zymosan/metabolism , Zymosan/pharmacologyABSTRACT
Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated whether immunization with P. brasiliensis antigens fractionated by anionic chromatography on fast protein liquid chromatography (FPLC) could elicit protective immunity. BALB/c mice were immunized by subcutaneous injection of either 10 microg fractions 0 (F0), II (FII) or III (FIII) in the presence of 100 microg of Corynebacterium parvum and 1 mg of Al(OH)(3) and challenged with pathogenic P. brasiliensis strain. Mice immunized with F0 presented cellular and humoral immune responses with significant production of IFN-gamma, and high levels of IgG2a and IgG3 isotypes. Immunization with FII induced significant production of IFN-gamma and IL-10 associated with high levels of IgG1 and IgG2a. It was demonstrated that immunization with F0 or FII promoted significant decrease of organ colony-forming units (CFUs) in the lung after challenge infection without fungi dissemination to the spleen or liver. In contrast, FIII immunized mice develop a progressive disseminated disease to spleen and liver presented significant levels of INF-gamma, IL-10 or TGF-beta associated with high production of IgG1 and IgG2a with low production of IgG2b and IgG3 after challenge infection. Taken together, these findings suggest that antigens of F0 and FII are reliable vaccine candidates against the paracoccidioidomycosis.
Subject(s)
Antigens, Fungal/immunology , Paracoccidioides/immunology , Paracoccidioidomycosis/immunology , Paracoccidioidomycosis/prevention & control , Animals , Antibodies, Fungal/biosynthesis , Cell Division/drug effects , Cytokines/biosynthesis , Female , Fluorescent Antibody Technique , Immunization , Immunoglobulin G/biosynthesis , Mice , Mice, Inbred BALB C , Paracoccidioidomycosis/pathology , Spleen/cytology , Spleen/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the main mode of protection against this fungal infection. We investigated in vitro the response of peripheral blood mononuclear cells (PBMC) from paracoccidioidomycosis (PCM) patients presenting different clinical forms to antigenic fractions from P. brasiliensis yeast cell lysate (PbAg). These fractions designated F0 to FV were obtained using anion-exchange chromatography on a FPLC system. Our studies showed variation in the cellular responses induced by different antigenic fractions. The fraction F0 caused significant decrease in cellular proliferation, granuloma formation, accompanied by significant elevation in the production of IL-10. The fractions FII and FIII increased in vitro granuloma formation associated with high production of TNF-alpha. Besides that, FII and FIII evoked decrease in NO production but not F0 that induced very high levels, among patients with PCM from acute form. The findings suggest that P. brasiliensis antigenic components participate in the modulation or activation of PBMC response in PCM, and IL-10 and NO could be important in the regulation of in vitro granuloma formation.
Subject(s)
Antigens, Fungal/immunology , Leukocytes, Mononuclear/immunology , Nitric Oxide/metabolism , Paracoccidioides/immunology , Antigens, Fungal/isolation & purification , Cell Division , Granuloma/etiology , Granuloma/immunology , Humans , Immunity, Cellular , In Vitro Techniques , Interleukin-10/analysis , Leukocytes, Mononuclear/metabolism , Paracoccidioidomycosis/immunology , Tumor Necrosis Factor-alpha/analysisABSTRACT
Gp43, the major 43-kDa antigenic glycoprotein of Paracoccidioides brasiliensis, or its 15-amino acid inner peptide (P10), induces a T-CD4(+), Th1 cellular immune response which protects BALB/c mice from intratracheal infection by virulent yeast forms. We investigated whether DNA vaccination using the gp43 gene could elicit protective immunity against P. brasiliensis. Animals immunised intramuscularly (i.m.) or intradermally (i.d.) with plasmid DNA containing the gp43 gene induced a specific, long lasting humoral and cellular immune response. A mixed Th1/Th2 cellular immune response in DNA-immunized mice was modulated in vivo by IFN-gamma and was protective in BALB/c mice. A significant decrease in the lung colony forming units (CFUs) and reduced, or no dissemination to the spleen and liver of immunised mice were observed.
Subject(s)
Antigens, Fungal/immunology , Fungal Vaccines/immunology , Paracoccidioides/immunology , Vaccines, DNA/immunology , Animals , Antigens, Fungal/genetics , COS Cells , Female , Immunization , Interferon-gamma/physiology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Paracoccidioidomycosis/pathology , Paracoccidioidomycosis/prevention & controlABSTRACT
Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were fractionated in a fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that could elicit cell reactivity and antibody recognition in human paracoccidioidomycosis (PCM). PbAg fractions were eluted by 20 mM Tris-HCl solution (pH 9.6) with an increasing gradient up to 1 M NaCl. The FPLC system was able to resolve 7 fractions, enumerated from 0 to VI, according to the elution on the NaCl gradient. The analysis of each fraction on SDS-PAGE showed that fractions 0 to V were constituted by multiple protein bands with molecular mass ranging from 18 to 114 kDa. Large amounts of nucleic acids were evidenced in fraction VI, as revealed by agarose gel stained with ethidium bromide. Sera from PCM patients presenting different clinical forms contained antibodies that recognized antigens in all fractions with the exception of fraction VI as detected by ELISA. Further studies were designed to investigate the capacity of these fractions to induce cell proliferation. It was demonstrated that fractions III and V (200 and 450 mM NaCl, respectively) stimulated a significant proliferative response of peripheral blood mononuclear cells, while fraction 0 induced the lowest proliferative response among patients with PCM, in either acute, acute treated, or chronic forms.
