Biomedical subjects

José Daniel Lopes

Publications and source records attributed to José Daniel Lopes.

16 recordsLinked to original sources

PubMed2007

Cloning, characterization and expression of a calnexin homologue from the pathogenic fungus Paracoccidioides brasiliensis.

We report the cloning of a Paracoccidioides brasiliensis cDNA, here named PbCnx, encoding the homologue of the endoplasmic reticulum molecular chaperone calnexin. Calnexin specifically recognizes monoglucosylated glycoproteins in the endoplasmic reticulum, thus being an essential component of the complex that interacts with the folded state of nascent secreted glycoproteins. The PbCnx open reading frame was found in a 1701 base pair (bp) fragment that encodes a 567 amino acid protein with an estimated mass of 62 680 Da. Northern and Southern blot hybridizations showed that PbCnx is encoded by a single, or a low number of, gene copies. PbCnx contains the hallmark KPEDWD motifs that are found in all members of the calnexin/calreticulin family proteins. A cDNA-encoding PbCnx was overexpressed as recombinant protein in Escherichia coli. The purified recombinant PbCnx was recognized by 6 out of 10 sera from PCM patients, a result that rules out its possible consideration for further use in diagnosis. Using confocal microscopy with anti-PbCnx mouse serum against yeast forms, a cytoplasmic staining pattern was observed.

Amino Acid Sequence↗

PubMed2006

Diagnosis of Paracoccidioidomycosis by detection of antigen and antibody in bronchoalveolar lavage fluids.

Paracoccidioidomycosis (PCM) is a systemic infection caused by the fungus Paracoccidioides brasiliensis and is believed to be the leading cause of fungal pulmonary infection. In this study, we used an inhibition enzyme-linked immunosorbent assay to diagnose pulmonary PCM based on the detection of 43-kDa and 70-kDa molecules in bronchoalveolar lavage fluids. The results were compared with results obtained by classical methods for antibody detection.

Antibodies, Fungal↗

PubMed2006

Granuloma formation in vitro requires B-1 cells and is modulated by Paracoccidioides brasiliensis gp43 antigen.

The mechanisms that determine granuloma formation and the significance of this type of inflammatory response in the pathogenesis of fungal diseases such as paracoccidioidomycosis are far from fully understood. We developed a granuloma model in vitro using beads to evaluate the role of isolated mouse peritoneal macrophages and B-1 cells. We also investigated granuloma formation in the presence of gp43, the main antigenic component of Paracoccidioides brasiliensis, which is secreted exocellularly. To determine whether B-1 cells, macrophages, or both, participate in granuloma formation, peritoneal cells from Xid mice, which lack B-1 cells, were used. Granuloma-like structures were not formed with Xid peritoneal cells or with cells from wild type mice that had their peritoneal and pleural cavities irradiated before the cultures were established. Granulomas were observed either when total adherent peritoneal cells or when isolated B-1 cells were added to macrophage cultures. The data strongly suggest that an interaction of B-1 cells and macrophages plays an important role in granuloma-like formation in this experimental model and that the presence of gp43 strongly stimulates this response.

Animals↗

PubMed2005

In vivo and in vitro effect of killed Propionibacterium acnes and its purified soluble polysaccharide on mouse bone marrow stem cells and dendritic cell differentiation.

Among the effects exerted by Propionibacterium acnes, a most relevant one is its capacity to modulate the Th1/Th2 cellular immune response. This effect depends on the induction and activation of antigen presenting cells, mainly dendritic cells (DCs), whose number is increased in the peripheral blood of animals treated with this bacterium. A soluble P. acnes polysaccharide (PS) extract also acts on DCs, modulating a Th1 immune response. These data led us to investigate the role of P. acnes and its soluble PS on murine bone marrow (BM) DCs. Bone marrow cells were analyzed by flow cytometry, showing an increase of stem cells and DCs in P. acnes- or PS-treated animals. Culturing in the presence of granulocyte monocyte colony stimulating factor (GM-CSF) increased the in vitro differentiation and maturation of these cells into BM-derived DCs (CD11c+ and MHC class II+). Maturation of DCs was determined by increased CD80 and CD86 expression, IL-4 and IL-12 production, reduction in phagocytic capacity and increase in the antigen presenting ability to primed or naïve T lymphocytes. These data indicate that P. acnes as well as its PS can modulate BM stem cells, originating mature DCs, which are important mainly at the initial antigen contact.

Animals↗

PubMed2005

B-1 cells are pivotal for in vivo inflammatory giant cell formation.

The mechanisms that govern giant cell (GC) formation in inflammatory, neoplastic and physiologic conditions are far from being understood. Here, we demonstrate that B-1 cells are essential for foreign-body GC formation in the mouse. GCs were analysed on the surface of glass cover slips implanted into the subcutaneous tissue of the animals. It was demonstrated that GCs are almost absent on cover slips implanted into the subcutaneous tissue of BALB/c or CBA/N X-linked immunodeficient mice. As these animals do not have B-1 cells in the peritoneal cavity, they were reconstituted with B-1 cells obtained from cultures of adherent mouse peritoneal cells. Results showed that in B-1-reconstituted animals, the number of GCs on the implant surface surpassed the values obtained with preparations from wild animals. In animals selectively irradiated (pleural and peritoneal cavities) to deplete these cavities of B-1 cells, GCs were also not formed. Enriched suspensions of B-1 cells grown in culture were labelled with [(3)H]-tymidine and injected into the peritoneal cavity of naive mice before implantation of glass cover slips. After 4 days, about 17% of mononuclear cells had their nuclei labelled, and almost 70% of GCs had one or more of their nuclei labelled when analysed by histoautoradiographic technique. A few GCs expressed an immunoglobulin M when analysed by immunostaining and confocal microscopy. Overall, these data demonstrate that B-1 cells are pivotal in the mechanisms of foreign-body GC formation in the mouse.

Animals↗

PubMed2005

Diagnosis of neuroparacoccidioidomycosis by detection of circulating antigen and antibody in cerebrospinal fluid.

Neuroparacoccidioidomycosis (neuroPCM) is the central nervous system infection by the fungus Paracoccidioides brasiliensis. Its diagnosis is a difficult task that depends on neuroimaging techniques such as computed tomography and magnetic resonance imaging. However, the detection of circulating P. brasiliensis antigens in body fluids by inhibition enzyme-linked immunosorbent assay (inh-ELISA) has provided encouraging results. In this study, 14 cerebrospinal fluid (CSF) and 11 serum samples of patients with neuroPCM were analyzed by inh-ELISA for detection of circulating glycoprotein antigens of 43 kDa (gp43) and 70 kDa (gp70). Circulating gp43 and gp70 antigens were detected in all CSF samples from patients with neuroPCM at mean concentrations of 19.3 and 6.8 mug/ml, respectively. In addition, both gp43 and gp70 antigens were detected in 10 of 11 serum samples analyzed at mean concentrations of 4.6 and 4.0 mug/ml, respectively. By immunodiffusion test, CSF samples were determined to be negative in 13 of 14 samples. The detection of anti-gp43 and anti-gp70 antibodies by conventional ELISA showed positive results for all CSF samples, with titers ranging from 1:50 to 1:51,200. Therefore, the high sensitivity of the inh-ELISA technique in detecting gp43 and gp70 antigens in the CSF of neuroPCM patients strongly indicates that this assay can be considered as a powerful diagnostic tool. In addition, the finding of anti-gp43 and anti-gp70 antibodies in CSF samples by conventional ELISA also seems to be a promising diagnostic method for this special modality of PCM.

Antibodies, Fungal↗

PubMed2004

Down-regulation of dendritic cell activation induced by Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (Pb). The infection can evolve to different clinical forms that are associated to various degrees of suppressed cell-mediated immunity. With the recognition that DCs are able to initiate response in naïve T cells and that they also participate in Th cell education, the present study was undertaken to check whether DCs interact with P. brasiliensis, as well as to elucidate possible mechanisms and consequences of this interaction. Our results indicate that P. brasiliensis infection and purified gp43, its main antigenic component, lead to down-regulation of MHC-II and adhesion properties of immature DCs. The down-regulation was also observed in LPS-induced DC maturation. In addition, an inhibition of IL-12 and TNF-alpha production by both P. brasileinsis or gp43, was observed in LPS-induced DC maturation. These results suggest that protein, released in great amounts by the fungus, might be used, to reduce the effectiveness of the immune response.

Animals↗

PubMed2004

Interleukin-10 secreted by B-1 cells modulates the phagocytic activity of murine macrophages in vitro.

As demonstrated previously in our laboratory, B-1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage-like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B-1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B-1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild-type mice. Moreover, macrophages from wild-type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1 cells down-regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B-1 cells are one of the main sources of interleukin (IL)-10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL-10 production by cells from wild-type mice. When B-1 cells from IL-10 knock-out mice and macrophages from wild-type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B-1 cells can influence the effector functions of macrophages in vitro via IL-10 secretion.

Animals↗

PubMed2004

Detection of Paracoccidioides brasiliensis gp70 circulating antigen and follow-up of patients undergoing antimycotic therapy.

Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Central and South America, is caused by the dimorphic fungus Paracoccidioides brasiliensis and has a high prevalence in Brazil. Glycoproteins of 43 and 70 kDa are the main antigenic compounds of P. brasiliensis and are recognized by Western blotting by 100 and 96% of PCM patient sera, respectively. In the present study, an inhibition enzyme-linked immunosorbent assay (ELISA) was used to detect gp70 in different biological samples from patients with PCM. gp70 was detected in 98.76% of 81 serum samples, with an average concentration of 8.19 microg/ml. The test was positive for 100% of the patients with the acute and chronic unifocal forms of PCM and 98.43% of the patients with the multifocal chronic form, with average concentrations of 11.86, 4.83, and 7.87 microg/ml, respectively. Bronchoalveolar lavage fluid from 23 patients with pulmonary unifocal PCM and 14 samples of cerebrospinal fluid from patients with neurological PCM were also tested for gp70 detection, with the test showing 100% sensitivity and 100% specificity, with mean gp70 concentrations of 7.5 and 6.78 microg/ml, respectively. To investigate the potential of gp70 detection by inhibition ELISA for the follow-up of PCM patients during antimycotic therapy with itraconazole (ITZ), the sera of 23 patients presenting with the chronic multifocal form of PCM were monitored at regular intervals of 1 month for 12 months. The results showed a decrease in circulating gp70 levels during treatment which paralleled the reduction in anti-P. brasiliensis antibody levels. The detection of P. brasiliensis gp70 from the biological fluids of patients suspected of having PCM proved to be a promising method for diagnosing infection and evaluating the efficacy of ITZ treatment.

Adolescent↗

PubMed2004

Monitoring gp43 antigenemia in Paracoccidioidomycosis patients during therapy.

Paracoccidioidomycosis (PCM) is a systemic fungal disease that is particularly important among individuals living and working in rural areas of endemicity in Latin America. Detection of anti-Paracoccidioides brasiliensis antibodies is of limited value due to false-negative results. Detection of P. brasiliensis-gp43 circulating antigen is a practical approach for a specific diagnosis of the disease. In a previous study we described an inhibition enzyme-linked immunosorbent assay able to detect the 43-kDa P. brasiliensis antigen in sera of 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM. To investigate its potential application for the follow-up of PCM patients during treatment, antigen levels were monitored at regular intervals for up 8 to 12 months in serum samples from 23 patients. The results showed that treatment with itraconazole resulted in decreasing levels of circulating gp43 that were correlated with the reduction of anti-gp43 antibodies. It was also observed that by the end of 12 months of treatment gp43 levels were <5 microg/ml in all patients.

Adult↗

PubMed2003

Modulation of proliferation, differentiation and cytokine secretion of murine B-1b cells by proteins of the extracellular matrix.

At least three B cell subsets, B-1a (Ly-1B), B-1b and B-2, are present in the mouse periphery. B-1a and B1-b cells represent a small population in the adult spleen and are abundant in the peritonial and pleural cavities. It has been demonstrated in our laboratory that B-1b cells spontaneously proliferated in stationary cultures of adherent peritonial cells. Further, that these cells migrate to a non-specific inflammatory focus. Based on these findings, it was investigated whether components of the extracellular matrix (ECM) might selectively influence the adherence, proliferation and cytokine production of these cells in vitro. Results showed that collagen induced a higher level of B-1b cells differentiation into adherent phagocytic cells. It was observed that only fibronectin induced higher level of proliferation than other matrix components. The analysis of cytokine production has shown that the presence of laminin stimulated B-1b cells led to high levels of IL-10 production and fibronectin and collagen induced the production of high levels of TNF-alpha. The combination of fibronectin, collagen and laminin induced higher levels of IL-1beta. These results demonstrate that differentiation, proliferation and cytokine production by B-1b cells are markedly influenced by ECM components.

Animals↗

PubMed2003

Morphological characterization of mouse B-1 cells.

At least three B cell subsets, B-1a, B-1b and B-2 are present circulating peripherally in the mouse. In these animals, B-1 cells constitute a minor fraction of B cells in spleen and are absent in lymph nodes although they represent the main B cell population in peritoneal and pleural cavities. Currently these cells are identified by a surface phenotypic repertoire; they express Mac-1, IgM(high), and B220(low). B-1a cells express CDS. The aim of this work emerged from the fact that the morphology of B-1 cells is not fully characterized. Here we identified B-1 cells using colloidal gold immunocytochemical assays and purified B-1 cells from supernatants of adherent peritoneal cell cultures by a magnetic bead technique. These techniques lead us to demonstrate that, in mice, either B-1a or B-1b cells have a unique morphology distinct from that of B-2 cells.

Animals↗

PubMed2003

Immunity and hypersensitivity to gp43 antigen in susceptible and resistant mice infected with Paracoccidioides brasiliensis.

Pathogenic mechanisms underlying paracoccidioidomycosis are still poorly understood. A well-established murine model of resistance (mouse lineage A/Sn) and susceptibility (lineage B10.A) to P. brasiliensis pulmonary infection was here employed to compare immune response to gp43, the major antigenic component of the fungus. Mice were infected and their cellular and humoral immunity to gp43 were investigated for up to 16 weeks. In both mouse strains, challenge with gp43 indistinguishably evoked a typical immediate-hypersensitivity response, followed by a 24-h late-phase reaction consistent with the same type of immunological activation. IL-4 was detected in cultures of gp43-stimulated lymph node cells only in susceptible animals 2 weeks post-infection, while IL-5 was found throughout the study in both mouse strains. IL-10 appeared in the supernatants of stimulated cells from resistant and susceptible animals in increasing amounts as infection advanced. Conversely, interferon (IFN)-gamma was produced under gp43 stimulation only by cells from A/Sn animals. The humoral response was characterized by low levels of anti-gp43. Titration of IgG isotypes, however, revealed a predominance of IgG1. IgG2a levels were highest in resistant animals, whereas IgG2b levels were highest in susceptible mice. In conclusion, immunity induced by gp43 exhibits common features in A/Sn and B10.A phenotypes, such as immediate hypersensitivity, late phase reaction and high levels of IL-10, but some differences between the strains are also seen.

Animals↗

PubMed2003

Detection of circulating gp43 antigen in serum, cerebrospinal fluid, and bronchoalveolar lavage fluid of patients with paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is an important systemic fungal disease, particularly among individuals living and working in rural areas of endemicity in Latin America, who, without antifungal therapy, may develop fatal acute or chronic infection. For such patients, the detection of antibody responses by immunodiffusion is of limited value due to false-negative results. In contrast, the detection of Paracoccidioides brasiliensis gp43 circulating antigen may represent a more practical approach to the rapid diagnosis of the disease. Accordingly, an inhibition enzyme-linked immunosorbent assay (inh-ELISA) was developed for the detection of a 43-kDa P. brasiliensis-specific epitope incorporating a species-specific murine monoclonal antibody. With sera from patients with acute and chronic forms of the disease (n = 81), the overall sensitivity of the test was found to be 95.1%, while specificity was found to be 97.5% compared to that with normal human sera from blood donors (n = 93) and sera from patients with other chronic fungal infections (histoplasmosis [n = 33] and cryptococcosis [n = 20]). The inh-ELISA detected circulating antigen in 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM according to the patient's clinical presentation. Cerebrospinal fluid from 14 patients with neuroparacoccidioidomycosis and 13 samples of bronchoalveolar lavage fluid from patients with pulmonary unifocal PCM were also tested for gp43 detection, with the test showing 100% sensitivity and specificity. This novel, highly specific inh-ELISA represents a significant addition to the existing tests for the diagnosis of PCM.

Antibodies, Monoclonal↗

PubMed2002

Characterization of B-1b cells as antigen presenting cells in the immune response to gp43 from Paracoccidioides brasiliensis in vitro.

Antigen presentation is an essential stage in the development of immune response to a specific antigen. This response can lead to the production of antibodies and/or effector T lymphocyte activation. Macrophages, dendritic cells and B-lymphocytes, among others, act as antigen presenting cells. B-lymphocytes capture antigenic particles through a surface receptor of IgM nature. The interaction IgM-antigen leads to endocytosis of the complex and antigen processing which culminates in presentation of the antigen on the cell surface associated with a class II MHC molecule. At least three B cell subsets, B-1a (Ly-1B), B-1b and B-2, are present in the mouse periphery. B-1a and B1-b cells represent a small population in the adult spleen and are abundant in the peritonial and pleural cavities. It has been demonstrated in our laboratory that B-1b cells spontaneously proliferated in stationary cultures of adherent peritonial cells. Further, that these cells migrate to a non-specific inflammatory focus. Based on these findings, we investigated whether these cells are antigen presenting cells in vitro using as antigenic stimulus gp43 from Paracoccidioides brasiliensis. Results showed that B1-b cells express constitutively high levels of class II MHC and costimulatory molecules inducing an efficient proliferation of gp43 sensitized T lymphocytes.

Animals↗

PubMedDate not supplied

GP43 from Paracoccidioides brasiliensis inhibits macrophage functions. An evasion mechanism of the fungus.

Macrophages constitute one of the primary cellular mechanisms that impairs parasite invasion of host tissues. The phagocytic and microbicidal properties of these cells can be modulated by specific membrane receptors involved in cell-microorganism interactions. Gp43, the main antigen secreted by Paracoccidiodes brasiliensis (Pb), the causative agent of Paracoccidioidomycosis, is a high mannose glycoprotein. The role played by gp43 in the pathogenesis of the disease is not completely known. Here, we describe the influence of this molecule on the interaction between peritoneal murine macrophages and Pb. Phagocytosis of Pb, live or heat-killed, by adherent peritoneal cells from both, B10.A (susceptible) and A/Sn (resistant) mice, was evaluated. Addition of different concentrations of gp43 to the culture medium inhibited, in a dose-dependent pattern, phagocytosis of live or heat-killed Pb by peritoneal macrophages from both B10.A and A/Sn mice. Gp43 also inhibits phagocytosis of zymosan particles but did not interfere with the uptake of opsonized sheep red blood cells. It was also shown that both gp43 and heat-killed Pb have an inhibitory effect on the release of NO by zymosan stimulated macrophages. Finally, we demonstrated that gp43 inhibits the fungicidal ability of macrophages from both lineages. Based on these data, it is suggested that gp43 can be considered one of the evasion mechanisms for the installation of primary infection in susceptible hosts.

Animals↗