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Biomedical subjects

J D Lopes

Publications and source records attributed to J D Lopes.

At least 19 recordsLinked to original sources
PubMed2001

Immunostimulatory DNA from Paracoccidioides brasiliensis acts as T-helper 1 promoter in susceptible mice.

Th1 immune responses afford protection against some pathogens like the fungus P. brasiliensis (P.b.), etiological agent of Paracoccidioidomycosis (PCM). It is well known that nonmethylated CpG sequences from bacterial DNA have immunomodulatory properties and can be used as a Th1-promoting adjuvant. By analyzing the available gene sequences of P.b. we observed a high number of unmethylated CpG dinucleotides. In a murine model of the PCM infection, the isogenic mouse strain known to be susceptible presents a predominant Th2 pattern. In order to access the possibility of the genomic DNA to act as a Th1-promoting adjuvant, in vitro assays were made and indicated a significant increase in phagocytosis when the macrophages were stimulated with DNA from P.b. and in vivo assays of a decreased production of antibodies antigp43, the main antigen of the PCM system. The analysis of the antibody isotypes and the cytokine production suggested a Th1 modulation in the susceptible animals. Thus, when mice were infected with fungus plus synthetic oligodeoxynucleotide (ODN), made from the available sequence of gp43, a decrease in the fungus dissemination was observed. Results herein described suggest that genomic DNA from P.b. could have a immunostimulatory function as a Th-1-promoting adjuvant in susceptible mice.

Adjuvants, Immunologic↗
PubMed2001

Levels of specific antigen (gp43), specific antibodies, and antigen-antibody complexes in saliva and serum of paracoccidioidomycosis patients.

The present study analyses human immunoglobulin G (IgG) antibodies directed against the Paracoccidioides brasiliensis exoantigen, gp43, as well as the presence of gp43-IgG immune complexes (ICs) in 31 samples of saliva and serum from 19 patients with paracoccidioidomycosis (PCM) and 12 normal donors. Additional analysis of secretory IgA (sIgA) was performed on the same saliva samples. Consistent with previous findings, a significant increased specific IgG level was observed in PCM patients' saliva and serum (P < 0.05). The analysis of serum gp43 and gp43-IgG IC demonstrated a higher level in patients with PCM (P < 0.05); however, this difference was not statistically significant with regard to gp43 and gp43-IgG in saliva when compared to the healthy donors. A high level of sIgA in saliva of PCM patients compared to that of normal donors was also observed (P < 0.05). Patients exhibiting low levels of serum IgG but with high titres of IC were observed, thus strengthening the idea of the necessity to use more than one marker for diagnosis and treatment monitoring of PCM. This is the first report of sIgA in PCM patients' saliva and may be indicative of a protective role in neutralizing antigens on mucosal surfaces.

Adult↗
PubMed2001

Mouse B-1 cell-derived mononuclear phagocyte, a novel cellular component of acute non-specific inflammatory exudate.

At least three B cell subsets, B-1a, B-1b and B-2, or conventional B cells are present in the mouse periphery. Here we demonstrate that B-1 cells spontaneously proliferate in stationary cultures of normal adherent mouse peritoneal cells. B-1 cells were characterized by morphology, immunohistochemistry and flow cytometry. IgM was detected in the supernatants of these cultures. We demonstrated that the major cell population analyzed expresses the B-1b phenotype. When these cells were transferred to a new culture, a large proportion of them adhere to the plastic surface, and spread as bipolar cells endowed with the capacity to phagocytose via Fc and mannose receptors. Flow cytometry analysis of these adherent cells demonstrated that the great majority of them share both B-220 and Mac-1 antigens. Nevertheless, 45% of them were exclusively Mac-1(+). Finally, when they were labeled in vitro with [(3)H]thymidine and transferred to the peritoneal cavity of naive mice, they migrate to a non-specific inflammatory focus induced by a foreign-body implant. These data demonstrate that B-1 cells, mainly B-1b cells, not only proliferate and differentiate into a mononuclear phagocyte in vitro, but also that they exit the peritoneal cavity and migrate to a non-specific inflammatory milieu.

Animals↗
PubMed2001

The low efficiency of dendritic cells and macrophages from mice susceptible to Paracoccidioides brasiliensis in inducing a Th1 response.

In the present study we evaluated T cell proliferation and Th lymphokine patterns in response to gp43 from Paracoccidioides brasiliensis presented by isolated dendritic cells from susceptible and resistant mice. T cell proliferation assays showed that dendritic cells from susceptible mice were less efficient than those from resistant mice. The pattern of T cell lymphokines stimulated by dendritic cells was always Th1, although the levels of IL-2 and IFN-gamma were lower in T cell cultures from susceptible mice. To determine whether different antigen-presenting cells such as macrophages and dendritic cells stimulated different concentrations of Th1 lymphokines, the production of IFN-gamma and IL-2 was measured. It was observed that dendritic cells were more efficient than macrophages in stimulating lymphoproliferation in resistant mice. However, no significant difference was observed for IFN-gamma or IL-2 production. When cells from susceptible mice were used, macrophages were more efficient in stimulating lymphoproliferation than dendritic cells, but no difference was observed in the production of Th1 cytokine. Taken together, these results suggest the lower efficiency of dendritic cells and macrophages from B10.A mice in stimulating T cells that secrete Th1 lymphokines in vitro, an effect that may be involved in the progression of the disease in vivo.

Animals↗
PubMed2001

Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line.

The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, São Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis), as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA) and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified.

Adenocarcinoma↗
PubMed2000

Anti-idiotypic antibodies in patients with different clinical forms of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Patients with PCM show a wide spectrum of clinical and pathological manifestations depending on both host and pathogen factors. Two clinical forms of the disease are recognized: the acute or juvenile form and the chronic or adult form. The major antigenic component of the parasite is a glycoprotein of 43 kDa (gp43). All patient sera present antibodies against gp43 (anti-gp43) and, as demonstrated before by our group, spontaneous anti-idiotypic (anti-Id) antibodies (Ab2) can be detected in patient sera with high titers of anti-gp43. Since it has been postulated that anti-Id antibodies may have a modulating function, we decided to purify and characterize anti-Id antibodies in this system. The possible correlation of Ab2 titers with different clinical forms of disease was also verified. Results showed that purified human anti-Id antibodies (human Ab2) recognized specifically the idiotype of some murine monoclonal anti-gp43 (17c and 3e) but not others (40.d7, 27a, and 8a). Spontaneous anti-Id antibodies were found in all clinical forms of disease. The majority of patients (88%, n = 8) with the acute form of PCM had high titers of Ab2. However, among patients with the multifocal chronic form of the disease, only 29% (n = 14) had high titers of Ab2; 70% (n = 10) of patients with the unifocal chronic form had low titers of Ab2. A correlation between Ab2 titers and anti-gp43 titers was observed before and during antimycotic treatment. Our results suggest that titers of anti-Id antibodies correlate with the severity of PCM in humans.

Adult↗
PubMed1999

Subcutaneous infection of mice with Paracoccidiodes brasiliensis induces a peculiar pattern of inflammatory and immune responses.

It is well established that resistance or susceptibility to Paracoccidioidis brasiliensis infection in mice is under strict host's genetic control. Mice from A/Sn strain inoculated by the ip route are resistant to fungal infection while infection induced in mice from B10.A strain results in a fatal disease. The early cellular events of infection in both strains are characterized by a marked neutrophilic infiltration that is more prominent in B10.A mice. A peculiar characteristic of the Paracoccidioides brasiliensis-mouse model is that the subcutaneous (sc) inoculations of the fungus either in resistant (A/Sn) or susceptible (B10.A) mice is self-curing and tums mice from the B10.A strain able to express typical DTH reaction to fungal antigens, as observed in A/Sn mice. Here we report the investigation on the early events of the inflammatory response induced by the inoculation of live fungus into the hind footpad of A/Sn (resistant) and B10.A (susceptible) mice. The influence of neutrophils on the inflammatory response and antibody titers or DTH response to gp43, the major fungal antigen, was also evaluated. Results showed a different course of the inflammatory response induced by fungal inoculation in A/Sn and B10.A mice. Neutrophil depletion before infection differently influenced the kinetics of the inflammatory process in both mice strains but did not modify the fungal load in the lesions. In neutrophil depleted mice from both strains, a decrease in DTH response and an increase in total antibody titers to gp43 were observed. The significant increase in the fungal load in lesions seen in nude mice indicates that the self-limited infection evoked by fungal inoculation into the subcutaneous tissue is a T-cell dependent phenomenon. The implications of these observations in the pathogenesis of paracoccidioidomycosis are discussed.

Animals↗
PubMed1998

Pattern of immune response to GP43 from Paracoccidioides brasiliensis in susceptible and resistant mice is influenced by antigen-presenting cells.

Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis. The infection can evolve to different clinical forms that are associated with various degrees of suppressed cell-mediated immunity. In the murine model, A/Sn and B10.A isogenic strains of mice are known to be resistant and susceptible, respectively, to this fungal infection. Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated the importance of two antigen-presenting cells (APCs), macrophages and B cells, in the development of the immune response to P. brasiliensis. In resistant mice, purified gp43, the main antigenic component of P. brasiliensis, seems to have been preferentially presented by macrophages and stimulated Th1 lymphokine production. On the other hand, in susceptible animals gp43 was distinguishably presented by B cells, which led to stronger activation of Th2 subsets. Moreover, T cells from resistant mice responded as those from susceptible animals when stimulated by gp43 presented by APCs from susceptible mice and vice versa, indicating that there are no significant differences in the T cell repertoires from A/Sn and B10.A mice. When T cells from F1 (A/Sn x B10.A) mice were stimulated by gp43 presented by APCs from A/Sn or B10.A, impaired behavior of B10.A macrophages in activating Th1 cells and a B10.A B cell tendency to stimulate T cells that secrete higher levels of IL-10 were observed. Taken together, our results suggest that APCs may be implicated in the outcome of P. brasiliensis infection.

Animals↗
PubMed1998

Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43,000 D (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with PCM. Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus. As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH). Results show not only the expected presence of anti-Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization. Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c. AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a. It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies. These data suggest that the immune response to P. brasiliensis can be spontaneously modulated by the idiotypic network.

Animals↗
PubMed1996

Monoclonal antibodies against the 43,000 Da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis.

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin. Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction. Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells. Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity. Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Animals↗
PubMed1994

Anti-idiotypic monoclonal antibody AB3, reacting with the primary antigen (CEA), can localize in human colon-carcinoma xenografts as efficiently as AB1.

BALB/c mice were immunized with anti-idiotypic monoclonal (MAb) antibody (anti-Id or Ab2) directed against an AB1 MAb anti-carcinoembryonic (CEA) in order to obtain AB3 MAbs (anti-anti-Id). AB3 MAbs were shown to recognise the primary antigen (CEA) and one of them was tested extensively in vitro and in vivo. This AB3 MAb was shown to bind specifically to CEA on frozen sections of a human colon carcinoma by immunoperoxidase. Scatchard plot analyses showed that the affinity of this AB3 was of the same order of magnitude as the AB1. In vivo experiments, in nude mice bearing CEA-producing human colon-carcinoma xenografts showed that up to 30% of the intravenously injected dose of 125I-labelled AB3 were localized per gram of tumour tissue. Furthermore, calculation of the ratios of AB3 concentration in the tumour over those in normal organs such as lung, liver, kidney, spleen and bone gave relatively high values similar to results obtained with AB1. All together our results show that AB3 can localize as efficiently and specifically in the tumour as AB1, despite the fact that the mice from which it was derived were immunized with a mouse MAb (AB2) and had never been exposed to CEA.

Animals↗
PubMed1994

Binding of Paracoccidioides brasiliensis to laminin through surface glycoprotein gp43 leads to enhancement of fungal pathogenesis.

Extracellular matrix protein laminin binds specifically to yeast forms of Paracoccidioides brasiliensis and enhances adhesion of the fungus to the surface of epithelial Madin-Darby canine kidney cells in vitro. Immunoblotting of fungal extracts showed that the gp43 glycoprotein is responsible for adhesion. This was confirmed by binding assays using purified gp43, with a Kd of 3.7 nM. The coating of P. brasiliensis yeast forms with laminin before injection into hamster testicles enhanced the fungus virulence, resulting in a faster and more severe granulomatous disease. These results indicate that interaction of fungi with extracellular matrix elements may constitute a basis for the evolution of fungal infection toward regional spreading and dissemination.

Adhesiveness↗
PubMed1994

Monoclonal antibody capture enzyme immunoassay for detection of Paracoccidioides brasiliensis antibodies in paracoccidioidomycosis.

Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM.

Animals↗
PubMed1994

Characterization of glycoprotein gp43, the major laminin-binding protein of Paracoccidioides brasiliensis.

We have demonstrated that laminin mediates the adhesion of P. brasiliensis to monolayers of epithelial cells through specific binding to the surface glycoprotein gp43. This binding seems to be related to the fungal pathogenesis. We now report the confirmation of these findings by scanning electron microscopy and show that some isolates that do not secrete gp43 do express the protein as seen by studying whole cell extracts. These results confirm the ability of these strains to produce paracoccidioidomycosis but should not be used for serological purposes since the absence of gp43 in exoantigens may lead to false negative results.

Animals↗
PubMed1993

Immunological and functional characterization of proteins of the Mycobacterium tuberculosis antigen 85 complex using synthetic peptides.

As tuberculosis re-emerges as an important health problem worldwide, new drugs, better diagnostic tests and vaccines are being sought. In order to identify potentially useful peptides for the development of a synthetic vaccine against tuberculosis, immunological and functional studies were performed using proteins of the antigen 85 complex. Western blot (immuno-blot) analysis and a lymphoproliferation study was used to investigate the B- and T-cell immune response of tuberculosis patients, healthy household contacts and normal controls to proteins of the Mycobacterium tuberculosis antigen 85 complex. Peptides derived from the 85A amino acid sequence were synthesized and used in fibronectin-binding and in ELISA assays. A peptide with the sequence CQPACRKAGCQTYKWEC bound to radiolabelled fibronectin in a time-dependent manner and was recognized by human sera in ELISA. This peptide was identified as a potential component of a synthetic vaccine against tuberculosis.

Amino Acid Sequence↗
PubMed1992

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.

Animals↗